Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue

doi:10.1016/S0167-4781(00)00054-3

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Volume 1491, Issues 1–3, 25 April 2000, Pages 229-239

https://doi.org/10.1016/S0167-4781(00)00054-3 Get rights and content

Abstract

Copper homeostasis in mammals is maintained by the balance of dietary intake and copper excretion via the bile. Sheep have a variant copper phenotype and do not efficiently excrete copper by this mechanism, often resulting in excessive copper accumulation in the liver. The Wilson disease protein (ATP7B) is a copper transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile. To investigate the role of ATP7B in the sheep copper accumulation phenotype, the cDNA encoding the ovine homologue of ATP7B was isolated and sequenced and the gene was localised by fluorescence in situ hybridisation to chromosome 10. The 6.3 kb cDNA encoded a predicted protein of 1444 amino acids which included all of the functional domains characteristic of copper transporting P-type ATPases. ATP7B mRNA was expressed primarily in the liver with lower levels present in the intestine, hypothalamus and ovary. A splice variant of ATP7B mRNA, which was expressed in the liver and comprised approximately 10% of the total ATP7B mRNA pool, also was isolated. The results suggest that ATP7B is produced in the sheep and that the tendency to accumulate copper in the liver is not due to a gross alteration in the structure or expression of ATP7B.

Introduction

Copper is one of at least 12 heavy metals essential for normal biological function [1]. It facilitates electron transfer reactions by a number of enzymes, such as lysyl oxidase, superoxide dismutase, cytochrome c oxidase and dopamine-β hydroxylase. These enzymes have critical roles in connective tissue crosslinking, antioxidant defense, cellular respiration and catecholamine biosynthesis, respectively [2]. Despite this essential requirement, excess copper mediates free radical production resulting in the direct oxidation of cellular components. Therefore, organisms have evolved efficient transport and homeostatic mechanisms to maintain intracellular copper at the required level [3].

In humans and other mammals a balance between intestinal copper absorption and hepatic copper excretion maintains the copper content of the body [4]. The liver is the major organ involved in the regulation of overall copper status, with excess copper being excreted from the body through the bile [4]. The importance of the biliary excretion pathway is illustrated by the human copper toxicosis disorder Wilson disease (WD). Patients with this disease cannot excrete copper into the bile, resulting in hepatic copper accumulation and if untreated, fatal copper toxicosis [2]. The gene that is defective in WD patients has been cloned and encodes a copper transporting P-type ATPase (ATP7B) [5], [6], [7]. ATP7B plays a dual role in copper homeostasis within the hepatocyte. One role is to excrete excess copper from the cell via the bile [8], [9]. ATP7B also functions in the biosynthetic pathway, delivering copper to the secretory pathway for incorporation into secreted cuproenzymes, particularly ceruloplasmin [10]. The biliary excretion of copper and the production of functional ceruloplasmin is restored in the Long Evans Cinnamon (LEC) rat, a model of WD, by the introduction of the ATP7B cDNA [11], [12]. ATP7B mRNA is present primarily in the liver and in reduced amounts in other tissues, for example in kidney, brain, placenta, lung, heart and intestine [5], [6]. The role of ATP7B in extrahepatic tissues has not been established but may relate to the supply of copper to ceruloplasmin or other cuproproteins.

Sheep, as a species, display a variant copper phenotype when compared with other mammals. Copper deficiency in sheep is a relatively common agricultural problem and was first recognised over 50 years ago [13]. The condition generally occurs as a result of copper interacting with other dietary components, particularly molybdenum and sulfur, resulting in a decrease in copper absorption [14]. Paradoxically, sheep are also very susceptible to copper toxicosis. The process of biliary excretion of copper is reduced in sheep, and unlike most mammals, is not responsive to dietary copper intake [15]. Consequently, supplementation with moderate dietary levels of copper, to prevent deficiency can result in elevated hepatic copper concentrations, liver failure and death of the animal [16]. Numerous studies have indicated that lysosomes are the primary site of copper accumulation within the sheep liver [16], [17], [18]. All breeds of sheep have a reduced ability to excrete copper in the bile and will accumulate excess copper in the liver. However, there are breed-specific differences in the amount of dietary copper required for hepatic accumulation and the development of copper toxicosis [19]. This variation between breeds reflects differences in the efficiency of copper absorption in the intestine rather than differences in the efficiency of biliary copper excretion [20].

