[Analysis of Asparagine Deamidation and Its Impact on γS-Crystallin Aggregation].

Abstract

Nonenzymatic posttranslational modifications of proteins occur under physiological conditions, leading to structural alterations and functional impairments that contribute to age-related diseases and reduced efficacy of antibody drugs. Among these modifications, deamidation of asparagine (Asn) residues in crystallins, a major lens protein, is implicated in the development of age-related cataracts. This study investigated the conditions that promote Asn deamidation and analyzed its impact on γS-crystallin aggregation. Molecular dynamics simulations and quantum chemical calculations revealed that deamidation propensity is influenced by the main-chain conformation on the N-terminal side of Asn. Specifically, the activation barrier for deamidation decreased by approximately 9-20 kJ/mol when the α-hydrogen and the amide hydrogen of the Asn main chain adopted a syn-periplanar conformation, This finding enabled identification of backbone structures susceptible to deamidation. However, residues prone to deamidation and those that significantly affect protein structure upon modification do not always overlap. To further explore these effects, deamidation-mimetic mutants of γS-crystallin (N14D, N37D, N53D, N76D, N143D) were constructed. Structural and aggregation analyses revealed that deamidation at Asn37 significantly reduced thermal stability and promoted aggregation of γS-crystallin. These findings enhance our understanding of the aggregation mechanisms underlying cataract formation and may contribute to the development of novel preventive and therapeutic strategies for cataracts and other age-related diseases associated with nonenzymatic posttranslational modifications of proteins.