Oxidation of apolipoprotein A-I by myeloperoxidase impairs the initial interactions with ABCA1 required for signaling and cholesterol export

Abstract

A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.

Fig. 1.

ABCA1-dependent cholesterol efflux activities of apoA-I oxidized by MPO. (A) [³H]Cholesterol efflux from ABCA1-transfected BHK cells was measured after a 2-h incubation with 3 µg/ml of apoA-I, apoA-I exposed to MPO in the presence of H₂O₂ and either NaCl (MPO-Cl) or NaNO₂ (MPO-NO₂), or apoA-I exposed to reagent HOCl or ONOO⁻. Oxidation reactions were carried out at the indicated ratio (mol:mol) of oxidant to apoA-I. (B) [³H]Cholesterol efflux was measured during 2-h incubations with the indicated concentrations of control apoA-I or apoA-I treated with a 25:1 ratio (mol:mol) of oxidant to apoA-I. [³H]Cholesterol efflux is expressed as % of total medium and cell [³H]cholesterol that is released into the medium. Results are means ± SD of three determinations, and are representative of more than six independent experiments.

Fig. 2.

Product yields of (A) chlorotyrosine, (B) nitrotyrosine, (C) methionine sulfoxide, and (D) hydroxytryptophan in apoA-I exposed to HOCl, MPO-H₂O₂-chloride system, ONOO⁻, or MPO-H₂O₂-nitrite system. ApoA-I (10 μM) was exposed to HOCl (solid bars), ONOO⁻ (empty bars), H₂O₂ in the MPO-chloride system (single-line shaded bars), or MPO-nitrite system (crossing-line shaded bars) at molar ratio of 25:1 (oxidant/apoA-I) for 60 min at 37°C in phosphate buffer (100 μM DTPA, 20 mM sodium phosphate, pH 7.4). After the reaction was terminated with L-methionine, a tryptic or Glu-C digest of oxidized apoA-I was analyzed by LC-ESI-MS and MS/MS, and the oxidized peptides were detected and quantified using reconstructed ion chromatograms of precursor and product peptides as described in the Methods section. Peptide sequences were confirmed using MS/MS. Results are from three independent experiments (mean ± SD).

Fig. 3.

Binding of MPO-oxidized apoA-I to ABCA1. BHK cells transfected with ABCA1 were incubated for 2 h at 37°C with 1 (A) or 2 (B) µg/ml ¹²⁵I-apoA-I minus (None) or plus 2 (A) or 10 (B) µg/ml unlabeled control apoA-I (Ctrl) or apoA-I treated with reagent HOCl or ONOO⁻ or with MPO and H₂O₂ in the presence of either 100 mM NaCl (MPO-Cl) or 100 μM NaNO₂ (MPO-NO₂). Oxidation reactions were carried out at a 25:1 (A) or 50:1 (B) (mol:mol) ratio of oxidant to apoA-I. Cells were treated with the cross-linker DSP, detergent-solubilized ABCA1 was immunoprecipitated and isolated by reduced SDS PAGE, and ¹²⁵I-apoA-I was visualized by phosphorimaging (A) and quantified (B). Results are representative of five similar experiments. Values in (B) are the mean ± SD of 4–6 incubations from two experiments (*P < 0.001).

Fig. 4.

Stimulation of JAK2 autophosphorylation by MPO-oxidized apoA-I. BHK cells transfected with ABCA1 were incubated for 15 min without (None) or with 10 µg/ml apoA-I (Control), or apoA-I treated with HOCl, MPO-H₂O₂-NaCl (MPO-Cl), ONOO⁻, or MPO-H₂O_2-NaNO₂ (MPO-NO₂). Oxidation reactions were carried out at a 25:1 [mol:mol] ratio of oxidant to apoA-I. Tyrosine-phosphorylated JAK2 (P-JAK2) was detected by immunoblotting. Immunoblots were stripped of antibody, and reprobed with a total JAK2 antibody (JAK2). Results are representative of three experiments.

