Cleavage of the C-terminal serine of human αA-crystallin produces αA1-172 with increased chaperone activity and oligomeric size

This study aimed to study the oligomeric size, structure, hydrodynamic properties, and chaperone function of the C-terminally truncated human alpha A-crystallin mutants with special emphasis on alpha A(1-172) which is the cleavage product of the Ser(172)-Ser(173) bond, unique to human lenses and constituting a major part of alpha A-crystallin. Various truncated forms of human alpha A-crystallins were prepared by site-directed mutagenesis. The proteins were expressed in Escherichia coli BL21(DE3) pLysS cells and purified by size exclusion column chromatography. Molecular masses and the other hydrodynamic properties were determined by dynamic light scattering measurements. The secondary and tertiary structural changes were assessed by far- and near-UV CD spectra measurements, respectively. Chaperone activity was determined by using ADH, insulin, and beta L-crystallin as the target proteins. alpha Alpha(1-172) exhibited a significant increase in oligomeric size, i.e., 866 kDa by light scattering measurements as compared to 702 kDa in alpha A-wt. alpha Alpha(1-172) and alpha A-wt had similar secondary structure, but the former exhibited slightly altered tertiary structure. The most interesting observation was that alpha Alpha(1-172) behaved as a 28-46% better chaperone than alpha A-wt. The oligomeric size and structure of alpha Alpha(1-168) were similar to those of alpha A-wt, while the chaperone activity was decreased by 12-23%. alpha Alpha(1-162), on the other hand, had an oligomeric size of 400 kDa, a decrease in chaperone activity of 80-100%, and significantly altered secondary and tertiary structures. The data show that the overall chaperone function of alpha A-crystallin will be significantly improved by the presence of the major truncated product alpha Alpha(1-172). This will be beneficial to the lens undergoing oxidative stress. Since alpha Alpha(1-168) and alpha Alpha(1-162) are present only in small amounts, their effect would be minimal.