Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Tyrosine 192 in Apolipoprotein A-I Is the Major Site of Nitration and Chlorination by Myeloperoxidase, but Only Chlorination Markedly Impairs ABCA1-dependent Cholesterol Transport

2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês

10.1074/jbc.m411484200

ISSN

1083-351X

Autores

Baohai Shao, Constanze Bergt, Xiaoyun Fu, Pattie Green, John C. Voss, Michael N. Oda, John F. Oram, Jay W. Heinecke,

Tópico(s)

Cholesterol and Lipid Metabolism

Resumo

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO-). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO-. Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr192 of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO-, suggesting that solvent accessibility accounted in part for the reactivity of Tyr192. The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr192. Both ONOO- and MPO nitrated Tyr192 in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr192 is the predominant site of nitration and chlorination when MPO or ONOO- oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease. High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in humans, indicating that MPO oxidizes HDL in vivo. In the current studies, we used tandem mass spectrometry to identify the major sites of tyrosine oxidation when lipid-free apolipoprotein A-I (apoA-I), the major protein of HDL, was exposed to MPO or peroxynitrite (ONOO-). Tyrosine 192 was the predominant site of both nitration and chlorination by MPO and was also the major site of nitration by ONOO-. Electron paramagnetic spin resonance studies of spin-labeled apoA-I revealed that residue 192 was located in an unusually hydrophilic environment. Moreover, the environment of residue 192 became much more hydrophobic when apoA-I was incorporated into discoidal HDL, and Tyr192 of HDL-associated apoA-I was a poor substrate for nitration by both myeloperoxidase and ONOO-, suggesting that solvent accessibility accounted in part for the reactivity of Tyr192. The ability of lipid-free apoA-I to facilitate ATP-binding cassette transporter A1 cholesterol transport was greatly reduced after chlorination by MPO. Loss of activity occurred in concert with chlorination of Tyr192. Both ONOO- and MPO nitrated Tyr192 in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr192 is the predominant site of nitration and chlorination when MPO or ONOO- oxidizes lipid-free apoA-I but that only chlorination markedly reduces the cholesterol efflux activity of apoA-I. This impaired biological activity of chlorinated apoA-I suggests that MPO-mediated oxidation of HDL might contribute to the link between inflammation and cardiovascular disease. Many lines of evidence indicate that high density lipoprotein (HDL) 1The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; ClY, chlorotyrosine; DTPA, diethylenetriaminepentaacetic acid; ESI, electrospray ionization; MS, mass spectrometry; NO2Y, nitrotyrosine; SDSL-EPR, site-directed spin label electron paramagnetic resonance spectroscopy; HPLC, high pressure liquid chromatography; LC, liquid chromatography; BHK, baby hamster kidney.1The abbreviations used are: HDL, high density lipoprotein; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; ClY, chlorotyrosine; DTPA, diethylenetriaminepentaacetic acid; ESI, electrospray ionization; MS, mass spectrometry; NO2Y, nitrotyrosine; SDSL-EPR, site-directed spin label electron paramagnetic resonance spectroscopy; HPLC, high pressure liquid chromatography; LC, liquid chromatography; BHK, baby hamster kidney. protects the artery wall from atherosclerosis. One important pathway involves HDL apolipoproteins that remove cellular cholesterol and phospholipids by an active transport process mediated by ATP-binding cassette transporter A1 (ABCA1) (1Oram J.F. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 720-727Crossref PubMed Scopus (207) Google Scholar, 2Wang N. