Hl. Schwartz et al., High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase, J MOL BIOL, 287(5), 1999, pp. 983-999
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.