Lysine Residues Direct the Chlorination of Tyrosines in ... · Lysine Residues Direct the...

Lysine Residues Direct the Chlorination of Tyrosines in YxxK Motifs

of Apolipoprotein A-I when Hypochlorous Acid Oxidizes HDL

Constanze Bergt1, Xiaoyun Fu1, Nabiha P. Huq2, Jeff Kao2, Jay W. Heinecke1

1Department of Medicine, University of Washington, Seattle, WA 98195

2Department of Chemistry, Washington University, St. Louis, MO 63110

Address correspondence to: Jay Heinecke, Division of Metabolism, Endocrinology and

Nutrition, Box 356426, University of Washington, Seattle, WA 98195, email:

[email protected].

Abbreviations: DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ESI, electrospray

ionization; HDL, high density lipoprotein; MALDI, matrix assisted laser desorption ionization;

MS, mass spectrometry; PBS, phosphate buffered saline; TFA, trifluoroacetic acid; TOF, time-

of-flight; TOCSY, total correlation spectroscopy.

Running title: Regiospecific chlorination of tyrosine residues

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Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on December 3, 2003 as Manuscript M309046200 by guest on January 14, 2020

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SUMMARY

Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated

levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid

(HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus,

HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein

oxidation in humans. One important target of the myeloperoxidase pathway may be high density

lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether

activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass

spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The

major site of chlorination was a single tyrosine residue located in one of the protein’s YxxK

motifs (Y, tyrosine; K, lysine; x, non-reactive amino acid). To investigate the mechanism of

chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid

sequences YKxxY, YxxKY, or YxxxY. MS/MS analysis demonstrated that chlorination of

tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the

substrate was KxxY or YxxK. NMR and MS analyses revealed that the Nε amino group of lysine

was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine

chlorination. Molecular modeling of the YxxK motif in apolipoprotein A-I demonstrated that

these tyrosine and lysine residues are adjacent on the same face of an amphipathic α-helix. Our

observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed

to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently

damage proteins when they destroy microbial proteins during infection or damage host tissue

during inflammation.

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INTRODUCTION

Protein oxidation has been implicated in the pathogenesis of diseases ranging from

ischemia-reperfusion injury to atherosclerosis as well as in the aging process itself (1). However,

most studies of protein oxidation have focused on the vulnerability of individual amino acid side

chains, including the phenolic group of tyrosine and the thiol groups of methionine and cysteine.

Remarkably little is known about the influence of nearby residues or of specific sequence motifs

on the susceptibility of protein-bound amino acid chains to oxidation.

One example of such effects is the oxidation of specific cysteine residues in tyrosine

phosphatases by hydrogen peroxide (2). This reaction, which affects phosphatase activity, is

thought to rely on a high local concentration of positively charged amino acid side chains to

stabilize the reactive anionic form of the thiol. Another example is the apparent effect of acidic

and basic amino acid residues on protein nitrosation (3). However, an extensive study of model

proteins oxidized by peroxynitrite revealed few obvious common features among the tyrosine

residues that were targeted for nitration (4). Recently, histidine and cysteine residues at the metal

binding site have been identified as primary targets during metal catalyzed oxidation of ß-

amyloid peptides and iron regulatory protein 2 (5,6).

Neutrophils, monocytes and some population of macrophages use the heme enzyme

myeloperoxidase (7-9) to produce hypochlorous acid (HOCl), a potent cytotoxic oxidant (10,11).

Cl- + H2O2 + H+ → HOCl + H2O (Equation 1)

HOCl plays a critical role in destroying microbial pathogens (12,13). However, it can also react

with proteins, lipids, and nucleic acids in host tissues, contributing to inflammatory damage

(14,15).

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Because proteins are a major target for HOCl, the acid’s reactions with amino acids and

peptides have been widely studied (16-21). HOCl readily oxidizes the sulfur-containing amino

acids cysteine and methionine, producing disulfides, oxyacids, sulfoxides, and compounds in

which sulfur is cross-linked to nitrogen (22-24). However, other reactive species, such as

hydroxyl radical and peroxynitrite, can also generate oxygenated sulfur products (25,26), which

therefore cannot serve as definitive markers for HOCl-induced damage. In contrast, in vitro and

in vivo studies have shown that 3-chlorotyrosine is a specific product of myeloperoxidase

(13,27,28).

Chlorination of the phenolic ring of tyrosine may have physiological relevance because both

active myeloperoxidase, the enzyme that generates HOCl, and elevated levels of 3-

chlorotyrosine have been detected in human atherosclerotic lesions (8,9,14,29). One important

target might be high density lipoprotein (HDL). HDL is believed to inhibit atherosclerosis by

mobilizing excess cholesterol from cells of the artery wall, but oxidation of HDL has been

proposed to alter its biological properties, thereby contributing to the pathogenesis of

atherosclerosis (30,31). Previous studies have shown that methionine and phenylalanine residues

in apolipoprotein A-I, the major protein in HDL, are oxidized by reactive intermediates (32-35).

Tyrosine residues are converted to o,o’-dityrosine by tyrosyl radical (36). However, little is

known about the vulnerability to chlorination of specific tyrosine residues in apolipoprotein A-I.

HOCl also reacts with primary amino groups, producing primary (RNClH) and secondary

(RNCl2) chloramines (37,38).

HOCl + RNH2 → RNClH + H2O + HOCl RNCl2 + H2O (Equation 2)

Studies with free α-amino acids have shown that they form unstable α-amino chloramines,

which are deaminated and decarboxylated into aldehydes (39-41). In contrast, HOCl yields stable

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chloramines at the primary ε-amino group of lysine, which lacks an adjacent carboxylic acid

(19). It has been suggested that N-centered radicals deriving from chloramines could contribute

to protein fragmentation and tissue damage (42).

Both HOCl and chloramines react with aromatic and unsaturated compounds to form

chlorinated products (7,37,43). HOCl is in equilibrium with molecular chlorine, and this potent

electrophile has been proposed to mediate the chlorination of free tyrosine (28). It remains to be

established whether this reaction pathway is relevant to the chlorination of protein-bound

tyrosine residues. N-Terminal chloramines have been proposed to be intermediates in the

formation of 3-chlorotyrosine in synthetic peptides (27,44). However, little is known about the

factors that control the site-specific chlorination of tyrosine residues in proteins. In the current

study, we use HDL, synthetic peptides and tandem mass spectrometric analysis to investigate the

role of HOCl and chloramines in tyrosine chlorination.

