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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 Determinants¹

James A. Dromey^*, Sarah M. Weenink^*, Günther H. Peters, Josef Endl, Patrick J. Tighe, Ian Todd and Michael R. Christie^2,*

^* Department of Medicine, Guy’s, King’s, and St. Thomas’ School of Medicine, King’s College, London, United Kingdom; Center for Biomembrane Physics(MEMPHYS), Department of Chemistry, Technical University of Denmark, Lyngby, Denmark; Roche Diagnostics, Penzberg, Germany; and Division of Immunology, School of Molecular Medical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham, United Kingdom

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 Asn⁸⁵⁸, Glu⁸³⁶, and Trp⁷⁹⁹ 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.

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