Copper-dependent trafficking of Wilson disease ... - Semantic Scholar

© 2000 Oxford University Press

Human Molecular Genetics, 2000, Vol. 9, No. 13 1927–1935

Copper-dependent trafficking of Wilson disease mutant ATP7B proteins John R. Forbes and Diane W. Cox+ Department of Medical Genetics, 8–39 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada Received 18 January 2000; Revised and Accepted 19 June 2000

We have previously developed a functional assay in yeast for the copper transporter, ATP7B, defective in Wilson disease (WND). Analysis of WND variant ATP7B proteins revealed that several were able to completely, or nearly completely, complement a mutant yeast strain in which the ATP7B ortholog CCC2 was disrupted, indicating that these ATP7B proteins retained copper transport activity. We analyzed the intracellular localization of these active WND ATP7B variant proteins using transient transfection of Chinese hamster ovary cells and triplelabel immunofluorescence microscopy, as a second possible aspect of defective function. Two ATP7B variants, Asp765Asn and Leu776Val, which have normal copper transport activity in yeast, retained partial normal Golgi network localization, but were predominantly mislocalized throughout the cell. Asp765Asn and Leu776Val proteins were capable of only partial copper-dependent redistribution. WND variant protein Arg778Leu, which has defective function in yeast, was extensively mislocalized, presumably to the endoplasmic reticulum. ATP7B variant proteins Gly943Ser, which has nearly normal function in yeast, and CysProCys/Ser (mutation of the conserved CysProCys motif to SerProSer), inactive in yeast, were localized normally but were unable to redistribute in response to copper. Localization data from this study, combined with functional data from our yeast studies, provide a biochemical mechanism that can explain in part the variable biochemical features of WND, in particular the normal holoceruloplasmin levels observed in some patients. Our data have direct implications for WND diagnosis, indicating that decreased serum ceruloplasmin concentration is not likely to be observed with certain genetic variants of WND. INTRODUCTION Wilson disease (WND) is an autosomal recessive disorder of copper transport with a worldwide frequency of ∼1 in 30 000. WND is characterized by chronic liver and/or neurological +To

disease, sometimes accompanied by kidney damage (1). The WND gene (ATP7B), mapping to 13q14.3 (2), has been cloned, and encodes a copper transporting P-type ATPase (ATP7B) (3,4). On the basis of northern blots, ATP7B is expressed primarily in the liver and kidney. The main biochemical phenotype of WND is hepatic copper accumulation due to impaired biliary copper efflux (1,5). Normal adults typically have 250 µg/g, which can approach 3000 µg/g (1,5). Hepatic copper levels vary among normal individuals and WND patients depending on dietary copper intake and bioavailability, as well as genetic factors (5–8). The serum ceruloplasmin level, currently measured immunologically, or less commonly by oxidase activity, is often the first step diagnostic biochemical marker of WND (1,5,8). Apoceruloplasmin biosynthesis is normal in WND patients, but copper incorporation into the protein during biosynthesis is impaired such that patients have reduced circulating holoceruloplasmin levels (5,9,10). Total ceruloplasmin (holo- plus apo-ceruloplasmin) levels vary considerably in normal individuals. This is due to factors such as pregnancy, acute inflammation, infection and oral contraception (5). Genetic variation of the ceruloplasmin gene may also influence circulating ceruloplasmin levels. Although total ceruloplasmin levels vary, there is usually little apo-ceruloplasmin found in the plasma of normal individuals (1,5). WND patients usually have low plasma holo-ceruloplasmin levels; however, this is not always the case (1,5). Some WND patients have circulating holo-ceruloplasmin levels within the normal range. Hepatic copper homeostasis, mediated by ATP7B, is dependent on both copper transport activity, and localization to the correct intracellular compartment. ATP7B plays a dual functional role in the hepatocyte. One role is biosynthetic, delivering copper to apo-ceruloplasmin within the Golgi network (1,9,11,12). The other role of ATP7B is to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile (1,13,14). ATP7B is localized in the trans-Golgi network of hepatocytes under low copper conditions, redistributes to cytoplasmic vesicles when cells are exposed to elevated copper levels, and then recycles back to the trans-Golgi network when copper is removed (15,16). Copperdependent redistribution and recycling of ATP7B probably occur continuously in the presence of either low or high copper concentrations as was observed with ATP7A (15–17). The observable steady-state localization of ATP7B is altered

