The C-terminal cysteines in zinc transporter 8 (ZnT8) form 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 conformational 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 utilized 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 heterogeneous. Epitope spreading of ZnT8A during the natural history of preclinical T1D remains unclear and requires further investigation in larger studies.