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EXPERIMENTAL PROCEDURES

Materials

Sodium hypochlorite (NaOCl), trifluoroacetic acid (TFA), and HPLC grade CH3CN and

methanol were obtained from Fisher Scientific (Pittsburgh, PA). Phosphate buffered saline

(PBS), free and acetylated amino acids, and α-cyano-4-hydroxycinnamic acid were purchased

from Sigma (St. Louis, MO). Peptides AcGYKRAYE (YKxxY), AcGEYARKY (YxxKY), and

AcGEYAREY (YxxxY) were prepared by the Protein and Nucleic Acid Chemistry Laboratory,

Washington University (St. Louis, MO). Peptides AcPYSDELRQRLAARLE-NH2 (Yxxxxx),

AcPYSDELKQRLAARLE-NH2 (YxxxxK), AcPYSDEKLQRLAARLE-NH2 (YxxxKx),

AcPYSDKELQRLAARLE-NH2 (YxxKxx), AcPYSKDELQRLAARLE-NH2 (YxKxxx) and

AcPYKSDELQRLAARLE-NH2 (YKxxxx) were synthesized by GenScript Corporation (Scotch

Plains, NJ). Purity of the peptides was confirmed by HPLC and mass spectrometric analysis.

Methods

HDL isolation. Plasma was prepared from blood anticoagulated with EDTA collected

from healthy adults that had fasted overnight. HDL (density 1.125-1.210 g/mL) was prepared by

sequential ultracentrifugation from plasma and was depleted of apolipoprotein E and

apolipoprotein B100 by heparin-agarose chromatography (45).

Oxidation reactions. Reactions were carried out at 37 oC in PBS (10 mM sodium

phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.4) supplemented with 100 µM peptide or 1 mg/ml

HDL protein. Reactions were initiated by adding oxidant and were terminated by the addition of

10- to 50-fold molar excess of L-methionine. Concentrations of HOCl and H2O2 were

determined spectrophotometrically (ε292 = 350 M -1 cm-1 and ε240 = 39.4 M-1cm –1) (46,47). The

pH dependence of reactions was determined using buffers (100 mM) composed of phosphoric

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acid, monobasic sodium phosphate and dibasic sodium phosphate and 100 mM NaCl. Protein

was determined using the Lowry assay (BioRad; Hercules, CA).

HPLC analysis of peptides. Peptides were separated at a flow rate of 1 mL/min on a

reverse-phase column (Beckman ODS, 4.6 x 250 mm) using a Beckman HPLC system

(Fullerton, CA) with UV detection at 215 nm. The peptides were eluted using a gradient of

solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in 90% CH3CN, 10% H2O). Solvent B

was increased from 10% to 50% over 25 min.

Isolation of peptide-bound chloramines. AcGEYARKY (100 µM) was incubated with

HOCl (100 µM) in PBS (pH 7.4) for 2 min at room temperature and the reaction mixture was

subjected immediately to HPLC without methionine addition. The methionine-sensitive

oxidation product was collected and stored on ice for further analysis.

Oxidation of lipid-associated peptides. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC) associated peptides were prepared using a peptide to DPPC ratio of 1:20 (mol/mol).

DPPC was dissolved in ethanol and dried under nitrogen. PBS was added and the mixture was

vortexed vigorously and incubated for 15 min at 37 ºC. Vesicles were aliquoted and incubated

with 25 µM peptides for 20 min at 37 ºC. Peptides were oxidized with HOCl in PBS at 37 ºC

(0.5 and 1, mol oxidant/mol peptide, lipid-free and DPPC-associated, respectively). The reaction

was stopped with excess methionine after 30 min.

Proteolytic digestion of proteins. Native or HOCl modified HDL was incubated overnight

at 37 oC with sequencing grade modified trypsin (Promega, Madison, WI) at a ratio of 25:1

(w/w) protein:trypsin in 50 mM NH4HCO3, pH 8. Digestion was halted by acidification (pH 2-3)

with TFA.

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Electrospray ionization mass spectrometry (ESI-MS). LC-ESI-MS analyses were

performed in the positive ion mode with a Finnigan Mat LCQ ion trap instrument (San Jose, CA)

coupled to a Waters 2690 HPLC system (Milford, MA)(48). Synthetic peptides were separated at

a flow rate of 0.2 mL/min on a reverse-phase column (Beckman ODS; 2.1 x 250 mm) using a

gradient of solvent A (0.2% HCOOH in H2O) and solvent B (0.2% HCOOH in 80% CH3CN,

20% H2O). Solvent B was increased from 10% to 50% over 25 min. Tryptic digests of HDL

were separated at a flow rate of 0.2 mL/min on a reverse-phase column (Vydac C18 MS; 2.1 x

250 mm). The electrospray needle was held at 4500 V. Nitrogen, the sheath gas, was set at 80

units. The collision gas was helium. The temperature of the heated capillary was 220oC.

Matrix assisted laser desorption ionization (MALDI) time of flight (TOF) MS. MALDI

TOF-MS analyses were performed on a Voyager DE-STR (PerSeptive Biosystems, Foster City,

CA) in the reflectron mode using delayed ion extraction (49).The accelerating voltage was 2.5

kV, the delay time was 300 nsec, the mirror to accelerating voltage ratio was 1.12, and the low

mass gate was 400 Da. Spectra (100 shots) were acquired with a laser power of 1850. α-Cyano-

4-hydroxycinnamic acid (10 mg/mL) in 0.1% TFA:CH3CN (1:1, v/v) was used as matrix.

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NMR Spectroscopy. NMR spectra were obtained on the peptide and the chloramine

intermediate (3.6 mM and 0.5 mM, respectively) in 0.5 mL of PBS (10% D2O, 90% H2O, pH

7.4). The spectra were acquired on a Varian Unity INOVA-600 spectrometer (599.749 MHz for

1H) and processed using VNMR software (Varian, Palo Alto, CA). 1-D NMR experiments were

carried out at 4 °C using presaturation or hard-pulse WATERGATE sequences for water

suppression (50). Spectra were obtained under the following conditions: pre-acquisition delay =

0.5 sec, acquisition time = 1.416 sec (16k complex data points), pulse width = 7.8 µsec, spectral

width = 5650.1 Hz and 2 msec 15G/cm field-gradient pulses.

Total correlation spectroscopy (TOCSY) experiments for peptides and peptide-bound

chloramines were recorded using an MELV-17 mixing sequence of 100 ms flanked by two 2

msec trim pulses with 256 t1 and 2048 t2 data points. After two-dimensional Fourier

transformation, the spectra resulted in 2048 x 2048 data points that were phase and baseline

corrected in both dimensions.

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RESULTS

HOCl Preferentially Chlorinates a Single Tyrosine Residue in Apolipoprotein A-I.

