Oxidation regulates the inflammatory properties of the murine S100 proteinS100A8

The myeloid cell-derived calcium-binding murine protein, S100A8, is secrete d to act as a chemotactic factor at picomolar concentrations, stimulating r ecruitment of myeloid cells to inflammatory sites, S100A8 may be exposed to oxygen metabolites, particularly hypochlorite, the major oxidant generated by activated neutrophils at inflammatory sites. Here we show that hypochlo rite oxidizes the single Cys residue (Cys(41)) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 mu M) converted 70-80% of S100 A8 to the disulfide-linked homodimer, The mass was 20,707 Da, 92 Da more th an expected, indicating additional oxidation of susceptible amino acids (po ssibly methionine). Phorbol 12-myristate 13-acetate activation of different iated HL-60 granulocytic cells generated an oxidative burst that was suffic ient to efficiently oxidize exogenous S100A8 within 10 min, and results imp licate involvement of the myeloperoxidase system. Moreover, disulfide-linke d dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to rec ruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala(4 1)S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the che motactic hinge domain. Disulfide-dependent dimerization may be a physiologi cally significant regulatory mechanism controlling S100A8-provoked leukocyt e recruitment.