The ubiquitously expressed MURR1 protein is absent in canine copper toxicosis
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 This genomic in-frame deletion of approximately 13 kb (unpublished data) causes a predicted truncated MURR1 protein of 94 amino acid residues.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′- CAT GGC GGC GGG CGA GCT TG-3′ and antisense primer 5′- 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)
- et al.
Isolation and characterization of a human liver cDNA as a candidate gene for Wilson disease
Biochem Biophys Res Commun
(1993) - et al.
Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae
J Biol Chem
(1997) - et al.
Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion
Gastroenterology
(2000) - et al.
Human short-chain L-3-hydroxyacyl-CoA dehydrogenase: cloning and characterization of the coding sequence
Biochem Biophys Res Commun
(1996) - et al.
Biochemical characterization of the human copper transporter Ctr1
J Biol Chem
(2002) - et al.
Alteration in hepatocyte lysosomes in experimental hepatic copper overload in rats
Gastroenterology
(1993) - et al.
Lysosomal defect of hepatic copper excretion in Wilson's disease (hepatolenticular degeneration)
Gastroenterology
(1973) - et al.
Wilson disease and Menkes disease: new handles on heavy-metal transport
Trends Genet
(1994) - et al.
Absorption of 64Cu, 66An, 90Mo and 59Fe from ligated segments from the rat gastrointestinal tract
J Nutr
(1965) - et al.
Genetic disorders of copper transport
Am J Physiol
(1999)
The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene
Nat Genet
The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene
Nat Genet
Inheritance of copper toxicosis in Bedlington terriers
Am J Vet Res
Inherited copper toxicosis in Bedlington terriers: Wilson's disease (hepatolenticular degeneration)
Am J Pathol
Cited by (132)
COMMD1, a multi-potent intracellular protein involved in copper homeostasis, protein trafficking, inflammation, and cancer
2021, Journal of Trace Elements in Medicine and BiologyIdentification of the COMM-domain containing protein 1 as specific binding partner for the guanine-rich RNA sequence binding factor 1
2020, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :A unifying mechanism, which would explain the function of these proteins in these events is lacking, but recent studies suggested the possibility that regulation of the ubiquitin pathway may constitute the functional basis for the biological activity of many COMMD containing proteins [12]. Originally, COMMD1 was discovered as regulator of hepatic copper excretion [15–17] but later on additional experimental data suggested a connection to the ubiquitin-proteasome system [18]. In fact, COMMD1 has been shown to regulate ubiquitination of its protein interaction partners and targets them for proteasomal degradation [18].
Liver Toxicity
2018, Veterinary Toxicology: Basic and Clinical Principles: Third EditionCOMMD1 in copper homeostasis
2018, Clinical and Translational Perspectives on WILSON DISEASE