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.
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.
Mauch
L
L
Department of Molecular Biology, ELIAS Entwicklungslabor, Freiburg, Germany.
Abney
C C
CC
Berg
H
H
Scherbaum
W A
WA
Liedvogel
B
B
Northemann
W
W
D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000097:blood*; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D003922:Diabetes Mellitus, Type 1 / Q000175:diagnosis / Q000276:immunology*; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology
D001323:Autoantibodies; D000939:Epitopes; D011994:Recombinant Proteins; D004247:DNA; D005968:Glutamate Decarboxylase