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (215) Google Scholar, 3Joyce C. Freeman L. Brewer Jr., H.B. Santamarina-Fojo S. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 965-971Crossref PubMed Scopus (98) Google Scholar, 4Singaraja R.R. Brunham L.R. Visscher H. Kastelein J.J. Hayden M.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1322-1332Crossref PubMed Scopus (221) Google Scholar, 5Aiello R.J. Brees D. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 972-980Crossref PubMed Scopus (131) Google Scholar). Lipid-laden macrophages represent the cellular hallmark of the atherosclerotic lesion, and ABCA1 plays a critical role in removing cholesterol from macrophages in vivo (1Oram J.F. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 720-727Crossref PubMed Scopus (207) Google Scholar, 2Wang N. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (215) Google Scholar, 3Joyce C. Freeman L. Brewer Jr., H.B. Santamarina-Fojo S. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 965-971Crossref PubMed Scopus (98) Google Scholar, 4Singaraja R.R. Brunham L.R. Visscher H. Kastelein J.J. Hayden M.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1322-1332Crossref PubMed Scopus (221) Google Scholar, 5Aiello R.J. Brees D. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 972-980Crossref PubMed Scopus (131) Google Scholar). The most abundant apolipoprotein in HDL is apolipoprotein A-I (apoA-I), which accounts for ∼70% of the total protein content. Lipid-poor apoA-I promotes cellular efflux of cholesterol and phospholipids exclusively by the ABCA1 pathway (1Oram J.F. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 720-727Crossref PubMed Scopus (207) Google Scholar, 2Wang N. Tall A.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1178-1184Crossref PubMed Scopus (215) Google Scholar, 3Joyce C. Freeman L. Brewer Jr., H.B. Santamarina-Fojo S. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 965-971Crossref PubMed Scopus (98) Google Scholar, 4Singaraja R.R. Brunham L.R. Visscher H. Kastelein J.J. Hayden M.R. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1322-1332Crossref PubMed Scopus (221) Google Scholar, 5Aiello R.J. Brees D. Francone O.L. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 972-980Crossref PubMed Scopus (131) Google Scholar). Several other mechanisms, including the ability of HDL to inhibit low density lipoprotein oxidation, reduce lipid hydroperoxides, and transport oxidized lipids to the liver for excretion, may also be cardioprotective (6Parthasarathy S. Barnett J. Fong L.G. Biochim. Biophys. Acta. 1990; 1044: 275-283Crossref PubMed Scopus (633) Google Scholar, 7Francis G.A. Mendez A.J. Bierman E.L. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6631-6635Crossref PubMed Scopus (133) Google Scholar, 8Garner B. Waldeck A.R. Witting P.K. Rye K.A. Stocker R. J. Biol. Chem. 1998; 273: 6088-6095Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 9Christison J. Karjalainen A. Brauman J. Bygrave F. Stocker R. Biochem. J. 1996; 314: 739-742Crossref PubMed Scopus (69) Google Scholar, 10Marsche G. Hammer A. Oskolkova O. Kozarsky K.F. Sattler W. Malle E. J. Biol. Chem. 2002; 277: 32172-32179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 11Navab M. Ananthramaiah G.M. Reddy S.T. Van Lenten B.J. Ansell B.J. Fonarow G.C. Vahabzadeh K. Hama S. Hough G. Kamranpour N. Berliner J.A. Lusis A.J. Fogelman A.M. J. Lipid Res. 2004; 45: 993-1007Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar, 12Getz G.S. Reardon C.A. Curr. Opin. Lipidol. 2004; 15: 261-267Crossref PubMed Scopus (145) Google Scholar, 13Francis G.A. Biochim. Biophys. Acta. 2000; 1483: 217-235Crossref PubMed Scopus (109) Google Scholar, 14Bergt C. Oram J.F. Heinecke J.W. