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| Article Authors: | H L Schwartz; J M Chandonia; S F Kash; J Kanaani; E Tunnell; A Domingo; F E Cohen; J P Banga; A M Madec; W Richter; S Baekkeskov |
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| Article Title: | High-resolution autoreactive epitope mapping and structural modeling of the 65 kDa form of human glutamic acid decarboxylase. |
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| Reference ID: | 1014039 |
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| 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. |
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| Affiliations: | Departments of Microbiology/Immunology and Medicine, Hormone Research Institute, San Francisco, CA, 94143-0534, USA. |
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| Date: | 1999 |
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| Reference Type: | Literature |
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| PubMed ID: | 10222205 |
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| Journal: | J Mol Biol |
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| Journal Volume: | 287 |
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| Article Pages: | 983-99 |
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| Journal ISSN: | 0022-2836 |
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| Article Chemical List: | Antibodies, Monoclonal;Isoenzymes;Recombinant Proteins;Glutamate Decarboxylase |
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| Article MeSH List: | Amino Acid Sequence; Antibodies, Monoclonal(metabolism); Binding Sites; Epitope Mapping(methods); Glutamate Decarboxylase(chemistry; genetics; immunology); Humans; Isoenzymes(chemistry; genetics; immunology); Models, Molecular; Molecular Sequence Data; Mutagenesis; Protein Conformation; Recombinant Proteins(genetics; metabolism); Sequence Homology, Amino Acid |
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| Curation Last Updated: | 2013-05-28 21:38:25 |
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