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 Tyr(192) 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 Tyr(192). 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 Tyr(192). Both ONOO(-) and MPO nitrated Tyr(192) in high yield, but unlike chlorination, nitration minimally affected the ability of apoA-I to promote cholesterol efflux from cells. Our results indicate that Tyr(192) 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) is the major carrier of lipid hydroperoxides in plasma, but itis not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 ؎ 178 mol/mol Tyr) than that in circulating HDL (104 ؎ 11 mol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.

High density lipoprotein (HDL) isolated from human atherosclerotic lesions and the blood of patients with established coronary artery disease contains elevated levels of 3-chlorotyrosine. Myeloperoxidase (MPO) is the only known source of 3-chlorotyrosine in vivo, indicating that MPO oxidizes HDL in humans. We previously reported that Tyr-192 is the major site that is chlorinated in apolipoprotein A-I (apoA-I), the chief protein in HDL, and that chlorinated apoA-I loses its ability to promote cholesterol efflux from cells by the ATP-binding cassette transporter A1 (ABCA1) pathway. However, the pathways that promote the chlorination of specific Tyr residues in apoA-I are controversial, and the mechanism for MPO-mediated loss of ABCA1-dependent cholesterol efflux of apoA-I is unclear. Using site-directed mutagenesis, we now demonstrate that lysine residues direct tyrosine chlorination in apoA-I. Importantly, methionine residues inhibit chlorination, indicating that they can act as local, proteinbound antioxidants. Moreover, we observed near normal cholesterol efflux activity when Tyr-192 of apoA-I was mutated to Phe and the oxidized protein was incubated with methionine sulfoxide reductase. Thus, a combination of Tyr-192 chlorination and methionine oxidation is necessary for depriving apoA-I of its ABCA1-dependent cholesterol transport activity. Our observations suggest that biologically significant oxidative damage of apoA-I involves modification of a limited number of specific amino acids, raising the feasibility of producing oxidation-resistant forms of apoA-I that have enhanced anti-atherogenic activity in vivo. High density lipoprotein (HDL)3 protects against atherosclerosis by removing cholesterol from cells of the artery wall (1, 2). Apolipoprotein A-I (apoA-I), which accounts for ϳ70% of the total protein in HDL, promotes cholesterol and phospholipid efflux largely by an active transport process mediated by ATP-binding cassette transporter A1 (ABCA1). However, oxidation of apoA-I severely impairs cholesterol efflux by the ABCA1 pathway (3-5). Moreover, HDL is chlorinated in human atherosclerotic lesions and the blood of subjects with established coronary artery disease, suggesting that apoA-I oxidation might promote atherogenesis.One potential pathway for apoA-I oxidation involves myeloperoxidase (MPO), a heme protein expressed by macrophages in human atherosclerotic tissue (6 -8). MPO secreted by phagocytes uses hydrogen peroxide (H 2 O 2 ) and chloride (Cl Ϫ ) to generate the powerful oxidant hypochlorous acid (HOCl). HOCl converts tyrosine to 3-chlorotyrosine (8 -10), and MPO is the only known source of this halogenated amino acid during acute inflammation in mice (11), indicating that MPO oxidizes HDL in vivo. We previously showed that MPO or HOCl targets tyrosine residue 192 (Tyr-192) when it chlorinates apoA-I, regardless of whether the protein is free or associated with HDL (12, 13). Moreover, apoA-I loses its ability to remove cholesterol from cells as it becomes oxidized in this manner (3-5, 13), i...

Background:Oxidation of apolipoprotein A-I by myeloperoxidase has been proposed to deprive HDL of its cardioprotective effects. Results: Tyrosine 192 is the major site of chlorination in apoA-I in both plasma and lesion HDL isolated from humans. Conclusion: Chlorination of apolipoprotein A-I by myeloperoxidase may contribute to generation of a dysfunctional form of HDL in vivo. Significance: Quantifying apolipoprotein A-I chlorination might help diagnose and perhaps treat human cardiovascular disease.

Rationale The efflux capacity of HDL with cultured macrophages associates strongly and negatively with CAD status, indicating that impaired sterol efflux capacity might be a marker—and perhaps mediator—of atherosclerotic burden. However, the mechanisms that contribute to impaired sterol efflux capacity remain poorly understood. Objective To determine the relationship between myeloperoxidase-mediated oxidative damage to apoA-I, the major HDL protein, and the ability of HDL to remove cellular cholesterol by the ABCA1 pathway. Methods and Results We quantified both site-specific oxidation of apoA-I and HDL’s ABCA1 cholesterol efflux capacity in control subjects and subjects with stable coronary artery disease or acute coronary syndrome. The CAD and ACS subjects had higher levels of chlorinated tyrosine-192 and oxidized methionine-148 than the control subjects. In contrast, plasma levels of MPO did not differ between the groups. HDL from the CAD and ACS subjects was less able to accept cholesterol from cells expressing ABCA1 than HDL from control subjects. Levels of chlorinated tyrosine and oxidized methionine associated inversely with ABCA1 efflux capacity and positively with atherosclerotic disease status. These differences remained significant after adjusting for HDL-cholesterol levels. Conclusions Our observations indicate that MPO may contribute to the generation of dysfunctional HDL with impaired ABCA1 efflux capacity in humans with atherosclerosis. Quantification of chlorotyrosine and oxidized methionine in circulating HDL might be useful indicators of the risk of cardiovascular disease that are independent of HDL-cholesterol.