(Received for publication, August 9, 1996, and in revised form, April 23, 1997)
From the Department of Medicine, ¶ Department of
Chemistry, and
Department of Molecular Biology and Pharmacology,
Washington University, St. Louis, Missouri 63110
Activated human phagocytes employ the
myeloperoxidase-H2O2-Cl
system to convert L-tyrosine to
p-hydroxyphenylacetaldehyde (pHA). We have explored the
possibility that pHA covalently reacts with proteins to form Schiff
base adducts, which may play a role in modifying targets at sites of
inflammation. Because Schiff bases are labile to acid hydrolysis, prior
to analysis the adducts were rendered stable by reduction with
NaCNBH3. Purified pHA reacted with
N
-acetyllysine, an analog of protein
lysine residues. The reduced reaction product was identified as
N
-acetyl-N
-(2-(p-hydroxyphenyl)ethyl)lysine
by 1H NMR spectroscopy and mass spectrometry. The compound
N
-(2-(p-hydroxyphenyl)ethyl)lysine
(pHA-lysine) was likewise identified in acid hydrolysates of bovine
serum albumin (BSA) that were first exposed to myeloperoxidase,
H2O2, L-tyrosine, and
Cl
and then reduced with NaCNBH3. Other
halides (F
, Br
, I
) and the
pseudohalide SCN
could not replace Cl
as a
substrate in the
myeloperoxidase-H2O2-L-tyrosine
system. In the absence of the enzymatic system, pHA-lysine was detected in reduced reaction mixtures of BSA, L-tyrosine, and
reagent HOCl. In contrast, pHA-lysine was undetectable when BSA was
incubated with L-tyrosine and HOBr, peroxynitrite, hydroxyl
radical, or a variety of other peroxidases, indicating that the
aldehyde-protein adduct was selectively produced by HOCl. Human
neutrophils activated in the presence of tyrosine also modified BSA
lysine residues. pHA-lysine formation required L-tyrosine
and cell activation; it was inhibited by peroxidase inhibitors and
catalase, implicating myeloperoxidase and H2O2
in the reaction pathway. pHA-lysine was detected in inflamed human
tissues that were reduced, hydrolyzed, and then analyzed by mass
spectrometry, indicating that the reaction of pHA with proteins may be
of physiological importance. These observations raise the possibility
that the identification of pHA-lysine in tissues will pinpoint targets
where phagocytes inflict oxidative damage in vivo.
Hypercholesterolemia and hyperglycemia are two important risk factors for atherosclerotic vascular disease (1, 2). A wealth of evidence indicates that low density lipoprotein (LDL),1 the major carrier of blood cholesterol, must be oxidatively modified to trigger the pathological events of atherosclerosis (3-7). Aldehydes derived from oxidized LDL lipids play a critical role in mediating many of these events. Diabetic vascular disease may similarly result from covalent modification of vascular wall and plasma proteins by glucose, which in its open chain form possesses an aldehyde moiety (8-10). Thus, reactive aldehydes derived from oxidized lipids and reducing sugars may be of central importance in atherogenesis.
Despite widespread interest in the potential importance of reactive
aldehydes in the pathogenesis of disease (3-12), little is known
regarding the nature of the covalent adducts formed between aldehydes
and proteins in vivo. Most studies have relied on
immunohistochemical methods to detect aldehyde-modified proteins
(13-16), and the exact structure(s) of the cognate epitope(s) is
unknown. Indeed, the only well characterized protein-bound aldehydes
in vivo are those generated by the reaction of reducing
sugars with amino groups, such as glucoselysine (17), fructoselysine
(17), pentosidine (18), and
N-(carboxymethyl)lysine (19).
One potential pathway for vascular injury involves myeloperoxidase, a heme protein secreted by phagocytes (7, 20-23). Myeloperoxidase uses H2O2 generated by phagocytes to produce diffusible cytotoxic oxidants (20, 21, 24-29). Active myeloperoxidase is a component of human atherosclerotic lesions, and the enzyme co-localizes with macrophages in transitional lesions (22). Immunohistochemical studies suggest that proteins modified by myeloperoxidase are present in atherosclerotic tissue (30). We have recently shown that 3-chlorotyrosine, a specific product of myeloperoxidase, is present at elevated levels in human atherosclerotic aorta and in LDL recovered from atherosclerotic aortic intima (23). Thus, myeloperoxidase may contribute to lipoprotein oxidation in the artery wall.
