Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling

Myers, M. A., Davies, Janet M., Tong, J. C., Whisstock, J., Scealy, M., Mackay, I. R., & Rowley, M. J. (2000) Conformational epitopes on the diabetes autoantigen GAD65 identified by peptide phage display and molecular modeling. Journal of Immunology, 165(7), pp. 3830-3838.

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Abstract

The major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing tile PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive width MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.

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ID Code: 87923
Item Type: Journal Article
Refereed: Yes
Keywords: autoantigen, epitope, glutamate decarboxylase, peptide library, pyridoxal 5 phosphate, amino acid sequence, article, autoimmune disease, binding site, human, insulin dependent diabetes mellitus, molecular model, phage display, priority journal, protein conformation, protein domain, sequence analysis, sequence homology, Antibodies, Monoclonal, Antigen-Antibody Reactions, Autoantigens, Binding Sites, Antibody, Coliphages, Diabetes Mellitus, Type 1, Epitope Mapping, Humans, Isoenzymes, Models, Molecular, Molecular Sequence Data, Peptide Fragments, Protein Structure, Tertiary, Pyridoxal Phosphate, Sequence Alignment, Sequence Homology, Amino Acid
DOI: 10.4049/jimmunol.165.7.3830
ISSN: 0022-1767
Copyright Owner: Copyright 2000 The American Association of Immunologists
Deposited On: 30 Sep 2015 05:45
Last Modified: 30 Sep 2015 05:45

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