Está en la página 1de 5

In Vitro Filament-like Formation upon Interaction

between Lens -Crystallin and L-Crystallin

Promoted by Stress
Orly Weinreb,1 Anke F. van Rijk,1 Ahuva Dovrat,2 and Hans Bloemendal1
PURPOSE. To determine whether -crystallin is capable of forming filament-like structures with other
members of the crystallin family.
METHODS. Water-soluble crystallins were isolated from calf lenses and fractionated into -, H-, L-,
and -crystallins according to standard procedures. Chaperone-like activity of -crystallin was
determined in control and UV-Airradiated lenses by the heat-induced aggregation assay of Lcrystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament
formation was examined by transmission immunoelectron microscopy using specific antibodies
directed against the crystallins. Involvement of intermediate filament constituents was excluded by
the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril
interaction test using thioflavin T (ThT) was also performed.
RESULTS. At the supramolecular level heating at 60C has no effect on the morphologic appearance
of -crystallin as observed by transmission electron microscopy. Moreover -crystallin obtained
from UV-Airradiated lenses shows a virtually identical shape. However, heating in the presence of
L-crystallin results in the formation of filament-like -hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against - or L-crystallin. Parallel
experiments with -crystallin derived from UV-Airradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to
show that the UV-light treatment affected the chaperone-like capacity of -crystallin, as
revealed by a diminished ability to inhibit in vitro denaturation of L-crystallin. To exclude the
presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were
also tested. These latter experiments were negative. The filamentous nature of the hybrids was
further confirmed by the results obtained with the ThT assay earlier applied for the detection
of amyloid fibrils.
CONCLUSIONS. Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called rods, may
result from stress-induced interaction between -crystallin and other lens constituents such as
L-crystallin. Because the hybrid formation is enhanced when -crystallin from UV-Airradiated
lenses is used as one of the two components of the hybrid, one can only speculate that this
formation may be one of the factors leading to UV-A cataract. (Invest Ophthalmol Vis Sci. 2000;
wo members of the small heat shock protein family,1,2
A- and B-crystallin, possess molecular chaperone
properties.3 A decade ago it became apparent that these
two polypeptides, which form 800-kDa aggregates in the lens,

From the 1Department of Biochemistry, University of Nijmegen,

The Netherlands; and 2B. Rappaport Faculty of Medicine, TechnionIsrael Institute of Technology, Haifa, Israel.
Supported by a Marie Curie Research Training Grant, Biotechnology, European Commission Science Research Development (Bio.4CT96 to 5121) (OW) and by Dr. Endre Balazs and The Biomatrix
Institute (HB).
Submitted for publication November 12, 1999; revised April 11,
2000 and June 12, 2000; accepted July 5, 2000.
Commercial relationships policy: N.
Corresponding author: Hans Bloemendal, Department of Biochemistry, University of Nijmegen, PO Box 9101, 6500 HB Nijmegen,
The Netherlands.
Investigative Ophthalmology & Visual Science, November 2000, Vol. 41, No. 12
Copyright Association for Research in Vision and Ophthalmology

also exist in a variety of other tissues.4 6 Current studies

indicate that small heat shock proteins like B-crystallin are
able to interact with intermediate filaments in response to
stress and to function as molecular chaperones.710 Earlier
ultrastructural observations showed that crude fractions from
chicken lens consisted of 5- to 6-nm-thick core filaments and
irregularly sized globular particles 15 to 20 nm in diameter
called beaded filaments.11 It was noted that the beads had
dimensions that were similar to native -crystallin.12 Moreover,
two proteins with molecular masses of 115 and 49 kDa, respectively (named filensin and phakinin), have been localized
in the beaded filament fraction of the lens with the aid of
immunoelectron microscopy.13,14 However, the question still
remains whether or not other lens proteins may be involved in
the formation of filamentous structures. In this report we
demonstrate that water-soluble -crystallin has the ability to
form, in response to heat stress, in vitro filament-like structures


Weinreb et al.

IOVS, November 2000, Vol. 41, No. 12

filtration on a Sephacryl S-300 (Pharmacia-LKB, Uppsala, Sweden) HR column.16

Chaperone-like Activity
The chaperone-like activity of -crystallin from control and
UV-irradiated lenses was determined by the heat-induced aggregation assay of L-crystallin at 60C.3 The proteins were
dissolved in a solution of 20 mM sodium phosphate, 100 mM
Na2SO4, 10 mM EDTA, at pH 6.9. The assay was performed at
a concentration of 0.25 mg/ml substrate protein and 0.05
mg/ml -crystallin.

