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

Srinivasagan Ramkumar, Xingjun Fan, Benlian Wang, Sichun Yang, Vincent M. Monnier

Research output: Contribution to journalArticle

5 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3595-3604
Number of pages10
JournalBiochimica et Biophysica Acta - Molecular Basis of Disease
Volume1864
Issue number11
DOIs
StatePublished - Nov 2018
Externally publishedYes

Keywords

  • Age related cataract
  • Copper oxidation
  • Cysteine disulfide
  • Human gamma D crystallin

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology

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