Coupling of Insulin Secretion and Display of a Granule-resident Zinc Transporter ZnT8 on the Surface of Pancreatic Beta Cells

Abstract

Edited by Jeffrey E. PessinThe islet-specific zinc transporter ZnT8 mediates zinc enrichment in the insulin secretory granules of the pancreatic beta cell. This granular zinc transporter is also a major selfantigen found in type 1 diabetes patients. It is not clear whether ZnT8 can be displayed on the cell surface and how insulin secretion may regulate the level of ZnT8 exposure to extracellular immune surveillance. Here we report specific antibody binding to the extracellular surface of rat insulinoma INS-1E cells that stably expressed a tagged human zinc transporter ZnT8. Flow cytometry analysis after fluorescent antibody labeling revealed strong correlations among the levels of ZnT8 expression, its display on the cell surface, and glucose-stimulated insulin secretion (GSIS). Glucose stimulation increased the surface display of endogenous ZnT8 from a basal level to 32.5% of the housekeeping Na ؉ /K ؉ ATPase on the cell surface, thereby providing direct evidence for a GSIS-dependent surface exposure of the ZnT8 self-antigen. Moreover, the variation in tagged-ZnT8 expression and surface labeling enabled sorting of heterogeneous beta cells to subpopulations that exhibited marked differences in GSIS with parallel changes in endogenous ZnT8 expression. The abundant surface display of endogenous ZnT8 and its coupling to GSIS demonstrated the potential of ZnT8 as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations.

Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

“…Zn transporter (ZNT)-family proteins except ZNT1 and ZNT10 (1)(2)(3) localize to specific subcellular compartments and perform unique functions (4,5). ZNT2, ZNT3, and ZNT8, which localize to regulatory secretory vesicles/granules in specialized cells, play pivotal roles in the specific physiological functions of these cells (6)(7)(8)(9)(10)(11)(12)(13) in distinct manners (14)(15)(16); conversely, ZNT5, ZNT6, and ZNT7, which localize to the compartments of the early secretory pathway in generic cells, play general but key roles in the cells (17)(18)(19). One of these roles of ZNT proteins is their critical function of supplying zinc to numerous proteins, including several secretory, extracellularly membrane-bound or organelle-resident enzymes biosynthesized in the compartments (hereafter zinc ectoenzymes), for their activation (20,21).…”

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

“…Reducing the serum-to-ZnT8⅐GFP ratio in the binding mixture reduced the multipeak profile to a single complex peak (Fig. 2B, red line), which was shifted leftward from the unbound ZnT8⅐GFP peak position (blue line) with an increase of 170 kDa, corresponding to the formation of an ZnT8A-ZnT8⅐GFP complex at a 1-to-1 binding stoichiometry as described previously (21). The formation of ZnT8A-ZnT8⅐GFP complex was confirmed by anti-GFP and anti-mouse-IgG immunoblotting (Fig.…”

“…This zinc transporter is unique in its tissue-specific expression in pancreatic islets (29), mostly restricted to ␤-cells and to a lesser extent to non-␤ endocrine cells (30 -32). We showed recently that glucose stimulation increases ZnT8 display on the surface of INS-1E cells (21). The secretion-coupled ZnT8 surfacing may enforce a vicious cycle of ␤-cell dysfunction and/or destruction during T1D progression (33).…”

“…We showed recently that the granular ZnT8 is trafficked to the surface membrane following insulin secretion (see Fig. 1A), thereby exposing its transmembrane domain (TMD, amino acids 60 -266)) to serum ZnT8A recognition (21). By comparing the incidences of anti-CTD and anti-full-length ZnT8 (TMDϩCTD) autoantibodies in 307 human sera from T1D diabetic and healthy control subjects (22), we detected a subclass of anti-TMD ZnT8A in the disease population.…”

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

“…HEK293 cells with or without ZnT8 overexpression were fixed, permeabilized, and resuspended in PBS plus 5% BSA at a density of 1 ϫ 10 6 /ml as described previously (37). Aliquots of 100 l of cell suspension were stained with mAb20 or mAb39 in 3-fold serial dilutions for 2 h at room temperature and then Anti-ZnT8 mAbs stained with Alexa Fluor 647-conjugated donkey anti-mouse secondary antibody in a 1:1000 dilution with 5% BSA (Thermo Fisher Scientific, catalog no.…”