Tyrosine 192 in Apolipoprotein A-I Is the Major Site of Nitrationand Chlorination by Myeloperoxidase, but Only ChlorinationMarkedly Impairs ABCA1-dependent Cholesterol Transport*

Received for publication, October 8, 2004, and in revised form, November 29, 2004Published, JBC Papers in Press, November 30, 2004, DOI 10.1074/jbc.M411484200

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

From the ‡Department of Medicine, University of Washington, Seattle, Washington 98195, the §Department of BiologicalChemistry, University of California, Davis, California 95616, and ¶Children’s Hospital Research Institute,Oakland, California 94609

High density lipoprotein (HDL) isolated from humanatherosclerotic lesions and the blood of patients withestablished coronary artery disease contains elevatedlevels of 3-nitrotyrosine and 3-chlorotyrosine. Myeloper-oxidase (MPO) is the only known source of 3-chloroty-rosine in humans, indicating that MPO oxidizes HDL invivo. In the current studies, we used tandem mass spec-trometry to identify the major sites of tyrosine oxidationwhen lipid-free apolipoprotein A-I (apoA-I), the majorprotein of HDL, was exposed to MPO or peroxynitrite(ONOO�). Tyrosine 192 was the predominant site of bothnitration and chlorination by MPO and was also themajor site of nitration by ONOO�. Electron paramag-netic spin resonance studies of spin-labeled apoA-I re-vealed that residue 192 was located in an unusuallyhydrophilic environment. Moreover, the environment ofresidue 192 became much more hydrophobic whenapoA-I was incorporated into discoidal HDL, and Tyr192

of HDL-associated apoA-I was a poor substrate for nitra-tion by both myeloperoxidase and ONOO�, suggestingthat solvent accessibility accounted in part for the reac-tivity of Tyr192. The ability of lipid-free apoA-I to facili-tate ATP-binding cassette transporter A1 cholesteroltransport was greatly reduced after chlorination byMPO. Loss of activity occurred in concert with chlori-nation of Tyr192. Both ONOO� and MPO nitrated Tyr192

in high yield, but unlike chlorination, nitration mini-mally affected the ability of apoA-I to promote choles-terol efflux from cells. Our results indicate that Tyr192 isthe predominant site of nitration and chlorination whenMPO or ONOO� oxidizes lipid-free apoA-I but that onlychlorination markedly reduces the cholesterol efflux ac-tivity of apoA-I. This impaired biological activity of chlo-rinated apoA-I suggests that MPO-mediated oxidation ofHDL might contribute to the link between inflammationand cardiovascular disease.

Many lines of evidence indicate that high density lipoprotein(HDL)1 protects the artery wall from atherosclerosis. One impor-

tant pathway involves HDL apolipoproteins that remove cellularcholesterol and phospholipids by an active transport process me-diated by ATP-binding cassette transporter A1 (ABCA1) (1–5).Lipid-laden macrophages represent the cellular hallmark of theatherosclerotic lesion, and ABCA1 plays a critical role in remov-ing cholesterol from macrophages in vivo (1–5). The most abun-dant apolipoprotein in HDL is apolipoprotein A-I (apoA-I), whichaccounts for �70% of the total protein content. Lipid-poor apoA-Ipromotes cellular efflux of cholesterol and phospholipids exclu-sively by the ABCA1 pathway (1–5).

Several other mechanisms, including the ability of HDL toinhibit low density lipoprotein oxidation, reduce lipid hydroper-oxides, and transport oxidized lipids to the liver for excretion,may also be cardioprotective (6–14). Methionine and phenyl-alanine residues in apoA-I are oxidized by reactive intermedi-ates (8–10, 15–17), but it is unclear if oxidation of these resi-dues affects the ability of the apolipoprotein to remove choles-terol from cells. Whether specific tyrosine residues in apoA-Iare also vulnerable to oxidation in vivo is unclear, although itstyrosine residues are readily converted to o,o�-dityrosine bytyrosyl radical in vitro (7).

Macrophages might be an important source of oxidantsthat damage HDL. One pathway involves myeloperoxidase, aphagocyte heme protein that is expressed at high levels inhuman atherosclerotic tissue (19, 20). The enzyme uses hydro-gen peroxide (H2O2) for oxidative reactions in the extracellularmilieu (21–24). The major end product at plasma concentra-tions of chloride ion (Cl�) is generally thought to be hypochlo-rous acid (HOCl).

Cl� � H2O2 � H� 3 HOCl � H2O

REACTION 1

LDL isolated from human atherosclerotic lesions contains ele-vated levels of 3-chlorotyrosine, a product characteristic ofHOCl (25–27), indicating that myeloperoxidase is one pathwayfor oxidizing lipoproteins in the human artery wall.

Myeloperoxidase can also produce nitrogen dioxide radical(NO2

� ), a reactive species that converts tyrosine to 3-nitroty-rosine (28–30). We have recently shown that myeloperoxi-dase nitrates HDL in vitro (31). The reaction probably in-

* This work was supported by National Institutes of Health GrantsAG021191, HL18645, HL75381, HL55362, DK02456, HL073996,HL30086, HL030086, and HL77268 and the Donald W. Reynolds Foun-dation. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

� To whom correspondence should be addressed: Division of Metabo-lism, Endocrinology, and Nutrition, Box 356426, University of Wash-ington, Seattle, WA 98195. E-mail: heinecke@u.washington.edu.

1 The abbreviations used are: HDL, high density lipoprotein; ABCA1,

ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; ClY,chlorotyrosine; DTPA, diethylenetriaminepentaacetic acid; ESI, elec-trospray ionization; MS, mass spectrometry; NO2Y, nitrotyrosine;SDSL-EPR, site-directed spin label electron paramagnetic resonancespectroscopy; HPLC, high pressure liquid chromatography; LC, liquidchromatography; BHK, baby hamster kidney.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 7, Issue of February 18, pp. 5983–5993, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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volves direct one-electron oxidation of nitrite (NO2�) by

compound I, a complex of myeloperoxidase and H2O2.

NO2� � compound I 3 NO2

� � compound II

REACTION 2

LDL isolated from atherosclerotic lesions is enriched in 3-ni-trotyrosine (32), indicating that myeloperoxidase or other path-ways that generate reactive nitrogen species also operate in theatherosclerotic artery wall.

Peroxynitrite (ONOO�) is another nitrating species thatmight be important in oxidizing lipoproteins during inflamma-tion (18, 33, 34). It is generated when nitric oxide (NO) reactswith superoxide (O2

.).

O2� � NO 3 ONOO�

REACTION 3

Like nitrogen dioxide radical, ONOO� generates 3-nitroty-rosine when it reacts with tyrosine residues.

We have shown that Tyr192 is the single major site of chlo-rination in apoA-I of HDL exposed to HOCl in vitro (35). Tyr192

resides in an YXXK motif, and molecular modeling demon-strates that these tyrosine and lysine residues are adjacent onthe same face of an amphipathic �-helix in apoA-I. HOCl reactsrapidly with the � amino group of lysine to form long livedchloramines (23, 24, 35). Studies with synthetic peptides dem-onstrate that lysine residues can direct the regiospecific chlo-rination of tyrosine residues by a reaction pathway involvingchloramine formation, suggesting that the site-specific chlori-nation of apoA-I requires the participation of a nearby lysineresidue (35).

