The C-terminal cysteines in zinc transporter 8 (ZnT8) forms an independent epitope for autoantibody binding in newly-diagnosed type 1 diabetes.

Autoantibodies to zinc transporter 8 (ZnT8A) are important diagnostic and predictive markers for type 1 diabetes (T1D). Current methods to detect ZnT8A use C-terminal ZnT8 (aa268-369). Previous research identified three major ZnT8A epitopes [R325, W325, and a four residue confirmational epitope (R332/E333/K336/K340)], but the human-specific cysteine region has not been investigated. We sought to examine ZnT8A binding to three C-terminal cysteines and confirm previously known ZnT8A epitopes. Assay buffer constituent (Tween-20) and site-directed mutagenesis of radiolabelled-ZnT8 antigen were utilised to compare ZnT8A binding in R325-ZnT8 and W325-ZnT8 monomeric radiobinding assays (RBAs). Truncated ZnT8 (D360X) and cysteine-to-serine mutations (C361S, C364S, and C368S) were compared with wild-type R325-ZnT8/W325-ZnT8 RBAs in 71 newly-diagnosed T1D cases. Samples were indexed to controls and the median reduction in binding (MRB, %) was compared using Wilcoxon signed-ranked tests. Unbiased hierarchical clustering analysis was used to review effects of all ZnT8 mutations. A higher Tween-20 concentration reduced ZnT8A binding (p<0.0001). All cysteine region mutations reduced 325-agnostic ZnT8A binding towards wild-type R325-ZnT8 (MRB range -37.0% to -66.4%, p<0.0001) and W325-ZnT8 (MRB range -32.1% to -43.8%, p<0.0001). Mutating the cysteine region nearly abolished binding (>75%) in 7.7%-36.8% of R325-ZnT8/W325-ZnT8 positive ZnT8A, and in 10.5-13.2% of subjects studied, reduced binding below that of major ZnT8A epitopes. The cysteine region of C-terminal ZnT8 constitutes an independent epitope for ZnT8A, but mature ZnT8A responses in newly-diagnosed T1D are heterogenous. Epitope spreading of ZnT8A during the natural history of pre-clinical T1D remains unclear and requires further investigation in larger studies.