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. Oxidative modification has been linked to changes in the function of a number of proteins with examples of both loss and gain of function (1-12). The oxidative modification of low density lipoprotein (LDL), 1 for example, is believed to play a crucial role in the initiation and progression of atherosclerosis (13-16). Oxidatively modified LDL, but not native LDL, is taken up by macrophages via scavenger receptors that are not regulated by cellular cholesterol content to produce cholesterolladen foam cells. Although the exact mechanisms of LDL oxidation that are proatherogenic in vivo are not known, myeloperoxidase (MPO)-mediated modification appears to be a likely contributor to the process (17). MPO is a heme peroxidase that is produced by various phagocytes and utilizes hydrogen peroxide and chloride to generate chlorinating oxidants like HOCl to kill pathogens (18). Human monocytes utilize this enzyme and the co-substrates hydrogen peroxide and nitrite to generate nitrating oxidants capable of initiating lipid peroxidation and protein nitration (19). Importantly recent reports confirm that MPO promotes both protein nitration and lipid peroxidation in vivo (20 -22) and that both MPO and nitrotyrosine levels in blood are strong predictors of an increased risk of cardiovascular disease (23)(24)(25).High density lipoprotein (HDL) also has an important role in atherosclerosis. Unlike LDL, however, HDL has antiatherosclerotic effects related to the maintenance of vascular homeostasis (26 -28). One major antiatherosclerotic function of HDL is its participation in the reverse cholesterol transport process by acting as an acceptor of free cholesterol taken from peripheral tissues for ultimate delivery to the liver and other steroidogenic tissues.Several laboratories have reported that the oxidation of HDL in vitro alters its cholesterol acceptor function with both enhancement and inhibition in efflux noted, depending upon the oxidation system used (29 -34). Until recently, whether or not the major protein component of HDL, apolipoprotein A-I (apoA-I), was oxidized in vivo and if so by which pathway(s) were unknown. We have recently showed that apoA-I is a selective target for nitration and chlorination in human serum and within human atherosclerotic lesions (35).

Site-specific Nitration of Apolipoprotein A-I at Tyrosine 166 Is Both Abundant within Human Atherosclerotic Plaque and Dysfunctional

“…We demonstrated previously that apoA-I recovered from human atherosclerotic lesions is heavily oxidized (7)(8)(9)18), a finding that others have replicated (6,19,20). Proteomic identification of oxidatively modified sites within apoA-I isolated from atherosclerotic lesion by immunoprecipitation with apoA-I-specific antibodies identified a number of preferred residues for MPO-and NO-derived oxidant PTMs (8,9).…”

“…Nitrated at Position 166-We first reported that apoA-I was a preferential target of protein nitration within atherosclerotic lesions, and in our later studies, NO 2 -Tyr 166 -apoA-I was identified as a preferred site of nitration using proteomics studies (7,8). We initially sought to develop an mAb that specifically recognized this site-specific nitrated apoA-I to better study its abundance and particle distribution.…”

“…at sites of inflammation and cardiovascular disease (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Inflammation and oxidative stress are widely touted as playing a role in the pathogenesis of cardiovascular disease; however, studies aimed at understanding the functional consequences of modifications of proteins through these processes have proven difficult.…”

“…Proteomic identification of oxidatively modified sites within apoA-I isolated from atherosclerotic lesion by immunoprecipitation with apoA-I-specific antibodies identified a number of preferred residues for MPO-and NO-derived oxidant PTMs (8,9). In addition to apoA-I tyrosine 192, for which mutagenesis studies by our group failed to demonstrate changes in biological activity, tyrosine 166 of apoA-I was reported as being of particular interest because this site was also shown to be within a region we identified as a "solar flare," a solvent-exposed region on nascent HDL that we predicted interacts with lecithin-cholesterol acyltransferase (LCAT) (12).…”

Chlorotyrosine promotes human aortic smooth muscle cell migration through increasing superoxide anion production and ERK1/2 activation

“…It catalyzes the two-electron peroxidation of chloride to generate hypochlorous acid (HOCl) [2], a potent chlorinating oxidant, capable of chlorinating tyrosine into chlorotyrosine. Levels of chlorotyrosine are increased in atherosclerotic tissues [3] and the blood of in patients with inflammatory conditions including atherosclerosis [4,25,26]. The chlorination of apolipoprotein A-I was found to impair its cardioprotective ability to remove excess cellular cholesterol from lipid-laden macrophages in the artery wall [5].…”