Recent studies demonstrate that HDL is chlorinated andnitrated in human atherosclerotic lesions (31, 36, 37), indicat-ing that myeloperoxidase is one pathway for oxidizing HDL invivo. Tandem mass spectrometric (MS) analysis identified my-eloperoxidase as a component of lesion HDL (36), suggestingthat the enzyme and lipoprotein interact in the artery wall.Moreover, 3-chlorotyrosine and 3-nitrotyrosine were also de-tected in circulating HDL (31, 36, 37). Levels of these oxidizedamino acids were elevated in HDL isolated from the blood ofhumans with established coronary artery disease, raising thepossibility that circulating levels of chlorinated and nitratedHDL represent a novel marker for clinically significant athero-sclerosis (31, 36, 37). Tyrosine chlorination and nitration maybe physiologically significant because HDL or apoA-I exposedto HOCl or the myeloperoxidase system is less able to removecholesterol from cultured cells by the ABCA1 pathway (36, 37).

Remarkably little is known about the factors that control thesite-specific nitration and chlorination of tyrosine residues inproteins. In the current study, we use apoA-I, synthetic pep-tides, and tandem mass spectrometric analysis to investigatethe role of reactive nitrogen and chlorine species in modifyingapoA-I and altering its ability to remove cholesterol from cells.Our observations indicate that Tyr192 is the predominant siteof nitration as well as chlorination in apoA-I. However, onlychlorination of apoA-I markedly impairs ABCA1-dependentcholesterol transport by the oxidized apolipoprotein.

EXPERIMENTAL PROCEDURES

Materials

Myeloperoxidase (donor:hydrogen peroxide, oxidoreductase; EC1.11.1.7) was isolated by lectin affinity and size exclusion chromatog-raphies from human neutrophils (38, 39) and stored at �20 °C. Purifiedenzyme had an A430/A280 ratio of 0.8 and was apparently homogeneouson SDS-PAGE analysis; its concentration was determined spectropho-tometrically (�430 � 0.17 M�1 cm�1) (40). Sodium hypochlorite (NaOCl),trifluoroacetic acid, acetonitrile (CH3CN), and methanol were obtained

from Fisher. All organic solvents were HPLC grade. Peptides Ac-GYKRAYE (YKXXY), AcGEYARKY (YXXKY), and AcGEYAREY (YX-XXY) were prepared by the Protein and Nucleic Acid Chemistry Labo-ratory, Washington University (St. Louis, MO). Purity of the peptideswas confirmed by HPLC and mass spectrometric analysis.

Methods

HDL and ApoA-I Isolation—Blood collected from healthy adults whohad fasted overnight was anticoagulated with EDTA to produce plasma.HDL (density 1.125–1.210 g/ml) was prepared from plasma by sequen-tial ultracentrifugation and was depleted of apolipoprotein E and apo-lipoprotein B100 by heparin-agarose chromatography (41). ApoA-I waspurified to apparent homogeneity from HDL (41). Protein was deter-mined using the Lowry assay (Bio-Rad) with albumin as the standard.

Oxidation Reactions—Reactions were carried out at 37 °C in phos-phate buffer (20 mM sodium phosphate, 100 �M diethylenetriaminepen-taacetic acid (DTPA), pH 7.4) containing 5 �M apoA-I. For the myeloper-oxidase-H2O2-nitrite system, the reaction mixture was supplementedwith 50 nM myeloperoxidase, 100 �M nitrite, and the indicated concen-tration of H2O2. For the myeloperoxidase-H2O2-chloride system, thereaction mixture was supplemented with 50 nM myeloperoxidase and100 mM NaCl. Reactions were initiated by adding oxidant and termi-nated by adding 2.5 mM methionine. ONOO� was synthesized fromnitrite and H2O2 under acidic conditions, and peroxynitrous acid wasstabilized by rapidly quenching the reaction with an excess of sodiumhydroxide (42). Concentrations of ONOO�, HOCl, and H2O2 were de-termined spectrophotometrically (�302 � 1670 M�1 cm�1, �292 � 350 M�1

cm�1, and �240 � 39.4 M�1 cm�1, respectively) (42–44).Carbon dioxide reacts rapidly with ONOO� to form ONO2CO2

�,whose reactivity differs from that of ONOO� (45). In preliminary ex-periments, we determined that adding 25 mM NaHCO3 to the reactionmixture failed to alter yields of oxidized amino acids in apoA-I whenONOO� was the reactant. This probably reflects the presence of bicar-bonate in the phosphate buffer used for oxidation reactions.

HPLC Analysis of Peptides—Peptides were separated at a flow rateof 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 at280 nm. The peptides were eluted using a gradient of solvent A (0.06%trifluoroacetic acid in H2O) and solvent B (0.05% trifluoroacetic acid in90% CH3CN, 10% H2O). Solvent B was increased from 10 to 45% over50 min.

Proteolytic Digestion of Proteins—Native or oxidized apoA-I was in-cubated overnight at 37 °C with sequencing grade modified trypsin(Promega, Madison, WI) at a ratio of 25:1 (w/w) protein/trypsin in 100mM NH4HCO3, pH 7.8 (35). Digestion was halted by acidification (pH2–3) with trifluoroacetic acid.

Liquid Chromatography-Electrospray Ionization Mass Spectrometry(LC-ESI-MS)—LC-ESI-MS analyses were performed in the positive ionmode with a Finnigan Mat LCQ ion trap instrument (San Jose, CA)coupled to a Waters 2690 HPLC system (Milford, MA) (46, 47). Syn-thetic or tryptic digest peptides were separated at a flow rate of 0.2ml/min on a reverse-phase column (Vydac C18 MS; 2.1 � 250 mm) usinga 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 forsynthetic peptides or to 45% over 66 min for tryptic digest peptides fromapoA-I. The electrospray needle was held at 4500 V. Nitrogen, thesheath gas, was set at 80 units. The collision gas was helium. Thetemperature of the heated capillary was 220 °C.

Production of Recombinant Human ApoA-I—Individual Cys substi-tution mutations within apoA-I cDNA were created by primer-directedPCR mutagenesis or the Mega-Primer PCR method (48). Mutationswere verified by dideoxy automated fluorescent sequencing. ApoA-I wasexpressed using the pET-20b (Novagen, Inc., Madison, WI)-based vectorpNFXex in Escherichia coli strain BL21 (DE-3) pLysS and isolated witha His-Trap chelating column (Amersham Biosciences) (49). During theisolation procedure, expressed proteins were maintained in 3 M guani-dine hydrochloride, 20 mM phosphate, 0.5 M NaCl (pH 7.4). Elutedprotein was dialyzed extensively against Tris-buffered saline (150 mM

NaCl, 20 mM Tris, pH 8) supplemented with 1 mM benzamidine and 1mM EDTA and then filter-sterilized. ApoA-I preparations contained nodetectable phospholipid.

Site-directed Spin Labeling of ApoA-I with Methionine Thiosulfon-ate—ApoA-I labeling was performed with 5 mg of cysteine-substitutedapoA-I, as described (50). ApoA-I was loaded onto a 1-ml His-Trapchelating column preloaded with 0.1 M NiSO4, extensively washed,derivatized on column with methionine thiosulfonate, and eluted. The

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labeled protein was dialyzed extensively against Tris-buffered salinesupplemented with 1 mM benzamidine and 1 mM EDTA.

