Myeloperoxidase Impairs ABCA1-dependent Cholesterol Efflux through Methionine Oxidation and Site-specific Tyrosine Chlorination of Apolipoprotein A-I*

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, protein-bound 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.

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)(4)(5)13), indicating that Tyr chlorination might be important for this impaired activity.
The pathways that promote the MPO-dependent oxidation of specific residues in apoA-I and loss of its ABCA1 activity are controversial. The mechanism we have proposed is based on the observation that Tyr-192 lies in an YXXK motif (Y ϭ Tyr, K ϭ Lys, X ϭ unreactive amino acid) and therefore is adjacent to a Lys residue on the same face of an amphipathic ␣-helix (12,14). Moreover, HOCl reacts rapidly with the ⑀ amino group of lysine to form longlived chloramines (8,12). Using synthetic peptides, we demonstrated that lysine residues can direct the regiospecific chlorination of Tyr residues by a pathway involving chloramine formation. In this model, the site-specific chlorination of Tyr-192 in apoA-I requires the participation of a nearby lysine residue (12).
An alternative proposal is that MPO must bind directly to the region of apoA-I containing Tyr-192 to promote site-specific chlorination of the residue (5). Based on studies of mutated apoA-I, it was also concluded that tyrosine chlorination is not a prerequisite for loss of ABCA1 transport activity when MPO oxidizes apoA-I (15).
Methionine sulfoxide (Met(O)) has also been detected in circulating HDL (16). ApoA-I can reduce lipid hydroperoxides to alcohols in concert with oxidation of Met residues to Met(O) (17,18). HDL is the major carrier of lipid hydroperoxides in the blood of humans (19), suggesting that this pathway may be physiologically relevant. The alkyl thiol of Met is the most HOCl-reactive moiety in the 20 common amino acids (20,21), but the role of oxidation of Met residues in the impaired cholesterol efflux activity of MPO-oxidized apoA-I has received surprisingly little attention.
Met(O) residues in proteins are reduced to methionine by a family of intracellular enzymes termed methionine sulfoxide reductases (22). In the current studies, we used mutated apoA-I and methionine sulfoxide reductase to probe the role of specific amino acids residues in Tyr chlorination and the loss of ABCA1-cholesterol efflux activity that occurs when apoA-I is exposed to MPO.

EXPERIMENTAL PROCEDURES
Isolation of MPO, PilB, and ApoA-I-MPO (EC 1.11.1.7) was isolated from human neutrophils (23). A truncated gene of PilB of Neisseria gonorrhoeae expressed in Escherichia coli was purified as described (24). Individual substitution mutations within human apoA-I cDNA were introduced by primer directed PCR mutagenesis or by the Mega-Primer PCR method and expressed using the pNFXex bacterial expression vector (25,26). All mutations were verified by dideoxy automated fluorescent sequencing.
Efflux of Cellular Cholesterol-BHK cells expressing mifepristone-inducible human ABCA1 were radiolabeled with [ 3 H]cholesterol. ABCA1 expression was induced by incubating cells for 20 h in medium supplemented with 10 nM mifepristone (31). Efflux of [ 3 H]cholesterol was measured after a 2-h incubation in medium without or with apoA-I (31).