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1928 Human Molecular Genetics, 2000, Vol. 9, No. 13

depending on copper levels. Copper-dependent localization probably represents a post-translationally inducible switch from a primarily biosynthetic role for ATP7B in the trans-Golgi network, to a primarily excretory role involving membrane vesicles, under conditions of copper overload (16,18). The Menkes disease protein, ATP7A, is a copper transporting P-type ATPase very similar to ATP7B, sharing ∼57% amino acid identity (3,19–21). Studies on the intracellular localization of ATP7A in mammalian cells, done prior to those of ATP7B, revealed a copper-dependent trafficking event similar to ATP7B, except that ATP7A moves from the transGolgi network to the plasma membrane following copper stimulation (22). There have been reports attempting to correlate the disease severity of Menkes disease mutations with the intracellular localization of the corresponding ATP7A variant proteins (23–25). These data suggest that movement from the trans-Golgi network and localization to the plasma membrane is essential for copper efflux by ATP7A/Atp7a (23–25). However, these studies were lacking functional data on the ATP7A protein variants analyzed, and could not determine conclusively whether the Menkes disease phenotype observed in cell lines is caused by defective ATP7A localization alone, or whether the phenotype was caused by defects in ATP7A copper transport activity. We have previously used complementation of the yeast ccc2 mutant which lacks Ccc2p, the yeast ortholog of ATP7B (26), as a functional assay for WND mutations found within the transmembrane domain of ATP7B (18). Analysis of WND variant proteins revealed that several were able to completely, or nearly completely, complement the defective high-affinity iron uptake phenotype of ccc2 mutant yeast cells, indicating that these ATP7B protein variants retained normal, or only partially reduced, copper transport activity (18). We hypothesized that ATP7B variant proteins retaining all, or most, copper transport activity, may be rare normal variants not yet found on normal chromosomes, or variant proteins with normal copper transport activity that are mislocalized in hepatocytes. Secondly, we hypothesized that ATP7B protein variants retaining at least some copper transport activity, that are unable to undergo copper-dependent trafficking from the trans-Golgi network to the vesicular compartment, or are mislocalized, would not mediate adequate biliary copper efflux, leading to hepatic copper accumulation. However, ATP7B protein variants such as these could still transport sufficient copper into the Golgi network for incorporation into ceruloplasmin. This report describes the copper-dependent localization, and mislocalization, of WND variant ATP7B proteins, that we have previously assayed for function (18). The goal of this work was to attempt to understand the interrelation between ATP7B intracellular localization, copper-dependent trafficking and copper transport function, with respect to WND pathogenesis. To do so, we have analyzed the intracellular localization of ATP7B variant proteins associated with WND, using transient transfection of Chinese hamster ovary (CHO) cells and triple-label immunofluorescence microscopy. We can distinguish variants in which copper transport is disrupted from those in which intracellular trafficking is impaired.

RESULTS Specificity of antibodies The antibody used for immunofluorescence was affinitypurified anti-ATP7B.N60, which recognizes the copperbinding domain of ATP7B. This antibody can detect the ATP7B protein expressed in yeast on immunoblots while the pre-immune serum cannot. Anti-ATP7B.N60 antibody was able to recognize endogenous ATP7B protein expressed in HepG2 cells by immunofluorescence. Pre-incubation of the antibody with 5 µg of total cell protein extract from yeast expressing ATP7B completely eliminated the signal, whereas the same amount of protein from control yeast extracts did not. Additionally, anti-ATP7B.N60 gave no specific signal by immunofluorescence in HeLa, or CHO cells, which express ATP7A but not ATP7B (15,22). These data demonstrate that anti-ATP7B.N60 antibody was specific for ATP7B and did not cross-react with ATP7A in this application. Copper-induced trafficking of ATP7B Endogenous ATP7B, detected in HepG2 cells by immunofluorescence using anti-ATP7B.N60 antibodies, gave distinct Golgi network localization in standard medium, and redistributed almost entirely to cytoplasmic vesicles on addition of 500 µM CuSO4 to the growth medium, consistent with previous studies (15,16). The presence of endogenous ATP7B made interpretation of results more difficult. Transient transfection of ATP7B expression vector into HeLa cells, which do not express ATP7B, followed by immunofluorescent detection, revealed distinct Golgi localization of ATP7B, with no background staining visible in non-transfected cells (data not shown). However, ATP7B did not visibly redistribute to cytoplasmic vesicles in response to copper and remained localized to the Golgi membrane region at all copper concentrations used. HeLa cells were therefore judged unsuitable for studies of ATP7B copper-dependent trafficking. For analysis of normal and WND variant ATP7B proteins we used CHO cells, which have been used extensively for studies of ATP7A localization (17,22,23,27,28). Triple-label immunofluorescence analysis of CHO cells transiently transfected with ATP7B constructs was performed using ATP7B specific polyclonal antibodies generated in our laboratory, monoclonal antibodies against a protein marker of the Golgi network (Golgi-58K protein), and [([(succinimidyl)oxy]carbonyl)methyl]-4-methylcoumarin-6-sulfonic acid (AMCA-S) fluorophor-labeled concanavalin A (ConA), a lectin which stains the endoplasmic reticulum. Anti-ATP7B and Golgi-58K antibodies were detected with secondary antibodies labeled with fluorescein isothiocyanate (FITC) or Cy3 fluorophors, respectively. These were visualized in the same cell using the appropriate fluorescent filters such that assignment of the subcellular localization of transfected ATP7B protein could be made. Transfection of ATP7B expression vector into CHO cells followed by indirect immunofluorescent detection revealed expression and perinuclear or juxtanuclear staining of normal ATP7B, co-localizing with Golgi-58K marker antibody under copper-limited conditions (Fig. 1). Addition of copper chelator