The 10 amphipathic helices in apolipoprotein A-I, HDL’s major protein, are thought to

play essential roles in lipid binding, lipoprotein stability, and reverse cholesterol transport

(51,52). Five of the 7 tyrosine residues in apolipoprotein A-I are located in amphipathic helices

(Fig. 1A). It is noteworthy that 3 of those tyrosines reside in a YxxK motif (Y29xxK32,

Y192xxK195, and Y236xxK239; Y = tyrosine, K = lysine, x = other amino acids). Importantly, the

helical wheel representation of amphipathic helices predicts that tyrosine and lysine residues in

this motif will lie next to each other on the same face of the α-helix (Fig. 1B) (51,52). We

therefore used HDL to determine whether HOCl preferentially chlorinates specific tyrosine

residues in proteins.

LC-ESI-MS analysis of the trypsin digest of apolipoprotein A-I detected peptides that

collectively covered ~ 80% of the protein’s sequence and included all 7 peptides predicted to

contain tyrosine. To determine which tyrosine residues can be chlorinated, we oxidized HDL

with HOCl and used reconstructed ion chromatograms to detect: i) each of the peptides that

contained tyrosine; and ii) any tyrosine-containing peptides that had gained 34 amu (addition of

1 chlorine and loss of 1 hydrogen).

We exposed HDL to HOCl (80:1, mol/mol, oxidant:HDL particle) in a physiological

buffer (138 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate) at neutral pH for 120 min at

37°C and then terminated the reaction with a 20-fold molar excess (relative to oxidant) of

methionine. Because the average HDL3 particle contains 2 mol of apolipoprotein A-I (7 tyrosine

residues, 243 amino acids) and 1 mol of apolipoprotein A-II (8 tyrosine residues, 154 amino

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acids), the ratio of oxidant to substrate (mol:mol) was ~ 30:1 for apolipoproteins A-I and A-II,

3:1 for tyrosine residues, and 1:8 for total amino acids.

After digesting unmodified and oxidized HDL with trypsin, we analyzed the resulting

peptides. LC-ESI-MS and MS/MS analysis detected 3 tryptic peptides whose mass corresponded

to the mass of the precursor peptide plus 34 amu, which suggests the formation of 3-

chlorotyrosine (Table 1). The 3 peptides came from residues 189-195 ([LAEYHAK + 34 amu +

2H]2+, m/z 433.2), residues 227-238 ([VSFLSALEEYTK + 34 amu + 2H]2+, m/z 710.9), and

residues 28-40 ([DYVSQFEGSALGK + 34 amu + 2H]2+, m/z 717.9). Using LC-ESI-MS/MS

analysis, we confirmed each peptide’s sequence and showed that its tyrosine had been targeted

for chlorination (data not shown). Quantification of the ion current of each precursor and product

peptide using reconstructed ion chromatograms (Table 1) indicated that the major tyrosine

oxidation product was LAE(ClY)HAK (Fig. 2). Importantly, the tyrosine residue in this region of

apolipoprotein A-I is located in a YxxK motif. The other chlorinated peptides were present at

much lower levels. These findings indicate that HOCl chlorinates 3 of the 7 tyrosines in

apolipoprotein A-I (Table1) and that the major oxidation product (~ 50% 3-chlorotyrosine) was

located in the YxxK motif of LAEYHAK. The other 2 tyrosines were chlorinated in much lower

yield (< 6% 3-chlorotyrosine); one of these tyrosine residues was also located in a YxxK motif

(VSFLSALEEYTK[K], where [K] represents a lysine residue removed by trypsin). These

observations suggest that the amino group of lysine might direct protein oxidation to specific

sites.

Lysine Residues Direct the Regiospecific Chlorination of Tyrosine in Peptides.

To explore the influence of lysine residues on the chlorination of nearby tyrosine

residues, we investigated the reaction of HOCl with 3 model peptides: AcGYKRAYE,

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AcGEYARKY, and AcGEYAREY (YKxxY, YxxKY, and YxxxY). We included lysine in 2 of

the peptides because it has a primary ε-amino group that forms a long-lived chloramine when

exposed to HOCl (19), and chloramines can react with phenolic groups (37) such as the one in

tyrosine. The 3 peptides contained the same amino acids (acetyl-G, 2Y, K, R, A, E), including

glutamic acid (E), but the latter replaced lysine (K) in YxxxY. Glutamic acid was included

because its negative charge ensures aqueous solubility. Arginine was included because its

positive charge promotes peptide ionization during mass spectrometric analysis. The acetyl

group on the N-terminus prevented the primary amine from forming a chloramine.

We exposed each peptide to HOCl (1:1, mol/mol) in a physiological buffer for 30 min at

37°C, terminated the reaction with methionine (which reacts rapidly with HOCl), and analyzed

the reaction products by HPLC with UV detection, LC-ESI-MS, and LC-ESI-MS/MS. When the

substrate was YKxxY or YxxKY, HPLC with UV detection revealed a high yield (~ 55%, mol

product/mol oxidant) of one major product and smaller yields of a minor product (Fig. 3A,B;

Table 2). In contrast, oxidation of YxxxY generated four minor products that individually

accounted for only ~ 11% of the total oxidant (Fig. 3C, peaks 1-4; Table 2). LC-ESI-MS analysis

of the peptides YKxxY and YxxKY incubated in buffer alone demonstrated doubly charged ions

at mass-to-charge ratio (m/z) 464.8, the anticipated m/z of the doubly protonated unmodified

peptide [peptide + 2H]+2. LC-ESI-MS analysis of the two reaction products derived from YKxxY

and the two derived from YxxKY revealed ions of m/z 481.8, corresponding to the masses of the

doubly protonated peptides plus 34 amu [peptide + 34 amu + 2H]+2. These observations suggest

that each peptide loses a hydrogen atom and gains a chlorine atom when it is exposed to HOCl (–

1 amu, + 35 amu), which is consistent with chlorination of the phenolic ring of one of each

peptide’s two tyrosine residues. LC-ESI-MS analysis of YxxxY incubated in buffer alone

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demonstrated a singly charged ion of m/z 929.3, the anticipated m/z of the unmodified peptide

[peptide + H]+. Two of its oxidation products (Fig. 3C, peak 1 and peak 2) exhibited ions at m/z

963.3 [peptide + 34 amu + H]+, suggesting that each contained a single chlorine atom. In

contrast, the other two (Fig. 3C, peak 3 and peak 4) exhibited ions of m/z 997.3 [precursor

peptide + 68 amu + H]+, suggesting that HOCl oxidizes YxxxY into a mixture of peptides that

have 3-chlorotyrosine at either the first or last position or into a peptide that contains 3,5-

dichlorotyrosine residue at one position.

