Deamidation Promotes AGE-Modifications in Human Lens γS-Crystallin.

Abstract

Deamidation and advanced glycation end products (AGEs) are among the major post-translational modifications (PTMs) in eye lens proteins. Due to deamidation and AGE formation, proteins may aggregate and scatter light, contributing to lens aging and cataract formation. So far, the relation between the two PTMs remains poorly understood. γS-crystallin (γSC), a major subtype of γ-crystallin, undergoes significant modifications through deamidation, especially at the surface-exposed asparagine residues, N14, N76, and N143. In this study, deamidation of γSC was mimicked by mutating asparagine residues to aspartic acid residues. The deamidation mimics were then incubated with a glycating mixture, and AGE formation in proteins was evaluated by LC-MS/MS. Results indicate that deamidation promotes the formation of both non-cross-linking (CML or CEL) and cross-linking AGEs (GOLD, MOLD, or pentosidine) in lysine residues. AGE formation in arginine residues (e.g., MG-H3) is mostly unaffected. Comparative analysis shows that N14D, N143D, and N14DN76DN143D (triple deamidated, TD) consistently accumulated more AGEs than native γSC. Oxidation with 2 mM GSSG led to increased disulfide-linked cross-linking in deamidated γSC. Upon glycation, the deamidated and oxidized γSC accumulated more AGEs than deamidated γSC; however, the specific AGE levels followed the same trend as deamidated γSC. The results suggest that deamidation promotes AGE formation in γSC, and further oxidation makes it even more susceptible to AGE modifications. The combined interdependent effects of deamidation, oxidation, and AGE modifications could therefore contribute to protein cross-linking and aggregation during lens aging and cataract formation.