Fig. 5.

Binding of MPO-oxidized apoA-I to ABCA1-expressing cells. ABCA1-transfected BHK cells were incubated for 2 h with 1 µg/ml ¹²⁵I-apoA-I minus or plus the indicated concentrations of unlabeled control apoA-I (Control) or apoA-I oxidized with HOCl or ONOO⁻ or with MPO plus H₂O₂ in the presence of either NaCl (MPO-Cl) or NO₂ (MPO-NO₂). Cell-associated ¹²⁵I-apoA-I was then measured. Oxidation reactions were carried out at a 25:1 [mol:mol] ratio of oxidant to apoA-I. Results are means ± SD of three determinations and are representative of two independent experiments.

Fig. 6.

Stabilization of ABCA1 protein by MPO-oxidized apoA-I. J774 macrophages loaded with cholesterol were incubated for 20 h with 0.5 mM 8-Br-cAMP followed by a 4-h incubation with (+) or without (−) 8-Br-cAMP minus apoA-I (None) or plus 10 µg/ml apoA-I treated with the indicated molar ratios of oxidants. The membrane content of ABCA1 was measured by immunoblotting. Results represent those from three independent experiments.

Fig. 7.

Solubilization of phospholipid vesicles by MPO-oxidized apoA-I. Control or oxidized apoA-I samples were added to a solution of DMPC vesicles to give a final DMPC/apoA-I ratio of 2:1 (w/w) at a final protein concentration of 0.17 mg/ml. The experiment was performed at 24.5°C with monitoring of absorbance at 325 nm. Oxidation reactions were carried out at a 25:1 [mol:mol] ratio of oxidant to apoA-I. Results represent two independent experiments.

Fig. 8.

Binding of MPO-oxidized apoA-I to egg PC SUV. Oxidation reactions were carried out at a 25:1 [mol:mol] ratio of oxidant to apoA-I. SUV (0.2 mg/ml egg PC containing 170 pmol/ml of [³H]cholesterol) were incubated with 25 μg/ml control or oxidized [¹⁴C]apoA-I for 1 h at room temperature. The mixture was then subjected to size-exclusion chromatography on a Superdex 200 column that was eluted with 10 mM Tris buffer at a flow rate of 1 ml/min with collection of 1 ml fractions. Radioactivity was determined by liquid scintillation counting. A: Elution profiles of [³H]Cholesterol of SUV (empty square) and [¹⁴C]apoA-I of lipid-free apoA-I (solid square); B: elution profiles of [¹⁴C] control (empty circle) or MPO-Cl modified (solid circle) apoA-I after binding to vesicles; C: binding capacity of oxidized apoA-I to egg PC SUV. Results are the average and ranges of two independent experiments. Abbreviations: apoA-I, apolipoprotein A-I; MPO, myeloperoxidase; PC, L-α-phosphatidylcholine; SUV, small unilamellar vesicles.

Fig. 9.

Model for lipid export by the ABCA1 pathway and its impairment by MPO. A:ABCA1 has intrinsic lipid translocase activity that generates phospholipid- (PL) and cholesterol-rich domains that protrude from the plasma membrane. B: The initial interaction of apoA-I with ABCA1 or other sites stimulates autophosphorylation of JAK2. C: Activation of JAK2 increases the binding of apoA-I to sites on ABCA1 that facilitate interactions with the lipid domains that the transporter generates. D: These lipid domains are solubilized when they interact with apoA-I, liberating nascent, discoidal particles of HDL. Inset: Schematic structure of lipid-free apoA-I showing the location of tyrosine 192 (Y192) and 2 of the 3 methionine (M86 and M148) residues in the 4-helix bundle in the N-terminal domain. Previous studies (31, 32) demonstrated that MPO selectively targets those residues for chlorination (Y192) and oxidation (M86, M148). Abbreviations: ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; JAK2, Janus kinase 2; MPO, myeloperoxidase.