Evidence of Highly Conserved β-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was approximate to 4x accelerated in LEGSKO lens. Most cysteine disulfides in -crystallins (except A4 in human) were highly conserved in mouse and human and could be generated by oxidation with H2O2, whereas -crystallin oxidation selectively affected C23/42/79/80/154, D42/33, and S83/115/130 in human cataracts, and B79/80/110, D19/109, F19/79, E19, S83/130, and N26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in C; Cys42, Cys33 in D, and Cys83, Cys115, and Cys130 in S. In conclusion, the -crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in -crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.