Reactive cysteine residues in the oxidative dimerization and Cu2+ induced aggregation of human γD-crystallin: Implications for age-related cataract

Abstract

Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu²⁺-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.

Keywords: Age related cataract; Copper oxidation; Cysteine disulfide; Human gamma D crystallin.

Fig. 1.

Diamide oxidation of human γD-crystallin. (A) 1 mg/ml of recombinant hγD in Chelex treated 50mMK + /PO4 buffer pH 7.4 was incubated with 10 μl diamide at 37 °C for 3 h. Dimer formation was analyzed by 12% SDS-PAGE and destained overnight. The image was scanned under greyscale using Epson scanner. M-Protein marker, lane 1 -non-oxidized hγD, lane 2 -oxidized hγD. (B) Dimerization was confirmed by western blot using hγD polyclonal antibody and the blot was developed using X-ray film using ECL solution the developed film was scanned under greyscale using EPSON scanner, lane 1 -non-oxidized hγD, lane 2 -oxidized hγD. For both SDS-PAGE and western blot samples were derived from the same experiment and processed in parallel. (C) Tandem mass spectrum of disulfide formation between Cys111-Cys111 in hγD dimer. Both Cys 109 residues are carbami-domethylated. The series b and y ions with A and B represent the fragment ion from peptide DCSCLQDR (A) and DCSCLQ (B), respectively. The precursor ion m/z is 859.8151 (2+).

Fig. 2.

Western blot of dimer formation by diamide oxidation of recombinant γD-crystallin and cysteine mutants in Chelex treated 50 mM potassium phosphate (pH 7.4). Protein was incubated with 10 μM of diamide at 37 °C for 3h and disulfide bond formation was analyzed by western blot using hγD polyclonal antibody and the blot was developed using X-ray film using ECL solution the developed film was scanned under greyscale using Epson scanner. For both reduced and non-reduced blot samples were derived from the same experiment and blots were processed in parallel.

Fig. 3.

Western blot analysis of γD crystallin oxidized in the presence of αB crystallin. Human γD crystallin and αB crystallin in the ratio of 1:1 and 1:2 were incubated 60 min at 37 °C and mixture was oxidized by diamide. The formation of dimer by γD crystallin was determined by western blot analysis using γD crystallin polyclonal antibody and the blot was developed using X-ray film using ECL solution the developed film was scanned under greyscale using Epson scanner.

Fig. 4.

Typical mass spectrum and analysis of individual cysteine residues involved in disulfide formation. The peptide AEFSGECSNLADR from human gamma D crystallin covering cysteine residue 111 was analyzed in human cataract level V lens nucleus using data deposited into the ProteomeXchange Consortium bank from a previous study [7].

Fig. 5.

Effect of Cu²⁺ ions in the aggregation of hγD crystallin WT and cysteine mutants. (A) Turbidity assays of γD crystallin WT and single cysteine mutants (50 μM) in the absence or presence 1,3 and 5 equivalent of Cu²⁺. (B) Turbidity assays of γD crystallin WT and multiple cysteine mutant in the (50 μM) in the absence or presence of 1, 3, and 5 equiv. of Cu²⁺. In all experiments temperature was 37 °C, and the absorbance at 405 nm was measured 3 h after the addition of the metal ion. Turbidity assays were run six times (n = 6) for each condition.

Fig. 6.

Effect of Cu²⁺ ions in the aggregation of glutathionylated γD crystallin WT. (A) Western Blot analysis of hγD incubated with GSSG using monoclonal GSH antibody. Antibody and the blot was developed using X-ray film using ECL solution the developed film was scanned under greyscale using Epson scanner 1: γD crystallin WT alone, 2 & 3: γD crystallin reacts with GSSG as indicated by the appearance of an immunoreactivity band. (B) Turbidity assays (n = 6) of hγD crystallin, glutathionylated (Glu) hγD (50 μM) incubated without or with 1, 3 and 5 equivalent of Cu²⁺ for 60 min.

Fig. 7.

Effect of αB crystallin and αB crystallin minichaperone tested at 5 mg/ml on copper mediated aggregation of hγD crystallin. A: αB crystallin totally suppressed aggregation. B: αB minichaperone suppressed equally well except in presence of 5 equivalent Cu²⁺.

Fig. 8.

Top four dimeric models of human γ-D crystallin with intermolecular Cys111-Cys111. These models were ranked in order using the protein-protein docking software ClusPro [17].