The human zinc transporter ZnT8 provides the granules of pancreatic β-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,β structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.

• Klíčová slova
• C-terminal domain, ZnT8, diabetes type 2, nickel, zinc, zinc transporter,
• MeSH
• beta-buňky metabolismus   MeSH
• diabetes mellitus 2. typu genetika   patologie   MeSH
• inzulin genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix genetika   MeSH
• lidé MeSH
• molekulární konformace MeSH
• nikl chemie   MeSH
• proteinové domény genetika   MeSH
• transportní proteiny chemie   genetika   ultrastruktura   MeSH
• vztahy mezi strukturou a aktivitou * MeSH
• zinek chemie   MeSH
• zinkový transportér 8 chemie   genetika   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inzulin MeSH
• nikl MeSH
• SLC30A8 protein, human MeSH Prohlížeč
• transportní proteiny MeSH
• zinc-binding protein MeSH Prohlížeč
• zinek MeSH
• zinkový transportér 8 MeSH

BACKGROUND: Testing for autoantibodies against the zinc transporter ZnT8 (ZnTA) is becoming routine in pediatric diabetes. However, available data are inconclusive when focusing on adult-onset diabetes, including autoimmune diabetes, which does not require insulin at diagnosis (LADA). BASIC PROCEDURES: We examined the ZnTA prevalence and titers and matched them with the clinical phenotype and PTPN22 genotypes of Czech LADA patients who were positive for GADA and/or IA2A and had a fasting C-peptide level >200 pmol/L at diagnosis as well as HNF4A-, GCK- or HNF1A-MODY patients and healthy controls. MAIN FINDINGS: Most LADA patients were negative for ZnTA, and the sensitivity of the assay was only 18-20% for patients with LADA-like progression to insulinotherapy compared to healthy controls. In LADA patients, there was no association between the ZnTA and PTPN22 risk genotypes. LADA patients positive for ZnTA had a lower BMI than those positive for other autoantibodies alone. Importantly, MODY patients were completely negative for ZnTA, and the levels of ZnTA in MODY patients were similar to those in healthy controls. CONCLUSIONS: ZnTA quantification did not improve LADA diagnosis. However, positivity for ZnTA can be used as a negative MODY pre-diagnostic criterion even in the region of Central and East Europe, where other islet cell autoantibodies are common in MODY patients.

• Klíčová slova
• Adult-onset autoimmune diabetes, BMI, LADA, LYP, PTPRN, Zinc transporter ZnT8,
• MeSH
• autoprotilátky krev   imunologie   MeSH
• biologické markery krev   MeSH
• diabetes mellitus 1. typu krev   farmakoterapie   genetika   imunologie   MeSH
• diabetes mellitus 2. typu krev   farmakoterapie   genetika   imunologie   MeSH
• fenotyp MeSH
• genotyp MeSH
• hypoglykemika terapeutické užití   MeSH
• inzulin terapeutické užití   MeSH
• lidé středního věku MeSH
• lidé MeSH
• progrese nemoci MeSH
• tyrosinfosfatasa nereceptorového typu 22 genetika   MeSH
• zinkový transportér 8 krev   imunologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• autoprotilátky MeSH
• biologické markery MeSH
• hypoglykemika MeSH
• inzulin MeSH
• PTPN22 protein, human MeSH Prohlížeč
• SLC30A8 protein, human MeSH Prohlížeč
• tyrosinfosfatasa nereceptorového typu 22 MeSH
• zinkový transportér 8 MeSH

Insulin is produced and stored inside the pancreatic β-cell secretory granules, where it is assumed to form Zn2+-stabilized oligomers. However, the actual storage forms of this hormone and the impact of zinc ions on insulin production in vivo are not known. Our initial X-ray fluorescence experiment on granules from native Langerhans islets and insulinoma-derived INS-1E cells revealed a considerable difference in the zinc content. This led our further investigation to evaluate the impact of the intra-granular Zn2+ levels on the production and storage of insulin in different model β-cells. Here, we systematically compared zinc and insulin contents in the permanent INS-1E and BRIN-BD11 β-cells and in the native rat pancreatic islets by flow cytometry, confocal microscopy, immunoblotting, specific messenger RNA (mRNA) and total insulin analysis. These studies revealed an impaired insulin production in the permanent β-cell lines with the diminished intracellular zinc content. The drop in insulin and Zn2+ levels was paralleled by a lower expression of ZnT8 zinc transporter mRNA and hampered proinsulin processing/folding in both permanent cell lines. To summarize, we showed that the disruption of zinc homeostasis in the model β-cells correlated with their impaired insulin and ZnT8 production. This indicates a need for in-depth fundamental research about the role of zinc in insulin production and storage.

