In vitro filament-like formation upon interaction between lens alpha-crystallin and beta L-crystallin promoted by stress

PURPOSE. To determine whether alpha -crystallin is capable of forming filam ent-like structures with other members of the crystallin family. METHODS. Water-soluble crystallins were isolated from calf lenses and fract ionated into alpha-, betaH-, betaL-, and gamma -crystallins according to st andard procedures. Chaperone-like activity of alpha -crystallin was determi ned in control and UV-A-irradiated lenses by the heat-induced aggregation a ssay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmissi on immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was exclude d by the results of Western blot analysis, which were all negative. Moreove r, the in vitro amyloid fibril interaction test using thioflavin T (ThT) wa s also performed. RESULTS. At the supramolecular level heating at 60 degreesC has no effect o n the morphologic appearance of alpha -crystallin as observed by transmissi on electron microscopy. Moreover alpha -crystallin obtained from UV-A-irrad iated lenses shows a virtually identical shape. However, heating in the pre sence of betaL-crystallin results in the formation of filament-like alpha b eta -hybrids as demonstrated by immunoelectron microscopy using specific an tibodies directed either against alpha- or betaL-crystallin. Parallel exper iments with alpha -crystallin derived from UV-A-irradiated lenses showed ev en more pronounced filamentous structures, compared with the controls. None theless, we were able to show that the UV-light treatment affected the chap erone-like capacity of alpha -crystallin, as revealed by a diminished abili ty to inhibit in vitro denaturation of betaL-crystallin. To exclude the pre sence of cytoskeletal contamination in the crystallin preparations, vimenti n antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obta ined with the ThT assay earlier applied for the detection of amyloid fibril s. CONCLUSIONS. Crystallin hybrids have previously been detected in the water- soluble lens crystallin fraction. Our findings indicate that such endogenou s hybrids, formerly called "rods," may result from stress-induced interacti on between alpha -crystallin and other lens constituents such as betaL-crys tallin. Because the hybrid formation is enhanced when alpha -crystallin fro m 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 lead ing to UV-A cataract.