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SUMMARYAutoantibodies to 65 kDa glutamic acid decarboxylase (GAD65) are produced in many patients with autoimmune polyendocrine syndrome type II (APS-II) or stiff-man syndrome (SMS) and are heterogeneous in their epitope specificities, recognizing both conformational and linear determinants. Major linear epitopes of GAD, which are recognized by autoantibodies in a minority of these patients, occur in the N-terminal and C-terminal regions. We have investigated antibody recognition of the N-and Ctermini of GAD65 in relation to their structural features as an approach to understanding what modifications to the native GAD structure may occur that facilitate the generation of antibodies specific to linear epitopes in these regions during the autoimmune pathogenesis. A monoclonal antibody specific to the N-terminus of GAD65 bound both native and denatured GAD in ELISA, whereas monoclonal and polyclonal antibodies specific to the C-terminus of GAD bound only denatured GAD. These antibodies were epitope mapped using random peptide phage-display libraries and the epitopes related to a previously proposed structural model of GAD65. This has led us to propose that the α -helical secondary structure of the C-terminus of GAD65 must be denatured to generate linear epitopes. In contrast, the N-terminus is both surface exposed and linear in the native structure, but may be masked by membrane interactions, which must be broken to facilitate recognition by B cells.

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Glutamic acid decarboxylase-65 (GAD-65) is a major target for autoantibodies and autoreactive T cells in patients with insulin-dependent diabetes mellitus (IDDM). Autoantibodies to GAD are also found in patients with stiff man syndrome (SMS) or polyendocrine autoimmunity (PE). The epitope specificities of autoantibodies to GAD in IDDM and SMS have been well documented, but the locations of autoantibody epitopes of GAD in PE patients have not been mapped. Thus, the properties of anti-GAD antibodies in PE patients (with or without diabetes) were investigated. The ability of PE serum antibodies to inhibit the binding of the mouse monoclonal antibody, GAD-6, to native GAD in ELISA was determined. For PE patients without diabetes, levels of inhibition of GAD-6 binding ranged from 0% to almost 70% and were unrelated to the level of binding of serum antibodies to GAD (P = 0.351) or to the functional affinities of these antibodies. This suggests differences in the epitope specificities of anti-GAD antibodies in different patients. Levels of inhibition were also unrelated to clinical condition. SMS antibodies showed similar levels of inhibition of GAD-6 binding. Similar analysis was applied to PE patients with diabetes and levels of inhibition of GAD-6 binding to GAD were determined. These ranged from 0% to 80%, and levels of inhibition were similar in samples taken before or after diabetes onset. There was no significant difference between anti-GAD antibodies from PE patients with or without diabetes in the range of abilities to inhibit GAD-6 binding to GAD, although the highest levels of inhibition were given by sera from non-diabetic patients. This raises the possibility of differential expression of subsets of anti-GAD antibodies in progressive versus slow or non-progressive anti-islet autoimmune responses. Serum antibodies of PE and SMS patients did not inhibit the binding of antibodies specific for the extreme C-terminus of GAD, indicating that this is not the site of the epitopes for the patients' antibodies or for GAD-6.

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