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.
Highlights
From the ‡Department of Pathology, ʈCenter for Proteomics, §Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44120; ¶State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; **Fairview High School, Fairview, Ohio; ‡‡Department of Ophthalmology, the Huichang County People’s Hospital, Jiangxi, China; §§Department of Ophthalmology, Ganzhou City People’s Hospital, Jiangxi, China
Using the Isotope-coded Affinity Tag labeling Experiments (ICAT) labeling technique to probe all existing disulfides and their quantitative change relative to an appropriate control, we asked the novel question of how do crystallin disulfides found in human cat
The role of cysteine oxidation into disulfides in the pathogenesis of age-related cataract has been a topic of intense research for many years [29, 30]. It is a well accepted participant mechanism in the pathogenesis of age-related cataracts, in nuclear cataractogenesis [31,32,33] despite an earlier report claiming, based on Raman spectroscopy, that crystallin disulfide crosslinks are not associated with aging and cataract formation in human lens [34]
Summary
LEGSKO mouse cataract and age matched wild type control lenses were collected as previously described [21], and processed using the same procedure as for human lenses, except that the whole lens was used rather than the nucleus because of its small size. Analysis of Dimethyl Labeled Proteins—In addition of carbamidomethylation of Cys and oxidation of Met residues, light and heavy dimethylation (with the mass shift of 28.0313 and 32.0475 Da, respectively) at peptide N-terminal and Lys residues were set as variable modifications for Mascot Demon searching of dimethyl labeling samples. Carbamidomethylation of Cys, as well as the oxidation of Met residues was set as variable modifications Both light and heavy ICAT labeled peptides with the mass shift of 227.1270 and 236.1571 Da, respectively, were first identified using Mascot Demon searching algorithm, and confirmed by manually examination, especially for the peptides having multiple Cys residues and mix-labeled with iodoacetamide (IAM) and ICAT. Statistical significance of differences in mean values was assessed by repeated-measures ANOVA or Student’s t test. p values of Ͻ0.05 were considered statistically significant
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