Itabe H, Yamamoto H, Suzuki M, Kawai Y, Nakagawa Y, Suzuki A, Imanaka T, Takano T
Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-01, Japan.
J Biol Chem. 1996 Dec 27;271(52):33208-17. doi: 10.1074/jbc.271.52.33208.
Oxidatively modified low density lipoprotein (OxLDL) is known to be involved in atherogenesis. We have previously developed a murine monoclonal antibody, FOH1a/DLH3, which recognized oxidatively modified lipoproteins as well as foam cells in human atherosclerotic lesions (Itabe, H., Takeshima, E., Iwasaki, H., Kimura, J., Yoshida, Y., Imanaka, T., and Takano, T. (1994) J. Biol. Chem. 269, 15274-15279). The antigen of this monoclonal antibody was formed by peroxidation of phosphatidylcholine (PC), and the antigenic oxidized PC (OxPC) derivatives are thought to form complexes with polypeptides including apolipoproteins. OxLDL was measured by a sensitive sandwich enzyme-linked immunosorbent assay using the monoclonal antibody and anti-human apolipoprotein B antibody, in which antigenic OxPC competed with OxLDL. When antigenic activities of PC analogs were tested by the competition assay, 1-palmitoyl-2-(9-oxononanoyl) PC (9-CHO PC) and the hydroperoxide of egg PC potently inhibited the detection of OxLDL. 1-Palmitoyl-2-linoleoyl PC was oxidized with ferrous ion and ascorbic acid, and the antigenic products were purified from the OxPC extracts on high pressure liquid chromatography columns and subsequently analyzed by laser desorption mass spectrometry. Molecular weight determination and retention times of high pressure liquid chromatography suggest that one of these products was 9-CHO PC. Other products are thought to be 8-carbon aldehyde, dihydroxy, and ketohydroxy derivatives of PC. When a C-terminal 16-mer synthetic peptide of the 70-kDa peroxisomal membrane protein was simply incubated with 9-CHO PC, it was found to be reactive in a sandwich enzyme-linked immunosorbent assay using FOH1a/DLH3 and an anti-peptide antiserum. These results suggest that the anti-OxLDL monoclonal antibody FOH1a/DLH3 reacts with several oxidized products of PC including aldehyde derivatives of PC, which covalently modify polypeptides.
氧化修饰的低密度脂蛋白（OxLDL）已知参与动脉粥样硬化的发生。我们之前开发了一种鼠单克隆抗体FOH1a/DLH3，它能识别氧化修饰的脂蛋白以及人类动脉粥样硬化病变中的泡沫细胞（伊田博、竹岛惠、岩崎浩、木村纯、吉田洋、今中俊、高野哲（1994年）《生物化学杂志》269卷，第15274 - 15279页）。这种单克隆抗体的抗原是由磷脂酰胆碱（PC）过氧化形成的，并且抗原性氧化PC（OxPC）衍生物被认为会与包括载脂蛋白在内的多肽形成复合物。使用该单克隆抗体和抗人载脂蛋白B抗体，通过灵敏的夹心酶联免疫吸附测定法来检测OxLDL，其中抗原性OxPC与OxLDL竞争。当通过竞争测定法测试PC类似物的抗原活性时，1 - 棕榈酰 - 2 -（9 - 氧代壬酰基）PC（9 - CHO PC）和鸡蛋PC的氢过氧化物能有效抑制OxLDL的检测。将1 - 棕榈酰 - 2 - 亚油酰基PC用亚铁离子和抗坏血酸氧化，然后在高压液相色谱柱上从OxPC提取物中纯化抗原性产物，并随后通过激光解吸质谱法进行分析。高压液相色谱的分子量测定和保留时间表明这些产物之一是9 - CHO PC。其他产物被认为是PC的8 - 碳醛、二羟基和酮羟基衍生物。当将70 kDa过氧化物酶体膜蛋白的C末端16聚体合成肽简单地与9 - CHO PC一起孵育时，发现在使用FOH1a/DLH3和抗肽抗血清的夹心酶联免疫吸附测定中它具有反应性。这些结果表明，抗OxLDL单克隆抗体FOH1a/DLH3与PC的几种氧化产物反应，包括PC的醛衍生物，这些醛衍生物会共价修饰多肽。