The best characterized product of myeloperoxidase is hypochlorous acid
(HOCl), which is generated from chloride ion (Cl) in a
two-electron oxidation reaction (Equation 1; Refs. 21, 24, and 25).
![]() |
(Eq. 1) |
We have recently demonstrated that activated phagocytes also employ the
myeloperoxidase-H2O2-Cl system to
convert L-tyrosine to the amphipathic aldehyde,
p-hydroxyphenylacetaldehyde (pHA). At physiological
concentrations of L-tyrosine and Cl
, pHA is
the major product of phagocyte activation (28). In the current studies
we examine the ability of pHA to covalently modify proteins and
identify the reduced Schiff base between the aldehyde and the
N
-amino moiety of lysine residues.
Characterization of this reaction demonstrated that the reduced
Schiff base,
N
-(2-(p-hydroxyphenyl)ethyl)lysine
(pHA-lysine), is acid-stable and serves as a specific marker of
protein modification by myeloperoxidase. pHA-lysine was detected by
mass spectrometry in inflamed human tissues, raising the possibility
that reactive aldehydes generated by myeloperoxidase covalently modify
proteins in vivo.
Materials
D2O, L-[13C6]lysine, L-[13C6]tyrosine, and HBr in n-propyl alcohol were purchased from Cambridge Isotopes, Inc. L-[14C]Tyrosine was purchased from NEN Life Science Products. HPLC solvents were purchased from Baxter. Fatty acid-free bovine serum albumin (BSA), and crystalline catalase (from bovine liver, thymol-free) were purchased from Boehringer Mannheim. Pentafluoropropionic acid anhydride and heptafluorobutyric acid anhydride were obtained from Pierce. Sodium phosphate, ethyl acetate, H2O2, and NaOCl were purchased from Fisher. Peroxynitrite was a generous gift from Monsanto Corp. (St. Louis, MO). All other materials were purchased from Sigma except where indicated.
Methods
General ProceduresMyeloperoxidase (donor:hydrogen
peroxide, oxidoreductase, EC 1.11.1.7) was isolated
(A430/A280 ratio of 0.6)
and stored as described previously (26, 42). Enzyme concentration was determined spectrophotometrically (430 = 170 mM
1 cm
1; Ref. 43). Human
neutrophils were isolated by buoyant density centrifugation (28). Cell
experiments were performed in medium A ((Hanks' balanced salt
solution, pH 7.2 (magnesium-, calcium-, phenol red-, and
bicarbonate-free); Life Technologies, Inc.) supplemented with 100 µM diethylenetriaminepentaacetic acid (DTPA)). Ionomycin and phorbol myristate acetate were prepared in ethanol and dimethyl sulfoxide, respectively; the final content of each vehicle in cell
experiments was
0.2% (v/v). NaOCl concentration was determined spectrophotometrically (
292 = 350 M
1 cm
1; Ref. 44). Buffers were
Chelex-100 (Bio-Rad) treated and supplemented with 100 µM
DTPA to remove redox-active metals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described by Laemmli (45). Protein content was measured by the method of Lowry
et al. (46) with BSA as the standard. Amino acid analyses were performed at the Washington University School of Medicine Protein
Chemistry Core Laboratory. Pre-column derivatization of amino acid
hydrolysates with 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate was followed by high performance liquid chromatography (HPLC)
with ultraviolet detection (47, 48). HOBr was prepared and quantified
the day of use as described (49).
NaOCl (1:1, mol/mol, NaOCl/tyrosine) was
added dropwise with constant mixing to 2 mM
L-tyrosine dissolved in ice-cold 20 mM sodium
phosphate, pH 7.0. The solution was then warmed to 37 °C for 60 min
and immediately used for experiments. Preparations were analyzed by
reverse phase HPLC prior to use and routinely were 95% pure
(28).
HPLC analysis of pHA
was performed utilizing a C18 column (Beckman µPoracil, 5-µm resin,
4.6 × 260 mm) equilibrated with solvent A (5% methanol, 0.1%
trifluoroacetic acid, pH 2.5). Products were monitored by absorbance
(A276) and eluted at a flow rate of 1 ml/min
with a nonlinear gradient generated with solvent B (90% methanol,
0.1% trifluoroacetic acid, pH 2.5) as follows: 0-35% solvent B over
10 min; isocratic elution at 35% solvent B for 20 min; 35-100%
solvent B over 10 min.