Human Molecular Genetics, 2000, Vol. 9, No. 13 1929

Figure 1. Composite image, showing the copper-dependent subcellular localization of ATP7B. CHO cells were transiently transfected with an ATP7B expression construct. Each row represents the same cells. Examples of typical cells were selected from 50–100 examined for each variant. Two to three hours prior to processing for triple-label immunofluorescence microscopy, cells were treated with either the copper chelator BCS or copper chloride at the indicated concentrations. ATP7B protein was detected with an affinity-purified rabbit polyclonal antibody against its copper binding domain. The Golgi network was detected with mouse monoclonal Golgi-58K antibody (early Golgi). The endoplasmic reticulum was detected with fluorescent ConA. Microscopy was performed using a 100× lens with oil immersion.

bathocuproine disulfonate (BCS) to the medium, to limit copper levels prior to immunofluorescence, gave more distinct localization to the Golgi network than seen in standard medium. Negligible background staining was observed in untransfected cells. Addition of copper to the growth medium caused a shift in ATP7B localization from the Golgi network to cytoplasmic vesicles that were distinct from the endoplasmic reticulum in transfected CHO cells (Fig. 1). Extracellular copper at a final concentration of 250 µM was sufficient to stimulate robust redistribution of ATP7B protein in CHO cells and was used in subsequent experiments. Residual Golgi network localized ATP7B protein was evident in coppertreated transfected cells. Localization of ATP7B WND variant proteins The ATP7B WND variant proteins analyzed in this study were Asp765Asn (D765N), Leu776Val (L776V) and Gly943Ser (G943S), each of which had normal, or nearly normal, capability to complement ccc2 mutant yeast, indicating normal copper transport activity (18). Arg778Leu (R778L) was chosen as a representative of a severely defective, but not inactive, WND variant ATP7B protein. A protein with the CysProCys (CPC) motif mutated to serine (both serines, CPC/S) was chosen as an example of an ATP7B variant protein completely unable to transport copper in our complementation assay. Met769Val (M769V) had temperature-dependent activity in our yeast complementation assay (no complementation at 37°C). These mutations are all found in the transmembrane domain of ATP7B in patients with WND. D765N, M769V, L776V and R778L are found in the fourth membrane-spanning segment. G943S and

Figure 2. Composite image, showing the copper-dependent subcellular localization of WND variant ATP7B proteins. Each row represents the same cells. CHO cells were transiently transfected with variant ATP7B expression constructs. Variant proteins are designated by mutation. Two to three hours prior to processing for triple-label immunofluorescence microscopy, cells were treated with either the copper chelator BCS or copper chloride at the indicated concentrations. ATP7B protein was detected with an affinity-purified rabbit polyclonal antibody against its copper binding domain. The Golgi network was detected with mouse, monoclonal, Golgi-58K antibody. The endoplasmic reticulum was detected with fluorescent ConA. Microscopy was performed using a 100× lens with oil immersion.

