The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific,zinc-regulated zinc transporter in mice

The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans.     

Zinc deficiency and its inherited disorders -a review

Zinc is an essential trace element required by all living organisms because of its critical roles both as a structural component of proteins and as a cofactor in enzyme catalysis. The importance of zinc in human metabolism is illustrated by the effects of zinc deficiency, which include a diminished immune response, reduced healing and neurological disorders. Furthermore, nutritional zinc deficiency can be fatal in newborn or growing animals. While zinc deficiency is commonly caused by dietary factors, several inherited defects of zinc deficiency have been identified. Acrodermatitis enteropathica is the most commonly described inherited condition found in humans. In several of the few cases that have been reported, this disorder is associated with mutations in the hZIP4 gene, a member of the SLC39 family, whose members encode membranebound putative zinc transporters. Mutations in other members of this family or in different genes may account for other cases of acrodermatitis in which defects in hZIP4 have not been detected. Another inherited form of zinc deficiency occurs in the lethal milk mouse, where a mutation in ZnT4 gene, a member of the SLC30 family of transmembrane proteins results in impaired secretion of zinc into milk from the mammary gland. A similar disorder to the lethal milk mouse occurs in humans. In the few cases studied, no changes in ZnT4 orthologue, hZnT4, were detected. This, and the presence of several minor phenotypic differences between the zinc deficiency in humans and mice, suggests that the human condition is caused by defects in genes that are yet to be identified. Taking into account the fact that there are no definitive tests for zinc deficiency and that this disorder can go undiagnosed, plus the recent identification of multiple members of the SCL30 and SLC39, it is likely that mutations in other genes may underlie additional inherited disorders of zinc deficiency.     

A novel member of a zinc transporter family is defective in acrodermatitis enteropathica

The rare inherited condition acrodermatitis enteropathica (AE) results from a defect in the absorption of dietary zinc. Recently, we used homozygosity mapping in consanguineous Middle Eastern kindreds to localize the AE gene to an approximately 3.5-cM region on 8q24. In this article, we identify a gene, SLC39A4, located in the candidate region and, in patients with AE, document mutations that likely lead to the disease. The gene encodes a histidine-rich protein, which we refer to as "hZIP4," which is a member of a large family of transmembrane proteins, some of which are known to serve as zinc-uptake proteins. We show that Slc39A4 is abundantly expressed in mouse enterocytes and that the protein resides in the apical membrane of these cells. These findings suggest that the hZIP4 transporter is responsible for intestinal absorption of zinc.     

Identification of a unique splice site variant in SLC39A4 in bovine hereditary zinc deficiency, lethal trait A46: An animal model of acrodermatitis enteropathica

Lethal trait A46, also known as bovine hereditary zinc deficiency, Adema disease, and hereditary parakeratosis, is an autosomal recessive disorder first described in 1964, with a clinical presentation similar to that of acrodermatitis enteropathica (AE) in humans. The molecular basis of the defect has not been previously identified. Recently, the basic defect in AE was found to lie in SLC39A4. We report the characterization of the bovine ortholog of SLC39A4 and identification of a unique splice site variant within this gene in affected animals. The mutation leads to exon skipping, leaving the coding region in frame. The gene product is predicted to lack two critical motifs, which lie in adjacent transmembrane domains implicated in the formation of a pore responsible for the transport of zinc. While further functional studies are warranted, this unique variant is likely to be responsible for the impaired zinc absorption in this disease.     

Zn2+-stimulated endocytosis of the mZIP4 zinc transporter regulates its location at the plasma membrane

Zinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis.     

