Characterization of hydroxyl radical modified GAD65: A potential autoantigen in type 1 diabetes

Glutamic acid decarboxylase-65 (GAD65) is an immunological marker of type 1 autoimmune diabetes. High titre of autoantibodies against GAD65 (GAD65Abs) have also been detected in some other autoimmune diseases. In search of a potential immunological marker of type 1 diabetes, in vitro GAD65 was modified by hydroxyl radical followed by the study of structural and conformational perturbed protein by different spectroscopic techniques (UV, fluorescence and CD) and thermal denaturation profile. Binding studies of circulating autoantibodies from diabetic groups (type 1 and type 2) with native and reactive oxygen species (ROS) modified GAD65, exhibited high recognition of type 1 diabetic serum autoantibodies with modified antigen (p0.001) over unmodified GAD65. Binding specificity of isolated IgG of patients (n=17) from each diabetic group and control group (n=10) was checked by inhibition enzyme linked immunosorbent assay (ELISA) and quantitative precipitin titration assay. Relative affinity of ROS-GAD65Abs for modified and native GAD65 was in the order of 1.5610-6 and 2.7210-7 M, as calculated by Langmuir plot. In coherence, ROS oxidation of GAD65 causes conformational perturbation, generating highly immunogenic unique neoepitopes that may be one of the factors in antigen-driven induction of type 1 diabetes autoantibodies that can serve as a potential marker in early diagnosis/prognosis of the disease.