Human Molecular Genetics, 2001, Vol. 10, No. 4 361-370
© 2001 Oxford University Press
Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase
1Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, VIC 3052, Australia and Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood, VIC 3125, Australia, 2Centre for Microscopy and Microanalysis, 3Department of Physiology and Pharmacology and Centre for Molecular and Cellular Biology, University of Queensland, St Lucia, QLD 4072, Australia
Received 27 September 2000; Revised and Accepted 21 December 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Wilson disease is an autosomal recessive inherited disorder characterized by an impaired ability to excrete copper into the bile, which results in an accumulation of copper to toxic levels in the liver and subsequent damage to this organ due to the redox properties of this metal (1,2). The gene defective in Wilson disease (ATP7B, WND) has been cloned and encodes a copper-transporting P-type ATPase (3–5). This gene is expressed primarily in the liver (3,4), although WND mRNA is present in lower amounts in other tissues such as kidney, brain and placenta. The Wilson protein (WND) closely resembles the protein product (MNK) of the Menkes gene (ATP7A, MNK) (6,7). WND and MNK are transmembrane proteins that function to transport copper across cellular membranes, and have features characteristic of P-type ATPases, such as an ATP binding domain, and a conserved aspartic acid residue, which is transiently phosphorylated during the transduction of copper across cellular membranes (6,7). In addition, MNK and WND have six copper binding sites at their N-termini (8,9).
Several studies have shown that WND is located within the trans-Golgi network (TGN) (10–14), but in response to elevated copper levels, it relocalizes to an intracellular, vesicular compartment (10,15). A recent study of rat liver sections showed predominant TGN localization of Wnd in copper-deficient rats, and a concentration of Wnd near the bile canalicular membrane in copper-loaded rats (15). The presence of WND in a perinuclear region and near the plasma membrane of Long–Evans cinnamon (LEC) rat hepatocytes in vivo following injection of WND cDNA into LEC rat livers has also been reported (12), and copper ATPase activity has been detected in the plasma membrane fraction of rat hepatocytes (16). In the TGN, WND is thought to deliver copper to secreted copper-requiring enzymes such as ceruloplasmin. WND is also believed to play a role in copper excretion into bile, but the specific mechanisms and details of WND involvement in this process remain to be clarified.
Animal models are valuable for both clinical and scientific studies aimed at understanding the aetiology and pathology of diseases, and for developing more effective diagnostic and therapeutic strategies. An established animal model of Wilson disease is the LEC rat, which does not express Wnd (17,18). An additional animal model for Wilson disease is the toxic milk mouse. Toxic milk (tx) is an autosomal recessive mutation in mice which causes hepatic accumulation of copper that commences in the third postnatal week, and by 6 months the copper concentration can be 100-fold greater than that of the normal adult (19). This gradual accumulation of copper in the liver resembles that seen in patients with Wilson disease. In addition, pups are born copper deficient and the milk produced by mutant mothers is low in copper, resulting in the death of pups (19). Recently, we described a point mutation in the WND gene homologue (Wnd) of the tx mouse, which was predicted to cause a methionine to valine alteration in the putative eighth transmembrane domain of the encoded Wnd product. It was proposed that this mutation is responsible for the tx phenotype (20).
In this study, the cDNA encoding Wnd from normal and tx mice was expressed from cDNA constructs in Chinese hamster ovary (CHO) cells. The effect of the tx mutation on Wnd localization and on its ability to confer copper resistance upon transfected CHO cells was examined. Co-localization experiments with Wnd and MNK, endosomal and lysosomal markers were carried out, in addition to ultrastructural analyses, to define the nature of the copper-induced intracellular destination of Wnd.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Localization and copper-dependent redistribution of wild-type (Wnd-wt) and mutant (Wnd-tx) Wnd in CHO cells
To enable a detailed characterization of the intracellular location and function of Wnd-wt and Wnd-tx, CHO cell lines that stably expressed these proteins were generated. CHO-K1 cells were transfected with either pCMB98 or pCMB124, which encoded wild-type (Wnd-wt) and mutant (Wnd-tx) Wnd proteins, respectively, in a low copy eukaryotic expression vector (21). For each expression construct, several clones that expressed the Wnd protein were identified by immunofluorescence and clonally purified, and one of each was chosen for further analysis. These clones were designated CHO/Wnd-wt and CHO/Wnd-tx. The size and expression level of Wnd in each clone was examined by western blot analysis using the anti-Wnd antibody. Both clones produced a protein that was detected by the antibody and was of the expected size (
165 kDa) (Fig. 1). This antibody had previously detected a protein of the same size from mouse liver (22), which confirmed the specificity of the antibody for the Wnd protein. However, in all of the CHO/Wnd-tx clones obtained, the expression level of Wnd-tx was reduced compared with Wnd-wt (Fig. 1). Ponceau staining of the membrane confirmed equivalent loading of total cellular protein from the parental CHO-K1 control, CHO/Wnd-wt and CHO/Wnd-tx cell lines (data not shown).
