title: Mapping of epitopes for autoantibodies to the type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modeling: overlap of antibody and T cell determinants.
abstract: IA-2 is a major target of autoimmunity in type 1 diabetes. IA-2 responsive T cells recognize determinants within regions represented by amino acids 787-817 and 841-869 of the molecule. Epitopes for IA-2 autoantibodies are largely conformational and not well defined. In this study, we used peptide phage display and homology modeling to characterize the epitope of a monoclonal IA-2 Ab (96/3) from a human type 1 diabetic patient. This Ab competes for IA-2 binding with Abs from the majority of patients with type 1 diabetes and therefore binds a region close to common autoantibody epitopes. Alignment of peptides obtained after screening phage-displayed peptide libraries with purified 96/3 identified a consensus binding sequence of Asn-x-Glu-x-x-(aromatic)-x-x-Gly. The predicted surface on a three-dimensional homology model of the tyrosine phosphatase domain of IA-2 was analyzed for clusters of Asn, Glu, and aromatic residues and amino acids contributing to the epitope investigated using site-directed mutagenesis. Mutation of each of amino acids Asn(858), Glu(836), and Trp(799) reduced 96/3 Ab binding by >45%. Mutations of these residues also inhibited binding of serum autoantibodies from IA-2 Ab-positive type 1 diabetic patients. This study identifies a region commonly recognized by autoantibodies in type 1 diabetes that overlaps with dominant T cell determinants.
authors.affiliation: Department of Medicine, Guy's, King's, and St. Thomas' School of Medicine, King's College, London, United Kingdom.
authors.forename: James A
authors.initials: JA
authors.lastname: Dromey
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Weenink
authors.forename: Günther H
authors.initials: GH
authors.lastname: Peters
authors.forename: Josef
authors.initials: J
authors.lastname: Endl
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Tighe
authors.forename: Ian
authors.initials: I
authors.lastname: Todd
authors.forename: Michael R
authors.initials: MR
authors.lastname: Christie
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001323:Autoantibodies / Q000097:blood / Q000737:chemistry*; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017104:Bacteriophage M13; D001666:Binding Sites, Antibody / Q000235:genetics; D002999:Clone Cells; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D018984:Epitopes, T-Lymphocyte; C096322:ICA512 autoantibody; D019151:Peptide Library