Está en la página 1de 336

EXS87

Chitin and Chitinases
Edited by P. Jolles and R.A.A. Muzzarelli

Springer Basel AG

Editors
Prof. Dr. P. Jolles
Laboratoire de Chimie
des Substances Naturelles
URA C.N.R.S. No. 401
Museum National d'Histoire Naturelle
63, rue Buffon
F-75005 Paris
France

Prof. R.A.A. Muzzarelli
Center for Innovative Biomaterials
Faculty of Medicine, University
Via Ranieri 67
1-60100 Ancona
Italy

Library of Congress Cataloging-in-Publication Data
Cbitin and Chitanases / edited by P. Iolles and R. A. A. Muzzarelli.
p. cm. -- (EXS ; 87)
Includes bibliographical references and index.
ISBN 978-3-0348-9760-0
ISBN 978-3-0348-8757-1 (eBook)
DOI 10.1007/978-3-0348-8757-1
1. Chitin. 2. Chitinase. 3. Chitosan. 1. Iolles, Pierre, 1927-.
II. Muzzarelli, Riccardo A.A., 1937III. Series.
QP702.C5C47 1999
573.7'74--dc21
Deutsche Bibliothek Cataloging-in-Publication Data
Cbitin and chitinases / ed. by P. Jolles and R. A. A. Muzzarelli. Basel ; Boston; Berlin: Birkhăuser, 1999
(EXS; 87)
87. Chitin and chitinases. - 1999
EXS. - Basel ; Boston; Berlin: Birkhăuser
Friiher Schriftenreihe
Fortlaufende BeiI. zu: Experientia
The publisher and editor can give no guarantee for the inforrnation on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other
sources of reference in each individual case.
The use of registered names, trademarks etc. in this publication, even if not identified as such, does
not imply that they are exempt from the relevant protective laws and regulations or free for general
use.
This work is subject to copyright. All rights are reserved, whether the whole or part of the material
is concemed, specifically the rights of translation, reprinting, re-use of illustrations, recitation,
broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind
ofuse, permission ofthe copyright owner must be obtained.
© 1999 Springer Basel AG
Originally published by Birkhauser Verlag in 1999
Softcover reprint of the hardcover Ist edition 1999
987654321

Contents
List of Contributors .

VII

Preface . . . . . . . .

XIII

Riccardo A. A. Muzzarelli
Native, industrial and fossil chitins.

1

Chitin synthesis
Regula A. Merz, Markus Horsch, Lars E. Nyhten and Dora M Rast
Biochemistry of chitin synthase . . . . . . . . . . . . . . . . . . ..

9

Jose Ruiz-Herrera andAlfredo D. Martinez-Espinoza
Chitin biosynthesis and structural organization in vivo .

39

M Henar Valdivieso, Angel Duran and Cesar Roncero
Chitin synthases in yeast and fungi . . . . . . . . . . .

55

Jeroen Bakkers, Jan W. Kijne and Herman P. Spaink
Function of chitin oligosaccharides in plant and animal development

71

Subba Reddy Palli and Arthur Retnakaran
Molecular and biochemical aspects of chitin synthesis inhibition

85

Hildgund Schrempj
Characteristics of chitin-binding proteins from streptomycetes. .

99

Chitinases
Daizo Koga, Masaru Mitsutomi, Michiko Kono and
Masahiro Matsumiya
Biochemistry of chitinases . . . . . . . . . . . . .

111

Jon D. Robertus and Arthur E Monzingo
The structure and action of chitinases .

125

Bernard Henrissat
Classification of chitinase modules. .

137

Graham W. Gooday
Aggressive and defensive roles for chitinases .

157

Alfredo Herrera-Estrella and Ilan Chet
Chitinases in biological control . . . . . . . .

171

VI

Contents

Lee A. Hadwiger
Host-parasite interactions: elicitation of defense responses
in plants with chitosan . . . . . . . . . . . . . . . . . .

185

Klaus-Dieter Spindler and Margarethe Spindler-Barth
Inhibitors of chitinases . . . . . . . . . . . . . . . . .

201

Gilles Bleau, FrMeric Massicotte, Yannick Merlen and
Chantale Boisvert
Mammalian chitinase-like proteins . . . . . . . . .. . . . . . . . 211
Mohammed Shahabuddin and Joseph M Vinetz
Chitinases ofhuman parasites and their implications
as antiparasitic targets. . . . . . . . . . . . . . . . . . . . . . . . . 223
Riccardo A.A. Muzzarelli
Analytical biochemistry and dinical significance of
N-acetyl-ß-D-glucosaminidase and related enzymes. . . . . . . . . 235

Exogenous chitosans
Riccardo A. A. Muzzarelli, Monica Mattioli-Belmonte,
Armanda Pugnaloni and Graziella Biagini
Biochemistry, histology and dinieal uses of chitins and chitosans
in wound healing . . . . . . . . . . . . . . . . . . . . . .
Saburo Minami, Yoshiharu Okamoto, Koji Hamada, Yukio
Fukumoto and Yoshihiro Shigemasa
Veterinary practice with chitin and chitosan . . . . . .
Seiichi Tokura, Hiroshi Tamura and Ichiro Azuma
Immunologieal aspects of chitin and chitin derivatives
administered to animals. . . . . . . . . . . . . ..

. 251

. . . . . 265

. . . . . . . 279

Riccardo A. A. Muzzarelli
Clinical and biochemieal evaluation of chitosan for
hypercholesterolemia and overweight control. . . .

. . . 293

Ida Genta, Paola Perugini, Franca Pavanetto, Tiziana Modena,
Bice Conti and Riccardo A. A. Muzzarelli
Microparticulate drug delivery systems. . . . . . . . . . . . .

305

Raul G. Cuero
Antimicrobial action of exogenous chitosan

315

Subject index . . . . . . . . . . . . . . . .

335

1-60100 Ancona.O. e-mail: Olimpa@aal. TX 77446. Campo Charro s/n. Italy . The Netherlands. Hiroshima 731-3355. Italy Raul G. Texas A & M University Systems. e-mail: boisvertc@ere. Hokkaido University. e-mail: bleaug@ere. Box 4079.ca Ilan Chet. Department ofPharmaceutical Chemistry. Instituto de Microbiologia Bioquimica. boul. Leiden University. NL-2333 AL Leiden. Institute for the Immunological Sciences. CARC. Höpital Saint-Luc. Prairie View A & M University. Montreal. Room 219. Japan Jeroen Bakkers. P. Centre Hospitalier de l'Universite de Montreal. Japan Ida Genta. 264. Center for Innovative Biomaterials. Faculty ofMedicine. Asa Zoological Park. Sapporo 060. Asa-Kita.umontreal. Departement d'Obstetrique-Gynecologie.nl Graziella Biagini.leidenuniv. Wassenaarseweg 64. Centre Hospitalier de l'Universite de Montreal. Canada H2X lPl. Israel Bice Conti. Avda. boul. USA. Quebec. Rene-Levesque est. Italy Gilles Bleau. University ofPavia. Spain Yukio Fukumoto.ca Chantale Boisvert. Rehovot 76100. Consejo Superior de Investigaciones Cientificas/ Universidad de Salamanca. E-37007 Salamanca. Montreal. Quebec. Prairie View. Via Ranieri 67. Box 12. Rene-Levesque est.umontreal. University of Pavia. Faculty of Agriculture. Cuero. Canada H2X IP1. Institute of Molecular Plant Sciences. Otto Warburg Center for Agricultural Biotechnology. 264. O.1-27100 Pavia. e-mail: BAKKERS@rulbim. The Hebrew University of Jerusalem. Department of Pharmaceutical Chemistry.1-27100 Pavia.com Angel Duran. Departement d'Obstetrique-Gynecologie.List of Contributors Ichiro Azuma. Edificio Departamental. Höpital Saint-Luc. P. Viale Taramelli 12. Viale Taramelli 12. University.

Martinez-Espinoza. Mexico.edu Koji Hamada.cnrs-mrs.cinvestav.cinvestav. Fisheries Research Laboratory.ira. Department of Molecular and Cell Biology.u-tokyo. Foresterhill. Shizuoka 431-0211. Faculty of Agriculture. e-mail: bemie@afmb. Quebec. Centre Hospitalier de l'Universite de Montreal. Centro de Investigaci6n y Estudios Avanzados. Japan. Departamento de Ingenieria Genetica.jp Alfredo D. Zollikerstrasse 107. Rene-Levesque est. USA. Department ofPlant Biology. Japan. Irapuato 36500. Omu. e-mail: massicottef@ere. UK. Laboratory of Biochemistry.VIII List of Contributors Graham W Gooday. Yamaguchi 753-8515.mx Markus Horsch. Höpital Saint-Luc. F-13402 Marseille Cedex 20. Maisaka. University of Zurich. A. 264. Switzerland Jan W Kijne. Department of Biological Science. Centro de Investigaci6n y de Estudios Avanzados deI Instituto Politecnico Nacional.nl Daizo Koga.ac. Yamaguchi University. CH-8008 Zürich. Institute of Molecular Plant Sciences. France. Washington State University.ac.mx Frederic Massicotte.ira. Leiden University. e-mail: KlJNE@rulbim. University of Aberdeen.gooday@abdn. Canada H2X IP1. e-mail: amkono@honga. Pullman. Japan Bemard Henrissat.w. Ohmu Agricultural Mutual Aid Association. Institute of Medical Sciences. 31 Chemin Joseph Aiguier. 36500 Irapuato. Gto.leidenuniv. Mexico.uk Lee A. NL-2333 AL Leiden. ofPlant Pathology. Apartado Posta1629.wsu.yamaguchi-u.ac.fr Alfredo Herrera-Estrella. Architecture et Fonction des Macromolecules Biologiques CNRS. Departement d'Obstetrique-Gynecologie. Unidad Irapuato. WA 99164-6430. Wassenaarseweg 64.jp Michiko Kono.ca . e-mail: amartinez@irapuato. Aberdeen AB25 2ZD. The Netherlands. Gto.umontreal. Monbetsu 098-1702.ecc. e-mail: chitosan@mail. Hadwiger.p. Montreal. Unidad Irapuato. Dept. e-mail: g. 629. The University ofTokyo. boul. Faculty of Agriculture. e-mail: aherrera@irapuato. e-mail: koga@agr.

Kanagawa 252-8510. Canada H2X IP1. Department of Chemistry and Biochemistry.agr. Switzerland. Rene-Uvesque est.agr. University of Zurich. Spring House. University. 1-60100 Ancona. e-mail: remerz@botinst. e-mail: amonzingo@mail. Nyhlen. Faculty of Agriculture. Viale Taramelli 12. Laboratory ofMarine Products Uzilization.jp Subba Reddy Palli. Nihon University. boul.List of Contributors IX Masahiro Matsumiya. Via Ranieri 67. Merz. Tottori 680-8553. Montreal.O.ac. Faculty of Agriculture. Department ofVeterinary Surgery. e-mail: muzzarelli@popcsi. Italy Arthur F. A. Japan. e-mail: okamoto@pear. TX 78712. University of Zurich. College ofBioresource Sciences. email: matsumiya@brs. 4-101 Koyama-Minami. Laboratory of Food Chemistry. Muzzarelli.ca Regula A. 264. Department of Applied Biological Sciences. Japan. Quebec. Italy Yannick Merlen.tottori-u. Saga University. Department ofVeterinary Surgery. Department of Plant Biology.unizh. University ofPavia. PA 19477. Via Ranieri 67. Department ofMarine Science and Resources.utexas. Japan. Saga 840-8502.jp Tiziana Modena.jp Masaru Mitsutomi. Box 904. CH-8008 Zürich. Höpital Saint-Luc. Center for Innovative Biomaterials. Tottori 680-8553. CH-8008 Zürich. USA . University. Austin.ac. Tottori University. Center for Innovative Biomaterials. Zollikerstrasse 107. Rohm and Haas Research Laboratories. Centre Hospitalier de l'Universite de Montreal.jp Monica Mattioli-Belmonte. 1-60100 Ancona.it Lars E.unian. Departement d'Obstetrique-Gynecologie. Italy. e-mail: mitsutom@cc. Department of Pharmaceutical Chemistry. Japan.ac. 4-101 Koyama-Minami. 727 Norristown road. e-mail: merleny@ere. Monzingo. Zollikerstrasse 107. University of Texas. Department of Plant Biology.edu Riccardo A. e-mail: minami@pear. Faculty of Agriculture. Tottori University. USA. Faculty ofMedicine. Faculty ofMedicine. P. Fujisawa.nihon-u.saga-u.ac.umontreal.tottori-u. Switzerland Yoshiharu Okamoto. Institute of Cellular and Molecular Biology.1-27100 Pavia.ch Saburo Minami.

Ontario P6A 5M7. Robertus. e-mail: mshahabudd@nih.de Mohammed Shahabuddin. Italy Dora M. Laboratory ofParasitic Diseases. Marie. Center for Innovative Biomaterials. USA. National Institutes of Health. Instituto de Microbiologia Bioquimica. Department of Pharmaceutical Chemistry. Medical Entomology Section. Unidad Irapuato.gov Yoshihiro Shigemasa.ira.gc. FB Biologie/Chemie. Institute of Cellular and Molecular Biology.edu Cesar Roncero. lrapuato 36500.1-27100 Pavia. Avda.utexas. Department of Materials Science. University ofPavia. Bethesda. Department of Chemistry and Biochemistry. Great Lakes Forestry Centre.ch Arthur Retnakaran. TX 78712. University of Texas. Japan .unizh. Departamento de Ingenieria Genetica. University ofPavia. 4 Center Drive MSC 0425. Campo Charro s/n. Austin. e-mail: jruiz@irapuato. Centro de Investigaci6n y de Estudios Avanzados deI Instituto Politecnico Nacional. Canada. E-37007 Salamanca.cinvestav. 4-101 Koyama-Minami. Mexico. Sault Ste. Box 490. Tortori University. University. Natural Resources Canada. 1219 Queen Street East. e-mail: crm@gugu.uni-osnabrueck.1-27100 Pavia.es Jose Ruiz-Herrera. Department of Pharmaceutical Chemistry.O. Viale Taramelli 12. Pavanerto.usal. Rast.mx Hildgund Schrempf. Tottori 680-8552. Consejo Superior de Investigaciones Cientificas/ Universidad de Salamanca. Barbarastrasse 11. Germany. e-mail: aretnak@nrcan. Italy Armanda Pugnaloni. National Institute of Allergy and Infectious Diseases. Apartado Postal 629. 1-60100 Ancona. Zollikerstrasse 107. Italy Paola Perugini. e-mail: schrempf@mail. Edificio Departamental.x List of Contributors F.ca Jon D. e-mail: jrobertus@mail. e-mail: rast@botinst. MD 20892-0425. CH-8008 Zürich. University of Zurich. Room 219. Canadian Forest Service. Via Ranieri 67. Spain. Faculty ofEngineering. Universität Osnabrück. USA. Switzerland. D-49069 Osnabrück. Department of Plant Biology. P. Viale Taramelli 12. Faculty ofMedicine. Gto.biologie.

Suita. Vinetz. Faculty of Engineering. Avda. Heinrich-Heine-Universität Düsseldorf. e-mail: spindler@rz. The Netherlands. e-mail: klaus-dieter. Galveston. Suita. Spaink. WHO Collaborating Center for Tropical Diseases. Leiden University. Henar Valdivieso. Keiller 2. Japan M. E-37007 Salamanca. e-mail: SPAINK@rulbim. 301 University Blvd. Universitätsstr. Germany. 1. Room 219.de Hiroshi Tamura. Consejo Superior de Investigaciones Cientificasl Universidad de Salamanca. Abteilung Allgemeine Zoologie.spindler@biologie.und Entwicklungsphysiologie. Wassenaarseweg 64..de Margarethe Spindler-Barth. NL-2333 AL Leiden.uni-ulm. Edificio Departamental. Japan Seiichi Tokura. TX 77555-0609.uni-duesse1dorf. Albert-Einstein-Allee 11-13. Faculty of Engineering.1eidenuniv. Universität Ulm. Instituto de Microbiologia Bioquimica.nl Klaus-Dieter Spindler. Kansai University and HRC. Spain Joseph M. Osaka 564-8680. Department of Pathology and Division of Infectious Diseases. Osaka 564-8680. USA . D-40225 Düsseldorf. Institute of Molecular Plant Sciences. Kansai University and HRC. Campo Charro s/n.138. Lehrstuhl fiir Hormon.List of Contributors XI Herman P. D-89069 Ulm. University ofTexas Medical Branch. Germany.

and the chitin synthases of yeasts and fungi. Some enzyme inhibitors are also mentioned. after a short presentation of chitin in the environment. Newly characterized mammalian chitinase-like proteins are presented. Some chitin-binding proteins are reviewed. also produce chitinases for their defence. are also approached. Aspects concerning N-acetyl-ß-D-glucosaminidases. is the most abundant nitrogen-bearing organic compound found in nature. Exciting applications are discussed in aseries of chapters that emphasize the fact that chitosan applications based on its biological significance often depend on its biodegradability. The second part is devoted to chitinases. January 1999 Pierre Jolles and Riccardo A. chitin biosynthesis and structural organization in viva. but some organisms deprived of chitin. which split the ß-I . The agricultural. is devoted to chitin biosynthesis. Successive1y. microspheres and liposomes. are also discussed in relation with their growing medical importance. The third part ofthe book is devoted to chitosan. a family of deacetylated chitins. the insoluble polymer of N-acetylglucosamine. We have selected the most recent and sophisticated chitin-related advances in life sciences. The first part of this book. we discuss the biochemistry of chitin synthase and the state of knowledge of chitin synthesis in vitra. food. present in insect exoskeletons. Chitin-containing organisms produce chitinases. These aspects are reviewed in aseries of chapters.4-glucosidic bonds of chitin as. We are confident that this book will provide a stimulating background for further fruitful research on chitin in the biochemical and biological area. lysozymes also do. such as a wide variety ofbacteria and higher plants.Preface Chitin. in a less pronounced manner. films. crustacean shells and fungal cell walls. fibers. cosmetic and pharmaceutical industries more and more frequently use this polysaccharide in the form of threads. gels. Biochemical. and approached chitin from an original standpoint: the prompt and enthusiastic response of the colleagues invited to collaborate is gratefully acknowledged. enzymes releasing N-acetylglucosamine monomers from chitin. and biochemical aspects of inhibitors of chitin synthesis. A. The role of chitin oligosaccharides in plant morphogenesis. Muzzarelli . structural and evolutionary aspects conceming chitinases are discussed.

and degree of acetylation. is widely distributed among invertebrates. morphogenetic and defensive or aggressive purposes. and fossil chitin is not frequently encountered. for instance in the insect cutiele and tendon (a-chitin) and in the pen ofCephalopoda (ß-chitin). degree of covalent bonding to other wall components. The molecular order of chitin explains its physiologieal role and tissue characteristics. solubility and reactivity are different. Chitin is found in exoskeletons. cuttlefish bone. whereas in a-chitin they are antiparallel. Chitin is ubiquitous in fungi: the chitin in fungal walls varies in crystallinity. Countless living organisms continuously synthesize and degrade chitin enzymatically. by P. Today chitins and chitosans from different animals are eommercially available.A. Being convinced that this book will attract many readers not fully acquainted with chitin. The grasping spines of Sagitta are made ofpure a-chitin because they should be suitably hard to hold a prey.A. the eggshells of nematodes and rotifers. It is found as a-chitin in the calyces of hydrozoa. the radulae of mollusks and the cutieies of arthropods. I deern it appropriate to provide abrief introduction. Muzzarelli Center for Innovative Biomaterials. The polymorphie forms of chitin differ in packing and polarities of adjacent chains in successive sheets. typieally elose to 0. peritrophic membranes and the cocoons of insects. In the areas of fisheries. The reader is referred to a large body of information on chitin available in a number ofbooks: a selection is listed below [1-20].10 13 kg) of chitin are synthesized and degraded each year in the biosphere. with . scientists and industry people were urged to upgrade chitin in order to exploit renewable resourees and to alleviate waste problems. industrial and fossil chitins Riccardo A. (1-4)-linked 2-acetamido-2-deoxy-ß-D-glucan. Via Ranieri 67. Muzzarelli @ 1999 Birkhäuser Verlag BasellSwitzerland Native. for nutritional. textiles. At least 10 gigatons (1. in the ß-form all chains are aligned in parallel manner. Faculty ofMedicine. Jolles and R. and pogonophora tubes. Also. squid pen. their environmentally friendly behaviour permits industrial exploitation ofthe huge chitinous biomasses generated by fishing activities and biotechnology. Italy Summary. Chitin. Chitin isolates differ from eaeh other in many respeets. Chemically modified chitins are important in the light of their biochemical significance in medicine and crop protection. among whieh are degree of aeetylation. food and ecology. University. I-60100Ancona. elemental analysis.Chitin and Chitinases ed. Chitin is promptly metabolized in sediments. A.90. and as ß-chitin in the shells of brachiopods and mollusks. mainly glucans.

chitin contains nitrogen. As a point of difference from other abundant polysaccharides. dimethylacetamide containing 5-9% LiCl (DMAclLiCl) and N-methyl-2pyrrolidinonelLiCl are systems where chitin can be dissolved up to 5%. so that mesomorphic properties may be expected at a sufficiently high concentration ofpolymer. obtained by moisture uptake from DMAclLiCl solutions. supported by hydrogen bonds mainly through the acetamido group. reveals a cholesteric structure. The main chain of chitin is rigid at room temperature. Chitin hydrolysates can be prepared byadding chitin to concentrated HCl at 4°C and stirring at 40°C. Excess acid is then removed with ion-exchange resin. Colloidal chitin is being used since the 1950s for the study of chitinases. In the wet state it is degraded by a number of microorganisms. Muzzarelli nitrogen content typically c10se to 7%. In general. Ethanol-containing calcium chloride. The solubility of chitin is remarkably poorer than that of cellulose because of the high crystallinity of chitin. Chitin is easily hydrolyzed by acids. and the product is resuspended to prepare the so-called colloidal chitin. the major component. but is stable to dilute alkali. in warm concentrated alkali it is oxidized by air. The removal of the acetyl group is a harsh treatment usually performed with concentrated NaOH. is the structural unit of native chitin. which produce chitinolytic enzymes or other enzymes with unspecific activity towards chitin.A. but chitin isolates have lower values due to partial random depolymerization occurring during chemical treatment and depigmentation steps. and N/C ratio 0. Chitobiose. and polydispersity. Circular dichroism of Congo red bound to the chitin films.40. molecular size. Isolated chitin is a highly ordered copolymer of2-acetamido-2-deoxY-ßD-glucose. Chitosans Chitosan indicates a family of deacetylated chitins. and 2-amino-2-deoxy-ß-D-glucose. having an organization similar to that naturally occurring in the chitin cutic1e. Commercial chitosans may contain insoluble highly acetylated fractions that come from the core of the . Polydispersity may vary depending on such treatments as powder milling and blending of various chitin batches. chitosans have a nitrogen content higher than 7% and a degree of acetylation lower than 0. which remains stable for several weeks when stored at 4°C.A. O-(2-amino-2-deoxy-ß-D-glucopyranosyl)-(1-4)-2amino-2-deoxy-D-glucose. Bound water is also apart of the structure.2 R. Protection from oxygen. The average molecular weight of chitin in vivo is probably in the order of the MDa. is necessary in order to avoid undesirable reactions such as depolymerization and generation of reactive species.146 for fully acetylated chitin. with a nitrogen purge or by addition of sodium borohydride to the alkali solution.

two chains pass through the unit cell with an antiparallel packing arrangement. yield more soluble polymers. The presence of a prevailing number of2-amino-2-deoxyglucose units in a chitosan facilitates bringing the polymer into solution by salt formation. glyoxylate. among others. The chemical modifications of chitin and chitosan. On chitosan. are water-soluble: formate. Countless chitin deri- . together with N-carboxymethyl chitosan. and the degree of acetylation is around 0. the latter being preferred when glucans are to be dissolved. The latter compound has been and still is one of the protagonists of chitin chemistry. for instance for chitosan ascorbate. easily cultured on simple nutrients. chitin lends itself to many reactions. soluble in water. citrate. both have found applications in a variety of fields.3. Chitin and chitosan derivatives In the past. malate. The acetyl groups in the acid-soluble fractions are randomly distributed. whilst the insoluble fractions contain relatively long sequences of acetylated units. The following salts. Chitosan is present in the cell wall ofMucorales and can be isolated from the accompanying glucans by extraction with either acetic acid or alkali. acetate. the pK ofthe chitosan amine being 6. the reactions of the primary amino group and primary and secondary hydroxyl groups can be easily performed. pyruvate. The latter have higher biodegradability in animal bodies and physical properties of interest for applications in the solid state or in solution. Despite the alteration due to deacetylation. The crystal structures of salts and derivatives have also been determined. carried out under mild conditions in order to protect glycoside and acetamido linkages. known as glycol chitin: this material was probably the first derivative to find practical use and to be recommended as a substrate for lysozyme. industrial and fossil chitins 3 granules submitted to heterogeneous deacetylation. Chitosan can be reacetylated with acetic anhydride to obtain water-soluble partially reacetylated chitin. affording a wide choice of modified chitins. Chitosan can be obtained from fungi. Main hydrogen bonds are 03···05 (intramolecular) and N2··· 06 (intermolecular).10. Chitin treated with NaOH yields alkali chitin. The final molecular weight is in the order of 500 kDa. glycolate and ascorbate. The reaction of alkali chitin with sodium monochloroacetate gives O-carboxymethyl chitin sodium salt. chitosan from crab tendon possesses a crystal structure showing an orthorhombic unit cello The unit cell comprises four glucosamine units. As soon as the molecular association is prevented or depressed. lactate.Native. chitin has been often considered as an intractable biopolymer due to the difficulties encountered in dissolving and reacting it. Chitosan is a primary aliphatic amine that can be protonated by selected acids. a widely used unstable intermediate which reacts with 2-chloroethanol to yield 0-(2-hydroxyethyl) chitin.

indicating substitution of chitin by more resistant organic compounds [23-25].4 R. and desert sand. Because those chitinases seem to be particularly stable and effective under deep-sea conditions. but fossils of crustacea were found to contain only traces of chitin. A huge number of mineralized skeletal structures containing chitinprotein complexes settle into deep-sea sediments: there. millipeds occur as calcified remains. regioselective synthesis affords chitin sulfates endowed with anti-HIV-l activity [22]. but also provides delicate structures and . they are promptly degraded in other environments such as ocean sediments. such as in the case of insect eyes. it is possible that phosphate salts provide protection from degradation. but some of the most spectacular examples of soft part preservation involve replication in calcium phosphate: apatite minerals inhibit the decay of organic compounds. Fossil chitin Whilst chitinous materials are relatively resistant to degradation under certain conditions. coleoptera fossils were recovered from buried peat. chitin associations with calcium phosphate are being studied today for bone regeneration purposes. Interestingly. and insect wings from amber. Terrestrial arthropods have a fossil record that reaches back to 420 megayears ago (Upper Silurian): their remains are preserved as cuticle fragments. Chitin is not generally preserved as such. the present trend is to exert control over the modification reactions in order to achieve the best performance or to enhance the biological significance. chitin yields 6-oxychitin. and no chitinlike microfibrils were detected by electron microscopy. and therefore preserves the highest fidelity in detail.A. which use it as a nutrient. For example. Replication of soft tissues is more rapid in calcium phosphate than in any other mineral.A. Muzzarelli vatives have so far been prepared. Conclusion Chitin is amply distributed in the biosphere where it constitutes the exoskeletons of many organisms. Detection of chitin in fossils is not frequent: there are reports of fossil chitin in pogonophora. extraction of organic matter is performed by fungi. Arachnids were found preserved by precipitation of silica. sediments contain little chitin. The fossil cuticles revealed alkanes and alkenes. algae and bacteria. a fully water-soluble compound [21]. Extracellularly secreted enzymes hydrolyze the organic polymers of the skeletal remains colonized by the microorganisms. AIthough proteins have a short survival time even within CaC03 crystals. upon regiospecific oxidation. for instance suspension in seawater.

Cosani A.Native. Muzzarelli BB (1998) Structural and functional versatility of chitins. eRC Press. London Muzzarelli RAA. AlT. New York Neville AC (1975) Biology of the arthropod cuticle. Gooday GW (eds) (1986) Chitin in Nature and Technology. Chitin nitrogen is recyded by biodegradation operated by microbial genera which exist in just about every conceivable environment. Rome (contract no. Peter MG (eds) (1997) Chitin Handbook. Atec. chitosan and related enzymes. Tokyo Jeuniaux C (1963) Chitine et chitinolyse. Goods manufactured from chitin are environmentally important items. Stanic V. 2. Oxford Muzzarelli RAA (1996) Chitin Chemistry. In: JC Salamone (ed) The Polymerie Materials Encyclopedia. Technomic. Chandrkrachang S (1996) Chitin and Chitosan. Univ Minnesota Press. St Paul Stevens WF.00032. San Diego Zikakis JP (1984) Chitin. In: C Bucke (ed) Methods in Biotechnology. Academic Press. Rao MS. 98. London Muzzarelli RAA (1985) In: GO Aspinall (ed) The Polysaccharides. Massachusetts Institute ofTechnology Press. Grottammare Muzzarelli RAA. Ramos V (1998) Enzymatic depolymerization of chitins and chitosans. Cambridge. Pectin and Chitin. Chitin therefore represents a widely distributed organic compound of nitrogen. USA Japanese Society for Chitin and Chitosan (1995) Chitin and Chitosan Handbook. NewYork. Muzzarelli RAA. Academic press. Lancaster. Bangkok Wood WA. Voss-Foucart MF (1991) Chitin biomass and production in the marine environment. Val. Amsterdam Domard A. Boca Raton FL. chitosan solutions are used to recover proteins and to preserve cereal seeds. Jeuniaux C. MA Muzzarelli RAA. and tendons of crustaceans. Carbohyd Polym 39: 361-367 . Jacques Andre. Pergamon. Masson. novel hyaluronan-like regiospecifically carboxylated chitin. New York. Terbojevich M (1999) 6-0xychitin. Plenum. 3 Muzzarelli RAA. Atec. Haris PI (eds) (1998) New Biomedical Materials -Applied and Basic Studies lOS Press. The nitrogen present in chitin imparts unique properties to this dass of polysaccharides. Biochem Syst Eco119: 347-356 Muzzarelli RAA (1977) Chitin. 161: Lignin. Jeuniaux C. "Progetto Finalizzato Materiali Speciali Tecnologie Avanzate 11". Humana Press. industrial and fossil chitins 5 tissues such as the flying apparatus of insects.PF34). Pariser ER (eds) (1978) Proceedings ofthe First International Conference ofChitin/Chitosan. Marcel Dekker. European Chitin Society. In: S Dumitriu (ed) Structural Diversity and Functional Versatility ofPolysaccharides. p 312-314 Muzzarelli RAA (ed) (1996) Chitin Enzymology. 569-594 Muzzarelli RAA. Paris Jeuniaux C. Roberts G (eds) (1996) Advances in Chitin Sciences. Gihodo Shuppan Co. Lyon Coosen MFA (ed) (1996) Applications of Chitin. Academic Press. Kellogg ST (eds) (1988) Methods in Enzymology. vol. or to prepare biomaterials such as wound dressings and biodegradable packaging. Acknowledgements This chapter was prepared with the financial contribution of the Italian National Research Council. References 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Chapman D. Springer. Muzzarelli C. Berlin Richards AG (1951) The Integument ofArthropods. Vol. Grottammare Muzzarelli RAA.

Evershed RP (1998) Biodegradation of the chitin-protein complex in crustacean cuticle. Carbohydr Res 306: 427 -433 23 StankiewiczAB. Yoshida T. Kurita K. Uryu T (1998) Regioselective synthesis of sulfated polysaccharides: specific anti-HIV-l activity of novel chitin sulfates. Yamamoto N. Series 707. Muzzarelli 22 Nishimura SI. Nakashima H. Stankiewicz BA. Shinada K. Analyst 123: 139-145 . HofCHJ. Briggs DEG. BierstedtA. Tokura S. Mastalerz M.A. ACS Symp. Evershed RP (1998) Quantitative and qualitative analysis of chitin in fossil arthropods.A. Kai H. American Chemical Society. Briggs DEG.6 R. Org Geoehem 28: 67-76 24 Stankiewicz AB (ed) (1998) Nitrogen Containing Maeromoleeules in Biosphere and Geosphere. Flannery MB. Philadelphia 25 BierstedtA.

Chitin synthesis .

Introduction The pivotal role chitin plays in the life cyc1es of arthropods as well as fungi and some other microorganisms (for recendy described examples. not referred to anymore in this review). but often not as to interpretation (for examples. non-allosteric activation or priming of CS. Rast Department ojPlant Biology. Indeed. and "Structure ofCS" (the putative UDPGlcNAc-binding domain ofCS. entomologists. Switzerland Summary. "Regulation of CS" (cooperativity and allostery. And even with these. cation requiremement and intermediates. molecular cell biologists.Chitin and Chitinases ed. . product). Lars E. latency). Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Biochemistry of chitin synthase Regula A. earlier papers cited in these publications are. 1). "Concerted action of CS and enzymes of chitinolysis". with some exceptions. ever since the first description ofthe enzyme synthesizing chitin as an N-acetylglucosaminyltransferase using the uridine diphosphate (UDP)-activated monomer as the sugar donor ([9]. by P. there is overall agreement only as to phenomenology.for researchers in academia and industry alike. This article compiles the papers dealing with the biochemistry of chitin synthase (CS) published during the last decade. Nyhlen and Dora M. parasitologists and scientists representing other disciplines as well. A. see [10. and points out some firmly entrenched ideas and tenets of CS biochemistry that have become of age without hardly ever having been critically re-evaluated. A. "Inhibition of CS". University ojZürich. JollE" and R. chitin synthase (CS) has remained both an attractive and an intriguing enzyme for biochemists. Despite this broad interest and the copious bibliography dealing with CS obtained through the four decades ofinvestigations. identification ofCS polypeptides. The prospects are outlined of obtaining a refined three-dimensional (3D) model ofthe catalytic site of CS for biotechnological applications. eH-8008 Zürich. 11]. "Multiplicity of CSs". glycoconjugation). Fig. Merz. Zollikerstrasse 107. only a comparatively modest insight has been gained into the basic biochemical characteristics of the enzyme. mycologists. Markus Horsch. provides up-to-date information on the state ofknowledge and understanding of chitin synthesis in vitra. The subject is dealt with under the headings "Components of the CS reaction" (educt. see [1-3]) and the vast array of technological applications of this polymer have been known for a very long time and been dealt with in many books [4-8].

16). namely GlcNAc-glucosyldiacylglycerol [25] and dolicholdiphosphate-linked chito-oligomers with a maximum chain length of n = 8 [26. Components of the CS reaction Educt. such as a chito-oligomer with n ~ ca. there are reports of the formation of GlcNAc-glycolipids acting as intermediates of chitin synthesis. A. and a lipid intermediate or a lipid acceptor is generally held not to be involved_ Nevertheless. R 1 = H [9]. The presence ofMi+.10 R. thence. cation requirement and intermediates The obligate substrate for the enzyme is UDP-N-acetylglucosamine (UDPGlcNAc). C~H HO~ UDP uridine-5'-diphosphoN-acetyl-a-glucosamine (UDPGlcNAc) [chitin1n chitin synthase (eS) UDP [chitinln+1 Figure 1. [14. Merz et al. UDP-N-acetyl-Dglucosamine: chitin 4-ß-N-acetylglucosaminyltransferase. protein (. The latter appear to be transferred to a polypeptide acceptor and.eCS. see [24]). The significance of this requirement has not been elucidated. [12. EC 2. exogenous chitin does not act as a 'primer' either (for a discussion ofthe term.15]).eCS. this article). No inference is made by the scheme as to the mechanism of chain elongation (cf. . CS (= R 2 . 18-22].13]). to act as GlcNAc-acceptor substrates for CS ([14. 17]). [16. 10 (higher analogues are insoluble). there is no need for a soluble GlcNAc acceptor.1. in contrast to the original view (see Fig_ 1. but presumably reflects an essential role of CS for a divalent metal cation in substrate binding_ Chain assembly occurs without the formation of free chito-oligomers [23]_ Also.27]. or priming UDPGlcNAc-transferase (. Mn2+ or C02+ (depending on enzyme species) is indispensable for CS activity [1.4. Sum equation for the reaction catalyzed by chitin synthase (CS. R=H). 15].

yet producing a low degree of inhibition. a requirement for an enzyme that catalyzes the removal of (part ot) the polymer chain to produce pure chitin. for good activity. Hence. 1. as weIl as to harbour the enzyme protein in a liposome that obviously mimics natural conditions (see [41)). such as phospholipids [1. The generally low specificity of glycerophosphatides and the vastly different responses of CSs of different origin to individual representatives of these cast doubt.a situation turning the systematic name of the enzyme into a definitional oddity. for definitions. the experimental evidence implies that CS acts on a covalently-bound primer. might be the main reason. CS ME complex. 29-32] and some strongly amphipathic lipid-linked sugars [32-37] stimulate CS activity (for earlier ref's for the effect of lipids on CS. The chitin synthesized by DIG-solubilized CS (16S) has been described as consisting of lozenge-shaped particles about 10 nm wide and some 50-100 nm long [12]. as lateral aggregates of spindlelike segments of similar dimensions [30. see [38)). Amphiphiles. which. R j = priming polypeptide). inasmuch as it effects solubilization ofthe enzyme. as quite generally occurs with other detergentlenzyme combinations [39]. DIG interacts with CS also in a more specific manner. beyond the fact that. producing a pattern of stimulation/inhibition. 1. The CS-inhibitory potency ofthese is particularly pronounced in the case of the respective glycoforms not carrying a terminal glucose at the side-chain residue. In any case. the enzyme apparently needs to be bound to a membrane.Biochemistry of chitin synthase 11 Fig. R 1 = CS) . It is not known whether the presumptive primed-CS educt and the UDPGlcNAc binding site for the iterating events of chitin chain elongation concern distinct regions of the same polypeptide or are situated in two different. 42] or as extremely slender (width . physically c10sely associated polypeptides [CS multienzyme (ME) polypeptide vs. With the 3ß-sterol glycoside digitonin (DIG) the situation appears to be different. additionaIly. However. according to the definition ofthe enzyme. Product To reiterate what is known of the chemical structure of chitin lies outside the scope ofthis artic1e. but the supramolecular organization ofthe product deserves comment. there is. see "Nonallosteric activation or priming of CS" and "Structure of CS"]. why DIG has remained the agent of choice for solubilizing CS. As is also evident from Fig. as do other. is chitin (Fig. on their direct involvement in the CS reaction. The ability of DIG not only to bind to genuinely membrane-bound CS efficiently and quite specificaIly. for an hypothesis. structurally c10sely related spirostanol glycosides [40]. 1. the mode of action of CS cannot be assessed adequately without a consideration of co-occurring chitinolytic activity (see section "Concerted action of CS and enzymes of chitinolysis"). however. refer to [28]. either a natural or a suitable artificial one. the process being preceded by a stimulatory action of the saponin. 33.

Transglycosylating chitinases. 3 and text). Merz et a1. unpublished results). Indeed. In either case. co-occurring specific lytic enzymes could restrict chain length. for further examples. prevention of further chain elongation must be an extemally determined. up to 2 f. yield single. particularly chitin-modifying entities. should be discounted (see also [30]. M. see [35]) are artefactual. evidence for a temporal gap between the CS-polymerization reaction and the assembly of the polymer chains into microfibrils has been obtained also by another experimental approach [44].M. have not been investigated to any extent. 2a. An experimentally based model that encompasses restriction of the chain 1ength and a remodelling of chitin chains in a controlled manner is presented under section "Concerted action of CS with the enzymes of chitinolysis". long-time incubation yielded configurations of thick fibres consisting of twisted 5 -1 O-nmfibrils. the reaction would. prec1ude further addition by the specific polymerase concemed [24].E. this may result in a simple capping ofthe growing end or in a cross-linking reaction. D. Obviously. Rast.12 R. 2. Besides. while thin fibrils were no longer discemible (Fig. the possibility cannot be exc1uded that the extreme lengths observed of chitin fibrils synthesized in vitra after short-time incubation in the absence of chitinase (Fig. On the other hand. M. From a mechanistic point ofview. Mayer. Indeed. L. it is highly unlikely that CS catalyses the synthesis of a fibrillar product. the notion that the generation and crystallization of nascent chitin occur simultaneously and are very tightly coupled [43]. corresponding to one or two mo1ecu1ar chains) and very 10ng (severa1 hundred nm. purified 16 S-CS ex chitosomes display virtually no chitinase activity (tested with chromogenic and fluorogenic chitotetraoside analogues as the substrates. rather. as in situ the chain length of newly synthesized chitin is likely to be restricted by co-occurring chitinase (see Fig. C. too. on Ockham 's razor.A. Depending on the structure ofthe residue transferred.46]. controlled event [24]. and section "Inhibition of CS"). Therefore. . of 1-2 nm. the extent to which crystallization of chitin occurs to form fibrils depends largely on the physicochemical composition of the enzyme's microenvironment and the presence of other enzymes. whereas only very thin long fibrils were present after reacting CS with substrate and activators for short times. thus. Rast. Termination of the chain in polysaccharide synthesis can occur in principle by transfer of a hetero-group to its growing end and. hexosaminidases as well as ß-glucosidase and ß-glucanases are probably involved [47]. monomolecular chitin chains. Nyhlen and D. for example ß-glucan [45. Thus. which could be caused by the long incubation times used. Altematively. unpublished). and the enzymes effecting cross-linking between chitin and co-occurring polymers in situ. Horsch.llll) fibri1s emanating from the 16 S partic1es [35]. the formation of thick fibrils observed in some studies represents probably an artefactual postsynthesis event. Reports are not available of heterotransferases specifically acting at the nonreducing end of nascent chitin.

The enzyme preparation (from Agaricus bisparus mycelium) consisted of 16 S-CS obtained by dissociation of gradient-purified chitosomes (peak fraction) with DIG. Influence of the duration of incubation of CS with activators and substrate on the supramolecular structure of the product synthesized in vitra.Biochemistry of chitin synthase 13 Figure 2. refer to [35]. . Sampies were negatively stained after reaction times of (a) 6 min and (b) 25 min. For methodological details.

in fact. This may in part be due to contaminating chitinase and HexNAc'ase acting on firmly bound traces of chitin from the entrapment step. Since the intracellular concentrations of free GlcNAc appear to be extremely low. never been observed to become completely abolished in the presence of saturating [GlcNAc] [23. In view of the additional evidence provided by Horsch et al. see [52]) results from its competition with GlcNAc or UDPGlcNAc at the effector-binding site. and (ii) a single-bonded oxo-function present at C(1). [13] upon isolation of the enzyme by gel filtration of a microsomal fraction. [23] for the existence of a genuine allosteric site of CS for GlcNAc as well as of the observation that the activation constant (GlcNAc) is lower with 16 S-CS than with 105 S-CS [23. therefore. Merz et al. since neither of the chitinolytic enzymes would be separated from CS under the purification scheme followed in that study (see [53. The generally accepted view whereupon the substrate kinetics of CS are govemed solely by features ofhomotropic-heterotropic regulation calls for a critical reevaluation. [53. digestion of this with chitinase and extraction of the enzyme into buffer must be considered an experimental artefact.35]. [UDPGlcNAc] plots has. 14 Regulation of es Co-operativity and allostery Homotropic-heterotropic regulation with GlcNAc acting as an allosteric activator is a firmly established tenet of the biochemistry of CS. Thus. its validity has recently been revisited [23. it is a reasonable hypothesis that its source is situated at the cell surface. which is preferentially a hydrogen bond donor. 16 S-CS ex chitosomes or 16 S-CS ex walls) and regardless ofwhether GlcNAc was present.48-50. that may be equatorially spaced off but must not be a-anomeric.35. A. namely. where GlcNAc is likely to be generated through the action of enzymes effecting lysis of chitin. entrapment of CS into its reaction product.54]).R. the notion is to be discounted. irrespective ofthe type of enzyme preparation used (105 S chitosomal CS.50]. In the absence of any evidence for more than one type of allosteric site of CS. Accumulation at low substrate concentrations of soluble products (which would not K: . which is also the locus operandi ofCS. substrate kinetic curves could reasonably well be parameterized on the basis of the Monod mathematical model for co-operative ligand binding. the almost complete loss ofGlcNAc stimulation of CS reported by Kang et al.in contrast to what is required by the theory of allostery. The same holds for the suggestion [51] that the CS inhibitory action ofnikkomycin (for essential structural features. 55] . that GlcNAc would be but a poor substitute at an effector site for UDPGlcNAc [49.47]). since the sigmoidicity of the CS activity vs. however. The stereochemical requirements of the allosteric site of CS for ligand binding are: (i) an aminoglucopyranose skeleton with the amino function acetylated.48]. chitinase and ß-N-acetylhexosaminidase (HexNAc'ase.

EC 2. areaction proceeds under these conditions that enables CS to change from a truly latent into a fully active state even in the absence of GlcNAc. namely.186) and glycogen synthase proper (GS. [59]). The approach to the steady-state rate is slow. substrate and effector concentrations as weH as the most sensitive analytical method presently available [23.4. Considering the nature ofthe reactants.l. GG is a self-glucosylating protein that primes glycogen synthesis. Thus. since free chito-oligomers were not found as reaction intermediates (tested using a variety of purified enzyme. The next section presents an hypothesis for this hitherto unexplained kinetic idiosyncrasy of the CS reaction. both ofwhich are subunits ofthe glycogen synthase complex (ca. the curve on the one side extrapolating towards a steady rate of synthesis at the start of the incubation and towards a steady rate of synthesis on the other. whence the second.Biochemistry of chitin synthase 15 be detected by the filtration assay) as the cause ofthis phenomenon can be excluded. These kinetics are independent of the length of the enzyme 's preexposure to the protease and also not an artefact that would be due to the formation of soluble chitooligomers [23]. the efficient synthesis of glycogen requires the sequential action of two UDPGlc-transferases. Deceleration of the activation rate with decreasing enzyme concentration and vice versa. 15S.48].4. Hence. since this represents the only polysaccharide synthase for which more information is available regarding the mechanism of reaction (for reviews. the type of GlcNAc linkage generated by this putative activation or priming reaction.1. a glycogen initiator synthase termed glycogenin (GG.M. Since knowledge of the molecular structure of CS is quite lirnited (see "Structure of CS"}. to turn to the synthesis of glycogen as a paradigm. Therefore.11). linear phase of the reaction is reached. M. Horsch and D. It is useful. The first. Rast. indicates that this selfglycosylation might be intermolecular. unpublished data). the site at which it takes place and the mechanism whereby it causes chitin chain formation can presently only be speculated upon. nonlinear phase of the reaction is characterized by a gradual increase in the rate of product accumulation with time. That the frequency of enzyme activation shows a linear dependence on [UDPGlcNAc] is in line with this conclusion. it may be assumed that two different UDPGlcNAc-binding sites/two different enzymes are involved in de novo chitin synthesis. It effects auto-glucosylation by first gen- . EC 2. therefore. CS and UDPGlcNAc only. Non-allosteric activation or priming o/es In the absence of GlcNAc and at low concentrations of UDPGlcNAc the CS overall reaction is absolutely dependent on proteolytic activation and kinetically displays two distinct phases ([23. the conclusion thus appears unavoidable that there occurs an autoglycosylation prior to chitin chain formation of the CS system. 56]).58]). furthermore. see [57.

in the formation of hyaluronic acid (containing a chito-oligosaccharide residue at its reducing end [69]). see [70]). (2) some of the differential properties of GG vs.16 R. (3) an analogous priming function ofthe DG42 protein. The as yet hypothetical enzyme that would generate covalently-bound chito-oligosaccharide as a primer for es sensu stricto is hereafter referred to as 'chitogenin' (Fig.0).5 vs. [61]). GS. (iv) GG is not inactivated by UDP-pyridoxal (a specific affinity label for GS). but of polysaccharide synthesis in general (for references. (v) GG is not activated by glucose-6-phosphate. and (4) the evidence accumulating in recent years that a priming protein may be a fundamental property not only ofthe GS system. see [60] for amino acid sequence] and then successively adding further glucose units to finalIy generate an enzyme-bound oligomer of a-I. 103 X smaller (this means that at low substrate concentrations only the GG component of the GS complex is operative and. in an analogous manner. erating a glucosyl-tyrosine linkage [Glc(I-O)TyrI94. A. es). It is upon the maltosaccharide acceptor structure that GS performs chain elongation to afford the polysaccharide. I. whereas the following elongation events proceed with Mg 2+ (GS is not activated by Mn2+ and is. for the chitin synthesizing system: (I) the particular kinetics of the CSreaction that proceeds in the absence of GlcNAc (as stated above). and effector). generates a storage pool ofprimed GS substrate that is available for glycogen synthesis proper as conditions become favourable for this (that is high [UDPGlc].37. essentially unaffected by either cation).7175]. (iii) the first glucosyl transfer is Mn2+-dependent. (vii) the time-course approach of the GS overall reaction to constant rate is sigmoidal if the primer has to be generated first. Merz et al. catalyzing the synthesis of an array of chito-oligosaccharides [68]. a potent effector of GS. (iii) and (v)-(vii) have similarly been described to occur between different CS-preparations.64-67]: (i) GG activity is only displayed in the presence of high concentrations of various salts (GS is not active under such conditions). 8. Latency Limited proteolysis generally greatly stimulates es activity [32. Four lines of argument may lead one to suggest that a situation corresponding to that of the GGIGS system could exist.4-linked Glc units (n averaging 8. The self-glucosylation appears to be intermolecular.63]. (ii) the pH optimum ofGG is higher than that ofGS (ca. particularlY those concerning items (ii). (vi) the Km (UDPGlc) of GG is ca. thence. as the rate departs from constancy and falls away upon lowering the enzyme concentration [62. This indicates that most of a cell's es pool may be present in a 'classical' proenzyme form prior to product deposition at the proper time and . in fact. 7. The GG reaction has certain other properties that clearly distinguish it from that catalysed by GS [57.62. R j =t:. and hyperbolic when exogenous primer is provided.

truly latent UDPGlcNAc-transferase species generating a product that efficiently enhances the overall rate of chitin synthesis. The CHS4-product has no homology with any known protease. inc1uding limited proteolysis. for the gene product of CHS4 (= CAL2/CSD4/SKT5. see [81]). additional mechanisms must exist that pro- . for new nomenc1ature. In view ofreasons (i)-(vi) below. The deduced AA sequences of chito-oligosaccharide synthases of various origins show high similarities to those offungal CSs [69]. Concerted action of CS and enzymes of chitinolysis Although manipulation of CS activity by allosteric and non-allosteric activation. The most telling example in the present context may be CSIII. 78]. Cooperation in efficient chitin synthesis of two CSs differentially regulated with respect to limited proteolysis and cation requirement woulel.and Mg 2+-mediated stimulation of CSIII does not directly concern csm proper. as holds. this enzyme is hypothesized here to be 'chitogenin' (see section "Non-allosteric activation or priming of CS"): (i) (ii) (iii) (iv) (v) (vi) There exist some suggestive analogies between the chitin and the glycogen synthesis systems. but a tightly associateel. 'chitogenin' would simply be perceived as an activator of CSIII. Phenomenologically. finally. is sufficient to allow some control ofthe enzyme's activity in vitra. its activity becomes greatly increased if the (detergent-extracted) membrane preparation harbouring it is treated with trypsin in the presence of UDPGlcNAc prior to allosteric stimulation of chitin synthesis by GlcNAc. also obviate the need to entre at a change in the cation specificity of CSIII proper upon preincubation of the enzyme preparation with protease in the presence ofUDPGlcNAc [21].78]. Some ofthe UDPGlcNAc provided at the trypsin pre-incubation step (in the absence of GlcNAc) undoubtedly becomes reacted upon anel.Biochemistry of chitin synthase 17 site [76]. which is an essential component ofthe CSIII-complex. This is generally held not to be zymogenic and displays a preference of C0 2+ over Mg2+ as the activating cation [78-80]. the possibility has to be considered that the trypsin. however. and its significance remains elusive [21. inasmuch as the trypsin-dependent activity is favoured by an increase in [Mg 2+] and as there seemingly occurs areversal in the metal preference ofthe re action [21]. incorporated into acid-insoluble product (representing phase 1 of the CS overall reaction). attest to the still puzzling nature of the phenomenon. Recent papers do. 77. CSIII is presumably a multi enzyme complex [21. is zymogenic and appears to be involved in substrate processing [78]. for example. Nevertheless. thence.22. This protease treatment also has a bearing on the metal requirement of the CS-system concerneel.

potentially providing a donor for transglycosylation reactions (adapted from [47]).16).1. which consists of several HexNAc'ases ~ Figure 3. probably covalent binding to wall components [47. (2) remodelling of nascent as weil as of preformed. vide for a tight regulation of chitin synthesis in vivo. 54]. encompassing mechanisms of nonallosteric activation (including partiallatency) as well as allosteric stimulation (see the respective sections ofthis review). The presence in some CS species (100 S. A. The chitinolytic enzyme species ofparts (a) and (b) are isozymes differing in substrate chain length preference and some other properties [53]. The hypothesis is based on premises (i)-(viii): (i) (ii) (iii) (iv) (v) The topology ofthe site ofaction ofthe chitin synthesizing system at the cell surface. with increasing amounts of shorter chito-oligomers and N. the correct balance between the activity of an appropriately regulated chitinolytic system and that ofthe CS system appears paramount in vivo.18 R.14) and ß-N-acetylhexosaminidase (HexNAc'ase. see [84]). which could conceivably serve as a region of complexation for the non-catalytic high-affinity chitin binding domain of chitinase [90].1. Merz et al.2.53. . chitinase and HexNAc'ase is depicted in Figure 3. simultaneously. at the transcriptional as well as at the posttranslational level [77]. and (4) HexNAc'asemediated c1eavage of the glycosidic bond of chitobiose resulting in the generation of G1cNAc available for CS-activation and.as well as 16 S-size forms) of a chito-oligosaccharide residue.87-89]. The characteristics ofthe constitutive complex chitinolytic system of growinglbranching hyphae. The co-occurrence in log-phase hyphae of genuinely wall-associated CS.1. Part (a) depicts an integrated tripie enzyme system consisting ofCS (EC 2. chitinase and HexNAc'ase. not only in filamentous as weIl as in yeast growth offungi [47. namely hydrophobic bonding in an amphiphilic environment and very tight. 83].41]). the periplasmic space as weIl as the wall fabric itself(references in [23. Concerning these. The regulatory properties of CS. partly crystalline chitin through the combined transglycosylatinglhydrolysing activity of chitinase and HexNAc'ase. A biochemical model for the controlled synthesis and catabolism of chitin in situ. (3) progressive lysis of chitin. The integrated action of (a) and (b) encompasses four steps: (1) de novo synthcsis of chitin by activated CS.52). any ofwhich being held in the wall compartment by two types of interaction. and controlled action of CS with respect to cooccurring enzymes at the site of product deposition. encompassing the plasma membrane. Examples are phosphorylation/dephosphorylation as mediated by the Ca2+-calmodulin system [82. chitinase (EC 3.4.85]. A speculative scheme for the controlled synthesis and lysis of chitin during hyphal growth through the concerted action ofCS. An indication is given in the graph of the approximate spatial arrangement of the enzymes at the cell surface. but probably also in other biological systems effecting a quasi-simultaneous synthesis and degradation of chitin [86].2. Part (b) illustrates the transglycosylating activity and the tandem action of chitinase and HexNAc 'ase in the hydro lysis of chitin. besides an N-glycoconjugated partial structure [41. interaction of CS-secretory vesicles with components of the cytoskeleton (for references. EC 3.N' -diacety1chitobiose ('chitobiose') becoming available for chitinase and HexNAc'ase associated with CS.

. UDPGlcNAc..-0 marks the nonreducing end and .Biochemistry of chitin synthase 19 ~ .t/~ ~ /~ cf (a) o .-0 "'~ ...GlcNAc.. Legend see p.:. The active sites 01 the enzymes are marked as ~ (chitin synthase). . site of hydrolytic attack of chitinase. 18 . ~ (chitinase)...7 points to the reducing end 01 the polymer./1 ~ . reducing GlcNAc units generated through the action 01 chitinase and HexNAc'ase are shown as 0---. To better iIIustrate transglycosylation...~-7 (O)-{!~-<:..~ . Figure 3.>. ~ (HexNAc'ase).. .. allosteric site 01 ChS lor GlcNAc... the GlcNAc residues of two chains between which the event is depicted to take place are tagged differently ( • • • and -0-0-0-). 00. chitobiose.' ". l>-O. the sign .

Fig. as opposed to HexNAc'ase. ability to degrade preformed chitin and response to treatment with proteases [53. 56. A. 92]. and part (b) both the remodelling of chitin through the combined hydrolysing/transglycosylating action of chitinase and the final breakdown of the polymer. U. thus generating the CS effector GlcNAc to provide for full capacity binding of UDPGlcNAc and. affinity to polysaccharide gels. 1. not meant to be single. (vi) The tandem action of the binary chitinolytic system in effecting the breakdown of chitin to chiobiose and GlcNAc [47. however. namely. and two types of chitinases (A. chito-oligosaccharides conjugated with protein or lipid. (vii) The transg1ycosy1ating activity of genuinely wall-associated as well as of soluble fungal chitinases and HexNAc'ases ([47. separate events. chitinase and HexNAc' ase co-occur [53]. Part (a) of the model concerns the synthesis of chitin. R j =I:. the time-Iapse of the preceding step overlapping with the onset ofthe following: (1) Polymer synthesis by CS (presumed to be a multi enzyme complex with a chitin initiator synthase as a subunit. a moiety generated by a chitin initiator synthase (Fig. de novo synthesis of chitin.4-linked partial structures present in individual chitin chains can be of a threefold origin. postulated to be (loose1y) associated with CS by van der Waals forces and carbohydrate recognition. this can use as alternative acceptors ß-1.CS. 54. B) differing in pH optimum. thence. unpublished data). Mayer. These are.54]. according to the substrate chain length specificity of chitinase (n ~ 4. see [47]). see "Non-allosteric activation of CS") that encompasses the reducing end . There are three major implications inherent in the biochemical scheme presented for the concerted action of CS and chitinolytic enzymes in logphase hyphae: (i) The ß-l.20 R. (2) Restriction as weH as extension of the chain length of chitin synthesized de novo by the action of chitinase. one of which is fairly strongly associated with CS (16 S forms. D. Thus.CS) upon activation by limited proteolysis and allostery. (viii) The observation that in each size-type preparation of chitinase -large and small-particu1ate as well as soluble ones . the attack gradually resulting in an increased production of chito-oligomers as the preferred substrates of a proteolyticaHy activatable chitinase (type A). R j . for example. but to occur quasi-simultaneously. and thus effect cross-linking. (4) Hydrolysis ofthe end product of chitinase action by HexNAc'ases. Merz et al.M.91]. Rast. (3) Rearrangement by chitinase (type B) also ofpreformed chitin. C. ex 100 S-CS as well as ex walls). Sennhauser. 1. hypothesized to be 'chitogenin'.4-linked GlcNAc residues of the required length that are covalently bound to other polymers. the controlled deposition of chitin in a growing wall can be hypothesized to proceed in four steps.

see [41]). With chito-oligomer synthases. inasmuch as reduction of the length of eS-attached chitin to that ofa short oligomer restores some ofthe catalytic potential of the enzyme molecule concemed and as the concomittant breakdown ofpreformed chitin yields an ample supply ofthe effector GlcNAc to temporarily maintain some further polymer synthesis at this site. the complex structure-activity relationships observed with numerous analogues. the large differences exhibited by them in inhibitory potency with es species having a different cation preference (Mg 2 + vs. 3] has its main function. HexNAc' ase appears intrinsically well befitted for such a function. thus. Thus. kinetically behave as excellent competitive inhibitors of es. refer to [23. With its generally high leaving group/acceptor tolerance (for reaction mechanism. besides that of providing for a dynamic remodelling of preformed chitin. 71. (iii) eonceming the mediator function between the lysis and the synthesis of chitin assigned to the HexNAc'ase of the tripie enzyme system represented by part (a) ofthe scheme.47. however. if the enzyme. were also to chemically modify es and. the transfer ofGlcNAc to es would be particularly effective if the association between the two enzymes were such that the active site ofthe former was positioned in dose proximity to the allosteric site of the latter. Despite the wide use ofNPs as a tool in chitin biochemistry. For a discussion of the biochemical model of the controlled chitin metabolism in hyphal growth in the physiological context. to generate a covalently linked acceptor structure. nikkomycins and polyoxins. the inhibitory potency ofNPs appears to be quite moderate. . The mediator role of HexNAc' ase could be even more sophisticated. It is this situation. stretches generated de novo by es sensu stricto. see [47]). as undoubtedly must also the displacement of the enzyme from the source of its substrate at the cell surface to the adjacent area of the developing wall (for potential mechanisms ofUDPGlcNAc release on to the protoplast. and residues attached by transglycosylating chitinase at the nonreducing. 50 J. namely. (ii) Entrapment of es into its product [13. Inhibition of es Some nudeoside-peptides (NPs).1M [52.1M [68]. reported Ki-values are 2:: 0. provided with its substrate by a nearby chitinase. e0 2 +) as well as the non-competitive behaviour of nikkomycin with a mutant es [51] have remained unexplained mechanistically. that is the acceptor end of a growing chain. the DG42 protein has K/s of ca. 53]. 93-98].Biochemistry of chitin synthase 21 of the polymer.50] reduces its activity.1 J. where the mural nonzymogenic chitinolytic system [part (b) of Fig.

for example. are almost ineffectual). there was no inhibition ofthe activity of 100 S-CS exA. Tyr26 1). In analogy to the situation with glycogenin (see [58]). Merz et a1. potently (K j values ~ 1 J. In view of these observations and the finding that the CS reaction is biphasic (see "Non-allosteric activation or priming of CS"). (i) With the stilbene derivative Calcofluor White M2R (CFW). The complete lack of structural similarity between PCNB and UDPGlcNAc. makes it highly improbable that it could 'compete' as a monomerlc substrate in the polymerization reaction.A. however. which was weakly inhibited. 15]. Nevertheless. The CSmodulatory activity of these agents depends not only on their concentration but also on the presence of organic solvents as weH as the type oftest enzyme preparation used. In contrast to this. even in the presence of solvent for the ligand.1M CFW. while co-occurring chitin deposition was strongly affected [27].1M. competition of PCNB with UDPGlcNAc for a non-carbohydrate reactant is therefore an obvious alternative. indeed. Considerlng the chemical reactivity of PCNB (cf. No definite answer can be given yet because most of the data reported were based on CS preparations that were highly active (achieved either by high enzyme and/or high substrate concentrations and in the presence of saturating GlcNAc. such as hexachlorophenol and pentachlorophenol. Certain compounds that are structurally largely different from NPs and PCNB and also from each other influence the activity of CS according to a stimulation/inhibition pattern [see examples (i) and (ii) below]. the available experimental evidence indicates that PCNB interferes with the first priming rather than an iterating chain elongation reaction. bisporus (up to 20 J. at low substrate concentrations and with short incubation times. 4 and 5. A similar situation has been reported for the Mn 2 +-dependent chito-oligosaccharide synthase presumably catalysing the priming reaction for CS [14. the question arises whether glycosylation of the putative CS-primer protein or chitin chain elongation represents the primary target of PCNB and NPs. This situation evidently prec1udes a reliable assessment of inhibitor constants and a truly mechanistic interpretation of the data. whereas with CS systems values are usually in the 1-10 JlM range. whereafter there occurred a sharp dec1ine to nearly zero residual activity . result by interference at a chain elongation step. best with highly purified CS preparations. reconstituted chitosomes). whence steady-state conditions are reached quickly). inhibition of chitin synthesis by NPs might. [99]). Its inhibitory efficacy is highest in the absence of GlcNAc. [100]) and further experimental data (the inhibitor/CS complex is not disrupted upon gel filtration or density gradient centrifugation in high gravitational fields.22 R. and competitively [99]. PCNB does not interfere with the proteolytic activation of the CS system [99]. Pentachloronitrobenzene (PCNB) is another agent that inhibits CS specifically (other chlorobenzenes. this could conceivably be the predicted invariant tyrosine of the catalytic site (see Figs.

1M in the absence of solvent). 10-7 M to micelIes and aggregates therefrom above the critical micellar concentration [101. unlikely to act by a single mode. 16 S-CS ex chitosomes was slightly stimulated (some 30% at 1 J.1M) and then gradually inhibited (with 150 ca. Since all polyenes bind to membrane sterol. although in this case the CS-inhibitory efficacy ofCFW was more than 10 times lower. [14C]AMB methyl ester or [14C]MEB did not become disrupted by gel filtration or density gradient centrifugation). Iso-values were in the 100-J. 40]. since aqueous solutions of PMAs consist of a variety of potentially reactive species. amphotericin B (AMB) stimulated enzyme activity (byabout 150-170%) and caused no inhibition at all. (ii) In the case ofthe polyene macrolide antibiotics (PMAs).1M) [99]. cf. among which particularly weH with ergosterol [101. but only the heptaene AMB and ring-size analogues therefrom produce a sizable response [34. appears to be an hypothesis worthy of consideration.1M with the same enzyme isolate [99]. 99] is intrinsically even greater than in the case of CFW. The latter response occurred also with 16 S-CS ex walls from Mucor rouxii [30].. CFW is. itself producing some stimulation).1M). namely intercalation between polysaccharide chains. when present in mixed micelIes together with CS (that is as liposomes. 102]. Thus.Biochemistry of chitin synthase 23 at 100 J.1M range [40]. as the cause of the stimulatory action of heptaenic polyenes and specific binding of the monomeric form at 'the active site ofCS as the reason for inhibition. rather than a biophysical event clearly separated from the polymerization step (see also section "Product"). A superposition of mimicking a lipid intermediate (see "Educt. Under the same conditions (this means applied at 100 J. nystatin and mepartricine B (MEB). whereas in the presence of solvent (2 % dimethylformamide.1M). . [41]). ranging from single molecules at ::. 102]. the complexity of the interaction with CS that underlies their modulatory effect on the activity ofthe enzyme [40. there was 50% net inhibition at 1 J. cation requirement and intermediates"). hence. bisporus. while 16 S-CS ex walls displayed only inhibition (with Kapp 5 J. as generally assumed when claiming crystallization of the polymer into fibres to be a biochemical. the CS/polyene interaction concerned has probably not the character of a simple membrane perturbance. sonicated in buffer. 8 J. but using 100 S-CS exA. ca. The CS-modulatory activity of these polyenes is undoubtedly a consequence oftheir strongly binding to the enzyme (the complexes generated upon incubation of 100 S-CS with AMB. exhibited a pattern of very slight stimulation followed by net inhibition with chitosomes from M rouxii.

24

RA. Merz et al.

Multiplicity of CSs
The unequivocal assignment of an enzyme to CS EC 3.2.1.16 requires critical
identification of the product by direct means, for example, a combination of
assessment of soluble products afforded by chemical or enzymic hydro lysis
and X-ray di:ffraction analysis of the presumptive chitin. Whereas this holds
for the CSs that were described a long time ago, for example those of Saccharomyces cerevisiae [12], Schizophyllum commune [45] andM rouxii [30, 33],
and recently also that of Saprolegnia monoica [103], other CSs have often
been named such simply by the behaviour of the product generated under
standard assay conditions, namely, measurement of the incorporation of the
glycosyl residue ofUDPGlcNAc into filter-retainable material that is insoluble in dilute acid or alkali and sometimes additionally by identification of
chitobiose upon digestion of the deposit with chitinase. On this and similar
CS assays alone, however, other UDPGlcNAc-transferases, such as those
performing elongation at the nonreducing ends of the glycan side chains of
N-glycoproteins, producing hyaluron-like polysaccharides ([69,104], see
also [105]), synthesizing the backbone of complex lipo-chito-oligosaccharides [106,107], or possibly priming the CS system (see Fig. I, R j "# CS),
would eventually qualify as CSs as weIl (if studied with marginally purified
enzyme preparations). Indeed, the NodC N-acetylglucosaminyltransferase
has been explicitly referred to as CS [108].
Conversely, the available experimental evidence is insufficient to rule
out the possibility that the carbohydrate skeleton of Nod factors primarily
originates from the catalytic action of the bacterial analogue of CS, namely a
UDPGlcNAc-transferase involved in the synthesis of peptidoglycan that is
probably also present in the crude membrane fractions serving as the source
of the NodC enzyme, followed by a subsequent modification of the product
through the combined hydrolytic and transglycosylating activity of co-occurring lysozyme [47,54,56,92]. A similar caveat as to the concept of de novo
synthesis ofthe backbone ofNod factors by NodC has been presented earlier,
likewise because of the insufficient purity of the enzyme preparation used to
identify the presumptive NodC UDPGlcNAc-transferase [107].
Although more critical for experiments in vivo, another source of error
may come from assessing the purported chitin product with CFW, a fluorocbromic agent intercalating between polysaccharide chains (for references,
see [43, 109]). The CFW test is unspecific, since it affords positive staining
not only with chitin but also with other glycans, inc1uding various ß-glucans and mannoproteins as weIl as bacterial exopolysaccharides [110].
Moreover, unequivocal interpretation of results is complicated by the fact
that the dye also has an effect on the activity of CS itself, inasmuch as it can
inhibit or stimulate CS (see "Inhibition ofCS").
The limited specificity of the indirect CS assays is of a particular concem,
when enzyme preparations used to identify the enzyme and to assess its 'typical' characteristics are crude microsomal or mixed-membrane fractions, or

Biochemistry of chitin synthase

25

extracts obtained from the membrane-bound species simply by solubilization
with DIG. This is frequently the case. Therefore, some of the apparent discrepancies reported for the properties of CS can reflect genuine differences
between enzyme species of different ceHular or organismic origins, but could
as weH derive from a misidentification of the UDPGlcNAc-transferase in
question. It may explain the bewildering multiplicity of CSs reported hitherto, representing five different classes [111-117], some of which, however,
have been found to be nonessential for chitin synthesis in vivo.
Structure of es

Despite the wealth of articles on CS published during the last decade,
knowledge of the structural properties of the enzyme is surprisingly smaH
and mainly concems amino acid (AA) sequences deduced from cloned
chitin synthesis genes (CHSs; cf., [81]), some ofwhich, however, code for
UDPG1cNAc-transferases not catalysing the defining reaction ofCS (see
Fig. 1, and "Multiplicity ofCSs"). Figure 4 represents a summary ofthese reports. Based on this as weH as some additional information from other
ß-glycosyltransferases, a three-dimensional model of the catalytic site of
CS has been established (Fig. 5). Further, there are various papers reporting the isolation of CS proteins (see below for references). However, the
evidence for a particular one to, indeed, represent a CS polypeptide is less
than compelling, and the few AA sequence data obtained by direct analysis of one of these are, therefore, of doubtful value. Figure 6 displays the
key evidence for the unequivocal identification of a genuine CS polypeptide.
The putative UDPGlcNAc-binding domain oles

A large number of derived AA sequences (ca. 70) for CS are known from
fungal genes (cf. [112]; formore recentexamples, see [116,118,119]). In
cases where the fuH gene has been sequenced, this codes for up to 1500
AA residues. However, either for CS, or for homologous proteins, there
does not exist structural information that goes beyond the primary level.
The assignment of gene sections that are highly likely to represent protein
domains with catalytic function, therefore, relies fuHy on predictive analyses. A first clue as to the catalytic domain was found through multiply
aligning the derived CS sequences and singling out a section of about 450
residues that display high consensus among aH genes known so far [120].
Furthermore, structure prediction studies for CS and related sequences
using hydrophobic cluster analysis [17] and knowledge-based homology
modeHing [120] independently concluded that the protein fold is of the
altemating ßI a type and gave a selection of AA residues that are invariant

26

R. A. Merz et al.

ClI' •• 51'

.re •• t

CR.;'

~"cr Cliit

I'bUo !lode
.IDol. dlO"~

.tnw

h&.A
cblatl exoW
21>gU.

Con.ln.I'U1
a~

:a.'WI

21>gU. ••
~bgu

•••

-

-210 . ··--·-220 •• -- ---

-. ----- - -110. ----- - - -200. --- - -

W.

Wh

-

-·-no. ------300.-

W.

CHI ••p
p •• t.

etLI~

"\101' CII••

rbUo nod.C
Mal. 4g4a
n;rpy ha ...

-

rbiae .ICO.

2bau
Co"' ....."'.

2bau ....
::iIbgu ••

.3b9u ...

-----uo...9

Wh

•210.--·-

. ..

-

·-270.----·· 210.······.,0.···

",0

Pli

WM
PIZ

CHI . . p

,.. •• t CM.a
uucr CHI'
:rhilo Q04C

.10.1.. dgt:a
It.rpy hall.

rtllM .xoM

2bau
Con •• alu.
:iIIbgy. D\Ia

::Ib9\a ..
lbgu: . . .

-

' 00.--

...

- -

--·---110.----·ull

'20 .... -----"0.-- . . .
all

--

---"0. -ul3

Figure 4. Multiple alignment of deduced amino acid sequences for largely different ß-glycosyltransferases with the UOPGlc-binding subdomain of a phage T4 ONA-modifying ß-glucosyltransferase (POB-code 2bgu) as the structural and functional homology template. The
sequence tags used are: CRS ssp, consensus secondary structure prediction for chitin synthases
(
, a-helix; VWIv, ß-strand; T, turn; [120]; yeast CRS2, chitin synthase 2 from Saccharomyces cerevisiae [125]; neuer CHS4, chitin synthase 4 from Neurospora crassa [126], rhilo
nodC, rhizobial nodulation factor from Rhizobium loti [127]; xenla DG42, hyaluronan synthase
from Xenopus laevis [128]; strpy hasA, hyaluronan synthase from Streptococcus pyogenes [129];
rhime exoW, succinoglycan synthase from Rhizobium meliloti [130]; 2bgu, ß-glucosyltransferase from the bacteriophage T4 [122]; consensus, consensus colouring as in Figure 5; 2bgu
num, sequence nurnbering of 2bgu; 2bgu ss, secondary structure of 2bgu (
, a-helix;
WM, ß-strand); 2bgu sse, secondary structure elements of2bgu as assigned by [124]. Amino
acids are coloured within similarity groups, that is white, Gly, Pro, Ala; grey, Ser, Thr; light red,
Asn, Gin; red, Asp, Glu; blue, Lys, Arg; black, Phe, Tyr, Trp, His; yellow, Cys; green, Ile, Leu,
Val, Met.

Biochemistry of chitin synthase

27

Figure 5. An hypothesis for the 3D structure of the catalytic site of CS, based on the data
presented in Fig. 4 and a space-filling model ofthe UDPG1c binding subdomain ofthe DNAmodifying ß-glucosyltransferase from the phage T4 (residues 179- 342, inc\uding the uridinediphosphoryl part of its ligand UDPG1c; [143]). Code 2bgu of the Brookhaven National
Laboratory PDB, Upton, NY, USA; reproduced using the program RasMol by R. Sayle, Glaxo
Research & Development, UK). The amino acids are coloured according to the degree of
consensus within the alignment of 2bgu with various other ß-glycosyltransferases listed in
Figure 4, that is red, strictly conserved in a11 sequences; light red, conserved in at least four
sequences inc\uding 2bgu; green, conserved in at least three sequences, but dissimilar in 2bgu.

in aH CRS genes and in other genes coding for ß-glycosyltransferases. The
latter study has proposed either a (ß/akbarrel fold or a structure similar
to the nucleotide-binding (Rossmann) fold for CS, using QSLAVE, an
expert system ofprotein fold prediction [121]. A fold similar in topology
to the Rossmann fold, separated by a deep central eIeft that forms the
binding site for the DDP-sugar substrate, was indeed found for a ßglycosyltransferase from the phage T4 [122]. This structure was classified together with glycogen phosphorylase into a glycosyltransferase
superfamily of protein folds on purely 3D-structural analogy criteria
[123, 124].
The functional analogy as weH as the good agreement of the phage ßglucosyltransferase (2bgu) fold with the structure predictions for CS and
other ß-glycosyltransferases lead us to establish the working hypothesis
that these proteins all belong to this same fold superfamily. Thus, a match
for one ofthe phosphate binding loops ßl1/alO of2bgu [124] can be local-

28

R. A. Merz et al.

ized in the genes of all classes of CRSs and of related ß-glycosyltransferases (Fig. 4). Furthermore, the alignment can be extended to the whole
2bgu half-domain that is responsible for the binding of the UDP-sugar
moiety by matching the secondary structure elements (Fig. 4, 2bgu sse)
with the secondary structure prediction for the CS sequences (Fig. 4, CRS
ssp). Within the alignment of the UDP-sugar binding domain of 2bgu,
pairwise homologies are in the 24-30%AA identity range for such different enzyme sequences as those for a S. cerevisiae class I CS (Fig. 4; for
sequence originator citations, see caption to the figure), a N. crassa c1ass
IV CS, a nodulation factor from R. loti, and two hyaluronan synthases,
fromX laevis and from S. pyogenes. Sequence homologies ofthe R. meliloti succinoglycan synthase (rhime exoW) and 2bgu with any ofthe other
sequences are, however, not evident on a residue-to-residue basis (8-12 %
id. range).
A localization in the 2bgu subdomain structure (Fig. 5) of the 9 residues
that are conserved in all aligned sequences (Figs. 4, 5; deep red consensus
colouring) leaves 4 which make direct contact with the UDP-sugar ligand
moiety, namely Asn215 (Asp in all others), Tyr261, Arg269 (or Lys) and
Glu272 (or GIn). Also, Trp341 is strictly conserved and may playa key
structural role in the hinge region between the two subdomains of the fold.
Furthermore, the ligand-binding surface ofthe subdomain hosts many residues that are conserved in at least 3 ofthe prediction sequences (Figs. 4, 5;
green consensus colouring) and those that additionally are similar in 2bgu
(Figs. 4, 5; light red consensus colouring). The N-terminal extensions of
the prediction sequences represent 150-200 residues that are, in terms of
length and predicted secondary structure, in perfect accord with the N-terminal subdomain of 2bgu. Since this appears not to make c10se contacts to
the substrate ligands in the model case and - therefore not unexpectedly since sequence homology in this region is less pronounced among the
various ß-glycosyltransferase sequences, a full alignment with 2bgu is not
suggested at this juncture.
Identification 0/ es polypeptides

A recently developed new procedure for the purification of CS [88,89],
relying on the selective adsorption of chitosomes to concanavalin A
(ConA)-gel and selective desorption of the enzyme from this, yields the
purest active CS preparation described hitherto, with components identified separately as a genuine CS polypeptide vs. contaminant proteins (Fig. 6;
[41, 89]) - regardless of whether obtained from a mixture of membranes
and wall fragments [13], from unspecified microsomes [22, 131, 132], a
mixed-membrane fraction [32] or chitosomes [37, 99, 133-135]. Thus,
there exists a 60-kDa polypeptide representing a UDPGlcNAc-transferase
with the defining properties of CS, as assessed by the standard test (Fig. 6,

Biochemistry of chitin synthase

A

29

B

c

0

Mr
[kDa]
200

116
97
66
45

31
21
Figure 6. Identification of a 60-kDa CS polypeptide: SDS-PAGE patterns (silver staining) of
three different preparations of active CS obtained by subjecting chitosomal CS to affinity
chromatography (AC) procedures. A Gradient-purified chitosomes (peak fraction; isolated
accordingto [136]; D, asA, purified by heparin AC (as described by [89], B, asA, obtained upon
removal of contaminating proteins by conventional ConA-AC and desorption ofthe CS that had
remained tighly bound to the column packing by a mixture of methyl-a-mannopyranoside and
NaCI (each 0.5 M) applied under batch conditions (for experimental details, refer to [41, 88]);
C, as B, but desorption effected with NaCI only (0.5 M). Band C: the protein bands at 32 kDa
and below are due to ConA leakage from the AC colurnn.

lane C). The identity of this with a CS polypeptide was eonfirmed further
by eomparison ofthe SDS polyacrylamide gel eleetrophoresis (PAGE) pattern ofthe isolate with that ofan aetive CS preparation eolleeted upon lipose1eetive affinity ehromatography (AC) of ehitosomes on heparin (Fig. 6,
lane D), whieh also displays the 60-kDa band. The relevanee for chitin synthesis of the additional 57 -kDa band in the latter preparation ean only be
speeulated upon. Sinee desorption ofCS from ehitosome-loaded ConA-gel
with MeMan/DIG, instead of MeMan/NaCl (Fig. 6, lane B), yields the
same sharp 57-kDa band, besides the 60-kDa polypeptide [41, 89], and
sinee the 57-kDa band is also present in the SDSIPAGE pattern of a CS
isolate obtained by AC of ehitosomes on wheat germ agglutinin (WGA,
[89]), the polypeptide concerned might represent a more hydrophobie
subunit ofthe (as yet hypothetical) CS multienzyme complex (see sections
"Components of the CS reaction" and "Non-allosteric activation or priming of CS").
The fact that ConA affinity columns display leakage of various species
of ConA, of which the 32-kDa component is the most prominent (Fig. 6,
lanes B,C; [41, 89]; see also [137, 138]), casts doubt on the claim by

mainly because their isolation protocol involved ConA-AC.N'. the lectin affinity ofwhich is annihilated in the presence of N. may appear discouragingly impressive.signifying N-glycosylation of CS. that is catalyse the generation of the GlcNAc-acceptor/priming substrate and perform chitin chain elongation. but would by no means be shorter for any other polysaccharide synthase catalysing a cell surface-Iocated product: (i) the identity of those polypeptides of the chitin synthesizing system that unequivocally represent UDPGlcNAc-transferases effecting the formation of chitin de novo. Moreover. the 60-kDa polypeptide present in the SDS-PAGE electropherograms of CS purified by fast protein liquid chromatography stains weIl with the periodic acid-Schiff reagent. (iv) the possible involvement of a lipid intermediate. however. but differential requirement of CSs for a divalent cation as a cofactor. Outlook The list ofthe major gaps in present-day knowledge ofthe biochemistry of CS. summarized under items (i)-(ix) below.and WGA-fractions were somewhat variable. Indeed. Glycoconjugation of es Based not only on indirect but also on direct evidence.and 16 S CS exist as glycoforms [41. there occurs a specific adsorption ofthe enzyme to the saccharide binding site ofConA .N" '-triacetylchitotriose . (iii) the cause of the absolute. 87-89. Merz et al. (v) the origin of the bound chitin (or chito-oligomer) serving as the primer substrate for CS EC . the amino-terminal sequence (12 amino acids) ofthe putative 30-kDa CS protein was reported to be identical with that of the corresponding stretch of ConA [139] and displays homology to none of the corresponding AA sequences derived from cloned CRS genes.pointing to the presence of a chito-oligomer residue in the glycan side chain of CS. 99].indicating fucosylation of the enzyme species concemed at the C-6 position of the N-proximal GlcNAc moiety. both 100 S. (ii) the distinctive physicochemical features of CS polypeptides. Machida and Saito [132] to have purified CS to homogeneity said to be represented by a single 30-kDa polypeptide. Thus. Quantitatively. qualitatively. they do demonstrate a complex glycoconjugation ofpart ofthe ceIl's CS pool. AC on lentillectin (LL) affords a binding fraction (accounting for some 60 % oftotal activity) . and AC of the same enzyme isolate on WGA likewise yields a binding fraction (about 50% oftotal). A. the results of the analysis by high performance anion exchange chromatography/pulsed amperometric detection of the carbohydrate monomer constituents generated by trifluoroacetic acid-hydrolysis ofthe LL.30 R.

Besides. it would allow a truly rational design of es inhibitors as potential specific biocidal agents for use in agriculture and medicine [140. 3064.1.H. .93).16. It is also a p1easure to thank Prof. University of British Co1umbia. (vii) es . Acknowledgements The senior author's research is supported by project no. for photographic work. of course. as required for understanding growth of any organism relying on chitin as a building block ofits exoskeleton (refer to "Introduction" for examples). Towers. 141]. The availability of this information would evidently benefit also the investigations into any of the other problem areas listed above [(iii)-(ix)]. would be that of a glycoform. The very limited data available on the structure ofthe chitin-synthesizing system has undoubtedly represented the decisive obstacle to a speedy progress ofthe research into the reaction mechanism ofeS and the interaction of this with enzymes catalysing the concerted remodelling and breakdown of chitin in vivo.4. 143]). The dearth of structural knowledge of es has been a barrier also to an adequate streamlining of the enormous efforts invested during the last decade into unravelling the molecular biology of chitin synthesis amI. Although this is clearly a goal that realistically cannot be reached in the near future. besides various types of spectroscopy performed with pure es entities or relevant fragments thereof. since glycoconjugation undoubtedly has a bearing on the activity and the cellular integration of es. Jauch. G. insertion at the reducing. The ideal 3D model of es. and mo1ecular modelling can now be performed also with glycosylated enzymes (für examples.N.Biochemistry of chitin synthase 31 2. therefore. 31-39699. (viii) the types of enzymes responsible for chain-Iength restriction of chitin by simple capping or cross-linking the nascent product with co-occurring polymers to which this becomes bound in situ. as it has on all proteins that enter the secretory pathway (see [142. also an impediment to efficiently exploit the biotechnological potential of the enzyme. U. (vi) the mechanism of chain elongation (processing at the nonreducing end vs. the chances appear fair of finally attaining even this.a polypeptide containing two catalytic domains or a multi enzyme complex?. enzyme-bound end of the growing chain). for reading the manuscript and Mr. Vancouver. since efficient automated techniques for the carbohydrate analysis of glycoproteins are becoming routine [143]. eonsidering some very recent advances in es biochemistry pointed out in this review and using as the strategy a combination of protein and enzyme technology as well as molecular biology methods. see [144]). the prospect of thus gaining a refined 3D model of the catalytic site( s) of es is good. University of Zürich.1 of the Swiss Commission for Techno10gy and Innovation (joint program with Novartis) and the Swiss National Science Foundation (grant no. and (ix) the significance of (partial) latency of es.

Mattia E. Exp Parasitol89: 195-204 Muzzarelli RAA (1977) Chitin. New York. Roberts G (1996) Advanees in Chitin Scienee. Brown RM. European Chitin Society. T MacRae. J Gen Microbiol139: 2117 -2122 Leal-Morales CA. European Chitin Society. abundance and stability of chitin synthetases 1 and 2 from Saeeharomyees eerevisiae. Pergamon Press.32 R. Jaques Andre. Bracker CE. Desouza W. Exp Myeol4: 175-180 Horst MN (1983) The biosynthesis of crustacean chitin. MG Peter (eds): Chitin handbook. Mierobiol140: 2207-2216 Choi W-J. Brown DH (1957) The synthesis of chitin in cell-free extracts of Neurospora erassa. Confirmation ofthe zymogenic nature ofthe enzyme. J Biol Chem 228: 729-742 Cabib E (1987) The synthesis and degradation of chitin. London Muzzarelli RAA (1993) Chitin enzymology. Rast DM (1996) Stereochemical requirements of chitin synthase for ligand binding at the allosteric site for N-acetylglucosamine. Ancona Glaser L. Peter MG (1997) Chitin handbook. J Bioehern 119: 659-666 Horsch M. London. Mayer C. 59-76 Robyt JF (1979) Mechanisms involved in the biosynthesis of polysaccharides. and assembly. J Baeterioll77: 1419-1424 Gay L. Girard V. 337 . Cantino EC (1980) The glycolipid involved in chitin synthesis by zoospores of Blastocladiella emersonii is a monoglucosyldiacylglycerol. Sburlati A. Alan R. vol 2. Henrissat B (1995) Multidomain architecture of ß-glycosyl transferases: implications for mechanism of action. Liss. In: AH Warner. synthesis. Cabib E (1994) The use of divalent cations and pH for the determination of specific yeast chitin synthetases. Mills GL. 51-61 Horst MN (1989) Molecular and cellular aspects of chitin synthesis in larval Artemia. Croom Helm. Muzzarelli RAA. Lyon Muzzarelli RAA. Purification of an enzyme by entrapment in the reaction product. Anal Bioehern 219: 368-372 Choi W-J. Cabib E (1994) Chitin synthase 3 from yeast has zymogenic properties that depend on both the CALl and the CAL3 genes. Au-Young J. Adv Enzymol 59: 59-10 I Ruiz-Herrera J (1992) Fungal eell wall: structure. European Chitin Society. Ancona. Travassos LR (1998) Triehomonas vaginalis and Triehomonas loetus . Elango N. Chanzy H. Trends Bioehern Sei 4: 47-49 Saxena IM. New York. J Bagshaw (eds): Cell and moleeular biology 01Artemia development. voll. ET Kaiser (eds): Synthetie peptides: approaehes to biologieal problems. Jeuniaux C. Robbins pw.Arisawa M. Areh Bioehern Biophys 223: 254-263 . Gillin FD (1991) Chitin synthase in encysting Entamoeba invadens. Plenum Press. In:JP Tarn. In: RAA Muzzarelli. J Biol Chem 253: 4419-4425 Kang MS. A. Boca Raton FL Duran A. Proe Natl Aead Sei USA 91: 4727-4730 Uchida Y. Shimmi 0. Ancona Muzzarelli RAA (1996) Chitin enzymology. Merz et al. J Biol Chem 259: 14966-14972 Horst MN (1989) Glycosylation of exogenous peptide acceptors by larval brine shrimp microsomes. Atec. Isolation and characterization of polyprenol-linked intermediates from brine shrimp microsomes. Schermuly G (1997) Chitin synthesis in lower organisms. Grottamare Domard A. References 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Das S. Alviano CS. Bulone V. Bioehern J 280: 641-647 Mulisch M. Sudoh M.344 Kneipp LF. Jr. Eur J Bioehern 237: 476482 Stoddart RW (1984) The biosynthesis olpolysaeeharides. Andrade AFB.expression of chitin at the cell surface. Fevre M (1993) Synthesis in vitro of crystalline chitin by a solubilized enzyme from the cellulosic fungus Saprolegnia monoiea. Cabib E (1984) Isolation of chitin synthetase from Saeeharomyees eerevisiae. Fevre M. Cabib E (1978) Solubilization and partial purification ofyeast chitin synthetase. Angluster J. CRC Press. Yamada-Okabe H (1996) Characterization of chitin synthase 2 of Saeeharomyees eerevisiae II: both full size and processed enzymes are active for chitin synthesis. Bartnicki-Garcia S (1994) Subcellular localization. Geremia RA.

Nyhlen LE. Simons K (1975) Solubilization ofmembranes by detergents. J Biol Chern 270: 1170-1178 47 Horsch M. Rast DM (1997) ß-N-Acetylhexosaminidase: a target forthe design ofantifungal agents. Bartnicki-Garcia S (1991) Purification and characterization of 16 Schitin synthetase particles from cell walls of Mucor rouxii. Bartnicki-Garcia S (1997) The properties and 10calization of Saprolegnia rnonoica chitin synthase differ from those of other fungi. Wesseis JGH (1994)The linkage of(l3)-ß-glucan to chitin during cell wall assembly in Saccharornyces cerevisiae. Rast DM (1983) Isolation and properties of chitin synthetase from Agaricus bisporus mycelium. 45-52 28 NC-IUB (1989). Biochirn Biophys Acta 415: 29-79 40 Furter R (1985) Die Chitinsynthese des dimorphen Zygomyceten Mucor rouxii. Pharrnacol Ther 76: 187-218 48 Horsch M. Cabib E (1995) Architecture ofthe yeast cell wall. Bracker CE (1980) Dissociation of chitosomes by digitonin into 16-S subunits with chitin synthetase activity. J Bacteriol163: 1180-1185 44 Vermeulen CA. Nomenc1ature for multienzymes. Klis FM. Humphreys A. University of Zürich 41 Merz RA. Horsch M. NewYork. J Biol Chern 246: 4008-4016 50 Ruiz-Herrera J. Microbiology 143: 2473-2483 33 Ruiz-Herrera J. In: RAA Muzzarelli. Chanzy H (1994) An electron microscope and electron diffraction study of the effect of Calcofluor and Congo red on the biosynthesis of chitin in vitro. European Chitin Society. Evidence for a transient non-crysta11ine state ofchitin. vol 18. chitin. Fevre M. Ruiz-Herrera J. Todoriki S. EurJ Biochern 158: 411-415 45 HartlandRP. Food Hydrocolloids 1: 353358 43 Roncero C. Proc NatlAcad Sei USA 78: 1233-1236 35 Hänseler E. Nyhlen LE. Trinci APJ (1990) Chitin synthesis in Fusariurn grarninearurn and its inhibition by edifenphos (Hinosan). Mayer C. Amsterdam. Phytochernistry 52: 213 . Rast DM (1993) Allosteric activation of chitin synthetase by N-acetylglucosamine: a mechanistic study. Leal-Morales CA. Lopez-Romero E. nystatin. Saito M (1994) Phospholipid requirements ofmembrane-bound chitin synthase from Absidia glauca. Biochirn Biophys Acta 745: 121-133 36 Hänseler E. Biochirn Biophys Acta 629: 201-216 34 Rast DM. SietsrnaJH. Persson J. PhD thesis. In: RAA Muzzarelli (ed): Chitin enzyrnology. and chitosan. 47 -56 49 McMurrough I. Exp Mycol 7: 17 . J Gen Microbiol137: 615-620 30 Bartnicki-Garcia S. Plenum Press. and other polyene antibiotics on chitin synthase. Robbins pw. Gay L. Yeast 10: 1591-1599 46 Kollar R. Goosey MW.240 32 Leal-Morales CA. Elsevier.226 42 Bartnicki-Garcia S (1987) Chitosomes and chitin biogenesis. Bartnicki-Garcia S (1977) Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii. The linkage between chitin and ß(1 ~ 3)-glucan. Rast DM (1999) Interaction between chitosomes and concanavalin A. Ruffuer HP. J Biol Chern 252: 33383343 51 Cubero B. C Jeuniaux. Flores-Martinez A. Rast DM (1983) Dissociation and reconstitution of chitosomes. Vermeulen CA. Exp Mycol15: 11-25 38 Gooday GW (1983) The microbial synthesis of cellulose. Bartnicki-Garcia S (1971) Properties of a particulate chitin synthetase from Mucor rouxii. Arch Biochern Biophys 310: 6-15 31 Machida S. Bracker CE. 85-127 39 Helenius A. Bartnicki-Garcia S. Ashwell G. FEMS Microbiol Lett 115: 235 .30 37 Lending CR. Robson GD. Hamamatsu S. Petrakova E. Bartnicki-Garcia S (1981) Effects of amphotericin B.Biochemistry of chitin synthase 33 27 Horst MN (1986) Lipid-linked intermediates in crustacean chitin synthesis. In: ME Bushell (ed): Progress in Industrial Microbiology. Eur J Biochern 185: 485-486 29 Binks PR. Duran A (1985) Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. Mol Gen Genet 240: 9-16 . Cerda-Olmedo E (1993) Chitin synthetase mutants of Phycornyces blakesleeanus. Wesseis GH (1986) Chitin biosynthesis by a fungal membrane preparation. Ancona. Sennhauser U. GW Gooday (eds): Chitin in nature and technology.

Rast DM (1997) High-pH anion-exchange chromatography analysis of hydrolysis and transglycosylation catalyzed by chitinolytic enzymes. Bioehem Biophys Res Commun 174: 204210. Peter MG (eds): Chitin handbook. Lomako J. In: NAR Gow. Whelan WJ.4-glucan-protein. Smythe C. Mol Mierobiol4: 197-207 73 Silverman SJ. Chapman & Hall. London. Adams DJ (1994) Characterization of chitin synthase from Botrytis cinerea. Cabib E (1991) Proteinase Bis. Eur J Bioehem 176:391-395 68 Semino CE. Horsch M. Cohen P (1989) The amino acid sequence ofrabbit skeletal musc1e glycogenin. Areh Bioehem Biophys 319: 293 . FEBS Lett 352: 222-226 62 Alonso MD. Lomako WM. Gooday GW (1991) A complex chitinolytic system in exponentially growing mycelium of Mueor rouxii: properties and function. Robbins PW (1990) Isolation ofa chitin synthase gene (CHS1) ftom Candida albieans by expression in Saeeharomyees eerevisiae. Ancona. A. Roach PJ (1995) Mechanism of glycogenin self-glucosylation.EurJBioehem 185: 119-125 61 Alonso MD. Cohen P (1988) Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal musc1e. Fevre M (1989) Chitin synthase activity from Neoeallimastix frontalis. 52 Decker H. Zähner H. Fiedler H-P (1991) Structure-activity relationships of the nikkomycins. Merz et al. J Gen Mierobiol89: 146-154 56 Mayer C. In: RAA Muzzarelli. FEMS Mierobiol Lett 149: 279-284 76 Gooday GW (1995) Cell walls. Hebraud M. Bioehem Biophys Res Commun 46: 1206-1212 65 Krisman CR (1973) A possible intermediate in the initiation of glycogen biosynthesis. Gooday GW (1975) A kinetic study of a solubilized chitin synthetase preparation from Coprinus einereus.34 R. Lomako WM. 43-61 . SteinraufLK. Ann NY Aead Sei 210: 81-89 66 Krisman CR. indeed. Furter R. Milling RJ. Barengo R (1975) Aprecursor of glycogen biosynthesis: a-l. Cohen P (1987) Identification ofthe 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin. Girard V. Robbins PW (1995) Synthesis of"Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42. Foster SG. University of Zürich 55 De Rousset-Hall A. Cohen P (1991) The discovery of glycogenin and the priming mechanism for glycogen biogenesis. Rothschild A. J Biol Chem 270: 15315-15319 63 Cao Y. J Gen Mierobiol 137: 2797-2810 54 Mayer C (1997) Eine mechanistische Analyse von Enzymen der Chitinlyse bei Pilzen unter der Verwendung von Lysozym als dem Paradigma.298 64 Krisman CR (1972) A possible intermediate in the initiation of glycogen biosynthesis. an anaerobic rumen fungus. Arisawa M. Lomako J. Proe NatlAead Sci USA 92: 3498-3501 69 Semino CE. J Gen Mierobiol135: 279-283 72 Au-Young J. König WA. Tandecarz JS (1997) Initiation of starch biosynthesis: purification and characterization of UDP-glucose:protein transglucosylase from potato tubers. Whelan WJ (1995) Catalytic activities of glycogenin additional to autocatalytic self-glucosylation. 345-351 57 Smythe C. 74 Causier BE. Takagi M. Eur J Bioehem 169: 497 . Heitsch H. Yamada-Okabe H. Raimondi A. Mierobiology 140: 2199-2205 75 Tatsuno K. European Chitin Society. Proe NatlAead Sci USA 93: 4548-4553 70 Bocca SN. Campbell DG. Whelan WJ (1995) A new look at the biogenesis of glycogen. Specht CA. Smythe C. Preiss J (1994) Properties ofcarbohydrate-free recombinant glycogenin expressed in an Eseheriehia eoli mutant lacking UDPglucose pyrophosphorylase activity. GM Gadd (eds): The growingjungus.502 60 Campbell DG. Lomako J. Robbins PW (1996) Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis ofNod-like chitin oligosaccharides during early embryogenesis. EurJ Bioehem 52: 117-123 67 Pitcher J. Lomako WM. J Gen Mierobiol13 7: 1805 -1813 53 Rast DM. Sudoh M (1997) Properties ofyeast expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth. Eur J Bioehem 200: 625-631 58 Alonso MD. PhD thesis. Plant Physiol Bioehem 35: 205-212 71 Gay L. not required for chitin synthetase 1 function in Saeeharomyees eerevisiae. Shaw JA. FASEB J9: 1126-1137 59 Pitcher J.

Robbins PW (1991) Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. Antimicrob Agents Chemother 35: 170-173 96 Ramirez-Ramirez N. Mio T. agene of Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4. Osmond BC (1990) Chitin synthase I and chitin synthase 11 are not required for chitin synthesis in vivo in Saccharomyces cerevisiae. Santos B. Arch Microbiol130: 195-197 94 Furter R. Arisawa M (1995) Isolation of canCHSIA. J Gen Microbiol138: 97102 86 Kramer KJ. University of Zürich 90 Kuranda MJ. FEMS Microbiol Lett 150: 95 -1 00 84 Gow NAR (1995) Tip growth and polarity. Yeast 13: 795-807 79 Bulawa CE. a variant gene of Candida albicans chitin synthase. Subramanyam C (1997) A putative role for calmodulin in the activation of Neurospora crassa chitin synthase. In: NAR Gow. Silverrnan SJ (1994) Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo. Kramer KJ (1985) Mechanism of chitin hydrolysis by the binary chitinase system in insect moulting fluid. Zähner H. Microbiology 141: 2673-2679 99 Rodewald R (1990) 16 S-Chitinsynthetase aus Agaricus bisporus. and polyoxin A: a comparison. Horsch M. European Chitin Society. Kirsch DR. Jacques Andre. Lai MH. University of Zürich 100 Renner G. Furter R. CosT. Shaw JA (1992) Chitinase and chitin synthase I: counterbalancing activities in cell separation of Saccharomyces cerevisiae. Lyon. voll. 87 Merz RA. Physical state. Roncero C (1997) Characterization ofCHS4 (CAL2). Rast DM (1993) Lectin affinity chromatography of chitin synthetase. J Biol Chem 266: 19758-19767 91 Fukamizo T. Reinigung und Hemmbarkeit durch antifungale Agenzien. PhD thesis. Silverman SJ. Rast DM (1996) Chitosomes and concanavalin A. In: A Domard. Arch Microbiol148: 280-285 83 Suresh K. Inhibition of chitosomal chitin synthetase and growth of Mucor rouxii by nikkomycin Z. Otto H-H. Ruiz-Herrera J (1987) Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin. C Jeuniaux. Lopez-Romero E (1993) Nikkomycin-resistant mutants of Mucor rouxii . G Roberts (eds): Advances in chitin seience. Koga D (1986) Insect chitin. J Bacteriol176: 5857-5860 98 Sudoh M.Biochemistry of chitin synthase 35 77 Choi W-J. RAA Muzzarelli. 203. DuranA. voll. voll. DuranA (1991) CALl. Duran A. Takagi M. Proc Natl Acad Sei USA 87: 7424-7428 80 Valdivieso MH. 102-107 89 Merz RA (1997) Die chitosomale Chitinsynthase von Mucor rouxii: Lektinaffinität und hämolytische Aktivität. GM Gadd (eds): The growing fUngus. synthesis. Rast DM (1985) A comparison ofthe chitin synthase-inhibitory and antifungal efficacy of nuc1eoside-peptide antibiotics: structure-activity relationships.physiological and biochemical properties. Antonie van Leeuwenhoek64: 27-33 97 Gaughran JP. SchaUer E. Lyon. Ruffuer HP. J Cell Bio1114: 101-109 81 Cabib E (1994) Nomenc1ature ofgenes related to chitin synthesis. 108-113 93 Müller H. Rast DM (1981) Metabolic products of microorganisms. Chapman & Hall. Cabib E. In: A Domard. Ancona. Insect Biochem 16: 851-877. Horsch M. Ruffuer Hp. London. Nagahashi S. Guiterrez-Corona F. Nguyen P-T (1985) Die Fungizide Hexachlorbenzol und Pentachlomitrobenzol und ihre Stoffwechselwege. C Jeuniaux. FEMS Microbiol Lett 28: 205-211 95 Cabib E (1991) Differential inhibition of chitin synthetases I and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins. 277-299 85 Cabib E. Rast DM (1996) Assessment of the mode of action of chitinases with anion exchange chromatography/pulsed amperometric detection. . Jaques Andre. Shaw JA. 13 7 -146 88 Merz RA. Yamada-Okabe H. degradation and metabolic regulation. Cabib E (1994) Are yeast chitin synthases regulated at the transcriptional or the posttranslationallevel? Mol Cell Bio114: 7685-7694 78 TrillajA. PhD thesis. Toxicol Environ Chem 10: 119-132. RAA Muzzarelli (eds): Advances in chitin seience. a gene required for activity ofchitin synthase 3 in Saccharomyces cerevisiae. Watanabe M. Mol PC. In: RAA Muzzarelli (ed): Chitin enzymology. Yeast Newsl XLIII: 58 82 Martinez-Cadena G. Insect Biochem 15: 141-145 92 Mayer C. nikkomycin X.

Chanzy H. Robbins pw. J Mol Bio1231: 735-752 122 Vrielink A. Johnston AWB. Gooday Gw. Levins TA. J Med ~tMycoI34: 117-125 119 Nino-Vega GA. Blundell TL (1993) Alignment and searching for common protein folds using a data bank ofstructural templates. Winter KR. Downard J (1997) Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding. Iartchouk N. Nature Struct Biol2: 117-120 . Fungal Gen Bio120: 153-167 115 Borgia PT. Fungal Gen Biol 22: 199-208 117 Miyazaki A. Dworkin M. Bulawa CE (1996) The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Poirrette AR. Culp DW. Holsters M (1994) The NodC protein of Azorhizobium caulinodans is an N-acetylglucosarninyltransferase. J Biol Chem 271: 22945-22948 105 Varki A (1996) Does DG42 synthesize hyaluronan or chitin? A controversy about oligosaccharides in vertebrate development. Buurman ET. Robbins pw. EMBO J 13: 3413 . Mol Microbiol19: 443-453 109 Pringle JR (1991) Staining ofbud scars and other cell wall chitin with Calcofluor. Riggle PJ. A. Liu Y. Mergaert p. Szaniszlo PJ. Freemont PS (1994) Crystal structure of the DNA modifYing enzyme ß-glucosyltransferase in the presence and absence of the substrate uridine diphosphoglucose. Rüger W. Merz et al. Borgia PT (1996) The chsD and chsE genes of Aspergillus nidulans and their roles in chitin synthesis. Fungal Genet Bio120: 193-203 116 XoconostIe-Cazares B. Geelen D. In: RAA Muzzarelli (ed): Chitin enzymology. Winter KR. Specht CA. Rice DW. Holden DW (1995) A multigene family related to chitin synthase genes ofyeast in the opportunistic pathogen Aspergillus jUmigatus. Mol Gen Genet 246: 353-359 114 Specht CA. Price NJ. Proc NatlAcad Sei USA 89: 519-523 112 Bulawa CE (1993) Genetics and molecular biology of chitin synthesis in fungi. Robbins pw. Iartchouk N. Sowdhamini R (1996) A fold prediction for the catalytic domain of chitin synthases. Gay L. San-Blas G. Methods Enzymo1194: 732-735 110 Ramaswamy S. a gene coding for a class IV chitin synthase in Ustilago maydis. Italy. Young R.or ergosterol-containing phosphatidylcholine monolayers. Bulawa CE.3422 123 Artymiuk PJ. Peng M. J Bacteriol 179: 2863-2871 111 Bowen AR. Robbins PW (1992) Classification offungal chitin synthases. Tancrede P (1997) The effect of aggregation state of amphotericin-B on its interactions with cholesterol. Chen-Wu JL. Willett P (1995) ß-Glucosyltransferase and phosphorylase reveal their common theme. Ruiz-Herrera J (1997) Umchs5. vol 2. Riggle P J. Economou A. Specht CA. Mendoza L. Van Montagu M. 447-458 121 Johnson MS. Momany M. Downie JA (1996) The C-terminal domain of the Rhizobium leguminosarum chitin synthase NodC is important for function and determines the orientation ofthe N-terminal region in the inner membrane. J Gen Appl Microbiol43: 333-340 118 Karuppayil SM. Szaniszlo PJ (1996) Identification ofthe conserved coding sequence of three chitin synthase genes in Fonsecaea pedrosoi. Girard V. Schoonejans E. Ootaki T (1997) Multiple genes for chitin synthase in the zygomycete fungus Phycomyces blakesleeanus. 101 Bolard J (1986) How do the polyene macrolide antibiotics affect the cellular membrane properties? Biochim Biophys Acta 864: 257-304 102 Barwicz J. Fevre M (1992) Characterization of chitin synthase from the cellulosic cell wall fungus Saprolegnia monoica. Leon C. Atec Edizioni. Yeast 14: 181-187 120 Horsch M. Koltin Y. Driessen HPC. Exp Mycol16: 8-21 104 Watanabe K. Proc Natl Acad Sei USA 91: 2669-2673 107 Carlson RW. Proc NatlAcad Sei USA 93: 4523-4525 106 Geremia RA. Stacey G (1994) The biosynthesis ofrhizobiallipo-oligosaccharide nodulation signal molecules.36 R. Aufauvre-Brown A. Liu Y. Annu Rev Microbiol47: 505-534 113 Mellado E. Overington JP. Dodge CL. Mol Plant-Microbe Interact 7: 684-695 108 Barny M-A. Chem Phys Lipids 85: 145-155 103 Bulone V. Rhodes JC. Yamaguchi Y (1996) Molecular identification of a putative human hyaluron synthase. Gow NAR (1998) Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis.

