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• W2093897417 abstract "Reactive oxygen species generated by activated neutrophils can cause oxidative stress and tissue damage. S100A8 (A8) and S100A9 (A9), abundant in neutrophil cytoplasm, are exquisitely sensitive to oxidation, which may alter their functions. Murine A8 is a neutrophil chemoattractant, but it suppresses leukocyte transmigration in the microcirculation when S-nitrosylated. Glutathione (GSH) modulates intracellular redox, and S-glutathionylation can protect susceptible proteins from oxidative damage and regulate function. We characterized S-glutathionylation of A9; GSSG and GSNO generated S-glutathionylated A8 (A8-SSG) and A9 (A9-SSG) in vitro, whereas only A9-SSG was detected in cytosol of neutrophils activated with phorbol myristate acetate (PMA) but not with fMLP or opsonized zymosan. S-Glutathionylation exposed more hydrophobic regions in Zn2+-bound A9 but did not alter Zn2+ binding affinity. A9-SSG had reduced capacity to form heterocomplexes with A8, but the arachidonic acid binding capacities of A8/A9 and A8/A9-SSG were similar. A9 and A8/A9 bind endothelial cells; S-glutathionylation reduced binding. We found little effect of A9 or A9-SSG on neutrophil CD11b/CD18 expression or neutrophil adhesion to endothelial cells. However, A9, A9-SSG and A8/A9 promoted neutrophil adhesion to fibronectin but, in the presence of A8, A9-mediated adhesion was abrogated by glutathionylation. S-Glutathionylation of A9 may protect its oxidation to higher oligomers and reduce neutrophil binding to the extracellular matrix. This may regulate the magnitude of neutrophil migration in the extravasculature, and together with the functional changes we reported for S-nitrosylated A8, particular oxidative modifications of these proteins may limit tissue damage in acute inflammation. Reactive oxygen species generated by activated neutrophils can cause oxidative stress and tissue damage. S100A8 (A8) and S100A9 (A9), abundant in neutrophil cytoplasm, are exquisitely sensitive to oxidation, which may alter their functions. Murine A8 is a neutrophil chemoattractant, but it suppresses leukocyte transmigration in the microcirculation when S-nitrosylated. Glutathione (GSH) modulates intracellular redox, and S-glutathionylation can protect susceptible proteins from oxidative damage and regulate function. We characterized S-glutathionylation of A9; GSSG and GSNO generated S-glutathionylated A8 (A8-SSG) and A9 (A9-SSG) in vitro, whereas only A9-SSG was detected in cytosol of neutrophils activated with phorbol myristate acetate (PMA) but not with fMLP or opsonized zymosan. S-Glutathionylation exposed more hydrophobic regions in Zn2+-bound A9 but did not alter Zn2+ binding affinity. A9-SSG had reduced capacity to form heterocomplexes with A8, but the arachidonic acid binding capacities of A8/A9 and A8/A9-SSG were similar. A9 and A8/A9 bind endothelial cells; S-glutathionylation reduced binding. We found little effect of A9 or A9-SSG on neutrophil CD11b/CD18 expression or neutrophil adhesion to endothelial cells. However, A9, A9-SSG and A8/A9 promoted neutrophil adhesion to fibronectin but, in the presence of A8, A9-mediated adhesion was abrogated by glutathionylation. S-Glutathionylation of A9 may protect its oxidation to higher oligomers and reduce neutrophil binding to the extracellular matrix. This may regulate the magnitude of neutrophil migration in the extravasculature, and together with the functional changes we reported for S-nitrosylated A8, particular oxidative modifications of these proteins may limit tissue damage in acute inflammation. IntroductionNeutrophils play important roles in innate immune host defense against invading pathogens by producing a respiratory burst that generates reactive oxygen species (ROS) 2The abbreviations used are: ROSreactive oxygen