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1488-1490Crossref PubMed Scopus (25) Google Scholar). Methionine and phenylalanine residues in apoA-I are oxidized by reactive intermediates (8Garner B. Waldeck A.R. Witting P.K. Rye K.A. Stocker R. J. Biol. Chem. 1998; 273: 6088-6095Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 9Christison J. Karjalainen A. Brauman J. Bygrave F. Stocker R. Biochem. J. 1996; 314: 739-742Crossref PubMed Scopus (69) Google Scholar, 10Marsche G. Hammer A. Oskolkova O. Kozarsky K.F. Sattler W. Malle E. J. Biol. Chem. 2002; 277: 32172-32179Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 15Panzenboeck U. Raitmayer S. Reicher H. Lindner H. Glatter O. Malle E. Sattler W. J. Biol. Chem. 1997; 272: 29711-29720Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Panzenbock U. Kritharides L. Raftery M. Rye K.A. Stocker R. J. Biol. Chem. 2000; 275: 19536-19544Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17Bergt C. Oettl K. Keller W. Andreae F. Leis H.J. Malle E. Sattler W. Biochem. J. 2000; 346: 345-354Crossref PubMed Scopus (47) Google Scholar), but it is unclear if oxidation of these residues affects the ability of the apolipoprotein to remove cholesterol from cells. Whether specific tyrosine residues in apoA-I are also vulnerable to oxidation in vivo is unclear, although its tyrosine residues are readily converted to o,o′-dityrosine by tyrosyl radical in vitro (7Francis G.A. Mendez A.J. Bierman E.L. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6631-6635Crossref PubMed Scopus (133) Google Scholar). Macrophages might be an important source of oxidants that damage HDL. One pathway involves myeloperoxidase, a phagocyte heme protein that is expressed at high levels in human atherosclerotic tissue (19Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1103) Google Scholar, 20Sugiyama S. Okada Y. Sukhova G.K. Virmani R. Heinecke J.W. Libby P. Am. J. Pathol. 2001; 158: 879-891Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar). The enzyme uses hydrogen peroxide (H2O2) for oxidative reactions in the extracellular milieu (21Harrison J.E. Shultz J. J. Biol. Chem. 1976; 251: 1371-1374Abstract Full Text PDF PubMed Google Scholar, 22Foote C.S. Goyne T.E. Lehrer R.I. Nature. 1983; 301: 715-716Crossref PubMed Scopus (226) Google Scholar, 23Hurst J.K. Barrette Jr., W.C. Crit. Rev. Biochem. Mol. Biol. 1989; 24: 271-328Crossref PubMed Scopus (219) Google Scholar, 24Klebanoff S.J. Clark R.A. The Neutrophil: Functional and Clinical Disorders. North-Holland Pub. Co., New York1978Google Scholar). The major end product at plasma concentrations of chloride ion (Cl-) is generally thought to be hypochlorous acid (HOCl). Cl−+H2O2+H+→HOCl+H2OReaction 1 LDL isolated from human atherosclerotic lesions contains elevated levels of 3-chlorotyrosine, a product characteristic of HOCl (25Hazen S.L. Heinecke J.W. J. Clin. Invest. 1997; 99: 2075-2081Crossref PubMed Scopus (744) Google Scholar, 26Domigan N.M. Charlton T.S. Duncan M.W. Winterbourn C.C. Kettle A.J. J. Biol. Chem. 1995; 270: 16542-16548Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 27Hazen S.L. Hsu F.F. Crowley J.R. Mueller D.M. Heinecke J.W. J. Clin. Invest. 1996; 98: 1283-1289Crossref PubMed Scopus (231) Google Scholar), indicating that myeloperoxidase is one pathway for oxidizing lipoproteins in the human artery wall. Myeloperoxidase can also produce nitrogen dioxide radical (NO·2), a reactive species that converts tyrosine to 3-nitrotyrosine (28Klebanoff S.J. Free Radic. Biol. Med. 1993; 14: 351-360Crossref PubMed Scopus (195) Google Scholar, 29Eiserich J.P. Hristova M. Cross C.E. Jones A.D. Freeman B.A. Halliwell B. van der Vliet A. Nature. 1998; 391: 393-397Crossref PubMed Scopus (1344) Google Scholar, 30Gaut J.P. Byun J. Tran H.D. Lauber W.M. Carroll J.A. Hotchkiss R.S. Belaaouaj A. Heinecke J.W. J. Clin. Invest. 