Bracker CE. ASM News 64: 31-39 142 Rudd PM. Gooday GW (1984) Studies on the purification ofchitin synthase from Coprinus cinereus. Trends Microbiol 3: 98 -104 141 Kurtz MB (1998) New antifungal drug targets: avision for the future. Terzaghi EA. sensitive sequencing ofoligosaccharides from glycoproteins. Sander C (1995) Evolutionary link between glycogen phosphorylase and a DNA modifying enzyme. Nucleic Acids Res 18: 6690 128 Rosa F. Tkacz JS (1995) The fungal cell wall as a drug target. Lopez-Romero E. J Biol Chem 268: 19181-19184 130 Glucksmann MA. Jonas E. Exp Mycol 2: 173-192 137 Marikar Y. Yarden 0 (1996) CHS-4. Ruiz-Herrera J (1978) Isolation of chitosomes from taxonomically diverse fungi and synthesis of chitin microfibrils in vitro.52 135 Flores-MartinezA. Bartnicki-Garcia S (1989) Sedimentation properties of chitosomal chitin synthetase from the wild-type strain and the 'slime' variant of Neurospora crassa. Sentandreu R (1987) Separation of chitosomes and secretory vesicles from the 'slime' variant of Neurospora crassa. Dawid IB (1988) Accumulation and decay of DG42 gene products follow a gradient pattern duringXenopus embryogenesis. Martinez JP. Curr Opin Biotechnol8: 488-497 144 Rudd PM. J Biochem Biophys Meth 27: 127-132 139 Carrington DM. Juranville J-F (1993) Reduced binding capacity of concanavalinA-sepharose after treatment with chaotropic agents. Cabib E (1988) Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae. Saito M (1993) Purification and characterization ofmembrane-bound chitin synthase. a class IV chitin synthase gene fromNeurospora crassa.Biochemistry of chitin synthase 37 124 Holm L. Gil ML. Reyes E. Martinez JP. Walker GC (1993) Family of glycosyl transferases needed for the synthesis of succinoglycan by Rhizobium meliloti. Grunz H. Slater ML. Arch Microbiol149: 156-162 134 Martinez JP. Gimenez G. Dwek RA (1997) Rapid. J Biol Chem 268: 1702-1707 133 Ruiz-Herrera J. Opdenakker G. EMBOJ 14: 1287-1293 125 Silverman SJ. Specht CA. Nature 313: 64-67 140 Georgopapadakou NH. Robbins PW. BartnickiGarcia S (1990) Protein composition ofpurified chitosomes of Mucor rouxii.297 132 Machida S. Rebbert ML. Dev Bio1129: 114-123 129 DeAngelis PL. Zachariah B. Biochim Biophys Acta 990: 45 . Campbell ID. Sargent TD. Downing AK. Bracker CE. J Bacteriol 175: 70337044 131 Montgomery GWG. Sburlati A. Casanova M. Anal Biochem 201: 306-310 138 Fountoulakis M. J Gen Microbiol130: 291. Proc NatlAcad Sei USA 85: 4735-4739 126 Beth DinAB. Dwek RA (1995) The effect of variable glycosylation on the functional activities of ribonuclease. Biochim Biophys Acta 1248: 1-10 . Ruiz-Herrera J. Exp Mycol 14: 160-168 136 Bartnicki-Garcia S. Weige1 PH (1993) Molecular cloning. Papaconstantinou J. Crit Rev Biochem Mol Bio132: 1-100 143 Rudd PM. Winkles JA. Michaels GS. Mol Gen Genet 250: 214-222 127 Collins-Emerson JM. Adams DJ. Scott DB (1990) Nucleotide sequence of Rhizobium loti nodC. Woods RJ. plasminogen and tissues plasminogen activator. Jarnrich M. identification. Dwek RA (1997) Glycosylation: heterogeneity and the 3D structure of proteins. Basu D (1992) Leaching of concanavalin A during affinity chromatographic isolation of cell surface glycoproteins from human fetal neurons and glial cells. Reuber TL. and sequence of the hyaluronan synthase gene from group A Streptococcus pyogenes. Hanke DE (1985) Polypeptide ligation occurs during posttranslational modification of concanavalin A. Auffret A. Bracker CE. Wormald MR.

Chitin. is immersed in a matrix of proteins and other polysaccharides.A. Introduction Throughout evolution. (ii) transport of the chitin molecules to the extracellular space. keep the microfibrils separate. synthesis of chitin occurs as the result of a transglycosylation reaction catalyzed by membrane-bound enzymes collectively called chitin synthetases. and deposition of different substances.Chitin and Chitinases ed. Many organisms utilize chitin as a structural component of the protective cell walls or exoskeletons which surround them. These utilize the nuc1eotide uridine diphosphate N-acetylglucosamine (UDPGlcNAc) as the corresponding sugar donor. many organisms have utilized chitin as a structural component. the basic biochemical mechanisms appearing to be rather uniform. its transport to the exocellular space where it crystallizes in the form of microfibrils. and provide support to tensions. Gto. These structures are light and resistant composites with specific structural and mechanical properties which allow them to fulfill their protective role. Mexico Summary. In all the systems studied so far. The resulting supramolecular structure acquires viscoelastic mechanical properties. Unidad lrapuato. in the form of microfibrils. Martinez-Espinoza Departamento de Ingenier{a Genetica. Centro de Investigacion y de Estudios Avanzados deI Instituto Politecnico Nacional. lrapuato 36500. Apartado Postal 629. (iv) crystallization ofthe unmodified chitin which is covered by the rest of the components. The resulting structures adopt specific forms which are conserved during growth and are transmitted in a hereditary fashion. The cementing compounds protect chitin from chemical attack. Synthesis of these complex structures involves the following steps: (i) synthesis of chitin either intracellularly or at the interphase with the extracellular medium.A. its most common modifications and its association with other molecules in order to give rise to the protective structures which surround the organisms. Jolles and R. (v) maturation of the composite through formation of secondary covalent bonds among its components. Nevertheless. . preventing fracture. Chitin microfibrils provide the high strength which allows them to resist tensions and modulus. In the following pages we will describe the current general ideas on the mechanisms involved in chitin biosynthesis in vivo. by P. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Chitin biosynthesis and structural organization in vivo Jose Ruiz-Herrera and Alfredo D. (iii) chemical modification of part of the noncrystallized chitin and association with other molecules. the specific mechanism by which the polymer is synthesized in vivo by the different organisms appears to have selective characteristics.

Martinez-Espinoza Cytological basis of chitin biosynthesis Because of its insolubility it has been generally accepted that chitin is deployed in the extracellular space. accumulating in eytoplasmie vesicles during its transit to the eell surface. depending on the method of eell breakage. eonelusive evidence supports the location of the enzyme in the Golgi complex. chitin synthetase follows the exocytie route of membrane-bound proteins. 14]. Ruiz-Herrera and A. Nevertheless. including the wall.40 J. Moreover. Chitin synthetase activity may be detected in almost all membranous fractions from fungal homogenates. as already suggested several years aga [13. soluble precursors and the polymerizing enzyme chitin synthetase itself are synthesized intracellularly. In this sense. by eleetron-microseopie immunoassays. These include (i) findings from electron-mieroscopie autoradiography that chitin synthesis by permeabilized cells of M. bona fide reports demonstrate intracellular synthesis of chitin in fungi. cerevisiae in a fraetion eorresponding probably to the Golgi. where separation of cell-free extracts by isopycnic sedimentation revealed that the low-density membranes containing chitin synthetase aetivity eould beeome aggregated with membranous fraetions of higher density depending on the buffer used [6]. it may be concluded that. which agrees with a membranous location of the enzymes. rouxii oceurs in the cytoplasm. and that UDPGlcNAc is not compartmentalized. is relevant. and (iii) the immunolocalization of chitin synthetase 3 in whole cells of S. location of enzymatic activity in the different membranous fraetions may be considered to be due to an artifaetual aggregation of enzyme-carrying membranes with other particulate fraetions. protozoa and invertebrates [1-3]. Further results have settled this issue in adefinite way. Its relative accumulation in a vesieular form and in its target membrane may explain part of the eonflieting data existing in the early literature. Evidence for this has been described in the fungus Mucor rouxii. from where it is transferred to the eell surface in the form of vesicles [1]. but accessible to the enzyme in the cytosol [4]. (ii) studies which reveal. D. Aecording to all these data. whereas in the protozoan Eufolliculina uhligi chitin synthetase is aceumulated in cortieal vesicles. but not in the cytosol. the description of contradictory results on the loeation of chitin synthetase in homogenates from the yeast Saccharomyces cerevisiae. In arthropods. the loealization of chitin synthetase in apical vesicles (ehitosomes) of Neurospora crassa hyphae [11].9]. Comparison of the predicted amino acid sequences of all chitin synthetase genes cloned thus far demonstrates the existence of a hydrophobie domain loeated towards their C-termini [5]. and not in the plasmalemma [10]. demonstrating the intraeellular location of chitin synthetase in fungi. and not in the plasmalemma [12]. . either plasmalemma [7] or mierovesicles [8. Nevertheless.

Chitosomes are made of protein and lipids in a ratio of 1. Purified chitosomes incubated with UDPGlcNAc and activators synthesize very fine microfibrils which crystallize in their interior. demonstrating that they contain over 80% of the total chitin synthetase from the cells. . and their chemical and enzymatic uniqueness (see below) all silenced initial criticism suggesting that chitosomes might be artifacts ofthe vesiculation oflarger membranes. and the absence of phosphatidylserine in chitosomal membranes [16-18]. extending the fungal results to arthropods. Among polar lipids. polar lipids being more abundant than neutraiones [16. 500 kDa. This composition was quantitative and qualitatively different from the gross of the cell membranes. ID. These microfibrils associate at later periods of the synthetic reaction in the form ofvery thick and short microfibrils [24]. 2).0 nm thick [15] (Fig. lA. 17]. E). 1 C). kinked or straight. In sections they appear surrounded by a thin membrane 6. the fact that harsh or mild cell breakage procedures all gave rise to identical structures. Nevertheless. 14]. All these data helped to identify chitosomes as the conveyors responsible for the transport of chitin synthetase to the cell surface. that is accepting GlcNAc residues from UDPGlcNAc at the cytosolic face. These yields. phosphatidylcholine and phosphatidylserine were the most prominent ones. suggesting that the catalytic polypeptide is active in the form of a multiproteic complex [22]. massive data have accumulated indicating that the structures where chitin synthetase is accumulated in the cytosol are a kind of specialized microvesicles-denominated chitosome [3. whose width has been calculated to correspond to the association of two GlcNAc chains [23]. 16S chitosomal subunits synthesize extreme1y fine chitin microfibrils. in arthropods and protozoa chitin synthetase is accumulated in the Golgi apparatus and specialized vesicles. and eventually breaking apart the chitosomal membrane [15] (Fig. whereas sterols were the most abundant neutral lipids. appearing either coiled. The results reveal that chitosomes synthesize chitin microfibrils through an asymmetrie mechanism. Purified chitosomes appear in the form of spheroidal structures measuring 40-70 nm in diameter (Fig. In fungi. Similar images were obtained during electron microscopic studies of chitin microfibril synthesis by homogenates from the flour beetle Tribolium castaneum [20]. and de1ivering chitin moleeules at the inner face (see Fig. Chitosomes have been purified to a high degree from fungi representatives from the main taxa. 13.4-2.5-7. The most relevant differences were the abundance of ergosterol and glycolipids. Chitosomal subunits are enriched in a few polypeptides.Chitin biosynthesis and structural organization in vivo 41 Cellular location of chitin synthetases and their transport to the sites of chitin biosynthesis: the fungal chitosome As described above. Protein composition of chitosomes is also unique [19]. which conserved enzymatic activity [21]. Treatment of chitosomes with digitonin gave rise to their dissociation in the form of 16S subunits with an M r ca.0. B).

cerevisiae . 2]. In this sense it is important to indicate that in crustaceans and protozoa evidence exists that synthesis of chito-oligomers associated with proteins occurs intracellularly. showing a microfibril bundle crystallized inside a chitosome. Magnification bars: A. is unknown (Fig. these complexes being secreted in a postsynthetic reaction [1. (0) Microfibrils made after incubation of S. O. it has been reported that chitin synthetase is made in the endoplasmic reticulum (ER) and follows the normal exocytic route to the outer surface of the cell in S. Although the origin of chitosomes is uncertain. cerevisiae chitosomes with substrate and activators. C. (E) same as (0). Martinez-Espinoza Figure I. (C) Ultrathin section of chitosomes from M rouxii. B. (A) Negatively stained (uranyl acetate) chitosomes isolated from Mucor rouxii. or once the chitosomes fuse with the plasmalemma. 100 nm. whether the chitin biosynthetic process in whole cell occurs during the transit of the chitosomes to the surface.42 1. 50 nm. Ruiz-Herrera and A. and chitin synthesis in vitro. 25 nm. 0. E. 2). (B) Negatively stained chitosomes from Saccharomyces cerevisiae. Fungal chitosomes.

.. UOPGlcNAc ! UDP / E~ ~ 1 F G ~ 0- ~ 1 - n. chains are synthesized and extruded at the surface of the plasmalemma.. Mg'+. CH.. (0) Crystallization of chitin microfibrils in the ß-configuration..oH Figure 2. Scheme representing chitin synthesis by chitosomes and other chitin-synthesizing microvesic\es in vivo and in vitro.OH NHCOCH. 0 NHCOCH... In (E)... GIcNAc. (C) Representation of the vectorial mechanism involved in (B).. E) Alternate pathways of chitin synthesis in vivo. Notice that chains contain reducing and nonreducing ends represented by open rectangles or filled circ\es. CH. (B) Intrusion of chitin chains into the microvesicle. .. OH 0 _____ .43 Chitin biosynthesis and structural organization in vivo B ® D + Protease... H~ b~o CH.... -~ 'O~H CH....OH NHCOCH... In (D)... (D.. (F) Arrangement of chains in antiparallel fashion. (A) Schematic representation of transmembrane chitin synthetase in the microvesic\es.. - ..----O~OH NHCOCH..OH NHCOCH....Q 0 0 ä --=<i CH.----------------.. chitin chains are made inside microvesic\es.oH H~O~O---------..... NHCOCH..

the polypeptidic composition of chitosomes is unique [19]. tubes of pogonophores. only one being present in nature. In crustaceans. chitin appears to be synthesized in Golgi cistemae associated with proteins which are secreted further on [1]. second. ß chitin. The most abundant. is present in arthropods. whereas in other organisms the size and packing of the chains is more variable. has been identified in the pen of the squid Loligo. different mechanisms appear to operate. two chains would run in one direction. Chitin crystallization and physicochemical characteristics Although the mechanisms for the deployment of chitin in the exocellular structures appears to be different in the several organisms analyzed. 28]). third. chitin synthetase amino acid sequences deduced from cloned chs genes lack a canonical signal peptide [5]. In insects chito-oligomers are made intracellularly. and it is assumed that sugar chains are extruded to the cell wall space. three different crystalline polymorphie forms of chitin exist under natural conditions. made ofparallel chains. D. chaetae from Aphrodite. [29]. where chains are antiparallel. some reasonable doubts exist for these generalizations. as weIl as by nonbonding interactions between the pyranose rings. loricae from some protozoans and the spines of several diatoms.44 1. a universal plasmalemmal enzyme. Also variable is the association of chitin with other components. a chitin. all of them finally lead to its crystallization in the form of microfibrils. In contrast to cellulose. In fungi. In fungi. it is probable that we may apply the same basic mechanisms described for the vesicle transit in general [24]. Ruiz-Herrera and A. no evidence for an association of chitin with proteins during the biosynthetic process has been described. Nevertheless. chitosomes have no additional enzymatic activities besides chitin synthetase [6]. in the opposite direction. whereas in ciliates similar chitoproteins are secreted through an exocytic process involving cortical vesicles [2]. and another. except for the absence of bonding between the sheets in ß chitin. Martinez-Espinoza [12]. where two different crystalline forms exist. First. H . These forms differ in the packing and polarity of the GlcNAc chains.N bonds. fungi and the cysts of Entamoeba (review in [27. and then transported across the plasma membrane to the extracellular space where they become associated with lectin-type proteins [25. chitin microfibrils are usually made of 20-400 sugar chains associated through hydrogen bonding. In animals and protozoa. and is not fullyknown. 26]. According to data from Blackwell et al. In the third form. chitosomes have no adenosinetriphosphatase (ATPase) activity.. both a and ß chitins consist of arrays of sheets or chains stabilized by C = o· . This form has been reported in cocoon fibers ofthe beetle Ptinus and the stornach of Loligo. and fourth. This characteristic . Regarding the mechanisms involved in chitin synthetase traffic and delivery. y chitin.

Through determination of the intrinsic viscosity values of solutions of chitin in N. Oue to its insolubility chitin hardly yields to the normal procedures for molecular weight determination. 2E-G). since solvation of ß or y chitins in HCI or lithium thiocyanate solutions.N-dimethylacetamide-5 % LiCI. First. such as viscoelasticity. The nascent chitin. but varied in stiffness. its modifications and its association with other compounds occurs outside the permeability barrier of the cell. respectively. Unit cell type and dimensions of both chitins are very different. The reason why chitin can crystallize in three different forms remains unknown. posing the problem of how the energy for the reactions involved is made available to the enzymes responsible for the processes. the mechanical properties of the three forms are different. leading to their distinct X-ray difIraction patterns. but becomes resistant and crystalline when it is dried [37]. and therefore the skeletal structures. In vivo crystallization. Nascent chitin can be exposed to several chemical modifications. 36]. 40]. but not the preformed one. ciliates [34] and fungi [35. Analysis ofthe tensile mechanical properties ofthe three forms of chitin revealed that all of them behaved as viscoe1astic polymers. strength and extensibility by several orders of magnitude. but not synthesis of chitin microfibrils. leading to its swelling. reverts them to a chitin [30]. is very sensitive to chitinase [38. reactivity and resistance . their properties being sensitive to wetting [30]. that is differential labelling of chitin and their terminal residues in a synthetic reaction. probably coadjuvating to their different roles. The polymer thus synthesized is very sensitive to chitinase and not crystalline. the fact that in two forms the sugar chains are antiparallel indicates that the processes of synthesis and assembly into microfibrils cannot be simultaneous and must be separated in time (Fig. Second. Some of them change different and important properties ofthe polysaccharide. The idea that synthesis and crystallization of chitin are nonsimultaneous processes rests on different pieces of evidence.Chitin biosynthesis and structural organization in vivo 45 explains the introduction of water molecules in the crystals of ß chitin. 39] and chitin deacetylase [32. is interfered by Calcofluor white and Congo red in algae [33]. but it is possible that the process is affected by the presence of some compounds during crystallization of the polymer. U sing a different approach. molecular weights ranging from 0. All these data reveal the existence of a gap between chitin polymerization and essembly. a Chitin was the stiffest and displayed the highest maximum tensile strength.8 x 10 6 were calculated for different crustacean chitins [31]. Nevertheless the phenomenon has several consequences.8 to 1. Chemical modifications of chitin in the extraceUular space Chitin crystallization in the form of microfibrils. we calculated that chitin molecules of M rouxii are made of over 2000 sugar residues [32].

since it is necessary for the expansion of the cell walls and exoskeletons. Sacculina rotundata and Neptunes sanguinolentus. Substantial evidence exists indicating that chitosan biosynthesis occurs by the deacetylation of chitin. Chitosan was originally reported in the walls of the sporangiophores and mycelium of Phycomyces blakesleeanus [47]. cerevisiae it has been suggested that chitinases playa role in the separation of daughter and mother cells after budding [45]. where spore germination is blocked by allosamidin. Chitosan has been found in several insect species such as Oecophila longinoda. which may playa role in the linkage of chitin with glucan. rather than by de novo biosynthesis involving the action of a specific polymerase. It was suggested that the gene encodes for a peptidoglycan deacetylase. but only UDPGlcNAc has been detected in the cytosol of fungi [40]. This enzyme was later on found to lead to the synthesis of chitosan when cellfree extracts of M.46 J. cerevisiae [49]. in S. has been obtained [32]. It can account for up to 30% of the vegetative cell wall of some zygomycetes. No possible nucleotide precursors made of glucosamine. Accordingly. crustaceans including Lepas sp. Evidence for the participation in the process of a particulate. otherwise the organisms would remain encased into rigid structures. Presumably due to the insolubility of preformed chitin. rouxii containing a mixture of chitin synthetase and chitin deacetylase were incubated with UDPGlcNAc [40]. indicating the universality of the mechanism for chitosan biosynthesis. Martinez-Espinoza to the harmful conditions of the medium [3]. which weaken chitin in an ordered way and facilitate expansion. rather than a soluble form of the deacetylase. and later on in the cell walls ofthe yeast and mycelial forms of M. Deacetylation of chitin probably occurs in its nascent state.. and in vegetative walls of Schizosaccharomyces pombe [50]. 42]. is of vital importance. a specific inhibitor of chitinases [42]. constituting a characteristic component of the cell walls of this fungal group. One of these modifications. Another role of chitinases is cell division. Chitin deacetylases with M r ranging in the order of 35-42 kDa have also been described in Arthropoda [46]. It has been also shown that chitinases have a transglycosidase activity. In fungi. The gene coding for chitin deacetylase from M. . and its sequence revealed strong similarities to several rhizobial nodB genes. Ruiz-Herrera and A. An enzyme able to deacetylate soluble derivatives ofchitin was found by Araki and Ito [51] in Mucor. rouxii has been cloned [52]. thus participating in the macromolecular arrangement of the wall [44]. and some Arachnida and Myriapoda [46]. D. chitinolysis. Anax inmaculifrons and Apis cerana indica. Chitosan is also present in a variety of fungi. An example is Mucor spp. rouxii [48]. the latter is a poor substrate of chitin deacetylase. cell wall expansion utilizes the turgor pressure exerted by the protoplasm as the driving force [43]. this important modification involves the partial and careful degradation of chitin by chitinases [41. It is also present in the ascospore walls of S.

and can associate with additional substances such as lipo- . thus acquiring different properties. Removal ofN acetyl groups makes a polysaccharide resistant to chitinases. but nevertheless it has significant biological importance. In P. Association of chitin with other components Once in the extracellular space. chitin may associate chemically with other components of the cell walls or exoskeletons. blakesleeanus it was observed that fluorescent wheat germ lectin (specific for GlcNAc) bound only to the tip of stage I sporangiophores. However. This results suggests that nascent chitin synthesized at the apex becomes deacetylated to chitosan as it moves to the lateral walls of the sporangiophore. which make it appropriate as a cementing compound.groups. providing hardness and rigidity.3). it is thought that chitosan does not playa structural role. its ability to neutralize anionic charges. This reaction is very important. Of these. inc1uding extreme cases. indicating that the compound is indeed made through the deacetylation of chitin [53]. chitosan formation allows stretching of the cutic1e. as in the physogastric queens oftermites [46]. these products form reactive a-quinone or p-quinonemethide derivatives which cross-link proteins and chitin via Michael-type conjugate addition and Schiff's base formation with free amino or OH. Also important are the mechanical properties of chitosan. Sc1erotization involves the formation of adducts of chitin and proteins with the oxidation products of diphenolic substrates. cerevisiae lack chitosan. the most important ones appear to be N-acetyldopamine and N-ß-alanyldopamine (Fig. Due to its physical properties (easy hydration). The resulting chitin-protein complexes gain stability. In arthropods. In this way covering ofthe chitin microfibrils with chitosan protects the integrity ofthe cell wall [32. Chitosanases are less abundant than chitinases in normal habitats. where it covers the chitin microfibrils [54]. Proteins then "harden" or sc1erotize. Association of chitin with proteins has been described in a large number of invertebrates (reviewed in [56]). and perhaps its capacity to accumulate important ions within the wall. Neverthe1ess in viva and in vitra data obtained by double label revealed that the most important associations between phenolic compounds and chitin occur through noncovalent bonds.Chitin biosynthesis and structural organization in vivo 47 That deacetylation of chitin is the process for chitosan synthesis in viva is supported by two lines of evidence. In insects these properties allow movement and limit expansion [41]. Through oxidation. Proteins form covalent links with chitin. but pliable and flexible. normal functions of insect integument depend on sc1erotization. the lectin bound to the whole sporangiophore. giving rise to structures with higher strength. Disruptants in the gene coding for chitin synthetase 3 from S. when they were treated with a substance that degrades chitosan (HN0 2 ). 55]. which in the case ofinsects stabilize the sc1erotized cutic1e [56a].

(B) N-ß-alanyldopamine.6 glucans and the C6 of GlcNAc [63].4 linkage between the reducing end of GlcNAc and the nonreducing end of ß-l. Two phenolic compounds involved in sc1erotization of insect integument. Martinez-Espinoza A 0 11 HO '-':::: HO ~ B NH-C-CH 3 0 11 HO '-':::: HO ~ NH-C-CH 2-CH 2-NH 2 Figure 3. In Ustilago maydis. Chitin can also associate with the proteins called arthropodins or sclerotins to create stable glycoprotein conjugates.48 1.3-glucan [64]. In Zygomycetes which do not contain . D. which provide impermeability properties to the exoskeletons. These associations can also be found in the cyst walls of Rhizopoda and Ciliata [57]. evidence exists that chitin also associates with glycoproteins. the shells of inarticulate Brachiopoda and Mollusca. (A) Nacetyldopamine. such as galactomannan [41] and glucans [62]. Chitin-protein aggregates provide a substrate for calcium and silica deposition. These associations occur through different covalent linkages. In fungi. a linkage involving the glucosereducing end of ß-l. as well as the envelopes of eggs. for example with the involvement of basic amino acids [62]. A substantial number of wall proteins are resistant to extraction with detergents and can be rendered soluble only after treatment with chitinase [59]. Chitin also forms conjugates with carotenoids. 30% ofthe protein was extracted by the action of hydrolases [60]. This mineralization process seems to be common in crustaceans and mollusks. where it provides rigidity and ensures stability to the exoeskeletons [41. 58]. These associations transform formerly soluble glucans into insoluble derivatives [65]. Reports exist which document the association of chitin with other components inc1uding polysaccharides. cysts and masticatory organs of different invertebrates [41]. and a ß-l. probably through aspartyl or histidyl residues [57]. Ruiz-Herrera and A. It has been suggested that mannoproteins regulate the formation of cross-links between polymers [61]. proteins and waxes. giving color to tissues in insects and crustaceans [41].

65]. 4). Organization of chitin in the form of coherent composites Cell walls and exoskeletons are not made by the simple and disordered aggregation of different chemical compounds. The resulting structures behave like composites. chitin in the form of microfibrils is immersed in a matrix of proteins and other polysaccharides [3. Accordingly. They adopt specific forms. . some of them extreme1y elaborate. part of the chitin is the substrate for important modifications. regeneration starts with the deposition of chitin microfibrils around the cell. preventing fracture. light and resistant with specific structural and mechanical properties which allow them to fulfill their protective role [66]. chitin associates with chitosan. the chitin microfibrils providing them with high strength to resist tensions and modulus (Fig. As described. around which protective structures are made. true composites.Chitin biosynthesis and structural organization in vivo 49 glucans in their cell walls (except for the spores). Elaboration of the supramolecular structure of these composites involves the association of their components in an orderly fashion with the formation of a three-dimensional complex structure. it may be suggested that chitin serves as the organizer or the anchor. Although chitin is not the most abundant component in the cell walls and exoskeletons. (iv) The unmodified chitin crystallizes and is covered by the rest of the components. and forms interactions with other polymers such as polyuronides [55. (iii) In its noncrystalline stage. whereas chitin digestion originates the complete destruction of the structures. The resulting supramolecular structure acquires viscoelastic mechanical properties. In fungal protoplasts. The resulting structure matures into a composite which provides protection to the organism. 59]. which are conserved during the growth ofthe organism and are transmitted in a hereditary fashion. and provide support to tensions. upon which proteins start to be deposited [59]. and associates with other molecules. They are in fact coherent structures. it is easy to understand that its association with other chemical components is of great importance. Since chitin is the main skeletal component of these structures. Similarly. removal of the rest of the chemical components leaves a ghost that retains the original shape. keep the microfibrils separate. (ii) Chitin molecules are transported to the extracellular space. The cementing compounds protect chitin from chemical attack. lorica formation by ciliates involves primarily the association of chitin microfibrils into a structure over which other components are deposited later on. We can summarize the steps giving rise to either cell walls or exoskeletons as follows: (i) Chitin molecules are synthesized either intracellularly or at the interphase with the extracellular medium.

Acknowledgments Original work from the authors was partially supported by Consejo Nacional de Ciencia y Tecnologia and CONCYTEG. (C) Positive replica ofthe cyst wall from Entamoeba invadens. O. Chitin was stained with colloidal gold-tagged wheat germ lectin. 1. rouxii showing the microfibrillar arrangement of chitin in the wall. rouxii. voll. is member ofthe Centro lnternacional de Ciencias. Walker AN (1993) Crustacean chitin synthesis and the role of the Golgi apparatus: in yiyo and in vitro studies. References Horst MN. voll . (0) Fragment of isolated Neurospora crassa cell wall stained with ferritin-labelled wheat germ lectin.118 2 Mulisch M. Markmann-Mulisch U (1993) Chitin synthesis in the ciliated protozoan Eufolliculina uhligi.50 1. In: RAA Muzzarelli (ed): Chitin enzymology. 109. Cuernavaca. Grottammare. Martinez-Espinoza Figure 4. Alkali-treated wall shows the presence ofrandomly-oriented chitin microfibrils on the outer surface. Ruiz-Herrera and A. 119. (A) Section of a gerrninating spore of M . Mexico. are National Investigators. Grottammare. Atec. 100 nm. Magnification bar. In: RAA Muzzarelli (ed): Chitin enzymology. Atec. Scherrnuly G. E. R. M. 1. H. H. o. (8) Tangential section of a germinating spore of M . Mexico. and A. R.128 . Walther M. Chitin in the cell walls of fungi and cyst wall of amoeba.

Bracker CE. Lopez-Romero E. Ruiz-Herrera J (1981) Lipid analysis of chitosomes. BartnickiGarcia S (1990) Protein composition of purified chitosomes of Mucor rouxii. Sjollema KA. Bracker CE (1980) Dissociation of chitosomes by digitonin into 16S subunits with chitin synthetase activity. Arch Med Res 26: 315 . Bartnicki-Garcia S (1987) Intracellular localization of UDP-Nacetylglucosamine in Neurospora crassa wild-type and slime mutant strains. Ruiz-Herrera J. Exp Mycolll: 278-286 5 Ruiz-Herrera J. A Zabza. Atec. Bracker C. Nyhlen LE. Lopez-Romero E. Can J Microbiol31: 1120-1126 18 Lopez-Romero E. Rast DM (1983) Dissociation and reconstitution of chitosomes. Rast DM (1985) Cellular and chitosomal lipids of Agaricus bisporus and Mucor rouxii.63 25 Retnakaran A.Chitin biosynthesis and structural organization in vivo 51 3 Ruiz-Herrera J (1992) Fungal cell wall: structure. 162 9 Leal-Morales CA. Further R. Cambridge. Cerbon J. Bracker CE. Bracker CE (1979) Chitosomes and chitin synthesis. Gimenez G. 547-565 26 Retnakaran A. J Gen Microbiol 130: 1193 -1199 11 Sietsma JH. Beth Din A. voll. Exp Mycol 5: 349356 17 Weete JD. Biochim Biophys Acta 629: 201-216 22 Lending C. Ruiz-Herrera J. Bartnicki-Garcia S (1991) Purification and characterization of l6S chitin synthetase partic1es from cell walls of Mucor rouxii. Biochim Biophys Acta 745: 121-133 24 Rothman JE (1994) Mechanisms of intracellular protein transport. Proc NatlAcad Sei USA 72: 3952-3955 8 Hanson B. Snyder M (1997) Targeting ofchitin synthase 3 to polarized growth sites in yeast requires Chs5p and My02p. Bartnicki-Garcia S (1976) Structure and transformation of chitin synthetase partic1es (chitosomes) during microfibril synthesis in vitro. Oberlander H (1993) Control of chitin synthesis in insects. Cabib E (1975) Chitin synthetase zymogen is attached to the yeast plasma membrane. MacDonald A (1988) Biosynthesis and deposition of chitin in insects and interference with this system as a means of contro!. Xoconostle-Cazares B (1995) Molecular and genetic control of chitin biosynthesis in fungi. Abstracts Annual Meeting oJ the Soeiety Jor Complex Carbohydrates. Cambridge University Press. Ruiz-Herrera J. In: JH Burnett. FEMS Microbiol Lett 30: 369-372 19 Flores-Martinez A. Bartnicki-Garcia S (1984) Sedimentation properties of chitosomes from Mucor rouxii. Protoplasma 122: 178-190 7 Duran A. Wroclaw. Ruiz-Herrera J (1984) Localization of chitin synthase in Mucor rouxii by an autoradiographic method. Martinez-Ramon A. ExpMycol15: 11-25 23 Hanseier E. Exp Mycol14: 160-168 20 Cohen E (1982) In vitro chitin synthesis in an insect: formation and structure of microfibrils. Hanseier E. synthesis and assembly. Marple S (1983) The localization of chitin synthetase in yeast. FL 4 Martinez JP. Wroc1aw Technical University Press.321 6 Ruiz-Herrera J. Bowers B. Nature 372: 55 . J Cell Biol136: 95-110 13 Bartnicki -Garcia S. Grottammare. AP J Trinci (eds): Fungal walls and hyphal growth. Bartnicki-Garcia S. In: RAA Muzzarelli (ed): Chitin enzymology. Amsterdam. Bracker CE. In: C Nombela (ed): Microbial cell wall synthesis and assembly. In: F Sehnal. 89-99 . Elsevier. Martinez Jp. CRC Press. Flores-Martinez A. Yarden 0 (1996) The localization of chitin synthase in membranous vesicles (chitosomes) in Neurospora crassa. Microbiology 142: 1591-1596 12 Santos B. Ruiz-Herrera J (1985) Sterol composition of chitosomes from yeast cells of Mucor rouxii: comparison with whole cells. Ziv V. Monzon E. ProcNatlAcadSci USA 85: 8516-8520 10 Sentandreu R. Leal-Morales CA. Bartnicki-Garcia S (1988) Localization of chitin synthetase in cell-free homogenates of Saccharomyces cerevisiae: chitosomes and plasma membrane. Eur J Cell Bio116: 289-294 21 Ruiz-Herrera J. Boca Raton. 113 -120 15 Bracker CE. DL Denlinger (eds): Endocrinological Jrontiers in physiological insect ecology. 149-168 14 Ruiz-Herrera J (1984) The role of chitosomes in the apical growth offungi. chitin synthesizing microvesic1es from Mucor rouxii. Proc Natl Acad Sei USA 73: 4570-4574 16 Hemandez J.

In: RAA Muzzarelli. Carabez-Trejo A. ER Pariser (eds): Proceedings of the First International Conference on Chitin!Chitosan. Ruiz-Herrera J (1980) Identification of the structural component in the cyst wall of Entamoeba invadens. Sentandreu R. J Gen Microbiol 138 : 97 -1 02 46 Aruchami M.741 29 Blackwell J. Biochim Biophys Acta 58: 102-119 49 Briza P. O'Donell WR (1992) What are the roles of chitinases in the growing fungus? FEMS Microbiol Lett 100: 387-392 43 Ruiz-Herrera J. Polachek I. NewYork. Mulisch M. GW Gooday (eds): Chitin in nature and technology. 7-16 45 Cabib E. In: RAA Muzzarelli. Cambridge 28 Arroyo-Begovich A. In: RAA Muzzarelli. Ruiz-Herrera and A. J Gen Microbiol 130: 2095 . Plenum Press. J Biol Chem 263: 1156911574 50 Sietsma JH. Oxford 42 Gooday G. In: RAA Muzzarelli. Grottarnmare. 108-123 30 Hepburn HR. Shaw JA (1992) Chitinase and chitin synthase 1: counter-balancing activities in cell separation of Saccharomyces cerevisiae. Gowri N. Cambridge. Eur J Biochem 158: 411-415 38 Molano J. Cabib E (1979) An endochitinase from wheat germ: activity on nascent and preformed chitin. Martinez-Espinoza 27 Muzzarelli RAA. Exp Mycolll: 128-140 33 Herth W (1980) Ca1cofluor white and Congo red inhibit chitin microfibril assembly of Poteriochromonas: evidence for a gap between polymerization and microfibril formation. RP Elander. composition and structure of cell walls of filamentous and yeast-like forms of Mucor rouxii. J Gen Microbiol 129: 1577-1582 36 Vannini GL. 124-143 31 Rutherford FA.21 02 41 Muzzarelli RAA (1977) Chitin. Cambridge. J Cell Bio187: 442-450 34 Herth W. Nickerson WJ (1962) Isolation.and ßchitins by X-ray diffraction. Massachusetts Institute ofTechnology. Ruiz-Herrera J (1987) Biosynthesis of chitosan in membrane fractions from Mucor rouxii by the concerted action of chitin synthetase and a particulate deacetylase. Sentandreu R (1983) Calcofluor white alters the assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells. ER Pariser (eds): Proceedings ofthe First International Conference on Chitin/Chitosan. 1. Duran A. Atec. C Jeuniaux. Pancaldi S (1983) Effects ofCongo red on cell wall synthesis and morphogenesis in Saccharomyces cerevisiae. J Parasitol 66: 735 . In: DK Arora. Cambridge. vol. In: RAA Muzzarelli (ed): Chitin enzymology. Biochim BiophysActa 702: 233-236 40 Davis LL. Zhu W. Plant Sei Lett 31: 9-17 37 Vermeulen CA. Massachusetts Institute of Technology. J Gen Microbiol128: 2667 -2674 . Massachusetts Institute ofTechnology. Plenum Press. Pergamon Press. Ellinger A. The second outer layer consits of chitosan. Massachusetts Institute of Technology. Marce1 Dekker. KG Mukerji (eds): Handbook 0/ applied mycology. 182-192 32 Calvo-Mendez C. Chandler HD (1978) Tensile mechanical properties and transconformational changes of chitins. Ruiz-Herrera J. Rico H. Silverman SJ. Wesseis JGH (1986) Chitin synthesis by a fungal membrane preparation. Bartnicki-Garcia S (1984) Co-ordination of chitosan and chitin synthesis in Mucor rouxii. J Biol Chem 254: 4901-4907 39 Lopez-Romero E. vol 4. Austin PR (1978) Marine chitin properties and solvents. Breitenbach M (1988) Chemical composition of yeast ascospore wall. C Jeuniaux. In: RAA Muzzarelli. New York. Poli F. Biochim Biophys Acta 13: 1-9 48 Bartnicki-Garcia S. Winnkler G.52 J. 263-268 47 Kreger DR (1954) Observations on cell walls of yeast and some other fungi by X-ray diffraction and solubility tests. ER Pariser (eds): Proceedings of the First International Conference on Chitin! Chitosan. Donini A. Bartnicki-Garcia S (1982) The inhibitory protein of chitin synthetase fromMucor rouxii is a chitinase. Minke R. Pariser ER (eds) (1978) Proceedings ofthe First International Conference on Chitin!Chitosan. 107-120 35 Elorza MV. GW Gooday (eds): Chitin in nature and technology. Zugenmaier P (1986) Comparison of chitin fibril structure and assembly in three unicellular organisms. NewYork. Wesseis JGA (1990) The occurrence of glucosaminoglycan in the cell wall of Sehizosaccharomyces pombe. Sundara-Rajulu G (1985) Chitin deacetylases in invertebrates. D. Evidence for a transient non-crystalline state of chitin. 281-312 44 Sietsma JH (1993) The role of chitin synthesis and modification in the fungal morphogenesis. Gardner KR (1978) Determination of the structure of a. Martinez JP (1992) Chitin biosynthesis in fungi.

Yeast 8: 1089-1099 54 Herrera-Estrella L. ER Pariser (eds): Proceedings 01 the First International Confrrence on Chitin/Chitosan. Berlin. Breitenbach M (1992) DITIOI (CSD2. the last stronghold ofvitalism. Salinas-Reyes E (1996) Structure and chemical composition of the cell walls from the haploid yeast and mycelial forms of Ustilago maydis. Wesseis JGH (1977) The hyphal wall of Mucor mucedo. Briza P. Sentandreu R (1994) Structural organization of the components of the cell wall from Candida albicans. 5 -I 0 56a Peter MG. Ellinger A. Keller R (1986) Structural studies on sclerotized insect cuticle. I-li . Stucka R. Gopal P. In: JFVVicent. 281-312 60 Ruiz-Herrera J. Polyanionic polymers. Morrneneo S. Jeuniaux C (1985) Chitinoproteic complexes and mineralization in mollusk skeletal structures. Microbiology 140: 1513 -1523 63 Surarit R.79 66 Gordon JE (1980) Biomechanics. JD Currey (eds): The mechanical properties 01 biologica/ materials. differentiation and sexuality. Ruiz-Herrera J (1994) Biogenesis of the cell wall. Kegel G. Sentandreu R (1988) Wall mannoproteins of the yeast and mycelial cells and polydispersity oftheir mannan moieties. Vanaclocha P. J Gen Microbiol134: 1723-1730 64 Kollär R. Shepherd MC (1988) Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans. GW Gooday (eds): Chitin in nature and technology. In: RAA Muzzarelli. Hoppe Seyl Z 331: 269-276 58 Poulicek M. Funga/ Genet Bio/20: 133 -142 61 Elorza MV. Cabib E (1995) Architecture ofthe yeast cell wall. Plenum Press. Exp Mycol7: 362-369 55 Datema R. Ende H. Puertes I. Bouriotis V (1993) cDNA cloning of a chitin deacetylase from Mucor rouxii. Grottammare. Ashwell G. M Meihardt (eds): The mycota l. In: R Muzzarelli. Peträkova E. In: RAA Muzzarelli. C Jeuniaux.Chitin biosynthesis and structural organization in vivo 53 51 Araki Y. Marcilla A. Plenum Press New York. In: JG Wesseis. Biochem Biophys Res Commun 56: 669-675 52 Kafetzopoulos D. C Jeuniaux. 21-28 57 Stegmann H (1963) Nature ofchitin in the cuttle bone. Font-de-Mora J. Carabez-Trejo A. Growth. GW Gooday (eds): Chitin in nature and technology. Schuster T. Ruiz-Herrera J (1983) Light response in Phycomyces blakesleeanus: evidence for roles of chitin biosynthesis and breakdown. SpringerVerlag. The linkage between chitin and ß (I ~ 3) glucan. J Biochem 80: 611-619 56 Jeuniaux Ch (1978) Distribution and quantitative importance of chitin in animals. Leon CG. 7-12 59 Sentandreu R. CALI) a cell cycle regulated yeast gene required for synthesis of chitin in cell wall and chitosan in spore walls. Int Rev Cytol 104: 37 . Cambridge. Feldemann H. voll. Voss-Foucart MF. Vournakis J. In: RAA Muzarelli (ed): Chitin enzymology. Massachusetts Institute ofTechnology. Atec. 431-436 53 Pammer M. J Gen Microbio/134: 2393-2403 62 Ruiz-Herrera J. Cambridge. Thireos G. Mormeneo S. New York and London. Robbins PW. J Biol Chem 270: 1170-1178 65 Wesseis JGH (1986) Cell wall synthesis in apical growth. 1. Iranzo M. Cambridge University Press. Ito E (1974) A pathway of chitosan formation in Mucor rouxii: enzymatic deacetylation of chitin.

The polysaccharide chitin is an important structural component of the cell walls of many fungi. responsible for the synthesis of the primary septum that separates mother and daughter cells. and CSIII. the two cells separate in an asymmetrie al fashion so that most of the chitin remains in the mother cell in a structure called the bud scar. Henar Valdivieso. responsible for the formation ofthe ring (bud scar) where most ofthe cell wall chitin is located. which inc1udes ScCHS3. is also randomly located around the wall ofthe mother cell. In this way. Chitin synthesis is directly governed by an enzymatic activity called chitin synthase (CS).Ä. Finally.Chitin and Chitinases ed.is the most extensively studied. the so-called primary septum. is formed. Angel Dunin and Cesar Roncero Instituto de Microbiologia Bioquimica. a thin disklike structure.3)glucanchitin [2] and ß(1. but the fungal cell wall . a new deposition of chitin occurs in a centripetal fashion in the furrow of the invaginating plasma membrane. General aspects of fungal chitin Chitin synthesis has been described in several systems. highly similar to ScCHS3. . although in a very small percentage of its total amount. while a less conspicuous structure . which are important for the maintenance of wall integrity. Chitin is a minor but essential structural polymer of the wall of Saccharomyces cerevisiae vegetative cells. Universidad de Salamanca.Ä. cerevisiae that CSIII activity also depends on the products of other genes. Spain Summary. several chitin synthase (CHS) genes have been also identified in other fungi. respectively. IV and V.remains on the surface of the daughter cell (for a review see [1]). It is mainly located in a ring that constitutes the neck between the mother and the growing daughter budo At cytokinesis. 11 and III in terms of sequence similarity. The rest are defined as two CHS classes. involved in repair functions at the end of cytokinesis. most of them are similar to ScCHS 1 and ScCHS2 genes and are c1assified in chitin synthases c1asses I. by r Jolles and R. although it has been shown in S. While CHS c1ass V genes have been only identified in filamentous fungi and their functions are unknown. CHS2 and CHS3. Cell separation requires partial degradation ofthe chitin remaining in the mother cell by the action of a periplasmic acidic chitinase. The use ofthe budding yeast Saccharomyces cerevisiae as a biological model allowed the identification of three distinct chitin synthase activities: CSI.the birth scar . are involvcd in the synthesis ofmost chitin in yeast cells. forming part of ß(1. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Chitin synthases in yeast and fungi M.particularly the yeast cell wall . E-37007 Salamanca. Chitin. and this closes the gap between mother and daughter cells. These chitin synthases differ not only in functions but also in catalytic properties. Consejo Superior de Investigaciones Cientificasl Universidad de Salamanca and Departamento de Microbiologia y Gem!!tica. class IV genes.6)glucan-chitin [2] covalently bound complexes. CSII. The catalytic subunit of each of these activities is encoded by separated genes. To date. CHSI.

14]. . Until 1986. For more extensive reviews on yeast chitin synthesis see [13. Later on (see below). haploid cells of complementary mating types form projections (shmoos) toward each other under the stimulus of the corresponding sexual pheromone.4G1cNAc]n + 2n UDP. that is mating and sporulation. even after its solubilisation with detergents.that were indeed responsible for these two new activities was reported: chitin synthase 11 (CSII) [10] and chitin synthase III (CSIII) [11. although it is also present in intracellular vesic1es called "chitosomes" that have been proposed to deliver chitin synthase to the plasma membrane [6]. Valdivieso et al. 12]. this forms a complex with a dityrosinerich layer that is responsible for the resistance of the ascospore. H. The work ofBulawa et al. cation dependence and optimum pH. In addition. Chitin synthase activity is located at the plasma membrane [5] where chitin synthesis occurs. has been described (see below for further details). cerevisiae differ significantly with respect to their zymogenic behaviour. During conjugation. Chitin synthases Chitin synthases use uridine-diphospho-N-acetylglucosamine (UDPG1cNAc) as substrate and catalyse the reaction 2n UDPG1cNAc ~ [G1cNAc-ß-l. Chitin synthesis occurs as a transmembrane process in a vectorial way in which the substrate is located on the intracellular side and the product is extruded to the outside of the membrane. During this process. were detected independently. Finally. during sporulation a layer of chitosan is formed in the ascospore walls [4]. In cell extracts from chsl strains two different chitin snythase activities called "chitin synthase 2" [9] and "chitin synthase 11" [7]. another chitin synthase activity had to be responsible for in vivo chitin synthesis. very specific chitin synthase competitive inhibitors. differential inhibition of these CSs by polyoxin D and nikkomycins.CHS2 and CHS3 . chitin is synthesised and is deposited mainly in the subapical portion of the shmoo [3]. it was thought that only one chitin synthase activity exists in yeast. Obviously. between 120 and 170 G1cNAc units in length [7]. The in vitro activity of chitin synthase from many fungi is stimulated by controlled pretreatment of the enzyme preparation with a protease. Chitin synthesis also takes place in other events of the S. This is why this activity has been traditionally considered as zymogenic [1]. where the polysaccharide is located. respectively. The chitin synthesised in vivo and in vitro is quite uniform in size.56 M. cerevisiae life cyc1e. The three CSs described so far in S. [8] described a mutant defective in in vitro zymogenic chitin synthase activity that had neither a growth defect nor a reduced rate of chitin synthesis. the existence of two different genes .

Indeed. the efficiency of zygote formation in crosses in which the two complementary strains were chsl was identical to that found in the control experiment [20]. This and other results raise the possibility that the temporal and spatial regulation of chitin synthesis carried out by CSI may be mediated by the mobilisation of an endosomal pool of CHSI enzyme [21]. Therefore. cerevisiae CHSI gene encodes a protein of 130 kD and constitutes the structural gene for chitin synthase I (CSI) (Tab. when chsl cells were grown in minimal medium (more precisely.Chitin synthases in yeast and fungi 57 Chitin synthase I The S. Treatment of S.!! cells with the complementary pheromone (a-factor) promotes a three. After the gene (CHSI) had been cloned. Chs 1p resides partly in the plasma membrane and partly in the chitosomes. 18]. When grown on complex media. which confer inducibility on CHSI transcription. or by adding sorbitol as an osmotic stabiliser. the possibility that CSI might be activated by acidic culture conditions cannot be ruled out.to fourfold increase in chitin content [3].21]. and hence in its absence septum formation may be impaired during cell separation. The CHSI gene may playa role in mating. These data point to the notion that Chs 1p counterbalances the physiological lytic effect that chitinase exerts at the time of cytokinesis to separate mother and daughter cells. However. its disruption demonstrated that it was not involved in chitin synthesis in vivo and that it was not an essential gene. cerevisiae . 1). It has been shown that chitosome production is blocked in an endocytosis mutant (end4-I). Addition to the medium ofthe chitinase inhibitor allosamidin or disruption ofthe CTSI gene. CSI levels and the product of CHSI (Chslp) remain constant during vegetative growth in synchronised cells [18. these lysed buds were seen to have a hole located in the centre of the birth scar [15]. which codes for an acidic chitinase [16]. When observed under electron microscopy. . CHSI is not responsible for chitin synthesis during mating [19]. upon a-factor treatment the levels of CHSI messenger RNA (mRNA) rise rapidly and considerably although transiently [17. However. Some ehs1strains do not show lysis in acidic medium. also al1eviated the phenotype. CHSI has four pheromone response elements in the upstream portion of the gene. perhaps as arepair enzyme during the wall lysis that must occur for the fusion of mating cells. in addition to CSTI the product of at least another unknown gene (named SCSI for suppressor of chitin synthesis) must be involved in this phenotype [16]. CSI is the major CS activity detected in vitra (it accounts for more than 90%). However. the growth rates of the wild-type and isogenic chsl were indistinguishable. small refractile buds were produced [8]. However. It therefore seems that this activity is not regulated by synthesis and degradation. This phenotype could be prevented by buffering the growth medium. in poorly buffered media).

28] [10.25. sporulation and by calcofluor Posttranslational activator of chitin synthase III.28] CHS3 Regulated transcriptionally and posttranslationally Structural gene for chitin synthase 11 Primary septum No [8.21] Transcription activated by mating factor CHS2 In cytokinesis (repair function) References Other comments Structural gene for chitin synthase I Time or place of chitin synthesis No Essential Proposed enzymatic function CHSI Gene Table 1.27. required for fusion in mating As above but not the ascospore chitosan layer Ascospore chitosan layer? As in CHS3 but not the ascospore chitosan layer Not determined Asin CHS3 Catalytic component of chitin synthase III Required for chitin synthase III Related to CHS4 Required for chitin synthase III Required for chitin synthase III? Required for chitin synthase III No No No No No No CHS4 SHCl CHS5 CHS6 CHS7 Glycoprotein.22.Budding neck ring and lateral wall Glycoprotein. ~ '"o f: ~ ~ . 54.21.18.55] [22.:r: 00 . required for proper Chs3p localization [lA. in mating.] [22] [20.18. 18. 56] [12. unp.17. Genes involved in yeast cell wall chitin synthesis v.15. Trilla. transcription activated in mating. posttranslationally in vegetative cells. g. regulated ascospore chitosan layer Transcription induced during sporulation [22] Required for Chs3p localization.

It has been suggested that the similar regions are related to common cata1ytic functions. The fact that CSI and CSII perform the same biochemical reaction in vitro but seem to have different roles in vivo suggests that these enzymes must be strict1y regulated. reviewed in [22]). The CHS2 gene was sequenced and compared with CHSI. However. 25. Initial observations indicated that no defined primary septa could be observed in strains lacking a functional CHS2 gene [10]. 26]. 24]. which permitted detection of this new activity in extracts from wild-type cells[9]. it appears that all the information required for membrane localisation. contain large vacuoles and in some cases are multinucleated [25]. In addition.t1 cells transformed with a multicopy-plasmid genomic library [10]. small deletions in the homologous regions are deleterious to enzymatic activity and function [23. The conclusion is that the CHS2 gene must be involved in primary septum formation and. Disruption of CHS2 produces several interesting phenotypes. CSI and CSII differ in cation specificity and in their optimum pH values. Instead.Chitin synthases in yeast and fungi 59 Chitin synthase 2 A chitin synthase II (CSII) activity. for both synthases most or all ofthe nonhomologous region can be deleted with little effect on their enzymatic activity in vitro or on their function in vivo.25]. CSII proved to be more sensitive to polyoxin D and NaCI than CSI. As mentioned above. whose levels are approximately 5% of those of CSI. CSII shares some properties with CSI: it is located at the plasma membrane and it is stimulated in vitro by N-acetylglucosamine and by partial proteolysis. also in some related final step of cytokinesis. However. subsequent work demonstrated that the viability of chs2L1 spores ranges from 0 to 90%. perhaps. CSII is involved in the .t1 cells aggregate in clumps. The predicted amino acid sequence of Chs2 was very similar to that of Chs 1 (42% identity in the carboxyl two-thirds of the protein. Thus. indicating that regulation of these truncated proteins has been properly achieved. As was the case for CHSI. are misshapen and abnormally large. chs2. depending on the strain background and on the germination media used [11. However. overexpression of CHS2 in Schizosaccharomyces pombe led to the expression of a CS activity with characteristics similar to those of the activity observed in Saccharomyces cerevisiae. Since no chs2 mutant was available. thick amorphous septa are formed at the neck region between mother and daughter cells. whi1e the amino-terminal regions may be invo1ved in regulation or localisation of the respective enzymes. enzymatic activity and function resides in the homologous regions of CSI and CSII. CHS2 was originally reported to be an essential gene [10].t1 mutants [9]. and chitin is overproduced in up to twofold amounts in comparison with the wild-type level [11. On the other hand. the CHS2 gene was cloned by detecting an increase in the CS activity of chsl. was identified in chsl.

31]. Chs2p degradation seems to occur at the vacuole [28]. but its sequence was seen to be more similar to that ofthe SeCHS2 gene. These two domains have been shown to be essential for CS activity in the case ofthe Chs2 enzyme [24. Chs2p localises to the neck only at the end of mitosis [28]. since mutants in neck filaments are able to form septumlike structures at abnormallocations [29]. 28].60 M. Furthermore. although it has been suggested that the 10-nm neck filaments could be involved in this function. formation of the primary septum (Tab. CHS2 and Candida albicans CHSI genes. H. Two short conserved sequences (Gln-Asp-Arg and Gln-Arg-Arg-Arg-Trp) are present in all chitin synthases. which was . CRS1 and CHS2 homologues in other fungi Using degenerate oligonucleotides corresponding to conserved sequences of CHSl. and it has been proposed to be essential [33]. Valdivieso et al. peaking at the time of septation [18. It is not known what mechanism restricts the localisation of Chs2p at this part of the membrane. However. it must be active immediately before cytokinesis. The CaCHS2 gene. The C. It is therefore concluded that CSII is only present in actively growing cells. As well as this temporal regulation. cerevisiae chsl mutant [32]. This temporal regulation seems to be achieved by transcriptional and posttranscriptional mechanisms. In fact. CSII must be spatially regulated so that it will only be functional at the mother-bud neck. Chs2 protein and the level of CSII activity are cell cycle-regulated. 22]. 28]. Furthermore. in agreement with a cyclic synthesis-degradation mechanism of control. sporulating and stationary-phase cells [18. It is important to keep in mind that for CSII no physiological activator has yet been identified and that there is no evidence suggesting the conversion from a zymogenic precursor to the active form in vivo. therefore. It has been suggested that these classes are taxonomically relevant. Chs2 protein has a very rapid turnover rate [18. All the above results argue against the previously proposed hypothesis that suggested that chitin synthases would be uniformly distributed throughout the membrane and would be proteolytically activated when and where required [1]. CHS2 mRNA. 1). a recently cloned isoform ofthis enzyme is able to complement the morphologic defect of an S. therefore. CHS2 transcription and CSII activity have been shown to be abolished in pheromone-treated. cerevisiae mutant [35]. which were included in three different classes according to the presence of some signature domains [30]. albicans CaCHSl gene was cloned by complementation of the S. Most species had several CS gene homologues. polymerase chain reaction (PCR) techniques were employed to analyse genomic DNA from taxonomically different fungal species. it is likely to be responsible for septum formation [34]. whether the genes of each group have similar functions in vivo remains to be elucidated. 27.

However. Colonies are minute. However. shows a Calcofluor staining pattern similar to that ofthe wild-type strain [33] and does not have reduced virulence [34]. but it is necessary for normal hyphal growth and organisation [42]. is inhibited by polyoxin D and is activated after trypsin treatment when overexpressed in S. the mycelia are not deficient in chitin synthesis. chsDA. have aberrant hyphae and reduced CS activity [47]. fumigatus. mutants grow as weIl as the wild-type [39]. Rhizopus oligosporus chsl and chs2 play some role in the hyphal stage of growth but not in the late stage of spore formation [49]. In other filamentous fungi. These data made AnCHSB a eandidate for the functional homologue of SeCHS2. so far no physiological role has been assigned to these genes. CS activity levels and the chitin content of the mycelial cells. although the gene nomenclature used is slightly different [44-46]. Several CS genes have been eloned in the filamentous fungus Aspergillus nidulans. The Cachs2A. also a dimorphie fungus.Chitin synthases in yeast and fungi 61 eloned by PCR and whose sequenee is very elose to that of the S. However. but its absence has no effeet on morphology or growth rates in comparison with those ofthe wild-type [43]. In Ustilago maydis. implicating these genes in this process. Deletion of this gene produces severe growth defects that eannot be remedied by the presence of sorbitol [39]. 38]. mutants grow slowly. The AnCHSC gene is expressed in the mycelium. and display a strong degree of branching and disorganised lateral walls. do not conidiate. virulence and dimorphie behaviour are not affected in the deletant strains [37. chsAA. Therefore. mating. as is the case for some Oomycetes [50] and for the fission yeast Sehizosaccharomyces pombe [30]. CS homologue genes have also been found in organisms that do not have crystalline chitin in their cell walls. two CS genes (Umchsl and Umchs2) were eloned by PCR. several chitin synthase genes have been isolated. Neurospora crassa chs-l A. has a 20-50% reduction in chitin synthesis in hyphae. but only in a few cases has some in vivo function been assigned. Both genes are expressed in the yeast and the mycelium phases. mutants have normal morphology even though they have redueed levels of CS activity and increased sensitivity to Edifenphos [48]. but chsAA. double mutants displaya remarkable deerease in the effieiency of conidia formation [40]. cerevisiae mutants defective in CSI or CSII aetivities turned out to be able to synthesise chitin in vivo. Their deletion leads to a reduction in growth rates. cerevisiae [41]. cerevisiae CHSl gene. is indueed at the yeast-mycelium transition [36]. chs-2A. strain grows normally. the AnCHSB-encoded enzyme does not contribute to the rigidity of the cell wall. Similar results have been obtained in A. Chitin synthase 3 Since S. it was evident that at least one more CS . The AnCHSB gene encodes a chitin synthase activity that requires Mg ++. However.

Transformation with a multicopy plasmid carrying the CHS3 gene restored the wild-type level of this new activity [12]. overexpression of either CHSl or CHS2 in Saccharomyces cerevisiae or Schizosaccharomyces pombe cells results in increased activity [8. These results confirmed that Chs3 is the structural gene for CSIII. the presence ofNi++ cations inhibits CSI and CSII but does not affect CSIII [52]. CSIII requires the presence of substrate during protease treatment in order to be activated to some extent [53]. The second screening was designed to isolate mutants defective in the incorporation of exogenous 3H-glucosamine into 3H-chitin [22]. a specific function was attributed to CSI. the zymogenic property of CSIII is not simple. activity had to be present in S. respectively. 10]. CHS61CSD3 and CHS7 (see below). First. the activity responsible for chitin synthesis in vivo. Thus. 1) was initially cloned by complementation of the Calcofluor resistance phenotype ofthe chs3 mutant [12]. Additionally. Five complementation groups were isolated in these screenings: CHS3ICALlICSD2. It was shown that chs3 mutants are defective in basal CS activity when assayed in the presence of Mg ++. 12]. they displayed a 10-fold decrease in the amount of chitin present in their cell walls during vegetative growth and mating. T. There are some differences in the characteristics ofthe CS enzymes. two different approaches were used to isolate chitin-deficient mutants. dUlOl and kti2 mutants. Valdivieso et al. cerevisiae cells. whereas that of CHS3 does not ([12]. including trypsin and chemotrypsin. isolated in screenings searching for mutants defective in ascospore cell wall synthesis and in a search for mutants resistant to the Kluyveromyces lactis killer toxin. turned out to belong to the CHS3 group [13]. it was confirmed that CSII catalyses the formation of the primary septum . The CHS3 sequence shares limited but significant homology with the carboxyl portion of CHSl and CHS2. To study chitin synthesis rather than chitin synthases. cerevisiae and other fungi by interfering with the process of chitin synthesis [51]. The first consisted ofthe isolation of Calcofluorresistant mutants [19]. Cos. The CHS3 gene (Tab. CHS3 deletion is not lethaI and prornotes a phenotype identical to that of the original chs3 mutant. the cation dependence of the three chitin synthases is different. 11. Several proteases. These mutants had no apparent defect in growth or morphology and had wild-type levels ofCSI and CSII activities. By the use of appropriate disruptants or mutants that permitted the analysis of the function of each of the chitin synthases in the absence of the other two. H. activate CSI as well as CSII in vitro [1. while simple treatment ofCSIII with trypsin inactivates the enzyme [7. unpublished).62 M. Calcofluor is a fluoro chrome that exerts an antifungal action on S. This result implies that Chs3 must interact with some regulatory components indispensable for activity. CSII and CSIII. CHS4ICAL2ICSD4. Third. While CSI and CSIII have a preference for Mg ++. CSII shows the highest activity when assayed with Co++. 9]. However. Second. CHS5ICAL3.

(C) Chs3p is highly polarized to the base ofnew buds during vegetative growth. B) Chitin synthesis takes place at the base ofthe new bud during vegetative growth (A) or at the base ofthe shrnoo during a-factor treatment (B). Chitin is shown under fluorescence microscopy after staining with calcofluor during 90 min. Strains defective in any of these genes have severely reduced levels of chitin in their cell walls (see [13] and Tab. CHS6 (CSD3) and eHS7. CHS5 (CAL3). 1 for a summary).1) during mating [19] and is required for the synthesis ofthe chitosan layer ofthe ascospore cell wall [27].Chitin synthases in yeast and fungi 63 A • ~' Figure I. Further work with these mutants led to the isolation of four new genes: CHS4 (SKT5 = CSD4 = CAL2). . Chs3p is observed after immunofluorescence staining. eSIII proved to be responsible for the synthesis of a chitin ring that forms at the base of an emerging bud (see Fig. eSIII is also responsible for the synthesis of the chitin that forms at the base ofthe shmoo projection (see Fig. where chitin synthesis takes place. Regulation of eSIII in Saccharomyces cerevisiae The strategies used to obtain mutants deficient in chitin synthesis in vivo [19. 22] allowed the isolation of several other mutants in addition to the already described chs3. As indicated above. (A. eSI has arepair function that facilitates separation between mother and daughter cells [15] (see Tab. 1). 1) and the chitin interspersed in the cell wall [25]. Chitin synthesis is localized during cell cycIe. [25].

the firstworking hypothesis was that these genes would act as regulators of this activity. this transport is impaired.64 M. its function is significantly different from that of Chs5p. but its absence can be compensated by proteolytic treatment of membrane extracts [53. This protein is required for functional CSIII activity in vitro. This hypothesis is in c1ear agreement with the evidence indicating that CSIII activity is regulated at posttranslationallevel [28]. indeed. AB0033I 0). 54]. unpublished data) despite the transcriptional regulation of CHS3 expression observed during the cell cyc1e [27]. Chs5p is also involved in mating. Chs4p also has an important role during mating. has been also recently implicated in Chs3p positioning [56]. and consequently chitin synthesis is defective. overexpression of Chs4p results in a significant increase in CSIII activity [22. which interacts physically with Chs4p and septins. 54]. None ofthem seems to be involved in the expression of CHS3 [20. currently. in the process of cellular fusion. This restricted position of Chs3p also depends on septins. and all the available evidence indicates that they act as regulatory subunits of CSIII activity. Ace. as seen in the c1ear defect in this activity observed in chs5 null mutants [20]. but lacks any role in the synthesis of the chitosan ascospore layer. Beyond these general ideas. Chs4p has another important function in CSIII activity. a fair amount of information about the role of CHS4 and CHS5 gene products in the regulation of CSIII activity is available.54]. specifically. specifically in the formation of mating pairs. CHS5 encodes a protein with a fibronectin type III domain that is involved in the polarised transport of Chs3p to the division site with the participation ofMy02p [20. Valdivieso et al. In addition. Bni4p [56]. Since CHS3 encodes the catalytic subunit ofCSIII [12]. The model evolving from these studies suggests that Chs5p acts in the polarised transport of secretory vesic1es. Recent findings also suggest that there are different regions of Chs4p involved in the localisation or activation of CSIII [56]. inc1uding Chs3p. The exact way in which Chs4p acts in chitin synthase activity is not yet known. The CHS4 gene product. while Chs4p is specifically involved in the localisation ofChs3p at the base ofthe mother-bud neck. although the evidence obtained to date suggests a role ofthis protein in the correct folding ofChs3p in the membrane [54]. Cos. Chs4p. This suggests that Chs4p can act as a direct regulator of CSIII activity (recall that Chs4p interacts physically with Chs3p). however. It has recently been shown that the C. Interestingly. It is tempting to speculate that the role of Chs4p in chitin synthesis during vegetative growth would be assumed during sporu- . 55]. but its function remains to be tested. In the absence ofthis protein. T. H. which mark the localisation for Chs3p through an intermediate protein. These data point to a general role of Chs5p in the polarised transport ofproteins during the Saccharomyces life cyc1e. Apparently. correct positioning ofChs3p at the septum site is required for CSIII to function properly. albicans genome contains a CHS4 homologue (EMBL.

this volume). The reason for this is unknown.4% idendity) that is induced during sporulation [22]. but their CSIII activity is not significantly affected [22. The cloning and characterisation of CaCHS3 [57] andNcCHS4 [58]. In yeast. the CHS3 gene product is not essential but is involved in the synthesis of . In comparison with these genes. This strategy led to the isolation of the ScCHS3 homologues shown in Figure 2 (see figure legend for references). 62].. a highly homologous protein (43. Bakkers et al. it is probable that all fungal CS genes would have derived from a class IV CS ancestor (see J. either Saccharomyces or Candida. two ScCHS3 homologues. CHS3 homologues and function of CSIII activity in other fungi Bowen's c1assification of fungal chitin synthase genes [30] defines three different CHS classes (see above) that exclude the ScCHS3 gene and its possible homologues. and since these genes are more related to class IV CS than to any other CS classes. The lower part of the tree represents a new class of chitin synthases. the upper one included ScCHS3 and all the genes with homologies greater than 50% in this region. but this gene is not involved in chitin biosynthesis [62]. clearly related to class IV [59] and so far described only in filamentous fungi. This philogenetic alignment clearly identified two perfectly separated clusters. probably indicating the evolutionary separation of these two groups of organisms and that in agreement with the different functionality of class IV chitin synthases between yeast and fungi (see below). but very preliminary evidence hints at a role for Chs6p in Chs3p localisation [62]. class V. there is no clear answer to this question. The tree also separates yeast class IV chitin synthases from those of filamentous fungi. All these genes are assumed to code for class IV chitin synthases. However. CHS6 has a very conserved (43. It is noteworthy that CHS homologues have also been identified in beings ranging from bacteria (nodC genes) to higher organisms (DG42 in Xenopus) [22].3% identity) homologue. What is the function of class IV CS in fungi? Unfortunately. This clearly indicates that the catalytic domain of CS (the most conserved part) is very ancient. its function must be significantly different from that Chs4p or Chs5p. partly because only four class IV CS genes have been characterised so far. This figure was obtained using the Clustal algorithm and comparing equivalent regions of all the proteins (from amino acids 1002 to 1107 of ScCHS3). very little is known about CHS6 and its gene product. Confirmation of this hypothesis awaits further work. Null chs6 mutants lack chitin.Chitin synthases in yeast and fungi 65 lation by Shc1p. allowed the design of specific primers for the amplification ofthis gene in other organisms. The function of CHS7 in chitin synthesis has not yet been determined. YKR027. as can be concluded from the characteristic phenotype observed in chs6 mutants.

66

M. H. Valdivieso et al.

-r-t

C.CHS3
SoCHS3
YlCHS3
AgCHS3

-

ScCHS3

I

KlCHS3
HpCHS3
MgCHS3

I

NcCHS4

I

I

ThCHS3
AnCHSE

.

CnCHS3
McCHS3
AfCHSE
AnCHSD
PbCHS3-1
PbCHS3-2

30

25

20

15

10

0

Figure 2. Philogenetic alignment of dass IV and V chitin synthases. The origin of the sequences
is as folIows: SoCHS3 (Swaniomyces occidentalis), YlCHS3 (Yarrowia lipolytica),AgCHS3 (Ahsbya gosypii), SeCHS3 (8. cerevisiae), K1CHS3 (Kluyveromyces lactis), HpCHS3 (Hansenulla
polymorpha), ThCHS3 (Trichoderma harzianum), McCHS3 (Mucor circinelloides) and PbCHS31 (Phycomyces blakesleeanus) are from T. Cos and C. Roncero, (unpublished data), CaCHS3 (C
albicans) [57], MgCHS3 (Magnaesfera griseus) (Specth, c., EMBL Acc.AF020528), NcCHS4
(Neurospora crassa) [58], AnCHSD (Aspergillus nidulas) [61], CnCHS3 (Criptococcus neoformans) (Specth, c., EMBL Acc. AF021318), AjCHSE (AspergillusjUmigatus) [46], and PbCHS32 (Phycomyces blakesleeanus) (A. Perez-Eslava, unpublished data).

most (90-95%) ofthe cellular chitin. However, the biological relevance of
this chitin synthesis is not yet clear since it has been shown that SeCHS3 is
only important for proper maturation of the spore wall [27] and that
CaCHS3 could play some role in virulence [34, 60].
In fungi, NcCHS4 [58] and AnCHSE [40, 61] have been characterised.
Mutants in these genes displayed a minor decrease in chitin content and no
obvious phenotypes during vegetative growth. However, some evidence in
both fungi indicates that class IV es in fungi serves as auxiliary or redundant
enzymes. It is conceivable that the new fungal class V es could assume the
role of yeast class IV in chitin synthesis. However, some work with A. nidulans chsD mutants indicate that this gene, although involved in chitin synthesis, cannot account for the majority ofchitin synthesis in this fungi [40, 61].
Obviously, much more work is required in the characterisation of class
IV and V chitin synthases in fungi, and the emergence in the near future of
new classes of chitin synthases is quite possible.

Chitin synthases in yeast and fungi

67

Acknowledgement
We thank all members of A. Duran 's lab for communicating unpublished results and for their
support during the course ofthis work. Special thanks are due to E. Mellado for helpful COffiments on Aspergillus chitin synthases, to A. Perez-Eslava for the Phyeomyees sequence and to
N. Skinner for correcting the English version. This work was supported by grants CICYT
BI095-0500, Junta de Castilla y Le6n SA13/97 and a EUROFAN 11 project.

References
1 Cabib E, Roberts R, Bowers B (1982) Synthesis of the yeast cell wall and its regulation.
Annu Rev Biochem 51: 763 - 793
2 Kollar R, Reinhold BB, Petrakova E, Yeh HJ, Ashwell G, Drgonova J, Kapteyn JC, Klis FM,
Cabib E (1997) Architecture of the yeast cell wall. Beta (1-7 6)-glucan interconnects
mannoprotein, beta(1 -7 )3-glucan, and chitin. J Biol Chem 272: 17763-17775
3 Schekman R, Brawley V (1979) Localized deposition of chitin on the yeast cell surfacc in
response to mating pheromone. Prae Natl Acad Sei USA 76: 645-649
4 Briza P, Ellinger A, Winkler G, Breitenbach M (1988) Chemical composition of yeast
ascospore wall. The second outer layer consists of chitosan. J Biol Chem 263:
11569-11574
5 Duran A, Bowers B, Cabib E (1975) Chitin synthase zymogen is attached to the plasma
membrane. Prae NatlAead Sei USA 72: 3952-3955
6 Leal-Morales CA, Bracker CE, Bartnicki-Garcia S (1988) Localization of chitin synthetase
in cell-free homogenates of Saeeharamyees cerevisiae: chitosomes and plasma membrane.
Proc NatlAcad Sei USA 85: 8516-8520
7 Orlean P (1987) Two chitin synthases in Saccharomyces cerevisiae. J Biol Chem 262:
5732-5739
8 Bulawa CE, Slater M, Cabib E, Au-Young J, Sburlati A, Adair WL Jr, Robbins PW (1986)
The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in
vivo. Ce1l46: 213-225
9 Sburlati A, Cabib E (1986) Chitin synthase 2, a presumptive participant in septum formation in Saccharamyces cerevisiae. J Biol Chem 261: 15147-15152
10 Silverman SJ, Sburlati A, Slater ML, Cabib E (1988) Chitin synthase 2 is essential for
septum formation and cell division in Saccharomyces cerevisiae. Prac Natl Acad Sei USA
85: 4735-4739
11 Bulawa CE, Osmond BC (1990) Chitin synthase I and chitin synthase 11 are not required
for chitin synthesis in vivo in Saccharamyces cerevisiae. Prac Natl Acad Sei USA 87:
7424-7428
12 Valdivieso MH, Mol PC, Shaw JA, Cabib E, Duran A (1991) CALl, a gene required for
activity of chitin synthase 3 in Saccharomyces cerevisiae. J Cell Biol 114: 101-109
13 Cid VJ, DuranA, dei Rey F, Snyder Mp, Nombela C, Sanchez M (1995) Molecularbasis of
cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev 59: 345-386
14 Orlean P (1997) Biogenesis of yeast cell wall and surface components. In: JR Pringle, JR
Broach and EW Jones (eds): The molecular and cellular biology ofthe yeast Saccharamyces,
vol3. Cell cycle and cell biology, 229-362. Cold Spring Harbor Laboratory Press, NewYork
15 Cabib E, SburiatiA, Bowers B, Silverman SJ (1989) Chitin synthase I, an auxiliary enzyme
for chitin synthesis in Saccharomyces cerevisiae. J Cell Bioll 08: 1665 -1672
16 Cabib E, Silverman SJ, Shaw JA (1992) Chitinase and chitin synthase 1: counterbalancing
activities in cell separation of Saceharamyces cerevisiae. J Gen Micrabiol138: 97 -1 02
17 Appeltauer U, Achstetter T (1989) Hormone-induced expression of the CHSI gene from
Saccharomyces cerevisiae. Eur J Biochem 181: 243 - 24 7
18 Choi W, Santos B, Duran A, Cabib E (1994) Are yeast chitin synthases regulated at the
transcriptional or the posttranslationallevel? Mol Cell Bio114: 7685-7694
19 Roncero C, Valdivieso MH, Ribas JC, Duran A (1988) Isolation and characterization of
Saccharomyces cerevisiae mutants resistant to calcofluor white. J Bacteriol 170:
1950-1954
20 Santos B, Duran A, Valdivieso MH (1997) CHS5, a gene involved in chitin synthesis and
mating in Saccharomyces cerevisiae. Mol Cell Bio117: 2485-2496

68

M.H. Valdivieso et al.

21 Ziman M, Chuang JS, Schekman RW (1996) Chslp and Chs3p, two proteins involved in
chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic
pathway. Mol Biol Ce1l7: 1909-1919
22 Bulawa CE (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu Rev
Microbiol47: 505-534
23 Uchida Y, Shimmi 0, Sudoh M, Arisawa M, Yamada-Okabe H (1996) Characterization of
chitin synthase 2 of Saccharomyces cerevisiae 11: both full size and processed enzymes are
active for chitin synthesis. J Biochem 119: 659-666
24 Ford RA, Shaw JA, Cabib E (1996) Yeast chitin synthases 1 and 2 consist of a non-homologous and dispensable N-terminal region and a homologous moiety essential for function.
Mol Gen Genet 252: 420-428
25 Shaw JA, Mol PC, Bowers B, Silverman SJ, Valdivieso MH, Duran A, Cabib E (1991) The
function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. J Cell Biol
114: 111-123
26 Baymiller J, McCullough JE (1993) Identification of a Saccharomyces cerevisiae mutation
that allows cells to grow without chitin synthase I or 2. Curr Genet 23: 102 -1 07
27 Pammer M, Briza P, Ellinger A, Schuster T, Stucka R, Bteintenbach M (1992) DITIOI
(CSD2, CAU), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls
and chitosan is spore walls. reast 9: 1089-1099
28 Chuang JS, Schekman RW (1996) Differential trafficking and timed localization of two
chitin synthase proteins, Chs2p and Chs3p. J Cell Biol135: 597-610
29 Slater ML, Bowers B, Cabib E (1985) Formation of septum-like structures at locations
remote from the budding sites in cytokinesis-defective mutants of Saccharomyces
cerevisiae. J Bacteriol162: 763-767
30 Bowen AR, Chen-Wu JL, Momany M, Young R, Szaniszlo JP, Robbins PW (1992) Classification offungal chitin synthases. Proc NatlAcad Sei USA 89: 519-523
31 Nagahashi S, Sudoh M, Ono N, Sawada R, Yamaguchi E, Uchida Y, Mio T, Takagi M,
Arisawa M, Yamada-Okabe H (1995) Characterization of chitin synthase 2 of Saccharomyces cerevisiae. J Biol Chem 270: 13961-13967
32 Au-Young J, Robbins PW (1990) Isolation ofa chitin synthase gene (CHSl) from Candida
albicans by expression in Saccharomyces cerevisiae. Mol Microbiol4: 197-207
33 Gow NAR (1994) Growth and guidance of the fungal hypha. Microbiology 140 : 3193 - 3205
34 Mio T, Yabe T, Sudoh M, Satoh Y, Nakajima T, Arisawa M, Yamada-Okabe H (1996) Role
of three chitin synthase genes in the growth of Candida albicans. J Bacteriol 178:
2416-4219
35 Sudoh M, Watanabe M, Mio T, Nagahashi S, Yamada-Okabe H, Takagi M, Arisawa M
(1995) Isolation ofcan CHSIA, a variant gene ofCandida albicans chitin synthase. Microbiology 141: 2673-2679
36 Chen-Wu JL, Zwicker J, Bowen AR, Robbins PW (1992) Expression of chitin synthase
genes during yeast and hyphal growth phases of Candida albicans. Mol Microbiol 6:
497-502
37 Gold SE, Kronstad JW (1994) Disruption of two genes for chitin synthase in the phytopathogenic fungus Ustilago maydis. Mol Microbioll1: 897 -902
38 Xoconostle-Cazares B, Leon-Ramirez C, Ruiz Herrera J (1996) Two chitin synthase genes
from Ustilago maydis. Microbiology 142: 377-387
39 Yanai K, Kojima N, Takaya N, Horiuchi H, OhtaA, Takagi M (1994) Isolation and characterization of two chitin synthase genes from Aspergillus nidulans. Biosci Biotech Biochem
58: 1828-1835
40 Motoyama T, Fujiwara M, Kojima N, Horiuchi H, Ohta A, Takagi M (1997) The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions
in conidia formation. Mol Gen Genet 253: 520-528
41 Tatsuno K, Yamada-Okabe H, Takagi M, Arisawa M, Sudoh M (1997) Properties ofyeast
expressed Aspergillus nidulans chitin synthase B which is essential for hyphal growth.
FEMS Microbiol Lett 149: 279-284
42 Borgia PT, Iartchouk N, Riggle PJ, Winter KR, Koltin Y, Bulawa CE (1996) The chsB gene
of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal
Genet Bio120: 193-203
43 Motoyama T, Kojima N, Horiuchi H, Otha A, Takagi M (1994) Isolation of a chitin synthase
gene (chsC) of Aspergillus nidulans. Biosci Biotechnol Biochem 58: 2254-2257

Chitin synthases in yeast and fungi

69

44 Mellado E, Specht CA, Robbins PW; Holden DW (1996) Cloning and characterization of
chsD, a chitin synthase-like gene of Aspergillus fumigatus. FEMS Microbiol Lett 143:
69-76
45 Mellado E, Aufauvre BA, Gow NA, Holden DW (1996) The Aspergillus fumigatus chsC and
chsG genes encode dass III chitin synthases with different functions. Mol Microbiol 20:
667-679
46 Aufauvre-Brown A, Mellado E, Gow NA, Holden DW (1996) Aspergillus fomigatus chsE:
a gene related to CHS3 of Saccharomyces cerevisiae and important for hyphal growth and
conidiophore development but not pathogenicity. Fungal Genet Biol 21: 141-152
47 Yarden 0, Yanofsky C (1991) Chitin synthase 1 plays a major role in cell wall biogenesis in
Neurospora crassa. Genes Dev 5: 2420-2430
48 Din AB, Yarden 0 (1994) The Neurospora crassa chs-2 gene encodes a nonessential chitin
synthase. Microbiology 140: 2189-2197
49 Motoyama T, Sudoh M, Horiuchi H, OhtaA, Takagi M (1994) Isolation and characterization of two chitin synthase genes of Rhizopus oligosporus. Biosci Biotech Biochem 58:
1685-1693
50 Mort-Bontemps M, Gay L, Fevre M (1997) CHS2, a chitin synthase gene from the
oomycete Saprolegnia monoica. Microbiology 143: 2009-2020
51 Roncero C, Duran A (1985) Effect of Calcofluor white and congo red on fungal wall
morphogenesis: in vivo activation ofchitin polymerization. J Bacteriol170: 1945-1949
52 Choi W; Cabib E (1994) The use of divalent cations and pH for the determination of specific
yeast chitin synthases. Anal Biochem 219: 368-372
53 Choi W; Sburlati A, Cabib E (1994) Chitin synthase 3 from yeast has zymogenic properties
that depend on both the CALl and CAL3 genes. Proc Natl Acad Sei USA 91: 4727 -4730
54 Trilla JA, Cos T, Duran A, Roncero C (1997) Characterisation of CHS4 (CAL2), a gene of
Saccharomyces cerevisiae involved in chitin biosynthesis and allelic to SKT5 and CSD4.
Yeast 13: 795-807
55 Santos B, Snyder M (1997) Targeting ofchitin synthase 3 to polarized growth sites in yeast
requires Chs5p and My02p. JCell Biol136: 95-110
56 DeMarini DJ, Adams AEM, Fares H, De Virgilio C, Valle G, Chuang JS, Pringle JR (1997)
A septin-based hierarchy of proteins required for localized deposition of chitin in the
Saccharomyces cerevisiae cell wall. J Cell Biol139: 75-93
57 Sudoh M, Nagahashi S, Doi M, OhtaA, Takagi M,Arisawa M (1993) Cloning ofthe chitin
synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition.
Mol Gen Genet241: 351-358
58 Din AB, Specht CA, Robbins PW; Yarden 0 (1996) chs-4, a c1ass IV chitin synthase gene
from Neurospora crassa. Mol Gen Genet 250: 214-222
59 Mellado E, Aufauvre-Brown A, Specht CA, Robbins PW; Holden DW (1995) A multigene
family related to chitin synthase genes of yeast in the opportunistic pathogen Aspergillus
jumigatus. Mol Gen Genet 246: 353-359
60 Bulawa CE, Miller DW, Henry LK, Becker JM (1995) attenuated virulence of chitindeficient mutants of Candida albicans. Proc NatlAcad Sei USA 92: 10570-10574
61 Specht CA, Liu Y, Robbins PW; Bulawa CE, Iartchouk N, Winter KR, Riggle PJ, Rhodes JC,
Dodge CL, Culp DW; Borgia PT (1996) The chsD and chsE genes of Aspergillus nidulans
and their roles in chitin syntheiss. Fungal Genet Bio120: 153 -167
62 Trilla JA, Duran A, Roucero C (1999) Chstp, a new protein involved in the control ofprotein export from the ER that is specifically engaged in the regulation of chitin synthesis in
Saccharomyces cerevisiol. J Cell Biol; in press

Chitin and Chitinases
ed. by ~ Jolles and R.AA Muzzarelli
© 1999 Birkhäuser Verlag Basel/Switzerland

Function of chitin oligosaccharides in plant
and animal development
Jeroen Bakkers, Jan W. Kijne, Herman P. Spaink
Leiden University, Institute ofMolecular Plant Sciences, Wassenaarseweg 64,
NL-2333 AL Leiden, The Netherlands
Summary. In plant development chitin oligosaccharides have been studied intensively as part
of the communication between leguminous plants and Rhizobium bacteria. The Rhizobium
bacteria synthesize and secrete lipochitin oligosaccharides (LCOs) to induce the development
of a root nodule, in which the bacteria will infiltrate to start a symbiotic relation with the plant.
Here we will give an overview ofthe biosynthetic route used by the bacteria to synthesize these
LCOs. Perception by the plant will also be discussed as weil as early responses to the LCOs. By
working with the genes from the biosynthetic route, other genes were identified that share
homology with the ehitin synthase genes from Rhizobium. These genes are now isolated from
human, mouse, chick, Xenopus and zebrafish and can be divided into three classes. They are
mainly expressed during early development at the same stage as chitin oligosaccharide synthase
activity can be detected. A controversy has been risen about their biochemical activity and will
be further discussed here.

Introduction
Lipoehitin oligosaeeharides (LCOs) are signal moleeules that were diseovered during the study of the root nodulation proeess in leguminous
plants. Nitrogen-fixing root nodules are the result of an association of
plants with baeteria belonging to the family of Rhizobiaeeae, eommonly
ealled rhizobia. LCOs produeed by rhizobia are key factors in the speeific
reeognition proeess that underlies the formation of infection threads and
root nodules [1-3]. In the absence ofbacteria these LCOs in nanomolar
eoneentrations are able to elicit all the early events normally oeeurring in
the nodulation process such as root hair deformation, preinfection thread
formation, induction of eell divisions and expression ofnodulation-related
plant genes. Here we will give an overview ofthe present knowledge ofthe
biosynthesis and biologie al role of LCOs and related compounds in plant
and also animal development.
Structure and biosynthesis of lipochitin oligosaccharides
The biosynthesis of the LCOs by rhizobia has been studied in great extent
and reviewed in detail by many authors [4-6]. In response to flavonoids
present in the exudate of host plants, rhizobia secrete metabolites, which

72

J. Bakkers et al.

were initially detected due to their ability to elicit root hair deformation,
one of the earliest events in the nodulation process. These metabolites,
named nodulation (Nod) factors, have been purified from a large number
of rhizobial strains. Their chemical structure has been elucidated using
nuclear magnetic resonance and mass spectrometry [2, 7]. Most Nod
factors characterized so far share a common structure, which consists of an
oligosaccharide backbone of ß-l,4-linked N-acetylglucosamine (GlcNAc)
residues, with a fatty acid group attached to the nitrogen ofthe nonreducing
terminus (Fig. I). For this reason these compounds are named lipochitin
oligosaccharides. All rhizobial strains examined produce a mixture of different LCOs. They vary in (i) the presence of additional groups on the reducing or nonreducing terminus of the chitin oligosaccharide backbone,
(ii) the type of acyl chain present on the nonreducing terminus and (iii) the
length of the oligosaccharide backbone. These variations are major determinants of host specificity (for reviews see [5, 8, 9]). examples of host
range-determining modifications are a sulfate group in LCOs of Sinorhizobium meliloti and the O-acetyl group in LCOs of R. leguminosarum
biovar viciae. Mutant strains that lack the sulfate group on their LCOs no
longer nodulate alfalfa roots, the natural host for S. meliloti [1, 10]. LCOs
of R. leguminosarum biovar viciae that lack the O-acetyl group on the nonreducing terminus lost their mitogenic activity in vetch [2]. Also, the presence of special polyunsaturated fatty acids in the LCOs of R. leguminosarum and S. meliloti was shown to be important for these strains to

m
~

~o~o"'o~o\.

~o~o~
ID
/
/

o=c
/,

/,

LI:.LIo:iI

0

OH

~_~o

o=c,

CHs

n

Figure 1. Structure of an LCO molecule with known modifications. The LCO shown here
contains a C 18: 4 fatty acyl group. Different fatty acyl groups have been reported to be present
on LCOs, varying from C16:0 to C20:4. Ac, acetyl; Ara, arabinosyl; Cb, carbamoyl; Fuc,
fucosyl; Me, methyl; S, sulfate.

it was concluded that synthesis of chitin oligosaccharides starts at the reducing terminus. containing a common acyl chain and lacking other modifications of the oligosaccharide backbone [2].Function of chitin oligosaccharides in plant and animal development 73 nodulate their natural host plants [2. coli leads to the synthesis of chitin oligosaccharides which are N-deacetylated at the non-reducing-end residue [17]. the NodC protein can convert uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) into metabolites that migrate as chitin oligosaccharides in thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) systems and are degradable by chitinases. When expressed in E. Since GlcNAc and pnp-GlcNAc also stimulate fungal chitin synthases. It has been shown that the NodC protein synthesizes chitin oligosaccharides by a processive mechanism. The addition of the acyl chain to the terminally N-deacetylated chain oligosaccharides produced by NodC and NodB is mediated by NodA. and higher concentrations of GlcNAc do not affect chain length ofthe chitin oligosaccharides. 16]. since short chitin oligosaccharides are not used as substrates for elongation. Information on the biochemical function of nodulation proteins has mainly come from structural analysis of LCOs produced by strains carrying mutations in nodulation genes and by analysis of LCOs after introduction of cloned nodulation genes of one rhizobial strain into another. it was suggested that a similar mechanism is involved in the synthesis of fungal chitin. It is therefore most likely that NodC does not require lipid-linked intermediates in the synthesis of chitin oligosaccharides. 15]. In this way the functions ofthe nodA. The NodB protein has been characterized as a chitin deacetylase. showing that this component can be used by NodC as a starter for chitin oligosaccharide synthesis [4. It was shown by mass spectrometry that these metabolites are indeed linear oligosaccharides of ß-l. Band C genes have been characterized. The bacterial nodulation (nod) genes are responsible for the synthesis and secretion of the LCOs. NodA-dependent acylation of chitin oligosaccharide back- . Several compounds that inhibit the synthesis of prenyl-linked oligosaccharides or recycling of prenyl carriers do not affect the synthesis of chitin oligosaccharides by NodC [4. This was confirmed by studies on the incorporation ofp-nitrophenyl-ß-N-acetylglucosamine (pnp-GlcNAc) in chitin oligosaccharides by NodC.4-linked GlcNAc residues with a degree of polymerization of 2 to 5 [14. Since GlcNAc can be incorporated by NodC only at the reducing terminus. followed by the characterization of nod gene-dependent metabolites produced by such strains. 11-13]. Expression of nodABC in Rhizobium is sufficient for the synthesis of the core of the LCO. 15]. Insight in the biochemical function of nodulation proteins has also been obtained by expressing nodulation genes in Escherichia coli. This activity is needed for the deacetylation of the nonreducing terminal GlcNAc residue in order to allow a fatty acid to be coupled to the resulting free amino group. coli. The combined expression of nodC together with nodB in E. It was also shown that purified recombinant NodB protein can deacetylate chitin oligosaccharides [18].

Acceptors for acetylation by NodL are GlcNAc. Indeed. coli. 13. using acetyl-coenzyme A (acetyl-CoA) as acetyl donor [23]. which are N-deacetylated at the nonreducing terminal position. Many other modifications of LCOs have been characterized. opposite to the protoxylem poles ofthe vascular bundle [1. as suggested by the inductions of preinfection thread structures in the outer cortex of vetch roots by mitogenic LCOs in the absence ofbacteria [3]. These preinfection thread structures are composed of so-called cytoplasmic bridges in cells of the outer cortex. When LCOs in nanomolar concentrations are applied externallyon the plant root. chitosan oligosaccharides and LCOs. The NodL protein. The NodZ protein. chitin oligosaccharides. which are radially aligned. Besides their role in the formation of the root nodule primordia. bones has been demonstrated in vitro with extracts of E. 2. after overexpression in E. mainly by mass spectrometry. The lowest Km values were observed with chitin oligosaccharides. it has been shown by expression patterns of cell cycle genes that these outer cortex cells reenter the cell cycle but remain in the G2 phase. 25]. chitin tetra-. Of all tested substrates. Bakkers et al. they can elicit in the root cortex the formation ofnodule primordia [1. LCOs are also involved in the bacterial infection process. The formation of cytoplasmic bridges is preceded by polarization of the cell in which the nucleus moves to the center of the cell just as in cells that are about to divide. after overexpression in E. re- . namely the position were young root hairs emerge. meliloti nodA and nodB and extracts of S. many groups have studied their effect on plant development. has been shown to have fucosyl transferase activity using guanosine 5'-diphosphate-a-L-fucose (GDP-fucose) as fucosyl donor. The inner cortex cells that form the nodule primordium continue the cell cycle. penta. An example of a modification at the nonreducing terminus is the addition of an O-acetyl group at C6 of the nonreducing GlcNAc residue by the NodL protein. the primordia are induced only at certain positions in the plant roots. 24]).and hexamers were shown to be the preferred substrates by the NodZ protein [22]. meliloti cells [19-21]. Several factors involved in the signaling cascade induced by LCOs have been identified. Furthermore. giving the impression of cytoplasmic treads that cross the outer cortex. as in plants that are infected by rhizobia. An example of a modification at the reducing residue is the presence of a fucosyl group on the LCOs of Bradyrhizobium japonicum.74 J. Perception of lipochitin oligosaccharides by leguminous plants Since the identification ofLCOs. coli strains expressing S. 25]. coli. and probably many more are still to be discovered (reviewed in [9. has been shown to have acetyltransferase activity. The presence of an O-fucosyl residue at C6 of the reducing GlcNAc residue is due to the activity of the NodZ protein. These modifications are normally divided in two main groups depending on whether they are at the reducing or nonreducing terminus of the chitin oligosaccharide. 2.

For example. Already I h after the addition of nanomolar concentrations of the LCOs isolated from R. With a delay of 10 min. no LCO receptor with specificity and high binding efficiency towards LCOs has been characterized. Chitin oligosaccharides and plant development Schlaman et al. Current research interest has been focusing on how LCO signal perception and transduction are achieved. The factor was identified as uridine and may act as a morphogen in plant roots at picomolar concentrations [28]. For this effect the O-acetylation on the nonreducing terminus of the chitin oligosaccharide . which is important for the induction of a nodule primordium. isolated from the central root cylinder (the stele). is the deformation of root hairs.Function of chitin oligosaccharides in plant and animal development 75 sulting in mitosis [26]. or a host plant may have a number of different LCO receptors each with a different specificity towards LCOs and chitin oligosaccharides. the root hairs start to swell [29]. which makes plant cells respond according to their position within this gradient. At the same time an increase in the cytoplasmic pR in the root hair can be observed [32]. A good candidate for such a factor. It is also possible that these receptors are present at different places of the plant root. stimulates cell division in pea root explants [27]. which can recognize a small range of different LCOs as produced by its symbiotic Rhizobium strain. which do not induce the formation of a nodule primordium. Though binding sites for LCOs are found [34]. Two hours later. It has been suggested that O-acetylation is not essential for recognition of LCOs in root epidermal cells but is required for a response by cells ofthe inner root cortex [35]. in certain root hairs. Interestingly. meliloti LCO is not necessary for plasma membrane depolarization but only for inducing cortical cell divisions [31]. the increase in the intercellular pR in root hairs of alfalfa can also be induced by LCOs that lack the sulfate group. 31]. [36] have shown that O-acetylated chitin oligosaccharides in combination with uridine can induce cell divisions when delivered inside the root cortex of vetch by microballistic targeting. Since some ofthese early events can be induced by other factors. This effect is not observed when LCOs ofheterologous strains are added. This root hair deformation is preceded by a cascade of responses starting with the depolarization of the plasma membrane already after 15 s of LCO addition [30. a regular oscillation of spikes of intracellular calcium of root hairs is detected [33]. after application of LCOs on the plant roots. Most likely this is achieved by a gradient of a plant-derived factor. it was found that the O-acetyl group at the nonreducing terminus of the S. The final result apparently is determined by the position of the cells in the cortex. The first morphological event to be observed. Each plant may have its own specific LCO receptor. a new tip will grow out of the swelling. leguminosarum bv viciae to the roots of vetch. it is not clear what the role of these events is for LCO signaling.

either separately or in combination. In contrast. LCOs of rhizobia species that induce cell divisions in the outer cortex elose to the epidermis only contain common fatty acids [37].5 ng of the NodZ protein resulted in the development of embryos without normal tail structures. Bakkers et al. has a strong effect on the plant growth and also leaf shape [42]. This is in agreement with the finding that special unsaturated fatty acids are present in the LCOs of rhizobia species that induce cell divisions in the inner cortex. This indicates that NodA and NodB interfere with the synthesis of plant moleeules important in plant morphogenesis. suggesting that the chitinase releases a signal with structural homology with LCOs [40]. When LCOs or chitin oligosaccharides are added to suspension-cultured tomato cells. These effects . a rapid and transient alkalinization of the culture medium occurs [38]. suggesting that tomato cells recognize the chitin oligosaccharide by a receptor and release a signal upon stimulation. suggesting a role in early embryogenesis [41]. a nonhost of rhizobia. This could mean that the fatty acyl group has a ftmction in the delivery ofthe chitin oligosaccharide backbone inside the plant root. which share a structural homology with chitin oligosaccharides. In carrot plants this chitinase (ep3) is expressed in the tissue surrounding the developing carrot embryo. These compounds labeled by 14C acetate or 35S sulfate were very hydrophobic and migrated on TLC like LCOs. a carrot temperature-sensitive somatic embryogenesis mutant can be rescued by the addition of achitinase purified from wild-type cultures [39]. Injection of zebrafish embryos in the one-cell stage with the rhizobial NodZ fucosyl transferase protein caused severe developmental defects in those embryos [44]. In agreement with this is the finding that chitinase-degradable compounds could be extracted from flowers of Lathyrus plants [43]. and (ii) the fatty acyl moiety ofthe LCO is not necessary for the induction of cell divisions in the inner root cortex. Microinjection inside the developing embryo of 0. Also. The NodZ protein used was shown to modifY chitin oligosaccharides very efficiently by transferring a fucosyl group on the reducing terminus [22].76 1. Microinjection of 10 times less NodZ protein gave rise to less severe aberrant tail morphologies. These results implicate that (i) a receptor is present in/on the cells of the root cortex of vetch that recognizes an O-acetylated chitin oligosaccharide and upon activation induces cell divisions. Chitin oligosaccharides in vertebrate development Chitin oligosaccharides playa role not only in plant development but also in vertebrate development. There are several indications that chitin oligosaccharides are involved in plant developmental processes other than nodulation. was found to be necessary. In tobacco plants expression of the Rhizobium nodA and nodB genes. The same mutant could also be complemented by the addition of purified LCOs in nanomolar concentrations.

By incubating chitin oligosaccharides and NodZ protein in the presence ofradiolabeled GDP-fucose. containing the nodZ gene under control of promoter active during early embryo development. A sequence from zebrafish has been identified that shares high homology with the Xenopus DG42 gene and also with the Rhizobium nodC gene. the radio label is transferred to the chitin oligosaccharide and can be easily detected by HPLC or TLC [22. 2) [48]. This chitin oligosaccharide synthase activity could also be detected using other assays. a polymer consisting of altemating units of ß-I.3-linked glucuronic acid (GlcA). Microinjection of athermo inactivated NodZ protein had no effect. The Xenopus DG42 gene and also the zebrafish sequence fall into a large vertebrate gene family (Fig. four from Xenopus and one from zebrafish. Expression of the gene in an in vitro transcription/translation system resulted in chitin oligosaccharide synthase activity that could be reduced by immunoprecipitation by the DG42 antiserum. two members from chicken. Additionally. Up to now three members of this family have been identified from human as well from mouse.Function of chitin oligosaccharides in plant and animal development 77 were not only observed when the protein was injected but also when a plasmid. 46]. The question is whether they are involved in synthesizing chitin oligosaccharides or whether they are involved in synthesizing hyaluronan. when protein extracts of zebrafish and carp embryos of gastrulation and neurulation stages are incubated with UDP_[14C]GlcNAc. In this system no hyaluronan synthase activity could be detected [50]. fungal chitin synthase genes and a bacterial hasA [46]. (ii) The NodZ protein has been used as an efficient tool in the detection and identification of chitin oligosaccharides. (i) A Catharanthus roseus plant cell suspension culture gives a very rapid alkalinization response when chitin oligosaccharides are applied [44] as described for other plant cell suspension cultures [38]. For a long time now there has been a controversy about the true biochemical function ofthe members of this gene family [49]. The same group showed that overexpression ofthe Xenopus DG42 gene in mouse 3T3 cells resulted in chitin oligosaccharide synthase activity in these cell extracts and no increased hyaluronan synthase activity compared to control trans- . Microinjection of the DG42 antiserum into zebrafish embryos of a singlecell stage had a similar effect on the development as the injection of the NodZ protein. In all assays used. Microinjection of the preimmune serum had no effect on zebrafish development [44]. immunoprecipitation of the protein extract with antiserum raised against a protein named DG42 of Xenopus leavis can reduce the chitin oligosaccharide synthase activity. 44]. was injected [45].4-linked GlcNAc and ß-l. This would implicate that in zebrafish embryos a DG42-like protein is involved in the synthesis of chitin oligosaccharides whose function is very important for normal embryonic development. In vitro experiments with the Xenopus DG42 gene by different groups gave different results. The DG42 protein is very homologous to the rhizobial NodC protein and to proteins from the c1ass III of fungal chitin synthases [47]. chitin oligosaccharides are synthesized [44.

which makes it even more difficult to explain the controversy. 100 90 80 70 60 50 40 30% I m[ I I I I I I I chas3 hhas3 mhas3 xhas3 hhas2 mhas2 TI rhas2 chas2 xhas2 I[ zDG42 hhas1 mhas1 xDG42 xhas-rs nodC fbfa chs3 Figure 2. Mus musculus (mouse) mhas3 (MMU86408). The jbfa gene of Stigmatella aurantiaca is included for its high homology towards nodC. H. . M. loti nodc (X52958). Homology tree ofthe genes homologous to the rhizobial nodC gene. S. M. musculus mhasl (D82964). In the figure is shown which genes belong to the vertebrate class I. Homo sapiens (human) hhas3 (HSU86409). X leavis xhas-rs (AFOI5780). a progressive sequence is used as described by [59].78 1. These results were obtained using Saccharomyces cerevisiae [51. but it is not known whether it is involved in synthesizing chitin oligosaccharides [58]. It is not known whether the hyaluronan synthase activity can be inhibited by the DG42 antiserum as reported for the chitin synthase activity. cerevisiae chs3 (M73697). sapiens hhasl (HSU59269). sapiens hhas2 (HSU54804). X leavis xhas3 (AFOI5778). The gene is involved in fruiting body formation. For the alignment. X leavis xhas2 (AFOI5779). aurantiaca fbfa (ZI1601). 11 or III as described in the text. 52] as weIl as rabbit kidney and human osteosarcoma cells [53]. G. Gallus gallus (chick) chas3 (AFOI5777). formed cells [46]. H. Rattus norvegicus (rat) rhas2 (AF008201). The accession numbers for the genes listed above are: S. Bakkers et al. Other groups reported that overexpression of the Xenopus DG42 gene resulted in induction of the hyaluronan synthase activity. R. gallus chas2 (AFOI5776). musculus mhas2 (MMU52524). Danio rerio (zebrafish) zDG42 (DRU53223). Unfortunately. The scale bar shows the percentage of homology at the branch points. these authors never tested the presence of chitin oligosaccharides in their system. Xenopus leavis xDG42 (X52958).

Function of chitin oligosaccharides in plant and animal development 79 In spite of this controversy several authors have designated all the members of this gene family. It has already been suggested that chitin oligosaccharides might act as primers for hyaluronan synthesis [46]. The NodZ enzyme can only use oligosaccharides containing at least two GlcNAc residues but preferentially more at the reducing end [22]. induding DG42. only show induction ofhyaluronan synthesis depending on the cell type in which they are expressed.54]. since the experiments in zebrafish show a strong correlation between the presence of a DG42-like protein and that of chitin oligosaccharides. induce the synthesis ofhyaluronan in vitro and induce the formation ofhyaluronidase-sensitive pericellular coats in vivo [48. In this model chitin oligosaccharides could regulate hyaluronan synthesis. induding DG42. This is also the case for the extra gene identified in Xenopus (xhas-rs). as has been found for the synthesis of glycogen and starch. Results similar to those with NodZ protein have been obtained by injection ofa chitinase in zebrafish embryos [55]. Mouse. whereas expression of dass 111 members can be detected only in adult tissue. However. Only for Xenopus DG42 has the expression pattern in the developing Xenopus embryo been shown by in situ hybridization and by immunohistochemical localization [56]. when expressed in mammalian cells. All this indicates that there is a difference in biological function between members of the different dasses. They do not induce the formation of pericellular coats in any cell type tested. human and Xenopus have one gene from each dass except for Xenopus dass I. This corresponds very weH with the effects on tail development found in microinjection experiments done in zebrafish embryos. A possible explanation would be that during vertebrate development different forms of hyaluronan or chitin are needed for anormal development. members of dass I. Expression of the dass I and 11 members seems to be restricted to early embryo development. The gene family can be divided into three dasses based on sequence comparison. In agreement with this is the difference in expression pattern. which does not show any in vitro hyaluronan synthase activity in any cell type tested [48]. which has two genes (DG42 and xhas-rs) [48]. What might be a common theme in plant nodule formation . as hyaluronan synthase (has) genes [48]. It would be too simple to state that the chitin oligosaccharide synthase activity observed would be due to an artifact introduced by the in vitro experiments. Conclusion All the data reviewed above indicate that chitin oligosaccharides or chitin oligosaccharide derivatives play an important role during plant and animal development. Xenopus DG42 expression moves as a wave or gradient through the Xenopus embryo. Members of the dass 11 and III from human and mouse. The protein is present for a longer time in posterior regions where the tail is formed.

Mai1let F. all the different cell types present in the tail in a later stage can be formed. Spaink HP.80 J. Thomas-Oates JE (1996) Rhizobium leguminosarum bv trifolii praduces lipochitin oligosaccharides with nodE-dependent highly unsaturated fatty acyl moieties. vo129. Reinhold VN. van Spransen PC. Glushka J. Lugtenberg BJJ (1991) A novel highly unsaturated fatty acid moiety of lipooligosaccharide signals determines host specificity of Rhizobium. J Biol Chem 271: 22563-22569 8 Denarie J. Science 257: 70-72 4 Kamst E. since most genes are expressed during embryogenesIs. Cells of the root cortex that are induced to form a preinfection thread or a nodule primordium must go from a differentiated state back into an undifferentiated state and reenter the cell cycle. Kafetzopoulos D (1998) Biosynthesis and secretion of rhizobial lipochitin-oligosaccharide signal molecules. Faucher C. It has been suggested that this G 1 ~ G 2 transition is related to animal cell migration. These cells must remain in an undifferentiated state and are characterized by their high migration rate. Lugtenberg BJJ. Bloemberg Gy. the tailbud. Ann Rev Biochem 65: 503-535 . Stacey G (1994) The biosynthesis ofrhizobiallipooligosaccharide nodulation signal molecules. the posterior region where transcripts of the Xenopus DG42 gene can be detected last. This would suggest that chitin oligosaccharides are important in giving cells the potential to migrate and inducing cells to be in an undifferentiated state. Bakhuizen R. Spaink H. Plenum Press. Debelle F. Kijne JW (1992) Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipo-oligosaccharides of Rhizobium. since this is the only state in which the plant nucleus can move around freely and cytoplasmic rearrangements occur [57]. Haverkamp J. can be regarded as a center for multipotent cells. Spaink Hp. preferably without background activities for hyaluronan or chitin synthase. In: Biswas BB and Das HK (eds): Subcellular biochemistry. Prome JC (1996) Rhizobium lipo chitooligosaccharide nodulation factors: signaling moleeules mediating recognition and morphogenesis. Sheeley DM. From these cells. Roche p. Tak T. Lugtenberg BJJ. Mol Plant-Microbe Int 7: 684-695 7 van der Drift KMGM. Nature 354: 125-130 3 van Brussel AAN. Kennedy EP. This must be done by comparing both activities for the different genes in one and the same system. and development of the vertebrate embryo? To begin with the latter. Plant-microbe-interactions. Truchet G. More effort has to be put in clearing up the controversy between the chitin and hyaluronan synthase activity observed for the DG42 class of genes. van Brussel AAN. An electrospray ionization and collision-induced dissociation tandem mass spectrometric study. Nature 344: 781. Prame JC. A suitable test system would be E. van Brussel AAN. eoli or an in vitra transcriptionltranslation system. Crit Rev Plant Sei 15: 559-582 6 Carlson RW. Bakkers et al.784 2 Spaink HP. References 1 Lerouge P. Tak T. York WS. Geiger 0. Price NPJ. New York 5 Spaink HP (1996) Regulation of plant morphogenesis by lipo chitin oligosaccharides. Denarie J (1990) Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. The strong genetics of the zebrafish system could help in solving the regulation of and biological role for the different classes.

