Myeloperoxidase: an oxidative pathway for generating dysfunctional high-density lipoprotein

Abstract

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by artery wall macrophages triggers atherosclerosis, the leading cause of cardiovascular disease. Conversely, high-density lipoprotein (HDL) retards atherosclerosis by promoting cholesterol efflux from macrophages by the membrane-associated ATP-binding cassette transporter A1 (ABCA1) pathway. HDL has been proposed to lose its cardioprotective effects in subjects with atherosclerosis, but the underlying mechanisms are poorly understood. One potential pathway involves oxidative damage by myeloperoxidase (MPO), a heme enzyme secreted by human artery wall macrophages. We used mass spectrometry to demonstrate that HDL isolated from patients with established cardiovascular disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of MPO. When apolipoprotein A-I (apoA-I), the major HDL protein, was oxidized by MPO, its ability to promote cellular cholesterol efflux by ABCA1 was impaired. Moreover, oxidized apoA-I was unable to activate lecithin:cholesterol acyltransferase (LCAT), which rapidly converts free cholesterol to cholesteryl ester, a critical step in HDL maturation. Biochemical studies implicated tyrosine chlorination and methionine oxygenation in the loss of ABCA1 and LCAT activity by oxidized apoA-I. Oxidation of specific residues in apoA-I inhibited two key steps in cholesterol efflux from macrophages, raising the possibility that MPO initiates a pathway for generating dysfunctional HDL in humans.

Figure 1. HDL isolated from human atherosclerotic lesions and humans with established cardiovascular disease contained elevated levels of 3-chlorotyrosine

Human atherosclerotic tissue was obtained at surgery from subjects undergoing carotid endarterectomy. Plasma was obtained from healthy humans and humans with established coronary artery disease. HDL was isolated by sequential ultracentrifugation. Oxidized amino acids isolated from hydrolyzed HDL proteins were quantified by isotope dilution GC/MS with selected ion monitoring. Reproduced with permission from (29).

Figure 2. Chlorination of Tyr192 and Met oxidation are necessary for depriving apoA-I of its cholesterol efflux activity

(A) [³H]cholesterol efflux was measured in ABCA1-transfected BHK cells incubated with the indicated concentrations of wild-type (WT) or Tyr192Phe mutant apoA-I. (B) WT or Tyr192Phe mutant apoA-I was oxidized by the MPO-H₂O₂-chloride system (25:1, mol/mol, H₂O₂/apoA-I). Where indicated, apoA-I was incubated with the methionine sulfoxide reductase PilB. Reproduced with permission from (41).

Figure 3. Oxidation may alter the remodeling pathway of apoA-I

The conversion of lipid-free apoA-I to a lipid-associated form has been proposed to involve remodeling around the protein’s hairpin loops (21, 46). Based on this model, we suggest that MPO could inactivate the ABCA1 activity of apoA-I by oxidizing residues in or near its loop regions, which serve as hinges when the protein unfolds and refolds.

Figure 4. 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). Reproduced with permission from (56).

Figure 5. Mutation of Met148 to Leu protects apoA-I from the oxidative loss of LCAT activity

rHDL was prepared with wild-type (WT) or mutated apoA-I. (A) LCAT activity of Met-mutated rHDLs exposed to a 30-fold molar ratio of HOCl. (B) LCAT activity of Met-, Trp-, and Tyr-mutated rHDLs exposed to a 40-fold molar ratio of HOCl. DeltaW (ΔW), deletion all four Trp residues of apoA-I. 3M/L, Met86Leu/Met112Leu/Met148Leu mutant apoA-I. Reproduced with permission from (56).

Figure 6. Model of Met148 oxidation in apoA-I in LCAT inactivation

Oxidation of Met148 disrupts the central loop-like structure and affects the conformation of apoA-I. Modifying Met148 might disrupt the proper alignment of the hydrophobic face of repeat 6 in apoA-I, and then disrupt the maintenance and stability of the helix-bilayer and helix-helix interactions, which are key structural requirements for LCAT activation.

Figure 7. Oxidation of apoA-I by MPO inhibits two steps of reverse cholesterol transport

Oxidation of lipid-free apoA-I by MPO impairs the protein’s ability to transport cellular cholesterol by the ABCA1 pathway. Oxidation of lipid-associated apoA-I by MPO impairs its ability to activate LCAT.