Abstract
The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment 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 hypochlorite oxidizes the single Cys residue (Cys41) of S100A8. Electrospray mass spectrometry and SDS-polyacrylamide gel electrophoresis analysis indicated that low concentrations of hypochlorite (40 microM) converted 70-80% of S100A8 to the disulfide-linked homodimer. The mass was 20,707 Da, 92 Da more than expected, indicating additional oxidation of susceptible amino acids (possibly methionine). Phorbol 12-myristate 13-acetate activation of differentiated HL-60 granulocytic cells generated an oxidative burst that was sufficient to efficiently oxidize exogenous S100A8 within 10 min, and results implicate involvement of the myeloperoxidase system. Moreover, disulfide-linked dimer was identified in lung lavage fluid of mice with endotoxin-induced pulmonary injury. S100A8 dimer was inactive in chemotaxis and failed to recruit leukocytes in vivo. Positive chemotactic activity of recombinant Ala41S100A8 indicated that Cys41 was not essential for function and suggested that covalent dimerization may structurally modify accessibility of the chemotactic hinge domain. Disulfide-dependent dimerization may be a physiologically significant regulatory mechanism controlling S100A8-provoked leukocyte recruitment.
Highlights
The myeloid cell-derived calcium-binding murine protein, S100A8, is secreted to act as a chemotactic factor at picomolar concentrations, stimulating recruitment of myeloid cells to inflammatory sites
We recently demonstrated S100A8 secretion from LPSactivated murine macrophages [9], and in the absence of a secretion signal sequence, Rammes et al [13] proposed a novel tubulin-dependent pathway for S100A8/A9 release from activated human monocytes which may provide a paradigm for the secretion of other S100 proteins
S100A8 was isolated as a soluble product of activated murine spleen cells [14] and in the picomolar range stimulates myeloid cell chemotaxis in vitro [15] and a sustained leukocyte recruitment, with mononuclear cell infiltration following an early influx of neutrophils, in vivo [15, 16]
Summary
RPMI 1640 and fetal bovine serum were purchased from Life Technologies, Inc. Hanks’ balanced salt solution (HBSS), HBSS (without CaCl2 and MgCl2), bovine serum albumin, sodium hypochlorite, dimethyl sulfoxide (Me2SO), phorbol 12-myristate 13-acetate (PMA), Nethylmaleimide (NEM), and Triton X-100 were purchased from Sigma. S100 proteins were treated with increasing concentrations of hypochlorite (10 –100 M) for 15 min on ice before an equal volume of 2 ϫ SDS-PAGE sample mixture (Ϯ100 mM DTT) was added, and samples were heated (100 °C, 3 min) and analyzed by 10% SDS-PAGE and silver staining or Western blotting using anti-S100A8. An equal volume of 2ϫ SDS-PAGE sample mixture was added, and samples were analyzed by 10% non-reducing SDS-PAGE and silver staining or Western blot analysis using anti-S100A8. Specific pathogen-free female Balb/c mice (6 – 8 weeks) were injected intraperitoneally with 1 ml of S100A8 (final concentration 10 g per mouse), or analogues, or vehicle control solution, sacrificed after 16 h (maximal leukocyte recruitment occurred between 16 and 24 h [32]), and cells harvested by peritoneal lavage with 5 ml HBSS, 0.38% citrate, washed twice, and counted
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