Mol Microbiol16: 1123-1136 12 Demont N. Prome Je. de Blank C. Lerouge P. Schell J (1993) Rhizobium NodB protein involved in nodulation signal synthesis is a chitooligosaccharide deacetylase. Vasse J. Spaink HP (1997) Bacterial nodulation protein NodZ is a chitin oligosaccharide fucosyltransferase which can also recognize related substrates of animalorigin. Nature 351: 670-673 26 Yang WC. Röhrig H. de Billy F. Dordrecht. Pilling J. D'Haeze W. Vasse J. Plant Cell 6: 1415-1426 . Spaink HP (1995) Mass spectrometric analysis of chitin oligosaccharides produced by Rhizobium NodC protein in Escherichia coli. Kondorosi A (1996) Tbe role ofN od signal structures in the determination ofhost specificity in the Rhizobium-Iegnme symbiosis. Denarie J.29223 16 Kamst E. Long SR (1994) Biosynthesis of Rhizobium meliloti 1ipooligosaccharide Nod factors: NodA is required for anN-acyltransferase activity. Proc Natl Acad Sei USA 94: 4336-4341 23 Bloemberg Gy. Spaink HP (1994) Nodulation protein NodL of Rhizobium leguminosarum O-acetylates lipo-oligosaccharides. Lugtenberg BJJ. Lerouge p. Geelen D. Camut S. World J Microbiol Biotechnol 12: 137-149 10 Roche p. Barlier I. Proc Natl Acad Sei USA 91: 3122-3126 22 Quinto C. Hirt H. Curr Opinion Plant BiolI: 353-359 25 Truchet G. Lugtenberg BJJ. Schell J. L6pez-Lara IM. Kluwer Academic Pub1ishers. Kondorosi E. de Billy F. Thomas-Oates JE. van Kammen A. Thomas-Oates JE (1995) Host specificity of Rhizobium leguminosarum is determined by the hydrophobicity of highly unsaturated fatty acyl moieties of the nodulation factors. John M (1994) Biosynthesis of lipooligosaccharide nodulation nactors . Holsters M (1995) Biosynthesis of Azorhizobium caulinodans Nod factors: study of the activity ofthe NodABC proteins by expression ofthe genes in Escherichia coli. Prome JC. Wijfjes AHM. J Biol Chem 268: 20134-20142 13 Spaink Hp. Camut S. Mol Micrubiul11: 793-804 24 Bladcrgroen MR.Function of chitin oligosaccharides in plant and animal development 81 9 Schultze M. Geremia R. Meskiene I. Quaedvlieg NEM. Lugtenberg BJ. Mol Plant-Microbe Int 8: 155-164 14 Kamst E. Bakkers J. cortical cell division and nodule organogenesis on alfalfa roots. ProcNatlAcadSei USA 91: 8418-8422 20 Ritsema T. Schmidt J. Biochem 38: 4045-4052 17 Spaink HP (1994) The molecular basis of the host specificity of the Rhizobium bacteria. Lugtenberg BJJ. Wieneke U. J Bacteriol179: 4053-4055 21 Rihrig H. J Bacterioll77: 6282-6285 15 Mergaert P. Spaink HP (1999) Chitin oligosaccharide synthesis by Rhizobia and zebrafish embryos starts by glycosyl transfer to 04 ofthe reducing-terminal residue. Prome JC (1993) Role ofthe Rhizobium meliloti nodF and nodE genes in the biosynthesis of lipo-oligosaccharidic nodulation factors. Bisseling T (1994) Rhizobium Nod factors reactivate the cell cyde during infection and nodule primordium formation. Harteveld M. Franssen H. Wieneke U. van Montagu M. 119-126 11 Bloemberg Gy. chitin fragments and N-acetylglucosamine in vitra. Roche P. In: H Hennecke and DPS Verma (eds): Advances in molecular genetics ofplant-microbe interactions. Blok-Tip L. van Brussel AAN. J Biol Chem 270: 29217 . a su1phated lipo-oligosaccharide signal of Rhizobium meliloti elicits hair deformation. Haverkamp J. Thomas-Oates JE. Palcic MM. van der Drift KMGM. Truchet G (1991) NodRm 1. van der Drift KMGM. Spaink HP (1997) Acyl-acy1 carrier protein is a donor of fatty acids in the NodA-dependent step in biosynthesis oflipochitin oligosaccharides by rhizobia. Thomas-Oates JE. van der Drift KMGM. Spaink IIP (1998) Genes and signal molecules involved in the rhizobiaLeguminoseae symbiosis. but the cycle is only completed in primordium formation. Kijne Jw. Lugtenberg BJJ. Haverkamp J. Kamst E. Denarie J. Prome JC. Aurelle H. Debelle F. Lugtenberg BJJ. Lugtenberg BJJ. Prome D.Rhizobium NodA protein is involved in N-acylation of the chitooligosaccharide backbone. Maillet F. Anton LeeuwenhoekInt J Gen M65: 81-98 18 John M. Spaink HP (1995) A central domain of Rhizobium NodE protcin mediates host specificity by determining the hydrophobicity of fatty acyl moieties of nodulation factors. Bakkers J. Schmidt J. Proc Natl Acad Sei USA 90: 625-629 19 Atkinson EM. Denarie J (1991) Sulphated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Bloemberg Gy. Faucher C. Bloemberg Gy. Thomas-Oates JE. Hindsgaul 0.

Wasco W (1991) Chitin and nodulation. Schell J (1993) Alteration of plants growth and development by Rhizobium nodA and nodB genes involved in the synthesis of oligosaccharide signal molecules. Kluwer Academic Publishers. Cullimore JV (1997) Identification of a high affinity binding site for lipo-oligosaccharidic NodRM factors in the microsomal fraction ofMedicago cell suspension cultures. a cell division factor in pea roots. MeDonald JA (1998) Charaeterization and moleeular evolution of a vertebrate hyaluronan synthase gene family. Heidstra R. Sautter C (1997) Chitin oligosaeeharides ean induee eortieal eell division in roots of Vieia sativa when delivered by ballistie mierotargeting.30 I 33 Ehrhardt DW. Gisel AA. Geiger 0. Plant Ce1l5: 615-620 41 van Hengel AJ. Plant Ce1l4: 425 -433 40 De Jong AJ. Plant J 10: 295 .368 38 Staehelin C. Wiemken A. Bakkers et al. Spaink Hp. New Phytol133: 25-43 30 Ehrhardt DW. Bakkers J. Schripsema J. de Vries SC (1998) Expression patern ofthe carrot EP3 endoehitinase genes in suspension cultures and in developing seeds. Röhrig H. Truchet G (1994) Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induetion of plant symbiotie developmental responses. Maillet F. J Mora. Regenass M. WijfjesAHM. Debelle F. 27 Libbenga KR. WE Newton (eds): New horizons in nitrogenflXation. Robbins Pw. Granado J. Dordrecht. J Biol Chem 273: 1923-1932 . Proe Natl Aead Sei USA 94: 7982-7986 45 Semino CE. Plant J 7: 939-947 32 Felle HH. Kondorosi E. Bisse1ing T (1996) Nod factor induced host responses and mechanisms ofNod factor perception. Bono JJ. van Kammen A. Terzi M. Stroband HWJ. de Koster CC. Lugtenberg B. Demont N. Bogers RJ. In: R Palacios. Raimondi A. Ann NY Aead Sei 842: 49-54 46 Semino CE. Prome JC. Felix G. Robbins PW (1996) Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of nod-like chitin oligosaeeharides during early embryogenesis. Proe Natl Aead Sei USA 91: 2196-2200 39 De Jong AJ. Debilly F. Kijne J. Kondorosi A. Development 124: 4887 -4895 37 Spaink HP (1995) The moleeular basis of infeetion and nodulation by rhizobia: the ins and outs of sympathogenesis. de Vries S (1992) A carrot somatic embryo mutant is rescued by ehitinase.82 1. Kondorosi A. Mellor RB. Bloemberg GY. van Brussel AAN. de Vries S (1993) A plant somatic embryo mutant is reseued by rhizobiallipooligosaecharides. Lo Schavio F. Spaink H. Spaink HP. van Iren F. van Kammen A. Quaedvlieg N. Schraag-Lamers MF (1973) The role ofhormones and gradients in the initiation of cortex proliferation and nodule formation in Pisum sativum L Planta 114: 19-39 28 Smit G. Muller J. Plant J 4: 651-658 43 Spaink HP. Stacey G. Nature 353: 710 48 Spieer Ap. Denarie J. Boiler T (1994) Pcreeption of Rhizobium nodulation faetors by tomato cells and inaetivation by root ehitinases. Plant Mol Bio129: 869-873 29 Heidstra R. Specht CA. Annu Rev Phytopathol 33: 345 . Hendriks T. Guzzo F. Lugtenberg BJJ (1993) The function of the rhizobial nodABC and nodFEL operons in the biosynthesis of lipo-oligosaccharides. Schultze M (1996) Rapid alkalinization in alfalfa root hairs in response to rhizobiallipochitooligosaccharide signals. Atkinson EM. Robbins PW (1998) Expression of Rhizobium chitin oligosaeeharide fucosyltransferase in zebrafish embryos disrupts normal development. Bisseling T. Hartog MY. Kondorosi E. Long SR (1996) Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Vandekerekhove J. van Kammen A. Seienee 256: 998-1000 31 Felle HH. Spaink HP (1997) An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish. Ritsema T. Proe Natl Aead Sei USA 93: 4548-4553 47 Bulawa CE. Mol Plant Microbe Interact 10: 132-134 35 Ardourel M. Lo Sehavio F. Schultze M (1995) Nod signal-induced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications ofthe lipochitooligosaccharide. Potrykus I. Ranjeva R. Plant Ce1l6: 1357-1374 36 Schlaman H. Kijne J. Koncz C. Plant Physiol117: 43-53 42 Sehmidt J. Long SR (1992) Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti nod factars. John M. Kijne JW (1995) Uridine. Bloemberg G. Terzi M. Cordewener JHG. Ce1l85: 673-681 34 Niebel A. Allende ML. Broughton WJ. 165 -170 44 Bakkers J. Wieneke U. Spaink Hp. Wais R. Semino CE.

Proc NatlAcad Sei USA 93: 4523-4525 50 Semino CE. Achyuthan AM. avertebrate hyaluronan synthase. PhD thesis. Sargent TD. Schairer HU (1996) Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases. Winkles JA. J Biol ehern 271: 22945-22948 55 Semino CE. Doolittle RF (1987) Progressive sequence alignment as aprerequisite to correct phylogenetic trees. The Netherlands 58 Silakowski B. Jamrich M. Michaels GS. Gruuz H. Rebbert ML. Robbins PW (1995) Synthesis of "Nod"-like chitin oligosaccharides by the Xenopus developmental protein DG42. DeAngelis PL (1998) Enzymological characterization of recombinant Xenopus DG42. Kreil G (1996) Cells expressing the dg42 gene from early xenopus embryos synthesize hyaluronan. Leiden University. Dawid IB (1988) Accumulation and decay ofDG42 gene products follow a gradient pattern duringXenopus embryogenesis. in press 56 Rosa F. J Biol ehern 273: 4976-4981 53 Meyer MF. Yamaguchi Y (1996) Molecular identification of a putative human hyaluronan synthase. J Biol ehern 271: 23657-23660 52 Pummill PE.Function of chitin oligosaccharides in plant and animal development 83 49 Varki A (1996) Does DG42 synthesize hyaluronan or chitin: a controversy about oligosaccharides in vertebrate development. Jonas E. Devel Bio1129: 114-123 57 Bakhuizen R (1988) The plant cytoskeleton in the Rhizobiurn-legurne syrnbiosis. Allende ML (1999) Temporal synthesis and degradation of chitin oligosaccharides during embryonic zebrafish development. Proc Natl Acad Sei USA 92: 3498-3501 51 DeAngelis PL. Achyuthan AM (1996) Yeast-derived recombinant DG42 protein ofXenopus can synthesize hyaluronan in vitro. Pospiech A. J Mol Evo125: 351-360 . Proc NatlAcad Sei USA 93: 4543-4547 54 Watanabe K. Neumann B. J Bacteriol178: 6706-6713 59 Feng DF.

8]. Muzzarelli © 1999 Birkhäuser Verlag BaseVSwitzerland Molecular and biochemical aspects of chitin synthesis inhibition Subba Reddy Palli 1 and Arthur Retnakaran 2 1 2 Rahm and Haas Research Laboratories. it can result in the blocking of cutic1e formation. by P. is a ß-I . Marie. Chitin synthesis can be blocked during the various steps by a variety of antibiotics. With the advent of biotechnology and the availability of both complementary DNA and antibody probes. . 727 Norristown road. po. 1219 Queen Street East. and the arthropod form. it is possible to develop high throughput assays for discovering new chemieals that can block chitin formation. which degrades diacetylchitobiose into G1cNAc monomers.Chitin and Chitinases ed. Chitin synthase is thought to have evolved into two types. genes are sequentially expressed and repressed by developmental hormones. alkaloids and hormone analogs.A. Introduction Chitin synthesis is c10sely associated with specific glycoproteins.1. It is synthesized in two different ways: in fungi the chitin synthase enzyme occurs as an inactive zymogen in vesicles called chitosomes and requires proteolytic activation. Po. During the molting process in arthropods. Chitin is degraded by three different chitinases.4. the endochitinase that degrades chitin into oligosaccharides of differing chain lengths. in arthropods this enzyme is membrane-bound and catalyzes the addition of G1cNAc units to a dolichol carrier. Jolles and R. Box 904. Spring House. and the covalent binding of chitin to proteins has been well documented in insects as well as in fungi [1-5]. which has been well characterized in fungi [6]. the exochitinase that degrades oligosaccharides into diacety1chitobiose and chitobiase. Great Lakes Forestry Centre.16). This enzyme has the conserved amino acid sequence essential for chitin biosynthesis in yeasts [7. inc1uding chitin synthesis. Canadian Forest Service. the cell wall of fungi and in various components of diverse invertebrates. Inhibition of chitin synthesis as well as degradation can both result in deleterious effects that are often similar. The enzyme chitin synthase (chitin-UDP-acetylglucosaminyltransferase. which is membranebound [10]. USA Natural Resources Canada. the fungal form. metabolie inhibitors. fungal pathogens and helminthic parasites. PA 19477. Box 490. When these hormones or their analogs are administered temporally out of sequence. one of which is the human hyaluronan synthase. Chitin synthesis inhibitors as well as inhibitors of chitin degradation that produce similar effects are promising agents for controlling insect pests. 2. Ontario P6A 5M7.4-linked aminopolysacharide homopolymer of GlcNAc that occurs as a glycoprotein in the exoskeleton of arthropods. E. C. Canada Summary. has probably undergone sequential gene duplication and divergence during evolution and at present is manifested in different forms in diverse species. Sault Ste. which occurs as an inactive zymogen requiring proteolysis for activation [9].A. Chitin. insect growth regulators.

some ofthe newer analogs such as chlorfluazuron. (iii) blocking ecdysone metabolism resulting in ecdysone accumulation. this class of compounds does not inhibit fungal chitin synthase. In addition to chitin synthase inhibition. One such compound is buprofezin. linked with uridine triphosphate (UTP). R.86 S. A wide variety of chitin synthesis inhibitors belonging to different chemical groups have been reported in the literature. it serves as the site of action for most of these inhibitors. Retnakaran Inhibition of chitin synthesis: biochemical aspects Chitin biosynthesis is a complex process consisting of aseries of enzymatic steps beginning with a glucose unit which is converted to N-acetyl glucosamine (GlcNAc). However. The first compound in this series is diflubenzuron (Dimlin). polymerized into chitin and covalently linked to proteins to form chitin microfibrils in the cuticle. 35]. (v) blocking chitin synthase activity by an active metabolite ofthe parent benzoyl- . including chitin synthesis. Palli and A. and some of the more common ones are listed in Table 1. (ii) inhibition ofthe protease that activates chitin synthase. One of the largest groups of insect growth regulators that deserve special attention is the benzoylphenylureas. There are a variety of compounds commonly referred to as insect growth regulators that adversely interfere with the normal growth process. and in crustaceans it is mineralized with CaC0 3 deposition in the cuticular canals [20]. transported within the cell in combination with dolichol phosphate. which in turn digests nascent chitin. Several types of bacterial and fungal antibiotics have been shown to block chitin synthesis by inhibiting specific enzymes or interfering with intracellular functions of the Golgi apparatus and the endoplasmic reticulum. which is difficult to explain. 37]. which stimulates chitinase. which has been shown to interfere with the formaton of the mitotic apparatus but manifests its effects by inhibiting chitin formation. although the evidence is not unequivocal. Benzoylphenylureas have long since been considered to be inhibitors ofthe enzyme chitin synthase in arthropods. The entire sequence of events is schematically represented in Figure 1 [1-15]. which has been extensively studied ever since it was serendipitously discovered by scientists at the Philips-Duphar company in 1970. It is then sclerotized [17 -19]. lufenuron and novaluron have been found to be effective against a variety ofinsects [36. hexaflumuron. Recently. various other modes of action have been suggested for benzoylureas such as (i) inhibition of transport of UDP-GlcNAc across biomembranes. and since its discovery it has spawned the development of a whole array ofnew analogs [31. It appears therefore that since the synthesis of chitin occurs in the epidermal cell. The process of chitin formation can be blocked at any one of these biosynthetic steps by various inhibitors [21-23]. (iv) blocking ofthe conversion of glucose to fructose-6-phosphate. Certain metabolic inhibitors that block dihydrofolate reductase have been shown to inhibit chitin formation by interfering with nucleic acid synthesis.

A schematic representation of an epidermal cell showing the generalized pathway of chitin biosynthesis in insects_ The entry of trehalose from the hemolyrnph and the sequential changes in the cytosol.87 Molecular and biochemical aspects of chitin synthesis inhibition Microvilli with Plasma Membrane Plaques . N-Acetylglucosam. processing in the Golgi apparatus and transport through vesicles culminating in exocytosis into the assembly zone are indicated.ne-6-p t Glucosamine-6-p r Fructose-6-p~ Glucose-6-p t ==-========-4-4-=-=-====-=-ß-D-Glucose ~ Hemolymph ~ Trehalose Figure 1. . Dolichol-p . endoplasmic reticulum (ER). . Protein-asn Oligosacch-protein-transferase t t ER Dolichol-Oligosaccharide Dolichol-p-p-NAGA-NAGA-NAGA Nucleus Cytosol NAGA-1-p t .

. Avermectin Inhibits chitin formation in Artemia [27]. 4. Cyromazine Inhibits dihydrofolate reductase and lndirectly inhibits normal chitin interferes with nuc1eic acid synthesis deposition in Musea [28]. Insect Growth Regulators 11. Chitin-protein complex not formed in the blue crab [14]. Inhibits cutic1e formation and chitin biosynthesis in Nilaparvata [30]. Palli andA. Brefeldin Disrupts the glycosylation function ofGolgi. widely considered to be an inhibitor ofthe arthropod chitin synthase. indicating protein synthesis is essential [24]. Buprofezin Interfercs with the mitotic apparatus. Inhibits G1cNAc incorporation into chitin in Tribolium [26]. B. Polyoxin-D Structural analog ofUDP-GlcNAc and competitive1y inhibits chitin synthase. Inhibitors of chitin biosynthesis Inhibitor Mode of action Effect A.88 S. Iunicamycin Prevents transfer of G1cNAc to dolichol phosphate and prevents glycosylation. 2. concurrent protein synthesis is essential for chitin formation. effective on helminth parasites. Abnormal chitin formation in An S-triazine inhibitor of dihydroLucilia [29]. 5. 6. Monensin Inhibits glycosylation in the endoplasmic reticulum during the process of extracellular secretion (Palade pathway is blocked). Blocks chitin synthesis in Triatoma [11]. in the epidermis.R. Inhibits G1cNAc uptake in Plodia cells. Prevents chitin complexing with protein for vesic1e formation enroute to cutic1e in the blue crab [14]. Metabolie Inhibitors 9. C. 3. Inhibits chitin biosynthesis in Chilo [25]. Inhibits chitin synthesis in numerous insects [31]. Diflubenzuron (and analogs) It is the harbinger of all benzoylphenylureas. Aminopterin 10. 7. Antibioties 1. 8. Indicates that and inhibits protein synthesis. Retnakaran Iable 1. 12. Puromycin A nuc1eoside antibiotic that interferes Inhibits chitinlprotein synthesis in with transfer RNA (tRNA) function the blue crab [16]. Antiparasitic drug that prevents chitin synthesis. Nikkomycin Structural analog ofUDP-GlcNAc and is a more powernd chitin synthase inhibitor than polyoxin-D. Cyc10heximide Binds to subunit ofribosomes and prevents protein synthesis. folate reductase. Dolichol pathway is blocked.

we need to examine the hormonal control of insect development. Molting hormone expresses and represses molting cyc1e genes and is released at a precise time frame. we are now embarking on the development of designer methods of targeting specific synthetic pathways as weIl as regulatory steps in chitin biosynthesis as control methods of the future. inc1uding parasitic nematodes. Insect development and metamorphosis are orchestrated by aseries of temporally regulated endocrine secretions. Inhibits cutic1e formation in Plodia wing discs [32]. To understand the mode of action of some of these analogs. Several key hormones such as the prothoracicotrophic hormone (PTTH). Alkaloids 13. especially the molting process. Absence of chitin in vertebrates makes it an attractive target for control of invertebrate pests. With the advent of recombinant DNA technology. Binds to tubulin and prevents tubulin assembly into microtubules and blocks mitosis. Inhibition of chitin synthesis: molecular aspects It is becoming increasingly evident that many of the metamorphic events especially in insects are regulated by developmental hormones that act at the molecular level very often by inducing the expression of transcription factors that down. Cutic1e is not formed in Plodia wing discs [32]. phenylurea and (vi) blocking the binding of chitin to cuticular proteins [38. Hormones! Analogs 15. the microfilarial sheath appears to con-tain chitin [41]. stable and persist in the epidermis. Chitin has been shown to be present in the eggshells of nematodes.Molecular and biochemical aspects of chitin synthesis inhibition 89 Table 1 (continued) Inhibitor D. Chitin is synthesized in its absence during the intermolt stage in Manduca [33]. and many benzoylphenylureas adversely interfere with egg development [40]. 20-Hydroxyecdysone 16. and benzoylphenylureas such as hexaflumuron have been found to be effective in controlling the filaria! parasite [42].or upregulate other functions.39]. Dibenzoyl hydrazines that are agonists of 20-hydroxyecdysone. Tebufenozide (RH-5992) andanalogs Mode of action Effect Combines with tubulin and inhibits the assembly of microtubules resulting in blocking mitosis. In addition to the eggshell. Colcemid E. 20-hydroxyecdysone (20E) and . Hormone analogs that have been shown to inhibit chitin synthesis probably do so by acting in such a fashion. Persistence during intermolt stage prevents chitin synthesis [34]. Vinblastine 14.

The mRNAs for ecdysone-induced transcription factors CHR75 and CHR3 are undetectable during the intermolt periods. arthropods in general and insects in particular have to periodically replace the exoskeleton by molting to accommodate for growth. fumiferana and found to be members of the steroid hormone receptor superfamily. R. upon reaching the target tissue binds to its receptor and initiates a sequence of events. including chitin. Ecdysteroids then downregulate the intermolt synthetic activities such as chitin synthesis and initiate the molting process. the mRNA levels increase during 20E peaks (Fig. fumiferana development. whose protein products in turn induce the transcription of "late" genes and suppress the transcription of"early" genes. Because of the rigid exoskeleton. The complementary DNAs (cDNAs) for the two isoforms of ecdysone-induced early Choristoneura hormone receptor 75 (CHR75). [45] showed the induction of a sequence of specific puffs in the polytene chromosomes of the salivary glands in Chironomus tentans and Drosophila melanogaster in response to 20E that reflected the molting events. pupal and adult stages [43]. The two components ofthe ecdysone-receptor complex. that is ecdysone receptor or EcR [46] and ultraspiracle or USP (47) were cloned from C. characterized or weB studied [48-51]. In most insects. Retnakaran juvenile hormone (JH) play important regulatory roles. Aseries of pioneering experiments by Clever and Karlson [44] and Ashburner et al. 2). Developmental Northem hybridization analysis showed that EcR messenger RNAs (mRNAs) are present throughout C. [45] hypothesized that ecdysone binds to its receptor. As outlined in Asburner's model [45]. Choristoneura fumiforana. the two isoforms ofearly-Iate gene Choristoneura hormone receptor 3 (CHR3) and the late genes such as dopadecarboxylase have been cloned. The molting cycle consists of an intermolt period when the larva feeds and grows and a molt period when the old cuticle is degraded and in its place a new one is synthesized. the products of ecdysone induced early genes can repress the transcription of late genes such as chitin synthase. resulting in the inhibition of chitin synthesis. where the mRNA levels increase during the prepual peak of 20E. During the last 2 decades this hypothesis has become the central paradigm for explaining the mode of action of ecdysone. larval. including the spruce budworm. However. like other steroid hormones. The PTTH in turn stimulates the prothoracic glands to secrete ecdysteroids into the hemolymph. Palli and A.90 s. When the larva reaches a critical weight. an ecdysteroid peak is observed in the hemolymph at the middle of each of the embryonic. Ashburner et al. During the intermolt period the insect actively synthesizes components for the new cuticle. and the mRNA levels increase during the 20E peaks. and the ecdysone-receptor complex induces the transcription of"early" genes. proprioreceptors send signals to the neurosecretory cells of the brain to release PTTH. 20E. USP mRNAs are maintained at constant levels throughout development in all tissues except in the larval midgut of the sixth instar. CHR3 and .

. Schematic diagram of ecdysteroid titers. The entire cuticle.A_' . transported and cross-linked.. : ..A __...91 Molecular and biochemical aspects of chitin synthesis inhibition 20E : :'. In order for this to happen. CHR75 [50] and CHR3 [48] are based on published results. Figure 2. USP.. 38]. (Chilindegradalionl _ _ _ _ . 20E.. . . chitin synthesis decreases as CHR3 and CHR75 mRNA levels increase..._____ . CHR75B gene products are prime candidates that can act as repressors of late gene activity.. expression of ecdysone receptor complex (ECR and USP). CHR3.... . In the spruce budworm chitin synthesis occurs during the first 48-h period of each larval stadium as shown by incorporation of 14C-GlcNAc by isolated abdominal epidermis in vitro [34.. . Electron microscopic studies of cuticle development showed that newly molted sixth instar spruce budworm larvae had a thin cuticulin layer overlaid on a dense epicuticle layer.. USP [47]. . The ecdysteroid titers for 5 th instar and 6 th instar were measured as described in Palli et al.._______.A ___. . concomitant with the ascent of the 20E peak at each molt.: :: •• • :: : :. is fully formed by 48 h after ecdysis . --____. The mRNA profiles ofEcR [46]. .A_L-"__. CHR75.. . 20 hydroxyecdysone... chitin has to be synthesized. Choristoneura hormone receptor 75. including chitin deposition and sclerotization._'--. . . EcR."1_ r4th Insw I5th Insw I 6th Instar I Pupe EcR USP CHR75 CHR3 ~~::~ns7o"!=~~nl A_L-_.-. Chitin synthesis and degradation were measured during three to six larval instars [34].":'s:~I~star:-:-lr~' ••••~ JDS:.. ecdysone·induced transcription factors (CHR75 and CHR3) mRNA.. . ultraspiracle. Di!'P'!Ulie o--:E::-m':"bryo--l"'. The chitin synthesis and degradation for embryo and early instars are extrapolated from the chitin synthesis and degradation patterns during the last three larval instars.. As shown in Figure 2. A ____ A_ _ AL-. Choristoneura hormone receptor 3. The ecdysteroid titers for embryo and early larval instars are extrapolated from the ecdysteroid titers of other lepidopteran insects.... chitin formation and chitin degradation during deve10pment of the spruce budworm Choristoneura fumiferana. ecdysone receptor. [43]. The rest of the cuticle is elaborated during the early part of the stadium. A _____ A_ Chilinase .'.

and this is probably true of all insects. blocking embryonic development. Table 2.apple. Some of the commercial applications ofthese inhibitors are summarized in Table 2. resulting in the mortality of the insect at moult. Alsystin. For the most part they are effective as larvicides. beet.s. primarily the benzoylphenylureas. Penfluron Treated females lay sterile eggs [52] Spodoptera littoralis (Egyptian cotton Diflubenzuron. Retnakaran 92 [51]. RH5992.58] Diflubenzuron. R. Novaluron tomato. Since cDNA and antibody probes are currently available for several genes involved in the molting cascade. It follows therefore that if 20E or any compound that mimics 20E is applied early in the stadium when 20E is absent. leafworrn) . It is conceivable that 20E or RH-5992 elicit ecdysone-induced transcription factors such as CHR3 and CHR75. giving the same end result as inhibition of chitin synthesis.pest on cotton. and upon ingestion they block chitin formation. whose gene products in turn repress the transcription of the enzyme chitin synthase. Alsystin. It is also possible that 20E or its analog RH-5992 evokes ecdysone-inducible transcription factors which in turn upregulate the transcription of chitinases. Chitin synthesis inhibitors used for controlling insect pests of agriculture. alfalfa Contact and feeding toxicity to different stages [36. Palli and A. forestry and public health Pest species Inhibitor used Activity Anthonomus grandis (cotton boll weevil) . They also act as ovicides. This is precisely what happens to larval epidermis isolated from newly ecdysed larvae (before 48 h) and cultured in vitro in the presence of 20E or its stable analog. Application of chitin synthesis inhibitors: commercial use Chitin synthesis inhibitors. Penfluron. we should be able to inhibit chitin synthesis. Penfluron . We have observed in the spruce budworm that chitin synthesis occurs at the beginning of each stadium before the appearance of the ecdysteroid peak. pear Provides excellent larvicidal control [57. Other chitin synthesis inhibitors whose mode of action is not understood at the present mayaIso act similarly to RH-5992 by interfering with the normal developmental gene expression cascade.a severe pest of cotton Diflubenzuron. which can degrade nascent chitin. 53-56] Laspeyresia pomonella (coddling moth) . it is possible to design high-throughput screening assays for discovering new compounds with different chemistries that can block chitin synthesis by interfering with the cascade of gene expression during the molting process. resulting in the inhibition of chitin synthesis. have been considered to be "soft insecticides" belonging to the group of biorational control agents often referred to as insect growth regulators (IGRs).

Alsystin Very good control shredder) .hardwoods low dosages [71. . Sitophilus and Teflubenzuron.oak [73. especially oak low dosages [69.vector for malaria in Central America Culex pipiens fatigans (house Diflubenzuron.pine and control [68] other conifers Lymantria dispar (gypsy moth) Diflubenzuron.Molecular and biochemical aspects of chitin synthesis inhibition 93 Table 2 (continued) Pest species Inhibitor used Anticarsia gemmatalis (velvet bean caterpillar) .lemon. Alsystin [63.vector for California encelphalitis virus Anopheles albimanus . 82] Hexaflumuron. Diflubenzuron. Alsystin Excellent control at very hardwoods. Ovicidal and larvicidal Oryzaephilus.vector for filarial parasite Excellent control [80] Reticulitermes jlavipes (termite) wooden structures Bait control [81. Acanthoscelides. 72] Croesia semipurpurana (oak leaf Diflubenzuron.vector for yellow fever virus Penfluron Aedes triseriatus (encephalitis mosquito) . orange sis [67] Thaumetapoea pityocampa (pine Diflubenzuron Excellent larvicidal processionary caterpillar) . 62] Excellent control Lycoriella maU (mushroom fly)Diflubenzuron.malarial mosquito . Penfluron mosquito) . Alystin.various stored grains Ingestion and contact Bemisia tabaci (cotton whitefly) Novaluron> Chloreffects on larvae [36] cotton fluazuronITeflubenzuron Aubeonymus mariaefranciscae (sugar Hexaflumuron Adult and embryonic beet weevil) .70] Malacosoma disstria (forest tent Diflubenzuron Excellent control at very caterpillar) .soybean Diflubenzuron Activity Excellent control [59. Alsystin Ovicidal and larvicidal potato beetle) .74] Haematobia irritans (horn fly) Diflubenzuron Ovicidal control by feedrange cattle-biting fly through method [75] Stomoxys calcitrans (stable fly) -live Diflubenzuron stock biting fly Diflubenzuron Ovicidal by surface treatment and feed through [76] Ovicidal and larvicidal at dosages as low as 51 ppb [77] Excellent control [78] Diflubenzuron Excellent control [79] Aedes aegypti (yellow fever mosquito) Diflubenzuron. control [65] Rhyzopertha species (stored product Chlorflubenzuron insects) .64] mushroom Tribolium.60] Leptinotarsa decemlineata (Colorado Diflubenzuron.potato control [61.sugar beet effects [66] Aonidiella aurantii (California red Buprofezin Suppresses embryogenescale) . Plodia. Triflumuron. Lufenuron .

and it has been shown that the material is broken down by various microbial agents and does not accumulate in the soil [83]. benzoylureas have varying degrees of adverse effects on crustaceans as weH as beneficial insects and must be used with care.7 mg/I Application of chitin synthesis inhibitors: ecological aspects The benzoylphenylureas adversely interfere with chitin formation in all arthropods and therefore are detrimental to many nontarget arthropods. Ecological consequences of some chitin synthesis inhibitors [84] Inhibitor Crustacean Fish Bees Parasitoids Chlorfluazuron Daphnia (48 h) EC so 0.R. predators and pollinators are minimal. Marilyn Scott for help with the typing and Bill Tomkins for the illustration. Palli andA.4Ilg/L carp (48 h) > 100 Ilglbee LC so > 300 mg/I rainbow trout > 100 Ilglbee (96 h) LC so > 100 mg/I very little effect Flufenoxuron rainbow trout LC so > 100 mg/L very little effect Hexaflumuron Daphnia (96 h) EC so O. Since most of the formulations have to be ingested to be effective. The fears that spray runoff into streams might cause widespread mortality of nontarget species have not been realized. topical effects on parasites.9 1lg!L Flucycloxuron Daphnia (48 h) EC so 4. A representative list of IGRs that inhibit chitin formation and their salient environmental effects [84] are listed in Table 3. The environmental fate of diflubenzuron. By virtue of its arthropod specificity. the harbinger of all benzoylphenylureas. Retnakaran Table 3. Acknowledgments The authors thank Karen Jamieson for editorial help.6 mg/L rainbow trout (96 h) > 320 mg/I very little effect > 100 Ilglbee extoparasites affected > 381lglbee moderately harmful > 1000 Ilglbee very little effect toxic moderately harmful carp (48 h) > 2. including crustaceans. has been extensively investigated.IIlg/L Tilapia LC so > 3 mg/I Lufenuron Teflubenzuron - carp (96 h) > 500 mg/I Triflumuron Buprofezin Daphnia (48 h) EC so 225 1lg!L Daphnia (3 h) EC so 50. .94 S.

75-115 Porcheron P.272 . CSIRO Editorial Services. Elsevier. London Kramer KJ. Hackman RH (1985) Synthesis and deposition ofchitin in larvae oftheAustralian sheep blowfly Lucilia cuprina. NewYork. University Kebangsaan (Malaysia) Press. Ruiz-Herera J (1978) Isolation of chitosomes from taxonomically diverse fungi and synthesis of chitin microfibrils in vitra. Yamaguchi Y (1996) Molecular identification of a putative human hyaluronan synthase. J Biol Chem 273: 1923-1932 Watanabe K. LI Gilbert (eds): Integument. 75-138 Horst MN (1990) Concurrent protein synthesis is required for in vivo chitin synthesis in postmolt blue crabs. 109-118 Locke M (1998) Epidermis. Arch Insect Biochem Physiol38: 44-52 Ziegler A (1996) Ultrastructural evidence for transepithelial calcium transport in the anterior sternal epithelium of the terrestrial isopod Porce/lio scaber (Crustacea) during the formation and resorption ofCaC03 deposits. Comprehensive insesct physiology. In: K Binnington. 583-610 Bulawa CE (1993) Genetics and molecular biology of chitin synthesis in fungi. 141-168 Sugumaran M. In: RAA Muzzarelli (ed): Chitin enzymology. Oberlander H (1991) Ecdysteroid-stimulated synthesis and secretion ofan acetyl-D-glucosamine rich glycopeptide in a lepidopteran cell line derived from imaginal dises. Academic Press. Microscopic anatomy of invertebrates llA: insecta. voll. 123-140 Sugumaran M (1991) Molecular mechanisms of sclerotization. CSIRO Editorial Services. European Chitin Society. Arthropoda. Moriniere M.22948 Bartnicki-Garcia S. MacDonald JA (1998) Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family. J Exp Zoo1256: 242-254 Anderson SO (1991) Sclerotization. Springer. Choristoneurafumiferana Clem (Lepidoptera: Tortricidae). biochemistry and pharmacology. Adv Enzymol59: 59-101 Hackman RH (1984) Cuticle biochemistry. AG Matiksty. Wroclaw. In: J Bereiter-Hahn. Walker AN (1993) Crustacean chitin synthesis and the role ofthe Golgi apparatus: in vitro and in vivo studies. J Biol Chem 271: 22945 . Generation ofN-acetylmethionyl catechol adducts during tyrosinase-catalyzed oxidation of catechols in the acetylmethionine. In: F Sehnal. Archiv Insect Biochem Physiol2: 251-263 Horst MN. MacDonald A (1988) Biosynthesis and deposition of chitin in insects and interference with this system as a means of contro!. DL DenIinger (eds): Endocrinologicalfrontiers on physiological insect ecology. MP Abdullah (eds): Chitin and chitosan: the environmentally friendly modern materials. respiration and circulation. Pergamon Press. vol3. WMW Muda. Oxford. Int J Biol Macromol 22: 137-144 Hepburn HR (ed) (1976) The insect integument. 4. Neville AC (1998) The role of pH. Wroclaw Tcchnical University (Polant) Press. Berlin. Cell Tissue Res 284: 459-466 Retnakaran A. In: K Binnington. Melbourne. A Retnakaran (eds): Physiology of the insect epidermis. In: MB Zakaria. Annu Rev Micrabiol47: 505-534 Spicer AP. Bangi. Exp Mycol2: 173-178 Vardanis A (1979) Characteristics of the chitin-synthesizing system of insect tissue.Molecular and biochemical aspects of chitin synthesis inhibition 95 References 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Richards AG (1951) The integument of arthropods. University of Minnesota Press. Ancona. A Retnakaran (eds): Physiology of insect epidermis. A Zabza. 547-565 Coats JR (1982) Insecticide mode of action. Dziadik-Turner C. Bracker CE. Coudouel N. Reyes E. Melbourne. Koga D (1985) Chitin metabolism in insects. Arch Insect Biochem Physiol 16: 257 . temperature and nucleation in the formation of cholesteric liquid crystal spherulites from chitin and chitosan. KS Richards (eds): Biology ofthe integument. In: GA Kerkut. WileyLiss. Minneapolis Murray SB. Amsterdam Cabib E (1987) The synthesis and degradation of chitin. Nelson E (1998) Model sclerotization studies. 1-10 RetnakaranA. Biochem Biophys Res Commun 105: 312-319 RetnakaranA (1995) Chitin formation in the spruce budworm. Biochim Biophys Acta 588: 142-147 Quesada-Allue LA (1982) The inhibition of insect chitin synthesis by tunicamycin.

Tissue Cell 17: 131140 29 Binnington KC. A Retnakaran (eds): Physiology ofinsect epidermis. 307 . Pestic Biochem Physiol42: 242-247 26 Cohen E. Primavera M. W Junk. A Retnakaran (eds): Physiology ofthe insect epidermis. 147-163 39 Retnakaran A. Retnakaran A (1991) Epidermis: a biologically active target for metabolie inhibitors. Lambert D. J Helminthol70: 269-270 43 PaUi SR. 46-54 34 Retnakaran A. Wright JE (1987) Control of insect pests with benzoylphenylureas. PaUi SR. Exp Cell Res 20: 623-626 45 Ashburner M. Pestic Biochem Physiol 37: 249-253 27 Calcott PH. Spindler-Barth M. at the biochemical and ultrastructuralleveL In: WF Stevens. Fujota T (1992) Inhibition of N-acetylglucosamine incorporation into the cultured integument of Chilo suppressalis by diflubenzuron. MS Rao.96 S. Oberlander H (1993) Control of chitin syntbesis in insects. 205-282 32 Oberlander H. a novel benzoylphenyl urea. Oxford 36 Ishaaya I.R. induced by certain insecticides and biological inhibitors. CSIRO Editorial Services. buprofezin. Asian Institute of Technology. Mansour Y. Chihara C. PaUi andA.334 30 Uchida M. RH-5992. Karlson P (1960) Induktion von Puff-Veränderungen in den Speicheldrüsenchromosomen von Chironomus tentans durch Ecdyson. Brighton Crop Protection Conference: Pests and Diseases 1996. Tomkins WL. vol 12. Piessens WF (1985) Chitin synthesis and sheath morphogenesis in Brugia malayi microfilariae. GranettJ. In: RAA Muzzarelli (ed): Chitin enzymology. J Exp Zoo1278: 37 -44 38 Retnakaran A (1986) Chitin biosynthesis in insects and its disruption as a means of pest controL In: R Muzzarelli. C Juniaux. Sugimoto R (1985) Inhibition of cuticle deposition and chitin biosynthesis by a new insect growth regulator. Brownwright A (1996) The regulation of chitin synthesis and deposition in an insect. Ranjit MR. Europ J Entomol92: 325-332 44 Clever U. Atec. J Antibiot 37: 253-259 28 Binnington KC (1985) Ultrastructural changes in the cuticle of the sheep blowfly. Richards G (1974) Temporal control ofpuffing activity in polytene chromosomes of Drosophila melanogaster. In: K Binnington. Plenum Press. a chitin-synthesis inhibitor. in Nilaparvata lugens StäL Agr Biol Chem 49: 1233-1234 31 Retnakaran A. A Retnakaran (eds): Chitin and benzoylphenyl ureas. on the spruce budworm. Melbourne. In: JE Wright. Asai R. CSIRO Editorial Services. Cryan JR (1997) Lufenuron. Retnakaran A (1995) Age specific effects of a nonsteroidal ecdysone agonist. Nishimura K. Parasitol Res 76: 283-288 41 Fuhrman JA. Meltze P. Leach CE (1983) Inhibition of cuticle production in imaginal discs of Plodia interpunctella cultured in vitro: effects of colcemid and vinblastine. Dr. suppressing developing stages oflepidopteran. Yablonski S. the spruce budworm. Grottammare 40 Spindler KD. Retnakaran 25 Nakagawa Y. Primavera M. Dordrecht. In: K Binnington. Dash AP (1996) The effect of chitin synthesis inhibitors on development of Brugia malayi in Aedes aegypti. white fly and leafininer pests. Pergamon Press. Bangkok. Mendelson Z. interrupts development of Drosophila melanogaster.174-182 35 RetnakaranA. LI Gilbert (eds): Insect control. Ennis T (1984) Insect growth regulators. vol2. Comprehensive insect physiology biochemistry and pharmacology. In: GA Kerkut. Cassida JE (1990) Insect and fungal chitin synthetase activity: specificity of lectins as enhancers and nucleoside peptides as inhibitors. GW Gooday (eds): Chitin in nature and technology. Choristoneurafumiferana (Lepidoptera: Tortricidae). NewYork. Lynn DE. Londershausen M (1990) Chitin metabolism: a target for drugs against parasites. J Insect Physiol29: 47-54 33 Riddiford LM (1991) Hormonal control ofsequential gene expression in insect epidermis. Kurihara N. Matsutani M. Fatig RO III (1984) Inhibition of chitin metabolism by avermeetin in susceptible organisms. S Chandrkrachang (eds): Chitin and chitosan: environmentally friendly and versatile biomaterials. Horowitz AR (1996) Novaluron (MCW275). Mol Biochem Parasitol17: 93 -104 42 Mohapatra R. Melbourne. 8C-2: 1013-1020 37 Wilson TG. Lucilia. Cold Spring Harbor Symp Quant Bio138: 655-662 .

Zeit Pjlanzenkrank Pjlanzenschutz 85: 328-333 65 Oberlander H. Sohi SS. Da Silva RFP. Plenum Press. Dev Genet 22: 169-179 48 Palli SR.153-177 53 Abbassy MA. Krell P J. Dhadialla TS. J Econ Entomol67: 760-762 61 Cooper RM. J Econ Entomol76: 536-566 62 Tamaki G. Moffitt HR. Lyon. Silhacek DL. vol 2. EI-Rafai ARM. BrownwrightA. In: EP Lioyd. an ecdysone inducible gene and a member ofthe steroid hormone receptor superfamily. Tomkins WL. Ladd TR. In: I Ishaaya. NewYork. Tamaki G (1983) Effects of chitin-synthesis inhibitors on oviposition by treated adults and on subsequent egg hatch of the codling moth Cydia pomonella (Lepidoptera: Olethreutidae). Palli SR. Berlin. Lindquist RK. Phytoparasitica 9: 133-138 55 EI-Guindy MA. Abdel-Sattar MM (1983) The joint action of mixtures of insecticides or of insect growth regulators and insecticides on susceptible and diflubenzuron-resistant strains of Spodoptera littoralis Boisd. J Stored Prod Res 33: 1-6 66 Perez-Farinos G. Ishaaya I (1997) Current status and future perspectives of the use of Insect Growth Regulators for the control of stored product insects. Salama MA (1980) Selective effects of diflubenzuron and trifluron (SIR 8514) and diflubenzuron-resistance in Spodoptera littoralis Boisd. Dev Genet 20: 36-46 51 Retnakaran A. Sohi SS. Mantey KD. Villavaso EJ (1983) Boll weevil sterility. RL Ridgeway. Shaaya E. Retnakaran A (1996) Cloning and developmental expression of Choristoneura hormone receptor 3. Tood JW (1974) Response of soybean insects to foliar applications of a chitin synthesis inhibitor TH 6040. Palli SR. Primavera M. 1-24 57 Burts EC (1983) Effectiveness of a soft-pesticide program on pear pests. Chauvin RL. Ladd TR. Ladd TR. Smagghe G. Insect Biochem Mol Bio126: 485-499 49 Palli SR. Gill SK (1997) Chitinprotein complex system in insects. Dev Genet 17: 319-330 47 Perera SC.202 63 Cantelo WW (1979) Lycoriella mali: control in mushroom compost by incorporation of insecticides into compost. CanEnt 115: 1659-1662 59 Greene GL (1975) Pest management ofthe velvet-bean caterpillar in a soybean ecosystem. Heinrichs EA. Proc World Soybean Res Conf 1975: 602-610 60 Turnipseed SG. J Econ Entomol72: 703-705 64 Kalberer P. Ladd TR. Retnakaran A (1995) Cloning and developmental expression of the ecdysone receptor from spruce budworm. WC Cross (eds): Cotton insect management with special reforence to the boll weevil. Choristoneurafomiferana. Castanera P (1998) Effects oftopical application of Hexaflumuron on adult sugar beet weevil Aubeonymus mariaefranciscae on embryonic development: pharmakokinetics in adults and embryos. J Econ Entomol 75: 936-941 58 Moffitt HR. Horowitz AR (1998) Insecticides with novel modes of action: an overview. 110-118 52 Wright JE. Jacques Andres. Ladd TR. Can Ent 116: 197 . Vogel E (1978) Control of sciarids (Lycoriella auripila) in mushroom cultures (Agaricus bisporus) by diflubenzuron in the casing soi!. Sohi SS. GAP Roberts. Eliyahu M (1981) The residual contact toxicity of BAY SIR 8514 to Spodoptera littoralis larvae. Choristoneura fumiferana: cloning cDNA and developmental expression of mRNA. Ashry M. Marco V. Palli SR. Archiv Insect Biochem Physiol35: 33-44 50 Palli SR. Tirry L. D Degheele (eds): Insecticides with novel modes of action: Mechanisms and application. Simeonet DE (1983) Timing applications for SIR 8514 for control of the Colorado potato beetle (Coleoptera: Chrysomelidae) on potatoes. Ricci AR.Molecular and biochemical aspects of chitin synthesis inhibition 97 46 Kothapalli R. Cook BJ. Pestic Sci 14: 246-252 56 Ishaaya I. KM Varum (eds): Advances in chitin science. Cress D. Retnakaran A (1998) The ultraspiracle of the spruce budworm. Mantey KD (1984) Diflubenzuron: differential toxicity to larvae of the Colorado potato beetle (Coleoptera: Chrysomelidae) and its internal parasite Doryphorophaga doryphoae (Diptera: Tachinidae). Pestic Biochem Physiol 61: 169-182 . Retnakaran A (1997) Cloning and characterization of a new isoform of Choristoneura hormone receptor 3 from the spruce budworm. In: A Domard. Retnakaran A (1997) Cloning and developmental expression of Choristoneura hormone receptor 75: a homologue of the Drosophila E75A gene. Med Fac Landbouww Rijkuniv Gent 45/3: 721-726 54 Ascher KRS. Springer.

Rouland C. In: I Ishaya. Bordereau C (1998) Efficacy of hexaflumuron against the fungus-growing termite Pseudocanthotermes spiniger. Kunz SE (1980) Testing immature laboratory-reared stable flies and horn flies for susceptibility to insecticides. J Econ Entomol69: 728-730 76 Schmidt CD. Gaaboub IA. J Agric Sei Camb 97: 87-96 78 Hsieh MYG. Polgar LA (1998) Novel-type insecticides: specificity and effects on non-target organisms. Mechanism and applications. Tomkins B. J Econ Entomol89: 1156-1160 82 Peppuy A. Carter Mw. 188-259 . W Junk. Lee ML. Smith L. In: I Ishaaya. J Econ Entomol69: 655-658 81 Su N-Y. Norland RL (1974) Insect growth regulators: evaluation procedures and activity against mosquitoes. CFS Bi-mon Res Notes 2: 5-6 75 Wright JE. Berlin. A Retnakaran (eds): Chitin and benzoylphenyl ureas. Mosquito News 34: 278-282 79 Mulla MS.6-difluorobenzoyl) urea (DiflubenZUfon. D Degheele (eds): Insectieides with novel modes of action: mechanism and application. Seegmiller RE (1987) Environmental fate and properties of 1-(4-chlorophenyl)3-(2. Tomkins W (1982) Effectiveness ofmoult-inhibiting insect growth regulators in controlling the oak leaf shredder. a new biologieal. J Econ Entomol67: 329-332 80 Miura T. In: JE Wright. Retnakaran 67 De Cock A. Granett J (1979) Control of forest tent caterpillar Malacosoma disstria (Lepidoptera: Lasiocampidae) with Dimilin. Can Ent 117: 363-369 74 Retnakaran A. Robert A. Takahashi RM. Delbecque Jp. Dunbar DM (1975) TH 60-40: laboratory and field trials for control of gypsy moths. Schefrahn RH (1996) Comparative effects 01' two chitin-synthesis inhibitors Hexaflumuron and Lufenuron in a bait matrix against subterranean termites (Isoptera: Rhinotermitidae). Berlin.98 S. 141-204 84 Dorvan B. Mulligan FS III (1976) Effects of the growth inhibitor Dimilin on hatching ofmosquito eggs.846 73 RetnakaranA. J Econ Entomol78: 702-703 77 Saleh MS. Pestic Sei 54: 22-26 83 Booth GM. Pali i and A. Springer. Agricultura 49: 269-299 69 Abdel-Monem AR. Springer. Mumma RO (1981) Comparative toxicity of some molt inhibiting insecticides to the gypsy moth. D Degheele (eds): Insectieides with novel modes of actoin. non-toxic and non-contaminating insecticide. Smith L (1976) Greenhouse evaluation ofPH 60-50 activity on the forest tent caterpillar. Dordrecht. Kassem SHMI (1981) Larvicidal effectiveness ofthree controlledrelease formulations of Dursban and Dimilin on Culex pipiens (L) and Aedes aegypti (L). J Econ Entomol74: 176-179 70 Granett J. Degheele D (1998) Buprofezin: a novel chitin synthesis inhibitor affecting specifically planthoppers. CFS Bi-mon Res Notes 32: 2 72 Retnakaran A. Grant GG (1985) Control ofthe oak-Ieafshredder Croesia semipurpurana (Kearfott) (Lepidoptera: Tortricidae) by aerial application of diflubenzuron. Leca JL. Steelman CD (1974) Susceptibility of selected mosquito species to five chemicals which inhibit insect development. Alder De. J Econ Entomol68: 99-102 71 Retnakaran A. Can Ent 111: 841. Schaefer CH. Darwazeh HA. 74-91 68 Robredo FJ (1980) Control ofthe pine processionary moth with Dimilin. DIMILIN®). R. Harris RL (1976) Ovicidal activity of Thompson-Hayward TH 6040 in the stable fly and horn fly after surface contact by adults. Whitmore RC. whiteflies and scale insects. Dr.

and their N-terminal amino acids were determined. Germany Summary. Several motifs. the N-terminal domain (12 kDa) ofthe S. The structural parameters inducing the recognition and possible loosening of a-chitin or of a. erythraeus (now Saccharopolyspora erythraea) [9] and S. Barbarastraße 11. D-490690snabrück. nearly every Streptomyces species is chitinolytic. like a glue. CHBs were purified. Muzzarelli © 1999 Birkhäuser Verlag BaselfSwitzerland Characteristics of chitin-bin ding proteins from Streptomycetes Hildgund Schrempf FB Biologie/Chemie. . small chitin-binding proteins (CHBs) which lack enzymatic activity. Cellvibrio [3]. Streptomycetes are mycelia. Universität Osnabrück.and ß-chitin. They produce a wide range of antibacterial and antifungal compounds. including the relative location and spacing of four tryptophan residues.and ß-chitin are at present being investigated. lividans [10].and spore-forming Gram-positive bacteria abundant in soil. In contrast. olivaceoviridis exochitinase can be used to detect a. griseus [7]. and several of its chitinases were purified [12]. Vibrio [5]. Introduction Chitin can be hydrolysed by chitinolytic enzymes produced by different bacteria. which were then sequenced. S. Our recent studies have revealed that the chitinolytic systems of S. S. fungi and plants. The deduced CHB land CHB2 proteins contain 20 land 200 amino acids.7% ofwhich are identical. S. In addition to a few species of the bacterial genera Aeromonas [1]. Comparative studies of various generated mutant CHB 1 proteins led to the conclusion that mainly onc of the exposcd tryptophan residues directly contributed to the interaction with chitin. olivaceoviridis was shown to degrade chitin very efficiently [11]. a highly specific and rapid assay was developed to visualize the location of crystalline a-chitin within native sampies by fluorescence or confocallaser microscopy. Jolles and RA A. respectively. 77. S. by P. Serratia [4]. a-chitin. S.Chitin and Chitinases ed. specifically target and invade. but not ßchitin or other polysaccharides. olivaceoviridis. many Streptomyces strains have been shown to secrete formerly unknown. The characteristics of these chitin-binding proteins (CHB s) have been investigated in more detail. In addition. Some chitinases were enriched from Streptomyces antibioticus [6]. During growth in the presence of chitin-containing substrates. reticuli and several other investigated streptomycetes comprise the synthesis of a formerly unknown type of extracellular small proteins (18-19 kDa) targeting specifically a-chitin. The affinity of CHB 1 to crab shell chitin is two times higher than that of CHB2. plicatus [8]. are conserved in CHB 1 and CHB2. they hydrolyse a number of macromolecules. induding chitin. Using CHB doupled with FITC (fluoresceine isothiocyanate). Bacillus [2]. Deduced oligonucleotides were used to identifY the corresponding genes.

was purified to homogeneity.e. The dissociation constants (K d ) for CRBI and CRB2 have been determined to be 0. More detailed binding studies revealed that CRB I has a binding capacity ofO.11 pM and 0. Properties of chitin-binding proteins Neither CRB displays any cata1ytic or antifungal activity. The purified CRBs were subjected to Edman degradation. The CRB1 protein.100 H. .7-kDa protein which was shown to adhere strongly to the insoluble substrate and was hence named CRB I. consisting of crystalline (type I) and amorphous regions]. Weaker adsorption of CRBs has been found with colloidal chitin.063 pM (i.7% amino acids with CRB1 (Fig.2 mg protein/ mg purified crab shell powder). The NHrterminal amino acids were used to deduce the respective oligonucleotides. but by high concentrations of guanidine hydrochloride [13]. which was deduced from the S. The cross-reacting protein is predominantly associated with the insoluble substrate and. ehbl [13] and ehb2 [14]. which was named CRB2. including crab shell powder. Using antibodies raised against CRB 1. retieuli also secretes a protein (18-19 kDa) that cross-reacts with CRBl. The protein could neither be released by detergents nor by increasing concentrations of NaCI (up to 1 M). CRBs do not interact with crystalline bacterial cellulose [chains arranged in parallel orientation (cellulose I)]. 1). The chitin-binding protein produced by S. plant-derived cellulose [Avicel. which were subjected to DNA sequence analysis. The protein was purified to homogeneity by consecutive chromatography. 2). resulted in the identification of the corresponding genes. this is derived from native crab shell a-chitin after treatment with acids.27 pM. when cultivated with chitin. From these results we concluded that both proteins recognise and interact highly specifically with a-chitin. we found that. diatom spike. squid pen. e. 1. The proteins adhere neither to ß-chitin from various sources (i. S. retieuli. ciliates) nor to carboxychitin or chitosan. either (Fig. like CRB I. respectively [14. as well as further cloning. can on1y be re1eased by guanidine hydrochloride. and its structure is probably predominantly amorphous. contains 201 amino acid residues as wen as an N-termina1 signal peptide of 30 amino acids. It shares 77. 15]. The deduced S. e. Rybridisations of genomic libraries and sublibraries. The proteins interact strongly with various types of a-chitins.037 pM) of this capacity. olivaeeoviridis ehbl gene. Schrempf Identification of the chitin-bin ding proteins During growth with chitin. 0. olivaeeoviridis secretes on 18. CHB2 has about half (i. retieuli CRB2 protein has a length of200 amino acids and comprises a 30-amino acid signal peptide. S. or mureine from Eseheriehia eoli.

left and C. middle and C. and inspeeted by eonfoeal mieroscopy.-------------- Position of aminoacids A CH8I C R :B2 eRBl CR\!2 B 99 PS~GPADGRICSAGNTSPAO~DS 134 a'psGG~.. the positions of the tryptophan residues are numbered. left) binding only to the surface of the substrate.-----.32 ----------------------------------------.. . (A) Hydrophobicity plot of CHBL (B) The deduced CRBI was aligned with the dedueed CHB2 protein_Aromatie amino aeid residues are marked by a dot.P"~~.. left) or a eonidiophore from Aspergillus proliferans (B.-.1.-. A B c Figure 2 . middle) and ß-chitin from squid (C. Controls were performed with erab shell powder using FITC-labelled WGA (A.GG PAaGPA~ROLCNAGLGQ'sO~sAPa'P8GAA••"KY'GG CH81 C882 Figure 1. right). Crab shell powder (B. B) under ultraviolet (UV) light using a light microscope (C). and inspected by eonfocal microseopy (A. or with the nonbinding FITC-labelled ß-lactoglobulin (A. right) were treated with FITC-labelled CRBl.

Rowever. Consequently. the protein W57 lacks one exposed W-residue. Therefore their role may correspond to that ofthe CRBI residues. the mutant protein retains one exposed and three buried Wresidues. Thus the secondary structure ofthe mutant W134L protein must significantly differ from that of CRB 1. As the replacement ofW57 by a tyrosine (Y) residue also leads to the formation ofthe mutant protein W57Y. they were individuaIly changed to leucine. . as weIl as the slightly lower affinity to anti-CRB 1 antibodies suggest that the conformational change in Wl84 is less pronounced than that in W134L. The data obtained by fluorimetry indicate that W134L has two buried and two exposed Wresidues. too. or with a chitin-binding lectin (39 kDa) secreted by human macrophages [19]. The number and relative position of the tryptophan residues within CRB2 correspond to those of CRB 1 [14]. I). compared with CRB I. the presence of three buried W-residues can be calculated for CRB 1 and the mutant protein W57. The latter are due to aromatic residues (primarily tryptophan and tyrosine) stacked along the face of pyranose rings [16. The significant reduction of the binding efficiency ofW57L to a-chitin can be primarily attributed to the lack of one exposed W-residue. olivaceoviridis exochitinase (see below) [18]. including a chitin binding domain from the S. Our spectroscopical investigations suggested an involvement of tryptophan residues in the interaction of CRBI with a-chitin [13]. The CD and fluorescence spectra. Therefore it can be assumed that the Wl84-residue also plays a part in the specific interaction of CRB 1 with a-chitin [15]. The CD spectra and the affinity studies with antibodies suggest that there is comparatively little conformational change of the mutant protein W57L. glycosylated matrix protein (30 kDa) binding to chitin. according to the hydrophobicity plot of the deduced protein. whose affinity to a-chitin is even slightly lower than that ofW57L. In order to explore the role of tryptophan (W) residues within CRBI in more detail (Fig. this is also indicated by its considerably reduced affinity to anti-CRB I antibodies. No common amino acid motifs were identified among the deduced CHBs and chitin binding domains from several chitinases. The CD spectrum suggests that the secondary structure of the mutant protein W134L diverges from that of CRBl. Schrempf Targeting a-chitin: the role of individual tryptophan residues Previous studies indicated that the binding of proteins to carbohydrates is govemed by hydrogen bonding and hydrophobic interactions.102 H. in some cases to tyrosine residues. 17]. The novel CRBs do not share amino acid identities with a new Anopheles gambiae type I peritrophic. Contrary to CRB I. A clustering of cysteine and glycine residues typical of WGA (wheat germ agglutinin) is also missing in the deduced CRBs. no conclusion can be drawn about the contribution ofthe W134-residue to the binding affinity of CRB 1 to a-chitin. it can be concluded that the W57residue is essential and cannot be replaced by the other aromatic Y-residue. Based on the fluorescence spectra.

which vary as to the length of their N-acetylglucosamine chains and the degree of crystallinity. molluscs. It is interesting that the CBP21 protein deduced from the corresponding gene shares 45. The Streptomyces hyphae adhere closely to the chitin-containing substrates. harveyi to chitin [23]. nematodes. The marine bacterium Vibrio parahemolyticus secretes a large lectin (134 kDa) called chitovibrin that shows a high atImity to swollen a-chitin prepared from crab chitin. including glucans. canescens. and fungi differs considerably [21]. Neurospora crassa). as weIl as longer chitooligomers (> dp 9). . coelicolor A3(2). but not in the presence of chitobiose or glucose. In their natural habitat. Aspergillus proliferans. lividans. followed by colloidal and regenerated chitin [20]. see be1ow) that. S. the consistency of the chitinous layers in organisms like arthropods (comprising crustaceans and insects). We showed by confocal microscopy (using CHBI labelled with fluorescence dye. citrojluorescens. see below) deeper layers of crab chitin or of the fungal weIl wall containing a-chitin (Fig. in the course of the elose interactions. streptomycetes encounter different a-chitin types. 3). It binds most strongly to ß-chitin (from squid). S. which perhaps mediate the specific attachment of V. and in dependence on the presence of accessory inorganic compounds or proteins. ineluding strains of S. cell-associated proteins (55 and 150 kDa) lacking catalytic activities were discovered. S. or with living or autoelaved mycelia from chitin-containing fungi (i.Characteristics of chitin-binding proteins from streptomycetes 103 Occurrence of chitin-binding protein! homologues Several other Streptomyces species. S. S. tendae secrete homologues of CHB 1 and CHB2 during cultivation with insoluble chitin. parvulus. S. We also detected proteins cross-reacting with anti-CHBI from several other chitinolytic bacteria. rimosus and S. Within Vibrio harveyi. e. Recently a 21-kDa protein (CBP21) was found to be secreted by Serratia marcescens 2170. The biological role of chitin-binding proteins Detailed investigations revealed that Streptomyces strains secrete the CHBs only when grown with ground crab shells. coelicolor Müller. Within fungal cell walls chitin is embedded in several types of other polysaccharides. S. worms. As a consequence. albus. CHBs invade (in contrast to wheat germ agglutinin [WGA]. vinaceus.3 % amino acid identity with the Streptomyces CHB 1. It has been speculated that chitovibrin plays a part in the adhesion to chitin [22]. and the secreted CHBs seem to act like a glue. S. It is thus assumed that binding constants ofCHB 1 and CHB2 and of the different chitin types vary and consequently affect the contact to CHB-producing Streptomyces strains.

treated with anti-eRBl antibodies and analyzed under UV light (A. The use of chitin-binding proteins for the in situ localisation of a-chitin Fluorescent dyes. the most commonly used. left) or electron (B. it requires considerable technical skill and time. B. and other compounds containing GleNAc. Schrempf Figure 3. Thus the detection of Calcofluor-binding material does not prove the presence of chitin. right). WGA (wheat germ agglutinin) is a plant lectin consisting of 171 amino acids. and the location ofthe bound electrondense particles be determined by transmission electron microscopy [29]. Streptomyces reticuli growing with chitin (crab shell powder) (A) or mycelia from Aspergillus proliferans (B) were inspected by light (A. Although this method provides the highest resolution currently obtainable.104 R. Second. . it has several drawbacks. Calcofluor. including cellulose and exopolysaccharides from Rhizobium strains [27]. Sectioned cells can be treated with gold-conjugated WGA. First. It recognises and binds to N-acetylglucosamine (GlcNAc) and exhibits an expecially high affinity for ß(1 ~ 4)-linked GlcNAc oligomers which are composed ofthree or more residues [28]. fluorescein isothiocyanate (FITC) or rhodamine can be coupled with WGA and used for binding assays [30]. To achieve a more rapid assay. Congo red [25] and primulin [26] interact with chitin and other polysaccharides. it also interacts weakly with N-acetylneuraminic acid. left) microscopy. WGA is not absolutely specific for GlcNAc. such as calcofluor [24]. N-acetylgalactosamine. was found to bind to many ß-linked polysaccharides [24].

FITC-Iabelled CHB 1 is most suitable to detect the relative position of a-chitin in native sampies (including native a-chitin-containing fungi. and the released chitobiose can be enzymatically cleaved to GlcNAc.or ß-chitins from various sources. However. only high concentrations ofurea and guanidine hydrochloride led to its release. Neither standard physiologica conditions nor elevated NaCI and detergent concentrations allowed the removal of the bound Streptomyces exochitinase (59 kDa). its sensitivity is relatively low. These data reve- . The predicted catalytic domain belongs to family 18 of glycosyl hydrolases [35] and shares high amino acid identity with the deduced Bacillus circulans chitinase D [2]. unlike the 47 -kDa truncated form. In the course of cultivation. but does not adhere to colloidal chitin. A protein domain recognizing lr- and ß-chitin S. insects and crustaceans) by fluorescence microscopy [l3. mediates a very specific and strong binding to crystalline a. we could demonstrate that the 59-kDa chitinase. it can be treated with a chitinase. and it cannot be applied to ascertain the location of chitin within biological material [31]. 32]. Thus the novel chitin-binding protein has proven to be superior to any of the other known methods to rapidly detect the relative location of crystalline a-chitin. the 59-kDa chitinase is specifically proteolytically processed to a 47-kDa truncated form which retains the catalytic and the FnIII domain. a central region containing an FnIII module [33] and an N-terminal region [34]. too. whereas FITC-marked CHB I invades its deeper layers [32]. but lacks the N-terminal part. The CHB l-labelled protein is therefore weH suited.Characteristics of chitin-binding proteins from streptomycetes 105 In order to quantify chitin. Although this is a highly reliable method to definitely identify chitin. Since CHB 1 binds neither to chitotriose or chitoligomers. Moreover. nor to chitin of low crystallinity (situated at the tips of fungi). the 59-kDa enzyme hydrolyses crab chitin and even better the chitin within the fungal cell wall. None of the above described methods can discriminate between polymer chains arranged in a parallel (ß) and in an antiparallel (a) fashion. By immunological studies. shells or cuticles of arthropods. It is also expected that a gold-conjugated CHB 1 could be utilised for an analysis of sections through cells. The 59-kDa enzyme was shown to adhere very strongly to crystalline chitin and to efficiently hydrolyse native crab and fungal a-chitin. to estimate the width of chitin layers. The amino acids deduced from the exo-chiO 1 gene consist of a C-terminally located catalytic domain. this is only possible by X -ray diffraction spectra of purified chitin [21]. it can in future be used to study the crystallisation process of chitin within various organisms. olivaceoviridis secretes an exochitinase (exo-chiOl) of 59 kDa. With confocal laser microscopy we found that FITC-Iabelled WGA binds more strongly to the surface of chitin.

The chitin-binding domain of the S. tryptophan residues playa key part in the interaction with insoluble chitin. olivaceoviridis exochitinase shares no significant similarity with the above-described CHBs or with chitin binding domains from other chitinases identified in various organisms. 4). .106 H. squid. Lemme for supporting the writing of the manuscript and to D. olivaceoviridis exo-chi gene encoding the binding domain led to the generation of mutant genes. Müller for taking the photographs. Acknowledgements I am grateful to M. As control. al that a strong adhesion of the large form of the enzyme is aprerequisite for effective hydro lysis of the crystalline structure of chitin. In addition. right) was incubated with the 59 kDa exochitinase containing the l2-kDa binding domain. An exchange of individual tryptophan codons within part of the S. As with CHBI. it can be concluded that the NHrterminal domain (12 kDa) ofthe 59-kDa enzyme is a chitin-binding domain (Fig. and subsequently to mutated proteins with varying degrees ofbinding capacity. left and ß-chitin. The work was financed by the Deutsche Forschungsgemeinschaft (DFG). a-Chitin (crab shell. All sampies were treated with antibodies raised against the exochitinase and inspected microscopically under UV (A) or visual (B) light. a-chitin from crab (middle) was treated with the truncated exochitinase (47 kdA) lacking the binding domain. Schrempf A B Figure 4.

J Bioehem 105: 484-489 10 Miyashita K. are homologues of family 18 glycosyl hydrolases secreted by human macrophages. and the 39-kDa human cartilage glycoprotein. Zhu BCR. McPherson SA. Appl Environ Mierobiol51: 504-509 5 Soto-Gil RW. Pergamon Press. Zyskind JW (1984) Cloning of Vibrio harveyi chitinase and chitobiase genes in Eseheriehia eoli. a chitinase. Ohnishi K. Sawada Y (1991) Molecular cloning and characterization of chitinase genes from Streptomyees lividans 66. Muijsers AO. J Mol Bio1226: 15-22 17 Quiocho FA (1986) Carbohydrate-binding proteins: tertiary structures and protein-sugar interactions. Fujii Y. Menge U. Taiyoji M. Appl Environ Mierobiol60: 4284-4288 24 Pringle JR. Appl Environ Mierobiol57: 2426-2428 2 Watanabe T. Hrebicek M. Fujii T. J Baeteriol174: 408-414 3 Wynne EC. Becirevic A. In: JP Zikakis (ed): Chitin. Zeltins A. Donker-Koopman WE. Albright C. Appl Environ Mierobiol 52: 1362-1367 4 Fuchs RL. Suzuki K. Maley F (1974) Purification and properties of an endo-ß-N-acetylglucosaminidase from Streptomyees griseus. Diekmann H (1985) The chitinase system of Streptomyees sp. J Biol Chem 249: 811-817 8 Robbins pw.Characteristics of chitin-binding proteins from streptomycetes 107 References 1 Chen JP. Glyeoeonj J 11: 526 23 Montgomery MT. Kirchman DL (1994) Induction of chitin-binding proteins during the specific attachment ofthe marine bacterium Vibrio harveyi to chitin. Diekmann H (1992) Chitinaes of Streptomyees olivaeeoviridis and significance ofprocessing for multiplicity. amylase and pectinase. Ikenada T (1989) Purification and characterization of chitinase produced by Streptomyees erythraeus. Biosci Bioteehnol Bioehem 62: 128-135 21 Muzzarelli RAA (1977) Chitin. Drahos DJ (1986) Cloning of a Serratia marceseens gene encoding chitinase. 209-223 6 Jeuniaux C (1966) Chitinases. Boot RG. Schrempf H (1997) Specific interaction of the Streptomyees chitin-bindin protein CHB 1 with a-chitin: the ro1e of individual tryptophan residues. 565-602 . Methods Enzymol8: 644-650 7 Tarentino AL. Aerts JMFG (1998) Chitotriosidase. Hinz p. Benfield B (1988) Cloning and expression of a Streptomyees plieatus chitinase (chitinase-63) in Eseheriehia eoli. ATCC 11238 and its significance for funga1 cell wall degradation. London. In: C Guthrie. Mol Mierobiol13: 807-819 14 Kolbe S. Watanabe T (1998) Chitin binding protein (CBP21) in the culture supematant of Serratia marceseens 2170. a chitin-binding lectin. Breves R. Drubin DG. Tryptophan residues in the periplasmic ma1todextrin receptor for active transport and chemotaxis. SchrempfH (1994) The novellectin-like protein CHB I is encoded by a chitin-inducible Streptomyees olivaeeoviridis gene and binds specifically to a-chitin offungi and other organisms. Au FL. SchrempfH (1995) Binding and substrate specificities ofa Streptomyees olivaeeoviridis chitinase in comparison with its proteolytically processed form. ehitosan and related enzymes. Adams AEM. Chang MC (1991) Cloning and expression ofa chitinase gene from Aeromonas hydrophila in Eseheriehia eoli. J Baeteriol174: 3450-3454 13 Schnellmann J. StriklandA. Blaak H. J Biol Chem 263: 443-447 9 Hara S. Laine RA (1994) Chitovibrin: a chitin-binding lectin from Vibrio parahemolytieus. Pemberton JM (1986) Cloning of a gene cluster from Cellvibrio mixtus which codes for cellulase. Fischer S. San Diego. Eur J Bioehem 229: 132-139 19 Renkerna GH. Mega T. Academic Press. Nikaidou N. Ann Rev Bioehem 55: 287 . J Gen Mierobiol137: 2065-2072 11 Beyer M.315 18 B1aak H. Academic Press. GR Fink (eds): Guide to yeast geneties and moleeular biology. Appl Mierobiol Bioteehnol23: 140-146 12 RomagueraA.564 16 Spurlino JC. Oxford 22 Gildemeister OS. Oyanagi W. Eur J Biochem 251: 504-509 20 Suzuki K. Eur J Bioehem 246: 557 . Suzuki M. Yamamura Y. Nagayama F. Tanaka H (1992) Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology ofthe enzyme to other prokaryotic chitinases and class III plant chitinases. chitinase. Rodseth LE. Schrempf H (1998) The Streptomyees retieuli a-chitin-binding protein CHB2 and its gene. Haarer BK (1991) Immunofluorescence methods for yeast. Quiocho FA (1992) Atomic interactions in protein-carbohydrate complexes. Mierobiology 144: 1291-1297 15 Zeltins A.

Sburlati A. Cabib E (1991) The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle. Broekaert WF (1993) Structure and function of chitin-binding proteins. Anal Biochem 231: 287-294 33 Bork P. Bowers B. Silverman SJ. Lee H-I. Schrempf H (1993) Characteristics of an exochitinase from Streptomyces olivaceoviridis. Duran A. Au-Young J.108 H. Mol PC. Doolittle RF (1992) Proposed acquisition of an animal protein domain by bacteria. Cabib E. Donini A. Brawley V (1979) Localized deposition of chitin on the yeast cell surface in response to mating pheromone. Proc Natl Acad Sei USA 76: 645-649 27 Glazebrook J. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo. Eur J Biochem 214: 659-669 35 Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarity. Cabib E (1980) Distribution of chitin in the yeast cell wall: An ultrastructural and chemical study. putative protein domains and relationship to other chitinases. its corresponding gene. Proc Natl Acad Sei USA 89: 8990-8994 34 Blaak H. Cell 56: 661-672 28 Raikhe1 NY. J Cell Bio185: 199-212 31 Bulawa CE. Lee WA Jr. Pancaldi S (1983) Effects ofCongo Red on wall synthesis and morphogenesis in Saccharomyces cerevisiae. Henrissat B. Annu Rev Plant Physiol Plant Mol Biol 44: 591-615 29 Shaw JA. Valdivieso MH. Schrempf 25 Vannini GL. Ce1l46: 213-225 32 Zeltins A. Walker GC (1989) A novel exopolysaccharide can function in place ofthe Calcofluor-binding exopolysaccharide in nodulation of alfalfa by Rhizobium meliloti. Robbins PW (1986) The S. Slater M. Plant Sei Lett 31: 9-17 26 Schekman R. Schrempf H (1995) Visualization of a-chitin with a specific chitin-binding protein (CHB 1) from Streptomyces olivaceoviridis. Bowers B. Biochem J 280: 309-316 . Poli F. J Cell Biol 114: 111-123 30 Molano J. Walter S. Schnellmann J.

Chitinases .

and the optimum and stability conditions for chitinase activity are described. In this chapter.Chitin and Chitinases ed. 5]. College o/Bioresource Sciences. Thus. The seaweed chitinases also playa role in defence sirnilar to plant chitinases [2. chitinases are used for defense against plant pathogens and pests [1]. these living organisrns produce and use chitinase for their own specific and biological purposes. Department 0/Marine Science and Resources. Chitinases of 40-90 kDa and sorne as high as 120 kDa have been obtained frorn rnollusks. Introduction In higher plants. Japan Summary. Furthermore. by r Jolles and R. Nihon University. hydrolytic mechanisms such as inversion and retention of the substrate are discussed in relation to allosamidin inhibition. chitinase acts by degrading the exoskeletal chitin in the cuticle OT shell for ecdysis [4. and Masahiro Matsumiya 4 J Laboratory 0/ Biochemistry. Faculty 0/Agriculture. In insects and crustaceans. 8]. chitinases are found in other organisrns [6]. Furthermore. Yamaguchi University. Japan 3 Fisheries Research Laboratory. Kanagawa 252-8510. Michiko Kono 3 . chitinase was even found in rnarnrnals [7 -11]. Saga University. arthropods and sorne vertebrates such as fishes. Maisaka. Recently. Shizuoka 431-0211. Chitinases are found in many organisms. Physicochemical properties Molecular size Chitinases found in higher plants and seaweeds (algae) have a rnolecular weight of about 30 kDa. the physicochemical properties of chitinases such as molecular size are compared among organisms. Japan 4 Laboratory 0/Marine Products Utilization.A. The University o/Tokyo. 3]. Fujisawa. kinetic behaviors are discussed together with their biological functions. Masaru Mitsutorni 2. Yamaguchi 753-8515. Department 0/Applied Biological Sciences. and their properties seem to be c10sely related to their biological function. Japan 2 Laboratory 0/Food Chemistry. considering their c1assification based on amino acid sequence. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Biochemistry of chitinases Daizo Koga 1.A. Faculty 0/Agriculture. A wide range of rnole- . Fish and rnammals also use chitinase for defense [6. Department 0/ Biological Science. Microorganisrns produce chitinase to digest the chitinous nutrient or to partially hydrolyze the chitinous cell wall for cell proliferation [6]. Saga 840-8502. Faculty 0/Agriculture. In particular. arnphibians and rnammals.

4.7-9. the soluble substrates glycol chitin and N-acetyl chito-oligosaccharides were used instead of chitin. it is remarkable that class III plant chitinases [27] and the chitinases from Bacillus licheniformis [28] found in a hot spring show a resistance to high temperatures such as 80°C. 27]. Of course. Rowever. such as pR 4-6 toward the N-acetyl chito-oligosaccharides [21. The optimum pR seems to be dependent on the substrate used. Koga et al.8-7. pR 4. Therefore. Enzyme activity OptimumpH The optimum pR's ofchitinases are pR 4-9 for higherplants and algae. the chitinases from insects such as the silkworm are not very stable . Some chitinases from higher plants such as carrot [22] and from insects such as the tobacco homworm [23] and silkworm [21] are glycoproteins.0-10.5-8. The chitinase with a high chitin-binding ability would show two optimum pRs in the reaction with glycol chitin.5-8.5 for animals and pR 3. the chitinases from the silkworm [21] and plant yam [26] showed two optimum pR values such as 4 and 8-10 toward glycol chitin. The enzymes from microorganisms such as Aeromonas [24] and Rhizopus [25] were reported to be glycoproteins. Therefore. acidic and basic chitinases are present in these organisms. On the other hand. cular sizes from 30 to 120 kDa is observed in bacteria and fungi. The optimum pR of chitinase toward glycol chitin was observed at a slightly alkaline pR or at two pRs compared with the short substrates such as the N-acetyl chito-oligosaccharides and their derivatives.8 in microorganisms. Stability Regarding thermal stability. crustaceans. For analytical purposes. a certain yam chitinase shows only one optimum pR at 4 even during the re action with glycol chitin [27].0 for microorganisms. Isoelectric point Chitinases have the following wide range ofpI values: 3. Some of these small chitinases may possibly be processed from a larger enzyme by limited proteolysis [18-21]. For example. mollusks and fishes. the two optimum pRs do not necessarily come from the substrate glycol chitin. these chitinases show only one optimum pR in the acidic pR range. This fact may be due to the chitin-binding ability of chitinase or to the existence of another chitin binding domain.112 D.0 in higher plants and algae.3 in insects. and 3.

The chitinase enhanced by Cu 2 +was found in some fishes [36-38] and microorganisms such as Pseudomonas aeruginosa [39]. the small and compact chitinase may be thermally more stable. this chitinase hydrolyzes the ß-l. The inhibitor has a structure similar to the intermediate of the substrate such as an oxazoline ring that may be formed between the carbonyl oxygen of the N-acetyl group and the C-l of N-acetylglucosamine during hydrolysis. Inhibitor and activator Allosamidin was first reported to be a specific inhibitor against insect chitinase [29]. Considering that insect chitinases are generally larger than plant chitinases. Allosamidin inhibits chitinase competitively.4 linkage between C-l of N-acetylmuramic acid and C-4 of N-acetylglucosamine in the cell wall of grampositive bacteria such as Micrococcus lysodeikticus (luteus). [32] and S. Its K i value is about 0. Allosamidin is produced by Streptomyces sp.1 pM. Reaction mechanism of chitinase Comparison with similar enzymes Lysozyme (EC 3. allosamidin was found to bind to plant chitinase such as hevamine [34] and inhibit plant chitinase such as yam chitinase H with ID 50 (dose for 50% inhibition) of 44. Recently. Streptomyces sp. the optimum temperature is weIl related to the thermal stability. Therefore. Certain plant chitinases such as hevamine also have lysozyme activity in addition to chitinase activity. allosamidin and its derivatives inhibit only chitinases belonging to family 18 ofthe glycosyl hydrolases but not to family 19. Regarding metal ions. prawn [30] and microorganisms such as Piromyces communis [31]. These plant chitinases belong to family 18 of the glycosyl hydrolases.7 pM. there are two types ofchitinases: one is inhibited and the other enhanced. In the case ofCu 2 +. [35]. this microorganism produces two types of chitinases: one is allosamidin-sensitive and the other is resistant [32]. chitinase is commonly inhibited by Hg2+ and Ag+. However. 25°C. Therefore. So far allosamidin and its derivatives have been found only to inhibit certain chitinases from the silkworm [29].17) is weIl known as a chitinolytic enzyme.4 linkage .Biochemistry of chitinases 113 above 40°C [21] because the insect grows at ca.2. Accordingly. olivaceoviridis [33]. Interestingly. which corresponds to the K 1 value [27]. Insects hydrolyze their own cutic1e chitin in vivo during ecdysis.1. insect chitinase does not require stability against such high temperatures. whereas plants must degrade other organisms (pathogens and pests). Hen egg white lysozyme hydrolyzes the ß-l.

. Choanephora cucurbitanum [41] and Phascolomyces articulosus [41] and in plants such as bean [42].2. However. amino acid sequence and three-dimensional structure. the water should enter from the other side to form the Panomer and retain the configuration at C-I. the formation of such an oxazoline ring may be dependent on the three-dimensional structure. On the other hand. If this oxazoline ring is formed. there are two types of chitinases [47].or random-type chitinase for degradation of the exoskeletal chitin in insects [5] and crustaceans [49]. these chitinases produce the Panomers. allosamidin inhibited family 18 chitinases such as the silkworm chitinase [21] and a cIass III yam chitinase [27]. hen egg white lysozyme (family 22 of glycosyl hydrolases) produces the p anomer [50] but is not inhibited by allosamidin [29]. One produces the N-acetyl chito-oligosaccharides containing N-acetylglucosamine at the reducing end and is from family 18. In fact. Furthermore. However.1. which is different from the lysozyme at the hydrolytic site of the substrate [40]. we can now distinguish these enzymes by investigating the products as to whether the reducing end and nonreducing end are N-acetylglucosamine or glucosamine [46]. In fact. N-acetylglucosaminidase or N-acetylhexosaminidase is an exo-type chitinolytic enzyme and hydrolyzes the chitin oligosaccharide from the nonreducing end to release a monomeric Nacetylglucosamine [5. Anomer formation There are two methods ofwater ingress to perform hydrolysis. This may be related to inhibition by allosamidin. the oxazoline ring cannot form. Such chitinases with lysozyme activity were also found in microorganisms such as Pseudomonas aeruginosa [39]. whereas the other is from family 19. between the C-I of N-acetylglucosamine and C-4 ofN-acetylmuramic acid. This table suggests that family 18 chitinases act in the retaining mechanism. two anomeric forms at C-l such as a and p were found in the hydrolytic reaction as shown in Table 2.132) and chitinase because the chitin used as the substrate for chitinase is usually partially deacetylated. Koga et al. It is also difficult to distinguish between chitosanase (EC 3. As show in Table I. since it has a conformation similar to an oxazoline ring as mentioned in the section on inhibition. whereas family 19 chitinases act in the inverting mechanism. If steric hindrance occurs during the reaction. the oxazoline ring would be formed in the substrate between the carbonyl oxygen of the N-acetyl group and C-I of the N-acetylglucosamine.114 D. However. the configuration may invert to the a anomer. pea [43]. As an intermediate. These chitinolytic enzymes are different from chitinase in antigenicity. As shown in Tables 3A and H. chitosanases show that glucosamine is necessarily observed at the reducing end or at the nonreducing end [46]. 48]. sweet orange [44] and Wasabi [45]. These exo-type enzymes act synergistically with an endo. In such a case.

oeo0 a. . ehitinase C-l [59] Streptomyces griseus HUT 6037 nO. e.l0S-24 ehitinase II [46] Aeromonas hydrophila Aeromonas sp. reducing end residue. _. 00 e0 eo0 oe0 oeo0 o ehitinase [56] Bacillus circulans WL-12 00 eo0 oeo0 eeo0 eeeo0 o ehitinase D [58] Bacillus circulans WL-12 ehitinase [57] Streptomyces griseus eOO0 eeo0 eoe0 ee0 oeo0 e00 00 000 e0 o ~ oeeo0 oeo. 12'. Family 19 Family 18 Table 1. Produets ofpartially N-aeetylated ehitosan hydrolyzed by ehitinases ()()()e()0 ooeoo0 oeoo0 00e00 00.•••• os 00 e0 oe0 eo0 ee0 oee0 eeo0 •••0 eeeo0 0 •••0 o eoo0 00 e0 e00 oe0 oeo0 o ehitinase Al [58] o . ~ t. oa. 00 000 o Pokeweed ehitinase PLC-A [47] VI -- CD '" f e.GleNAe. o 00 a. c:. GleN.

. whereas other chitinases do not. sorne chitinases are able to hydrolyze the trisaccharide. Anomeric configuration of N-acetylglucosamine moiety at reducing end Organism a-Anomer formation (Inverting mechanism) Plant chitinase barley bean yam Microorganism chitinase Streptomyces griseus HUT 6037 Other enzyme papaya ß-Anomer formation (Retaining mechanism) Plant chitinase cucumber rubber tree Enzyme (dass. Kogaetal. chitinase (dass II. and the other is an exo-type and produces monosaccharides and tetrasaccharides. There are also two types of chitinases that hydrolyze the pentasaccharide [52]: one is an endo-type and produces disaccharides and trisaccharides. Chitinase hydrolyzes these oligosaccharides in several patterns [51]. sorne details are surnrnarized in Tables 3A and B. family 19) [60] chitinase (dass I. Such oligosaccharides are usually hydrolyzed in an endo or randorn mann er. Table 2. N-acetyl chito-oligosaccharides were used as soluble substrates instead of chitin. Therefore. family 18) chitinasel lysozyme Microorganism chitinase Bacillus circulans WL-12 chitinase Al and D (family 18) Streptomyces griseus chitinase (family 18) Other enzyme hen (egg-white) human Comments lysozyme (family 22) lysozyme [63] [50] inhibited by allosamidin [61] [34] [64] [62] not inhibited by allosamidin [50] [50] Splitting pattern Not rnuch information is available on splitting patterns.116 D. However. To investigate the splitting pattern. however. it is generally better to say that chitinase is a randorn-splitting hydro lase. family) Ref. family 19) [61] chitinase E (dass IV. not inhibited by allosamidin [62] family 19) chitinase C-I (family 19) similar to dass IV plant chitinase lysozyme chitinase (dass III) hevamine (dass III.

084/sec 0.23 mg/mI not available 0.68/sec [21] [65] [66] [27.. '" r:t '< e.07 mM Km G1cNAcs_6 (pH 6.5) 0. 5. N-acetyl chito-oligosaccharide ofn-mer ofGlcNAc.743/sec/mM 2.07/sec 0.049-0..381 mg/mI 0.017 mM 0.044-0.68 mg/mI not available 0. carboxymethyl chitin. Kinetic parameters l:l:I o' g- -.5) Glycol chitin (pH 4) (pH 8) G1cNAc3_6 (pH 4) Glycol chitin (pH 4) (pH 8) G1cNAc4_6 (pH 4) Substrate Red algae (Gigartina mikamii) chitinase GI chitinase H class III (family 18) Plants Cabbage (30 kDa) Tomato Yam chitinase E class CV (family 19) A.08/sec [36] [30] [67] [67] [21] [23] 2.05-0..249-0..005 mM 1.51] Ref..6-502/sec/mM 1. '" '"<> !l> () a es 0 .059/sec 1.5) 0.3 ml/mg/seg 0. CM-chitin.134 mg/mI not available G1cNAc3_6 2./Km 0.7) Prawn (family 18) Glycol chitin (pH 9) G1cNAc3_6 Glycol chitin (pH 9) G1cNAc3_6 Glycol chitin (pH 9) GlcNAc3_6 CM-chitin (pH 7.408 mg/mI 0.424-0.829/sec 0. Chitinase Table 3..984 mI/mg/sec 1.30/sec 3.591/sec 0.80 mI/mg/sec 1.60 mI/mg/sec 7.323 mg/mI 0.9/sec kcat orVrnax G1cNAc.63 mI/mg/sec 0.033-2.83 mI/mg/sec 0.139 mM Animals Silkworm 65 kDa (family 18) Glycol chitin (pH 4) G1cNAc4_6 (pH 4) 76}lM 0.629/sec 0.l .639 mg/mI 0.88-0.30-41.37 mI/mg/sec 0.7) Chitin Glycol chitin (pH 6.7 mI/mg/sec 13.25 mI/mg/sec 0.99/sec 1.116-2.15 mg/mI not available 0. N-acetylglucosamine.24 mg/mi 0. G1cNAcn .023 mg/mI not available G1cNAc3_6 88kDa (family 18) Tobacco homworm 50 kDa (family 18) 62kDa (family 18) 75kDa (family 18) Krill Antarctic Krill North America Glycol chitin (pH 5.72/sec endo splitting inhibited by allosamidin random splitting [23] endo splitting 13.518 mg/mI 0.0.2 mg/mI [23] endo splitting 8.4 mI/mg/sec 3. () ..38-2.6 mg/mI..3 mg/mI 1.395/sec 3.11-0.5) GlcNAc4_6 (pH 6.84/sec/mM 0. endo splitting not inhibited by allosamidin endo splitting [27] except for G1cNAcs inhibited by allosamidin endo splitting [27] not inhibited by allosamidin endo splitting [2] Comment endo splitting inhibited by allosamidin endo splitting inhibited by allosamidin endo splitting 0. Japanese eel (stornach) GlcNAc3_6 (pH 5.7) (family 19) Glycol chitin (pH 5) Chitin (pH 5..645/sec 0.9/sec/mM kc.7 mg GlcNAclhlml 0.

0 mg/mi 1.0) 4Mu-GlcNAcz (pR 7.0) chitosan (pR 4.0) colloidal chitin (pR 5.904 x 10 3 jlmol/min/jlmol enzyme 7. carboxymethyl.5) colloidal chitin (pR 5. 4-methylumbelliferyl.5) 4MU-GlcNAc3 (pR 5.63 mg/mi 0.0) squid chitin (pR 7.8 jlmol/min/mg 2.0) CM-chitin (pR 5.4 mg/mi 0. (continued) [73] [41] [41] [53] [32] [71] [72] [28] [70] [69] [68] Ref.5) CM-chitin (pR 4.33 mg/mi 2.S IS Chi42 2.1) pNp-GlcNAc2 endo splitting Comment CI C3 B 3.0) glycol chitin (pR 5. CM.0) pNp-GlcNAc2 (pR 6.47 mg/mi 2. ~ ~ ~ E ):J 00 - .8 mg/mi 34.3 mg/mi 0.0) 4MU-GlcNAc2 (pR 6.3jlM 5jlM 0.1 jlmol/min/mg 3.0) glycol chitin (pR 5.1 jlM/min 0. anaerogenes Vibrio alginolyticus R-8 B.85 mg/mi pNp-GlcNAc2 (pR 6.8 mg/mi Km colloidal chitin (pR 5.0) pNp-GlcNAc2 (pR 6.0) II 3.67 x 10 3 jlmol/min/jlmol enzyme 1.0) 4MU-GlcNAc2 (pR 5.5) regenerated chitin (pR 5) colloidal chitin (pR 5. Chitinase Table 3.5 jlMlmin endo splitting hydrolyzes GlcNAc2 hydrolyzes GlcNAc2 inhibited by allosamidin not inhibited by allosamidin endo splitting endo splitting endo splitting endo splitting endo splitting O.11 mM 111 IV ChiB Chi63 ende splitting endo splitting exo splitting 1.056 mg/mi 16jlM 18jlM 0.8 jlmol/min/mg k cat orVrnax 2. 4MU.0) regenerated chitin (pR 5) colloidal chitin (pR 5.8 mg/mi 1.2) Substrate pNp.50mM 0.33 mM 6.56 mg/mi 0. Myrothecium verrucaria Phascolomyces articulosus Choanephora cucurbitarum Streptomyces kurssanovii Streptomyces sp.1 jlM squid chitin (pR 7.80 mg/mi 0. AJ9463 Clostridium paraputrificum Streptomyces plicatus Serratia marcescens BJL2000 Bacillus licheniformis X-7u Microorganisms Aeromonas hydrophyla subsp.9 jlmol/min/mg 46 jlmol/min/mg ende splitting 0.77mM 0. para-nitrophenyl.5jlM 0.0) glycol chitin (pR 5.

the chitinases with small Km values can be expected to have high affinity for chitin. Such chitinases are found in microorganisms such as Bacillus licheniformis [28]. the coexistence of chitinase and . and may have strong antipathogenic activity. The kinetic parameters are summarized in Tables 3 A and B. On the other hand. colloidal chitin. Such wide-ranging values may be due to the solubility of the substrate in the reaction solution. In fact. Yam chitinase H belongs to dass III (family 18). It may be postulated that such insects produce an exo-type chitinolytic enzyme such as N-acetylglucosaminidase that hydrolyzes the N-acetyl chito-oligosaccharides produced from the old cutide chitin by insect chitinase [5]. On the other hand. plant chitinases from yam have different values [27]. which measures the maximum velocity. whereas yam chitinase G has low antipathogenic activity.47-2. for the short substrates such as the N-acetyl chito-oligosaccharides.38-2. chitinases such as yam chitinases E and H have high antipathogenic activity.23 mg/mI for insect chitinases. some chitinases can do the transglycosylation reaction.24 mg/mI for plant chitinases. Therefore. whereas yam chitinase G has a large value. This enzyme might have such a chitin binding domain. Km values could not be obtained from the insect chitinases of the tobacco homworm [23] and silkworm [21]. Yam chitinases E and H have small Km values. is almost the same as kcat' but its value equals kcat x (total concentration ofthe enzyme). Furthermore. Km values are 0. whereas the short substrates are the products. Perhaps the long substrates are the real substrates for chitinases. Yam chitin ase E belongs to dass IV and has another chitin binding domain in the N-terminal region.6 mg/mI for crustacean chitinases.3-1. CM -chitin (carboxymethyl chitin) and glycol chitin differ from those for short substrates such as the N-acetyl chito-oligosaccharides. The parameter Vmax. 0. 0. insect chitinase does not necessarily hydrolyze small oligosaccharides.85 mg/mI for microbial chitinases. For the long substrates.Biochemistry of chitinases 119 Transglycosylation reaction Like lysozymes. the smaller the Km value. The k cat value indicates the rate constant for the reaction from the enzyme-substrate complex to enzyme and product. The Km value is especially interesting in relation to the affinity for chitin. Therefore. therefore.023-0. Furthermore. 0. Kinetics The Km value indicates the dissociation constant of an enzyme-substrate complex. For example. there are different types of chitinase in each organism. thermoviolaceus [54] and Nocardia orientalis [55]. the former chitinases have a high affinity to chitin compared with the latter enzyme. Streptomyces kurssanovii [53]. which is the reverse ofhydrolysis. S. the stronger the affinity toward the substrate. The kinetic parameter values for long substrates such as chitin.

References 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Stintzi A. Jo DH (1997) Chitinolytic enzymes from the gizzard and the chyme of the broiler (Gallus gallus L. Wilson KS. Ready MP.8 Aresolution structure of hevamine. Matsumiya M. which shares identity with chitinases. OppenheimAB. the substrate for insects is only the cuticle chitin. Infect Immun 63: 4770-4773 Desouza MM. whereas the substrate for plants is the chitin of pathogens and pests. 211-219 Kramer KJ. vol3. Wiedermann-Merdinoglu S. Structure 2: 1181-1189 Terwisscha van Scheltinga AC. Biotech Leu 19: 981-984 Henrissat B. J Mol Bio1225: 565-567 Terwisscha van Scheltinga AC.257 Perrakis A. Murray MK (1995) An estrogen-dependent secretory protein. and its complex with an inhibitor. Legrand M. Glycobiol6: 627-634 Rehli M. Lee WJ. seeds. aplant chitinase/lysozyme. Genomics 43: 221-225 Han BK. GAF Roberts. Fritig B (1993) Plant "pathogenesis-related" proteins and their role in defense against pathogens.3 Aresolution. J Gen Microbiol130: 1359-1366 . Gooday G (1984) Properties of chitinase activities from Mucor mucedo: evidence for a membrane-bound zymogenic form. Aplant needs to produce the chitinase that is able to degrade the exoskeletal chitin of the invaders. Lyon. Andreesen R (1997) Molecular characterization ofthe gene for human cartilage gp-39 (CHI3Ll). Adams DJ (1995) Chitinase activity in human serum and leukocyte. synthesis degradation and metabolic regulation. Dauter Z. Vorgias CE (1994) Crystal structure of a bacterial chitinase at 2. Steijn JY. Kalk KH. Structure 2: 1169-1180 Humphreys AM. Hennig M. Vad K (1993) Plant chitinases. KM Varum (eds): Advances in chitin science. 75-115 Kramer KJ. thus requiring wide substrate specificity far the chitins of various orgamsms. That is. Koga D (1986) Insect chitin: physical state.788 Collinge SB. Tews I. JMB 262: 243 . Pilet PE. Endocrinology 136: 2485-2496 Overdijk B. Mochizuki A (1998) Purification and properties of chitinase from a seaweed. Integument. Pergamon Press. Biochimie 75: 687-706 Sekiguchi J. is expressed in a temporally and regionally specific manner in the sheep oviduct at the time of fertilization and embryo development. Koga et al. Dijkstra BW (1996) The 1. the exo-type chitinolytic enzyme has not been reported in plants and algae [2]. Odds FC (1996) Chitinase levels in guinea pig blood are increased after systemic infection withAspergillusfumigatus.). Jacques Andre. and analysis of the conserved sequence and structure motifs of glycosyl hydro lase family 18. Robertus JD (1992) Crystallization ofan endochitinase from Hordeum vulgare L. There is also another reason to consider: insects need only hydrolyze their own cuticle chitin. vol2. Prasad V. biochemistry and pharmacology. Geoffroy P. Koga D (1985) Chitin metabolism in insects. the chitinases from plants and algae may even hydrolyze these small substrates. Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Dziadik-Turner C. Kragh KM. Nielsen KK. Oxford. Dijkstra BW (1994) Crystal structures ofhevamine. Gigartina mikamii. Krause Sw. Rausmussen U. Biochem J 293: 781. aplant defence protein with chitinase and lysozyme activity. Kauffmann S.120 D. Plant J 3: 31-40 Hart PJ. Heitz T. and not exogenous chitin. In: A Domard. Chet I. Fisheries Sei 61: 876-881 Matsumiya M. Insect Biochem 16: 851-877 Flach J. LI Gilbert (eds): Comprehensive insect physiology. In: GA Kerkut. Miyauchi K. Therefore. respiration and circulation. Beintema JJ. Mochizuki A (1995) Distribution of chitinolytic enzymes in seaweeds. Nikkelsen JD. Jolles P (1992) What's new in chitinase reseach? Experientia 48: 701-716 Escott GM. a member of the chitinase protein family and marker for late stages ofmacrophage differentiation.

Insect Biochem 13: 295-305 24 Ueda M. integument and pupal haemolymph of Manduca sexta L. vo12. Matsumoto S. Kimura S. FEBSLett411: 161-163 41 Balasubramanian R. Isogai A. Matsumoto S. Agric Biol Chem 51: 471-476 30 Koga D. Plant Ce1l4: 425-433 23 Koga D. J Antibiotics 40: 296-300 36 Kono M. Horiuchi H. Menge U. 10S-24. Dijkstra BW (1995) Stereochemistry of chitin hydro lysis by aplant chitinase/lysozyme and x-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis. Onodera R (1996) Purification and characteristics of cytosolic chitinase from Piromyces communis OTS 1. Cordewener J. Takaya N. J Bacteriol174: 7398-7406 26 Tsukamoto T. Mizuki K. Chang W-T (1997) Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Insect Biochem Mol Bio127: 759-767 22 Oe Jong AJ. Henrissat B. Uchiumi Y. J Bacteriol174: 3450-3454 20 Radwan RH. In: RAA Muzzarelli (ed): Chitin enzymology. Suzuki A. Appl Environ Microbiol63: 380-386 40 Bokma E. Beintema JJ (1997) Hevamine. cleaves peptidoglycan between the C-l of N-acetylglucosamine and C-4 of N-acetylmuramic acid and therefore is not a lysozyme. Jeronimus-Stratingh M. Bull Coll Agr fet Med. Kono M. Horino-Matsushige M. Arakane Y. No. Ide A. Isogai A. Can J Microbiol38: 331-338 . Vandekerckhove J. Hirai N. Takiguchi Y. Agric Biol Chem 54: 2505-2512 31 Sakurada M. Biochemistry 34: 15619-15623 35 Sakuda S. Atec. Nagamatsu Y (1997) Purification and characterization of Bombyx mori chitinases. Jilka J. II. Kawaguchi T. Van Kanunen A. Nippon Suisan Gakkaishi 53: 131-136 38 Matsumiya M. Agric Biol Chem 48: 931-939 27 Koga D (1996) Comparative biochemistry of insect and plant chitinases. Sakuda S. Arai M (1995) Purification and some properties ofsix chitinases from Aeromonas sp. Shimizu C (1987) Purification and some properties of chitinase from the stornach ofred sea bream Pagrus major. FEMS Microbiol Lett 120: 31-36 21 Koga D. Allosamidin. Grottammare. Imoto T (1984) Purification and some properties of chitinases from yam. Zhou Z-Y. Oe Vries SC (1992) A carrot somatic embryo mutant is rescued by chitinase. Suzuki A (1987) Search for microbial insect growth regulators. a novel insect chitinase inhibitor. Koga D (1990) Purification and some properties of chitinase from the stomach of Japanese eel.Biochemistry of chitinases 121 19 RomagueraA. Terzi M. Kojima N. Matsui T. Ide A. FujiwaraA. Mochizuki A (1995) Purification and some properties of chitinase from the stornach of common mackerel Scomber japonicus. Takagi M (1992) Purification of two chitinases fromRhizopus oligosporus and isolation and sequencing ofthe encoding genes. Biosci Biotech Biochem 59: 2162-2164 25 Yanai K. Diekmann H (1992) Chitinases of Streptomyces olivaceoviridis and significance of processing for multiplicity. a chitinase from the rubber tree Hevea brasiliensis. Komatani K. Ohta A. Breves R. Plattner HJ. Lo Schiavo F. Tomita Y. Ajisaka K. Enzyme MicrobTechnol15: 412-417 34 Terwisscha van Scheltinga AC. Bender S. Yagashita K. Sakuda S. Plattner HJ. Biochim Biophys Acta 1078: 404-410 29 Koga D. Diekmann H (1994) The 92-kDa chitinase of Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus. Penaeusjaponicus. van Koningsveld GA. Nihon Univ 52: 131-136 39 Wang S-L. Shimizu C (1990) Kinetics of a chitinase from a prawn. Isogai A. TschechA. Manocha MS (1992) Cytosolic and membrane-bound chitinases oftwo mucoraceous fungi: a comparative study. Ide A (1987) Specific inhibition of Bombyx mori chitinase by allosamidin. Anguillajaponica. Ishibashi T. Shimizu C. Dioscorea opposita THUNB. Matsui T. Matsui T. Yamada Y (1993) Purification ofallosamidin-sensitive and insensitive chitinases produced by allosamidin-producing Streptomyces. FEMS Microbiol Lett 137: 75-78 32 Wang Q. Morgavi Dp. Biosci Biotech Biochem 57: 467-470 33 RomagueraA. 85-94 28 Takayanagi T. Shimahara K (1991) Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u. Diekmann H (1993) Protoplast formation by a mycelase from Streptomyces olivaceoviridis and purification of chitinases. Kramer KJ (1983) Insect endochitinases: glycoproteins from moulting fluid. Agric Biol Chem 54: 973-978 37 Kono M. Koga D. Sasaki Y. Annand S. Kalk KR.