speciesGSHglutathioneGSNOS-nitrosoglutathioneGSSGoxidized glutathioneSNOS-nitrosothiolAAarachidonic acidPMAphorbol 12-myristate 12-acetatefMLPN-formyl-Met-Leu-PheLCliquid chromatographyRP-HPLCreverse phase-high performance liquid chromatographyESIelectrospray ionizationMSmass spectrometryCIcell indexILinterleukinPBSphosphate-buffered salineANS1-anilino-8-naphthalene sulfonic acidA8S100A8A9S100A9VCAM-1vascular cell adhesion molecule-1ICAM-1intercellular adhesion molecule 1. via the NADPH oxidase complex and the myeloperoxidase system (1Smith J.A. J. Leukocyte Biol. 1994; 56: 672-686Crossref PubMed Scopus (752) Google Scholar, 2Hampton M.B. Kettle A.J. Winterbourn C.C. Blood. 1998; 92: 3007-3017Crossref PubMed Google Scholar). However, excessive ROS can lead to oxidative stress, inflicting damage to cells and tissues, thereby amplifying inflammation (3Ryter S.W. Kim H.P. Hoetzel A. Park J.W. Nakahira K. Wang X. Choi A.M. Antioxid. Redox Signal. 2007; 9: 49-89Crossref PubMed Scopus (934) Google Scholar). ROS also fine-tunes the inflammatory responses, depending on the circumstances of production and amounts produced (4Hultqvist M. Olsson L.M. Gelderman K.A. Holmdahl R. Trends Immunol. 2009; 30: 201-208Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar), and can induce reversible or irreversible oxidative modifications to Cys thiols of susceptible proteins. Reversible modifications may protect proteins against permanent oxidative damage and/or modulate their functions, but excessive ROS can lead to permanent loss of function and/or cell apoptosis and necrosis that may promote pathogenesis (5Niwa T. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007; 855: 59-65Crossref PubMed Scopus (46) Google Scholar).Glutathione (GSH) is an abundant anti-oxidant in cells and plays a critical role in protection from oxidative damage (6Richie Jr., J.P. Skowronski L. Abraham P. Leutzinger Y. Clin. Chem. 1996; 42: 64-70Crossref PubMed Scopus (213) Google Scholar). Protein S-glutathionylation, the disulfide coupling of a GSH moiety to Cys residues, is the prevalent S-thiolation reaction in biological systems (7Giustarini D. Rossi R. Milzani A. Colombo R. Dalle-Donne I. J. Cell. Mol. Med. 2004; 8: 201-212Crossref PubMed Scopus (247) Google Scholar, 8Ghezzi P. Romines B. Fratelli M. Eberini I. Gianazza E. Casagrande S. Laragione T. Mengozzi M. Herzenberg L.A. Herzenberg L.A. Mol. Immunol. 2002; 38: 773-780Crossref PubMed Scopus (87) Google Scholar), regulating numerous physiological processes (9Dalle-Donne I. Rossi R. Giustarini D. Colombo R. Milzani A. Free Radic. Biol. Med. 2007; 43: 883-898Crossref PubMed Scopus (380) Google Scholar). This modification may be driven by oxidative/nitrosative stress in the presence of endogenous GSH but can also persist under basal conditions and in reducing environments (10Lind C. Gerdes R. Hamnell Y. Schuppe-Koistinen I. von Löwenhielm H.B. Holmgren A. Cotgreave I.A. Arch. Biochem. Biophys. 2002; 406: 229-240Crossref PubMed Scopus (276) Google Scholar, 11Fratelli M. Demol H. Puype M. Casagrande S. Eberini I. Salmona M. Bonetto V. Mengozzi M. Duffieux F. Miclet E. Bachi A. Vandekerckhove J. Gianazza E. Ghezzi P. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 3505-3510Crossref PubMed Scopus (483) Google Scholar). It can occur via thiol-disulfide exchange with oxidized GSH (GSSG) or reaction of oxidative thiol intermediates such as S-nitrosothiols (SNO) with GSH (12Ghezzi P. Free Radic. Res. 2005; 39: 573-580Crossref PubMed Scopus (227) Google Scholar). Removal of GSH is promoted by reduced thiols and changes in intracellular redox or is catalyzed by enzymes such as thioredoxin and glutaredoxin (13Holmgren A. Johansson C. Berndt C. Lönn M.E. Hudemann C. Lillig C.H. Biochem. Soc. Trans. 2005; 33: 1375-1377Crossref PubMed Scopus (354) Google Scholar); reversibility is a key determinant of its physiological relevance in regulating protein function (14Shelton M.D. Mieyal J.J. Mol. Cells. 