Cell-Surface Autoantibody Targets Zinc Transporter-8 (ZnT8) for In Vivo ß-Cell Imaging and Islet-Specific Therapies.

Type 1 diabetes (T1D) is a disease in which autoimmune attacks are directed at the insulin-producing ß-cell in the pancreatic islet. Autoantigens on the ß-cell surface membrane are specific markers for molecular recognition and targets for engagement by autoreactive B lymphocytes, which produce islet cell surface autoantibody (ICSA) upon activation. We report the cloning of an ICSA (mAb43) that recognizes a major T1D autoantigen, ZnT8, with a subnanomolar binding affinity and conformation specificity. We demonstrate that cell-surface binding of mAb43 protects the extracellular epitope of ZnT8 against immunolabeling by serum ICSA from a patient with T1D. Furthermore, mAb43 exhibits in vitro and ex vivo specificity for islet cells, mirroring the exquisite specificity of islet autoimmunity in T1D. Systemic administration of mAb43 yields a pancreas-specific biodistribution in mice and islet homing of an mAb43-linked imaging payload through the pancreatic vasculature, thereby validating the in vivo specificity of mAb43. Identifying ZnT8 as a major antigenic target of ICSA allows for research into the molecular recognition and engagement of autoreactive B cells in the chronic phase of T1D progression. The in vivo islet specificity of mAb43 could be further exploited to develop in vivo imaging and islet-specific immunotherapies.

Novel autoantibodies to the ß-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune destruction of insulin-producing ß-cells in pancreatic islets. Seroconversions to islet autoantibodies (IAbs) precede the disease onset by many years, but the role of humoral autoimmunity in the disease initiation and progression are unclear. In the present study, we identified a new IAb directed to the extracellular epitopes of ZnT8 (ZnT8ec) in newly diagnosed patients with T1D, and demonstrated immunofluorescence staining of the surface of human ß-cells by autoantibodies to ZnT8ec (ZnT8ecA). With the assay specificity set on 99th percentile of 336 healthy controls, the ZnT8ecA positivity rate was 23.6% (74/313) in patients with T1D. Moreover, 30 children in a longitudinal follow up of clinical T1D development were selected for sequential expression of four major IAbs (IAA, GADA, IA-2A and ZnT8icA). Among them, 10 children were ZnT8ecA positive. Remarkably, ZnT8ecA was the earliest IAb to appear in all 10 children. The identification of ZnT8ec as a cell surface target of humoral autoimmunity in the earliest phase of IAb responses opens a new avenue of investigation into the role of IAbs in the development of ß-cell autoimmunity.

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

Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic ß-cells. Loss-of-function mutations in ZnT8 are associated with protection against type-2 diabetes (T2D), but the protective mechanism is unclear. Here, we developed an in-cell ZnT8 assay to track endogenous ZnT8 responses to metabolic and inflammatory stresses applied to human insulinoma EndoC-ßH1 cells. Unexpectedly, high glucose and free fatty acids did not alter cellular ZnT8 levels, but proinflammatory cytokines acutely, reversibly, and gradually down-regulated ZnT8. Approximately 50% of the cellular ZnT8 was localized to the endoplasmic reticulum (ER), which was the primary target of the cytokine-mediated ZnT8 down-regulation. Transcriptome profiling of cytokine-exposed ß-cells revealed an adaptive unfolded protein response (UPR) including a marked immunoproteasome activation that coordinately degraded ZnT8 and insulin over a 1,000-fold cytokine concentration range. RNAi-mediated ZnT8 knockdown protected cells against cytokine cytotoxicity, whereas inhibiting immunoproteasomes blocked cytokine-induced ZnT8 degradation and triggered a transition of the adaptive UPR to cell apoptosis. Hence, cytokine-induced down-regulation of the ER ZnT8 level promotes adaptive UPR, acting as a protective mechanism that decongests the ER burden of ZnT8 to protect ß-cells from proapoptotic UPR during chronic low-grade inflammation.

Water molecules mediate zinc mobility in the bacterial zinc diffusion channel ZIPB.