TsukamotoT. Borders CL. Fukunaga Y. Wu CJ. Watanabe T (1995) The action of Bacillus circulans WL12 chitinases on partially N-acetylated chitosan. Kuwahara T (1995) Purification and characterization of novel chitinases from Streptomyces griseus HUT 6037. Berdis E. purification. Nikaidou N. Yamagami T. 273-284 47 Mitsutomi M. Tanaka A. Mitsutomi M (1990) Action pattern of Streptomyces griseus chitinase on partially N-acetylated chitosan. Arch Biochem Biophys 344: 335-342 61 IseH B. In: A Domard. Biochemistry 8: 694-700 51 KogaD.122 D. In: RAA Muzzarelli (ed): Chitin enzymology. Matsushita M. Fukamizo T. Miyamoto K. Planta 200: 289-295 45 Yamamoto Y. Penaeus japonicus. Agric Biol Chem 53: 3121-3126 52 Koga D (1996) Biological functions and properties of chitinase isozymes from yam. Honda Y. Fisheries Science 61: 725 .726 50 Dahlquist FW. vol2. Hearn CJ.3-glucanases differentially regulated during development and in response to fungal infection. FEBS LeU 382: 186-188 62 Fukamizo T. 70-77 53 Stoyachenko IA. Goto S (1990) Action pattern of Aeromonas hydrophila chitinase on partially N-acetylated chitosan. Sung HY (1992) Purification and properties of chitinase from cabbage. Wilder MN. Varlamov VP. 42 BolIerT. Boller T. Biochem Internat 28: 707-715 . RAA Muzzarelli. KM Värum (eds): Advances in chitin science. Watanabe T (1996) Action pattern ofmicrobial chitinases and chitosanases on partially N-acetylated chitosan. Plant Physiol 87: 325-333 44 Mayer RT. Lo HF. properties and possible function. Furukawa K. J Ferment Bioeng 80: 153-158 60 Hollis T. Grottarumare. Moriwaki M. GAF Roberts. Henrissat B. Matsui T. Kogaetal. McColIum TG. Inamori Y (1993) Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520. Ohtakara A. Kidoh H. IdeA (1989) Kinetics ofchitinase from yam. Jacobson G. Hayashi Y. Biosci Biotech Biochem 59: 311-313 63 Ohno T. Mauch F. Ueda M. Goto S (1995) Comparative biochemistry of chitinases-anomeric form of the reaction products. Arai M. Ide A. Matsunaga H. Jacques Andre. Watanabe T. vol 2. Neuhaus 1M. Fukamizo T. vol 1. McDonald RE. J Bacteriol178: 5065-5070 64 Armand S. Hoshika H. Gehri A. In: A Domard. Jacques Andre. tail fan and hemolymph ofKuruma prawn Penaeus japonicus during the molt cyc1e. C Jeuniaux. Vogeli U (1983) Chitinase in bean leaves: induction by ethylene. Koga D. Dioscorea opposita THUNB. Marcotte E (1997) Kinetic analysis ofbarley chitinase. Agric Biol Chem 54: 871-877 57 Ohtakara A. Nanjo F. Biosci Biotech Biochem 60: 194-199 49 Kono M. Davankov VA (1994) Chitinases of Streptomyces latrssanavii: purification and some properties. Heyraud A. Biosci Biotech Biochem 59: 529-531 59 Mitsutomi M. Purification and characterization of two chitinases and two ß-l. Boller T (1988) Antifungal hydrolases in pea tissue. Lyon. Hata T. Kono M (1996) Purification and characterization of ß-N-acetylhexosaminidase from the liver of a prawn. Koga D (1995) Chitinolytic enzyme activities in the hepatopancreas. Endo H. Minoura K. Sakai K. Planta 157: 22-31 43 Mauch F. hen and papaya lysozymes. Agric Biol Chem 54: 3191-3199 58 Mitsutomi M. Watanabe T (1996) A modular family 19 chitinase found in the prokaryotic organisms Streptomyces griseus HUT 6037. Utsumi T. Hata T. Armand S. Gey C. Ando A. GAF Roberts (eds): Advances in chitin science. Henrissat B (1994) Stereochemical course of the hydrolysis reaction catalyzed by chitinases Aland D from Bacillus circulans WL-12.FEBSLett343: 177-180 65 Chan CT. Biochim Biophys Acta 923: 302-309 56 Mitsutomi M. Atec. Sueshige N. Appl Environ Microbiol59: 620-622 55 Usui T. Tomita H. Niedz RP. Mitsutomi M. J Ferment Bioeng 80: 148-152 46 Mitsutomi M. Ishido Y (1987) Transglycosylation reaction of a chitinase purified from Nocardia orientalis. Raftery MA (1969) The specificity ofhuman. Tomita H. Armand S. Tanaka H (1995) Purification and characterization of chitinase secreted by cultured Wasabijaponica cells. Lyon. Doostdar H (1996) Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L). Watanabe T (1997) Mode ofaction offamily 19 chitinases. Henrissat B (1996) Plant chitinases use two hydrolytic mechanisms. UchiyamaA. Carbohydr Polym 24: 47-54 54 Tsujibo H. 250-255 48 Koga D. Aoyagi H. Hadwiger LA.

sequencing and expression of the gene encoding Clostridium paraputrificum chitinase Chi Band analysis ofthe functions ofnovel cadherin-like domains and a chitin-binding domain. J Bacteriol 179: 7306-7314 72 Robbins pw. Shimada M. Kimura T. anaerogenes A52. Benfield B (1988) Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Esherichia coli. Ando A. Vessey JC (1973) Chitinase activity in Lycopersicon esculentum and its relationship to the in vivo lysis of Verticillium albo-atrum mycelium. Albright C. Izumida H. Deshpande MV (1993) Purification and characterization ofan endo chitinase from Myrothecium verrucaria. Fujii T. Suzuki M. Ohta M.Biochemistry of chitinases 123 66 Pegg GF. Microbiology 142: 1581-1589 71 Morimoto K. Comp Biochem Physiol 105B: 673-678 68 Yabuki M. dyelabelled chitin derivative adapted for the assay of krill chitinase. J Biol Chem 263: 443-447 73 Vyas P. Yamashita M (1986) Purification and characterization of chitinase and chitobiase produced by Aeromonas hydrophila subsp. Yamagishi M. J Ferment Bioeng 82: 598-600 70 Brurberg MB. Physiol Plant Pathol 3: 207-222 67 Saborowski R. Nes IF. Eijsink VGH (J 996) Comparative studies of chitinases A and B from Serratia marcescens. Miwa T (1996) Purification and properties of two chitinases from Vibrio alginolyticus H-8. Nishijima M. Wirth SJ. Sakka K. J Gen Appl Microbiol32: 25-38 69 Ohishi K. J Gen Appl Microbiol39: 91-99 . Wolf GA (1993) A soluble. Karita S. Vetter R-AH. Buchholz F. Ohmiya K (1997) Cloning. Amatatsu T. Mizushima K.

171. by P. have evolved to hydrolyze this important polysaccaride. Despite any significant sequence homology with lysozymes. Muzzarelli © 1999 Birkhäuser Verlag BaseVSwitzerland The structure and action of chitinases Jon D. Among the enzymes involved in breakdown of sugar-based polymers are cellulases. These have been cataloged. Monzingo Institute of Cellular and Molecular Biology. chitosanases and lysozymes. and higher plants. They have a bilobal structure with a high a-helical content.A. crucial to the catalytic mechanism. Glycohydrolase family 18 enzymes. Austin. Introduction Polysaccharide metabolism is a very ancient and widespread biological activity. based on amino acid sequences. Because of its central role in energy flux it is not surprising that a wide range of enzymes have evolved over time to catalyze these reactions.Chitin and Chitinases ed. but are unrelated to family 18 proteins. and include a Glu residue. Several families of enzymes. and Serratia marcescens [6]. it has representatives among bacteria. 130 Chi-ci Chi-th Chi-aa Chi-sm LSIGGWTYSPNF LSIGGWTWSTNF LSIGGWTWSTNF PSIGGWTLSDPF 170 FDGIDIDWEYPED FDGIDIDWEYPAD FDGIDIDWEYPAD FDGVDIDWEFPGG Family 19 contains primarily enzymes from plant sourees. as shown below for the enzymes from Coccidioides immitis [2. USA Summary. Department of Chemistry and Biochemistry. fungi. A section of signature . together with family 46 chitosanases. of distincdy different structure. chitinases. In general these chitinases act through a retaining mechanism in which ß linked polymer is c1eaved to release a ß anomer product. In general. Members of this family are related to one another by amino acid sequence. chitinases. the family 19 enzymes operate through an inverting mechanism. Jolles and R. Trichoderma harzianum [4]. The structures reveal that the different enzyme groups arose from a common ancestor glycohydrolase antecedent to the procaryotic/eucaryotic divergence. 18 and 19. Family 18 contains several runs of conserved amino acids. Family 19 chitinases are found primarily in plants but some are found in bacteria. the sequence numbers correspond to Coccidioides immitis. TX 78712. are similar to severallysozymes inc1uding those from T4-phage and from goose. Robertus and Arthur F. University ofTexas. These signature sequences are found in the active site. Chitinases are found primarily in two ofthe families of glycohydrolases. structural analysis reveals that family 19 chitinases. are characterized by an eight-fold alß barrel structure.A. Chitin is second only to cellulose in biomass and it is an important component of many cell wall structures. although there are some representatives from bacterial sourees. 3]. Aphanocladium album [5]. into nearly 50 families of glycohydrolases [1].

potato. In a similar fashion. the sequence differences are reflected in differences in tertiary structure as weIl. as will be developed below. Glu 67 and Glu 89 are important residues in the mechanism of action of family 19 chitinases. F. and pea. The eight strands of the sheet bend into barrel structure with the helices forming a ring toward the outside. 3] is presently being refined in our laboratory and exhibits the same a/ßbarrel configuration.8 A [14]. eight strands of parallel ß sheet are laid down with an a helix as the "return stroke". As will be developed below. Figure 2A shows aribbon drawing of the protein revealing a mixture of secondary structure. Robertus and A. It should be noted that the bacterial enzyme contains 561 residues. The nature of these conserved residues justifies the notion that the barley chitinase is a reasonable model for the other enzymes of family 19 where . Hordeum vulgare [7].8 Aresolution [13] and laterrefined to 1. An analysis of the protein shows that many hydrophobie residues conserved in the family 19 family form a core for the protein. while residues 148 to 561 form the chitinase a/ßbarrel domain. and one three-stranded ß sheet. Pisum sativum [10]. and that the two families are themselves quite dissimilar. an eight-stranded a/ß barrel.126 1. 60 70 80 90 100 Chi-hvKREVAAFLAQTSHETTGGWATAPDGAFAWGYCFKQERGASSDYCTPSAQWPCAPGK Chi-stKRElAAFFAQTSHETTGGWASAPDGPYAWGYCFLRERGNPGDYCPPSSQWPCAPGR Chi-atKKEVAAFFGQTSHETTGGWATAPDGPYSWGYCFKQEQNPASDYCEPSATWPCASGE Chi-psKRElAAFLGQTSHETTGGWPTAPDGPYAWGYCFLREQNP-STYCQASSEFPCASGK It is clear that the members of each family are homologs. in both cases. That is. Monzingo sequence for the family 19 chitinases is shown as represented by barley. A backbone representation of the Serratia enzyme is shown in Figure 1. The structure was originally solved at 2. Structure of family 19 chitinases The only member of this family for whieh an X-ray structure has been solved is Hordeum vulgare (barley). Arabidopsis thaliana [9]. The numbering refers to the barley sequence. Solanum tuberosum [8]. those polar residues conserved within the family tend to line the large cleft in the enzyme which is presumed to be the substrate binding and catalytic site. The amino (N) terminal 147 residues form a distinct chitin anchoring domain. D. The enzyme core ofthese enzymes is. including 10 a-helical segments. Structure of family 18 chitinases The three-dimensional structures of several family 18 chitinases have been solved. The X-ray structure of the chitinase cloned from the pathogenic fungus Coccidioides immitis [2. including a bacterial chitinase from Serratia marcescens [11] and the plant enzyme hevamine [12].

nonpolar residues in the core control folding and those residues responsible for enzyme activity are conserved in the active site. that is deacetylated chitin. Structure of family 46 chitosanases In addition to chitinases. The presence of the charged amino groups renders chitosan a polycationic polymer. which hydrolyze chitosan. this appears at the lower left of the chitinase barrel domain. Glycohydrolase family 46 is a small one. The chitosanase frorn Streptomyces has been crystallized. The overall folding ofthis . and Glu 315 is the catalytic acid. The N-terminal 147 residues of the Serratia enzyme form a chitin-anchoring domain. which is filled with side chains. there exists a family of enzymes called chitosanases. like the handle on a mirror. The view is down the core of the barrel. These protein sequences are related to one another but show no significant sequence homology to other glycohydrolases. say 20-60%. Chitosan resernbles chitin except that a fraction of the sugar residues. and the X-ray structure solved to 2. This enzyme is a representative of the farnily 18 chitinases. but not all. Many.The structure and action of chitinases 127 N Figure 1. The active site is on the top of the barrel. A ribbon drawing of the chitinase from Serratia marcescens. farnily 18 chitinases have such an anchoring domain.4 Aresolution [17]. containing to date only a chitosanase from Streptomyces NI74 [15] and one from Bacillus circulans [16]. are glucosamine. It is an a /ß barrel structure with eight parallel strands of sheet and eight return helices.

These enzymes are representatives of glycohydrolase families 22. containing a single a helix. 14]. had been added to during the evolution toward modem glycohydrolases.24 and 23. A comparison of enzyme structures Our analysis ofbarley chitinase. It is roughly 100 to 150 amino acids long and contains a number ofhelices and sheets which occupy the same position and orientation in space. and it was initially proposed that the two molecules were unrelated. F. Robertus and A. Monzingo protein resembles that of the family 19 chitinases in the sense of having a pronounced bilobal structure with a pronounced substrate binding c1eft. For example. family 46 [17]. that despite the lack of sequence similarity HEWL and T4L were almost certainly related by divergent evolution [22. and their active sites appeared to bind substrate in a similar fashion. the five proteins shared a common core structure. the comparison was extended to inc1ude GEWL. Our analysis also showed that the ancient core protein. The folds ofHEWL and T4L had been compared previously. It bears no structural resemblance to the family 18 chitinases. Our analysis involved a comparison in which a-helical and ß-sheet secondary structural elements were superimposed in a least-squares sense [18]. proteins of 238 and 164 amino acids. led us to the hypothesis that the overall folding of these two glycohydrolases bore some resemblance to that of three other glycohydrolases [18]. bacteriophage T4. but the folding pattern is the same for all the cores. despite lacking any significant sequence homology. . Even though the proteins differed in size and complete folding pattern. a pairwise superposition of chitosanase and T4L. from goose [21]. The three eukaryotic members of our comparison possess an N-terminal domain. based on structural comparisons. The eukaryotes also have aC-terminal domain of about 40 residues. In our study we compared all possible pairs ofthe five enzymes. family 19 [13. respectively. T4L [20] and GEWL. The larger cores in enzymes like chitinase have inserts in loop regions. These were the lysozymes from hen egg white (HEWL) [19]. they shared several major secondary structural elements. containing the substrate binding and catalytic site. respectively.128 J. consistent with their lack of obvious amino acid sequence similarity [20]. D. Later. which also appeared to have a similar core structure [21]. revealed that 106 residues in various secondary structural elements occupied essentially the same relationships in space and that they differed by a root mean square (rms) distance of only 3. It was later proposed. and these comparisons led to the conc1usion that. inc1uding three a helices in its length of roughly 80 residues. The prokaryotic enzymes have a larger C-terminal domain. while the prokaryotic members of this superfamily lack such a structure. The core consists of a bilobal globular domain with an elongated polysaccharide binding site between the lobes. and chitosanase. of around 50 residues. 23].7 A.

Representatives of a glycohydrolase superfamily. prokaryotes modified the core hydro lase by adding a relatively large Cterminal domain. After the split. It shows that glycohydro- Figure 2. (C) chitosanase (family 46) and (D) phage lysozyme (famiIy 22). The top row shows two eukaryotic enzymes. It is not c1ear how these modifications improve the enzymes.The structure and action of chitinases 129 This analysis led us to conc1ude that the 150-residue conserved core of the glycohydrolases represents an ancient ß glycohydrolase which has existed since before the prokaryotic/eukaryotic split. The eukaryotes modified the core protein by adding a small N-terminal and a small C-terminal domain. Figure 2 shows a panel of four ofthe enzymes to illustrate the conserved cores and added units of these glycohydrolases. but presumably they increased stability or allowed a wider range of substrate affinities to evolve. the amino terminal domains are shown in dark shading and carboxy-terminal domains in lighter shading. the added domains are in wider ribbons. whereas the lower row shows two prokaryotes. (A) barley chitinase (family 19) and (B) goose lysozyme (family 23). In each case the ancient and conserved central core is shown as a thin gray ribbon. .

The mechanism was hypothesized to be an inverting one. Monzingo lase families 19. This result is consistent with similar work showing that the chitinase from Dioscorea opposita (yam) proceeds with inversion ofproduct [29].22. Conversion of either of these acids to the corresponding amide eliminates measurable activity. The importance of these two residues to catalysis has since been confirmed by site-directed mutagenesis [28]. This binding mode. Mechanisms of chitinase action Chitinases act by hydrolytically cleaving the ß-glycosidic linkages between GlcNAc residues. this has been described in detail elsewhere [18]. the amino acid sequences. It is reasonable to assume that the family 19 chitinases all work in this way. but this will only be revealed when enough tertiary structural information is available to see beyond the linear structures. The threedimensional structure of the core is much more conservative than the amino acid sequence and it has diverged for the different families of enzymes over time. In fact. The hydrolytic profile for hexasaccharides by barley chitinase suggests the preferred binding of substrates may be at sites B-G [25]. because the space between the "second carboxylate". As indicated in Figure 4. Hydrolysis would occur between sugars in sites D and E. . and it has been hypothesized to contain at least six sugar binding subsites labeled A-F. the secondary structural elements are anchored by conserved hydrophobic contacts in which a Phe residue in one protein may be replaced by a Met in another. The details of glycohydrolase mechanisms have been reviewed extensively [24]. which serves the same role. In general. Glu 67 as the catalytic acid and Glu 89 as a base. D. or to other ancient superfamilies. is shown in Figure 5. Robertus and A. hexasaccharides are cleaved into two trisaccharides.130 1. this hydrolysis can occur in one oftwo ways.24 and 46 are in fact all distantly related. and the susceptible glycosidic bond demanded that an attacking water be interposed [14]. a convention developed for hen lysozyme [26. although differing in identity. the inverting mechanism proceeds through a positively charged oxocarbonium intermediate which has a distorted geometry. 27]. It may be that other families of glycohydrolases also belong to this. For example. either with retention of anomeric configuration in the product or with inversion. together with the catalytic residues. from the nonreducing end [14]. The substrate binding cleft ofbarley chitinase is an extensive one. have retained certain structurally important patterns which maintain the ancestral folding pattern. This inverting mechanism was confirmed using nuclear magnetic resonance (NMR) to follow the anomeric hand of the product sugars which were a [25]. F.23. that is. This is illustrated in Figure 3. it assurnes a roughly "half-chair" configuration compared with the chair conformation of the other sugars. Two carboxylates were hypothesized to be responsible for the catalysis. Glu 89.

another family 19 enzyme [30]. it gave apparent Km values of 33 mM. whereas Glu 67 acts as an acid to protonate 04 of the leaving sugar.131 The structure and action of chitinases oy ~~­ GluA GluA o~ HO i-.0 ~ OH 1. and k cat is 35 min. a fluorescent substrate.1• These values are consistent with those measured for yam chitinase.N'. 19 and 6 mM.33 min. The Km for (GlcNAc)4 is 3 mM. in which the product is the a anomer. respectively. has been examined kinetically [25].1• The pR profile for the enzyme shows that activity is cQntrolled by a base with a PKa of . The single displacement mechanism involves Glu 89 acting as a base to polarize the attacking water moleeule. It revealed that the n-acetylglucosamine monomer does not bind to any measurable degree while the dissociation constants for the dimer. Inherent fluorescence ofthe enzyme was used to monitor the dissociation constant for binding of natural substrate polymers. The upper path corresponds to a retaining mechanism where the ß-glycosidic linkage is preserved in the form of a product which initially exhibits the ß-anomeric configuration. (G1cNAc)4 is cleaved almost exclusively into dimers of (GlcNAc)z. Substrate cleavage patterns show that (G1cNAc)6 is cleaved into (G1cNAc)3 and also cleaved asymmetrically to (G1cNAc)4 and (G1cNAcb with almost equal efficiency. 4-methylumbelliferyl ß-N.N"-triacety1chitotrioside was used in a simple and convenient assay to characterize kinetic parameters of the enzyme [25]. The lower path is an inverting mechanism. trimer and tetramer are 43. Barley chitinase. GluA o~ . Mechanisms of glycohydrolases. and the k Cat was 0. H Figure 3. the archetypal enzyme of the family 19 chitinases. In addition.

. Analysis ofkinetic and dissociation constants proves that the mechanism ofbarley chitinase is consistent with aBi-Bi kinetic model for hydrolysis.•••.. hydro lysis is taken to occur between sugar-binding subsites labeled D and E.-J .. .o 0NH-<- OoooooHOt C )->m 0 H TYR 123 OO~ ~OOH ° D NH-<" ASN 199 "\ HO OJ. and on kinetic data. 3.' 0 GLN~NHi o HO NH// LYS 86 I . A model can be constructed based on the observed binding of polysaccharide to hen lysozyme. It appears that this family of enzymes operates by a retaining . with (GlcNAc)4 and water as substrates and two (GlcNAc)2 moieties as products [25].. By convention....9 (Glu 67)..67 / Hot 0j-O E GLU89 ~NH ° i{0W .. F. Robertus and A.9 (Glu 89) and an acid with a pK a of6. Family 18 chitinases have not been studied as extensively as those from family 19.. D.0. Hypothetical binding of a chitin polymer to barley chitinase... Monzingo t{ OH ASN oH B HO /LYS165 + H3 0 .0 :' .:// / ...-NH2 ••••• O(~H.H2N~ + NH2 ci ° NH~ Hot )-NH ° OH ° OH OH Figure 4.l32 1./ ~. ° ° ~ HNhG211 00". It has not been possible to observe binding of substrate or analogs crystallographically to this family 19 chitinase.

that Glu 204 and Asp 200 may be the catalytic residues for the chitinase from Bacillus circulans [34]. the National science Foundation and by grants from the Foundation for Research and the Welch Foundation. This view is supported by the crystal structure of a complex between the chitinase called hevamine and the inhibitor allosamidin which contains an oxazoline moiety which mimics the transition state of the reaction [36]. First there is bond breaking between the D and E sugars involving protonation of the leaving group alcohol and stabilization of the positively charged intermediate by a second carboxylate [19. As shown in Figure 4. cts2) from the fungus Coccidioides immitis. Kirkland TN. the N-acetyl group at position 2 of the scissile sugar may itself facilitate the reaction via formation of a transient oxazolinium intermediate [36. 32]. Lafay JF (1992) Primary structure of a chitinase-encoding gene (chi 1) from the filamentous fungus Aphanocladium album: similarity to bacterial chitinases. This intermediate is then attacked by a solvent molecule which replaces the leaving sugar group. Zhu Y. Gene 167: 173-177 3 Yang C.Cox RA (1996) Molecular cloning and characterization ofthe Coccidioides immitis complement fixationlchitinase antigen. Llobell A. 32]. It has also been suggested that the stable juxtaposition of a carboxylate and an oxocarbonium ion is unlikely and that a transient covalent intermediate may occur in the double displacement mechanism [35]. 33]. based on site-directed mutations. Acknowledgements This work was supported by grants from the National Institutes of Health (GM 30048). the mechanism is thought to be a double displacement type. Benitez T. Gene 120: 243-248 . In the case of glycohydro1ases like lysozyme. Distortion of the D sugar by interactions between the enzyme and substrate is thought to playamajor role in transition state stabilization [19]. these chitinases act by an inverting mechanism involving action of enzyme-supplied acid and base. References 1 Henrissat B. In summary. Magee DM. the family 18 chitinases are basically eight-stranded a/ß barrels which operate by a retaining catalytic mechanism. this might generally be expected to invo1ve two catalytic residues and to proceed through a geometrically distorted oxocarbonium intermediate. perhaps with substrate assistance. Curr Genet 27: 83-89 5 Blaiseau PL. Cole GT (1995) Isolation and characterization oftwo chitinaseencoding genes (ctsl. Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similaritis. Biochem J 293: 781-788 2 Pishko EJ. The family 19 chitinases are bilobal structures with an ancient core structure of a helices and a three-stranded ß sheet. It has been suggested. Infect Immun 64: 1992-1997 4 Garcia I. de la Cruz J.The structure and action of chitinases 133 mechanism [31. That is. It has also been suggested that the retaining mechanism of family 18 chitinases may involve substrate assistance. Lora KM. Pintor-Toro JA (1994) Cloning and characterization of a chitinase (chit42) ~cDNA from the mycoparasitic fungus Trichoderma harzianum.

Hadwiger LA (1995) Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding. James MNG. Nature Struct Biol3: 155-162 18 Monzingo AF. Mundy J (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. Dijkstra BW (1996) The 1. J Mol Bio1262: 243-257 13 Hart PJ. Robertus JD (1993) Crystal structure of an nedochitinase from Hordeum vulgare L. seeds. Robertus JD (1994) SIRAS Structure of an anti-fungal chitosanase from Streptomyces N174. Horovitz 0. Nature 206: 757 -761 20 Matthews BW. Leah R. Johnson LN.3 Aresolution. Brzezinski R. Koga 0. Arch Biochem Biophys 344: 335-342 26 Blake CCF. Svendsen I. Prac NatlAcad Sei USA 71: 4178-4182 21 Grutter MG. Noguchi K. Plant Mol Biol 28: 105-111 11 PerrakisA. Yanagi M. Gene 140: 103-107 16 Ando A. J Mol Biol 248: 402-413 15 Masson J-Y. Remington SJ (1974) The three dimensional structure ofthe lysozyme from bacteriophage T4. Brzezinski R (1994) Primary sequence of the chitosanase from Streptomyces sp. Nucleic Aeids Res 16: 5210 9 Samac DA. Marcotte EM. Sielecki AR. Fujii T (1992) Primary structure of chitosanase produced by Bacillus ctrculans MH-K1. chitosanases and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Pfluger HO. Hironaka CM. Jensen A. Monzingo 6 Brurberg MB. Ernst SR. Robertus JD (1997) Kinetic analysis ofbarley chitinase. Nature 290: 334-335 24 Sinnott ML (1990) Catalytic mechanisms of enzymic glycosyl transfer. J Biol Chem 226: 1564-1573 8 Gaynor JJ (1988) Primary structure of an endochitinase mRNA from Solanum tuberasum. Remington SJ (1981) Common precursor of lysozymes ofhen egg-white and bacteriophage T4. Vorgias CE (1994) Crystal structure ofa bacterial chitinase at 2. Weaver LH. Skrivers K (1997) Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L. Yallaly PE.). Honda. Dauter Z. fungal infection and the elicitor chitosan. Shah DM (1990) Isolation and characterization of the genes encoding basic and acidic endochitinase in Arabidopsis thaliana.134 1. Biosei Biotech Biochem 59: 311-313 . Tommerup H. Hennig M. D. Nature 282:875-878 28 Andersen MD. Monzingo AF. Robertus JD. Monzingo AF. Matthews BW (1983) Goose lysozyme structure: an evolutionary link between hen and bacteriophage lysozymes? Nature 303: 828-831 22 Rossmann MG. Koenig DF.8 Aresolution. Wilson KS. Phillips DC (1979) X-ray crystallography ofthe binding ofthe bacterial cell wall trisaccharide NAM-NAG-NAM to lysozyme. Culley 0. Grutter MG. Structure 2: 1169-1180 12 Terwisscha van Scheltinga AC. Kagawa Y. Plant Physiol93: 907-914 10 Chang MM. Yabuki M. Hart PJ. Sykes BD. seeds at 1. Chem Rev 90: 1171-1202 25 Hollis T. Chet I. Marcotte EM. Robertus JD (1995) The refined crystal structure of an endochitinase from Hordeum vulgare L. strain N174 and comparison with other endoglycosidases. Ernst SR. Hollis T. Biochem J322: 815-822 29 Fukamizo T. Hirata H. Fukamizo T. J Mol Biol105: 75-96 23 Matthews B\V. Sarma VR (1967) Crystallographic studies ofthe activity ofhen egg-white lysozyme. Sarma VR (1965) Structure of hen egg-white lysozyme. Shinoyama H. J Mol Bio1229: 189-193 14 Hart PJ. Robertus JD (1996) Chitinases. Tews I. J Gen Appl Micrabiol38: 135-144 17 Marcotte E. FEMS Micrabiol Leu 124: 399-404 7 Leah R. North ACT. Phillips DC. Monzingo AF. Mair GA. OppenheimAB. Nature Struct Biol3: 133-140 19 Blake CCF. Anderson WF.8 Aresolution structure of hevamine. Denis F. Ready Mp. Robertus and A. North ACT. F. Phillips DC. Eijsink VG. Nes IF (1994) Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification ofthe gene product. Prac Roy Soc Ser B 167: 378-388 27 Kelly JA. Goto S (1995) Comparative biochemistry of chitinases-anomeric form of the reaction product. aplant chitinase/lysozyme and analysis of the conserved sequence and: structure motifs of glycosyl hydrolase family 18. Mair GA. Argos P (1976) Exploring structural homology ofproteins.

Fujii T. J Biol ehem 268: 18567-18572 35 Withers SG. Dijkstra BW (1995) Stereochemistry of chitin hydrolysis by a plant chitinase/1ysozyrne and X-ray structure of a complex with allosamidin: evidence for substrate assisted cata1ysis. Rupitz K. J Am ehem Soc 112: 5887 -5889 36 Terwisscha van Scheltinga AC. Aebersold R (1990) Unequivoca1 demonstration of the invo1vement of a glutamate residue as a nuc1eophi1e in the mechanism of a "retaining" glycosidase. Heyraud A. Henrissat B. Sueshige N. Miyashita K. Ide A (1989) Kinetics of chitinase from yam. Davies GJ.The structure and action of chitinases 135 30 Koga D. Agric Biol ehem 53: 3121-3126 31 Annand S. Henrissat B (1994) Stereochemica1 course ofthe hydrolysis reaction cata1yzed by chitinases Al and D from Bacillus circu1ans WL-12. Sakai H. Biochemistry 34: 15619-15623 . Antony R. Kobori K. Utsumi T. Annand S. Levitt M (1976) Theoretica1 studies of enzyme reactions: die1ectric. Gey C. Tanaka H (1993) Identification of glutamic acid 204 and aspartic acid 200 in chitinase Al of Bacillus circulans WL12 as essential residues for chitinase activity. Warren J. Vorgias CE. e1ectrostatic and steric stabi1ization ofthe carbonium ion in the reaction of1ysozyrne. Tsukamoto T. Dioscorea opposita THUNB. Watanabe T. FEBS Lett 343: 177-180 32 Drouillard S. Street IP. Uchida M. Isogai A. Biochem J328: 945-949 33 Warshel A. J Mol Biol103: 227-249 34 Watanabe T. Annand S. Kempton JB. Henrissat B (1997) Serratia marcescens chitobiase is a retaining glycosidase uti1izing substrate acetamido group participation. Kalk KR. Tomita H.

Similarly to their catalytic domains. This chapter presents a uni:IYing classification system of the various chitinase modules that combines specific features of their sequences. prokaryotes and viruses (Tab. Similarly. Catalytic domains Family 18 This is the largest chitinase family. In addition to a vast majority . Chitinases frequently displayamodular structure featuring a catalytic domain attached to one or several ancillary noncatalytic domains whose function is often chitin binding. Although chitinases are classified under a single IUB-MB enzyme number (EC 3.4-glycosidic bonds between the N-acetylglucosamine residues of chitin. the noncatalytic domains of chitinases can be grouped into a number of distinct families. when subjected to independent sequence analysis.Chitin and Chitinases ed. once identified for one member. Classification of chitinases modules based on sequence similarities Chitinases are the glycoside hydrolases that hydrolyse the ß-l . Gene cloning and DNA sequencing have allowed the determination of a massive number of amino acid sequences of chitinases during the last 10 years. early comparisons oftheir amino acid sequences have revealed that their catalytic domains could be grouped in two different families [3]. it has been shown that the molecular mechanism is conserved within the families [8]. F-13402 Marseille Cedex 20.A. 31 Chemin JosephAiguier.14). France Summary. three-dimensional structures and reaction mechanisms. Muzzarelli © 1999 Birkhäuser Verlag BasellSwitzerland Classification of chitinases modules Bemard Henrissat Architecture et Fonction des Macromolecules Biologiques CNRS. Jolles and R. Gene cloning and DNA sequencing have allowed the determination of a massive number of amino acid sequences of chitinases during the last 10 years. Since sequence conservation is indicative of structural similarities.2. with about 180 members found in eukaryotes.A. The sections below list the various families of chitinase catalytic and noncatalytic domains known so far. that is families 18 and 19 of the classification of glycoside hydrolases based on amino acid sequence similarities [4-6]. 1). by P. One of the features of the classification of glycoside hydrolases in families of related proteins is that. the catalytic residues can be deduced for the other members as these residues are invariant in each family [4]. this classification system also correlates with the structural and mechanistic features of these enzymes [7].1.

2.2.1.14 3.2.2. Classification of chitinases and related enzymes in families of related catalytic domains (compiled in February 1998) Organism ECno.1.1.2.2.14 3.14 3.2.2.14 3.1. Alteromonas sp.1. Bos taurus Bos taurus unknown 3.14 3.14 3.2.1.2.14 P36910 Bombyx mori n.1.1.14 P41684 3.14 3.1. P37531 Bacillus thuringiensis chitinase SE2 putative chitinase chitinase-like protein 1 chitobiase Beta vulgaris 3.2.2. Aeromonas sp.2.14 3.1.2.1.14 3. Swiss-Prot EMBLI GenBank Glycoside hydrolase family 18 chitinase chitinase chitinase chitinase 1 chitinase 2 (3 repeats) chitinase A chitinase chitinase 1 chitinase 2 chitinase 3 chitinaseA chitinase C chitinase 1 chitinase 2 Acanthocheilonema viteae Acanthocheilonema viteae Autographa californica nuclear polyhedrosis virus Aedes aegypti Aedes aegypti Aeromonas caviae Aeromonas sp.1.2.2.1.14 3.1.14 3.2.14 3.30 Q01458 Anopheles stephensi Anopheles stephensi Anopheles stephensi Aphanocladium album Arabidopsis thaliana Arabis gemmifera Arabis glabra Arabis lyrata Aspergillus nidulans Aspergillus nidulans Bacillus circulans Bacillus circulans AF026491 AF026492 U09139 031818 063139 063139 063139 013762 AB004557 AF026495 AF026496 AF008575 AF026493 AF026494 AF026497 AF026498 AF026499 X64104 M34107 AB006070 AB006071 AB006072 D87063 087895 M57601 chitinase 1 chitinase 2 chitinase 3 chitinase 1 chitinase 2 chitinase 3 chitinase 1 chitinaseA chitinase chitinase chitinase chitinase chitinase chitinase AI Anopheles gambiae U14638 L42010 L22858 P32470 P19172 U71214 026185 U89796 S66038 U86876 AFOI1373 M95770 .2.14 3.1.1.14 3.14 3.14 3.2.1.14 3.2.1.1.1.14 3.2.1.2.14 3.2.2.14 3.2.1.1.14 3.14 3.1.1.14 3.1. IOS-24 Aeromonas sp.1.1.2.2.d.1.14 3.1.1.14 P32823 P20533 P27050 089568 010594 Anopheles gambiae Anopheles gambiae chitinase C 1 chitinase 0 Bacillus circulans chitinase unknown protein chitinase Bacillus licheniformis 3. IOS-24 Alteromonas sp.1.2. Anopheles freeborni Anopheles freeborni 3.138 B.d.14 3.2.14 Bacillus subtilis n.1.2. Henrissat Table I.2.14 3.14 3.14 3.2.14 3. 10S-24 Aeromonas sp.2.1.2.

n.2.14 3.2.14 X83426 Z46802 U36490 Ul5800 Ul5801 Un030 P36908 X70660 ABOO1874 Z68924 P54196 L41663 .3 ORF ZK938.2.3 ORFR09D1.3 ORF C08H9. Z49913 Caenorhabditis elegans n.8 ORF R09DI.2.1 ORF R09D1.d.1.d.14 ORFC08H9.1.2.d.1 ORFTl3H5. AF016419 Z81062 Z78412 Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Swiss-Prot EMBLI GenBank U59689 U59690 AF026152 U42835 Z54342 Z54342 Z70035 Z70035 Z70035 Z70035 Z70035 Z70035 Z70035 Z70035 U70858 Z66524 n.d.1.d.1 (2 repeats) concanavalin B putative narbonin chitinase 1 chitinase 2 chitinase 3 Organism ECno.1.6 ORFR09D1. Bos taurus Brugia malayi Brugia pahangi Brugia pahangi Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans unknown Q28042 3.1.1.14 chitinase Chelonus sp.8 ORF M176. n.2.d.d.d.14 3.1.2.6 ORFF07GII.9 ORFTOIC4.139 Classification of chitinases modules Table 1 (continued) oviduct glycoprotein chitinase chitinase chitinase chitinase CHT I ORF C04F6. Dl6639 M73689 n.1.2. n. n.6 (2 repeats) ORFTl9H5.14 3. n.10 ORF C08H9.14 3.d.d.d.d. n.d.14 3.14 Y13233 Y13234 chitinase chitinase Choristoneura fomiferana nuc1eopolyhedrovirus Geer arietinum chitinase B chitinase chitinase 1 Clostridium paraputrijieum Clostridum thermocellum Coecidioides immitis P49347 P46876 P40953 P40954 3.8 ORF R09D1.14 Ul0422 chitinase chitinase Chironomus tentans Chironomus tentans 3. venom 3.2.14 P29030 3.d.d. Z54342 Z54342 n.14 3. n.14 3.7 ORF R09D1.d.10 ORF R09D1.2.d. n.2.14 3.d. n.1.12 ORF C08H9.1.1.9 ORF F15A4.1.d.1. n.11 ORF R09D1.d. n. n. Z47745 Canavalia ensiformis Canavalia ensiformis Candida albieans Candida albieans Candida albieans unknown unknown 3.2. n.1.2.2. n.1.2.14 3.14 n. QI1174 n.

14 3.2.1.1.2.1.14 chitinase oviduct glycoprotein Kurthia zopjii 3.2.2.1.2.1. M84214 ORF3 Cucumis sativus n.14 D63702 Macaca mulatta unknown U87259 U86877 U07025 P09805 X07127 .2.1.2.1.1. 3.1.2.2.2.14 U59304 X90562 YI1391 Z46825 U67265 P23472 chitobiase Homo sapiens 3.14 3.14 chitinase Glossina morsitans Glycin max Helicoverpa zea nuc1ear polyhedrosis virus Hevea brasiliensis chitinase Homo sapiens 3.2.1.96 P36912 L06331 endo-N-acetylglucosaminidase F3 Flavobacterium meningosepticum Flavobacterium sp.1.14 U29615 YHEB pro tein endo-N-acetylglucosaminidase chitinase putative narbonin chitinase 3.2.1.14 n.2.d.1.14 AF026500 chitinase 2 Drosophila melanogaster chitinase 3 Drosophila melanogaster 3.2.14 P17541 U60807 M24365 chitinase ORF 1 Cucumis sativus n.14 P13656 U18997 chitinase Escherichia coli Ewingella americana endo-N-acetylglucosaminidase Fl Flavobacterium meningosepticum 3.96 P36911 M80793 endo-N-acetylglucosaminidase F2 Flavobacterium meningosepticum 3.1.1.d.1.2.2.1.1.2.1.14 3.2.1. Swiss-Prot EMBLI GenBank chitinase 2 Coccidioides immitis 3.2.30 Q01459 M95767 gp-39 protein Homo sapiens unknown P36222 M80927 oviduct glycoprotein Homo sapiens unknown Q12889 U09550 YKL-39 precursor Homo sapiens U49835 chitinase chitinase Hyphantria cunea Janthinobacterium lividum unknown 3. M84214 chitinase 1 Drosophila melanogaster 3.1.14 AF026503 n.d.2.14 3. 3.1.2.14 Killer toxin Kluyveromyces lactis 3.14 U78318 chitinase-like chitinase chitinase chitinase chitinase AF026501 U78319 U78320 Enterobacter agglomerans 3.2.1.96 P80036 3.2.14 U58514 U58515 chitinase (chitoriosidase) Homo sapiens 3.1. Henrissat Table 1 (continued) Organism ECno.14 3.14 unknown 3.140 B.96 P36913 L06332 3.14 P54197 L41662 chitinase Coccidioides immitis Cucumis sativus 3.14 AF026502 chitinase 4 Drosophila melanogaster Drosophila melanogaster 3.2.2. U13825 Entamoeba dispar Entamoeba histolytica Entamoeba invadens 3.d.2.1.1.

2.2.2.2.1. U42580 chitinase chitinase 1 Parthenocissus quinquifolia chitinase 2 chitinase 3 chitinaseA chitinase chitobiase chitinase chitinase I chitinase II chitinase III chitinase ORFD9481.d.1.2.14 chitinase Serratia marcescens 3.2.14 3.1. 010363 Oryza sativa 3.14 3.1.1.2.1.2.2.14 3.1.2.2.2.14 3.14 3.2.14 3.1.1.Classification of chitinases modules 141 Table 1 (continued) chitinase oviduct glycoprotein chitinase chitinase CHIT42 BRP39 protein ECF-L precursor oviduct glycoprotein secretory protein YM-l chitinaseA Organism ECno.1.1.1.2.2.1.14 3. 3.2.14 chitinaseA chitinase B putative chitinase chitinaseA Serratia marcescens 3.14 3.14 chitinase Serratia marcescens n.1.7 Penaeus japonicus Penaeusjaponicus Phaseolus angularis 3.14 Mus musculus unknown Mus musculus unknown unknown Q62010 Mus musculus Mus musculus Swiss-Prot EMBU GenBank unknown U02270 032218 X89212 AF027497 X93035 D87757 032137 M94584 3.2.14 3.1.14 3. Manduca sexta Mesocricetus auratus Metarhizium anisopliae Metarhizium anisopliae 3.14 P29060 Orgyia pseudotsugata multicapsid polyhedrosis virus n.14 3.1.14 3.2.d.1.2.1.1.2.1.1.14 3.1.14 P36362 unknown Q60557 3. 3.1.d.2.14 unknown Q28542 unknown P36718 3.14 3.1.14 3.1.1.2.2.2.14 Z11563 Ul4639 Ul4639 L42021 U75930 P23473 D84250 P29024 Q01460 P29025 P29026 P29027 P29028 D89751 AB008027 011335 D49953 M95768 010154 010157 010158 D87894 M74070 U28373 L38484 L41660 P07254 P11797 L01455 X15208 Z48674 AF014950 .2.14 AB003195 Ul6719 M59903 U42580 Paramecium bursaria Chlorella virus 1 n.30 3.d.14 Nicotiana tabacum chitinase B chitinase Nicotiana tabacum chitinase chitinase (putative) Onchocerca volvulus chitinase III a oviduct glycoprotein oviduct glycoprotein chitinase (GI-1181344) ORF (GI-1181344) Onchocerca volvulus Ovis aries Papio anubis Paramecium bursaria Chlorella virus 1 Penaeusjaponicus Psophocarpus tetragonolubus Rattus norvegicus Rhizopus niveus Rhizopus oligosporus Rhizopus oligosporus Rhizopus oligosporus Saccharomyces cerevisiae Saccharomyces cerevisiae Serratia marcescens Sesbania rostrata Stenotrophomonas maltophilia n.2.

2.14 M94105 M94106 Viciafaba Vicia narbonensis Vicia pannonica Vicia sativa Vigna unguiculata Vigna unguiculata Glycoside hydrolase family 19 Allium sativum chitinase 1 Allium sativum chitinase 2 Arabidopsis thaliana chitinase unknown unknown unknown unknown unknown chitinase (gene FI8019.a.2.14 3.1.14 3.1.2.14 3.2.1.2.27) Arabidopsis thaliana 3.1.1.1.2.2.a.32) Arabidopsis thaliana 3.2. 3.14 n.2.1.2.2.2.1.1.2.14 unknown Dl4536 U19900 unknown Q28990 3.96 P36909 Q05638 Pl1220 P04067 Streptomyces lividans Streptomyces olivaceoviridis Streptomyces plicatus Streptomyces plicatus Streptomyces thermoviolaceus Sus scrofa Sus scrofa chitinase chitinase chitinase chitinase chitinase chitodextrinase chitinaseA narbonin Trichoderma hamatum Trichoderma hamatum putative narbonin putative narbonin putative narbonin narbonin narbonin narbonin-like chitinase chitinase chitinase PRM 3 protein Viciafaba Viciafaba Trichoderma harzianum Trichoderma harzianum Vibrio furnissii Vibrio jUrnissii Vibrio harveyi Viciafaba 8wiss-Prot EMBLI GenBank AL021411 D13775 D12647 X71080 M82804 K02182 3.14 3.2.14 P51614 Zea mais unknown Z46827 Q41660 Z46834 Z25536 Z33641 Z46835 X88801 X88802 Z68123 882314 3.31.1.1.14 U43490 n.2. Henrissat Table 1 (continued) chitinase chitinase chitinase A chitinase C exo-chitinase chitinase-63 end-N-acetylglucosaminidase H chitinase heparin-binding glycoprotein oviduct glycoprotein Organism ECno.1.1.14 3.1. T01024. Streptomyces coelicolor Streptomyces erythaeus 3.1.2.14 3.14 3.1.2.14 unknown Z71415 U88560 L14614 X80006 L42548 U41418 U81496 Z46910 Vitis vinifera unknown 3.14 3.14 P48827 3.142 B.2.1.14 P14529 Streptomyces lividans 3.14 ACOO2333 . 3.14 ACOO2333 Arabidopsis thaliana 3.14 Z25683 ACOO2333 chitinase (gene FI8019.1.1.1.1.28) chitinase (gene F18019.1.2.2.14 3.2.14 3.2.

1.1.d.2.2.2.33. FI8019. Arabidopsis thaliana 3.14 3.d.14 Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Beta vulgaris Beta vulgaris Beta vulgaris Brassica napus 3.14 ACOO2333 Arabidopsis thaliana 3.2.2.2.1. 3. n.14 3. T01024.2.34.2.d.2.2.1.2 chitinase 3 chitinase 4 chitinaseA chitinase B chitinase chitinase 4 chitinase SP2 chitinase Organism ECno.2.2.1.1. n.14 3.2.2.1.1.1.1.14 3.4 ORF C51E3.14 3.14 n.14 3.14 3.2.1.14 chitinase B4 chitinase-25 ORFC08B6.8 ORF RI0Dl2.1.14 chitinase 1 chitinase chitinase chitinase Cynodon dactylon Daucus carota Daucus carota 3.1.1.1 chitinase 2.14 3.2.14 chitinase Citrus sinensis 3.14 3.29) chitinase B chitinase CHIV chitinase lA chitinase 1B chitinase 2 chitinase 2.14 chitinase Coix lachryma-jobi 3.1.14 3.7 ORFT26F2.d.14 3.2.1.2. FI8019.l5 ORFT05H4.14 3.2.14 ACOO2333 Arabidopsis thaliana 3.30) chitinase (gene T01024.14 3.1.32.d.14 3.14 3.1.2.2.1.1.2.2.1.14 3.2.14 3.1. n.2.1.1. n.31) chitinase (gene T01024.2.2.2.1.14 3.1.14 3.d.1 ORFT26H2.14 Arabidopsis thaliana 3.1.2.1.2. n.1.1 chitinase 1B chitinase Brassica napus Brassica napus chitinase chitinase chitinase Chenopodium amaranticolor Chenopodium amaranticolor 3.14 Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Caenorhabditis elegans Castanea sativa Chenopodium amaranticolor Chenopodium amaranticolor Daucus carota Swiss-Prot EMBU GenBank P19171 Q06012 Q06013 P42820 Q06209 Q09023 M38240 Y14590 Q06012 Q06013 Q06014 X82329 X82330 Q06015 Q06016 X56890 X56891 X79301 A23392 L25826 U21848 X61488 M95835 Z72502 Z78410 Z81109 AFOI6452 Z82054 Z82054 U48687 D45182 D45183 D45184 D45181 Z70032 P15326 AF020766 U52845 U52847 U52848 .2.1.14 3.Classification of chitinases modules 143 Table 1 (continued) chitinase (gene FI8019.1.14 ACOO2333 Arabidopsis thaliana 3.

14 3.1.14 3.14 3.14 3.14 3.14 3.2.1.2.14 3.1.14 P80052 Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Haemophilus injluenzae Helianthus annuus Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare Linum usitatissimum Lycopersicon chilense Lycopersicon esculentum Lycopersicon esculentum Lycopersicon esculentum Lycopersicon esculentum Lycopersicon esculentum Lycopersicon esculentum Medicago sativa Medicago truncatula Nicotiana Nicotiana Nicotiana Nicotiana tabacum tabacum tabacum tabacum Nicotiana tabacum Nicotiana tabacum P44187 P1l955 P23951 Q40114 Q05539 Q05540 Q05538 Q05537 P08252 P17513 P17514 P24091 P29059 U60197 U78888 Z68152 U32821 U96640 X15349 U02287 X78671 X78672 L34210 L34211 U94847 Ll9342 U30465 A32906 Z15141 Z15139 Z15140 Z15138 U83591 YI0373 X16938 M29869 M29868 X51599 X64518 844869 Oryza sativa 3.14 3.1.1.2.1.1.14 AF013581 ABOO3194 D16222 D16223 L37289 L40338 X56063 .14 3.14 3.2.2.1.1.2.2.1.1.14 n.14 3.1.2.14 3.1.2.14 3.1.1.1.2.2.2.14 3.14 3.2.2.1.14 3.14 3.2.1.1.14 3.2. Henrissat Table 1 (continued) chitinase chitinase chitinase chitinase ORFHI1415 chitinase chitinase chitinase chitinase chitinase chitinase 26 chitinase 33 chitinase chitinaseA chitinase chitinase chitinaseA chitinase B chitinase C chitinase D chitinase chitinase chitinase chitinase chitinase chitinase chitinase chitinase Organism ECno.14 3.144 B.2.2.1.2.1.14 3.14 A16119 A21091 AFOO1500 chitinase Oryza sativa 3.1.14 3.14 3.1. 3.1.2.2.2.2.2.1.2.d.14 3. 8wiss-Prot EMBLI GenBank Dioscorea japonica 3.2.1.14 3.1.2.14 3.2.2.14 AFOO1501 AF013580 chitinase chitinase chitinase chitinase chitinase chitinase Oryza sativa chitinase chitinase chitinase Nicotiana tabacum Nicotiana tabacum Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa 3.14 3.14 3.1.2.14 3.2.1.14 3.1.14 chitinase chitinase Oryza sativa 3.1.2.1.14 3.2.1.1.2.14 3.2.2.1.2.2.1.14 3.1.2.1.14 3.1.

14 3.1.145 Classification of chitinases modules Table 1 (continued) chitinase chitinase chitinase chitinase chitinase 1 chitinase 2 chitinase chitinase chitinase chitinase 4 chitinase 5 chitinase chitinase chitinase chitinase chitinase chitinase chitinase chitinase 2 chitinase 6 chitinase 8 chitinase B chitinase C chitinase chitinase chitinase chitinase chitinase chitinase chitinase chitinase chitinase I chitinase 2 chitinase 3 Swiss-Prot EMBLI GenBank Organism ECno.2.2.14 chitinase C chitinase Streptomyces griseus Theobroma cacao 3.14 3.14 3.1.14 chitinase 4 chitinase ChtA4 Solanum tuberosum 3.14 3.14 3.2.2.14 3.1.2.1.1.14 3.14 3.1.1.1.1.14 3.14 3.2.2.14 3.14 3.1.2.2.2.14 3.2.14 3.2.1.1.14 3.1.14 3.14 3.2.14 Solanum tuberosum 3.2.14 chitinase chitinase Triticum aestivum Triticum aestivum 3.1.1.14 3.2.1.1.14 3. Populus sp.1.2.2.2.2.14 3.1.1.2.2.2.2.1.1.1.1.1.14 3.2.2.2.14 3.14 3.1.1.2.1.2.1.1.1.1.14 X87109 Z29961 3.1.1.2.14 U02286 Z29962 X54367 Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa Persea americana Petunia hybrida Phaseolus vulgaris Phaseolus vulgaris Phaseolus vulgaris Picea glauca Pinus strobus Pinus taeda Pisum sativum Pisum sativum Pisum sativum Pisum sativum Pisum sativum Populus sp.1.2.14 3.14 3. Populus trichocarpa Populus trichocarpa Sambucus nigra Sambusu nigra Solanum tuberosum Solanum tuberosum Solanum Solanum Solanum Solanum tuberosum tuberosum tuberosum tuberosum Solanum tuberosum Solanum tuberosum Solanum tuberosum 3. Oryza sativa 3.1.14 3.1.14 P24626 P25765 P29021 P06215 P27054 P36361 P36907 P21225 P21226 P21227 PI6579 PI6061 P29031 P29032 P05315 P52403 P52404 P52405 P52406 Z78202 X51427 M13968 X57187 S43926 L42467 U57410 U31309 X63899 L37876 L37876 UOl661 M25337 X59995 X59995 Z46948 Z46950 X07130 AF024537 XI4133 X67693 U49969 U49970 U02605 U02606 U02607 U02608 AF024538 ABOO9289 U30324 X76041 X95000 .2.2.14 3.14 3.14 3.14 3.2.14 3.2.2.2.14 3.1.2.2.1.2.2.14 3.1.

The three-dimensional (3D) structures of several members of this family have been determined: examples include Serratia marcescens chitinase A [14].1.1. unknown.2. such as mammalian oviduct glycoproteins and several putative proteins found during the systernatic sequencing of the nematode Caenorhabditis elegans (Tab. Although the sequence similarity between these three proteins is small. d. the chitinase frorn Hevea brasiliensis [15.1.. 16] and the endo-ß-N-acetylglucosaminidases from Flavobacterium meningosepticum [17] and Streptomyces plicatus [18]. none.2.1. Henrissat Table 1 (continued) chitinase chitinase chitinase 1 chitinase 4 chitinase chitinase chitinase B chitinase chitinase chitinaseA chitinase B Organism ECno.2.2. ORF. EC number not available.146 B.2.1.1.14 3. and it is instead the acetamido group . one protonating the glycosidic oxygen and the other providing nucleophilic assistance and leading to the formation of a glycosyl-enzyme intermediate. the number of repeats is indicated.2. 1).14 3. of chitinases.1.1.96).1.14 3.1. Retaining glycosidases usually perform their catalysis utilising two carboxylic catalytic residues. they share a similar (ß/a)g fold (Fig. this family contains enzymes such as chitodextrinase.1. no enzymatic activity. which have no catalytic activity [9.2.2. have a carboxylic residue which protonates the glycosidic oxygen in the first step of catalysis. 10]. n. For a detailed description ofthe 3D structures of chitinases. open reading frame. this volume. implying a double displacement rnechanism [11-13]. Ulmus americana Urtica dioica Vigna unguiculata Vigna unguiculata Vitis vinifera Vitis vinifera Vitis vinifera Zeamays 3.14 3. like other glycoside hydrolases. A number ofproteins ofunknown function are also found in this family.30) and endo-Nacetylglucosaminidases (EC 3. see Robertus and Monzingo. unknown enzymatic activity.2.1.14 Zeamays Zeamays Zeamays 3. an exochitinase.2.14 3. An interesting case is the occurrence of several plant proteins such as concanavalin Band narbonin.14 3. these enzymes are unusual in that they lack an enzymic nucleophile. When a particular module is known to be present in multiple copies in a protein.14 3. Chitinases offamily 18.. However.14 Swiss-Prot EMBLI GenBank Pll218 P51613 P29022 P29023 L22032 M87302 X88800 X88803 U97521 U97522 Z54234 L16798 LOO973 M84164 M84165 n. Chitinases offamily 18 have been found to operate with overall retention ofthe anorneric configuration at the cleavage point. a. several di-N-acetyl chitobiases (EC 3.2. enzymatic activity not determined.14 3.1. 1).14 3.2.2.

Interestingly. 19. fibronectin type III modules: I TTG. module wl: ICTN. most have a modular structure with one or several noncatalytic domains which can be found indifferently at their N. cellulose binding domain type II: I EXG. chitin ase of Hevea brasiliensis [15. Some members of family 18 display an extremely large and complex modular structure such as the chitinase 2 of Aedes aegypti with three chitin . module 10 ofhuman fibronectin [38]. chitin ase of Hordeum vulgare [24.21]. cellulose binding domain ofthe Cex xylanase of Cellulomonas fimi [35]. hevein of Hevea brasiliensis [40] . Although several chitinases of family 18 comprise only a catalytic domain. Several proteins listed in Table 1 are probably noncatalytic. cellulose binding domain of the cellulase Z of Erwinia chrysanthemi [33]. of the substrate that provides intramolecular nucleophilic assistance to the departure of the aglycone. chitin binding domain family 2: IHEY. glycoside hydrolase family 19: 2BAA. chitin binding domain family 3: IAIW.or C-termini. presumably resulting in an oxazolinium ion intermediate [12. 16]. lack the protonating catalytic residue [9]. N-terminal domain of Serratia marcescens chitinase [14]. 25] . Ribbon representation of the main fold of the various modules identified in chitinases or homologous to chitinase modules.147 Classification of chitinases modules l hvq Ihev laiw 2baa l ttg lexg lctn Figure I. G1ycoside hydro lase family 18: I HVQ. the family 18 proteins devoid of catalytic activity. The figure was prepared using the MOLSCRIPT program [41]. narbonin and concanavalin B. as they also lack the catalytic residue. A number of these noncatalytic domains function as chitin binding domains.

Catalysis by inverting glycosidases involves a single nucleophilic substitution by a water moleeule. operates by inversion of configuration [26]. is made of more than two modules and contains a tandem repeat of two N-terminal chitin binding domains upstream of its catalytic domain. Although the first examination ofthe structure had led to the proposal that the enzyme worked by a retaining lysozymetype mechanism [24]. Besides its catalytic domain. or the YHEB protein of Escherichia coli. The largest modular sequence (2025 residues) in this family is that encoded by the F07G 11. Contrary to family 18.148 B. 1). . Enzymes acting on somewhat similar substrates (N-acetylglucosaminidases. Related enzymes So far chitinases have been found exclusively in the two families mentioned above. For instance. Henrissat binding domains and three catalytic domains. the chitinase of Urtica dioica. The two catalytic residues of barley chitinase are strictly invariant in family 19. only one enzyme. lysozymes and chitosanases) can be found in other glycoside hydrolase families. this huge protein contains 12 copies of a module found only in the nematode genome. lysozymes in families 22-25 and chitosanases in family 46 [4-6]. elegans (Tab. One of these residues protonates the glycosidic oxygen. many family 19 chitinases displaya modular structure with an N-terminal chitin binding domain homologous to wheat germ agglutinin. it is now clear that this enzyme. 1). Its global fold is completely different from that observed in family 18.9 gene of the nematode C. which contains several members with a large number of modules. indicating majordifferences in the geometry of their active sites compared with family 18 enzymes. However. Several chitinases of family 19 are made of a single catalytic domain with no ancillary domain. which bears no less than five putative polysaccharide binding domains and one catalytic domain. Family 19 This family counts more than 130 members.23]. A detailed description of the 3D structures of barley chitinase is found in Robertus and Monzingo. 25]. a putative protein from Haemophilus injluenzae and several putative proteins found in the genome of C. as the structure is essentially composed of a-helices (Fig. The 3D structures ofthe chitinase from barley has been determined [24. like all other studied family 19 chitinases. while the other activates the water molecule. elegans (Tab. this volume. which are all from plant origin except for a chitinase from Streptomyces griseus [22]. 1). Family 19 chitinases have been found to perform their hydrolytic reaction with inversion of the anomeric configuration at the site of cleavage [13.22. The catalytic machinery is made of two carboxylic catalytic residues separated by a distance greater than that of retaining enzymes [27]. N-acetylglucosaminidases can be found in families 3 and 20.

not associated with a catalytic domain). The function ofthese modu- . this module binds strongly to cellulose. some family II cellulose binding domains of cellulases have been shown to bind chitin [34].Classification of chitinases modules 149 Noncatalytic domains Sequence analysis of cellulases and chitinases has revealed that the catalytic domains of these enzymes form a number of different families [2-6. pullolanases. 1). Given the resemblance between the two target polysaccharides. It is like1y that the bacterial modules were initially acquired from an animal source and were then spread further between distantly related bacteria by horizontal transfers [36]. modules belonging to the third family have also been found in several cellulases and a serine protease. 2). Whereas the first two families contain modules found only in chitinases or in nonhydrolytic proteins such as wheat germ agglutinin or insect intestinal mucin. 3D structures have been determined for members of the second family (Fig. There are chitin binding proteins that exist as such (e. The precise function of polysaccharides binding domains has been carefully evaluated only in a few cases. 29-32]. 1). g. it is not surprising that some cellulose and chitin binding domains display cross-specificity [31]. and the function of most of these domains has been inferred from sequence data. Cellulose binding domains Cellulose binding domains have been c1assified in a number of families [2. this volume. these fibronectin type IIIlike modules occur as tandem repeats (Tab. the cellulose binding domains of cellulases and chitin binding domains of chitinases have been c1assified in several families based on sequence similarities [2. 2). acetyl xylan esterases and arabinofuranosidases. polyhydroxybutyrate depolymerases. and its 3D structure has been solved [33] (Fig. The 3D structure of the cellulose binding domain of the xylanase Cex of Cellulomonas fimi has been solved [35] (Fig. Several chitinases contain modules which are c1early homologous to cellulose binding domains of family II (Tab. and these are reviewed by Schrempf. exomaltopentaohydrolases). 2). Fibronectin type lII-like modules Several discrete modules displaying significant sequence similarity with fibronectin type III modules have been identified in a number of bacterial depolymerases (cellulases. inc1uding chitinases [36] (Tab. In the cellulase Z of Erwinia chrysanthemi. The affinity of the corresponding modules of chitinases for cellulose or chitin has not been investigated. 29-32]. In several instances. xylanases. Similarly. Interestingly. Chitin binding domains There are three families of chitin binding domains identified so far (Tab. 2). and the corresponding modules of chitinases most likely have an identical fold. 1). 28]. This family comprises over 50 other members which are found in cellulases.

14 hevein Arabidopsis thaliana unknown P43082 chitinase SP2 chitinase 25 Beta vulgaris 3.14 chitinase Gossypium hirsutum 3.14 3.2.2.2.14 3.3 (3 repeats) Caenorhabditis elegans chitinase I B Castanea sativa chitinase Chenopodium amaranticolor Chenopodium amaranticolor chitinase n.2. U86876 unknown (GI-1049446) (l0 repeats) Caenorhabdits elegans n.14 3.1.14 chitinase U83591 P08252 YI0373 XI6938 .2.1.14 3.1 (2 repeats) Caenorhabditis elegans ORF Tl3H5. Henrissat Table 2.2.2. U39678 chitinase chitinase Hyphantria cunea 3.1.1.d.1. 3.14 chitinase Medicago truncatula 3.14 AF02649I chitinase 2 (3 repeats) Aedes aegypti 3. AF016419 n.2.1.2.2.14 P80052 U78888 hevein (minor) Hevea brasiliensis unknown P02877 unknown P80359 chitinase Hordeum vulgare 3.1.14 Gossypium hirsutum 3.14 P1l955 XI5349 root-specific lectin (4 repeats) Hordeum vulgare none P15312 M29280 killer toxin Kluyveromyces lactis 3.1.2.d.2.14 U60197 hevein (major) Hevea brasiliensis M36986 chitinase 3.1.14 P42820 L25826 Brassica napus chitinase B4 Brassica napus 3.1.1.2.1.1.2.1.2.14 3.2.2.2. Swiss-Prot EMBLI GenBank Chitin-binding domains Family I chitinase 1 Aedes aegypti 3.2.14 Q09023 Q06209 M95835 X61488 ORF F07G 11.1. Classification of noncatalytic modules of chitinases in families of related sequences (compiled in February 1998) Organism ECno.1.14 P09805 X07127 chitinase C chitinase Lycopersicon esculentum Q05538 ZI5140 Medicago sativa 3.14 chitinase 3.d.1.1.d.1.2.14 D84250 D98751 tachycitin Tachypleus tridentatus unknown D85756 inscct intestinal mucin IIMI4 (5 repeats) Trichoplusia ni unknown AFOO0605 Chitin-binding domains Family 2 chitinase B Arabidopsis thaliana 3.9 (2 repeats) Caenorhabditis elegans n.1.150 B. U70858 Z66524 U48687 D45181 D45182 ORF TOl C4.1.14 3.1.1.2.2.14 Nicotiana tabacum 3.d.2.14 PI9171 M38240 UOl880 D45183 D45184 chitinase Chenopodium amaranticolor Chenopodium amaranticolor chitinase Dioscorea japonica 3.14 chitinase Manduca sexta Penaeus japonicus chitinase Penaeus japonicus P36362 U86877 U02270 3.2.1.14 AF026492 putative chitinase Bombyxmori n.

2.2.14 unknown unknown 3.1. Populus sp.1.14 3.1.14 3.2.14 none 3.14 3.14 P24091 P29059 Pl1219 X51599 X645 18 P24626 X54367 Nicotiana tabacum Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa Oryza sativa 3.2.14 3.14 3.14 Solanum tuberosum Solanum tuberosum Solanum tuberosum Solanum tuberosum Theobroma cacao Vigna unguiculata Vitis vinifera Vitis vinifera Pl12l8 P51613 M87302 X88800 Z54234 U97521 U97522 .14 3.2.1.1.1.2.1.2.2. 8wiss-Prot EMBLI GenBank Nicotiana tabacum 3.14 3.14 P16061 P29031 P29032 P05315 P52403 P52404 P52405 P52406 P09762 P09761 M13968 X57187 843926 L42467 L37876 X63899 U01661 M25337 X59995 X59995 agglutinin 1 (4 repeats) agglutinin 2 (4 repeats) Triticum aestivum none P10968 X07130 U02605 U02606 U02607 U02608 X13497 X13497 U30324 M25536 Triticum aestivum none agglutinin 3 (4 repeats) Triticum aestivum none P02876 P10969 M25537 J02961 chitinase chitinase Triticum aestivum 3.14 3.14 3.1.2.1.14 3.1.1. Solanum tuberosum Solanum tuberosum Solanum tuberosum M24504 Dl6223 L37289 X56787 X56063 P25765 Z78202 L41872 P06215 P27054 P36361 P21226 P36907 P21227 P16579 3.2.1.14 3.Classification of chitinases modules 151 Table 2 (continued) chitinase chitinase agglutinin (4 repeats) chitinase chitinase chitinase chitinase chitinase 1 chitinase 2 chitinase antifungal chitin-binding protein chitinase Organism ECno.14 3.14 3.1.2.14 3.l4 3.2.14 3.1.2.2.2.2.14 3.2.2.2.1.14 3.1.14 X76041 Ulmus americana 3.1.2.14 L22032 chitinase (2 repeats) chitinase 1 chitinase Urtica dioica chitinase chitinase Vitis vinifera 3.1.1.l.2.1.2.1.14 Persea americana 3.1.2.2.2.1.2.2.1.14 chitinase 4 chitinase 5 chitinase chitinase chitinase chitinase chitinase 6 chitinase 8 Phaseolus vulgaris Phaseolus vulgaris chitinase B chitinase C chitinase chitinase 1 chitinase 2 chitinase 3 chitinase 4 wound-induced protein wound-induced protein chitinase Populus trichocarpa Populus trichocarpa Picea glauca Pisum sativum Pisum sativum Pisum sativum Populus sp.2.1.14 3.1.2.2.1.2.1.2.14 3.14 Pharbitis nil unknown Phaseolus vulgaris 3.14 3.1.14 3.1.1.14 3.1.2.

14 3.14 Vibrio jurnissii Vibrio harveyi n.1.14 Streptomyces lividans Streptomyces coelicolor 3. 3. Swiss-Prot EMBLI GenBank Zea mays Zea mays 3.a.2.2.2.2.1.14 3.2.14 Bacillus thuringiensis 3.1.1.14 3.14 3.14 3. Streptomyces plicatus P11797 P27050 U89796 PI1220 P36909 Q05638 D63702 M82804 AF014950 D13775 Dl2647 AL021411 X71080 .2.14 3.1.2.2.2.1.2.2.14 3.1.1.1.14 3.1.d.14 P29022 P29023 M84164 M84165 P32823 U09139 D31818 D13762 AB004557 D63139 Chitin-bin ding domains Family 3 Aeromonas caviae chitinase A (2 repeats) chitinase (2 repeats) Aeromonas sp.14 P20533 P27050 P13656 D63139 D63139 M57601 Dl0594 U71214 ABOO1874 U18997 U07025 Streptomyces griseus 3.2.1.1.2.1.2.1.2.1.1. chitinase 1 (2 repeats) Aeromonas sp.1. 10S-24 chitinase AI Bacillus circulans chitinase D Bacillus circulans chitinase Bacillus licheniformis chitinase B Clostridium paraputrijicum YHEB protein (5 repeats) Escherichia coli chitinase (2 repeats) Janthinobacterium lividum chitinase Kurthia zopjii chitinase B chitinase C chitodextrinase chitinase A 3.14 3.1. chitinaseA Alteromonas sp.2.14 Streptomyces olivaceoviridis n.14 Stenotrophomonas maltophilia 3.152 B.2.1. 10S-24 chitinase 3 (2 repeats) Aeromonas sp.a.2.2.14 3.14 3.1.1.1. Henrissat Table 2 (continued) chitinase A chitinase B Organism ECno.14 3.14 n.1.2.14 3.14 U89796 U42580 Serratia marcescens Cellulose-binding domains type 11 chitinase Bacillus thuringiensis chitinase (GI-1181344) Paramecium bursaria Chlorella virus 1 chitinase C Streptomyces lividans chitinase Streptomyces coelicolor Fibronectin type III-like modules chitinase AI (2 repeats) Bacillus circulans Bacillus circulans chitinase D D63702 X15208 3.1.14 3.1.1.2.2. 3.2.14 3.14 AB009289 U41418 U81496 3.1. chitinase C Alteromonas sp.1.2.2.14 3.1.14 P36909 Dl2647 AL021411 P20533 M57601 Dl0594 U71214 chitinase chitinase Bacillus licheniformis 3.2.1.14 3.2. 10S-24 chitinase 2 (2 repeats) Aeromonas sp.14 Streptomyces lividans 3.14 3.2.2.14 chitinase (2 repeats) chitinase 63 chitinaseA chitinase A chitinase C chitinase (2 repeats) exo-chitinase Kurthia zopjii 3.14 3.2.1.2.2.1.

5. and is found in several other chitinases (Tab.1.1.2.2. enzymatic activity not determined.14 AB004557 n. 2. d. 010363 U75930 3. none.1.14 3.1.14 3. a. module xl). circulans chitinaseAI significantly reduces the activity of the enzyme on chitin.1 of the nematode C..a.2. F07G 11.2. the number of repeats is indicated. .2.9 and TO 1C4..80 residues conserved in several chitinases which is found in two copies in a cellulase from Clostridium thermocellum (Tab. unknown enzymatic activity. When a particular module is known to be present in multiple copies in a protein. Aeromonas caviae 3. Similarly. elegans (Tab.1.14 U59304 U67265 n. chitinase (putative) chitodextrinase chitinaseA Swiss-Prot EMBLI GenBank P32823 P41684 U09139 013762 L22858 3. open reading frame. [37] have shown that the deletion ofthe fibronectin type III-like module of B. This module shows some distant structural similarities with fibronectin type III domains albeit with significant differences [14] (Fig. ORF. Watanabe et al. It does not bear any sequence similarity to typical type III domains offibronectin or with the fibronectin type III-like domains described in the preceding section.1. An example is a stretch of.1. n.2. There are probably several other types of chitinase modules left to identify.14 P07254 LOl455 3.2.2. The function of all these modules remains to be discovered. unknown. Autography californica nuclear polyhedrosis virus Choristoneura jitmiferana nucleopolyhedrovirus Enterobacter agglomerans chitinaseA Helicoverpa zea nuclear polyhedrosis virus Orgyia pseudotsugata nuclear polyhedrosis virus Serratia marcescens Modules xl chitinase C Alteromonas sp.153 Classification of chitinases modules Table 2 (continued) Moduleswl chitinaseA chitinaseA chitinase chitinase chitinase chitinase Organism EC no.14 U72030 3. no enzymatic activity.1.14 U41418 AF014950 Vibrio jitrnissii n. les in depolymerases is not known. genes FIOG2.14 Alteromonas sp. EC number not available. Other modules An N-tenninal module was identified during the detennination of the 3D structure of chitinase A of Serratia marcescens [14]. but not on soluble substrates. 1). module wl). The 3D structure of the human fibronectin type III module is known [38] (Fig.14 3. Stenotrophomonas maltophilia 3. 1) have a family 18 module preceded by multiple repeats which are found only in the nematode genome. I).d.2.2.1.

Geremia RA. chitinases 1 and 2 of Drosophila melanogaster belong to family 18. circulans belongs to family 18. 11 and IV chitinases belong to family 19. g. However. in particu1ar in family 18. Henrissat B. A great deal of confusion exists in the naming of chitinase genes and the resulting encoded proteins: for instance. Watanabe for many useful discussions and help throughout the years. Implications for mechanism. Acknowledgements I would like to thank Drs. and the corresponding proteins wou1d be Chi I8a. Miller RC. the occurrence of chitinases in two clearly different families (families 18 and 19) reflects convergent evolution [4]. whereas classes III and V form family 18. 11 and III of Rhizopus oligosporus all belong to fami1y 18. Prot Seq Data Anal 3: 523 . g. References I Saxena 1. chitinase D of Lycopersicon esculentum is a member of family 19. J Bacteriol 177: 1419-1424 2 Gilkes NR. Kilbum DG. Microhiol Rev 55: 303-315 3 Henrissat B (1990) Wcak scquencc homologies among chitinases detected by c1ustering analysis. W. Another advantage ofthis classification is that it allows enzymes of different specificity (e. while chitinase D of B. exochitinase. chitinases I. Chitinase genes and proteins wou1d be more convenientIy named after their family classification (e. Henrissat B (1995) Multiple domain architecture of ß-glycosyl transferases. chitodextrinase. The classification in families ofrelated protein modules allows evolutionary analysis to be carried out on each independent module (see for instance [36] for an analysis of the fibronectin type III modules).4-glycanases: sequence conservation. these could be named chi18a. Brown RM Jr. Inversely. M. Class I. P. Coutinho with the preparation of Figure 1 is gratefully acknowledged. Similarly. Warren RAJ (1991) Domains in microbial ß-l. whereas chitinases 1 and 2 of Oryza sativa belong to family 19. When an organism produces multiple genes encoding chitinases from the same family. Chi 18b and so on. shows that some modern chitinases have been subjected to complex evolutionary events. Fevre M. and offers insights into the divergent evolution of enzyme families [4]. an important step in e1ucidating their structure and function. Davies. di-N-acetyl chitobiases and endo-N-acetylglucosaminidases) to be grouped in a single family (family 18). chi18b and so on. The help of Dr. Henrissat Concluding remarks The classification of chitinase modules based on the similarities of their amino acid sequences combines the structural features of enzymes with their 3D structures [39]. The highly modular structure of some chitinases. chi18 or chi19 for the genes.526 4 Henrissat B (1991) A c1assification of glycosyl hydrolases based on amino acid sequence similarity. Bioehern J280: 309-316 . function and enzyme families. G.154 B. Dijkstra and T. and ChiI8 or ChiI9 for the proteins). B. J.

Beguin P. Wakarchuk Ww. Schlesier B. Pfluger HD. Davies GJ (1997) Structural and sequence-based classification of glycosyl hydrolases. Marcotte E. Henrissat B (1996) Plant chitinases use two different hydrolytic mechanisms.4-glucanases and ß-l . Vorgias CE (1994) Crystal structure of a bacterial chitinase at 2. Dijkstra BW (1994) Crystal structure ofhevamine. An "inactivated" chitinase from seeds of Canavalia ensiformis. J Mol Bio1262: 243-257 17 Van Roey P. Dijkstra BW. seeds at 1. Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Robertus JD (1997) Kinetic analysis of badey chitinase.8 Aresolution structure of hevamine. Structure 3: 449-457 19 Drouillard S. Hata T. Schlesier B (1995) Crystal structure of concavalin B at 1. Honda Y. Armand S. Isogai A. Monzingo AF. a plant chitinase/lysozyme. Hollis T. Wilson KS. Nikaidou N. Pfeffer-Hennig S. Monzingo AF. Boiler T. Warren RAJ. J Biol Chem 267: 12559-12561 9 Hennig M. Ready Mp. Armand S. Henrissat B. FEBS Lett 343: 177-180 12 Terwisscha van Scheltinga AC. a plant defence protein with chitinase and lysozyme activity. Van Roey P (1995) Crystal structure of endo-ß-N-acetylglucosaminidase H at 1. Goto S (1995) Comparative biochemistry of chitinase. Miller RC Jr. Kalk KH. Anomeric form of reaction products. Jansonius JN. Perrakis A. Henrissat B (1997) Serratia marcescens chitobiase is a retaining glycosidase utilizing substrate acetamido group participation. Mitsutomi M. Dauter Z.4-xylanases. J Am Chem Soc 119: 7954-7959 22 Ohno T. Fukamizo T. J Mol Bio1229: 189-193 25 Hart PJ. Henrissat B. Arch Biochem Biophys 344: 335-342 27 McCarter JD. PlummerTH. Evidence for substrate assisted catalysis. Guan C. Gilkes NR. Terwisscha van Scheltinga AC. Oppenheim AB. Davies G. Biosci Biotechnol Biochem 59: 311-313 24 Hart PJ. Claeyssens M. Watanabe T (1996) A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT6037. Oppenheim A. Nature Struct Biol3: 638-648 21 Tews I. Acta Cryst D5!: 177 -189 II Armand S. Wilson KS. Bairoch A (1996) Updating the sequence-based classification of glycosyl hydrolases.8 Aresolution. Biochemistry 33: 13989-13996 18 Rao V. Biochem J 328: 945-949 20 Tews I. Dauter Z. Neuhaus JM. Dijkstra BW (1996) The 1. Withers SG (1992) Stereoselective hydrolysis catalyzed by related ß-I . Tews I. Terwisscha van Scheltinga AC. Biochem J 293: 781. Hennig M. Dauter Z. Tomita H. Day PJ. Biochem J316: 695-696 7 Henrissat B. Vorgias CE (1996) Bacterial chitobiase structure provides insight into catalytic mechanism and the basis of Tay-Sachs disease. seeds. J Bacterioll78: 5065-5070 23 Fukamizo T. Dijkstra BW (1997) Substrate-assisted catalysis unifies 2 families of chitinolytic enzymes. J Mol Bio1248: 402-413 26 Hollis T. Nong VH (1995) Crystal structure ofnarbonin at 1. and analysis of the conserved sequence and structure motifs of glycosyl hydrolase family 18.Classification of chitinases modules 155 5 Henrissat B. Dijkstra BW (1995) Stereochemistry of chitin hydrolysis by a plant chitinase/lysozyme and x-ray structure of a complex with allosamidin. Armand S. Watanabe T. Kilburn DG. Structure 2: 1169-1180 15 Terwisscha van Scheltinga AC. Chet I. and its complex with an inhibitor.3 Aresolution. Biochemistry 34: 15619-15623 13 Iseli B. Vorgias C. Kalk KH. Perrakis A.9 Aresolution: active-site geometry and substrate recognition. Withers SG (1994) Mechanisms of enzymatic glycoside hydrolysis Curr Opin Struct Biol4: 885-892 . Koga D.65 A resolution. FEBS LeU 382: 186-188 14 Perrakis A. Tarentino AL (1994) Crystal structure ofendo-ß-N-acetylglucosaminidase F I.8 A resolution. and alß-barrel enzyme adapted for a complex substrate. Armand S. Curr Opin Struct Biol7: 637-644 8 Gebier J. Beintema JJ. J Mol Bio1254: 237 -246 10 Hennig M. Ernst SR. Gey C. Structure 2: 1181-1189 16 Terwisscha van Scheltinga AC. Robertus JD (1993) Crystal structure of an endochitinase from Hordeum vulgare L.788 6 Henrissat B. Wilson KS. Wilson KS. Henrissat B (1994) Stereochemical course ofthe hydrolysis re action catalyzed by chitinases AI and D from Bacillus circulans WL-12. Heyraud A. Rao V. Robertus JD (1995) The refined crystal structure of an endochitinase from Hordeum vulgare L. Wilson DB.

Mornon JP (1989) Cellulase families revealed by hydrophobie cluster analysis. Barras F. Kay LE. Carver JP.8994 37 Watanabe T. Miller RC Jr. Rodriguez-Romero A. Claeyssens M. Ito Y. Arreguin B (1993) Hevein: NMR assignment and assessment of solution-state folding for the agglutinin-toxin motif. Lee Hl. Gene 81: 83-95 29 Coutinho JB. Driscoll PC. Ann Rev Microbiol 50: 183-212 33 Brun E. Cao B. Lemesle L. Campbell ID (1992) Biochemistry 31: 2068-2073 39 Davies G. Blackledge MJ. Harvey TS (1995) Solution structure of a eellulose-binding domain from Cellulomonas jimi by nuclear magnetie resonanee speetroseopy. Gilkes NR. Henrissat 28 Henrissat B. Harris-Brandts M. Muhandiram DR. Broekaert WF (1993) Structure and function of chitin-binding proteins. Protein Eng 7: 117-123 35 Xu GY. Tanaka H (1994) The roles ofthe Cterminal domain and type III domains of ehitinase Al from Bacillus circulans WL-12 in chitin degradation. Mardon HJ. Warren RAJ. Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. Warren RAJ.7009 36 Bork P. Gilkes NR. Kilburn DG. Kilburn DG (1994) An internal eellulosebinding domain mediates adsorption of an engineered bifunetional xylanase/eellulase. Hashimoto M. Biochemistry 34: 6993 . Doolittle RF (1992) Proposed aequisition of an animal protein domain by bacteria. J Bacteriol176: 4465-4472 38 Baron M. Gilkes NR. MainAL. Henrissat B (1995) Structures and mechanisms of gIyeosyI hydrolases. Yamada T. J Appl Cryst 4: 946-950 . Ong E. Kilburn DG. Proc Natl Acad Sei USA 89: 8990. Tomme P. Marion D (1997) Solution structure ofthe cellulose-binding domain ofthe endoglueanase Z seereted by Erwinia chrysanthemi. Structure 3: 853-859 40 Andersen NH. Miller RC Jr (1992) The binding of Cellulomonas jimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats. Sekine S. Biochemistry 32: 1407-1422 41 Kraulis PJ (1991) MOLSCRlPT: a program to produce both detailed and schematie plots of protein structures. Annu Rev Plant Physiol Plant Mol Bio144: 591-615 31 Tomme P.156 B. Boyd J. Moriaud F. Mol Microbiol6: 1243-1252 30 Raikhel Nw. Adv Microb Physiol 37: 1-81 32 Warren RAJ (1996) Microbial hydrolysis of polysaccharides. Biochemistry 36: 16074-16086 34 Tomme P. Gans P. Warren RAJ.

Aberdeen AB25 2ZD. University 0/Aberdeen. Pathogens and predators of chitinous organisms characteristically produce chitinases [1. some some few some few some few ? .Chitin and Chitinases ed. 999 Birkhäuser Verlag Basel/Switzerland Aggressive and defensive roles for chitinases Graham W Gooday Department 0/ Molecular and Cell Biology. Examples discussed include enzymes of insect and algal viruses. Jolles and R. At least some vertebrates. whereas hosts to chitinous pathogens. of bacterial and fungal pathogens of fungi and insects. also may utilise chitinases in their defence against pathogenic fungi and some parasites.19 18 18 18 18 18 Morphogenetic Defence Aggression 2 >8 2-10% many few many many many many many aIl? aIl? many some ? many all? all all all all many ? ? ? ? all ? ? ? ? * An exception is the family 19 chitinase of Streptomyces griseus HUT6037. Chitinases playamajor defensive role in all plants against attack by fungi. Salivas of some invertebrate predators have chitinolytic activity which may be involved in their attack on their prey. Muzzarelli @ . These chitinases play roles in penetration of fungal cell walls. notably plants but perhaps also vertebrates inc1uding humans. 2]. UK Summary. Foresterhil/. and of parasitic protozoa. A. and perhaps also against attack by insect pests. incIuding fish and humans. This account concems their aggressive and defensive roles. and of exoskeIetons and peritrophic membranes of arthropods. of yeast killer toxin plasmids. Table 1. Chitinases are produced by a wide variety ofpathogenic and parasitic microbes and invertebrates during their attack on chitin-containing organisms. Introduction Chitinases and related enzymes have many roles in an increasingly wide range of different biological systems. by P. Roles for chitinases Group Chitinase family Nutritional Plasmids Viruses Archaea Eubacteria Protists Plants Fungi Nematodes Arthropods Molluscs Other invertebrates Fish Amphibians/reptiles Birds Mammals 18/19 18 ? 18* 18 18. The plant chitinases form a very large and diverse assemblage of enzymes from two families of glycosyl hydrolases. A. Institute 0/Medical Sciences.

respectively. californica NPV from which the chitinase gene and the adjacent cathepsin gene had been disrupted was less pathogenic to larvae ofthe cabbage looper. 3]. Chitinases ofboth families are discussed in this chapter (Tab. was that after . a potent inhibitor of most family 18 chitinases. Trichoplusia ni. 18 and 19. leading to retention or inversion. The insect cell cultures (Spodoptera jrugiperda. if produced.158 G. however. Killer toxins of some strains of Kluyveromyces lactis and Pichia acaciae have chitinase activity [6. Comparative DNA sequence analysis suggests that there has been lateral gene transfer relatively recently. perhaps from S. and its amino acid sequence shows high homology to those of some bacterial chitinases. family 19 chitinases are unable to hydrolyse some model substrates. 1). but on infection an enormous increase in chitinase activity was observed [8]. The two families have different hydrolytic mechanisms. lactis the toxin is a trimeric protein. but the insects still died. This enzyme is encoded by the virus genome. Autographa californica nuclear polyhedrosis virus (AcMNPV) is a baculovirus that infects some lepidoptera and is used for biological control of insect pests. Chitinases fall into two unrelated families of glycosyl hydrolases. Astrain of A. A dramatic difference. distinguished by their amino acid sequences [4]. and/or localised weakening of. but may involve its binding to. one corresponding to the catalytic domain of family 18 chitinases and another corresponding to the cysteine-rich chitin binding domain offamily 19 chitinases and lectins [6]. with the intracellular y-subunit responsible for killing a susceptible cell of Saccharomyces cerevisiae. at a low activity. As a result. such as 4-methylumbelliferyl glycosides. shown by the inhibition of activity by allosamidin. and are resistant to inhibition by allosamidin.W Gooday produce chitinases to defend themselves [2. The significance of this chitinase activity remains unclear. this enzyme could aid to penetration of the host caterpillar's peritrophic membrane. Plasmids of some strains of yeasts encode toxins that kill other strains. Chitinases encoded by plasmids and viruses Viruses and plasmids are mobile packages of genes. and in several cases they encode chitinase genes. Chitin-deficient yeast cells are resistant to the toxin. 7]. fall armyworm) used to grow the virus produced their own chitinases. expecially Serratia marcescens chitinase A. while the a-subunit has exochitinase activity essential for the action of toxin. The amino acid sequence of the a-subunit has striking simi1arities to other chitinases in two regions. of the anomeric configuration [5]. marcescens as it is itself an insect gut pathogen. The original rationale for looking for a chitinase activity in this baculovirus was that. the susceptible yeast cell surface to facilitate the uptake of the y-subunit. which suggests that it has arisen by fusion of portions of two genes of different origins. In K.

produces six inducible chitinoIytic enzymes when grown on chitin. as with some bacterial. [9]). whereas several proteins with chitinase activity and several with chitosanase activity were identified in disrupted particles of Chlorella virus CVK2 [11]. its major significance is in aiding release of viruses from the dead host. photographed 7 days after in feet ion with Autographa californica nucleopolyhedrovirus: M. Trichoplusia ni.4-Nacetylglucosaminidases acting in exo-type fashion to hydrolyse chitin to . although this baculovirus chitin ase may aid penetration of the peritrophic membrane ofthe insect host. W has died and liquefied. mock-infected. D. and one to bacterial chitosanases [10]. Trichoderma harzianum. four endochitinases and two ß-I . infected with wild-type virus. Caterpillar M is healthy.Aggressive and defensive roles for chitinases 159 Figure I. D. Possee (cf. death the insects infected by the chitinolytic virus were totally liquefied. infected with virus with chitinase and cathepsin genes disrupted. These enzyme activities presumably are associated with entry and/or eventual lysis ofthe algal cell wall. 1). Thus. protozoan and microfilarial chitinases discussed later. D has died and dried up. Larvae of cabbage looper. Two viruses ofthe green al ga Chlorella also produce chitinases. W. Photograph from R. whereas those infected by the mutant strain were dry cadavers [9] (Fig. which contains chitin. The most investigated. Microbial attack on fungi A number of chitinolytic mycoparasitic fungi are being investigated for their potential as biocontrol agents against fungal plant pathogens [12]. Analysis of the genome of Chlorella virus PBCV-l revealed the sequences of three genes with strong resemblance to microbial chitinases.

compared with only the exotype activities when grown with Sclerotium rolfsi. marcescens chitinase A. they can provide nutrients both directly in the form of amino sugars and indirectly by exposing other host material to enzymatic digestion. and in the case of G. gliotoxin [15]. or of the chitinase itself. virens. Chitinolytic bacteria can be pathogens of fungi. a pathogen of crayfish [22]. Metarhizium anisopliae. The direct involvement of chitinase activity in the antifungal activity is suggested by the demonstration of control of plant disease caused by R. the causative agent ofinternal stipe necrosis ofthe commercial mushroom Agaricus bisporus. and sometimes by N-acetylglucosamine. and acaropathogens. and so are likely to be . They are usually induced by chitin and its oligomers. rolfsii by application to infected plants of Escherichia coli bearing a plasmid expressing the gene for S. Different host fungi induce different patterns ofthe enzymes by T harzianum. Examples incIude the Oomycete Aphanomyces astaci. A mushroom-derived strain of this bacterium appeared to produce only one chitinase. Hirsutella spp. Isolates of this bacterium produced four chitinolytic enzymes when grown on chitin as sole carbon source [17]. and there has been much interest in their use as biocontrol agents. such as Ewingella americana. this volume). Beauveria bassiana. with its toxic secondary metabolite. both endochitinase and exo-type enzyme activities were induced. The antifungal activities of chitinases of mycoparasitic fungi. notably ßglucanases [14]. entomopathogens.160 G. marcescens from soils harbouring plant pathogenic fungi also have potential as biocontrol agents [18]. The direct roles of chitinases in the case of the entomopathogens and acaropathogens remains uncIear. in particular against insect pests [20. Strains of the strongly chitinolytic bacterium S. 21]. Most of these pathogens produce chitinases. coli with a plasmid lacking the promoter for expression of the gene [19] (see Herrera-Estrella and Chet. Aspergillus flavus and Nomuraea rileyi [24. [26]. Paecilomyces lilacius. and could not grow with chitin as sole carbon source [16]. solani and S. a pathogen of nematode eggs [23]. but produced this constitutively. 25].W Gooday N-acetylglucosamine [13]. Chitinolytic bacteria such as Enterobacter agglomerans are also being investigated as biocontrol agents against fungal plant pathogens. There is good evidence that these enzymes are directly involved in mycoparasitism. Chitinolytic attack on invertebrate exoskeletons Many fungi are pathogens of chitinous invertebrates. These chitinases can have three roles: they can aid the penetration of the host exoskeleton. leading to the lysis of the cell walls of host fungi. A range of other antifungal chemicals also act synergistically with these chitinases [14]. but not of a strain of E. such as T harzianum and Gliocladium virens work synergistically with those of other lytic enzymes. When grown with Rhizoctonia solani. and they can aid the release of progeny pathogens.

protozoa and filarial nematodes where the pathogen's endogenous chitinolytic activities appear to aid penetration of the peritrophic membrane. has turned the chitinolysis by Serratia to its advantage by developing a symbiotic relationship with S. Flies with mutations in two genes. two virulent isolates had high chitinase activities. than those of other strains. The sugar-beet root maggot. Often. W Gooday. and especially by synergistic action of chitinase and protease. marcescens [31]. In the case of N rileyi. thuringiensis required to kill caterpillars and midge larvae [30]. including the eggs. whereas an avirulent isolate had low activity [25]. are more susceptible than the wild type to infection via the gut by the bacterium S. deficient in chitinase and protease. marcescens. Thus chitinase is formed during the . There is evidence that malarial and trypanosome parasites use chitinases to penetrate the chitin-rich peritrophic membranes formed around the blood meals oftheir insect vectors [32]. are chitinolytic. was much less pathogenic to the flies. liquefaciens and S. Most isolates of B. the chitinase inhibitor. thuringiensis. chitin also has a major structural role in the ephemeral protective lining ofmany insect guts. Sampson and G. Treatment of isolated peritrophic membranes with chitinases leads to their perforation [28]. Further evidence for the direct involvement of the bacterial chitinases and virulence comes from the observation that feeding of aHosamidin. N. cut and miniature. These bacteria become embedded in the inner puparial surface and aid the emergence of the adult fly by their digestion of the chitin of the puparium. causing lesions ofthe tanner crab [27]. markedly increased the dose of B. That the chitin of the peritrophic membrane is a site of attack by pathogenie bacteria is suggested by experiments with Drosophila melanogaster [29]. however. unpublished observations). Also a mutant bacterial strain. chitinolytic enzymes would aid proteolytic enzymes and mechanieal pressure in penetration of the cuticle. Chitinolytic attack on the peritrophic membrane As weH as being a component of insect exoskeletons. It seems likely that when they are produced in high activity. Addition of exogenous chitinase aids the pathogenesis ofinsects by Bacillus thuringiensis [30]. the peritrophic membrane and of other internaiorgans. however. That the cut and miniature mutations lead to defieiencies in chitin content was demonstrated by showing that pupal shells from the mutant strains were more readily digested by Serratia chitinase. There are examples of bacteria. The symbiotic bacteria are present in all deve10pmental stages. and there is a positive correlation between chitinase activity in different strains and host mortality (M. there appears to be little or no correlation between chitinase activity and virulence. however.Aggressive and defensive roles for chitinases 161 produced by the fungus on the arthropod cuticle. A bacterial example of a chitinolytic bacterium eroding a chitinous exoskeleton is Photobacterium sp.

as the parasites undergo a more complex developmental process in the insect.162 G. rather than in degradation of the peritrophic membrane. The filarial nematode. and they produce a range of chitinase isozymes. it injects saliva via the salivary papilla into its prey at one point. donovani [37]. from the posterior salivary glands. In model infections in gerbils a major antigen of the B. The susceptible flies are infected with these intracellular bacteria. involving invasion of the cardiac valve. aiding penetration of the trypanosomes. are covered by a chitin-rich coat which is formed by stretching of the original eggshell. As discussed for protozoan parasites. contains a . Microfilariae. 36]. This saliva. Leishmania species. has mosquitoes as its vectors between mammalian hosts.41]. A more complicated involvement of chitinase with infection of a vector is seen with the implication of chitinase from maternally inherited bacteria in susceptibility oftsetse flies to infection with trypanosomes. or they may aid the penetration ofthe mosquito gut peritrophic membrane. Resistance of refractory tsetse flies (lacking the bacterial infection) is ascribed to killing of the trypanosomes in the gut by a lectin. Cultures of the trypanosomatids. as this correlated with the lack of damage to the cardiac valve in blood-fed infected flies [36]. In addition. The precise nature ofthe involvement of chitinases in infection is less clear. Gooday maturation of ookinetes. invertebrate parasites may use chitinases to aid penetration of chitinous hosts [28]. malayi microfilariae is a nematode chitinase [39]. This suggestion is supported by the observation that chitinase secretion was inhibited by incorporation of haemoglobin or blood in the growth medium of Leishmania major. Chitinases in saliva and venom As soon as the octopus Eledone cirrhosa has captured a crab. The involvement of chitinase in this development was shown by its inhibition by allosamidin. but additonally the chitinolytic bacteria may weaken the peritrophic membrane. They may playa role in the regulation of stretching of the chitinous sheath. 34]. 33. This gives the possibility of constructing adeletion mutant lacking chitinase activity to determine the role of the enzyme in infection.w. the invasive form of Plasmodium gallinaceum which penetrates the peritrophic membrane of the host mosquito [28. also produce chitinases [35. Brugia malayi. but the roles for the enzymes are unclear [40. sometimes one of the eye sockets. produced during infection of the mammal. and it may be at this stage that the chitinase plays a role. mosquito gut proteases potentiated malarial chitinase activity. It is suggested that bacterial chitinolysis releases amino sugars that inhibit the lectin-trypanosome binding and thus results in survival of the trypanosomes [38]. The microfilariae are the infective form for the insect vector. which consequently blocked malaria parasite transmission [34]. A secretory chitinase has been cloned and characterised from L.

S. [43]. A chitinase has also been characterised as a major component in the nonparalysing venom of the endoparasitic wasp ehelonus sp. Chitinases are major components of plant "pathogenesis-related proteins" induced following attack by potential pathogens or treatment with ethylene [3. In this way the octopus reproducibly recovers about half of the fresh weight of the crab (M. very sensitive to N-acetylchito-octaose. < 12. Plant cells are very responsive to specific oligosaccharides. with other defensive activities in the same molecule as the chitinase. with nascent chitin being very susceptible to enzymic lysis. The plant's chitinolytic activities will also have an indirect defence action by releasing oligosaccharide elicitors from fungal chitin. including those of N-acetylglucosamine. 44]. another group of pathogenesis-related proteins that attack the glucans cross-linked with chitin in the fungal cell wall [46]. with very low molecular sizes. Thus exposure to chito-oligosaccharides can elicit a variety of responses. with pI values of > 9 and 5.400 Da as estimated by size exclusion chromatography [42]. They have the specific 28S ribosomal RNA (rRNA) N-glycosidase activity characteristic ofribosome-inactivating proteins. A putative receptor. which rapidly diffuse throughout the crab and serve very specifically to digest the musculoskeletal attachments. as resolved by isoelectric focussing. causing rapid lysis offungal hyphal tips and germinating spores [45-47]. but its functional significance is unclear. Plant defensive chitinases All plants produce a range of chitinases which form at least four structural groups: classes I. including synthesis of antifungal phytoalexins. Some plant chitinase molecules are bifunctional. has been characterised from rice cells [51]. This mimics what occurs during moulting. The meat is chopped up with the beak.Aggressive and defensive roles for chitinases 163 potent mix of chitinases and proteinases [42]. and allows the octopus to remove the "meat" cleanly from within the carapace. This is a consequence of most potential pathogenic fungi having chitin as a key structural component of their hyphal walls. 11. The saliva contains at least two chitinases.6. and class III of family 18 [3].. This small size may aid the passage ofthe enzymes throughout the body of the prey. Some of these chitinases have direct antifungal activity. Three class III endochitinases produced by cell cultures of Trichosanthes kirilowii also have ribosome-inactivating activity [52]. An endochitinase with insect a-amylase inhibitor activity has . and collects in the octopus's crop. induction of antifungal chitinases and induction of K+ and er release. as the eggs do not appear to contain chitin. Grisley et al. The antifungal activity of the plant chitinases is synergistically potentiated by ß-glucanases. extracellular alkalinization and H 2 O2 synthesis [48-50]. and IV of family 19. The venom is injected into the eggs of lepidopteran prey. unpublished results).

fungi. Coix lachryma-jobi [53]. but even a weak activity may synergistically potentiate other antibacterial activities. This presumably is an adaptation of the ubiquitous production of chitinases as defence enzymes in plants. Pest attack. Nevertheless. which suggests that this may be the site of action of the plant chitinases. Experiments with spruce cells have led to the suggestion that the host's defensive responses are attenuated by degradation of chito-oligosaccharide elicitors by host chitinases [50]. which suggests that they may have an antibacterial role. but they mayaiso act against arthropod pests. so we can expect to see major developments in this area. 44.we are trying to improve on complex multilayered and tightly regulated defence systems that have developed during plant evolution. Results to date have been mixed. Some insectivorus plants produce chitinases which aid in digestion of their prey [56].W Gooday been characterised from seeds of Job's tears. causes induction of pathogenesis-related proteins. like fungal attack. Transgenic tobacco plants expressing a chitinase from tobacco homworm. some ofthe results look promising and some early field trials are taking place. as might be expected . Root protein extracts degraded the insect's peritrophic membrane. Diaprepes abbreviatus [54]. Many plant chitinases have lysozyme activity [3]. which have to exist in the presence ofthe plants' defence mechanisms [58]. There has been much interest in the possibility of increasing the natural defences of crop plants against fungal and pest attack by creating transgenic plants expressing chitinase genes from bacteria. Insects feeding on the transgenic plants were also more sensitive to the effects of B. plants and insects [3. Roots with the highest induced chitinase activities tended to be more resistant to attack by the weevil. Most plant roots are infected by mycorrhizal fungi. Manduca sexta. 47]. and extracts from more resistance strains showed greater damage. as assessed by feeding damage and growth ofthe pest larvae [55]. Thus chitinases were induced to different extents in roots of a range of citrus hybrids when they were fed on by a root weevil. thuringiensis toxin. Most attention has been paid to the antifungal activity of plant chitinases. Heliothis virescens. as the activity is usually only weak and confined to the plant cell's vacuole. Vertebrate defensive chitinases? There is increasing evidence that chitinases may have defence roles against pathogenic fungi and parasites in animals.164 G. 21. or they may locally suppress expression ofthese enzymes. This has been questioned [57]. It has also been suggested that changes in fungal wall composition resulting from host chitinolysis may aid in the exchange of nutrients between the symbionts [59]. These fungi may have walls that are resistant to the hosts' chitinases. showed increased resistance to attack by tobacco budworm. Blood of many vertebrates has .

which may have a defence role against potential fungal pathogens. (b) Same cells viewed with brightfield optics. The turbot plasma chitinase also has antifungal activity. In turbot. Scale bar represents 10 llm. Note activity associated with distinct cell inclusions (shown by arrow. (d) Same cells viewed with brightfield optics. but associated with nuclei in red blood cells (R). 2). but more dispersed throughout cytoplasm in white blood cells CL). Note activity throughout cells in Iymphocytes (L) and thrombocytes (T). Serum of ruminants and some other mammals is rich in chitinase activity [61]. probably lysosomes) in red blood cells (R). (c) N-Acetylglucosaminidase activity shown as fluorescence after incubation in 4-methyl-umbelliferyl-N-acetylglucosamine. there are chitinase activities in white and red blood cells and in plasma. Cytochemical localization of chitinolytic enzymes in blood cells of turbot. (a) Chitinase activity shown as fluorescence after incubation in 4-methylumbelliferyldiacetylchitobiose. This enzyme is a marker for .Aggressive and defensive roles for chitinases 165 Figure 2. chitinolytic activities. Human blood also has a chitinase activity which may have a defence function. with leucocytes being especially rich [60] (Fig. Activity has been detected in serum [62] and also in granulocyte-rich homogenates of leukocytes [63]. Scophthalmus maximus. This chitinolytic activity of leucocytes may have a defence role in digestion of ingested chitinous pathogens of fish such as microsporidia. Fluorescence is also apparent as haloes around red blood cells and their nuclei. From [60]. Scophthalmus maximus.

References 1 Gooday GW (1994) Physiology of microbial degradation of chitin and chitosan. Eur J Biochem 199: 483 -488 7 McCracken DA. leading to the cloning ofits gene [65. Howard SC. Nielsen KK. Chitin was originally thought of as being just an inert structural component. Upon infection with the fungal pathogen. Rasmussen V. Henrissat B (1996) Plant chitinases use two different hydrolytic mechanisms. Kluwer. BiochemJ280: 309-316 5 Iseli B. O'Donnell RW. Microbiology 140: 425-431 8 Hawtin RE. Neuhaus J-M. 279-312 2 Gooday GW (1996) Aggressive and defensive roles for chitinases. as its substrate hydrolytic activities were those of the animal rather than those of the fungus. 56. Virology 213: 673-685 . as most patients have a greatly elevated activity secreted by their macrophages [64]. Bolen PL (1994) The linear-plasmid-encoded toxin produced by the yeast Pichia acaciae: characterization and comparison with the toxin of Kluyveromyces lactis. Chitinase has been purified from sampIes from these patients. Pos see RD (1995) Identification and preliminary characterization of a chitinase gene in the Autographa californica nuc1ear poyhedrosis virus genome. In: RAA Muzzarelli (ed): Chitin enzymology. Stark MJR (1991) Kluyveromyces lactis toxin has an essential chitinase activity. Mikkelsen JD. and derivatives such as glycolchitin. Kitts PA. Gooday Gw. Plant J 3: 31-40 4 Henrissat B (1991) A c1assification of glycosyl hydrolases based on amino acid similarity. that of processing glucosaminoglycans. Chappell LH. Boiler T. ranging from chitin itself. but we are beginning to realise that it and its oligomers are used in a variety ofways throughout biology that require precise enzymic tailoring ofthe saccharide chains. however. FEBS Lett 382: 186-188 6 Butler AR. Gooday GW. Dordrecht. Martin VJ. Aspergillus fumigatus. ConcIusions Work with diverse biological systems over the past 20 years has led us to a realisation of the very wide occurrence of the enzymes classified as chitinases by their potential for hydrolysing various substrates. Zanotto MA. King LA. Atec. vol 2. Gooday Gaucher's disease. In: C Ratledge (ed): Biochemistry ofmicrobial degradation. as opposed to the alternative suggestion. comes from experiments with a similar enzyme in guinea pigs. Grottammare. 68]. Thus we can look forward to an exciting future for chitinase research. the serum levels of chitinase in circulation of pathogen-free guinea pigs increased about threefold over 4 days [67]. Kragh KM. Stark MJR. Treatment with antifungal drugs diminished the increase. to N-acetylglucosamine oligomers and their fluorogenic and chromogenic glycosides. These enzymes have been put to a remarkably wide range of uses [1. Amold K.w. Martin VJ. Vad K (1993) Plant chitinases. As yet. Ayres MD. 66]. 125-134 3 Collinge DB.166 G. The authors attributed this increase to the host's enzyme. Some evidence in favour. Armand S. there is no evidence for a direct defence role for this enzyme.

Bromel MC. Appl Environ Microbiol 61: 1720~ 1726 18 Kobayashi DY. Zarkowska T. Physiol Plant 35: 140~146 23 Dackman C. J Invert Path 54: 394~403 26 Chernin L. 165~184 13 Haran S. Cabib E. Hayes CK. Isamilov Z. FEMS MicrobiolLett 157: 189~194 17 Chernin L. Miller LH (1991) Malaria parasite chitinase and penetration ofthe mosquito peritrophic membrane. Microbiology 144: 2189~2194 31 Iverson KL. Can J Microbiol 43: 440~446 27 B Baross JA. J Invert Path 61: 81 ~ 84 25 El-Sayed GN. Pos see RD (1997) Liquefaction of Autographa californica nucleopolyhedrovirus-infected insects is dependent on the integrity ofvirus-encoded chitinase and cathepsin genes. Que Q. Roberts DW (1993) Entomopathogenic isolates of Metarhizium anisopliae. Chet I. B Söderström (eds): The mycota IV. Microbiology 140: 623~629 15 Di Pietro A. Hayes CK. Agaricus bisporus. Muthukrishan S (1997) Insect chitinases: molecular biology and potential use as pesticides. Gooday GW (1998) Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects. Beauveria bassiana and Aspergillus flavus produce multiple extracellular chitinase isozymes. Soil Biol Biochem 27: 1479~1487 19 Shapira R. Van Etten JL (1996) Analysis of 94 kb of the Chlorella virus PBCV-l 330-kb genome: map positions 88 to 182. Virology 216: 102~123 11 Yamada T. Freeman TP (1984) Bacterial symbionts in the sugar beet root maggot Tetanops myopaejormis (von Roder). Hiramatsu S. Proc Natl Acad Sei USA 88: 2807 ~281O 29 Flyg C. Gerson U. Clarke BB (1995) Isolation of the chitinolytic bacteria Xanthomonas maltophilia and Serratia marcescens as biological control agents for summer patch disease ofturfgrass. Phytopathology 79: 1246~1249 20 Khachatourians GG (1996) Biochemistry and molecular biology of entomopathogenic fungi. characterization and synergistic antifungal activity in combination with gliotoxin. Schickler H. Harman GE (1993) Endochitinase from Gliocladium virens: isolation. Songsri P. Chet I (1996) Differential expression of chitinases of Trichoderma harzianum during mycoparasitism. Virology 230: 361~368 12 Chet I. In: DT Wicklow. Morita RY (1978) Incidence. Kuzio JA. Riba G (1989) Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus Nomuraea rileyi. J Fish Res Bd Can 35: 1141~ 1149 28 Huber M. In: DH Howard. Human and animal relationships. Broadway RM. Gooday GW. Thomas CA. Arnold K. Harman GE (1994) Synergistic interactions between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition ofspore germination. Berlin. Oppenheim A. Appl Environ Microbiol47: 22~27 . Anderson AW. Mol Plant Phytopathol83: 308~313 16 Inglis PW. Nordbring-Hertz B (1989) Fungal parasitism of the cyst nematode Heterodera schachtii: infection and enzymatic activity. Unestarn T (1975) Properties of extracellular enzymes from Aphanomyces astaci and their relevance in the penetration process of crayfish cuticle. Coudron TA. Sztejnberg A (1997) Chitinolytic activity of the acaropathogenic fungi Hirsutella thompsonii and Hirsutella necatrix. King LA. JD Miller (eds): The mycota VI. Haran S. Virology 238: 243~253 10 Lu Z. Ignoffo CM. Peterbauer C. Chet I (1995) Chitinolytic Enterobacter agglomerans antagonistic to fungal pathogens. Rock DL. Berlin. Springer. Tester PA. 331~363 21 Kramer Kl. Li Y. Mol Plant Phytopathol86: 980~985 14 Lorito M. Guglielmoni M. Insect Biochem Mol Bio127: 887 ~900 22 Söderhall K.Aggressive and defensive roles for chitinases 167 9 Hawtin RE. Hadar Y (1997) Fungal antagonists and mycoparasites. Chet I. Staples RC. a recently discovered pathogen of the mushroom. Mozes-Koch R. Boman HG (1988) Drosophila genes cut and miniature are associated with the susceptibility to infection in Serratia marcescens. Inbar J. Fujie M (1997) Alternative expression of a chitosanase gene produces two different proteins in cells infected with Chlorella virus CVK2. Peberdy JF (1997) Production and purification of a chitinase from Ewingella americana. Environmental and microbial relationships: Springer. Kutish GF. Lorito M. Oppenheim AB (1995) Control of plant diseases by chitinase expressed from cloned DNA in Escherichia coli. microscopy and etiology of exoskeleton lesions in the tanner crab Chionectes tanner. FEMS Microbiol Ecol 62: 20 1~ 208 24 St Leger RJ. Gafni A. Genet Res 52: 51 ~ 56 30 Sampson MN. Ordentlich A.

