The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes
- Karen T. Elvers1,
- Ivey Geoghegan1,
- Debbie K. Shoemark2,
- Vito Lampasona3,
- Polly J. Bingley1 and
- Alistair J.K. Williams1⇓
- 1School of Clinical Sciences, Learning and Research, University of Bristol, Southmead Hospital, Bristol, U.K.
- 2School of Clinical Sciences, University of Bristol, Bristol, U.K.
- 3Genomic Unit for the Diagnosis of Human Pathologies, Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy
- Corresponding author: Alistair J.K. Williams, .
Cysteines are thought integral to conformational epitopes of islet antigen-2 (IA-2) autoantibodies (IA-2A), possibly through disulfide bond formation. We therefore investigated which cysteines are critical to IA-2A binding in patients with newly diagnosed type 1 diabetes. All 10 cysteines in the intracellular domain of IA-2 were modified to serine by site-directed mutagenesis, and the effects of these changes on autoantibody binding in comparison with wild-type control were investigated by radiobinding assay. Mutation of the protein tyrosine phosphatase (PTP) core cysteine (C909) in IA-2 caused large reductions in autoantibody binding. In contrast, little or no reduction in binding was seen following substitution of the other cysteines. Modification of the core cysteine (C945) in IA-2β also greatly reduced autoantibody binding. Lysine substitution of glutamate-836 in IA-2 or glutamate-872 in IA-2β resulted in modest reductions in binding and identified a second epitope region. Binding to IA-2 PTP and IA-2β PTP was almost abolished by mutation of both the core cysteine and these glutamates. The core cysteine is key to the major PTP conformational epitope, but disulfide bonding contributes little to IA-2A epitope integrity. In most patients, at disease onset, >90% of antibodies binding to the PTP domain of IA-2 recognize just two epitope regions.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1590/-/DC1.
- Received November 14, 2011.
- Accepted July 8, 2012.
- © 2013 by the American Diabetes Association.
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