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PubMed1997

Identification and analysis of mutations in the Wilson disease gene (ATP7B): population frequencies, genotype-phenotype correlation, and functional analyses.

Wilson disease (WD) is an autosomal recessive disorder characterized by toxic accumulation of copper in the liver and subsequently in the brain and other organs. On the basis of sequence homology to known genes, the WD gene (ATP7B) appears to be a copper-transporting P-type ATPase. A search for ATP7B mutations in WD patients from five population samples, including 109 North American patients, revealed 27 distinct mutations, 18 of which are novel. A composite of published findings shows missense mutations in all exons-except in exons 1-5, which encode the six copper-binding motifs, and in exon 21, which spans the carboxy-terminus and the poly(A) tail. Over one-half of all WD mutations occur only rarely in any population sample. A splice-site mutation in exon 12 accounts for 3% of the WD mutations in our sample and produces an in-frame, 39-bp insertion in mRNA of patients homozygous, but not heterozygous, for the mutation. The most common WD mutation (His1069Glu) was represented in approximately 38% of all the WD chromosomes from the North American, Russian, and Swedish samples. In several population cohorts, this mutation deviated from Hardy-Weinberg equilibrium, with an overrepresentation of homozygotes. We did not find a significant correlation between His1069Glu homozygosity and several clinical indices, including age of onset, clinical manifestation, ceruloplasmin activity, hepatic copper levels, and the presence of Kayser-Fleischer rings. Finally, lymphoblast cell lines from individuals homozygous for His1069Glu and 4 other mutations all demonstrated significantly decreased copper-stimulated ATPase activity.

Adenosine Triphosphatases↗

PubMed1997

Haplotype and mutation analysis in Japanese patients with Wilson disease.

Wilson disease (WD), an autosomal recessive disorder of copper transport, is characterized by impaired biliary excretion and by impaired incorporation of copper into ceruloplasmin. Toxic accumulation of copper causes tissue damage, primarily in the liver, brain, and kidneys. The gene for WD (ATP7B) has been cloned, and the protein product is predicted to be a copper-transporting P-type ATPase with high amino acid identity with that for Menkes disease, an X-linked disorder of copper transport. Mutation screening in WD patients has led to the identification of at least 40 mutations. In addition, haplotype analysis using three dinucleotide-repeat markers, D13S314, D13S301, and D13S316, has been a useful indicator of specific mutations. We have determined haplotypes for the patients and their parents and sibs, in 21 unrelated WD families from Japan. Twenty-eight different haplotypes were observed on 42 WD chromosomes. In all the patients, the ATP7B coding sequence, including the intron-exon boundaries, was screened for mutations, by SSCP, followed by direct-sequence analysis of the shifted fragments. We identified 13 mutations, of which 11 mutations are novel, including 7 mutations-1 insertion, 4 deletions, and 2 missense mutations-in the coding region. The mutations reported in previous studies are 2299insC and Arg778Leu. Two patients were shown to have the 2299insC mutation, which has occurred in many different haplotypes in several populations, indicating a mutation hot spot. Primer-extension analysis of ATP7B mRNA has revealed multiple transcription start sites. Four of the novel mutations (three 1-bp changes and one 5-bp deletion) occur in the 5' UTR and may result in altered expression of the WD gene.

Adenosine Triphosphatases↗

PubMed2002

Rapid and robust screening of the Menkes disease/occipital horn syndrome gene.

Menkes disease and occipital horn syndrome (OHS) are allelic neurogenetic disorders of copper transport associated with mutations in an X-linked gene, ATP7A. This gene encodes a copper-transporting P-type ATPase. The spectrum of mutations at the Menkes/OHS locus is estimated to include 1% chromosomal rearrangements and 15-20% large deletions, with the remaining defects involving small alterations. There is a compelling need for a rapid and reliable molecular diagnostic approach for patients and families impacted by these conditions. In addition to testing suspected affected males, carrier screening of females in Menkes/OHS families and prenatal evaluation of at-risk pregnancies will be enhanced by the wider availability of robust mutation analysis for this large (23-exon) locus. Here we describe a stepwise approach to mutation screening for these disorders that successfully identified molecular alterations in over 95% of our patient population (n = 49). This genomic DNA-based technique employs multiplex PCR, heteroduplex analysis, and direct sequencing, in a serial fashion. This approach should find application in molecular diagnostic laboratories in the United States and other countries. Currently, only a single European center provides commercial testing for unknown mutations in Menkes/OHS patients, even though these disorders occur worldwide.

