Elsevier

Gene

Volume 236, Issue 2, 20 August 1999, Pages 251-257
Gene

Cloning and expression analysis of the sheep ceruloplasmin cDNA

https://doi.org/10.1016/S0378-1119(99)00276-0Get rights and content

Abstract

The cDNA encoding sheep ceruloplasmin (sCP) was isolated from a sheep liver cDNA library. The cDNA contig was 3530 nucleotides in length and encoded a protein of 1048 amino acids. The deduced amino acid sequence showed a high degree of conservation (87%) when compared to the human ceruloplasmin (hCP) sequence. Northern blot analysis of sheep tissue revealed that the sheep ceruloplasmin gene (sCP) was expressed primarily in the liver, but low levels of mRNA were detected in the hypothalamus, spleen and uterus. No sCP mRNA was detected in the cortex, heart, intestine or kidney. Expression was not significantly affected by hepatic copper content. Northern blot analysis of sheep liver during development demonstrated little sCP expression during fetal life, but significant levels of mRNA were observed after birth. Significantly, the developmental expression pattern of sCP was closely correlated with that of the sheep Wilson disease gene (sATP7B), suggesting that the expression of the two genes may be coordinated to ensure that copper is supplied to apoceruloplasmin. Overall, the structure and expression of sCP appeared similar to other mammals, suggesting that abnormalities in CP were not responsible for the unusual sheep copper phenotype.

Introduction

Ceruloplasmin (CP), a multicopper oxidase, is a blue α2-glycoprotein containing six atoms of copper per molecule with an apparent molecular mass of approximately 130 kDa. The protein occurs as a single polypeptide chain and accounts for 60–95% of the plasma copper of vertebrates (Cousins, 1985, Evans and Wiederanders, 1967). CP is synthesized in the hepatocyte, and the incorporation of copper to generate the active holoprotein occurs in the secretory compartment of the cell (Sato and Gitlin, 1991, Terada et al., 1995). If copper is not available during biosynthesis, an unstable apoprotein, which lacks oxidase activity, is produced and secreted into the plasma (Gitlin et al., 1992). CP appears to be multifunctional and is thought to be involved in copper transport, iron metabolism and antioxidant defense (Linder, 1991). A clear link between iron metabolism and CP function was established with the recent finding that mutations in CP result in the genetic disease systemic hemosiderosis (Harris et al., 1995, Yoshida et al., 1995).

CP is expressed primarily in the liver with lower levels observed in non-hepatic tissues such as the placenta, uterus and testis (Aldred et al., 1987, Fleming and Gitlin, 1990). Developmental regulation of CP expression in the mammalian liver has been reported. CP mRNA is present at low levels prior to birth, then rapidly increases to adult levels after parturition (Bingle et al., 1991, Gutteridge and Stocks, 1981). Serum copper levels, which are low prior to birth, rise to adult levels when CP synthesis commences. Hepatic copper levels, which are elevated in the fetal liver, rapidly decrease to adult levels as a result of the production of CP and the initiation of biliary excretion. It is thought that these changes are brought about by the maturation of the homeostatic capacity of the liver (Balistreri, 1996, Srai et al., 1986).

A decreased level of plasma CP is a key diagnostic feature of the genetic disorder Wilson disease (WD). Most Wilson patients have low or absent serum ceruloplasmin, due to reduced copper incorporation into apoceruloplasmin in the hepatocyte (Gibbs and Walshe, 1979, Sato and Gitlin, 1991). The primary characteristic of Wilson disease is the progressive accumulation of copper in the liver, caused by a defect in biliary excretion of copper from this organ (Frommer, 1974, Gibbs and Walshe, 1980, Scheinberg and Gitlin, 1952). The production and flow volume of bile in Wilson patients are similar to control values (Frommer, 1974), implying that the defect involves transport of copper from the hepatocyte to the bile rather than bile production per se. The gene defective in WD patients has been cloned and encodes a cation transporting P-type ATPase, termed the Wilson disease protein (ATP7B or WND) (Bull et al., 1993, Tanzi et al., 1993, Yamaguchi et al., 1993), that is required for incorporation of copper into apoceruloplasmin (Terada et al., 1998).

Copper metabolism in sheep differs from that observed in other mammals. Copper deficiency is relatively common and was first recognized over 50 years ago (Underwood, 1977). Paradoxically, sheep are also very susceptible to copper toxicosis. Biliary copper excretion is greatly reduced in sheep compared with other mammals, and is not responsive to dietary copper intake (Weber et al., 1980). In this respect, sheep have been postulated as an animal model of WD (Danks, 1995, Howell et al., 1984). However, in contrast to WD patients, CP levels in sheep plasma have been shown to be comparable to the normal human range (McCosker, 1968).

We are investigating the molecular mechanisms of copper transport in sheep and possible reasons for the reduction of biliary copper excretion. Here, we report the cloning and sequencing of the sheep CP cDNA (GenBank Accession No. AF134814) and its expression pattern in tissue and the developing liver.

Section snippets

Construction of a sheep liver cDNA library

Total RNA was isolated from the liver of an adult sheep (Merino) using a modified guanidinium hydrochloride protocol (Paynter et al., 1990). Poly(A) RNA was purified from total RNA using an oligo(dT) purification system (Boehringer Mannheim). Double-stranded cDNA was synthesized using an oligo(dT) primer or random primers according to the Promega Universal Riboclone cDNA Synthesis System protocol and packaged in a λgt11 vector (Promega). The library contained approximately 1.6×106 independent

Cloning and sequence analysis of the sheep CP mRNA

A sheep liver cDNA library was screened with hCP cDNA probes. Positive phage clones were purified, and the inserts were subcloned. A contig of 3530 bp was generated and sequenced, and the nucleotide and predicted amino acid sequences were deposited into GenBank (Accession No. AF134814). An open reading frame (ORF) of 3144 bp was identified and sequenced on both strands. This ORF encoded a protein of 1048 amino acids (Fig. 1). The N-terminal region of the protein contained a potential signal

Conclusions

  • 1.

    A sCP cDNA contig of 3530 bp was isolated, which predicted a protein of 1048 amino acids. The amino acid sequence showed a high degree of conservation with human ceruloplasmin, particularly in relation to the amino acids involved in copper binding.

  • 2.

    A single mRNA transcript of approximately 3.7 kb was observed by Northern blot analysis. Expression levels were highest in the liver, with lower levels observed in the uterus, hypothalamus, spleen, testis and ovary.

  • 3.

    Expression of sCP in the liver was not

Acknowledgements

We thank Dr S. La Fontaine for critical reading of the manuscript and express our sincere appreciation for the support and advice of our laboratory colleagues. P.J.L. is an NHMRC Dora Lush Postgraduate Scholar. This work was supported in part by a Block grant from the National Health and Medical Research Council of Australia.

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