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The intracellular location of Wnd-wt and Wnd-tx in the presence and absence of added copper was examined by immunofluorescence using anti-Wnd antibodies. In basal, low copper conditions, the Wnd protein expressed from both wild-type and mutant cDNA constructs had a perinuclear location, consistent with location in the TGN (Fig. 2). This result was consistent with previous studies (10–15,23), which demonstrated a TGN location for WND, and thus also indicated that the mutant protein was not mislocalized, in contrast with human WND containing the common H1069Q mutation that incorrectly localized to the endoplasmic reticulum (24). When the cells were incubated for 2 h in media containing 200 µM copper (in the form of CuCl2), Wnd-wt was detected in a punctate, vesicular pattern throughout the cytoplasm, in addition to the perinuclear region (Fig. 2). In contrast, Wnd-tx remained in the perinuclear region with elevated copper levels. Incubation of cells in media containing higher copper concentrations (400 and 600 µM) also had no effect on the localization of the mutant protein (data not shown).
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Co-expression of Wnd (Wnd-wt and Wnd-tx) and MNK in CHO cells
The perinuclear location of Wnd was similar to that observed for the MNK protein (25,26). However, the cellular redistribution of these two proteins differs in response to elevated copper levels in that MNK becomes redistributed throughout the cytoplasm and plasma membrane (25,26), whereas Wnd relocalizes to a cytoplasmic vesicular compartment. To confirm that MNK and Wnd were similarly located at the TGN under basal copper conditions and differentially located in response to increased copper, both proteins were overexpressed in the same cell. The cell line 615D is a CHO-K1-derived cell line that stably expresses the MNK protein tagged with an exofacial c-myc epitope tag (27). This cell line was transiently transfected with pCMB98. The MNK protein was detected with the monoclonal anti-myc antibody followed by the Alexa Fluor 488 goat anti-mouse IgG conjugate (green), and Wnd was detected using the rabbit-derived anti-Wnd antibody followed by the Alexa Fluor 568 goat anti-rabbit IgG conjugate (red). Evidence that the antibodies were specific for their respective proteins came from the fact that there were a small proportion of cells in the population that did not express MNK but did transiently express Wnd, and in which there were no green fluorescent signals produced by excitation at 488 nm. Similarly, in cells that expressed MNK but not Wnd, there were no red fluorescent signals produced by excitation at 568 nm (data not shown).
Under basal copper conditions (–Cu) both antibodies produced very similar perinuclear signals which clearly overlapped (Fig. 3A, i–iii) indicating that, in the same cell, MNK and Wnd were co-localized in the TGN. The c-myc antibody also detected small MNK-containing vesicles throughout the cytoplasm, whereas such vesicles were not detected by the anti-Wnd antibody. With the addition of 200 µM copper (+Cu), the number of MNK-containing vesicles, which were of uniform size and morphology, increased throughout the cytoplasm and MNK was also detected at the plasma membrane (Fig. 3A, iv) as previously observed (Fig. 2) (25,26). In contrast, Wnd-wt redistributed to a cytoplasmic compartment that comprised larger, more irregularly shaped vesicles, with minimal occupation at the plasma membrane (Fig. 3A, v), as seen in Figure 2. There appeared to be little or no overlap between MNK and Wnd-containing vesicles (Fig. 3A, vi). Therefore, these proteins appeared to redistribute to distinct cellular compartments in response to increased intracellular copper. Note that there was reduced staining of MNK at the plasma membrane with the c-myc antibody (Fig. 3A) compared with that observed in this (Fig. 3B) and previous studies with anti-MNK antibodies. This is possibly due to the recognition of a single c-myc epitope per MNK molecule by the c-myc antibody, compared with the recognition of multiple epitopes derived from the entire MNK N-terminal metal binding region by the other MNK antibodies used (Fig. 3A versus B).
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Wnd-tx was transiently expressed in the MNK-expressing cell line, 600-5#3. MNK was detected with a sheep-derived anti-MNK antibody followed by fluorescein isothiocyanate (FITC)-conjugated anti-sheep IgG, and Wnd-tx was detected using the rabbit-derived anti-Wnd antibody followed by Texas Red-conjugated anti-rabbit IgG. Control experiments were carried out in which the anti-MNK antibody was tested on cells expressing only Wnd, and the anti-Wnd antibody was tested on cells expressing only MNK (data not shown). A specific signal could not be detected in either case, indicating that the antibodies were specific for their respective proteins. In the same cell, again both MNK and Wnd-tx were localized to the TGN under basal copper conditions (Fig. 3B, i–iii). However, in elevated copper, Wnd-tx remained in the TGN (Fig. 3B, v and vi), whereas MNK redistributed throughout the cytoplasm and to the plasma membrane (Fig. 3B, iv and vi), with some residual MNK-derived TGN staining overlapping with Wnd-tx.
To identify the compartment to which Wnd-wt relocated in response to copper, co-localization experiments were carried out using markers that are known to localize to early or late endosomal compartments and lysosomes. These markers included antibodies directed against the early endosomal autoantigen EEA1 (28), against the late endosomal markers, lysosomal glycoprotein and lysobisphosphatidic acid (LBPA) (29), and LysoTracker (Molecular Probes), which is a fluorescent probe which labels acidic organelles, such as lysosomes. However, co-localization of Wnd-wt with these markers could not be demonstrated (data not shown).