Adenosine Triphosphatases↗

PubMed2001

Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase.

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (WND:) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The tx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell. Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multi-vesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of tx mice.

Adenosine Triphosphatases↗

PubMed1997

Developmental expression of the mouse mottled and toxic milk genes suggests distinct functions for the Menkes and Wilson disease copper transporters.

Menkes disease and Wilson disease are human disorders of copper transport caused by mutations in distinct genes encoding similar copper-transporting P-type ATPases. These genes are expressed in different adult tissues in patterns reflecting disease manifestations. The mouse homologues for the Menkes (MNK) and Wilson (WND) disease genes are the mottled (Atp7a) and toxic milk (Atp7b) genes, respectively. Using RNA in situ hybridization we describe the distribution of mottled and toxic milk transcripts during mouse embryonic development. The mottled gene is expressed in all tissues throughout embryogenesis and is particularly strong in the choroid plexuses of the brain. Mottled expression in the liver is in contrast to the prior observation of absent or very low expression in the adult liver. Expression of the toxic milk gene is significantly more delimited, with early expression in the central nervous system, heart and liver. Later in gestation, toxic milk transcript is clearly seen in the liver, intestine, thymus and respiratory epithelium including nasopharynx, trachea and bronchi. In lung, toxic milk expression is restricted to bronchi, while mottled expression is diffuse. Hepatic expression of both toxic milk and mottled is in the parenchyma, as opposed to blood cells. These results suggest that the mottled gene product functions primarily in the homeostatic maintenance of cell copper levels, while the toxic milk gene product may be specifically involved in the biosynthesis of distinct cuproproteins in different tissues.

Adenosine Triphosphatases↗

PubMed1998

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network.

The human X-linked recessive disorder of copper metabolism, Menkes disease, is caused by a defect in the MNK ( ATP7A ) gene which encodes a transmembrane copper-transporting P-type ATPase (MNK). MNK is an important component of the mammalian copper transport pathway, and previous studies in cultured cells have localized MNK to the final compartment of the Golgi apparatus, the trans -Golgi network (TGN). At this location, MNK is predicted to supply copper to copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. In this study, three di-leucine motifs and a cluster of four acidic amino acids within the C-terminal region of MNK were investigated as candidate signals necessary for steady-state TGN localization. In vitro mutagenesis of the human MNK cDNA and immunofluorescence detection of mutant forms of MNK expressed in cultured cells demonstrated that the di-leucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. We suggest that this di-leucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Our data, along with the recent demonstration that the third transmembrane region of MNK functions as a TGN targeting signal, suggests that MNK localization to the TGN may be a two-step process involving TGN retention via the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.

Adenosine Triphosphatases↗

PubMed2000

The Menkes copper transporter is required for the activation of tyrosinase.

Menkes disease is an X-linked recessive copper deficiency disorder caused by mutations in the ATP7A (MNK) gene. The MNK gene encodes a copper-transporting P-type ATPase, MNK, which is localized predominantly in the trans-Golgi network (TGN). The MNK protein relocates to the plasma membrane in cells exposed to elevated copper where it functions in copper efflux. A role for MNK at the TGN in mammalian cells has not been demonstrated. In this study, we investigated whether the MNK protein is required for the activity of tyrosinase, a copper-dependent enzyme involved in melanogenesis that is synthesized within the secretory pathway. We demonstrate that recombinant tyrosinase expressed in immortalized Menkes fibroblast cell lines was inactive, whereas in normal fibroblasts known to express MNK protein there was substantial tyrosinase activity. Co-expression of the Menkes protein and tyrosinase from plasmid constructs in Menkes fibroblasts led to the activation of tyrosinase and melanogenesis. This MNK-dependent activation of tyrosinase was impaired by the chelation of copper in the medium of cells and after mutation of the invariant phosphorylation site at aspartic acid residue 1044 of MNK. Collectively, these findings suggest that the MNK protein transports copper into the secretory pathway of mammalian cells to activate copper-dependent enzymes and reveal a second copper transport role for MNK in mammalian cells. These findings describe a single cell-based system that allows both the copper transport and trafficking functions of MNK to be studied. This study also contributes to our understanding of the molecular basis of pigmentation in mammalian cells.