The human ZIP4 transporter has two distinct binding affinities and mediates transport of multiple transition metals

Zinc is the second most abundant transition metal in the body. Despite the fact that hundreds of biomolecules require zinc for proper function and/or structure, the mechanism of zinc transport into cells is not well-understood. The ZIP (Zrt- and Irt-like proteins; SLC39A) family of proteins acts to increase cytosolic concentrations of zinc. Mutations in one member of the ZIP family of proteins, the human ZIP4 (hZIP4; SLC39A4) protein, can result in the disease acrodermatitis enteropathica (AE). AE is characterized by growth retardation and diarrhea, as well as behavioral and neurological disturbances. While the cellular distribution of hZIP4 protein expression has been elucidated, the cation specificity, kinetic parameters of zinc transport, and residues involved in cation translocation are unresolved questions. Therefore, we have established a high signal-to-noise zinc uptake assay following heterologous expression of hZIP4 in Xenopus laevis oocytes. The results from our experiments have demonstrated that zinc, copper(II), and nickel can be transported by hZIP4 when the cation concentration is in the micromolar range. We have also identified a nanomolar binding affinity where copper(II) and zinc can be transported. In contrast, under these conditions, nickel can bind but is not transported by hZIP4. Finally, labeling of hZIP4 with maleimide or diethylpyrocarbonate indicates that extracellularly accessible histidine, but not cysteine, residues are required, either directly or indirectly, for cation uptake. The results of our experiments identify at least two coordination sites for divalent cations and provide a new framework for investigating the ZIP family of proteins.     

Extreme Population Differences in the Human Zinc Transporter ZIP4 (SLC39A4) Are Explained by Positive Selection in Sub-Saharan Africa

Extreme differences in allele frequency between West Africans and Eurasians were observed for a leucine-to-valine substitution (Leu372Val) in the human intestinal zinc uptake transporter, ZIP4, yet no further evidence was found for a selective sweep around the ZIP4 gene (SLC39A4). By interrogating allele frequencies in more than 100 diverse human populations and resequencing Neanderthal DNA, we confirmed the ancestral state of this locus and found a strong geographical gradient for the derived allele (Val372), with near fixation in West Africa. In extensive coalescent simulations, we show that the extreme differences in allele frequency, yet absence of a classical sweep signature, can be explained by the effect of a local recombination hotspot, together with directional selection favoring the Val372 allele in Sub-Saharan Africans. The possible functional effect of the Leu372Val substitution, together with two pathological mutations at the same codon (Leu372Pro and Leu372Arg) that cause acrodermatitis enteropathica (a disease phenotype characterized by extreme zinc deficiency), was investigated by transient overexpression of human ZIP4 protein in HeLa cells. Both acrodermatitis mutations cause absence of the ZIP4 transporter cell surface expression and nearly absent zinc uptake, while the Val372 variant displayed significantly reduced surface protein expression, reduced basal levels of intracellular zinc, and reduced zinc uptake in comparison with the Leu372 variant. We speculate that reduced zinc uptake by the ZIP4-derived Val372 isoform may act by starving certain pathogens of zinc, and hence may have been advantageous in Sub-Saharan Africa. Moreover, these functional results may indicate differences in zinc homeostasis among modern human populations with possible relevance for disease risk.     

Analysis of zinc transporter,<Emphasis Type="Italic"> hZnT4</Emphasis> (<Emphasis Type="Italic">Slc30A4</Emphasis>), gene expression in a mammary gland disorder leading to reduced zinc secretion into milk

Zinc deficiency, causing impaired growth and development, may have a nutritional or genetic basis. We investigated two cases of inherited zinc deficiency found in breast-fed neonates, caused by low levels of zinc in the maternal milk. This condition is different from acrodermatitis enteropathica but has similarities to the "lethal milk" mouse, where low levels of zinc in the milk of lactating dams leads to zinc deficiency in pups. The mouse disorder has been attributed to a defect in the ZnT4 gene. Little is known about the expression of the human orthologue, hZnT4 (Slc30A4). Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4, carried out on control cells and cells from unrelated mothers of two infants with zinc deficiency, showed no differences. The hZnT4 gene was highly expressed in mouthwash buccal cells compared with lymphoblasts and fibroblasts. The hZnT4 protein did not co-localise with intracellular free zinc pools, suggesting that hZnT4 is not involved in transport of zinc into vesicles destined for secretion into milk. This observation, combined with phenotypic differences between the "lethal milk" mouse and the human disorder, suggests that the "lethal milk" mouse is not the corresponding model for the human zinc deficiency condition.     