Ultrastructural analysis of Wnd localization in CHO cells
To further investigate the compartment to which Wnd localizes in high copper, the distribution of the Wnd protein was then examined at an ultrastructural level using the anti-Wnd antibodies on ultra-thin frozen sections of CHO/Wnd-wt cells. In cells cultured in basal medium, specific gold particle labelling was observed on vesicular and tubular profiles concentrated in the perinuclear area of the cell close to the Golgi complex (Fig. 4). The morphology of the labelled elements and the close proximity to clathrin-coated buds suggested that Wnd is concentrated in the TGN, consistent with the immunofluorescence studies. Other membrane systems, such as the plasma membrane, the endoplasmic reticulum and the mito-chondria, generally showed very low background labelling. The effect of copper treatment on the distribution of Wnd in CHO/Wnd-wt cells was then examined. Incubation of cells for 2–4 h in 200 µM copper prior to preparation for electron microscopy caused a decreased labelling of tubulovesicular elements in the Golgi area of the cell, but a dramatic accumulation of the Wnd protein in large multivesicular structures with the morphology of late endosomes (Fig. 5). Labelling for the Wnd protein was predominantly concentrated on the limiting membrane of the putative late endosomes rather than the internal membranes.
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Copper resistance characteristics of clones expressing Wnd-wt and Wnd-tx
Previously we demonstrated that overexpression of MNK in CHO cells could confer copper resistance upon these cells (26). We also demonstrated that overexpression of MNK and Wnd-wt in fibroblasts from a Menkes patient was able to restore copper transport activity to these cells (22). However, the latter experiment was carried out at low media copper concentrations. To determine whether Wnd-wt was able to confer resistance to high copper concentrations upon CHO cells, and to ascertain the effect of the tx mutation on the resistance phenotype, colony survival assays were carried out using the stable cell lines (Fig. 6). For each cell line, the percentage survival was determined that represents the number of colony-forming units (c.f.u.) present at a given copper concentration, as a percentage of the number of c.f.u. present in basal copper conditions. The parental CHO-K1 cell line was included as a control. Wnd-wt conferred copper resistance to CHO-K1 cells to at least 190 µM copper (Fig. 6) and was consistent with the resistance profile conferred by overexpression of MNK as previously demonstrated (26). In contrast, CHO/Wnd-tx was as sensitive to copper as the parental CHO-K1 cell line, with an inability of both to survive copper concentrations beyond 30 µM (Fig. 6). Similar results were obtained over several independent experiments. The level of Wnd-wt expression was higher than that of Wnd-tx (Fig. 1), and it was not possible to obtain clones that expressed approximately the same amount of Wnd protein. However, this difference in protein expression cannot account for the lack of resistance of CHO/Wnd-tx since clearly there was overexpression of the correct-sized Wnd-tx protein, but the survival profile of the cell line was identical to that of the parental CHO-K1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study, the murine Wnd protein was overexpressed in CHO cells to determine the effect of the tx mutation on the trafficking and function of Wnd, to investigate the degree to which the intracellular locations of Wnd and MNK overlap and to define the nature of the Wnd-wt-containing compartment in the absence and presence of copper. The localization of Wnd-wt to the TGN under basal copper conditions was consistent with the results obtained from previous studies on human hepatoma cells (10,11,14,23), WND-transfected LEC rat hepatocytes (12,13), normal rat hepatocytes (15), copper-resistant human hepatoblastoma cells (30), Menkes patient fibroblasts (22), normal rat (15) and human (31) liver sections, and the mammary gland of non-lactating mice (32). These data are consistent with a role for WND in the delivery of copper to ceruloplasmin, which is thought to occur in the Golgi apparatus (13,33).
Reports on the effect of copper on the intracellular distribution of WND have been variable. Two studies have reported the absence of copper-induced redistribution of WND (14,34). Other in vitro studies have reported copper-induced movement of WND to a cytoplasmic vesicular compartment, but not to the plasma membrane in HepG2 cells (10), whereas in normal primary rat hepatocytes (15) and in polarized hepatoma cells (23) WND relocated to cytoplasmic vesicles and to the plasma membrane. In vivo studies have demonstrated the presence of WND both at the TGN and at or near the plasma membrane of hepatocytes following direct injection of a WND expression plasmid into LEC rat livers (12), and a predominance of WND-containing vesicular structures near the bile canalicular membrane in liver sections from a copper-loaded normal rat (15).