Adenosine Triphosphatases↗

PubMed1996

Helicobacter pylori genes hpcopA and hpcopP constitute a cop operon involved in copper export.

We previously reported that a gene hpcopA isolated from Helicobacter pylori is associated with copper transport. In the present study, the DNA upstream of the hpcopA was cloned and the nucleotide sequence analyzed. An open reading frame coding for 124 amino acids was predicted, which was connected in frame to the hpcopA. The deduced protein sequence exhibits striking homology with known copper-transporting P-type ATPases. Disruption of this ORF renders H. pylori hypersensitive to copper present in growth media, indicating that it encodes the N-terminal amino acid sequence of the hpCopA protein. Measurement of copper content in the wild-type and hpcopA-disrupted cells showed that the copper content was increased in the mutant cells, further supporting that the previous proposal that the gene hpcopA is involved in copper export. In addition, the cop operon consisting of the genes hpcopA and hpcopP was identified by primer extension and reverse transcription-polymerase chain reaction. Our results indicate that the genes for copper import and export are located in separate operons within H. pylori.

Adenosine Triphosphatases↗

PubMed1997

Mutation analysis and expression of the mottled gene in the macular mouse model of Menkes disease.

The gene for Menkes disease, an X-linked disorder of copper transport, has recently been identified and shown to encode a copper-transporting P-type ATPase. The macular mutant mouse has been proposed as an animal model for Menkes disease. In the present study, we report the finding of a missense mutation in the mottled gene of the macular mouse. A single base change, T to C, at nucleotide position 4223, is predicted to result in an amino acid change from serine to proline at residue 1382 in the eighth transmembrane domain. This mutation differs from the 6-bp deletion we find in brindled cDNA. With validation of macular as an animal model of Menkes disease, we compared mottled gene expression in the intestine, kidney, and brain of macular and normal mice. In Northern analyses an 8.3-kb transcript was detected in the intestine, kidney, and brain of both normal and macular mice, with the level of transcript in macular approximately 80% that of normal. In situ hybridization studies revealed that the mottled gene was clearly expressed in intestinal epithelial cells, Paneth cells, and renal proximal tubular cells of both normal and macular mice. In normal brain, mottled gene expression was most intensely observed in the choroid plexus, in Ammon's born and the dentate gyrus in the hippocampus, in Purkinje cells, and the granular layer of the cerebellum. The intensity and localization of the signals in the brain of macular mice were similar to those of the controls. The distribution of expression of mottled is correlated with cells and tissues showing histopathology or abnormal copper sequestration in macular and other mutants.

Adenosine Triphosphatases↗

PubMed2002

Molecular mechanism of copper transport in Wilson disease.

Wilson disease is an autosomal recessive disorder of copper metabolism. The Wilson disease protein is a putative copper-transporting P-type ATPase, ATP7B, whose malfunction results in the toxic accumulation of copper in the liver and brain, causing the hepatic and/or neurological symptoms accompanying this disease. The cytosolic N-terminal domain (approximately 70 kDa) of this ATPase comprises six heavy metal-associated domains, each of which contains the conserved metal-binding motif GMTCXXC. The N-terminal domain (Wilson disease copper-binding domain [WCBD]) has been expressed, purified, and characterized using various techniques. The WCBD binds six atoms of copper in the +1 oxidation state competitively, and with a greater affinity than all other metals. The copper atom is coordinated by two cysteines in a distorted linear geometry. Copper binds the WCBD in a cooperative manner and induces secondary and tertiary conformation changes. Zinc binding to the WCBD has also been characterized by circular dichroism spectroscopy and shown to produce conformational changes that are completely different from those induced by copper. The phosphorylation/nucleotide-binding domain of ATP7B has also been expressed and characterized and shown to be capable of binding ATP but lacking ATPase activity. A peptide corresponding to the sixth transmembrane domain of ATP7B has been constructed and shown to undergo secondary conformational changes upon binding a single atom of copper. Finally, a chimeric protein consisting of the WCBD and truncated ZntA, a zinc-transporting ATPase lacking the N-terminal domain, has been constructed and analyzed for metal ion selectivity. These results suggest that the core determines the metal ion specificity of P-type ATPases, and the N-terminal metal-binding domain may play a regulatory role.