CPC/C are found in the fifth and sixth transmembrane segments, respectively. The latter has not been reported in patients. The ATP7B protein variants D765N and L776V ATP7B were partially localized to a perinuclear location, likely the trans-Golgi network, of transfected CHO cells in the absence of copper (Fig. 2). The majority of D765N and L776V protein was distributed throughout the cell in virtually all transfected cells. Addition of copper to the growth medium resulted in some redistribution of both D765N and L776V proteins to cytoplasmic vesicles; however, the type of staining typical of dispersion in endoplasmic reticulum remained prominent. Therefore, the D765N and L776V WND mutations result in initial mislocalization of the ATP7B variant proteins, as well


Table 1. Summary of localization and functional data for ATP7B WND mutant proteins ATP7B mutant protein

CopperGolgi network Endoplasmic reticulum dependent localizationa mislocalizationb redistribution

Normal or near normal function in yeast (18) D765N

Partial

Extensive

Partial

L776V

Partial

Extensive

Partial

G943S

Normal

Moderate

Negligible

Impaired function in yeast (18) R778L

Not determined Very extensive Not determined

M769Vc

Normal

None

Normal

CPC/S

Normal

Slight

Negligible

aPerinuclear

localization compatible with Golgi marker, Golgi-58K. appearance in cytoplasm compatible with endoplasmic reticulum. cTemperature-sensitive function. bGranular

Figure 3. Composite image, showing the copper-dependent subcellular localization of Wilson disease variant ATP7B proteins, as stated. Conditions are as outlined in the legend for Figure 2.

variant protein’s localization. The R778L mutation clearly disrupts normal localization of ATP7B protein. G943S and CPC/S variant ATP7B proteins were predominantly localized to the perinuclear region of the Golgi network under copper-limited conditions when expressed in CHO cells (Fig. 3). Addition of copper to the growth medium resulted in very little or no visible redistribution of either variant protein to cytoplasmic vesicles. These data indicate that the G943S and CPC/S mutations result in variant proteins that do not redistribute in response to copper. G943S and CPC/S proteins exhibited slightly more endoplasmic reticulum staining compared with normal ATP7B protein, which is not shown in the images presented in Figure 3. Our localization results, together with previous functional data, are summarized in Table 1. DISCUSSION

as impaired capacity to redistribute to cytoplasmic vesicles when stimulated by copper. The M769V variant ATP7B protein was localized to the position of the Golgi network under copper-limited conditions, and redistributed to cytoplasmic vesicles in response to copper in a manner indistinguishable from normal ATP7B when transfected into CHO cells (Fig. 2). There was no excessive endoplasmic reticulum associated M769V staining compared with normal ATP7B in transfected CHO cells. These data indicate that the M769V mutation had no effect on ATP7B localization, or copper-induced trafficking. R778L variant ATP7B protein was predominantly and very extensively localized throughout the cell, in the distribution pattern of the endoplasmic reticulum, when expressed in CHO cells (Fig. 3). This phenotype was observed in virtually all transfected cells. The endoplasmic reticulum associated R778L staining was so strong that it could not be determined with confidence whether copper addition had any effect on the

We have previously analyzed the effect of WND-associated amino acid substitutions on ATP7B function using a yeast complementation assay (18). The goal of the present study was to determine any potential changes in the copper-dependent localization of ATP7B protein due to the effect of single amino acid substitutions found in WND patients, and for which we have functional data, in order to gain further insight into WND pathology. ATP7B was localized to the Golgi network in HepG2 cells and transiently transfected CHO cells under copper-limited conditions. Addition of copper to the extracellular medium resulted in the redistribution of ATP7B to cytoplasmic vesicles in both CHO and HepG2 cells, but not in HeLa cells. These results confirm those previously reported (15,16,29) and demonstrate that Golgi localization under low copper conditions and copper-induced redistribution of ATP7B is maintained in CHO cells. We have used Golgi here in a broad sense, but evidence suggests that ATP7A and -B localize to the trans-Golgi region. A recent study, claiming localization of ATP7B to late endosomes rather than Golgi network did not document copper concentration in the


