Glutamate decarboxylase (GAD) is an
autoantigen associated with the autoimmune disorders Type-1 diabetes (T1D) and
stiff-person syndrome (SPS). The
protein, being an essential
enzyme involved in the production of the inhibitory
neurotransmitter γ-
aminobutyric acid, exists in two
isoforms, GAD67 and GAD65. Both
isoforms may be targeted by
autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67
autoantibodies, whereas T1D mainly is associated with the presence of GAD65
autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67
peptides covering the complete
protein sequence by modified
peptide enzyme-linked
immunosorbent assay in order to identify potential GAD67
epitopes using two
monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear
epitopes located at the N-terminal end of GAD67. The
epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to
amino acids 14-23 and 91-99. Fine mapping of the
epitopes revealed that antibody reactivity was related to
amino acid side-chain functionality, rather than
amino acid side-chain specificity. Additionally, results suggested that non-contact
amino acids in the
epitope structure were essential for antibody reactivity. The exact role of these
amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the
epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67
epitopes and may be a first step in the development of
peptide-based
therapeutics against SPS.