The susceptibility of sheep to copper toxicosis has been compared to that observed for patients with Wilson disease [2], [21]. The current study was undertaken to determine whether alterations in the structure or expression of ATP7B could explain the copper accumulation phenotype of sheep. In this report we describe the cloning, localisation, sequence and expression pattern of the ovine homologue of ATP7B (GenBank accession number AF032881). We also report the isolation of a novel form of ovine ATP7B (GenBank accession number AF118225).

Section snippets

Construction of a sheep liver cDNA library

Total RNA was isolated from the liver of an adult sheep (Merino), which had not received supplementary dietary copper, using a modified guanidinium hydrochloride protocol [22]. Poly(A) RNA was purified from total RNA using an oligo(dT) purification system (Boehringer Mannheim). Double stranded cDNA was synthesised using an oligo(dT) primer or random primers according to the Promega Universal Riboclone cDNA Synthesis System protocol. Two cDNA libraries were constructed in a λgt11 vector

Isolation of the sheep ATP7B homologue

A sheep liver cDNA library constructed in λgt11 was screened with mouse ATP7B cDNA probes. Positive phage clones were plaque purified and the cDNA inserts were subcloned and sequenced. A contig of 6325 bp was generated from three overlapping cDNA clones (GenBank accession number AF032881) and a 4332 bp ORF was identified, which encoded a predicted protein of 1444 amino acids. Comparison of the predicted sheep ATP7B amino acid sequence with that of human ATP7B (Fig. 1) indicated a high level of

Discussion

This is the first report of the primary structure of sheep ATP7B and the expression pattern of the corresponding mRNA. This analysis was undertaken to determine if alterations in the structure or expression of sheep ATP7B underlie the unusual copper metabolism of this species.

The size and quantity of the ATP7B mRNA detected in sheep liver RNA preparations by Northern blot analysis and the frequency of ATP7B cDNA clones isolated from the sheep liver library, were similar to that reported for the

Acknowledgments

We are grateful to Dr C. Bottema for providing the chromosome 10 BAC clone, Mr Andrew Grimes for helpful technical advice and Dr Sharon La Fontaine for critical reading of the manuscript. We express our sincere appreciation for the advice and support given by our laboratory colleagues. P.J.L. is a National Health and Medical Research Council Dora Lush Postgraduate Scholar. This work was supported in part by the National Health and Medical Research Council of Australia, the Australian Research

References (45)

Y. Yamaguchi et al.
Biochem. Biophys. Res. Commun.
(1993)
K. Gibbs et al.
Lancet
(1980)
Y. Murata et al.
Biochem. Biophys. Res. Commun.
(1995)
K. Terada et al.
FEBS Lett.
(1999)
K. Terada et al.
J. Biol. Chem.
(1998)
W.W. Saylor et al.
J. Nutr.
(1980)
J.S. Kumaratilake et al.
J. Comp. Pathol.
(1989)
P.J. Lockhart et al.
Gene
(1999)
P.J. McCosker
Res. Vet. Sci.
(1968)
I.H. Hung et al.
J. Biol. Chem.
(1997)

J.R. Forbes et al.

Am. J. Hum. Genet.

(1998)

O. Bakke et al.

Cell

(1990)

M. Mertz

Science

(1981)

D.M. Danks, in: C.R. Scriver, A.L. Beaudet, W.M. Sly, D. Valle (Eds.), The Metabolic and Molecular Basis of Inherited...

C.D. Vulpe et al.

Annu. Rev. Nutr.

(1995)

M.C. Linder, Biochemistry of Copper, Plenum, New York,...

P.C. Bull et al.

Nature Genet.

(1993)

R.E. Tanzi et al.

Nature Genet.

(1993)

I.H. Scheinberg et al.

Science

(1952)

E.J. Underwood, Trace Elements in Human and Animal Nutrition, 4th edn., Academic Press, New York,...

J. Mason

Ir. Vet. J.

(1990)

K.M. Weber et al.

Aust. J. Agric. Res.