N-Acetyl-pHA-lysine was isolated by
reverse phase HPLC with the following gradient: 0-10% solvent B over
5 min; isocratic elution at 10% solvent B for 20 min; 10-100%
solvent B over 10 min. Under these conditions,
N
-acetyl-pHA-lysine is base-line
resolved from pHA-lysine, free pHA, p-hydroxyphenylethanol
and L-tyrosine.
Production of the protein-bound Schiff base was routinely quantified by reducing the products, subjecting them to acid hydrolysis, and then monitoring for the presence of the stable marker, pHA-lysine (see below). Reactions were carried out under the conditions indicated in the figure legends. Schiff base adducts were reduced by addition of 10 mM NaCNBH3 and incubation for the indicated times at 37 °C. Where indicated, 100 mM ammonium acetate, pH 7.2, was included in the reaction mixture during reduction to scavenge free pHA.
Isolation of Membrane-associated and Soluble ProteinsWhole
human blood anticoagulated with EDTA (5 mM) was diluted
with phosphate-buffered saline (PBS, 10 mM sodium
phosphate, pH 7.4; Sigma) supplemented with 0.1 mM DTPA and
then modified with myeloperoxidase as indicated in the legend to Fig.
6. Following reduction with 10 mM NaCNBH3 in
the presence of 100 mM ammonium acetate, the cells were
pelleted by centrifugation at 5000 × g for 15 min at
4 °C, washed twice with PBS supplemented with 0.1 mM
DTPA, and then homogenized on ice with a tight fitting Potter-Elvehjem homogenizer. The cell lysate was fractionated into soluble and membrane-associated fractions by centrifugation at 100,000 × g for 1 h at 4 °C, delipidated with two sequential
extractions with water-washed diethyl ether (1:1; v/v), and
L-[13C6]tyrosine (300 nmol; a
marker of protein content) and
N-acetyl-[13C6]pHA-lysine
(20 pmol) were added as internal standards. Following HBr hydrolysis
and solid phase extraction on a C18 column, the content of pHA-lysine
was determined by stable isotope dilution GC/MS as described below.
Preparation of N
L-[13C6]Tyrosine
(2 mM) in 20 mM sodium phosphate, pH 7.0, was
first converted to [13C6]pHA by addition of
NaOCl as described above.
N-Acetyllysine (4 mM) was
then added, the mixture was incubated at 37 °C for 4 h, and the
Schiff base was reduced by overnight incubation with 10 mM
NaCNBH3.
N
-Acetyl-[13C6]pHA-lysine
was isolated by reverse phase HPLC as described above.
Protein (~250 µg) solutions were dried under vacuum in 2-ml glass reaction vials. Following the addition of 13C-labeled internal standards and 0.5 ml of 6 N HBr supplemented with phenol (1%, w/v), samples were alternately evacuated and purged five times with argon gas. The argon-covered solution was hydrolyzed at 120 °C for 24 h. The protein hydrolysate was diluted to 2.0 ml with 0.1% trifluoroacetic acid and applied to a C18 column (Supelclean LC-18, 3 ml, Supelco Co.) equilibrated with 0.1% trifluoroacetic acid. The column was washed with 2 ml of 0.1% trifluoroacetic acid, and pHA-lysine was recovered with 2 ml of 20% methanol in 0.1% trifluoroacetic acid.
Samples were evaporated to dryness under either anhydrous N2 or vacuum prior to derivatization. n-Propyl esters were prepared by the addition of 200 µl of 3.5 M HBr in n-propyl alcohol followed by heating at 65 °C for 30 min. Propylated products were dried under N2, and pentafluoropropionyl (PFP) derivatives were generated by addition of 50 µl of pentafluoropropionic acid anhydride (Pierce) in ethyl acetate (1:3; v/v) and heating for 1 h at 65 °C. Heptafluorobutyryl derivatives were prepared by adding 50 µl of heptafluorobutyric acid anhydride/ethyl acetate (1:4, v/v) and heating for 30 min at 65 °C.