2008; 25: 332-346PubMed Google Scholar).Altered levels of S-glutathionylation in some proteins are associated with pathologies such as hyperlipidemia (15Niwa T. Naito C. Mawjood A.H. Imai K. Clin. Chem. 2000; 46: 82-88Crossref PubMed Scopus (97) Google Scholar), diabetes (16Al-Abed Y. VanPatten S. Li H. Lawson J.A. FitzGerald G.A. Manogue K.R. Bucala R. Mol. Med. 2001; 7: 619-623Crossref PubMed Google Scholar), Friedreich's ataxia (17Piemonte F. Pastore A. Tozzi G. Tagliacozzi D. Santorelli F.M. Carrozzo R. Casali C. Damiano M. Federici G. Bertini E. Eur. J. Clin. Invest. 2001; 31: 1007-1011Crossref PubMed Scopus (150) Google Scholar), diabetes, atherosclerosis, and cancer (14Shelton M.D. Mieyal J.J. Mol. Cells. 2008; 25: 332-346PubMed Google Scholar), and identification of targets and their functional consequences may be clinically important. Some targets include transcription factors (Jun, NF-κB), enzymes (creatine kinase, human immunodeficiency virus-1 protease), and cytoskeletal proteins (actin, tubulin), all of which influence critical pathways in growth, differentiation, and metabolism (7Giustarini D. Rossi R. Milzani A. Colombo R. Dalle-Donne I. J. Cell. Mol. Med. 2004; 8: 201-212Crossref PubMed Scopus (247) Google Scholar, 12Ghezzi P. Free Radic. Res. 2005; 39: 573-580Crossref PubMed Scopus (227) Google Scholar). Phagocytosis induces a respiratory burst in monocytes, resulting in S-thiolation of glyceraldehyde 3-phosphate and concomitant loss of activity (18Ravichandran V. Seres T. Moriguchi T. Thomas J.A. Johnston Jr., R.B. J. Biol. Chem. 1994; 269: 25010-25015Abstract Full Text PDF PubMed Google Scholar). Glutathionylation of the p50 and p65 subunits of NF-κB, a transcription factor with pivotal roles in inflammation and proliferation, inhibits its binding to promoter regions of genes (19Pineda-Molina E. Klatt P. Vázquez J. Marina A. García de Lacoba M. Pérez-Sala D. Lamas S. Biochemistry. 2001; 40: 14134-14142Crossref PubMed Scopus (340) Google Scholar, 20Qanungo S. Starke D.W. Pai H.V. Mieyal J.J. Nieminen A.L. J. Biol. Chem. 2007; 282: 18427-18436Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). S-Thiolated actin was generated in PMA-activated neutrophils (21Chai Y.C. Ashraf S.S. Rokutan K. Johnston Jr., R.B. Thomas J.A. Arch. Biochem. Biophys. 1994; 310: 273-281Crossref PubMed Scopus (195) Google Scholar) and may be important in cell spreading and cytoskeletal organization (22Fiaschi T. Cozzi G. Raugei G. Formigli L. Ramponi G. Chiarugi P. J. Biol. Chem. 2006; 281: 22983-22991Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar).S100A8 (A8, MRP 8, calgranulin A) and S100A9 (A9, MRP 14, calgranulin B) are highly expressed in neutrophils (comprise ∼40% of cytoplasmic proteins (23Edgeworth J. Gorman M. Bennett R. Freemont P. Hogg N. J. Biol. Chem. 1991; 266: 7706-7713Abstract Full Text PDF PubMed Google Scholar)) and induced in numerous cells, including monocytes (24Berntzen H.B. Fagerhol M.K. Scand. J. Clin. Lab. Invest. 1990; 50: 769-774Crossref PubMed Google Scholar), macrophages (25Hsu K. Passey R.J. Endoh Y. Rahimi F. Youssef P. Yen T. Geczy C.L. J. Immunol. 2005; 174: 2318-2326Crossref PubMed Scopus (86) Google Scholar, 26Endoh Y. Chung Y.M. Clark I.A. Geczy C.L. Hsu K. J. Immunol. 2009; 182: 2258-2268Crossref PubMed Scopus (54) Google Scholar, 27Xu K. Yen T. Geczy C.L. J. Immunol. 2001; 166: 6358-6366Crossref PubMed Scopus (72) Google Scholar), and keratinocytes (28Grimbaldeston M.A. Geczy C.L. Tedla N. 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A9 may also modulate the resolution of inflammation by suppressing macrophage activity after phagocytosis of apoptotic neutrophils (38De Lorenzo B.H. Godoy L.C. Novaes e Brite R.R. Pagano R.L. Amorim-Dias M.A. Grosso D.M. Lopes J.D. Mariano M. Immunobiology. 