The long-term objective of this continued application is to identify the role of beta cell autoantigens in type 1 (insulin-dependent) diabetes mellitus. The investigator's previous research resulted in the discovery of the 64K antigen, the cloning and demonstration that it was a novel glutamic acid decarboxylase (GAD) isoform, GAD65. The GAD65 cDNA was used to develop a novel radiobinding assay using 35S-or 3H-GAD65 prepared by coupled in vitro transcription translation (IVTT), an approach now also proven to be applicable to other cloned autoantigens such as IA-2, 21-hydroxylase and transglutaminase. The conformation dependent GAD65Ab complicates the epitope analysis since deletion mutants cannot be used. They therefore developed molecular hybrids between GAD65 and GAD67 and employed both natural variants of GAD65 (mouse, rat, human) and variants created by site-directed mutagenesis swapping GAD65 with GAD67 residues. Preliminary data demonstrate that progression to type 1 diabetes is associated with GAD65Ab directed to unique antibody epitopes and that healthy individuals with GAD65Ab tend to have multiple species reactive GAD65Ab. Furthermore, children with new onset type 1 diabetes have GAD65Ab which are uniquely directed to a C-terminal GAD65 epitope controlled by 2-3 amino acid residues.
The specific aims are to test the hypotheses 1) that the GAD65Ab response in healthy individuals is directed to the N-terminal end of GAD65 demonstrating antibody epitope spreading not associated with progression; 2) that the progression to type 1 diabetes is associated with GAD65Ab to the middle portion and C-terminal end of GAD65, demonstrating a maturation of the antibody response towards unique C-terminal amino acid residues, and 3) that epitope-specific GAD65Ab are associated with HLA and other putative diabetes genes and marks the specific loss of beta cells in individuals diagnosed with diabetes. They have identified the C-terminal end conformational epitope and will determine genetic and other factors which are associated with the development of type 1 diabetes in relation to the change in GAD65Ab affinity of isotype and subtype-specific GAD65Ab. These studies will lead to a better understanding of the importance of multiple antibody positivity in relation to genetic predisposition and factors accelerating or decelerating disease progression. The epitope-specific GAD65Ab in individuals progressing or not to type 1 diabetes will therefore be analyzed in relation to IA-2Ab and insulin autoantibodies (IAA). The significance of this research is to understand the mechanisms of the early immune reaction to GAD65 and the subsequent development of GAD65Ab which may either result in progression to type 1 diabetes and loss of beta cells or antibody epitope spreading without progression to diabetes.
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