title: Localization of nitration and chlorination sites on apolipoprotein A-I catalyzed by myeloperoxidase in human atheroma and associated oxidative impairment in ABCA1-dependent cholesterol efflux from macrophages.
abstract: We recently reported that apolipoprotein A-I (apoA-I), the major protein component of high density lipoprotein, is a selective target for myeloperoxidase (MPO)-catalyzed nitration and chlorination in both and serum of subjects with cardiovascular disease. We further showed that the extent of both apoA-I nitration and chlorination correlated with functional impairment in reverse cholesterol transport activity of the isolated lipoprotein. Herein we used tandem mass spectrometry to map the sites of MPO-mediated apoA-I nitration and chlorination in vitro and in vivo and to relate the degree of site-specific modifications to loss of apoA-I lipid binding and cholesterol efflux functions. Of the seven tyrosine residues in apoA-I, Tyr-192, Tyr-166, Tyr-236, and Tyr-29 were nitrated and chlorinated in MPO-mediated reactions. Site-specific liquid chromatography-mass spectrometry quantitative analyses demonstrated that the favored modification site following exposure to MPO-generated oxidants is Tyr-192. MPO-dependent nitration and chlorination both proceed with Tyr-166 as a secondary site and with Tyr-236 and Tyr-29 modified only minimally. Parallel functional studies demonstrated dose-dependent losses of ABCA1-dependent cholesterol acceptor and lipid binding activities with apoA-I modification by MPO. Finally tandem mass spectrometry analyses showed that apoA-I in human atherosclerotic tissue is nitrated at the MPO-preferred sites, Tyr-192 and Tyr-166. The present studies suggest that site-specific modifications of apoA-I by MPO are associated with impaired lipid binding and ABCA1-dependent cholesterol acceptor functions, providing a molecular mechanism that likely contributes to the clinical link between MPO levels and cardiovascular disease risk.
authors.affiliation: Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
authors.forename: Lemin
authors.initials: L
authors.lastname: Zheng
authors.forename: Megan
authors.initials: M
authors.lastname: Settle
authors.forename: Gregory
authors.initials: G
authors.lastname: Brubaker
authors.forename: Dave
authors.initials: D
authors.lastname: Schmitt
authors.forename: Stanley L
authors.initials: SL
authors.lastname: Hazen
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Smith
authors.forename: Michael
authors.initials: M
authors.lastname: Kinter
mesh_terms: D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000378:metabolism; D001161:Arteriosclerosis / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002384:Catalysis; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D015227:Lipid Peroxidation; D008264:Macrophages / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D002784:Cholesterol; D009195:Peroxidase