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In Vitro Filament-like Formation upon Interaction between Lens -Crystallin and ßL-Crystallin Promoted by Stress

¹ From the Department of Biochemistry, University of Nijmegen, The Netherlands; and ² B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.

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 -, ßH-, ßL-, and -crystallins according to standard procedures. Chaperone-like activity of -crystallin was determined in control and UV-A–irradiated lenses by the heat-induced aggregation assay of ßL-crystallin. 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 60°C has no effect on the morphologic appearance of -crystallin as observed by transmission electron microscopy. Moreover -crystallin obtained from UV-A–irradiated lenses shows a virtually identical shape. However, heating in the presence of ßL-crystallin results in the formation of filament-like ß-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against - or ßL-crystallin. Parallel experiments with -crystallin derived from UV-A–irradiated 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 ßL-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 ßL-crystallin. Because the hybrid formation is enhanced when -crystallin from UV-A–irradiated 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.

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