Elsevier

Gene

Volume 250, Issues 1–2, 30 May 2000, Pages 67-76
Gene

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes

https://doi.org/10.1016/S0378-1119(00)00167-0Get rights and content

Abstract

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25 kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT–PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.

Introduction

Parathyroid hormone (PTH) is the principal agent of hypercalcaemia in terrestrial vertebrates. It is produced by the parathyroid gland which first evolved in the amphibia and which, therefore, may be considered to have been a specific development required for successful evolution of a terrestrial way of life by primitive tetrapod vertebrates. In spite of the absence of a parathyroid gland in aquatic vertebrates, they are able to maintain plasma calcium levels within strict limits regardless of high or low ambient calcium concentrations. Hypocalcaemic factors, including calcitonin and stanniocalcin, have been well characterised, but there is little substantiated evidence of a PTH-like hypercalcaemic factor in fish, although immune cross-reaction with antisera to human PTH have given positive results (Fraser et al., 1991, Harvey et al., 1987, Kaneko and Pang, 1987). In 1987 a second peptide, which is similar in amino acid sequence to PTH at the N-terminus, was isolated from human tumour cells (Burtis et al., 1987, Moseley et al., 1987, Strewler et al., 1987) and identified as the agent of humoral hypercalcaemia of malignancy (HHM). This peptide, parathyroid hormone-related protein (PTHrP), as well as causing hypercalcaemia by interaction at the common PTH/PTHrP receptor in bone and kidney in chicken and mammals, is now recognised as a pluripotent peptide involved normally in a number of physiological systems, including cardiovascular tone, epidermal integrity, transplacental calcium transport, immune system, lactation in mammals, neural functions and as a differentiating factor in embryonic development (Ingleton and Danks, 1993) Using antisera to the N-terminus 1–16 human PTHrP, we showed that immunoreactive PTHrP (irPTHrP) could be detected in plasma and pituitaries of a teleost fish, the sea bream (Danks et al., 1996) an elasmobranch, the dogfish (Ingleton et al., 1995) and in the saccus vasculosus of sea bream (Devlin et al., 1996). These observations suggested that fish have a PTHrP-like molecule which may be an hypercalcaemic factor in aquatic species, especially when considered in the light of the report by Parsons et al. (1979), in which an extract of fish pituitaries caused rapid hypercalcaemia in test species; the kinetics of the reaction were similar to those of PTHrP and different from the slower action of PTH in mammals.

The gene for PTHrP has been cloned in several mammals, including man (Suva et al., 1987, Yasuda, 1989a), mouse (Mangin et al., 1990), rat (Yasuda et al., 1989b), dog (Rosol et al., 1990) and chicken (Schermer et al., 1991, Thiede and Rutledge, 1990). The PTHrP gene of all species is larger and more complex than that of PTH and in humans gives rise to three splice variants and the translation products can be further modified by enzyme action. Nevertheless the similarities between the amino acid sequences of the N-terminus of PTH and PTHrP suggest a common evolutionary origin with the chromosome location of the human PTHrP gene on the short arm of chromosome 12 (Mangin et al., 1988, Suva et al., 1989) and PTH on chromosome 11, a proposed precursor of chromosome 12 (Comings, 1972).

As an essential step towards understanding the evolution and functions of hypercalcaemic factors, we have cloned the PTHrP gene in the Japanese puffer fish (Fugu rubripes, L) and present details of its genomic organisation, expression and gene linkage.

Section snippets

Isolation and sequencing of PTHrP in Fugu rubripes

During routine Fugu cosmid library sequence scanning, a 589 bp DNA fragment was identified with 9e−06 homology to rat PTHrP. This fragment was subcloned, purified and used as a probe to screen an unamplified high-density gridded Fugu cosmid library (MRC HGMP Resource Centre, UK). Five positive cosmids were identified and confirmed as overlapping clones by analysis of their SacI restriction digestion patterns and secondary screening with the PTHrP probe described above. The presence of PTHrP was

Results and discussion

Initially, five overlapping cosmids were isolated which contained Fugu PTHrP (155E01, 169M02, 061D18, 006G15, 057G14). Using the sequence data generated from analysis of all the cosmids, it was possible to deduce the identity and order of the genes flanking PTHrP (Fig. 1). The 5′ flanking genes are thymopoietin (TMPO) and l-lactate dehydrogenase (LDHB) and the 3′ flanking genes are a novel G-protein coupled receptor and potassium voltage-gated channel protein (KCNA1). This contig represents a

Conclusions

(1) This paper reports for the first time the isolation, expression and genomic sequencing of a gene for PTHrP from a teleost, confirming the existence of a potential hypercalcaemic factor in an aquatic species. Moreover, the wide tissue distribution of PTHrP in Fugu suggests that in common with tetrapods, it may have multiple functions.

(2) The substantial divergence in the organisation of PTHrP in Fugu and that of tetrapods implies that the emergence onto land exerted considerable evolutionary

Acknowledgements

D.M.P was in receipt of a sabbatical grant from the Fundação de Ciência e Tecnologia. This work was supported by EC grant FAIR CT96-1773 and an MRC project grant (G.E and M.C).

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    The sequence of Fugu PTHrP reported in this paper has been deposited in the EMBL databank under Accession No. AJ249391. Shotgun sequence fragments from the five cosmids reported in this paper have been deposited in the EMBL GSS databank under Accession Nos. AL139430–AL139782, inclusive.

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