We used tandem mass spectrometry (MS/MS) to confirm the sequence of each precursor

peptide and to identify the amino acid residue that was amenable to chlorination (Fig. 4). MS/MS

analysis of YxxKY revealed a series of b-ions (b2, m/z 228.8; b5, m/z 619.2; b6, m/z 747.3) and y-

ions (y1, m/z 181.9; y4, m/z 537.2; y5, m/z 700.3) consistent with the predicted sequence

AcGEYARKY (Fig. 4A). MS/MS analysis of the major oxidation product of YxxKY (Peak 1;

Fig. 3B) demonstrated a series of b-ions and y-ions that had gained 34 amu (b5 + 34, m/z 653.1;

b6 + 34, m/z 781.3; y5 + 34, m/z 734.2; y6 + 34, m/z 863.4). However, the masses of ions b2 (m/z

228.9), y1 (m/z 182.0), and y4 (m/z 537.2) were unchanged (Fig. 4B). These observations indicate

that exposing YxxKY to HOCl generates a major oxidation product that contains a chlorine atom

on its first tyrosine residue (AcGEClYARKY; ClYxxKY). MS/MS analysis of peak 2 (Fig. 3B),

the minor product of YxxKY oxidation, demonstrated y1, y4, and y5 ions that had gained 34 amu

(y1 + 34, m/z 216.0; y4 + 34, m/z 571.2; y5 + 34, m/z 734.3) and ions that were unaffected (b4, b5,

and b6). These observations indicate that AcGEYARKClY (YxxKClY) is the minor product

when YxxKY is oxidized with HOCl. The sequences of the YxxKY peptide and its major and

minor oxidation product were confirmed using MALDI-TOF-MS analysis with post source

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decay (data not shown). These observations indicate that HOCl converts YxxKY into a single

major oxidation product, ClYxxKY.

We used this analytical approach to confirm the sequences of YKxxY and YxxxY and to

identify the residues that are chlorinated when these peptides are exposed to HOCl (data not

shown). MS/MS analysis revealed that the major and minor product of YKxxY oxidation were

respectively AcGYKRAClY (YKxxClY) and AcGClYKRAY (ClYKxxY). Oxidation of YxxxY

produced a different pattern of products: peaks 1 and 2 were monochlorinated on a single

tyrosine residue (AcGEClYAREY and AcGEYAREClY), whereas peaks 3 and peak 4 had two

chlorine atoms on a single tyrosine residue (AcGECl2YAREY and AcGEYARECl2Y).

Our results indicate that HOCl chlorinates tyrosine residues in peptides containing the motif

KxxY or YxxK with high yield and that chlorination is regiospecific for the tyrosine residue two

residues away from a lysine residue (Table 2). In contrast, only small amounts of

monochlorotyrosine and dichlorotyrosine form in YxxxY, which lacks a lysine residue (Table 2).

Moreover, we found no evidence for chlorination of both tyrosines in YxxxY. Instead, each

tyrosine residue was oxidized to either the monochlorinated or dichlorinated derivative (and the

other was left unchlorinated). These findings suggest that lysine plays a regiospecific role in the

chlorination of tyrosine in peptides.

YxxKY Reacts with HOCl to Produce a High Yield of Chlorotyrosine.

To characterize the reaction of HOCl with peptides, we incubated YxxKY with HOCl

(1:1, mol/mol) in a physiological buffer at neutral pH and 37ºC, and identified the reaction

products by HPLC. Peptide chlorination (monitored as production of both ClYxxKY and

YxxKClY) was complete by 60 min. At an equimolar ratio of oxidant and peptide, the product

yield exceeded 85% (Fig. 5A). It was maximal when the ratio of oxidant to peptide was ≤1, and

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it decreased at higher ratios (Fig. 5B). Chlorination of tyrosine in the peptides was optimal under

alkaline conditions, suggesting that hypochlorite (ClO-, the conjugate base of HOCl, pKa 7.5)

was the chlorinating agent or that deprotonation of the lysine ε amino group (pKa 10.0) favored

the reaction (Fig. 5C).

We also investigated the effects of proposed antioxidants on the chlorination of YxxKY

(Fig. 5D). At equimolar concentrations, both vitamin C and methionine completely blocked 3-

chlorotyrosine formation. In contrast, trolox, a water-soluble form of vitamin E (53), failed to

inhibit peptide chlorination. These findings indicate that compounds that react rapidly with

HOCl (54) but not radical scavengers, might be able to inhibit protein chlorination by HOCl.

Regiospecific Chlorination of Tyrosine Requires Peptide-bound Lysine.

To assess whether lysine can direct tyrosine chlorination when it is free in solution rather

than incorporated into a peptide, we incubated HOCl with N-acetyl-tyrosine alone (1:1, mol/mol)

or with mixtures of free amino acids representing the residues in YKxxY, YxxKY, or YxxxY.

The amino acids were acetylated on the Nα-amino group to mimic a peptide bond. Oxidation of

N-acetyl-tyrosine alone with HOCl generated N-acetyl-3-chlorotyrosine and N-acetyl-3,5-

dichlorotyrosine, as determined by HPLC with UV detection (Fig. 6A) and ESI-MS analysis

(data not shown). However, the yield of 3-chlorotyrosine was much lower (~ 10%, mol/mol,

oxidized amino acid/oxidant) than that obtained when HOCl oxidized the peptides YKxxY and

YxxKY (~ 55%). When a mixture of N-acetylated (Ac) amino acids identical to that found in

YxxxY (Ac-Tyr, Ac-Gly, Ac-Glu, Ac-Ala, Ac-Arg) was oxidized with HOCl, we observed the

same low product yields of N-acetyl-chlorotyrosine and N-acetyl-dichlorotyrosine that were

obtained by oxidizing YxxxY (Fig. 3C) or N-acetyl-tyrosine alone (Fig. 6B). When the mixture

of N-acetylated amino acids representing those in YxxKY and YKxxY (Ac-Tyr, Ac-Gly, NαAc-

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Lys, Ac-Arg, Ac-Ala and Ac-Glu) was exposed to HOCl, neither N-acetyl-3-chlorotyrosine nor

N-acetyl-3,5-dichlorotyrosine were detected (Fig. 6C). These observations strongly suggest that

lysine and tyrosine must be incorporated into a peptide for chlorination of the tyrosine to be

efficient.

Chloramines Mediate the Regiospecific Chlorination of Peptide-bound Tyrosine.

To determine whether the formation of lysine chloramine is a necessary step in the

regiospecific chlorination of peptides, we incubated YxxKY with an equimolar amount of HOCl

and immediately analyzed the reaction mixture. For these studies, we omitted methionine from

the mixture because alkylated thiols rapidly reduce chloramines (37,55). HPLC analysis revealed

a single peak of material (Fig. 7A, retention time 20 min) that was absent in the reaction

mixtures exposed to HOCl and then methionine (Fig. 3B). This product could be collected and

detected after it was again subjected to HPLC (Fig. 7B), indicating that it was reasonably stable

under our analytical conditions. Moreover, methionine converted it back to its precursor peptide

(Fig. 7C). These observations indicate that HOCl converts YxxKY into a stable intermediate that

can be converted back to the precursor peptide by methionine.

