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

Abstract

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 (p < 0.001) 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 1.56 × 10^{− 6} and 2.72 × 10^{− 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.

• Autoantibodies
• type 1 diabetes
• ROS-GAD₆₅
• reactive oxygen species

Acknowledgements

This study was supported by the Aligarh Muslim University authorities who provided the essential facilities for carrying out this research. Authors are thankful to Dr. Hussein Eissa Kaal, Faculty of Medical Sciences, Al-Gomail, 7th of April University, Zawia, Libya for giving valuable support during manuscript preparation.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.