medium, which can be appreciable in the absence of chelators (30). The exact nature of the cytoplasmic vesicles into which ATP7B is redistributed in response to copper remains to be elucidated. Recent studies done using human and rat livers and hepatocytes have suggested that the ATP7B-containing vesicles are derived, as expected, from the Golgi network (16,31). The CHO cells used in our study do not normally express ATP7B and there is no background due to detection of endogenous ATP7B protein in immunofluorescence experiments using ATP7B-specific antibodies. However, CHO cells are not physiologically relevant cells in terms of ATP7B biology. ATP7B is expressed primarily in the liver and kidney, and finding a biologically relevant cell line that does not express endogenous ATP7B is difficult. Hepatocytes from the Long– Evans cinnamon (LEC) rat, in which Atp7b (32) is partially deleted, or the recently described Atp7b knockout mouse strain (33) grown under conditions to maintain cellular polarity could be useful reagents for this type of study. Obtaining hepatocytes from WND patients for immunolocalization studies is not feasible. The rarity of most WND mutations is such that the likelihood of obtaining hepatocytes homozygous for a given mutation is small, and the procedure required to obtain such hepatocytes is invasive. Therefore, CHO cells provide a good experimental system to detect differences in ATP7B localization due to the effect of amino acid substitutions found in WND patients, as we have done in this study. CHO cells have been used extensively to study the effect of Menkes disease and copper-binding domain mutations on the subcellular localization of ATP7A variant proteins (17,22,23,27,28). Determining the precise subcellular localization of proteins transiently or stably transfected into a heterologous cell line can be difficult due to over-expression-induced artifacts. Overexpression of ATP7B protein variants did not present a major problem during our study. We observed that normal ATP7B protein was observed throughout the cell, compatible with distribution in the endoplasmic reticulum, in ∼10–20% of transfected cells. This endoplasmic reticulum-associated mislocalization of normal ATP7B was observed in cells that stained very strongly with our anti-ATP7B antibodies, suggestive of mislocalization due to over-expression. Therefore, very strongly staining cells, which were easily distinguished from the remaining transfected cell population for both normal and mutant ATP7B protein variants, were not included in our analysis. The images presented in this paper are representative of the remaining transfected cell population which expressed ATP7B protein variants at an apparently moderate level, and in which the we expect the localization of transfected proteins to be minimally influenced by over-expression. Even so, precise assignment of the subcellular localization of ATP7B protein variants will require future studies using analysis by confocal or electron microscopy of stably transfected cell lines which have been carefully screened for low levels of ATP7B protein expression to further reduce the potential for over-expressioninduced localization artifacts. The abnormal localization of ATP7B throughout the cell occurs with the same distribution as shown by staining of the endoplasmic reticulum. The presumably misfolded variant protein could also be present in lysosomes or proteasomes, pathways for degradation of misfolded protein. The WND mutation D765N is a rare mutation reported in patients of Italian descent (34). L776V was originally not clas-

sified with certainty as a mutation or normal variant due to the conservative nature of the amino acid substitution (35). Both D765N and L776V variant proteins were found to complement ccc2 mutant yeast in a manner indistinguishable from normal ATP7B (18). Immunofluorescent detection of these variant proteins transiently transfected into CHO cells revealed that both proteins were localized in part to the Golgi network and were capable of redistribution to cytoplasmic vesicles when stimulated by copper. However, D765N and L776V proteins also exhibited extensive localization similar to the endoplasmic reticulum of CHO cells suggesting that the proteins were mislocalized, probably due to protein misfolding and retention in the endoplasmic reticulum. D765N and L776V should be considered disease-causing mutations. The WND mutation R778L is a mutation commonly found in patients of Asian descent and, in its homozygous form, is associated with a severe, early onset of WND with hepatic presentation (36,37). Functional analysis of this variant protein in our yeast assay revealed a severe defect in the ability of R778L variant protein to complement ccc2 mutant yeast, indicative of impaired copper transport function (18). Our localization data have revealed extensive mislocalization of the variant protein to the endoplasmic reticulum in CHO cells. R778L mutation likely causes the variant ATP7B protein to misfold and be retained in the endoplasmic reticulum and/or lysosomes, in addition to reducing its copper transport ability (18). These data are consistent with available phenotypic data (early disease onset) for R778L homozygotes (36,37). The mutation M769V was originally not classified definitively as either a WND mutation or rare normal variant (35). Since the original publication, M769V has been found in more WND patients supporting its designation as a diseaseassociated mutation (D.W. Cox, unpublished data). Functional data obtained from our yeast complementation analysis revealed that the variant protein was able to fully complement ccc2 mutant yeast at 30°C, but was profoundly temperature sensitive, unable to complement at 37°C (18). We concluded that M769V is a disease-causing mutation in patients. Our immunofluorescence data have revealed that M769V protein is localized to the Golgi network of CHO cells and redistributes to cytoplasmic vesicles when incubated with copper in a manner indistinguishable from normal ATP7B protein at 20°C. Little endoplasmic reticulum localization was detected (no more than normal ATP7B) for M769V in CHO cells suggesting that the variant protein was not severely misfolded at 20°C. G943S is a mutation originally found in the Bangladeshi population and was associated with later onset WND in one homozygous patient (35). Functional data revealed that the G943S protein had a slight but statistically significant reduction in its ability to complement ccc2 mutant yeast, indicative of slightly reduced copper transport capacity, consistent with the late onset disease presentation (18,35). G943S appears to be normally localized to the Golgi network of CHO cells, but appears to be non-responsive to copper, exhibiting no obvious copper-induced redistribution. The G943S substitution is found within the fifth transmembrane segment, and the Ser substitution may prevent putative transmembrane domain conformational changes required to initiate trafficking out of the Golgi network in response to copper or may disrupt a hypothetical membrane-targeting signal. CPC/S is an ATP7B


