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Ranatensin-HL: A Bombesin-Related Tridecapeptide from the Skin Secretion of the Broad-Folded Frog, Hylarana latouchii.
Lin Y
,
Chen T
,
Zhou M
,
Wang L
,
Su S
,
Shaw C
.
???displayArticle.abstract??? Bombesin-related peptides are a family of peptides whose prototype was discovered in amphibian skin and which exhibit a wide range of biological activities. Since the initial isolation of bombesin from Bombina bombina skin, diverse forms of bombesin-related peptides have been found in the skins across Anura. In this study, a novel bombesin-related peptide of the ranatensin subfamily, named ranatensin-HL, was structurally-characterised from the skin secretion of the broad-folded frog, Hylarana latouchii, through combination of molecular cloning and mass spectrometric methodologies. It is composed of 13 amino acid residues, pGlu-RAGNQWAIGHFM-NH₂, and resembles an N-terminally extended form of Xenopus neuromedin B. Ranatensin-HL and its C-terminal decapeptide (ranatensin-HL-10) were chemically synthesised and subjected to in vitro smooth muscle assays in which they were found to display moderate stimulatory effects on rat urinary bladder and uterus smooth muscles with EC50 values in the range of 1-10 nM. The prepro-ranatensin-HL was highly homological to a bombesin-like peptide from Rana catesbeiana at both nucleotide and amino acid levels, which might provide a clue for the taxonomic classification of ranid frogs in the future.
Figure 1. Region of reverse phase HPLC chromatogram of the skin secretion of Hylarana latouchii. The elution position/retention time of the pharmacologically-active peptide (ranatensin-HL), is indicated (arrow).
Figure 2. MS/MS fragmentation sequencing spectra of the bioactive peptide (ranatensin-HL) identified in the HPLC fraction of Hylarana latouchii skin secretion. Panel shows predicted singly and doubly charged b-ions and y-ions arising from MS/MS fragmentation. Observed ions are underlined and in bold typeface.
Figure 3. National Centre for Biotechnology Information-Basic Local Alignment Search Tool Protein Database (NCBI-BLASTp) analysis of the mature peptide amino acid sequence of ranatensin-HL. (a) Ranatensin-HL and bombesin precursor (Rana catesbeiana) amino acids 46–58 showing 100% identity. (Accession number: ACO51610.1); (b) Ranatensin-HL and neuromedin-B precursor (Xenopus laevis) amino acids 43–54 showing 92% identity (Accession number: NP 001079342.1); however, the C-terminal 10-amino acid of ranatensin-HL and the putative mature neuromedin-B peptide [22] showed 100% identity. The predicted mature peptide domain is boxed.
Figure 4. Nucleotide and open-reading frame translated amino acid sequences of cloned cDNA encoding the biosynthetic precursor of ranatensin-HL. The putative signal peptide is double-underlined, the mature peptide sequence is single-underlined and the stop codon is indicated by an asterisk. The proteolytic cleavage sites for the mature peptide are boxed.
Figure 5. NCBI-BLAST analysis of the open-reading frame nucleotide and amino acid sequences of the ranatensin-HL biosynthetic precursor. (a) Ranatensin-HL ORF nucleotides 1–378 and bombesin precursor mRNA (Rana catesbeiana) nucleotides 63–440 showing 97% identity. (Accession number: BT081479.1); (b) Ranatensin-HL ORF amino acid 1–125 and bombesin precursor (Rana catesbeiana) amino acid 1–125 showing 94% identity. (Accession number: ACO51610.1).
Figure 6. Comparison of myotropic effects of synthetic ranatensin-HL and ranatensin-HL-10 on isolated rat urinary bladder and uterus smooth muscles. (a) Dose-response curves of ranatensin-HL and ranatensin-HL-10 using rat urinary bladder smooth muscle preparations. EC50 values were determined as 19.2 and 63.8 nM, respectively; (b) Dose-response curves of ranatensin-HL and ranatensin-HL-10 using rat uterus smooth muscle preparations. EC50 values were determined as 5.4 and 70.9 nM, respectively. Each data point represents the mean and standard error of five determinations.
Figure 7. Sequence comparison of the precursors of bombesin-related peptides from different species of the Ranidae family. HL, Hylarana latouchii (this study); RP, Rana pipiens; RS, Rana shuchinae; SV, Sanguirana varians; OG1-OG3, Odorrana grahami. The sequences of mature peptide are underlined. The processing sites of the precursor for releasing mature peptides are boxed. The second possible processing sites are circled in some sequences. Gaps are introduced to optimise the sequence homology.
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