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2. Mapping of epitopes for autoantibodies to the Type 1 diabetes autoantigen IA-2 by peptide phage display and molecular modelling: Overlap of antibody and T-cell determinants.

Author

A. Dromey, James, Weenink, Sarah M., Peters, Günther H.J., Endl, Josef, A. Dromey, James, Weenink, Sarah M., Peters, Günther H.J., and Endl, Josef

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 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.

Published
2004

6. A continuous central motif of invariant chain peptides, CLIP, is essential for binding to various I-A MHC class II molecules.

Author

Weenink, Sarah M., Milburn, Peter J., and Gautam, Anand M.

Abstract

Invariant chain (Ii) associates with MHC class II molecules and performs a number of crucial functions in antigen presentation. A nested set of class II-associated Ii peptides (CLIP) has been isolated, comprising the Ii sequence between residues 82 and 107. Recently, X-ray crystallographic analysis has revealed that residues 87–101 occupy the HLA-DR3 peptide-binding groove. Based on our previous results, Lee and McConnell have also proposed a model for the binding of CLIP to various mouse I-A molecules in the binding groove. CLIP sequences are able to bind many MHC class II molecules but the molecular basis of this promiscuity has not yet been resolved. We have shown recently that CLIP binding to I-A class II molecules is generally tolerant to side chain substitutions, suggesting that the backbone structure of CLIP may provide the features critical for its interaction with class II. In pursuit of this, backbone stereochemical disruptions by serial Dalanine substitutions in CLIP86–104 have been used in competitive binding assays to I-A class II molecules. These studies have revealed that the phylogenetically conserved central continuous region, CLIP91–99, is intolerant to such configurational substitutions. Experiments with truncated and frame-shift analogues of CLIP showed that for effective binding to class II, the sequence element CLIP90–100 must be incorporated into a peptide of 13 or more residues including at least three residues N-terminal to this motif. Additionally, it appears that different I-A molecules accommodate CLIP in different binding frames. These investigations of the relationship between the structure and binding of CLIP analogues lead us to propose that there is a general backbone motif of a periodic nature within the CLIP sequence that minimizes deleterious contacts and allows promiscuous binding to class II molecules. [ABSTRACT FROM AUTHOR]

Published
1997
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7. 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

Author

Dromey, Jame A., Weenink, Sarah M., Peters, Gunther H., Endl, Josef, Tighe, Patrick J., Todd, Ian, Christie, Michael R., Dromey, Jame A., Weenink, Sarah M., Peters, Gunther H., Endl, Josef, Tighe, Patrick J., Todd, Ian, and Christie, Michael R.

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 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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