In vitro filament-like formation upon interaction between lens alpha-crystallin and betaL-crystallin promoted by stress.
OBJECTIVE
To determine whether alpha-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 alpha-, betaH-, betaL-, and gamma-crystallins according to standard procedures. Chaperone-like activity of alpha-crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-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 degrees C has no effect on the morphologic appearance of alpha-crystallin as observed by transmission electron microscopy. Moreover alpha-crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alphabeta-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha-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 alpha-crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-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 alpha-crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha-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.
Weinreb
O
O
Department of Biochemistry, University of Nijmegen, The Netherlands.
van Rijk
A F
AF
Dovrat
A
A
Bloemendal
H
H
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D003459:Crystallins / Q000378:metabolism* / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D018882:Heat Stress Disorders / Q000378:metabolism* / Q000473:pathology; D007908:Lens, Crystalline / Q000378:metabolism / Q000528:radiation effects; D016253:Microscopy, Immunoelectron; D018832:Molecular Chaperones / Q000378:metabolism; D009924:Organ Culture Techniques; D011489:Protein Denaturation; D014466:Ultraviolet Rays
D003459:Crystallins; D003598:Cytoskeletal Proteins; D018832:Molecular Chaperones