To investigate the nature of the reaction intermediate, we subjected the unmodified

peptide and the methionine-sensitive oxidation product to 1-D and 2-D NMR analyses (Fig. 8).

High-resolution 1H-NMR analysis of the unmodified peptide revealed the lysine ε NH2 and ε

CH2 proton resonances were at 7.5 ppm and 2.85 ppm, respectively (Fig. 7A, left panel). The

assignment of these protons was confirmed by TOCSY (Fig. 8A, right panel). The methionine-

sensitive oxidation product lacked the ε NH2 protons and exhibited a marked downfield shift of

the ε CH2 resonance (3.55 ppm; Fig. 8B, right and left panels). These observations strongly

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suggest that the ε NH2 group in the methionine-sensitive oxidation product had been modified by

the addition of a strongly electron-withdrawing group(s).

We used MALDI TOF-MS analysis to confirm the identity of the methionine-sensitive

intermediate derived from YxxKY. The HPLC-purified intermediate displayed three major ions

of m/z 628.3, m/z 962.2, and m/z 996.2, corresponding to the masses of the singly protonated

precursor peptide, the protonated peptide plus 34 amu, and the protonated peptide plus 68 amu

(Fig. 9A). The peaks of material that had gained 34 amu and 68 amu demonstrated the isotopic

clusters characteristic of monochlorinated and dichlorinated compounds, respectively. Post

source decay analysis of the protonated precursor peptide (m/z 628.3) showed an unmodified

lysine residue (K; Fig. 9B). In contrast post source decay analysis of the ions at m/z 962.2 and

m/z 996.2 demonstrated that the lysine residue had gained 34 amu (K + 34 amu; Fig. 9C) and 68

amu (K + 68 amu; Fig. 9D). These observations strongly suggest that HOCl converts the Nε

amino group of YxxKY into a monochlorinated and/or dichlorinated intermediate. The mixture

of chlorinated and non-chlorinated species detected by MALDI TOF-MS likely reflects a loss

and/or exchange of chlorine atoms during ionization.

To determine whether lysine Nε chloramine is an intermediate in the reaction pathway

that generates peptide-bound 3-chlorotyrosine, we oxidized the YxxKY peptide with an

equimolar amount of HOCl and used HPLC to monitor the progress curve of the reaction (Fig.

10). Immediately following the addition of oxidant, the reaction mixture contained

approximately equal amounts of the unmodified peptide and its chloramine. As the reaction time

increased, both the unmodified peptide and the lysine Nε-chloramine disappeared in concert with

the appearance of peptide-bound 3-chlorotyrosine. The stoichiometry of the reaction was ~ 1 mol

of product peptide per 0.5 mol chloramine intermediate. Collectively, these observations provide

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strong evidence that chloramines derived from the Nε-amino group of lysine are crucial

intermediates in the regioselective chlorination of peptide-bound tyrosine.

Lysine Residues in YxxK and YxxxK Motifs Promote the Chlorination of Tyrosine in Lipid-free

and Lipid-associated Peptides.

Molecular modeling indicates that tyrosine and lysine residues separated by 3 other

amino acids (YxxxK) reside on the same side of an α-helix (51,52), raising the possibility that

the phenolic group of tyrosine and the Nε amino group of lysine could come close enough to

interact chemically. Because apolipoprotein A-I contains only one tyrosine residue in a YxxxK

motif, it is difficult to assess whether lysine directs tyrosine chlorination by studying this

molecule. We therefore synthesized the model peptide AcPYSDELRQRLAARLE-NH2

(Yxxxxx), which duplicates helix 7 of apolipoprotein A-I. This helix contains tyrosine 166,

which was not chlorinated by HOCl under our experimental conditions (Table 1; peptide 161-

171) and does not reside in a YxxK/KxxY motif (Fig. 1). AcPYSDELRQRLAARLE-NH2 is

predicted to adopt an α-helical conformation ~ 40% of the time (56), suggesting that it should

approximate the secondary structure of the intact apolipoprotein.

After acetylating the peptide’s N-terminus to prevent the primary amine from forming a

chloramine, we exposed it to HOCl and quantified its chlorination by LC-ESI-MS. In parallel

studies, we attempted to mimic the lipid environment that envelops apolipoprotein A-I in HDL

by incubating the peptide with DPPC vesicles before exposing it to HOCl. When we exposed

Yxxxxx to HOCl in a physiological buffer for 30 min at 37 °C, we detected very little

chlorination of either the lipid-free or lipid-associated peptide (Fig. 11).

To explore the role of the YxxxK motif in promoting tyrosine chlorination, we prepared

variants of the Yxxxxx peptide. In this case, the lysine residue was separated from the tyrosine

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residue by 0, 1, 2, 3, or 4 amino acids (YKxxxx, YxKxxx, YxxKxx, YxxxKx, YxxxxK). After

we exposed these peptides to HOCl, LC-ESI-MS analysis revealed that YKxxxx, YxKxxx,

YxxKxx, and YxxxKx yielded progressively greater amounts of chlorinated peptide (Fig. 11).

The level of chlorination declined when the tyrosine and lysine residues were separated by 4

amino acids (YxxxxK). MS/MS analysis confirmed that tyrosine was always the site of

chlorination. When the peptides were lipid-associated, those containing YxxK or YxxxK motifs

were most susceptible to chlorination, perhaps because of increased α-helical structure. These

observations indicate that lysine residues permit the chlorination of tyrosine in synthetic peptides

that mimic a region of apolipoprotein A-I that is normally resistant to chlorination. Chlorination

of lipid-associated peptides was maximal when the lysine residue was 2 or 3 amino acid residues

away from the tyrosine residue.

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DISCUSSION

Our results provide strong evidence that lysine residues are involved in the regiospecific

formation of 3-chlorotyrosine when peptides or proteins containing tyrosine are exposed to

HOCl. They also indicate that chloramines are central to the reaction pathway. Thus, specific

amino acid sequences—YxxK and KxxY motifs—directed the regioselective chlorination of

synthetic peptides by HOCl in high yield. Moreover, the single major site of 3-chlorotyrosine

formation in apolipoprotein A-I of HDL resided in a YxxK motif. Remarkably, 50% of the

tyrosine residues in this YxxK motif were chlorinated when the mole ratio of HOCl to protein-

bound amino acid residues in HDL was 0.125, indicating that this region of the protein was

targeted selectively for oxidation. Collectively, these observations indicate that the proximity of

tyrosine to lysine, which is able to form chloramines, is likely to be one important factor that

determines the sites at which HOCl can chlorinate tyrosine residues in proteins.