Ultrastructural analysis in this study confirmed that Wnd-wt was concentrated in the TGN, consistent with the immunofluorescence studies, but in elevated copper it was redistributed to large, multivesicular structures resembling late endosomes, with minimal occupation at the plasma membrane. Large, WND-containing vesicular structures, possibly derived from vesicle fusion, were also observed in polarized hepatocytes exposed to elevated copper (23). However, based on the lack of colocalization of Wnd with endosomal/lysosomal markers in this study (EEA1, lysosomal glycoprotein, LBPA and LysoTracker) and by Hung et al. (10) (transferrin receptor, lamp-1 and FITC–dextran), these structures could not be definitively identified. Therefore, the specific compartment to which Wnd redistributes with elevated copper remains to be identified and may, as suggested (10), represent a novel, as yet uncharacterized compartment of the cell into which copper is transported. Although the observations made in this study are subject to the caveats of in vitro experiments, including the lack of polarization of CHO cells, with the exception of copper-induced Wnd membrane localization, the data obtained are consistent with Wnd localization and function as demonstrated in polarized cell systems and in vivo. Therefore, CHO cells, due to their robustness and ease of manipulation, and as demonstrated for MNK (26,27,35,36), are a useful cell line for studying and rapidly assessing the functional effects of WND patient mutations (37) and for dissecting the molecular signals and pathways directing the function and copper-responsive sorting of WND.
The co-expression of Wnd-wt and MNK in the same cell showed that they were both located at the TGN in basal copper conditions. At the TGN, MNK is also thought to deliver copper to secreted cuproenzymes, such as lysyl oxidase. With elevated copper levels, based on the distinct morphology of MNK and Wnd-containing vesicles as evident by immunofluorescence and immunogold-labelling (26, and this study), it was clear that MNK and Wnd-wt relocated to separate compartments, with MNK redistributed throughout the cytoplasm on smaller, uniformly sized vesicles and at the plasma membrane, whereas Wnd-wt relocated to large, irregularly shaped cytoplasmic vesicles to produce a more punctate pattern of cytoplasmic staining than has been observed with MNK. This punctate staining pattern was reflected by the large, endosomal-like structures observed by immunogold-labelling with the anti-Wnd antibody. In contrast to the lack of co-localization of Wnd with endosomal markers (10, and this study), MNK was recently shown to co-localize with the transferrin receptor to peripheral and perinuclear endosomes (27). When MNK and Wnd-tx were co-expressed in the same cell, MNK redistributed in elevated copper, but Wnd-tx remained in the TGN. These results clearly indicate that MNK and Wnd are sorted to distinct destinations in the cell and follow separate trafficking pathways in response to elevated copper, and that the copper-responsive sorting of Wnd is directly or indirectly disrupted by the tx mutation. This difference in re-localization patterns of MNK and Wnd may reflect the distinct roles of these two proteins within the cell.
In this study, we demonstrated that overexpression of Wnd-wt in CHO cells conferred levels of resistance to copper similar to that conferred by MNK. We suggested that copper resistance resulting from overexpression of MNK was due to the presence of MNK at the plasma membrane from where excess copper was extruded from the cell, either by active efflux from the plasma membrane or by fusion of copper-containing vesicles with the membrane (26,38). Copper resistance mediated by Wnd-wt may result from copper sequestration into the late endosome-like vesicles, as suggested for copper-resistant human hepatoblastoma cells that overexpress WND (30). Our data are consistent with a recently proposed model whereby WND molecules are recycled back to the TGN from vesicles, while the copper-containing vesicles devoid of WND are further targeted to the canalicular membrane for excretion of copper into bile (15). An alternative model proposes both direct copper excretion by WND from the apical membrane of hepatocytes, and copper sequestration into vesicles followed by fusion with the apical membrane to release their contents into bile (23). The specific mechanisms of copper delivery from vesicles to the plasma membrane will require further investigation.
The tx mutation causes an alteration of a highly conserved methionine residue to valine within the putative eighth transmembrane region of Wnd (20). In contrast with Wnd-wt, overexpression of the mutant protein, albeit at lower levels, did not confer copper resistance on CHO cells. In addition, Wnd-tx was correctly localized to the TGN under basal copper conditions, but did not relocalize in response to elevated copper levels. It is possible that the tx mutation disrupts a critical signal required for trafficking or vesicular sorting of the protein in response to copper, which in turn leads to the inability of Wnd-tx to confer copper resistance since Wnd-containing vesicles cannot leave the TGN. However, recently it was demonstrated that WND with mutation of the conserved CPC motif to SPS localized normally in CHO cells, but was unable to redistribute in response to copper and was inactive in a yeast copper transport activity assay (37). In addition, several mutations within structurally and functionally distinct regions of MNK, either identified in patient cells or introduced into the cDNA, and which have been predicted or shown to disrupt the copper transport activity of the protein, also prevent the copper-induced redistribution of the protein (39,40). Based on these data, it is likely that the inability of Wnd-tx to relocalize in elevated copper is related to the loss of copper transport function of this protein resulting from the tx mutation. Therefore, copper-responsive sorting of Wnd is likely to be dependent on an active Wnd molecule. The details of the relationship between copper transport activity and copper-induced trafficking remain to be elucidated, but as we have suggested (39), conformational changes associated with the transduction of copper across cellular membranes may be required to expose sorting signals or to facilitate interactions that induce vesicular trafficking.