variant with mutations (Cys-Ser) in the CPC motif conserved in CPx-type ATPases. Due to its conservation, and functional analogy to ion binding sites in P-type ATPases, this motif is considered to be critical for ATP7B function, perhaps acting as a copper binding site within the transmembrane domain (38,39). Indeed, mutation of the CPC motif resulted in a nonfunctional ATP7B protein completely unable to complement ccc2 mutant yeast (15,18,40) even when expressed from a multicopy vector (18). Perhaps mutations in this motif prevent copper binding to the transmembrane domain of ATP7B required for copper transport, and also required as a trafficking initiation signal. The CPC/S mutation may prevent putative transmembrane domain conformation changes that may be required to allow copper-induced redistribution. Studies done previously by others on the common WND mutation, His1069Gln (H1069Q), revealed a defect in intracellular localization of the H1069Q variant ATP7B protein (41). H1069Q was mislocalized and degraded in the endoplasmic reticulum of transfected Menkes disease patient fibroblasts when the cells were incubated at 37°C prior to analysis of the protein localization by immunofluorescence microscopy, but was localized normally to the trans-Golgi network at 28°C. However, at 28°C the intracellular localization of H1069Q protein was found to be insensitive to extracellular copper addition, leading to its retention in the Golgi network under high copper conditions. Since H1069Q protein could not mediate copper efflux from the fibroblasts as did normal ATP7B (at 28 and 37°C), these data suggest that copperdependent redistribution of ATP7B is required for copper efflux. However, the H1069Q mutation also results in impaired ability of the ATP7B variant protein to complement ccc2 mutant yeast, suggesting defective copper transport function (15,42). Based on these data, the effect of H1069Q on ATP7Bdependent copper efflux could not be specifically ascribed either to its inability to traffick in response to copper or to its apparent defect in copper transport function. Therefore, the physiological significance of copper-dependent redistribution of ATP7B could not be established. The current model of ATP7B-mediated biliary copper efflux involves copper transport into cytoplasmic vesicles which then fuse with the plasma membrane to allow net copper efflux across the hepatocyte apical plasma membrane and into the bile canaliculus (16,31). Since the proteins D765N, R778L and L776V are largely mislocalized through the cell, these proteins would not be expected to mediate effective copper efflux, even when copper transport activity is retained (i.e. D765N, L776V) thereby causing WND as seen in patients carrying these mutations (35,36). Similarly, G943S likely causes WND in patients carrying this mutation (35) because of an inability to redistribute from the Golgi to cytoplasmic vesicles, rather than by defective transport activity, thus preventing effective biliary copper efflux. Taken together, these data support the requirement for ATP7B copper-dependent redistribution as a requirement for its role in hepatic copper efflux. Hypotheses have been made to explain normal holo-ceruloplasmin levels seen in some WND patients in the absence of normal ATP7B function. One hypothesis is that copper may be incorporated into ceruloplasmin by diffusion when hepatic cytosolic copper levels reach saturation. Ceruloplasmin is copper loaded by ATP7B within the membrane-enclosed lumen of the trans-Golgi network (10,11). Ionic copper

