A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells

Abstract

The islet-specific zinc transporter ZnT8 is a major self-antigen found in insulin granules of pancreatic β-cells. Frequent insulin secretion exposes ZnT8 to the cell surface, but the humoral antigenicity of the surface-displayed ZnT8 remains unknown. Here we show that a membrane-embedded human ZnT8 antigen triggered a vigorous immune response in knock-out mice. Approximately 50% of serum immunoreactivities toward ZnT8 were mapped to its transmembrane domain that is accessible to extracellular ZnT8 antibody (ZnT8A). ZnT8A binding was detected on live rat insulinoma INS-1E cells, and the binding specificity was validated by a CRISPR/Cas9 mediated knock-out. Applying established ZnT8A assays to purified serum antibodies from patients with type 1 diabetes, we detected human ZnT8A bound to live INS-1E cells, whereas a knock-out specifically reduced the surface binding. Our results demonstrate that ZnT8 is a cell surface self-antigen, raising the possibility of a direct involvement in antibody-mediated β-cell dysfunction and cytotoxicity.

A change in the zinc ion concentration reflects the maturation of insulin-producing cells generated from adipose-derived mesenchymal stem cells

“…Many reports indicate that the ZnT family carries Zn 2+ from the cytoplasm to organelles or extracellular regions, while the ZIP family carries Zn 2+ retrodirectionally 21,30–32 . Among them, ZnT8 expression is detected mainly in insulin secretory granules 33–35 . Some T1DM cases are caused by an autoimmune disorder against ZnT8 36–41 .…”

Down-regulation of the islet-specific zinc transporter-8 (ZnT8) protects human insulinoma cells against inflammatory stress

“…Our results indicate that ZnT8 is temporally and spatially regulated to modulate zinc, ER, and insulin homeostasis. Our recent findings also showed that GSIS promotes ZnT8 trafficking to the surface membrane (60), where ZnT8 becomes a surface autoantigen recognized by autoantibodies that arise from autoimmunity in T1D (33,61). Hence, emerging evidence suggests cross-talk among multiple regulatory pathways intersecting at ZnT8.…”

“…We tested two recently developed anti-ZnT8 mAbs (mAb20 and mAb39). Although both mAbs had a subnanomolar binding affinity (33), only mAb20 had a significant signal-to-noise ratio when endogenous ZnT8 in EndoC-␤H1 cells was probed at a saturating mAb concentration (1:600 dilution). Assay calibration showed a linear increase in the HRP readout with a cell number that was proportional to the amount of endogenous ZnT8 (Fig.…”

Highly specific monoclonal antibodies for allosteric inhibition and immunodetection of the human pancreatic zinc transporter ZnT8

“…A ZnT8 homodimer is referred to as ZnT8 hereafter. Immunization of ZnT8-KO mice with purified human ZnT8 in proteoliposomes yielded serum antibodies that exhibited a ϳ100-fold increase in antibody titers compared with titers of two commercial antibodies directed to linear peptide sequences from the N-and C-terminal domain (NTD and CTD), respectively (32). Splenocytes of immunized mice were isolated and fused with myeloma cells to establish immortalized hybridomas.…”

“…All experiments with mice were approved by the institutional animal care and use committee of the Johns Hopkins University School of Medicine. Serum antibody titering and validation of anti-ZnT8 specificity by CRISPR/Cas9 ZnT8 knockout were described previously (32). Following a final immunization boost, spleens were isolated.…”