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 (ÁOH) 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. V C 2015 IUBMB Life, 67(10): [746][747][748][749][750][751][752][753][754][755][756] 2015

Colorimetric and Fluorometric Profiling of Advanced Glycation End Products

“…Thus, it is highly significant to develop assays for the detection of AGEs, which can enable rapid and facile diagnosis. The most widely used methodologies for the measurement of AGEs are immunochemical methods such as immunohistochemistry, enzyme-linked immunosorbent autoantibody assay, 13,14 method also have been explored for the sensing of AGEs. 19−21 However, most of these laboratory-based methods are cumbersome and require trained personnel, preanalytical sample preparation, as well as sophisticated instrumentation to perform the assay.…”

“…Thus, it is highly significant to develop assays for the detection of AGEs, which can enable rapid and facile diagnosis. The most widely used methodologies for the measurement of AGEs are immunochemical methods such as immunohistochemistry, enzyme-linked immunosorbent autoantibody assay, , and bioanalytical methods such as fluorescence spectroscopy, UV–visible spectroscopy, surface plasmon resonance, boronate affinity chromatography, high-performance liquid chromatography (HPLC), liquid chromatography–mass spectroscopy (LC/MS), and fluorescent phenylboronate gel electrophoresis. − Biochemical techniques such as nitroblue tetrazolium assay and thiobarbituric acid colorimetric method also have been explored for the sensing of AGEs. − However, most of these laboratory-based methods are cumbersome and require trained personnel, preanalytical sample preparation, as well as sophisticated instrumentation to perform the assay. Herein, we propose a facile approach for the colorimetric detection of AGEs using cationic polythiophenes (PTs) and aptamers (Apts).…”

Impact of wedelolactone in the anti‐glycation and anti‐diabetic activity in experimental diabetic animals

“…In in vivo studies, the rats were injected with STZ intraperitoneally at a dosage of 70 mg/kg of body weight (13). Blood sera were separated by centrifuging it for 10 min at 3,000g and aliquots were stored at 220 8C for further use (17).…”

“…The animals were taken care of in standard laboratory with 12-h light-dark cycle at 25 6 1 8C and were given 2-week time period for acclimatization. After 2 weeks, 40 rats were injected with STZ at a dosage of 70 mg/kg of body weight intraperitoneally (13…”

Recent advances in detection of AGEs: Immunochemical, bioanalytical and biochemical approaches

“…AGE competitive ELISA allows rapid detection and estimation of AGE protein adducts. The determination of AGEs is similarly done as it is done for reactive oxygen species (ROS) protein adducts . The quantification of AGE adducts in protein samples is done by comparing its absorbance with a known AGE‐BSA standard curve.…”