Glycoxidation of low-density lipoprotein generates cytotoxic adducts and elicits humoral response in type 2 diabetes mellitus

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 Nϵ-(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.