RESULTS
The YXXK Motif Directs Tyr Chlorination in ApoA-I-To determine whether lysine residues in the YXXK motif direct the regiospecific chlorination of Tyr residues in proteins, we used site-directed mutagenesis to engineer a series of mutations in the cDNA of human apoA-I. After isolating the wild-type or mutant apoA-I proteins, we exposed them to reagent HOCl or the MPO-H 2 O 2 -Cl Ϫ system. Reactions were initiated by adding oxidant and terminated by adding Met, a scavenger of HOCl. After digesting the oxidized apoA-I with trypsin, we used LC-ESI-MS and reconstructed ion chromatograms of precursor and product peptides to quantify the yields of chlorinated tyrosine peptides. This approach detected all seven peptides predicted to contain Tyr.
As with apoA-I isolated from human HDL (12, 13), Tyr-192 was the major site of chlorination in recombinant wild-type apoA-I (Fig. 1A). In the K195R mutant, Tyr-192 chlorination was markedly decreased, suggesting that Lys-195 plays a critical role in directing the chlorination of Tyr-192. Tyr-166, which does not reside in a YXXK motif in apoA-I, was chlorinated in low yield when the protein was exposed to HOCl or the complete MPO system (Fig. 1B). However, when Glu-169 was mutated to lysine, chlorination of Tyr-166 increased 20-fold (Fig. 1B). These results demonstrate that lysine residues located in the YXXK motif can direct tyrosine chlorination in apoA-I.
Methionine Residues in the MXXK Motif Inhibit Tyrosine Chlorination-Protein-bound Met residues have been proposed to act as local scavengers of H 2 O 2 (32). To determine whether Met residues might similarly scavenge oxidants derived from HOCl, we mutated glutamic acid 198 (which lies 2 residues away from the Lys-195 that directs chlorination of  to Met. In this mutant, Tyr-192 chlorination was dramatically decreased (Fig. 1C), suggesting that Met residues can inhibit Tyr chlorination by scavenging lysine chloramines.
Tyr-115, which also resides in a YXXK motif in apoA-I, was chlorinated poorly (Fig. 1D) by HOCl or the complete MPO system. When we mutated Met-112 to Ala, however, HOCl generated a high yield of chlorinated Try-115 (data not shown). Thus, chlorination of Tyr-115 appears to be inhibited by the adjacent Met residue in wild type apoA-I. When Met-112 was mutated to Lys, chlorination of Tyr-115 also increased markedly (Fig. 1D). Collectively, these observations suggest that protein-bound Met residues that reside in MXXY or KXXM motifs can inhibit Tyr chlorination by scavenging lysine chloramines.

Methionine Sulfoxide Reductase Converts Met(O) to Methionine in Oxidized
ApoA-I-ApoA-I contains 3 Met residues, but it is unknown how oxidizing them with HOCl affects apoA-I-mediated cholesterol efflux by the ABCA1 pathway. When we exposed apoA-I to HOCl or the MPO system and then digested it with trypsin or Glu-C, LC-ESI-MS/MS analysis showed that each Met had been targeted for oxidation to Met(O) (Fig. 2, A and B). In contrast, methionine sulfone was not detected in oxidized apoA-I. When apoA-I was first exposed to the complete MPO system and then incubated with the methionine sulfoxide reductase PilB, Met(O) was converted back to methionine (Fig. 2C). We next compared the effects of oxidation on the ability of apoA-I and Tyr-192 3 Phe apoA-I to remove cholesterol from cells by the ABCA1 pathway. Whereas H 2 O 2 alone had no effect, oxidation by the complete MPO system significantly decreased the cholesterol efflux promoted by apoA-I or Tyr-192 3 Phe apoA-I ( Fig. 3B and Fig. 4A). However, the mutant protein appeared somewhat resistant to oxidative inactivation by MPO when the concentration of H 2 O 2 was high. Similar results were observed when apoA-I was directly oxidized with HOCl (Fig. 4B). After apoA-I was exposed to the complete MPO system with increasing H 2 O 2 concentrations (Fig. 4A) or increasing HOCl concentrations (Fig. 4B), incubating apoA-I with PilB to reduce Met(O) back to Met partially restored its ability to promote cholesterol efflux at all oxidant concentrations. Similar partial protective effects of the Tyr-192 3 Phe substitution or methionine sulfoxide reduction were observed when cellular cholesterol efflux was monitored over a range of apoA-I concentrations (Fig.  3B). Thus, neither inhibition of Tyr-192 chlorination nor reduction of Met(O) alone markedly protected apoA-I from oxidative inactivation.
We next determined the effect of a combination of Met(O) reduction and the Tyr-192 3 Phe mutation on the ability of oxidized apoA-I to promote cholesterol efflux by the ABCA1 pathway. Remarkably, when the mutant protein was first exposed to increasing H 2 O 2 concentrations in the complete MPO system and then incubated with PilB, its ability to promote cholesterol efflux was nearly the same as that of native apoA-I (Fig. 4A). This was true even at low concentrations of apoA-I (Fig. 3B). We obtained similar results after exposing Tyr-192 3 Phe to HOCl (Fig. 4B). Our observations strongly suggest that two oxidation events, chlorination of Tyr-192 and oxidation of Met residues, account for most of the decrease in the ability of apoA-I to promote cholesterol efflux by the ABCA1 pathway.