Adenosine Triphosphatases↗

PubMed2000

Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance.

The accumulation of cisplatin is decreased in many cisplatin-resistant cell lines, and an active efflux pump for cisplatin exists in some of them, but it has not yet been identified. In this study, we transfected the copper-transporting P-type ATPase cDNA (ATP7B) into human epidermoid carcinoma KB-3-1 cells. The transfectant, KB/WD cell line, which overexpressed the P-type ATPase, ATP7B, was resistant to both cisplatin (8.9-fold) and copper (2.0-fold). The accumulation of cisplatin in KB/WD cells was lower than in mock-transfected KB/CV cells, and the efflux of cisplatin from KB/WD cells was enhanced compared with KB/CV cells. KB/WD cells were sensitive to other heavy metals, such as antimony, arsenate, arsenite, cadmium, and cobalt. ATP7B was overexpressed in cisplatin-resistant prostate carcinoma PC-5 cells but not in the parental PC-3 cells and the revertant PC-5R cells. ATP7B may be involved in cisplatin resistance in some tumors.

Adenosine Triphosphatases↗

PubMed2002

Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans.

Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron-poor host environment. In Saccharomyces cerevisiae, high-affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis in the trans-Golgi compartment requires a copper-transporting P-type ATPase, ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct from a previously described C. albicans copper-transporting P-type ATPase, CaCrp1, which appears to be specifically involved in copper detoxification. Regulation of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate that it is required for high-affinity iron import, making it the bona fide CCC2 homologue of C. albicans. Remarkably, in a mouse model of systemic infection, the Caccc2Delta strain displayed robust proliferation and no significant reduction in pathogenicity, suggesting the existence of alternative mechanisms of iron uptake from host tissues. We identify haemin and haemoglobin as potential iron sources that can be used by C. albicans in a CaCcc2-independent manner.

Adenosine Triphosphatases↗

PubMed1998

Identification of three novel mutations and a high frequency of the Arg778Leu mutation in Korean patients with Wilson disease.

Four mutations--R778L, A874V, L1083F, and 2304delC--in the copper-transporting enzyme, P-type ATPase (ATP7B), were identified in Korean Patients with Wilson disease. Arg778Leu, the most frequently reported mutation of this enzyme, was found in six of eight unrelated patients studied, an allele frequency of 37.5%, which is considerably higher than those in other Asian populations. The novel single nucleotide deletion, 2304delC, was found in one patient. Since a mutation at cDNA nucleotide 2302 (2302insC) had been previously described, this region of the ATP7B gene may be susceptible to gene rearrangements causing Wilson disease.

Adenosine Triphosphatases↗

PubMed2000

Molecular analysis of Wilson disease in Taiwan: identification of one novel mutation and evidence of haplotype-mutation association.

Wilson disease (WND) is caused by a deficiency of the copper-transporting enzyme, P-type ATPase (ATP7B). Twelve different mutations have previously been identified in Taiwan Chinese with Wilson disease. We, herein, report another 4 missense mutations, 1 of which is novel. We did haplotype analysis of Taiwanese WND chromosomes, using three well characterized short tandem repeat markers (haplotype was assigned in the order of D13S314-D13S301-D13S316). Association correlation was found between the mutations and their respective haplotypes. Haplotype-deduced pedigree analysis was shown to be helpful in the mutation analysis of WND chromosomes and in the molecular assessment of both pre-symptomatic WND patients and carriers. Given the complexity and heterogeneity of the mutation spectrum of ATP7B, we suggest that haplotype analysis should be performed before full-scale mutation analysis.