2002; 109: 1311-1319Crossref PubMed Scopus (197) Google Scholar). We have recently shown that myeloperoxidase nitrates HDL in vitro (31Pennathur S. Bergt C. Shao B. Byun J. Kassim S.Y. Singh P. McDonald T.O. Brunzell J. Chait A. Oram J.F. O'Brien K. Geary R.L. Heinecke J.W. J. Biol. Chem. 2004; 279: 42977-42983Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The reaction probably involves direct one-electron oxidation of nitrite (NO2−) by compound I, a complex of myeloperoxidase and H2O2. NO2−+compound I→NO2+compound IIReaction 2 LDL isolated from atherosclerotic lesions is enriched in 3-nitrotyrosine (32Leeuwenburgh C. Hardy M.M. Hazen S.L. Wagner P. Oh-ishi S. Steinbrecher U.P. Heinecke J.W. J. Biol. Chem. 1997; 272: 1433-1436Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar), indicating that myeloperoxidase or other pathways that generate reactive nitrogen species also operate in the atherosclerotic artery wall. Peroxynitrite (ONOO-) is another nitrating species that might be important in oxidizing lipoproteins during inflammation (18Souza J.M. Daikhin E. Yudkoff M. Raman C.S. Ischiropoulos H. Arch. Biochem. Biophys. 1999; 371: 169-178Crossref PubMed Scopus (279) Google Scholar, 33Beckman J.S. Koppenol W.H. Am. J. Physiol. 1996; 271: C1424-C1437Crossref PubMed Google Scholar, 34Beckman J.S. Beckman T.W. Chen J. Marshall P.A. Freeman B.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1620-1624Crossref PubMed Scopus (6644) Google Scholar). It is generated when nitric oxide (NO) reacts with superoxide (O2⋅¯). O2−+NO→ONOO−Reaction 3 Like nitrogen dioxide radical, ONOO- generates 3-nitrotyrosine when it reacts with tyrosine residues. We have shown that Tyr192 is the single major site of chlorination in apoA-I of HDL exposed to HOCl in vitro (35Bergt C. Fu X. Huq N.P. Kao J. Heinecke J.W. J. Biol. Chem. 2004; 279: 7856-7866Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Tyr192 resides in an YXXK motif, and molecular modeling demonstrates that these tyrosine and lysine residues are adjacent on the same face of an amphipathic α-helix in apoA-I. HOCl reacts rapidly with the ϵ amino group of lysine to form long lived chloramines (23Hurst J.K. Barrette Jr., W.C. Crit. Rev. Biochem. Mol. Biol. 1989; 24: 271-328Crossref PubMed Scopus (219) Google Scholar, 24Klebanoff S.J. Clark R.A. The Neutrophil: Functional and Clinical Disorders. North-Holland Pub. Co., New York1978Google Scholar, 35Bergt C. Fu X. Huq N.P. Kao J. Heinecke J.W. J. Biol. Chem. 2004; 279: 7856-7866Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Studies with synthetic peptides demonstrate that lysine residues can direct the regiospecific chlorination of tyrosine residues by a reaction pathway involving chloramine formation, suggesting that the site-specific chlorination of apoA-I requires the participation of a nearby lysine residue (35Bergt C. Fu X. Huq N.P. Kao J. Heinecke J.W. J. Biol. Chem. 2004; 279: 7856-7866Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Recent studies demonstrate that HDL is chlorinated and nitrated in human atherosclerotic lesions (31Pennathur S. Bergt C. Shao B. Byun J. Kassim S.Y. Singh P. McDonald T.O. Brunzell J. Chait A. Oram J.F. O'Brien K. Geary R.L. Heinecke J.W. J. Biol. Chem. 2004; 279: 42977-42983Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 36Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. Geary R.L. Oram J.F. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13032-13037Crossref PubMed Scopus (376) Google Scholar, 37Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. J. Clin. Invest. 2004; 114: 529-541Crossref PubMed Scopus (636) Google Scholar), indicating that myeloperoxidase is one pathway for oxidizing HDL in vivo. Tandem mass spectrometric (MS) analysis identified myeloperoxidase as a component of lesion HDL (36Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. Geary R.L. Oram J.F. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13032-13037Crossref PubMed Scopus (376) Google Scholar), suggesting that the enzyme and lipoprotein interact in the artery wall. Moreover, 3-chlorotyrosine and 3-nitrotyrosine were also detected in circulating HDL (31Pennathur S. Bergt C. Shao B. Byun J. Kassim S.Y. Singh P. McDonald T.O. Brunzell J. Chait A. Oram J.F. O'Brien K. Geary R.L. Heinecke J.W. J. Biol. Chem. 2004; 279: 42977-42983Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 36Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. Geary R.L. Oram J.F. