Novel intra- and inter-molecular sulfinamide bonds in S100A8 produced by hypochlorite oxidation

Hypochlorite is a major oxidant generated when neutrophils and macrophages are activated at inflammatory sites, such as in atherosclerotic lesions. Mu rine S100A8 (AS) is a major cytoplasmic protein in neutrophils and is secre ted by macrophages in response to inflammatory stimuli. After incubation wi th reagent HOC1 for 10 min, similar to 85% of AS was converted to 4 oxidati on products, with electrospay ionization mass spectrometry masses of m/z 10 354, 10388, 10354 +/- 1, and 20707 +/- 3. All were resistant to reduction b y dithiothreitol. Initial formation of a reactive Cys sulfenic acid interme diate was demonstrated by the rapid conjugation of 5,5-dimethyl-1,3-cyclohe xanedione (dimedone) to HOC1-treated AS to form stable adducts. Matrix-assi sted laser desorption-reflectron time of flight peptide mass fingerprinting of isolated oxidation products confirmed the mass additions observed in th e full-length proteins. Both Met(36/73) were converted to Met(36/73) sulfox ides. An additional product with an unusual mass addition of m/z 14 (+/-0.2 ) was identified and corresponded to the addition of oxygen to Cys(41), con jugation to various E-amines of Lys(6), Lys(34/35), or Lys(87) with loss of dihydrogen and formation of stable intra- or inter-molecular sulfinamide c ross-links. Specific fragmentations identified in matrix-assisted laser des orption-post source decay spectra and low energy collisional-induced dissoc iation tandem mass spectroscopy spectra of sulfinamide-containing digest pe ptides confirmed Lys(34/35) to Cys(41) sulfinamide bonds. HOCI oxidation of mutants lacking CyS41. (A1a(41)S100A8) or specific Lys residues (e.g. Lys( 34/35), Ala(34/35)S100A8) did not form sulfinamide cross-links. HOCI genera ted by myeloperoxidase and H2O2 and by phorbol 12-myristate 13-acetate-acti vated neutrophils also formed these products . In contrast to the disulfide linked dimer, oxidized monomer retained normal chemotactic activity for ne utrophils. Sulfinamide bond formation represents a novel oxidative cross-li nking process between thiols and e-amines and may be a general consequence of HOCI protein oxidation in inflammation not identified previously. Simila r modifications in other proteins could potentially regulate normal and pat hological processes during aging, atherogenesis, fibrosis, and neurogenerat ive diseases.