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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;
41:38933897)
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. h.bloemendal@bioch.kun.nl
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
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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

RESULTS
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.

METHODS
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).

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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

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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.

CONCLUSION
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-

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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
cataract.

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

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