Mutation of R116C results in highly oligomerized alpha A-crystallin with modified structure and defective chaperone-like function.

An autosomal dominant congenital cataract in human is associated with mutation of Arg-116 to Cys (R116C) in alpha A-crystallin. To investigate the molecular basis of cataract formation, rat alpha A-crystallin cDNA was cloned into pET-23d(+), and the site-directed mutants S142C (similar to wild-type human alpha A) and R116C/S142C or R116C (similar to human R116C variant) were generated. These were expressed in E. coli and the recombinant alpha A-crystallins purified by Sephacryl size-exclusion chromatography. The chaperone-like function of mutant R116C determined at 37 degrees C with insulin and alcohol dehydrogenase as target proteins was about 40% lower than those of wild-type and mutant S142C. Based on size-exclusion chromatography data, the oligomeric size of the R116C mutant was about 2000 kDa at 25 degrees C, 1400 kDa at 37 degrees C, and 900 kDa at 45 degrees C. In comparison, alpha A-wild-type and alpha A-S142C ranged from 477 to 581 kDa. Heat stability studies corroborated the effect of temperature on the dynamic quaternary structure of the R116C mutant. Circular dichroism spectra showed secondary and tertiary structural changes, and ANS fluorescence spectra showed loss of surface hydrophobicity in the R116C mutant. These findings suggest that the molecular basis for the congenital cataract with the alpha A-R116C mutation is due to the generation of a highly oligomerized alpha A-crystallin having a modified structure and decreased chaperone-like function.