The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26
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 h...
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2025-03-01
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| author | Kate Halverson-Kolkind David C. Thorn Martin Tovar-Ramirez Eugene Shakhnovich Larry David Kirsten Lampi |
| author_facet | Kate Halverson-Kolkind David C. Thorn Martin Tovar-Ramirez Eugene Shakhnovich Larry David Kirsten Lampi |
| author_sort | Kate Halverson-Kolkind |
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| description | 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. |
| format | Article |
| id | doaj-art-8beeb0685bfa4629923ef03d6a357aa5 |
| institution | OA Journals |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-03-01 |
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| series | Biomolecules |
| spelling | doaj-art-8beeb0685bfa4629923ef03d6a357aa52025-08-20T02:11:22ZengMDPI AGBiomolecules2218-273X2025-03-0115340210.3390/biom15030402The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26Kate Halverson-Kolkind0David C. Thorn1Martin Tovar-Ramirez2Eugene Shakhnovich3Larry David4Kirsten Lampi5Biomaterials and Biomedical Science, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USADepartment of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USABiomaterials and Biomedical Science, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USADepartment of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, MA 02138, USAChemical Physiology & Biochemistry, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USABiomaterials and Biomedical Science, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USAThe 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.https://www.mdpi.com/2218-273X/15/3/402eye lenscrystallinprotein structureglutathionylationprotein oxidationmass spectrometry |
| spellingShingle | Kate Halverson-Kolkind David C. Thorn Martin Tovar-Ramirez Eugene Shakhnovich Larry David Kirsten Lampi The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26 Biomolecules eye lens crystallin protein structure glutathionylation protein oxidation mass spectrometry |
| title | The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26 |
| title_full | The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26 |
| title_fullStr | The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26 |
| title_full_unstemmed | The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26 |
| title_short | The Eye Lens Protein, γS Crystallin, Undergoes Glutathionylation-Induced Disulfide Bonding Between Cysteines 22 and 26 |
| title_sort | eye lens protein γs crystallin undergoes glutathionylation induced disulfide bonding between cysteines 22 and 26 |
| topic | eye lens crystallin protein structure glutathionylation protein oxidation mass spectrometry |
| url | https://www.mdpi.com/2218-273X/15/3/402 |
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