Tissue Collection and ProcessingResidual material from human tissues obtained for clinical indications was stored on ice and processed within 30 min of collection. Tissue samples were mixed 1:1 (v/v) with reduction buffer (10 mM NaCNBH3, 100 mM ammonium acetate, 1 mM NaN3, 10 µg/ml catalase, 50 mM sodium phosphate, pH 7.4) and incubated at 37 °C for 1 h. Preliminary experiments confirmed that no additional pHA-lysine was generated under these conditions when samples were supplemented with 20 nM myeloperoxidase and 100 µM H2O2. All procedures involving human tissue were approved by the Washington University Human Studies Committee.
Reduced tissue samples were delipidated employing a single phase extraction mixture of H2O/methanol/water-washed diethyl ether (1:3:7, v/v/v) as described previously (23). The protein pellet was washed twice with 10% trichloroacetic acid at 0 °C under a fume hood and then subjected to acid hydrolysis, solid-phase extraction on a C18 minicolumn, and derivatization for GC/MS analysis.
Mass Spectrometric AnalysisAmino acids were quantified using stable isotope dilution GC/MS in the negative ion chemical ionization mode. Samples were analyzed with a Hewlett-Packard 5890 gas chromatograph interfaced with a Hewlett-Packard 5988A mass spectrometer with extended mass range. Injector, transfer line, and source temperatures were set at 250, 250, and 150 °C, respectively. High resolution mass spectrometry was performed with a VG-ZAB SE double-focusing mass spectrometer. The resolution was set at 10,000 with perfluorokerosine as the reference.
Amino acids were quantified as their n-propyl, per-PFP
derivatives using selected ion monitoring. pHA-lysine was monitored using the base peak at mass-to-charge ratio (m/z) 726 (M HF)
, another major fragment ion at
m/z 706 (M
2HF)
, and their
corresponding isotopically labeled internal standard ions at
m/z 732 and 712. L-Tyrosine was monitored using
the base peak at m/z 367 (M
PFP)
,
another major fragment ion at m/z 495 (M
HF)
, and their corresponding isotopically labeled
internal standard ions at m/z 373 and 501. L-Lysine was monitored using the base peak at
m/z 460 (M
HF)
, another major fragment
ion at m/z 440 (M
2HF)
, and their
corresponding isotopically labeled internal standard ions at
m/z 466 and 446.
Quantification was based on an external calibration curve constructed with each authentic compound and its isotopically labeled internal standard. To ensure that no interfering ions co-eluted with the analyte, the ratio of ion currents of two characteristic ions of each compound and its internal standard were routinely monitored. All amino acids were base-line separated and co-eluted with 13C-labeled internal standards. The limit of detection (signal/noise >10) was <1 pmol for all compounds.
NMR StudiesAnalyses were performed at 25 °C in
D2O/H2O (1:9, v/v) with a Varian Unity-Plus 500 spectrometer (499.843 MHz for 1H) equipped with a Nalorac
indirect detection probe. 1H chemical shifts were
referenced to external sodium
3-(trimethylsilyl)-propionate-2,2,3,3,d4 in
D2O. The amide proton of
N-acetyl-pHA-lysine is not visible at
neutral pH due to rapid exchange. To facilitate structural assignment
N
-acetyl-pHA-lysine was acidified with
DCl (Cambridge Isotopes Inc.) until exchange of the amide proton was
inhibited. For proton and total correlation spectroscopy (TOCSY)
experiments, the intense water signal was attenuated by transmitter
pre-irradiation. The proton NMR spectrum of
N
-acetyl-pHA-lysine was recorded at
25 °C from 64 transients under the following conditions:
pre-acquisition delay = 2 s, acquisition time = 1.89 s (37,760 complex data points), pulse width = 7 µs (80° flip angle), and spectral width = 10,000 Hz. The free
induction decay was processed with a line broadening apodization of 1.0 Hz. For TOCSY eight transients were collected for each of 200 t1 domain increments. A 10-ms mixing period was
employed resulting in cross peaks for only the strongest scalar
couplings (geminal and vicinal). The acquisition time was 0.256 s in
t2 (2048 complex data points) and 0.050 s in
t1 (200 data points). TOCSY data was processed
by the hypercomplex method with Gaussian weighting in both
t1 and t2 dimensions.
Digital signal processing was employed to suppress artifacts arising
from the intense water resonance.
We initially studied the reaction
of pHA with N-acetyllysine, a model
compound for free amino groups on proteins, to facilitate the isolation
and characterization of products. Reactions were carried out at pH 7.4 and 37 °C in a phosphate-buffered physiological salt solution.
