Structural analysis of oxidatively modified protein as a oxidative stress probe

作为氧化应激探针的氧化修饰蛋白的结构分析

基本信息

批准号：
13660122
负责人：
UCHIDA Koji
金额：
$ 2.11万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660122/
关键词：
Oxidized cholesterol esters Low density lipoprotein Lipid peroxidation Oxidative modification of protein Reactive aldehydes Monoclonal antibody Lysine adduct Oxiative stress 免疫化学 NMR解析

项目摘要

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 (apo B). 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 apo B. 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 Cu^<2+> produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol (5-OVC) as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-OVC peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde leve … More ls was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester-core aldehydes, and (ii) the increase in the amounts of apo B-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester-core aldehydes were further converted to their 7-ketocholesterol and apo B-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody (mAb2A81) 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 mAb2A81 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. Less

已经提出，血浆低密度脂蛋白（LDL）在可以在动脉粥样硬化中产生泡沫细胞之前经过氧化物的修饰。 LDL的氧化产生了各种反应性醛产物，它们与LDL载脂蛋白B-100（APO B）共价结合。 In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autooxidation of LDL and Characterized their covalent binding to the lysine residuals of LDL apo B. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human动脉粥样硬化病变。 LDL与Cu^<2+>的过氧化产生了9-氧酮烯酰胆固醇（9-onc）和5-氧化羟基胆固醇（5-OVC），作为主要的氧化胆固醇酯。我们观察到，9-ONC和5-OVC的水平在12小时达到峰值，此后显着降低。 The reduction of the core aldehyde level … More ls was accomplished by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester-core aldehydes, and (ii) the increase in the amounts of apo B-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester-core aldehydes were further converted to their 7-ketocholesterol and APO B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占9剂修饰蛋白的单克隆抗体（MAB2A81），发现它广泛识别为蛋白质结合的蛋白质结合的胆固醇酯核核醛醛。琼脂糖凝胶电泳，然后对氧化的LDL进行免疫印迹分析清楚地证明了抗原结构的形成。此外，对人主动脉的动脉粥样硬化病变的免疫组织化学分析表明，具有MAB2A81的免疫反应性材料确实存在于病变中，其中强烈的免疫反应性主要位于巨噬细胞衍生的泡沫细胞中，并在巨噬细胞的泡沫细胞中，并具有静脉内膜的增厚植物膜的增厚。这项研究的结果表明，胆固醇酯核醛与LDL的结合可能代表脂蛋白氧化修饰的过程。较少的