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13032-13037Crossref PubMed Scopus (376) Google Scholar, 37Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. J. Clin. Invest. 2004; 114: 529-541Crossref PubMed Scopus (636) Google Scholar). Levels of these oxidized amino acids were elevated in HDL isolated from the blood of humans with established coronary artery disease, raising the possibility that circulating levels of chlorinated and nitrated HDL represent a novel marker for clinically significant atherosclerosis (31Pennathur S. Bergt C. Shao B. Byun J. Kassim S.Y. Singh P. McDonald T.O. Brunzell J. Chait A. Oram J.F. O'Brien K. Geary R.L. Heinecke J.W. J. Biol. Chem. 2004; 279: 42977-42983Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 36Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. Geary R.L. Oram J.F. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13032-13037Crossref PubMed Scopus (376) Google Scholar, 37Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. J. Clin. Invest. 2004; 114: 529-541Crossref PubMed Scopus (636) Google Scholar). Tyrosine chlorination and nitration may be physiologically significant because HDL or apoA-I exposed to HOCl or the myeloperoxidase system is less able to remove cholesterol from cultured cells by the ABCA1 pathway (36Bergt C. Pennathur S. Fu X. Byun J. O'Brien K. McDonald T.O. Singh P. Anantharamaiah G.M. Chait A. Brunzell J. Geary R.L. Oram J.F. Heinecke J.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13032-13037Crossref PubMed Scopus (376) Google Scholar, 37Zheng L. Nukuna B. Brennan M.L. Sun M. Goormastic M. Settle M. Schmitt D. Fu X. Thomson L. Fox P.L. Ischiropoulos H. Smith J.D. Kinter M. Hazen S.L. J. Clin. Invest. 2004; 114: 529-541Crossref PubMed Scopus (636) Google Scholar). Remarkably little is known about the factors that control the site-specific nitration and chlorination of tyrosine residues in proteins. In the current study, we use apoA-I, synthetic peptides, and tandem mass spectrometric analysis to investigate the role of reactive nitrogen and chlorine species in modifying apoA-I and altering its ability to remove cholesterol from cells. Our observations indicate that Tyr192 is the predominant site of nitration as well as chlorination in apoA-I. However, only chlorination of apoA-I markedly impairs ABCA1-dependent cholesterol transport by the oxidized apolipoprotein. Myeloperoxidase (donor:hydrogen peroxide, oxidoreductase; EC 1.11.1.7) was isolated by lectin affinity and size exclusion chromatographies from human neutrophils (38Heinecke J.W. Li W. Daehnke H.L.D. Goldstein J.A. J. Biol. Chem. 1993; 268: 4069-4077Abstract Full Text PDF PubMed Google Scholar, 39Hope H.R. Remsen E.E. Lewis Jr., C. Heuvelman D.M. Walker M.C. Jennings M. Connolly D.T. Protein Expr. Purif. 2000; 18: 269-276Crossref PubMed Scopus (30) Google Scholar) and stored at -20 °C. Purified enzyme had an A430/A280 ratio of 0.8 and was apparently homogeneous on SDS-PAGE analysis; its concentration was determined spectrophotometrically (ϵ430 = 0.17 m-1 cm-1) (40Morita Y. Iwamoto H. Aibara S. Kobayashi T. Hasegawa E. J. Biochem. (Tokyo). 1986; 99: 761-770Crossref PubMed Scopus (64) Google Scholar). Sodium hypochlorite (NaOCl), trifluoroacetic acid, acetonitrile (CH3CN), and methanol were obtained from Fisher. All organic solvents were HPLC grade. Peptides AcGYKRAYE (YKXXY), AcGEYARKY (YXXKY), and AcGEYAREY (YXXXY) were prepared by the Protein and Nucleic Acid Chemistry Laboratory, Washington University (St. Louis, MO). Purity of the peptides was confirmed by HPLC and mass spectrometric analysis. HDL and ApoA-I Isolation—Blood collected from healthy adults who had fasted overnight was anticoagulated with EDTA to produce plasma. HDL (density 1.125-1.210 g/ml) was prepared from plasma by sequential ultracentrifugation and was depleted of