Preparation of Lipid-associated ApoA-I—Discoidal HDL was pre-pared by a modified method originally described by Nichols et al. (51,52). Equal volumes of Tris-buffered saline (pH 8) supplemented with16.3 mM 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and Tris-buffered saline (pH 8) supplemented with 22 mM sodium cholate werevortexed and incubated at 37 °C until clear. Spin-labeled apoA-I wasthen 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 bygradient ultracentrifugation. The size of lipidated discoidal HDL wasconfirmed by gradient gel electrophoresis (51).

EPR Spectroscopy—EPR measurements were carried out in aJEOL X-band spectrometer fitted with a loop-gap resonator (53).Purified, spin-labeled protein (final concentration �5 mg/ml proteinin Tris-buffered saline, pH 8) was placed in a sealed quartz capillarycontained in the resonator. Spectra of samples were obtained by asingle 60-s scan over 100 gauss at a microwave power of 2 milliwattsand a modulation amplitude optimized to the natural line width(1.5–2.5 gauss) (54). Accessibility of the spin-labeled sites to polar ornonpolar relaxers (20 mM chromium oxalate or oxygen in equilibriumwith atmospheric levels, respectively) was measured at room temper-ature (20–22 °C) from power saturation measurements (55).The contrast function � was determined from ln(collision fre-quencynonpolar/collision frequencypolar) (50).

Cell Culture and Cholesterol Efflux—Baby hamster kidney (BHK)cells expressing mifepristone-inducible human ABCA1 were generatedas described previously (56). Cellular cholesterol was labeled by adding1 �Ci/ml [3H]cholesterol (PerkinElmer Life Sciences) to the growthmedium. Twenty-four hours later, strong expression of ABCA1 wasinduced by incubating the cells for 20 h with Dulbecco’s modified Ea-gle’s medium containing 1 mg/ml bovine serum albumin and 1 nM

mifepristone (56). To measure cholesterol efflux, mock- or ABCA1-transfected cells were incubated with Dulbecco’s modified Eagle’s me-dium/bovine serum albumin without or with apoA-I or HDL. After 2 h,the medium and cells were assayed for [3H]cholesterol as described (56).Cholesterol efflux mediated by apoA-I was calculated as the percentageof total [3H]cholesterol (medium plus cells) released into the mediumafter subtracting the value obtained with Dulbecco’s modified Eagle’smedium/bovine serum albumin alone. BHK cells incubated with nativeor oxidized apoA-I demonstrated no changes in morphology and cellprotein or cholesterol content per well.

RESULTS

Myeloperoxidase and Reagent ONOO� Nitrate All of the Ty-rosine Residues in Lipid-free ApoA-I, but Tyrosine 192 Is theMain Target—We previously showed that Tyr192 in apoA-I isthe single major site of chlorination when HDL is exposed toHOCl (35). To determine whether reactive nitrogen species alsoselectively target this amino acid residue, we exposed lipid-freeapoA-I to either the myeloperoxidase-H2O2-nitrite system orreagent ONOO�. Oxidation reactions were carried out in the

absence of chloride ion (to prevent chlorination reactions) andat neutral pH in phosphate buffer containing the metal chela-tor DTPA. We used various ratios of oxidant (H2O2 or ONOO�,mol/mol, oxidant/apoA-I) for 60 min at 37 °C and then termi-nated the reaction with a molar excess (relative to oxidant) ofmethionine. Because apoA-I contains 7 tyrosine residues and243 amino acids, a 10:1 ratio of oxidant to apoA-I gave a �1:1ratio of oxidant to tyrosine residues and a 1:24 ratio of oxidantto total amino acids.

We first showed that LC-ESI-MS analysis of the trypticdigest of native apoA-I detected peptides that collectively cov-ered �80% of the protein’s sequence and included all sevenpeptides predicted to contain tyrosine. To determine whichtyrosine residues had been nitrated, we used reconstructed ionchromatograms to detect (i) each of the peptides that containedtyrosine and (ii) any tyrosine-containing peptides that hadgained 45 atomic mass units (addition of one NO2 group andloss of one hydrogen). We quantified product yields using theion current of each precursor and product peptide and recon-structed ion chromatograms.

LC-ESI-MS and MS/MS analysis of the tryptic digest ofoxidized apoA-I detected seven peptides whose mass corre-sponded to the mass of the precursor peptide plus 45 atomicmass units, suggesting the formation of nitrated amino acids.We also detected one peptide that had gained 61 atomic massunits, suggesting the addition of both a nitro group and anoxygen atom (45 � 16 atomic mass units). Using LC-ESI-MS/MS analysis, we confirmed each peptide’s sequence (Ta-ble I) and showed that its tyrosine had been targeted fornitration (Fig. 1; peptide LAEYHAK containing Tyr192). Amethionine-containing peptide (WQEEMELYR) had an oxy-gen on its methionine residue (Met � 16) and had beennitrated on its tyrosine residue (Tyr � 45). No peptidescontaining 3-nitrotyrosine together with an unoxidized me-thionine residue were identified with the MPO system. How-ever, we did detect WQEEMEL(NO2Y)R when apoA-I wasexposed to reagent ONOO�.

When apoA-I was exposed to either the myeloperoxidase-H2O2-nitrite system or reagent ONOO� (Fig. 2), the predomi-nant tyrosine oxidation product was LAE(NO2Y)HAK. For themyeloperoxidase-H2O2-nitrite system, nitration of Tyr192 wasoptimal at a molar ratio of 10:1 (50 �M H2O2) of oxidant toapoA-I (Fig. 2A). Approximately 50% of Tyr192 was nitratedunder those conditions; the yield of 3-nitrotyrosine decreased atlower or higher concentrations of H2O2. For ONOO�, nitrationincreased in a hyperbolic manner with increasing concentra-

TABLE ILC-ESI-MS detection of peptides containing 3-nitrotyrosine in lipid-free apoA-I exposed to the

myeloperoxidase-H2O2-nitrite systemApoA-I was exposed to the myeloperoxidase-H2O2-nitrite system (10:1, mol/mol, oxidant/apoA-I) for 60 min at 37 °C in phosphate buffer (100 �M

DTPA, 20 mM sodium phosphate, pH 7.4). After the reaction was terminated with L-methionine, a tryptic digest of apoA-I was analyzed byLC-ESI-MS and MS/MS. Nitrated peptides were detected and quantified using reconstructed ion chromatograms of precursor and productpeptides. Peptide sequences were confirmed using MS/MS. Results are representative of those from three independent experiments.

Residues SequencePrecursor, m/z Product, m/z

Yielda

(M � H)� (M � 2H)2� (M � 45 � H)� (M � 45 � 2H)2�

%

13–23 DLATVYVDVLK 1235.4 618.3 1280.5 640.9 2228–40 DYVSQFEGSALGK 1400.5 701.2 1445.3 723.5 1697–106 VQPYLDDFQK 1252.5 627.1 1297.4 649.3 11

108–116 WQEEMELYRb 1283.4 642.5108–116 WQEE(M�16)ELYRc 1299.4 650.3 1344.4 673.0 15161–171 THLAPYSDELR 1301.5 651.6 1346.3 673.8 13189–195 LAEYHAK 831.3 416.6 876.4 438.8 53227–238 VSFLSALEEYTK 1386.5 694.0 1431.4 716.4 16

a mol of product peptide/(mol of precursor peptide � mol of product peptide) � 100.b No 3-nitrotyrosine was detected in this peptide when the methionine residue was not oxidized.c Methionine was oxidized to methionine sulfoxide (16).

