Novel Intra- and Inter-molecular Sulfinamide Bonds in S100A8 Produced by Hypochlorite Oxidation
- Publication Type:
- Journal Article
- Citation:
- Journal of Biological Chemistry, 2001, 276 (36), pp. 33393 - 33401
- Issue Date:
- 2001-09-07
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Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Murine S100A8 (A8) is a major cytoplasmic protein in neutrophils and is secreted by macrophages in response to inflammatory stimuli. After incubation with reagent HOCl for 10 min, ∼85% of A8 was converted to 4 oxidation products, with electrospay ionization mass spectrometry masses of m/z 10354, 10388, 10354 ± 1, and 20707 ± 3. All were resistant to reduction by dithiothreitol. Initial formation of a reactive Cys sulfenic acid intermediate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohexanedione (dimedone) to HOCl-treated A8 to form stable adducts. Matrix-assisted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in the full-length proteins. Both Met36/73 were converted to Met36/73 sulfoxides. An additional product with an unusual mass addition of m/z 14 (±0.2) was identified and corresponded to the addition of oxygen to Cys41, conjugation to various ε-amines of Lys6, Lys34/35, or Lys87 with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide cross-links. Specific fragmentations identified in matrix-assisted laser desorption-post source decay spectra and low energy collisional-induced dissociation tandem mass spectroscopy spectra of sulfinamide-containing digest peptides confirmed Lys34/35 to Cys41 sulfinamide bonds. HOCl oxidation of mutants lacking Cys41 (Ala41S100A8) or specific Lys residues (e.g. Lys34/35, Ala34/35S100A8) did not form sulfinamide cross-links. HOCl generated by myeloperoxidase and H 2O2 and by phorbol 12-myristate 13-acetate-activated neutrophils also formed these products In contrast to the disulfide-linked dimer, oxidized monomer retained normal chemotactic activity for neutrophils. Sulfinamide bond formation represents a novel oxidative cross-linking process between thiols and amines and may be a general consequence of HOCl protein oxidation in inflammation not identified previously. Similar modifications in other proteins could potentially regulate normal and pathological processes during aging, atherogenesis, fibrosis, and neurogenerative diseases.
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