Identification of Glutamic Acid Decarboxylase Autoantibody Heterogeneity and Epitope Regions in Type I Diabetes

Glutamic acid decarboxylase (GAD) is an autoantigen of the islet cell antibodies (ICAs) present in type I diabetes. GAD autoantibodies are also found in patients with stiffman syndrome and in certain ICA-positive individuals who rarely develop diabetes on long-term follow-up. This latter subset of ICA has been termed restricted or beta-cell-specific ICA because the antibodies react with only the beta-cells of the islet. By immunoprecipitation of recombinant GAD₆₅ and GAD₆₇ protein and protein fragments, 83% of sera from individuals with new-onset diabetes or prediabetes (n = 30) had GAD₆₅ autoantibodies, but only 26% had GAD₆₇ autoantibodies. In contrast, all restricted ICA sera (n = 6) had both GAD₆₅ and GAD₆₇ autoantibodies. In both types of sera, the binding of GAD₆₇ autoantibodies could be blocked by preincubation of the serum with GAD₆₅ and GAD₆₇, but the binding of GAD65 autoantibodies could not be blocked by preincubation with GAD₆₇. The titer of GAD₆₅ autoantibodies was much higher in the restricted ICA sera (titer > 1:1,000) than in the sera from individuals with new-onset diabetes or prediabetes (titer < 1:100) and was reflected by the greater amount of GAD₆₅ protein immunoprecipitated by restricted ICA sera (2.61 ± 1.39 U) compared with sera from individuals with new-onset diabetes (0.51 ± 0.34 U). The restricted ICA sera immunoprecipitated equimolar amounts of GAD₆₅ protein fragments, suggesting a non-conformational or linear epitope; epitope mapping localized the major epitope region to amino acids 361–442 and a second minor epitope region to amino acids 1–195. For the monoclonal antibody GAD₆, the GAD₆₅ epitope region localized to amino acids 529–585. In contrast, the sera from individuals with new-onset diabetes or prediabetes immunoprecipitated equal amounts of full-length GAD₆₅ protein and a GAD₆₅ fragment containing amino acids 188–585, but did not immunoprecipitate smaller GAD₆₅ protein fragments, which suggests an epitope(s) dependent on protein conformation. These results suggest that subsets of GAD autoantibodies exist and indicate a heterogeneity in the immune response to GAD.