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tions of oxidant (Fig. 2B). At a molar ratio of 5:1 or 50:1 (25 or250 �M ONOO�) of oxidant to apoA-I, �40% or �90% of Tyr192

residues were nitrated, respectively. When �50% of the Tyr192

residues were nitrated by ONOO� (10:1, mol/mol, oxidant/protein), Tyr192 and to a lesser extent Tyr236 were the predom-inant sites of nitration, and a much lower level (6%) of nitra-tion was observed in the other tyrosine residues (Fig. 2B). Incontrast, when �50% of Tyr192 residues were nitrated by my-eloperoxidase (10:1, mol/mol, oxidant/protein), there was a sig-nificant level (10–20%) of nitration of all of the other tyrosineresidues in apoA-I (Fig. 2A and Table I). These findings indi-cate that myeloperoxidase and high concentrations of ONOO�

nitrate all 7 tyrosines in apoA-I, that the predominant nitra-tion product generated by both sources of reactive nitrogenspecies is Tyr192 in peptide LAEYHAK, and that reagentONOO� is a more selective nitrating agent than the myeloper-oxidase-H2O2-nitrite system when �50% of Tyr192 residues inapoA-I are nitrated.

Myeloperoxidase Preferentially Chlorinates Tyrosine 192 inLipid-free ApoA-I—To determine whether Tyr192 is the majorsite of chlorination when the myeloperoxidase system oxidizesapoA-I, we exposed the lipid-free apolipoprotein to myeloper-oxidase, chloride ion, and various ratios of oxidant (mol/mol,H2O2/apoA-I) for 60 min at 37 °C and then terminated thereaction by adding 2.5 mM methionine. To prevent nitration, allreactions were carried out in buffer that lacked nitrite.

LC-ESI-MS and MS/MS analyses of a tryptic digest of theoxidized protein confirmed that Tyr192 was the predominantsite of chlorination by the myeloperoxidase-H2O2-chloride sys-

tem (Fig. 3A). A low level of chlorination was also observed atTyr29, Tyr115, Tyr166, and Tyr236. The patterns for tyrosinechlorination by the enzymatic system and HOCl were similar(Fig. 3, compare A and B). Chlorination of �50% of Tyr192

required a 50:1 ratio (mol/mol) of oxidant to protein for theenzymatic system or reagent HOCl. These findings indicatethat Tyr192 in peptide LAEYHAK is the major chlorination sitein apoA-I by the myeloperoxidase-H2O2-chloride system. More-over, chlorination of apoA-I tyrosine residues by myeloperoxi-dase is much more selective than nitration.

Oxidation by Reactive Nitrogen or Chlorine Changes the Ap-parent Mr of ApoA-I—Analytical SDS-PAGE under reducingconditions detected proteins of increased apparent Mr inApoA-I that had been modified by either the myeloperoxidase-H2O2-nitrite system or the myeloperoxidase-H2O2-chloride sys-tem (Fig. 4A). A broad band of material (45–60 kDa) wasapparent in apoA-I that had been exposed to the myeloperoxi-dase-H2O2-chloride system. Species with modestly increased ordecreased apparent mass of �28 kDa were also observed whenapoA-I was exposed to nitrating or chlorinating species gener-ated by myeloperoxidase (Fig. 4A), perhaps due to conforma-

FIG. 1. MS/MS identification of the major site of tyrosine ni-tration in lipid-free apoA-I exposed to the myeloperoxidase-H2O2-nitrite system. MS/MS analysis of (LAEYHAK � H)� (m/z831.4) and (LAE(NO2Y)HAK � H)� (m/z 876.3) in apoA-I oxidized withmyeloperoxidase. Lipid-free apoA-I (5 �M) was exposed to H2O2 (10:1,mol/mol, oxidant/protein) for 60 min at 37 °C in phosphate buffer (20mM sodium phosphate, 100 �M DTPA, pH 7.4) supplemented with 50 nM

myeloperoxidase and 100 �M nitrite. After the reaction was terminatedwith L-methionine, apoA-I was digested with trypsin, and the peptideswere analyzed with LC-ESI-MS/MS. FIG. 2. Identification of nitrated tyrosine residues in lipid-free

apoA-I exposed to the myeloperoxidase-H2O2-nitrite system orreagent ONOO�. ApoA-I (5 �M) was exposed to the myeloperoxidase-H2O2-nitrite system or ONOO� at the indicated molar ratio of oxidant/protein for 60 min at 37 °C in phosphate buffer. The myeloperoxidasesystem was supplemented with 100 �M nitrite. A tryptic digest ofapoA-I was analyzed by LC-ESI-MS and MS/MS. Nitrated peptideswere detected and quantified using reconstructed ion chromatograms ofprecursor and product peptides. Peptide sequences were confirmedusing MS/MS. Results are representative of those of three independentexperiments.

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tional changes or intramolecular cross-linking. Catalase or theheme protein inhibitor azide blocked the alterations in Mr

induced by both the nitrating and chlorinating enzymatic sys-tems (data not shown).

Reagent ONOO� yielded apoA-I species with apparent mo-lecular mass values of �60 kDa, which were similar to thoseobserved in apoA-I exposed to reactive nitrating species gener-ated by the enzymatic systems (Fig. 4, compare A and B).Reagent HOCl, but not ONOO�, produced a broad band ofmaterial (45–60 kDa) and apoA-I species with a modest in-crease or decrease in apparent molecular mass (Fig. 4B). Theseobservations suggest that reactive nitrogen and chlorine spe-cies can induce conformational changes and aggregation orcross-linking of apoA-I.

Tyrosine 192 of Lipid-free ApoA-I Resides in a HydrophilicEnvironment—The aromatic ring of tyrosine’s side chainmakes the amino acid residue strongly hydrophobic, but thepolar hydroxyl group can form strong hydrogen bonds withwater and other polar molecules. Tyrosine can thus inhabitboth hydrophobic and hydrophilic environments. To investi-gate the hydrophobicity of the local environments of tyrosine

residues in apoA-I, we used site-directed spin label EPR spec-troscopy (SDSL-EPR) (50). To incorporate a spin label at aparticular site of interest, we took advantage of the absence ofendogenous cysteines in apoA-I and the sulfhydryl-specific re-activity of methionine thiosulfonate. The apoA-I cysteine sub-stitutions Y18C, Y29C, Y115C, Y166C, Y192C, and Y236Cwere bacterially expressed, purified, and labeled as described(50). By examining the accessibility of the spin-labeled sites topolar or nonpolar relaxers (chromium and oxygen, respec-tively), we quantified the hydrophobicity of the environmentsurrounding the examined residues, as represented by the con-trast function �.