entering this compartment by diffusion would have to pass through the membrane, a feat that is thermodynamically unfavorable for a charged, hydrophilic cation, and which would require a large concentration gradient (43). Since most of the cytosolic copper in WND or experimentally copper-loaded hepatocytes is bound by metallothioneins (44–47) and likely metallochaperones (48), there would not likely, or rarely, be a large enough concentration of ionic copper to drive passage by diffusion through the trans-Golgi network membrane. Therefore, copper incorporation into ceruloplasmin by this mechanism is unlikely. This is supported by studies on the LEC rat which show that in the absence of functional ATP7B, there is no significant copper incorporation into ceruloplasmin even in the presence of highly elevated hepatic copper levels (49–51). During these LEC rat studies, holo-ceruloplasmin levels were observed to increase in the rat serum immediately preceding onset of liver failure. Since the reappearance of plasma holoceruloplasmin was accompanied by plasma markers of liver failure and breakdown, the likely cause of ceruloplasmin copper loading in the LEC rat was the disruption of intracellular membranes. By analogy, some WND patients may exhibit a rise in circulating holo-ceruloplasmin levels independent of ATP7B function at the onset of liver failure. Changes in ATP7B function and localization due to allelic variation of the ATP7B gene can explain in part the variation of diagnostic biochemical parameters in patients with WND, in particular, normal holo-ceruloplasmin levels observed in patients. As described, several of the WND variants that were analyzed for function (G943S, D765N, L776V) had normal or nearly normal function assessed by our yeast complementation assay (18). These data combined with our immunofluorescence data have direct relevance to the diagnosis of WND. Mutations such as G943S, that result in ATP7B proteins with transport activity that are restricted to the Golgi network, would be predicted to incorporate copper into ceruloplasmin but not effectively mediate copper efflux. In one Pakistani compound heterozygote for this mutation and another unidentified mutation, the serum ceruloplasmin is indeed normal at 290 g/l, normal 200–400 g/l) (D.W. Cox, unpublished data). Similarly, mutations such as D765N and L776V that apparently have full activity, and are substantially, but not completely, mislocalized, may result in enough Golgilocalized protein to incorporate copper into ceruloplasmin, while ineffectively mediating copper efflux. Therefore, WND patients carrying these mutations, or mutations that effect ATP7B function and/or localization in a similar manner even in compound heterozygous form, may have normal, or borderline normal, holo-ceruloplasmin levels, and their diagnosis may be missed if emphasis is placed on ceruloplasmin for initial screening. These data are also of importance for the diagnosis of presymptomatic sibs of an individual affected with WND. Since WND is treatable, early detection, monitoring, and treatment of pre-symptomatic patients is critical to prevent irreversible liver damage requiring transplant (1,5). This is best accomplished by molecular testing for haplotype sharing within the proband’s family or by direct mutation detection in addition to biochemical tests (1,52–54). Most WND mutations are rare and most patients are compound heterozygotes (1,37). Consequently, precise genotype–phenotype correlation for individual mutations is difficult. Although functional and localization data can provide a


likely mechanism to explain variation in the biochemical features of WND, the overall phenotype of the disease in patients cannot be readily explained. Patients may accumulate different amounts of hepatic copper at different rates, dependent on the amount and bioavailability of copper ingested, which may affect WND progression (55). Genetic factors likely play a role as well. For example, allelic variation of proteins such as canalicular multi-organic anion transporter (56) may reduce copper accumulation in the absence of normal ATP7B function. There may be allelic variation in protective genes such as those for metallothionein (57). Since bile is the main route of copper excretion, allelic variants of bile transporters may alter copper accumulation. These are only a few of the potential genetic loci that might modify the overall WND phenotype. MATERIALS AND METHODS Cell culture Cell lines used in this study were HepG2, HeLa and CHO cells originally obtained from the American Type Cell Culture (Mannassas, VA) resource. Cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, and 100 U/ml each of penicillin and streptomycin. All cell culture reagents were obtained from Gibco Life Technologies (Burlington, Ontario). Cells were maintained at 37°C in a 5% CO2 atmosphere. Antibodies For production of a rabbit polyclonal antibody against the copper binding domain of ATP7B, purified glutathione S-transferase fusion protein containing the ATP7B copper binding domain was obtained from Michael DiDonato (Hospital for Sick Children, Toronto, Ontario) (58). The protein was dissolved in sterile phosphate-buffered saline (4.3 mM Na2HPO4, 1.4 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl) containing 2% sodium dodecyl sulfate. For each of two rabbits (New Zealand White; Vandermeer Rabbits, Sherwood Park, Alberta) used, 1 ml of protein solution was emulsified with an equal volume of Freund’s complete adjuvant (Sigma Aldrich, Oakville, Ontario). This mixture was injected into the rabbits using one 0.5 ml intramuscular injection, and four 0.25 ml subcutaneous injections. A total of ∼375 µg of protein was injected each time. The injections were repeated three weeks later using Freund’s incomplete adjuvant (Sigma). A second booster injection was performed 10 days later. Three weeks later the rabbits were anesthetized, exsanguinated and then euthanized. Antiserum was collected by centrifugal removal of clotted red blood cells. Antibodies were titred against ATP7B protein expressed in yeast by western blotting. Animal housing and animal handling was done by technicians of the Health Sciences Laboratory Animal Services, at the University of Alberta, in accordance with the Health Sciences Animal Welfare committee guidelines. Affinity-purification of the antiserum was done as previously described (59). Purified antibody is designated ‘antiATP7B.N60’. Secondary antibody was goat anti-rabbit IgG monoclonal antibody conjugated to FITC fluorophor (Jackson Laboratories, Bar Harbour, ME). Endoplasmic reticulum was detected