To date only one study has attempted immunohistochemical analysis of WND localization in the liver of a Wilson disease patient to discover lack of WND expression (31). However, support for the above model comes from immunohistochemical studies of Wnd localization in the mammary glands of normal and tx mice (32). In non-lactating normal and tx mice, Wnd is located in a perinuclear region of luminal epithelial cells, and this localization remains unchanged in lactating tx mice. In contrast, in lactating normal mice Wnd showed a dispersed, granular, cytoplasmic location. This localization remained unchanged with copper loading of this mouse but, with copper loading of the lactating tx mouse, the mutant Wnd appeared to mislocalize to a region between the nucleus and cytoplasm, a location that was clearly different to that of the normal mouse. It was hypothesized that an additional important function of Wnd is the transport of copper into milk in the mammary gland, and that lactation has the effect of increasing intracellular copper levels, thus leading to Wnd relocation to increase copper transport into milk. The production of copper-deficient milk by lactating tx mice is likely to be due to lack of function and trafficking of Wnd-tx.
Animal models of human diseases are important in helping to derive a more complete understanding of such diseases, which ultimately will lead to better management strategies. The identification of the tx mutation (20) and the analysis of its effects on the function and intracellular distribution of Wnd in this study have confirmed that the tx mouse is a true model of Wilson disease, and has provided an important insight into the molecular basis for the phenotype of the tx mouse. The disruption of copper transport activity and copper-induced relocalization of Wnd account for the hepatic copper accumulation phenotype of these mice. This study has also provided compelling evidence that MNK and Wnd follow different pathways within the cell in response to copper, which is likely to reflect their distinct roles within different cell types. In addition, there is an accumulating body of evidence for a relationship between the copper transport activity and copper-induced redistribution of copper ATPases such as MNK and WND, which will provide important insights into the molecular basis of phenotypes displayed by Menkes and Wilson disease patients. Clarification of the details of such a relationship is likely to contribute to the development of more effective treatment strategies for these patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture and transfection experiments
CHO-K1 cells and derivatives were maintained in basal media (1.5 µM Cu) which consisted of Eagle’s Basal medium (BME; Trace Biosciences), supplemented with 10% fetal calf serum (FCS; Trace Biosciences), L-proline at a final concentration of 20 mM and 0.2% (w/v) bicarbonate. For transient transfections CHO-K1 cells were seeded onto glass coverslips at a density of 1 x 104 cells per coverslip and grown overnight at 37°C in 5% CO2. The following day, cells were transfected using the Superfect Reagent (Qiagen) according to the manufacturer’s protocols. Cells were allowed to recover for 24 h to allow expression of the protein from the plasmid. Where the effect of copper was tested, copper was added to the media (as CuCl2) at the desired concentration for 2–4 h before cells were fixed and processed for immunofluorescence. For stable transfections CHO-K1 cells were transfected with pCMB98 or pCMB124, which carry normal and tx mouse Wnd cDNAs, respectively. Liposome-mediated transfection was carried out using Superfect Reagent (Qiagen) and recommended protocols, followed by selection in G418 (Life Technologies) for 2 weeks. Screening of G418-resistant transfectants for those that expressed the Wnd protein was carried out by indirect immunofluorescence.
Production of the mouse Wnd cDNA construct
The full-length Wnd cDNA was assembled from three overlapping subclones. The first of these, clone 
12, represented the first 1.5 kb of the cDNA. This clone was derived from a BALB/c liver cDNA library (Clontech) and contained 162 bp of 5' untranslated sequence, followed by coding sequence to the first EcoRI site (nucleotide 1285) (20). The remainder of the cDNA was amplified from mRNA in two sections using RT–PCR as described previously (20). The middle section (nucleotides 1016–3320) overlapped with the 5' clone to allow the use of a unique ApaI site for cloning. The 3' section (nucleotides 2874–4545) harboured a unique KspI site that was used for ligation with the remainder of the cDNA. The cDNA containing the full-length open reading frame was cloned as an XbaI–XhoI fragment into the low copy number vector pWSK29 (41), and the plasmid was designated pCMB94. The cDNA then was cloned as an SpeI–SalI fragment into the expression vector pCMB77 (35) under the control of the cytomegalovirus promoter and this expression plasmid was designated pCMB98.
For the construction of the plasmid carrying the tx mutation, the 3' section of Wnd (nucleotides 2874–4545) was amplified as before, this time using mRNA isolated from a tx mouse liver. This region was used to replace the corresponding region of the normal cDNA from the plasmid pCMB94 to yield a full-length Wnd cDNA clone carrying the tx mutation, which was designated pCMB123. The cDNA then was subcloned as an SpeI–SalI fragment into the pCMB77 expression vector to generate the plasmid pCMB124.
The sequence of the cDNAs was confirmed using Sequenase Version 2.0 DNA Sequencing kit (US Biochemical). All other DNA manipulations were carried out using standard techniques (42).
Antibody production
Primary Wnd antibodies were prepared by immunizing rabbits with a fusion protein that was generated and purified using the QIAexpress System (Qiagen). The fusion protein comprised 275 N-terminal amino acids of Wnd which spanned part of MBS 2 and all of MBS 3 and 4, fused to a 6xHis tag. The Wnd IgG fraction was prepared by sodium sulfate precipitation as described (43) and a proportion of this preparation was subjected to further affinity-purification, also as previously described (43).