apolipoprotein E and apolipoprotein B100 by heparin-agarose chromatography (41Mendez A.J. Oram J.F. Bierman E.L. J. Biol. Chem. 1991; 266: 10104-10111Abstract Full Text PDF PubMed Google Scholar). ApoA-I was purified to apparent homogeneity from HDL (41Mendez A.J. Oram J.F. Bierman E.L. J. Biol. Chem. 1991; 266: 10104-10111Abstract Full Text PDF PubMed Google Scholar). Protein was determined using the Lowry assay (Bio-Rad) with albumin as the standard. Oxidation Reactions—Reactions were carried out at 37 °C in phosphate buffer (20 mm sodium phosphate, 100 μm diethylenetriaminepentaacetic acid (DTPA), pH 7.4) containing 5 μm apoA-I. For the myeloperoxidase-H2O2-nitrite system, the reaction mixture was supplemented with 50 nm myeloperoxidase, 100 μm nitrite, and the indicated concentration of H2O2. For the myeloperoxidase-H2O2-chloride system, the reaction mixture was supplemented with 50 nm myeloperoxidase and 100 mm NaCl. Reactions were initiated by adding oxidant and terminated by adding 2.5 mm methionine. ONOO- was synthesized from nitrite and H2O2 under acidic conditions, and peroxynitrous acid was stabilized by rapidly quenching the reaction with an excess of sodium hydroxide (42Beckman J.S. Chen J. Ischiropoulos H. Crow J.P. Methods Enzymol. 1994; 233: 229-240Crossref PubMed Scopus (961) Google Scholar). Concentrations of ONOO-, HOCl, and H2O2 were determined spectrophotometrically (ϵ302 = 1670 m-1 cm-1, ϵ292 = 350 m-1 cm-1, and ϵ240 = 39.4 m-1 cm-1, respectively) (42Beckman J.S. Chen J. Ischiropoulos H. Crow J.P. Methods Enzymol. 1994; 233: 229-240Crossref PubMed Scopus (961) Google Scholar, 43Morris J.C. J. Phys. Chem. 1966; 70: 3798-3805Crossref Scopus (795) Google Scholar, 44Nelson D.P. Kiesow L.A. Anal. Biochem. 1972; 49: 474-478Crossref PubMed Scopus (813) Google Scholar). Carbon dioxide reacts rapidly with ONOO- to form ONO2O2CO2−, whose reactivity differs from that of ONOO- (45Lymar S.V. Jiang Q. Hurst J.K. Biochemistry. 1996; 35: 7855-7861Crossref PubMed Scopus (313) Google Scholar). In preliminary experiments, we determined that adding 25 mm NaHCO3 to the reaction mixture failed to alter yields of oxidized amino acids in apoA-I when ONOO- was the reactant. This probably reflects the presence of bicarbonate in the phosphate buffer used for oxidation reactions. HPLC Analysis of Peptides—Peptides were separated at a flow rate of 0.5 ml/min on a reverse-phase column (Vydac C18 MS, 4.6 × 250 mm) using a Beckman HPLC system (Fullerton, CA) with UV detection at 280 nm. The peptides were eluted using a gradient of solvent A (0.06% trifluoroacetic acid in H2O) and solvent B (0.05% trifluoroacetic acid in 90% CH3CN, 10% H2O). Solvent B was increased from 10 to 45% over 50 min. Proteolytic Digestion of Proteins—Native or oxidized apoA-I was incubated overnight at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI) at a ratio of 25:1 (w/w) protein/trypsin in 100 mm NH4HCO3, pH 7.8 (35Bergt C. Fu X. Huq N.P. Kao J. Heinecke J.W. J. Biol. Chem. 2004; 279: 7856-7866Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Digestion was halted by acidification (pH 2-3) with trifluoroacetic acid. Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-ESI-MS)—LC-ESI-MS analyses were performed in the positive ion mode with a Finnigan Mat LCQ ion trap instrument (San Jose, CA) coupled to a Waters 2690 HPLC system (Milford, MA) (46Fu X. Kassim S.Y. Parks W.C. Heinecke J.W. J. Biol. Chem. 2003; 278: 28403-28409Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 47Fu X. Kassim S.Y. Parks W.C. Heinecke J.W. J. Biol. Chem. 2001; 276: 41279-41287Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). Synthetic or tryptic digest peptides were separated at a flow rate of 0.2 ml/min on a reverse-phase column (Vydac C18 MS; 2.1 × 250 mm) using a gradient of solvent A (0.2% HCOOH in H2O) and solvent B (0.2% HCOOH in 90% CH3CN, 10% H2O). Solvent B was kept at 2% for 8 min, increased to 10% in 1 min, and then increased to 35% over 36 min for synthetic peptides or to 45% over 66 min for tryptic digest peptides from apoA-I. The electrospray needle was held at 4500 V. Nitrogen, the sheath gas, was set at 80 units. The collision gas was helium. The temperature of the heated capillary was 220 °C. Production of Recombinant Human ApoA-I—Individual Cys substitution mutations within apoA-I cDNA were created by primer-directed PCR mutagenesis or the Mega-Primer PCR method (48Ryan R.O. Forte T.M. Oda M.N. Protein Expr. Purif. 