Isolation of a low molecular weight zinc binding ligand from human milk

A low molecular weight zinc binding compound from human milk has been purified by ultrafiltration, gel filtration, and ion-exchange chromatography. Evidence is provided that this compound is citrate. A higher amount of citrate-bound zinc was found in human milk than in cow's milk. It is suggested that the therapeutic value of human milk for patients with the genetic disorder of zinc metabolism acrodermatitis enteropathica (AE) derives from a greater content of bioavailable zinc citrate in human than in cow's milk.     

Ileal Paneth cells and IgA system in rats with severe zinc deficiency: An immunohistochemical and morphological study

Summary Morphological abnormalities in Paneth cells occur in patients with acrodermatitis enteropathica, a chereditary disease associated with zinc deficiency; furthermore, rat Paneth cells contain large amounts of zinc. This study was conducted to assess the effect of severe zinc deficiency in Sprague-Dawley rats on various parameters of Paneth cells. Morphology at both the light microscopical and ultrastructural levels, Paneth cell numbers per crypt and the intracellular distribution of lysozyme were not altered by zinc deficiency. A weak correlation (r=+0.38,P=0.05) was noted between ileal zinc concentration and numbers of IgA-containing Paneth cells per crypt. These findings indicate that the morphological abnormalities noted in human Paneth cells in patients with acrodermatitis enteropathica cannot be reproduced by experimental severe zinc deficiency in rats. Furthermore, these generally negative findings suggest that the severe diarrhoea often associated with zinc deficiency is not attributable to abnormalities induced in Paneth cells by zinc deficiency.     

A histidine-rich cluster mediates the ubiquitination and degradation of the human zinc transporter, hZIP4, and protects against zinc cytotoxicity

Zinc is an essential nutrient. Genetic evidence for this nutritional requirement in humans is the zinc deficiency disease, acrodermatitis enteropathica. This disorder is caused by mutations in hZIP4 (SLC39A4), a zinc importer required for zinc uptake in enterocytes and other cell types. Studies in mice have demonstrated that levels of the mZIP4 mRNA are reduced by elevated dietary zinc, resulting in a decreased abundance of the ZIP4 protein at the plasma membrane. Moreover, studies in cultured cells have demonstrated that low micromolar concentrations of zinc stimulate the endocytosis of the mZIP4 protein resulting in a reduction in cellular zinc uptake. In this study, we demonstrate an additional level of hZIP4 regulation involving ubiquitination and degradation of this transporter in elevated zinc concentrations. Mutational analysis identified a cytoplasmic histidine-rich domain that was essential for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity. However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is distinct from zinc-stimulated endocytosis.     

A mouse model of acrodermatitis enteropathica: loss of intestine zinc transporter ZIP4 (Slc39a4) disrupts the stem cell niche and intestine integrity

Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.     

Genome Wide Identification,Phylogeny and Expression of Zinc Transporter Genes in Common Carp

Background

Zinc is an essential trace element in organisms, which serves as a cofactor for hundreds of enzymes that are involved in many pivotal biological processes including growth, development, reproduction and immunity. Therefore, the homeostasis of zinc in the cell is fundamental. The zinc transporter gene family is a large gene family that encodes proteins which regulate the movement of zinc across cellular and intracellular membranes. However, studies on teleost zinc transporters are mainly limited to model species.

Methodology/Principal Findings

We identified a set of 37 zinc transporters in common carp genome, including 17 from SLC30 family (ZnT), and 20 from SLC39 family (ZIP). Phylogenetic and syntenic analysis revealed that most of the zinc transporters are highly conserved, though recent gene duplication and gene losses do exist. Through examining the copy number of zinc transporter genes across several vertebrate genomes, thirteen zinc transporters in common carp are found to have undergone the gene duplications, including SLC30A1, SLC30A2, SLC30A5, SLC30A7, SLC30A9, SLC30A10, SLC39A1, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7 and SLC39A9. The expression patterns of all zinc transporters were established in various tissues, including blood, brain, gill, heart, intestine, liver, muscle, skin, spleen and kidney, and showed that most of the zinc transporters were ubiquitously expressed, indicating the critical role of zinc transporters in common carp.