Electron Microscopy
Protein samples from the heating assay were also analyzed by
electron transmission microscopy. Samples were negatively
stained with uranyl acetate (1% v/v). The in vitro filament
formation of -crystallin from control and UV-treated lenses
with L-crystallin was followed by immunoelectron microscopy using antibodies against vimentin, A-, B-, and L-crystallin. The grids were examined with a transmission electron
microscope (Jeol TEM1210, Tokyo, Japan) using 70 to 80 kV.

Thioflavin T Interaction Assay

Fluorometric determinations were carried out using the thioflavin T (ThT) interaction assay at excitation and emission of
450 and 482 nm, respectively.17

Electron Microscopy

FIGURE 1. Electron micrographs of (A) -crystallin obtained from control lenses (bar, 200 nm); (B) -crystallin from UV-Atreated lenses
(bar, 200 nm); (C) L-crystallin obtained from control lenses separately
heated at 60C (bar, 500 nm). Complexes were visualized by negative
staining with uranyl acetate.

Micrographs of mixtures of - and L-crystallin, obtained from

control lenses and -crystallin from UV-Atreated lenses, which
were separately heated at 60C, are shown in Figure 1. Apparently heating has no effect on -crystallin obtained from the
control lenses (Fig. 1A), because comparison with previously

with one other crystallin, namely L-crystallin. This filament

formation is enhanced by UV-A irradiation.

UV-A Irradiation
Lenses, excised from 2- to 4-year-old bovine eyes, were irradiated in special organ culture glass vessels described previously
by Dovrat and Weinreb.15 Briefly, a 400 W UV lamp (Vilber,
Lourmat Cedex, France) contained a filter that provided radiation of 33 J/cm2 for 75 minutes at 365 nm.

Fractionation of Crystallins
Lenses were dissected under a binocular stereomicroscope.
The lens cortex was homogenized in 100 mM Tris buffer at pH
7.5 and spun at 4C at 13,000g for 30 minutes. The supernatant
comprises the water-soluble lens fraction. Separation of this
fraction into -, H-, L-, and -crystallin was carried out by gel

FIGURE 2. Immunogold labeling with antiA-crystallin of samples

from the heating assay. 0.05 mg of -crystallin from control lenses
incubated with 0.25 mg L-crystallin at 60C. F, filament-like chains;
A, labeling (bar, 500 nm).

IOVS, November 2000, Vol. 41, No. 12

Filament-like Formation and Crystallin Interaction


FIGURE 3. (A) Filament samples

from the heating assay. -Crystallin,
0.05 mg, from UV-Atreated lenses
incubated with 0.25 mg L-crystallin
at 60C (bar, 500 nm); (B) filaments
at higher magnification (bar, 100
nm). F, filament-like structures; long
arrows in (B), labeling with antiAcrystallin.

published electron micrographs of nonheated -crystallin revealed no detectable morphologic differences.18 Normal
-crystallin consists of molecules having an apparent spherical
structure, with a diameter of approximately 17 nm. Likewise
-crystallin obtained from irradiated lenses shows a similar
shape, albeit the size is somewhat smaller (Fig. 1B). After
incubation at 60C, L-crystallin (Fig. 1C) lost its irregular
spherical shape when compared with nonheated -crystallin as
described earlier.18
The in vitro filament formation was also examined by
immunoelectron microscopy (Fig. 2). AntiB- and antiLcrystallin labeling yielded identical results (not shown). The
formation of filament-like structures can be observed after the
heating assay at 60C using 0.05 mg -crystallin obtained from

FIGURE 4. Chaperone-like activity of

water-soluble -crystallin fraction obtained from the cortex of control and
UV-Airradiated bovine lenses determined by heating assay at 60C with
0.25 mg of L-crystallin and 0.05 mg
of -crystallin.

control lenses with 0.25 mg L-crystallin. The identical experiment with -crystallin from irradiated lenses (Figs. 3A, 3B)
showed more pronounced filament-like structures compared
with the control. The results show that UV-A irradiation promotes the filament-like formation. Experiments with anti-vimentin were negative showing that no intermediate filament
component was involved in the crystallin hybrid formation.

Chaperone-like Activity
The chaperone-like activity determined with the aid of the
protein scattering at 360 nm is depicted in Figure 4. It can
be seen that this property of the water-soluble -crystallin
was affected by UV-A light. Compared with controls (curve
II), -crystallin derived from UV-Airradiated lenses revealed


Weinreb et al.