DISCUSSION
In the current study, we engineered mutations in apoA-I to determine whether the YXXK motif plays a critical role in Tyr chlorination and whether chlorination of Tyr-192 accounts for the loss of ABCA1-dependent cholesterol efflux that is seen when apoA-I is exposed to MPO. Studies with a series of mutations provided strong evidence that YXXK can direct the regiospecific chlorination of Tyr residues in apoA-I. Indeed, we blocked Tyr-192 chlorination with the Lys-195 3 Arg mutation and chlorinated normally resistant Tyr residues by creating the KXXY motif in the protein. Virtually identical results were observed with either the complete MPO system or reagent HOCl. These observations strongly support the proposed role of the YXXK motif (12) and argue against the hypothesis that the selective chlorination of Tyr-192 requires MPO to interact directly with the apolipoprotein (5).
We previously noted that two other Tyr residues in apoA-I (Tyr-115 and Tyr-236) that reside in YXXK motifs are resistant to chlorination (13). Importantly, Tyr-115 lies close to Met-112 in a YXXM motif. When we mutated Met-112 to Ala, which does not react with HOCl, the yield of chlorinated Tyr-115 in apoA-I exposed to MPO increased. When we introduced a Met residue 2 residues away from the Lys residue in apoA-I, Tyr-192 chlorination was inhibited. Our results strongly support the hypothesis that protein-bound Met residues act as local antioxidants (32) by scavenging chlorinating intermediates.
Met oxidation might affect Tyr chlorination by additional mechanisms. The ␣-helical structure of apoA-I depends critically upon hydrophobic amino acids, which form the lipid-associating face of the amphipathic helix (14). Oxygenation markedly decreases the hydrophobicity of Met. By disrupting the secondary structure of the apolipoprotein, Met oxidation could alter the ability of nearby Lys residues located in the YXXK motif to promote the site specific chlorination of apoA-I.
A key question is whether or not Tyr chlorination impairs the ability of apoA-I to promote ABCA1-dependent cholesterol efflux. We had noted the strong linear association between the extent of Tyr-192 chlorination and loss of biological activity, which suggests that Tyr oxidation might be an important contributor (3,13). However, recent studies of a mutant form of apoA-I in which Phe replaced all 7 Tyr residues led to the proposal that Tyr chlorination is irrelevant to the loss of ABCA1-dependent cholesterol efflux that occurs when MPO oxidizes apoA-I (15).
To address this issue, we measured the cholesterol efflux activity of apoA-I containing a Phe substituted for Tyr-192, which makes this residue completely resistant to chlorination. In contrast to the previous report with murine macrophages (15), we found using ABCA1-transfected BHK cells that this mutation had a small but reproducible protective effect against apoA-I inactivation by either HOCl or the MPO system. The difference between our study and the previous one may reflect the fact that our transfected BHK cells express much higher ABCA1 levels than murine macrophages, allowing us to detect modest changes in apoA-I activity.
Our observation that protein-bound Met is a potent local scavenger of   HOCl led us to investigate the role of Met oxidation in the biological activity of apoA-I. When we exposed apoA-I to HOCl or to H 2 O 2 with the complete MPO system, we observed near-quantitative oxidation of the 3 Met residues in apoA-I. Met oxidation was completely reversed by treatment with PilB, a methionine sulfoxide reductase that reduces both the (R) and (S) epimers of Met(O) (22). Remarkably, when the Tyr-192 3 Phe mutant was exposed to HOCl or the complete MPO system and then incubated with the methionine reductase PilB, its ability to promote cholesterol efflux by the ABCA1 pathway was almost completely restored. These observations indicate that Met oxidation and Tyr chlorination together, in contrast to Met oxidation alone (33), are necessary for depriving apoA-I of its cholesterol efflux activity. This synergism between the Tyr-192 3 Phe mutation and Met(O) reduction implies that the functional defect induced by oxidation of apoA-I arises from a cooperative interaction between oxidation of Tyr-192 and one or more Met residues. Alternatively, it is possible that other structural modifications in oxidized apoA-I are involved in the loss of ABCA1 activity. It is noteworthy that Tyr-192 resides in the middle of the random coil loop that changes its conformation when it encounters lipid, driving the remodeling of apoA-I to its lipid-associated form (34). Chlorination of this Tyr residue, in concert with Met oxidation, may stabilize a conformational variant that fails to either associate strongly with lipid or interact productively with ABCA1.
In conclusion, oxidation by MPO impairs the ability of apoA-I to promote cholesterol efflux by the ABCA1 pathway, suggesting that this oxidative process might contribute to foam cell formation and atherogenesis. Studies with mutant forms of apoA-I strongly support the proposal that specific amino acid sequences direct the regioselective chlorination of Tyr. Moreover, oxidation of Met and Tyr residues plays a critical role in the oxidative inactivation of the protein. Our observations raise the possibility that modified forms of apoA-I that are resistant to oxidation might be especially anti-atherogenic in vivo.