High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase

J Mol Biol. 1999 Apr 16;287(5):983-99. doi: 10.1006/jmbi.1999.2655.

Abstract

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarboxylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmunity affecting its major sites of expression, GABA-ergic neurons and pancreatic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specific amino acid residues which form autoreactive B-cell epitopes in this molecule. Detailed mapping of 13 conformational epitopes, recognized by human monoclonal antibodies derived from patients, together with two and three-dimensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phosphate-binding middle domain (residues 201-460) and to dialkylglycine decarboxylase in the C-terminal domain (residues 461-585). Six distinct conformational and one linear epitopes cluster on the hydrophilic face of three amphipathic alpha-helices in exons 14-16 in the C-terminal domain. Two of those epitopes also require amino acids in exon 4 in the N-terminal domain. Two distinct epitopes reside entirely in the N-terminal domain. In the middle domain, four distinct conformational epitopes cluster on a charged patch formed by amino acids from three alpha-helices away from the active site, and a fifth epitope resides at the back of the pyridoxal 5'-phosphate binding site and involves amino acid residues in exons 6 and 11-12. The epitopes localize to multiple hydrophilic patches, several of which also harbor DR*0401-restricted T-cell epitopes, and cover most of the surface of the protein. The results reveal a remarkable spectrum of human autoreactivity to GAD65, targeting almost the entire surface, and suggest that native folded GAD65 is the immunogen for autoreactive B-cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / metabolism
  • Binding Sites
  • Epitope Mapping / methods*
  • Glutamate Decarboxylase / chemistry*
  • Glutamate Decarboxylase / genetics
  • Glutamate Decarboxylase / immunology*
  • Humans
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / immunology
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Protein Conformation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Antibodies, Monoclonal
  • Isoenzymes
  • Recombinant Proteins
  • Glutamate Decarboxylase