Conclusions

To some extent, examination of gene families with detailed phylogenetic or orthology analysis could verify the authenticity and accuracy of assembly and annotation of the recently published common carp whole genome sequences. The gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp zinc transporters provides an important genomic resource for future biochemical, toxicological and physiological studies of zinc in teleost.     

Plasma zinc and copper in primary and secondary immunodeficiency disorders

Plasma copper and zinc concentrations were measured in 58 patients with a laboratory-confirmed primary or secondary immunodeficiency. Patients with severe combined immunodeficiency, collagen vascular disease with depressed cell-mediated immunity, and acquired or congenital acrodermatitis enteropathica had mean plasma zinc concentrations substantially below the lower limit of normal. In contrast, patients with primary humoral and polymorphonuclear leukocyte defects had normal plasma zinc concentrations. Patients with primary polymorphonuclear leukocyte defects had a mean plasma copper concentration substantially above the upper limit of normal. Those subjects with primary humoral immunity defects also had significantly elevated plasma copper concentrations in comparison to controls. Plasma copper concentrations in patients with severe combined immunodeficiency or acrodermatitis enteropathica were normal. Cutis laxa patients had low plasma zinc and copper concentrations. These data demonstrate that zinc and copper homeostasis are altered in come immunodeficiency disorders and may be important factors in host defenses. Since it is known that cellular immunity is impaired by zinc deficiency, patients with primary and secondary immunodeficiency states with appropriately documented mild or severe zinc deficiency should receive zinc supplements.     

Discovery and importance of zinc in human nutrition

The present explosion in knowledge of zinc has been the result of several factors, the major ones being the recognition of the important role of zinc in human health and diseases, its vital functions in biochemical reactions, and the technological advances that make it feasible to quantitate this essential trace element in biological fluids. Deficiency of zinc in humans due to nutritional factors and several disease states has now been recognized. The high phytate content of cereal proteins is known to decrease the availability of zinc; thus, the prevalence of zinc deficiency is likely to be high in a population consuming large quantities of proteins. Alcoholism, malabsorption, sickle cell anemia, chronic renal disease, and chronically debilitating diseases are now known to be predisposing factors for zinc deficiency. A severe deficiency of zinc such as that seen in patients with acrodermatitis enteropathica may be life-threatening. A spectrum of clinical manifestations ranging from mild to severe degrees has now been recognized in human zinc deficiency states. Zinc appears to be involved in many biological functions including DNA synthesis. Roles for zinc in enzymatic functions, cell membranes, and immunity are now well established.     

Serum zinc levels in heterozygous carriers of the gene for acrodermatitis enteropathica

Summary Three cases of acrodermatitis enteropathica (a. e.) from two nonrelated families are described. Two siblings had characteristic symptoms of a. e. in childhood. Both survived to adulthood without treatment, at which time the clinical picture became uncharacteristic of a. e. Even so, the serum zinc levels confirmed the diagnosis in both cases. The third case showed classic symptoms of a. e.; the patient had a greatly reduced serum zinc level and responded at once to treatment with zinc sulphate.Heterozygous carriers of the gene for a. e. have often had slightly reduced serum zinc levels. The value of this test could probably be improved by corresting the normal range of serum zinc for factors known to influence this, such as the patient's age and serum albumin level. The normal range ought also to be corrected for diural and postprandial variations of serum zinc.     

Zinc transport by fibroblasts from patients with acrodermatitis enteropathica

Acrodermatitis enteropathica (AE) is a zinc deficiency disease. To date, the only defect has been demonstrated in the gut. We have investigated zinc uptake in fibroblasts established from four unrelated patients with AE using normal skin fibroblasts as controls. Zinc content of AE and control cells was similar (0.3 fmol/cell). Zinc accumulation over 24 h from a complete culture medium was similar in both normal controls and mutant cells. The fraction of zinc removed by Pronase treatment remained constant at 50 pmol/micrograms DNA, whereas the zinc remaining after Pronase treatment accumulated rapidly for 8 h, then more slowly. Analysis of binding data showed no significant difference between AE and control cells, with apparent Ka values of 4-6 X 10(6) M-1 and between 1 and 2 X 10(8) receptors/cell. Analysis of Pronase resistant data showed no difference between the control and the mutant cells with apparent Km values of 0.2-0.3 microM and Vmax values of 17-19 pmol/micrograms DNA/h. No difference in zinc efflux rates was detected. We conclude that the defect that underlies acrodermatitis enteropathica is either not expressed in fibroblasts or cannot be detected under these experimental conditions.     