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.

Genetic, Functional, and Immunological Study of ZnT8 in Diabetes.

Zinc level in the body is finely regulated to maintain cellular function. Dysregulation of zinc metabolism may induce a variety of diseases, e.g., diabetes. Zinc participates in insulin synthesis, storage, and secretion by functioning as a "cellular second messenger" in the insulin signaling pathway and glucose homeostasis. The highest zinc concentration is in the pancreas islets. Zinc accumulation in cell granules is manipulated by ZnT8, a zinc transporter expressed predominately in pancreatic α and ß cells. A common ZnT8 gene (SLC30A8) polymorphism increases the risk of type 2 diabetes mellitus (T2DM), and rare mutations may present protective effects. In type 1 diabetes mellitus (T1DM), autoantibodies show specificity for binding two variants of ZnT8 (R or W at amino acid 325) dictated by a polymorphism in SLC30A8. In this review, we summarize the structure, feature, functions, and polymorphisms of ZnT8 along with its association with diabetes and explore future study directions.

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

Solute carrier family 30 member 8 (SLC30A8), encoding the pancreatic zinc transporter ZnT8, is a susceptibility gene for type 2 diabetes (T2D). Reducing ZnT8 transport activity or down-regulating its cellular expression is hypothesized to be an antidiabetogenic strategy mimicking the protective effect of SLC30A8 haploinsufficiency in humans. However, research tools to inhibit ZnT8 activity and measure cellular ZnT8 levels are not available. Here, we report the identification of two anti-ZnT8 mAbs applicable to addressing these unmet needs. Both mAbs exhibited subnanomolar affinities for human ZnT8 and were selective against homologous zinc transporters with distinct cross-species reactivities and epitope recognition. We showed that antigen-binding fragments (Fabs) protected ZnT8 from unfolding and inhibited ZnT8-mediated zinc transport in proteoliposomes. Negative-stain EM revealed a ternary binding complex of a ZnT8 monomer and two different Fabs at a 1:1:1 stoichiometry. Moreover, dual bindings of two different mAbs to a single ZnT8 protein multiplied the individual anti-ZnT8 specificities, enabling quantification of cellular ZnT8 levels by homogeneous time-resolved fluorescence (HTRF). Our results demonstrate the utilities of the two generated mAbs as allosteric inhibitors and highly specific biosensors of human ZnT8.

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

The islet-specific zinc transporter ZnT8 is a major self-antigen found in insulin granules of pancreatic ß-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 ZnT8 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 ZnT8 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 ZnT8 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 ß-cell dysfunction and cytotoxicity.

Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform.

Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity (n = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.

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

The 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 self-antigen 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.

Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes.

Zinc is a critical element for insulin storage in the secretory granules of pancreatic beta cells. The islet-specific zinc transporter ZnT8 mediates granular sequestration of zinc ions. A genetic variant of human ZnT8 arising from a single nonsynonymous nucleotide change contributes to increased susceptibility to type-2 diabetes (T2D), but it remains unclear how the high risk variant (Arg-325), which is also a higher frequency (>50%) allele, is correlated with zinc transport activity. Here, we compared the activity of Arg-325 with that of a low risk ZnT8 variant (Trp-325). The Arg-325 variant was found to be more active than the Trp-325 form following induced expression in HEK293 cells. We further examined the functional consequences of changing lipid conditions to mimic the impact of lipid remodeling on ZnT8 activity during insulin granule biogenesis. Purified ZnT8 variants in proteoliposomes exhibited more than 4-fold functional tunability by the anionic phospholipids, lysophosphatidylcholine and cholesterol. Over a broad range of permissive lipid compositions, the Arg-325 variant consistently exhibited accelerated zinc transport kinetics versus the Trp-form. In agreement with the human genetic finding that rare loss-of-function mutations in ZnT8 are associated with reduced T2D risk, our results suggested that the common high risk Arg-325 variant is hyperactive, and thus may be targeted for inhibition to reduce T2D risk in the general populations.

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.