• Klíčová slova
• insulin, pancreatic islets, proinsulin, zinc ions, znt8, β-cells,
• MeSH
• beta-buňky metabolismus   ultrastruktura   MeSH
• chemická frakcionace MeSH
• cytoplazmatická granula metabolismus   MeSH
• exprese genu * MeSH
• glukosa metabolismus   MeSH
• inzulin genetika   metabolismus   MeSH
• krysa rodu Rattus MeSH
• Langerhansovy ostrůvky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• průtoková cytometrie metody   MeSH
• zinek metabolismus   MeSH
• zinkový transportér 8 MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosa MeSH
• inzulin MeSH
• messenger RNA MeSH
• Slc30a8 protein, rat MeSH Prohlížeč
• zinek MeSH
• zinkový transportér 8 MeSH

AIMS: The prevalence of autoantibodies to zinc transporter 8 (ZnT8) in Czech children at the onset of Type 1 diabetes mellitus and dynamic changes in ZnT8 autoantibody levels during disease progression were studied. The value of ZnT8 autoantibody measurements in diagnosis of Type 1 diabetes was assessed. METHODS: Serum samples from 227 children with newly diagnosed Type 1 diabetes and from 101 control children without diabetes were analysed in a retrospective cross-sectional study. One hundred and seventy-one samples from 116 of the patients with diabetes were analysed in a follow-up study at (median) intervals of 1, 3, 5 and 10 years after onset of Type 1 diabetes. ZnT8 autoantibodies were measured using a bridging enzyme-linked immunosorbent assay, while antibodies to glutamic acid decarboxylase, insulinoma antigen 2 and insulin were measured by radioimmunoassays. RESULTS: ZnT8 autoantibodies were detected in 163/227 (72%) of children at Type 1 diabetes onset and in 1/101 (1%) of the control subjects. Sixteen out of 227 (7%) patients with Type 1 diabetes were antibody negative based on three antibodies (glutamic acid decarboxylase, insulinoma antigen 2 and insulin). This false-negative rate was reduced to 10/227 (4.4%) (P < 0.05) after inclusion of ZnT8 autoantibody measurements. Of the children, 142/227 (63%) were positive for at least three antibodies and the most common combination was insulinoma antigen 2, glutamic acid decarboxylase and ZnT8. ZnT8 autoantibody levels decreased over time after Type 1 diabetes onset and the presence and level of ZnT8 autoantibodies correlated with IA-2 autoantibodies. CONCLUSIONS: A ZnT8 autoantibody enzyme-linked immunosorbent assay showed 72% disease sensitivity and 99% specificity at Type 1 diabetes onset. Measurements of ZnT8 autoantibodies are important for Type 1 diabetes diagnosis and should be included in the panel of autoantibodies tested at the onset of Type 1 diabetes.

• MeSH
• autoprotilátky krev   MeSH
• časové faktory MeSH
• diabetes mellitus 1. typu krev   epidemiologie   imunologie   MeSH
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• předškolní dítě MeSH
• proteiny přenášející kationty imunologie   MeSH
• průřezové studie MeSH
• séroepidemiologické studie MeSH
• studie případů a kontrol MeSH
• věk při počátku nemoci MeSH
• zinkový transportér 8 MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• autoprotilátky MeSH
• proteiny přenášející kationty MeSH
• SLC30A8 protein, human MeSH Prohlížeč
• zinkový transportér 8 MeSH

Type 1 diabetes mellitus (T1 DM) is caused by autoimmune-mediated and idiopathic beta-cell destruction of the pancreatic islets of Langerhans resulting in absolute insulin deficiency. Susceptibility to T1 DM is influenced by both genetic and environmental factors. It is generally believed that in genetically susceptible individuals, the disease is triggered by environmental agents, such as viral infections, dietary factors in early infancy, or climatic influences. Many candidate genes for diabetes have been reported; those within the Major Histocompatibility Complex being among the most important. The most common autoantigens are insulin, glutamic acid decarboxylase 65, insuloma-associated antigen 2, and zinc transporter ZnT8. The destruction of beta-cells is mediated mainly by cellular mechanisms; antibodies only seem to reflect the ongoing autoimmune processes and are not directly involved in the tissue damage. They, however, appear prior to the onset of insulin deficiency which makes them suitable for use in the prevention of the disease.

• Klíčová slova
• T, and NKT cells., autoantigens - autoantibodies - HLA alleles - insuline - B,
• MeSH
• autoantigeny genetika   imunologie   MeSH
• diabetes mellitus 1. typu genetika   imunologie   MeSH
• Langerhansovy ostrůvky imunologie   MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• autoantigeny MeSH