Abstract

Aims/hypothesis Insulinoma-associated protein 2 (IA-2) is a major target of autoimmunity in type 1 diabetes. When first detected, IA-2-autoantibodies commonly bind epitopes in the juxtamembrane (JM) domain of IA-2 and antibody responses subsequently spread to the tyrosine phosphatase domain. Definition of structures of epitopes in the JM domain, and genetic requirements for autoimmunity to these epitopes, is important for our understanding of initiation and progression of autoimmunity. The aims of this study were to investigate the contribution of individual amino acids in the IA-2 JM domain to antibody binding to these epitopes and the role of HLA genotypes in determining epitope specificity.
Methods Regions of the JM domain recognised by autoanti- bodies were identified by peptide competition and inhibitory effects of alanine substitutions of residues within the JM re- gion. Antibody binding was determined by radioligand bind- ing assays using sera from patients genotyped for HLA-DRB1 and -DQB1 alleles.
Results Patients were categorised into two distinct groups of JM antibody reactivity according to peptide inhibition. Inhibition by substitutions of individual amino acids within the JM domain differed between patients, indicating heteroge- neity in epitope recognition. Cluster analysis defined six groups of residues having similar inhibitory effects on anti- body binding, with three clusters showing differences in pa- tients affected or unaffected by peptide. One cluster demon- strated significant differences in antibody binding between HLA-DRB1*04 and HLA-DRB1*07 patients and within DRB1*04 individuals; antibody recognition of a second clus- ter depended on expression of HLA-DQB1*0302. Conclusions/interpretation The results identify amino acids contributing to distinct epitopes on IA-2, with both HLA-DR and HLA-DQ alleles influencing epitope specificity.