Covalent binding of oxidized cholesteryl esters to protein - Implications for oxidative modification of low density lipoprotein and atherosclerosis
Covalent binding of oxidized cholesteryl esters to protein - Implications for oxidative modification of low density lipoprotein and atherosclerosis
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DOI：
10.1074/jbc.m212426200
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发表时间：
2003-06-06
影响因子：
4.8
通讯作者：
Uchida, K
中科院分区：
文献类型：
--
作者：
Kawai, Y;Saito, A;Uchida, K
有人提出，血浆低密度脂蛋白（LDL）在动脉粥样硬化形成泡沫细胞之前会发生氧化修饰。LDL的氧化产生多种反应性脂代谢产物，其与LDL载脂蛋白B-100（apoB）共价结合。在本研究中，调查的机制，有助于LDL的修改，我们分析了氧化胆固醇酯在LDL的自氧化过程中产生的，其特征在于它们的共价结合的LDL载脂蛋白B的赖氨酸残基。此外，我们提出了一种单克隆抗体特异性赖氨酸结合氧化胆固醇酯，并确定其生产在人类动脉粥样硬化病变。LDL与Cu ~（2+）的过氧化反应产生9-氧壬酰胆固醇（9-ONC）和5-氧戊酰胆固醇作为主要的氧化胆固醇酯。我们观察到9-ONC和5-氧代戊酰胆固醇的水平在12小时达到峰值，此后显著降低。核心醛水平的降低伴随着（i）游离7-酮基胆固醇和7-酮基胆固醇酯核心醛的形成和（ii）apoB结合胆固醇和7-酮基胆固醇的量增加，表明胆固醇酯核心醛进一步转化为其7-酮基胆固醇和apoB结合衍生物。为了检测蛋白质结合的9-ONC，我们提出了针对9-ONC修饰的蛋白质的单克隆抗体2A 81，并发现其广泛识别蛋白质结合的胆固醇酯核心醛。琼脂糖凝胶电泳后，免疫印迹分析的氧化LDL清楚地表明抗原结构的形成。此外，免疫组化分析的动脉粥样硬化病变从人主动脉表明，免疫反应物质与mAb 2A 81确实存在于病变，其中强烈的免疫反应主要位于巨噬细胞源性泡沫细胞和动脉壁增厚的新生内膜。本研究的结果表明，胆固醇酯核心醛与LDL的结合可能代表了脂蛋白氧化修饰的共同过程。
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.