Site-specific oxidation of apolipoprotein A-I impairs cholesterol export by ABCA1, a key cardioprotective function of HDL

Abstract

The mechanisms that deprive HDL of its cardioprotective properties are poorly understood. One potential pathway involves oxidative damage of HDL proteins by myeloperoxidase (MPO) a heme enzyme secreted by human artery wall macrophages. Mass spectrometric analysis demonstrated that levels of 3-chlorotyrosine and 3-nitrotyrosine - two characteristic products of MPO - are elevated in HDL isolated from patients with established cardiovascular disease. When apolipoprotein A-I (apoA-I), the major HDL protein, is oxidized by MPO, its ability to promote cellular cholesterol efflux by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway is diminished. Biochemical studies revealed that oxidation of specific tyrosine and methionine residues in apoA-I contributes to this loss of ABCA1 activity. Another potential mechanism for generating dysfunctional HDL involves covalent modification of apoA-I by reactive carbonyls, which have been implicated in atherogenesis and diabetic vascular disease. Indeed, modification of apoA-I by malondialdehyde (MDA) or acrolein also markedly impaired the lipoprotein's ability to promote cellular cholesterol efflux by the ABCA1 pathway. Tandem mass spectrometric analyses revealed that these reactive carbonyls target specific Lys residues in the C-terminus of apoA-I. Importantly, immunochemical analyses showed that levels of MDA-protein adducts are elevated in HDL isolated from human atherosclerotic lesions. Also, apoA-I co-localized with acrolein adducts in such lesions. Thus, lipid peroxidation products might specifically modify HDL in vivo. Our observations support the hypotheses that MPO and reactive carbonyls might generate dysfunctional HDL in humans. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Figure 1. Cholesterol efflux activities of lipid-free apoA-I oxidized with HOCl, ONOO⁻, MPO-H₂O₂-chloride, or MPO-H₂O₂-nitrite

ApoA-I (5 μM) was incubated with the indicated concentrations of HOCl, ONOO⁻, or H₂O₂ for 60 min at 37 °C in phosphate buffer. The reaction was terminated by adding methionine. Where indicated, the system was supplemented with 50 nM myeloperoxidase (MPO) and 100 μM nitrite (NO₂⁻) or 100 mM NaCl (NaCl). (A, B) [³H]Cholesterol-labeled ABCA1-transfected BHK cells were incubated for 2 h with native (0 μM oxidant), HOCl-oxidized, ONOO⁻-oxidized, or MPO-H₂O₂-oxidized apoA-I (5 μg/mL). (C) [³H]Cholesterol-labeled ABCA1-transfected BHK cells were incubated with the indicated concentration of apoA-I for 2 h. ApoA-I was incubated with MPO and 0 μM oxidant (Ctrl), 125 μM H₂O₂ plus 100 μM nitrite (NO₂), or 100 mM NaCl (NaCl). At the end of the incubation, [³H]cholesterol efflux to the acceptor apolipoprotein was measured (81).

Figure 2. Site-specific chlorination of tyrosine residues in apoA-I exposed to HOCl or the MPO-H₂O₂-NaCl system (A) and nitration of tyrosine residues in apoA-I exposed to ONOO⁻ or the MPO-H₂O₂-NaNO₂ system (B)

ApoA-I (10 μM) was exposed to HOCl (A, solid bars), ONOO⁻ (B, solid bars), or H₂O₂ in the MPO-chloride system (A, single-line, shaded bars) or MPO-nitrite system (B, single-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 digest of oxidized apoA-I was analyzed by MS and tandem MS, and the oxidized peptides were detected and quantified, using reconstructed ion chromatograms of precursor and product peptides. Product yield (%) = peak area of product ion/sum (peak area of precursor ion + peak areas of product ions) × 100. Peptide sequences were confirmed using tandem MS. Results are from 3 independent experiments (mean ± SD) (83).

Figure 3. Cholesterol efflux activities of apoA-I (WT) and Tyr192Phe (Y192F) apoA-I exposed to the MPO system and reduced by PilB

(A) [³H]Cholesterol-labeled ABCA1-transfected BHK cells were incubated with the indicated concentration of apoA-I (WT) or Tyr192Phe apoA-I (Y192F) for 2 h. At the end of the incubation, [³H]cholesterol efflux to the acceptor apolipoprotein was measured. (B) ApoA-I or Tyr192Phe apoA-I (5 μM) was oxidized by the MPO-H₂O₂-NaCl system (25:1, mol/mol, H₂O₂/apoA-I) for 60 min at 37 °C in phosphate buffer (100 μM DTPA, 20 mM sodium phosphate, pH 7.4). The reaction was terminated by adding methionine. Where indicated, apoA-I was incubated with the methionine sulfoxide reductase PilB. [³H]Cholesterol-labeled ABCA1-transfected BHK cells were incubated with the indicated concentration of lipoproteins, and cholesterol efflux was measured at the end of the incubation. Results are means ± SD of 3 determinations and are representative of 3 independent experiments (84).

Figure 4

Structures of physiologically relevant reactive carbonyls and carbonyl adducts detected by mass spectrometry.

Figure 5. Cholesterol and phospholipid efflux activities of acrolein-modified apoA-I

ApoA- I (25 μM) was incubated with the indicated concentrations of acrolein (A) or with 500 μM acrolein (B) for 24 h at 37°C in 50 mM sodium phosphate buffer (pH 7.4) containing 100 μM DTPA. Reactions were initiated by adding acrolein, and terminated by adding a 20-fold molar excess (relative to acrolein) of aminoguanidine. (A) [³H]Cholesterol- or phospholipid-labeled ABCA1-transfected BHK cells were incubated for 2 h with 5 μg/ml of native (0 μM acrolein) or acrolein-modified apoA-I. (B) [³H]Cholesterol-labeled ABCA1-transfected BHK cells were incubated with the indicated concentrations of untreated or acrolein-treated apoA-I (20:1, mol/mol, acrolein/protein) for 2 h. At the end of the incubation, [³H]cholesterol efflux to the acceptor apolipoprotein was measured. Results represent those from 2 independent experiments (120).

Figure 6. Impact of carbonyl modification on apoA-I’s ability to promote cholesterol efflux by ABCA1

ApoA-I (5 μM) was incubated with 250 μM glyoxal, methylglyoxal, glycolaldehdye, MDA, or HNE (A), or with the indicated concentrations of MDA for 24 h (B and D), or with 250 μM MDA for the indicated times (C) at 37°C in 50 mM sodium phosphate buffer (pH 7.4) containing 100 μM DTPA. Reactions were initiated by adding carbonyl, and terminated by adding a 20-fold molar excess (relative to carbonyl) of aminoguanidine. (A, B, and C) [³H]Cholesterol-labeled ABCA1-transfected BHK cells were incubated for 2 h with 3 μg/mL of control (0 μM carbonyl) or carbonyl-modified apoA-I. (D) [³H]Cholesterol-labeled ABCA1- transfected BHK cells were incubated with the indicated concentrations of untreated or MDA- treated apoA-I (20:1 or 50:1, mol/mol, MDA/protein) for 2 h. At the end of the incubation, [³H]cholesterol efflux to the acceptor apolipoprotein was measured. Results represent 2 independent experiments (128).