Abstract

This study elucidates the immunological implications of methylglyoxal (MGO)-modified low-density lipoprotein (LDL) in type 2 diabetes mellitus (T2DM) patients. Under in vitro modifications, MGO altered the tertiary structure of LDL. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) and phenanthrenequinone assays confirmed lysine and arginine residues as main targets of MGO in LDL. High-performance liquid chromatography (HPLC) and Liquid chromatography-mass spectrometry (LC-MS) studies confirmed the generation of N^ϵ-(carboxymethyl) lysine in the modified protein. Comet assay showing increased tail length of DNA in lymphocytes inferred the cytotoxicity of MGO-LDL. The easy penetration of MGO-LDL into the nucleus is possibly a consequence of its reduced size, postmodification, as observed from hydrodynamic radii studies in Dynamic light scattering (DLS) experiments. MGO-LDL was found to be more immunogenic, as compared to native LDL, in immunological studies conducted on experimental rabbits. Our results reflect the presence of neo-antigenic determinants on modified LDL. Competitive inhibition enzyme-linked immunosorbent assay suggested the presence of neo-epitopes with marked immunogenicity eliciting specific immune response. Binding studies on purified immunoglobulin G confirmed the enhanced and specific immunogenicity of MGO-LDL. Studies on interaction of MGO-LDL with the circulating auto-antibodies from T2DM patients showed high affinity of serum antibodies toward MGO-LDL. This study suggests a potent role of glycoxidatively modified LDL in the generation of auto-immune response in T2DM patients.

中文翻译：

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低密度脂蛋白的糖氧化产生细胞毒性加合物并引起2型糖尿病的体液反应

摘要

这项研究阐明了甲基乙二醛（MGO）修饰的低密度脂蛋白（LDL）在2型糖尿病（T2DM）患者中的免疫学意义。在体外修饰下，MGO改变了LDL的三级结构。2,4,6-三硝基苯磺酸（TNBS）和菲醌测定证实了赖氨酸和精氨酸残基是LDL中MGO的主要靶标。高效液相色谱法（HPLC）和液相色谱-质谱（LC-MS）研究证实n的代^ε-（羧甲基）赖氨酸在修饰的蛋白质中。彗星试验显示，淋巴细胞中DNA的尾巴长度增加，提示MGO-LDL具有细胞毒性。从动态光散射（DLS）实验中的流体动力学半径研究可以看出，MGO-LDL容易渗透到核中可能是其尺寸减小，后修饰的结果。在对实验兔进行的免疫学研究中，与天然LDL相比，发现MGO-LDL具有更高的免疫原性。我们的结果反映了修饰的LDL上新抗原决定簇的存在。竞争性抑制酶联免疫吸附试验表明，新表位的存在具有显着的免疫原性，可引起特异性免疫反应。对纯化的免疫球蛋白G的结合研究证实了MGO-LDL的增强和特异性免疫原性。MGO-LDL与来自T2DM患者的循环自身抗体相互作用的研究表明，血清抗体对MGO-LDL具有高度亲和力。这项研究表明，糖氧化修饰的LDL在T2DM患者自身免疫应答的产生中具有强大作用。