Ahmad Saheem, Uddin Moin, Habib Safia, Shahab Uzma, Alam Khursheed, Ali Asif
Department of Biochemistry, J.N. Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, India.
Department of Biosciences/Biochemistry, Integral University, Lucknow, UP, India.
J Clin Transl Endocrinol. 2014 Jun 25;1(3):66-72. doi: 10.1016/j.jcte.2014.05.002. eCollection 2014 Sep.
Non-enzymatic glycation of DNA both and results in generation of free radicals, known as glycoxidation. Glycoxidation leads to structural perturbation of DNA resulting in generation of neo-antigenic epitopes having implication in autoimmune disorders like diabetes mellitus. In this study human placental DNA was glycated with methylglyoxal (MG) and lysine (Lys) in the presence of Cu and its auto-antibody binding was probed in Type 1 diabetes patients.
Glycation was carried out by incubating DNA with MG, Lys and Cu for 24 h at 37 °C. Carboxyethyl deoxyguanosine (CEdG) formed in glycation reaction was studied by LC-MS and the pathway for Amadori formation was studied by ESI-MS techniques. Furthermore, binding characteristics of auto-antibodies in diabetes patients were assessed by direct binding, competitive ELISA and band shift assay.
DNA glycation with MG, Lys and Cu results in the formation of CEdG (marker of DNA glycation) which was confirmed by LC-MS. The intermediate stages of glycation were confirmed by ESI-MS technique. Serum from diabetes patients exhibited enhanced binding and specificity for glycated DNA as compared to native form.
Glycation of DNA has resulted in structural perturbation causing generation of neo-antigenic epitopes thus recognizing auto-antibodies in diabetes.
DNA的非酶糖基化作用会产生自由基，即糖氧化反应。糖氧化会导致DNA结构紊乱，进而产生新抗原表位，这与糖尿病等自身免疫性疾病有关。在本研究中，在铜存在的情况下，用人胎盘DNA与甲基乙二醛（MG）和赖氨酸（Lys）进行糖基化反应，并检测1型糖尿病患者中其自身抗体的结合情况。
通过将DNA与MG、Lys和铜在37℃孵育24小时进行糖基化反应。采用液相色谱-质谱联用（LC-MS）研究糖基化反应中形成的羧乙基脱氧鸟苷（CEdG），并采用电喷雾电离质谱（ESI-MS）技术研究阿马多里产物的形成途径。此外，通过直接结合、竞争性酶联免疫吸附测定（ELISA）和凝胶迁移试验评估糖尿病患者自身抗体的结合特性。
用MG、Lys和铜对DNA进行糖基化反应会形成CEdG（DNA糖基化的标志物），这通过LC-MS得到证实。糖基化的中间阶段通过ESI-MS技术得到证实。与天然形式相比，糖尿病患者的血清对糖基化DNA表现出更强的结合能力和特异性。
DNA糖基化导致结构紊乱，产生新抗原表位，从而在糖尿病中识别自身抗体。