Reverse phase HPLC analysis of the complete reaction mixture after
reduction with NaCNBH3 revealed a single major product (Fig. 1; retention time 14.0 min). Formation of the
compound required the presence of both pHA and
N
-acetyllysine (Fig. 1). In the
absence of reduction, a compound with an identical retention time was
observed; however, this product slowly decomposed under the acidic
conditions employed for HPLC. The acid lability of the nonreduced
compound suggested initial formation of a Schiff base between pHA and
the N
-amino group of
N
-acetyllysine.
Reduction of the imine with NaCNBH3 would then yield the
acid-stable adduct (Scheme I). This hypothesis is strongly supported by
structural analysis of the reduced form of the product (see below)
which demonstrated that it was the
N
-acetyl derivative of
N
-(2-(p-hydroxyphenyl)ethyl)lysine
(pHA-lysine).
To determine the structure of the compound, the reaction mixture was
reduced with NaCNBH3, and HPLC-purified material was derivatized and subjected to GC/MS analysis. A single major peak of
material was apparent in the total ion chromatogram (Fig.
2, left panel). The negative ion chemical
ionization mass spectrum of the n-propyl ester, per-PFP
derivative of the compound (Fig. 2, right panel) was
consistent with the proposed structure of pHA-lysine (Fig. 2,
inset). The compound demonstrated a low abundance ion on
selected ion monitoring at m/z 746, the anticipated
m/z of the molecular ion (M), that co-eluted with
the major ions seen in the mass spectrum of the compound (data not
shown). GC/MS analysis of the n-propyl ester,
per-heptafluorobutyryl derivative of the compound also exhibited a mass
spectrum consistent with the proposed structure; ions were observed at
m/z 896 (M
), m/z 876 (M
HF)
, m/z 856 (M
2HF)
,
m/z 832 (M
HF
CO2)
, m/z 698 (M
CF3CF2CF2CHO)
and
m/z 579 (M
CF3CF2COO-C6H4-CH2CH2)
.
NMR spectroscopy was performed to confirm the structure of the compound
(Fig. 3). Both the chemical shifts and integrated peak
areas of the 1H NMR spectrum were consistent with the
structure of N-acetyl-pHA-lysine (Fig.
3, inset). To verify the proton assignments, TOCSY was
employed to identify scalar couplings between resonances (Fig.
4). The sequential order of 2,3-bond H-H couplings
observed in the TOCSY experiment established the structure of the
compound as N
-acetyl-pHA-lysine.
Collectively, these studies indicate that pHA forms a Schiff base with
the free amino group of N
-acetyllysine
and that the structure of the reduced product is N
-acetyl-pHA-lysine (Scheme I).
pHA Generated by the Myeloperoxidase-H2O2-Cl
Preliminary
experiments utilizing L-[14C]tyrosine
demonstrated that BSA was covalently modified by an
L-tyrosine-derived product in the presence of the complete
myeloperoxidase-H2O2-Cl system,
as assessed by SDS-PAGE and subsequent autoradiography (data not
shown). To determine whether the Schiff base adduct between pHA and the
-amino group of lysine accounted in part for this reaction, BSA was
exposed to the myeloperoxidase-H2O2 system
supplemented with physiological concentrations (50) of L-tyrosine (100 µM) and Cl
(100 mM). Following incubation, the protein was reduced with NaCNBH3, acid-hydrolyzed, and the content of pHA-lysine in
the amino acid hydrolysate determined by stable isotope dilution GC/MS. In the presence of the complete
myeloperoxidase-H2O2-Cl
system,
protein-bound pHA-lysine was formed (Table I). Synthesis of the adduct required the presence of myeloperoxidase,
H2O2, L-tyrosine, and
Cl
and was inhibited by the H2O2
scavenger catalase (Table I). Addition of either azide or cyanide, two
heme protein inhibitors, resulted in inhibition of pHA-lysine
synthesis, consistent with the reaction requiring myeloperoxidase. The
Cl
dependence of the enzymatic reaction was consistent
with HOCl (or enzyme-bound hypochlorite; Refs. 51 and 52) as an
intermediate in the formation of pHA and pHA-lysine.
