Biophysical changes in methylglyoxal modified fibrinogen and its role in the immunopathology of type 2 diabetes mellitus

Abstract

Methylglyoxal (MG), a highly reactive dicarbonyl metabolite gets generated during glucose oxidation and lipid peroxidation, which contributes to glycation. In type 2 diabetes mellitus (T2DM), non-enzymatic glycosylation of proteins mediated by hyperglycemia results in the pathogenesis of diabetes-associated secondary complications via the generation of AGEs. Under in vitro conditions, MG altered the tertiary structure of fibrinogen. High-performance liquid chromatography (HPLC) and liquid chromatography mass spectroscopy (LCMS) studies confirmed the generation of N-(carboxymethyl) lysine, N-(carboxyethyl) lysine, hydroimidazolone, pentosidine and argpyrimidine in the modified protein. The altered fibrinogen structure upon glycation was further confirmed by confocal microscopy and nuclear magnetic resonance spectra (NMR). MG-Fib was found to be more immunogenic, as compared to its native analogue, in the immunological studies conducted on experimental rabbits. Our results reflect the presence of neo-antigenic determinants on modified fibrinogen. 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 (IgG) confirmed the enhanced and specific immunogenicity of MG-Fib. Studies on interaction of MG-Fib with the circulating auto-antibodies from T2DM patients showed high affinity of serum antibodies toward MG-Fib. This study suggests a potent role of glycoxidatively modified fibrinogen in the generation of auto-immune response in T2DM patients.