Rocky Strollo1,2, Chiara Vinci1,2, Mayda H Arshad1, David Perrett3, Claudio Tiberti4, Francesco Chiarelli5, Nicola Napoli2,6, Paolo Pozzilli7,8, Ahuva Nissim9. 1. Centre for Biochemical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK. 2. Endocrinology & Diabetes, University Campus Bio-Medico, via Alvaro del Portillo 21, 00128, Rome, Italy. 3. BioAnalysis, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK. 4. Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy. 5. Department of Pediatrics, University of Chieti, Ospedale Policlinico, Chieti, Italy. 6. Division of Bone and Mineral Diseases, Washington University in St Louis, St Louis, MO, USA. 7. Endocrinology & Diabetes, University Campus Bio-Medico, via Alvaro del Portillo 21, 00128, Rome, Italy. p.pozzilli@unicampus.it. 8. Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK. p.pozzilli@unicampus.it. 9. Centre for Biochemical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK. a.nissim@qmul.ac.uk.
Abstract
AIM/HYPOTHESIS: Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. METHODS: oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). RESULTS: MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. CONCLUSION/ INTERPRETATION: These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.
AIM/HYPOTHESIS: Insulin is the most specific beta cell antigen and a potential primary autoantigen in type 1 diabetes. Insulin autoantibodies (IAAs) are the earliest marker of beta cell autoimmunity; however, only slightly more than 50% of children and even fewer adults newly diagnosed with type 1 diabetes are IAA positive. The aim of this investigation was to determine if oxidative post-translational modification (oxPTM) of insulin by reactive oxidants associated with islet inflammation generates neoepitopes that stimulate an immune response in individuals with type 1 diabetes. METHODS: oxPTM of insulin was generated using ribose and various reactive oxygen species. Modifications were analysed by SDS-PAGE, three-dimensional fluorescence and MS. Autoreactivity to oxPTM insulin (oxPTM-INS) was observed by ELISA and western blotting, using sera from participants with type 1 or type 2 diabetes and healthy controls as probes. IAA was measured using the gold-standard radiobinding assay (RBA). RESULTS: MS of oxPTM-INS identified chlorination of Tyr16 and Tyr26; oxidation of His5, Cys7 and Phe24; and glycation of Lys29 and Phe1 in chain B. Significantly higher binding to oxPTM-INS vs native insulin was observed in participants with type 1 diabetes, with 84% sensitivity compared with 61% sensitivity for RBA. oxPTM-INS autoantibodies and IAA co-existed in 50% of those with type 1 diabetes. Importantly 34% of those with diabetes who were IAA negative were oxPTM-INS positive. Altogether, 95% of participants with type 1 diabetes presented with autoimmunity to insulin by RBA, oxPTM-INS or both. Binding to oxPTM-INS was directed towards oxPTM-INS fragments with slower mobility than native insulin. CONCLUSION/ INTERPRETATION: These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.
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