|
To establish the specificity of pHA-lysine as a marker for
protein modification by myeloperoxidase, we examined the ability of a
variety of in vitro oxidation systems to generate the adduct on BSA incubated in the presence of plasma concentrations of
L-tyrosine (Table I). Modification of protein lysine
residues by pHA was monitored by reducing the reaction products,
subjecting them to acid hydrolysis, and quantifying pHA-lysine
formation in the amino acid hydrolysate. Significant levels of
pHA-lysine were detected in BSA exposed to reagent HOCl in the presence
of L-tyrosine (Table I). HOBr could not replace HOCl. There
was no detectable formation of pHA-lysine when other halides
(Br, I
, F
) or
SCN
replaced Cl
in the
myeloperoxidase-H2O2-tyrosine system.
Furthermore, pHA-lysine was undetectable in BSA incubated with
L-tyrosine and the following in vitro oxidation
systems: a hydroxyl radical generating system (copper plus
H2O2), lactoperoxidase plus
H2O2, horseradish peroxidase plus
H2O2, or peroxynitrite (Table I). Collectively,
these results demonstrate that pHA-lysine production is a highly
specific marker for protein modification by the
myeloperoxidase-H2O2-Cl
-tyrosine
system.
To determine the quantitative significance of
pHA-lysine relative to other potential protein adducts, BSA was
incubated with HPLC-purified [14C]pHA. The modified
protein was then reduced with NaCNBH3, acid-precipitated, washed, hydrolyzed in HBr, and the amino acid hydrolysate analyzed by
HPLC and scintillation spectrometry. Over 80% of the radioactivity recovered in the amino acid hydrolysate co-migrated with authentic pHA-lysine on reverse phase HPLC (Fig. 5). The identity
of the radiolabeled compound as pHA-lysine was confirmed by GC/MS
analysis. Amino acid analysis demonstrated that lysine was the major
target for covalent modification by pHA. Incubation of BSA (1 mg/ml) with purified pHA (1 mM) in the presence of
NaCNBH3 resulted in the consumption of 34% of total
L-lysine residues in the protein. Small but consistent
losses of L-arginine (~6%) were also observed and may
account for the late eluting product seen in Fig. 5.
Schiff bases are in equilibrium with their parent aldehyde and amino moieties. In the presence of NaCNBH3, the formation of pHA-lysine is enhanced by the reduction of existing Schiff bases. To estimate the number of protein lysine residues modified in the absence of a reducing agent, we incubated BSA with pHA and then stabilized the Schiff base adduct by reduction with NaCNBH3 in the presence of high concentrations of ammonium acetate to scavenge non-reacted pHA. Amino acid hydrolysis confirmed that lysine was a major target for covalent modification by pHA under these conditions (Table II). The loss of L-lysine residues was accompanied by the appearance of a product that co-eluted with L-glycine on amino acid analysis and that likely represents pHA-lysine.
|
Previous studies revealed that ~90% of the pHA generated
by activated neutrophils partitioned into the membrane fraction of the
cells due to the amphipathic nature of the aldehyde (28). We therefore
determined whether myeloperoxidase-generated pHA preferentially
modifies membrane-associated proteins. Whole blood was diluted with PBS
and incubated with myeloperoxidase, H2O2, and
physiological concentrations of L-tyrosine and
Cl. After isolation of cells by centrifugation, the
extent of pHA-lysine formation was determined in amino acid
hydrolysates prepared from membrane-associated and soluble proteins.
Substantial amounts of the adduct were generated on both
membrane-associated and soluble proteins (Fig. 6,
left panel). Selected ion monitoring GC/MS analysis demonstrated that the major ions expected for pHA-lysine co-eluted with
those of synthetically prepared
[13C6]pHA-lysine (Fig. 6, right
panel). Generation of pHA-lysine required myeloperoxidase,
H2O2, L-tyrosine, and protein.
These results suggest that the amphipathic nature of pHA permits the aldehyde to react with cellular proteins in both lipid and aqueous environments. The relative enrichment of pHA-lysine in
membrane-associated proteins may reflect high local concentrations of
pHA or the location of the proteins at the interface between the
intracellular milieu and extracellular space where free pHA is
generated by myeloperoxidase.
