Autoimmune response to AGE modified human DNA: Implications in type 1 diabetes mellitus

Abstract

Aims: Non-enzymatic glycation of DNA both in vivo and in vitro 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 Cu²⁺ and its auto-antibody binding was probed in Type 1 diabetes patients.

Methods: Glycation was carried out by incubating DNA with MG, Lys and Cu²⁺ 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.

Results: DNA glycation with MG, Lys and Cu²⁺ 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.

Conclusions: Glycation of DNA has resulted in structural perturbation causing generation of neo-antigenic epitopes thus recognizing auto-antibodies in diabetes.

Keywords: Auto-antibody; DNA; Diabetes mellitus; Glycation; MethylGlyoxal.

Figure 1

a) Full scan LC-MS spectral analysis of synthesized CEdG standard. b) Full scan LC-MS spectral analysis of hydrolyzed modified human DNA (1 mg/ml). c) Full scan LC-MS spectral analysis of hydrolyzed native human DNA (1 mg/ml).

Figure 2

a) Full scan ESI-MS spectral analysis of native human DNA (1 mg/ml) of HPLC resolved products. b) Full scan ESI-MS spectral analysis of modified human DNA (1 mg/ml) of HPLC resolved glycated products.

Figure 3

Direct binding ELISA of serum antibodies from diabetes mellitus (DM) patients to native human DNA (□) and MG-Lys-Cu²⁺ glycated human DNA (■). Serum from normal human subjects (NHS) served as control. The microtitre plates were coated with the MG-Lys-Cu²⁺ glycated human DNA (2.5 μg/ml). p < 0.001 vs native human DNA.

Figure 4

Band shift assay of IgG isolated from diabetes type 1 patient serum with MG-Lys-Cu²⁺ modified human DNA(a) and native human DNA(b). Varying concentrations of IgG were incubated with a constant amount of DNA (0.5 μg) for 2 h at 37 °C and overnight at 4 °C. Electrophoresis was carried out on 0.8% agarose gel for 2 h at 30 mA. Lane 1 contains native (or modified) human DNA while lanes 2–5 contain native or MG-Lys-Cu²⁺ modified human DNA with 20, 40, 60 and 80 μg of IgG from type 1 diabetes patient.