Structural analysis of oxidatively modified protein as a oxidative stress probe

有研究认为，在动脉粥样硬化中，血浆低密度脂蛋白(LDL)在产生泡沫细胞之前要经过氧化修饰。低密度脂蛋白氧化生成各种反应性醛类产物，与低密度脂蛋白载脂蛋白B-100(Apo B)共价结合。在本研究中，我们分析了在低密度脂蛋白自氧化过程中产生的氧化胆固醇酯，并表征了它们与低密度脂蛋白载脂蛋白B赖氨酸残基的共价结合。此外，我们还提出了一种针对赖氨酸结合的氧化胆固醇酯的单抗，并测定了其在人类动脉粥样硬化病变中的产生，以探讨低密度脂蛋白的修饰机制。低密度脂蛋白与Cu2+的过氧化反应生成了主要的氧化胆固醇酯：9-氧酮酰胆固醇(9-ONC)和5-氧代戊酰胆固醇(5-OVC)。我们观察到，9-ONC和5-OVC的水平在12h达到峰值，此后显著下降。核心缩醛…的还原更多的LS伴随着(I)游离的7-酮胆固醇和7-酮胆固醇酯芯醛的形成，以及(Ii)载脂蛋白B结合的胆固醇和7-酮胆固醇的数量的增加，这表明胆固醇酯核心醛进一步转化为它们的7-酮胆固醇和载脂蛋白B结合的衍生物。为了检测蛋白结合的9-ONC，我们提出了针对9-ONC修饰蛋白的单抗(MAb2A81)，发现它能广泛识别蛋白结合的胆固醇酯核心醛。琼脂糖凝胶电泳和免疫印迹分析氧化的低密度脂蛋白清楚地显示了抗原性结构的形成。此外，对人动脉粥样硬化病变的免疫组织化学分析表明，病变内确实存在mAb2A81免疫反应物质，其中强烈的免疫反应主要定位于巨噬细胞源性泡沫细胞和动脉壁增厚的新生内膜。这项研究的结果表明，胆固醇酯核醛与低密度脂蛋白的结合可能代表了脂蛋白氧化修饰的共同过程。较少

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