Western blot analysis
Western blot analysis was carried out as described previously (21). The primary antibody consisted of a sodium sulfate-precipitated preparation of anti-Wnd antibodies directed against the Wnd N-terminus.
Indirect immunofluorescence
Immunofluorescence analysis of CHO-K1 cells and derivatives was carried out essentially as described previously (25). In general, cells were cultured on 13 mm glass coverslips for 48 h unless otherwise specified. Where appropriate, copper chloride was added to the growth medium to a final concentration of 200 µM for 2–4 h, unless otherwise specified. The primary antibody consisted of either a sodium sulfate-precipitated or an affinity-purified preparation of anti-Wnd antibodies. The secondary antibody consisted of FITC-conjugated sheep antibodies to rabbit IgG (Silenus).
For co-localization studies either the MNK-expressing CHO cell line 600-5#3 (26) or a CHO clone that expressed a c-myc-tagged MNK protein (615D) (27) were used. For co-localization studies in the cell line 600-5#3 (26), mouse Wnd was detected using the affinity-purified rabbit anti-Wnd antibody as the primary antibody, followed by Texas Red-conjugated anti-rabbit IgG (ICN Pharmaceuticals) as the secondary antibody. MNK was detected using affinity-purified anti-MNK antibodies raised in sheep followed by FITC-conjugated anti-sheep IgG (Silenus). In the 615D cells, MNK was detected with an affinity-purified monoclonal anti-myc antibody (9E10; Boehringer Mannheim) as the primary antibody and the Alexa Fluor 488 goat anti-mouse IgG conjugate (Molecular Probes) as the secondary antibody. In these cells, Wnd was detected with the sodium sulfate-precipitated preparation of anti-Wnd antibodies as the primary antibody followed by the Alexa Fluor 568 goat anti-rabbit IgG conjugate (Molecular Probes) as the secondary antibody.
Electron microscopy
Cells of the stable clones CHO/Wnd-wt (either untreated or treated for 2–4 h with 200 µM CuCl2) were fixed in 8% paraformaldehyde in 0.1 M phosphate buffer pH 7.35 for 1 h at room temperature. They were then washed with 0.2 M phosphate buffer, scraped from the culture dish and pelleted at 10 000 r.p.m. in a microfuge. The cells then were resuspended in warm gelatin (10% in phosphate buffer) and repelleted at maximum speed in the microfuge. After cooling, the gelatin-embedded cells were infiltrated with PVP-sucrose overnight at 4°C and processed for frozen sectioning as described previously (44). Ultra-thin frozen sections (60–80 nm) were labelled, stained and viewed (Jeol 1010; Centre for Microscopy and Microanalysis, University of Queensland) according to published techniques (44). The antibody consisted of the sodium sulfate-precipitated preparation of anti-Wnd antibodies.
Colony survival assay
Cells were seeded into 3 cm wells at 100 cells per well and were allowed to attach overnight. For each cell line tested, appropriate amounts of copper (0, 30, 60, 95, 125, 160 and 190 µM) as CuCl2 were then added to wells in triplicate, and colonies were allowed to form over the following 7 days. The colonies were washed with phosphate-buffered saline, fixed with 90% methanol/10% formaldehyde, rinsed with water and stained with Giemsa stain (Sigma) diluted 1 in 10 with water. The visible colonies in each well were counted and the mean number of colonies for each triplicate set of wells was determined. For each cell line, the mean number of colonies in basal media (no added CuCl2) was taken as 100% survival, and the survival of each cell line at increasing copper concentrations is represented as a percentage of the number of colonies present with no added CuCl2.
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ACKNOWLEDGEMENTS |
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We thank Rosario Reyes for invaluable technical support, Natalie Barnes for assistance with transient transfections, Professor Jean Gruenberg, University of Geneva, for kindly providing the late endosomal antibody markers, and Loreta Ambrosini and Michael Petris for helpful scientific discussions and critical reading of the manuscript. This work was supported by grants from the National Health and Medical Research Council of Australia and the International Copper Association. M.B.T. was a Helen Schutt postgraduate scholar.
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FOOTNOTES |
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+ These authors contributed equally to this work
§ To whom correspondence should be addressed at: Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Melbourne Campus, Building L, 221 Burwood Highway, Burwood, VIC 3125, Australia. Tel: +61 3 9251 7329; Fax: +61 3 9251 7328; Email: jmercer@deakin.edu.au
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Danks, D.M. (1995) The metabolic and molecular basis of inherited disease. In Scriver, C.R., Beaudet, A.L., Sly, W.M. and Valle, D. (eds), Disorders of Copper Transport. McGraw-Hill, New York, NY, pp. 2211–2235.
2 Menkes, J.H. (1999) Menkes disease and Wilson disease: two sides of the same copper coin. Part II: Wilson disease. Eur. J. Paed. Neurol., 3, 245–253.
