Biomolecules (Mar 2025)

The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26

  • Kate Halverson-Kolkind,
  • David C. Thorn,
  • Martin Tovar-Ramirez,
  • Eugene Shakhnovich,
  • Larry David,
  • Kirsten Lampi

DOI
https://doi.org/10.3390/biom15030402
Journal volume & issue
Vol. 15, no. 3
p. 402

Abstract

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The oxidation of cysteines in crystallins is a major age-related modification associated with cataract formation. The purpose of this research was to determine the susceptibility of γS-crystallin to glutathionylation-induced oxidation and disulfide bond formation. Recombinantly expressed wild-type human γS-crystallin and four cysteine-to-serine mutants were reduced and incubated for up to 2 days with oxidized glutathione. Following incubation and alkylation, the overall degree of glutathionylation and disulfide bond formation were determined by whole-mass measurement. Tryptic digests were also analyzed by LC-MS/MS to identify specific sites of S-glutathionylation and disulfide linkages. We determined that C22, C24, and C26 undergo glutathione-mediated disulfide interchange with each other, with C24 being most susceptible to oxidation and mixed disulfide formation. Our data suggest C24 is S-glutathionylated sequentially with C22 and C26 participating in disulfide exchange reactions, yielding a major species with a single glutathionylation at C24 and a disulfide between C22 and C26. The results imply that as glutathione levels are depleted in aged lenses, γS-crystallin undergoes stepwise oxidation reactions and disulfide shuffling, which may contribute towards its aggregation and cataract formation.

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