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

The smaller isoform of the GABA-synthesizing enzyme, glutamic acid decarbox ylase 65 (GAD65), is unusually susceptible to becoming a target of autoimmu nity affecting its major sites of expression, GABA-ergic neurons and pancre atic beta-cells. In contrast, a highly homologous isoform, GAD67, is not an autoantigen. We used homolog-scanning mutagenesis to identify GAD65-specif ic amino acid residues which form autoreactive B-cell epitopes in this mole cule. Detailed mapping of 13 conformational epitopes, recognized by human m onoclonal antibodies derived from patients, together with two and three-dim ensional structure prediction led to a model of the GAD65 dimer. GAD65 has structural similarities to ornithine decarboxylase in the pyridoxal-5'-phos phate-binding middle domain (residues 201-460) and to dialkylglycine decarb oxylase in the C-terminal domain (residues 461-585). Six distinct conformat ional and one Linear epitopes cluster on the hydrophilic face of three amph ipathic 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 d omain, four distinct conformational epitopes cluster on a charged patch for med 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 s ite and involves amino acid residues in exons 6 and 11-12. The epitopes loc alize 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 i s the immunogen for autoreactive B-cells. (C) 1999 Academic Press.