Autoantigenic reactivity of diabetes sera with a hybrid glutamic acid decarboxylase GAD67-65 molecule GAD67(1-101)/GAD65(96-585)

Glutamic acid decarboxylase (GAD) is a major autoantigen in insulin-dependent diabetes mellitus (IDDM). Two GAD isoforms exist, GAD65 and GAD67, which differ mostly in the first 100 amino acids of the amino terminus. IDDM sera are predominantly reactive with GAD65 but autoepitopes have been localised only to regions of GAD65 highly homologous with GAD67. In this study we investigated the contribution of the amino terminus to the IDDM epitope on GAD65, in order to test whether this region of GAD could explain the difference in reactivity between GAD65 and GAD67, A recombinant hybrid GAD molecule consisting of amino acids 1-101 of GAD67 and 96-585 of GAD65 was constructed and a truncated GAD65 was also constructed consisting of amino acids 98-585 of GAD65, The reactivity with the hybrid GAD molecule, GAD65 and GAD67, and truncated GAD65 was examined by radioimmunoprecipitation using 50 IDDM sera with known reactivity to purified porcine brain GAD, Over 90% of the IDDM sera were reactive with the hybrid GAD molecule confirming that the amino terminus of GAD65 does not contribute to the autoepitope and that the IDDM epitope is localised to the middle and carboxyl terminal domains of GAD65. Furthermore, evidence is presented that autoantibodies to GAD65 in IDDM sera react with an epitope formed on a dimeric configuration of the molecule.