Antibodies to post-translationally modified insulin in type 1 diabetes.
OBJECTIVE
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.


CONCLUSIONS
These data suggest that oxPTM-INS is a potential autoantigen in individuals with new-onset type 1 diabetes.
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.|Endocrinology & Diabetes, University Campus Bio-Medico, via Alvaro del Portillo 21, 00128, Rome, Italy.
Rocky
R
Strollo
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.|Endocrinology & Diabetes, University Campus Bio-Medico, via Alvaro del Portillo 21, 00128, Rome, Italy.
Chiara
C
Vinci
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.
Mayda H
MH
Arshad
BioAnalysis, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.
David
D
Perrett
Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy.
Claudio
C
Tiberti
Department of Pediatrics, University of Chieti, Ospedale Policlinico, Chieti, Italy.
Francesco
F
Chiarelli
Endocrinology & Diabetes, University Campus Bio-Medico, via Alvaro del Portillo 21, 00128, Rome, Italy.|Division of Bone and Mineral Diseases, Washington University in St Louis, St Louis, MO, USA.
Nicola
N
Napoli
Endocrinology & Diabetes, University Campus Bio-Medico, via Alvaro del Portillo 21, 00128, Rome, Italy. p.pozzilli@unicampus.it.|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.
Paolo
P
Pozzilli
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.
Ahuva
A
Nissim
D000293:Adolescent; D017668:Age of Onset; D000595:Amino Acid Sequence; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology* / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007328:Insulin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D007330:Insulin Antibodies / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D011869:Radioligand Assay
D000939:Epitopes; D007328:Insulin; D007330:Insulin Antibodies
Insulin autoantibodies; Oxidative stress; Post-translational modification; Reactive oxygen species; Type 1 diabetes