Modifying apolipoprotein A-I by malondialdehyde, but not by an array of other reactive carbonyls, blocks cholesterol efflux by the ABCA1 pathway

Abstract

Dysfunctional high density lipoprotein (HDL) is implicated in the pathogenesis of cardiovascular disease, but the underlying pathways remain poorly understood. One potential mechanism involves covalent modification by reactive carbonyls of apolipoprotein A-I (apoA-I), the major HDL protein. We therefore determined whether carbonyls resulting from lipid peroxidation (malondialdehyde (MDA) and hydroxynonenal) or carbohydrate oxidation (glycolaldehyde, glyoxal, and methylglyoxal) covalently modify lipid-free apoA-I and inhibit its ability to promote cellular cholesterol efflux by the ABCA1 pathway. MDA markedly impaired the ABCA1 activity of apoA-I. In striking contrast, none of the other four carbonyls were effective. Liquid chromatography-electrospray ionization-tandem mass spectrometry of MDA-modified apoA-I revealed that Lys residues at specific sites had been modified. The chief adducts were MDA-Lys and a Lys-MDA-Lys cross-link. Lys residues in the C terminus of apoA-I were targeted for cross-linking in high yield, and this process may hinder the interaction of apoA-I with lipids and ABCA1, two key steps in reverse cholesterol transport. Moreover, levels of MDA-protein adducts were elevated in HDL isolated from human atherosclerotic lesions, suggesting that lipid peroxidation might render HDL dysfunctional in vivo. Taken together, our observations indicate that MDA damages apoA-I by a pathway that generates lysine adducts at specific sites on the protein. Such damage may facilitate the formation of macrophage foam cells by impairing cholesterol efflux by the ABCA1 pathway.

Figures

FIGURE 1.

Structures of carbonyls and carbonyl adducts.

FIGURE 2.

Impact of carbonyl modification on ability of apoA-I to promote cholesterol efflux by ABCA1. ApoA-I (5 μm) was incubated with 250 μm glyoxal, methylglyoxal, glycolaldehyde, 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–C, [3H]cholesterol-labeled ABCA1-transfected BHK cells were incubated for 2 h with 3 μg/ml of control (0 μm carbonyls) 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. HNE was added to apoA-I in ethanol. Control experiments showed that the same final concentration of ethanol (∼1 μl per 200 μl) increased the efflux activity of native apoA-I by ∼10%. Results represent two independent experiments.

FIGURE 3.

MALDI-TOF-MS of carbonyl-modified apoA-I. ApoA-I was exposed to the indicated concentrations of carbonyl for 24 h, as described in the legend to Fig. 2. After the reaction was terminated with aminoguanidine and reaction products reduced by NaBH4, intact proteins were desalted and analyzed by MALDI-TOF-MS. The masses of the intact native or carbonyl-modified proteins were determined, using myoglobin and bovine serum albumin as the internal standards.

FIGURE 4.

Precursor loss and product yields of N-propenal-Lys (K+54), N-propenal-Trp (W+54), and N-propenal-His (H+54) in MDA-modified apoA-I. ApoA-I was exposed to a 20-fold molar ratio of MDA, as described in the legend to Fig. 2. A mixture of 5 μg of modified or control apoA-I and 2.5 μg of 15N-labeled apoA-I was digested with trypsin or Glu-C. A, following LC-ESI-MS/MS analysis, the ratio of precursor peptides derived from modified or control apoA-I relative to 15N-labeled peptides derived from 15N-labeled apoA-I was determined from extracted ion chromatograms. Precursor peptide loss was calculated as described under “Experimental Procedures.” B, product yields of K+54, W+54, and H+54 were calculated from the ratio of peak area of product peptide from modified apoA-I to that of the corresponding 15N-labeled peptide from 15N-labeled apoA-I, as described under “Experimental Procedures.” The N-propenal adducts were identified by LC-ESI-MS/MS analysis. N-t, N-terminal amino group. Results represent three independent experiments.

FIGURE 5.

MS/MS analysis of N-propenal-Lys (K+54) and Lys-MDA-Lys (K+36+K) in MDA-modified apoA-I. ApoA-I was incubated in buffer alone (A and C) or exposed to a 20-fold molar excess of MDA (B and D), as described in the legend to Fig. 2. A Glu-C peptide digest was analyzed by LC-ESI-MS/MS, as described under “Experimental Procedures.” A, MS/MS spectrum of [LYRQKVEPLRAE + H]+ (m/z 1501.8) where K is Lys118. B, MS/MS spectrum of [LYRQ(K+54)VEPLRAE + H]+ (m/z 1555.8), where K is Lys118. C, MS/MS spectrum of [KAKPALE + H]+, where the first K is Lys206 and the second K is Lys208 (m/z 756.5). D, MS/MS spectrum of [KAK(K+36+K)PALE + H]+, where the first K is Lys206 and the second K is Lys208 (m/z 792.5, Lys206+Lys208+36).

FIGURE 6.

Product yield of Lys-MDA-Lys (K+36+K) and cross-linking map between Lys residues in MDA-modified apoA-I. ApoA-I was exposed to a 50-fold molar ratio of MDA, as described in the legend to Fig. 2. A mixture of control or modified apoA-I (5 μg) and 15N-labeled apoA-I (2.5 μg) was digested with trypsin or Glu-C. Following LC-ESI-MS/MS analysis, the product yields of Lys-MDA-Lys (K+36+K) between Lys residues were calculated from the ratio of peak area of product peptide from modified apoA-I to that of the corresponding 15N-labeled peptide from 15N-labeled apoA-I, as described under “Experimental Procedures.” All cross-linked adducts were confirmed by LC-ESI-MS/MS analysis. A, distant cross-linking map between Lys residues in MDA-modified apoA-I; B, product yield of adjacent cross-linking (bottom panel) and product yield of distant cross-linking (top panel).

FIGURE 7.

Hexagon model of MDA-modified C terminus of apoA-I. A, location of Lys residues modified in high yield by MDA in the C-terminal region of lipid-free apoA-I. B, energy minimization model of repeats 7–10 of apoA-I cross-linked by MDA. C, hexagon model of cross-links between C-terminal lysine residues in MDA-modified apoA-I. D, close-up view of C-terminal in the energy minimization model of apoA-I cross-linked by MDA.

FIGURE 8.

Quantification of MDA-modified proteins in HDL isolated from plasma and atherosclerotic tissue of humans. A, HDL (from healthy plasma, 0.2 mg of protein/ml) was exposed to indicated concentrations of MDA as described in the legend to Fig. 2. B, plasma was obtained from healthy humans. Human atherosclerotic tissue was obtained at surgery from subjects undergoing carotid endarterectomy. HDL was isolated from plasma and tissue by ultracentrifugation, and equal amounts of HDL protein were added to each well, and then MDA-modified proteins were quantified by enzyme-linked immunosorbent assay using the MDA2 monoclonal antibody (RLU/ms = relative light units/ms). Each value shown is the average of triplicate measurements as described under “Experimental Procedures.”

FIGURE 9.

Summary of amino acid residues modified by MDA, HNE, or methylglyoxal in lipid-free apoA-I. The product yields of MDA-Lys (K+54) and Lys-MDA-Lys (K+36+K) (red), HNE-His (H+158, purple), or methylglyoxal-Arg (R+54 and R+72, yellow) were determined by isotope dilution MS. High yield cross-linking between lysine residues by MDA are indicated (red arc).