McDonald RE. Kaslow DC (1993) Chitinase: a novel target for blocking parasite transmission? Parasitol Today 9: 252-255 33 Shahabuddin M.168 G. Plant J 12: 347-356 52 Shih NR. Messer G (1992) Leishmania infections damage the feeding mechanism of the sand fly vector and implement parasite transmission by bite. Inhibition of fungal growth by combination of chitinase and ß-l . Kaslow DC (1994) Plasmodium: parasite chitinase and its role in malaria transmission. Comp Biochem Physiol95 B: 311-316 43 Krislman A. Toppan A. J Biol Chem 269: 20971-20976 44 Graham LS. Shewry PR (1989) Purification and characterization of an insect a-amylase inhibitor/endochitinase from seeds of Jacob's Tears (Coix lachryma-jobi). Dalamagas D (1995) Structure and function of a family of chitinase isozymes from Brugian microfilariae. Amold K. Girbes T. Boiler T (1988) Antifungal hydrolases in pea tissue. Heam CJ.3-glucanase. Hebe G. Plant J72: 1057-1083 45 Broekaert WF. Gene 208: 315-322 38 Welbum SC. Gooday GW (1993) Rickettsia-like organisms and chitinase production in relation to transmission of trypanosomes by tsetse flies. Chet I (1993) Cytology of infection of 35S-bean chitinase transgenic canola plants by Rhizoctonia solani . Lane WS. Jacobson RL. Can J Microbiol39: 318-328 48 Yamada A. Aikawa M. Kodama 0. Richardson M. Perler FB (1992) Transmission-blocking antibodies recognise microfilarial chitinase in brugian Iymphatic filariasis. Broglie K. tobacco and wheat. Shibuya N. Proc R Soc Lond B 245: 121-126 36 Schlein Y. Physiol Mol Plant Pathol33: 319-331 46 Mauch F.W Gooday 32 Shahabuddin M.cytochemical aspects of chitin breakdown in vivo. Piessens WF. Physiol Plant 94: 164-173 . Biochim Biophys Acta 993: 260-266 54 Mayer RT. Doostdar H (1995) Citrus rootstock responses to herbivory by larvae of the sugarcane roostock borer weevil (Diaprepes abbreviatus). Proc Natl Acad Sci USA 89: 1548-1552 40 Fuhrman JA. Maudlin I. Exptl Parasitol 80: 672-680 41 Fuhrman JA (1995) Filarial chitinases. Plant Physiol87: 936-942 47 Benhamou N. Esquerre-Tugaye (1988) Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment. Shlomai J (1991) Chitinase secreted by Leishmania functions in the sandfly vector. Shibuya N (1997) Identification of a high-affinity binding protein for Nacetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labelling. Kaku H. Berdis E. extracellular alkalinization and H2 0 2 synthesis of Picea abies cells. Plan ta 203: 470-479 51 Ito Y. Sticklen MB (1994) Plant chitinases. cloning and characterisation of new chitinase stored in active form in chitin-lined venom reservoir. Toyoshima T. enzymatic and antifungal properties of chitinases from thom-apple. Van Parijs J. Peumans WJ (1988) Comparison of some molecular. Hager A (1997) Cleavage of chitinous elicitors from the ectomycorrhizal fungus Hebeloma crustuliniforme by host chitinases prevents induction of K+ and Clrelease. Parasitol Today 11: 259-261 42 Grisley MS (1993) Separation and partial characterization of salivary enzymes expressed during prey handling in the octopus Eledone cirrhosa. Iglesias R (1997) Bifunctional plant defence enzymes with chitinase and ribosome inactivating activities from Trichosanthes kirilowii cell cultures. Mauch-Mani B. Parasitology 107: 141-145 39 Fuhrman JA. Shapiro JP. Jackman AP. Proc Natl Acad Sei USA 89: 9944-9948 37 ShakarianAM. Nair PN. McDonald KA. Exp Parasitol79: 85-88 34 Shahabuddin M. Dwyer DM (1998) The Ld Chtl gene encodes the secretory chitinase ofthe human pathogen Leishmania donovani. Proc Natl Acad Sci USA 90: 4266-4270 35 Schlein Y. Physiol Mol Plant Pathol33: 409-417 50 Slazer P. Smith RF. Kaslow DC (1993) Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease. Lee J. Akatsuka T (1993) Induction of phytoalexin formation in suspension-cultured rice cells by N-acetylchitooligosaccharides. Jones D (1994) Isolation. Biosei Biotech Biochem 57: 405-409 49 Roby D. Jacobson R. Allen AK. Plant Sei 130: 145-150 53 Ary MB. Broglie R. McCollum TG.

Chitin Chitosan Res (Japan) 3: 233-243 . Renkema GH. Aerts JMFG (1994) Marked elevation of plasma chitotriosidase activity.Aggressive and defensive roles for chitinases 169 55 Ding XF. 'Iransgenet Res 7: 77 -84 56 Gooday GW (1990) The ecology of chitin degradation. Slettengren K (1979) Bovine serum chitinase. New Phytol131: 255 . A novel hallmark of Gaucher disease. Strijland A. a novel member ofthe chitinase farnily ofproteins. Mijsers AO. Infect Immun 63: 4770-4773 64 Hollak CEM. Adv Microbiol Ecolll: 387 -430 57 Düring K (1993) Can lysozymes mediate antibacterial resistance in plants? Plant Mol Biol 23: 209-214 58 Hodge A. van Zonneveld AJ. Van Steijn GJ (1994) Human serum contains a chitinase: identification of an enzyme formerly described as 4-methylumbelliferyl-N-acetyl-ß-chitotetraoside hydrolase (MU-TACT hydrolase). J Biol Chem 270: 26252-2656 67 Overdijk B. Aerts JMFG (1995) Cloning of a cDNA encoding chitotriosidase. J Clin Irrvest 93: 1288-1292 65 Renkema GH. Lind J. Glycobiology 6: 627-634 68 Gooday GW (1997) The many uses of chitinases in nature.261 59 Sauter M. Wang XR. Johnson LB. Morgan TD. Eur J Biochem 100: 455-460 62 Overdijk B. Donker-Koopman WE. Odds FC (1996) Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus jUmigatus. Gopalakrishnan B. Van Weeley S. Hager A (1989) The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies. J Biol Chem 270: 2198-2202 66 Boot RG. Van Steijn GJ. Kramer Kl. Gooday Gw. Adams DJ (1995) Chitinase activity in human serum and leukocytes. Alexander IJ. Muthukrishnan S (1998) Insect resistance of transgenic tobacco expressing an insect chitinase gene. and Pinus sylvestris L. J Fish Bio140: 919-927 61 Lundblad G. a human chitinase produced by macrophages. Planta 179: 61-66 60 Manson FDC. root systems challenged with pathogenic and ectomycorrhizal fungi. Glycobiology 4: 797 -803 63 Escott GM. Aerts JMFG (1995) Purification and characterization ofhuman chitotriosidase. Fletcher TC (1992) Localization of chitinolytic enzymes in blood cells ofturbot. White FF. Boot RG. Van Oers MHJ. Elander M. Scophthalmus maximus. Gooday GW (1995) Chitinolytic enzymes of Eucalyptus pilularis Sm.

providing large numbers of generally near-identical plants in one vicinity.Ä. It is generally recognized that biological control agents are safer and more environmentally sound than is reliance on the use of high volumes of pesticides. they are receiving attention in regard to their development as biopesticides or chemical defense proteins in transgenic plants and microbial biocontrol agents. Box 12. Unidad Irapuato.and bacteria-producing chitinases exhibit antagonism against fungi. environmentally friendly control alternatives. Insect pathogenic fungi have considerable potential for the biological control ofinsect pests. an ecologically unnatural situation. Mexixo 2 The Hebrew University of Jerusalem. It is. Apartado Postal 629. Control of plant pests by the application of biological agents holds great promise as an alternative to the use of chemicals. Rehovot 76100. In general the amount of pesticides released into the environment has risen about 1900 % in the 50-year period between 1930 and 1980 [2].Chitin and Chitinases ed. monoculture is the norm. and economic ifthere is a crop yield and a quality response above and beyond the cost of the chemicals and their application. Yet the use of pesticides has also resulted in significant costs to public health and the environment. The public concern over the harmful effects of chemical pesticides on the environment and human health has enhanced the search for safer. Entomopathogenic fungi apparently overcome physical barriers of the host by producing multiple extracellular enzymes inc1uding chitinolytic enzymes. 36500. disease and so on which today still destroy almost 33 % of all food crops.. Irapuato. Israel 1 Summary. The use of pesticides is considered effective if they achieve the desired biological result.A. which help to penetrate the cutic1e and facilitate infection. In this sense. Such agricultural practice enables us to continue to provide foodstuffs for the world's ever-increasing population. P. nematode. the role of chitinases in biological control and their potential use in the improvement of biocontrol agents and crop plants by genetic engineering is analyzed in view of recent findings. It is clear that this sort of agriculture cannot be sustained if the price for this success is unacceptable destruction of the . however. Pesticides are applied in agricultural systems for the purpose of protecting plants from injury by insects. biological control of some soil-borne fungal diseases has been correlated with chitinase production. Gto. Muzzarelli © 1999 Birkhäu5er Verlag Ba5el/Switzerland Chitinases in biological control Alfredo Herrera-Estrella 1 and Han Chet2 Centro de Investigacion y Estudios Avanzados. and inhibition of fungal growth by plant chitinases has been demonstrated. which is inherently unstable and offers considerable opportunity for the invasion of crops by plant pests. Faculty ofAgriculture. Introduction In modern agriculture. weeds and diseases [1]. In this chapter. Jolle5 and R. 0. by P. Due to the importance of chitinolytic enzymes in insect. Fungi. Otto Warburg Centerfor Agricultural Biotechnology. and fungal growth and development.

vegetables and ornamental crops. weeds and diseases [1]. Control ofplant pests by the application ofbiological agents holds great promise as an alternative to the use of chemicals. yeasts and fungi. or antagonist. Representing two extreme views of the concept. On the other hand. accomplished naturally or through manipulation ofthe environment. we find the following definitions: "Biological control [of plant pathogens] is their control by one or more organisms. This has led to a renewed interest in the discovery. Physiological role of chitinases Chitinases have been detected in a great variety of organisms. or by mass introduction of one or more antagonists" [6]. Biotechnology. in conjunction with conventional breeding programs. In this regard. The term "biological control" will be used in this more restricted sense throughout this text. we have the classical concept: "Biological control is the deliberate use of one organism to control another" [7]. plants resistant to insects and microbial pathogens. Chet environment [3]. it is long lasting and further investments in control are not necessary. herbicide-tolerant cultivars. and . environmentally dangerous pesticides. development and refinement of biological control agents. In this way. among others. since there are currently not many alternatives to agricultural practices such as monoculture. Herrera-Estrella and I. crustaceans. It is generally recognized that biological control agents are safer and more environmentally sound than is reliance on the use of high volumes of pesticides and other antimicrobial treatments. Classical biocontrol. This includes. evenjust to maintain current human populations the need remains for scientists to continually seek for new. there is an equally great or greater need for biological control of pathogens that presently go uncontrolled or are only partially controlled by these "traditional" means [5]. effective and environmentally friendly ways of controlling pests. but also because ifthe introduced biocontrol agent becomes properly established. We can now look at micoorganisms with inhibitory activity against pathogens as potential sources of genes for disease resistance. there has been intensive research in agricultural biotechnology aimed at plant protection. and biopesticides to use as biological control agents [4]. Different meanings have been given to the term "biological control". could make significant contributions to sustainable agriculture. Such efforts have followed classical plant pathology screening strategies but have also begun to utilize the methodologies made available through molecular biology.172 A. which require repeated application. such as insects. However. it differs from the use of pesticides. Besides. is an outstanding method of pest control not only because it eliminates the use ofpowerful. when effective. including those that contain chitin. host. This definition provides us with a broad concept that includes such notions as cultural practices and disease resistance. disease-free clones of fruits.

chitinases are involved in molting and digestion. germination. [18] showed that chitinase is present during spore swelling. Insects periodically sheed their old cuticles and resynthesize new ones. chitinases found in the gut have a digestive function in addition to their role in breaking down chitin present in the gut lining [11]. Demonstration of lysosomal chitinases was based on sedimentation studies. Sahai et al. Chitinases had no apparent function in intracellular digestion since they were synthesized shortly before auto lysis in gills [19]. Evidence for the association of chitinases and chitin synthases comes from parallel behavior of the two activities during spore germination in Mucor mucedo [13]. during exponential growth in Mucor rouxii [14] and Candida albicans [15]. kinetic and biocidal properties of chitinases has been stimulated by their possible involvement as defense agents against chitinous pathogenic or pestiferous organisms such as fungi and insects. This enzyme is passively released into the wall when metabolic activity stops in senescing cells. fungal chitinases have been implicated in apical growth. liberation of spores. Resistance to organisms can be imparted by the degradation ofvital structures such as the peritrophic membrane or cuticle of insects. The process of autolysis of mature fruiting bodies of Coprinus lagopus is accomplished by the action of chitinases which are formed shortly before spore release begins [19]. Chitinase activity was localized intracellularly in vacuoles together with other lytic enzymes. 21]. Thus. The model offungal cell wall growth proposed by Bartnicki-Garcia [12] envisages the role played by lytic enzymes in maintaining a balance between wall synthesis and wall lysis during hyphal apical growth. In arthropods. Often larvae will ingest the old cuticle. chemieal. The products ofhydrolysis are recycled for the synthesis ofthe new cuticle. These data led to the suggestion that chitinases are associated with hyphal branching rather than autolytic wall turnover. providing plasticity to the apex and permitting insertion of nascent chitin into the wall. the cell wall of fungal pathogens or by liberation of compounds that subsequently elicit other defense responses [22]. It has been described that some autolytic enzymes including chitinases are bound to subapical walls of Neurospora crassa and Aspergillus nidulans [20. and from the finding ofa chitinase activity in the same cell fraction as chitin synthase in M. such as bacteria. cell separation and budding. This process is mediated by the elaboration of chitinases in the molting fluid that accumulates between the old cuticle and the epidermis. higher plants and vertebrates. Failure to localize chitinase at the hyphal tips suggests its possible lack of involvement in apical growth. Apparently. 17]. spore swelling and germination.Chitinases in biological control 173 also organisms that do not contain chitin. sporangium formation and response to mechanical injury in Choanephora cucurbitarum and four other Zygomycetes fungi. . mucedo [16. Considerable interest in the physical.

but their precise role(s) in host infection is unclear [30]. the nematode Brugia malayi utilizes a chitinase to break down a protective chitinous extracellular sheath and/or the peritrophic membrane to gain entry into the mosquito host [28. the peritrophic membrane and exoskeleton of insects act as physicochemical barriers to environmental hazards and predators. Baculoviruses also contain genes for chitinases. Autographa californica nuclear polyhedrosis virus (AcMNPV) carrying the M sexta chitinase complementary DNA (cDNA) under the control of the polyhedrin gene promoter. hydrolytic enzymes used by insects. entomopathogenic fungi apparently overcome these kinds of barriers by producing multiple extracellular enzymes. Choristoneura fumiferana. as weH as some Entomophtorales for insect control. where it does not normally OCCUf. Larvae of spruce budworm.. including chitinolytic and proteolytic enzymes that help to penetrate the cuticle and facilitate infection [23 . died more rapidly when exposed to a chitinase-Bacillus mixture than when exposed to the enzyme or bacterium alone [32-34]. A Manduca sexta chitinase has been shown to increase the killing rate of a recombinant baculovirus [31]. However. Nevertheless. and this effect was correlated with enzyme levels.26]. sexta larvae. mortality of gypsy moth (Lymantria dispar) larvae was enhanced when chitinase was combined with B. The larvicidal activity of a nuclear polyhedrosis virus toward gypsy moth larvae was increased fivefold when it . In another study. jrugiperda) in approximately three quarters of the time required for the wild-type virus to kill the larvae [31]. Bacterial chitinolytic enzymes have been used to enhance the activity of microbial insecticides including Bacillus thuringiensis and a baculovirus. The majority of these fungi OCCUf in the Deuteromycotina and Zygomycotina. In that study a recombinant nonocc1uded baculovirus. Chet Chitinases in insect control Insect pathogenic fungi have considerable potential for the biological control of insect pests of plants. Aschersonia aleyrodis and Verticillium lecanii. Herrera-Estrella and I. In this sense. 29]. expressed the chitinase. chitinase was found in the hemolymph. thuringiensis compared with treatment with the bacteria alone. This enzyme was secreted into the medium when insect celliines were infected with the virus. Some insect venoms also contain chitinolytic enzymes that might serve to facilitate the entry of venomous components into prey [27]. Many attempts have been made to exploit the Deuteromycotina fungi Metarhizium anisopliae. fungi and other organisms for molting or barrier penetration are potentially useful in pest management because their physiological action is to destroy vital structures such as the exoskeleton or peritrophic membrane of insects. Bauveria spp. Similarly. Nomurae rileyi. When the recombinant virus was injected in M. Chitinases have also been used in mixing experiments to increase the potency of entomopathogenic microorganisms. The recombinant baculovirus expressing the chitinase killed larvae of fall armyworms (S.174 A.

caused no mortality. tritici [47. redoiens and Gauemannomyces graminis var. B. marcescens was effective in reducing disease incidence caused by Scleratium rolfsii and Rhizoctonia solani [45. The modified Pseudomonas strain was shown to control the pathogens F. anisopliae. occurred when 0. The anti- . In that case. Chitinase gene expression in entomopathogenic fungi is believed to be controlled by a repressor-inducer system in which chitin or the oligomeric degradation products serve as inducers [24]. No mortality of the nymphal stages of the rice brown plant hopper. Similarly.09% w/v Streptomyces griseus chitinase was added to an artificial diet [38]. The importance of chitinase activity was further demonstrated by the loss of biocontrol efficacy in Serratia marcescens mutants in which the chiA gene had been inactivated [44]. 46]. Biological control of some soil-borne fungal diseases has been correlated with chitinase production [39].48]. bacterial chitinases were ineffective in assays in which insects were fed a diet containing the enzymes. any development aimed to diminish this problem will be useful. the destruction of crop plants by fungal pathogens is a serious problem worldwide that annually leads to losses of about 15% [3]. Serratia and Streptomyces chitinases at 1-2 % levels in the diet of the merchant grain beetle. Virulent isolates of N. Chitinases in the control of phytopathogenic fungi Even with intensive fungieide use. and the plant symbiont Rhizobium meliloti.3)-glucanase have been demonstrated [42. In other studies. Hence. 43]. especially if at the same time it helps to decrease the strong fungieide application.Chitinases in biological control 175 was coadministered with bacterial chitinase [35]. Chitinases appear to be involved in the penetration of host cuticle by entomopathogenic fungi [25. facilitating entry of the pathogens into the hemocoel of susceptible insects [36]. inhibition of fungal growth by plant chitinases and dissolution of fungal cell walls by a streptomycete chitinase and ß-(1. chitinases and ß-Nacetylglucosaminidases are secreted when the entomopathogens M. 41]. chitinase genes from S. oxysporum f sp. However. bacteria-producing chitinases and/or glucanases exhibit antagonism in vitra against fungi [40. 37]. 26. Oryzaephilus mercator. chitinases were supposed to cause perforations in the gut peritrophic membrane. In this regard. Nilaparvata lugens. bassiana and Verticillium lecanii are grown on insect cuticles [24]. Molecular techniques have also facilitated the introduction ofbeneficial traits into rhizosphere competent and model organisms to produce potential biocontrol agents. rileyi exhibit substantially higher levels of chitinase activity than avirulent strains at the time of cuticle penetration [23]. marcescens have been expressed in Pseudomonas sp. A recombinant Escherichia coli expressing the chiA gene from S.

Penetration of the host mycelium takes place aparently by partial degradation ofits cell wall [57. Herrera-Estrella and 1. have a broad host range extending to wide taxonomic groups. followed by nutrient utilization by the parasite [50]. in biotrophic parasitism the development of the parasite is favored by a living rather than a dead host structure [50]. The potential for the use of Trichoderma species as biocontrol agents was suggested 67 years ago by Weindling [54]. Biotrophic mycoparasites tend to have a more restricted host range and in many cases produce specialized structures (haustoria) to absorb nutrients from their host [52]. There are a number of examples of fungi that parasitize plant pathogens. Necrotrophic mycoparasites are those that kill the host cells before. Talaromyces jlavus and Sporidesmium sclerotivorum [5. Several species of Trichoderma spp. 55].3)-glucanases.50. (iii) secretion of extracellular enzymes. toxins or hydrolytic enzymes in such proportions as to cause death and destruction of their host [52]. who was the first to demonstrate the parasitic activity ofmembers ofthis genus toward pathogens such as Rhizoctonia solani [54. Other mycoparasites reported to have some potential for biocontrol are Ampelomyces quisqualis. Microscopic observations [59. and it has been shown to attack a range of economically important soil-borne plant-pathogenic fungi. solani [61.176 A. According to Barnett and Binder [51] mycoparasites can be divided into biotrophic and necrotrophic. The level of hydrolytic enzymes produced differs for each host parasite interaction analyzed. This phenomenon correlates with the ability of each . or just after. chitinases. 62]. solani hyphal tips treated with cell-free nodule extracts [49]. Instead. and are relatively unspecialized in their mode of parasitism. have been tested as biocontrol agents. invasion and use the nutrients released. produced and secreted mycolytic enzymes responsible for the partial degradation ofthe host's cell wall. Pythium nunn. The use of mycoparasites is a promising alternative for disease control by biological means. Chet fungal activity of the transgenie Rhizobium during symbiosis on alfalfa roots was verified by lysis of R. (ii) recognition of the host by the parasite. The antagonistic activity of necrotrophs is due to the production of antibiotics. Trichoderma species and Gliocladium virens have probably been studied more extensively. among them Trichoderma harzianum has proved to be more effective [56]. These mycoparasites tend to be more aggressive and destructive than biotrophs. Coniothyrium minitans.53]. Results supporting this hypothesis have shown that indeed Trichoderma produces extracellularly a complex set of ß-(1. 58]. Of these only a few have been studied to any extent with the aim of biological control. (iv) hyphae penetration and (v) lysis of the host or their combination. 60] lead to suggest that Trichoderma spp. The parasitic process by Trichoderma apparently includes (i) chemotropic growth. lipases and proteases when grown on cell walls of R. Mycoparasitism is defined as a direct attack on a fungal thallus. Laetisaria arvalis.

consisting of six distinct enzymes. They reported the isozymes to be 37. cinerea cell walls in vitro. and four endochitinases of 52. harzianum was grown under inducing conditions increased up to 42-fold in multicopy strains as compared with the nontransformed strain. The purification and characterization of three chitinases from T. Expression of the gene (ech42) is strongly induced during fungus-fungus interaction. 33 and 31 kDa. This observation supports the idea that recognition is an important factor in the mycoparasitic activity of Trichoderma. whereas gene disruptants . 68]. The system is apparently composed of two ß-(1. A second endochitinase and a ß-(1.4)-N-acetylglucosaminidases of 102 and 73 kDa. harzianum was reported by De la Cruz et al. The effect ofthe cell wall-degrading enzymes on the host has been observed using different microscopy techniques. the specificity of Trichoderma cannot be simply explained by a difference in enzyme activity.4)-N-acetylglucosaminidase encoding genes from Trichoderma have been cloned [67. The complexity and diversity of the chitinolytic system of T. but this effect was heightened in the presence of either of the other two isoenzymes [64]. Interaction sites have been stained by fluoresceinisothiocyanate-conjugated lectins or calcofluor. with either S. The level of extracellular endochitinase activity when T. Since the report of the purification of this enzyme the corresponding gene has been cloned [66]. harzianum involves the complementary modes of action of six enzymes. Recently we have analyzed the role of the T. All the chitinolytic enzymes were induced and secreted during growth of Trichoderma on chitin as the sole carbon source. respectively. Several transgenic T. harzianum endochitinase Ech42 in mycoparasitism by genetic manipulation of its coding gene ech42 [69]. harzianum was recently found to be more complex [65]. However. the chitinolytic system of T. harzianum strains carrying multiple copies of ech42 as weIl as the corresponding gene disruptants were generated. respectively. all of which might be required for maximum efficiency against a broad spectrum of chitin-containing plant pathogenic fungi. Its expression is apparently repressed by glucose and may be affected by other environmental factors such as light and nutritional stress and may even be developmentally regulated [66]. However. 33 and 42 kDa. rolfsii or R. The appearance of fluorescence indicated the presence of localized cell wall lysis at points of interaction between the antagonist and its host. solani the parasite hyphae contacted their host and enzymatically digested their cell walls [57]. Probably the most interesting individual enzyme ofthe system is the 42-kDa endochitinase because ofits ability to hydrolyze B. since the nonantagonistic Trichoderma isolates produce lower but significant levels of lytic enzymes [63].Chitinases in biological control 177 Trichoderma isolate to control a specific pathogen. respectively. 42. [64]. Electron microscopy analysis has shown that during the interaction of Trichoderma spp. Only the purified 42-kDa chitinase hydrolyzed Botrytis cinerea purified cell walls in vitro.

Increased levels of chitinase activity induced in C. as compared with the nontransformed control strains. higher levels of chitin synthase may be present in P. transgenic tobacco plants were generated which constitutively expressed a bean endochitinase gene under the control of the cauliflower . In an attempt to increase its effectiveness. In a successful case. rolfsii. culture filtrates of P. cucurbitarum may culminate in enhanced plasticity of the host cell wall and a limited incorporation of a chitin precursor at the penetration site due to degradation of nascent chitin by chitinase. Light and scanning electron microscopy studies have shown inpushing of the susceptible host cell wall and enzymatic erosion of the resistant host cell wall by the advancing infection hyphae [73]. Phoscolomyces articulosus when attacked by P. However. Two transformants showed increased constitutive chitinase activity and expressed a protein of the expected size (58 kDa). Manocha [72] has proposed that metabolic shifts favoring chitinase occur in the susceptible host. 30% higher degradation ofthe chitin content in the R. When evaluated in dual cultures against the phytopathogenic fungus S. Numerous plant chitinase genes or cDNAs have been cloned. thus indicating strict regulatory control of these lytic enzymes. Similarly. solani cell walls was observed during interaction with the overexpressing Trichoderma than with the wild type. Chitinases as defense and transgenes in plants The role ofplant chitinases in disease resistance is well documented [75]. when quantified by transmission electron microscopy. In greenhouse experiments. no differences in the efficacy of the gene disruptants to control Rhizoctonia solani or Sclerotium rolfsii were observed. articulosus because of deposition of chitin and papilla formation at the penetration sites [72].178 A. as compared with the nontransformed control [70]. Herrera-Estrella and I. Interestingly. virginiana. Furthermore. both showed higher antagonistic activity. which is characteristic ofa biotrophic mycoparasite [74]. An extracellular chitinase produced by Myrothecium verrucaria inhibits germination and germ tube elongation of the groundnut rust fungus Puccinia arachidis . Other necrotrophic mycoparasites also secrete chitinases. Penetration of fungal hosts by the biotrophic mycoparasite Piptocephalis virginiana [72] occurs by both mechanical and enzymatic mechanisms. T. Choanophora cucurbitarum and chitin synthase in the resistant host. By contrast. multicopy transformants allowed about 10% lower disease incidence. harzianum was transformed with plasmid pSL3chiAII containing a bacterial chitinase gene from S. ehet showed practically no activity. virginiana contained only negligible levels of chitinases and chitosanases. marcescens under the control of the CaMV35S promoter. Acremonium obclavatum produces and secretes a chitinase in vitro which inhibits germination ofuredospores ofthe peanut rust [71].

suggesting that stored-produet inseets have evolved to overeome plant ehitinases.g/g). by constitutive coexpression in transgenic tobacco was analyzed. Hybrid plants were generated by erossing trans genie parental lines exhibiting strong constitutive expression of CaMV35S enhancerl RCHI0 and CaMV 35S double promoter/AGLUI gene fusions. a eDNA eneoding the major molting fluid chitinase of Manduca sexta was eharaeterized. The activity of this gene is apparently tightly regulated by hormones. These data led to the suggestion that combinatorial expression of antifungal genes could be an effective approach to engineering enhanced crop proteetion against fungal disease [77]. resulting in enhancement of resistance of the host plant to fungal pathogens [78-80]. Unfortunately. Based on the tight developmental and hormonal regulation of the chitinase expression. the role ofvarious chitinases in mediating plant resistanee to inseets is less well understood. When a tobaceo chitinase gene was expressed in high levels in Nicotiana sylvestris. Furthermore. [11] found that transgenie rice plants expressing relatively high levels of a rice chitinase have no detrimental effects on the growth of the fall armyworm Spodoptera jrugiperda. respectively. Although the sueeessful use of plant chitinases for controlling fungi is well documented. Evaluation of disease development in these hybrids. not all eases have been sueeessful. nicotianae infection as wild-type plants [81]. showed that combination of the two transgenes gave substantially greater proteetion against the fungal pathogen Cercospora nicotianae than either gene. and either the disease deve10pment was delayed or they were not affected at all [76]. heterozygous for each transgene. no reports of successful use of aplant chitinase in controlling insect pests are available. theyare susceptible to inseet attaek. the authors suggested that plants eonstitutively expressing it might be resistant to inseets that feed on them because exposure to this enzyme might damage the gut lining. the transgenic plants were still as susceptible to C. the possible functional interactions between two different hydrolytie enzymes. recently Kramer et al. in spite of the substantial levels of chitinases found in cereal grains (10-100 p. The same group generated ehimeric gene eonstructs carrying the M sexta chitinase under the control of single or double CaMV 35S promoter which were introduced into tobaeeo and tomato plants. the riee RCHIO basic chitinase and the aifaifaAGLUI acidic glucanase. and homozygous selfed progeny. Leaves from transformed . The M sexta chitinase gene is not expressed during larval feeding behavior. However. In other work. In fact. it is switched on only during a narrow time frame just prior to larval-larval and larval-pupal molting. both positively and negatively [82]. The transgenie tobacco plants were less suseeptible to infeetion by Rhizoctonia solani.Chitinases in biological control 179 mosaie virus 35S promoter. There are many other examples of the introduction of chitinase genes into plants under the control of constitutive promoters. In other very interesting work.

These data led to the suggestion that insect chitinases are potential host plant resistance factors in transgenic plants and might be more potent than chitinases from other sources. In: D Hornby (ed): Biological control 01soU-borne plant pathogens. Boca Raton. Streptomyces and Hordem species were incorporated into a diet at a 1-2 % level and fed to neonate beetle larvae. 1-14 4 Chet 1(1993) Preface. Wiley-Liss. the data summarized in this chapter allow us to envisage chitinases as an important factor in the development of improved agents and novel strategies for biological control. 445-463 2 Hall FR (1991) Pesticide application technology and integrated pest management (IPM). New York. Further work on cloning and characterization of chitinases will provide us the tools and understanding needed to make better use of these genes. The potential of chitinases is likely to be enhanced by combining them with other bioactive peptides and lytic enzymes. Schell J (1993) The impact of biotechnology on plant breeding. In: I Chet (ed): Biotechnology in plant disease control. 84]. Annu Rev Phytopathol31: 53-80 . as is found in natural systems [22. Faull JL. International. In: D pimentel (ed): Handbook olpest management in agriculture. The enormous potential of genetic engineering will allow us to combine the natural responses of plants with transgenes of microbial and/or insect chitinases. References 1 Powell KA. or how to combine increases in agricultural productivity with an improved protection of the environment. Wiley-Liss. CRC Press. In: I Chet (ed): Biotechnology in plant disease control. Oxon. To determine whether the Manduca chitinase from transgenic tobacco and several chitinases from other sources were directly toxie to insects.B. special emphasis should be made of the use of combinatorial strategies. Chet and control plants were excised and fed to first instar larvae of the tobacco budworm. all larvae consuming the insect chitinase-supplemented diet died a few days after egg hatch [83]. Recombinant Manduca chitinase from transgenie tobacco and chitinase from Serratia.180 A. 135-163 3 Logemann J. Thus. Herrera-Estrella and I. whereas that on the chitinase transformed leaves was only 177 mg. After 3 weeks. Renwick A (1990) The cornrnercial and regulatory challenge. Whereas growth and mortality of larvae consuming the bacterial and plant chitinase-supplemented diets were the same as those of larvae consuming the untreated diet. Concluding remarks All together. such as glucanases.A.XV 5 Cook RJ (1993) Making greater use ofintroduced microorganisms for biological control of plant pathogens. the total mass of surviving larvae on control plants was 966 mg. a beetle feeding study was conducted using purified enzymes. NewYork. other bioactive peptides and improved microbial biocontrol agents. a reduction of more than 80 % [83]. C.

J [nvert Pathol 58: 415-426 27 Krishnan A. In: JH Bumett. McIntosh WH (1986) Roles of chitinases in fungal growth. Bristol.84 26 St Leger RJ. J Gen Mierobiol 137: 2797-2810 15 Barret-Bee K. voll. Roberts DW (1991) Entomopathogenic isolates of Metarhizium anisopliae. J Biol Chem 269: 20971-20976 28 Huber M. NewYork. Humphreys AM. Kaslow D (1993) Transmission blocking activity of a ehitinase inhibitor and activation of malarial parasite chitinase by mosquito protease. Beauveria bassiana and Aspergillus flavus produce multiple extracellular ehitinase isozymes. APJ Trinci (eds): Fungal walls and hyphal growth. White F (1997) Chitinases for insect control. Lowell J. [nseet Bioehem 16: 851. Hamilton M (1984) The detection and analysis of chitinase activity from the yeast form of Candida albieans. Matile P (1970) Role of chitinase and other lysosomal enzymes of Coprinus lagopus in the autolysis of fruiting bodies. Furter R. C Jeuniaux.384-414 23 El-Sayed GN. J Gen Mierobiol130: 1359-1366 18 Sahai AS. Curr Mierobiolll: 187-190 17 HumphreysAM. Cabib E. Taylor and Francis. 167-176 8 Cabie E (1987) The synthesis and degradation ofchitin. Exp Myeol17: 55-69 19 Iten W. Nair PN. In: N Carozzi. 185 -193 12 Bartnicki-Garcia S (1973) Fundamental aspects of hyphal morphogenesis. Plenum Press. EB Cowling (eds): Plant disease: an advaneed treatise. Miller LH (1991) Malaria parasite ehitinase and penetration of the mosquito peritrophic membrane. Cooper RM. Aikawa M. London. 265-278 22 Boller T (1987) Hydrolytic enzymes in plant disease resistance. New York. J Gen Mierobiol 61: 301-309 20 Mahadevan PR. Adv Enzymol59: 59-101 9 Gooday G (1990) The ecology of chitin degradation. Proe Natl Aead Sei USA 88: 2807 . Charnley AK (1986) Cuticle-degrading enzymes of entomopathogenic fungi: regultion of production of ehitinolytie enzymes. Toyoshima T. Microb EcollO: 387-431 10 Kramer Kl. Coudron TA. 83-91 14 Rast DM. Ignoffo CM (1989) Chitinolytic activity and virulence associated with native and mutant isolates ofthe entomopathogenic fungus. J [nvert Pathol 61: 81. GW Gooday (eds): Chitin in nature and teehnology. EW Nester. Gooday GW (1984) Phospholipid requirement ofmicrosomal chitinase fromMueor mueedo. J Baeteriol101: 941-947 21 Rosenberger RF (1979) Endogenous Iytic enzymes and wall metabolism. Cooper RM. Proe Natl Aead Sci USA 90: 4266-4270 .Chitinases in biological control 181 6 Baker KF. Manocha MS (1993) Immunofluorescence study of zygomycetous fungi with two chitin-binding probes. Muthukrishnan S. In: JG Horsfall.2810 29 Sahabuddin M. In: T Kosuge. Charnley AK (1991) Characterization of chitinase and chitobiase produeed by the entomopathogenie fungus Metarhizium anisopliae. cloning and eharacterization of a new ehitinase stored in active form in chitin-lined venom reservoir. synthesis. J Gen Mierobiol 132: 1509-1517 25 St Leger RJ. 24 St Leger RJ. Smith RJ (1977) Managing weeds with pathogens. degradation and metabolie regulation. Horsch M. Cambridge. Jones D (1994) Isolation. In: 1M Ashworth. J Gen Mierobiol130: 1857 -1861 16 Humphreys AM. Paul. 245-267 13 Gooday Gw.877 11 Kramer Kl. MN 7 Templeton GE. Cambridge University Press. Gooday GW (1991) A complex ehitinolytie system in exponentially growing mycelium of Mueor rouxii properties and function. Gooday GW (1984) Properties ofchitinase activities fromMueor mueedo: evidence for membrane-bound zymogenic form. (eds): Plant-mierobe interactions: moleeular and genetie perspeetives. MacMillan. Koga D (1986) Insect chitin: physical state. Staples RC. New York. Mahadkar UR (1970) Role of enzymes in growth and morphology of Neurospora erassa: cell wall bound enzymes and their possible role in branching. JE Smith (eds): Mierobial differentiation. St. In: RAA Muzzarelli. Balasubramanian R. J [nvert Pathol 54: 394-403 . Nomuraea ileyi. The American Phytopathological Society. Cambridge University Press. Cook RJ (1974) Biological control of plant pathogens. Academic Pres. M Koziel (eds): Advanees in inseet eontrol: the role oftransgenie plants.

Comp Biochem Physiol79B: 339-348 38 Powell KS. Heidelberg. Howard SC. Hadar Y (1997) Funga1 antagonista and mycoparasites. Can J Microbiol39: 269-275 53 Adams PB (1990) The potential ofmycoparasites for biological control ofplant diseases. Trends Biotech 10: 392-394 46 Shapira R. Gatehouse AMR. Kroha MJ. International. J Invert Pathol24: 344-348 35 Shapiro M. Robertson JL (1987) Enhancement of baculovirus activity on gypsy moth (Lepidoptera: Lymantriidae) by chitinases. Possee RD (1994) The complete DNA sequence of Autographa californica nuclear polyhedrosis virus. Annu Rev Phitopathol28: 59. Insect Biochem Mol Bio125: 255-265 32 Lysenko 0 (1976) Chitinase of Serratia marcescens and its toxicity to insects. RJ Cook (eds): Biological control oJplant diseases: progress and challenges Jor the Juture. Potgieter HJ. Saikurnar KV. Sahai AS (1993) Mechanisms of recognition in necrotrophic and biotrophic mycoparasites. In: DT Wicklow.B. Söderstrom (eds): The mycota IV: environmental and microbial relationships. Entomol Exp App166: 119-126 39 Buxton EW. Muthukrishnan S. New York. Mol Plant-Microbe Interact 6: 293-298 50 Chet I.72 54 Weindling R (1932) Trichoderma lignorum as a parasite of other fungi. Chet I (1993) Biological control of soilborne plant pathogens by a ß-1 . Gatehouse JA (1993) Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests.3)-glucanase. Lopez-Ferber M. Kuzio J. Barak Z. Springer. Mauch F. Can Entomol108: 3225-3233 34 SmirnoffWA (1974) Three year ofaerial field experiments with Bacillus thuringiensis plus chitinase formulation against the spruce bud worm. J Cell Biochem 13A: 171 48 Sundheim L (1992) Effect of chitinase encoding genes in biocontrol Psseudomonas spp.165-184 51 Barnett HL. Oxon. GC Papavizas. Bedbrook IR (1986) Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens. Ordentlich A. In: EC Tjamos. Ignoffo CM (1989) Levels of chitinolytic activity during development ofthree entomopathogenic fungi. Soil Biol Biochem 25: 1211-1221 41 Gay PA. In: D Hornby (ed): Biological control oJsoil-borne plant pathogens. Binder FL (1973) The fungal host-parasite relationship. sp. Nilaparvata lugens and Nephotettix cinciteps. pisi. Khalifa 0. Suslow TV. Chet I (1992) C10ned chitinases in funga1 plant-pathogen control strategies. Phytopathology 22: 837-845 55 Chet I (1990) Biological control of soil-borne plant pathogens with fungal antagonists in combination with soil treatments. Spence KD (1978) The peritrophic membrane: ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugata. Inbar J. Preisler HK. Vögeli U. Phytopathology 82: 1074 42 Schlumbaum A. Nature 324: 365-367 43 Skujins JJ. Plenum. Chet 30 Ayres MD. Kramer KJ (1995) Baculovirus-mediated expression of a Manduca sexta chitinase gene: properties of the recombinant protein. AnnAppl Bio155: 83-88 40 Fridlender M. Ward V (1965) Effects of soil amendment with chitin on pea wilt caused by Fusarium oxysporum f. 15-25 . EMBO J 5: 467 -473 45 OppenheimAB.3-g1ucanase-producing Pseudomonas cepacia. Chet I (1993) Expression of a Serratia marcescens chitinase gene in Rhizobium meliloti during symbiosis on alfalfa roots. 331-333 49 Sitrit Y. A1exander M (1965) Disolution of funga1 cell walls by a streptomycete chitinase and ß-(1. Tuzun S (1992) Antagonistic effect of chitino1ytic bacteria against toxin producing fungi. Phytopathology 79: 1246-1249 47 Sundheim L (1990) Biocontro1 of Fusarium oxysporum with a chitinase encoding gene from Serratia marcescens on a stab1e p1asmid in Pseudomonas. Grady KL. J Invert Pathol32: 12-24 37 Coudron TA. C.182 A. Oppenheim AB. J Invert Pathol27: 385-386 33 Morris ON (1976) A 2-year study ofthe efficacy of Bacillus thuringiensis-chitinase combinations in spruce budworm (Choristoneurafomiforana) contro!. Virology 202: 586-605 31 Gopalakrishnan B.A. Annu Rev Phytopathol 11: 273-292 52 Manocha MS. Kapu1nik Y. Herrera-Estrella and I. Hilder VA. Arch Biochem Bioph 111: 358-364 44 Jones IDG. Oppenheim AB (1989) Control of plants diseases by chitinases expressed from cloned DNA in Escherichia coli. Chet I. Cleve1and TE. Adang MJ. Inbar J. J Econ Entomol80: 1113-1116 36 Brandt CR. Boller T (1986) Plant chitinases are potent inhibitors offunga1 growth.

Chitinases in biological control 183 56 Chet I (1987) Triehoderma . Chet I. Biol Control 3: 101-103 71 Manocha MS. PhD thesis dissertation. Chet I. and chitosanase activities in germinating spores of Piptoeephalis virginiana. Holliday M. Garcia I. Schickler H. In: 0 Petrini. Herrera-Estrella A (1991) Induction and secretion of hydrolytic enzymes by the biocontrol agent Triehoderma harzianum. Phytopathology 73: 85-88 58 Elad Y. Oppenheim A. Broglie RM (1991) Transgenic plants with enhanced resistance to the fungal pathogen. Manocha MS (1986) Proteinase. Oppenheim A. Mauvais J. Myeologia 78: 157-163 75 Graham LS. Eur J Bioehem 206: 859-867 65 Haran S. Henis Y (1983) Ultrastructural studies ofthe interaction between Triehoderma spp. 137-160 57 Elad Y. Schickler H. Chet I (1993) Hyphal interactions between Triehoderma harzianum and Rhizoetonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Bioteehnology 12: 807-812 .application. Goldman GH. Scanning electron microscopy and fluorescens microscopy. Kubicek CP (1996) Molecular cloning and expression of the nagl gene (N-acetyl-ß-D-glucosaminidase-encoding gene) from Trichoderma harzianum PI. Henis Y (1982) Degradation of plant pathogenic fungi by Triehoderma harzianum. Proe Natl Acad Sei USA 91: 10 903 -1 0907 67 Lim6n MC. Curr Genet 30: 325-331 69 Carsolio C (1997) Role ofthe endochitinase Ech42 in the mycoparasitism by Triehoderma harzianum. Maher EA. mode of action and potential as a biocontrol agent of soilborne plant pathogenic fungi. Amsterdarn. Golesorki R (1981) Host-parasite relations in a mycoparasite. GB Ouellette (eds): Host wall alterations by parasitiejungi. Lora JM. Hidalgo-Gallego A. Hayes CI<. Chet I. Benitez T. De la Cruz J. CINVESTAV. Llobell A. Lorito M. VII. St. B Schippers. Can J Mierobiol28: 719-725 64 De la Cruz J. Chet I (1993) Increased constitutive chitinase activity in trasformed Triehoderma harzianum. M Gerlagh. Logemann S. Gutierrez A. a Triehoderma harzianum endochitinase gene expressed during mycoparasitism. A Tempel (eds): Biotie interaetions and soil-borne disease. 42-61 70 Haran S. Dixon RA. chitinase. and plant pathogenic fungi. Jacobs D. Baker R (1980) Mechanism ofbiological control in soil suppresive to Rhizoetonia solani. Scienee 254: 1194-1197 77 Zhu Q. Myeol Res 99: 441-446 66 Carsolio C. Chet I. Llobell A (1992) Isolation and characterization of three chitinases from Triehoderma harzianum. Pintor-Toro JA. Phytopathology 70: 404-412 61 Geremia R. Jimenez B. Appl Environ Mierobiol64: 1442-1446 63 Elad Y. Van Montagu M. Light and scanning electron microscopy of interactions of Piptoeephalis virginiana with host and nonhost species.3-g1ucanolytic system ofthe biocontrol agent Triehoderma harzianum. Phytopathol Z 107: 168-175 59 Benharnou N. Balasubramanian R (1994) Fungal chitinases: their properties and roles in morphogenesis. Rhizoetonia solani. Van Montagu M. In: ABR Beemster. Wiley. Can J Bot 72: 1057-1083 76 Broglie K. Paul. Herrera-Estrella A (1998) Analysis of the ß-l. Mexico. Harman GE. Barak R. Biddle 0. J Plant Dis Prot 94: 431-444 73 Manocha MS. Knowlton S. Cressman R. NewYork. In: I Chet (ed): Innovative approaehes to plant disease eontrol. 181-186 62 Väzquez-Garcidueiias S. Henis Y (1983) Parasitism of Triehoderma spp. MA Ruissen. 81-90 72 Manocha MS (1987) Cellular and molecular aspects of fungal host-mycoparasite interaction. Benitez T. on Rhizoetonia solani and Sclerotium rolfoii. Herrera-Estrella A (1994) Characterization of eeh-42. Irapuato. Pintor-Toro JA (1995) Primary structure and expression pattern ofthe 33-kDa chitinase gene from the mycoparasitic fungus Triehoderma harzianum. Mycologia 73: 976-987 74 Balasubramania R. GJ Bollen. Boyle P. Lora JM. Curr Genet 28: 478-483 68 Peterbauer CK. Lamb CJ (1994) Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Leal-Morales CA. Elsevier. Sticklen MB (1994) Plant chitinases. mycoparasitism and control of pathogenic fungi. Masoud S. Chet I (1995) New components of the chitinolytic system of Triehoderma harzianum. Pe'er S. Phytopathology 83: 1062 -1 071 60 Liu DA. APS Press.

San Diego. R Wu (eds): Transgenie plants. Anuratha CS. Muthukrishnan S (1998) Insect resistance of transgenic tobacco expressing an insect chitinase gene. Johnson LB. Herrera-Estrella and I. Biotechnology 13: 686-691 80 Vierheilig H. Muthukrishnan S (1993) Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta.629 . Chet 78 Broglie RM. Hayes CK. by the root pathogen. Meins F (1991) High level of expression of a tobacco chitinase gene in Nicotiana sylvestris. Boller T. Insect Biochem Mol Bio123: 6911-701 83 Ding X. Mol PlantMicrobe Interact 6: 261-264 81 Neuhaus JM. Alt M. Susceptibility of transgenic plants to Cercospora nicotianae infection. Potrykus I. 265-276 79 Lin W. Peterbauer C. Choi H. Hinz U. Morgan TD. Transgen Res 7: 77-84 84 Lorito M. Chet I (1993) Production of transgenic plants with enhanced resistance to microbial pathogens. expressing different forms of N tabacum chitinase. sylvestris plants.184 A. In: SD Kung. Kramer KJ. Neuhaus J-M. Corpuz LM. Flores S. Harman GE (1994) Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Datta SK (1995) Genetic engineering ofrice for resistance to sheath blight. Plant Mol Bio116: 141-151 82 Kramer KJ. Wiemken A (1993) Colonization oftransgenic N. Academic Press. Gopalakrishnan B. and by the mycorrhizal symbiont. Rhizoctonia solani. Broglie K. Datta K. Muthukrishnan S. Ahl-Goy P. Roby D. White FF. Glomus mosseau. Wang X. Microbiology 140: 623 .

Jolles and R. Both of these manipulations can result in the rapid activation of a subset of genes called PR (pathogenesis-related) genes. . USA Summary. Each plant species resists most ofthe fungal. which pathologists understand is at the heart of disease resistance. Pullman. often single dominant genes. Plant pathogens known to be incompatible on a given plant species can elicit strong disease resistance responses. Such genetic traits. generally regarded as the genes that functionally develop disease resistance. The region of origin of aplant species is usually inhabited by plant genotypes that can still prevent the pathogen/plant species niche. intense incompatibility response in the host are able to tolerate the adversity of the plant environment long enough to reproduce.A. intense induction of an entire set ofPR genes. have been transferred to commercially suitable crop plants. Genetics and biochemistry of plant pathogen interactions To better understand the function of chitosan in inducing disease resistance in plants. Pathogens that have ingeniously found a niche for surviving on a given plant species are certainly the exception to the rule of incompatibility. thereby enabling the control of disease resistance by external chitosan applications. bacterial and viral pathogens in nature. 0/Plant Pathology. The plant's response can be manipulated genetically by the transfer of"R" genes (single dominant genes for race-specific disease resistance) or by treatment with elicitors such as chitosan. it is useful to state briefly what constitutes natural disease resistance.A. a gene-for-gene interaction [1]) and thus any loss of the avirulence gene function resuits in a susceptible reaction. Washington State University. Genes controlling a qualitative potential for resistance (R genes) against such pathogens have often been acquired from the center of origin of the plant species of interest. There appear to be multiple modes by which chitosan can increase PR gene function. Thus pathogens that do not signal a rapid.e. Hadwiger Dept. The resistance (race-specific resistance) afforded the plant by the single R genes often provides short-lived proteetion in commercial agriculture because its function depends on an interaction with a specific gene (avirulence gene) in the pathogen (i.Chitin and Chitinases ed. Muzzarelli © 1999 Birkhäuser Verlag BasellSwitzerland Host-parasite interactions: elicitation of defense responses in plants with chitosan Lee A. whereas an adapted compatible pathogen generates a weaker response and thus can more readily infect the plant tissue. WA 99164-6430. by P. A novel strategy for controlling PR gene expression proposes to transform plants with a chitosan-inducible gene promoter linked in line with a single signal gene capable of rapid. including activating cell surface or membrane receptors and internal effects on the plant's DNA conformation that in turn influence gene transcription. The plant's defense response against pathogens can be elicited by numerous external signals.

the term "pathogenesis related" (PR) has become accepted to define both the gene and its protein product. however. Signals composed of chitin and chitosan and other elicitor compounds will be reviewed. The gene-für-gene interaction between the Pto gene from tomato that encodes a serine-threonine kinase and the AvrPto gene from the Pseudomonas syringe pv. the host response must in turn signal changes in the pathogen that result in suppression of growth or replication by the pathogen. Chitosan can elicit increases in the PR proteins and thus can elicit a defense response resembling nonhost disease resistance. The predicted proteins of these genes provide clues to therr biochemical functions. generated by the plant against all of the other challenging avirulent pathogens undoubtedly implicates many more interacting genes [3] and is thus very stable to single gene changes in the pathogen and resembles. Race-specific resistance: its link witb tbe induction of PR proteins The gene for gene interactions between the R genes and their corresponding Avr genes have been rewarding genetic interactions to study. As the DNA sequences of the R genes became available. however. The Pto gene is a member of a clustered gene family [7]. nonhost resistance is so inclusive that any recessive traits remain hidden. tomato pathogen [6] has provided some valuable insights. The only traits defined in the nonhost resistance response are those identified in terms of the specific pattern of gene expression occurring as the plant resists an incompatible pathogen. nonhost resistance. at a lower intensity and after an initial delay. some ofthese R genes were cloned [2]. the visible symptoms ofthe interaction and the mode by which the products come into contact with each other. biochemically. and thus their resistant counterparts are not identifiable with standard recombinant mapping. 5]. a reasonable scenario of the interaction between the challenging pathogen and the host tissue must implicate the release of a signal. In contrast. Following the altered transcription. The other members include (i) Fen kinase. many ofthese response genes expressed in nonhost resistance are the same as those activated as a result ofthe proper gene-für-gene interaction in race-specific resistance. these interactions were analyzed in the context of their predicted sequences.186 1. the understanding of the entrre disease resistance process remains incomplete. A more complicated disease resistance. In generating resistance. which is 87% similar . A. the reception of the signal and the conveyance of the signal to a target capable of altering transcription. The dominant R genes controlling race-specific resistance and their recessive alleles occur within genotypes within a given plant species and thus can be identified and mapped with the aid of genetic crosses. Hadwiger Recently. Fortunately. the resistance induced by chitosan [4. Because the same response genes are often activated in a susceptible interaction.

the predicted proteins of the R genes currently sequenced from plants can be grouped into potentially related classes of function. Nonhost resistance: investigations of other signals One might anticipate a myriad of such plant-pathogen signaling when examining the more complicated and stable nonhost resistance. Other R genes that encode proteins with leucine repeats and or nucleotide binding sites may eventually be found to participate in their own linkage to transcriptional changes. hypersensitive cell death and reduced bacterial populations. kinase domains and some combinations ofthese [9]. prepared from the mycelium of the Phytophthora sojae. nucleotide binding sites. interest has centered on the events of anion flux. however. a pathogen of soybeans [10]. Such processes have been examined in the incompatible interaction between parsley cells in culture. which is a serine-threonine kinase possibly acting downstream of Pto. making it resemble the class of R genes discussed above. Components of this system are encoded by Hrp genes in many bacterial pathogens of plants. membrane anchoring sequences.Host-parasite interactions: elicitation of defense responses in plants with chitosan 187 to Pto. respectively. (ii) Prf. which contains leucine-rich repeats and a nucleotide binding site. and consequently are usually found to be necessary in the expression of the hypersensitivity response. The features of these proteins include leucine-rich repeats. leucine zippers. Inhibitors of the elicitor-stimulated ion . mammalian interleukin I-like receptors. In an effort to examine the earliest detectable changes that occur in the plant/nonpathogen interaction. The presence of the host Pto gene and the pathogen-derived PtoAvr gene in the same plant resulted in the activation of resistance responses such as phenol accumulations. since the transcription of the defense response genes in a given plant species is induced by almost every plant pathogen or by any foreign cell. 10]. and an elicitor. within 2-20 min. The possibility that at least two gene products of the clustered Pto gene family can indeed interact was demonstrated in the yeast two-hybrid system [7]. More recently the Pto kinase has been shown to interact with proteins that bind a cis-element of a PR gene [8] thus possibly linking the signaling by the avirulence gene to activation of the disease resistance response. oxidative burst and targeted protein phosphorylation [9. The need to confirm the existence of a pathogen cell to plant cell transport has been bypassed by transferring the PtoAvr gene direct1y to the host plant. the symptomology often associated with resistance against bacterial pathogens. (iii) Pti I. the AvrPto gene product is not endowed with a cell-tocell transport domain. It has been proposed that the interaction of AvrPto with Pto may stimulate Pto kinase activity and trigger a phosphorylation cascade. This elicitor stimulated Ca++ uptake and hydrogen peroxide increases in the parsley cells and culture solution. On the pathogen side. Interestingly. Pseudomonads are known to possess a type III protein secretion system.

This 0. some ofwhich are large enough (heptamers) to induce both phytoalexins and PR proteins as weIl as inhibit the growth of the pathogen. These fragments were also recovered from the pea-Fusarium interaction [19]. Enzymes of the plant host can also function to release from the fungus chitin. Single nucleotide exchanges in nip 1 alleles were sufficient to dictate the difference between virulent or avirulent phenotypes. however. especially mono-. The transfer of the nip 1 gene to a "virulent" race of the pathogen converted that pathogen to an "avirulent" strain. A. Endopolygalacturonases have been shown to activate plant defense responses [13]. in animal systems DNA damage can be a direct effect ofO. a small phytotoxic protein is secreted by R. chitosan fragments capable ofinducing defense responses [18. This interaction is associated with a resistance reaction. action [11]. PGIP has been found in walls ofvarious plants and may serve as a receptor for fungal polygalacturonases [16. Another example of a fungal enzyme serving as an elicitor of the plant defense response is a DNase from the fungal pathogen Fusarium solani f . Examples of other components signaling defense responses Proteins In the interaction between barley and the fungus. is proposed to be generated by an NAD(P)H (nicotinamide adenine dinucleotide phosphate. The details of such a cascade in plants have not been resolved. Altematively. Pure preparations of pea chitinase and ß-glucanase can combine to release chitosan fragments from Fusarium solani. 19]. Hadwiger fluxes blocked the oxidative burst (O~) which was proposed to be the essential element ofthe signal cascade leading to phytoalexin (small antifungal compounds induced in the plant) production. the plant produces a polygalacturonase-inhibiting protein (PGIP) [16] which is also involved in determining the accumulation of elicitor active oligogalacturonide. Rhynchosporium secalis. These fragments must be 10-14 sugar units long to be elicitors [15].and trigalacturonic acid may even assist parasitism by providing nutrition to the pathogen. di. reduced form) oxidase. secalis strains carrying the nip 1 gene (an avirulence gene) when in the presence of barley lines with the R gene.188 1. an activation that results from the enzymatic release ofresponse-eliciting oligogalacturonide fragments from the plant's own cell wall [14]. To further complicate the interaction. the smaller fragments. 17]. Enzymes A plant can also be a target for a fungal enzyme. Rrsl [12].

Recently. phaseoli (Fsph). this complex may signal a cascade of events leading to the eventual activation of genes controlling secondary pathways en route to phytoalexin synthesis. Glucan elicitors Glucan elicitors were first derived from Phytophthora megasperma f sp. Nicotianae [22]. Other fungal proteins called elicitins are secreted by Phytophthora species [21]. there was an increase in the levels of some PR proteins in the transformed plants that were not detected in nontransformed plants. Also. pisi. ß cryptogein. The cell membrane is the hypothesized initial point of contact that sets off a signal transduction cascade leading to PR protein production. This elicitor was purified to homogeneity. One class consists of enzymes that degrade the pectin or pectate components of the cell wall. Subsequent evaluations of such wall fragments produced subfractions with phytoalexin-eliciting activity. F solani f sp. This DNase adequately induces the nonhost resistance response of peas to immunize the tissue against its true pathogen. the primary structure was determined and the structure was confirmed by chemical synthesis. As with other receptors. Within 6 h after inoculation with Fsph the catalytic activity of the fungal enzyme is detected inside the pea nuclei. The second class involves extracellular fungal products in which the protein component can be subjected to denaturation or proteinase digestion without any apparent effect on elicitor activity. Reports from two laboratories indicate protein(s) exists in soybean membranes with a high affinity far the hepta-ß-glucoside elicitor [15. Consequently. glycinea walls by heat or acid extraction. tobacco has been transformed with the gene encoding the elicitin. which provided the recipient plant with an increased level of resistance to the tobacco pathogen Phytophthora parasitica var. The transformed plant was normal in growth even though the elicitin accumulated in the plant tissue and would have been expected to interact with the cell membrane. pea tissue treated with the enzyme accumulates RNA homologous with PR genes within 3 h [20]. These proteins induce cell death and tissue necrosis. Importantly. In tobacco it appears that elicitin plays an important role in the determination of avirulence for those species of Phytophthora for which tobacco is a natural host. primarily in tobacco and radish. 25]. the carbohydrate moiety is credited with possessing the elicitor activity. Glycoprotein elicitors Glycoproteins have been divided into two classes [23].Host-parasite interactions: elicitation of defense responses in plants with chitosan 189 sp. . A well-characterized elicitor isolated from fungal cell walls is a branched hepta-ß-glucoside [24].

with both radio and immunolabeled chitosan that this e1icitor entered the plant cell. chitosan was also seen to be residual in the region of the cell wall and the plasma membrane [28]. chitosan-altered DNA circular dichroism spectra and migration of restriction-digested DNA on a chitosan-containing ge1 [4]. Taken together. solani f sp. The degree of induction increases with the degree of chitosan polymerization up to several thousand.within the DNA sugar-phosphate backbone [30. DNAIchitosan precipitations. Chitosan induced accumulations of the phytoalexin pisatin at levels as low as 16 p. Recently. plant responses such as callose synthesis can be triggered by the polycationic chitosan as it localizes to the bound surface ofprotoplasts. There was an early indication that chitosan had a potential dual role in inducing a defense response as weil as directly inhibiting fungi. with maximum activity starting with the chitosan heptamer [18]. alterations of the DNA melting profile.g/ml [4]. First. however. pisi (susceptible reaction) [4]. More chitosan was recovered from the interaction between pea tissue and the incompatible pathogen. phaseoli (resistance reaction). The potential of chitosan to cause DNA degradation in pea tissue has also been reported [30]. chitosan was evaluated as an inducer of defense responses in plants. It was determined.190 L. Altemately. Correspondingly. Thus there are at least two modes of action proposed for chitosan in living cells. in suspension-cultured plants and protoplasts. than between pea and F. Pea endocarp tissue treated with radiolabeled chitosan was fractionated into microsomal membrane. F. The phospholipid head groups on the plasma membrane were suggested as a possible point of interaction [29]. 32]. Kashige et al. solani f sp. respectively of the label taken up by the cell within 5 h [30]. . The membrane association was c1early shown by the Kaus laboratory following chitosan treatments to protoplasts [29]. these reports suggest that DNA may be a cellular target of chitosan. Second. The affinity of chitosan with DNA was demonstrated by DNA retention on chitosan columns. Kauss et al. [31] showed that chitosan oligomers in the presence ofCu++ cause single strand breaks in pBR322 plasmid DNA. 5% and 19%. Hadwiger Elicitation of defense responses with chitosan Because the positively charged chitosan was known to be a component of fungal walls and polyamines were known to increase the template activity of chromatin and induce the synthesis of the phytoalexin pisatin in peas [26]. chloroplast and nuc1ear components that contained 6%. Additionally. A. The acetylation of chitosan would both reduce its interaction with fatty acids and its affinity for the P0 4 . phytoalexin production and fungal inhibition is size-related. [29] observed by laser scanning microscope that chitosan is bound to the surface of protoplasts and induces the synthesis of callose. chitosan has been found to directly inhibit the germination and growth of an array of fungi [27]. This induction potency of chitosan is reduced by N-acetylation.

fully deacetylated chitosan exhibited only low H2 0 2 activity. It has an absolute requirement for Ca++ in the J. does not require induction and is activated by chitosan. 35] (Fig. A DP of 5000 produced maximum callose increases. Chitosan fragments up to DP 14 are almost inactive. develops when the plasma membrane deposits callose-rich accumulations at sites adjacent to sites of fungal penetration. the increase does not appear to result from the de novo synthesis of ß-I. I B). a morphological defense structure. increasing Ca++ uptake or plasma membrane depolarization alone appear insufficient for the induction of callose [32]. suggesting that part ofthe action was related to the molecule's ability to bind broad areas of the membrane surface. Also the papillae. chitosan is known to induce defense responses associated with the en- . Another cell surface action of chitosan may be in eliciting H20 2 as demonstrated with nitrous acid-cleaved chitosan fragments (DP 26) [32]. I).Host-parasite interactions: elicitation of defense responses in plants with chitosan 191 in intact pea endocarp and/or tobacco leaf tissue. 33] and/or their promoters [34. thereby causing an influx of external Ca++ into the cello However. chitosan can induce a set of genes known as disease resistance response (DRR) or pathogenesisrelated (PR) genes [5.43 as the DP increases from 90 to 2500 DP when the chitosan elicitor is totally deacetylated. We propose to utilize the chitosan-inducible promoters to express structural genes [20] capable ofinducing immunity to true pathogens (Fig.3-glucan synthase. suggesting that Ca++ uptake may lead to an increase in cytoplasmic Ca++ and thereby trigger callose synthesis [29. Chitosans differing in degree of polymerization (DP) and N-acetylation differ in their efficiency to induce callose synthesis. Because a modification ofthe enzyme may occur to increase the catalytic activity and appearance of callose within 20 min of the application of chitosan. A potential application of chitosan may reside in its ability to activate defense gene promoters. Highly acetylated chitosan (chitin) oligomers required much higher levels to elicit a lower concentration of H20 2 • In addition to the catalytic increases in callose synthesis and H20 2 . 36]. Interestingly. This synthase responds to chitosan with a catalytic increase [29].3-glucan synthase.3-glucan synthase was due to an increased influx of Ca++ ions and that the activity increased by a direct and reversible interaction of this ion with the enzyme. Callose (microgram pachyman equivalents) increases from 1.85 J. It was proposed that the chitosan-promoted cellular increase in ß-I .06 to 5. comparable DPs increase callose from 0. Similar treatments to plant cells required higher concentrations of chitosan. The action of chitosan was also linked to increased leakage of cell constituents. the membrane-Iocated enzyme ß-I .lg pachyman equivalents. possibly because of the reductions due to wall binding and exclusions oflarger chitosan polymers [29].lM range.82 to 2. The formation of callose was proposed to be important in plant/pathogen interactions since callose deposition in the walls of surviving cells is an early event in wound healing. Chitosan binds rapidly (within Imin) to protoplasts. When the chitosan is 23% acetylated. One ofthe PR proteins.

chitosan will activate PR gene promoters and thus should activate the expression of transferred structural genes (e. Chitosan is also directiy antifungal and can activate ß-l .PR PROTEINS TRANSGENIC PLANT Indu tion of neighboring cells Figure 1. when challenging pea tissue. the fungal DNase gene) linked to such promoters. Some components ofnatural (A) and proposed elements of chitosan-assisted disease resistance (B). apathogen of bean.3 glucan synthase CALLOSE DISEASE RESISTANCE Transcription of some PR genes PLANTCELL B. releases a DNase and chitosan both of which can induce some PR genes [35]. PROPOSED CHITOSAN-ASSISTED DISEASE RESISTANCE Chitosan Callose induction DISEASE RESIST AN CE I 1---+10. an enzyme required for callose formation.3-g1ucan synthase. SOME COMPONENTS OF NATURAL DISEASE RESISTANCE FUNGALPAmOGEN -1~~::~-ciiiTIi&\N-===:'~ Direct antifungal action Activation of _ ß 1. Extemally applied chitosan will proposedly increase DNase production. Hadwiger 192 A. The DNase protein can enter plant cells and mayaiso translocate to neighboring cells. phaseoli (Fsph). In interactions such as between Phytophthora infestans and potato. an enzyme shown to promote complete immunity in peas to a pea pathogen [20]. providing additional defense potential [36]. thus the extemal application of chitosan initiates the expression ofthe DNase gene. g. . the pathogen does not have a chitinous wall and is unlikely to release the chitosan elicitor.L. sp. In the proposed chitosanassisted disease resistance. Fusarium so/ani f.A.

Organelles ofthe cytosol capable ofreplicating such as mitochondria. Computer-modeled chitosan octamer is a straight rod that easily fits in the minor groove of DNA. Our current knowledge is that chitosan reaches the nucleus [30]. Nuclear proteins such as histones and transcription factors would likely compete with the foreign chitosan molecule for sites on the DNA. Chitosan mode 0/ action Since chitosan confronts plant cell walls. and aggregation of the positive elements of the gene promoters. is difficult to decipher. . chitinase and ß-glucanase [37-40]. Chitosan's mode of inducing defense responses is not solely a product of its cationic nature. Chitosan heptamers have the size and positive charges capable of interacting with nuc1ear DNA in several ways. the cytosol and the nuc1eus [28]. Chitosan's targets within the membrane enable it to alter membrane function [36]. is associated with limited DNA degradation and induces defense gene promoters in many plant species [35]. (iii) antifungal cysteine-rich peptides called thionins orplant defensins [41. since polygalactosamine when compared with polyglucosamine (chitosan) is a poor elicitor of pisatin accumulation [30]. whereas the distorted rod of a galactosamine octamer does not. chloroplasts and nuc1eus possess polyanionic DNA. Especially vulnerable to such competition would be histone H1 molecules that are essential for the intranuc1eosomal assembly of chromatin and histone H5 that suppresses transcription.42] and (iv) peroxidases that are directly antimicrobial or capable of generating phenolic polymers such as lignin [10. All of these can be induced in plant tissue following chitosan treatments. Furthermore. (ii) hydrolytic enzymes such as RNase. chitosan oligomers less than six sugar units long progressively lose their pisatin-eliciting activity [43]. thus the DNA alteration is probably both weak and repairable. Other shorter polyamines such as spermidine. Chitosan is a poor mutagen.Host-parasite interactions: e1icitation of defense responses in p1ants with chitosan 193 hanced transcription/translation of DRR or PR plant defense genes [5]. Because of the complexity of chromatin and the assembly of regulatory proteins on the polymerase complex. cell membranes. Some of these genes encode (i) enzymes for secondary pathways en route to phytoalexins (antifungal compounds) [4]. 30]. enhanceosomes [45]. all ofwhich contain some negatively charged compounds. the predicted effect of chitosan on the "poised states of chromatin" [44]. one must conc1ude that chitosan may have multiple cellular targets. putrescine and cadaverine require much higher concentrations to elicit pisatin production [26].

The action of chitosan on inhibiting the germination and growth of plant pathogenic fungi was first reported on an array of fungi that did not inc1ude those with a predominance of chitosan as a natural component of the fungal cell wall [27]. Chitin from fungal walls or crab shells induced a heavy lignification response at 2 p. Chitosan also possesses antifungal activity against Rhizopus stolonifer. Lodging is the toppling over of mature wheat that can significantly reduce recoverable yield. More recently.0 mg/mI) applied to tomato leaves or roots markedly reduced the number of root lesions caused by a vascular wilt pathogen. and increased the formation of physical barriers in infected root tissue. both seedlings and plants approaching maturity contain slightly elevated levels oflignin. Glucosamine or polymers of N-acetylglucosamine less than six sugars in length were ineffective in inducing this response. plants from chitosantreated seeds have been observed to suffer less extensive lodging.2. The antifungal action of chitosan has also been utilized to reduce the production of aflatoxin on maize [49]. an adversity that could be lessened with larger stern diameters and lignin-reinforced structural compounds. Benhamou and Theriualt [46] found that chitosan (0. A. Seedlings developing from chitosan-treated wheat seeds have been observed to possess increases in stern diameter and plumper roots. Under conditions that promote a wheat stem-breaking disease. Oligomer size plays a .g/wound. caused by the fungal pathogen Pseudocercosporella herpotrichoides. Chitin as an elicitor Pearce and Ride [50] have observed that both chitin and chitosan effectively elicit the lignification response in wounded wheat leaves. chitosan has activity in controlling Pythium aphanidermatum on cucumber plants. Oligomers consisting of3-5 N-acetylglucosamine residues with variations via sulfur. suggesting that the induction response may not be exclusively based on the density of positive charges. Hadwiger Practical aspects of chitosan as an agricultural entity Chitosan has been approved by the Food and Drug Administration of the USA as a wheat seed treatment [34]. which is also a postharvest fungal pathogen [48]. suggesting that the benefits of chitosan applied to seeds can be translocated to other parts of the developing plant.g/ wound compared with chitosan that required 5 p.194 L. sugar and acetyl derivations play vital roles in the nodulation signaling between Rhizobia and plant hosts [51]. [48] have reported a potential use of chitosan in the postharvest preservation of fruits and vegetables. Fusarium oxysporum. additional plant pathogenic fungi have been added to the chitosan-inhibited list [34]. Specifically. Applications of chitosan to wheat seeds have been shown to influence the level of lignin in nearly mature wheat plants.5 . EI Ghaouth et al. Further.

It is not known if comparable amounts of chitinase are generated at the host-parasite interface.Host-parasite interactions: elicitation of defense responses in plants with chitosan 195 definitive role in the induction ofplant defense responses [15. chitosanase and ß-glucanase [52] capable of generating chitinous fragments [19]. Immunity may require combinations ofhydrolytic enzymes. The two simplest explanations of this antifungal action are that the enzyme either weakens the hyphal tip and the exposed plasma membrane can burst. These efforts have improved the plant's resistance to some pathogens [53. Since challenged plants were first examined for glycosidic enzymes such as chitinase. The role of chitinases is variable in the overview of all plantpathogen interactions. The molecular basis ofthe antifungal activity of chitosan may reside in its ability to inhibit RNA and DNA synthesis in Fusarium solani [4]. when obtained. whereas Botrytis cinerea hyphal growth was not affected by concentrations of pure enzyme as high as 320 Jlgml. 19. since chitinases appear to be present throughout plant tissue. all of these large molecules are capable of eliciting defense responses to levels comparable to heptamer or octamer size elicitors. tobacco and wheat have similar antifungal properties. Chitosan oligomers are generated in interactions between plants and fungi with chitinous walls. there have been many attempts to manipulate oligomer release by engineering plants with the genes encoding the respective hydrolytic enzymes linked with constitutive promoters. Commercial development of chitosan Chitosan is one of the few natural microbial inhibitors/defense gene inducers that can be produced in quantities that enable economic applications to crop plants [34]. since the chitosan-mediated inhibition of F. solani germination and growth is not accompanied by visible hyphal tip lysis. or the enzyme generates oligomers with antifungal properties. Purified chitinases [56] have been shown to be potent inhibitors of fungal growth [57]. germinating seed as it . Chitosan can be prepared in molecular weights exceeding a million. All three enzymes inhibited the germination of Trichoderma hamatum and Phycomyes blakesleeanus spores at 8 . 18. oligomers with less than five sugars do not substantially elicit phytoalexin accumulations [18]. chitinases from thorn apple. 54] but not others [55]. For example.1.1. Interestingly. Applications of chitosan to wheat seeds have been shown to influence yield and the accumulative levels of lignin in the adult wheat plants [30]. Some direct benefit may be attributable to chitosan's antimicrobial properties at the site of the coated. Only a few fungi are extremely sensitive to chitinases alone [58]. As indicated above. was short of immunity.43]. The wheat enzyme did not produce the same oligosaccharide pattern when digesting chitin. The larger molecules may be digested to smaller oligomers by hydrolysis ofthe N-acetylglucosamine segments of chitosan. The increased level of resistance.32 Jlgml.

chitosan elicits a defense reaction in lodgepole pine that is expressed as monoterpene accumulations in the phloem [64].196 L. Leaves ofthe developing seedling increasingly accumulated lignin precursors. a benefit that consequently improves yield [34]. Chitosan elicits other defense responses Chitosan and its derivatives. also accumulates in tomato in response to wounding and insect damage. the toppling of the plants (lodging) is markedly reduced. radicis-lycopersici infection of roots [60. Chitosan at concentrations of 12 and 37 mg I-I reduced plant mortality. A restriction of fungal growth to the epidermis and the outer cortex. a decrease in pathogen viability and the formation of wall appositions at sites of attempted penetration were observed. and lignin as the chitosan concentration was increased.5 times the control when applied to cut ends ofthe tomato stern [63]. Thus it is not clear ifthe chitosan oligomer is the primary signal inducing defense responses in distal parts ofthe plant. Hadwiger contacts pathogen-infested soil. 61]. The occurrence of seed-bome F. These barriers contained callose. The treatment of tomato roots via cut stern openings of decapitated plants with native chitosan and ß-glucan elicitors caused a significant decrease in R OXYsporum f sp. induce the synthesis of proteinase inhibitor I in detached young tomato plants [62]. which often causes some obstruction of water transport in the plant.I ) can activate a 48-kDa myelin basic protein kinase to levels 2. Chitosan treatments of wheat seeds also induce resistance to Fusarium graminearum [59]. A. Cleaved chitosan was three times more potent than the other oligosaccaride inducers. Likewise. When the wheat seeds are treated with chitosan (500 p.gml. Interestingly. Chitosan (125 p. Wheat grown under optimal conditions of moisture and mild temperature can incur a wheat stem-breaking disease caused by the fungal pathogen Pseudocercosporella herpotrichoides. Further. chitosan triggers the release of linolenic acid from membranes and its conversion to jasmonic acid.1 significantly improved seed germination and seedling vigor. especially nitrous-cleaved chitosan. Although chitosan is directly antifungal to this pathogen.g g-l of seed) prior to planting. Chitosan at 4 mgml. it is difficult to obtain accurate determinations of the final oligomer size transported. the ultrastructural observations suggested that chitosan 's mode of action was to sensitize the plant to respond more rapidly and efficiendy to attack. pectin and phenolic compounds (probably lignin). Although radioactivity from radiolabeled chitosan applied to seeds can be detected in the upper portion of the developing plant. . root rot symptoms and yield loss attributable to Fusarium oxysporum f sp. there was a wheat varietal difference in response to the chitosan seed treatment. graminearum was reduced by more than 50%. radicis in greenhouse-grown tomatoes [60]. The proteinase inhibitor. a factor in the plant's defense against insects.

New York. Trends Microbioll: 136-141 4 Hadwiger LA. Frederick RD. Salmeron 1M. Hahlbrock K. EMBO J 14: 4168-4177 13 Bruce BJ. Thus the importance of chitin and chitosan signals in agriculture is becoming obvious despite gaps in our understanding of their individual modes of action. Tbe role of pectic fragments of the plant cell wall in elicitation by a fungal endopolygalacturonase. Culley DE (1993) Nonhost resistance genes and race specific resistance. Aplant protein converts a fungal pathogenesis factor into an elicitor of plant defense responses. MCGraw-Hill. Caprarai C. from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Cervone F. Ann Rev Phytopathol 9: 275-296 2 Michelmore R (1996) Flood warning-resistance genes unleashed. Plant Physiol90: 542-548 15 Hahn MG. In: T Kosuge. Bellincampi D. Nature Genet 14: 376-378 3 Hadwiger LA. De Lorenzo G. Darvill A. Doares SH. Physiol Plant Pathol 23: 163 -173 6 Ronald P. Holub E (1997) La dolce vita: a molecular feast in plant-pathogen interactions.Rosahl S. O'Neil RA. Proc Natl Acad Sei USA 94: 514-519 12 Rohe M. Bucheli P. Martin GB (1996) Initiation ofplant disease resistance by physical interaction of AvrPto and Pto kinase. Van Houten B (1997) Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Albersheim P (1989) Roles of cell wall constituents in plant-pathogen interactions. primarily chitosan. Carland FM. Proc Natl Acad Sei USA 94: 4800-4805 11 Yakes FM.Host-parasite interactions: elicitation of defense responses in plants with chitosan 197 Conclusions Chitin and its derivatives. Forrest R. Martin GB (1997) The Pto kinase conferring resistance oftomato bacterial speck disease interacts with proteins that bind a cis-element of pathogenesis-related genes. Scheel D (1997) Elicitor-stimulated ion fluxes and 0. Darvill A. Beckman 1M (1980) Chitosan as a component of pea-Fusarium solani interactions. Biochemical Soc Trans 22: 394-397 . Science 274: 2060-2063 8 Zhou J. EW Nester (eds): Molecular and genetic perspectives. 131-181 16 DeLorenzo G. Plant Physiol66: 205-211 5 Loschke DC. Desiderio A. West CA (1982) Elicitation of casbene synthetase activity in castorbean. Tschope M. Hermann H. NIP I from the barley pathogen. Devoto A. Staskawicz BJ (1992) The cloned avirulence gene avrPto induces disease resistance in tomato cultivars containing the Pto resistance gene. Schmidt B. vol 3. References I Flor RH (1971) Current status of the gene-for-gene concept. constitute part of the natural signaling in host-pathogen interactions. Nuss L et al (1994) Polygalacturonase PGIP and oligogalacturonides in cell-cell communication. Leckie F. JBacterio1174: 1604-1611 7 Tang X. Chitosan can induce immunity in the plant to its true pathogens and all or portions of the nonhost disease resistance response. XXXIII. EMBOJl6: 3207-3218 9 Dangl J. Hadwiger LA. Gierlich A. Colling D. Hahn MG. Tang X. Zhou J. Jia Y. Hahn M. Clark AJ. Wagoner W (1983) Comparison of mRNA populations coding for phenylalanine arnmonia lyase and other peptides from pea tissue treated with biotic and abiotic phytoalexin inducers. Halterman DA. Cell 91: 17-24 10 Jabs T. Rhynchosporium secallis determines avirulence on host plants ofthe Rrsl resistance genotype. Knogge W (1995) Tbe race-specific elicitor. Albersheim P (1989) Host-pathogen interactions. Cervone F. PlantPhysiol69: 1181-1188 14 Cervone F.

P Sandford (eds): Chitin and chitosan. Plant Physiol53: 52-63 27 Allan C. Exper Mycol3: 285-287 28 Hadwiger LA.198 L. In: R Ranjeva. In: A Domard. Lyon. Ossowski p. JacquesAndre. Hadwiger LA (198) Chitosan oligomers from Fusarium solani/pea interactions. Plant Physiol 67: 170-175 29 Kauss H. GAF Roberts (eds): Advances in chitin seience. Wagoner W (1986) Chitosan both activated genes in plants and inhibits RNA synthesis in fungi. Eddy R (1974) Mode ofpisatin induction. Lyon. 209-214 34 Hadwiger LA (1995) Chitosan as crop growth regulator. 102-109 36 Kohle H. Aymes S. Parsons MA (1995) Fusarium solani DNase is a signal for increasing expression of nonhost disease resistance response genes. T Anthonsen. 227-236 35 Hadwiger LA. von Broembsen S. C Jeuniaux. Huet J-C. O'Donohue MJ. Miake F. WMW Muda. A. Hosick HL (1997) Chitosan heptamer alters DNA induces defense genes in plants and induces the accumulation of gene p53 product in anima1 cells.341-11. Albersheim P. Plant Ce1l3: 137-147 18 Kendra DF. hypersensitivity. Beckman IM. and pisatin production. Gayler KR (1996) Elicitins: proteins in search ofa role? Austral Plant Pathology 25: 148-157 22 Tepfer D. Waldmann T. KM Varum (eds): Advances in chitin seience. Christian D. Springer. Ohtakara A. Klosterman S. Exp Mycol8: 276-281 19 Kendra DF. Kauss H (1985) Chitosan-elicited callose synthesis in soybean cells as a Ca++-dependent process. Chang M-M. physical properties and applications. Mitsutomi M. Victory S. J Biol Chem 249: 11. In: A Domard. Elsevier Applied Science. vol2. Watanabe K (1994) Structure-activity relationships in the induction of single-strand breakage in plasmid pBR322 DNA by amino sugars and derivatives. Increased template activity and dye-binding capacity of chromatin isolated from polypeptidetreated pea pods. Pilotti A. chitinase/ß-glucanase digestion of spore1ings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance. Siegrist AI (1997) Partial acetylation of chitosan and a conditioning period are essential for elicitation ofH2 0 2 in surface-abraded tissues from various plants. Physiol Mol Plant Pathol45: 215-230 20 Hadwiger LA. Berlin. Perez V. Pernollet J-C (1998) Phytophthora resistance through production of a fungal protein elicitor (ß-cryptogein) in tobacco. NewYork. Vigon C. Carbohydr Res 257: 285-291 32 Kauss H. Linberg B (1984) Comparison of the structures and elicitor activities of a synthetic and a mycelial-derived hexa (ß-D-glucopyranosyl)-D-glucitol. Chiang C. Jeblick W. AM Boudet (eds): Signal perception and transduction in higher plants. Fristensky BW. 94-101 33 Hadwiger LA. JacquesAndre. In: MB Zakaria.345 25 Schmidt SE. 119-138 31 Kashige D. Blaschek W. Horovitz C (1989) The molecular biology of chitosan in plant/pathogen interaction and its application of agriculture. Brimacombe JS. Penerbit Universiti Kebangsaan. Hahn MG (1991) A specific. Poten F. Quader H (1990) Ca++ as a signal in the induction of callose synthesis. Mol Plant-Microb Interact 8: 871-879 21 Grant BR. Plant-microbe interactions 3:87-130 24 Sharp JK. Malaysia Bangi. Kendra DF. GAF Roberts. GW Gooday (eds): Chitin in nature and technology. Friel P. Plant Physiol77: 544-551 . Adams MJ (1981) Localization of fungal components in the pea-Fusarium interaction detected immunochemically with anti-chitosan and antifungal cell wall antisera. MPMI 11: 64-67 23 Anderson AI (1989) The biology of glycoproteins as elicitors. In: G Skjak-Braek. Ebel J (1987) Specific binding of a fungal glucan phytoalexin elicitor to membrane fractions from soybean Glycine max. biochemistry. Jafri A. 117 -131 30 Hadwiger LA. Ebert D. Boutteaux C. Garegg p. Domard A. Hadwiger LA (1979) The fungicidal effect of chitosan on fungi ofvarying cell wall composition. Plenum. Chang M-M. sources chemistry. In: RAA Muzzarelli. Yamaguchi Y. Proc Natl Acad Sei USA 84: 41174121 26 Hadwiger LA. vol2. MP Abdullah (eds): Chitin and chitosan: the versatile environmentally jriendly modem materials. London. Jeblick W. Hadwiger LA (1984) Characterization ofthe smallest chitosan oligomer that is maximally antifungal to Fusarium solani and elicits pisatin formation in Pisum sativum. Hadwiger 17 Cheong JJ. high-affinity binding site for the hepta-ß-glucoside exists in soybean membranes.

Fedoreyeva LI. Ahl-Goy P.3-glucanases differentially regulated during development and in response to fungal infection. Hadwiger LA. Asselin A (1992) Antifungal activity of chitosan on two postharvest pathogens of strawberry fruits. Nucleic Acids Res 23: 1782-1789 45 Carey M (1998) The enhanceosome and transcription synergy. J Agr Sei 117: 165 -169 50 Pearce RB. sp. Biddle P. Angers p. Hinz U. J Agric Food Chern.3glucanase gene. Broglie R (1991) Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani Science 254: 1194-1197 54 Zhu Q. Plant Mol Bio116: 141-151 56 Mauch F. Hadwiger LA (1980) Glycosidic enzyme activity in pea tissue and pea-Fusariurn solani interactions. Larnb CI (1994) Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in trangenic tobacco. Kuyama H (1994) Chitosan polymer sizes effective in inducing phytoalexin accumulation and fungal suppression are verified with synthesized oligomers. Elsevier Applied Sci. Osbom RW (1995) Plant defensins: Novel antimicrobial peptides as components of the host defense system. Hadwiger LA (1990) Characterization of a disease resistance response gene in pea inducible by Fusarium solani. Plan ta 193: 470-472 41 Broekaert WF. PA Sanford. Physiol Mol Plant Path 41: 33-52 47 EI Ghaouth A. Arul J. Physiol Plant Pathol20: 119-123 51 Downie JA (1994) Signaling strategies for nodulation of legumes by Rhizobia. Masoud S. Ride IP (1982) Chitin and related compounds as elicitors of the lignification response in wounded wheat leaves. Holliday M. Osuji G. fungal infection and the elicitor. I. Phytopathology 82: 398-492 49 Cuero R. Maher EA. Culley DE. Mauvais CJ. Hadwiger LA (1995) Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding. Hanawalt PC (1995) Presence of negative torsion tension in the promoter region of the transcriptionally poised dihydrofolate reductase gene in vivo. Grenier I. Huynh QK (1991) Purification and characterization of an antifungal chitinase from Arabidopsis thaliana. Hadwiger LA (1993) Nucleotide sequence of a pea ß-l. Boller T (1988) Antifungal hydrolases in pea tissue. Beintema JJ. AmI J. In: m Brines. Ogawa T. Plant Physioll01: 1121-1122 38 Chang M-M. Nature 324: 365-367 58 Verburg IG.Host-parasite interactions: elicitation of defense responses in plants with chitosan 199 37 Chang M-M. Horowitz D. Theriualt G (1992) Treatment with chitosan enhances resistance oftomato to the crown and root rot pathogen Fusariurn oxysporurn f. Cammue BPA. Plant Physiol 108: 13531358 42 Chiang CC. Flores S. Yakovlev GI (1994) High sequence similarity between a ribonuclease from ginseng calluses and fungus-elicited proteins from parsley indicates pathogenesis-related proteins are RNases. London. Cressman R. Mol Plant-Microbe Interact 7: 531-533 44 Ljungman J. BollerT (1986) Plant chitinases are potent inhibitors of fungal growth. Chet IM. Beckman IM. Plant Physiol95: 450-455 59 Reddy MVB. Hadwiger LA (1991) The Fusariurn solani-induce expression of a pea gene farnily encoding high cysteine content proteins. AmI J. Dixon RA. Mol Plant-Microb Interact 3: 78-85 40 Moiseyer GP. Couture L (1999) Chitosan treatment ofwheat seeds induces resistance to Fusariurn grarninearurn and improves seed quality. chitosan. JP Zikakis (eds): Advances in chitin and chitosan. Bio/Technology 12: 807-812 55 Neuhaus I. Meins F (1991) High levels expression ofa tobacco chitinase gene in Nicotiana sylvestris. Plant Physiol66: 199-204 53 Broglie K. radicis-lycopersici. in press . Mauch F. Trends Microbiol2: 318-324 52 Nichols EI. Purification and characterization of two chitinases and two ß-l. Duffus GE. 440-452 48 EI Ghaouth A. Susceptibility of transgenetic plants to Cercospora nicotianae infection. Terras FRG. Asselin A (1992) Potential use of chitosan in postharvest preservation offruits and vegetables. Pettit R (1991) Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents. Vogeli U. Culley D. Mol Plant-Microbe Interact 4: 324331 43 Hadwiger LA. Plant Mol Bio128: 105-111 39 Chiang CC. Ce1l92: 5-8 46 Benharnou N. Knowlton S. Plant Physiol 87: 325333 57 SchlumbaumA.

sp. A. Hadwiger 60 Benhamou N. Plan ta 197: 89-102 61 Lafontaine PJ.200 L. Benhamou N (1995) Chitosan treatment: an emerging strategy for enhancing resistance of green house tomato plants to infection by Fusarium oxysporum f sp. Ryan CA (1997) Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in response to systemin and oligosaccharide elicitors. Ryan CA (1986) Biotic elicitors of defense reactions in lodgepole pine. Ann Rev Cell Biol3: 295-317 63 Stratmann JW. Radicislyeopersici. Proe Natl Aead Sei USA 94: 11085-11089 64 Miller RH. Bioeontrol and Teehnology 10: 111-114 62 Ryan CA (1987) Oligosaccharide signaling in plants. Phytoehemistry 25: 611-612 . radicis-Iyeopersiei. Berryman AA. Lafontaine PJ (1995) Ultrastructural and cytochemical characterization of elicitor-induced structural responses in tomato root tissues infected by Fusarium oxysporum f.

by the X-ray crystallographic analysis of hevamine/inhibitor complexes by Scheltinga et al. Universitätsstr.und Entwicklungsphysiologie. Abteilung Allgemeine Zoologie. Lehrstuhlfiir Hormon. D-89069 Ulm.7]. 11] not only for agricultural pests but also for the prevention of insect-transmitted diseases [12] and as antiparasitic drugs [5-8.g. interference with chitin metabolism is considered as a suitable target for pesticides [5 . 999 Birkhäuser Verlag BasellSwitzerland Inhibitors of chitinases Klaus-Dieter Spindler 1 and Margarethe Spindler-Barth 2 I Universität Ulm. Introduction Interest in chitinase inhibitors focuses mainlyon two aspects: First. A role in glycoprotein and glycolipid metabolism is also discussed. fungi. However. Less complex compounds are therefore possible as lead structures for the development of agents interfering with chitinase. since chitinase activities are markedly increased in patients with Gaucher's disease [27]. D-40225 Düsseldorf.01-70 pM). inhibitors are useful tools to elucidate the mechanism of enzymatic catalysis [1. therr many essential physiological functions such as defence mechanisms. and the spatial arrangement of the allosamizoline moiety are important for inhibition. which has been demonstrated successfully by the application of chitin synthesis inhibitors as insecticides for nearly 30 years [5. chitinases are present in vertebrates [23]. In addition. In this case chitinases may be involved in defence mechanisms against pathogens [24-26]. Germany 2 Heinrich-Heine-Universität flüsseldorf. These differences cannot be caused by the catalytic centers of family 18 and 19 chitinases. in all arthropods). Jolles and R. morphogenesis and reproduction in plants [16-19]. 7]. Germany Summary. There is a pronounced species specificity in chitinase inhibition by ailosamidin: halfmaximal values are often in the range ofO. respective1y) and amoeba (0. styloguanidin and divaient cations.Chitin and Chitinases ed. being lower in nematodes (0. nutrition and molting in arthropods [20-22] and hatching in nematodes [13] must be taken into account. [3. 9]. 10-15]. a broad range of applications for inhibitors seems possible as agents against fungi [8. since chitin does not occur in vertebrates. including humans. Due to the widespread occurrence of chitinases. In this review we describe inhibition of chitinases from bacteria. if chitinase inhibitors are used. 4]. Most information is available for ailosamidin. Muzzarelli @ . for example. 2] as shown. A. 10] or nematodes [10. whose important structural features necessary for inhibition are known. At least one N-acetylailosamine sugar must be present. by P. .l-l pM (e.A. plants and animais by allosamidin and its derivatives. 0.01 pM) and quite divergent in fungi (0. cyc1ic peptides.002-0. Albert-Einstein-Allee 11-13. Second.0002 pM. insects and ticks [5-7. 1.048.

allosamidin (Fig. Until recently it was assumed that only members of family 18 chitinases. Spindler and M. Despite the fact that a considerable amount of information on plant chitinases is available. 1). for example the chitinase isoenzyme Ib from Phaseolus. which are characterized by an a. an enzyme of the family 19 enzyme was also found in Streptomyces griseus [36]. by Sakuda et al. Structural fonnulae of allosamidin and its demethylated fonns and of styloguanidin . because this type of chitinase is predominant in plants [31]. 30]. The compound is active in a broad variety of different animals. many enzymes have been isolated and the corresponding genes c10ned [31]. R 1 = H 3:R1 =R1=Br OH OH O OH HO~~~O~~O~'l ~ ~ OH Ac OH A N. The highly susceptible enzymes from root extracts from Pinus and Eucalyptus [35] might also be family 19 chitinases. Spindler-Barth Allosamidin The most prominent and best-characterized chitinase inhibitor.202 K. 1) was isolated 10 years aga from the culture broth of Streptomyces sp. whereas the isoenzyme type Ia from the same species is not inhibited [10]. But this is not strictly true.·-· N CI Styloguanidines 1: R 1 = R 1 = H 2: R 1 = Br. Recently. bacteria and fungi [2.D. since some subtypes of family 19 chitinases are inhibited by allosamidin.-barrel structure of the catalytic centre [32-34] are affected. [28].R1 = H DidemethylaUosamidin: R 1 = R 1 = H Figure I.2 'AI Allosamidin: R 1 = R 1 = CH 3 DemethylaUosamidin: R 1 = CH3. where chitinases ofboth families N H 2 N --------# ~N NH·· H- HzN A. it is unknown in most cases whether these enzymes are inhibited by allosamidin or not.p. although considerable speciesspecific differences in sensitivity are observed (Tab.

17 0.6 46. data from independent investigations are presented or different substrates were used. 0.48 0.1. 48 35 35 35 35 35 35 Plants Phaseolus vulgaris (type Ib) Pinus sylvestris Eucalyptus pilularis 1 0.3.7.1. Since in this species both an allosamidin-sensitive and an -insensitive clone has been described [37] it would be interesting to see whether sensitivity correlates with a certain enzyme family. 0. and .045 58 70 63 63 39 11. 0. 0.002.56.01. 0. Fungi Candida albicans K i value (j.49 34. 6 10 35 35 Protozoa Entamoeba invadens 0. occur simultaneously. 0.29. 29 37 3.1.048 10 0.1M) Bacteria Serratia marcescens Streptomyces griseus Streptomyces sp.1 0. 0.69 10 Kluyveromyces lactis Trichoderma sp.0011 15 Nematodes Onchocerca gibsoni 0.01 0.9 0. Inhibition of chitinases by allosamidin Taxon Species IC so value (j. Saccharomyces cerevisiae Neurospora crassa Paxillus involutus Pisolitus tinctorius Suillus variegatus Boletinus claviceps Armillaria ostoyae Heterobasidion annosum Haemonchus contortus Ticks Boophilus microplus Crustacea Artemia salina Penaeusjaponicus Insects Bombyx mori Chironomus tentans (celIline) Luci/ia cuprina Spodoptera litura 0.1M) Reference 0.1 46 64 0.01.02 0. because its structure is too complicated to allow large-scale synthesis.2 0.44 61.44 10 60 If more than one value per line is given.09. 0.15.2.22 65 46. 0.0002 11 0.32 0. Allosamidin itself is not suitable. 0.23 37 62 0. Due to the selective inhibition of endochitinases.203 Inhibitors of chitinases Table I.8 0.6 0. allosamidin is a lead for the development of new pesticides.27.24 0.29 0.5 3. 0. 1. 20.025 0.

These essential features were obtained by inhibition studies using an insect enzyme [44] and can vary in some aspects according to the source of enzyme used. 4. revealed the essential structural features of the molecule necessary for enzyme inhibition: 1) At least one N-acetylallosamine sugar must be present for efficient inhibition. 30.2 IC so 49 1. 3) The spatial arrangement of the allosamizoline moiety is important for inhibition. 4) If one sugar is omitted and the arrangement of the cyclitol residue is changed. the influence of methyl groups at the allosamizoline moiety is given in Table 2. X-ray analysis ofthe three-dimensional (3D)-structure of the plant enzyme hevamine [3] and chitinase A from Serratia marcescens [47] revealed that both family 18 enzymes bind allosamidin in the catalytic groove.6 0. As an example.3 0. 46]. D. Influenee of methyl groups at the allosamizoline moiety on allosamidin ehitinase aetivity Taxon Species Allosamidin K.61 Demethylallosamidin K. 29.5 10.d.1 bibliographie referenee.7 12. [7] Saccharomyces cerevisiae [7] Crustaeea Artemia salina [46] Inseets Bombyx mori [7] Chironomus tentans (eeIlline) [46] All data are given in }IM. 2) Glucosamine can replace the allosamine moiety without a negative effect on inhibitory potential.2 1. Spindler and M.08 2. whereas the inhibition of the bacterial enzyme from Serratia marcescens is impaired if one sugar moiety is lacking [44].2 0.204 K. Baeteria Serratia marcescens [46] Fungi Candida albicans [7] Trichoderma sp. 45. the insect enzyme is inhibited to the same extent whether or not an N -acetylallosamine moiety is omitted. Spindler-Barth the compound is not stable under field conditions.8 18.05 0. which is in line with the competitive type of inhibition which is Table 2.6 2. Species-specific variation ofthe inhibition by allosamidin derivatives is also reported from Sakuda [8]. n. K. chemically synthesized [8.17 Didemethylallosamidin 1.7 .2 1.3 55. 38-44] or isolated from the culture broth of Streptomyces [8.0 206. [] = IC so 0. the inhibitory effect is diminished further. IC so 1. For example. Tests conducted with a variety of different allosamidin derivatives.4 0.

Inhibitors of chitinases

205

observed in most cases [30]. For the Serratia enzyme, the presence of an
additional binding site was assumed. An indication for differences in the
catalytic centre and presumably also for the mechanism ofhydrolysis is the
fact that sometimes a noncompetitive or mixed-typed inhibition by allosamidin is observed [35], even for a family 18 chitinase [48].
Experiments were performed with diacetylchitobiose or triacetylchitotriose instead of two units of N-acetyl-D-allosamine. Moreover, the allosamizoline ring system was replaced by N-phenylcarbamate, epoxybutyl,
histidineamide or formylpyrrolidine groups. In all these cases, three to four
orders of magnitude higher concentrations were necessary for inhibition
[30].
Cyclic peptides
Two years aga a novel chitinase inhibitor, the dipeptide cyclo(L-Arg-DPro), was isolated ftom the marine bacterium Pseudomonas sp. [49]. The
compound is active against chitinases ftom Bacillus sp. [49], Saccharomyces and Candida [50]. Since the Saccharomyces enzyme is also inhibited
by allosamidin, it would be interesting to test whether chitinases ftom other
species such as insects and nematodes are affected by this dipeptide.
In the case of the Bacillus chitinase, the inhibitory effect is the same
whether L- or D-Pro is present in the moleeule, but a change in the optical
configuration ofL- to D-Arg impairs the efficiency of chitinase inhibition.
In contrast, for the yeast enzyme the inhibitory potential is identical for
both optical isomers of Arg and Pro. The type of inhibition is not yet determined, and the binding site of the two optical isomers is also unknown.
The cyclic dipeptide inhibits cell separation in Saccharomyces, although
less efficiently than aIlosamidin. In the dimorphie yeast Candida albicans,
the change into the filamentous form is prevented, indicating a functional
role of chitinase in the elongation process. This is an important observation, because only the filamentous form is pathogenic, but not the yeast
form. Since the compound has no growth-inhibiting activity as tested on
various microorganisms, and since it is not cytotoxic to various vertebrate
celliines, it seems weIl suited also for medical applications.

Styloguanidin
Three c10sely related chitinase inhibitors were isolated ftom the marine
sponge Stylotella aurantium [51] (Fig. 1). Until now only the inhibition of
chitinase ftom a bacterium, Shewanella sp. (2.5 ]lg/disk) and indirectly by
a bioassay in an arthropod, the bamacle (10 ppm), is reported. In the latter
species the settlement of the ftee-swimming cypris larvae is impaired,
making this inhibitor a putative antifouling agent. Unfortunately nothing is

206

K.D. Spindler and M. Spindler-Barth

known so far about the type of inhibition, the specificity of these compounds, the types of hydrolases being affected and about the stability of
these alkaloids in nature. In addition, due to the complex structure largescale synthesis or mass-isolation from sponges seems impossible.
Other inhibitors
Several divalent ions were tested as chitinase inhibitors with inconsistent
results. Cu z+, Znz+ and Hg z+ inhibit chitinases from bacteria [52,53], some
[54,55] but not all [56] insects and two crustacean species [53, 57] only at
concentrations of 1 mM. If inhibition by these ions is observed in arthropods, it is not due to complex formation with SH groups, since SHblocking agents do not influence chitinase activity [53-55]. Some plant
chitinases are inhibited by divalent ions [58]. A unique feature is the activation of chitinase from the marine bacterium Alteromonas by Na+, K+, Ca2+
and Mg z+ [59].

Conclusions
With the isolation of allosamidin [28], the first potential and sensitive
chitinase inhibitor was available. Its efficient inhibitory action on enzymes
from various taxa, which is restricted mainly to family 18 and some family
19 chitinases, makes this compound a promising lead for the development
of a new group of pesticides. Three approaches are currently being pursued:
1) Investigation of the structure-activity relationship between allosamidin
derivatives and inhibitory action to reduce the complexity of the lead
structure, as already demonstrated successfully.
2) Elucidation of the 3D structure of the catalytical centre of various
chitinases, which supports directed drug design.
3) High-throughput screening for compounds interfering with chitinase
activity obtained either from relevant pests or by recombinant gene
expression, as already demonstrated successfully.
References
I Flach J, Pilet P-E, Jolles P (1992) What's new in chitinase research? Experientia 48:
701-716
2 Milewski S, O'Donnell RW, Gooday GW (1992) Chemical modification studies of the
active centre of Candida albicans chitinase and its inhibition by allosamidin. J Gen
Micribiol138: 2545-2550
3 Terwisscha van Scheltinga AC, Armand S, Kalk KH, Isogai A, Henrissat B, Dijkstra BW
(1995) Stereochemistry of chitin hydrolysis by aplant chitinase/lysozyme and X-ray structure of a complex with allosamidin: evidence for substrate assisted catalysis. Biochemistry
34: 15619-15623.

