Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken - PubMed (original) (raw)
Gene structure, transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken
Pedro L C Pinheiro et al. BMC Evol Biol. 2010.
Abstract
Background: Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.
Results: The PTH-L gene is present throughout the vertebrates with the exception of placental mammals. Gene structure of PTH and PTH-L seems to be conserved in vertebrates while PTHrP gene structure is divergent and has acquired new exons and alternative promoters. Splice variants of PTHrP and PTH-L are common in Xenopus and chicken and transcripts of the former have a widespread tissue distribution, although PTH-L is more restricted. PTH is widely expressed in fish tissue but from Xenopus to mammals becomes largely restricted to the parathyroid gland. The N-terminal (1-34) region of PTH, PTHrP and PTH-L in Xenopus and chicken share high sequence conservation and the capacity to modify calcium fluxes across epithelia suggesting a conserved role in calcium metabolism possibly via similar receptors.
Conclusions: The parathyroid hormone family contains 3 principal members, PTH, PTHrP and the recently identified PTH-L. In teleosts there are 5 genes which encode PTHrP (2), PTH (2) and PTH-L and in tetrapods there are 3 genes (PTHrP, PTH and PTH-L), the exception is placental mammals which have 2 genes and lack PTH-L. It is hypothesized that genes of the PTH family appeared at approximately the same time during the vertebrate radiation and evolved via gene duplication/deletion events. PTH-L was lost from the genome of eutherian mammals and PTH, which has a paracrine distribution in lower vertebrates, became the product of a specific endocrine tissue in Amphibia, the parathyroid gland. The PTHrP gene organisation diverged and became more complex in vertebrates and retained its widespread tissue distribution which is congruent with its paracrine nature.
Figures
Figure 1
Multiple sequence alignment of the Xenopus and chicken 1-34 PTH family members mature peptide N-terminal region with teleost (Takifugu and zebrafish) and mammals (human and mouse). Conserved amino acid positions identified in all vertebrates are indicated by "*" and percentage of sequence similarity in comparison with human PTH and PTHrP and Takifugu PTH-L is given. The typical three amino acid motifs characteristic of each PTH family member in positions 8 to 10 are indicated in black. % similarity to first sequence is indicated on the right. Accession number of the sequences used were: Human (PTH, AAH96144.1; PTHrP, AAA60216); Mouse (PTH, NP_065648; PTHrP, CAC39218.1); Zebrafish (PTHA, NP_998115.1; PTHB, NP_998114.1; PTHrPA, AAY87956.1; PTHrPB, AAY87957.1; PTH-L, CU856139); Takifugu (PTHA, CAG26460.1; PTHB, CAG26461.1; PTHrPA, CAB94712.1; PTHrPB, CAG26459.2; PTH-L, CAG26462.1).
Figure 2
Gene organization of the vertebrate PTH-like family members. Exons are represented by boxes and lines indicate introns. Coding (E1 to E4) and non-coding (E1' to E'3) exons are numbered and annotated in bold and italics, respectively. Dotted-filled boxes represent the mature coding regions and black lines box the signal peptide sequence. Arrows represent alternative splice isoforms identified in Xenopus and chicken and previously reported in human. The general organization of the conserved vertebrate PTH gene structure is represented and the size of vertebrate PTHrP and PTH-L precursors is given (amino acids). The length of the chicken PTH-L precursor was predicted in silico and is indicated in italics. Dashed lines indicate incomplete structures that were not confirmed in silico or amplified by RT-PCR. The start of the mature peptide (+1) and the size of the signal peptide for all vertebrate PTH family members is indicated. The localization of the human PTHrP promoter regions (P1, P2 and P3) and the chicken PTHrP putative promoter sites (P1', P2' and P3') and TATA box consensus sequence within the region of P3' are shown. The figure is not drawn to scale and Takifugu A structure was taken from Power et al. [42].
Figure 3
Consensus phylogenetic tree of Xenopus and chicken PTH family members using the Neighbor Joining method [60]and 1000 bootstraps replicates with the complete amino acid precursor sequence in Mega3.1 software [61]with the settings pairwise deletion, p-distance model and 222 informative sites. Xenopus and chicken PTH family members are in italics and the sequence of human GIP (HsaGIP, NP_004114) was used as outgroup. Human (NP_848544), mouse (NP_444486) and zebrafish (NP_991140) TIP39 mature protein sequences were included for comparative purposes. The accession numbers of other sequences utilized for tree construction are indicated in Figure 1 and seabream PTHrP is AAF79073.
Figure 4
Short-range gene linkage comparisons of the PTH family members in the Takifugu, Xenopus, chicken and human genomes. Genes are represented by closed boxes and the size of the chromosome region analysed is given underneath. Genes were named using HUGO and lines indicate chromosome/scaffold segments. The vertebrate PTH family members are in bold and conserved flanking genes identified within the homologue regions are underlined. The PTH gene is localized in Xenopus scaffold_235 and in chicken chromosome 5 and two conserved genes ARNTL and BTBD10 were identified. The Xenopus and chicken PTHrP maps to scaffold_766 and chromosome 1, respectively and the gene MRPS35 was found in close proximity in all vertebrate regions analysed. PTH-L and SFRS3 genes map to Xenopus scaffold_169 and to chicken chromosome 26. SFRS3 was not linked to Takifugu PTH-L and is present on human chromosome 6 which lacks PTH-L. For simplicity, only genes with correspondence across species are represented. The figure is not drawn to scale.
Figure 5
Expression of chicken (open bars) and Xenopus (closed bars) PTH family genes as determined by q-PCR. Gene specific primers were used to amplify PTH and PTH-L transcripts and PTHrP spliced isoforms from several tissues. The number of amplified transcripts is presented in relation to 18S copy number and data is presented as mean ± S.E. (n = 2 to 3 for Xenopus and n = 3 for chicken except for pituitary where n = 1).
Figure 6
Calcium fluxes (water to blood side) in Xenopus abdominal skin and chicken 16 to 18 day old embryo CAM (shell to embryo side) after the addition of 10 nM, N-terminal (1-34) PTH, PTHrP and PTH-L to the basolateral membrane site. Human PTH (1-34) and salmon Luteinizing hormone-releasing hormone (LHRH) were used as positive and negative controls respectively. Results are shown as mean ± SEM and the "*" indicates statistical significance compared to control (time 0) (p < 0.05).
References
- Canario AVM, Fuentes J, Guerreiro PM, Power DM. In: Novel aspects of PTHrP physiopathology. Luparello C, editor. Nova Science Publishers; 2007. Parathyroid hormone and related peptides in fish: From sequence to function; pp. 27–40.
- Guerreiro PM, Renfro JL, Power DM, Canario AVM. The parathyroid hormone family of peptides: structure, tissue distribution, regulation, and potential functional roles in calcium and phosphate balance in fish. Am J Physiol Regul Integr Comp Physiol. 2007;292(2):R679–696. - PubMed
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