Covalent Binding of Oxidized Cholesteryl Esters to Protein: IMPLICATIONS FOR OXIDATIVE MODIFICATION OF LOW DENSITY LIPOPROTEIN AND ATHEROSCLEROSIS

Yoshichika Kawai,Akiko Saito,Noriyuki Shibata,Makio Kobayashi,Satoshi Yamada,Toshihiko Osawa,Koji Uchida

doi:10.1074/jbc.m212426200

Abstract

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.

Highlights

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis
The formation of lipid peroxidation products bound to proteins in vascular lesions, such as the atherosclerotic lesion, is a phenomenon common in most, if not all, types of vascular damage associated with oxidative stress
The possibility that aldehydic molecules generated from lipid peroxidation play a role in the pathogenesis of atherosclerosis is suggested by the facts that (i) the level of reactive aldehydes increases in plasma in relation to extensive aortic atherosclerosis, (ii) high concentrations of aldehydes can be generated during the oxidation of LDL phospholipids, (iii) the structural and functional changes associated with the in vitro oxidation of LDL can be produced by the direct interaction of LDL with aldehydes, and (iv) the reaction of aldehydes with a critical number of lysine residues of the LDL apolipoprotein B-100 (apoB) produces internalization by the scavenger receptor of human monocyte-macrophages and subsequent intracellular accumulation of the lipoprotein-derived cholesteryl ester

Summary

Covalent Binding of Oxidized Cholesteryl Esters to Protein

IMPLICATIONS FOR OXIDATIVE MODIFICATION OF LOW DENSITY LIPOPROTEIN AND ATHEROSCLEROSIS*□S. It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The primary products of lipid peroxidation, lipid hydroperoxides [6], can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals, giving rise to the formation of short-chain, unesterified aldehydes [6, 7] or a second class of aldehydes still esterified to the parent lipid These esterified aldehydes are commonly termed core aldehydes [8]. Based on the fact that the cholesteryl ester- and 7-ketocholesteryl ester core aldehydes of varying chain length were identified in human atheromas [13, 14], we raised a monoclonal antibody directed to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION