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Currently, two transporter protein families (the CDF family encoded by the SLC30 gene and the protein (ZIP) family encoded by the SLC39 gene) have been found to be involved in the absorption and transport of zinc ions in the body. The CDF family is divided into three subfamilies, and its members are widely found in prokaryotes and eukaryotes. So far, 10 CDF family members (ZnT1~10) have been discovered in mammals. ZnT8, also known as SLC30A8 (solute carrier family 30, member 8), is mainly located in pancreatic islet β cells, mediates the transport of zinc ions from the cytoplasm to vesicles, and participates in the regulation of insulin synthesis, storage and secretion. Studies have shown that ZnT8 is closely related to the occurrence and development of diabetes, and the single nucleotide polymorphism (SNP) of its gene rs13266634 (R325W) is significantly associated with type 1 diabetes (T1DM) and type 2 diabetes (T2DM).
The ZnT8 protein is localized in the form of a dimer on the insulin secretion/storage vesicle membrane of β cells and regulates zinc transport between the cytoplasm and vesicles. The protein monomer consists of 369 amino acids and contains 6 transmembrane α-helices (I~VI), in which the loop between domains IV and V is rich in histidine. The C and N termini of the protein are both located in the cytoplasm. The C terminus is composed of two α helices (aa 284~292, aa 327~341) and three β sheets (aa 299~308, aa313~321, aa345~352). The first two β-sheets form a hairpin structure. The polymorphic amino acid at position 325 of the C-terminus is located on the far side of the membrane and is fully exposed to the cytoplasm. Human ZnT8 protein may contain the following sites: three PKC phosphorylation sites, a casein kinase II phosphorylation site, a tyrosine kinase phosphorylation site, and a cAMP- and cGMP-dependent protein kinase phosphorylation site, two glycosylation sites and N-tetradecane acylation site. These putative sites provide clues for the study of the molecular mechanism of ZnT8 biological functions. ZnT8 is highly conserved in species evolution, and the structure and function of ZnT8 proteins in humans and mammals are very similar, suggesting that this protein plays an important role in the zinc transport process. A variety of factors are involved in regulating intracellular zinc content and maintaining it within an appropriate range. ZnT8 protein is an important regulatory factor. ZnT8 relies on the H+ concentration difference caused by the proton pump on the vesicle membrane. Every time it transfers out 2 H+, it transfers 1 Zn2+ in the reverse direction, causing Zn2+ to accumulate in the vesicles and participate in the maturation and storage of insulin. Experiments on rat insulinoma INS-1E cells showed that when blood sugar levels are normal, only zinc supplementation measures are taken to increase the level of extracellular zinc ions, but the total intracellular zinc content cannot be increased accordingly, while overexpression of ZnT8 protein can promote cellular uptake and Store zinc ions and increase the total intracellular zinc content. Furthermore, the ZnT8 protein may play a key role in insulin secretion stimulated by elevated blood glucose. When blood glucose levels are normal, although ZnT8 overexpression can increase the total intracellular zinc content, insulin secretion does not increase significantly. When blood glucose levels rise, ZnT8 overexpression can significantly increase insulin secretion, which is almost twice that of non-overexpressing ZnT8 protein β cells. The proton pump on the secretory/storage vesicle membrane of pancreatic islet β cells consumes ATP to transport H+ from the cytoplasm into the vesicle cavity, resulting in a concentration difference of H+ inside and outside the vesicle membrane. With the help of this concentration difference, ZnT8 transports out 2 H+ and simultaneously transfers into 1 Zn2+. The 2 zinc ions and 6 insulin molecular monomers form a hexamer and are stored in the insulin secretion/storage vesicle cavity of β cells.
Figure 1. ZnT8 topology and structural models based on YiiP, a bacterial homolog.(Source: Daniels MJ, et al. 2020)
When the ZnT8 protein mutates, its zinc ion transport function is reduced, zinc homeostasis in vesicles and cytoplasm is destroyed, β-cell function is damaged, and insulin secretion is insufficient. The concentration of zinc in the vesicles decreases, the amount of insulin hexamers it participates in forming is significantly reduced, the storage level of vesicular insulin is reduced, and the insulin secreted out of the cell under external high glucose stimulation is correspondingly insufficient. Decreased zinc concentration within the vesicles may also lead to an increase in the ratio of proinsulin to insulin within the vesicles. Proinsulin cannot be completely converted into insulin during exocytosis, and the function of β-cells in secreting insulin is impaired. The mechanism of this action is not yet clear, but it may be related to the decrease in the expression activities of β cytohormone converting enzymes 1 and 2 and carboxypeptidase E after the zinc ions in the vesicles are reduced, and the changes in pH and calcium ion concentration in the vesicles. The ZnT8 protein's zinc ion transport capacity is reduced, resulting in the inability of zinc ions to be transported to vesicles in time, and it can also destroy the zinc homeostasis in the cytoplasm. Excessive zinc ions accumulate in the cytoplasm, and when they reach or exceed the toxicity threshold, they can induce massive β-cell death. The mechanism may be related to the rapid decrease in intracellular ATP levels. ZnT8 protein can also serve as an antigen to cause T cell-mediated autoimmune reactions characterized by β-cell damage, and even induce T1DM. The possible mechanism by which ZnT8 causes autoimmune damage to β cells is that the ZnT8 epitope is presented by MHC class I molecules and expressed on the surface of β cells. CD8+ Tc cells recognize and kill β cells through the perforin-dependent pathway and/or the Fas/FasL pathway. β cells may express MHC class II molecules after being jointly induced by the cytokines IFN-γ and TNF-α, thereby presenting the ZnT8 epitope to CD4+T cells, which secrete a variety of cytokines and activate macrophages to exert cytotoxic effects ultimately damage β cells. In addition, ZnT8 can also activate B cells to participate in inducing β cell death. Studies have found that there is a considerable proportion of B cells in the damaged area of the islet. Experiments on non-obese diabetic (NOD) mice have shown that intravenously infused B cells can form ectopic follicular areas in pancreatic islets after being acted upon by B lymphocyte chemical inducers (BLC). B cells can produce specific ZnT8 antibodies (ZnT8A), which can persist in the prodromal stage of T1DM. The concentration of antibodies increases with age. The mechanism of β cell damage needs further study.
ZnT8 gene polymorphisms and T1DM antigen and antibody specificity The ZnT8 gene is located on q24.11 of human chromosome 8, with eight exons and seven introns, spanning 37kb. The DNA coding sequence is located in the adjacent (sequence) group AC027419, and the transcription start site is in the expressed sequence tag (EST) of BM565086. Studies have found that the ZnT8 gene rs13266634 (R325W) single nucleotide polymorphism has nothing to do with T1DM susceptibility in Caucasians, but is closely related to T1DM antigen and antibody specificity. The human ZnT8 gene rs13266634 (R325W) single nucleotide polymorphism determines the amino acid residue polymorphism at position 325. The C and T alleles encode arginine and tryptophan respectively. In addition, the rs16889462 single nucleotide polymorphism can undergo non-synonymous substitution, that is, when the second nucleotide base in the codon encoding arginine or tryptophan changes from C to A, the amino acid at position 325 to glutamine. ZnT8 has three conformational epitopes, two are related to arginine and tryptophan at position 325 of the C-terminus, and the other is not related to the amino acid at position 325. According to surveys, about 30% of diabetic patients can carry out autoimmune reactions to arginine at position 325, 15% to 20% react to tryptophan, and 30% react to non-325 amino acid epitopes. The mechanism by which ZnT8 induces T1DM is related to both cellular and humoral immune responses. However, the tertiary structure of ZnT8 protein is difficult to maintain in the extramembranous environment, so it is difficult to conduct in vitro research on the immune response induced by it. It is currently believed that ZnT8 may mainly damage β cells through T cell-mediated cellular immunity, thereby causing T1DM. The pathogenic role of ZnT8-specific antibodies is still unclear. ZnT8 gene polymorphism determines the specificity of ZnT8 antibody (ZnT8A). ZnT8A does not show cross-reactivity to other human zinc transporters, and it also does not show cross-reactivity to mouse ZnT8, which has 82% genetic sequence similarity with humans. The proportion of new-onset T1DM patients who can produce antibodies to ZnT8 is 60% to 80%, that of T2DM patients is less than 3%, and that of patients with T1DM-related autoimmune diseases is about 30%. The sensitivity of ZnT8A is also higher, and T1DM signature antibodies (glutamic acid decarboxylase, GADA), protein tyrosine phosphatase IA2 (IA2A), insulin antibodies (IAA) and islet cytoplasmic autoantibodies (ICA)) are negative the proportion of ZnT8A-positive patients was approximately 26%. The combined measurement of ZnT8A, GADA, IA2A and IAA can increase the detection rate of autoimmune reactions to 98%. Therefore, ZnT8A can be used alone as a marker of T1DM progression, or it can be used in combination with IAA, GADA or IA2A to predict the occurrence of individual T1DM.
Research has found that the ZnT8 gene is a new T2DM susceptibility gene. The C allele (R325) of the ZnT8 gene is associated with an increased risk of T2DM. In the intravenous glucose tolerance test, diabetic patients carrying CC homozygotes have reduced insulin sensitivity compared with patients carrying TT homozygotes or heterozygotes. The mechanism by which ZnT8 gene variation ultimately leads to T2DM may be related to the weakened zinc ion transport function of ZnT8 protein. ZnT8 protein mutation causes an imbalance of zinc ion concentration in vesicles and cytoplasm, leading to impairment of insulin secretion function of β cells, and the disease may progress to T2DM when the disease worsens. In addition, the C allele may be a type of thrifty gene and is closely related to the occurrence of T2DM. Phenotypic expression of thrifty genes can stimulate insulin secretion and promote energy conversion and storage. When food is abundant, thrift genes are highly expressed, and basal and postprandial insulin levels continue to rise. Prolonged hyperinsulinemia and insulin resistance will eventually damage pancreatic beta cells, leading to decompensation of beta cell secretion function and hyperglycemia, if not treated in time, hyperglycemia toxicity and hyperlipidemia toxicity can aggravate the damage of β cell function, and eventually cause T2DM. ZnT8 gene polymorphisms are associated with an increased risk of T2DM, but not all ZnT8 risk gene carriers will develop T2DM. Other disease-related genes, diet and lifestyle are also important causative factors. In addition, ZnT8 gene polymorphisms are not associated with diabetes in late adolescents (MODY), but are associated with an increased risk of post-transplant diabetes and gestational diabetes in allogeneic kidney transplant recipients.
Reference
| Target | Cat. No. | Product Name | Host | Isotype | Application | |
| SLC30A8 | DCABH-13499 | Anti-SLC30A8 monoclonal antibody | IgG | WB, ELISA | Inquiry | |
| CABT-L2081 | Rabbit Anti-Human SLC30A8 Polyclonal Antibody | IgG | WB | Inquiry | ||
| DPABH-27011 | Anti-SLC30A8 (internal region) polyclonal antibody | IgG | WB | Inquiry | ||
| CABT-L6308 | Human Anti-Human ZnT-8 monoclonal antibody, clone U0 | IgG | ELISA | Inquiry | ||
| CABT-L6307 | Human Anti-Human ZnT-8 monoclonal antibody, clone U3 | IgG | ELISA | Inquiry | ||
| CABT-B8539 | Anti-SLC30A8 (aa 268-359) monoclonal antibody, clone 926140 | IgG2b | ELISA, IHC | Inquiry |
| Target | Cat. No. | Product Name | Expression System | Tag/Conjugate | Application | |
| SLC30A8 | DAG-WT1343 | Recombinant ZnT8 | HEK293 cells | His | ELISA, CLIA, CG | Inquiry |
| DAG-ZNT8I | Recombinant Human Zinc transporter 8 (Insect cells) [His] | Sf9 Cells | His | N/A | Inquiry | |
| DAGC025 | Recombinant Human Zinc transporter 8 [His] | Yeast | His | ELISA | Inquiry | |
| DAG-ZNT8 | Recombinant Human ZnT8 | E. coli | TBD | Immunoassay | Inquiry | |
| CDBP2704 | Human SLC30A8 blocking peptide | N/A | Unconjugated | BL | Inquiry |
| Target | Cat. No. | Product Name | Size | Species Reactivity | Application | Detection Sample | |
| SLC30A8 | DEIABL438 | Zinc Transporter 8 (ZnT8) Autoantibody ELISA Kit | 96T | Quantitative | Serum | Inquiry | |
| DEIA-FN1421 | Human SLC30A8 (Zinc transporter 8) ELISA Kit | 96T | Quantitative | serum, plasma, cell culture supernatants, tissue homogenate | Inquiry |
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