A Novel Mutation (f71l) in aA-Crystallin Associated With Age-Related Cataract Results in Defective Chaperone-Like Function Despite Unaltered Structure

Abstract

Purpose: : We identified a genetic variation (F71L) in the exon-2 of CRYAA gene in three unrelated sporadic cases among 450 age-related cataracts patients but not in 200 normal non-cataractous controls. Therefore, the purpose of this study was to investigate the impact of F71L mutation on structural and functional properties of A-crystallin.

Methods: : We have constructed, expressed in E.coli, and purified human recombinant wild-type and F71L A-crystallin. We have characterized the chaperone function of F71L A-crystallin by light scattering method using a battery of target proteins. In addition, we have examined the hydrophobicity, secondary, tertiary and the quaternary structure by biophysical methods.

Results: : Size exclusion chromatography studies revealed that F71L mutation had no significant effect on the apparent molecular mass of A-crystallin oligomeric complexes. Intrinsic tryptophan fluorescence and far- and near-UV CD spectra indicated that F71L missense mutation did not significantly affect the secondary and tertiary structures of A-crystallin. The ANS fluorescence emission spectra suggested no changes in surface hydrophobicity due to the F71L conversion. While the mutant A displayed almost complete loss (90%) of chaperone-like activity (CLA) by measuring protection against heat-induced aggregation of carbonic anhydrase, it showed approximately 50% less protection in heat-induced aggregation of βL-crystallin. The CLA of the F71L mutant was decreased approximately 35% in heat-induced aggregation of γ-crystallin, and varied with other client proteins.

Keywords: cataract • genetics • protein structure/function