FIGURE 5. Fluorescence determination using Thioflavin T (ThT) interaction with samples obtained from the heating assay, measured at
excitation of 450 nm and emission of 482 nm. ThT concentration was
250 nM. The reaction buffer contained 50 mM glycine-NaOH at pH 6.0.
Bars, SD (in four experiments).

a decreased ability to inhibit L-crystallin denaturation in

vitro (curve III). Curve IV represents L-crystallin in the
absence of -crystallin. Furthermore, -crystallin obtained
from control and UV- irradiated lenses did not denature
during 30 minutes of incubation at 60C (compare the
coinciding curves Ia and Ib). These results are consistent
with previous reports that described decreased chaperonelike activity of -crystallin on UV-B irradiation.19 21

ThT Interaction Assay

The results of the ThT test are depicted in Figure 5 This assay,
in which fibrils convert to a -sheet configuration in vitro, has
previously been successfully applied for detection of amyloid
fibrils.17 It can be seen that heated L-crystallin or heated
L-crystallin plus -crystallin from control lenses produced a
10 times higher fluorescence value than heated -crystallin
obtained from control and UV-irradiated lenses alone. The
amount of fluorescence increased when heated L-crystallin is
assembled with -crystallin from UV-treated lenses.

Previously Slingsby et al.22 suggested a new model for crystallin
assembly in lens fiber cells. In the highly hydrated solution-like
region of the lens, it is envisaged that weak interaction between subunits such as those of -crystallin will occur, forming
elements of a network with dynamic branching. An open gel
structure would maintain proteinprotein interactions at a
high concentration, covering the more prominent hydrophobic regions and preventing random aggregation of subunits. This may possibly explain the present observation that
(heated) L-crystallin assembles with -crystallin, resulting in
filament-like structures. It cannot be excluded that one or more
of UV-Aprovoked alterations23 are related to the ability of
water-soluble -crystallin to form filaments in vitro more effi-

IOVS, November 2000, Vol. 41, No. 12

ciently than with -crystallin derived from control lenses. The
in vitro filament-like chains identified by electron microscopy
after irradiation have a high degree of morphologic similarity to
the -hybrids that have been described previously after reconstitution of the dissociated total mixture of the watersoluble crystallins.18 Dhir et al.24 have recently shown by in
vitro UV-A irradiation of recombinant A-crystallin that sensitized photooxidation can occur in amino acids other than Trp
in the presence of kynurenine or 3-hydroxykynurenine with
effects similar to, albeit smaller than, direct UV-B photooxidation. In the old lens, other types of sensitizers may be operative, such as advanced glycation end products (AGE). Finley et
al.,25 studying the photooxidation sites in bovine A-crystallin,
found that in addition to Trp, Met and His were photooxidized.
Their conclusion is that the N-terminal region of A-crystallin is
exposed to an aqueous environment and is in the vicinity of
Trp from neighboring subunits. Albeit we did not try to identify
the exact site of photooxidation being beyond the aim of our
study, it might well be that particularly AGE could play a role
as sensitizer because we used adult bovine lenses. Besides, the
relatively large amount of NAD(P)H in bovine lens could also
initiate photochemical processes as it does in human and
rabbit lens cells.26 Furthermore, the ThT interaction assay,
which is used as a method for the demonstration of -sheet
conformation and which appeared previously to be a useful
tool for detection of amyloid fibrils in vitro,17 provided additional evidence for possible -crystallin filament formation
(Fig. 5). According to Levine,27 it is very likely that both the
-sheet conformation and the aggregation state provide the
environment to stabilize the long wavelength ThT fluorescent
complex, regardless of the identity of the participating peptides. Therefore, at least some of the endogenous filament-like
structures that have been demonstrated in the lens may result
from interaction of -crystallin with other proteins such as
L-crystallin under stress conditions. This might provide a clue
regarding the processes leading to the development of UV

We thank Wilfried W. de Jong for fruitful discussions and Lucio
Benedetti for advice concerning electron microscopy.

