Accumulative deamidation of human lens protein γS-crystallin leads to partially unfolded intermediates with enhanced aggregation propensity

Abstract

Age-related cataract is a major cause of blindness worldwide. Yet, the molecular mechanisms whereby large, light scattering aggregates form is poorly understood, because of the complexity of the aggregates isolated from human lenses. The predominant proteins in the lens are structural proteins called crystallins. The {gamma}S-crystallin is heavily modified in cataractous lenses by deamidation, which introduces a negative charge at labile asparagine residues. The effects of deamidation at asparagines, N14, N76, and N143, were mimicked by replacing the asparagine with aspartate using site-directed mutagenesis. The effects of these surface deamidations on the stability, unfolding, and aggregation properties of {gamma}S were determined using dynamic light scattering, chemical and thermal-denaturation, and hydrogen-deuterium exchange with mass spectrometry. We found that a small population of all the deamidation mimics aggregated directly into large light scattering bodies with a radius greater than 10 nm that contributed 14-60% of the total scattering intensity compared to 7% for WT under the same conditions. A possible mechanism was identified under partially denaturing conditions, where deamidation led to significantly more rapid unfolding and aggregation particularly for N76D compared to WT. The triple mutant was further destabilized, reflecting the enhanced aggregation properties of N14D and N143D. Thus, the effects of deamidation were both site-specific and cumulative. A-crystallin was ineffective at acting as a chaperone to prevent the aggregation of destabilized, deamidated {gamma}S. It is concluded that surface deamidations, while causing minimal structural disruption individually, progressively destabilize crystallin proteins, leading to their unfolding and precipitation in aged and cataractous lenses.