2009; 215: 341-347Crossref PubMed Scopus (29) Google Scholar). A9 suppressed A8-mediated NF-κB activation in murine bone marrow cells (39Vogl T. Tenbrock K. Ludwig S. Leukert N. Ehrhardt C. van Zoelen M.A. Nacken W. Foell D. van der Poll T. Sorg C. Roth J. Nat. Med. 2007; 13: 1042-1049Crossref PubMed Scopus (991) Google Scholar). Intracellularly, the heterocomplex transports arachidonic acid (AA) (40Klempt M. Melkonyan H. Nacken W. Wiesmann D. Holtkemper U. Sorg C. FEBS Lett. 1997; 408: 81-84Crossref PubMed Scopus (59) Google Scholar), which is implicated in NADPH oxidase activation (41Kerkhoff C. Nacken W. Benedyk M. Dagher M.C. Sopalla C. Doussiere J. FASEB J. 2005; 19: 467-469Crossref PubMed Scopus (129) Google Scholar). A9 enhances A8 binding to tubulin and A8-mediated microtubule polymerization in resting phagocytes. Phosphorylation of A9, mediated by p38 MAPK after phagocyte activation, may regulate cytoskeletal rearrangements that promote migration, a property supported by the impaired transendothelial migration of A9−/− neutrophils (42Vogl T. Ludwig S. Goebeler M. Strey A. Thorey I.S. Reichelt R. Foell D. Gerke V. Manitz M.P. Nacken W. Werner S. Sorg C. Roth J. Blood. 2004; 104: 4260-4268Crossref PubMed Scopus (244) Google Scholar).A8 and A9 are readily oxidized by reactive oxygen/nitrogen species, which promote structural changes that alter their functions. Oxidation of murine A8 to disulfide-linked dimers (43Harrison C.A. Raftery M.J. Walsh J. Alewood P. Iismaa S.E. Thliveris S. Geczy C.L. J. Biol. Chem. 1999; 274: 8561-8569Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) or intermolecular sulfinamide-linked complexes (44Raftery M.J. Yang Z. Valenzuela S.M. Geczy C.L. J. Biol. Chem. 2001; 276: 33393-33401Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) abolished its chemotactic activity and the fugetactic activity of human A8 and A9 is regulated by oxidation (45Sroussi H.Y. Berline J. Palefsky J.M. J. Leukocyte Biol. 2007; 81: 818-824Crossref PubMed Scopus (54) Google Scholar, 46Sroussi H.Y. Berline J. Dazin P. Green P. Palefsky J.M. J. Dent. Res. 2006; 85: 829-833Crossref PubMed Scopus (34) Google Scholar). A8 and A9 are more susceptible to oxidation by HOCl than low density lipoprotein (47McCormick M.M. Rahimi F. Bobryshev Y.V. Gaus K. Zreiqat H. Cai H. Lord R.S. Geczy C.L. J. Biol. Chem. 2005; 280: 41521-41529Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and their exquisite capacity to scavenge ROS makes them primary candidates for protecting cells against oxidative stress in a localized environment. Recently, we showed that A8 and A9 are S-nitrosylated by the physiological NO donor S-nitrosoglutathione (GSNO) in vitro; A8 is more susceptible, and A8-SNO suppressed mast cell-mediated inflammation by reducing leukocyte adhesion and extravasation in the rat mesenteric microcirculation (48Lim S.Y. Raftery M. Cai H. Hsu K. Yan W.X. Hseih H.L. Watts R.N. Richardson D. Thomas S. Perry M. Geczy C.L. J. Immunol. 2008; 181: 5627-5636Crossref PubMed Scopus (86) Google Scholar). Moreover, both proteins are up-regulated in microvascular endothelial cells by proinflammatory mediators such as IL-1β or tumor necrosis factor-α (49Yen T. Harrison C.A. Devery J.M. Leong S. Iismaa S.E. Yoshimura T. Geczy C.L. Blood. 1997; 90: 4812-4821Crossref PubMed Google Scholar), and oxidative changes in the microcirculation during inflammation may alter functions of these proteins in endothelial cells.Here we show that A9 in neutrophils is S-glutathionylated after activation. S-Glutathionylated A8 (A8-SSG) and A9 (A9-SSG) were generated with GSNO and GSSG in vitro, whereas only A9-SSG was detected in neutrophils activated with PMA. The single Cys3 residue of A9, the site of GSH addition, is present only in the full-length form, which accounted for ∼72% of total A9 in neutrophils. Structural changes in A9-SSG apparently altered its affinity for complex formation with A8 but did not affect the capacity of A8/A9 to bind AA. A9 binding to endothelial cells is implicated in their activation (50Viemann D. Strey A. Janning A. Jurk K. Klimmek K. Vogl T. Hirono K. Ichida F. Foell D. Kehrel B. Gerke V. Sorg C. Roth J. Blood. 