We used synthetic peptides containing tyrosine to explore the mechanism by which

specific sequence motifs direct chlorination by HOCl. Because of our results with apolipoprotein

A-I and because primary amino groups form relatively stable chloramines, which can be potent

chlorinating reagents (37,38), we also included lysine residues in some of the peptides. When we

used the lysine-containing peptides YKxxY and YxxKY, the yield of 3-chlorotyrosine (relative

to HOCl) exceeded 65%. Moreover, a single tyrosine residue was preferentially chlorinated.

Significantly, a tyrosine residue located two amino acids away from a lysine residue was

chlorinated in much higher yield than a tyrosine immediately adjacent to a lysine. In contrast,

chlorination of tyrosine was not regiospecific in the peptide YxxxY, which lacked a lysine and

therefore a primary Nε amino group. Moreover, the product yield for chlorination of each

tyrosine residue in this peptide was <15%, demonstrating that YxxxY was a much poorer

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substrate for chlorination than the lysine-containing peptides. It is noteworthy that equal amounts

of 3-chlorotyrosine and 3,5-dichlorotyrosine formed in YxxxY (ClYxxxY, Cl2YxxxY, YxxxClY,

YxxxCl2Y) but that chlorination did not modify two different tyrosine residues in the same

molecule (ClYxxxClY). Also, we failed to detect tyrosine chlorination when we added HOCl to

a mixture of N-acetylated amino acids that mimicked the composition of the lysine-containing

YKxxY and YxxKY peptides. This suggests that lysine must be an integral part of a peptide to

direct the chlorination of tyrosine in high yield.

Primary amino groups react rapidly with HOCl to form chloramines (37,38,57).

Chloramines located on carbon atoms that lack a carboxylic acid group are long-lived and so

could be biologically relevant oxidizing intermediates (19,27,28,57). Indeed, our HPLC

procedure isolated a relatively stable oxidation product from the reaction of HOCl with peptide

YxxKY that was converted back to the precursor peptide by methionine. MALDI TOF-MS

analysis revealed the presence of three species in the methionine-sensitive oxidation product—

precursor peptide, monochloramine, and dichloramine. This suggests that molecular

decomposition and rearrangement during ionization by MALDI allow chlorine to be exchanged

among different species during the analysis (37,58). NMR analysis confirmed that the oxidized

intermediate was chlorinated on the Nε amino group of its lysine residue. Analysis of the

progress curve of the reaction of YxxKY with HOCl revealed that the initial step in the reaction

generated a high yield of peptide-bound chloramine, which disappeared as products containing

chlorinated tyrosine appeared. These observations provide strong evidence that the reaction

pathway for tyrosine chlorination involves the rapid initial formation of a chloramine on the ε

amino group of the lysine residue followed by a slower reaction in which the chloramine attacks

the aromatic ring of tyrosine. It is noteworthy that the stoichiometry of peptide chlorination

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implicates a dichloramine as the intermediate in the reaction pathway and that our NMR and MS

analyses detected dichloramines in the methionine-sensitive oxidation product. These

observations support the proposal that a dichloramine is the chlorinating intermediate in the

reaction pathway (58). Because the rate constant for the reaction of HOCl with lysine is

relatively high (K2 = 5 × 103 M-1s-1), the ε amino group of lysine (and the free amino group at the

N-terminus of proteins) together with the thiol residues of cysteine and methionine (K2 = 3.0

×107 M-1s-1 and 3.8 ×107 M-1s-1, respectively) might be initial primary targets when HOCl

oxidizes proteins (18,59).

It is noteworthy that 50% of the Tyr 192 residues in apolipoprotein A-I were chlorinated

when the mole ratio of HOCl to protein-bound amino acid residues of HDL was 1 to 8. The

reason why Tyr 192 is so much more amenable to chlorination than the two other tyrosine

residues (Tyr 115 and Tyr 236) that also reside in a YxxK domain might relate to differences in

spatial orientation, solvent accessibility, and the local amino acid environment. Importantly Tyr

115 lies in close proximity to Met 112. The alkylated thiol of methionine is extremely reactive

with HOCl, and protein-bound methionine residues have been proposed to act as local

antioxidants (60). Thus, Met 112 might scavenge HOCl in this region of apolipoprotein A-I.

Indeed, Met 112 is one of the primary targets for oxidation by HOCl in apolipoprotein A-I (34).

Another key factor controlling the reactivity of protein-bound amino acids is local

secondary structure. Apolipoprotein A-I contains 10 antiparallel amphipathic α-helices that

promote lipid association and are critical for removing cholesterol from cells (51,52). The helical

wheel representation of amphipathic helices predicts that tyrosine and lysine residues in the

YxxK (and KxxY) motif will lie next to each other on the same face of the α-helix (51). Both the

helical wheel representation (Fig. 1B) and the crystal structure of lipid-free apolipoprotein A-I

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(61) indicate that LAEYHAK, which includes the single tyrosine residue in apolipoprotein A-I

that was chlorinated in high yield (50%), contain the tyrosine and lysine residues in close

proximity. Importantly, the α-carbons of the two amino acid residues are separated by ~ 4.5 Å,

which suggests that the phenolic ring and primary amino groups of the tyrosine and lysine

residues in the YxxK/KxxY motif can interact chemically. The crystal structure of lipid-free

apolipoprotein A-I reveals that Tyr 192 lies in the middle of an amphipathic α-helix whereas Tyr

236 is located at the end of an amphipathic α-helix (61), where a less orderly structure might

hamper the interaction of the lysine and tyrosine residues and the production of 3-chlorotyrosine.

Molecular modeling indicates that tyrosine and lysine residues that are separated by 3

other amino acids also lie next to one another on the same face of an α-helix. Because their α-

carbons are separated by ~ 6 Å, their side chains might be able to interact chemically in

YxxxK/KxxxY motifs. However, only one tyrosine in apolipoprotein A-I resides in such a motif.

Therefore, it is difficult to use the intact molecule to explore the role of YxxxK/KxxxY in

tyrosine chlorination. Instead, we synthesized the model peptide AcPYSDELRQRLAARLE-NH2

(Yxxxxx) and exposed it to HOCl. In parallel studies, we determined whether lipid association

makes this peptide more amenable to chlorination. We selected this particular sequence because

it mimics a region of apolipoprotein A-I that contains a tyrosine residue that is resistant to

chlorination. When we exposed the peptide to HOCl, it was chlorinated in low yield.

