Mapping of an autoreactive epitope within glutamate decarboxylase using a diabetes-associated human monoclonal autoantibody and an epitope cDNA library.
Glutamate decarboxylase (GAD65) is a major autoantigen in insulin-dependent diabetes (IDDM) and the neurological disorder Stiff-Man-Syndrome (SMS). We derived a human monoclonal autoantibody (MICA 2) from peripheral blood of a patient newly diagnosed with IDDM, which reacted with GAD65 in Western blots. This indicated that a linear epitope is recognized by MICA 2. Using an epitope cDNA library we mapped the MICA 2 epitope to a contiguous stretch of 26 amino acids (506-531) in the C-terminus of GAD65. Neither blocking experiments with synthetic peptides nor analysis of overlapping decapeptides expressed as fusion proteins allowed us to further narrow down the epitope to the typical size of linear epitopes of 6-8 amino acids. We suggest that a miniconformational epitope provided by amino acids 506-531 is recognized by MICA 2, which withstands SDS gel electrophoresis without destruction or partially refolds during the Western blot procedure. A sequence homology with human heat shock protein 60 (HSP60) maps to this region of GAD65 but no cross-reactivity of MICA 2 with HSP60 occurred. Our data demonstrate that reactivity of an antibody in Western blots does not necessarily define a classic linear epitope of 6-8 amino acids and describe a new autoreactive epitope in GAD65 different from those reported for sera from patients with SMS.
Richter
W
W
Department of Internal Medicine 1, University of Ulm, Germany.
Northemann
W
W
Müller
M
M
Böhm
B O
BO
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000097:blood; D001324:Autoantigens / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology* / Q000276:immunology*; D018604:Epitope Mapping; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D016750:Stiff-Person Syndrome / Q000201:enzymology / Q000276:immunology
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D018834:Chaperonin 60; D018076:DNA, Complementary; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005968:Glutamate Decarboxylase