Myeloperoxidase targets apolipoprotein A-I, the major high density lipoprotein protein, for site-specific oxidation in human atherosclerotic lesions

Abstract

Oxidative damage by myeloperoxidase (MPO) has been proposed to deprive HDL of its cardioprotective effects. In vitro studies reveal that MPO chlorinates and nitrates specific tyrosine residues of apoA-I, the major HDL protein. After Tyr-192 is chlorinated, apoA-I is less able to promote cholesterol efflux by the ABCA1 pathway. To investigate the potential role of this pathway in vivo, we used tandem mass spectrometry with selected reaction monitoring to quantify the regiospecific oxidation of apoA-I. This approach demonstrated that Tyr-192 is the major chlorination site in apoA-I in both plasma and lesion HDL of humans. We also found that Tyr-192 is the major nitration site in apoA-I of circulating HDL but that Tyr-18 is the major site in lesion HDL. Levels of 3-nitrotyrosine strongly correlated with levels of 3-chlorotyrosine in lesion HDL, and Tyr-18 of apoA-I was the major nitration site in HDL exposed to MPO in vitro, suggesting that MPO is the major pathway for chlorination and nitration of HDL in human atherosclerotic tissue. These observations may have implications for treating cardiovascular disease, because recombinant apoA-I is under investigation as a therapeutic agent and mutant forms of apoA-I that resist oxidation might be more cardioprotective than the native form.

FIGURE 1.

Detection of chlorinated Tyr-192 peptide in HDL supplemented with plasma apoA-I and isotope-labeled apoA-I that had been oxidized by HOCl. Lipid-free apoA-I (3.5 μm) and [¹⁵N]apoA-I (3.5 μm) were exposed to 175 μm HOCl (50:1, mol/mol, HOCl:apoA-I) for 60 min at 37 °C in phosphate buffer (20 mm sodium phosphate, 100 μm DTPA, pH 7.4). After the reactive intermediates were quenched with l-methionine (5 mm), the reaction mixture was dialyzed against 10 mm sodium phosphate buffer, pH 7.4. Dialyzed HOCl-modified apoA-I (0.2 μg) and dialyzed HOCl-modified [¹⁵N]apoA-I (0.2 μg) were added to 10 μg HDL₃, and the protein mixture was digested with trypsin. The peptide digest was then analyzed with SRM on a Thermo TSQ triple quadrupole mass spectrometer. A, shown are ion chromatograms of precursor and chlorinated, unlabeled, and ¹⁵N-labeled peptides (LAEYHAK) containing Tyr-192. B, four transitions (b₂, y₄, y₅, and y₆) were selected to quantify the precursor and chlorinated, unlabeled, and ¹⁵N-labeled product peptides (lsqb]LAEY¹⁹²HAK + H]⁺ (m/z 831.4), [LAE(ClY¹⁹²)HAK + H]⁺ (m/z 865.4), [¹⁵N][LAEY¹⁹²HAK + H]⁺ (m/z 841.4), and [¹⁵N][LAE(ClY¹⁹²)HAK + H]⁺ (m/z 875.4)]. RT, retention time (min).

FIGURE 2.

Quantification of regiospecific chlorination of the Tyr residue in lipid-free and HDL-associated apoA-I exposed to HOCl or the MPO-H₂O₂-NaCl system. Lipid-free apoA-I (10 μm) (A) or HDL₃ (0.5 mg/ml, ∼12.5 μm apoA-I) (B) was exposed to 100 or 180 μm HOCl, respectively (striped bars) or to 100 or 180 μm H₂O₂, respectively, in the MPO-H₂O₂-chloride system (solid bars) for 60 min at 37 °C in phosphate buffer (20 mm sodium phosphate, 100 μm DTPA, pH 7.4). The MPO system contained 100 nm enzyme and 100 mm sodium chloride. The reaction was terminated with l-methionine. A tryptic digest of apoA-I or HDL₃ was analyzed by selected reaction monitoring mass spectrometry analysis. Four transitions were selected for each Tyr-containing precursor and product peptide to quantify the product yield of chlorinated tyrosine residues. The product yield of 3-chlorotyrosine was calculated as described under “Experimental Procedures.” Results are the means ± S.D. of three independent experiments, with triplicate determinations per experiment.