When we introduced a lysine residue into the peptide at a position that was 2 or 3 amino acids

away from the tyrosine residue (YxxKxx and YxxxKx), the lipid-free and lipid-associated

peptides were chlorinated in high yield. These observations suggest that the YxxxK/KxxxY

motif might also promote the chlorination of tyrosine residues in α-helices. We also observed a

significant level of chlorination when the lysine and tyrosine residues were separated by 4 amino

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acids (YxxxxK) and therefore would be expected to lie on opposite sides of an α-helix. It is

possible that chlorination was feasible because the side chains were long enough to permit the

phenolic ring of tyrosine and the primary amino groups of lysine to interact. Alternatively, a

conformational change might have brought the two groups together—synthetic peptides are very

flexible, and the ones we studied are predicted to reside in an α-helix only ~ 40% of the time

(56). In future studies, it will be important to determine whether lysine residues promote the

chlorination of tyrosine residues when the two are separated by 3 or 4 amino acids in a well-

defined α-helical structure.

Primary sequence and secondary structure alone do not determine the local structure of a

protein, however, and many other factors could correctly orient the Nε-chloramine of lysine (or

an Nα chloramine of the N terminus of a protein) to the phenolic ring of tyrosine. For example,

the tertiary structure might juxtapose residues that lie far apart in the primary sequence or

secondary structure. It is even possible that lysine residues in one protein could facilitate the

chlorination of tyrosine residues in a different but closely associated protein. Features of tertiary

structure might also explain why Tyr 29 of apolipoprotein A-I was chlorinated even though it

does not reside in a YxxK motif and why Tyr 115, which does lie in a YxxK motif, was not

chlorinated. For example, Tyr 29 lies in a globular region of apolipoprotein A-I and might

interact with a primary amino group in another region of the protein. In addition, there might be

a difference in solvent accessibility or an association of the side chains of Tyr 115 or its adjacent

lysine with lipid.

Tyrosine192 and 236 are located in amphipathic helices 8 and 10 of the C-terminal of

apolipoprotein A-I; these helices are essential for interactions with lipid (52,62). It will be

interesting to determine whether chlorination of these tyrosines alters the biochemical or

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biophysical properties of apolipoprotein A-I and its ability to mobilize cholesterol from cells. It

also will be important to determine whether chlorination of specific protein residues by

phagocytes is important for killing bacteria or damaging host tissue even though only ~ 1 in

1,000-5,000 tyrosine residues is modified (13,14,63,64). Presumably, loss of specific tyrosine

residues that are critical to protein integrity or function could be deleterious to bacteria or host

tissues despite the small absolute level of tyrosine chlorination.

Based on our findings, we propose that the primary ε amino group of lysine facilitates the

regioselective chlorination of tyrosine residues in proteins by a pathway involving a chloramine

intermediate. Modeling and structural studies indicate that tyrosine and lysine residues separated

by two amino acids are adjacent on the same face of an α-helix, suggesting that the YxxK motif

could direct protein chlorination if it resided in a α-helix. Consistent with this proposal, we

found that a single tyrosine residue in the 8th amphipathic α-helix of apolipoprotein A-I was the

major site of chlorination by HOCl and that this tyrosine resided in the YxxK motif. Moreover,

additional structural features might also juxtapose primary amino groups and tyrosine residues in

proteins to permit site-specific chlorination of tyrosine residues. It will be of great interest to

determine whether regiospecific chlorination can affect protein function and to investigate the

possible role of this reaction mechanism in the chlorination of proteins in vivo.

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ACKNOWLEDGMENTS

This work was supported by grants AG021191 and HL64344 from the National Institutes of

Health.

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FIGURE LEGENDS

Figure 1. Apolipoprotein A-I. (A) Primary sequence of apolipoprotein A-I and its 10 proposed

amphipathic helices (65).(B) Helical wheel representation of tryptic fragment 189-195

LAEYHAK in apolipoprotein A-I. Note that the tyrosine and lysine residue in the YxxK motif

are located on the same face of the α-helix. Modeling studies and the crystal structure of

apolipoprotein A-I indicate that the α-carbons of tyrosine and lysine are located ~ 4.5 Å from

each other in the motif (51,61).

Figure 2. MS/MS identification of the major site of tyrosine chlorination in apolipoprotein

A-I exposed to HOCl. MS/MS analysis of [LAEYHAK + H]+ (m/z 831.4) and [LAEClYHAK +

H] + (m/z 865.4) in HDL oxidized with HOCl. HDL was exposed to HOCl (80:1, mol/mol,

oxidant:HDL particle) for 120 min at 37°C in PBS (pH 7.4). After terminating the reaction with

L-methionine, HDL proteins were digested with trypsin, and the tryptic digest peptides were

subjected to analysis by LC-ESI-MS/MS.

Figure 3. HPLC analysis of YKxxY, YxxKY, and YxxxY exposed to HOCl. Peptide (100

µM) was incubated for 30 min at 37oC in PBS (A) or PBS supplemented with 100 µM HOCl

(B). Reactions were initiated by adding oxidant and terminated by adding L-methionine. The

reaction mixture was analyzed by reverse-phase HPLC and UV detection at 215 nm.

Figure 4. MS/MS analysis of YxxKY peptide (AcGEYARKY) and its reaction products.

The peptide was oxidized with HOCl as described in the legend to Fig. 3 and analyzed by LC-

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ESI-MS/MS. A, MS/MS spectrum of precursor YxxKY. B, MS/MS spectrum of product Peak 1

(Fig. 3B). C, MS/MS spectrum of product Peak 2 (Fig. 3B).

Figure 5. Reaction requirements for tyrosine chlorination by HOCl. YxxKY (100 µM) was

oxidized by incubating the peptide with 100 µM HOCl in PBS for 60 min at 37 °C. At the end of

the incubation, the reaction was terminated with methionine, and the reaction products were

quantified using reverse-phase HPCL with monitoring of products at 215 nm. Where indicated,

the time of the reaction (A), the concentration of HOCl (B), or the pH of the reaction mixture (C)

were varied, or the indicated concentration of antioxidant (D) was included in the reaction

mixture prior to the addition of HOCl.

Figure 6. HPLC analysis of N-acetyl-tyrosine exposed to HOCl. N-acetyl-tyrosine (Ac-Tyr)

alone or Nα-acetylated amino acids (Ac-amino acid) mimicking the amino acid composition of

the YxxKY and YKxxY or YxxxY peptides were incubated with 100 µM HOCl in PBS for 60

min at 37oC. The reaction was terminated with L-methionine and the mixture was subjected to

HPLC analysis on a reverse-phase column with monitoring of products at 280 nm. (A) Ac-Tyr

alone (200 µM). (B) Amino acid composition of YxxxY (200 µM Ac-Tyr, 200 µM Ac-Glu, 100

µM Ac-Ala, 100 µM Ac-Gly, 100 µM Ac-Arg). (C) Amino acid composition of YKxxY and

YxxKY (200 µM Ac-Tyr, 100 µM Ac-Ala, 100 µM Ac-Glu, 100 µM Ac-Gly, 100 µM Ac-Lys,

100 µM Arg).

