Methionine oxidation impairs reverse cholesterol transport by apolipoprotein A-I

Abstract

HDL protects against vascular disease by accepting free cholesterol from macrophage foam cells in the artery wall. This pathway is critically dependent on lecithin:cholesterol acyltransferase (LCAT), which rapidly converts cholesterol to cholesteryl ester. The physiological activator of LCAT is apolipoprotein A-I (apoA-I), the major HDL protein. However, cholesterol removal is compromised if apoA-I is exposed to reactive intermediates. In humans with established cardiovascular disease, myeloperoxidase (MPO) oxidizes HDL, and oxidation by MPO impairs apoA-I's ability to activate LCAT in vitro. Because a single methionine residue in apoA-I, Met-148, resides near the center of the protein's LCAT activation domain, we determined whether its oxidation by MPO could account for the loss of LCAT activity. Mass spectrometric analysis demonstrated that oxidation of Met-148 to methionine sulfoxide associated quantitatively with loss of LCAT activity in both discoidal HDL and HDL(3), the enzyme's physiological substrates. Reversing oxidation with methionine sulfoxide reductase restored HDL's ability to activate LCAT. Discoidal HDL prepared with apoA-I containing a Met-148-->Leu mutation was significantly resistant to inactivation by MPO. Based on structural data in the literature, we propose that oxidation of Met-148 disrupts apoA-I's central loop, which overlaps the LCAT activation domain. These observations implicate oxidation of a single Met in apoA-I in impaired LCAT activation, a critical early step in reverse cholesterol transport.

Fig. 1.

Loss of LCAT activation after HDL₃ is oxidized by MPO is associated with oxidation of Met-148 and Trp-72 of apoA-I. HDL₃ was incubated with the indicated molar ratios (oxidant:apoA-I) of HOCl or H₂O₂ in the complete MPO system. Reactions were initiated by adding oxidants and terminated by adding Met. LCAT activation by oxidized HDL₃ was monitored as the conversion of [³H]cholesterol to [³H]cholesteryl ester. Generation of oxidized peptides containing the indicated residues was quantified by MS and reconstructed ion chromatograms of proteolytic digests of apoA-I. Results are from three independent experiments.

Fig. 2.

Loss of precursor peptides of apoA-I in HDL₃ exposed to the complete MPO system. HDL₃ was exposed to a 20-fold molar ratio of H₂O₂ in the MPO system. A mixture of 7 μg oxidized HDL₃ or control HDL₃ and 2.5 μg of ¹⁵N-labeled apoA-I was digested with trypsin or Glu-C. After liquid chromatography-electrospray ionization-MS/MS (LC-ESI-MS/MS) analysis, the ratio of precursor peptides derived from apoA-I in oxidized HDL₃ or control HDL₃ relative to ¹⁵N-labeled peptides derived from ¹⁵N-labeled apoA-I was calculated from extracted ion chromatograms. Results are from two independent experiments.

Fig. 3.

Methionine sulfoxide reductase restores the LCAT activity of oxidized HDL. LCAT activity was determined in HDL₃ exposed to a 25-fold molar ratio of the indicated oxidant or the same oxidized HDL preparation after incubation with PilB, a bacterial methionine sulfoxide reductase with activity on both epimers of Met(O). Results are from two independent experiments (means ± SDs).

Fig. 4.

Mutation of Met-148 to Leu protects apoA-I from the oxidative loss of LCAT activity. rHDL was prepared with control or mutated apoA-I. (A) LCAT activity of native rHDLs. (B) LCAT activity of wild-type (WT) and ΔW apoA-I exposed to the indicated concentrations of HOCl. (C) LCAT activity of Met-mutated rHDLs exposed to a 30-fold molar ratio of HOCl. (D) LCAT activity of Met-, Trp-, and Tyr-mutated rHDLs exposed to a 40-fold molar ratio of HOCl. Results are from two independent experiments. DeltaW, deletion all four Trp residues of apoA-I. 3M/L, Met86Leu/Met112Leu/Met148Leu mutant apoA-I.