The contrast function � � ln(collision frequencynonpolar/col-lision frequencypolar) provides a measure of the hydrophobicityof the local environment of the labeled side chain (50). Wedetermined � for each derivatized apoA-I species under lipid-free conditions. Of the tyrosines examined, position 192 wasthe most accessible to the polar relaxer, with a � value of �3.08(Fig. 5). This low value indicates that position 192 is fullyexposed to the aqueous environment. In addition, we examinedthe solvent accessibility of the three lysine residues (Lys119,

FIG. 3. Identification of chlorinated tyrosine residues in lipid-free apoA-I exposed to the myeloperoxidase-H2O2-chloride sys-tem or reagent HOCl. ApoA-I (5 �M) was exposed to the myeloper-oxidase-H2O2-chloride system or HOCl at the indicated molar ratio ofoxidant/protein for 60 min at 37 °C in phosphate buffer. The myeloper-oxidase system was supplemented with 100 mM NaCl. A tryptic digestof apoA-I was analyzed by LC-ESI-MS and MS/MS. Chlorinated pep-tides were detected and quantified using reconstructed ion chromato-grams of precursor and product peptides. Peptide sequences were con-firmed using MS/MS. Results are representative of those from twoindependent experiments.

FIG. 4. SDS-PAGE of lipid-free apoA-I modified by reactivenitrogen and chlorine species. ApoA-I (5 �M for MPO and 25 �M forreagent HOCl or ONOO�) was exposed to the myeloperoxidase-H2O2-chloride system (MPO-Cl�), the myeloperoxidase-nitrite system (MPO-NO2

�), ONOO�, or HOCl at the indicated molar ratio of oxidant/proteinfor 60 min at 37 °C in phosphate buffer. The myeloperoxidase-nitritesystem and myeloperoxidase-chloride system were supplemented with100 �M nitrite and 100 mM NaCl, respectively. After the reaction wasterminated with methionine, apoA-I modified by the indicated condi-tions was subjected to electrophoresis under denaturing and reducingconditions.

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Lys195, and Lys238) that are situated within the YXXK motif ofapoA-I (35). Position 195 was in the most hydrophilic environ-ment, having a � value of �3.93, consistent with a residue fullyexposed to solvent. The positions of the other lysine residueshad significantly higher � values (�1.67 for residue 119 and�1.92 for residue 238). Thus, both Tyr192 and Lys195 are highlyexposed to the aqueous environment in lipid-free apoA-I.

To evaluate the effect of lipidation on the local environmentof the tyrosine residues in apoA-I, we prepared protein-lipid(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) complexesby the cholate dialysis method (51, 52), which were isolated byultracentrifugation and analyzed by SDSL-EPR spectroscopy(Fig. 5). In lipid-bound apoA-I, the � value for position 192 wassignificantly higher (�1.13) than in the lipid-free counterpart,indicating that this residue resides in a markedly more nonpo-lar environment upon lipid association. Lipidation also greatlyincreased the hydrophobicity of the local environment of posi-tion 195 (� of �0.80). These observations strongly suggest thatthe lipid association of apoA-I renders Tyr192 and Lys195 lessaccessible to aqueous solvent.

Tyrosine 192 in ApoA-I of HDL Is Resistant to Nitration butnot Chlorination—Accessibility to solvent may be one impor-tant factor controlling the susceptibility of tyrosine residues tonitration (57). Our SDSL-EPR studies suggested that lipidassociation greatly increases the hydrophobicity of the localenvironment around Tyr192. Thus, the residue is likely to be-come much less accessible to aqueous phase reactive interme-diates. To determine whether this process might affect thesusceptibility of Tyr192 to nitration or chlorination, we exposedplasma isolated HDL to myeloperoxidase or ONOO�, main-taining the ratio of oxidant to apoA-I used with lipid-free andlipid-associated apoA-I (�25:1 (mol/mol) for chlorination and10:1 (mol/mol) for nitration). Because the average HDL3 parti-cle contains 2 mol of apolipoprotein A-I (7 tyrosine residues,243 amino acids) and 1 mol of apolipoprotein A-II (8 tyrosineresidues, 154 amino acids), the ratio of oxidant to tyrosine was36% lower in HDL than in lipid-free apoA-I.

We previously showed that Tyr192 of apoA-I is chlorinated in

high yield when HDL is exposed to reagent HOCl (35). LC-ESI-MS and MS/MS analysis of a tryptic digest of HDL exposedto the myeloperoxidase-H2O2-chloride system revealed thatTyr192 of apoA-I was also the major site of chlorination byenzymatically generated HOCl (Fig. 6A). The product yield of

FIG. 5. EPR studies of spin-labeled apoA-I. Tyrosine residues 18,29, 115, 166, 192, and 236 of human apoA-I were each individuallymutated to cysteine residues, expressed in a bacterial system, andpurified by metal ion affinity chromatography. The cysteine residue ofisolated, lipid-free protein was derivatized with methanethiosulfonatenitroxide spin label, and the contrast function (�, an index of thehydrophobicity of the local environment) of each derivatized residuewas determined by SDSL-EPR spectroscopy. To determine the impactof lipidation on the local environment of each residue, spin-labeledapoA-I was incorporated into protein-lipid complexes by cholate dialy-sis, discoidal HDL particles were isolated by ultracentrifugation, and �was determined by SDSL-EPR.

FIG. 6. Identification of chlorinated and nitrated tyrosine res-idues in HDL-associated apoA-I exposed to the myeloperoxi-dase-H2O2-chloride system, the myeloperoxidase-H2O2-nitritesystem, or reagent ONOO�. Lipid-free apoA-I or HDL-associatedapoA-I (5 �M for the MPO system or 25 �M for ONOO�) was exposed tothe myeloperoxidase-H2O2 system or ONOO� for 60 min at 37 °C inphosphate buffer. The ratio of oxidant to apoA-I (mol/mol) was asfollows: MPO-H2O2-Cl�, 25:1; MPO-H2O2-NO2

�, 10:1; ONOO�, 10:1.The myeloperoxidase system was supplemented with 100 mM NaCl or100 �M nitrite. A tryptic digest of apoA-I was analyzed by LC-ESI-MSand MS/MS. Chlorinated and nitrated peptides were detected andquantified using reconstructed ion chromatograms of precursor andproduct peptides. Peptide sequences were confirmed using MS/MS.Results are representative of those from two independent experiments.

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3-chlorotyrosine at residue 192 was �80% of that observed inlipid-free apoA-I under our experimental conditions. Becausethe molar ratio of oxidant to apoA-I was �40% lower in HDLthan in lipid-free apoA-I, these observations suggest thatTyr192 reacts strongly with HOCl generated by myeloperoxi-dase when apoA-I is either lipid-free or associated with lipidsin HDL.

Tyr192 was also the major site of nitration when lipid-freeapoA-I was incubated with either the myeloperoxidase-H2O2-nitrite system or reagent ONOO� (Fig. 2). However, Tyr192 inapoA-I was not nitrated when HDL was exposed to the my-eloperoxidase-H2O2-nitrite system (Fig. 6B), although we diddetect a low level of nitration of Tyr18, Tyr29, Tyr100, andTyr236. When HDL was exposed to ONOO�, tyrosine residues18, 29, 115, 166, 192, and 236 of apoA-I were nitrated (Fig. 6C),and the overall product yield of 3-nitrotyrosine was markedlylower. These observations, in concert with the EPR studies oflipid-free and lipid-associated apoA-I, suggest that solvent ac-cessibility is a major factor controlling tyrosine nitration byreactive nitrogen species.