Figure 7. HPLC analysis of the methionine-sensitive reaction product formed in YxxKY

exposed to HOCl. (A) YxxKY peptide was oxidized with HOCl as described in the legend to

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Fig. 3 without the addition of methionine, and immediately subjected to HPLC analysis on a

reverse-phase column. The oxidized peptide eluting at 20 min was collected and reanalyzed by

reverse phase HPLC without further treatment (B) or was reduced with excess methionine and

then reanalyzed (C).

Figure 8. High-resolution 1H-NMR (A) and TOCSY (B) analysis of the methionine-sensitive

oxidation product formed in YxxKY exposed to HOCl. YxxKY peptide was oxidized with

HOCl as described in the legend to Fig.3, and its methionine-sensitive oxidation product was

isolated by HPLC on a reverse-phase column as described in Methods. Spectra were obtained on

precursor peptide or oxidized peptide solubilized in PBS (prepared with 10% D2O and 90% H2O,

pH 7.4).

Figure 9. MALDI TOF-MS and MS/MS analysis of the methionine-sensitive product

formed in YxxKY exposed to HOCl. YxxKY peptide was oxidized with HOCl as described in

the legend to Fig. 3, and its methionine-sensitive oxidation product was isolated by HPLC as

described in Methods. (A) The oxidation product was immediately analyzed by MALDI TOF-

MS using α-cyano-4-hydroxycinnamic acid as the matrix. MS/MS spectrum of (B) protonated

precursor peptide (ion of m/z 928.3, M + H), (C) protonated and chlorinated peptide (ion of m/z

962.2, M + H + 34 amu), (D) protonated and dichlorinated peptide (ion of m/z 996.2, M + H + 68

amu). Note that MS/MS analysis demonstrates the addition of 34 and 68 amu to the lysine

residue in the peptide.

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Figure 10. Progress curve of YxxKY oxidation by HOCl. YxxKY peptide was oxidized with

HOCl as described in the legend to Fig. 3 without the addition of methionine. The reaction

mixture was analyzed by reverse-phase HPLC and UV detection at 215 nm.

Figure 11. MS analysis of lipid-free and lipid-associated Yxxxxx, YxxxxK, YxxxKx,

YxxKxx, YxKxxx and YKxxxx exposed to HOCl. Peptide AcPYSDELRQRLAARLE-NH2

(Yxxxxx) mimics a region of apolipoprotein A-I that contains a tyrosine residue that is resistant

to chlorination (Table 1; peptide 161-171). Variants of the Yxxxxx peptide contained lysine

residues that were separated from the tyrosine residue by 0, 1, 2, 3, or 4 amino acids (YKxxxx,

YxKxxx, YxxKxx, YxxxKx, YxxxxK). Lipid-associated peptides were prepared by incubation

with DPPC vesicles (peptide:DPPC, 1:20, mol/mol). Peptides (25 µM) were exposed to HOCl

for 30 min at 37 oC in PBS. Reactions were initiated by adding oxidant and terminated by adding

L-methionine. The mol/mol ratios of peptide/oxidant were 0.5 and 1 for lipid-free and lipid-

associated peptides, respectively. The reaction mixture was analyzed by LC-ESI-MS. Peak areas

of the reconstructed ion chromatograms of ions at m/z corresponding to the native peptides [M +

2H]2+ and the chlorinated products [M + 34 + H]2+ were used for quantification. Results

represent the mean of two independent experiments.

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Table 1. LC-ESI-MS detection of peptides containing 3-chlorotyrosine in a tryptic digest of

HDL exposed to HOCl.

Residue Sequence Precursor, m/z Product, m/z

[M + H]+ [M + 2H]2+ [M + 34 + H]+ [M + 34 + 2H]2+ %Yield1

13-23 DLATVYVDVLK 1235.7 618.3 ud2 ud -

28-40 DYVSQFEGSALGK 1400.7 700.8 1434.7 717.8 2

97-106 VQPYLDDFQK 1252.6 626.8 ud ud -

108-116 WQEEMELYR* 1283.6 642.3 ud ud -

108-116 WQEEM(O)ELYR* 1299.6 650.3 ud ud -

161-171 THLAPYSDELR 1301.6 651.3 ud ud -

189-195 LAEYHAK 831.4 416.2 865.4 433.2 52

227-238 VSFLSALEEYTK 1386.7 693.9 1420.7 710.9 6

HDL was exposed to HOCl (80:1, mol/mol, oxidant:HDL particle) for 120 min at 37°C in PBS

(pH 7.4). A tryptic digest of HDL proteins was analyzed by LC-ESI-MS and MS/MS.

Chlorinated peptides were detected and quantified using reconstructed ion chromatograms of

precursor and product peptides. Peptide sequences were confirmed using MS/MS. Results are

representative of those found in three independent experiments.

1 (mol product peptide/mol precursor peptide) x 100

2 ud, undetectable

* HOCl oxidizes methionine (M) to methionine sulfoxide (34).

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37

Table 2. Formation of 3-chlorotyrosine and 3,5-dichlorotyrosine in YKxxY, YxxKY, and

YxxxY exposed to HOCl.

Motif Peptide Product Sequence % Product Yield1

YKxxY AcGYKRAYE Peak 1 AcGClYKRAYE 11

Peak 2 AcGYKRAClYE 60

YxxKY AcGEYARKY Peak 1 AcGEClYARKY 49

Peak 2 AcGEYARKClY 19

YxxxY AcGEYAREY Peak 1 AcGEClYAREY 10

Peak 2 AcGEYAREClY 11

Peak 3 AcGECl2YAREY 8

Peak 4 AcGEYARECl2Y 6

Peptides YKxxY, YxxKY, or YxxxY (100 µM) were incubated for 30 min at 37oC in PBS

supplemented with 100 µM HOCl. Reactions were initiated by adding oxidant and terminated by

adding methionine. Product yield was determined by HPLC with monitoring of A215. Peptides

were sequenced using LC-ESI-MS/MS.

1 (mol product peptide/mol HOCl) x 100


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0 25

Yxxxxx

YxxxxK

YxxxKx

YxxKxx

YxKxxx

YKxxxxM

otif

Chlorinated Product [%]

Lipid-freeLipid-associated

Fig. 11


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Constanze Bergt, Xiaoyun Fu, Nabiha P. Huq, Jeff L. Kao and Jay W. HeineckeA-I when hypochlorous acid oxidizes HDL

Lysine residues direct the chlorination of tyrosines in YxxK motifs of apolipoprotein

published online December 3, 2003J. Biol. Chem.

10.1074/jbc.M309046200Access the most updated version of this article at doi:

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