Abstract
Purpose :
Deamidation and glutathionylation of cysteines in crystallins are major age-related modifications associated with cataract formation. The purpose of this study was to test whether deamidation increases the rate of disulfide exchange between glutathionylated cysteines in γS and buried cysteines when γS is partially unfolded in guanidine.
Methods :
Wild type (WT) γS and triple mutant (TM) γS containing N14D, N76D, and N143D deamidation mimics were glutathionylated by incubation at 1.6 mg/ml concentration in 2 mM oxidized glutathione (GSSG) in 20 mM phosphate, 150 mM NaCl, 1 mM EDTA (pH 7) for 48 hours at 37°C. Disulfide exchange between sites of glutathionylation and buried cysteines was then initiated by the addition of 2.75 M guanidine and incubation at 30°C between 0.5-10 min. Loss of glutathione and introduction of disulfide bonds were determined by measuring whole protein masses using mass spectrometry. Experiments were performed in triplicate using two different preps of purified WT and TM γS.
Results :
Both WT and TM γS undergo a single glutathionylation and single disulfide bond introduction when incubated in 2 mM GSSG for 48 hours. Our previous studies showed that incubation in 2.75 M guanidine causes more rapid unfolding of TM γS than in WT γS. Treatment of glutathionylated γS under these conditions also causes a more rapid loss of glutathione. TM loses 60% of its glutathione after 1.5 min in 2.75 M guanidine, while WT γS loses only 40%. There were statistically significant differences in the rate of glutathione loss between WT and TM γS at 1, 1.5, and 2.5 min of unfolding in 2.75 M guanidine (p<0.03). Heating glutathionylated WT and TM γS at 60°C also initiates glutathione loss, but unlike guanidine treatment, causes precipitation of γS, while non-glutathionylated γS does not precipitate.
Conclusions :
Introduction of deamidations in γS resulted in more rapid disulfide exchange reactions in the protein due to its greater susceptibility to denaturation in guanidine. Glutathionylation is known to occur in aged cataractous lenses. Deamidation may destabilize γS and result in transient exposure of internal cysteines that undergo disulfide exchange at glutathionylation sites, locking the protein into non-native conformations that then precipitate. This model system will allow thorough examination of this process and investigation of the role of both deamidation and oxidation in cataract formation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.