2005; 105: 2955-2962Crossref PubMed Scopus (237) Google Scholar); significantly less A9-SSG and A8/A9-SSG bound HMEC-1 endothelial cells compared with the unmodified forms, likely due to differences in protein conformation. Moreover, unlike A8/A9, A8/A9-SSG did not induce neutrophil adhesion to fibronectin. We propose that glutathionylation may protect A9 from oxidative damage in activated neutrophils and may limit leukocyte exudation in inflammatory lesions.DISCUSSIONResponses of cells to oxidative stress typically involve changes in thiol content, and S-thiolation is an important protective mechanism that can regulate functions of susceptible proteins (9Dalle-Donne I. Rossi R. Giustarini D. Colombo R. Milzani A. Free Radic. Biol. Med. 2007; 43: 883-898Crossref PubMed Scopus (380) Google Scholar, 72Klatt P. Lamas S. Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (655) Google Scholar). A8 and A9, abundant in neutrophil cytosol, are extremely sensitive to oxidation by reactive oxygen/nitrogen species (43Harrison C.A. Raftery M.J. Walsh J. Alewood P. Iismaa S.E. Thliveris S. Geczy C.L. J. Biol. Chem. 1999; 274: 8561-8569Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 47McCormick M.M. Rahimi F. Bobryshev Y.V. Gaus K. Zreiqat H. Cai H. Lord R.S. Geczy C.L. J. Biol. Chem. 2005; 280: 41521-41529Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 48Lim S.Y. Raftery M. Cai H. Hsu K. Yan W.X. Hseih H.L. Watts R.N. Richardson D. Thomas S. Perry M. Geczy C.L. J. Immunol. 2008; 181: 5627-5636Crossref PubMed Scopus (86) Google Scholar), and oxidative modifications alter several functions (43Harrison C.A. Raftery M.J. Walsh J. Alewood P. Iismaa S.E. Thliveris S. Geczy C.L. J. Biol. Chem. 1999; 274: 8561-8569Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 44Raftery M.J. Yang Z. Valenzuela S.M. Geczy C.L. J. Biol. Chem. 2001; 276: 33393-33401Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 45Sroussi H.Y. Berline J. Palefsky J.M. J. Leukocyte Biol. 2007; 81: 818-824Crossref PubMed Scopus (54) Google Scholar, 73Sroussi H.Y. Köhler G.A. Agabian N. Villines D. Palefsky J.M. FEMS Immunol. Med. Microbiol. 2009; 55: 55-61Crossref PubMed Scopus (32) Google Scholar); we proposed that they may act as an oxidant sink. Human A8 and A9 are induced in neovessels at inflammatory sites (47McCormick M.M. Rahimi F. Bobryshev Y.V. Gaus K. Zreiqat H. Cai H. Lord R.S. Geczy C.L. J. Biol. Chem. 2005; 280: 41521-41529Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and are readily S-nitrosylated, and A8-SNO may modulate blood vessel homeostasis (48Lim S.Y. Raftery M. Cai H. Hsu K. Yan W.X. Hseih H.L. Watts R.N. Richardson D. Thomas S. Perry M. Geczy C.L. J. Immunol. 2008; 181: 5627-5636Crossref PubMed Scopus (86) Google Scholar). Hence, changes in intracellular/extracellular redox are likely to regulate their functional properties. Moreover, their dependence on IL-10 for induction in activated human monocytes/macrophages by TLR-3 and -4 ligands (26Endoh Y. Chung Y.M. Clark I.A. Geczy C.L. Hsu K. J. Immunol. 2009; 182: 2258-2268Crossref PubMed Scopus (54) Google Scholar) and their enhanced expression in various cells by anti-inflammatory agents such as corticosteroids (25Hsu K. Passey R.J. Endoh Y. Rahimi F. Youssef P. Yen T. Geczy C.L. J. Immunol. 2005; 174: 2318-2326Crossref PubMed Scopus (86) Google Scholar) implicates A8 and A9 in resolution of inflammation.S-Glutathionylation is a reversible modification that can regulate functions of numerous proteins (9Dalle-Donne I. Rossi R. Giustarini D. Colombo R. Milzani A. Free Radic. Biol. Med. 2007; 43: 883-898Crossref PubMed Scopus (380) Google Scholar, 72Klatt P. Lamas S. Eur. J. Biochem. 