Characterization of a linear epitope within the human pancreatic 64-kDa glutamic acid decarboxylase and its autoimmune recognition by sera from insulin-dependent diabetes mellitus patients

Abstract

A 2.0-kb cDNA coding for the full-length 64-kDa human glutamic acid decarboxylase (GAD64) was isolated from a pancreatic carcinoma cDNA library by oligonucleotide screening, polymerase-chain-reaction amplification and subsequently characterized by sequence analysis. Five overlapping fragments of GAD64 cDNA were constructed into the vector pH6EX3, allowing the highly efficient expression of corresponding fusion proteins with a histidine hexapeptide as an affinity ligand at their N-termini in Escherichia coli. The recombinant GAD64 fragments were analysed by Western blotting using sera from patients with early onset of insulin-dependent diabetes mellitus (IDDM). We found that at least 20% of the patients with an onset of IDDM have developed autoantibodies which can specifically recognize a linear antigenic epitope within the GAD64. With a selected IDDM serum, an antigenic epitope was localized in a region of 31 amino acids located at the C-terminus of GAD64, using epitope mapping techniques, and it was characterized. The possibility of using recombinant GAD64 for the development of an immunoassay for a predictive diagnosis of IDDM is discussed.