FIGURE 3.

On exposure to the MPO-H₂O₂-NaNO₂ system, Tyr-192 is the major nitration target in apoA-I, but Tyr-18 is the major target when apoA-I is associated with HDL. Lipid-free apoA-I (10 μm) (A and B) or HDL₃ (0.5 mg/ml, ∼12.5 μm apoA-I) (C and D) was exposed to 100 or 180 μm ONOO⁻, respectively (A and C), or to 100 or 180 μm H₂O₂, respectively, in the MPO-H₂O₂-nitrite system (B and D) for 60 min at 37 °C in phosphate buffer (20 mm sodium phosphate, 100 μm DTPA, pH 7.4). The MPO system was supplemented with 100 nm enzyme and 200 μm sodium nitrite. The reaction was terminated with l-methionine. A tryptic digest of apoA-I or HDL₃ was analyzed by isotope dilution SRM. Four transitions were selected for each Tyr-containing precursor and product peptide to quantify the product yields of nitrated tyrosine residues. Product yield of 3-nitrotyrosine was calculated as described under “Experimental Procedures.” Results are the means ± S.D. from three independent experiments.

FIGURE 4.

Quantification of the regiospecific chlorination and nitration of apoA-I in HDL isolated from human plasma. HDL was isolated by ultracentrifugation from plasma of apparently healthy subjects (n = 11). An extensively dialyzed mixture of HOCl-chlorinated and peroxynitrite-nitrated [¹⁵N]apoA-I (0.15 μg) was added to 10 μg of HDL before digestion. Levels of modified peptides were calculated from the ratio of the total peak area of four transitions of oxidized peptide of apoA-I from HDL relative to that of the corresponding modified [¹⁵N]peptides from oxidized [¹⁵N]apoA-I as described under “Experimental Procedures.” Chlorinated and nitrated tyrosine-containing peptides in the oxidized [¹⁵N]apoA-I standards were quantified by LC-ESI-MS-SRM analysis using reconstructed ion chromatograms of product and precursor peptides. Results are representative of those from two independent experiments.

FIGURE 5.

Tyr-192 is the major chlorination target, whereas Tyr-18 is the major nitration target in HDL isolated from human atherosclerotic lesions. HDL was isolated by ultracentrifugation from atherosclerotic lesions of carotid arteries harvested from humans (n = 8). Regiospecific oxidation of apoA-I was determined in tryptic and Glu-C digests of HDL, as described in the legends to Fig. 4. Results are representative of those from two independent experiments.

FIGURE 6.

Total levels of 3-chlorotyrosine and 3-nitrotyrosine in apoA-I of HDL isolated from human plasma and atherosclerotic carotid lesions. HDL was isolated from plasma and atherosclerotic tissue by sequential ultracentrifugation. Levels of individual 3-chlorotyrosine and 3-nitrotyrosine were quantified as described in the legends of Figs. 4 and 5. Total levels of 3-chlorotyrosine or 3-nitrotyrosine in apoA-I were calculated as the sum of individual levels of 3-chlorotyrosine or 3-nitrotyrosine at the 7 Tyr residues in apoA-I divided by 7. Results are representative of those from two independent experiments.

FIGURE 7.

Correlation of 3-chlorotyrosine with 3-nitrotyrosine levels in apoA-I of HDL isolated from human plasma and atherosclerotic lesions. Levels of 3-chlorotyrosine and 3-nitrotyrosine at Tyr-192 in apoA-I of HDL isolated from plasma (A) or lesions (B) were determined as described in the legends of Figs. 4 and 5. The coefficient of determination (R²) and p value were calculated by linear regression analysis.