Cleavage of the C-Terminal Serine of Human αA-Crystallin Produces αA1-172with Increased Chaperone Activity and Oligomeric Size

Abstract

This study aimed to study the oligomeric size, structure, hydrodynamic properties, and chaperone function of the C-terminally truncated human alphaA-crystallin mutants with special emphasis on alphaA1-172 which is the cleavage product of the Ser172-Ser173 bond, unique to human lenses and constituting a major part of alphaA-crystallin. Various truncated forms of human alphaA-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 betaL-crystallin as the target proteins. alphaAlpha1-172 exhibited a significant increase in oligomeric size, i.e., 866 kDa by light scattering measurements as compared to 702 kDa in alphaA-wt. alphaAlpha1-172 and alphaA-wt had similar secondary structure, but the former exhibited slightly altered tertiary structure. The most interesting observation was that alphaAlpha1-172 behaved as a 28-46% better chaperone than alphaA-wt. The oligomeric size and structure of alphaAlpha1-168 were similar to those of alphaA-wt, while the chaperone activity was decreased by 12-23%. alphaAlpha1-162, 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 alphaA-crystallin will be significantly improved by the presence of the major truncated product alphaAlpha1-172. This will be beneficial to the lens undergoing oxidative stress. Since alphaAlpha1-168 and alphaAlpha1-162 are present only in small amounts, their effect would be minimal.