Aims: Both in vivo and in vitro non-enzymatic glycation of DNA produces free radicals, or glycoxidation. Neo-antigenic epitopes, which play a role in autoimmune diseases like diabetes mellitus, are produced as a result of DNA structural alterations caused by glycolysis. In this study, autoantibody binding to human placental DNA was probed in Type 1 diabetes patients after it was glycated with methylglyoxal (MG) and lysine (Lys) in the presence of Cu2+.
Methods: DNA was lysed by incubating it for 24 hours at 37 °C with MG, Lys, and Cu2+. LC-MS and ESI-MS techniques were used to investigate the pathway for Amadori formation and the carboxyethyl deoxyguanosine (CEdG) formation during the glycation reaction. In addition, a direct binding, competitive ELISA, and band shift assay were used to evaluate the autoantibodies' binding properties in diabetics.
Results: CEdG, a marker of DNA glycation, is produced when DNA is glycated with MG, Lys, and Cu2+, as demonstrated by LC-MS. The ESI-MS method confirmed the intermediate glycation stages. When compared to the native form of DNA, serum from diabetic patients exhibited enhanced binding and specificity for glycated DNA.
Conclusions: DNA glycation has altered the structure, resulting in the generation of neo-antigenic epitopes that recognize diabetes-associated autoantibodies.
