A study on oxidative modification of low-density lipoprotein and the response of vascular endothelial cells

自由基引起低密度脂蛋白的氧化修饰，低密度脂蛋白在动脉粥样硬化形成的早期阶段起着关键作用。然而，氧化的低密度脂蛋白是如何启动动脉粥样硬化的确切机制，以及是什么导致内皮细胞内的异常信号仍不清楚。在这项研究中，我们对低密度脂蛋白是如何被氧化的，什么样的修饰对内皮细胞起着重要的作用进行了实验。首先，我们检测了低密度脂蛋白颗粒上的糖链结构，确定了八种类型的糖链。天然低密度脂蛋白和氧化低密度脂蛋白在结构上无显著差异。当我们通过添加酶来释放甘露糖时，观察到滞后期显著延长。其次，我们分析了低密度脂蛋白氧化产生的每个片段的氨基酸序列，发现各种血清蛋白如载脂蛋白A-I、纤维蛋白、α2-巨球蛋白、结合珠蛋白等与低密度脂蛋白非特异性结合。…最后，我们建立了一种用凝胶渗透层析法纯化被氧化低密度脂蛋白污染的低密度脂蛋白的方法，得到了氧化低密度脂蛋白的纯化片段。我们已经发现，氧化低密度脂蛋白刺激内皮细胞表达血管内皮细胞黏附分子-1。在动脉粥样硬化形成的各个早期阶段，VCAM-1对巨噬细胞的黏附起着关键作用。因此，我们进行了实验，以检测加入纯化的氧化型低密度脂蛋白是否会提高VCAM-1的信使RNA(MRNA)水平。首先，我们通过改变IL-1的浓度和孵育时间向内皮细胞中加入IL-1。结果表明，孵育2小时是诱导VCAM-1基因表达的最有效时间。最后，我们成功地找到了最合适的条件，并观察了加入氧化型低密度脂蛋白片段对VCAM-1mRNA的诱导。另外，本研究的结果是找到了一种估计载脂蛋白B-100(apoB-100)三维结构的方法。由于其巨大的尺寸和不可溶的性质，其三维结构一直很难推断。该方法是基于以下考虑而开发的，即在非极性环境中，亲水性较高的α-螺旋更容易相互结合并成束。建立了两个圆柱体模型，根据疏水性和氨基酸上的电荷计算了它们之间的相互作用力(E)。因此，得到了一系列E值，范围在383到7315之间。极高的E值被认为代表α-螺旋的区域。如果我们将这些区域紧密结合在一起，就可以确定5个α-螺旋束的候选区域。这些区域与其他人报道的apoB-100的脂结合基序匹配得很好。因此，我们的算法对预测apoB-100的高阶结构是有用的，正确使用该方法可能有助于识别膜蛋白潜在的脂结合结构域。较少

Radical species cause oxidative modification of low-density lipoprotein(LDL), which plays a key role in the very early stage of atherogenesis. However, the exact mechanism how oxidized LDL acts as an initiator of atherosclerosis and what causes abonormal signals in the endothelial cells are not still clear. In this study, we conducted experiments as how LDL is oxidized, what kind of modification plays an important role for the endothelial cells.First we examined the structure of sugar chain on the LDL particles and identified eight types of sugar chains. There was no significant difference between native LDL and oxidized LDL in the structure. When we released mannose by adding an enzyme, a significant prolongation of the lag phase was observed Second we analyzed the amino acid sequence of each fragment that was generated by oxidation of LDL, and found that various serum proteins such as apolipoprotein A-I, fibrin, α2-macrqglobulin, haptoglobin, etc. were non-specifically bound to LDL. … More Finally we established a method to purify thus contaminated LDL by using gel permeation chromatography, and obtained purified fragments of oxidized LDL.We already found that vascular endothelial adhesion molecule-1(VCAM-1) expressed on the endothelial cells by the stimulation of oxidized LDL. VCAM-1 plays a key role to adhere macophages in the every early stage of atherogenesis. So we conducted experiments to examine whether the level of messenger RNA(mRNA) of VCAM-1 would rise or not by adding purified oxidized LDL. First we added IL-1 to the endothelial cells by changing its concentration and incubation time. It was found that two hours incubation was the most effective time to induce mRNA of VCAM-1. Finally we succeeded in finding the most appropriate condition and observing the induction of mRNA of VCAM-1 by adding fragments of oxidized LDL.Another result of the study was to find a method to estimate three-dimensional structure of apolipoprotein B-100(apoB-100). Because of its extraordinary size and insoluble nature, the three dimensional structure has been difficult to deduce. The method was developed based on the consideration that sites of α-helices with higher hydrophilicity are more likely to combine with each other and form bundles in the non-polar environment. Two cylinder models were established and the interactive force (E) between them was calculated according to hydrophobicity and the charge on the amino acids. Consequently, a series of values of E ranging from 383 to 7315 was obtained. Extremely high E values were considered to represent regions of α-helices. If we combined the regions in close proximity to each other, 5 candidates for α-helix bundles were identified. These regions matched well with the lipid-binding motifs of apoB-100 as reported by others. Thus, our algorithm was useful in predicting the higher-order structure of apoB-100, and proper use of this method may enable identification of potential lipid-associating domains of membrane proteins. Less