Molecular and structural characterization of autoreactive epitopes in human glutamic acid decarboxylase (GAD 65)

The goal of this thesis is to gain a molecular understanding of the autoreactivity to glutamic acid decarboxylase 65 (GAD65), which plays a major role in two human diseases, type I diabetes and stiff-man syndrome. Homolog scanning mutagenesis was used to fine map distinct conformational and linear epitopes recognized by a panel of sixteen human monoclonal antibodies derived from patients and eight murine monoclonal antibodies derived from immunized Balb/c mice. The high resolution epitope mapping together with two- and three-dimensional structure prediction resulted in a model of the GAD65 dimer which has structural similarities to ornithine decarboxylase in the co-enzyme binding middle domain and to dialkylglycine decarboxylase in the C-terminal domain. A comparative study of GAD65 antibodies in prediabetic children, newly diagnosed individuals, and long-term diabetics revealed multiple epitopes are recognized throughout all disease stages and that waves of autoimmunity target different regions of the molecule during chronic inflammation. Amongst children who had recently developed GAD65 antibodies, multiple epitopes are recognized suggesting that epitope spreading is a very rapid event and/or that there are multiple co-dominant epitope regions in GAD65. The identified epitopes localize to multiple hydrophilic patches and reveal a spectrum of human autoreactivity to GAD65, which is not confined to one dominant region, but rather involves most of the surface of the folded protein, suggesting that GAD65 is mainly presented to B cells as a native rather than degraded antigen. Together, these findings establish a useful paradigm for the study of autoreactivity in other human autoimmune diseases.