To determine whether activated human neutrophils similarly modified protein lysine residues with pHA, BSA was incubated with phorbol ester-activated human neutrophils in a balanced salt solution supplemented with plasma concentrations of L-tyrosine. BSA was then reduced with NaCNBH3, subjected to acid hydrolysis, and analyzed for the presence of pHA-lysine (Table III). Selected ion monitoring revealed the presence of multiple ions with the expected retention time and m/z of pHA-lysine. Moreover, the negative ion chemical ionization mass spectrum of the neutrophil-generated product was essentially identical to that of authentic pHA-lysine (compare Fig. 7 and Fig. 2, right panel).
|
Covalent modification of lysine residues in BSA by neutrophil-generated
pHA required cells, L-tyrosine, and an activating stimulus
(Table III). Addition of superoxide dismutase to the reaction mixture,
which accelerates the conversion of superoxide anion into
H2O2 500-fold at neutral pH (53), caused a
2-fold increase in the yield of the adduct (Table III). Catalase
inhibited generation of pHA-lysine by activated human neutrophils,
indicating that H2O2 was required for the
reaction. Both azide and cyanide inhibited pHA-lysine formation,
consistent with a role of myeloperoxidase in aldehyde generation (28).
Collectively, these results indicate that activated human neutrophils
employ the
myeloperoxidase-H2O2-Cl system to
generate pHA, which then reacts with the free amino groups of proteins
to form a Schiff base.
Human plasma possesses highly efficient antioxidant defense mechanisms (54, 55). For example, oxidation of LDL by free metal ions is dramatically inhibited by the presence of <1% human plasma (55). To assess the potential physiological relevance of protein modification by neutrophil-generated pHA, we examined the effect of human plasma on pHA-lysine formation by phagocytes. Human neutrophils (4 × 106/ml) were first incubated in medium supplemented with 200 µM L-tyrosine in the absence of human plasma. Following incubation at 37 °C for 4 h, products were reduced with NaCNBH3 and the pHA-lysine content of total proteins determined by GC/MS analysis. Substantial quantities of pHA-lysine were formed on endogenous proteins of phorbol ester-stimulated neutrophils (440 ± 110 nM pHA-lysine; mean ± S.E., n = 3) compared with resting neutrophils (<0.1 nM pHA-lysine; n = 3). Thus, phagocyte activation resulted in covalent modification of endogenous neutrophil proteins by pHA. The cell-mediated reaction was stimulated nearly 2-fold by superoxide dismutase and was inhibited by NaCN, NaN3, or catalase. The presence of 20% human plasma markedly diminished adduct formation on total proteins in the reaction mixture; however, significant levels of pHA-lysine were still formed (6.4 ± 1.7 nM pHA-lysine; mean ± S.E., n = 3). Adduct formation required cell activation since minimal levels of pHA-lysine were generated in the absence of phorbol ester (0.11 ± 0.03 nM pHA-lysine; mean ± S.E., n = 3). Collectively, these results demonstrate that phagocyte activation results in covalent modification of proteins by the amino acid-derived aldehyde, pHA, through a peroxidase and H2O2-dependent mechanism. Furthermore significant levels of the adduct were still formed in the presence of human plasma. Thus, formation of the Schiff base adduct might occur in vivo, particularly within a protected environment where local antioxidant defense mechanisms might become compromised.
pHA-Lysine Is Present in Human Inflammatory FluidsTo
investigate whether pHA might react with proteins in vivo,
we searched for the presence of pHA-lysine in a variety of inflammatory fluids. Human tissue samples were obtained from an infected site (a
pilonidal cyst), sterile pus (a culture-negative peripancreatic abscess), and from an noninfectious inflammatory condition
(arthrocentesis fluid from an acutely inflamed gouty knee). Samples
from these sites were chosen because they should possess high numbers
of activated phagocytes and were likely sites of pHA formation.
Immediately following tissue collection, an aliquot of residual
specimen was removed, supplemented with inhibitors of the
myeloperoxidase-H2O2-Cl system
(azide and catalase), incubated with NaCNBH3 in the
presence of excess ammonium acetate to scavenge free aldehyde, and then prepared for GC/MS analysis. As shown in Fig. 8 for
abscess fluid, pHA-lysine was readily detected by selected ion
monitoring in amino acid hydrolysates prepared from each of the
tissues. These results demonstrate that pHA-modified proteins are
present in vivo.
