The Core Cysteines, (C909) of Islet Antigen-2 and (C945) of Islet Antigen-2β, Are Crucial to Autoantibody Binding in Type 1 Diabetes

Abstract

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.

Zinc transporter 8 (ZnT8) autoantibody epitope specificity and affinity examined with recombinant ZnT8 variant proteins in specific ZnT8R and ZnT8W autoantibody-positive type 1 diabetes patients

“…Consequently, they are currently the best markers of ongoing autoreactivity in both in subjects at genetic susceptibility to human leucocyte antigen (HLA) genes , as well as in newly diagnosed T1D patients . While the autoantibodies recognize discernible epitopes on insulin , GAD65 and IA‐2 , the B cell response against the ZnT8 protein is unique, as patients generate three variants of ZnT8 autoantibodies (ZnT8A). These autoantibody variants are directed specifically against epitope(s) that include arginine (R), tryptophan (W) or glutamine (Q) at amino acid (aa) position 325 and may be displayed either alone or in combination in T1D patients .…”

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

“…This in vitro observation could be a resemblance of the in vivo regulation of many receptor-type PTPs. Furthermore, the oxidation of the core cysteine (C909) to cysteic acid would block access of antibodies, which turns storage buffer a critical aspect in the stability of IA-2 ic [78]. For this reason, we decided to maintain the 2ME reducing conditions after purification.…”

Investigating the potential impact of post translational modification of auto-antigens by tissue transglutaminase on humoral islet autoimmunity in type 1 diabetes

“…For tTG-treated samples, in vitro translated [ 35 S]-methionine labeled antigen was incubated with 1 μM guinea pig TG2 enzyme (Sigma Aldrich Company Ltd., Gillingham, Dorset, U.K.), for 18 min at 37 °C in 100 mM Denver buffer, 3.33 nM CaCl 2 , and pH 7.3 according to a protocol previously described [ 11 ]. tTG-treated samples were desalted to remove calcium so that antigen: autoantibody binding was not affected by excess salt.…”