(1980)

Cited by (19)

Liver and Biliary System
2016, Jubb, Kennedy and Palmer's Pathology of Domestic Animals: Sixth Edition
Cellular multitasking: The dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance
2008, Archives of Biochemistry and Biophysics
Citation Excerpt :
In sheep (Ovis), two forms of ATP7B generated by alternate splicing of exon 1 have been identified [54,55]. The exon 1 of a shorter form, sATP7B, encodes an 18-amino-acid sequence, while in the longer form lATP7B, it is replaced by a 79-amino-acid sequence [55]. It has been hypothesized that the longer isoform might be associated with the additional role for ATP7B copper storage in cells to combat copper deficiency, which is common in sheep.
The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.
Trafficking of the copper-ATPases, ATP7A and ATP7B: Role in copper homeostasis
2007, Archives of Biochemistry and Biophysics
Citation Excerpt :
Unlike ATP7A, there have been no reports identifying ATP7B at the plasma membrane of non-polarized cells. Unexpectedly, two forms of ATP7B (sATP7B) were identified in sheep liver whereas all other mammals examined to date have only one [56]. The two forms of sATP7B were designated “normal” form (sATP7Ba) and “alternate” form (sATP7Bb) and differ only at the N terminus.
Copper is essential for human health and copper imbalance is a key factor in the aetiology and pathology of several neurodegenerative diseases. The copper-transporting P-type ATPases, ATP7A and ATP7B are key molecules required for the regulation and maintenance of mammalian copper homeostasis. Their absence or malfunction leads to the genetically inherited disorders, Menkes and Wilson diseases, respectively. These proteins have a dual role in cells, namely to provide copper to essential cuproenzymes and to mediate the excretion of excess intracellular copper. A unique feature of ATP7A and ATP7B that is integral to these functions is their ability to sense and respond to intracellular copper levels, the latter manifested through their copper-regulated trafficking from the transGolgi network to the appropriate cellular membrane domain (basolateral or apical, respectively) to eliminate excess copper from the cell. Research over the last decade has yielded significant insight into the enzymatic properties and cell biology of the copper-ATPases. With recent advances in elucidating their localization and trafficking in human and animal tissues in response to physiological stimuli, we are progressing rapidly towards an integrated understanding of their physiological significance at the level of the whole animal. This knowledge in turn is helping to clarify the biochemical and cellular basis not only for the phenotypes conferred by individual Menkes and Wilson disease patient mutations, but also for the clinical variability of phenotypes associated with each of these diseases. Importantly, this information is also providing a rational basis for the applicability and appropriateness of certain diagnostic markers and therapeutic regimes. This overview will provide an update on the current state of our understanding of the localization and trafficking properties of the copper-ATPases in cells and tissues, the molecular signals and posttranslational interactions that govern their trafficking activities, and the cellular basis for the clinical phenotypes associated with disease-causing mutations.
ATP7B mediates vesicular sequestration of copper: Insight into biliary copper excretion
2006, Gastroenterology
Background & Aims: The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Methods: Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. Results: In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. Conclusions: This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.
Restoration of copper metabolism and rescue of hepatic abnormalities in LEC rats, an animal model of Wilson disease, by expression of human ATP7B gene
2004, Biochimica et Biophysica Acta - Molecular Basis of Disease
Hepatic abnormalities in Long–Evans Cinnamon (LEC) rats, an animal model of Wilson disease (WD), were restored by the expression of the human ATP7B cDNA under the control of CAG promoter. Expression of ATP7B transcript and protein in the liver of the transgenic rats resulted in the restoration of biosynthesis of holoceruloplasmin and biliary copper excretion. Meanwhile, transgenic rats showed striking improvements in their hepatic abnormalities, i.e., rescue from fulminant hepatitis, late onset of hepatic cholangiofibrosis, suppression of hepatocellular carcinoma and much improved survival rates. Moreover, dramatic decreases were noted both in the levels of hepatic copper and iron in transgenic rats before the occurrence of hepatitis. These results indicated that the human ATP7B product compensated for the deficiency of the endogenous rattus protein and did function in intrahepatic copper transport by secreting copper into the plasma via incorporation into ceruloplasmin and by the excretion of copper into the bile, and that ATP7B is critical to hepatic dysfunctions in WD. This first successful transgenic rescue has important implications for the gene therapy of WD.
Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase
2002, Biochimica et Biophysica Acta - Molecular Basis of Disease
The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

View all citing articles on Scopus

View full text

Cloning, mapping and expression analysis of the sheep Wilson disease gene homologue

Abstract

Introduction

Section snippets

Construction of a sheep liver cDNA library

Isolation of the sheep ATP7B homologue

Discussion

Acknowledgments

Biochem. Biophys. Res. Commun.

Lancet

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

J. Nutr.

J. Comp. Pathol.

Gene

Res. Vet. Sci.

J. Biol. Chem.

Am. J. Hum. Genet.

Cell

Science

Annu. Rev. Nutr.

Nature Genet.

Nature Genet.

Science

Ir. Vet. J.

Aust. J. Agric. Res.