Elsevier

Journal of Hepatology

Volume 39, Issue 5, November 2003, Pages 703-709
Journal of Hepatology

The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis

https://doi.org/10.1016/S0168-8278(03)00380-5Get rights and content

Abstract

Background/Aims: Copper toxicosis (CT) in Bedlington terriers is an autosomal recessive disorder characterized by massive lysosomal copper accumulation in livers of affected dogs, and a defect in the biliary excretion of this metal. We propose that MURR1, the gene defective in canine CT, has a role in the regulation of copper excretion into bile during copper overload.

Methods: Polyclonal antibodies raised against full-length recombinant human MURR1 were used for immunoblot analysis and indirect immunofluorescence studies.

Results: Using Western blot analysis, these antibodies abundantly detected MURR1 as a 23 kDa protein in liver extracts of mice and dogs, but MURR1 was undetectable in the livers of affected Bedlington terriers. MURR1 was also detected in different tissues and cell lines; in cell lines the protein was found both in cytosol and membrane preparations. Consistent with this observation, indirect immunofluorescence staining revealed that in some cells MURR1 was associated with a vesicular compartment diffusely localized throughout the cell.

Conclusions: The genomic deletion in MURR1 results in complete absence of MURR1 protein. Based on the unanticipated subcellular localization, our results suggest a role for MURR1 in the regulation of vesicular copper sequestration during copper overload.

Introduction

The liver is the central organ of copper homeostasis in mammals, which is underscored by the existence of the copper overload disorder Wilson disease. In hepatocytes, the protein mutated in Wilson disease, ATP7B, transports copper into the secretory pathway for subsequent copper incorporation into ceruloplasmin and excretion of copper into bile [1], [2], [3], [4]. ATP7B is a resident TGN protein, which redistributes upon high copper levels to a cytoplasmic vesicular compartment. These copper-loaded vesicles probably fuse with the bile canalicular membrane thereby excreting the copper, and ATP7B returns to the TGN [5], [6]. However, the precise mechanism of cellular copper excretion is unknown.

Reduced biliary copper excretion is also seen in Bedlington terriers suffering from an autosomal recessive disorder, denoted copper toxicosis (CT) [7], [8], [9]. This disease is characterized by massive copper accumulation in the liver, leading to chronic hepatitis and finally to liver cirrhosis [10]. The accumulated hepatic copper is seen as electron-dense granules in the lysosomes and is mainly present centrilobularly [8], [11], [12]. However, in contrast to Wilson disease patient, the ceruloplasmin level is normal suggesting that copper intake and copper transport to the TGN are undisturbed in these dogs. We therefore propose that the gene defective in canine CT has a role in the regulation of vesicular copper excretion into bile during copper overload.

Recently, we identified a homozygous deletion encompassing exon 2 of the MURR1 gene in all Bedlington terriers with CT [13]. This genomic in-frame deletion of approximately 13 kb (unpublished data) causes a predicted truncated MURR1 protein of 94 amino acid residues. The function of the MURR1 protein is still unexplored. No homology with other proteins or known protein motifs was identified which may imply a putative function of MURR1. Remarkably, the MURR1 gene is restricted to bile-containing organisms, i.e. vertebrates [13]. To begin to address the role of MURR1 in canine copper toxicosis, a polyclonal antiserum against a MBP/MURR1 fusion protein was raised. Using this polyclonal anti-MURR1 antiserum we studied the tissue and subcellular distribution of the MURR1 protein. In addition, we determined the influences of different copper levels on expression of MURR1 in different cell lines.

Section snippets

Cloning and expression of recombinant MBP/MURR1

The complete coding region of human MURR1 was amplified from placenta cDNA by polymerase chain reaction (PCR) using sense primer 5′-CCC GGG CAT GGC GGC GGG CGA GCT TG-3′ and antisense primer 5′-GTC GAC TCA GTT AGG CTG GCT GAT CAG TG-3′. The primers contained a SmaI and a SalI site, respectively (underlined). The PCR product was cloned into the TA-cloning vector pCR2.1 (Invitrogen, Carlsbad, CA, USA), and an EcoRI/SalI fragment was ligated into the prokaryotic expression vector pMAL-c2X (New

Characterization of MURR1 antiserum

Polyclonal antibodies were generated by immunizing rabbits with a 70 kDa recombinant MBP/MURR1 protein. To investigate whether the produced antiserum contained MURR1 antibodies, immunoblot analysis was performed. In contrast to preimmune serum, antiserum of immunized rabbits contained antibodies that bound to both MBP and the MBP/MURR1 proteins (Fig. 1A). This immunoreactivity was completely abolished when the antiserum was preincubated with MBP/MURR1. When antiserum was preincubated with MBP,

Discussion

Canine copper toxicosis is caused by a deletion of exon 2 of the MURR1 gene [13]. We now show that this deletion is associated with a complete absence of MURR1 protein in livers of affected dogs. In addition, MURR1 protein is ubiquitously expressed. The amino acid sequence of MURR1 does not contain any cellular localization signals [13]. From crude subcellular fractionation experiments we concluded that MURR1 is mainly a cytosolic protein, although a small amount of MURR1 was also detected in

Acknowledgements

We are grateful to Dr. J.D. Gitlin for gifts of antibodies, Karen Duran for technical assistance, Jan Rothuizen for dog liver biopsies, Ellen van Binsbergen for help with the confocal microscope, and Ingrid Saarloos for kindly providing different cell lines. This work was supported by grants 902-23-252 and 902-23-254 from the Dutch Organization for Scientific Research (NWO) and the International Copper Association (TPT0551).

References (29)

  • P.C Bull et al.

    The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene

    Nat Genet

    (1993)
  • R.E Tanzi et al.

    The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene

    Nat Genet

    (1993)
  • G.F Johnson et al.

    Inheritance of copper toxicosis in Bedlington terriers

    Am J Vet Res

    (1980)
  • C.A Owen et al.

    Inherited copper toxicosis in Bedlington terriers: Wilson's disease (hepatolenticular degeneration)

    Am J Pathol

    (1982)
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