Inhibitors of chitinases

207

4 Terwisscha van Scheltinga AC, Baerends TRM, Ley JP, Dijkstra BO, Peter MG (1997)
Crystal structure of the plant chitinase/lysozyme hevarnine with tri-N-acetylchitotriosyl
L-histidinarnide. Ninth Eur Carbohydrate Symp Utrecht, 6-11 July 1997
5 Spindler K-D, Spindler-Barth M, Londershausen M (1990) Chitin metabolism: target for
drugs against parasites. Parasitol Res 76: 283-288
6 Londershausen M (1996) Approaches to new parasiticides. Pestic Sei 48: 269-292
7 Cohen E (1993) Chitin synthesis and degradation as targets for pesticide action. Arch Insect
Biochem 22: 245-261
8 Sakuda S (1996) Studies on the chitinase inhibitors, allosarnidins. In: RAA Muzzarelli (ed):
Chitin Enzymology, vol2. Atec, Grottarnmare, 203-212
9 Georgopapadakou NH, Tkacz JS (1995) The fungal cell wall as a drug target. Trends Micro-

biol3: 98-104
10 Londershausen M, Turberg A, Bieseler B, Lennartz M, Peter MG (1996) Characterization
and inhibition studies of chitinases from a parasitic blowfly (Lueilia cuprina), a tick
(Boophilus microplus), an intestinal nematode (Haemonchus contortus) and a bean
(Phaseolus vulgaris). Pestic Sei 48: 305-314
11 Gooday Gw, Brydon LJ, Chappell LH (1988) Chitinase in female Onchocerca gibsoni and
its inhibition by allosarnidin. Mol Biochem Parasitol29: 223-225
12 Shahabuddin M, Kaslow DC (1993) Chitinase: a novel !arget for blocking parasite transmission? Parasitol Today 9: 252-255
13 Fuhrmann JA (1995) Filarial chitinases. Parasitol Today 11: 259-261
14 Shahabuddin M (1995) Chitinase as a vaccine. Parasitol Today 11: 46-47
15 Villagomez-Castro JC, Lopez-Romero E (1996) Identification and partial characterization
of three chitinase forms in Entamoeba invadens with emphasis on their inhibition by
allosarnidin. Antonie van Leeuwenhoek 70: 41-48
16 Schlumbaum A, Mauch F, Vögeli U, Boller T (1986) Plant chitinases are potent inhibitors
offungal growth. Nature 324: 365-367
17 Swegle M, Kramer KJ, Muthukrishnan S (1992) Properties ofbarley seed chitinases and
release of embryo-associated isoforms during early stages of inhibition. Plant Physiol 99:
1009-1014
18 De Jong AJ, Cordewener J, Lo Schiavo F, Terzi M, Vandekerckhove J, van Kammen A, De
Vries SA (1992) A carrot somatic embryo mutant is rescued by chitinase. Plant Cell 4:
425-433
19 Leung DWM (1992) Involvement ofplant chitinase in sexual reproduction ofhigher plants.
Phytochem 31: 1899-1900
20 Spindler K-D (1983) Chitin: its synthesis and degradation. In: K Scheller (ed): The larval
serum proteins of insects. Thieme, Stuttgart, 135 -150
21 Kramer KJ, Dziadik-Turner C, Koga D (1985) Chitin metabolism in insects. In: GA Kerkut,
LI Gilbert (eds): Comprehensive insect physiology, biochemistry and pharmacology, vol3.
Pergamon Press, Oxford, 75-115
22 Kramer KJ, Koga D (1986) Insect chitin. Physical state, synthesis, degradation and metabolic regulation.lnsect Biochem 16: 851-877
23 Gooday GW (1996) Aggressive and defensive roles for chitinases. In: RAA Muzzarelli (ed):
Chitin enzymology, vol2. Atec, Grottarnmare, 125-134
24 Overdijk B, Van Steijn GJ (1994) Human serum contains a chitinase: identification of an
enzyme, formerly described as a 4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase (MU-TACT hydrolase) Glycobiol4: 797-803
25 Escott GM, Adams DJ (1995) Chitinase activity in human serum and leukocytes. Infect
Immun 63: 4770-4773
26 Renkema GH, Boot RG, Au FL, Donker-Koopman WE, StrijlandA, Muijsers AO, Hrebicek
M, Aerts JM (1998) Chitotriosidase, a chitiuase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of farnily 18 glycosyl hydrolases secreted
by human macrophages. Eur J Biochem 15: 504-509
27 Den Tandt WR, van HoofF (1996) Marked increase ofmethylumbelliferyl-tetra-N-acetylchitotetraoside hydro lase activity in plasma from Gaucher disease patients. J Inher Metab
Dis 19: 344-350
28 Sakuda S, Isogai A, Matsumoto A, Suzuki A, Koseki K (1986) The structure of allosarnidin,
a novel insect chitiuase inhibitor, produced by Streptomyces sp. Tetrahedron Lett 27:
2475-2478

208

K. D. Spindler and M. Spindler-Barth

29 Spindler K-D, Spindler-Barth M (1994) Inhibition of chitinolytic enzymes from Streptomyces griseus (bacteria), Artemia salina (crustacea), and a cell line from Chironomus
tentans (insecta) by allosamidin and isoallosamidin. Pestic Sci 40: 113-120
30 Ley J-p, Schweikart F, Peter MG (1997) Chitinase inhibitors. In: RAA Muzzarelli, MG Peter
(eds): Chitin handbook, Atec, Grottammare, 313-320
31 Graham LS, Sticklen MB (1994) Plant chitinases. Can J Bot 72: 1057-1083
32 Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence
similarities. Biochem J 280: 309-316
33 Monzingo AF, Marcotte EM, Hart PJ, Robertus JD (1996) Chitinases, chitosanases and
lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core.
Nature Struct Biol3: 133-140
34 Iseli B, Boiler T, Neuhaus J-M (1996) Functional analysis ofthe domains of a plant PR-3
chitinase. In: RAA Muzzarelli (ed): Chitin Enzymology, vol 2. Atec, Grottanunare,
135-142
35 Hodge A, Gooday GW, Alexander IJ (1996) Inhibition of chitinolytic activities from tree
species and associated fungi. Phytochem 41: 77-84
36 Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, Watanabe T (1996) A
modular family 19 chitinase found in the procaryotic organism Streptomyces griseus HUT
6037. J Bacteriol178: 5065 - 5070
37 Wang Q, Zhou Z, Sakuda S, Yamada Y (1993) Purification of allosamidin-sensitive and
-insensitive chitinases produced by allosamidin-producing Streptomyces. Biosci Biotech
Biochem 57: 467-470
38 Kinoshita M, Sakuda S, Yamada Y (1993) Preparation of N-monoalkyl and O-acyl derivatives of allosamidin, and their inhibitory activity. Biosei Biotech Biochem 57: 1699-1703
39 Terayama H, Kuzuhara H, Takahashi S, Sakuda S, Yamada Y (1993) Synthesis of a new
allosamidin analog, N,N'-diacetyl-ß-chitobiosyl allosamizoline, and its inhibitory activity
against some chitinases. Biosei Biotech Biochem 57: 2067-2069
40 Takahashi S, Terayama H, Kuzuhara H, Sakuda S, YamadaY (1994) Preparation ofa demethylallosamidin isomer having an N,N' -diacetylchitobiosyl moiety and its potent inhibition
against yeast chitinase. Biosei Biotech Biochem 58: 2301-2302
41 Blattner R, Fumeaux RH, Kemmitt T, Tyler P, Ferrier RJ, Tiden A-K (1994) Syntheses of
the fungicide/insecticide allosamidin and a structural isomer. J Chem Soc Perkin Trans I:
3411-3421
42 Corbett DF, Dean DK, Robinson SR (1994) Synthesis of pseudo-disaccharides related to
allosamidin. Tetrahedron Lett 35: 459-462
43 Blattner R, Gerard PJ, Spindler-Barth M (1997) Synthesis and biological activity of
allosamidin and allosamidin analogues. Pestic Sei 50: 312-318
44 Spindler-Barth M, Blattner R, Vorgias C, Spindler K-D (1998) Inhibition oftwo family 18
chitinases by various allosamidin derivatives. Pestic Sei 52: 47 -52
45 Nishimoto Y, Sakuda S, Takayama S, Yamada Y (1991) Isolation and characterization ofnew
allosamidins. J Antibiotics 44: 716-722
46 Spindler K-D, Spindler-Barth M, Sakuda S (1997) Effect of demethylation on the chitinase
inhibitory activity of allosamidin. Arch Insect Biochem Physiol 36: 223 - 227
47 Perrakis A, Tews I, Wilson KS, Vorgias CE (1996) Structural aspects on the catalytic
mechanism of chitinases, hevamine and chitobiases. "Far away and yet so elose?" In: RAA
Muzzarelli (ed): Chitin Enzymology, vol2. Atec, Grottammare, 109-122
48 McNab R, Glover LA (1991) Inhibition of Neurospora crassa cytosolic chitinase by
allosamidin. FEMS Microbiol Lett 82: 79-82
49 Izumida H, Imamura N, Sano H (1996) A novel chitinase inhibitor from a marine bacterium,
Pseudomonas spec. J Antibiotics 49: 76-80
50 Izumida H, Nishijima M, Takadera T, Nomoto AM, Sano H (1996) The effect of chitinase
inhibitors, cyelo(Arg-Pro) against cell separation of Saccharomyces cerevisiae and the
morphological change of Candida albicans. J Antibiotics 49: 829-831
51 Kato T, Shizura Y, Izumida H, Yokoyama A, Endo M (1995) Styloguanidines, new chitinase
inhibitors from the marine sponge Stylotella aurantium. Tetrahedron Lett 36: 2133-2136
52 Izumida H, Miki W, Sano H, Endo M (1995) Agar plate method, a new assay for chitinase
inhibitors using a chitin-degrading bacterium. J Mar Biotechnol2: 163 -166
53 Funke B, Spindler K-D (1989) Characterization of chitinase from the brine shrimp Artemia.
Comp Biochem Physiol94B: 691-695

Inhibitors of ehitinases

209

54 Spindler K-D (1976) Initial eharaeterization of ehitinase and ehitobiase from the integument of Drosophila hydei. Insect Biochern 6: 663-667
55 Boden N, Sommer U, Spindler K-D (1985) Demonstration and eharaeterization of
ehitinases in the Drosophila Ke-eelliine. Insect Biochern 15: 19-23
56 Zielkowski R, Spindler K-D (1978) Chitinase and ehitobiase from the integument of
Locusta rnigratoria: eharaeterization and titer during the fifth larval instar. Insect Biochern
8:67-71
57 Kono M, Matsui T, Shimizu C, Koga D (1990) Purification and some properties of ehitinase
from the liver ofa prawn, Penaeusjaponicus. Agric Biol Chern 54: 2145-2147
58 Chang CT, Hsueh YL, Sung HY (1996) Purifieation and properties of chitinase from
eabbage sterns with roots. Biochern Mol Biol Int 40: 417 -425
59 Hayashi K, Sato S, Tsujibo H, Orikoshi H, Imada C, Okami Y, Inamori Y, Hara S (1995)
Identifieation of the positions of disulfide bonds of chitinase from a marine bacterium,
Alterornonas sp. Strain 0-7. Biosci Biotechnol Biochern 59: 1981-1982
60 Kawazu K, Ohnishi S, Knzaki H, Kobayashi A (1996) A stable erude ehitinase solution from
Spodoptera litura pupae and a seareh for its inhibitors. Z Natur/arsch 51e: 738- 742
61 Somers PJB, Yao RC, Doo1in LR, MeGowan MJ, Fakuda DS, Mynderse JS (1987) Method
for the deteetion and quantitation of ehitinase inhibitors in fermentation broths: isolation
and inseet life cycle. Effect of A82516. J Antibiotics 40: 1751-1756
62 Diekinson K, Keer V, Hitehoek CA, Adams DJ (1989) Chitinase aetivity from Candida
albicans and its inhibition by allosamidin. J Gen Microbiol135: 1417-1421
63 Butler AR, O'Donnell RW, Martin VJ, Gooday Gw, Stark MJR (1991) Kluverornyces lactis
toxin has an essential chitinase activity. Eur J Biochern 199: 483 -488
64 Koga D, Mizuki K, Die A, Kono M, Matsui T, Shimizu C (1990) Kinetics of a ehitinase from
a prawn, Penaeusjaponicus. Agric Biol Chern 54: 2505-2512
65 Koga D, Isogai A, Sakuda S, Matsumoto S, Suzuki A, Kimura S, Die A (1987) Specifie
inhibition of Bornbyx rnori ehitinase by allosamidin. Agric Biol Chern 51: 471-476

Chitin and Chitinases
ed. by ~ Jolles and RA A. Muzzarelli
© 1999 Birkhäuser Verlag Basel/Switzerland

Mammalian chitinase-Iike proteins
Gilles Bleau, Frederic Massicotte, Yannick Merlen and Chantale Boisvert
Departement d 'Obstetrique-Gynecalagie, Centre Haspitalier de 1'Universite de Mantreal,
Hopital Saint-Luc, 264, baul. Rene-Levesque est, Mantreal, Quebec, Canada H2X 1P 1
Summary. Mammals express genes coding for proteins that show significant similarity to
chitinases of family 18 glycosyl hydrolases. These chitinase-Iike proteins have no chitinase
activity due to changes in critical residues in the putative active center. One of these is oviductin,
a high molecular weight glycoprotein most likely involved in fertilization and protection ofthe
tubal epithelium owing to its mucin character. Another is YKL-40 (HCgp39) produced in association with tissue remodeling. Such proteins could have a general function in morphogenesis.

Introduction

An intriguing concept is emerging for a newly discovered group of mammalian proteins characterized by sequence similarity to chitinases. While
chitin is not a polysaccharide found in mammals, all of the species studied
were shown to express chitinase-like proteins. In contradistinction to
family-18 glycosyl hydrolases to which they are related, these proteins are
devoid of chitinase activity due to amino acid substitutions in the region
corresponding to the active site in chitinases. However, their phylogenetic
conservation and their expression under specific conditions predict significant biological roles that need to be fully elucidated.
An impressive literature has rapidly accumulated describing a high
molecular weight glycoprotein secreted by the mammalian oviduct; we will
refer to this protein by the trivial name oviductin, also designated as
oviductal glycoprotein, oviduct specific protein and restrus-associated
oviductal glycoprotein [I]. The other most studied chitinase-like protein
will be conveniently referred to as YKL-40. It inc1udes human HC gp-39,
porcine gp38k, mouse BRP39 and bovine chitinase related protein-I
(CRPI). A different but c10sely related human protein, YKL-39, was
recently described; only sequence information is available on the murine
YM-l and eosinophil chemotactic factor (ECF-L precursor).
In this chapter, we will review the general structure of these proteins, the
striking similarity of their primary sequence as deduced from complementary DNA (cDNA) analysis, their tissue localization in relation to
control of biosynthesis and most interestingly their putative biological
functions.

which ranges from 76% to 99% upon pairwise species comparisons [12]. which has one consensus N-glycosylation site near the N-terminal. respectively) [2]. with stretches that are particularly weH conserved and that are unique to oviductins in some cases. For example. as weH as their absolute tissue specificity ofexpression. the high percentage of similarity over this region. Oviductins. Even though the biological function of oviductins is still uncertain. they are much smaHer proteins than oviductins. basicaHy the same protein with variations in structure from one species to the other. they are secreted as high molecular weight glycoproteins (reviewed in [4]). The presence of numerous sialic acid residues and of sulfate groups on O-linked sugars contribute to the charge heterogeneity of these negatively charged molecules (as detected by isoelectric focusing). By analogy to members of farnily-18 glycosyl hydrolases. could be involved in specific interactions with structures such as the zona peHucida. to which they firmly bind. Conserved residues in the deduced sequences of six weH-characterized oviductins from as many species indicate a high degree of identity (65%) distributed over the entire sequence. . these two proteins would adopt the structure of an (aß)8 barrel. unpublished). found only in oviductins. ovine and bovine species [5-9]. Also noteworthy are residues 201LGR203 and 237KSSA240. They exist as monomers. rhesus monkey [10-12] and cat (G. indicate that they are homologous proteins. highly hydrated conformation. General structural features of chitinase-like proteins YKL-40 andYKL-39 are ofrelatively smaH apparentmasses (40 and 39 kDa. baboon. 1t was shown that human YKL-40 is N-glycosylated [3]. some 40-60% ofthe M r consist of carbohydrate chains. whereas YKL-39. owing to their shorter polypeptide sequences as weH as major differences in glycosylation. is not a glycoprotein [2]. The very large differences in M r mainly result from the extent ofN. to -120-130 kDa in the human. Chitinase-like domain The region of the proteins oviductin and YKL-40 corresponding to their chitinase-like domain is presented in Figure 1. Bleau et al. Bleau. this should cause the protein to take an open. and up to -160-350 kDa in the hamster and mouse [13 -15]. since they do not form intermolecular disulfide bridges.and mostly O-glycosylation. While the core polypeptide contributes for 57-76 kDa. The absence of intermolecular disulfide bridges and the charges on the molecule likely prevent the formation of mucus gel. The M r ofthe secreted forms range from -75-100 kDa in the rabbit and the porcine. are more complex molecules. residues 139FFL 141 are conserved within the region corresponding to the catalytic center of chitinases. but are not encountered in YKL-40 nor in any of the other chitinase-like proteins. These stretches of conserved residues.212 G.

The open reading frame for human YKL-39 codes for a protein of385 amino acids with 61 % similarity to YKL-40 [2]. Identity M--LLL--GL -L--K---G.-TLASS-. bovine (AFOI1373) and porcine (U\9900) proteins.-. and arrow indicates the N-terminal ofthe secreted forms.--DE---YPE FN-LKERN-.EEL--AF--E A-LT--PRLL LSAAVS--P.- 1 386 383 Figure I. Identity NFGT-RFT-M LST---RE-F --S---.---VP-ATKG NQWV-Y-D-E SVK-K---LK 343 -LAY-E-C-F ---A-----.. Ident it y -EHFGGAMVW TLD-DD-RG.FCG-GPFPLV --LN--L--. corresponding to residues 1. U17988) and hamster (032218. 1).LKTLLS-GGW Oviductins YKL-40 prot. The region corresponding to the catalytic center in chitinases is in bold.A-KLVCYF-N WA-SRP--AS I-P-DLDPFL MG-----TGF --L-LLQ-C.-IO--YDIA.--GAP-SG-G -PG-FTKE-G 293 ---LG--.LR. .-S----R--F --S----LR.--YO-HG-W.200 . the similarity of these proteins with members of family-18 glycosyl hydrolase [16] is .KE--AEF. Identity FLAY-E-C-F ---A-K-WI. .-N-LK-RN-.. As described by many authors.---------Y Oviductins YKL-40 prot..-----G-.DK----TL. dots represent gaps introduced for optimal alignment of individual sequences.K L-MG-P--G.E ---LAGAMVW -LDLDDF--. There is no stretch of conserved residues specific for YKL-40.W---R----S --P-----FL 50 50 Oviductins YKL-40 prot . Comparison of conserved residues found in mammalian chitinase-Iike proteins. -KSSA--M-Y 245 I--HLDFI-. we assumed M at position 1).------L--.----L--S-. Oashes indicate lack of identity.LN-LK-RN-N LKTLLSVGGW 100 CTH----FA~ ~-------.345 -LAYYEICDF L-GA---R-. Identity -DRW-F-FL.-PG--TK--G Oviductins YKL-40 prot. bovine (016639.S---K----- Oviductins YKL-40 prot. mouse (X93035).-C----FPLT -AIKD-LAA-----GAMVW -LD-DD---. Identity WR-LG-P--K L-MG-P-YGR -F-LL--S-N -L-----GPA SPGKYTKQ-G 295 --RLGA-A-K L-MGIPTFG.S.. .---VP-A--G --W--Y---.----G---LL LS-A--AGK.150 -FG--RFS-I AS-T--R--F --SV-PFLR. were aligned using the program Pileup with invariant residues deduced using Pretty.LKTLLS-GGW 100 CTH-IYSFAN IS------T.SF-YKA---.AYKLVCY-TS WSQYREG-GS --PDA---FL M-------G.-E----F--.---GH-SPLF ----0----.---------.--FDGLDL-. with 63% identical residues. and the conserved N-glycosylation site is underlined.H-FDGLDLFF LYPGLRGSP.-----Y---- Oviductins YKL-40 prot.Mammalian chitinase-like proteins 213 Oviductins YKL-40 prot . asterisks identifY the conserved cysteines. Identity CTHL-FAFAS M---QIV--. Aligned sequences for oviductins include human (U09550).A-KLVCY--. mouse (032137). Chitinase-Iike region.386 for oviductins and complete protein for YKL-40. YKL-40 proteins from four species are also weil conserved (Fig.--------LL LS-A-----.-TYDFHG-WR ---GHHSPLF -GQ-D---DR -SN.-GFDGLDLAW --P--R .-----Y---.-------Y-.-YAV-Y 244 ----LDFI-. the sequences utilized for YKL-40 proteins include human (M80927).. U15048) proteins..--P--R---- Oviductins YKL-40 prot .-C----FPL. which suggests that this protein would interact with the same type of oligosaccharides as YKL-39 and chitotriosidase. 146 -FG--RF--..-QYVPYA--G KEW-GYD--. porcine (U43490) ovine (UI6719.194 -D------L. Identity LGR-LDFI-V LSYDLHGSWE --TGHNSPLF SL--O .EWND---Y-.

. Dashes represent the lack of identity.. DKQYFSTLI Bovine Porcine Identity SVPPFLRTHGFDGLDLAWISPGRR . despite the presence of an acidic residue at Oviductins Human SVISLLRTHDFDGLDLFFLYPGLRGSPMHDRWTFLFLI Mouse SVISFLRIHGFDGLDLFFLYPGLRGSPPHDRWNFLFLI Bovine Porcine Ovine SVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL SAIGLLRTHGFDGLDLFFLYPGLRGSPRRDRWNFLFLL SVIALLRTHGFDGLDLFFLYPGLRGSPARDRWTFVFLL Hamster SVVSFLRTHGFDGLDLFFLYPGLRGSPINDRWNFLFLI Identity S----LR-H-FDGLDLFFLYPGLRGSP--DRW-F-FL- Human SVPPFLRTHGFDGLDLAWLYPGRR . DKRHLTTLV SV-PFLR--GFDGLDLAW--P--R . where X and B are hydropathie and basic residues. .. which corresponds to a heparin binding site (X _r B-B-X+ 1-B-X+ 3 . The regions encompassing the putative active site in chitinases include those ofthe bacterium Serratia marcescens (L38484). the nematode Brugia malayi (M73689) and human chitotriosidase (U29615). DK----TL- YKL-39 Human SIILFLRNHNFDGLDVSWIYPDQK .. For example.. Sequences for oviductins and YKL-40 proteins are as in Figure 1. The Asp and Glu residues essential for the glycohydrolytic activity ofthe active center of chitinases (bold caracters) are marked by asterisks. striking: the residues that make up the identity sequence (Fig... . GRRDKR. and dots identifY gaps in the alignment.. Bleau et al. the yeast Saccharomyces cerevisiae (M74069. DKQHFTTLI Mouse SVAPFLRSYGFDGLDLAWLYPRLR . YKL-39 (U49835) and YM-I (M94584) are also presented. . 2) results in a total lack of enzymatic activity. respectively) reported in a variety of heparin-binding proteins [17].. malayi Human SAIAFLRKNNFDGFDLDWEYPVGVAEEHAKLVEAMKTA Identity -----------DG-D-D-E------------------- SAI RFLRKYSFDGLDLDWEYPGSQGSPAVDKERFTTLV * Figure 2... M74070). The conserved heparin binding site is underlined. where a major nonconservative substitution (replacement of one of the aspartic acid residues) in chitinaselike proteins (Fig. ENTHFTVLI YM-l Mouse SVIRFLRQYNFDGLNLDWQYPGSRGSPPKDKHLFSVLV Chitinases S. 1) are most often conserved in chitinases of this family. DKRHLTTLV VKEFLQTWKFFDGVDIDWEFPGGKGANPNLGSPQDGET B. Alignment of a region in chitinase-like proteins encompassing the putative active site of chitinases.. The deduced sequence of porcine YKL-40 contains a region.. marcescens S.. ........ this partially applies to the active center of chitinases.214 G. cerevisiae ASERPFDSAVVDGFDFDIENNNEVGYSALRTKLRTLFA YKL-40 SVPPFLRTHGFDGLDLAWLYPGWR .

porcine YKL-40 binds heparin strongly. and more than 60% for hamster). may counteract the effect of the acidic residue. accounts for the differences in the M r of core proteins and is coded by a single exon [12]. Mucin-like domain in oviductins The C-terminal portion of all known oviductins is rich in threonine and serine residues (27.8% T/S). 24% for the whole proteins).2% as part ofthe P/T/S region). The latter residue. but they both lack the additional basic residue at the position + 3 present in the porcine YKL-40. and nematode and insect chitinases. It is not known whether these sulfhydryl groups are free or whether they form intramolecular disulfide bonds. The hamster and human oviductins share the characteristic structural features that define epithelial secretory mucins [4]. Porcine oviductin and human YKL-40 might not bind heparin. which contributes to the basic character of this region. Some chitinases such as those of Bombyx mori (see Tab. information that would be useful in predicting the overall conformation of the proteins. length alleles with three or four imperfect repeats were observed in the case of Brugia malayi [21]. it also contains more proline residues (9. and . in human MUCI and MUC2 but not in mouse Muc 1 (reviewed in [19]). High numbers of similar repeats. Such a domain is absent in the other mammalian chitinaselike proteins. with tandemly repeated sequences rich in P/T/S (34% in their C-terminal portions. as weIl as in YKL39.6% T/S depending on the species) compared with the N-terminal portion (10. Of particular interest is the presence of four cysteine residues highly conserved in aIll 0 proteins from the six species (Fig.Mamma1ian chitinase-like proteins 215 the + 1 position. only porcine oviductin and human YKL-40 contain a similar consensus sequence with an acidic residue at + 1. an extensive allelic polymorphism is also observed. 2). Among oviductins [5] and members ofYKL groups (Fig. and allelic polymorphism in the number of tandem repeats was demonstrated in both species [13. the sugar residues in the PTS region contribute significantly to the M r of the fully glycosylated proteins. 1). They are extensively O-glucosylated (~40% O-linked oligosaccharides by weight for human. starting at position 366 of the mature secreted human protein. for example. 1) and Entamoeba histolytica [20] also contain repeated sequences. These conserved cysteines are also present in chitinases such as human chitotriosidase.7-11. YM-l and ECF-L. 18]. the hamster and human proteins contain tandemly repeated sequences of 15 residues (see Tab. are found in typical mucins. 1). This PTS region ofthe molecule.1-37. since oviductins with the shortest C-terminal portions have lower Mr(~90 kDa) than those with the longest C-terminal portions (M r of ~ 120 to up to ~ 350 kDa). either perfect or displaying small variations around a consensus. Interestingly. In addition to its core polypeptide sequence.

3.23]. This is best explained on the basis ofnonconservative substitutions of either one or both of the acidic amino acid residues (Fig.5 5/6/7 21 See text for references and other accession numbers. it was therefore considered to be a chitinase [26]. has conserved these catalytic residues and can hydrolyze chitin. Bleau et al.3 (GenBank accession no. Lack of chitin ase activity Using different substrates. Table I. 11. allelic polymorphism in the number of repeats. Chromosomallocalization The single-copy gene for human YKL-40 (HC gp-39) was localized to chromosome 1 [3]. This enzyme is responsible for the marked increase in chitotriosidase activity in the plasma of patients with Gaucher's disease and could be used in the diagnosis of this condition and to assess therapeutic modalities [27]. Typical or precise tandemly repeated sequences rich in S/T residues REPEATEDSEQUENCES No. human chitotriosidase.216 G. also has such substitutions and does not exhibit chitinase activity [25]. 5. 22. 1) shown to be essential for chitinase activity [24]. The corresponding region in a similar glycoprotein. DS47 from Drosophila melanogaster. OF REPEATS IN EACH ALLELE Chitinases Bombyxmori Brugia malayi Entamoeba histolytica PTTTTTTVK SETEAYDTDETEET SSESKHE and SSEVKPD 3 3/4 8 Mucins MUCI (human) Muc1 (mouse) MUC2 (human) GSTAPPAHGVTSAPDTRRAP DSTSSPVHSGTSSPATSAPE PTTPITTTTTVTPTPTPTGTQT 25 to > 125 16 50 to 100 Oviductins Human Hamster Mouse GEKTLTPVGHQSVTP GGETMTTVGNQSVTP SKTTTGI 3/4/5/5. U58515) and the human oviductin gene . many authors have observed that the mammalian chitinase-like proteins are devoid of glycolytic activity [2. which shows a high degree of sequence similarity to several chitinases and to the chitinase-like proteins. the gene for YKL-39 (HUMCHIT) to chromosome Ip13. This concept could be of significance in defining the biological function of oviductins as mucins [4] or sialomucins [22] possessing a chitinase-like domain. In contrast. Bombyx mori (U86876).

A higher level of expression of porcine YKL-40 was detected in nodulating smooth muscle cells compared with these cells in monolayers [23]. In contrast. This suggests a chitinase-like gene cluster in this region of the chromosome 1. 10. The biosynthesis of oviductins is confined to the epithelial secretory cells ofthe oviduct. These studies indicate a key role for estrogens in the control of oviductin production. Tissue specificity and control of expression Oviductins are tissue-specific. Long-term ovariectomy invariably results in virtually undetectable levels of oviductin mRNA and protein. interest focused on their potential control by cytokines: transforming growth factor ß (TGFß) can repress expression [3] but interleukin (IL)-l. 32]. and the protein is present in the oviduct even during early pregnancy [29]. the MG-63 osteosarcoma cell line (a murine mammary tumor) and low or undetectable level were reported for other tissues (reviewed in [2]). interestingly. Protein and/or mRNA for YKL-40 were also detected in the liver. infant brain and placenta [2].Mammalian chitinase-like proteins 217 was also mapped to chromosome I p 13 [18]. In contrast. tumor necrosis factor a (TNFa). oviductin messenger RNA (mRNA) levels in the hamster are fairly constant throughout the estrus cycle [13]. synoviocytes. as well as lung. Expression ofYKL-39 was also detected in chondrocytes and synovial cells. The physiological importance of this putative control mechanism remains to be demonstrated. maximum expression occurs around the time of ovulation with barely detectable levels during the luteal phase ofthe estrus cycle [5.22]. More recently. was mapped to chromosome 1q in the area of 1q31-1 qter [28]. or insulin-like growth factor-I (lGF-I) do not [3. mostly in the ampullary portion (reviewed in [4]). an effect that is antagonized by progesterone in all species except the hamster (reviewed in [4]). The production ofYKL-40 was originally observed as a heparin-binding protein secreted by cultured vascular smooth muscle cells ([23] and references therein) and bovine breast tissue during its involution [31]. the locus for human chitotriosidase. Estradiol replacement is a potent stimulus that increases the signals. 34]. In almost all ofthe species studied. YKL-40 (HC gp-39) was shown to be expressed in association with late stages of monocyte to macrophage differentiation [33. Given the cell types in which these proteins are produced. The possible participation of luteinizing hormone (LH) in increasing oviductin production would not be at the promoter level but wou1d rather reflect an effect on mRNA stability in cultured cells [30]. chondrocytes. heart. the enzyme responsible for plasma chitinase activity. whereas the other chitinase-like proteins are expressed in diverse tissues. Cloning and functional studies of the promoters should soon explain this major difference. the levels of expression correlate with the degree of morphological differentiation of a promyelocyte leukemia cell line induced .

the success rate of in vitro fertilization in humans.82 relative to the first in-frame start codon and 28 nucleotides downstream of a consensus TATA box [33]. which coincides and may be related with the profound changes in the morphology and secretory activity of the oviductal epithelium. a technique that completely bypasses any contribution by the oviduct. 18] is consistent with the antiadhesion properties of certain carbohydrate- . 34]. AF026552) and for CHI3L1 [33]. and an almost perfect estrogen responsive element (ERE) could most likely account for the estrogen-dependent expression of the protein (Y.218 G. While a facilitating role for oviductins in normal in vivo fertilization and embryonic development is possible. The 5' -flanking region of the gene CHI3L1 (coding for the human YKL-40) contains two transcription initiation sites. A role for oviductins in fertilization is a logical assumption. In most species. challenges the paradigm. Evidence supports an implication ofYKL-40 in tissue remodeling in cartilage [3]. The anatomical sites and the particular conditions under which the proteins are expressed provided the basic clues in formulating hypotheses. 5' -Flanking sequences are now available for the hamster oviductin gene (GenBank accession no. and ECF-L precursor (D87757) by T lymphocytes. 36 and reviewed in 12]. Bleau et al. The proposed role for oviductins in protecting the oviduct against invasion by pathogens [13. Biological functions The search for the biological roles of chitinases-like proteins is surely the most important issue in this area. For the hamster oviductin gene. This is substantiated by the increase in sperm binding and penetration of the zona pellucida of ovarian oocytes exposed to the protein [35. Interestingly. unpublished). Intemalization of the glycoprotein by the blastomeres occurs in many species (reviewed in [4]). but the importance of this intriguing phenomenon remains obscure. There is a paucity ofinformation on promoter ftmction for chitinase-like proteins. morphological reorganization of vascular smooth muscle cells [23]. with the major one at . tissue involution after cessation oflactation [31] and late macrophage differentiation [33. by phorbol myriotyl acetate (PMA) and vitamin D3 treatments [34]. the presence of an atypical TATA box is noteworthy. The deduced protein YM-l (M94584) would also be expressed by activated murine macrophages. oviductins are produced and secreted in large amounts around the time of ovulation. Promoter analyses will be critical to better understand the regulation of tissue specificity and temporal expression of chitinase-like proteins under diverse physiological and pathological conditions. since these proteins associate with the zona pellucida and/or the perivitelline space of ovulated oocytes. Bleau. Merlen and G. the association of ovine oviductin with F-actin in areas of blastomere-blastomere contact in earlyembryos suggests a role in early embryonic development [37].

Verhage HG. it was e1egantly demonstrated that human YKL-40 is a chitin-specific lectin [38]. As for YKL-40 and the other similar proteins. J Biol Chern 271: 19415-19420 3 HakalaBE. Cleaver BD.Mammalian chitinase-like proteins 219 binding proteins as weH as with the protective role generally attributed to mucins. The identification ofthe ligands is a key objective in attempting to unravel the physiological role of chitinaselike proteins. is a mammalian member of a chitinase protein family. Nancarrow CD. which is the natural ligand par excellence. could bind oviductin via its oligosaccharide moieties [4]. Hoshi H (1994) Purification and molecular cloning ofbovine oviduct-specific glycoprotein. a general strategy ought to be targeted at the zona pellucida. Brownlee AG (1996) Cloning and sequencing of a cDNA encoding an ovine oestrus-associated oviducta1 protein. Alvarez IM. Jaffe RC (1994) Complementary deoxyribonucleic acid cloning and molecular characterization of an estrogen-dependent human oviductal glycoprotein. Given the likely role ofYKL-40 in tissue remodeling. Endocrinology 136: 2485-2496 7 Marshall IT. we suggest that the oocyte and most interestingly the preimplantation embryo could be used as models to expand our concept of the physiological role of chitinase-like proteins in morphogenesis. the problem stands at a higher level of complexity due to the diverse cell types and conditions under which the proteins are secreted. ReckliesAD (1993) Human cartilage gp-39. is expressed in a temporally and regionally specific manner in the sheep oviduct at the time of fertilization and embryo development. Biol Reprod 55: 1305-1314 6 DeSouza MM. Trinh K. Potier M. Biol Reprod 50: 927-934 9 Oliphant G. and the structure of the sugar residues was elucidated in some species. These authors proposed that "He gp-39 may interact with so far unknown endogenous compounds with chitin-like motifs". Simmen FA (1996) Molecular cloning and characterisation of an estrogen-dependent porcine oviductal secretory glycoprotein. We suggested that the zona pellucida.and mucinlike domains: a lead in the search for the biological function of these oviduct-specific ZPassociating glycoproteins. Paquette Y. areminder of an ancestral protective chitinaseligand structure such as the shell and the bacterial cell wall. Merlen Y. In arecent publication. as well as to the ligand-containing structures that they recognize. The lack of chitinase activity led authors to propose functions and mechanisms based on a sugar-binding activity. Mol Reprod Dev 41: 384-397 5 Buhi WC. St-Jacques S. White C. Reprod Fert Dev 8: 305-310 8 Sendai Y. which shares identity with chitinases. Kikuchi M. Price PA (1996) Isolation and sequence of a novel human chondrocyte protein related to mammalian members of the chitinase protein family. Bleau G (1988) Characterization of an oviductal glycoprotein associated with the oviductal hamster oocyte. References 1 Robitaille G. Figueira WF. Biol Reprod 26: 537-543 10 Arias EB. Abe H. Ross PR (1982) Demonstration ofproduction and isolation ofthree sulfated glycoproteins from rabbit oviduct. J Biol Chern 268: 25803-25810 4 Malette B. Bleau G (1995) Oviductins possess chitinase. Choi I. Satoh T. Its biochemical composition is now well known. amajor secretoryproduct of articular chondrocytes and synovial cells. For oviductins. Murray MK (1995) An estrogen-dependent secretory protein. Biol Reprod 38: 687 -694 2 Hu B. Biol Reprod 51: 685-694 .

Tanaka H (1993) Identification of glutamic acid 204 and aspartic acid 200 in chitinase of Bacillus circulans WL-12 as essential residues for chitinase activity. 11 Jaffe RC. Rao CV (1997) A novel regulation of the oviductal glycoprotein gene expression by luteinizing hormone in the bovine tubal epithelial cells. Hum Genet 101: 205-207 29 Roux E. Lei ZM. van Weely S. Hoshi A. Bairoch A (1993) New Families in the classification of glycosyl hydrolases based on amino acid sequence sirnilarities. Venegas A. Bleau G. Onuma T. Golds EE (1990) Human synovial cells secrete a 39 kDa protein similar to a bovine mammary protein expressed during the non-Iactating period. Mavrogianis PA. a novel member of the chitinase family ofproteins. van Oers MHJ. Hiroi M. J Biol Chem 270: 2198-2202 27 Hollak CEM. McNulty DE. Bleau G (1995) Allelic polymorphism in the hamster oviductin gene is due to a variable number ofmucin-like tandem repeats. Mol Biochem Parasitol 85: 139-147 21 Arnold K. Houseweart C. Brenner RM. Biol Reprod 53: 1516-1526 23 Shackelton LM.32 18 Lapensee L. Slayden Ov. Araki Y (1995) Molecular cloning and characterization of a mouse oviduct-specific glycoprotein. Onuma T. Semino CE. Robbins PW. Biol Reprod 53: 285-294 16 Henrissat B. Biol Reprod 53: 345-354 15 Sendai Y. Malette B. Bleau et al. Kobori K. O'Day-Bowman MB. Hurley WL (1988) Isolation and caracterization of a novel 39 kilodalton whey protein from bovine mammary secretions collected during the nonlactating period. J Biol Chem 270: 13 076-13 083 24 Watanabe T. Arteriosclerosis 9: 21. J Biol Chem 268: 18567-18572 25 Kirkpatrick RB. Biochem J 268: 265-268 . Caplivski D. Rosenberg M (1995) An abundantly secreted glycoprotein from Drosophila melanogaster is related to mammalian secretory proteins produced in rheumatoid tissues and by activated macrophages. Weintraub HJR (1989) Molecular modeling of protein-glycosaminoglycan interactions. Paquette Y. Miyashita K. Mol Cel/ Endocrinol 131: 97-108 31 Rejman JJ. Fertil Steril 68: 702-708 19 Gendler SJ. Annu Rev Physiol57: 607-634 20 Delavega H. Biochem J 293: 781-788 17 Cardin AD. Donnelly KM. Muijsers AO. Matico RE. Mol Reprod Dev 46: 306-317 30 Sun T. Biochem Biophys Res Commun 150: 329-334 32 Nyirkos P. Samuelson J (1997) Cloning and expression of chitinases of Entamoebae. Sakai H. Boot RG. Kikuchi M. Kan FW (1997) Fate ofhamster oviductin in the oviduct and uterus during early gestation. Sendai Y. Mol Biochem Parasitol80: 149-158 22 DeSouza MM. Suzuki K. Spicer AP (1995) Epithelial mucins.220 G. Boomsma RA. Gene 153: 147-154 26 Renkema GH. Mann DM. Komiya H. Bleau G (1997) Allelic polymorphism and chromosomallocalization ofthe human oviductin gene (MUC9). Arias EB. Merlen Y. Verhage HG (1996) Regional distribution and hormonal control of estrogen-dependent oviduct-specific glycoprotein messenger ribonucleic acid in the baboon (Papio anubis). Araki Y (1995) Molecular characterization of a hamster oviduct-specific glycoprotein. Jujii T. Den Tandt WR (1997) Assigmnent ofhuman plasma methylumbelliferyl-tetra-Nacety1chitotetraoside hydrolase or chitinase to chromosome lq by a linkage study. Mol Reprod Dev 42:388-396 14 Suzuki K. Biol Reprod 57: 525-531 13 Paquette Y. Biol Reprod 55: 421-426 12 Verhage HG. Specht CA. Donker-Koopman WE. Aerts JMFG (1995) Purification and characterization of human chitotriosidase. Eichinger D. Jaffe RC (1997) Immunologie and molecular characterization of an estrogen-dependent glycoprotein in the rhesus (Macaca mulatta) oviduct. Schmidt A. Murray MK (1995) An estrogen-dependent sheep oviductal glycoprotein has glycan linkages typical of sialomucins and does not contain chitinase activity. Aerts JMFG (1994) Marked elevation of plasma chitotriosiase activity. Strickler JE. J Clin Invest 93: 1288-1292 28 Eiberg H. Hoshi H. Millis AJT (1995) Identification of a 38-kDa heparin-binding glycoprotein (gp38k) in differentiating vascular smooth muscle cells as a member of a group of proteins associated with tissue remodeling. Uchida M. Mavrogianis PA. Fuhrman JA (1996) Discrete transcripts encode multiple chitinase isoforms in Brugian microfilaria.

Mavrogianis PA. Genomics 43: 221-225 34 Kirkpatrick RB. Boot RG. Emery JG. a chitinase. Strijland A. Biol Reprod 54: 60-69 37 Murray MK.Mammalian chitinase-like proteins 221 33 Rehli M. Reuter LM. a member ofthe chitinase protein family and marker for late stages of macrophage differentiation. Verhage HG (1996) Association of oviduct-specific glycoproteins with human and baboon (Papio anubis) ovarian oocytes and enhancement ofhuman sperm binding to human hemizonae following in vitro incubation. a chitin-binding lectin. Rosenberg M (1997) Induction and expression of human cartilage glycoprotein-39 in rheumatoid inflammatory and peripheral blood monocyte-derived macrophages. Hrebicek M. Connor JR. Biol Reprod 52: 199-207 36 O'Day-Bowman MB. An<lreesen R (1997) Molecular characterization ofthe gene for human cartillage gp-39 (CHI3Ll). Aerts JMFG (1998) Chitotriosidase. EurJ Biochem 251: 504-509 . Muijsers AO. Donkerkoopman WE. Biol Reprod 51: 1126-1129 38 Renkema GH. Johnson DE. Lysko PG. Exp Cells Res 237: 46-54 35 Boatman DE. Au FL. Doods R. Messinger SM (1994) Early embryonie development in the Djungarian hamster (Phodopus sungorus) is accompanied by alterations in the distribution and intensity of an estrogen (E 2 )-dependent oviduct glycoprotein in the blastomere membrane and zona pellucida and its association with F-actin. Magnoni GE (1995) Identification of a sperm penetration factor in the oviduct ofthe golden hamster. Krause Sw. Fazleabas AT. are homologues of family 18 glycosyl hydrolases secreted by human macrophages. and the 39-kDa human cartilage glycoprotein.

National Institute ofAllergy and Infectious Diseases. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Chitinases of human parasites and their implications as antiparasitic targets Mohammed Shahabuddin 1 and Joseph M. malaria. TX 77555-0609. Until now.138. Galveston. the parasite may degrade the chitinous structures of the host to facilitate their further growth and transmission. MD 20892-0425. In most ifnot all cases. Vinetz 2 1 2 Medical Entomology Section. Bethesda. National Institutes of Health. In the latter. Most of these diseases are caused by protozoan or metazoan pathogenic parasites. which may suggest that chitin or chitinases are important for the function of the parasite at that stage. and Chagas' disease do not contain chitin. Department ofPathology and Division of Infectious Diseases. I). Pathogens causing a number ofhuman and animal diseases use chitin and chitinases in their life cycles. Recent studies indicate that each of these organisms has evolved to use chitin and chitinases differently and in a developmental stage-specific manner. In this chapter we will discuss the roles chitin and chitinases play in several animal diseases. they secrete chitinase enzymes to degrade the internal chitin-containing structures of their insect vectors [4]. These parasites also make chitinases. Some pathogens use chitinase to invade or exploit the chitin-containing structures of their host to establish successful infection or to be transmitted from one vertebrate to another via insect vectors. perhaps to modulate their chitinous structures. The parasites of leishmaniasis. USA Summary. and the predicted amino acid sequences show a great deal of diversity. Laboratory ofParasitic Diseases.Chitin and Chitinases ed. In some parasites.A. University of Texas Medical Branch. 4 Center Drive MSC 0425. a considerable number of medically important parasites have been shown to possess chitinase-like activities (Tab. This implies that the blocking ofthe chitin production or the chitinase activity in these parasites may block their development to . however. by ~ Jolles and R. Introduction Chitin and chitinases play important roles in the life cyde of several protozoan and metazoan parasites that infect humans. 301 University Blvd. USA WHO Collaborating Center for Tropical Diseases. Also.A. Some ofthese parasites contain chitin coats that protect them from the harsh conditions in the animal body or the environment. while in others the parasites interact with the chitin-containing struetures that they encounter during development. chitin is a component of the eyst wall of the intestinal pathogens Entamoeba [2] and Giardia [3].. The eggshell and the sheath that covers the outer surface of the microfilarial worms contain chitin [1]. the strategies used to clone the chitinase genes from various parasites and the usefulness of chitinases as preventive or therapeutic agents. chitin is a structural component of these organisms. Keil/er 2. the enzyme is produced at a particular stage of the parasite. Genes ofmany ofthese pathogenic parasites have been isolated.

The worms of Wuchereria bancrofti and Brugia malayi grow in the lymph glands and cause lymphedema or elephantiasis in humans.28] [43. The endemie range of B. Current estimates of the World Health Organization (1994) suggest that 100 million people are infected with lymphatic filariae of all types. found in both the Old and New World but with about 95% of all cases in Africa. Filarial worms Microfilarial nematodes cause a number of human diseases and are major health problems in several tropical countries. the current concept of transmission-blocking vaccines and how a chitinase can be deployed as a vaccine for humans. M. Microfilaria of Onchocerca volvulus are the primary cause of river blindness. The microfilarial parasites require an insect and avertebrate host to complete their life cycle. antigens or gene cloning Parasite Brugia malayi Brugia pahangi Oncocerca gibsoni Wuchereria bancrofti Acanthocheilonema viteae Entamoeba histolytica Entamoeba invadens Entamoeba dispar Leishmania donovani Leishmania major Leishmania braziliensis Leishmania infantum Trypanosoma lewisi T. Venezuela and most ofWestem Africa. bancrofti is distributed throughout the tropical regions of Asia. China.28] [31] [7] [31] [13] [13] [13] [34] [30. volvulus are Mexico. from India in the west to Korea in the east. Guatemala. W.224 M. unpublished results maturity and their transmission to the next host [4]. Shahabuddin and 1.46] [15] [15] [15] [47] [15] [15] [35.. we will ftrst summarize the putative biological roles of chitin and chitinase in some human pathogens. Vinetz et al. Vinetz Table I. List of parasite chitinases detected as enzymatic activity. Malayi is confined to South and Southeast Asia. We will also discuss how the genes for the different parasite chitinases have been cloned. . In this chapter.20] [7] [31] 1. brucei brucei Leptomonas seymouri Crithidia Jasciculata Plasmodium gallinaceum References for parasite chitinases as Enzyme activity Genecloned Antigen [43-45] [43-45. Wuchereria and Brugia parasites use mosquitoes. Other important foei of 0. M.6] [10] [31] [13] [32. Africa.33] [15] [15. the Paeific and isolated locations in the Americas.

11]. Cysts are sturdy and resist adverse environmental conditions. the worm emerges from this sheath by a process called exsheathment [5]. After ingestion. but the infection is more common in the tropics. in areas with poor sanitation. Cyst formation begins when the trophozoite rounds into a precyst in the intestine. It has been proposed that chitinase is essential for the emergence of the parasite from the cysts and establishment of a new infection. The insects ingest the microfilariae during the blood mea1. caused by L. A chitinase-like activity present in 0. Leishmania Leishmaniasis is a major threat in many tropical countries. It has been proposed that the microfilariae produce a chitinase-like enzyme to facilitate the exsheathment and further development of the worm [5]. The worm penetrates through the gut wall of the insect to reach the hemolymph. histolytica that expresses during the excystment process [13]. In an adverse environment. causing hemorrhage in the colon and bloody stoo1. The parasite is found all over the world. The cysts are shed with feces. the cyst wall is disrupted and the amoeba divides to form trophozoites. Brugia and Wuchereria microfilariae have a sheathlike structure that surrounds the parasite. however. Entamoeba Entamoeba histolytica is an intestinal protozoan parasite that causes amebic dysentery in humans. A chitinase gene has been detected in E. the eggshells of Onchocerca gibsoni and 0. Two types of infection caused by the parasites of genus Leishmania are commonly known: visceralleishmaniasis. volvulus contain chitin [9]. gibsoni is perhaps essential for hatching ofthe eggs [1. Antibodies against filarial chitinase apparently block the infection in some endemic areas [6-8]. The sheath of the microfilarial nematodes contains chitin [1]. histolytica forms cysts. The parasite colonizes in the large intestine and. which circulate in the peripheral blood or wander through subcutaneous tissues. The causative agent of cattle filariasis does not have a sheath. E. 12]. The adult worms in an infected human shed large numbers of microfilariae. The parasite then enters the thoracic muscle and passes through two to three stages of development or molting. in severe cases.Chitinases ofhuman parasites and their implications as antiparasitic targets 225 and Onchocerca parasites use blackfly vectors for transmission from one infected vertebrate to another. donovani. During each molting process. and cutaneous leish- . invades the tissue of the colon. The parasite secretes an impermeable outer cyst wall containing chitin [2. in a favorable environment of the small intestine. they mix with the drinking water.

It kills more than 2 million people worldwide every year. The developmentally regulated expression of the chitinase gene in Leishmania promastigotes is therefore important.226 M. helping the infected blood to be regurgitated at the site of biting [16]. When an infected fly feeds on an uninfected human. ugly-Iooking lesions. the disease caused by the parasites of genus Plasmodium. Immediately after blood feeding. It has been proposed that the PM protects the parasite from being damaged by the proteases secreted to digest the ingested blood. caused by L. are also encased in the endoperitrophic compartment. The parasites escape from the blood bolus and attach to the midgut epithelium. If the sandfly is heavily infected. it disrupts the PM. with the blood. In cutaneous leishmaniasis. Inside the PM. Vinetz maniasis. the fly regurgitates the contaminated blood from the midgut. When an infected sandfly bites a Leishmania-infected human. Second. The PM of the sandfly appears to be essential far the initial survival of the parasite in the fly stomach. helping the parasite to escape from the blood bolus [15]. because chitinase production at an earlier stage would damage PM formation in the stomach of the fly and disrupt the establishment of infection. is the most devastating insect-borne parasitic disease of humans. The promastigotes (sandfly stage) of Leishmania secrete chitinase when cultured in vitra. the cellular integrity of the valve is maintained by the chitinous lining. In the human. and the parasite enters the human blood at the biting site. it migrates toward the fore gut and damages the cardiac valve [15]. First. The parasites. It is proposed that the Leishmania chitinase plays a dual role in the transmission ofthe parasite. parasites invade internaiorgans such as spleen. causing enlargement of the organs. due to the damaged cardiac valve. a transient saclike structure. the lining is damaged and cellular assembly of the valve is disrupted. The PM gradually surrounds the blood meal [14]. In an uninfected sandfly. When PM formation was disrupted in the stomach. Plasmodium Malaria. is farmed in the stomach. The parasites are transmitted from one host to another by the bites of sandflies. In visceralleishmaniasis. M. The anterior ofthe PM begins to break 3 to 4 days after ingestion. tropica. amastigotes enter the stomach of the insect. the infection was abrogated [17]. liver and so on. the . Shahabuddin and J. the parasites transform into promastigotes and rapidly multiply. As the parasite further matures. it destroys the chitinous 1ining of the cardiac valve of the sandfly. the peritrophic matrix (PM). The parasite is transmitted from one human to another by the bite of mosquitoes. the parasite remains at the site of inoculation and causes severe. The cardiac valve regulates the unidirectional flow of the blood to the midgut during feeding and prevents regurgitation of blood from the stomach.

the parasite develops to the metacyeIic stage. 19].Chitinases of human parasites and their implications as antiparasitic targets 227 injected parasites first invade the liver cells and then infect red blood cells in a cyeIic manner. When the bug feeds on an infected animal. transmission of malaria is completely blocked [20]. Once in the hindgut. When a bug feeds. the parasite enters into the midgut and gradually become surrounded by the PM. The activated chitinase allows the parasite to cross the PM and facilitates further development ofthe parasite. If the chitinase activity in the mosqito midgut is inhibited. the parasite enters the body through skin damaged by the etching and scratching of the biting site. The bug feeds on humans as well as on other animals. The ookinetes invade the PM [18. The role of chitinase appears to be more complicated in the transmission of Trypanosoma than in that of Plasmodium and Leishmania. which is infectious to vertebrate hosts. The released sugars neutralize the activity of the lectins and help the parasite to survive [23]. is caused by Trypanosoma cruzi. The chitinase secreted by the RLO partially degrades the PM and releases D-glucosamine into the midgut. Plasmodium infection of erythrocytes results in the eIinical syndrome of malaria. After feeding. suggesting that chitinase is involved in both parasite maturation and transmission. a Rickettsia-like organism (RLO) of the tsetse also secretes chitinase in the midgut [22]. The feces of an infected bug contain large numbers of parasites. The enzyme has been detected in T lewisi and T brucei. as in sandflies. essentially limited to Central and South America. T brucei is transmitted by black flies. pulmonary and metabolic complications. 21]. It has been proposed that the lectins secreted in the midgut of the fly in response to the blood meal are toxic to the newly ingested parasite. The ability ofthe black fly to establish T brucei infection appeared to be positively related to its RLO load. The role of Trypanosoma chitinase in development and transmission remains to be understood. The zygotes then trans form into motile ookinetes. In mosquitoes. When a mosquito ingests gametocytes. Trypanosoma are flagellated protozoan parasites transmitted by the bite of infected kissing bugs such as Triatoma infestans. The ookinete stage ofthe parasite secretes a chitinase that is activated by the midgut proteases [20. sexual fertilization occurs. Some parasites develop as gametocytes after invading the red blood cells. The parasite escapes from the blood bolus and reaches the hindgut. . chitinous PM surrounds the ingested blood. it simultaneously defecates. cardiovascular. In addition to the procyc1ic stage of T brucei. Malaria is associated with lysis of erythrocytes leading to anemia and the adherence of infected erythrocytes to postcapillary endothelial cells (Plasmodium falciparum only) that can cause neurological. the parasite of cattle. Trypanosoma Chagas' disease.

and the resulting PCR fragments were used to screen complementary DNA (cDNA) library. MF 1 antigen was later shown to bind calcium. This chitinase was initially defined as the target antigen of a monoclonal antibody named MFI that recognized two proteins migrating on SDS polyacrylamide gel electrophoresis (PAGE) at 70 kDa and 75 kDa [24]. most recently. malayi 70-kDa and 75-kDa antigens [8. A discussion of some of these approaches follows. histolytica. This finding was confirmed when both parasiteproduced native MF 1 antigens had chitinase activity when tested on a glycol chitin activity gel [6]. The appearance of the MFI antigen correlated with the onset ofthe parasite's ability to infect the mosquito.27]. donovani and. The hatching of the filarial eggs and the exsheathment of the microfilaria are calcium-dependent processes. suggesting three possible relationships between the two proteins: (i) they were isoform products of two different but closely related genes. A variety of approaches have been used to clone these genes. The 70-kDa and 75kDa MFl antigens were partially purified by DE-52 chromatography of jird-derived microfilarial extracts and analyzed by microsequencing ofthe amino-termini and internal tryptic peptide fragments. Plasmodium gallinaceum. suggesting that the antigen plays an important role in these mechanisms [25]. malayi-infected jirds. Filarial chitinases The best-characterized filarial chitinase is that of B. the expression of the gene correlates with the development of the parasite to the mosquito stage [26]. In addition. A single full-Iength cDNA contained sequences with high similarity to several chitinases. (ii) they were products of differential splicing of coding messenger RNA (mRNA) from a single gene. Although Southern blot . either proteolysis or glycosylation. an avian parasite phylogenetically related to the human malaria parasite P. jalciparum (Tab. E. or (iii) they arose from differential posttranslational processing. W bancrofli and Onchocerca volvulus). malayi. malayi. Vinetz Cloning of parasite chitinase genes Chitinase genes have now been identified and cloned from a number of human parasites such as filarial nematodes (B. both the 70-kDa and the 75-kDa proteins had virtually identical profiles on high-pressure liquid chromatography (HPLC) chromatogram of tryptic digests of the isolated proteins. Furthermore. M.228 M. Interest in the MF 1 antigen as a vaccine candidate for human brugian lymphatic filariasis increased when it was found that sera from immunologically protected individuals contained antibodies that reacted with the same B. L. These amino acid sequences were used to design degenerate oligodeoxynucleotides for polymerase chain reaction (PCR) amplification. Administration ofMFl reduced microfilaremia in B. 1). Shahabuddin and J.

bancrofti. Finally. A chitinase from the other major causative agent oflymphatic filariasis. bancrofti larvae [29]. Irradiated L3 larvae were used to immunize jirds. A chitinase from Acanthocheilinema viteae. fungal and insect chitinases [13]. the antisera were used to probe a W. Subsequently. The functional significance of this repeat remains obscure but is likely not relevant to vaccine research. volvulus chitinase has not yet been explored [31]. it was later found that the mRNAs encoding transcripts for the 70-kDa and 75-kDa proteins differed only in an extra copy of a serine/ threonine-rich repeat [28]. bancrofti-endemic areas [7. The relation ofthe B. Purified 43-kDa protein was used to immunize rabbits. oligonucleotide primers based on the A. The PI sera preferentially recognized a 43-kDa antigen. Recombinant E.Chitinases ofhuman parasites and their implications as antiparasitic targets 229 analysis was unable to resolve this discrepancy. a filarial parasite of rodents. was identified in a similar way. e. 8]. malayi L3 larval 43-kDa antigen to the MF 1 microfilarial antigen has not yet been clearly defined. histolytica was cloned using degenerate oligonucleotides derived from consensus sequences of bacterial. with its presumed function related to encystation of the parasite within a cell wall. It was observed that 100 % of putatively immune (PI) (i. the N -terminus has acidic and hydrophilic domains composed ofmultiple tandemly arrayed seven-amino acid repeats similar to the repeats of other Entamoeba serine-rich proteins that displace the normally N-terminally located catalytic site toward the center of the open reading frame. W. was identified as a potentially protective immunogen using sera from patients from W. The rabbit antibodies to the 43kDa protein were found to react with intrauterine microfilariae as well as with tissues ofL3 larvae [7]. bancrofti genomic expression library. the chitinase from the human enteric pathogen E. the resultant immune sera reacted with an antigen subsequently shown to be a chitinase [30]. volvulus. malayi chitinase. Additionally. The function and transmission blocking-inducingltherapeutic potential ofthe 0. The resulting partial genomic DNA clone was found to encode a chitinase with 23% identity (47% similarity) to the B. This chitinase has putative substrate binding and catalytic and chitin binding domains similar to those previously described for bacterial and fungal chitinases. amicrofilaremic but with strong cellular and humoral responses to parasite antigens) but only 8% of infected microfilaremic subjects in endemie regions of the Cook Islands recognized a 43-kDa antigen from infective third-stage (L3) W. allowing resistance to environmental conditions [32]. Entamoeba chitinase A chitinase was demonstrated to be present in the reptilian parasite Entamoeba invadens. the filarial agent of onchocerciasis and river blindness. viteae chitinase were used to amplify a chitinase from 0. histolytica chitinase was demonstrated to bind chitin and to show endochitinase activity with chito-oligo- .

jalciparum ookinetes in vitra is difficult. Of interest. an L. gallinaeeum has been mostly used for chitinase studies.and endochitinase activity as assessed by the ability of promastigote culture supematants to hydrolyze both 4-methylumbelliferone-chitobiose and chitotriose. delays E. gallinaeeum and the human malaria parasite P. donovani chitinase has both exo. but cloning of the Plasmodium chitinase gene has been challenging. Vinetz saccharide substrates. histolytiea and E. dispar. Because the development of P. . gallinaeeum has been biochemically identified. Ld CHTl. the specific activity of the native L. its ookinetes can be obtained in sufficient quantities from in vitra cultures of gametocyte-containing chicken blood and ookinete chitinase activity can be readily detected [35]. Based upon these characteristics P. invadens encystation in vitra. while an encystation chitinase presumably functions as a cell wall-modifying enzyme during chitin synthesis. histolytiea chitinase. Plasmodium ehitinase The chitinase of the avian malaria parasite P. M. jalciparum. Allosamidin. A number of groups have unsuccessfully attempted to use degenerate oligonucleotide PCR to amplify the chitinase gene from both P. a potent inhibitor of chitinase.230 M. fungal and parasite chitinases. the consensus sequence identified by Shakarian and colleagues as the substrate binding site differs from other reported chitinases in that a cysteine substitutes for highly conserved serine or alanine at amino acid position 131. It is also unknown whether chitinase inhibition has an effect on the morphology. invadens has several chitinase isoforms [33]. but the effects of chitinase inhibition have not been studied in vivo for any of the Entamoebae in their natural hosts. was cloned by use of degenerate oligonucleotide PCR [34]. This substitution could be one reason for the enzyme 's slower kinetics. donovani chitinase gene. E. the chitinase of all three Entamoebae were found to be highly similar. pointing to the close evolutionary relationship of the members of this genus. gallinaeeum chitinolytic activity has been purified. Shahabuddin and J. life cycle or killing of Entamoeba cysts. P. especially E. The native L. P. jalciparum. histolytiea or the commensal E. Finally. but a similar finding has not been made for the pathogenic E. gallinaeeum is closely related to P. donovani chitinase seems to be substantially lower. While typical catalytic and chitin binding domains are encoded by Ld CHTl. dispar. no excysting chitinase has yet been demonstrated that would allow the parasite egress from the cyst. Compared with other bacterial. Leishmania ehitinase Similar to the E.

however. Chitinase activity in one peak is associated with an . Vinetz et al.Chitinases ofhuman parasites and their implications as antiparasitic targets 231 Two distinguishable peaks of endochitinase activity (as detected by hydrolysis of 4-methylumbelliferone ß-acetyl chitotrioside) can be separated by chromatography. The effects of chitinase inhibitor analogs remain to be investigated. a transmission-blocking vaccine against the vector stage would be more suitable. malayi conferred a moderate degree of protection to infected jirds [24]. Protein microsequencing of the . Detection of chitinase antibodies in the sera of immunologically protected individuals is a direct evidence that interfering with the chitinase activity would block the establishment of filarial infection. a vaccine can be prepared against the vertebrate stage of the parasite.. This observation suggested that chitinase could be a suitable candidate for an antifilarial vaccine. The hatching of filarial eggs and exsheathment of maturing nematodes in the human also suggests that a chitinase inhibitor would be a potential drug against microfilaria infection. The potent chitinase inhibitor allosamidin delays the cyst formation of Entamoeba invadens [32]. For some parasites. The relationship of the two chitinases whether they are products of the same gene with posttranslational proteolytic modification followed by multimerization.55-kDa doublet led to the cloning and sequencing of a single exon open reading frame that contained sequences typical of family-18 glycohydrolases. a chitinase inhibitor can act as a drug. a drug with chitinase inhibitory activity may be suitable. however. or whether they are products of two different genes . while in other cases. Jirds immunized with recombinant B. This hypothesis has been tested injirds. a putative substrate binding site. in the other with an . . In addition. Passive transfer ofmonoclonal antibodies to the MF I antigen of B. unpublished data). The strategies against the chitinase would. malayi chitinase also showed partial protection against microfilarial challenge. Whether this inhibitor can also block the emergence of the parasite from the cyst remains to be seen. For the modulation of the cyst wall in Entamoeba. The predicted amino acid sequence contains a signal sequence. when jirds were immunized with recombinant chitinase. a catalytic active site and a cysteine-rich chitin binding domain.is not clear at this time (I M.55-kDa doublet. be dependent on the parasites.21O-kDa protein. for others. they showed protection against filarial challenge. a suitable analog of allosamidin or other chitinase inhibitor can be a potential drug against Entamoeba infection. Chitinase as a target for antiparasitic strategies The expression of stage-specific chitinases by the medically important parasites suggests that chitinase could be a potential generalized target for blocking the development and transmission of these parasites.

Vinetz In the cases of malaria.when ingested by an insect with the parasite . the immune sera blocked development ofthe parasite in the mosquitoes. In this procedure. yeast and plants. Shahabuddin and J. In malaria. M. Several parasite surface antigens have been subsequently identified that showed potential as transmission-blocking targets [38]. The concept of a transmission-blocking vaccine is relatively new. this strategy has already shown potential. and because this requirement transpires within 24 h after blood ingestion. an activity that blocks antibody against Plasmodium chitinase would block parasite development. the immune sera . the catalytic active sites of the parasite chitinases are similar enough to the human chitinases/chitinase-like proteins that cross-reactive antibodies could be induced by vaccination [42]. Studies of the recently cloned parasite chitinases have opened a new vista of antiparasitic drug and vaccine research. parasite chitinases are poorly understood. 37]. Instead. Interfering with this enzyme in humans may cause unpredicted side effects. the vaccine would have to be against the insect vector stages. When an in vitro-transformed mosquito stage of Plasmodium was used to immunize monkey and chicken [36. The immune response in the host theoretically does not have an effect on the vertebrate and does not cure if the individual is infected. Therefore. it is important that the properties of the enzymes and the structures of the genes by understood in detail. The vertebrate host is immunized with purified native or recombinant proteins. although the overall amino acid sequence similarity is low. The attack blocks further development and transmission of the parasite. Structural studies to identify conformationally and immunologically unique regions of the chitinases will be critical for success in using chitinases as human vaccines.attacks the parasite when the antigen is expressed in the parasite. Compared with those ofbacteria. only transmission blocking would be possible. Recent cloning of the P. One potential problem of using parasite chitinases as drug or vaccine targets is their amino acid sequence and functional similarity to recently discovered human chitinases [39-41]. leishmaniasis and trypanosomiasis. Because chitinase is essential for parasite development in the mosquito. vector stage-specific antigens are targeted as vaccine candidates. . In addition. gallinaceum chitinase has opened the possibility of testing this hypothesis. as these organisms apparently express chitinase only at the insect stage. To fully exploit the potential of parasite chitinases.232 M. The function of the enzyme in humans is not clear but may be restricted to clearing fungal chitin from the body.

227 13 delaVega H. Aikawa M. Exp Parasitol79: 85-88 5 Fuhrman JA (1990) Biochemistry of microfilarial exsheathment. Eichinger D. Kaidoh T. Parasitology 115: 359-369 18 Sieber K. Caplivski D. Proc Natl Acad Sei USA 90: 4266-4270 21 Shahabuddin M. Gillin FD (1991) Chitin synthase in encystingEntamoeba invadens. Banks S. Perler FB (1992) Transmission-blocking antibodies recognize microfilarial chitinase in brugian Iymphatic filariasis. Aikawa M. Watawana L. Samuelson J (1997) Cloning and expression of chitinases of Entamoebae. Nutman TB (1994) Cloning and characterization of a potentially protective chitinase-like recombinant antigen from W. Keusch GT. Huber M. Arnold K. Arch Invest Med 21: 223 . Freedman DO. Dreyer G. Jacobson RL. Modi GB. Torii M. Kaslow DC (1995) Plasmodium gallinaceum: mosquito peritrophic matrix an the parasite-vector compatibility. Fuhrman JA et al (1995) Differential recognition of microfilarial chitinase. Exp Parasitol 81: 386-393 22 Welburn SC. Mol Biochem Parasitol85: 139-147 14 Peters W (1992) Peritrophic membranes. Proc Natl Acad Sei USA 89: 1548-1552 7 Raghavan N. Gooday GW (1993) Chitinolytic activities in Heligmosomoides polygyrus and their role in egg hatching. Exp Parasitol 70: 363-366 6 Fuhrman JA. Parasitology 107: 141-145 23 Welburn SC. Kaslow D (1994) Plasmodium: parasite chitinase and its role in malaria transmission. Piessens WF. Lane WS. Med fet Entomol8: 81-87 . Am J Trop Med Hyg 53: 289 . Robbins pw. Maudlin I. Lopez-Romero E. I'!fect Immun 62: 1901-1908 8 Dissanayake S. Exp Parasitol72: 145-156 19 Shahabuddin M. Gooday Gw. Mol Biochem Parasitol29: 223-225 11 Arnold K. Biochem J280: 641-647 3 Ward HD. Brydon LI. Shahabuddin M. by sera from patients with brugian and bancroftian filariasis. Ottesen EA. Yee CK. Toyoshima T. bancrofti. Mol Biochem Parasitol 17: 93 -1 04 2 Das S. Fitzgerald PC. Xu M. Shlomai J (1991) Chitinase secreted by Leishmania functions in the sandfly vector. Ghosh S. Pereira ME (1985) Identification of chitin as a structural component of Giardia cysts. Smith RF. Specht CA. Alroy J. Mol Biochem Parasitol58: 317 -323 12 Valdes J. Brydon LI. Wang S. Messer G (1992) Leishmania infections damage the feeding mechanism of the sandfly vector and implement parasite transmission by site. Aikawa M. Bull Inst Pasteur 92: 119-132 20 Shahabuddin M. Chappell LH. Kaslow DC (1994) Biology of the development of Plasmodium in the mosquito midgut: a molecular and cellular view. Unnasch TR.294 9 Brydon LI. Jacobson RL. Kaslow D (1993) Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease. Kurniawan L. Proc R Soc London Ser B: Biol Sei 245: 121-126 16 Schlein Y. Molyneux DH (1994) Midgut lectin activity and sugar specificity in teneral and fed tsetse. Heidelberg 15 Schlein Y. a transmission-blocking vaccine candidate antigen. Gooday GW (1993) Rickettsia-like organisms and chitinase production in relation to transmission of trypanosomes by tsetse flies. Mol Biochem Parasitol25: 267 -272 10 Gooday Gw. Proc Natl Acad Sei USA 89: 9944-9948 17 Pimenta PF. Piessens WF (1985) Chitin synthesis and sheath morphogenesis in Brugia malayi microfilariae. Infect Immun 49: 629-634 4 Shahabuddin M. Chappell LH (1988) Chitinase in female Onchocerca gibsoni and its inhibition by allosamidin. Chappell LH. Semino CE. King TP (1987) Chitin in egg shells of Onchocerca gibsoni and Onchocerca volvulus. Orozco E (1990) Antigens specific to pre-cysts and in vivo chitin synthetase activity in Entamoeba invadens. Pereira ST. Maudlin I. Lev BI. Springer. Kaslow D. Sacks DL (1997) A novel role for the peritrophic matrix in protecting Leishmania from the hydrolytic activities of the sand fly midgut.Chitinases ofhuman parasites and their implications as antiparasitic targets 233 References 1 Fuhrman JA. Southworth MW. Perler FB. Miller L (1991) The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a moncolonal antibody to ookinetes.

Nutman TB.234 M. Lee J. Vinetz 24 Canlas M. Renkema GH. a chitinase. ASM Press. Spielman A. M. Muijsers AO. Jacobson R (1992) Parasitol Today 8: 367 . J Biol Chem 271(32): 19415-19420 41 Boot RG. Parasite Immunol12: 213-228 28 Arnold K. Miller L (1991) Malaria parasite ehitinase and penetration of the mosquito peritrophic membrane. Proc NatlAcad Sei USA 88: 2807-2810 36 Gwadz R (1976) Suceessful immunization against the sexual stages of Plasmodium gallinaceum. Strijland A. Bianco AE (1996) Chitinase genes expressed by infective larvae ofthe filarial nematodes. Gene 177: 55 . J Biol Chem 270(44): 26252-26256 42 Vinetz JM. Exp Parasitol 90: 199-202 43 Fuhrman JA. Venegas A. Hamill B. Piessens WF (1984) A monoclonal antibody to surfaee antigens on microfilariae of Brugia malayi reduees mierofilaremia in infected jirds. Green I (1978) Malaria immunization in Rhesus monkeys. A vaccine effective against both the sexual and asexual stages of Plasmodium knowlesi. Fuhrman JA. Calvo-Mendez C. Lucius R (1996) Identifieation of ehitinase as the immunodominant filarial antigen recognized by sera ofvaeeinated rodents. Houseweart C. Rudin W. Beauregard K. Exp Parasitol80: 672-680 44 Southworth WM. Urioste SS. Dalamagas D (1995) Strueture and nmetion of a family of ehitinase isozymes from Brugian microfilariae. Gene 208: 315 . Am J Trop Med Hyg 36: 70-74 27 Kurniawan L. and the 39-kDa human cartilage glycoprotein. a chitin-binding lectin. Acanthocheilonema viteae and Onchocerca volvulus. Lamontagne L. Dwyer DM (1998) The Ld Chtl gene eneodes the seeretory chitinase of the human pathogen Leishmania donovani. Fuhrman JA. Fuhrman JA (1996) Diserete transeripts eneode multiple ehitinase isoforms in brugian microfilariae. Robbins pw. In: SL Hoffinan (ed): Malaria vaccine development. Mol Bioehern Parastiol 80: 149-158 29 Freedman DO. Mol Bioehern Parasitol78: 149-159 46 Sehlein Y. DC. Washington. Fuhrman JA (1996) Expression of recombinant microfilarial chitinase and analysis of domain funetion. JBiolChem271: 1441-1447 31 Wu Y. Goldstein JC. Perler FB (1996) Gene cloning and production of aetive recombinant Brugia malayi microfilarial chitinase. Piessens WF (1989) A stage-specific ealcium-binding protein from mierofilariae of Brugia malayi (Filariidae). Purtoma H. Trinh K. J Exp Med 148: 1311-1323 38 Kaslow DC (1996) Transmission-bloeking vaceines. Antonie Van Leeuwenhoek 70: 41-48 34 Shakarian AM. Oles A. Piessens WF (1990) Differential reeognition of mierofilarial antigens by sera from immigrants into an area endemie for brugian filariasis.58 45 Venegas A. Marti T. Am J Trop Med Hyg 33: 420-424 25 Fuhrman JA. Price PA (1996) Isolation and sequence of a novel human chondrocyte protein related to mammalian members ofthe chitinase protein family. Beauregard K. Abdulhayoglu N. Kaslow DC (1998) Plasmodium gallinaceum: use of anti sera to degenerate synthetic peptides derived from the aetive site of protozoal chitinases to characterize an ookinete-speeific chitinase. are homologues of family 18 glyeosyl hydrolases seereted by human maerophages. Mol Bioehern Parasitol52: 53-62 33 Villagomez-Castro JC. Figueira WF. Lopez-Romero E (1992) Chitinase aetivity in eneysting Entamoeba invadens and its inhibition by allosamidin. Se1eetive reeognition of a 43-kD larval stage antigen by infection-free individuals in an endemie area. Science 193: 1150-1151 37 Gwadz RW. Basundari E. Eur J Bioehern 251 (1-2): 504-509 40 Hu B. Adam R. Friedrich T. Mol Bioehern Parasitol35: 249-257 26 Fuhrman JA. Boot RG. a human chitinase produeed by maerophages. Hrebicek M. van Zonneveld AJ. Aerts JM (1998) chitotriosidase. Strijland A. Shahabuddin and 1. 181-227 39 Renkema GH. Cabib E. Kaltmann B. Mol Bioehern Parasitol75: 207-219 32 Villagomez-Castro JC.322 35 Huber M. Aerts JM (1995) Cloning of a cDNA eneoding chitotriosidase. Turner H. J Clin Invest 83: 14-22 30 Adam R. Wadee A. Au FL. Piessens WF (1987) Funetional and antigenie maturation of Brugia malayi mierofilariae. Donker-Koopman WE. Lopez-Romero E (1996) Identifieation and partial eharaeterization of three ehitinase forms in Entamoeba invadens with emphasis on their inhibition by allosamidin. Williams SA. Ottesen EA (1989) Proteetive immunity in bancroftian filariasis.

alkaloids and steroids. 1-60100Ancona. E. whose importance has been recently recognized. by P. The complex ofthe enzyme with the substrate chitobiose has been crystallized and characterized [5].Chitin and Chitinases ed. Sequencing has revealed an open reading frame encoding a protein of 885 amino acids. 3. cali. The reactions catalysed by these enzymes are shown in Figure 1. The enzymes from bacteria. hexosaminidase.2. sequenced and expressed in E. Comparison with the members of family 20 of glycosyl hydrolases revealed that N-acetyl-ß-D-glucosaminidase has a conserved central region which aligns with almost all members ofthis family and which comprises the catalytic domain [6]. however. The speetrophotometric determinations ofN-aeetyl-ß-D-glucosaminidase. Muzzarelli University o[Ancona.1. ß-N -acetylgalactosaminidase and glucocerebrosidase have not been so widely studied as the ß-N -aeetylhexosaminidases in baeteria.52. For instance. Human N-acetyl-ß-D-glucosaminidase.1. is widely distributed in animals.C. The term ß-N-acetylhexosaminidase. endo-ß-Nacetylglucosaminidase. Italy Summary. ß-N-Acetylhexosaminidase. A.A. A few words about nomenc1ature would be essential for understanding the bibliography and the content ofthis chapter. in particular for hypertension. EC 3.Ä. renal injuries and disorders. N-acetyl-a-D-glucosaminidase. most often done with 3-eresolsulphone phthaleinyl N-aeetyl-ß-D-glucosaminide. higher plants and microorganisms [1-4].2. The present chapter deals with the human N-acetyl-ß-D-glucosaminidase and related hydrolases. fungi and arthro· pods. Via Ranieri 67. The enzyme encoding gene from Serratia marcescens has been c1oned. On the other hand. have been reeently simplified and adapted to automatie instruments. covers today both enzymes known as chitobiase . Faculty o[Medicine. metals or other ligands in the enzyme. and their urinary and plasma determination is being adopted for the early detection of diseases before c1inical manifestation. depression and lysosomal storage diseases. Their bioehemieal role has been elueidated. There are no cofactors. in transglycosylation the tolerance for the acceptor structure may be strict.52. Jolles and R. involved in glycolipid and glycoprotein degradation. Center tor Innovative Biomaterials. The tolerance ofN-acetyl-ß-D-glucosaminidase for the aglycon moiety R is generally high with carbohydrate chains of glycoproteins and with phenolics. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Analytical biochemistry and clinical significance of N-acetyl-ß-D-glucosaminidase and related enzymes Riccardo A. the N-acetyl-ß-Dglucosaminidase from Escherichia cali is a monomeric protein with four domains and three disulphide bonds. fungi and crustaceans have been studied in considerable detail.

l. Endo-ß-Nacetylglucosaminidase.497 and 0. respectively [0.1 kDa [ca. since it varies from 31 % for A-l [8.5 mM. A. ß-N-Acetylgalactosaminidases.52). vaccine [17] and simian [18] sources. (b) Transglycosylating activity: under certain conditions. • the A form can be further refined into two forms.1. Band I. respectively. having K.1. • the molecular weight for A-1 is 52. human]. Enzyme characteristics Kidney N-acetyl-ß-D-glucosaminidase has been purified from human [8-13]. . however. EC 3. are distinctly recognized. A. Reactions catalyzed by ß-N-acetylhexosaminidase (EC 3. a single sugar or an oligosaccharide moiety. EC 3.5 mM. the aglycone partial structure of the ß-N-acetylhexosaminide represents a noncarbohydrate residue. A. EC 3. Rl HO ~ OH =H C> + HO HO-R l AcNH C> ~t OH + HO-R AcNH (b) Transglycosylation. the pH optimum is 4. In the current literature.627 mM. Acetate is a competitive inhibitor [also for the human enzyme]. the "old" names are still being used. human] to 17% for A-2 and the sialic acid is 6%and 1%. the enzyme ftmction as a transferase. human] • Ag + and Pb 2+ are the most potent uncompetitive inhibitors.29 and N-acetyl-ß-D-glucosaminidase EC 3.236 R. is a distinct enzyme that catalyses the hydro lysis of the N.6 and 8. canine [15].4] and pI 4. (a) Rydrolytic activity: R.97 • the optimum temperature for A and B is 50 and 42°C • the Km for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine for both isoforms is 0. Muzzarelli (a) Hydrolysis.53. equine [14]. or the carbohydrate side chain of a glycoprotein.3)-galactosyl-ß-l . with ROR' as the acceptor.1. l-ceramide.N' -diacetylchitobiosyl unit in high mannose glycopeptides containing the [Man(GlcNAc)2]Asn structure.30 (both outdated names) [7].2.4-glycosyl-ß-I. murine [16]. Rl~ H Figure 1.55 [4.2. values of 3. These exoglycosidases catalyse the hydrolysis of terminal nonreducing N-acetylglucosamine residues in N-acetyl-ß-glucosaminides. The characteristics of the baboon enzyme [10] are the following (values in parentheses are for human N-acetyl-ß-D-glucosaminidase): • it exists in three forms. A-l and A-2 • the carbohydrate content accounts for microheterogeneity. The term hexosaminidase is currently used to indicate enzymes that act on substrate G M2 gangliosides N-acetylgalactosaminyl-ß-l . 100 kDa.4-(N-acetylneuraminyl-a-2.2.96.1.2.2.5%.

this volume).00 and unit cell dimensions a = 63.45 pM for bovine kidney enzyme). None ofthese inhibitors shows sufficient antifungal activity to justify a practical use [7].5-lactam (K i = 0. and the space group is P21 with ß= 101. the nuc1eophilic attack takes place.036 pM) and nagstatin (K i = 0. The crystals of chitobiase from Serratia marcescens have the shape of prisms. marcescens. In the second step. mostly asparagines.017 pM). Vibrio harveyi and S. Mechanism of hydrolysis The reaction catalysed by glycoside hydrolases is a nuc1eophilic substitution at a saturated carbon. The loop area of the c1eft is formed by neutral polar residues. moderate [22].67 pM forbovine kidney enzyme)..1 A [20]. high. hybrid.2 A.2 A. 237 A total of 17 compounds are known today as true inhibitors. for instance from Alteromonas.. This endo H enzyme is commonly used in the analysis of glycoproteins and asparagine-linked oligosaccharides. The endo N-acetylglucosaminidase from Stigmatella aurantiaca has the following substrate specificity towards glycoasparagines: oligomannoside. The structure of endo H is very similar to that of N-acetyl-ß-D-glucosaminidase secreted by Flavobacterium meningosepticum. Lactones and lactams resemble the flattened ring of the transition state [19]. N-acetylglucosaminono-1. Then. The best inhibitors are structurally and mechanistically glycon-based analogues ofthe substrate and inc1ude N-acetylglucosaminono-1. N-acetylglucosaminono-1. . a glutamic acid unit protonates the oxygen to make it a good leaving group and deprotonates the acceptor [23]. b = 133. Several N-acetylhexosaminidases have been c10ned and sequenced. a carboxylate group leads to a glycosyl enzyme and to the removal of the aglycon group. c = 55.5-lactone (K i = 0. The 2-acetamido group vicinal to the glycosidic bond stabilizes the reaction intermediate through formation of an oxazoline (see Robertus and Monzingo. The endo N-acetylglucosaminidase from Streptomyces plicatus has dimensions 31 x 34 x 57 A and is a (aIß)8 barrel protein. a number of inhibitors are N-heterocycles: these bind to the active site in their protonated forms to counteract the negative charges [19] of the catalytic carboxylate. It c1eaves the bond between the two N-acetylglucosamine units of the chitobiose core and is specific for high-mannose asparagine-linked oligosaccharides. medium. the only major difference residing in the structure of one loop that imparts the ability to process glycoproteins having an a(1-6)-linked fucose substituent on the first N-acetylglucosamine unit [21]. Like nagstatin. leading to hydro lysis or transglycosylation.5-lactone oxime (K i 0.Analytical biochemistry and c1inical significance ofN-acetyl-ß-D-glucosaminidase . In a first step. The putative catalytic residues Asp 130 and Glu 132 are surrounded by several aromatic residues. complex.

3' -dichlorophenylsulfon phthalein (Chromogenic) and 4methylumbelliferone (fluorogenic) and a number of related compounds.238 R.0.6dichlorophenol. Fluorimetric procedure For instance. with E=42. have been examined as substrates [25.1 and 0. A number of other chromogenic substances have been developed.9 (all values for substrate 4-nitrophenyl N-acetyl-ß-D-glucosaminide).7 mM and in particular for humans between 0. The detection limit as low as 0. 4'-(6'diethylaminobenzofuranyl) phthalyl hydrazide-N-acetyl-ß-D-glucosaminide and o-aminophthalylhydrazido-N-acetyl-ß-D-glucosaminide. Muzzarelli Analytical assays The differences in substrate affinity are minor among the N-acetylhexosaminidases of different origins.11) with added 4-methylumbelliferyl-Nacetyl-ß-D-glucosaminide are incubated for 15 min at 25 oe under shaking. The microbial N-acetylglucosaminidase Km values are between 0. 3. 4-nitrophenyl-N-acetyl-ß-D-glucosaminide and 3-cresulsulphone phthaleinyl-N-acetyl-ß-D-glucosaminide. It is also sufficiently stable and soluble [24]. Stopping buffer (pH 10. which is determined by fluorimetry at 355 nm (ex) and 480 nm (ern) [17]. other chromophores of test substrates for the assay for N-acetyl-ß-D-glucosaminidase are 2-chloro-4-nitrophenol. and the absorptivity of product should be high. among which 4-amino-2. A microtiter plate version is the method of choice.5 mU/mI. A.000) atpH 5.005 mU/ml makes 4-methylumbelliferyl-ßN-acetylglucosaminide the preferred substrate over the other whose sens itivity is 0.4 and 4.4) is then added.5. . Two protocols for the determination of N-acetyl-ß-D-glucosaminidase with the two most popular substrates are the following.26]. in plants 0. A. N ew chemiluminogenic substrates. that is the product should absorb at visible wavelengths where absorbance from bilirubin and hemoglobin is negligible. Besides 4-nitrophenol. In fact it produces a green color (Ämax302 ~ 718 nm. The most popular substrates are 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide. in animals they range between 0.2 and 5.8 mM. Details are to be found in [27].1-0. The amount of N-acetyl-ß-Dglucosaminidase is proportional to 4-methylumbelliferone released from the substrate per unit time. The literature contains much information on the analytical assays being used to characterize and quantify N-acetyl-ß-D-glucosaminidase.1-0. 4-amino-2. milk sampies (10 ].6-dichlorophenyl N-acetyl-ß-D-glucosaminide satisfies the criteria for a good substrate.

l. such as serum. lymphocytes. However. are known to release mature lysosomal enzymes upon stimulation. which is a compromise between the enzyme optimum (5. The reaction is stopped at pH 10. Increased serum acivity is found in thyroiditis [29]. In the case ofurine sampies.. smooth-muscle cells) no mature forms of lysosomal enzymes are secreted. urine and tears. macrophages. The 3cresolsu1phone phthaleinyl N-acetyl-ß-D-glucosaminide. hepatocytes. Another source of mature forms are damaged cells. pH 4. the increased enzymatic activity results from metabolic derangements hut is independent of the development of microvascular complications [35]. New synthetic substrates that obviate these problems should be easily soluble in order to saturate the enzyme. B (basic).25. Increased activity ofN-acetyl-ß-D-glucosaminidase Increased activity of N-acetyl-ß-D-glucosaminidase may be indicative of acquired and genetic diseases and various forms of stress. Human lysosomal N-acetyl-ß-D-glucosaminidase is known to exist in at least five isoenzymic forms termed A (acidic). 239 Spectrophotometric procedure Enzyme sampie (5 p..Analytical biochemistry and clinical significance ofN-acetyl-ß-D-glucosaminidase . making unnecessary gel filtration of urine to eliminate urinary inhibitors. One unit ofN-acetyl-ß-D-glucosaminidase is defined as the amount of enzyme liberating I mmol of 4-nitrophenol (A 450 nm) in 1 min at 25°C [18]. Lysosomal enzymes are common in human body fluids. acromegaly [33] and cancer [34]. expecially urea.l. In fibroblasts and some other cell types (endothelial cells.5) at 37°C is incubated with 4-nitrophenyl-N-acetyl-ß-D-glucosaminide in citrate buffer (25 p. 100 mM. Lysosomal enzymes are synthesized as precursors ofhigher molecular weight and subsequently processed to mature forms.20) and the maximum for chromogenesis. however. and release a stable chromophore with a pK a value near the pH optimum of the enzyme.l) in citrate buffer (25 p. liver disease (including alcohol intoxication) [36. and therefore the N-acetyl-ß-D-glucosaminidase can be done routinely in view of its diagnostic significance [28]. 31]. and readings are made at 405 nm. also lends itselfto spectrophotometric determinations at 570 nm. In diabetes. 37]. pregnancy [36. 100 mM) for 20 min. and P (pregnant plasma). leukemia [32]. currentlyavailable as a test kit.3'-dichlorophenylsulfonphthaleinyl N-acetyl-ß-D-glucosaminide releases the chromophore chlorophenol red at pH 6. The substrate 3. This methodological disadvantage has in the past been the essential reason for a limited appreciation of N-acetyl-ß-D-glucosaminidase activity. and the N-acetyl-ß-D-glucosaminidase levels are indexed to creatinine to minimize the effects of diuresis [14]. diabetes mellitus [30. 38] and hypertension [39] appear to be the conditions mainly responsible for the increase in . The methods indicated above generally require gel filtration of urine to eliminate interfering and inhibitory substances. the urinary creatinine is also measured. 11 and 12 (intermediate). The test has been adapted to automatic analysers.

A. Serum N-acetyl-ß-D-glucosaminidase was clinically evaluated as a liver-function test [40]. A. Muzzarelli serum N-acetyl-ß-D-glucosaminidase achvlty: the serum intermediate isoenzymes land P are the main cause of the increase in total activity. Because of its high molecular weight it does not filter through the glomerular membrane. and systolic and diastolic blood pressure: a significant association between urinary Nacetyl-ß-D-glucosaminidase and blood pressure exists in normal young adults. it is important to keep in mind that urinary N-acetyl-ß-D-glucosaminidase may originate in macrophage-type cells [41]. and changes in urinary N-acetyl-ß-D-glucosaminidase may be evidence of early hypertensive disease [47]. urinary N-acetyl-ß-D-glucosaminidase activity increases. The urinary N-acetyl-ß-D-glucosaminidase changes in 980 young adults aged 18-32 was analysed in relation to age. sex. In urine. and is normally accepted as indicative ofkidney disease or damage. Secreted enzymes probably are involved in the physiological process of inflammation [42]. The increased serum N-acetyl-ß-D-glucosaminidase activities might be due to a decreased clearance of this enzyme by nonparenchymal cells of the liver and/or increased production of lysosomal enzymes. Hypertension Elevated serum N-acetyl-ß-D-glucosaminidase acttvlty is an effective indicator of future hypertension and may be related to functional and structural changes in the cardiovascular system [46]. released to serum from cells by exocytosis or due to breakdown of cells. The diagnostic value of the urinary N-acetyl-ß-D-glucosaminidase activity measurements was reviewed [44. This is a lysosomal enzyme widely distributed in human tissues. . [43] characterized N-acetyl-ß-D-glucosaminidase isoenzymes released from monocyte-derived macrophages when cultures were supplemented with an inflammatory agent such as zymosan. Clinical significance The determination of N-acetyl-ß-D-glucosaminidase is most relevant to pediatrics. race. nephrology and urology. ß-D-glucan and a-D-mannan. the major form normally excreted is tissuelike N-acetyl-ß-D-glucosaminidase A. a yeast cellwall product chiefly made oftwo carbohydrate polymers.240 R. In the presence of a glomerular lesion or tubular damage. Bourbouze et al. with traces of B and I. urinary N-acetyl-ß-Dglucosaminidase changes with increasing blood pressure may start early in life and mayaiso be evidence ofthe existence of early hypertensive disease. Since hypertension is increasingly recognized as beginning in childhood. 45]. However. possibly by activated liver macrophages.

Analytical biochemistry and clinical significance ofN-acetyl-ß-D-glucosaminidase . Once the obstruction has been alleviated or the nephrotoxic stimulus removed. In patients with cystic fibrosis on gentamicin inhalation. In the case of increased N-acetyl-ß-D-glucosaminidase excretion in stone patients. 241 Nephrology Tubular renal damage induced by crystals can trigger further stone formation. there is a positive correlation between N-acetyl-ß-D-glucosaminidase excretion and phosphate.4 ± 2.2 ± 2. but only 30-45% of insulin-dependent patients develop diabetic . respectively. in which case this enzyme proved to be of interest in assessing renal interference of this kind of antibiotic [57. the N-acetyl-ß-Dglucosaminidase/creatinine ratios are higher than in controls. remission of the disease restores normal concentrations. this ratio being indicative of one or more complications [49]. Several renal tubular enzymes such as a-glucosidase. and transient hypoxie periods and pathological oscillations in oxygen tension mayaiso cause tubular damage. uric acid. renal allograph rejection. sulphate. tubulointerstitial diseases.9 U/I and 4. Elevated urinary N-acetyl-ß-D-glucosaminidase has been reported in glomerulonephritis. In the diabetic patients. The increase in the Nacetyl-ß-D-glucosaminidase index or the changes in the isoenzyme pattern are also valuable early signs of an incipient rejection crisis after kidney transplantation [50-52]. however. The degree of damage is reflected bya characteristic increase in either the urinary protein content and enzymes in the urine. y-glutamyltransferase and N-acetyl-ß-Dglucosaminidase have been shown to be elevated in urine with obstructive uropathy as a result of nephrotoxic substances. indicative of a risk of renal toxicity [59]. Raised urinary excretion ofN-acetyl-ß-D-glucosaminidase is associated with proteinuria. N-acetyl-ß-D-glucosaminidase has proved most useful [56]. in particular impairment of renal growth and development of renal scarring. Among the many enzymes studied. the same can be said for aminoglycoside nephrotoxicity. but not in those with diabetic nephropathy [53]. Numerous studies have demonstrated the deleterious effects of vesicoureteral reflux on the structure of the kidneys. the enzyme levels return to normal. toxic renal injury and diabetes. Renal abnormalities can be detected via the determination ofthe enzyme in the amniotic fluid [55]. hypertensive nephrosclerosis and chronic pyelonephritis... Urinary N-acetyl-ß-D-glucosaminidase and proteinuria are weIl correlated at all levels of renal function in patients with glomerulonephritis. oxalate and creatinine excretion [48]. Most patients with diabetes mellitus display some histological renal lesions. Thus early detection ofthe initial damage is important. Many ofthe drugs widely used in intensive neonatal treatment are nephrotoxic. Thus. the enzymuria is a means of following tubular damage and recovery [54]. which are 10. 58]. a positive correlation betwen N-acetyl-ß-D-glucosaminidase concentration in urine and cumulative dose of nebulized gentamicin was found.1 U/I for N-acetyl-ß-DglucosaminidasesA and B.

some degree of histologieal. A significant relationship between cerebrospinal fluid N-acetyl-ß-D-glueosaminidase and serotonin was found [61]. . Because the N-aeetyl-ß-D-glucosaminidase aetivities correlate weIl with 24-h urinary free eortisol contents and less signifieantly with platelet imipramine binding. A. levels of urinary N-aeetyl-ß-D-glueosaminidase were signifieantly associated with severity of depressive symptoms. [60] investigated the faetors that aggravate renal damage after lithotripsy by measuring the urinary excretion of a tubular-eeIl-exeaped enzyme which is thought to be a marker for renal impairment. the authors assumed that urinary N-acetyl-ß-D-glueosaminidase activities were influenced by the density ofthe postsynaptie serotonin receptors. Lithotripsy Short-term follow-up studies have revealed that shock wave lithotripsy is a safe and effective treatment for urinary traet stones. morphologieal and funetional renal damage has been reported in experimental and clinical studies. The detection oftubulopathy caused by hypoxia and the estimation ofthe efficieney ofthe therapies are equally important. There was a significant elevation in the urinary excretion of N-aeetyl-ß-D-glueosaminidase immediately after lithotripsy in a group of patients who previously suffered from urinary traet infeetions. However. which may result in permanent renal damage in up to 40% of survivors [50]. Muzzarelli nephropathy [50]. baeteria hidden in the stones may appear after destruction of the stones and may aggravate renal damage caused by exposure to the shoek waves. N-aeetyl-ß-D-glucosaminidase index values have been proved to be valuable tools in the diagnosis of tubulopathies caused by either idiopathie respiratory distress syndrome in hypoxie preterms [50]. Results indicated that infeetions may be one of the risk faetors for the aggravation of renal damage after lithotripsy. Depression In bipolar patients. Sakamoto et al. It would be important to regularly screen the renal function ofpatients at greatest risk using a weIl established N-acetyl-ß-Dglucosaminidase or ß2-microglobulin assay.242 R. Furthermore. Acute renal failure is a weIl-known complieation of birth asphyxia. Early reeognition of acute renal failure is partieularly important in asphyxiated infants because it facilitates the administration of appropriate fluid and electrolyte replacement. A. transient hypoxie periods and pathologieal oseillations in oxygen tension mayaIso cause tubular damage. In spite of the administration of antibiotics.

Two human hexosaminidase genes encode proteins that form dimers A. A concentration increase of the enzyme in plasma of patients with Gaucher's disease was noted.0 that differ in their recognition and endocytosis by skin fibroblasts. bone deformities and excessive urinary excretion of heparan sulphate. dementia and death.74 mM for various a-D-glycosides.5.Analytical biochemistry and clinical significance ofN-acetyl-ß-D-glucosaminidase . Sanjilippo s syndrome Sanfilippo's syndrome is an inbom error of glycosaminoglycan metabolism characterized by mental retardation. Sandhoff's disease involves a deficiency or defect in the ß-subunit. whereas lack of hexosaminidase B leads to accumulation of oligosaccharides. paralysis. This genetic disorder is generated by the deficiency of the lysosomal a-Nacetylglucosaminidase [65]. accumulation of which cause blindness. Km 0. Purification of this enzyme from urine has been reported [66.14-0. optimum pH 4. Gaucher s disease Splenomegaly. The heterodimer A (a ß) has full activity against G M2 -type gangliosides.. Methylumbelliferyl tetra-N-acetylchitotretraoside hydrolase hase been reported in human plasma. The known mutations have been mapped into the hexosaminidase structure.. Tay-Sachs disease is caused by a deficient or defective hexosaminidase a-subunit. B and S of differing substrate specificities. anemia and deterioration of the skeleton are the major signs ofthis disease. failure to form the heterodimer or to bind the activation protein. it was also described as a chitotriosidase. Multiple forms exist with pI values 3. heparin and dermatan sulphate are competitive inhibitors. hepatomegaly. The fluorimetric deter- . The severity ofthe clinical course ofboth diseases corre1ates with enzyme activity. The characteristics of the enzyme are very similar to those of chitinase [62-64]. 67]: it is a glycoprotein with over 300 kDa molecular weight. instability within the lysosome. Various factors may cause these pathologies: poor folding. leading to lipidladen macrophages that secrete large quantities of hydrolases and cytokines. It is caused by a deficiency in activity ofthe lysosomal hydrolase glucocerebrosidase.2. The absence of hexosaminidase A causes G M2 accumulation. a-GlycosidicaIly linked N-acetylglucosamine units occur in heparan sulphate as weIl as in heparin. insolubility.1.3-6.45. 243 Tay-Sachs and Sandhoff diseases Deficiency of hexosaminidases causes lysosomal storage diseases. Heparan sulphate. Ee 3. The most severe infantile disease is generated by mutations around the active site [5].

FL. Yarnakawa T (1974) Multiple components of N-acetyl-ß-D-glucosaminidase. J Bioeh (Tokyo) 75: 495-507 . Vincentelli R.. HasilikA. in analogy with the protocol given above. Beutler E (1974) Studies on human N-acetyl-ß-Dglucosaminidase. Oppenheim A. References 1 Muzzarelli RAA (1997) Human enzymatic activities related to the therapeutical administration of chitin derivatives. Stanic V. Bioehem J 165: 49-53 13 ZuhlsdorfM. Awasthi YC. Mayer C. Imort M. Yoshida A. Yoshida A. Ramos V (1998) Enzymatic depolymerization of chitins and chitosans. In: JC Salamone (ed): The polymerie materials encyclopedia. Atec. Gene 170: 63-67 7 Horsch M. J Biol Chem 249: 2054-2057 12 Wiktorowicz JE. Beutler E (1974) Studies on human N-acetyl-ß-Dglucosaminidase: kinetic and structural properties. Dauter Z. Nature Struet Biol3: 638-648 6 Tews I. and consequent generation of hydrolysis products having high biological and biochemical significance forthe repair ofconnective tissues [69-72] (see Tokura et al. Grottammare 5 Tews I.A. In: C Bucke (ed): Methods in bioteehnology. Wilson KS. Sennhauser U.A. 320-325 3 Muzzarelli RAA. Cell Mol Life Sei 53: 131-140 2 Muzzarelli RAA (1996) Chitinases. J Biol Chem 249: 2043-2048 10 Srivastava SK. Marinkovic IN (1977) Purification oftwo N-acetyl-ß-D-glucosaminidase from human kidneys. The good c1inical results obtained in wound healing with the aid of modified chitosan dressings can be explained in terms of high susceptibility of certain modified chitosans to hydrolysis by lysozyme and N-acetyl-ß-D-glucosaminidase in vivo. London 4 Muzzarelli RAA (ed) (1996) Chitin enzymology. Boca Raton. Progetto Finalizzato Materiali Speciali per Tecnologie Avanzate 11. Beutler E (1974) Studies on human N-acetyl-ß-D-glucosaminidase. Vorgias CE (1996) Bacterial chitobiase structure provides insight into catalytic mechanism and the basis to Tay-Sachs disease.244 R. at 360 (ex) and 448 (em) nm. this volume). Wound healing Recent clinical data indicate that modified chitosans play an active role in wound healing. Shrivastava SK (1977) Purification and properties ofhuman kidney-cortex hexosaminidase A and B. Awasthi YC. Bioeh J 163: 133-140 9 Srivastava SK. J Biol Chem 249: 2049-2053 11 Shrivastava SK. Muzzarelli mination of N-acetyl-a-D-glucosaminidase is made with methylumbelliferyl-N-acetyl-a-D-glucosaminide [68]. Aeknowledgements The present work was carried out with financial support from the National Research Council of Italy. Von Figura K (1983) Molecular forms ofhexosaminidase and cathepsin D in serum and urine ofhealthy subjects and patients with elevated activity oflysosomal enzymes. Bioehem J213: 733-740 14 Seyama Y. Kurosky A. Vorgias CE (1996) N-Acetylglucosaminidase (chitobiase) from Serratia marceseens: gene sequence. Pharmaeol Ther 76: 187-218 8 Marinkovic DV. Rast DM (1997) ß-N-acetylhexosaminidase: a target for the design of antifungal agents. Awasthi YC. and protein production and purification in Scheriehia eoli. vo12. Perrakis A. CRC Press. Humnana Press.

[nt J Biochem 23: 239-251 19 Legler G (1990) Glycoside idrolases: mechanistic information from studies with reversible and irreversible inhibitors. Litthauer D. Structure 3: 449-457 22 Bourgerie S. Frier BM (1990) Urinary excretion of N-acetyl-ß-D-glucosaminidase in type-l diabetes. J Dairy Sei 74: 1539-1543 18 Beukes HAG. Ueno K. Chem Pharm Bull 39: 411-416 26 Sasamoto K. Gaedicke G. Sramkova J. Propok H.Analytical biochemistry and clinical significance ofN-acetyl-ß-D-glucosaminidase . Sears PM (1991) N-acetyl-ß-D-glucosaminidase etiologic agent and duration of clinical signs for sequential episodes of chronic clinical mastitis in dairy cows. 245 15 Reusch C. N-acetyl-ß-D-glucosaminidase. Stolba P (1991) N-acetyl-ß-D-glucosaminidase and albuminuria in acromegaly. Bengmark S (1983) Total fasting serum bile acids and N-acetyl-ß-D-glucosaminidase in alcoholic liver disease. Garcia-Diez JC. Sakssow A (1983) Isoenzyme pattern ofserum N-acetyl-ß-D-glucosaminidase in liver disease. Alhadetf MD. Ohkura Y (1991) Chemiluminiscence assay ofN-acetyl-ßD-glucosaminidase in urine using o-aminonaphthalylhydrazido-N-acetyl-ß-D-glucosaminidase. Oliver MD. Weschta E (1991) Enzyme activities ofurinary alanine aminopeptidase and N-acetyl-ß-D-glucosaminidase in healthy dogs. Clin ChimActa 136: 203-209 . Springer. subacute tyroiditis and silent tyroiditis. Cabezas JA. In: H Mattenheimer. Minowada J (1983) Isoenzyme studies in human leukemia. Dawes J. Unemura N. Tanaka K (1991) Renal tubular lesions induced by human BenceJones protein in the rat: N-acetyl-ß-D-glucosaminidase as a sensitive marker. Hormone Met 23: 285-287 34 Pluncinsky P. In: RAA Muzzarelli (ed): Chitin enzymology. Guan C.9 Aresolution: active-site geometry and substrate recognition. Zenko R. Dauter Z. Julien R (1994) Purification and characterization of an endo-N-acetyl-ß-D-glucosaminidase from the culture medium of Stigmatella aurantiaca DW4.. alcohol intoxication and pregnancy. Vorgias CE (1992) Crystallization of recombinant chitobiase from Serratia marcescens. Isaksson A. Clin Chem 37: 1696-1699 31 Patrick AW. Mcintyre CC. Harase C (1991) Urinary Nacetyl-ß-D-glucosaminidase activity in patients with Graves disease. Yamaji N (1995) Synthesis of 4-aminophenyl N-acetyl-ß-D-g1ucosaminidase derivatives and their application to the rate-assay ofN-acetyl-ß-D-glucosaminidase. vol 2. JVeter MedAss 38: 90-98 16 Yokota N. Naude RJ (1991) Properties of the isoenzyme forms A-I. III. Alwmark A. J Bact 176: 6170-6174 23 Tews I. Lambrechts H. Mutoh J. 213 . Ohkura Y (1991) A new chemiluminogenic substrate for N-acetyl-ß-D-glucosaminidase. Chern Pharm Bu1l39: 1317-1319 27 Simane ZJ (1992) N-acetyl-ß-D-glucosaminidase. [nt J Exp Path 72: 255-262 17 Wilson DJ. Cancer 58: 1484-1487 35 Miralles JM. A-2 and B of N-acetyl-ß-D-glucosaminidase purified from baboon kidneys. Okada K. Vorgias CE (1996) Structural studies on N-acetylglucosaminidase enzyme-inhibitor complexes. J Mol Bio1228: 696-697 21 Rao V. Marek J. Myburgh JA. Kosaka J. Potential indicator of early nephropathy. Ooshuizen MJ. Ishiyama M. Karamanos Y. Endocr Jpn 38: 303-308 30 Agardh CD. Herer PS. Van Roey P (1995) Crystal structure of endo-ß-N-acetylglucosaminidase H at 1. Chem Pharm Bu1l43: 266-270 25 Sasamoto K. Agardh E. Atec. Clin ChimActa 121: 373-378 36 Hultberg B. Corrales H. Hultberg B. Priem F (1991) A practical new assay for determining N-acetyl-ß-D-glucosaminidase activity in urine. Vocheser R.225 24 Kasai K. Leukemia Res 7: 611-619 33 Skrha J. U Burckhardt (eds): Urinary enzymes. Alhadetf JA (1986) Variant serum N-acetyl-ß-Dglucosaminidase as a biochemical marker ofmalignancy. Reglero A (1982) N-acetyl-ß-Dglucosaminidase and fucosidase activities in relation to glycosylated hemoglobin levels and to retinopathy in diabetes. Grard T. Clin ChimActa 203: 405-408 29 Nakamura S. Berlin.. Yamamoto Y. Diabetes Met 16: 441-447 32 Drexler HG. Hultberg B (1991) Association between urinary Nacetyl-ß-D-glucosaminidase and its isoenzyme patterns and microangiopathy in type-l diabetes mellitus. Enzyme 30: 166-171 37 Joelsson B. 118-125 28 Jung K. Howie AF. Oppenheim AB. Av Carbohydr Chem Biochem 48: 319-384 20 Tews I. Wilson KS. Grottammare. Isaksson A. Gullstrand P.

Diabetes Res Clin Practice 29: 99-105 50 Csathy L. Ganter K. Hesse A (1996) N-acetyl-ß-D-glucosaminidase excretion in calcium oxalate stone patients ans its relation to the risk of stone formation. Berenson GS (1996) Urinary Nacetyl-ß-D-glucosaminidase changes in relation to age. Maekawa M (1991) Examination ofaggravating factors ofurinary excretion ofN-acetyl-ßD-glucosaminidase after extracorporeal shock wave lithotripsy. Lipitska IY.lVephron 58: 205-209 . Modi N. Loko F.A. Novak A. sex. Zach MS (1998) Urinary N-acetyl-ß-D-glucosaminidase activity in patients with cystic fibrosis on long term gentamicin inhalation. Am L lVephro/18: 179-185 54 Carr MC. Yasumoto R. Cortona L. Nomura G (1995) Serum N-acetyl-ß-Dglucosaminidase activity in predicting the development ofhypertension. Elsevier. Peters CA. Biochem Med M 44: 247-251 41 Mauhuran DM. Price RG (1991) Variation ofthe isoenzymes of N-acetyl-ß-D-glucosaminidase and protein excretion in aminoglycoside nephrotoxicity in the rat. Erwa W. Kishimoto T. Ratfi F. Hypertension 25: 1311-1314 47 Agirbasli M. RetnikAB. Amsterdam 43 Bourbouze R. Bone IM (1998) Proteinuria and renal tubular damage. Radhakrishnamurthy B. Clin Chem 37: 583-584 53 Rustom R. Clin ChimActa 93: 409-417 39 Simon G. Scaramuzza A. Kiss L. Aliberti LM. Pocsi I (1995) Urinary N-acetyl-ß-D-glucosaminidase determination in newboms and children: methods and diagnostic applications. Ameno Y. Price RG (1990) The effect ofpolycythemia and hypoxia on urinary N-acetyl-ß-D-glucosaminidase activity in newboms. Clin Exper Hyper Theory Pract A4: 355-365 40 Severini G. Bhattach S. W Sly (eds): Lysosomes in biology and pathology. Tvodgorov MG. Titov VN (1991) Diagnostic value ofthe urine N -acetyl-ß-D-glucosaminidase activity measurements. race. Paraplegia 29: 324-329 52 Loko F. Nakatami T. Toxicol Pathol 26: 26-28 45 Babayeva Ni. Eber E. Scand J Urol lVephroI30:439-443 49 Lorini R. RT Dean. and diastolic and systolic blood pressure in a young adult biracial population. Bao W. Arch Dis Ch 66: 1147-1149 56 Csathy L. Altman S (1982) Increased serum N-acetyl-ß-D-glucosaminidase activity in human hypertension. Pocsi I. Dameron G. Adachi H. Bondiou MT. Zobel G. Bourbouze R (1991) Concentrations of N-acetyl-ß-Dglucosaminidase and its intermediate isoenzymes in serum of patients with renal transplants. Bertrand IM (1988) Aminoglycoside nephrotoxicity and urinary excretion ofN-acetyl-ß-D-glucosaminidase. Lowden JA (1985) The Iysosomal hexosaminidase isozymes. BiollVeonate 53: 253-259 58 Sanchez-Bemal C.246 R. Shenkin A. Hausler M (1991) Amniotic fluid Nacetyl-ß-D-glucosaminidase activity and renal abnormalities. Oommen A (1991) N-acetyl-ß-D-glucosaminidase in the localization of urinary-tract infection in patients with spinal cord injury. Lab Delo 1: 9-16 46 Hashimoto R. Balla G. Melzi d'Eril GV (1995) Increased urinary N-acetyl-ß-D-glucosaminidase excretion in young insulin-dependent diabetic patients. Robic D. Am J Hypertension 9: 157-162 48 Winter P. Hali-Miraftab H. Battisti 0. MandelI J (1991) Urinary levels ofrenal tubular enzyme N-acetyl-ß-D-glucosaminidase in relation to grade of vesicoureteral reflux. Erwa W. Muzzarelli 38 Lowden JA (1979) Serum N-acetyl-ß-D-glucosaminidase in pregnancy. Jiang X. Eur J Chem Clin Biochem 33: 575-587 51 Zacharia A. Riccaboni M. Clin Chim Acta 199: 185-194 44 Loeb WF (1998) The measurement of renal injury. Swank RT (1984) Macrophage as a secretory cello In: JT Dingle. Cell Biochem Funct 9: 209-214 59 Ring E. Tarquini M (1990) Clinical evaluation of serum N-acetyl-ß-D-glucosaminidase as a liver function test. Capurso L. Cabezas JA.A. Valenti G. Curr Topics Med Res 12: 229-288 42 Novak EK. Costigam M. Nishida H. J Urol 146: 654-656 55 Ring E. Clin Chim Acta 195: 77-84 57 Langhendries JP. Arch Dis Childhood 78: 540-543 60 Sakamoto W. Basha A. Wilde JL (1991) N-acetylß-D-glucosaminidase isoenzymes release from human monocyte-derived macrophages in response to zymosan and human recombinant interferon. Vlitos M. d'Annunzio G. Heimbach D. Hofmann H. Ohyama A. Kamizuru M. Koch M. Tsuruta M.