3 Tanzi, R.E., Petrukhin, K., Chernov, I., Pellequer, J.L., Wasco, W., Ross, B., Romano, D.M., Parano, E., Pavone, L., Brzustowicz, L.M. et al. (1993) The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genet., 5, 344–350.[Web of Science][Medline]
4 Bull, P.C., Thomas, G.R., Rommens, J.M., Forbes, J.R. and Cox, D.C. (1993) The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genet., 5, 327–337.[Web of Science][Medline]
5 Yamaguchi, Y., Heiny, M.E. and Gitlin, J.D. (1993) Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease. Biochem. Biophys. Res. Commun., 197, 271–277.[Web of Science][Medline]
6 Schaefer, M. and Gitlin, J.D. (1999) Genetic disorders of membrane transport. IV. Wilson’s disease and Menkes disease. Am. J. Physiol., 276, G311–G314.
7 Suzuki, M. and Gitlin, J.D. (1999) Intracellular localization of the Menkes and Wilson’s disease proteins and their role in intracellular copper transport. Pediatr. Int., 41, 436–442.[Web of Science][Medline]
8 DiDonato, M., Narindrasorasak, S., Forbes, J.R., Cox, D.W. and Sarkar, B. (1997) Expression, purification and metal binding properties of the N-terminal domain from the Wilson disease putative copper-transporting ATPase (ATP7B). J. Biol. Chem., 272, 33279–33282.
9 Lutsenko, S., Petrukhin, K., Cooper, M.J., Gilliam, C.T. and Kaplan, J.H. (1997) N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson’s and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat. J. Biol. Chem., 272, 18939–18944.
10 Hung, I.H., Suzuki, M., Yamaguchi, Y., Yuan, D.S., Klausner, R.D. and Gitlin, J.D. (1997) Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae. J. Biol. Chem., 272, 21461–21466.
11 Lutsenko, S. and Cooper, M.J. (1998) Localization of the Wilson’s disease protein product to mitochondria. Proc. Natl Acad. Sci. USA, 95, 6004–6009.
12 Nagano, K., Nakamura, K., Urakami, K.-I., Umeyama, K., Uchiyama, H., Koiwai, K., Hattori, S., Yamamoto, T., Matsuda, I. and Endo, F. (1998) Intracellular distribution of the Wilson’s disease gene product (ATPase7B) after in vitro and in vivo exogenous expression in hepatocytes from the LEC rat, an animal model of Wilson’s disease. Hepatology, 27, 799–807.[Web of Science][Medline]
13 Terada, K., Nakako, T., Yang, X.-L., Iida, M., Aiba, N., Minamiya, Y., Nakai, M., Sakaki, T., Miura, N. and Sugiyama, T. (1998) Restoration of holoceruloplasmin synthesis in LEC rat after infusion of recombinant adenovirus bearing WND cDNA. J. Biol. Chem., 273, 1815–1820.
14 Yang, X.-L., Miura, N., Kawarada, Y., Terada, K., Petrukhin, K., Gilliam, T.C. and Sugiyama, T. (1997) Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments. Biochem. J., 326, 897–902.
15 Schaefer, M., Hopkins, R.G., Failla, M.A. and Gitlin, J.D. (1999) Hepatocyte-specific localization and copper-dependent trafficking of the Wilson’s disease protein in the liver. Am. J. Physiol., 276, G639–G646.
16 Dijkstra, M., Veld, G.I.T., Berg, G.J.V.D., Muller, M., Kuipers, F. and Vonk, R.J. (1995) Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membrane. J. Clin. Invest., 95, 412–416.
17 Wu, J., Forbes, J.R., Chen, H.S. and Cox, D.W. (1994) The LEC rat has a deletion in the copper transporting ATPase gene homologous to the Wilson disease gene. Nature Genet., 7, 541–544. [Web of Science][Medline]
18 Yamaguchi, Y., Heiny, M.E., Shimizu, N., Aoki, T. and Gitlin, J.D. (1994) Expression of the Wilson disease gene is deficient in the Long-Evans Cinnamon rat. Biochem. J., 301, 1–4.
19 Rauch, H. (1983) Toxic milk, a new mutation affecting copper metabolism in the mouse. J. Hered., 74, 141–144.
20 Theophilos, M.B., Cox, D.W. and Mercer, J.F.B. (1996) The toxic milk mouse is a murine model of Wilson disease. Hum. Mol. Genet., 5, 1619–1624.
21 La Fontaine, S., Firth, S.D., Lockhart, P.J., Paynter, J.A. and Mercer, J.F.B. (1998) Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein. Plasmid, 39, 245–251.[Web of Science][Medline]
22 La Fontaine, S., Firth, S.D., Camakaris, J., Englezou, A., Theophilos, M.B., Petris, M.J., Howie, M., Lockhart, P.J., Greenough, M., Brooks, H. et al. (1998) Correction of the copper transport defect of Menkes patient fibroblasts by expression of the Menkes and Wilson ATPases. J. Biol. Chem., 273, 31375–31380.
23 Roelofsen, H., Wolters, H., Luyn, M.J.A.V., Miura, N., Kuipers, F. and Vonk, R.J. (2000) Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion. Gastroenterology, 119, 782–793.[Web of Science][Medline]
24 Payne, A.S., Kelly, E.J. and Gitlin, J.D. (1998) Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation. Proc. Natl Acad. Sci. USA, 95, 10854–10859.