Lysine Residues Fail to Direct the Regiospecific Nitration ofTyrosine in Peptides—Five of the 7 tyrosine residues in apoA-Ilie in amphipathic helices that are thought to play critical rolesin lipid binding, lipoprotein stability, and reverse cholesteroltransport (1, 58). The helical wheel representation of am-phipathic helices predicts that tyrosine and lysine residues in aYXXK motif will lie next to each other on the same face of the�-helix (58). The predominant site of nitration in apoA-I,Tyr192, resides in a YXXK motif.

To explore the influence of lysine residues on the nitration ofnearby tyrosine residues, we investigated the reactions of themyeloperoxidase-H2O2-nitrite system or ONOO� with threemodel peptides: AcGYKRAYE, AcGEYARKY, and AcGE-YAREY (YKXXY, YXXKY, and YXXXY). We previously usedthese peptides to demonstrate that lysine residues direct theregiospecific chlorination of tyrosine by HOCl (35). The threepeptides contained the same amino acids (acetyl-G, -2Y, -K, -R,-A, and -E), including glutamic acid (E), but the latter replacedlysine (K) in YXXXY. Glutamic acid was included because itsnegative charge ensures aqueous solubility. Arginine was in-cluded because its positive charge promotes peptide ionizationduring mass spectrometric analysis. The N-terminal acetylgroup prevented the primary amine from participating in thereaction pathway.

We exposed each peptide to the myeloperoxidase-H2O2-ni-trite system or reagent ONOO� in phosphate buffer supple-mented with DTPA for 60 min at 37 °C, terminated the reac-tion with methionine, and analyzed the reaction products byHPLC with UV detection, LC-ESI-MS, and LC-ESI-MS/MS.Oxidation of YXXXY with myeloperoxidase (4:1, mol/mol, oxi-dant/peptide) generated three products that individually ac-counted for 3–9% of the product yield (relative to total oxidant;Table II).

We used HPLC to quantify the product yield and MS/MS toidentify the residues that are nitrated when YXXXY, YXXKY,and YKXXY peptides are exposed to the myeloperoxidase-H2O2-nitrite system or ONOO�. HPLC analysis revealed thatthe relative product yields were similar when YXXXY, YKXXY,and YXXKY were exposed to reactive nitrogen species derivedfrom either system (Table II). MS/MS analysis (Fig. 7; YXXXYexposed to ONOO�) indicated that the three products of YX-XXY oxidized with myeloperoxidase and ONOO� were, respec-tively, AcGEYARE(NO2Y) (YXXXNO2Y), AcGE(NO2Y)AREY(NO2YXXXY), and AcGE(NO2Y)AREN(O2Y) (NO2YXXX-NO2Y). Similar results were observed with YKXXY and YX-XKY modified by myeloperoxidase or ONOO�; introduction of alysine residue into the peptide had little effect on either therelative product yields or the sites of tyrosine nitration (TableII). These observations indicate that lysine residues fail todirect the regiospecific nitration by myeloperoxidase orONOO� of tyrosine residues in synthetic peptides.

Oxidation of YXXXY, YKXXY, and YXXKY with HOCl pro-duced a different pattern of products (Table II). Oxidation ofYXXXY, which contains 2 tyrosine residues and lacks a lysineresidue, yielded small and approximately equal amounts of twooxidation products, suggesting the formation of chlorotyrosine(ClY). MS/MS analysis revealed that the two products weremonochlorinated on a single tyrosine residue (AcGE(ClY)AREYand AcGEYARE(ClY); ClYXXXY and YXXXClY). When thesubstrate was YKXXY or YXXKY, HPLC with UV detectionrevealed a high yield (�40%, mol of product/mol of oxidant) ofone major product and a smaller yield (9 and 17%, respectively,mol of product/mol of oxidant) of a minor product (Table II).HPLC and LC-ESI-MS/MS analysis of YXXKY exposed toHOCl demonstrated a major oxidation product that contained achlorine atom on its first tyrosine residue (AcGE(ClY)ARKY;ClYXXKY). When the substrate was YKXXY, the major oxida-tion product contained a chlorine atom on its second tyrosineresidue (AcGYKRA(ClY)E; YKXXClY). Our results are consist-ent with our previous observations (35) and confirm that HOClchlorinates tyrosine residues in peptides containing the motifKXXY or YXXK with high yield. Moreover, chlorination isregiospecific, because it targets the tyrosine residue that lies 2residues away from a lysine residue.

Chlorination but Not Nitration of ApoA-I Strongly Inhibitsthe Ability to Remove Cellular Cholesterol by the ABCA1 Path-way—We compared the effects of chlorination and nitration ofapoA-I on the protein’s ability to promote cholesterol effluxfrom cells, which occurs exclusively by an ABCA1-dependentprocess. For these studies, we used [3H]cholesterol-labeledBHK cells that were stably transfected with a mifepristone-inducible ABCA1 cDNA. ApoA-I was exposed to the same molratios of oxidants used for our experiments that quantifiedtyrosine chlorination and nitration (Figs. 2 and 3). TreatingapoA-I with increasing concentrations of HOCl progressivelyand dramatically impaired apoA-I-mediated cholesterol efflux

TABLE IIProduct yields of 3-nitrotyrosine and 3-chlorotyrosine in peptides exposed to the myeloperoxidase-H2O2-nitrite system, ONOO�, or HOCl

Peptide (25 �M for the MPO system and 100 �M for reagent ONOO� or HOCl) was incubated for 60 min at 37 °C in phosphate buffersupplemented with 50 nM myeloperoxidase, 100 �M H2O2, and 100 �M nitrite (MPO-H2O2-NO2

�), 500 �M ONOO�, or 125 �M HOCl. Reactions wereinitiated by adding oxidant and terminated by adding L-methionine. The reaction mixture was analyzed by reverse-phase HPLC and UV detectionat 280 nm. Results are representative of those observed in three independent experiments.

ProductMPO-H2O2-NO2

� ONOO� HOCl

YXXXY YKXXY YXXKY YXXXY YKXXY YXXKY YXXXY YKXXY YXXKY

NO2YXXXY 3 5 3 3 5 4YXXXNO2Y 7 9 11 6 7 9NO2YXXXNO2Y 9 7 6 11 6 5ClYXXXY 11 9 38YXXXClY 11 42 17

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from these ABCA1-expressing cells at a concentration of apoA-I(3 �g/ml) that was below the concentration required for maxi-mal efflux (Fig. 8A). Treating apoA-I with increasing concen-trations of ONOO�, however, inhibited cholesterol efflux onlymodestly (Fig. 8A). When loss of apoA-I activity was nearlymaximal (125 �M oxidant; 25:1, mol/mol, oxidant/protein), anestimated 40% of Tyr192 residues were chlorinated by HOCl,and an estimated 80% were nitrated by ONOO� (compare Fig.8A with Figs. 2 and 3).