2003; 27: 98-103Crossref PubMed Scopus (130) Google Scholar). Mutations were verified by dideoxy automated fluorescent sequencing. ApoA-I was expressed using the pET-20b (Novagen, Inc., Madison, WI)-based vector pNFXex in Escherichia coli strain BL21 (DE-3) pLysS and isolated with a His-Trap chelating column (Amersham Biosciences) (49Oda M.N. Bielicki J.K. Berger T. Forte T.M. Biochemistry. 2001; 40: 1710-1718Crossref PubMed Scopus (69) Google Scholar). During the isolation procedure, expressed proteins were maintained in 3 m guanidine hydrochloride, 20 mm phosphate, 0.5 m NaCl (pH 7.4). Eluted protein was dialyzed extensively against Tris-buffered saline (150 mm NaCl, 20 mm Tris, pH 8) supplemented with 1 mm benzamidine and 1 mm EDTA and then filter-sterilized. ApoA-I preparations contained no detectable phospholipid. Site-directed Spin Labeling of ApoA-I with Methionine Thiosulfonate—ApoA-I labeling was performed with 5 mg of cysteine-substituted apoA-I, as described (50Oda M.N. Forte T.M. Ryan R.O. Voss J.C. Nat. Struct. Biol. 2003; 10: 455-460Crossref PubMed Scopus (113) Google Scholar). ApoA-I was loaded onto a 1-ml His-Trap chelating column preloaded with 0.1 m NiSO4, extensively washed, derivatized on column with methionine thiosulfonate, and eluted. The labeled protein was dialyzed extensively against Tris-buffered saline supplemented with 1 mm benzamidine and 1 mm EDTA. Preparation of Lipid-associated ApoA-I—Discoidal HDL was prepared by a modified method originally described by Nichols et al. (51Nichols A.V. Gong E.L. Blanche P.J. Forte T.M. Biochim. Biophys. Acta. 1983; 750: 353-364Crossref PubMed Scopus (76) Google Scholar, 52Nichols A.V. Gong E.L. Blanche P.J. Forte T.M. Shore V.G. J. Lipid Res. 1987; 28: 719-732Abstract Full Text PDF PubMed Google Scholar). Equal volumes of Tris-buffered saline (pH 8) supplemented with 16.3 mm 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and Tris-buffered saline (pH 8) supplemented with 22 mm sodium cholate were vortexed and incubated at 37 °C until clear. Spin-labeled apoA-I was then added to the lipid dispersion and incubated for 1 h at 37 °C. Cholate was removed by extensive dialysis against Tris-buffered saline (pH 8). Discoidal HDL was separated from free lipid and protein by gradient ultracentrifugation. The size of lipidated discoidal HDL was confirmed by gradient gel electrophoresis (51Nichols A.V. Gong E.L. Blanche P.J. Forte T.M. Biochim. Biophys. Acta. 1983; 750: 353-364Crossref PubMed Scopus (76) Google Scholar). EPR Spectroscopy—EPR measurements were carried out in a JEOL X-band spectrometer fitted with a loop-gap resonator (53Hubbell W.L. Froncisz W. Hyde J.S. Rev. Sci. Instrum. 1987; 58: 1879-1886Crossref Scopus (135) Google Scholar). Purified, spin-labeled protein (final concentration ∼5 mg/ml protein in Tris-buffered saline, pH 8) was placed in a sealed quartz capillary contained in the resonator. Spectra of samples were obtained by a single 60-s scan over 100 gauss at a microwave power of 2 milliwatts and a modulation amplitude optimized to the natural line width (1.5-2.5 gauss) (54Chomiki N. Voss J.C. Warden C.H. Eur. J. Biochem. 2001; 268: 903-913Crossref PubMed Scopus (30) Google Scholar). Accessibility of the spin-labeled sites to polar or nonpolar relaxers (20 mm chromium oxalate or oxygen in equilibrium with atmospheric levels, respectively) was measured at room temperature (20-22 °C) from power saturation measurements (55Voss J. He

Referência(s)