2000; 267: 4928-4944Crossref PubMed Scopus (655) Google Scholar) including signaling and adhesion molecules, cytokines, and enzymes associated with inflammation (for review, see Ref. 14Shelton M.D. Mieyal J.J. Mol. Cells. 2008; 25: 332-346PubMed Google Scholar). Thus, glutathionylation of A8 and/or A9, particularly after phagocyte activation, may have important regulatory implications. However, A9 is expressed in two forms in neutrophils due to an alternate translation start site encoding Met5 (56Teigelkamp S. Bhardwaj R.S. Roth J. Meinardus-Hager G. Karas M. Sorg C. J. Biol. Chem. 1991; 266: 13462-13467Abstract Full Text PDF PubMed Google Scholar), and truncated A9* (A95–114) lacks the single Cys present in full-length A9 (Fig. 2A). Relative expression levels of these isoforms may be important because they are likely to have some functional distinctions, particularly as A9* would not be S-glutathionylated or S-nitrosylated. We first determined the relative levels of these isoforms in neutrophil cytosol and established that full-length A9 composed ∼70% and A9* composed ∼30% of total A9.S-Glutathionylated adducts of recombinant A8 and A9 were generated by GSNO or GSSG in vitro and confirmed by ESI-MS; Ca2+ or Zn2+ did not alter reactivity. Formation was reversed by GSH (not shown). Glutathionylation with GSSG likely occurred via thiol-disulfide exchange as proposed (74Gallogly M.M. Mieyal J.J. Curr. Opin. Pharmacol. 2007; 7: 381-391Crossref PubMed Scopus (369) Google Scholar). GSNO generated S-nitrosylated and S-glutathionylated A8 (48Lim S.Y. Raftery M. Cai H. Hsu K. Yan W.X. Hseih H.L. Watts R.N. Richardson D. Thomas S. Perry M. Geczy C.L. J. Immunol. 2008; 181: 5627-5636Crossref PubMed Scopus (86) Google Scholar), and because GSNO can promote S-glutathionylation via S-nitrosothiol intermediates (75Giustarini D. Milzani A. Aldini G. Carini M. Rossi R. Dalle-Donne I. Antioxid. Redox Signal. 2005; 7: 930-939Crossref PubMed Scopus (117) Google Scholar), we tested this possibility and confirmed generation of A9-SSG (Fig. 1B).Neutrophil activation generates superoxides via the NADPH oxidase complex, subsequently producing other ROS (76El-Benna J. Dang P.M. Gougerot-Pocidalo M.A. Elbim C. Arch. Immunol. Ther. Exp. (Warsz). 2005; 53: 199-206PubMed Google Scholar). Early studies of S-thiolated proteins in PMA-activated neutrophils using Tran35S-labeling demonstrated an ∼3–5% S-thiolation, predominantly glutathionylation, of a 14-kDa protein that increased over 30 min activation and was reduced by dithiothreitol. This is likely to be A9 because the protein was described as “the most abundant band in neutrophil extracts” (21Chai Y.C. Ashraf S.S. Rokutan K. Johnston Jr., R.B. Thomas J.A. Arch. Biochem. Biophys. 1994; 310: 273-281Crossref PubMed Scopus (195) Google Scholar). To analyze modifications of A8 and A9 after neutrophil activation, cytosolic proteins were separated by HPLC and analyzed by ESI-MS. Products likely corresponding to A9-SSG were generated by PMA or PMA plus ionomycin stimulation (Fig. 3, B and C), whereas none was separated from unstimulated cell lysates. Little co-localization of A9 with GSH was obvious in unstimulated neutrophils (Fig. 4A) but increased after PMA plus ionomycin stimulation (Fig. 4B), suggesting A9-SSG complexes. A9-SSG was not detected in the cytosol of neutrophils activated with opsonized zymosan or fMLP (Table 2), although in opsonized zymosan-activated cells, A9 and GSH co-localized around the plasma membrane and possibly the phagosomal membrane (Fig. 4D). Similarly, A9 associates with neutrophil phagosomes after phagocytosis of antibody- and complement-coated latex beads (77Burlak C. Whitney A.R. Mead D.J. Hackstadt T. Deleo F.R. Mol. Cell. Proteomics. 