The oxidized thiol proteome in aging and cataractous mouse and human lens revealed by ICAT labeling

Abstract

Age-related cataractogenesis is associated with disulfide-linked high molecular weight (HMW) crystallin aggregates. We recently found that the lens crystallin disulfidome was evolutionarily conserved in human and glutathione-depleted mouse (LEGSKO) cataracts and that it could be mimicked by oxidation in vitro (Mol. Cell Proteomics, 14, 3211-23 (2015)). To obtain a comprehensive blueprint of the oxidized key regulatory and cytoskeletal proteins underlying cataractogenesis, we have now used the same approach to determine, in the same specimens, all the disulfide-forming noncrystallin proteins identified by ICAT proteomics. Seventy-four, 50, and 54 disulfide-forming proteins were identified in the human and mouse cataracts and the in vitro oxidation model, respectively, of which 17 were common to all three groups. Enzymes with oxidized cysteine at critical sites include GAPDH (hGAPDH, Cys247), glutathione synthase (hGSS, Cys294), aldehyde dehydrogenase (hALDH1A1, Cys126 and Cys186), sorbitol dehydrogenase (hSORD, Cys140, Cys165, and Cys179), and PARK7 (hPARK7, Cys46 and Cys53). Extensive oxidation was also present in lens-specific intermediate filament proteins, such as BFSP1 and BFSP12 (hBFSP1 and hBFSP12, Cys167, Cys65, and Cys326), vimentin (mVim, Cys328), and cytokeratins, as well as microfilament and microtubule filament proteins, such as tubulin and actins. While the biological impact of these modifications for lens physiology remains to be determined, many of these oxidation sites have already been associated with either impaired metabolism or cytoskeletal architecture, strongly suggesting that they have a pathogenic role in cataractogenesis. By extrapolation, these findings may be of broader significance for age- and disease-related dysfunctions associated with oxidant stress.

Keywords: aging; cataractogenesis; disulfide; mass spectrometry; proteomics; reactive oxygen species.

Figure 1

Summary of the experimental design and analytical procedures that are used in this study. For in vitro modeling experiment, the sample identifications (T2B, T2C, T12B, and T12C) are listed under each oxidation condition.

Figure 2

Protein categorization based on molecular functions and shared protein informatics between human, mouse, and in vitro model. The protein functional categorization was achieved with the Panther classification system (www.pantherdb.org). (A) Human lens protein functional pie graph. (B) LEGSKO/WT mouse lens protein functional pie graph. (C) Functional pie graph from in vitro oxidation of the mouse lens protein extract. (D) Shared protein identity between each sample set.

Figure 3

Representative proteins and their cysteine residues that are involved in disulfide cross‐linking in lens structural proteins. (A) The disulfide bond ratio of the Cys167 in filensin (BFSP1) in human lens. (B) The disulfide bond ratio of the Cys290 in mouse BFSP1 in both LEGSKO mouse and in vitro oxidation by H₂O₂ compared to their control. (C) The disulfide bond ratio of the Cys65 in human phakinin (BFSP2). (D) The Cys217 in various actins (ACTB, ACTG1, and ACTBL2) and the Cys285 in various actins (ACTB and ACTG1) in aged normal and cataractous human lenses compared to young normal control. (E) The actin cysteine residue Cys285 and Cys217 in both LEGSKO mouse and in vitro oxidation by H₂O₂ compared to their control. (F) Cys1493 and Cys1616 in collagen 4a1, Cys1419, Cys1532, Cys1660, and Cys1653 in collagen 4a2, and Cys1626 in collagen 4a4 were oxidized in LEGSKO vs. age‐matched WT mice lenses. Cat‐II: grade II cataract; Cat‐II: grade III cataract; Cat‐IV: grade IV cataract; Cat‐V: grade V cataract. T2B, T2C, T12B, and T12C are in vitro oxidation sample pairs (see Fig. 1). All ICAT data are expressed as the ratio vs. young lenses pool, LEGSKO vs. age‐matched WT, or oxidized vs. nonoxidized lens protein extract. Standard errors were calculated from three biological replicates. One‐way ANOVA was used to compare the significance between groups, and P < 0.05 was considered significant. The significance level was marked by either “*, **“ or lower case letters and the significance value was shown inside the figures.

Figure 4

Representative enzymes whose cysteine residues are involved in disulfide cross‐linking. (A) The disulfide bond ratio of the Cys247 in GAPDH in human lens. (B) The disulfide bond ratio of the Cys271 in GAPDH in both LEGSKO mouse and in vitro oxidation by H₂O₂ compared to their control. In addition, Cys48 in GAPDH was also found close to 40 times oxidized in LEGSKO vs. age‐matched WT mice lenses. (C) The disulfide bond ratio of the Cys126 and Cys186 in ALDH1A1 in human lens. (D) The Cys126 and Cys187 (same motif as human Cys186) were found significantly oxidized in LEGSKO vs. WT and also positively associated with in vitro oxidation conditions. In addition, Cys370 oxidation was also detected in LEGSKO mice lenses. See Fig. 3 legend for labels. All ICAT data are expressed as the ratio vs. young lenses pool, LEGSKO vs. age‐matched WT, or oxidized vs. nonoxidized lens protein extract. Standard errors were calculated from three biological replicates. One‐way ANOVA was used to compare the significance between groups, and P < 0.05 was considered significant. The significance level was marked by either “*, **“ or lower case letters and the significance value was shown inside the figures.

Figure 5

Disulfide cross‐linking affects lens antioxidative proteins. (A) The disulfide bond ratio in of the Cys294 in glutathione synthase (GSS) in human lens. (B). The disulfide bond ratio of the Cys47 in PRDX6 in human lens. See Fig. 3 legend for labels. All ICAT data are expressed as the ratio vs. young lenses pool. Standard errors were calculated from three biological replicates. One‐way ANOVA was used to compare the significance between groups, and P < 0.05 was considered significant.

Figure 6

PARK7 (DJ‐1) protein was actively involved in disulfide cross‐linking. (A) The disulfide ratio of the Cys46 and Cys53 in human lens. (B) Similar results were also found in Cys46 and Cys53 oxidation in LEGSKO vs. WT and in vitro modeling samples. See Fig. 3 legend for labels. All ICAT data are expressed as the ratio vs. young lenses pool, LEGSKO vs. age‐matched WT, or oxidized vs. nonoxidized lens protein extract. Standard errors were calculated from three biological replicates. One‐way ANOVA was used to compare the significance between groups, and P < 0.05 was considered significant.