A diabetes-related epitope of GAD65: a major diabetes-related conformational epitope on GAD65.

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is a major autoantigen in type 1 diabetes, and most patients have serum antibodies reactive with conformational epitopes on the GAD65 molecule. The aims of this study were to prepare mutants of GAD65 to further localize the type 1 diabetes epitope in the region of the PEVKEK loop of GAD65 and to identify the particular amino acids within the epitope that are recognized by autoimmune diabetes sera.

Enzymatic characterization of a recombinant isoform hybrid of glutamic acid decarboxylase (rGAD67/65) expressed in yeast.

BACKGROUND AND AIMS: Glutamic acid decarboxylase (GAD, EC 4.1.1.15) catalyses the conversion of glutamate to gamma-aminobutyric acid (GABA). The 65 kDa isoform, GAD65 is a potent autoantigen in type 1 diabetes, whereas GAD67 is not. A hybrid cDNA was created by fusing a human cDNA for amino acids 1-101 of GAD67 to a human cDNA for amino acids 96-585 of GAD65; the recombinant (r) protein was expressed in yeast and was shown to have equivalent immunoreactivity to mammalian brain GAD with diabetes sera. We here report on enzymatic and molecular properties of rGAD67/65. METHODS: Studies were performed on enzymatic activity of rGAD67/65 by production of 3H-GABA from 3H-glutamate, enzyme kinetics, binding to the enzyme cofactor pyridoxal phosphate (PLP), stability according to differences in pH, temperature and duration of storage, and antigenic reactivity with various GAD-specific antisera. RESULTS: The properties of rGAD67/65 were compared with published data for mammalian brain GAD (brackets). These included a specific enzyme activity of 22.7 (16.7) nKat, optimal pH for enzymatic activity 7.4 (6.8), K(m) of 1.3 (1.3) mM, efficient non-covalent binding to the cofactor PLP, and high autoantigenic potency. The stability of rGAD67/65 was optimal over 3 months at -80 degrees C, or in lyophilized form at -20 degrees C. CONCLUSIONS: Hybrid rGAD67/65 has enzymatic and other properties similar to those of the mixed isoforms of GAD in preparations from mammalian brain as described elsewhere, in addition to its previously described similar immunoreactivity.

The PEVKEK region of the pyridoxal phosphate binding domain of GAD65 expresses a dominant B cell epitope for type 1 diabetes sera.

Molecular mimicry between the 65-kDa isoform of glutamic acid decarboxylase (GAD65) and the protein 2C (P2C) of Coxsackie B4 virus (CBV) may initiate human type 1 diabetes. GAD65 contains a motif that has a 6-amino acid identity with CBV-P2C (PEVKEK), whereas the weakly autoantigenic isoform, GAD67, contains PEVKTK. A human-derived monoclonal antibody (mAb) MICA3 reacts with a surface loop of GAD65 that includes PEVKEK, and mutagenic deletion of this loop was shown to reduce reactivity of GAD with the mAb by 70%. To establish that the PEVKEK motif on GAD65 contains a major epitope for diabetes sera and to identify the amino acids involved, mutants of nucleotides of GAD65 and GAD67 at sites in the PEVKEK motif were created and the expressed proteins used for radioimmunoprecipitation (RIP) tests with sera from patients with type 1 diabetes. A potent mouse mAb (GAD6) to GAD65, and a rabbit polyclonal antibody (AB108) to GAD67, were used to standardize the reactivity of the diabetes sera with the mutant molecules. Of 45 type 1 diabetes sera tested, 30 (67%) had an 80% or greater reduction of reactivity to GAD65(delta258-270) vs. intact GAD65. Various single-surface amino acids in the PEVKEK epitope region of GAD65 were mutated, but most molecules carrying these mutations reacted similarly to the parent molecule. However after point mutation of the equivalent motif of GAD67 (PEVKTK to PEVKEK), there was an increase in the reactivity of 12 of 49 (24%) type 1 diabetes sera tested; 7 of 8 sera reactive with GAD67 showed increased reactivity with GAD67(T273E), and 5 previously negative sera gained reactivity with GAD67(T273E). Thus, the PEVKEK motif on GAD65 contributes to serologic reactivity of type 1 diabetes sera. This favors the hypothesis that CBV infection causes type 1 diabetes by the process of viral mimicry with cross-reactivity to a critical epitope of GAD65.