252 70 Biagini G. Aerts JMFG (1995) Purufucation and characterization ofhuman chitotriosidase. Adams DJ (1995) Chitinase activity in human serum and leukocytes. J Biol Chem 270: 2198-2202 63 Escott GM. Van Weely S. Rizzoli C (1989) Reconstruction ofparodontal tissue with chitosan. Laukes C (1995) The association ofthe serotonin metabolite. Strijland A. Castaldini C.Analytical biochemistry and c1inical significance ofN-acetyl-ß-D-glucosaminidase . Grottammare. Moijsers AO. Filippini 0. Eur J Bioehem 80: 535-542 68 Den Tandt WR. Verhoek M (1996) Chitotriosidase: a human macrophage chitinase that is a marker for Gaucher disease manifestation. Biomaterials 10: 598-603 72 Muzzarelli RAA (1992) Depolymerization ofmethyl pyrrolidinone chitosan by lysozyme. Bertani A. 5-HIAA. Eur J Bioehem 80: 525-533 67 Von Figura (1977) Human N-acetyl-a-D-glucosaminidase. Activity towards natural substrates. Biagini G (1988) The biological activity of chitosan: ultrastructural study. In: RAA Muzzarelli (ed): Chitin enzymology. Purification and properties. Carbohydr Polymers 19: 29-34 . Renkema GH. Conti F.. Vasi V. 3-10 65 O'Brien JS (1972) Sanfilippo syndrome. Biomatenals 9: 247 . Biomaterials 12: 281-286 71 Muzzarelli RAA. Hollak CEM. Baldassarre V. Pugnaloni A. Neuropsyehobiology 32: 75-78 62 Renkema GH. Boot RG. Biagini G. 247 61 Garvey MJ. Infeet Immun 63: 4770-4773 64 Aerts JMFG. Donker-Kopman WE. Rizzoli C (1991) Wound management with Ncarboxybutyl chitosan. for the lysosomal enzyme N-acetyl-ß-D-glucosaminidase. vo12. Noyes R. Gazzanelli G. Proe NatlAead Sei USA 69: 1720-1722 66 Von Figura (1977) Human N-acetyl-a-D-glucosaminidase. Baldassarre V. Scharpe S (1993) Micromethod determination ofN-acetyl-a-D-glucosaminidase in human leucocytes. Atec. Muzzarelli RAA. Ferrara P. Woodman C. Boot RG. Int J Bioehem 25: 209-212 69 Muzzarelli RAA. Donke-Kopman WE..

Exogeneous chitosans .

preventing sickness or contributing to good health [1-7]. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Biochemistry. components of the intracellular matrix and connective tissue. by P. Chitin is degraded by enzymes such as lysozyme [4] N-acetyl-Dglucosaminidase and lipases [8]. scientists have reported that chitins show antibacterial. and the regeneration of bone.A. angiogenesis stimulation. Armanda Pugnaloni and Graziella Biagini Center for Innovative Biomaterials. Jolles and R. histology and clinical uses of chitins and chitosans in wound healing Riccardo A. Muzzarelli.A. Biodegradability. anticholesterolemic dietary foods. For the purpose of bone and soft tissue healing. Italy Summary. Faculty ofMedicine. antiinflammatory action. antimetastatic. In addition. Monica Mattioli-Belmonte. The resulting chitooligomers stimulate various cells. antiosteoporotic and immunoadjuvant activities. whose biological significance in the human body depends largely on the actions that certain hydrolases exert on them. Current biomedical applications are illustrated by the treatment ofleg ulcers. granulation and scar formation.Chitin and Chitinases ed.A. biocompatibility and capacity to promote the synthesis of hyaluronan are main characteristics of chitin-derived wound healing materials. 1-60100 Ancona. University ofAncona. Biodegradability Biodegradable and nontoxic materials capable of activating host defences to prevent infection and to accelerate the healing of the wound are highly desirable. indicating a large general potential of the polysaccharide in alleviating diseases. NO mayaiso playa role in the chemoenzymatic degradation process. cytokine production by macrophages and fibroblasts. keratan sulphate and chondroitin sulphate. besides their capacity to release glucosamine and N-acetylglucosamine monomers and oligomers. The healing process favoured by these materials is examined in terms of macrophage activation. Introduction Modified chitins have been administered to humans in the form of dressings for wounded soft and bone tissues. Evidence has been collected that significant portions of chitin-based dressings are depolymerised. while the released monomers are phosphorylated and incorporated into hyaluronan. biocompatibility and similarity to hyaluronan. antiuricemic. nerve and meniscus tissues. it would seem that the most relevant characteristics of chitins and chitosans are their biodegradability. cosmetics and items für the contrülled delivery üf drugs. and that oligomers . the use of skin substitutes.

a common amino sugar in the body which either enters the innate metabolic pathway to be incorporated into glycoproteins or is excreted as carbon dioxide [9]. Chitin sutures developed in the early 1980s undergo relatively rapid biodegradation in vivo: the resistance to traction oftheir knots falls to 74% after 5 days and 18% after 15 days.20) that is expected to last for 2 years or longer [10]. wound repair and metastasis that require massive cell movement and tissue reorganization. Overexpression ofDG42 in mouse cellS leads to the synthesis of chito-oligomers. inflammation. Methylpyrrolidinone . Many of the effects of hyaluronan are mediated through cell surface receptors. a nonsulphated glycosaminoglycan of D-glucuronic acid and N-acetylglucosamine. RHAMM and ICAM-l. and to have an important role in such processes as morphogenesis. or that the substrate is modified in situ [11]. adhesion and proliferation. is found in extracellular matrices of various connective tissues. 14]. The biodegadability is verified in many kinds of dressings and is independent of the degree of acetylation of the chitins. are further hydrolysed to N-acetylglucosamine. Muzzarelli et al. Biocompatibility The in vitro biocompatibility of wound dressings in terms of toxicity for fibroblasts has been assessed and compared with three commercial wound dressings made of collagen. particularly N -pentanoyl chitosan (degree of substitution < 0. producing chito-oligomers capable of acting as primers in the synthesis ofhyaluronan. cytokine release and stimulation of cell cycIe proteins. Hyaluronan synthesis Hyaluronan. A. Recent evidence is found for the presence of DG42 protein (a chito-oligomer synthase) during embryogenesis. intraperitoneal adhesion and intestinal strictures [12]. The hyaluronanmediated signals are transmitted. Thus chitooligomers can act as templates for hyaluronan synthesis [13. and hyaluronan synthase preparations also contain chitin synthase activities. three of which have been molecularly characterised. based on their similarity to hyaluronan: novel hyaluronanlike chitosans have therefore been developed recently [15]. These are important biochemical aspects that chitosans seem to be able to promote. at least in part.252 R. Increased knot resistance is cIaimed far N -acyl chitosans. resulting in a reduced risk of scar formation and related complications such as keloids. by activation of protein phosphorylation cascades. A. Hyaluronan has been shown to promote cell motility. In fresh wounds chitins seem to enhance its production. This last point implies that lysozyme is not the only enzyme involved in the degradation. alginate and gelatin. namely CD44.

Basically. Glutaraldehyde cross-linked chitosans are cytotoxic. Simultaneously. inflammatory cells from the surrounding tissue move towards the lesion site. however. polypropylene glycol. in contrast. the new epithelium forms and the wound is healed. and epithelial cells at the edge of the wound start filling in the area under the scab. In this chapter we focus on the complex mechanisms by which chitin and chitosans enhance the wound-healing process. and basic FGF induces fast wound c10sure [19]. 22]. The combination of basic fibroblast growth factor (FGF) and methylpyrrolidinone chitosan has an especially advantageous influence on impaired wound healing because the chitosan prevents excessive scar formation. guar gum and locust beam gum. Berscht et al. have been produced and clinically tested with good results. Several preclinical studies on chitin and chitosan biomaterials show that they are endowed with biochemical significance not encountered in cel- . resulting in the rebuilding of tissue and protection against infection [21. histology and clinical uses of chitins and chitosans in wound healing 253 chitosan and collagen were the most compatible materials [16.Biochemistry. fourfold less toxic than poly-L-Iysine. Wound healing Wound healing consists of a complex series of biochemical processes regulated by humoral factors and antiinflammatory mediators. fibroblasts appear and begin to produce collagen connective fibres that impart tensile strength to the regenerated tissue. growth factors and immunologie mediators. 17]. Thereafter. laminated with a gentamicin sulphate-impregnated polyurethane membrane. Mosbey proposes a wound-filling gel obtained from chitosan malate. the wound-healing process consists of three stages. Subsequently. Chitosan has been associated with other biopolymers and with synthetic polymer dispersions to produce wound dressings. Biosynthetic wound dressings with a drug delivery capability composed of a spongy sheet of chitosan and collagen. and most evidently with chitosan glutamate. First. sterilized in an autoc1ave [20]. The ranking of chitosan cytotoxicity was established by various laboratories to be chitosan hydrochloride > chitosan glutamate> glycol chitosan> chitosan lactate> methylpirrolidone chitosan. In the third and final phase. Chitosan hydrochloride is. Rat erythrocyte lysis was observed after 24 h of contact with soluble chitosan salts. numerous capillaries begin to form to supply the site with nutrients and oxygen. whose influence can be decisive particularly during the early phase oftissue rebuilding [23]. we will discuss the main medical applications of these versatile polysaccharides. methylpyrrolidinone chitosan caused only 35% inhibition [18]. Regulating factors inc1ude biochemical substances. [16] observed that after 72 h ofincubation with fibroblasts the chitosan salts inhibited cell growth by 70-80%.

Moreover. Polykaryocytes appear in granulation tissue in the early days of implantation of spongelike chitin in animals. lulose. Macrophage activation Macrophage activation is probably the main effect of the administration of chitin-based dressings. Ca. the healing process is faster and smooth scars are obtained. Zn. In general. There may be a elose relationship between the disappearance of granulation tissue and the appearance of polykaryocytes. More details about the complex process by which chitin and chitosan stimulate a variety of cells. This mechanism involves. Chitosan also has an in vivo stimulatory effect on both macrophage NO production and chemiotaxis [31-33). Key factors in the rebuilding of physiologically effective tissues exerted by chitosans are enhanced vascularisation and a continuous supply of chito-oligomers to the wound that stimulate correct deposition. Cytokine production by macrophages It is weIl known that granulation is accelerated by IL-l. In particular. chitin-based products provide improved healing of surgical wounds by first intention in all cases. The chito-oligomers are released by hydrolytic action of lysozyme and N-acetyl-ß-D-glucosaminidase. tumour necrosis factor-a (TNFa) or FGF [35]. Ag and pectin) that ensures that exudate is removed from the contact layer before it becomes saturated. Chitosan activates macrophages for tumoricidal activity and for the production of interleukin-l (IL-l). NO production plays a major role in diverse physiological functions and pathological conditions. A. They can be considered a primer on which anormal tissue architecture is organised. assembly and orientation of collagen fibrils. derived from macrophages. Muzzarelli et al.254 R. IL-l and TNFa produced by . and disappear within 4 weeks after surgery. are given below. at least in part. Many studies have been performed into the mechanisms of improved healing by chitins [27]. the production ofinterferon-y(IFN-y) [30). A. Macrophages obtained from laboratory animals are activated by chemically modified chitin [28-29]. starch and other polysaccharides [24]. These cells. and an extemallayer (alginate salts. According to Qin and Gilding [26] a wound dressing should inelude a contact layer (carboxymethyl chitosan or its Ag salts) that assists healing. chitosan shows immunopotentiating activity [29]. and favour the release of various proteins and regulatory factors involved in wound healing. Chitosan-based dressings also show the capacity to modulate peroxide production [34]. and are incorporated into the extracellular matrix components [25]. These data and the absence ofpolykaryocytes in controllesions suggest that chitin induces the formation of polykaryocytes. possess a yet unknown higher level of function.

Biochemistry. Peritoneal macrophages obtained from laboratory animals are activated by chitin [28. Chitinlchitosan items administered intravenously to mice become bound to macrophage plasma membrane mannose/glucose receptors that mediate their interiorisation. These responses may be common with microbial particulate components. oligosaccharides are unable to prime macrophages. 29]. The amount of IL-I in the exudate taken from around the areas where chitin nonwoven fabrics were implanted showed a twofold increase in comparison with that of control medium [37]. The mechanism for macrophage priming by the chitin particles involves the production of endogenous IFN y by NKl. even after oral administration. a higher level of priming is induced by chitin [30]. IL-8 is a potent neutrophil chemotaxin [38]. and accumulates in the articular tissue and bone [43]. It has been administered to human volunteers by intravenous. but chitosan heparin complex in vivo may be responsible for accelerated wound healing. In vivo histological studies in animal models also highly support the fact that the chitosan fibres stimulate migration ofthese cells [39-41].l + CHi cells. macrophages give a large oxidative burst when elicited with phorbol myristate acetate. generated by increased neutrophils in the lung. Within 3 days.1 +. They do not directly stimulate the production of IL-l. Morphological examination performed at the site of lesion treated with chitin shows an increase in vascularisation during wound healing [28]. Cytokine praduction by jibrablasts Chitins and chitosans do not enhance the rate of fibroblast proliferation in vitra. which leads to angiogenesis and migration of neutrophils. rather they induce IL-8 release from fibroblasts in vivo. intramuscular and oral routes for pharmacokinetic studies [42]. IL-6 and TNF a by fibroblasts. N-Acetylglucosamine is itself an antiinflammatory drug and is synthesised in the human body from glucose and incorporated into glycosaminoglycans and glycoproteins. This is further demonstrated by the respiratory distress syndrome produced by subcutaneous injection of high doses of chitosan in dogs. In mice preconditioned with a small dose of IFN y. especially macrophages to NK1. N-Acetylglucosamine diffuses very rapidly in most tissues and organs. This production is due to cell-cell interactions. histology and clinical uses of chitins and chitosans in wound healing 255 macrophages [36] are known to activate fibroblasts. They are then degraded enzymatically. Antiinflammatory action and angiogenesis stimulation Chitin accelerates the first phase of wound healing where inflammation is accompanied with infiltration of mononuclear and polymorphonuclear cells without any uncomfortable side effects such as high temperature and dolour. The .

The observed inflammatory accumulation at the site of contact with chitin sponge suggests that the excess collagen may be degraded by these chitin-induced inflammatory cells. epidermisation is observed with a minimum of convalescence. suggesting that N-acetylglucosamine may serve as a sub stratum for reinforcement of wounded tissues without excessive inflammatory reaction [44]. newly formed granulation tissue around chitin-nonwoven fabrie composite implants is actively invaded with new blood vessels. neutrophils and fibroblasts [46].256 R. and minimum scar formation remains in the wound. In contrast. Fibroblast proliferation increases at the site in contact with chitin sponge. Biomedical applications Leg ulcers The efficay of chitosan in the treatment of leg ulcers sterns from its antiinflammatory action and stimulation of epithelialization. litde histiocyte invasion and thick collagen fibres were observed in the controls. Chitins can induce healthy granulating tissue in the early stage of wound healing. In chitosan treatment of purulent digit disease. and might promote the proliferation of fibroblasts which produce fine collagen. Granulation and scar formation Granulating tissue can generally be divided into healthy and unhealthy granulating tissue. macrophages [45]. Several reports document an acceleration of tensile strength without an increase in collagen synthesis in the presence of chitin. the effects of chitin on the mechanism of formation of granulating tissue are still unclear. a phenomenon not observed in control experiments [37]. Evidence has been collected that chitin prornotes keratin production and facilitates rapid and effective regeneration of oral mucosa. Chitosan is superior to chitin in its effect on the acceleration of granulation tissue. Chitosans stimu- . recent studies show that in chitin-treated lesions many histiocytes are present and that fine collagen fibres are produced. epidermal cells. The healthy one develops only in the absence of foreign bodies (bacteria. Mild fibroblast activation is observed in the area close to the chitosan implant in experimental animals. A. Scar formation depends on the continued synthesis and catabolism of collagen. and is a serious problem in the wound-healing process. but synthesised collagen will be degraded to an appropriate amount in the subsequent healing stages by a variety of collagenases from granulocytes. Muzzarelli et al. without antibiotic administration [47]. A. The presence of histiocytes might be induced by chitin. however. debris). Therefore it is concluded that in chitin-treated wounds synthesis of collagen will accelerate in the early wound healing stage. However.

The treatment with methylpirrolidinone chitosan lasted for 14 months and led to complete healing [48]. aged 84. (C) complete healing. . Patient L. late the granulation process and epidermis formation. August 1995. (A) U1cer.. thus accelerating healing. the case ofL. Among the many patients treated so far.A.. (B) u1cer medicated with freeze-dried methylpirrolidinone chitosan sponge.A. August 1995.Biochemistry. aged 84. suffering from chronic venous insufficiency is reported in Figure 1. histology and c1inical uses of chitins and chitosans in wound healing 257 A B C Figure 1. October 1996.

chitosan and chondroitin 4. The differentiation processes inc1ude the expression offilaggrin and keratin and the formation of a structure similar to the basal lamina. A typical example is a skin substitute made of type I and type III collagen. and complete healing was obtained [49]. Selective preparative conditions generated asolid gel useful for this application. whieh is usually extremely difficult. Muzzarelli et al. This skin substitute inc1udes human fibroblasts on the "dermal side" and keratinocytes on the "epidermal" side. resulting in an organised matrix and limited formation of granulation and hypertrophie scar [9. which is known to take place in alginate gels and other encapsulation devices. once exposed to air during the last stage of culture. Cultured skin from human cells is extremely thin and needs mechanical support that can conveniently be provided by biopolymer complexes. For application in neurosurgery. A. the PCl2 cells attached successfully to the precipitated chitosan and respond to exposure to nerve growth factor (NGF) by extending neuritis. in which the epidermal layers inc1uding the stratum comeum are present. 20% chitosan and 8% glucosaminoglycan). The dermal substitute does not cause any antigenie incompatibility and allows controlled vascularisation and fibroblastic colonisation. macrocapsules and hollow fibres made of polyacrylonitrilepolyvinyl chloride (PAN-PVC) were filled with PC12 cell suspension in chitosan solution. Differentiation of neuronal cells was also supported by the chitosan matrix. The latter. The addition of chitosan also increases the cytotoxicity levels. The chitosan prevents extensive cell c1umping and necrosis. and provides good adhesion (better than collagen alone). Chitosan is indispensable for skin substitutes not only to provide insolubility but also to increase production of collagen and regulatory factors by fibroblasts. Results of an experimental . Nerve regeneration Thanks to its expanded structure chitosan is suitable as a matrix for anchorage-dependent mammalian cell encapsulation [52].and 6-sulphate (72% collagen. When microencapsulated with chitosan. Meniscus regeneration Chitosan has also been used to assist the spontaneous tissue repair of the meniscus. generates a skin similar to the natural one. The treatment periods were 63 -182 days. 51]. A.258 R. The cross-linking between the chitosan primary amine and the sulphate groups is essential in imparting insolubility and mechanieal resistance. 50. Skin substitutes Reacetylated chitin gels are also used to treat leg and decubitus ulcers in paraplegie subjects [25]. without proliferation problems.

The existence of osteoprogenitor (bone-producing) cells in the wound site offers the possibility of regenerating the periodontal. The polysaccharide is depolymerised by lysozyme and is no longer detectable 6 months after surgery. histology and clinical uses of chitins and chitosans in wound healing 259 study indicate that the natural polysaccharide is weH tolerated at the articular synovial level. which do not take place spontaneously in the meniscus. gave rise to intramembranous bone formation. Experimental studies on rabbit and sheep were performed in order to evaluate the possibility of improving bone tissue reconstitution with chitosan associated with calcium phosphate. Biodegradation of chitosan leads to controlled release of BMp. . The cationic nature and chelating ability ofthe methylpyrrolidinone chitosan apparently favoured mineralisation. In controllesions. Osteoinduction was also observed in rabbit endochondral bones [61]. Regeneration 0/ bone tissue Several studies dealing with reconstruction of periodontal tissue with chitosan were aprelude to the discovery ofthe osteoinductive properties of chitosan [54. Ultrastructural examination showed that bone osteoid formation was foHowed by mineralisation. It has been found that the number of bone-forming colonies is almost doubled in the presence of chitosan. Methylpyrrolidinone chitosan was found to be useful in apicectomy as well. which promoted bone regeneration. The pattern of bone regeneration has been studied in an osteoporotic experimental model with bone morphogenetic protein (BMP) linked to chitosan. Histological examination performed 60 days after surgery showed a considerable presence ofneoformed bone tissue as compared with control. stimulated by growth factor entrapped in the coagulum-polysaccharide mixture. periimplant and alveolar ridge bone tissue simply with the aid of chemical mediators from chitosan. None ofthe patients reported adverse effects over 3 years of observation [56]. Microscopic and histological analyses showed the presence of an osteogenic reaction moving from the rim of the surgical lesion towards the centre. Chitosan also stimulates the differentiation of osteoprogenitor cells and thereby facilitates the formation ofbone[57]. It also favours and stimulates the repair processes. dense fibrous tissue without the characteristic histoarchitecture of bone was observed. Surgical wounds from wisdom tooth avulsions were treated with freeze-dried methylpyrrolidinone chitosan. 58-60]. Bone defects surgicaHy produced in sheep and rabbit models have been treated with freeze-dried imidazolyl chitosan and methylpyrrolidinone chitosan [56.Biochemistry. 55]. Endostealperiosteal and bone marrow osteoblast-like precursors. Its initial angiogenic action appears to be effective enough to stimulate the repair ofthe meniscus by providing the latter with the necessary tissue elements and humoral factors [53].

2 ± 12. Thus. and the composition should have an acidic pH to enhance stability of the drug. timolol maleate. It seems that glucosamine 6-phosphate synthesis is the rate-controlling step. Ophthalmology N.509 to 0. Muzzarelli et al.001 ± 0.260 R.O-Carboxymethylchitosan is suitable as an excipient in ophthalmic formulations to improve the retention and bioavailability of drugs such as pilocarpine.875 ± 0. and are among the extracellular signals that can induce the osteoblastic phenotype. that is reduction of serum haemoglobin value. enabling it to enter the pathway of glycosaminoglycan synthesis [63]. neomycin sulphate and ephedrine. A. Therefore. BMPs are members of the transfonning growth factor ß (TGF ß) superfamily. Most of the drugs are sensitive to pH.1 to 68. Exogenous glucosamine can be transported into the cell and acted on by a kinase which phosphorylates it at the 6 position. Human erythropoietin is used for treatment of patients with anaemia nephrotic syndrome undergoing haemodialysis. administration of glucosamine as wen as chitosan (to be viewed as a delayed-release agent) in an possible ways is beneficial for wound healing [64]. the delivery should be made through an anion exchange resin that adjusts the pH at around 7. Autoc1aving the carboxymethylchitosan (CMC) . especially when rebuilding extra cellular matrix is necessary. Effects of oral delivery on wound healing and renal functionality Exogenous glucosamine is readily incorporated into hyaluronan by cultured fibroblasts. The nephrotic syndrome is often associated with hypercholesterolemy and anaemia. which is physiologically acceptable. Enhanced availability of exogenous glucosamine is beneficial for the synthesis of hyaluronan because endogenous glucosamine synthesis is insufficient to achieve the high concentration of UDP-N-acetyl glucosamine necessary to optimize synthase activity.0 g/l). but its administration must continue long tenn with the risk of many side effects. providing a synerglstlc effect on bone fonnation. which is difficult to eure. A. This important result also proves the validity of a biochemie al approach to the therapeutical correction ofvarious affiictions in the elderly [62]. as weil as reduced cholesterollevels [65]. Morphometric and morphological analyses show that bone tissue regeneration in a surgical bone defect is improved using this special chitosan. The oral administration of chitosan (for 12 weeks) to patients with chronical renal failure produced elevation of the serum haemoglobin level (from 58.227 m/I). Other expression ofimprovement of renal function were the reduced serum levels ofurea (from 75 ± 51 to 45 ± 19) and creatinine (from 1.1 ± 9.

The presence of mannitol. The resulting chito-oligomers stimulate various cells. Matsuhashi A.31 6 MaekawaA. Minami S. Shigemasa Y (1993) Enzymatic degradation of chitin and chitosan.PF34). histology and clinical uses of chitins and chitosans in wound healing 261 improves the ocular retention characteristics [66]. Asano T. Gooday GW (eds) (1986) Chitin in nature and teehnology. Boca Raton. Domard A (1995) Procede pour prolonger la duree de bioresorption d'un materiau implantable dans I'organisme a base de chitine ou de chitosane et materiau obtenu. This chapter was prepared under the auspices of the Italian National Research Council. Leger B. offering truly valuable medical items in the field of general medication.421 8 Sashiwa H.98. Saito K. Clin Mater 15: 259-265 10 Houard W. while the released monomers are phosphorylated and incorporated into hyaluronan. Bourgeois M. pore size and mechanical properties of a biomaterial designed for deep bum coverage. In: Salamone JC (ed): The polymerie materials eneyclopedia. keratan sulphate and chondroitin sulphate. Jeuniaux C. CRC Press. Japan Soe. and to Maria Wecla. plastic surgery. for providing experimental data and photographs.280. Roma (contract no. Damour 0 (1994) üptimization of thickness. drug carriers and immunostimulators. Grottammare 9 Berthod F. Conclusions The biological significance of chitosan biomaterials in the human body depends largely on the actions that certain hydrolases exert on them [68]. JP 03. Saintigny G. Plenum Press. Chretien F. components of the intracellular matrix and connective tissue. Chitosan solutions do not lend themselves to thermal sterilisation. for assistance in retrieving the bibliographie material. Marcel Dekker. Sapporo 5 Wada M (1995) Effect of chitin. Gekkan Fudo Kemikaru 11: 25 . FR Pat Appl 2 736 552 . Studies performed over the last few years have directed chitins and chitosans to the forefront of applied medical research. S. Hayek D. New York 2 Muzzarelli RAA (1993) In vivo biochemical significance of chitin-based medical items. Okamoto Y. Azuma I (eds) (1992) In: Chitin derivatives in life scienees. Mancini. Aeknowledgments The authors are grateful to Prof. Collombel C. chitosan intake on metabolism ofuric acid.00032. however. This preparation is particularly useful for pharmaceutical and ophthalmie compositions [67]. ltaly. JP 02. Mizuochi K (1989) Pharmaceuticals and food containing edible fibers of chitosan for hyperuricemia control. NewYork 3 Muzzarelli RAA (1996) Wound dressing materials.Biochemistry. In: RAA Muzzarelli (ed): Chitin enzymology (1993) 177-186. Wada M (1990) Food containing chitin or its derivatives for reduction ofblood and urine uric acid. Saimoto H. References I Muzzarelli RAA. Chitin.311. can be sterilised at 121°C for 15 min and then treated with sterile solution of a suitable acid (HCI). FL 4 Tokura S. Chatelin R.852 7 Mita N. University of Siena. wound healing. A chitosan suspension. sorbitol and glycerol seem to stabilise the sterilised solutions. Atec. In: S Dumitriu (ed): Polymerie biomaterials.

187-196 26 Qin Y. Kreuter J (1995) In vitro evaluation ofbiocompatibility of different wound dressing materials. Roberts GAF. Sagar BF (1998) Hydrogen peroxide generation by fungal/chitosans and their effects on the proliferation of murine L929 fibroblasts in culture. Petillo 0. Atec. Biotechnol Genetic Engin Rev 13: 383-420 25 Muzzarelli RAA. Muzzarelli et al.356. Myrvik QN (1997) Alveolar macrophage priming by intravenous administration of chitin particles. Azuma 1(1984) Immunological activity of chitin aud its derivatives. Saiki I. J Immunoll42: 1281-1286 36 Chang J. Infect Immun 65: 1734-1741 31 Peluso G. Sautin M. Jacques Andre. novel hyaluronan-like regiospecifically carboxylated chitin. Lyon 28 Nishimura K. Forsch CS. J Biol Chem 267: 21277-21280 24 Shigemasa Y. Biagini G (1993) Role aud fate of exogenous chitosaus in human wound tissues. Tanaka Y. Tokura S (1992) Application of chitin aud chitosau in small auimals. Potential role for nitric oxide in the regulation ofwound healing. 11 Muzzarelli RAA (1993) Biochemical significauce of exogenous chitins an chitosans in animals and patients. Eur Pat Appl 0. Tokura S. Robbins PW (1997) An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish. polymers of N-acetyl-D-glucosamine. Vaccine 2: 93-99 29 Nishimura K. Carbohydr Polym 19: 29-34 18 Carreno-Gomez B. Stroband H. Calabro D. Nies B. Avallone B. Int J Pharm 148: 231-240 19 Berscht PC. Ottemess IG. Matsuhashi A. Lewis VL (1998) Immunologic associations ofkeloids. Carbohydr Polym 20: 7 -16 12 McCarthy MF (1996) Glucosamine for wound healing. Proc NatlAcad Sei USA 93: 4523-4525 14 Bakkers J. Laskin DL.060 A2 21 Clark RAF (ed) (1996) The molecular and cellular biology ofwound repair. Liebendorfer A.262 R. in mice. Gilding KD (1995) Wound dressing. Cosani A. WO 96/13282 27 Domard A. In: CJ . Hamlyn PF. J Immunol136: 1283-1287 37 Okamoto Y. Med Hypoth 47: 273-275 13 Varki A (1996) Does DG42 synthesize hyaluronau or chitin? A controversy about oligosaccharides in vertebrate development. Nishi N. J Mat Sei Mater Med 6: 201-205 17 Muzzarelli RAA (1992) Depolymerization of methyl pyrrolidinone chitosan by lysozyme. A. In: RAA Muzzarelli (ed): Chitin enzymology. Nishimura S. A. Biomaterials 15: 583-600 20 Mosbey DT (1989) Wound filling compositions. J Biol Chem 268: 12231-12234 33 Wu KK (1995) Tnducible cyc100xygenase aud nitric oxide synthase. Kune JW. Terbojevich M (1999) 6-0xychitin. Carbohyd Polym 39: 361-367 16 Berscht PC. Surg Gynecol Obstet 175: 185-193 23 Heck DE. Matsuhashi A. Liebendorfer A. Adv Pharmacol 33: 179-207 34 Chung LY. Proc Nut! Acad Sei USA 94: 7982-7986 15 Muzzarelli RAA. Nies B. Varum KM (eds) (1997) Advances in chitin seience. Minami S. Duncau R (1997) Evaluation of the biological properties of soluble chitosan aud chitosau microspheres. Semino CE. Grottammare. Kunkel SL (1989) Monokine production by hypersensitive (Schistosoma mansoni EGG) and foreign body (Sephadex bead)-type granuloma macrophages. Schmidt RJ. Biomaterials 15: 12151220 32 Marletta MA (1993) Nitric oxide synthase structure aud mechanism. Shigemasa Y. Kreuter J (1994) Incorporation of basic fibroblast growth factor into methylpyrrolidinone chitosan fleeces aud determination of the in vitro release characteristics. Balsamo G (1994) Chitosau-mediated stimulation of macrophage function. Tokura S. Ranieri M. Tauigawa T. Gardner CR. Lawis AJ (1986) Interleukin I activates phosphate A z in rabbit chondrocytes: a possible signal forIL-I action. Higashi GI. Saimoto H. Minami S (1996) Applications of chitin and chitosan for biomaterials. Gilmau SC. Laskin JD (1992) Epidermal growth factor suppresses nitric oxide aud hydrogen peroxide production by keratinocytes. Foster La Metzger WY. Nishimura K. Nishi N. Azuma 1(1986) Bioactive chitin derivatives: activation of mouse peritoneal macrophages by O-carboxymethyl chitins. J Biomed Mat Res 39: 300-307 35 Chensue Sw. Muzzarelli C. NewYork 22 Placik OJ. Ambrosio L. Carbohydr Res 146:251-258 30 Shibata Y. Plenum Press.

NewYork. Biomaterials 14: 39-43 Klokkevold PR. Carbohydr Polym 8: 1-21 Ross GD. 70-78 Mori T. Ferioli G. Tournebise H (1995) Pansement pour plaies chroniques. Sparacca G. J Immunol 134: 3307-3315 Williams JD. PA Sandford.Biochemistry. Ueno K. Kennedy EB. Mattioli-Belmonte M. Tietz C. Biagini G. Simonelli C. Biomaterials 10: 598-603 Roussille G. Chretien F. Tokura S. Biomaterials 15: 1049-1056 Muzzarelli RAA. UTET. Biomaterials 15: 1075-1081 Muzzarelli RAA. Bemard GW (1996) Osteogenesis enhanced by chitosan (poly-N-acetyl glucosaminoglycan) in vitra. Biomaterials 14: 925-929 . Muzzarelli RAA (1998) Guarigione delle ulcere con metilpirrolidinone chitosano. Une T. New York Iida J. Saintigny G. Bicchiega V. Acta Derm Venereol73: 175-180 Zielinski BA. Mitzukoshi N. Biagini G. Biagini R. histology and clinical uses of chitins and chitosans in wound healing 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 263 Brine. Grandmontagne B. Med Hypoth 42: 323-327 Yano H. Mattioli-Belmonte M. Giardino R (1995) Osteoinduction in the presence of chitosancoated porous hydroxyapatite. Krajewski A. Zanolo G (1993) Phannacokinetics of glucosamine in man. Pugnaloni A. In: RAA Muzzarelli.a personal perspective. Castaldini C. Fratto G (1993) Osteoconduction exerted by N-methylpyrrolidinone chitosan used in dental surgery. Pugnaloni A. Berthin-Maghit M. Rizzoli C (1989) Reconstruction ofparodontal tissue with chitosan. l1ari P. Iriyama K. Tokura S. l1ari P. Lachmann PJ (1985) Membrane complement receptor type three (CR 3 ) has lectin-like properties analogous to bovine conglutinin and functions as a receptor for zymosan and rabbit erythrocytes as weH as a receptor for iCb. Plenum Press. Azuma I (1987) Stimulation of nonspecific host resistance against Sendai virus and Escherichia coli infection by chitin derivatives in mice. J Bioact Compat Polym 7: 130-148 Muzzarelli RAA. GW Gooday (eds): Chitin in nature and technology. en gel de chitine. Gordon S (1975) Secretion of a specific collagenase by stimulated macrophages. Biomaterials 18: 947-951 Muzzarelli RAA (1988) Carboxymethyl chitins and chitosans. notamment escarres. Bonnard M. Muzzarelli RAA. Mzccine 5: 270-274 Mancini S. Vandemark L. Filippini 0. Topley N. Barthet B (1991) Evaluation of a collagen-glycosaminoglycan complex as a dressing for gingival wounds. Giardino R. Ravaglioli A. C Jeuniaux. Baldassarre V. Immunology 58: 117-124 Setnikar I. Palumbo R. Okumura M. Damour 0. Alobaidi HM. Biagini G (1994) Stimulatory effect on bone formation exerted by a modified chitosan. Quigley HJ. FR 2 736 835 Damour 0. Castaldini C. Harber MJ (1986) Activation of human polymorphonuclear leucocytes by particulate zymosan is related to both its major carbohydrate components: glucan and mannan. Biagini G. Mie Med J 35: 53-56 Werb Z. Ishihara C. Aebischer P (1994) Chitosan as a matrix for mammalian cell encapsulation. Cain JA. Bellardini M. Berthod F (1994) A dermal substrate made of collagen-GAG chitosan for deep burn coverage: first clinical uses. Brunelli MA. Elsevier.. Arzneim Forsh Drug Res 43: 1109-1113 McCarty MF (1994) The neglet of glucosamine as a treatment for osteoarthritis . Castaldini C. Gandolfi MG. Collombel C (1993) Reconstruction of epidermis on a chitosan cross-linked collagen-GAG lattice: effect offibroblasts. Okamoto Y. Castaldini C (1993) Osteoconduction properties ofmethylpyrrolidinone chitosan in animal model. Fini M. Torino Domard A. Minami S. Matsuura M. Adickes ED (1986) Chitosan effect in vascular surgery. Fini M. J Bioact Compat Polym 10: 249-257 Muzzarelli RAA. Canali R. Gueugniard PY. In: S Mancini (ed): Irattato di Flebologia. Pugnaloni A. Clinical Mater 15: 273-276 Saintigny G. Karibian T. Zucchini C. tissue culture and tissue regeneration. Biagini G. Nishiwaki H. Biagini G. JP Zikakis (eds): Advances in chitin and chitosan. Kifune K (1985) Effects on N-acetyl-D-glucosamine on wound healing in rats. Rizzoli R (1992) Role ofN-carboxybutyl chitosan in the repair ofthe meniscus. J Mat Sei Mater Med 2: 208-211 Muzzarelli RAA. J Periodontol 67: 1170-1177 Mattioli-Belmonte M. Fujinaga T (1997) Effects of chitin and its derivatives on proliferation and cytokine production of fibroblasts in vitra. J Exp Med 142: 346-360 Malette WG.

264

R.A.A. Muzzarelli et al.

61 Borah C, Scott G, Wortham K (1992) Bone induction by chitosan in endochondral bones of
the extremities. In: C Brine, JP Zikakis, P Sandford (eds): Advanees in ehitin and ehitosan.
Elsevier, Amsterdam, 324-343
62 Muzzarelli RAA, Biagini G, Mattioli-Be1monte M, Talassi 0, Gandolfi MG, Solmi R,
Carraro S, Giardino R, Fini M, Nicoli-Aldini N (1997) Osteoconduction by chitosan-complexed BMP: morpho-structural responses in an osteoporotic model. J Bioaet Comp Polym
12: 321-329
63 Karze1 K, Domenjoz R (1971) Effects ofhexosamine derivates and uronic acid derivatives
on glicosaminoglycan metabolism offibroblast cultures. Pharmaeol5: 337-345
64 McCarthy MF (1996) Glucosamine for wound healing. Med Hypoth 47: 273-275
65 Jing SB, Ji D, Takiguchi Y, Yamaguchi T (1997) Effect of chitosan on renal function in
patients with chronic renal failure. J Pharm Pharmaeol 49: 721-723
66 Reed KW, Yen SF (1995) Compositions inc1uding O-carboxyalkyl chitosan and methods of
use in ophthalmics. WO 97/06782
67 Yen SF, Sou M (1997) Stabilized chitin derivative compounds. WO 97/07139
68 Muzzarelli RAA (1997) Human enzymatic activities related to the therapeutical administration ofchitin derivatives. Cell Biol Life Sei 53: 131-141

Chitin and Chitinases
ed. by P. Jolles and R.A.A. Muzzarelli
© 1999 Birkhäuser Verlag BasellSwitzerland

Veterinary practice with chitin and chitosan
Saburo Minamit, Yoshiharu Okamoto 1, Koji Hamada2, Yukio Fukumot0 3,
Yoshihiro Shigemasa4
1

2
3
4

Department olVeterinary Surgery, Faculty 01Agriculture, Tottori University, 4-101 KoyamaMinami, Tottori 680-8553, Japan.
OmuAgricultural MutualAidAssociation, Ohmu, Monbetsu 098-1702, Japan.
Asa Zoological Park, Asa-Kita, Hiroshima 731-3355, Japan.
Department 01 Materials Science, Faculty 01Engineering, Tottori University,
4-101 Koyama-Minami, Tottori 680-8552, Japan.

Summary. Dramatic effects of chitin and chitosan on wmmd healing were demonstrated in field
cases ofmany small animals (dogs and cats), food animals (338 cows) and 142 zoo animals. In
comparison with conventional therapy with irrigation and antibiotic administration to wound,
new treatment with chitin and chitosan permitted a substantial decrease in treatment frequency
with minimum scar formation.

Introduction

In recent years, much attention has been paid to various biological dressings derived from natural products due to their high compatibility with
various organisms. Since 1985, we have been developing chitin and chitosan products for veterinary use, and we have previously reported their
clinical effects on wound healing [1-5], their experimental effects on
wound healing [6-8] and their biological activities [9-24]. The biological
activities of these materials can be summarized as follows: complement
activation [23], polymorphonuclear cell (PMN) activation [15-17, 19,21,
22,24], fibroblast activation [9, 13, 14,20], cytokine production [13, 20],
giant cell migration [6, 11] and stimulation oftype IV collagen synthesis
[18]. Complement activation appeared to be induced by high-dose (200 mgl
kg) administration of chitosan to canine subcutaneous tissue. The dogs
administered chitosan experienced unexpected hemorrhagic pneumonia,
and 70% died within the first 7 days after administration [17]. The pneumonia was induced by the production of C5a, a well-known strong
chemotactic factor for PMN, that was itself induced by the complement
activation by chitosan. Both chitosan and chitin activate complement via an
alternative pathway [23]. The systemic activations induced by 1 mg/kg of
subcutaneous administration of chitosan or 1 mg/kg ofvenous administration of chitin can also be explained by the corresponding systemic
complement activation by these materials [22]. Furthermore, local accumulation of PMN caused by administration of chitosan or chitin is also
attributable to the local complement activation by these materials. On the

266

S. Minami et al.

other hand, fibroblasts were shown to produce interleukin 8 (lL-8) by
stimulation of chitin or chitosan [20]. IL-8 is also a fairly strong chemotactic factor for PMN.
We [19] demonstrated that chitin promoted the migration ofPMN more
strongly than chitosan, and the migration ofPMN corresponded to the concentration of chitin and chitosan. This phenomenon means that chitin and
chitosan are direct chemotactic factors for PMN. Furthermore, D-glucosamine and chito-oligosaccharides have much stronger chemotacticity than
chitosan. On the other hand, N-acetylchitooligosaccharides (GlcNAc)
whose degree of polymerization is below 5 are not a chemotactic factor for
PMN. GlcNAc6 showed chemotactic effect on PMN. Chitin and chitosan
are biodegraded in body tissue [10-12], so the chemotactic ability of
chitin would also rapidly disappear consequent to biodegradation. We have
previously investigated prostaglandin E 2 (PGE 2 ) production by these
materials in vivo and in vitro [24], and found a high concentration ofPGE 2
in exudate produced from chitin-implanted subcutaneous tissue. Furthermore, PMNs incubated with chitin or chitosan produced PGE2 [38]. These
data suggest that extended angiogenesis will be induced by the local increase ofPGE 2 level caused by migration ofPMNs. We also observed that
many giant cells appeared in the chitin- or chitosan-implanted areas, and
that large and marginal amounts, respectively, of type IV collagen and type I
collagen were synthesized in the chitin-implanted area [18]. These results
suggested that minimum scar formation would be induced by strong biodegradation of migrated giant cells and acce1eration of type IV collagen
synthesis. In the present chapter, we focus on the c1inical effects of chitin
and chitosan used in surgical treatment of various animal diseases.
Preparation of biomaterials

Chitin products
Chitipack S (Fig. 1): Squid pen ß-chitin (Nipp on Suisan Co., Ltd., Tokyo)
purified from Neon flying squid (Ommastrephes bartrami) was 9%
deacetylated; it had an average molecular weight of over 100,000 Da. A
1.5% (w/v) dispersion of chitin in water was frozen at - 20°C and then
freeze-dried for 24 h, resulting in a spongelike chitin product [25]. The
Chitipack S was a pure chitin product and consisted of 200 mg of ß-chitin
[commercialized in Japan by Eisai Co., Ltd. (Tokyo) since 1994].
Chitipack P (Fig. 2): Squid pen chitin was disaggregated (dispersed and
swollen) in water at 40°C or lower by use of a homogenizer or a mixer. The
mixture was poured into a predetermined amount of water to achieve
0.5-4.0 gl of chitin. The chitin suspension was poured onto a reinforcing
material (a nonwoven fabric of polyethyleneterephtalate (Du Pont Co.,
Ltd., San Francisco) by use of a suction-type paper-making apparatus of

Veterinary practice with chitin and chitosan

267

Figure 1. Chitin sponge-type commercial wound remedy in Japan (Chitipack S).
Figure 2. Chitin and nonwoven fabric composite commercial remedy in Japan (Chitipack P).
Figure 3. Chitosan cotton-type commercial wound remedy in Japan (Chitopack C).

the batch style, resulting in a composite sheet having a chitin layer. The
thickness of the composite sheet could be adjusted by acting on the concentration of the starting chitin suspension. The Chitipack P contained
144 mg of ß-chitin in one sheet [commercialized in Japan by Eisai Co., Ltd.
(Tokyo) since 1994].
Chitosan product

Chitopack C (Fig. 3): Chitosan flake (Flonac C, Kyowa Tecnos CO., Ltd.,
Chiba, Japan), which was 82% deaeetylated a-ehitin from erab shells
(average moleeular weight of 80,000, maximum 1.2% ash and 5 ppm heavy
metals as Pb, Cd and As) was pulverized into several grades between 3 and
90 pm with a mill (Ube Industries, Ltd., Ube, Japan, CF-400). The eottonlike ehitosan produet obtained was eomposed offibers 2-20 mm in length,
20-50 }lm in width and 3-15 pm in thiekness, and had an apparent
specifie gravity ofO.l-0.2 g/em 3 • Chitosan was dissolved with stirring in
a mixture of water and aeetie acid, and filtered twiee by the applieation of
pressure, and then defoamed by letting the solution stand overnight. The
dope was extruded from a spinneret, having 500 capillaries (0.1 mm diam.)
into a eoagulating bath eontaining a mixed solution of ethylene glyeol, iee
and potassium hydroxide. After eoagulation, the spun threads were passed
into a mixed solution of methanol and water. The resulting threads were

268

s. Minami et al.

stretched 1.15-fo1d in air, washed with water ovemight, treated with hot
water at 70-80°C for 3-5 hand immersed in methanol ovemight. The
material was reeled with a reeling machine and dried. The resulting chitosan threads were cut to a length of 1-2 cm, treated with a mixer, and dried,
resulting in a cottonlike chitosan product [commercialized in Japan by
Eisai Co., Ltd. (Tokyo) since 1993; 100 mg of chitosan in one pack].
Application to small animal clinic
Between 1992 and 1998, there were approximately 500,000 c1inical applications of chitosan and 300,000 of chitin in Japan [4]. The methods used
and the results obtained with these materials are summarized below.
Chitipack S
Wound dressing or filling agents: Skin defect with sores, and skin problems
arising as a result of traffic accidents was covered by Chitipack S (Figs.
4- 6). Wound cavity caused by abscess, surgical tissue defect due to oncotomy, hemiorrhaphy or other operations, and various types of trauma
inc1uding bite wound was filled by Chitipack S. Before application of

Figure 4. Tail skin trauma in a cat.
Figure 5. Wound covering performed with Chitipack S.
Figure 6. 2 weeks after first treatment, evident wound contraction was observed.

Veterinary practice with chitin and chitosan

269

Chitipack S, wounds were rinsed and disinfected with saline and povidoneiodine, and thereafter Chitipack S was exchanged every 4- 5 days until the
trauma and abscess healed. Antibiotic was administered systemically in
some animals which had systemic symptoms such as fever, anorexia and so
on, but was not done locally in all cases. In about 90% of all cases, good
healing developed. In the case of injuries, including traumas and abscesses,
healthy granulation tissue formed within I week after treatment. Skin
defects subsequently reepithelialized without scar formation or any functional disturbances. In cases in which granulation tissue did not develop,
general conditions were serious, and contamination of the wounds was
severe.
ChitipackP
Artificial skin: In the treatment of large skin defects due to skin tumor
resection, in which the resected ends could not be apposed by suturing,
Chitipack P was used as an artificial skin. Chitipack P was cut to about
70% of the wound area, and placed over the wound bed (Fig. 7). The
resected ends and Chitipack P were sutured using a continuous or interrupted suture pattern. The center site of Chitipack P did not fix to the

Figure 7. Large skin defect in a dog upon resection oflarge skin tumor. Chitipack P was placed
in the wound and was sutured to surrounding skin edge.
Figure 8. Ten days after operation, quite good wound contraction was observed after removal
of Chitipack P.
Figure 9. Severe hind leg trauma in a dog with automobile accident. Large skin defect and
severe tissue damage with dislocations ofmetatarsalphalangealjoints (long arrow). Application
of Chitopack C to wound surface (small arrows).

The dog was 2 years old and weighed 25 kg. Treatment is usually complicated. and reepithelialization within 3 -1 0 days in slightly or moderately wounded cases. Figure 9 shows severe trauma in the hind leg of dog caused by an automobile accident. Chitipack P was removed. Figure 9 shows the appearance ofthe leg upon first examination. including construction of normal subcutaneous tissue and regeneration of skin. with about half of the digits and their subcutaneous tissue. formation of granulating tissue. the wound rapidly contracted. Figure 10 shows a typical wound reaction to the application of chitosan. good wound contraction by reepithelialization. tendons. wound bed because granulation tissue invaded· into Chitipack P. a similar treatment was performed until the wound could be closed by suturing. In the case of large hemias.270 s. vessels and bones damaged with contamination (three metatarsus bones were visible and were dislocated at the metatarsophalangeal joints). The dog recovered walking ability. After I week. and granulating tissue formation. Complete recovery of the wounded leg with no functional disturbance and minimum scar formation was observed on 60 days post initial treatment (Fig. In the case where it was not possible to appose the wound. such as disappearance of purulence. and the wound was covered with sterile bandages and fixed with an aluminum splint. Our own . with a slight gait in his wounded leg. Chitipack P was rolled and inserted into the hemia cavity following reduction ofhemia content. and the wound was closed by suturing. The skin of the toes was widely defected. 8). Based on the results of many cases of trauma. Minami et al. were observed (Fig. and the dorsal skin of the leg was almost regenerated (Fig. The methods used and the results obtained with these materials are summarized below. Chitopack C was applied to the wound surface. After irrigation ofthe wound with saline. the characteristic biological reactions under chitosan treatment can be considered to be complete reconstruction of body tissue. All cases showed good response. Chitipack P was used as a filling agent. 12). surgical treatment was reduced from the usual2-3 h to a mere 30 min. 11). Thirty-three days after the initial treatment. Chitopack C There were approximately 1000 clinical applications of chitosan to contaminated skin and subcutaneous tissue trauma or abscess. Very fresh vascular granulation appeared in the wound area 12 days after the first examination. and was fixed to the surrounding tissue by sutures. Hernia treatment: In reduction of perineal hemia. if possible. and within 1 month in severely wounded ones. Application to pyothorax: Pyothorax is one of the most frequently encountered diseases in felines.

fresh glanulating tissue regenerated in the wounded site. two or three injections of chitosan (3 . Figure 11 .Veterinary practice with chitin and chitosan 271 Figure 10. Application to large animal clinic There were 338 clinical applications of chitin or chitosan to bovine surgical treatment. the cat had recovered vigor and appetite. After 33 days. treatment has usually consisted of simple pleuroclysis and injection of chitosan into the pleura after irrigation with physiological saline. The methods used and the results obtained with these materials are surnmarized below. In the present study. We have treated numerous cases of pyothorax. and a complete cure was indicated by X-ray findings.8 mg) was injected with 5 ml of saline into the pleural cavity. ChitipackP Umbilical hernia: Twelve bovine umbilical hernias that appeared to require reinforcement with artificial material due to their large hernia rings were . At 1 week post injection. the wound was almost healed.7 days interval by the symptom ofrecurrence of dyspnea). Twelve days after first treatment. After 60 days. Chitopack C (0. Figure 12. wound contraction and reepithelialization were observed. all cases recovered after either one.

Chitopack C Foot diseases: Two-hundred-fifty-eight cases of foot diseases were treated using Chitopack C. In the present study. and bandaged.8 times among successful cases). The method of the typical surgical procedure is as follows: The hemia sac was completely resected from the hemia ring. Chitipack P is the most suitable reinforcing material as an implant for cattle. and the mean treatment frequency was 3.1 times (2. and the mean treatment frequency was 3. and the mean treatment frequency was 3. Chitipack P was applied over the suture site and fixed to the abdominal wall by continuous suturing. The mean treatment frequency among the 6 cases was 8. After surgical debridement and irrigation with an antiseptic agent. Chitopack C was packed on to the debrided area. 20 with abscess and 18 with arthritis. after the hemia ring was closed by suturing. the l-week retreatment period required for Chitopack C represents a substantial improvement. Antibiotics were used in cases in which there was infection in the deeper tissue or in which there were systemic disorders. Chitopack C was applied to the trauma surface. Sixty out of 63 cases were cured (95%). were treated by Chitopack C. Minami et al. and because cattle generally weigh 500800 kg. Because 2 or 3 days are required for each retreatment using ordinary antibiotic ointment. the suturing can be expected to undergo considerable stress. and this combined with the treatment's success rate makes Chitopack C a suitable material for treatment of cattle foot diseases. The 15 failures were all cases of severe purulence with progressing bone and joint diseases in the infected foot.272 S.8 times among successful cases). and 5 of them were cured by the present treatment (83%). after which the swelling gradually disappeared without side effects. covered with cotton. and following disinfection by povidone-iodine solution. that is purulence or rupturing of the suture site.7 times among successful cases). The treatment procedures were as follows: To treat foot disease. In two of these cases the disease had recurred. and then the wound was covered with . Because the umbilical suture site locates at the base of the abdomen in standing cattle. and Chitopack C was reapplied weekly. and then the hemia ring was closed by the horizontal mattress sutures. Other surgical diseases: Sixty-eight cases.2 times (2.4 times (2.9 times among successful cases). treated. 243 out of 258 cases were cured. after rinsing diseased interdigital skin and surgical debridement of necrotic tissue. Temporary swelling of the implanted site was observed within 7 days. Among all foot diseases.1 times (2. 30 with trauma. resulting in the rupture of the hemia ring of the suture site by a typical surgical procedure (simple closure by suturing without reinforcing material). One-hundred-and-eighty-three out of 195 cases each having a sole ulcer were cured (94%). The bandage was changed. Six ofthe 63 cases had an interdigital nodule.

4 with abscess.3 times (12. and the mean treatment frequency was 9. Among cases with an abscess. Cases of arthritis with severe skin defect or with bone infection were also cured. Treatment of purulent bovine diseases could thus be greatly simplified by utilizing chitosan. Wound did not heal with antibiotic ointment treatment. the wound contracted rapidly and was cured within 2 weeks (Fig.Veterinary practice with chitin and chitosan 273 Figure 13. bandages. many surgical diseases showed good response to chitosan application.3 times. Figure 14.7 times (3. Some cases were treated only by aspirating discharge using a syringe and an 18-gage needle followed by injection of Chitopack C suspension to the aspirated cavity. Two of3 cases with arthritis were cured (67%). 29 of 30 (97%) were cured. Chronic udder trauma near nipple. Among cases with trauma. Thirteen of 15 cases with periarthritis were cured (87%). As described above. and the mean treatment frequency was 5. .3 among successful cases). and 4 with arthritis. and mean treatment frequency was 3. 20 were completely cured (100%). Figure 13 shows a case of chronic trauma in the udder skin. it did not heal. Antibiotic was applied systemically to the severe purulent cases. and the mean treatment frequency was 3.5 among successful cases).4 times (5.0 among successful cases). after irrigation of the infected region Chitopack C was inserted via the vent. 11 with trauma. The trauma was completely healed with a single application of Chitopack C. For treatment of arthritis or periarthritis. 14). Abscesses were also treated by direct application of Chitopack C to the wound after irrigation of the abscess cavity following incision. After a single application of Chitopack C. Although this wound was previously treated for about 2 months by direct application of an antibiotic ointment.

or suppurative or debrided wounds were paeked with it after rinsing with disinfeetant. The effeet of ehitosan was classified as exeellent (++) in 63 of 143 animals and good (+) in 58 animals.ofcases 3 1 9 9 1 2 1 10 5 1 10 1 4 19 23 3 5 6 29 12 9 1 6 4 1 1 1 4 I I Subtotal 11 25 44 121 Aves 10 10 16 18 2 2 2 4 22 37 62 143 Reptilia Total * Data were obtained at 16 zoos and an aquarium by 34 veterinarians. It should be noted that the evaluation was classified as no better than good (+). the animals were classified into 3 classes. and Lagomorpha whieh included 10 animals or more. 18 birds and 4 reptiles) at 16 zoos and an aquarium aeross Japan. Marsupialia. Camivore. even though they were severe wounds whieh healed very quiekly. A eure rate of 95%. Studies were earried out on 143 animals (121 mammals. Wounds were not healed in the remaining 21 animals or 15%. Minami et al. the dose of Chitopaek C was not greater than 50 mg/kg of body weight.274 S. Rodentia. Primates. Classification of case animals * Class Order Mammalia Marsupialia Edentata Chiroptera Primates Camivora Proboscidea Perissodactyla Hyracoidea Artiodactyla Rodentia Lagomorpha Family 2 1 Species 4 No. 37 families and 62 speeies (Tab. The therapeutie results were obtained in six groups of Artiodaetyla. 1). When drug effeets were evaluated aeeording to the classifieation of animals. and it was applied at intervals of several days to several weeks. 23 orders (11 in Mammaria. . the highest among all Table 1. Applications in wild and zoo animal clinics Chitopack C Chitopaek C was applied to the surfaee of wounds. The eure rate for reptiles was 75% (3/4). As a rule. The overall efficaey rate was 85%. a eure was effeeted in 107 of 121 mammals or 88%. but the eure rate for birds was 61% (11/18). 10 in Aves and 2 in Reptilia). The effeets of ehitosan were classified into 4 grades ranging from exeellent (++) to poor (-).

The wound was contracted and healed with several applications of Chitopack C to the wound surface. was achieved in Primates (21/22). When the effects of chitosan were compared between suppurative and nonsuppurative wounds. 17). mammals. in our study animals tended to refrain from such aets when treated with ehitosan. most were found to have had severe general emaeiation or severe suppuration eomplieated by periostitis. When unhealed animals were examined. Chitosan was not effeetive for the treatment of infeetions with Pseudomonas aeruginosa in birds. Figures 15 -17 show the dramatic effects of wound healing in a masked palm civet with a bite wound in his neck. Although animals generally liek or bite their wounds. woundhealing acceleration was confirmed in 84% of suppurative wounds and 96% of nonsuppurative ones. Figure 17. Application ofChitopack C to the wound.Veterinary practice with chitin and chitosan 275 Figure 15. Figure 16. Most birds had severe suppuration eomplieated by periostitis or osteomyelitis or both. The wound was extremely contraeted without purulence by several Chitopack C applieations (Fig. A masked palm civet with severe bite wound on his neck. .

Minami S. Shigemasa Y (1994) Migration of canine neutrophils to chitin and chitosan. Jpn J Equine Sei 2: 65-70 2 Minami S. Tokura S (1994) Polymeric N-acetyl-D-glucosamine (chitin) induces histionic activation in dogs. Saimoto H. Okumura M. Sasaki T. Shigemasa Y. In: M Yalpani (ed): Carbohydrates and carbohydrate polymers. Shigemasa Y. Saimoto H. Matsuhashi A. Mianmi S. Okamoto Y. Fujinaga T (1997) Effects of chitin and its derivatives on the proliferation and cytokine production of fibroblasts in vitro. Saimoto H. Grottammare. Minami S. Tanigawa T. Matsuhashi A (1991) A case of canker in a draft horse. Matsuhashi A. Shigemasa Y (1997) Chitin induces type IV collagen and elastic fiber in implanted nonwoven fabric ofpolyester. Okamoto Y. Tanaka Y. Sashiwa H. Minami S. J Vet Med Sei 56: 761-762 16 Usami Y. Matsuhashi A. Okamoto Y. Org Com WVC. Tokyo. Macromol Symp 120: 11-18 13 Tanigawa T. Sashiwa H. Okamoto Y.276 S. J Vet Med Sei 57: 765-767 8 Okamoto Y. Daimaru H. References 1 Minami S. Okamoto Y. Saimoto H. Carbohydr Pol 29: 295-299 19 Usami Y. Tanaka Y. Tanigawa T. YonagoActa Medica 35: 147-150 14 Minami S. Matsuhashi A. J Vet Med Sei 55: 739-742 7 Okamoto Y. Norrdin RW. Shigemasa Y (1994) Chitin and chitosan induce migration ofbovine polymorphonuclear cells. Matsuhashi A. Shigemasa Y (1995) Evaluation of chitin and chitosan on open wound healing in dogs. Mianami S. Matsuura M. Morimoto M. Saimoto H. Kumazawa NH. Sashiwa H. Sashiwa H. Matsuhashi A. Minami S. Shigemasa Y (1996) Chitosan inducing hemorrhagic pneumonia in dogs. Sashiwa H. JVet Med Sei 56: 1215 -1216 17 Minami S. Saimoto H. Tanaka Y. Sashiwa H. MM Brzeski. Nelson AW. Shigemasa Y (1995) The fate ofN-acetylD-glucosamine (chitin) in canine subcutaneous tissues. ATL Press. Okamoto Y. Saimoto H. Tanioka S. Tanioka S. PJ Bykowski (eds): Chitin world. Tanioka S. Okamoto Y. voll. In: RAA Muzzarelli (ed): Chitin enzymology. Hoya M. Matsuhashi A. Matsuhashi A. Shigemasa Y (1997) Biodegradation of chitin with enzymes and vital components. Ikuno I. Germany. Okamoto Y. Atec. Matsuhashi A. Ikebe Y. Ueno K. Wirtschaftsverlag NW. Miyatake K. Okamoto Y. Tanaka Y (1995) Polymeric N-acetyl-D-glucosamine (chitin) induces prostaglandin E 2 in dogs. Matsuhashi A. Tanigawa T. Sashiwa H. Tatara S. Minami S. JVet Med Sei 57: 377-378 15 Usami Y. Shigemasa Y (1995) Effect of chitosan on experimental abscess with Staphylococcus aureus in dogs. Mianmi S. Shibazaki K. Kumazawa NH. Minami S. Tomita T. Takamori Y. Shigemasa Y (1993) Enzymatic degradation of chitin and chitosan. Tokura S (1994) Application of polymeric N-acetyl-D-glucosamine (chitin) to veterinary practice. Kumazawa NH. Okamoto Y. Miyatake K. J Vet Med Sei 55: 743-747 4 Minami S. Matsuhashi A. In: ZS Karnicki. Matsuhashi A. Umemura T. Minami et al. (eds): Proceedings ofWorld Veterinary Congress. Southwood L. JVet Med Sei 57: 851-854 9 Okamoto Y. 275-282 5 Fukumoto Y. Tanioka S. Suzuki T. Minami S. Tanioka S. Carbohydr Pol 32: 115-122 20 Mori T. Okamoto Y. Shigemasa Y. 177 -186 11 Okamoto Y. Stashak TS. Tokura S. Carbohydr Pol 33: 33-38 10 Sashiwa H. Miyauchi Y. Matsumoto T. Matsuhashi A. Sashiwa S. Biomaterials 15: 947-951 . A Wojtasz-Pajak. Matsuhashi A (1992) Effect of chitin on the production of interleukin-l ß in human blood monocytes. Shigemasa Y. Kitamura Y. 141-152 3 Okamoto Y. 402-407 6 Okamoto Y. Saito K. Shigemasa Y. Oh-oka M. Okamoto Y. Kato K. In: A Takeuchi et al. Shimazu M et al (1995) Significance of clinical application of chitosan in zoo and wild animals. Tanioka S. Matsuhashi A. Tanaka Y. Shigemasa Y (1993) Effects of chitosan on wound healing. Saimoto H. Shigemasa Y (1995) Wound management II: new approach with "chitin-chitosan". Okamoto Y. Minami S. Sasano S. Shigemasa Y (1997) Effect of chitin on nonwoven fabric implant in tendon healing. Carbohydr Pol 29: 241-246 18 Minami S. Kumazawa NH. Matsuhashi A. Matsuhashi A. Tanigawa T. Tomita K. Tanioka S. Seitai Zairyo 13: 112-116 12 Saimoto H. Shigemasa Y (1997) Influence of chain length ofN-acetyl-D-glucosamine residues on direct and complement-mediated chemotactic activities for canine polymorphonuc1ear cells. Mt Prospect. Minami S. Minami S.

Shigemasa Y (1997) Systemic effect of chitin by intravenous administration in dog. Okamoto Y. Suzuki H. Saimoto H.Veterinary practice with chitin and chitosan 277 21 Minami S. Shigemasa Y (1998) Chitin and chitosan activate complement via the alternative pathway. 153 -164 . Mura-e R. Minami S (1998) Effect ofN-acetyl-Dglucosamine and D-glucosamine on canine polymorphonuc1ear cells in vitro. Matsuhashi A. Minami S. Okamoto Y. Sanekata T. Carbohydr Pol 33: 285-294 23 Minami S. Kato K. Matsuhashi A. Shigemasa Y (1997) Subcutaneous injected chitosan induces systemic activation in dogs. Mt Prospect. Okamoto Y. Matsuhashi A. Okamoto Y. Okamoto Y. Fujinaga T. In: M Yalpani (ed): Carbohydrates and carbohydrate polymers. Carbohydr Pol 36: 151-155 24 Usami Y. Masuda M. Tanioka S. ATL Press. Shigemasa Y (1993) Development of chitin and chitosan biomaterials. Carbohydr Pol 33: 243-249 22 Minami S. Sashiwa H. Shigemasa Y. Carbohydr Pol 36: 137-141 25 Tanioka S. Takayama T. Tokura S. Suzuki H.

Kansai University and HRC. A.0 in general tends to stimulate the immune system.4). Introduction Despite permanent aggression by bacteria and viruses. Osaka 564-8680.3-6. mouse peritoneal macrophage activation by chitosan is at a maximum at a degree of deacetylation of 70%. 1-3) [1].and intraresidual hydrogen bonds between acetamido groups and hydroxyl groups. even though biodegradable. The biodegradability and nontoxic properties of chitin stern from the capacity of the physiological system of animals to protect from infection. Macrophages are activated to various extents by chitin derivatives. Japan Institute for the Immunological Sciences. pure chitin isolated from other supporting materials such as protein might upset the immune system due to the presence of a rigid crystalline structure established by inter.Chitin and Chitinases ed. Therefore. Intraperitoneal injection ofN-acety1chitohexaose inhibits the growth of tumor cells and pathogens on a similar level as that of lentinan. Sapporo 060. every derivative of chitin should be assayed immunologically if biomedical applications are sought. As can be seen in Table 1. Hokkaido University. Jolles and R. Chitosan amino groups are recognized by the immune system. Japan Summary. These polysaccharides activate complement in analogy to zyrnosan. Accumulation of carboxyrnethyl chitin takes place in granulocytes and macrophages. Deacetylated chitin (30% deacetylation) and chitin sulfate stimulate the production of circulating antibodies. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Immunological aspects of chitin and chitin derivatives administered to animals Seiichi Tokura 1. Chitin derivatives. Suita. by P. Chitin did . Imino derivatives of chitosan mayaiso stimulate the immune system because chemie als bond uncommon to animal physiology. might stimulate the immune system when they carry unfamiliar chemical groups. an amino group with pK a higher than 9. However. and the resulting hydro lysis products become involved in normal metabolie cyc1es. Chitosans and carboxymethyl chitin ofvarious degrees of deacetylation or substitution were used to investigate the activation ofmouse peritoneal macrophages (Tabs. Lysozyme breaks down the bacterial cell wall. Therefore. Hiroshi Tamura 1 and Ichiro Azuma 2 1 2 Faculty ofEngineering. every derivative of chitin should be tested immunologically before its application as a biomedical material. Residual amino groups in chitosan also look unfamiliar to the immunological system despite the low pK a value for the amino group of chitosan (pK a 6.A. the enzymatic and immunologie al defense systems protect the animal body from infection.

CM-chitin promoted macrophage activation to the same extent as 70% deacetylated chitin.0 3.6 ±2.7· 61. 44.) % stasis (mean ± s. N. a Table 2.7 ± 5.5 ± 4.2 ±2.9 55.6 ±4.2 c 51.2· -5 -10 -15 98.9 C 48.3 c 78.) % stasis b (mean ± s.280 S.0±7.4±4.0 ± 6.D.1· 97. Significant difference from the control by Student's t-test (p < 0.9 ± 1.0 c 98. and hydroxyethyl-chitin was next.p.8 66.0±4.0 ± 1.3 b 97. b Each value is the ± standard error of six weHs in each group.2· 16.5 d 40.5 d 59.8 ± 6.3· 95.8 b 15..8" 24.1 ± 1.05).3· Chitosan 56.9 ± 1.8 ±4.7 ±0.2 ± 5.005).7 18.8 ± 3.7 ±2. 43. Tokura et al.6 58.3 b 44. p.1" 97.1 ±2.4· 70% DA-chitin -5 -10 -15 500 500 500 -5 -10 -15 500 500 500 98.3 Control Mice were injected intraperitoneally (i.0 c 55.2· 47.2 ± 1.g) % lysis (mean ± s.2" 24. with each polysaccharide at 3 days before harvest of macrophages. Table I.6· 62.D. The results were expressed as nanomoles ofHzOz per mg of ceH protein per 30 min.2 3.9±4.4 ± 6. Effect of chitin and its deacetylated derivatives on macrophage activation in vivo [1] Treatment" Timing (days) Dose (p.7 ± 3. a b not activate mouse peritoneal macrophages.6 ± 0.3 ± 2.2· 98.4" -1. not done. as seen in Table 2.9 33.9 b 43.6 0.0 ±0. DHP-chitin promoted scarce macrophage activation despite the bigger substituting group.2 ± 3.5 ± 0.3 ± 2.1· 31.6 ±4.6± 2.1 ± 2.5· 98.5 c 45.2 55.3 ± 2.8±3.0 ± 5.9· 19.7 ± 3.1· 37.8 ± 4.005).) HzO z releasee (nmolJmg protein) Chitin 30% DA-chitin -5 500 25.2 ± 3.0 ± 0.7· 33.D.1 ± 4.4 c N.3 ± 3.5 ± 5.e.4 Mice were injected i.0 c 1.3 ± 3.1 40. P-chitin and S-chitin did not activate mouse peritoneal macrophages. Effect of 6-0-substituted derivatives of chitin or chitosan on macrophage activation [1] Treatment" Dose (p. c Significant difference from the control (p < 0.e.2 2.2" 17.9 c 62.4· 500 500 500 9.2· 53.4 C 18.g) % Iysis b (mean ± s. The .5 ± 0.1 N.6 ± 7.4 ± 3.8 ± 2.0 d 22.7 49.0 e 3.5 ± 0.9 ± 3. e Macrophages were stimulated with 20 nM ofPMA for 30 min.0 53.6 ± 7.1" 34.5 ± 3.0 ± 5.05).9 ± 3.5 ± 1.2 ± 3. • Significant difference from the control (p < 0.2 ±4.6 22.3 e 58.3" 88.) HzOz release (nmolJmg protein) Chitin CM-Chitin P-Chitin S-Chitin Acetyl-chitin HE-chitin DHP-chitin CM-chitosan DHP-chitosan 70% DA-chitosan Pyran copolymer Contro! 500 500 500 500 500 500 500 500 500 500 500 2. d Significant difference from the control by Student's t-test (p < 0.e.7 d 19.4± 5.) with each polysaccharide at indicated days before harvest ofmacrophages.e.9 d 73.3 66.1 ± 2.9 ± 7.2 ± 3.2 ± 1.9 ± 3.6 ± 3.5 ± 0.1 c 59.8 ± 3.1 b 54.9 ± 0.5 c 38.5" 33.

6) 52 ± 8(1. Significant difference from the control (p < 0.8' 81.6) 835± 41(2.2) 15 (1.0) 1638 ± 50(4.9' 79.0' 87.9 ± 3.0 ± 1.1) 148 ± 67± 26± 53 ± 76± 215 ± 311 ± 46± 34(3.8 2.1 9.) % stasis (mean ± s.5' 29.4 ± 3. .4' 30.5) 1216 ± 135(3.4 1.0) 462 ± 483 ± 459 ± 490± 11 (1.0±0.6 3.2 ± 0.1 ± 1.9' 81.1) 362 ± 6(0.7 ± 1.0) Hartley guinea pigs were immunized in each foot pad with 200 mg ofBaA in Freund's incomplete adjuvant with 100 mg of each adjuvant.9' 87. .0' 2.e.0 3.6' 41.3 ± 3.e.3 4.7 3. with various doses of each polysaccharide at 3 days before harvest of macrophages.9± 1.0) 251 ± 8(0. a Table 4. p. a One unit ofthe antibody titre was defined as dilution number of antiserum needed to neutralise 10 units of amylase activity.6' 81.3 ± 3. The numbers in parentheses are stimulation indexes.4 1.1) 401 ± 16(1.5 ± 3.8' 25.2 ± 1.3 CM-chitin Pyran copolymer Control Mice were injected i.5) 3(0.2) 1393 ± 119(3.1) 12(1.2) 32(1. Cytolytic and cytostatic activity ofmacrophages induced by various doses of70% DAchitin and CM-chitin [1] Treatment a Dose (}lg) Numberof macrophages (x 106/mouse) % lysis (mean ± s.1' 38.2) 43(1.1 ± 4.2' 22.1 ± 3.0) 32 ± 7(0.2± 1.2) 15(1. Adjuvant activity of chitin derivatives on circulating-antibody formation to bacterial a-amylase (BaA) in guinea-pigs [2] Adjuvant Anti-BaA titre a (U) 2wks Water-insoluble derivatives Chitin Chitosan DHP-chitin DHP-chitosan Water-soluble derivatives DAC-30 DAC-70 CM-chitin CM-chitosan P-chitin S-chitin MDP Control(BaA ± FIA) 4wks 48 ± 11 (1.8) 10(1.6 b 9.7) 114(6.Immunological aspects of chitin and chitin derivatives administered to animals 281 Table 3.7) 38(4.6 ± 3.001).6) 14(1.) 70% DA-chitin 2500 500 100 20 2500 500 100 20 500 19.0' 77.4 84. The control group was immunized with 200 mg ofBaA alone in Freund's incomplete adjuvant.5' 80.3 47.8 ± 3.2 17. b Significant difference from the control by student's t-test (p < 0.9) 1192 ± 178(3.8 ± 3.05).6' 39. as calculated with the contro!.7) 28 ± 4(0.5 ± 2. The data are expressed as mean units ± standard error.4 ± 1.7 34.

as shown in Table 6. BCG-CWS.c o 1l « 0.20. Tokura et al.-----------------. free amino group seems to be the main factor in activating peritoneal macrophages.10 CIl . only chitosan induced further production of circulating antibodies for bacterial a-amylase (BaA) (Tab. As shown in the table.6. 0. 0... a slightly positive activity was observed on normal spleen by CM-chitin in spite of little serious value to cause allergy. Chitosans also induce delayed-type hypersensitivity to m-[4-(4' -Arsonophenyl-azo)-phenyl]-N-acetyl-L-tyrosine(ABAN-acetyl-tyrosine) in addition to the secretion of cytokines following macrophage activation a shown in Table 5 [2]. 0.05 10 100 x Reciprocal serum dilution 40 Figure 1. a hapten- . and dinitrophenyl-ovalbumine (DNP-OVA) induced anti-DNP serum in the presence of 70 % deacetylated chitin as shown in Figure 1 [2]. saline. whereas there was little difference among waterinsoluble chitin derivatives.15 E c ~ iii ~ c 0. Titration of mouse anti-DNP serum collected three weeks after the injection of 20 Jlg DNP-OVA in FIA(2). Dose-dependent macrophage activation is shown in Table 3.282 S. As the mitogenic activity stimulates the induction of B cell formation. Though a negative mitogenic activity was found by the administration of chitosan. . 4). 70% deacetylated chitin is a fairly strong effector for the activation of mouse peritoneal macrophages compared with CMchitin.. Three mice in each group were immunized with 50 Jlg OVA with or without 100 mg adjuvant in FIA two weeks before the injection ofDNP-OVA. As seen in Table 4. Although chitosan and CM-chitin were both found to accelerate mouse peritoneal macrophage activity. 70% deacety1ated chitin. The highest immunogenicity was obtained by the administration of 70% deacetylated chitosan. 30% deacetylated chitin and S-chitin were the best effectors among water-soluble chitin derivatives to stimulate the production of circulating antibodies.

9 13. Adjuvant activity of chitin derivatives on the induction of delayed-type hypersensitivity to azobenzenarsonate N-acetyltyrosine in guinea pigs [2] Skin reaction with ABA-BSA at 24 h Adjuvant 50 ]lg 100 ]lg Chitin DAC-30 DAC-70 P-chitin S-chitin (5.3 ± 0.:! 128 i5 (C) 64 32 16 0 12345 Time course of Immunization (Month) Figure 2.0 at 492 nm in the ELISA assay [3]. b The numbers in parentheses indicate the size of faint erythaema.2) (5.9) (6.5 ± 1.8 Control (ABA-Tyr + FIA) (4. .1 (7. .4 ± 0.3 ± 0.Immunological aspects of chitin and chitin derivatives administered to animals 283 Table 5.2 (6. Time course of immunization for rabbits.5 ± 0.9) (7. and skin reaction was measured 24 h after the intradermal injection of test antigen.6) 15.8 ± 0.4 ± 1. C oe( '5 c 0 :.4 ± 1.5) Hartley guinea pigs were immunized with 50 mg of ABA-N-acetyltyrosine (ABA-Tyr) in FIA with 100 mg of each adjuvant.7 12. Dilution of antiserum showed optimal dilution to give an absorbance of 1.W 13.2) (2.:::.0) MDP 22.4 21.0 ± 0. The control group was immunized with ABA-N-acetyltyrosine alone in FIA. The data are expressed as mean ± standard error of the skin reaction for four guinea pigs.0 ± 0.. a After 2 weeks. a skin test was performed with 50 and 100 mg of ABA-BSA.:::. 304 (A) 256 (8) E ::J Qj CIl :.9 ± 1.4 ± 1.3 ± 0.0±0.

0) 5953 ± 205 (1.6) 5953 ± 205 (1.0) 2957 ± 219 (1.1) 393 266 133 223 (3. CH3~ I C~-CH2~ Hapten (33 mol/IOD residues) H-N-(CH2)4. ['H] Thymidine (0.2) (1. The numbers in parentheses are stimulation ratios. Tokura et al.8) 4219± 237 (1.2) 10466 ± 190 (5.284 S.0) 250 50 10 2 250 50 10 2 DHP-chitin C57BLl6 158 165 101 54 (2.6) 15183 ± 1113 (4.9) 2869 ± 87 (0.0) Normal spleen cells (5 x 10 5 ) of BALB/C or C57BLl6 mice were incubated with mitogen or spleen cells alone for 72 h.0) 3575 ± 162 (1.5) 126058 ± 2247 (71.5) (2.2) (1.6) (1.7) 1432 ± 102 (0.N eH 3 o~~~~o~:'~o~ NHCOCH3 NHCOCH3 Scheme 1.1) 8901 ± 366 (5.9) 4150± 96 (1.1) 3563 ± 316 (1.3) (1.8) 2242 ± 89 (1.2) 3478± 296 (2. Mitogenic activity of chitin derivatives on normal spleen cells ofBALB/c or C57BLl6 mice [2] Mitogen Dose (Ilg mi-I) [H] Thymidine incorporation (Counts per min) BALB/C Expt 1 CM-chitin P-chitin ConA LPS Control Expt2 HE-chitin 5 10 3386± 1855 ± 2016± 1665 ± 5507 ± 3522 ± 2517 ± 2376± 250 50 10 2 250 50 10 2 DAC-70 250 50 10 2 5 10 ConA LPS Contro1 6065 ± 108 (3. Table 6.1) 3384± 186 (0.9) 1771 ± 169 (1.2) (1.6) 3760± 70 (1. Chemical structure ofhapten-bound CM-chitin.3) 2801 ± 288 (1.7) 1566 ± 177 (1.8) 3519 ± 303 (0. NHCOCH3 .5) 123836 ± 1818 (79.0) 6703 ± 279 (1.6) 2591 ± 408 (1.1) 3744± 279 (1.9) 88301 ± 4633 (23.4) 3822 ± 280 (2.0) 3325 ± 210 (0.5 }lCi) was added from 24 h until the end ofthe culture.

The specificity of produced antibody was studied. the result of intravenous injection of CM-chitin should be taken into consideration in the case of human administration. and in bone marrow on intravenous injection. The accumulation of 14C was observed mainly in spleen on per os administration. where A = MA concentration to reduce absorbance at 492 nm to 50% and B = MA analog concentration to reduce absorbance at 492 nm to 50%.Phenetbylamine Methoxyphenamine (O-Methoxymethamphetamine) p-Methoxymetbamphetamine p. The production ofhapten-specific antibody was found first in the presence of a strong immunoadjuvant such as Freund's complete adjuvant as shown in Figure 2. As the human organism has no ß-glycosidases in the intestine to hydrolyse chitin and chitosan. and the recognition of antibody was shown to be very limited for the hapten analogues. 14C-Iabelled CM-chitin was administered per os and intravenously to mouse.Methoxyamphetamine 13 20 266 < 0. but quite low sensitivity of antibody was found either for the carrier CM-chitin or for CMchitin oligomers [3]. bound CM-chitin saline solution was administered subcutaneously to rabbits every 2 weeks for 4 months (chemical structure of hapten-bound CM-chitin in Scheme 1).9 a 1 1 0. These observations would suggest that the accumulation ofCM-chitin .1 Ephedrine Methylephedrine N-Dimethylamphetamine p-Hydroxyephedrine Phentermine Mephentermine p. Fluorescent isothiocyanate (FITC)-labelled CM-chitin was incubated in the presence ofbone marrow extracted from mouse bone (Scheme 2). Phentermine Mephentermine Dlmelhylamphetamine O!J ~ yH'yH3 CH.6 2 0. The accumulation of radiolabelIed CM-chitin was further studied to detect the distribution ofCM-chitin among bone marrow cells.1 6.Immunological aspects of chitin and chitin derivatives administered to animals 285 Table 7. as shown in Figure 3.-NH- MABA(Hapten) Cross-reaction (%) was ca\culated from tbe equation. Cross-reaction ratios by metbamphetamine analogs [3] Compound Cross-reaction (%)' Methamphetamine Amphetamine Norephedrine p-Hydroxymetbamphetamine p. AlB X 100. There was little antibody production even after 4 months ofimmunization. as shown in Figure 4 [4].l. and macrophages accumulated fluorescence after 18 h.2 < 0.4 Methamphetamine O!J ~ yH3~ CH'-y-N-H CH. as listed in Table 7.Hydroxyamphetamine p-Hydroxynorephedrine 100 6.1 100 0. The fluorescence was first seen in the granulocytes after 8 h of incubation.1 < 0.-CH-N-(CH. Little fluorescence was observed in erythrocytes even after 98 h incubation.

~ Kidney Spleen • Lung 72h 1.O. a strong immunoadjuvant. colony stimulating factor secretion was positive both in vivo and in vitro. because specific accumulations ofCM-chitin to granulocytes and macrophages was observed among bone marrow components in addition to a slight mitogenic activity. Distribution of 14C-labeled CM-chitin on organs after intravenous and per os administrations to mice [4]. As shown in Table 8. Tokura et al. secretion of cytokines was observed upon mouse peritoneal administration of chitosan. secretions were positive . and the degree of deacetylation for maximum secretion of cytokines was 70% [5]. stimulation of the immune system seems to be almost the final stage in producing antibody on the administration of CM-chitin.V. Stomach lntestine Bone Marrow Brain 0 100 200 300 400 500 600 700 800 dpm I 100 mg of ti ue Figure 3. In further studies of chitosan administration.286 S. lnte tine Bone Marrow Brain 0 Liver 100 200 300 400 500 600 dpm I 100 mg of tissue 700 800 ~ Kidney Spleen • Lung 72h p. although the degree of secretion was not as much as that by B cell growth cell wall skeleton (BCG-CWS). However. But in the case of interferon and interleukin-l. is specific only for immune-related parts in the physiological pathway.

5% CO 2 t = 0.FITC-CM-chitin incubation with Microscopic observation Scheme 2. bottom photographs 98 h after incubation. 5 min) ~ 37' C.24. 18. ~ I) centrifugation 2) washed with PBS (twice) n V.200 rpm.}:ry ~ r V-/ thigh bone murine ~ rinse with PBS ~~o o( 0. Right side. fluorescent probe picture [4].96hr. ~ V-n centrifugation (1. . Figure 4. 50. 4. Left side. Top photographs were taken 18 h.f. bright field. Scheme for the incubation ofbone marrow cells with FlTC-CM-chitin and microscopic observation. 8. Staining of mouse bone marrow cells at implantation stage with FITC probe.

Recent data indicate that chitin and chitosan induce effective complement activation and IL-8 production from fibroblasts when applied in a wound.7 ± 1. The alternative pathway complement activation by these polysaccharides seems to be similar to that promoted by zymosan. Tumor necrosis factor (TNF) secretion was shown both in vivo and in vitro. b Incorporation of [3H] thymidine by bone marrow cells (1 x 105 cells) was assayed with CSF sampies (5%).5) ± 422 (2.0) Mice were injected intraperitoneally with each adjuvant at indicated times before collecting ofserum. Antiinfective activity was also induced by the administration of chitosan against Sendai virus and Escherichia coU as shown in Figure 5 [1].5 (7. similar to lymphokines. which are almost similar to those oflentinan.9 (1. Table 8. Tokura et al.7 ± 10.5 ± 1. C Bone marrow cells (1 x 105 cells ml.) DAC-70 200 BCG-CWS LPS (Salmonella) Control 200 100 -3 -6 -12 -24 -6 -3 2558 6232 6885 3721 4383 48319 2460 ± 262 (1. Results of immunochemical studies on chitosan and CM-chitin are summarized in Table 10 [25]. On the other hand.0) 69. N-acetylchitohexaose. a polysaccharide from the mushroomLentinus edodes.59 ± 374 (1.6) ± 116 (1.0) Colony formation C (colonies/10 5 bone marrow cells) 12. d Values in parentheses are stimulation ratios as calculated with control. Effect of DAC-70 on the induction of colony-stimulating factor (CSF) in vivo [5] CSF activity Adjuvant a Dose ()1g) Timing (h) [3H] Thymidine incorporation b (mean counts per minute ± s. a only in vivo (Table 9). Interleukin (lL-8) and C5a from the complement activation act on the polymorphonuclear cell migration to the wound site [26]. Since chitosan flocculates upon adsorption of heavy metals or organic materials in water.8) ± 506 (1.4)d 81 ±4. The proposed immunological behaviour of chitosan is shown in Scheme 3. a water-soluble chitin oligomer. They found inhibition of growth oftumour cells or pathogenic microbes in mice upon .5 (8.7) 9.8) ± 2754 (19.3 ± 0.7 (004) 78 ±3.0 ± 1.288 S.3 (7.5 (1.e.7) 69. These observations highlight the immunoadjuvant properties of chitosan. Chitosan microspheres were also applied to define the influence of the surface of solid chitosan. [32].0)d ± 450 (2.') were cultured with 5% CSF sampies for 7 days. was reported as an immunopotentiator by Suzuki et al. and the level of immunoadjuvant activity was found to be comparable to that of chitosan in liquid form [6]. chitosan administered as a solution may precipitate onto the surface of organs in vivo.7 (9.7) 3. The results were expressed as mean colonies from 105 bone marrow cells ± standard eITor of triplicate cultures in each group. The results were expressed as mean ± standard eITor of quadruplicate wells in eachgroup.

Comparison of cytokine production by DAC-70 and other adjuvants in mice [5] Cytokine Monokine CSF (in vivo) (in vitra) INF (in vivo) (in vitra) IL-l (in vitra) TNF (priming) (eliciting) Lymphokine CSF (in vitra) INF (in vitra) IL-2 (in vitra) MAF (in vitro) DAC-70 LPS MVE-2 BCG-CWS MDP + + + + + (18. MDp. Table 10. . eoli Sendai virus + + + + + + + + + + + + + + + ± + + + + + + + + + ± BCG-CWS.12) + (33)" .(33) + + + (7) + + (10) + (12) +(13) + (12) + (13) + + (32) + + " Nurnbers in parentheses are reference numbers. ABA. Biological activity terms ofDAC-70. 25) + + (26) + (11. BCG cell wall skeleton. Muramyldipeptide (N-acetylmuramyl-L-alanyl-Disoglutamine ).Immunological aspects of chitin and chitin derivatives administered to animals 289 Table 9. CM-chitin. azobenzenearsonate. BCG-CWS and MDP [25] Terms DAC-70 CM-chitin BCG-CWS MDP Adjuvant activities (a) Macrophage activations Cytolysis (in vivo) Cytostasis (in vivo) H2 0 2 release (in vivo) (in vitra) + + + + + + + + + ± (b) Delayed type hypersensitivity (ABA-N-acetyltyrosine) + (c) Antibody production Bacterial amylase (in vivo) Dinitrophenyl hapten (in vivo) + + (d) Cytokine production Interleukin-l (in vitro) Interleukin-2 (in vitro) Colony stimulating factor (in vivo) Macrophage activating factor (in vitro) Tumor necrosis factor (in vivo) Interferon (in vivo) (in vitro) Mitogenic activity (in vitra) Antitumor activity Suppression ofMeth-A Antiinfectious activity E.

70% DA-chitin (A...-. 70% DA-chitin (A.1 day ! (s.p.5 day - .1 day (s. coli (a).5 day ! ~ . chitosan (T). CM-chitosan (.p.05.) E. Each mouse was treated intraperitoneally (i. \''ts. Similarly. p < 0. a * * 8 (i.c. -0 . ·0 Time after infection (days) 2 b.4 x 10 6 . cfu E.0 4. with MDP-Lys (LI8) (e) at a dose of 100 Jlg per mouse I day before infection with 3.c. 0 v---v---v b * * 8 Figure 5. coli .) 100jlQ MDP-Lys(LlB) cn :::J .. Prospective activities of chitin derivatives and MDP-Lys(L-18) in mice infected subcutaneously (s..p.). with 500 mg of30% DA-chitin (\7). p. .) with chitin (6). '" ~ ~ Cl o . :". CM-chitin (./: .....). coli [I]...) 246 Time after infection (days) -. DHP-chitosan (0) and PBS (0) at 5 days before or s. c.. with 100 Jlg ofMDP-Lys (LI8) (e) one day before infection with 4.)500jlQ PS injection t . * Statistically different from contro! by X 2 method. )500I-lQ ~ E. coli .).).-0 . and DHP-chitin (0) at a dose of 500 mg per mouse or PBS alone (0) five days before or s._•.fr·..c. 6.c.~ 50 ctl .c.g o 100 t--J \0 ~ $P.11 •• _111 . (i.4X10 6 cfu infection (s.4X106 cfu infection (s.c..7 x 10 6 cfu E.cn ::J .) 100jlQ MDP-Lys(LlB) PS injection .. each mouse was treated i.e o 100 4 6 . coli (b).) with E..-0 ...~ 50 Cij 4..

Nishi N. Nishimura S-I. Tone Y. They thus concluded that the main target ofN-acetylchitohexaose was macrophages when N-acety1chitohexaose was administered intravenously and secretion of macrophage activating factor (MAF) was accelerated from T cells to suppress microbial growth [32]. Growth inhibition of tumour cells such as Sarcoma 180. Nishi N. Nishimura S-I. Takatori T (1987) Analytical Biochem 161: 117-122 . However. Nishi N. Tokura S. intraperitoneal injection ofN-acety1chitohexaose due to activation processes ofphagocytes and the immune system [27-31]. MM 46. but IL-2 secretion was not observed from spleen T cells. Meth A and 1ung carcinoma solid tumours was observed. Murata F. much lower concentations of oligomers are supposed to be supplied to the immune system from chitosan or chitin than in the case ofN-acetylchitohexaose.Immunological aspects of chitin and chitin derivatives administered to animals 291 Ag Scheme 3. Upon intravenous or intraperitoneal injection of N-acety1chitohexaose. Tokura S. They also treated macrophage with N-acetylchitohexaose to produce IL-1 in vitro. Nishimura S-I. acute influence is expected on the activation of the immune system due to the fluidity of low molecular weight N-acetyl chitohexaose. Azuma 1 (1984) Vaccine 2: 93-99 2 Nishimura K. References 1 Nishimura K. Azuma 1 (1985) Vaccine 3: 379-384 3 Tokura S. due to the slow rate ofhydrolysis in animal bodies. Proposed pathway of immunoresponse following administration of chitosan. Hasegawa 0. Saiki I.

Suzuki K. Tatewaki N. Tonio y. Aizawa K. Minami S. Ogawa H. Yasumoto K. Matsuhashi H. Yamamura Y (1975) Gann 70: 737 18 Saiki I. RudolfH. Yamawaki M. Mizel D (1981) J Immunol Methods 46: 211 21 Lowry OH. Capuis B (1970) Immunology 18: 501 25 Tokura S. Matsubara N. TsutsumiA. inpreparation 5 Nishimura K. Suzuki S. Okawa Y. PA Sandfonl. Otani T. London and NewYork. Azuma I (1987) In: MYalpani (ed): Industrial polysaccharides: genetic engineering. Shan TM. Nishi N. Elsevier Science Publishers. Tsutsumi A. Tanio Y. Nishimura K. Kabat EA. Imoto T. Azuma 1(1986) J Biomed Mater Res 20: 1359-1372 7 HackmanRH(1958)AustJBioISci7: 168 8 Hayashi K. Tokura S. Azuma I (1983) Infect Immun 40: 622 20 Pick E. Mikani T. Fujinaga T (1994) Pro-Vet 70: 32 27 Suzuki S. Tokura et al. Naruse R. Umemiya M. Suzuki K. Suzuki S. Yamamura Y (1963) J Biochem (Tokyo) 54: 477 23 Engval E (1980) Methods Enzymol70: 419 24 Brunner KT. Okumura H. Ishihara C. Tatewaki M. Carsten ME (1953) J Am Chem Soc 75: 4583 16 Schiffinan G. In: CJ Brine. Tokura S. Mikami T. Watanabe T. Tokoro A. Suzuki M (1990) Jpn J Cancer Res 81: 259 29 Tokoro A. Suzuki M (1991). Onoue K. Nishi N. Suzuki M (1988) Chem Pharm Bult 36: 784 30 Tokoro A. Azuma I (1986) Vaccine 4: 151-156 6 Nishimura K. Yamamura Y. Suzuki K. Nauel J. Suzuki M (1990) Microbiol Immunol34: 413 32 Suzuki S. JP Zikakis (eds): Advances in chitin and chitosan. Somorin 0 (1983) Polym J15: 485 10 Nishi N. Azuma 1(1982) Infect Immun 38: 58 19 Saiki I. Farr AL. Osaka Y. Kobayashi M. Randali RJ (1951) J Biol Chem 193: 265 22 Okada Y. 4 Tokura S. Okawa Y. Nishi N. Nishi N. Suzuki M (1990) Microbiol Immunol33: 357 31 Kobayashi M. Rosebrough HJ. 347 26 Okamoto Y. Sobotka H (1959) J Biol Chem 238: 1726 15 Eisen HN. Thompson W (1964) Biochemistry 3: 113 17 Azuma I. Nakashima S. Yamamura Y. Mikami T. Tokura S. Watanabe T. Tokuzen R. Tatewaki N. Tokura S (1984) Int J Biol Macromol6: 53 12 Wolform ML. Noguchi J. C Jeuniaux. structure/property relations and applications. Matsumoto T. Kamisango K. 96 . Elsevier Applied Science. Summers CG (1953) J Am Chem Soc 54: 1519 13 Matumoto K. Ukei S. GW Gooday (eds): Chitin in nature and technolgy. Seo H. Nishimura S-I. Ebina A. Saiki I. Plenum. Suzuki S. London and New York. Tokoro A. NewYork and London. Suzuki M (1986) In: RAA Muzzarelli.292 S. Tokushima Y. Nishimura S-I. Suzuki S. Une T. BeIman S. Funatsu M (1963) J Biochem 54: 381 9 Tokura S. Shiota H (1979) Polym J 11: 27 11 Nishi N. Azuma I (1983) Infect Immun 39: 1029 14 Tabachnick M. 485 28 Tsukada K. p. Yoshimoto T.

most of which is incorporated into mye1in. University ofAncona. namely. analysed statistically. is required for normal cell growth and proper membrane structure and function. cholesterol 7a-hydroxylase (a monooxygenase located in the endoplasmic reticulum). antiarthritic. and the use of chitins instead of chitosans. Dietary chitosan is effective on serum cholesterol and in atherosclerosis in normal and diabetic mice. such as gallstone disease. The most important elimination pathway for water-insoluble cholesterol is the conversion to water-soluble bile acids.A. and on management of hypertriglyceremia and hypercholesterolemia. I-60JOOAncona. Adrenals. biliary . Muzzarelli Center for Innovative Biomaterials. with the aid of cholestyramine and fibric acid.Chitin and Chitinases ed. After providing basic information on enzymes involved in cholesterol homeostasis. This chapter discusses several issues raised against the use of chitosan. the body reacts to interventions intended to lower the cholesterol concentration: the effect of cationic resins on cholesterol degradation is counteracted by a compensatory increase in cholesterol synthesis. Via Ranieri 67. Introduction Cholesterol. It also exhibits antiuicer. by ~ Jolles and R. It also directs attention to the unexplored areas of fungal and algal chitosans. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA-R). Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Clinical and biochemical evaluation of chitosan for hypercholesterolemia and overweight control Riccardo A. depletion of zinc and liposoluble vitamins. the most abundant sterol in the mammalian cell. further indicate that chitosan is effective to control overweight when associated to a diet. A. The flux of cholesterol and bile acis regulates the activity of the following enzymes: cholesterol: acyl coenzyme A transferase (ACAT). as well as advantages such as enhanced absorption of nutrients and competitive inhibition of lipases. corneal crystalline dystrophy and tumour proliferation [3].A. For instance. atherosclerosis. Bile acids are efficient suppressors ofthe HMG-CoA-R in vivo. The brain contains 10% of cholesterol (dry weight). Italy Summary. antihypertension and antiuricemic properties. gonads and placenta transform cholesterol into hormones [1-2]. Of course. and lends itself to the treatment of hypercholesterolemia in humans. The liver is the key organ in the maintenance of cholesterol homeostasis in the body. Unregulated accumulation of cholesterol is cytotoxic. The published human trials. There is therefore much interest in keeping the cholesterollevel under control and in limiting its ingestion. this chapter examines the effects of the ingestion of chitosan. and a failure to maintain homeostasis ofthe sterol results in a number ofpathologies.

'. cholestipol and other remedies Frequently applied drugs are bile acid sequestrants and inhibitors ofHMGCoA-R..:. + + ...d".. MuzzareJli ."" Mevalonate '. (iii) the general behaviour of chitosan might be partially reconducted to the behaviour ofits oligomers [5]..A. and bile acids. (-) deactivation. Acetate ! HMG-CoA e HMG-CoA . diversion or administration of bile acid-binding cationic resins leads to severalfold stimulation of both bile acid biosynthesis and cholesterol 7 ahydroxylase activity (Fig. In this chapter. which is recognized as a cytoprotective agent and a treatment for lower gastrointestinal tract disorder [4].. The hydrolysis of chitosan in the digestive tract appears to be of utmost importance because (i) the current definition of dietary fiber indicates that fibers should not be hydrolyzed by the digestive enzymes. Management of hypertriglyceremia and hypercholesterolemia Cholestyramine. ? + Cholesterol' e I ~ Cholesterol 7a-hydr oxy 1 ase 7a-OH-cholesterol + + Bile Acids Figure 1."? .. a . 1). cholesterol. Regulation of cholesterol 7a-hydroxylase and HMG-CoA reductase at different cholesterol precursors.A.. and several relevant topics will be discussed.. (ii) the more or less acetylated chitosans release N-acetylglucosamine and glucosamine.. a rate-limiting enzyme catalyzing the synthesis of mevalonate.294 R. the effects of the ingestion of chitosan will be examined. (+) activation.

Cholestyramine and cholestipol are synthetic ion-exchange resins approved for hyperlipidemia treatment. S. vomiting and other gastrointestinal disturbances besides cholestatic jaundice. Adverse effects are cutaneous flushing. dolichols and isoprenylated proteins. Studies done on various animal models have been recently reviewed [9. 10]. They are not approved by the U. heme A. Serum cholesterol was reduced by 52% in the chitosan-fed animals foIlowing 20 weeks of treatment. They are competitive inhibitors of the key regulatory enzyme of cholesterol biosynthesis. but not indicated in the management oflow HDL cholesterol. 295 precursor of all isoprenyl compounds inc1uding ubiquinones. cholesterol and other sterols). nausea. promoting the excretion of fatty materials. Dietary chitosan is effective on serum cholesterol and atherosclerosis in the hypercholesterolemic.Clinical and biochemical evaluation of chitosan for hypercholesterolemia . Their mode of action is complex and not fully established: they reduce cholesterol synthesis by suppressing HMGCoA-R activity and enhance LDL c1earance. These bile acid sequestrants lower LDL cholesterol 15-30% but raise HDL cholesterol only 3-5% and may raise triglyceride levels. Hypocholesterolemic action 0/ chitosan on animals The ingested chitosan salts react with fatty acids and bind additional lipids due to hydrophobic interactions (triglycrides. apo lipoprotein E-deficient mouse. Food and Drug Administration (FDA). DEAE-Dextran was also found effective in reducing intestinal fat absorption and speeding up bile turnover [6].. fluvastatin. sterols and triglycerides [8]. Cholestyramine sequesters bile salts in rats and alters intestinal morphology in humans. inc1uding cholesterol. The fibric acid derivatives reduce plasma cholesterol by 6-11 % and triglycerides by 22-43% [3]. which deve10ps high levels of blood cholesterol and atherosc1erosis without the need for dietary intervention.. and are the most commonly prescribed c1ass oflipid-Iowering agents. They are generaIly weIl tolerated. Nicotinic acid and derivatives reduce plasma levels of triglycerides and cholesterol and increase the plasma concentration of HDL cholesterol. Furthermore. Fibrates may be prescribed in combination with nicotinic acid or bile acid sequestrants. there is a significant increase in rat intestinal tumour induction by various agents when cholestyramine is added to the diet. They mayaiso reduce absorption of dietary cholesterol by reducing the activity of acylcholesterol acyltransferase in erythrocytes. or inhibitors of HMG-CoA-R. HMG-CoA-R inhibitors inc1ude lovastatin. Reductase inhibitors are effective in lowering LDL cholesterol [7]. provastatin and simvastatin. fatty and bile acids. and a great portion of these bound lipids are excreted rather than absorbed. and the area of plaque in the aortic arch of treated animals was 50% less than that of the . Bound triglycerides would escape hydro lysis by lipase.

These properties are related to the capacity to bind fatty acids. chitosan is able to form the corresponding complex salts that bind triglycerides. Interestingly. In no case was the body weight altered after 4 weeks of diet at 5% chitosan. chitosan improved lipid metabolism. cholesterol and triglycerides. Chitin and cellulose had no effect [12]. The existing in vitro data on the binding of fats to chitosan are of little use for those interested in the reactions taking place in the stomach and . fatty and bile acids. chitosan administration is associated with a diet. A. When chitin or chitosan in nylon net bags were administered orally to dogs. because diabetic subjects have elevated blood cholesterol and triglyceride levels caused by metabolic derangements. Chitosan exhibits anticholesterolemic. Bacterial flora seemed to influence the weight of chitosan. indicating that it is useful for diabetic complications. The hyperglycemic and hypolipidemic effects of chitosan were studied in normal and diabetic mice. cholesterol and other sterols. The plasma total cholesterollevel decreased to 77% on day 7 and subsequently to 54% on day 14. Muzzarelli controls. phospholipids and uric acid. but chitosan was degraded in the stomach and large intestine. The overall weight gain of the chitosan-fed mice was increased by 65% [11]. whereas in controls it was retarded. NSZ) [13]. significant hypoglycemic and hypocholesterolemic effects were also observed after chitosan administration. growth in the chitosan-fed animals was significantly enhanced. In NSZ mice. antiarthritic and antiuricemic properties. about 26% weight loss of the chitosan was observed in the presence of feces. antiulcer. and a great portion of these bound lipids are excreted [14-18].296 R. the latter including obese ones with hyperinsulinemia (KK-Ay) and lean ones with hypoinsulinemia (neonatal streptozocin-induced diabetic mice. and then recovered at the initial level on day 28. Total cholesterol in the plasma significantly decreased when chitosan was administered orally for 2 weeks. Chitosan-treated normal mice had significantly decreased blood glucose. the chitin did not undergo changes in weight and shape. When a chitosan bag was surgically placed in the large intestine for 24 h. In the presence of fatty acids. Furthermore. The fecal loss of bile constituents demands increased hepatic transformation of cholesterol into bile acids. A. Chitosan was released from the net into the gastrointestinal tract following the change to a gel in the stomach. In general. Management 0/ hypercholesterolemia in humans There is general agreement on the fact that orally administered chitosan lowers blood pressure and cholesterol in volunteers. Chitosan was therefore proposed for treatment of non-insulin-dependent diabetes mellitus. No effect was observed in KK-Ay mice. bile acids with consequent reduction of their enterohepatic recycling.

The lipid-Iowering effect of chitosan in obese subjects was recently assessed [25. [27] have shown the clinical importance of chitosan. 11.96 g chitosan each meal. the amount of corn oil bound to chitosan is currently taken by the producers as evidence of the efficacy of the product. Chitosan inhibited the putrefactive activity of the intestinal microbiota. respectively.27-3. over a 5-week period. and serum cholesterol and triglycerides were reduced in volunteers from 7. taurocholic acid and lipase were added.6 (vs. 297 duodenum due to the biochemical complexity of the digestive tract.3 mmol/l. Nevertheless. In general. The most recent study has involved 1000 volunteers with body mass index (kg' m-2) (BMI) over 25. The chitosan intake (biscuits) by healthy volunteers was found to produce a significant decrease ofthe fecal phenols. Chitosan was administered to adult males in the form ofbiscuit over a study period of 4 weeks [23].p-cresol and indole.Clinical and biochemical evaluation of chitosan for hypercholesterolemia . The results on humans show a favourable effect with a very low dose within a short period. 26]. this parameter ranging from 2. 3.5 placebo) for diastolic and systolic values. but responders reduced their weight significantly [19-22]. fat assimilation was reduced by 13-25% by chitosan (glycerol tri[I-14C]0Ieate test). Attempts at mimicking the in vivo situation were based on the following approach: chitosan (0. binds larger amounts of cholesterol and smaller amounts of bile acids.9 placebo) and 19. whereas the serm HDL-cholesterol level significantly increased when compared with the level for each of them before ingestion. 37°C).5 g) was preincubated with 10 mM HCl (2 h. Similarly.5 kg in the placebo group) and blood pressure reductions of 8. On the other hand. compared with cholestyramine.6 to 1. After 12 weeks of chitosan ingestion. these data are hardly useful for understanding the mechanism of action due to methodologicallimitations (for instance. it was realized that some subjects did not respond to the chitosan treatment.. for 12 weeks. the average total serum cholesterol level decreased .8 ml/g (wheat flour control1. Results were not reproducible. respectively. Renal failure patients undergoing hemodialysis were observed. When chitosan was given in the diet (3-6 g/day).22). were also reported in a randomized double-blind study (0. and the hydrolytic action of lipase on chitosan was observed in certain cases. in analogy with other polysaccharides [24]. In the course ofthis study.9 kg (2.81 to 10.2 mmol/l and 1.4 to 5. The lecithinase negative clostridia were the only microbiota significantly depressed by chitosan in humans after 2 weeks of chitosan intake. data have been produced on the absorption of cholesterol and bile acids on various chitosan-based products [20-23]: again. particle size and the identity ofthe other ingredients were not specified). then corn oil.. Jing et al. it can be said that chitosan. thus no conclusion could be drawn [19]. Weight reduction of 6.6 (vs. the total serum cholesterol level significantly decreased. thus reducing the risk of disease. 1000 kcal/day diet).

no stimulant action: chitosan is not a medicine.A. and chitosan is recognized as a safe compound. For example. Similar data were obtained by various authors.4 kg better than that for placebo groups. In some cases the scarce correspondence between the characteristics of chitosan and the existing regulations has represented an obstacle. Italy and Portugal. weight reduction is 4. Muzzarelli significantly to 5. is easy for the overweight to use and is a nonaddictive substance.40 mM. Vitamins could be prescribed to be taken at different hours by patients. Nevertheless.14 ± 4. no side effects. However. Therefore. Furda [34] has indicated the value of the association of chitosan with vitamins for clinical purposes. there is no risk of overdose. in other cases certain hypothetical problems have been raised. when analyzed statistically.A.82±2. The prescribed dose of chitosan is much lower than that used in animal tests. among which Veneroni et al. The published human trials. being nontoxic and deprived of activity on certain human enzymes involved in cholesterol synthesis.19mM from 10. orally administered chitosan leads to overweight reduction after a few weeks oftreatment. This section is abrief survey of the topics debated. indicate that chitosan groups showed greater weight loss than placebo groups. according to Lassus and Abelin [28]. some ofthe clinical reports cited above state that vitamin E level was not depressed. Liposoluble vitamins It has been remarked that chitosan could deprive the diet of liposoluble vitamins [33] that might be incorporated in the chitosan-fat aggregates. While the clinical trials with chitosan were directed to the abatement of cholesterollevels. Practically no change was observed in the serum HDL levels.298 R. later on it appeared that chitosan could be used as a diet integrator for the more general purpose of overweight control. [29-30] and Ventura [31]. . Hirano [32] has surveyed the many authorized applications of chitosan in the food area in Japan. England. A significant decrease in average serum lipoprotein level was also observed after 4 weeks of chitosan ingestion. as a point of difference from certain drugs. Immunopotentiating and anticancerlantimetastatic actions have also been documented. chitosan has met with difficulties and criticism by official organisms in several countries. For obese patients. Discussion Chitosan is presently on the market as a food additive or dietary integrator in several countries among which Japan.