Adenosine Triphosphatases↗

PubMed1998

Functional analysis of copper homeostasis in cell culture models: a new perspective on internal copper transport.

The movement of copper ions across membrane barriers of vital organs and tissues is a priority topic in nutrition and one for which there continues to be little understanding of the mechanism. Reports of membrane-bound, copper-transporting adenosine triphosphatases (Cu-ATPases) selective for copper ions have brought new focus to the problem and prompted fresh ideas. Using a cell culture model approach, we attempted to learn whether transport into and out of cells depends on a Cu-ATPase. Measurement of transport kinetics in fibroblasts, brain glial cells, neuroblastoma cells, and placental cells showed differences in the rates of copper uptake and response to sulfhydryl reagents. BeWo cells, a human choriocarcinoma placental cell line, behaved as did Menkes fibroblasts by avidly absorbing copper but not releasing copper to the immediate environment. Further tests showed that BeWo cells did not express the transcript for the membrane-bound Cu-ATPase that has been identified with Menkes syndrome. Transcript induction, however, was achieved by growing BeWo cells on porous filters that allowed apical and basolateral surfaces to form. With transcript expression, the cells showed a capacity to release copper into the medium. BeWo cells also synthesized a form of ceruloplasmin whose structure differed from that of the plasma protein and hence may be a product of a different gene. BeWo cells may also express the gene for Wilson disease, thus linking Menkes and Wilson proteins to maternal delivery of copper. We constructed a model in which both ATPases work in concert in a vesicle-based transport mechanism. The vesicle model may help us understand the transport of copper across the placenta and all cells in general.

Adenosine Triphosphatases↗

PubMed1995

A Synechococcus gene encoding a putative pore-forming intrinsic membrane protein.

A cyanobacterium, Synechococcus species PCC7942, has a gene encoding a copper-transporting P-type ATPase, which is located in the thylakoid membrane. At the 5'-upstream of this ATPase gene, we identified another gene, which was supposed to be implicated in a copper-transport process. This novel gene was found to encode a putative pore-forming membrane protein that belongs to a growing family of homologous intrinsic membrane proteins (the MIP family of proteins), which include the major intrinsic protein (MIP) from animal lens fibre junction membranes, the tonoplast intrinsic protein (TIP) from vacuolar membranes of higher plants, and the Escherichia coli glycerol facilitator (GlpF) in the cytoplasmic membrane. The deduced product, named SmpX (Synechococcus membrane protein), is highly homologous throughout its entire sequence to these intrinsic membrane proteins which were postulated to be pore-forming proteins involved in a variety of transport processes. The primary amino acid sequence of SmpX shares all properties characteristic for members of the MIP family. SmpX is more similar to the eukaryotic members (e.g., nodulin-26 from soybean) than to the prokaryotic ones.

Amino Acid Sequence↗

PubMedDate not supplied

Metabolic and molecular bases of Menkes disease and occipital horn syndrome.

Menkes disease and occipital horn syndrome (OHS) are related disorders of copper transport that involve abnormal neurodevelopment, connective tissue problems, and often premature death. Location of the gene responsible for these conditions on the X chromosome was indicated by pedigree analysis from the time of these syndromes' earliest descriptions. Characterization of an affected female with an X-autosomal translocation was used to identify the Menkes/OHS gene, which encodes a highly evolutionarily conserved, copper-transporting P-type ATPase. The gene normally is expressed in nearly all human tissues, and it localizes to the trans-Golgi network of cells. However, in over 70% of Menkes and OHS patients studied, expression of this gene has been demonstrated to be abnormal. Major gene deletions detectable by Southern blotting account for 15-20% of patients, and an interesting spectrum of other mutations is evident among 58 families whose precise molecular defects have been reported as of this writing. The center region of the gene seems particularly prone to mutation, and those that influence mRNA processing and splicing appear to be relatively common. Further advances in understanding the molecular and cell biological mechanisms involved in normal copper transport may ultimately yield new and better approaches to the management of these disorders.

Copper↗

PubMed1999

RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.

Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.

Alkenes↗