Model IgG Monoclonal Autoantibody-Anti-Idiotype Pair for Dissecting the Humoral Immune Response to Oxidized Low Density Lipoprotein
Model IgG Monoclonal Autoantibody-Anti-Idiotype Pair for Dissecting the Humoral Immune Response to Oxidized Low Density Lipoprotein
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DOI：
10.1089/hyb.2011.0058
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发表时间：
2012-04-01
期刊：
影响因子：
--
通讯作者：
Haskard, Dorian O.
中科院分区：
文献类型：
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作者：
Chang, Shang-Hung;Johns, Michael;Haskard, Dorian O.
越来越多的证据表明，抗氧化形式的低密度脂蛋白（oxLDL）的IgG自身抗体在动脉粥样硬化性疾病的病理生理中起作用。然而，对其结构和功能的认识不足是一个关键的空白。利用年老LDL受体缺乏的动脉粥样硬化小鼠，我们分离出一种新的抗oxLDL的IgG3k（命名为MAb LO1）。LO1与铜氧化LDL发生反应，但与天然LDL的反应最小。进一步分析表明，MAb LO1在体外也能与丙二醛偶联LDL （MDA-LDL）发生反应，MDA-LDL是铜氧化LDL制剂中已知的关键表位。通过筛选表达单链可变区抗体（scFv）的噬菌体文库，我们选择了一种抗独特型scFv（指定为H3），它可以中和MAb LO1与MDA-LDL的结合。H3与不相关的对照scFv C12之间的氨基酸替换表明，H3中的CDRH2、CDRH3和CDRL2中的残基都是MAb LO1结合的关键，这与H3上的构象表位涉及重链和轻链的情况一致。H3 CDRH2和CDRL2与主要LDL蛋白apoB的氨基酸序列比较，显示同源序列，表明H3与MDA-LDL上的MAb LO1结合位点具有结构相似性。免疫细胞化学染色显示MAb LO1结合小鼠和人类动脉粥样硬化病变的表位。因此，单抗LO1-H3组合为分析与动脉粥样硬化相关的单个IgG自身抗体的结构和功能提供了一个非常有前途的模型。
Increasing evidence implicates IgG autoantibodies against oxidized forms of low density lipoprotein (oxLDL) in the pathophysiology of atherosclerotic arterial disease. However, insufficient knowledge of their structure and function is a key gap. Using an elderly LDL receptor-deficient atherosclerotic mouse, we isolated a novel IgG3k against oxLDL (designated MAb LO1). LO1 reacts with copper-oxidized LDL, but minimally with native LDL. Further analysis showed that MAb LO1 also reacts in vitro with malondialdehyde-conjugated LDL (MDA-LDL), a known key epitope in copper-oxidized LDL preparations. By screening a phage library expressing single chain variable region antibodies (scFv), we selected an anti-idiotype scFv (designated H3) that neutralizes MAb LO1 binding to MDA-LDL. Amino acid substitutions between H3 and an irrelevant control scFv C12 showed that residues in the H3 CDRH2, CDRH3, and CDRL2 are all critical for MAb LO1 binding, consistent with a conformational epitope on H3 involving both heavy and light chains. Comparison of amino acids in H3 CDRH2 and CDRL2 with apoB, the major LDL protein, showed homologous sequences, suggesting H3 has structural similarities to the MAb LO1 binding site on MDA-LDL. Immunocytochemical staining showed that MAb LO1 binds epitopes in mouse and human atherosclerotic lesions. The MAb LO1-H3 combination therefore provides a very promising model for analyzing the structure and function of an individual IgG autoantibody in relation to atherosclerosis.