Covalent modification of proteins by reactive aldehydes is thought to play a critical role in vascular disease and inflammation (3-19). Our results indicate that pHA, a major product of L-tyrosine oxidation by phagocytes, reacts with the free amino group of lysine residues on proteins to form a Schiff base (Scheme I). The structure of the reduced adduct was established as pHA-lysine by mass spectrometric analysis and high resolution NMR spectroscopy. pHA-lysine was the major adduct detected in amino acid hydrolysates prepared from model proteins that were first exposed to pHA and then reduced with NaCNBH3 in the presence of ammonium acetate. This finding raises the possibility that the Schiff base adduct of pHA is a significant product of phagocyte activation. Indeed, we have identified pHA-lysine in acid hydrolysates prepared from inflamed human tissues that were treated with NaCNBH3 and ammonium acetate, strongly suggesting that pHA forms a Schiff base with proteins in vivo.
pHA-lysine was detected in amino acid hydrolysates of model proteins
exposed to either synthetically prepared pHA or to
L-tyrosine oxidized by either HOCl, the
myeloperoxidase-H2O2-Cl system,
or activated human phagocytes. These results implicate free pHA
generation by myeloperoxidase as a critical step in the reaction
pathway (28). Consistent with this proposal, addition of the
H2O2 scavenger catalase, and the peroxidase
inhibitors azide or cyanide, inhibited pHA-lysine generation by both
the purified enzymatic system and activated human neutrophils. The reduced Schiff base was detected in soluble and membrane-associated proteins of whole blood exposed to the myeloperoxidase system. Thus,
production of pHA at sites of inflammation may result in the covalent
modification of target proteins in a variety of chemical environments.
Myeloperoxidase catalyzes the incorporation of amines into proteins
through dichloramine intermediates (33). The structures of the products
formed by this reaction have not yet been identified. The potential
role of dichloramine intermediates in the generation of pHA-lysine
remains unknown. The conversion of L-tyrosine to pHA by
myeloperoxidase is Cl-dependent, and
pHA-lysine formation appears to be specific for HOCl or a reactive
intermediate derived from HOCl. In contrast, a variety of other halides
were unable to replace Cl
in the enzymatic system.
Moreover, HOBr, peroxynitrite, and hydroxyl radical could not
substitute for HOCl. These results suggest that pHA-lysine is a
specific marker for protein modification by HOCl. Detection of
pHA-lysine within human tissue should thus serve as a selective and
sensitive marker for proteins oxidatively damaged by myeloperoxidase.
The stable isotope dilution GC/MS assay we have developed is capable of
detecting pHA-lysine at the femtomole level, suggesting its utility for
assessing the role of protein modification by phagocyte-generated
aldehydes in a variety of inflammatory disease states.
The irreversible modification of proteins by lipid- and glucose-derived aldehydes has been implicated in the pathogenesis of diseases ranging from atherosclerosis to ischemia-reperfusion injury to diabetic vascular disease (3-19). The reversible covalent modification of proteins by Schiff base formation has received less attention. Schiff base formation by aromatic aldehydes has been proposed to modulate cytokine production in vivo (56), with potent effects on the immune system including antigen-induced T-cell proliferation, suppression of viral replication, and inhibition of solid tumor growth. One of the aldehydes employed in these pharmacological studies was pHA, which substantially enhanced T-cell proliferation in vitro and in vivo (56). These observations raise the possibility that phagocytes represent a physiological pathway for the production of aromatic aldehydes that regulate the immune system.
We have suggested that activation of phagocytes exposed to plasma
concentrations of free amino acids and Cl might result in
the generation of a family of low molecular weight, freely diffusible
aldehydes (28, 57). These selectively reactive amphipathic aldehydes
could then covalently modify susceptible target molecules. The
detection of pHA-lysine in proteins at sites of inflammation is
consistent with this hypothesis. Mass spectrometric studies quantifying
structurally defined aldehydes and their adducts in human and animal
tissues should further our understanding of the precise biochemical
mechanisms underlying oxidative damage in vivo, with
important implications for the role of activated phagocytes in the
pathogenesis of disease.
This paper is dedicated to the memory of Professor Herman Esterbauer, a pioneer in the study of reactive aldehydes in biology.
We thank Dr. George Sarris for providing human tissue and D. Mueller and S. Scotino for expert technical assistance. Gas chromatography-mass spectrometry experiments were performed at the Washington University School of Medicine Mass Spectrometry Resource. NMR spectroscopy was performed at the Washington University High Resolution NMR Facility.