We obtained similar results when we used the myeloper-oxidase-H2O2 system to oxidize apoA-I with chlorinating ornitrating intermediates (Fig. 8B). In this case, apoA-I ex-posed to increasing concentrations of H2O2 in the presence ofmyeloperoxidase and plasma concentrations of chloride ion(100 mM NaCl) markedly reduced the cholesterol efflux activ-ity of apoA-I, whereas the same concentrations of H2O2 aloneproduced only modest inhibition in the presence of nitrite andmyeloperoxidase (Fig. 8B). When loss of apoA-I activity wasnearly maximal (125 �M oxidant; 25:1, mol/mol, oxidant/pro-tein), an estimated 35% of Tyr192 residues were chlorinatedor nitrated by myeloperoxidase (compare Fig. 8B with Figs. 2and 3). Thus, although chlorination and nitration modifiedTyr192 to similar extents, chlorination was much more effec-

tive than nitration at preventing apoA-I from removing cel-lular cholesterol.

We constructed apoA-I concentration curves to evaluate theeffects of chlorination and nitration on the ability of apoA-I toremove cholesterol by the ABCA1 pathway. Oxidizing apoA-Iby the myeloperoxidase-H2O2-chloride system greatly reducedthe cholesterol efflux activity of apoA-I at all concentrations ofapoA-I examined (Fig. 8C). Oxidation by the myeloperoxidase-H2O2-nitrite system modestly decreased the apparent affinityof apoA-I for ABCA1 but did not inhibit the ability of apoA-I toremove cellular cholesterol above saturating concentrations.Thus, only chlorination reduced the maximum capacity ofapoA-I to remove cellular cholesterol.

We tested the possibility that specific antioxidants couldprevent the inhibitory effects of reactive intermediates gener-ated by the myeloperoxidase system. In the absence of my-eloperoxidase, treating apoA-I with H2O2 plus either chloride(Fig. 9) or nitrite (data not shown) had no effect on its choles-terol efflux activity. The severely impaired apoA-I activitycaused by the myeloperoxidase-H2O2-chloride system was sub-stantially or completely prevented when the peroxide scav-enger catalase, the heme poison azide, or the HOCl scavengersvitamin C or methionine were included in the reaction mixture

FIG. 7. MS/MS analysis of YXXXYpeptide (AcGEYAREY) and its reac-tion products. The peptide was oxidizedwith ONOO� as described in the legend toTable II and analyzed by LC-ESI-MS/MS.A, MS/MS spectrum of precursor YXXXY.B, MS/MS spectrum of product NO2YX-XXY. C, MS/MS spectrum of product YX-XXNO2Y. D, MS/MS spectrum of productNO2YXXXNO2Y.

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(Fig. 9). Although the inhibition was markedly lower, a similarpattern was observed when nitrite was substituted for chloride(data not shown). These observations indicate that scavengersof HOCl and H2O2 or inhibitors of heme proteins block theability of myeloperoxidase to impair the ability of apoA-I topromote cholesterol efflux from cells.

DISCUSSION

Our observations indicate that myeloperoxidase and ONOO�

selectively oxidize Tyr192 in lipid-free apoA-I, the major proteinin HDL. Tyr192 was the single major target for both chlorina-tion by the myeloperoxidase-H2O2-chloride system or HOCland nitration by ONOO�. It also was the predominant site ofnitration by myeloperoxidase. EPR studies of spin-labeledapoA-I demonstrated that residue 192 is located in an ex-tremely hydrophilic environment. This observation indicatesthat Tyr192 is highly accessible to polar solvents and raises thepossibility that this accessibility is one important factor con-trolling its reactivity. Moreover, upon incorporation into discoi-dal HDL, residue 192 was positioned in a much more hydro-phobic environment, likely at the lipid-water interface of theparticle. Notably, Tyr192 of HDL-associated apoA-I was a poor

substrate for nitration by both myeloperoxidase and ONOO�.Collectively, these observations indicate that lipid associationmarkedly changes the accessibility of Tyr192 to the aqueousenvironment and that this process in turn reduces the suscep-tibility of Tyr192 to nitration by myeloperoxidase and ONOO�.

Remarkably, the ability of apoA-I to promote ABCA1-de-pendent cholesterol efflux was dramatically impaired whenTyr192 was chlorinated. In contrast, nitration of Tyr192 hadlittle impact on this biological function. A recent study alsoshowed that nitration was much less effective than chlorina-tion in impairing the cholesterol efflux activity of apoA-I (37).These results indicate that Tyr192 is the major aromatic aminoacid targeted for oxidation when apoA-I is exposed to reactivechlorine and nitrogen species and that chlorinating and nitrat-ing intermediates exert different effects on the modified apo-lipoprotein’s ability to promote ABCA1-dependent cholesterolefflux. It is important to note that our observations do not provethat chlorination of Tyr192 is responsible for impairing theability of apoA-I to promote ABCA1-dependent cholesterol ef-flux, because other amino acids are also modified when apoA-Iis exposed to oxidizing intermediates (7–10, 15–17).

FIG. 8. Cholesterol efflux activities of lipid-free apoA-I and apoA-I oxidized with ONOO�, HOCl, myeloperoxidase-H2O2-chloride,or myeloperoxidase-H2O2-nitrite. ApoA-I (5 �M) was incubated with the indicated concentrations of HOCl, ONOO�, or H2O2 for 60 min at 37 °Cin phosphate buffer. The reaction was terminated by the addition of methionine. Where indicated, the system was supplemented with 50 nM

myeloperoxidase and 100 �M nitrite (NO2�) or 100 mM NaCl (NaCl). A and B, [3H]cholesterol-labeled ABCA1-transfected BHK cells were incubated

for 2 h with native (0 �M oxidant), ONOO�-oxidized, HOCl-oxidized, or MPO-H2O2-oxidized apoA-I (5 �g/ml). C, [3H]cholesterol-labeled ABCA1-transfected BHK cells were incubated with the indicated concentration of apoA-I for 2 h. ApoA-I was incubated with myeloperoxidase, 0 �M oxidant(Ctrl), and 125 �M H2O2 plus 100 �M nitrite (NO2) or 100 mM NaCl (NaCl). At the end of the incubation, [3H]cholesterol efflux to the acceptorapolipoprotein was measured.

FIG. 9. Effects of antioxidants onthe cholesterol efflux activity of lip-id-free apoA-I oxidized with the my-eloperoxidase-H2O2-chloride system.ApoA-I (5 �M) was incubated for 60 min at37 °C in phosphate buffer (Control).Where indicated, the system was supple-mented with 125 �M H2O2, 50 nM my-eloperoxidase (MPO), 100 mM NaCl (Cl�),catalase (Cat; 200 nM), azide (Az; 10 mM),vitamin C (Vit C; 10 mM), or methionine(Met; 10 mM). The reaction was termin-ated by the addition of methionine. [3H]-Cholesterol-labeled ABCA1-transfectedBHK cells were incubated for 2 h withapoA-I (5 �g/ml) modified under the in-dicated conditions for 2 h. At the end of theincubation, [3H]cholesterol efflux to theacceptor apolipoprotein was measured.

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Several factors might account for the differential abilitiesof chlorinated and nitrated apoA-I to remove cholesterol fromcells. One likely possibility is that apoA-I undergoes oxida-tive modifications distinct from tyrosine chlorination that areuniquely induced by HOCl. It is also possible that nitrationand chlorination of Tyr192 exert divergent effects on the abil-ity of apoA-I to promote cholesterol efflux from cells. Alter-natively, chlorination but not nitration might produce a con-formational alteration in apoA-I that reduces its interactionswith ABCA1 or its ability to acquire lipids. In future studies,it will be of interest to determine the specific molecularmechanism underlying the apoA-I chlorination-mediated im-pairment of ABCA1 activity.