2006; 5: 620-634Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Thus, A9 may associate with GSH in particular cell compartments. Little co-localization of A9 with GSH was obvious in neutrophils activated with fMLP (Fig. 4C), consistent with the apparent absence of A9-SSG in cytosol extracts from this population. Apart from actin (21Chai Y.C. Ashraf S.S. Rokutan K. Johnston Jr., R.B. Thomas J.A. Arch. Biochem. Biophys. 1994; 310: 273-281Crossref PubMed Scopus (195) Google Scholar), the subcellular localization of glutathione-protein adducts in activated neutrophils is virtually unexplored, although studies in other cells indicate their presence in various compartments (for review, see Ref. 78Martínez-Ruiz A. Lamas S. Cardiovasc. Res. 2007; 75: 220-228Crossref PubMed Scopus (153) Google Scholar). Our data suggest that A9-SSG may be generated in particular compartments depending on the stimulus and may have functional differences that warrant further investigation.Because of its relative abundance and stability, we focused on structural characterization and functional consequences of A9 glutathionylation. This modification did not alter its Ca2+ or Zn2+ binding affinities, although conformational changes indicated increased surface hydrophobicity upon Zn2+ binding (Fig. 5). Thus, in certain microenvironments, particularly after neutrophil activation that promotes A9 translocation from the cytosol to the membrane, glutathionylation of A9 may facilitate interactions with lipid environments within membranes (64van den Bos C. Roth J. Koch H.G. Hartmann M. Sorg C. J. Immunol. 1996; 156: 1247-1254PubMed Google Scholar) or with other hydrophobic components such as fatty acids (79Siegenthaler G. Roulin K. Chatellard-Gruaz D. Hotz R. Saurat J.H. Hellman U. Hagens G. J. Biol. Chem. 1997; 272: 9371-9377Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In a preliminary attempt to assess the ability of A8 and A9 to form complexes using cross-linking, we found that A9-SSG may have reduced capacity to form the heterocomplex but may preferentially form more higher order A9 homocomplexes (not shown). Several intracellular functions are dependent on A8/A9 heterocomplex formation (53Kerkhoff C. Klempt M. Kaever V. Sorg C. J. Biol. Chem. 1999; 274: 32672-32679Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 80Leukert N. Vogl T. Strupat K. Reichelt R. Sorg C. Roth J. J. Mol. Biol. 2006; 359: 961-972Crossref PubMed Scopus (130) Google Scholar), and differences in the Zn2+-bound conformation of A9-SSG and its propensity to form homocomplexes are likely to alter function. To test this, we examined AA binding (53Kerkhoff C. Klempt M. Kaever V. Sorg C. J. Biol. Chem. 1999; 274: 32672-32679Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) but found that binding capacities of A8/A9 and A8/A9-SSG were similar.Formation of thiol disulfides may protect key proteins including A9 from forming irreversible Cys adducts such as sulfenic and sulfonic acid derivatives during oxidative stress (81Poole L.B. Karplus P.A. Claiborne A. Annu. Rev. Pharmacol. Toxicol. 2004; 44: 325-347Crossref PubMed Scopus (494) Google Scholar). 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Med. 2003; 141: 110-120Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar, 85Vandal K. Rouleau P. Boivin A. Ryckman C. Talbot M. Tessier P.A. J. Immunol. 2003; 171: 2602-2609Crossref PubMed Scopus (190) Google Scholar). They are released from neutrophils during inflammatory responses induced by lipopolysaccharide (85Vandal K. Rouleau P. Boivin A. Ryckman C. Talbot M. Tessier P.A. J. Immunol. 2003; 171: 2602-2609Crossref PubMed Scopus (190) Google Scholar), IL-1β, and tumor necrosis factor (30Rammes A. Roth J. Goebeler M. Klempt M. Hartmann M." @default.
• W2093897417 created "2016-06-24" @default.
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• W2093897417 date "2010-05-01" @default.
• W2093897417 modified "2023-09-30" @default.
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