C-terminal clustering of autoantibody and T cell determinants on the structure of GAD65 provide insights into the molecular basis of autoreactivity

Objective: To gain structural insights into the autoantigenic properties of GAD65 in type 1 diabetes we analyzed experimental epitope mapping data in the context of the recently determined crystal structures of GAD65 and GAD67, to allow “molecular positioning” of epitope sites for Band T-cell reactivity. Research Design And Methods: Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs. Results: The C-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the C-terminal domain. Inclusion of epitope sites in the PLPand N-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAb to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T cell sequences restricted by HLA DRB1*0401 were aligned to solvent exposed regions of the GAD65 structure and co-localized within the two B-cell epitope clusters. The continuous C-terminal epitope region of GAD65 was structurally highly flexible, so differing markedly from the equivalent region of GAD67. Conclusions: Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of Band T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T cell reactivity.