Clinical and biochemical evaluation of chitosan for hypercholesterolemia . carboxyl ester lipase and phospholipase A2. Chitosan may act indirectly on carboxyl ester lipase (a bile salt stimulated lipase) by depressing the available bile salts [40]... Lipases and other hydrolases Pancreatic lipolytic activity originates from lipase and its cofactor colipase. It was confinned that the anti-Co albicans growth factor had been induced in the blood of mice. What is even more significant in these findings is that the chitosan-chelating activity is not large enough to deplete the organism of certain trace metal ions. showed that chitosan in rats does not reduce serum iron or hemoglobin [35]. and this iron-binding protein displayed as a C. which in any case occur as complexes. In order to induce the same factor in large amounts in the blood of mice. and therefore chitosan administration per os should be regarded as safe from this standpoint. whilst chitosan improves the intestinal transport by increasing the paracellular penneability of the intestinal epithelium. This result is indirect evidence that iron is not depleted as a consequence of chitosan administration [39]. Interestingly. 299 Trace metal ions The chelating ability of chitosan could represent a worry in tenns of depletion of iron. Similar to trypsin. carboxypeptidase B was only inhibited by polycarbophyl but not by chitosan and a chitosan salt. albicans growth inhibitor. the poly(acrylates) are FDA-approved. Administration of chitin or partially acid-degraded chitin in mice per os resulted in anti-Candida albicans growth factor. which could be identified to be a known factor. Induction of apo-transferrin took place in mouse blood. chitin was administered intraperitoneally to young mice. This subject was also addressed by other authors with similar conclusions [36]. [38] evaluated the potential of chitosan to inhibit the intestinal proteases trypsin and carboxypeptidase B. and to improve the intestinal transport of peptide drugs in a perfused intestinal model. A more substantial rmding came from research on mucoadhesive polymers that modulate the physiological barriers enhancing peptide drug absorption by reducing the metabolie activity of luminal and membrane-bound peptidase and proteases as weIl as by opening the intestinal intracellular junctions [37]. and its inhibition by the poly(acrylates) is due to their chelating properties for Zn and consequent depletion ofZn from the active site [38]. most of the metal ions present in foods are complexed by chelating agents more powerful than chitosan such as citric acid. in spite of their sequestering activity for Zn and metalloenzyme inactivation. Jennings et al. Carboxypeptidase B is a Zn-containing metal enzyme. Luessen et al. The substantial difIerence between the two biopolymers is that poly(acrylate) acts as a protease inhibitor. transferrin. . Moreover. which degrade peptide drugs.

By this means the advantages of chitosan over cholestyramine have been elearly focused. Chitosan provides a sustained-release form of glucosamine. and with a radiological survey of a BaS04 fi1led capsule. however. Oral ingestion (1 g/day) ofN-acetylglucosamine (NAG) or chitosan increase the serum concentration of NAG. These results confirm those about the efficacy of orally administered chitosan in the treatment ofpatients with osteoarthritis [46]. and plasma HDL cholesterol did not decrease. porcine pancreatic lipase and microbiallipase [42-43]. so that the chitosan capsule dissolves in the large intestine thus releasing its content. This mechanism was demonstrated with the release of insulin and the determination of its pharmacological availability. Blood glucose levels are not altered. Chitosan enhances the absorption of macromolecules across the intestinal epithelia. chitosan capsules were developed. where livers were smaller and yellowish. It is well known that the 2-monoglycerides and fatty acids are the partial hydro lysis products. Absorption 0/ nutrients Because of the increasing interest in targeting peptides and protein drugs to the colon. In the large intestine. an important property for the oral administration of . it might be possible that the amount of chitosan introduced with the diet would in part prevent lipases from hydrolyzing the lipids. but the nexus between chitosan and the enzyme has not been elucidated. For instance. Le Houx and Grondin [44] studied the consequences of chitosan administration on 3-hydroxy-3-methylglutaryl CoA reductase in rats fed a sterol diet. On the other hand. Experimental evidence from various laboratories indicates that orally administered chitosan is partially digested and absorbed. remains interestingly high [45]. The enzyme levels remained elose to the normal value with a 7. where proteolytic activities are lower than in other parts of the digestive system. 48 h after ingestion. A. eleared rapidly: in the case of chitosan the serum NAG concentration. yielded by pancreatic lipase.300 R. but dissolves in the small intestine. Studies on the lipase-chitosan system inelude wheat germ lipase [41]. suitable for absorption. This is particularly due to the action of hydrochloric acid in the stomach and the unspecific activities of enzymes present in saliva and gastric juice. other enzymes from microorganisms further digest chitosan. a sealed chitosan capsule containing insulin was coated with hydroxypropyl methyl cellulose phthalate: this enteric coating prevents dissolution of chitosan in the stomach. which is. A. the enzyme activity was overstimulated in the sterol + cholestyramine group. Muzzarelli If chitosan is a substrate for human lipase in vivo (as it is in vitro). with the detection of 6-carboxyfluorescein-vitamin B6 and salicylamide.5% chitosan formula.

It can then be said that chitin is ofplant origin. where the acrylic coating dissolves. and this would help in inc1uding chitin in the definition of dietary fiber. For instance. Aged patients had noticeable improvement of general conditions and working capacity.. The most important is that the cell wall of some of them [53] contains a large quantity of chitosan whose physicochemical properties can be controlled by acting upon the fermentation parameters. The cultivation ofthese algae has been proposed for the production ofhigh-quality chitin [50]. a pH-sensitive multicore microparticulate system. While algal chitins/chitosans have not been used c1inically so far. and lower rates of disease. Clinical analysis indicated a significant fall of the intoxication level in the patients [55]. The micropartic1es escape dissolution in the stornach and reach the intestinal region. 301 proteins and peptides. The naked chitosan microcores then adhere to the intestinal mucosa. The . Why not chitin? Herrera and Mata-Segreda [57] have reported that chitin binds more cholate than chitosan.. Algal and fungal chitosans The extracellular fibers of Cyclotella cryptica. Thalassiosira mentagrophytes and Thalassiosira fluviatilis are composed of pure chitin (15 % dry weight) [48-49]. Work in progress confirms these in vitra data. The release of the drug is completed as soon as chitosan is under the degradative action of colonic bacteria. The Poteriochramonas alga deposits its chitin fibers on the cell surface [51-52]. hepatic cirrhosis and cancer. they are important because botanists assimilate algae to plants [56]. consisting of mucoadhesive chitosan microcores entrapped in the enteric acrylic polymer Eudragit was developed [47]. These studies indicate that chitosan does not depress absorption ofnutrients. The cell wall of certain fungi has been considered as an alternative to crustacean shells. On the other hand. The extraction process is simple and produces litde waste [54]. The molecular weights of fungal chitosan show values in the range 100-450 kDa. fungal chitins/ chitosans provide the same advantages as animal chitinlchitosan. The antiinflammatory drug sodium dic10fenac was tested after pre1iminary work with fluorescein isothiocyanate-labeled bovine serum albumin. consequent to its adhesion to the intestinal surface. releasing the entrapped compound. There were several advantages of using these fungi to produce chitosan.Clinical and biochemical evaluation of chitosan for hypercholesterolemia . A chitinous material produced from higher Basidiomycetes is used orally as a sanitation and preventive remedy for c1inical treatment of liver and kidney insufficiency.

Minami S (1997) Fuodamental study on oral administration of chitin and chitosan in dogs. dietary supplements and immunostimulants. Usami M. New York 2 Luod E. In: A Domard. London 6 Pupita F. personal commuoication 12 Okarnoto Y. its fonctions and metabolism in biology and medicine. plasma triglyceride and coronary heart disease: pathophysiology and management. wound healing. Morimoto M. Pergamon.023 9 Muzzarelli RAA (1996) Chitosan-based dietary foods. Plenum. Carbohydr Polym 29: 309-316 10 Muzzarelli RAA. DeVincenzi V (1996) Chitosan-based dietary foods. Bjorkhem I (1995) Role of oxysterols in the regulation of cholesterol homeostasis: a critical evaluation: Acc Chem Res 28: 241-249 3 Patsch W. vol2. PI Haris (eds): New biomedical materialsapplied and basic studies. Seino Y (1995) Hypoglycemic and hypolipidemic effect of chitosan in normal and neonatal streptozotocin-induced diabetic mice. drug carriers. 874-896 8 Furda I (1980) Nonabsorbable lipid binder. Saimoto H. Biol Pharm BullI8: 1623-1625 . Nose M. chitin should be amorphous for oral administration. Tsuura Y. AS Nies. Jacques Andre. Ishida H. Sashiwa H. In particular. Acknowledgements The assistance of Maria Wecla in retrieving the bibliographic material and preparing the typescript is gratefully acknowledged.A. Barone A (1987) Action of DEAE-dextran on lipid digestion and absorption. In: MFA Goosen (ed): Applications 01 chitin. Technomic.A. Shigemasa Y. Lyon. Lancaster 11 Ormrord D. Rall Tw.302 R. 625-632 13 Miura T.223. US Patent 4. P Taylor (eds): The pharmacological basis 01 therapeutics. KM Varum (eds): Advances in chitin sciences. Oxford. GA Roberts. Can J Gstroenterolll: 120 5 Muzzarelli RAA (1998) Mangement of hypercholesterolemia and overweight by oral administration of chitosans. Of course. Gotto AM (1995) High-density lipoprotein cholesterol. Muzzarelli use of chitin would help in obviating criticism related to the chemical manipulation for chitosan production. In: D Chapman. Goldstein JL (1990) In: A Godman-Giman. Int J Obesity 113: 13-23 7 Brown MS. This work was performed with the financial contribution ofMURST. plastic surgery. Research carried out during the last few years has promoted chitins and chitosans to the forefront of applied activities intended to offer genuinely valuable medical items in the field of general medication. References 1 Bittman R (ed) (1997) Cholesterol. lOS Press. Conclusions The biological significance of chitosan in the human body depends on the actions that certain human hydrolases exert on it [58]. chitosan appears to be a most effective and tolerable biopolymer suitable for the management of hypercholesterolemia and overweight. Adv Pharmacol 32: 375-426 4 Russell AL (1997) Regarding glucosamine and the effect on the gastrointestinal system.

Acta Toxicol Ther 16: 215-230 27 Jing SB. vol2. Lyon. J Control Release 47: 15-23 39 Hino A. In: JC Salamone (ed): The polymeric materials encyclopedia. Wadstein J (1997) The effect of fatblocker Ll12 on animal fat resorption in humans. Ljlia G. Hara H. Boleyn K. Kotze AF. Kobayashi E (1995) Continuous andmassive intake of chitosan affects mineral and fatsoluble vitamin status in rats fed on a high-fat diet. Lehr CM. Suzuki M. Acta Toxicol Ther 17: 53 . Biagini G (1997) Medical and veterinary applications of chitin and chitosan. DC 37 Bochard G. 3. Helsinki-Stockholm 29 Veneroni G. a study of safety in obese patients. Stockholm. In: I Furda (ed): Unconventional sources of dietary fibers. Luessen HL. FL 16 Tokura S. Kawai Y et al (1993) Hypocholesterolemic effect of chitosan in adult males. Herbai Marketing Group. Nagyvary J (1993) Hypocholesterolemic effect. KM Varum (eds): Advances in chitin sciences. De Bernardi M. Mattioli-Belmonte M. Jacques Andre. Proc Soc Exp Biol Med 189: 13 . Bersh-Williford C (1983) Chitin and chitosan: influence on element absorption in rats. chitosan and oat gum in rats. PCT IntAppl WO 97 29. Verhoef JC. Atec. Marletta M (1996) Effect of a new chitosan dietary integrator and hypocaloric diet on hyperlipidemia and overweight in obese patients.237-258 33 Deuchi K. Watanabe T. Polycarbophyl and chitosan are potent enhancers of peptide-transport across intestinal mucosae in vitro. Gudmunson S. Boca Raton. Contos S. Lehr CM. De Bernardi M. a new dietary integrator. Junginger HE (1997) Mucoadhesive polymers in peroral peptide drug-delivery. In: RAA Muzzarelli (ed): Chitin enzymology. GAF Roberts. J Pharm Pharmacol 49: 721-723 28 Lassus A. Mattei G.20 36 Gordon DT. Sakai K (1997) Relevation ofblood transferrin level in mice administered intraperitoneally with a . Sweden 21 Heldman E. Thompson JL. 55-62 32 Hirano S (1996) Chitin bioteclmology applications. New York 15 Muzzarelli RAA (1996) Chitin. Japan 17 Muzzarelli RAA.Clinical and biochemical evaluation of chitosan for hypercholesterolemia . Matsumoto T. Kanauchi 0. Japan Soc Chitin. Tripodi S. MATS Medical AB. Abelin J (1994) Ll12 Biopolymer-fatblocker as a weight reducer in patients with moderate obesity. Tsuji K. Rentel CO. vol2. Junginger HE (1996) The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. In: S Dumitriu (ed): Polymeric biomaterials. Grottammare. Norway. Nakagawa Y. Contos S. 63-67 30 Veneroni G. Shizukuishi M. Veneroni F. 4. Proc Eur Soc Nuclear Med. CRC Press. Muzzarelli B. Anderson JW (1988) A comparison ofthe lipid-lowering and intestinal morphological effects of cholesteryramine. Marcel Dekker. Sato D et al (1995) Effect of dietary chitosan on faecal microbiota and faecal metabolites ofhumans. Atec. In: A Domard. Biosci Biotech Biochem 57: 1439-1444 24 Terada A. Matahira Y.. Grottamare. Lipids 18: 714-719 19 Anonymous (1997) Biocardio per os capsules. Os10. de Boer AG. Veneroni F. Yamaguchi T (1997) Effect of chitosan on renal function in patients with chronic renal failure. Fini M. Azuma I (eds) (1992) Chitin derivatives in lifo sciences. American Chemical Society. J Control Release 39: 131-138 38 Luessen HL. Guarino C. Colombo P (1995) Lipid-lowering effect of chitosan dietary integrator and hypocaloric diet in obese subjects.760 35 Jennings CD.70 31 Ventura P (1996) Lipid lowering activity of chitosan. Mikami T. Bridgs SR. AR Medicina. 20 Abelin J. Guarino C. BiotechnolAnn Rev 2. Effects of chitosan glutamate and carbomer on epithelial tight junction in vitro. Marletta M (1996) Effect of a new chitosan on hyperlipidemia and overweight in obese patients. Rossner S (1995) Fat binder. Sapporo.. de Boer AG. Biosci BiotechBiochem 59: 1211-1216 34 Furda I (1997) Multifunctional fat absorption and blood cholesterol reducing formulation containing chitosan and nicotinic acid. Suzuki S. 580-589 18 Nauss n. Ji D. Microb Ecol Health Dis 8: 15-21 25 Macchi D (1996) A new approach to the treatment of obesity: chitosan effects on body weight reduction and plasma cholesterollevels. Wood PJ. Preliminary report 23 Maezaki Y. Li L. Takiguchi Y. In: RAA Muzzarelli (ed): Chitin enzymology. 303 14 Muzzarelli RAA (1993) In vivo biochemical significance of chitin-based medical items. vol 2. Acta Toxicol Ther 17: 303-320 26 Sciutto AM. Tripodi S. Glasgow 22 Wadstein J (1998) Fat binding effects of chitosan in humans. Washington. Verhoef JC.

Grondin F (1993) Some effects of chitosan on liver function in the rat.304 R.A. Utet. vol2. Scand J Gastro- enter32: 261-267 41 Muzzarelli RAA. Alonso MJ (1997) Site-specific drug delivery using chitosan microparticles. In: A Domard. Prilutsky A (1997) Chitin Health Product "Mycoton" produced from fungi. physical properties and applications. C Jeuniaux. In: RAA Muzzarelli (ed): Chitin enzymology. Vila Jato JL. In: RAA Muzzarelli. T Anthonsen. chemistry. Mulisch M. Mata-Segreda JF (1996) Chitin binds more cholate than chitosan. Rudall KM (1967) Chitin fibers in the diatoms Thalassiosira fluviatilis and Cyclotella cryptica. Endocrinology 132: 1078-1084 45 Talent IM. Jacques Andre. Torino 57 Herrera Fp. GW Gooday (eds): Chitin in nature and technology. Zemskov V. 119-125 40 Sternby B. Lembi CA (1978) Localization of chitin in algal and fungal cell walls by light and electron microscopy. Jacques Andre. In: RAA Muzzarelli. Rev Biol Trop 44: 613-614 58 Muzzarelli RAA (1997) Human enzymatic activities related to the therapeutical administration of chitin derivatives. Boston. ER Pariser (eds): Proc 1" Int CorifChitin and Chitosan. Kaplan DL (1988) Fermentation. Lombardi SJ. processing and enzyme characterization for chitosan biosynthesis by Mucor rouxii. Yalpani M. KM Varum (eds): Chitin and chitosan. Arnzeim Forsh Drug Res 43: 1109-1113 47 Remunian-Lopez C. Grottamare. Jacques Andre. 600-607 48 Blackwell J. New York. Lyon. 319-332 54 White SA. Kong N (1978) A critical evaluation ofthe potential source of chitin and chitosan. Burdyukova V. Ilari PL (1995) Depolymerization of chitosan and substituted chitosans with the aid of a wheat germ lipase preparation. GAF Roberts. Clin Ther 18: 1184-1190 46 Setnikar I. J Marine Bio128: 383-385 49 Dweltz NE. Carbohydr Res 237: 325-332 44 Le Houx JG. MIT Press. Cosani A. Cell Mol Life Sei 53: 131-140 . GAF Roberts. Parker KD. Colvin JR (1968) Chitin fibers in the diatoms Thalassiosirafluviatilis. Palumbo R. Enz Micr Technoll7: 541-545 43 Pantaleone D. J Hystochem Cytochem 26: 782-791 53 Arcidiacono S. Farina PR. Plenum Press. KM Varum (eds): Advances in chitin science. 648-655 56 GerolaA (1994) Biologia vegetale. Zugenmaier P (1986) Comparison of chitin structure and assembly in three unicellular organisms. Portero A. Canali S. 64-78 51 Herth W. vol 2. Lyon. Fox JR. In: A Domard. Can J Chem 46: 1513-1521 50 Allan GG. Fulton I (1979) Production and isolation of chitosan from Mucor rouxii. Atec. New York. In: G Skjak-Braek. biochemistry. GAF Roberts. vol2. 107-120 52 Pearlmutter NL. Gracy RW (1996) Pilot study of oral polymerie N-acetyl-D-glucosamine as a potential treatment for patients with osteoarthritis. Terbojevich M (1996) Unspecific activities of lipases and amylases on chitosans. Lorenzo ML. Lyon. KM Varum (eds): Advances in chitin seiences. Schollar M (1992) Unusual susceptibility of chitosan to enzymic hydrolysis. Xia W. In: A Domard. Elsevier. Muzzarelli partially degraded chitin. Appl Environ Microbiol38: 323-328 55 Gorovoj L. Tomasetti M. P Sandford (eds): Chitin and chitosan: sources. Zanolo G (1993) Pharmacokinetics of glucosamine in man. 69-82 42 Muzzarelli RAA.A. Nilsson A (1997) Lipases in pancreatic enzyme supplements.

1-271 00 Pavia. Tiziana Modenal.and nanoparticles [4]. The main drug classes successfully encapsulated into chitosan microparticles are antiinflammatory. Franca Pavanetto 1. granules. University 01 Pavia. Chitosan was proposed as a drug carrier for mucosal administration in ocular. or microspheres that contain the drug dispersed or dissolved in a polymerie matrix. whereas "microspheres" are monolithic or matrix-type microparticles [5]. Microparticles may need to be cross-linked to retard their degradation in acidic media. gastroenteric and vaginal-uterine therapies based on its bioadhesive properties and biodegradability in vivo under the action ofhydrolases. as a binder and lubricant in wetgranulated tablets and as a stabilizing agent in emulsions [1. Examples are the delivery of acyclovir via ocular administration.3]. The distribution of the drug in the carrier system has profound consequenees on the release and degradation properties of microparticles. chemotherapeutic. Faculty 01 Medicine.Chitin and Chitinases ed. 1-60100 Ancona.A. nasal. Italy Center lor Innovative Biomaterials. yet cross-linking with glutaraldehyde introduces cytotoxic characteristics and depresses bioadhesion. xerogels and micro. Introduction In the pharmaceutical field the use of chitosan offers many advantages as an excipient for increasing the dissolution rate of poorly soluble drugs. Depending on process parameters and physicochemical properties of the drug. Viale Taramelli 12. antibiotic and anticaneer drugs [6]. This biopolymer and its derivatives are employed in the preparation of modified drug delivery systems such as implants. Alternative cross-linking approaches are discussesd along with the suitability of chitosan for the oral delivery of vaccines. chemical composition and manufacturing technique. and the delivery of 5-aminosalicylic acid to the colon. The term "microspheres". University 01Ancona. Particulate carriers have important potential applications for the loading and releasing of therapeutic molecules for oral mucosal and parenteral administration. Muzzarelli 2 1 Department 2 01 Pharmaceutical Chemistry. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Microparticulate drug delivery systems Ida Genta 1. Italy Summary. as an auxiliary substance in direct tableting. buccal.A. Bice Conti 1 and Riccardo A. is used to describe particulate delivery systems. irrespective of size. Paola Perugini 1. containing a drug core surrounded by a continuous coat of polymer. A. either "true microcapsules" are obtained. or "microparticles". pellets. by R Jolles and R. The term "microcapsule" indicates reservoir type devices. Via Ranieri 67. Recently chitosan and its derivatives have been studied for their .

efficient and reliable mucosal delivery systems expecially for poorly absorbable drugs such as peptides and proteins. Results from "in vivo" ocular administration of acyc1ovir-loaded chitosan microspheres to the rabbit eye showed prolonged high coneentrations of acyc1ovir. Bioadhesive microspheres spread on a large area of mucosa and decrease the rate of c1earanee ofthe drug from the mucosa. Mueociliary transport rates in volunteers have been studied [12. At present. potential mucoadhesive properties. Chitosan's mucoadhesive properties seem to be mediated by ionic interaetion between sialie acid residues in mucus and the polymer amino groups [8]. specific drug delivery to the colon is considered an important alternative for the treatment of serious local diseases such as Crohn's disease.306 I. as protective drug coatings. able to release the drug at a particular pR. Microspheres promote a transient widening of the tight junction between Caco-2 cells. The major drawback is that in contrast to what was believed in the past. nasal. The nasal absorption of insulin in rat and sheep models with the aid of a variety of chitosans has been performed according to a mechanism based on bioadhesion and a transient widening of the tight junctions of the nasal membranes. arthritis or inflammation [15]. There is increasing interest in the development of safe. Recent studies prove that chitosan has mueoadhesive properties and enhances the penetration of macromolecules across the intestinal and nasal barrier [7 -1 0]. Drug delivery to the colon Colon-selective drug delivery systems have been the foeus of increasing interest for the last decade. and open new perspectives in formulating bioadhesive dosage forms for mucosal administration (ocular. ulcerative colitis. no membrane of cellular damage was reported. On the other hand. thereby allowing larger moleeules to pass through the membrane [11]. 14]. [13] prepared chitosan microspheres for the ophthalmie administration of aeyc10vir for the treatment of localized or systemic Herpes simplex infection. careinomas and infections. such as asthma. thereby allowing a longer contact time with the absorptive epithelium. Genta et al. specific systemic adsorption in the eolonie region offers interesting possibilities for the treatment of diseases susceptible to diurnal rhythm. This is mainly due to the recently recognized importance of this region of the gastrointestinal tract for systemic therapy. Chitosan microspheres applied to the nasal mucosal membranes promote nasal peptide absorption into systemic circulation [12] and show no toxicity. buccal. the pR . gastroenteric and vaginal-uterine therapy). Genta et al. this characteristic is important for the formulation of targeted particulate drug carriers and for the administration of protein drugs. Drug delivery to the various regions of the gastrointestinal tract is based on the use of enteric polymers.

(v) drug solubility and diffusion through the chitosan gel. partially cross-linked with Eudragit. [19] studied the colon-specific delivery ofinsulin from chitosan capsules in rats. this crosslinking process is neither totally effective in preventing the early release of the encapsulated drug nor does it maintain the mucoadhesive properties of the polymer [7. The only inconvenience of these polymers is their high solubility in gastrointestinal fluids: this implies the need of cross-linking. The probable mechanism of drug release from these chitosan multicore microspheres at the solubility pR ofthe coating polymer could be understood as follows (Fig. (iii) the core/coat ratio. which is the time normally required to reach the colon (3-4 h). 16. The only limitation associated with this approach is the enormous variability in the gastric emptying of the dosage form depending on the quantity and kind of food consumed. furthermore. (iv) the microcore-coating interaction. Chitosans couple their specific degradability in the colon with their good adhesiveness to the gastrointestinal mucosa favouring the specific and persistent delivery of the local drug. the drug is rapidly released along the upper intestine before reaching the colon. Based on this idea.1): Once the microspheres reach the small intestine. the toxicity of aldehydes enormously limits the exploitation of these cross-linked microcapsules. thereby triggering the release of the entrapped drug. (ii) the size and swelling behaviour of chitosan microcores. swell upon contact with the basic release medium and form a ge1 through which the drug diffuses. chitosans have been evaluated for their susceptibility to c1eavage by these bacterial enzymes.Microparticulate drug delivery systems 307 of the proximal and transverse colon is more acidic than that in the small intestine. to preserve their integrity until they reach the colon. for instance with aldehydes. Bacterial hydrolases are in sufficient quantity to be exploited in colonic drug targeting. expecially chitinases. Despite this limitation. there are several factors which may affect the release ofthe drug: (i) the pH-dependent solubility ofthe Eudragit coating. Lorenzo-Lamosa et al. (vi) chitosan degradation in the colonic region [18]. Thus. 17]. Nevertheless. the chitosan microcores reach the colonic region where the chitosan undergoes a degradation process. these enteric coating formulations are the only commercialized products for the treatment ofthe ulcerative colitis with 5-aminosalicylic acid (5-ASA). . They showed improvement of insulin absorption from the rat colon. According to this explanation. Timed release systems have been also proposed for colonic drug delivery: they deliver drugs after a particular time. A more realistic strategy for targeting drugs to the colon uses the ecosystem ofthe specific microflora in the large intestine. After 3 -4 h. [16] proposed a novel multiparticulate system based on chitosan core microspheres coated with enteric polymers (Eudragit) to overcome the problem due to the high solubility of chitosan in the gastric cavity while avoiding chemical cross-linking with aldehydes. The chitosan microcores. thus leaving the chitosan microcores increasingly exposed to the release medium. Eudragit slowly and continuously dissolves over time. Tozaki et al.

Swelling of CS Degradation ofCS © © © @ Gastrie cavity © Small intestine © © @@ @ @ © ©©@ @ © © • © ©@ @ © © @© © © ©©@ © • Colon Figure I. 1. Scheme ofthe possible mechanism of drug release from the Eudragit microencapsulated chitosan (es) microcores.308 I. have revealed Figure 2. Scanning electron micrograph ofuncross-linked chitosan microspheres with mucin in aqueous solution. Genta et al. In this picture it is possible to observe mucin adhering to microparticle surface. In an in vitra study Genta et al. uncross-linked or cross-linked with glutaraldehyde. scanning electron microscopy and transmission electron microscopy observations ofmucin in contact with chitosan microspheres. Magnification 18000x. [17] showed that the bioadhesive characteristics of chitosan microspheres were depressed for glutaraldehyde crosslinked microspheres. . Dissolution ofEudragit 2.

Biodegradable particulate delivery systems may be useful in the field of vaccine therapy. Oral immunization is likely to offer advantages over the parenteral route: no pain for the patient. morphology and BSA-release. such as the ability to condense DNA and form small discrete particles in defined conditions [28. which sometimes are limitations in developing countries. Numerous experimental systems have demonstrated the ability of oral immunization to induce antibody secretion into the mucous which bathes these surfaces [31]. Several formulation parameters (chitosan molecular weight. which is administered orally. temperature and ultrasound by a passive absorption technique into degradable biopolymer matrices by taking advantage of their swelling behaviour. These mucosal antibodies are restricted almost entirely to the secretory form of IgA (sIgA). the results indicated the possibility of modifying the formulation to obtain the desired controlled release of insulin for convenient oral administration. [26] loaded insulin into a chitosanlcalcium alginate system. pH. Even though the load was low. [24] used BSA as a model to prepare chitosan gel micropartic1es for the controlled release of hydrophilie macromolecules. In addition. [25] measured the release of BSA from chitosan-alginate microcapsules and demonstrated that chitosan-alginate systems may be used for the delayed release of a protein acid. Hydrophilie chitosan nanoparticulate carriers have been proposed for the delivery of a model protein drug [30]. Chitosan presents favourable characteristics for gene delivery. The study demonstrates the possibility of incorporating biological macromolecules sensitive to organic solvents. an antibody type which is not effectively induced through conventional intramuscular or subcutaneous immunization. and a sustained release may be possible for macromolecules. partic1e size.310 I. The nanopartic1es incorporated polyethylene oxide in their structure for controlling the release of the loaded protein. and the logistics for mass immunization would be simpler. Remunan-Lopez et al. Hari et al. It was also shown that drugs passively adsorbed into such matrices are not necessarily released completely in the initial burst. Polk et al. oral immunization induces a vigorous immune response in the mucosal surfaces of various organs. Aydin and Akbuga [27] studied the release characteristics of salmon calcitonin from chitosan beads. type of acid and salt) were investigated with reference to encapsulation efficiency. Oral administration of antigens can be effective in this respect because the small intestine . Bovine serum albumin (BSA) was loaded by passive absorption from aqueous solution into preformed glutaraldehyde cross-linked chitosan microspheres [23]. except the polio vaccine. Genta et al. Parenteral vaccinations need highly trained personnel and sterilized equipment. 29]. no side effects . the most common site of entry of infectious agents.it would be more acceptable to the majority of patients. Currently used vaccines against viral infection consist of parenterally administered inactivated virus.

hormones and growth factors are now commercially available in large quantity. Over the past decade. cytokines. upon reaction with the amino groups responsible for the chitosanlmucin interaction. enzymes. 20]. various controlled release systems for peptide and protein drugs have been extensively explored [21]. Scanning electron micrograph of cross-linked chitosan microspheres (5% glutaraldehyde) with mucin in aqueous solution: the smooth microsphere surface is deprived of any interaction with the protein. that the protein was rejected from the microsphere surface (Fig. Protein delivery to other organs A wide range of proteins such as vaccines. Among them are the short in vivo half-lives and the side effects attributable to the multiple and high-dose injections. An interesting approach to maintain therapeutic levels is to deliver the proteins with the aid of biodegradable polymers [5. reduces the affinity of the polymer with mucin and depresses the mucoadhesive properties of chitosan micropartic1es. . Among them. but several problems are associated with the therapeutic use of protein drugs. This indicates that glutaraldehyde. 2 and 3). Magnification 5000 x.Microparticulate drug delivery systems 309 Figure 3. the most promising delivery approach is the encapsulation of protein within injectable microspheres composed ofbiodegradable polymers [22]. For this aim chitosans seem to be very useful because of their biomedical and hydrophilic properties. in addition to their biodegradability in vivo.

PF34). [23] loaded diphtheria toxoid by passive absorption into cross-linked chitosan microspheres. Roma. With red blood cells. Italy (contract no. but no information is available on their biocompatibility and adequacy as drug carriers.Microparticulate drug delivery systems 311 contains lymphoid aggregates. Other cross-linking agents have been described [39-42]. Preliminary immunization studies carried out on rats using diphtheria toxoid-loaded chitosan spheres have shown promise. the latter inc1uding a significant fraction of cells committed to the synthesis of IgA c1ass antibodies. Recent observations confirm extreme cytotoxicity of the glutaraldehyde cross-linking chitosan microspheres [34]. "Progetto Finalizzato Materiali Speciali per Tecnologie Avanzate 11". Glutaraldehyde cross-linked chitosan spheres were well tolerated by the living tissue and were not found to be degraded completely in 6 months in vivo in rat musc1e. in which antigens are incorporated into liposomes or polymer microspheres. 37] and pectins [38]. The IgA-committed B lymphocytes migrate through the mesenteric lymphonodes and into the blood circulation so to lead to the immunization induction also in the glandular tissues and the genitourinary and respiratory tracts [32].00032. 98. termed Peyer's patches. In addition. where antigens were gradually released from the particles to induce immune response [33]. the glutaraldehyde cross-linked chitosan microspheres were considerably more lytic than dextran. Jameela et al. however. Microencapsulation systems. It is obvious. the glutaraldehyde-re1ated toxicity of the products is a serious limitation of the model. and less membraneactive than soluble chitosan. Here antigenic materials trigger the c10nal expansion of specific B and T lymphocytes which then extravasate through the lymphoid drainage. were tested as the most plausible systems for oral vaccination. embedded along its antimesenteric aspect. the Peyer's patches are separated from the lumen ofthe gut by a layer of epithelial cells interspersed with a specialized type of phagocytic cells that actively internalize sampies from the lumen contents. that because the high reactivity of glutaraldehyde leads to chemical species of poorly understood formulae. Improved microsphere biocompatibility can be achieved with other reagents such as citric acid [35]. Glutaraldehyde is the cross-linking agent most frequently used to prepare chitosan microspheres. . Acknowledgements The present work was carried out with financial support from the Italian National Research Council. and pass them to the underlying lymphoid cells. 36. Peyer's patches are an organized lymphoid tissues which contains functional T and B lymphocytes. alginates [20. The micropartic1es were shown to be taken up by Peyer's patches.

Asti A. Montanari L (1998) Influence of glutaraldehyde on drug release and mucoadhesive properties of chitosan microspheres. de Boer GA. Takada M. Terare A. Hirochi K. US Patent 4. New York 5 Kissel T. University ofTexas. Kotze AF. Verhoef JC. Danti AG (1978) Pharmaceutical tablets containing chitin as a disintegrant. Muzzarelli BB (1998) Structural and functional versatility of chitins. Gcnta et al. deBoer AG. In: CG Gebelin (ed): Cosmetic and pharmaceutical applications 01 polymers. Yamamoto A. Junginger HE (1992) In vitro evaluation of mucoadhesive properties of chitosan and some other natural polymers. J Micraencaps 14 (6): 689-711 7 Lehr CM. Marcel Dekker. Farraj NF. Oda M. J Control Rel 6: 167-176 23 Jameela SR. IIIum L (1997) Chitosan as a nasal delivery system: the effect of chitosan solutions on in vitra and in vivo mucociliary transport rates in human turbinates and volunteers.2940 4 Muzzarelli RAA. Carbohydr Pol. Mason JDT. Koneberg R (1996) Injectable biodegradable microspheres for vaccine delivery. Puglisi G (1997) Bioadhesive microspheres for ophthalmie administration of acyclovir. Conti B. Suzuki I (1996) Chitosan capsules for colon specific drug delivery: improvement of insulin absorption from the rat colon. In: S Dumitriu (ed): Structural diversity and lunctional versatility 01polysaccharides. J Pharm Pharmacol49: 737 -742 14 Aspden T J. 51-87 6 Snheyla Kas H (1997) Chitosan: properties. Matsumoto T. Junginger HE (1997) Mucoadhesive polymers in peroral peptide drug delivery. Illum L. Lehr CM. Alonso MG (1998) Design of microencapsulated chitosan microspheres for colonic drug delivery. Lorenzo ML. J Contr Re145: 15-23 11 Edman P. Biol Pharm Bu1l17: 745-747 21 Langer R (1990) New methods of drug delivery. J ones NS. Perugini P. preparation and microparticulate systems. Muranishi S. J Biomat Sci Polymer Edn 6(7): 621-631 24 Remunan-Lopez C. Marcel Dekker. 54 ° . Mcleod AD (1995) Kinetic perspectives on colonic delivery. MisraA. Skaugrud 0.086. Davis SS (1994) Chitosan as a novel nasal delivery system for peptide drug. H Bernstein (eds): Microparticulate systems lor the delivery 01proteins and vaccines. J Pharmaceut Sei 86: 509-513 15 Tozer TN. Verhoef JC. Costantini M. Alonso MJ (1995) Deve10pment of new microencapsulated chitosan gel cores for the controlled release of hydrophilie macromolecules. Friend DR. Int J Pharm 72: 43 -48 8 Illum L. Chem Pharm Bull 30: 2935 . Pavanetto F. III: Effect of chitosan-glutamate and carbomer on ephithelial tight junctions in vitro. Proc 10 th Int Symp Microencaps. Schacht EH. Nagai T (1982) Directly compresssed tablets containing chitin or chitosan in addition to lactose or potato stareh. Nakayama A. Science 249: 1527-1533 22 Maulding H (1987) Prolonged delivery of peptides by microcapsules. Proc Int Symp Contr Rel Bioact Mater 23: 4422-4423 20 Miyazaki S. Austin Press. Fujita T. Ryden L (1992) Microspheres as a nasal delivery system for peptide drug. Vila-Jato JL. Austin. Lowe J. NewYork. References 1 Illum L (1998) Chitosan and its use as a pharmaceutical excipient. Proc Int Symp Contral Rel Bioact Mater 22: 2135-2136 13 Genta I. J Controlled Rel 52: 109-118 17 Genta I.335 3 Savyanagi Y. Nambu N. Hayashi K (1991) Cosmetic and pharmaceutical uses of chitin and chitosan. Luessen HL. Remunan-Lopez C.312 1. In: S Cohen. Plenum. Bouwstra JA. J Contr Re121: 165-172 12 Aspden Tl. Rentel CO. Pharm Res 11: 1186-1189 9 Borchard G. JayakrishnanA (1994) Crosslinked chitosan microspheres as carriers for prolonged delivery of macromolecular drugs. J Contr Re139: 131-138 10 Luessen HL. STP Pharma Sci5: 5-12 16 Lorenzo-Lamosa ML. Skaugrud (1995) The absence of chitosan toxicity when applied to nasal mucosa. Spadaro A. Conti B. Junginger HE (1996) The potential of mucoadhesive polymers in enhancing intestinal peptide drug adsorption. Sugiyama T. New York 19 Tozaki H. in press 18 Hirano S. Lehr CM. Attwood D (1994) Chitosan and sodium alginate based bioadhesive tablets for intraoral drug delivery. Pharm Res 15: 1326-1331 2 Bruscato FN. Bjork E.

Atec. In: RAA Muzzarelli. Dellacherie E (1994) Hemoglobin encapsulation in chitosanlcalcium alginete beads. Remunan-Lopez C. Steffan AM. J Appl Polymer Sci 59 (11): 1795-1801 27 Aydin Z. Ilari P. Muzzarelli C. Chandy T. Int J Pharm 148: 231-240 35 Zecchi V. Fell JT (1997) Hydroge1 beads based on amidated pectins for colonspecific drug delivery: the role of chitosan in modifYing drug release. Tomasetti M (1994) Tyrosinase-mediated quinone tanning of chitinous materials. I. Orienti I (1997) Controlled drug delivery systems. Florence A (1989) The uptake and translocation of latex nanospheres and microspheres after oral administration to rats. Aiedeh K. Orottammare. Remy JS (1998) Chitosan-based vector/DNA complexes for gene delivery: biophysical characteristics and transfection ability. August J. Terbojevich M (1999) 6-0xychitins. MO Peter (eds): Chitin handbook. Sharma CP (1996) Chitosan/calcium-alginate beads for oral delivery of insulin. Halbert W.Microparticulate drug delivery systems 313 25 Polk AA. Ooosen MFA (1984) Controlled release of albumin from chitosan-alginate microcapsules. Carbohydr Polym 24: 294-300 41 Muzzarelli RAA. Hammond CJ. Atec. Meulbroek JA. Orottarnmare. Co sani A. Richardson S (1996) Alginate/chitosan microporous microspheres for the controlled release of proteins and antigens. Roy K. Vila-Jato JL. Collett JH. Oilley RM. J Pharm Sci 83 (2): 178-185 26 Hari PR. Poncelet D. J Clin Immunol7: 731-737 32 Eldrige JH. Prac Int Symp Control Release Bioact Mater 23: 269-270 37 Huguet ML. Xia W. 415-422 40 Muzzarelli RAA. Alonso MJ (1997) Novel hydrophilic chitosanpolyethylene oxide nanoparticles as protein carriers. Langridge J. Arch Pharm Pharm Med Chem 331: 133-138 . Arnsden B. Walsh S. J Appl Polymer Sei 63: 125-132 31 Mestecky J (1987) The common mucosal immune system and current strategies for induction of immune responses in external secretions. De Angelis AA (1997) New chitosan chemical networks: some synthetic procedures. Zecchi V (1998) Controlled insulin release from chitosan microparticles. Carbohyd Polym 39: 361-367 42 Bugamelli F. In: RAA Muzzarelli. Pinotti M. 397-404 36 Heller J. Leong K (1996) DNA chitosan-nanospheres for gene delivery. Orally administered biodegradable microspheres target the Peyer's patches. Neufeld RJ. Duncan R. Oroboillot A. J Pharm Pharmacol 41: 809-812 34 Carreno-Oomez B. Staas JK. Zou SM. Lin-Shu Liu NOS. Duncan R (1997) Evaluation of the biological properties of soluble chitosan and chitosan microspheres. De Yao K. J Control Relll: 205-214 33 Jani PO. J Appl Polym Sei 51: 1427-1432 38 Munjeri 0. Orienti I. Raggi MA. Pharm Res 15: 1332-1339 30 Calvo P. Peng T. J Controlled Release 46: 273-278 39 Crescenzi V. Tice TR (1990) Controlled vaccine release in the gut-associated lymphoid tissues. Abkuga J (1996) Chitosan beads for the delivery of salmon calcitonin: preparation and release characteristics. novel hyaluronan-like regiospecifically carboxylated chitin. MO Peter (eds): Chitin handbook. Bettinger T. Prac Int Symp Contr Rel Bioact Mater 23: 401-402 29 Erbacher P. Int J Pharm 131: 101-103 28 Mao H.

Chitosan has been used as antimicrobial compound by its external application (exogenous) to the host. the antimicrobial action is influenced by intrinsic and extrinsic factors such as the type of chitosan (e.Ä. and environmental conditions (e. Aeromonas. algae and some bacteria. Chitosan antimicrobial action is more immediate on fungi and algae. The pure enzyme produced was tested direct1y for its biocontrol activity. Muzzarelli © 1999 Birkhäuser Verlag Basel/Switzerland Antimicrobial action of exogenous chitosan Raul G. 12-17. 17. has also been used to control growth of algae [19]. plain or derivative).25]. . and to inhibit viral multiplication in plants and in vitro [48. intrinsic and extrinsic) influencing chitosan as antimicrobial agent. 33]. g. 10. substrate chemical andJor nutrient composition. Chitinlchitosan antimicrobial activity has been reported by several authors [1. host natural nutrient constituency. Their differential antimicrobial effect is mainly exhibited in live host plants. 12-17]. showed that transgenic plants containing high constitutive levels ofbean endochitinase are more resistant to infection by the soil-borne pathogen. 28. [10] tested three different chitinase genes from Serratia. However. substrate water activity (A w ) and/or moisture). The antimicrobial activity of chitosan is well observed on a wide variety ofmicro-organisms inc1uding fungi. Chitosan.g. g. Rhizoctonia solani than are wild-type or control plants that lack the chimeric chitinase gene. they are also a source of nutrients. thus the antifungal effect ofN-carboxymethyl chitosan (NCMC) is different in vegetable as compared with graminea host. and Trichoderma. The cloned gene was then expressed in the nonrhizosphere bacteria Escherichia coU for biocontrol experiments. Introduction Chitin and chitosan [42] show antimicrobial activity along with the ability to resist environmental conditions. Induction of chitinase enzymes through genetic engineering as a contral alternative against plant pathogens seems to be very promising. to the substrate or media. the chitosan site of action is at the microbial cell wall. 15. The objective of this chapter is to present fundamental factors (e. for effective practical application.51]. The chitosanase and chitinase have been induced in plants as resistance mechanisms against pathogens. by ~ Jolles and R. followed by bacteria.Ä.Chitin and Chitinases ed. pentamer and heptamer chitosan units seem to have better antifungal action than larger units. Cuero Summary. Chet et al. as weH as its properties as a lytic enzyme. especiaHy fungi [12. Although both plain and derivative chitosans are effective as antimicrobial agents. At the same time. and to a physical surface containing microbial population [1. degree of chitosan polymerization. Broglie et al. there is a differential effect between them. [7].

20. Cuero and Lillehoj [19] demonstrated that NCMC exhibits both algistatic and algicidal effects by inhibiting total growth of the alga Anabaena sp. N-carboxymethylchitosan (NCMC) is obtained by N-carboxymethylation of chitosan. • • • • In addition to the nature and/or physicochemical structure of chitosan.26. many specific qualities. or 48 h after algal inoculation.316 R. 36. when applied to the media before algal inoculation. 16. 18. 37] • induction ofphenolic compounds and/or phytoalexins [15. which showed . polyelectrolyte complexes chelation of metals present in metalloenzymes alteration of the bacterial adhesion inhibition of the enzymes that link glucans to chitin prevention of nutrients permeation Effect of the nature of chitinlchitosan on antimicrobial activity The nature of chitinous material (e. NCMC's algicidal effect was shown in culture of Anabaena sp. and its antimicrobial action in vitra is more immediate or prompt than plain chitosan. plain or derivative). 28. moisture). Cuero Some mechanisms for antifungal activity of exogenous chitosans have been suggested: induction of phenylpropanoid and octadecanoid pathways induction of chitosanase [16a. 33] induction of chitosanase with many polypeptides [12-14. has a profound effect on its antimicrobial efficacy.37] effect of chitosans on the plant enzymes in relation to plant resistance against fungal pathogens [16.g. The chemical structure differences between the plain and derivative chitosan also has effects on their functional differences such as antimicrobial activity. as compared with native chitosan.G. 37] • induction ofmorphological and/or physiological changes [12. pR and water activity (a w )' Some mechanisms have been suggested to understand this antibacterial action [45]: • • • • • reaction with bacterial teichoic acids. temperature. g. 32a. inherent in the host and/or substrate that can intluence the antimicrobial activity of the polymer include environmental conditions (e. 23]. 21. incubated at 22°C under shaking conditions. 25. which mainly showed an algistatic effect at the concentrations used (> 1 nM). nutrient composition. which consists ofreacting the free amino groups of chitosan with glyoxylic acid to produce a soluble aldimine and then reducing the product with suitable reducing agent [42. 43. in vitra.28. thus: NCMC. All NCMCtreated algal cultures showed decreased biomass (40 mg/50 mllweek) as compared with algal culture without NCMC or control. even at lower concentration exhibits more potency than plain chitosan. 53].

7 25.317 Antimicrobial action of exogenous chitosan B Figure I. iron. and so on present in the media.05 % and 0. NCMC algistatic effect on an Anabaena sp.6 54. In another study.5 25 29. and the age of the algal culture [19] (Tab. in culture Controls Treatments no NCMC NCMC Time(days) 2 6 9 Biomass (mg/50 ml) Biomass (mg/50 ml) Biomass (mg/50 ml) 21.7 40.0 Algal cells incubated at 22°C under shaking conditions. Thus algal cells lyse through these holes and finally die. thus preventing nutrient availability to the microorganism. 1). could be due to its ability to chelate transition metal salts such as copper.3 48.6 . 12 nM 24 nM 39 nM 2 6 9 0 0 0 20 18 15. Electron microscopic view showing algicidal effects of chitosan: A.1 % concentration only. 13] also demonstrated a substantial antimicrobial effect of NCMC on toxigenicAspergillusflavus. Fig. 1).7 51. higher biomass (50 mg/50 ml/week). Both the algistatic and algicidal effects of exogenous chitosan also depend on the type of algae.0 30. NCMC. zinc. Source: [ 19]. Big punctures and/or holes were observed under electron microscopy in algal filaments treated with exogenous chitosan (Fig. Chitosan treated algal culture: PuncturedIHolel dead algal filaments [19]. as compared with native chitosan. Although NCMC was effective in inhibiting algal growth in liquid culture at higher concentrations (39 nM).0 42. This in vitro antimicrobial efficacy of the derivative chitosan. Cuero et al. B.1 %. The exogenous chitosan caused morphological changes to the algal filaments. which was effective at 0. 1. Control/no chitosan treatment: Normal algal filaments . [12. Cuero (unpublished data) inhibited Fusarium species in agar by using NCMC at concentrations between 0.0 42. lower NCMC concentrations (12 and 24 nM) were also effective in inhibiting algal growth.7 42. NCMC has also shown antiviral effect in vitro by inhibiting viral adsorption to the Table 1.

N-carboxymethylchitosan-N.50 0.21 0.22 0. 13-17. showed almost Table 2. Growth of Botrytis species in field-grown eggplants was similarly inhibited.67 0.318 R.48].21 0.66 (STDE) 0. natural host): (1) Exogenous chitosan from crustacean applied to a plant host seems to activate genes in the host RNA. NCMC functions at a wider pH range (3. thus causing lysis of the fungal cells.14. Several mechanisms have been suggested to explain the chitosan induced microbial resistance in vivo (e.17 0. 2). inhibited the propagation of the human immunodeficiency virus type 1 (HIV-l) in human CD4 cells and that ofRauscher murine leukemia virus (RLV) in murine fibroblasts [51].5 to 9) than native chitosan. jlavus was correlated with reduction of aflatoxin (Tab. thus inducing resistance to microbial pathogens coming in contact with the host.25. thus it is an ideal substance for in vitro studies. and consequently growth inhibition andlor death [1. unpublished data] Cuero et al. [13]. R. jlavus in field growing com and peanuts treated with chitosan.17 0. Plain chitosan seems to have a better molecular compatibility with the plant host andlor grain and fruit substrates than derivative chitosans such as NCMC. especially in live plants and in harvested grain and fruits.G.12. However. Mean Aspergillus flavus count in field grown corn.67 0. The effect of chitosan on growth inhibition of A. Cuero CD4 receptor and reverse transcription of the viral genome.G.17 0. Other reports have also shown marked antimicrobial activity of chitosan in growing plants as well as in harvested grain and fruits [1.22.00 0. as mentioned above. in vivo. The amphiphilic property ofNCMC is the reason for its pH versatility. The chitosan effect on inhibiting the toxigenic A. after single control treatments [12] Treatments Mean ASFL ASFLalone water alone chitosan alone ASFL + chitosan chitosan + ASFL ASFL+BSUB BSUB+ASFL LSD 3.33 0.O-sulfate (NCMCS). Cuero and Osuji [16a] achieved complete inhibition of toxigenic A.21 . jlavus also depends on the management of the extrinsic and intrinsic factors.g. exogenous chitosan induces microbial and plant chitosanase production. This enzyme has antifungal activity by hydrolyzing the chitosan in the cell wall ofthe microorganism.17 0. Cuero.36 0.28].26. native chitosan exhibits a more effective antimicrobial activity than NCMC. this will then induce the phenylpropanoid pathway and some enzymes responsible for triggering resistance mechanisms against pathogens [1.

Plain chitosan seems to induce chitosanase with more polypeptides than glycolchitosan. jlavus. Also. [15]. Cuero. Morphological changes of Aspergillus jlavus hyphae after chitosan treatment. it has been demonstrated that exogenous chitosan elicits chitinase in melon plants and Japanese radish. showed different levels ofphenolics (total phenols) enhancement at different water activities as compared with control. The results showed that native chitosan substrate induced chitosanase with a greater number of polypeptides than the derivative/synthetic glycolchitosan (Fig. 100% inhibition of A. Cuero (unpublished data) observed morphological changes such as weakening and swelling of A. unpub1ished data). the fungal toxin aflatoxin was also reduced to almost nil. jlavus growth in field-grown maize by treatment with chitosan. 25. 49]. euero. soybean. and consequently the fungal toxin (aflatoxin) was inhibited [15. to no growth within 72 h. 3). 2). jlavus hyphae. R. rice and black pine seeds during the germinating process [29. total phenols and unknown free phenolic compounds) after being applied to field peanut and maize plants. 26]. There . resulting in control of pathogenic and/or toxigenic fungus A. (R. 1988. The higher number of polypeptides seems to be advantageous because ofthe wider range of genes that can be elicited for antimicrobial action. Cuero et al. G. after treatment with exogenous chitosan (Fig.G.Antimicrobial action of exogenous chitosan 319 Figure 2. thus inducing antifungal activity in plants: (2) Exogenous chitosan from crustacean has also shown elicitation of phytoalexins and/or their precursors (free phenolic. 1988 (unpublished data) carried out a comparative electrophoretic study using both synthetic/derivative glycolchitosan or native chitosan as substrates to induce chitosanase in peanut seeds. consequently.

Different polypeptides horn two different Bacillus species. 1988. Cuero B nO. Levels of free phenolic acids were noticeably increased with incubation in combined chitosan + A. Induced by syntheticl derivative and native chitosan substrates.4 polypeptides.2 -2 .85 A w . and bound p-coumaric acid by chitosan in peanut seeds betwen 9 and 72 h: (3) The antimicrobial effect of exogenous chitosan also seems to be a result of its ability to react with proteins and essential nutritional elements used by microorganisms during growth. G. induced 7 polypeptides.1 B nO.7 .85) than at higher A w (0. 2 Bno. which increase with incubation with chitosan treatment. It has been demonstrated that chitosan chelates metals markedly [lla]. thus inhibiting availability of these nutrients to the microorganisms and causing slow growth and/or death of the organisms. unpublished data) were higher levels ofboth free phenolic compounds at lower A w (0. Fajardo et al.3 . Marked increases of phenolics occurred after 48 h of incubation. 4). [25] reported significant enhanced elicitation of free ferulic and p-coumaric acids.G.4 GLYCOLCHITOSAN AS SUBSTRATE CHITOSAN AS SUBST RATE Figure 3. Thinning and lysis of the algal . Cuero (unpublished data) also corroborated the induction of higher concentrations of phenolic compounds in tissue cultures ofpeanut (Fig. Left: Glycochitosan substrate. (R.3 . Right: Chitosan substrate. Chitosan also has the ability to immobilize enzymes relevant to food processing such as proteases [35]. flavus treatment at 0. induced 2.1 B nO.6 .1 . 2 -1 . Cuero.4 -5 .95).320 R. Chitosan is also believed to possess a fungistatic property due to its ability to induce morphological changes in the cell walls of Rhizopus stolonifer [13].

Seed treatment. Osuji (unpublished data) found inhibition of A. [17] reported control of A. 5: A. 3: B.:i At< 4 . thus inducing disease resistance responses [28. jlavus. 1995. When chitosan enters the host plant cells. as weIl as filamentous mal formation and flaccidity of the cell wall along with perforations ofthe algal filaments was observed in Anabaena sp. fruit and vegetable protection. logging and other symptoms of fungal infection were observed.90 A w after treatment with: 1: water. Cuero. [l3] also reported control of A.7%) in kerneis from chitosan-treated plants as compared to 30% in nontreated controls. chitosan was more effective in controlling the toxigenic fungi in peanut seeds. Chitosanase induction in peanut seeds at 0. fruit and vegetable protection Hadwiger et al.. Simultaneously. Similarly. subtilis # 1. R. subtilis #2. [30] observed a relationship between chitinase activities and resistance of seedlings to pathogens in Japanese radish. flavus and concomitant aflatoxin production in postharvest com kemeis after treatment with chitosan. .. Cuero and G. 2: B. unpublished data. rice. hulled rice and black pine seed. it triggers a sequence of reactions. [19](Fig.321 Antimicrobial action of exogenous chitosan 1 2 3 .g of chitosan per gram of seed on winter and spring wheat.. induction of systemic resistance to Fusarium crown and root rot in tomato plants was obtained after chitosan seed treatment [3]. However. • . 33]. The ability of chitosan to enzymatically and/or mechanically inhibit growth of fungi has effectively been used for practical applications such as seed treatment. 5 1IIjI. peas and lentils during a 5-year trial. G. aflatoxin was reduced to zero in kerneis from chitosan-treated plants as compared with 1104 p. subtilis # 1 + chitosan.G. aw.flavus and concomitant aflatoxin production in harvested com kerneIs treated with chitosan in the field during plant development. ta • 6 9' • ~ Figure 4. R. flavus growth in chitosan-coated com and peanut seeds. cells.glkg in nontreated controls . [28] reported seed and foliar treatments of field crops with commercial chitosan. They applied seed treatments ranging from 60-1000 p. Cuero et al. Plant yield increased 20-30%.1). The fungal population was almost nil (1. 6: B. Reduction of dampoff. 4: chitosan. soybean. Cuero et al. Hirano et al.

the fruits even increased in size as compared with untreated controls. carboxymethyl chitin was less active. such as the coating of fruits [22] and vegetables [8]. They carried out in vitro studies which showed that chitosan-coated strawberries were protected against decay-causing molds such as R. Although the antibacterial activity of chitosans has not been consistent and clear. phosphoryl chitin and some chitosan oligomers preared by nitrous acid deamination of partially deacetylated chitins inhibited bacterial growth. on the cell wall surface. Muzzarelli [43] and Mattioli-Belmonte [40] found in vitro that N-carboxymethyl and N-carboxybutyl chitosans are more effective bacteriostatic agents than other chitosans.G. However. . tomato. and may suppress bacterial growth. Autolysing enzymes. Cationic or amphoteric polyaminosaccharides interact with the bacterial cell wall. D-glucosamine hydrochloride did not show inhibitory activity. However. and cucumber) against postharvest fungal pathogens). the inhibition of growth might be caused by an optimum chitosan oligomer chain length. become lethaI substances when cationic compounds are present. sialic acid and muramic acid. The agricultural applications reported by [29] mainly concern grain coating but a range of applications are being developed. has been demonstrated by EI Ghaouth et al. They suggest that perhaps the inhibition ofbacterial growth is due to the cationic amino groups in these chitosan oligomers which combine.322 R. bell pepper. such as N-acetylmuramic acid. by electrostatic interaction with anionic components. Tanigawa et al. [23] and EI Ghaouth and Wilson [24]. Cuero The practical application of chitosan to protect fruits and vegetables against pathogens such as fungi in postharvest. and alter its structure as a consequence of polyelectrolyte complex formation and of disturbing action on the equilibria involving metal ions. which normally control bacterial division. Seedlings of eggplants were inoculated with the fungus Botrytis spp. was achieved by treating the plants with chitosan. before being transplanted into the field. They found that sulfuryl chitin. partially deacetylated chitins. thus it is possible that a similar mechanism is promoted by the strongly cationic chitosans. Complete protection offield grown egg-plant against Botrytis spp. They also reported protection of vegetables (e. [54] examined the antibacterial effect of water-soluble chitin derivatives. stolonifer. whereas N-trimethyl derivatives ofpartially deacetylated chitins inhibited bacterial growth more strongly than the partially deacetylated chitins. N-trimethyl derivatives of partially deacetylated chitins and chitosan oligomers in vitro. g. They also suggest that although a strict correlation between polysaccharide chain length and inhibition of bacterial growth was not found in his study. other authors have reported different antibacterial effects according the type of chitosan used.

An increase in phenolic compounds corresponds to an increase in some plant enzymes such as PAL [39. glutamate dehydrogenase (GDH) and glucose-6-phosphate dehydrogenase (G6PDH) were resolved by native polyacrylamide gel electrophresis (PAGE). Chitin oligosaccharides have been used as elicitors of chitinase activity in melon plants [49]. also demonstrated the effects of chitosan oligomers and chemically modified chitosan and chitin in triggering molecular signals. [37] in addition to chitinase. These enzymes were found in the growth medium and in subcellular parts. They reported hexamer and nonamer as the most efficient elicitors. flavus treatments inconsistently enhanced G6PDH activity. initially and near the end of the experiment. PAL is a key enzyme in the phenylpropanoid pathway in plants which produces phenolics inc1uding phytoalexin (antifungal compounds) [39. [26] demonstrated changes in isozymes and protein molecular weights in mature peanut seeds treated with chitosan and/or the fungus Aspergillus flavus.A. lysozyme and a-mannosidase activities in Rubus hispidus cultured in vitro. These results also concur with those ofother authors [28. Walker-Simmons and Ryan [57]. chitosan and A. 60]. Marked enhancement of phenolic compounds (phytoalexin pecursors) and chitosanase was reported after treating the germinating seeds with chitosan. induced phenylalanine ammonia-Iyase (PAL) along with accumulation of phenolic acids in cultured carrot cells treated with chitinase or fungal mycelial walls. personal communication]. Kurosaki et al. anodic perodixase (PRX) and shikimate dehydrogenase (SKD) were also determined. The disease resistance response in peas was correlated with increase in the activity of the fungal wall-hydrolyzing enzymes (e. endo-B-glucanase and endo-chitinase) and with de novo increases in the messenger RNA (mRNA) for and synthesis of phenylalanine ammonia lyase. The combined pre- . although their activities varied according the localization. [5] determined chitinase. Cinnamyl alcohol dehydrogenase (CAD). Enzymes involved with the synthesis of phenolic compounds were analyzed. After 48 h. These results corroborate earlier reports in pea-Fusarium pathogen interactions regulated by chitosan [28]. 60]. L. Fajardo et al. and receptors to activate plant defense responses in tomato leaves.Antimicrobial action of exogenous chitosan 323 Effect 01 chitosans on plant enzymes in relation to resistance to fongal pathogens Chitosans trigger expression of plant enzymes other than chitinase/chitosanase.g. Polyphenoloxidases (PPO). and also in tissue cultures [15]. The effect of chitosan on plant enzymes and/or defense response against pathogenic and/or toxigenic fungi has been demonstrated in vivo in germinating peanut seeds. Hadwiger. thus suggesting chitinase elicitation by chitin oligosaccharides as an important element of molecular communication in host-parasite interactions. Bemasconi et al.

except a slight inhibition after 4 weeks when the experiment was stopped. which are involved in plant-pathogen resistance mechanisms. He suggested that despite the structural similarities of the potential substrates. Rapid uptake (within 12 h) of NCMC by tomato plants has been reported [12].jlavus. . Solid chitosan tends to act slower and also to be less potent biologically.324 R. A. jlavus enhanced the activity of all GDH isozymes at 3 h in moderately susceptible cotyledonary tissues of peanut seed (Starr variety). Seed tissues also responded to A. jlavus invasion by inducing PRX enzymes. The tetramers of glucosamine (GlcN)4 and of N-acetylglucosamine (GlcNAC)4 were compared for their eliciting potency in Rubus protoplast suspensions. and through reduction ofthe fungal population [14. Liquid chitosan is readily or immediately uptaken by microbial and plant cells as compared with slow uptake of the solid (usually powder form). Kombrink et al. The uptake of chitosan by microbial cells has also been determined through induction of chitosanase enzymes and phenolic compounds (within 8 h) in vitra and/or tissue culture after treatments with exogenous chitosan. jlavus and chitosan + A. since rates equal or higher than 1% of the material were necessary to obtain adequate control ofthe target nematode [50]. The report also showed the reduced potency of the solid chitinous material. after treatment with chitinaceous elicitors. 15. Fajardo et al.G. [36] also reported detection of N-acetyl-ß-D-glucosaminidase and chitinase in parsley extracts. g. tannin and quinone) and to the shikimic acid pathway. jlavus treatments. Cuero (unpublished data) used powder and flaky chitosan to control toxigenic A. 17]. as compared with solutions of chitosan in acetic acid [34]. while a-amylase was not elicited [38]. Cuero sence of chitosan + A. Powdered chitinlchitosan or whole crab shell showed no efficacy. There are reports on the delayed action of solid chitinous material against nematodes in soil under greenhouse conditions. Type of chitosan: liquid or solid Use ofliquid chitosan as an antimicrobial agent is more effective than solid form. and chitosan + A. [26] also reported enhanced PPO and SKD activity after treatment with chitosan. and by observing death of algal cells [19] and fungal cells through staining with trypan and microscopy studies. respectively. The report indicated that xylosidase elicited the stronger activation in presence of(GlcNAc)4 or (GlcN) 4. The effectiveness of powdered chitosan against bacterial strains and fungi has been tested. There was no immediate inhibition of fungal growth. These enzyme activities are linked to synthesis of polyphenolic compounds (e. jlavus growing in maize and peanut kemels. N-acetyl-ß-D-glucosamidase(s) and chitinases are separate enzymes in parsley with distinct substrate specificities. and laminarase increased after glucosamine treatment only.

17] (Fig. This result is supported by Tronsmo et al. in agar media and B. which sometimes requires additional application. Isolation of inducible chitosanase from Bacillus spp. Chitosans prepared with acetic acid can be kept longer than 3 years even at room temperature without loosing antimicrobial activity. 17]. whose main antimicrobial mechanism seems to be induction of chitosanase only. jlavus produced chitosanase markedly [16. Bacillus. However. bacteria and fungi. this explains the higher efficacy of chitosan-acetate when applied to substrates such as seeds with high fungal contamination. subtilis.g. Perhaps. which includes crustacea. as compared with chitosan prepared with lactic acid which kept its full strength less than 3 years under room temperature. as compared with lactate chitosan. phenolic compounds and blocking nutrient availability of the microbial cells. 14. Streptomycetes) produce chitosan in their cell walls and exhibit antimicrobial activity [12. Many bacteria genera (e. 58].Antimicrobial action of exogenous chitosan 325 Also. bacteria. bacteria. 47 a. circulans in liquid media have been reported [20. Substantial antifungal activitity by bacterial and actinomycetes chitosanases induced by exogenous crustacean chitosan in germinating maize and peanut seeds was demonstrated. Fungal chitosan has also been reported as an antimicrobial agent [12. Fenton and Eveleigh [27] reported fungal chitosanase from Penicillium islandicum. Osuji. Serratia) and actinomycetes (e. the type of acid used in the preparation of chitosan solutions influences its antimicrobial activity.g. although some Bacillus species including a strain of B. 4). here I will mention only the most common sources of chitosan used for antimicrobial activity. Maize and peanut plants inoculated with A. Cuero and G. jlavus under field or laboratory conditions were treated with exogenous crustaceans chitosan alone or in duplex or tripie combina- . as compared with microbial chitosan. fungi. G. 16]. Crustacean chitosan seems to exhibit more diverse antimicrobial mechanisms. Chitosans prepared with acetic acid exhibit more immediate antifungal effects as compared with chitosans prepared with lactic acid [12. However. R. 5). Source of chitosan influencing its antimicrobial activity Source of chitosan is mainly found in crustacea. including induction of chitosanase. In agar plates. crustacean chitosan induced more chitosanase than the microbial chitosan. actinomycetes and fungi induced more chitosanase in germinating com and peanut seeds than crustacean chitosan. [56] who found growth inhibition of Fusarium oxysporum by crustacean chitosan with high viscosity and deacetylation percentage in vitro. algae and protozoa [32]. Chitosanase production was clearly demonstrated in SDS-PAGE (Fig. unpublished data]. 16]. The production of chitosanase corresponded with antifungal activity [12. depending upon the substrate and/or host where microorganisms are growing. 14. and also a strain of A.

Pseudomonas aeruginosa. However.G. Marked antifungal activity corresponded with high production of chitosanase by the microorganisms. The type of bacterial strain in mixtures 1 2 3 4 5 6 Figure 5. 2: B. and also in comparison with crustacean chitosanase. . unpublished data]. thus resulting in reduced enzyme production. the growth of A. subtilis tested. subtilis #2. 4: Chitosan. G. Aeromonas hydrophila. jlavus chitosanase in maize and peanuts after treatment with exogenous crustacean chitosan. the number of the enzyme polypeptides was influenced by the type of seed (e. Osuji. jlavus yielded more chitosanase than mixtures ofthe crustacean chitosan with any of the two bacterial strains of B. there is an antagonistic effect between fungal and bacterial chitosan in inducing chitosanase. antifungal activity was enhanced by microbial treatment in combination with the crustacean chitosan. 6: BS# 1 + chitosan. g. plus crustacean chitosan. jlavus. Cuero tion with bacteria B. R. 5: A. The production of chitosanase was also markedly higher in mixtures of Bacillus sp. 3: B. Chitosanase induction in corn seeds at 0. The best single antifungal treatment ws crustacean chitosan alone followed by Bacillus sp. jlavus over other microorganisms that corresponded with high chitosanase production by the toxigenic A. The most effective antimicrobial combined treatments were those carrying Bacillus sp. 1995. subtilis # 1. 16. Cuero.jlavus was completely inhibited. while inhibition ofbacterial growth was too inconsistent to be considered as definite results [12. 3) and that there is a synergistic effect between exogenous native chitosan and microbial chitosan in inducing chitosanase. monocotyledon or dicotyledon). In SDS-PAGE studies ofthe enzyme. Cuero and G. and also in combination with actinomycetes Streptomyces griseus. the peptide profile of the microbial chitosanase was different among all the different microorganisms used. However. Streptomyces showed the highest chitosanase production followed by Bacillus. licheniformis. However. jlavus completely. R. jlavus. and Aeromonas. G. Although crustacean chitosan alone inhibited growth of A. Cuero and Osuji [16] induced high production of A. Plain chitosan induced more chitosanase polypeptides than synthetic glycolchitosan in SDS-PAGE (Fig. unpubJished data.90 Aw after treatment with: 1: water. P jluorescens. resulting in great growth dominance of A.326 R. However. Mixtures of crustacean chitosan with fungus A.

Left: Discs containing the fungus were placed on water-agar. When two strains of B. G.327 Antimicrobial action of exogenous chitosan C. WATER CHlr Figure 7. there was no chitosanase induction.lAS ~ IN D II. subtilis (# 1 and #2) were independently mixed with crustacean chitosan.. the combination of one ofthe B. (WA+fR) Figure 6. subtilis strains (#2) with the crustacean chitosan yielded three times more chitosanase than the combination of B.I-IITOSA A. 1990. Chitosanase induction by Aspergillus jlavus in chitosan-agar. Fungal inoculum was placed in each disc (3). subtilis # 1 with the crustacean chitosan (Fig. Induction of chitosanase by Bacillus species in chitosan agar R. there was marked production of chitosanase. Cuero et al. 4). unpublished data. .()A! BJlCIII"$ S~~~I COHTIfOt. with crustacean chitosan also influences the induction of chitosanase."'. Right: Discs containing the fungus were placed on chitosan-agar.

Thus antifungal activity is more marked in dicotyledons (e. Also. soybeans. The antifungal activity is more marked in peanut seed host than in maize. peanuts.328 R. its duration period outside on the surface of the plant where some microorganisms reside is shorter than NCMC.g. 17]. circulans.) than in monocotyledons (e. The degree of antifungal activity in vegetables is between peanut and maize seeds [16. 17]. vegetables etc.G. Chitosan pentamer and heptamer are effective in inhibiting some fungal germination and growth. Chitooligosaccharides with varying degrees of polymerization displayed low antimicrobial activities against B. flavus growth in peanut and maize seeds as compared with chitosan with higher deacetylation. Cuero et al. 17]. This perhaps explains the sometimes short-lasting effet [12. flavus was found in peanut seeds than in maize [16. exogenous chitosan with lower deacetylation induced better antifungal activity and chitosanase production in bacteria and actynomycetes in peanut and maize seeds. which lasts longer outside on the surface ofthe plant. This antimicrobial activity corresponds with the production of chitosanase and/or phenolic compounds in the host. g. Tokura et al. Peanut and vegetable plant parts seem to uptake native chitosan faster than monocotyledons. Exogenous chitosan treatment with lower deacetylation exhibited better inhibition of toxigenic A. The best growth inhibition of A. and the need to reapply it frequently. 16. coli. Other workers also reported chitosan with a low percentage of deacetylation and high viscosity exhibiting antifungal activity against plant pathogens [56]. Thus the type of chitosan along with the their degree ofpolymerization can affect the antimicrobial activity of chitosan against microorganisms residing outside on the surface of the plant. The degree of antimicrobial activity corresponded to the degree of chitosanase production. Types of hosts (plants) and their concentration of hexosamine: Monocotyledons and dicotyledons The antimicrobial activity of exogenous chitosan also depends on the type of host where the microorganisms are growing. Cuero Degree of deacetylation and polymerization The antimicrobial activity of chitosan has been correlated with its degree of deacetylation and polymerization. Yalpani et al. Derivative NCMC us uptaken by the plant more slowly [16. Since native chitosan is quickly and readily uptaken by the plant. Both the antifungal and host-inducing resistance properties decreased proportionally as the degree ofpolymerization decreased [33]. and high activities against E. [55] conc1ude that chitosan oligomers block nutrient permeation through the cell wall. 17] of chitosan in host plants. maize). [12] demonstrated chitosan uptake by . Inhibition ofthe fungus Botrytis cinerea in tomato was also marked [16]. [59] reported different effects of chitosan derivatives with different degrees of polymerization.

seeds produce more phenolic compounds after chitosan treatments. and in the flowering stage of the plant [12. especially against fungi growing in p1ants with different pRs. as compared with fruiting and/or senescent p1ants. In aseparate investigation. this also corresponded with the levels of chitosanase extracted and with antimicrobial action. Growth of A. 15-17]. thus inhibiting funga1 growth. Cuero et al. NCMC functions at a wider pR range.flavus growth by NCMC in vitra under liquid conditions at pR changing from 5. Also. Cuero (unpublished data) found more hexosamine in peanut seeds than in maize.5 and 5 as an antimicrobial agent.1 to 9. [12] demonstrated effective suppression of A. during earlier germination periods. fruiting and senescence Chitosan and N-carboxymethyl chitosan exhibit better antifungal effects in germinating seeds. and also in field flowering p1ants. This enhances its versatility as an antimicrobial agent. Stage of plant development: germinating. . Extrinsic factors influencing chitosan antimicrobial activity pH It is weH established that pR is one of the factors influencing growth of microorganisms in a substrate and in any host. Cuero and Lillehoj [19] reported efficient algistatic and algicidal effects ofNCMC at different pRs ranging from 7. Perhaps this is due to the highest hexosamine content of the plant during germination and flowering. as compared with native chitosan.1. The function ofNCMC at a wider range of pR is a result of its amphiphilic property. the concentration of hexosamine in the fungus [11] and in the host influence the efficacy of exogenous chitosan as an antifungal agent in plants. affects the efficacy of the antimicrobia1 agent. thus interacting with exogenous chitosan and consequently inducing more endogenous chitosan to inhibit fungal growth. whi1e others are basic. which functions better at pR 5 or> 5. although it functions better at pRs between 3. as compared with senescent p1ants.5 (initial) to 3.5 (final). flowering. The fact that certain plant parts are acidic. Also. NCMC is more water-soluble than chitosan and has better chelation properties [46] than chitosan [lla].Antimicrobial action of exogenous chitosan 329 tomato plants within short time periods (12 h). flavus was marked1y inhibited in peanut and maize seeds when chitosan was app1ied in germinating seeds.

Concluding remarks The use of chitosans as antimicrobial agents is a clear example of successful biological control. Chitosanase from Bacillus sp.93 water activity (A w) than at 0. This antifungal activity corresponded with higher chitosanase production. are still both equally influenced by the water content of the host and/or substrate. Also.95 [17].80 Aw. The degree of the effect and mode of action of chitosan varies according to the microorganism targeted. unpublished data.G. including algae.85 Aw. 1 2 3 4 5 1. Chitosans exhibited better antifungal effects in maize and peanut seeds at 0. and the environmental conditions at the time of the interactions. R. bacteria.6 = PEANUT 6 Figure 8.90 or 0. chitosan showed better inhibition of A. is necessary for practical application of exogenous chitosan as an effective antimicrobial agent. Undoubtely. G. The responses of the microorganisms to the chitosan as an antimicrobial agent also depend upon the chemical makeup ofthe chitosan used. Cuero. A clear understanding of the biological activity of the different units andlor oligomers of the polymer chitosan. = .2. and the fundamental baseline of the interactions between intrinsic and extrinsic factors. protozoa and viruses. Cuero Water activitylwater content 0/ the host andlor substrate Water activity or water content of the substrate influenced the efficacy of chitosan on inhibiting fungal growth in monocotyledon and dicotyledon plant seeds [17].330 R. in corn and peanut at different water activities: 1 & 4 0.5. 2 & 5 = 0.3 = CORN 4. fungi. Although derivative chitosans such as NCMC tend to be more hydrophilie than native chitosan. jlavus growing in nonirrigated field peanut and maize plants than in irrigated plants [12]. chitosan is a versatile compound endowed with antimicrobial activity affecting growth and physiology of most microorganisms.

Wiley-Liss. Academic Press. Bordeaux. colleague and friend the late Dr. Vercellotti J (1978) Hexosamine biosynthesis and accumulation by fungi in liquid and solid media. In: F Fleurat-Lessard.459 Nov. Osuji G. J Agri Sei 117: 165-169 14 Cuero R.rzfth international working conference on stored-Produc. IT. Osuji G. 1998 17 Cuero R. Pettit R (199Ia) Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents. New York. Carbohydr Res 61: 529-543 11 a Cuero RG (1996) Enhanced heavy metal immobilization by a bacterial-chitosan complex in soil. J Zikakis (eds) Advances in chitin and chitosan. Osuji G. Duffus E. Food Biotechnol 5(2): 95103 . Toppan A (1986) Plant chitinases and lysozymes. Waniska R. US Patent N. Osuji G. Pilet P (1986) Purification of large amounts of lysozyme with chitinase activity from Rubus hispidus cultured in vitra. In: RCW Berkeley. References 1 Allan CR. P Ducom (eds): Proceedings ofthe. Phytopathology 84: 1432-1444 4 Berkely R (1979) Chitin. Elsevier Science Publishers. 3. 277 -282 15 Cuero R. Roby D. New York. 4119-4132 16 Cuero R. FoodAdditives and Contaminants 12(3): 479-483 16a Cuero R.Antimicrobial action of exogenous chitosan 331 Acknowledgements This chapter is written in memory of my dear former professor. 5. In: Biotechnology and plant disease control. I. Oppenheim A (1993) Genetic engineering of microorganisms for improved biocontrol activity. LaFontaine PJ. 205-236 5 Bemasconi P. Chapter 8: 39-156. C Jeuniaux. Plenum Press. and taxonomy of fungi. GW Gooday (eds): Chitin in nature and technology. A. In: RAA Muzzarelli. John Lacey. Broglie R. AnnuRevMicrobiol22: 87-108 3 Benhamou N. Fajardo J. Wiley-Liss. Protection. Osuji G (1993) Chitosanase induction in maize and peanuts: enzyme induction factors. Amsterdam. Plenum Press. Exper Mycol3: 285-287 2 Bartnicki-Garcia S (1968) Cell wall chemistry. jlavus mechanism for growth dominance over associated fungi and concomitant aflatoxin production. Osuji G. 234-236 7 Broglie K.830. Duffus E (1990) Aflatoxin control in postharvest com kemeIs: effects of chitosan and Bacillus subtilis. 353 lOChet I. who was a great microbiologist with incisive understanding of microbial control. 9-14. Page BBC. Hadwiger LA (1979) The fungicidal effect of chitosan on fungi of varying cell wall composition. Duffis E (1992) Enhancement ofphytoalexin and chitosanase by chitosan in germinating peanuts: biocontrol of toxigenic fungi and mycotoxins. DC Ellwood (eds): Microbial polysaccharides and polysaccharase. Barak Z. Chet (ed). Nicole M (1992) Induction of systemic resistance to Fusarium crown and root rot in tomate plants by seed treatment with chitosan. In: RAA Muzzarelli (ed): Chitin enzymology. France. water potential and biomass in tomate plants. Shepherd R (1997) Chitosan coating for inhibition of sc1erotinia rot of carrots. Chet I (1993) The role of cell wall degrading enzymes in fungal diseases resistance. NewYork. Behamon N. Jolles P. Duffus E (1992) N-carboxymethylchitosan: uptake and effect on chlorophyl production. Inc. 83-91 6 Bemasconi P. Inc NewYork.211-235 11 Cochrane TW. Biotech Lett 18(5): 511-514 12 Cuero RG. New Zealand J Crop Hort Sei 25: 89-92 9 Chet I (1993) Biotechnology in plant disease control. C Jeaniaux. In: RAA Muzzarelli. Biotechnol Lett 13(6): 441-444 13 Cuero R. Atec Grottammare. WashingtonA (1991) N-carboxymethylchitosan inhibition ofaflatoxin production: Role ofzinc. London. GW Gooday (eds) Chitin in nature and technology. September 19901: 279-290 18 Cuero R. P Sandford.. NewYork 8 Cheah LH. In: I Chet (ed) Biotechnology in plant disease contral. In: C Brine. chitosan and their degradative enzymes. Osuji G (1998) Effective Plant Biocontrol. Pilet PE. Wiley-Liss. Jolles P. morphogenesis. Esquerre-Tugaye MT. Osuji G (1995) Aspergillus jlavus-induced chitosanase in germinating com and peanut seeds. GW Gooday.

food processing. Wilson CL (1996) Bioactive coating for harvested commodities. Co Inc. Phytopathology 75: 774-777 30 Hirano S. and biotechnology aspects of chitin and chitosan: A review. and related enzymes. In: G Skjak-Braek. Iwahori S (1997) Effects of chitosan coating on the storage of peach. Poulicek M. Muzzarelli B. JP Zikakis (eds): Advances in Chitin and Chitosan. Atec. Gemma H. Grottammare. Wagooner LA (1983) Physiol Plant Pathol23: 163 40 Mattioli-Belmonte M. In: RAA Muzzarelli (ed): Chitin enzymology. Atec. Hayashi M. Cuero 19 Cuero R. Meyer R. Pettit R (1994) Effects of chitosan and Aspergillus jlavus in isozymes related to phenolic compounds synthesis and pro tein profiles of peanut seeds. Academic Press. 543-548 35 Knorr D (1986) Nutritional quality. Christian D. Waniska R. Asse1in A (1992) Potential use of chitosan in postharvest preservation of fruits and vegetables. Prac Nat Acad Sei USA 92: 4095-4098 22 Du J. chitosan. increases crop yoeld. In: JP Zikakis (ed) Chitin. Food BiotechnoI8(2&3): 213-228 27 Fenton DM. Amsterdam. and immobilization. In: JP Zikakis (ed): Chitin. Ryan CA (1995) Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoic pathway. Fristensky B. estimated from new data on chitin biomass and production. Waniska R. Amer Nat 111: 691-713 32 Jeuniaux C. Dubois R. Yamamoto T (1989) Chitinase activity of some seeds during their germination process and its induction by treating with chitosan and derivatives. New York. 440-452 24 EI Ghaouth AJ. J Japan Soc Hort Sei 66: 15 . Exp Mycol8: 276-281 33 Kendra D. Carbohydrates in Europe 19. Hadwiger LA (1984) Characterization ofthe smallest chitosan oligomer that is maximally antifunga1 to Fusarium solani and elicits pisatin formation in Pisum sativum. P Sandford (eds): Chitin and chitosan. Cuero R. Pettit R (1995) Phenolic compounds in peanut seeds: enhanced elicitation by chitosan and effects on growth and aflatoxin BI production by Aspergillusjlavus.G. pear and kiwifruit. Dauphin. Hahlbrock K. In: G Skjak-Braek. Riggleman R (1984) Chitosan. Gautier C. P Sandford (eds): Chitin and chitosan. Jr (1985) Encapsulation of potential biocontrol agents in an alginate-clay matrix. 743-747 31 Hildreth EL. Grottammare. Syrovetz T. Elsevier Applied Science. Domard A (1993) Tetramers of chitin (chitosan) as elecitors in Rubus protoplasts. Plant Ce/I Physiol 27(8): 1587-1591 38 Lienart Y. Hadwiger L (1989) Chitosan oligomers from Fusarium solani/pea interactions. JP Zikakis (eds) Advances in chitin and chitosan. Weiler EW. Witte B (1993) Properties and expression patterns of chitinases in potato and parsley. Janzen DH (1977) Why fruits rot. Eveleigh D (1981) Purification and mode of action of chitosanase from Penicillium islandicum. seed mold and meats spoil. Elsevier App Science. In: RAA Muzzarelli (ed): Chitin enzymology. Biotechnol Lett 4(4): 275-280 20 Davis B. chitinaselB-glucanase digestion of spore1ings and from fungal wall chitin actively inhibit fungal growth and enhance disease resistance. In: CJ Brine. Food BiotechnoI9(1&2): 59-78 26 Fajardo J. Hadwiger LA. chitosan and related enzymes. Process BiotechnoI21(3): 90-92 36 Kombrink E. Physiol Mol Plant Pathol35: 215-230 34 Kifune K (1992) Antimicrobial activity of chitosan derivatives. PA Sandford. Lumsden RD. Academic Press. 245-256 37 Kurosaki F. Elsevier. In: CJ Brine. 271-276 39 Loschke DC.332 R. Bussers JC (1989) Sources of chitin. NJ. a natural regulator on plantfungal pathogen interactions. Nishi A (1986) Induction of chitinase and phenylalanine ammonialyase in cultured carrot cells treated with fungal mycelial walls. PCT Pat Appl WO 96-13985 25 Fajardo J. T Anthonsen. Eveleigh D (1984) Chitosanases: occurrence. Elsevier. Connick WJ. New York. Tashiro N. NewYork. 3-11 32a Kendra FD. Lillehoj E (1990) N-carboxymethy1chitosan: Aigistatic and algicidal properties. Cuero R. Princeton. London. Marois JJ. December 1997 . Nishida T. T Antonsen. Kirsch C.22 23 EL Ghaouth. Muzzarelli RAA (1997) Chitin and chitosan in wound healing and other biomedical applications. N ew York 21 Doares SH. Ami AJ. Voss-Foucart MF. 292-301 29 Fravel DR. J Gen Microbiol126: 151-165 28 Hadwiger LA. PA Sandford.

Toppan A (1987) Chitosan oligosaccharides as elicitors of chitinase activity in melon plants. T Anthonsen. Biotechnol Bioeng 22: 885-896 45 Muzzarelli RAA. NewYork. Carbohydr Polym 9: 1-21 44 Muzzarelli RAA. P Sandfor (eds): Chitin and chitosan. In: J Bailey. Princeton. Ryan CA (1984) Proteinase inhibitor synthesis in tomate leaves.. 45-256 57 Walker-Simmons M. Biochem Biophys Res Commun 143(3): 885-892 50 Rodriquez-Kabana R. EIsevier Science Publishers B. Shigemasa Y (1992) Various biological effects of chitin derivatives. Matthews DE. Johnson F. A Nakamura (eds): New macromolar architecture and fonctions. P Sandford (eds): Chitin and chitosan. Saimoto H. In: C Brine. In: CJ Brine. T Anthonsen. Miyazaki S. Veno K. Antimicr Agents Chemoth 34: 2019-2023 46 Muzzarelli RAA (1988) Amphoteric derivatives of chitosan and their biological significance.Antimicrobial action of exogenous chitosan 333 41 Mauch F. Scarpini G (1980) Chelating. P Sandford. 206-215 55 Tokura S. Young RW (1989) Chitinous materials from blue crab for control ofroot-knot nematode. In: G Skjak-Braek. Sashiwa H. 181 . J Mansfield (eds): Phytoalexins. Elsevier Applied Science. In: G Skjak-Braek. Robinson LE (1992) Antimicrobial activity of some chitosan derivatives. Biochem Biophys Res Commun 174 (2): 489-491 52 Sneh B. Skaugrud 0. Henis Y (1971) Phytopathology 61: 1113-1117 53 St Angelo Al. Plant Science. P Sandord (eds): Chitin and chitosan. film-forming. Tarsi R.790 58 Yabuki M (1988) Characterization of chitosanase produced by Bacillus circulans MHKl. NewYork. 87-99 47 Muzzarelli RAA (1985) Chitin. Effect ofurea and enzymatic studies. In: G Skjak-Breaek. I. NewYork. Ruprecht RM (1991) N-carboxymethylchitosan-N. J Zikakis. v. Boller T (1988) Antifungal hydrolases in pea tissue. Mauch B. Grottamare. Amsterdam. Plant Physiol 88: 936-942 42 Muzzarelli RAA (1977) Chitin. Elsevier Applied Sciences. Azely F. Tanfani F. Oxford 43 Muzzarelli RAA (1988) Carboxymethylated chitins and chitosans. and coagulating ability ofthe chitosan-glucan complex fromAspergillus niger industrial wastes. In: RAA Muzzarelli (ed): Chitin enzymology. Nishi N (1996) Molecular weight dependent antimicrobial activity by chitosan. Smith DA (1982) Metabolism ofphytoalexins. In: GO Aspinal (ed): The polysaccharides. Pergamon. 308-319 48 Pospieszny H. Boube D. Elsevier Applied Sciences. Plant Physiol76: 787 . London. Vercellotti IR (1992) Preparation and use of food grade N-carboxymethylchitosan to prevent meat flavor deterioration. Berlin 56 Tronsmo A. New York 59 Yalpani M. 3: 417-450 47a Muzzarelli RAA (1989) Amphoteric derivatives of chitosan and their biological significance. Harman GE (1993) Use of chitin and chitosan in biological control ofplant diseases. Varaldo PE (1990) Antimicrobial properties of N-Carboxybutyl chitosan. Springer. In: M Kamachi. Tanaka Y. NewYork. Katan J. Neotropica 19(1): 53-74 51 Sosa MA. 711-722 54 Tanigawa T. Amsterdam. Elsevier. lAnthonsen. Koch JA. Gadelle A. Elsevier App Sciences. Atabekiw JG (1989) Effect of chitosan on the hypersensitive reaction ofbean to alfalfa mosaic virus. 543-548 60 Van Etten HD. Academic Press. Vercellotti Sv.O-Sulfate as an anti-HIV-l agent. P Sandford. Biagini G. Giovanetti E. JP Zikakis (eds): Advances oj chitin and chitosan. John Wiley & Sons. Atec. NJ. 62: 29-31 49 Roby D. Advances in chitin and chitosan. Filippini 0. In: G Charalambous (ed): Food science and human nutrition.

bacterial 279 cellulose 44 cellulose binding protein 149 Chagas' disease 223. agricultural 172 blackfly 225.48 Asburner's model 90 atherosclerosis 293 ATPase 44 avermeetin 88 Beeil formation 282 Beeil growth cell wall skeleton (BCG-CWS) 285 bacteria.55 cell wall. polymorphie form of 1 chitin. bacterial 282 Anax inmaculifrons 46 angiogenesis 255 animal clinic 274 antibody. colloidal 2.O-carboxymethy1chitosan 260 carboxypeptidase 299 carotenoid 48 CD44252 cell division 75 cell migration 80 cell wall 39.237 ß-N-acetylgalactosaminidase 236 ß-N -acetylgalactosaminidase. fossil 4 chitin. 119 N-acetyldopamine 47 N-acetylglucosamine 114. 39 chitin. chitinolytic 160 bacteria. cyst formation 231 allosamidin. supramolecular structure of 13. ophthalmie administration of 306 agriculture I 71 N-ß-alanyldopamine 47 alginate 311 allergy 282 allosamidin 46. 119. gycol 119 chitin. 148 N-acetyl-D-glucosaminidase 235. 24. non-competitive inhibition 205 Alteromonas 237 aminopterin 88 amoeba 50 a-amylase.4-linked N-acetylglucosamine (GleNAc) 72 N-acetylglucosaminidase 114.322 ABA-N-acetyl-tyrosine 282 Acanthocheilonema viteae 224 acetylation. 45 cancer 239 Candida albicans 299 carboxyl ester lipase 299 N. 119.265 a chitin 44. 202. regeneration of 259 bovine serum albumin (BSA) 310 Brachiopoda 48 brefeldin 88 Brugia malayi 224 Brugia pahangi 224 buprofezin 88 C5a 288 calcium 48 Calcofluor 62. colonic 301 benzoylphenylurea 86 bile acid 293 bile salt 299 biocompatibility 252 biodegradability 251 biological control 172 biopesticide 172 biotechnology.238. in biosphere 4 chitin.335 Subject index N-acetylchitohexaose 288 N-acetyl chito-oligosaccharide 112. 188. 230 allosamidin.251 N-acetyl-ß-D-glucosaminidase. 227 bone marrow 285 bone tissue. alkali 3 chitin. molecular weight of 2 chitin. tensile mechanical property of 45 chitin acetylase 45 chitin binding protein 149 . circulating 279 Aphrodite 44 apicectomy 259 Apis cerana indica 46 Arachnida 46 Arthropoda 46. urinary excretion of 241 endo-N-acetylglucosaminidase 146 N-acetylhexosaminidase 114.227 chitin 73. mixed-type inhibition 205 allosamidin. 113. concerted action of 18 ß-l. competitive inhibition 204 allosamidin. solubility of 2 chitin. 97 Calcofluor white (CFW) 22. 119 chitin.97 ßchitin 44. 97 ychitin 44 chitin. degree of 1 acromegaly 239 acyclovir.235 N-acetylmuramic acid 114. clinical use of 251 chitin.

46. reaction components of 10 chitin synthase. family 18 113. 224 chitinase. Brugia malayi 229 chitinase. microorganism 111 chitinase.57. reaction mechanism 113 chitinase. venom 162 chitinase. octopus 162 chitinase. 254 chitosan 2.114. family 19 114. algae 111 chitinase.202 chitinase. mammal 111 chitinase. Killer toxin 158 chitinase. plasmid 158 chitinase. glycoprotein 112 chitinase. inhibitor of 113.56. virus 158 chitinase action. encystation 230 chitinase. saliva 162 chitinase. 154 chitinase. role in transmission ofparasite 226 chitinase. 185. insect 111 chitinase. methylpyrrolidinone 252 chitosan heptamer 328 chitosan microsphere 308 chitosan oligomer 191 chitosan pentamer 328 chitosan salt 3 chitosan/mucin interaction 309 chitosan-alginate 310 chitosan-inducible gene 185 chitosanase 47. inhibition of 21 chitin synthase. algal 301 chitosan. 126. crustacean 111 chitinase. stage-specific 231 chitinase. 188. 63-65 chitin synthase. isoelectric point 112 chitinase. glycoconjugation of 30 chitin synthase. 126. filarial 228 chitinase. Acanthocheilonema viteae 229 chitinase. exo-type 116 chitinase. 3D model of 27 chitin synthase. splitting pattern 116 chitinase.57-59 chitin synthase 11 (CSII) 55.202 chitinase. fungi 112 chitinase. structure of 25 chitin synthase allostery 14 chitin synthase co-operativity 14 chitin synthase (CHS) gene 55. N-carboxymethyl 3 chitosan. plant 111. 230 chitinase. parasite 161. Plasmodium 162. 163 chitinase. c1ass IV 119 chitinase. 148 chitosanase. mollusk 111 chitinase. 164 chitinase. stability of 112 chitinase. 230 chitinase. 15 chitin synthase.58. c1ass III 119 chitinase. seaweed 111 chitinase. mechanism of 130 chitinase isofonn 230 chitinase like domain 212 chitinolysis 46 chitobiase 146 chitobiose 2. vertebrate 111. 18 chitin synthase.230 chitodextrinase 146 chito-oligomer 44. 165 chitinase. optimum pR 112 chitinase. 66. purification of 15 chitin synthase. kinetics 119 chitinase. fungal 301 chitosan.307 chitinase.61 chitin synthesis 18 chitin synthesis inhibitor 86 chitin synthetase 39 chitinase 45. 137. priming of 15 chitin synthase. nematode 162 chitinase. amino acid sequence 26 chitin synthase. arthropod 111 chitinase. c1ass 46 127 chitosome 41 chitotriose 230 . 85 chitin synthase I (CSI) 55. 4-methylumbelliferone. 230 chitinase. amino acid sequence of 137 chitinase.265 chitosan. human blood 165 Subject index chitinase.86 chitin chain elongation 10 chitin chain tennination 12 chitin deacetylase 46 chitin fibril 12 chitin microfibril 39 chitin suture 252 chitin synthase 56. endo-type 116 chitinase. c1assification of 137. 111. multiplicity of 24 chitin synthase. trypanosomatid 162 chitinase. amphibian 111 chitinase. mycoparasitic fungi 159 chitinase. regulation of 14 chitin synthase. latency of 16 chitin synthase. molecular size of 111 chitinase. bacteria 112 chitinase. activation of 14. Leishmania 226. 114. fungal 86 chitin synthase. transglycosylation reaction 119 chitinase.336 chitin biosynthesis 39.237 chitobiose. concerted action of 17. anomer fonnation 114 chitinase. mechanism of 146 chitinase. 58-60. 56. Entamoeba 229 chitinase. baculovirus 148 chitinase. fish 111. c1ass 11 114 chitinase. 62 chitin synthase III (CSIII) 55. activator 113 chitinase.235. 148.61. modular structure of 147 chitinase.

intestinal 300 erythrocyte 285 Escherichia coli 288 Eudragit 307 EuJolliculina uhligi 40 excystrnentprocess 225 exochitinasc 146 exoskeleton 39 exsheathment 225 extracellular matrix 254 fertilization 218 fibric acid 295 fibroblast 239. 160 galactomannan 48 gallstone disease 293 Gaucher's disease 243 gene delivery 310 Giardia 223 glucan 46 ß-I.91 egg.337 Subject index cholesterol 293 cholesterol7a-hydroxylase 294 cholestipol 294 cholestyramine 294 chondrocyte 217 chondroitin sulphate 261 Choristoneura hormone receptor 3 (CHR3) 90.55. hatching of 225.45. 286 cytosol 40 defense response 185 defense system 279 delivery.91 Choristoneura hormone receptor 75 (CHR75) 90.N-dimethy1acetamide 45 anti-dinitrophenyl serum 282 dinitropheny1-ova1bumine (DNP-OVA) 282 dipeptide cyclo(L-Arg-D-Pro) 205 diphtheria 311 disease resistance response 185 DNA 310 DNA degradation 190 drug delivery 305 dysentery.241 diatom 44 dietary fiber 294 diflubenzuron 86. comparison of 128 cpithelia. amebic 225 dystrophy. embryonic 218 DG42 77. cattle 225 filling agent 268 Flavobacterium meningosepticum 237 foot disease 272 fungi 40. basic 253 fibronectin type III-like module 149 filariasis.225 Entamoeba dispar 224 Entamoeba histolytica 225 Entamoeba invadens 50. 93 digitonin 41 N. 147 Congo red 2.6 glucan 48 ß-glucanase 188 glucosamine 260 a-glucosidase 241 ß-glucosidase 285 y-glutamyltransferase 241 glutaraldehyde 308. hapten-bound 282 colcemid 89 colon 307 colony stimulating factor 286 complement 279.228 eggshell 223 electron-microscopic autoradiography 40 elephantiasis 224 elicitor 187 endop1asmic reticulum (ER) 42 Entamoeba 223. 91 chromosome 1 217 chs gene 44 Ciliata 48 CM-chitin 119 CM-chitin.288 composite 39 concanava1in B 146. 311 glycohydrolase superfamily 129 glycol chitin 112 glycol chitosan 253 glycolipid 41 glycoprotein 85. 102 core structure 128 Crithidia Jasciculata 224 Crustacea 325 cuticle 47 cycloheximide 88 Cyclotella cryptica 301 cyromazine 88 cytokine 254. 224.60. 14C-Iabelled 285 CM-chitin.253. 229 Entamoeba.288 fibroblast growth factor. 88. oral 260 depression 242 dermal substitute 258 detergent 48 development. exo-type chitinolytic 114 enzyme structure.211 . 92.252 diabetes mellitus 239. cyst of 44 entopathogen 160 environment 171 enzyme.3 glucan 48 ß-I. corneal crystalline 293 ecdysone receptor (EcR) 90.

232 leishmaniasis. umbilical 271 hemia treatment 270 hevamine 113 hexosamine 329 histology 251 HMG-CoA reductase 294 host-parasite interaction 185 hyaluronan 77. microbial 300 lipase. visceral 225 lentinan 288 Leptomonas seymouri 224 leukemia 239 lipase 251. 286. porcine pancreatic 300 lipid.240 hypertriglyceremia 294 ICAM-l 252 immune system 279 immunoadjuvant 286 immunoassay. cutaneous 225 leishmaniasis.91 hypercholesterolemia 293 hypersensitivity. peritrophic 161 meniscus regeneration 25 metalloenzyme 316 MethA 291 mevalonate 294 Michael-type conjugate 47 Micrococcus lysodeikticus 113 microfibril 39 microfilaria. large 307 invertebrate 40 isothiocyanate.261 hyaluronan synthase (has) 79 hyaluronan synthesis 252 3-hydroxy-3-methylglutaryl CoA reductase 300 20-hydroxyecdysone(20E) 89. fluorescent 285 isozyme 323 juvenile hormone (JH) 90 keratan sulphate 261 kissing bug 227 large animal c1inic 271 lectin 101 lectin-type protein 44 Subject index leg ulcer 256 Leishmania 225 Leishmania braziliensis 224 Leishmania donavani 224 Leishmania in/antum 224 Leishmania major 224 leishmaniasis 223.251 macrophage 239. 41 Golgi cistemae 44 granulation 254 granulocyte 279 growth inhibition 291 HC gp-39 211 heavy metal 288 hemia. retaining 133 membrane. electron-microscopic 40 immunogenicity 282 infection 279 insect control 174 insect growth regulator 86 insect integument 47 insect vector 223 insulin 306 interferon 286 interleukin-l 254. exsheathment of 228 microsphere 288 microsphere. inverting 130 mechanism. bioadhesive 306 microvesicle 40 mineralization 48 MM46291 modulus 49 Mollusca 48 monensin 88 mosquito 224 mucin 215 mucoadhesive property 306 Mucor rouxii 40 mycoparasitism 176 Myriapoda 46 .226. delayed-type 282 hypertension 239. oviductal 211 glycoside hydrolase 137 Golgi apparatus 40.232 mannoprotein 48 mechanism. mouse peritoneal 279 macrophage activating factor (MAF) 291 macrophage activation 254 maize 325 malaria 223. 279 macrophage. 148. polar 41 lipochitin oligosaccharide (LCO) 71 lithium chloride 45 lithium thiocyanate 45 lithotripsy 242 Loligo 44 loricae 44 lymphedema 224 lysozyme 113.338 glycoprotein. neutral 41 lipid.299 lipase. 291 interleukin-2 291 interleukin-8 288 intestine.

protozoan 223 parasite. transmission of 231 pathogen. exocytic 40 Saccharomyces cerevisiae 40. development of 231 parasite. isopycnic 40 Sendai virus 288 Serratia marcescens 235.55-57. eggshell of 225 Onchocerca volvulus 224 ophthalmology 260 organ.237 sheath 225 sialic acid 306. regeneration of 270 . human 224 pathogen. 322 Onchocerca gibsoni 224 Onchocerca gibsoni. intestinal 223 pathogenesis-related gene 185 peanut 325 pectin 311 pentachloronitrobenzene 22 peptidoglycan deacetylase 46 peritrophic matrix (PM) 226 phagocyte 291 phenylpropanoid 316 phosphatidylcholine 41 phosphatidylserine 41 phospholipase A2 299 Phycomyces blakesleeanus 46 phytoalexin 316 plant.63 Sacculina rotundata 46 Sagitta 1 Sandhoff disease 243 Sanfilippo's syndrome 243 Sarcoma 180 291 scar formation 270 Schiff's base 47 Schizosaccharomyces pombe 46 sclerotin 48 sclerozation 47 sedimentation. chitin-binding 97 protein.147 nematode. chitinase-like 211 prothoracicotrophic hormone (PTTH) 89 protoplast. intestinal protozoan 225 parasite. 228 pogonophore 44 polio vaccine 310 poly(acrylate) 299 polyaminosaccharide 322 polyelectrolyte 316 polyene macro1ide 23 polyoxin-D 88 polypeptide 316 polysaccharide 47 Poteriochromonas 301 pregnancy 239 primulin 102 protein. fungal 49 protozoa 40 Ptinus 44 puromycin 88 pyothorax 270 o-quinone 47 p-quinonemethide 47 renal functionality 260 RHAMM 252 Rhizobiaceae 71 Rhizopoda 48 Rickettsia-like organism (RLO) 227 river blindness 224 route. masticatory 48 osteoinductive property 259 osteoporosis 259 overweight control 293 oviduct 211 oviductin 211 oxazolinium intermediate 133 oxocarbonium intermediate 133 6-oxychitin 4 parasite. artificial 269 skin. pathogenesis-related protein 163 plasmalemma 40 Plasmodium 226 Plasmodiumfalciparum 228 Plasmodium gallinaceum 224.339 Subject index nagstatin 237 narbonin 146. metazoan 223 parasite. emergence of 225 parasite. filarial 228 nephrology 241 Neptunes sanguinolentus 46 Neurospora crassa hyphae 40 nicotinic acid 295 nikkomycin 88 nitrite (HN02 ) 47 nitrogen content 2 nitrogen oxide 254 nodB gene 46 NodC 73 nodulation 73 NodZ 74 nonhost resistance 186 nuc1eoside-peptide 21 Oecophila longinoda 46 oligomer 288. 322 signal 187 silica 48 site-directed mutagenesis 130 skin.

104 tumor ce1l 288 tumor necrosis factor (TNF) 288 tumor necrosis factor-a 254 tumor proliferation 293 tunicamycin 88 turgor pressure 46 Subject index Ustilago maydis 48 ultraspirac1e (USP) 90. ecdysone-induced 91 transgene.253. 44 veterinary practice 265 Vibrio harveyi 237 vinblastine 89 vitamin.91 uranyl acetate 42 uridine diphosphate N-acetylglucosamine 39 vaccine candidate 228 vaccine therapy 310 vascularisation 255 vesic1e. apical 40 vesic1e.260 Wuchereria bancrofti 224 X-ray diffraction 45 YKL-40 213 zebrafish 76 zona pellucida 218 zoo animal c1inic 274 zygomycete 46 zymosan 279 . antimicrobial 172 Triatoma infestans 227 Tribolium castaneum 41 Trichoderma 176 Trypanosoma 227 Trypanosoma brucei 224 Trypanosoma cruzi 227 Trypanosoma lewisi 224 trypanosomiasis 232 trypsin 299 tryptophan 97.92 termite. microfilarial 223 wound dressing 253.plant 178 transglycosylation 39 transmission-blocking vaccine 224. antiparasitic 231 styloguanidin 205 substrate binding c1eft 130 16S subunit 41 synovial cell 217 tandem repeat 215 Tay-Sachs disease 243 tebufenozide (RH-5992) 89. 268 wound healing 251. 231. liposoluble 298 wall integrity 55 water activity 319 waxe 48 wheat germ agglutinin 102 wheat germ lectin 47 wheat germ lipase 300 worm. cortical 40. physogastric queen of 47 Thalassiosira jluviatilis 301 thyroiditis 239 trace metal ion 299 transcription factor. 232 treatment.340 skin substitute 258 spleen 282 spleen T cell 291 sporangiophore 46 spore germination 46 sterol 41 Stigmatella aurantiaca 237 strategy. filarial 224 worm.