title: Immuno-chemistry of hydroxyl radical modified GAD-65: A possible role in experimental and human diabetes mellitus.
abstract: The repertoire of known auto-antigens is limited to a very small proportion of all human proteins, and the reason why only some proteins become auto-antigens is unclear. The 65 kDa isoform of the enzyme glutamic acid decarboxylase (GAD-65) is a major auto-antigen in type I diabetes, and in various neurological diseases. Most patients with type I diabetes (70-80%) have auto-antibodies against GAD-65, which often appear years before clinical onset of the autoimmune diabetes. Thus, the aim of the study is to focus on the immunogenicity of GAD65 and its reactive oxygen species (ROS) conformer in STZ-induced diabetic rats and on human diabetic patients. In the present study, GAD-65 was modified by hydroxyl radical following Fenton's reaction. The modifications in the structure of the GAD-65 are supported by UV-vis and fluorescence spectral studies. Immunogenicity of both native and hydroxyl radical modified GAD-65 (ROS-GAD-65) was studied in experimental rabbits and was confirmed by inducing type I diabetes in experimental male albino rats using streptozotocin (45 mg/kg). We found that ROS-GAD-65 was a better immunogen as compared to the native GAD-65. A considerable high binding to ROS-GAD-65 was observed as compared to native GAD-65 in both the serum antibodies from diabetes animal models and as well as in the serum samples of type I diabetes. Hydrogen peroxide under the exposure of UV light produces hydroxyl radical (·OH) which is most potent oxidant, and could cause protein damage (GAD-65) to the extent of generating neo-epitopes on the molecule, thus making it immunogenic.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India.
authors.lastname: Moinuddin
authors.affiliation: Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India.
authors.forename: Nadeem A
authors.initials: NA
authors.lastname: Ansari
authors.affiliation: Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India.|Department of Biochemistry, King George Medical University, Lucknow, Uttar Pradesh, 226003, India.
authors.forename: Uzma
authors.initials: U
authors.lastname: Shahab
authors.affiliation: Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India.
authors.forename: Safia
authors.initials: S
authors.lastname: Habeeb
authors.affiliation: Department of Biochemistry, Faculty of Medicine, J.N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, 202002, India.|Department of Bio-Sciences, Integral University, Lucknow, Uttar Pradesh, 226026, India.
authors.forename: Saheem
authors.initials: S
authors.lastname: Ahmad
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000201:enzymology* / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000201:enzymology* / Q000276:immunology; D005260:Female; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000276:immunology; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D017665:Hydroxyl Radical / Q000737:chemistry*; D008297:Male; D010084:Oxidation-Reduction; D011817:Rabbits; D051381:Rats
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2
keywords: ELISA; OH radical; ROS-GAD-65; glutamic acid decarboxylase-65 (GAD-65); immunogenicity; streptozotocin; type I diabetes