25 Petris, M.J., Mercer, J.F.B., Culvenor, J.G., Lockhart, P., Gleeson, P.A. and Camakaris, J. (1996) Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J., 15, 6084–6095.[Web of Science][Medline]
26 La Fontaine, S., Firth, S.D., Lockhart, P.J., Brooks, H., Parton, R.G., Camakaris, J. and Mercer, J.F.B. (1998) Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1). Hum. Mol. Genet., 7, 1293–1300.
27 Petris, M.J. and Mercer, J.F.B. (1999) The Menkes protein (ATP7A; MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal. Hum. Mol. Genet., 8, 2107–2115.
28 Mu, F.-T., Callaghan, J.M., Steele-Mortimer, O., Stenmark, H., Parton, R.G., Campbell, P.L., McCluskey, J., Yeo, J.-P., Tock, E.P.C. and Toh, B.-H. (1995) EEA1, an Early Endosome-Associated Protein. J. Biol. Chem., 270, 13503–13511.
29 Kobayashi, T., Stang, E., Moerloose, P.D., Parton, R.G. and Gruenberg, J. (1998) A lipid antigen associated with the anti-phospholipid syndrome regulates endosome structure/function. Nature, 392, 193–197. [Medline]
30 Schilsky, M.L., Stockert, R.J., Kesner, A., Gorla, G.R., Gagliardi, G.S., Terada, K., Miura, N. and Czaja, M.J. (1998) Copper resistant human hepatoblastoma mutant cell lines without metallothionein induction overexpress ATP7B. Hepatology, 28, 1347–1356.[Web of Science][Medline]
31 Schaefer, M., Roelofsen, H., Wolters, H., Hofmann, W.J., Müller, M., Kuipers, F., Stremmel, W. and Vonk, R.J. (1999) Localization of the Wilson’s disease protein in human liver. Gastroenterology, 117, 1380–1385.[Web of Science][Medline]
32 Michalczyk, A.A., Reiger, J., Allen, K.J., Mercer, J.F.B. and Ackland, M.L. (2000) Defective localization of the Wilson disease protein (ATP7B) in the mammary gland of the toxic milk mouse and the effects of copper supplementation. Biochem. J., 352, 565–571.
33 Murata, Y., Yamakawa, E., Iizuka, T., Kodama, H., Abe, T., Seki, Y. and Kodama, M. (1995) Failure of copper incorporation into ceruloplasmin in the golgi apparatus of LEC rat hepatocytes. Biochem. Biophys. Res. Commun., 209, 349–355.[Web of Science][Medline]
34 Harada, M., Sakisaka, S., Kawaguchi, T., Kimura, R., Taniguchi, E., Koga, H., Hanada, S., Baba, S., Furuta, K., Kumashiro, R. et al. (2000) Copper does not alter the intracellular distribution of ATP7B, a copper-transporting ATPase. Biochem. Biophys. Res. Commun., 275, 871–876.[Web of Science][Medline]
35 Petris, M.J., Camakaris, J., Greenough, M., La Fontaine, S.L. and Mercer, J.F.B. (1998) A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network. Hum. Mol. Genet., 7, 2063–2071.
36 Strausak, D., La Fontaine, S.L., Hill, J., Firth, S.D., Lockhart, P.J. and Mercer, J.F.B. (1999) The role of GMXCXXC metal binding sites in the copper-induced redistribution of the Menkes protein. J. Biol. Chem., 274, 11170–11177.
37 Forbes, J.R. and Cox, D.W. (2000) Copper-dependent trafficking of Wilson disease mutant ATP7B proteins. Hum. Mol. Genet., 9, 1927–1935.
38 Camakaris, J., Petris, M.J., Bailey, L., Shen, P., Lockhart, P., Glover, T.W., Barcroft, C.L., Patton, J. and Mercer, J.F.B. (1995) Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux. Hum. Mol. Genet., 4, 2117–2123.
39 La Fontaine, S., Firth, S.D., Lockhart, P.J., Brooks, H., Camakaris, J. and Mercer, J.F.B. (1999) Intracellular localization and loss of copper-responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Moblo) and brindled (Mobr) mouse mutants. Hum. Mol. Genet., 8, 1069–1075.
40 Ambrosini, L. and Mercer, J.F.B. (1999) Defective copper-induced trafficking and localization of the Menkes protein in patients with mild and copper-treated classical Menkes disease. Hum. Mol. Genet., 8, 1547–1555.
41 Wang, R.F. and Kushner, S.R. (1991) Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene, 100, 195–199.[Web of Science][Medline]
42 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, NY.
43 Johnstone, A. and Thorpe, R. (1987) Immunocytochemistry in Practice. Blackwell Scientific Publications, Oxford, UK, pp. 50–54.
44 Parton, R.G., Way, M., Zorzi, N. and Stang, E. (1997) Caveolin-3 associates with developing T-tubules during muscle differentiation. J. Cell Biol., 136, 137–154.
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