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

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Abstract
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.

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