title: 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.
abstract: 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 ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H(2)O(2), whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-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 γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.
authors.affiliation: From the ‡Department of Pathology, xxf3@case.edu vmm3@case.edu.
authors.forename: Xingjun
authors.identifier: http://orcid.org/0000-0002-6477-4465
authors.initials: X
authors.lastname: Fan
authors.affiliation: ¶State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China;
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhou
authors.affiliation: ‖Center for Proteomics.
authors.forename: Benlian
authors.initials: B
authors.lastname: Wang
authors.affiliation: **Fairview High School, Fairview, Ohio;
authors.forename: Grant
authors.initials: G
authors.lastname: Hom
authors.affiliation: ‡‡Department of Ophthalmology, the Huichang County People's Hospital, Jiangxi, China;
authors.forename: Minfei
authors.initials: M
authors.lastname: Guo
authors.affiliation: §§Department of Ophthalmology, Ganzhou City People's Hospital, Jiangxi, China.
authors.forename: Binbin
authors.initials: B
authors.lastname: Li
authors.affiliation: ¶State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China;
authors.forename: Jing
authors.initials: J
authors.lastname: Yang
authors.affiliation: From the ‡Department of Pathology.
authors.forename: Dennis
authors.initials: D
authors.lastname: Vaysburg
authors.affiliation: From the ‡Department of Pathology, §Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44120; xxf3@case.edu vmm3@case.edu.
authors.forename: Vincent M
authors.initials: VM
authors.lastname: Monnier
mesh_terms: D000375:Aging / Q000378:metabolism*; D000818:Animals; D002386:Cataract / Q000378:metabolism*; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry; D004195:Disease Models, Animal; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000172:deficiency*; D006801:Humans; D066298:In Vitro Techniques; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods; D038204:beta-Crystallins / Q000737:chemistry* / Q000302:isolation & purification*
chemical_list: D004220:Disulfides; D038204:beta-Crystallins; D005978:Glutathione; D003545:Cysteine