The adaptive response to dietary zinc in mice involves the differential cellular localization and zinc regulation of the zinc transporters ZIP4 and ZIP5

The ZIP5 gene encodes a protein closely related to ZIP4, a zinc transporter mutated in the human genetic disorder acrodermatitis enteropathica. Herein, we demonstrate that mouse ZIP5 and ZIP4 genes are co-expressed in several tissues involved in zinc homeostasis (intestine, pancreas, embryonic yolk sac). However, unlike expression of the ZIP4 gene, which is induced during periods of zinc deficiency, ZIP5 gene expression is unaltered by dietary zinc. Immunohistochemistry localizes ZIP5 to the basolateral surfaces of enterocytes, acinar cells, and visceral endoderm cells in mice fed a zinc-adequate diet. However, this protein is removed from these cell surfaces and internalized during dietary zinc deficiency. In contrast, ZIP4 is induced and recruited to the apical surface of enterocytes and endoderm cells during zinc deficiency. In the pancreas, ZIP4 is expressed in beta-cells, whereas ZIP5 is expressed in acinar cells. These results suggest that the function of ZIP5 is antagonistic to that of ZIP4 in the control of zinc homeostasis; rather than functioning in the acquisition of dietary zinc, as does ZIP4, ZIP5 may function in the removal of zinc from the body. Thus, during periods when dietary zinc is replete, ZIP5 may function to remove zinc from the blood via the pancreas and intestine, the major sites of zinc excretion in mammals, whereas the acquisition of dietary zinc by intestinal ZIP4 would be minimal. In contrast, during periods of dietary zinc deficiency when secretion of zinc by the pancreas and intestine is minimized, ZIP5 is removed from the cell surface, and the intestinal uptake of zinc is augmented by induction of ZIP4.     

ZnT5 variant B is a bidirectional zinc transporter and mediates zinc uptake in human intestinal Caco-2 cells

Zinc is an essential micronutrient, so it is important to elucidate the molecular mechanisms of zinc homeostasis, including the functional properties of zinc transporters. Mammalian zinc transporters are classified in two major families: the SLC30 (ZnT) family and the SLC39 family. The prevailing view is that SLC30 family transporters function to reduce cytosolic zinc concentration, either through efflux across the plasma membrane or through sequestration in intracellular compartments, and that SLC39 family transporters function in the opposite direction to increase cytosolic zinc concentration. We demonstrated that human ZnT5 variant B (ZnT5B (hZTL1)), an isoform expressed at the plasma membrane, operates in both the uptake and the efflux directions when expressed in Xenopus laevis oocytes. We measured increased activity of the zinc-responsive metallothionein 2a (MT2a) promoter when ZnT5b was co-expressed with an MT2a promoter-reporter plasmid construct in human intestinal Caco-2 cells, indicating increased total intracellular zinc concentration. Increased cytoplasmic zinc concentration mediated by ZnT5B, in the absence of effects on intracellular zinc sequestration by the Golgi apparatus or endoplasmic reticulum, was also confirmed by a dramatically enhanced signal from the zinc fluorophore Rhodzin-3 throughout the cytoplasm of Caco-2 cells overexpressing ZnT5B at the plasma membrane when compared with control cells. Our findings demonstrate clearly that, in addition to mediating zinc efflux, ZnT5B at the plasma membrane can function to increase cytoplasmic zinc concentration, thus indicating a need to reevaluate the current paradigm that SLC30 family zinc transporters operate exclusively to decrease cytosolic zinc concentration.