1. van den Heuvel R, Hendriks W, Quax W, Bloemendal H. Complete
structure of the hamster A-crystallin gene: reflection of an evolutionary history by means of exon shuffling. J Mol Biol. 1985;185:
2. Bloemendal H, de Jong WW. Lens proteins and their genes. Progr
Nucleic Acids Res Mol Biol. 1991;41:259 281.
3. Horwitz J. -Crystallin can function as a molecular chaperone.
Proc Natl Acad Sci USA. 1992;89:10449 10453.
4. Bhat SD, Nagineni CN. B Subunit of lens-specific protein -crystallin is present in other ocular and non-ocular tissues. Biochem
Biophys Res Commun. 1989;158:319 325.
5. Dubin RA, Wawrousek EF, Piatigorsky J. Expression of the murine
B-crystallin gene is not restricted to the lens. Mol Cell Biol.
6. Kato K, Shinohara H, Kurobe N, Goto S, Inaguma Y, Ohshima K.
Immunoreactive A-crystallin in rat non-lenticular tissues detected
with a sensitive immunoassay method. Biophys Biochim Acta.

IOVS, November 2000, Vol. 41, No. 12

7. Nicholl ID, Quinlan RA. Chaperone activity of -crystallin modulates intermediate filaments assembly. EMBO J. 1994;13:945953.
8. Liang P, MacRae TH. Molecular chaperones and the cytoskeleton.
J Cell Sci. 1997;110:14311440.
9. Djabali K, de Nechaud B, Landon F, Portier MM. B-Crystallin
interacts with intermediate filaments in response to stress. J Cell
Sci. 1997;110:2759 2769.
10. Muchowski PJ, Valdez MM, Clark JI. B-Crystallin selectively targets intermediate filament proteins during thermal stress. Invest
Ophthalmol Vis Sci. 1999;40:951958.
11. Maisel H, Perry MM. Electron microscope observation on some
structural proteins of the chick lens. Exp Eye Res. 1972;14:712.
12. Benedetti EL, Dunia I, Bentzel CJ, Vermorken AJM, Kibbelaar M,
Bloemendal H. A portrait of plasma membrane specialization in
eye lens epithelium and fibers. Biochim Biophys Acta. 1976;457:
13. Ireland M, Maisel H. A family of lens fiber cell specific proteins.
Lens Eye Toxic Res. 1989;6:623 638.
14. FitzGerald PG, Gottlieb W. The Mr 115 KD fiber cell-specific
protein of a component of the lens cytoskeleton. Curr Eye Res.
1989;8:801 811.
15. Dovrat A, Weinreb O. Recovery of lens optics and epithelial enzymes after ultraviolet A radiation. Invest Ophthalmol Vis Sci.
16. Slingsby C, Bateman OA. Rapid separation of bovine -crystallin
subunits B1, B2, B3, A3 and A4. Exp Eye Res. 1991;51:21
17. Naiki H, Higuchi K, Hossokawa M, Takeda T. Fluorometric determination of amyloid fibrils in vitro using fluorescence dye, Thioflavine T. Anal Biochem. 1989;177:244 249.

Filament-like Formation and Crystallin Interaction


18. Bloemendal H, Zweers A, Benedetti EL, Walter H. Selective reassociation of the crystallins. Exp Eye Res. 1975;20:463 478.
19. Borkman RF, McLaughlin J. The molecular chaperone function of
-crystallin is impaired by UV photolysis. Photochem Photobiol.
1995;62:1046 1051.
20. Schauerte JA, Gafni A. Photodegradation of tryptophan residues
and attenuation of molecular chaperone activity in -crystallin are
correlated. Biochem Biophys Res Commun. 1995;212:900 905.
21. Ellozy AR, Ceger P, Wang RH, Dillon J. Effect of the UV modification of -crystallin on its ability to suppress nonspecific aggregation. Photochem Photobiol. 1996;64:344 348.
22. Slingsby C, Norledge B, Simpson A, et al. X-ray diffraction and
structure of crystallins. Prog Retinal Eye Res. 1997;16:329.
23. Weinreb O, van Rijk AF, Steely HT, Dovrat A, Bloemendal H.
Analysis of UV-A-related alterations upon aging of eye lens proteins
by mini-two-dimensional polyacrylamide gel electrophoresis. Ophthal Res. 2000;32(5):196 204.
24. Dhir P, Nila JA, Sun TX, Liang JJN. Photooxidized products of
recombinant A-crystallin and W9F mutant. Photochem Photobiol. 1999;69:329 335.
25. Finley EL, Busman M, Dillon J, Crouch RK, Schey KL. Identification
of: photooxidation sites in bovine -crystallin. Photochem Photobiol. 1997;66:635 641.
26. Atherton SJ, Lambert C, Schultz J, Williams N, Zigman S. Fluorescence studies of lens epithelial cells and their constituents. Photochem Photobiol. 1999;70:823 828.
27. Levine H. Thioflavin T interaction with synthetic Alzheimers disease -amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 1993;2:404 410.