A key question is why Tyr192 is so much more amenable tochlorination and nitration than the six other tyrosine residuesin apoA-I. SDSL-EPR analysis indicates that Tyr192 in lipid-free apoA-I lies in a random coil secondary structure, fullyexposed to aqueous solution. However, the secondary structureof this region of apoA-I undergoes a transition to an am-phipathic �-helix during discoidal HDL particle formation (50).Because solvent accessibility and loop structures are thought toaffect the ability of ONOO� to nitrate tyrosine (18, 57), weemployed SDSL-EPR to determine whether residues 18, 29,115, 166, 192, and 236 of lipid-free and lipid-associated apoA-Idiffer in their solvent accessibility (50). In spin-labeled lipid-free apoA-I, residue 192 resided in an extremely hydrophilicenvironment. In contrast, this residue was positioned in amuch more hydrophobic environment on discoidal HDLparticles.

These observations indicate that lipid association mark-edly affects the environment of residue 192 and stronglysuggest that Tyr192 in lipid-free apoA-I is highly accessible toaqueous solvent. Consistent with this possibility, Tyr192 inlipid-free apoA-I was the major nitration site for both my-eloperoxidase and ONOO�. When apoA-I was incorporatedinto reconstituted HDL particles, however, nitration of Tyr192

was markedly reduced.These observations suggest the following model (Scheme 1).

In lipid-free apoA-I, Tyr192 lies in a random coil structure in anenvironment that is highly accessible to aqueous solvent. Inthis environment, the residue reacts rapidly with the shortlived, highly reactive intermediates derived from ONOO� orthe myeloperoxidase-H2O2-nitrite system. When apoA-I is as-sociated with lipid, however, Tyr192 partitions into a morehydrophobic environment and is therefore unable to react with

nitrating intermediates generated in the aqueous phase bymyeloperoxidase and ONOO�. Thus, accessibility to solvent islikely to be an important feature controlling the nitration ofTyr192 in apoA-I.

In contrast to its behavior with nitrating species, Tyr192

was chlorinated in high yield in both lipid-free and lipid-associated apoA-I. This tyrosine resides in a YXXK motif, andwe have previously shown that HOCl reacts with lysine res-idues in peptides to form long lived chloramines that promotethe regiospecific chlorination of tyrosine (35). The helicalwheel representation of amphipathic helices predicts thattyrosine and lysine residues in the YXXK (and KXXY) motifwill lie next to each other on the same face of the �-helix (58),suggesting that lysine residues in the YXXK motif can beconverted to chloramines that then direct tyrosine chlorina-tion. In contrast to the free N� amino group of lysine (pKa

�10.5), which exists predominantly as the protonated NH3�

species at neutral pH, the chloramine derived from the lysineN� amino group is uncharged. Thus, this long lived speciescould potentially attack the phenolic group of tyrosine ineither a hydrophilic or hydrophobic environment. Moreover,Tyr192 lies in an �-helical structure when lipid-associated,which favorably positions it for interaction with Lys195.These observations suggest that the chloramine of Lys195 candirect the chlorination of Tyr192 of apoA-I in high yield inboth lipid-free and lipid-associated protein.

It has also been proposed that charged amino acid residueshelp direct tyrosine nitration by ONOO� (18, 57). We usedsynthetic peptides containing tyrosine and lysine to explore thepotential role of primary amines in promoting the regiospecificoxidation of tyrosine by reactive nitrogen species. These studiessuggest that Lys195 is unlikely to direct the regiospecific oxi-dation of Tyr192 by reactive nitrogen species. Instead, theTyr192 in apoA-I might be so much more amenable to nitrationthan the other 6 tyrosine residues because of differences insolvent accessibility, spatial orientation, and the local aminoacid environment.

Several lines of evidence indicate that myeloperoxidase-me-diated apoA-I oxidation may be sufficient to impair ABCA1-de-pendent cholesterol efflux from macrophage foam cells in thehuman artery wall. We showed that HDL isolated from humanatherosclerotic lesions and plasma from patients with estab-lished coronary artery disease contained elevated levels of both3-chlorotyrosine and 3-nitrotyrosine, which strongly implicatesmyeloperoxidase as a key contributor to HDL oxidation in vivo(31, 36). The level of tyrosine chlorination in lesion HDL rangedfrom 100 to 300 �mol of 3-chlorotyrosine/mol of Tyr, suggestingthat �1 in every 800 apoA-I molecules was chlorinated. It islikely, however, that there is a gradient in the degree of apo-lipoprotein oxidation between extracellular compartments andthe immediate pericellular environment. We previously re-ported that epitopes for 3-nitrotyrosine, a product of highlyreactive nitrogen dioxide radical, co-localize with myeloperoxi-dase and macrophages in human atherosclerotic tissue (31).Because the nitrogen dioxide radical is a short lived species,this finding implies that proteins in close proximity to macro-phages are selectively targeted for oxidative damage. More-over, apoA-I is poorly nitrated by both myeloperoxidase andONOO� when it is associated with HDL, raising the possibilitythat lipid-poor apoA-I, the biologically active ligand for ABCA1,is the major target for nitration in the artery wall. Thus,oxidation of apoA-I appears to be physiologically important,lipid-poor apoA-I may be the primary target for oxidation, andmicroenvironments depleted of antioxidants might enable oxi-dation to occur. One such environment surrounds tissue macro-phages, which generate high local concentrations of oxidants.

SCHEME 1. Proposed role of the local environment of Tyr192 inthe nitration of lipid-free and lipid-associated apoA-I.

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Our results suggest the following model for HDL oxidationand the functional impairment of ABCA-1-dependent choles-terol efflux in the human artery wall. Activated macrophagesuse a membrane-associated NADPH oxidase to produce highpericellular concentrations of superoxide and H2O2. Superox-ide reacts with NO to generate ONOO� in a diffusion-con-trolled reaction that may be most favorable near the plasmamembrane of the cells. Moreover, myeloperoxidase secretedby activated phagocytes uses H2O2 to convert chloride ionand nitrite into chlorinating and nitrating species. Thus,macrophage-dependent oxidation reactions are likely to behighly restricted in space by local changes in oxidant andenzyme concentrations. Modification of specific amino acidresidues in apoA-I impairs the ability of the apolipoprotein topromote cholesterol efflux from macrophages, contributing tothe formation of lipid-laden foam cells. In contrast, nitrationdoes not appear to markedly alter the ability of apoA-I toremove cholesterol from cells. It is important to note thatapoA-I promotes cholesterol efflux from cells by interactingwith ABCA1 at the plasma membrane of macrophages.

Our studies suggest that local, pericellular production ofreactive chlorinating and nitrating intermediates by phago-cytes is a physiological mechanism for oxidizing apoA-I. Thus,chlorination of apoA-I may play a critical role in inhibitingHDL function and reverse cholesterol transport duringatherogenesis.

Acknowledgments—We thank Will Driscoll for excellent technicalassistance. Mass spectrometric analyses were performed in the MassSpectrometry Resource (Department of Medicine, Universityof Washington).

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Nitration and Chlorination of Tyrosine Residues in ApoA-I 5993

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

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

doi: 10.1074/jbc.M411484200 originally published online November 30, 20042005, 280:5983-5993.J. Biol. Chem. 

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