using ConA, conjugated with AMCA-S fluorophor (Molecular Probes, Eugene, OR). Golgi-58K mouse monoclonal antibody, and sheep anti-mouse IgG monoclonal antibody conjugated to Cy3 fluorophor (Sigma) was used to detect the Golgi network. Transient transfection ATP7B cDNA and site-directed mutant variants were constructed as previously described (18). For transient transfection into mammalian cells, cDNAs were restriction enzyme digested with BamHI and SalI from pUC19 vector, gel purified and cloned into the mammalian expression vector pCDNA1 (Invitrogen, Carlsbad, CA) using BamHI and XhoI sites in the vector. Expression plasmids were isolated from 100 ml cultures of bacteria grown in luria broth medium containing 100 µg/ml carbenicillin using ion exchange chromatography (Midi Prep kit; Qiagen, Mississauga, Ontario) according to the manufacturer’s protocol. Prior to transfection, cells were plated to 40–50% confluence onto sterile glass coverslips, contained in six-well tissue culture plates, in 2 ml of medium. Cells grown overnight were transfected using 1.5 µg of plasmid DNA and 15 µl of Lipofectin reagent (Gibco BRL Life Technologies) according to the manufacturer’s protocols. The cells were incubated with the transfection mixture for 6–8 h followed by replacement with standard medium. Following overnight growth, medium was left unsupplemented, or was supplemented with 250 µM copper chloride or 50 µM copper chelator BCS. The cells were incubated for a further 2–3 h prior to immunofluorescence experiments. Triple-label indirect immunofluorescence microscopy Cells attached to coverslips were transferred to a new six-well plate and processed for immunofluorescence. Cells were rinsed twice with Tris-buffered saline (TBS) then fixed for 20 min at 4°C with 4% paraformaldehyde made in TBS. Fixed cells were rinsed twice with TBS then made permeable by incubation for 10 min in 0.5% Triton X-100 (membrane grade; Boehringer Mannheim, Laval, Quebec) made in TBS. Permeable cells were blocked for 30 min with 2% milk powder in TBS. Affinity-purified anti-ATP7B.N60 antibody was used as primary antibody, incubated for 2–3 h at room temperature, at a 1:100 dilution in blocking buffer. To detect the Golgi network Golgi-58K antibody was at a 1:100 dilution. After primary antibody incubation, cells were rinsed twice with TBS containing 0.5% Tween-20 (TBST) then washed three times for 5 min each in the same. Secondary antibody incubation was done for 1 h at room temperature in blocking buffer using a 1:100 dilution of goat anti-rabbit IgG–FITC to detect antiATP7B.N60. Sheep anti-mouse IgG–Cy3 was used at a 1:1000 dilution to detect Golgi-58 K antibody. To visualize the endoplasmic reticulum, AMCA-S-labeled ConA was added to the secondary antibody mixture at a final concentration of 100 µg/ ml. After secondary antibody incubation, cells were rinsed three times with TBST, then washed four times for 5 min each with TBST. Coverslips were then mounted onto slides using mounting media (Vectashield; Vector, Burlingame, CA) and sealed with clear nail polish. Microscopy was performed on a fluorescence microscope using a 100× objective lens and oil immersion (Leica, Willowdale, Ontario). Photographs were taken with 400 speed film using 20 and 40 s exposures.


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