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Identification of a novel starfish neuropeptide that acts as a muscle relaxant.
Kim CH
,
Kim EJ
,
Go HJ
,
Oh HY
,
Lin M
,
Elphick MR
,
Park NG
.
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Neuropeptides that act as muscle relaxants have been identified in chordates and protostomian invertebrates but little is known about the molecular identity of neuropeptides that act as muscle relaxants in deuterostomian invertebrates (e.g. echinoderms) that are ''evolutionary intermediates'' of chordates and protostomes. Here, we have used the apical muscle of the starfish Patiria pectinifera to assay for myorelaxants in extracts of this species. A hexadecapeptide with the amino acid sequence Phe-Gly-Lys-Gly-Gly-Ala-Tyr-Asp-Pro-Leu-Ser-Ala-Gly-Phe-Thr-Asp was identified and designated starfish myorelaxant peptide (SMP). Cloning and sequencing of a cDNA encoding the SMP precursor protein revealed that it comprises 12 copies of SMP as well as 3 peptides (7 copies in total) that are structurally related to SMP. Analysis of the expression of SMP precursor transcripts in P. pectinifera using qPCR revealed the highest expression in the radial nerve cords and lower expression levels in a range of neuromuscular tissues, including the apical muscle, tube feet and cardiac stomach. Consistent with these findings, SMP also caused relaxation of tube foot and cardiac stomach preparations. Furthermore, SMP caused relaxation of apical muscle preparations from another starfish species - Asterias amurensis. Collectively, these data indicate that SMP has a general physiological role as a muscle relaxant in starfish. Interestingly, comparison of the sequence of the SMP precursor with known neuropeptide precursors revealed that SMP belongs to a bilaterian family of neuropeptides that include molluscan pedal peptides (PP) and arthropodan orcokinins (OK). This is the first study to determine the function of a PP/OK-type peptide in a deuterostome. Pedal peptide/orcokinin (PP/OK)-type peptides are a family of structurally related neuropeptides that were first identified and functionally characterised in protostomian invertebrates. Here, we report the discovery of starfish myorelaxant peptide (SMP), a novel member of the PP/OK-type neuropeptide identified in the starfish Patiria pectinifera (phylum Echinodermata). SMP is the first PP/OK-type neuropeptide to be functionally characterised in a deuterostome.
Figure 1. The starfish Patiria pectinifera. The aboral side (a) and oral side (b) of an intact animal are illustrated. The position of the apical muscles on the inner surface of the aboral body wall of a dissected animal is shown in (c) marked by arrows. An extract of P. pectinifera containing peptidic materials relaxed apical muscle that was pre‐contracted with 1 μM acetylcholine (ACh); up and down arrows represent application of ACh and the extract, respectively (d).
Figure 2. Isolation, structure determination and pharmacology of purified myorelaxant peptide. Peak A was isocratically eluted with 20% acetonitrile/0.1% trifluoroacetic acid on RP‐HPLC (a), and an aliquot of purified peak A caused relaxation of the apical muscle (b). Purified peak A was identified as a peptide comprised of sixteen amino acid residues with a molecular mass of 1601.72 Da, which we have named starfish myorelaxant peptide or SMP (c). Comparison of chromatographic properties of native SMP (N) and synthetic SMP (S) on RP‐HPLC showed that native SMP and synthetic SMP with a free carboxy terminal have identical retention times on RP‐HPLC (d). The concentration‐dependent relaxing activity of SMP on the apical muscle of P. pectinifera. SMP with free carboxyl terminus and amidated carboxy terminus is SMP (●) and SMPamide (○), respectively. The effects of S1 (▲) and S2 (▵) from the starfish A. rubens and the molluscan neuropeptides FLRFamide (■) and FMRFamide (□) are shown to compare their activity with SMP. Each point represents the mean ± standard deviation determined from four separate experiments. ****p < 0.0001 for SMP (●) compared with S1/S2. The percentage relaxing activity was calculated by comparing each relaxation effect to the maximal contraction of the apical muscle by 1 μM ACh (e). Representative recording of the concentration‐dependent relaxing effect of SMP on P. pectinifera apical muscle pre‐contracted with 1 μM ACh (f).
Figure 3. Precursor of starfish myorelaxant peptide (SMP) in Patiria pectinifera. The DNA sequence of a transcript (lowercase, 1682 bases) encoding the P. pectinifera
SMP precursor (uppercase, 426 amino acid residues) is shown. The predicted signal peptide, the purified mature SMP (SMP
a) and three other variants (SMP
b, [Met3]‐SMP
a; SMP
c, [Met3, Glu16]‐SMP
a; SMP
d, SMP
a‐related octadecapeptide) are shown in blue, red, pink, orange and purple, respectively, and putative dibasic cleavage sites (KR) are shown in green. The asterisk shows the position of the stop codon. The SMP precursor protein comprises twelve copies of SMP and seven copies of SMP‐like peptides.
Figure 4. The expression levels for the starfish myorelaxant peptide (SMP) precursor transcript in various organs/tissues from P. pectinifera and the pharmacological effects of SMP on cardiac stomach and tube foot from P. pectinifera. Relative expression levels of SMP transcripts in each organ/tissue were normalized against the level of the EF1α gene as an internal control. Mean ± standard deviation (n = 3) are shown. Means denoted by the same letter did not differ significantly (p > 0.05) whilst different letters (a, b, c, d, e) at the top of the bars indicate statistically significant differences (p < 0.05) between tissues determined by one‐way anova followed by Duncan's Multiple Range test (a). SMP caused concentration‐dependent relaxation of the cardiac stomach (b) and tube foot (c) from P. pectinifera. The relaxing activity of SMP (●) was compared with S1 (▲) and S2 (▵). Each point represents the mean ± standard deviation determined from four separate experiments. Statistically significant difference between SMP and S1/S2 represents with ****p < 0.0001. The percentage relaxing activity was calculated by comparing each relaxation effect to the maximal contraction of cardiac stomach caused by 10 μM carbacol and of tube foot caused by 30 mM high‐potassium artificial seawater respectively. Representative recordings of the effects of SMP on cardiac stomach (d) and tube foot (e) preparations are shown.
Figure 5. Pharmacological effect of starfish myorelaxant peptide (SMP) on apical muscle from Asterias amurensis and identification of an SMP‐type precursor in Asterias rubens. (a). The concentration‐dependent relaxing activity of SMP (●) compared with S1 (▲) and S2 (▵) on the apical muscle of A. amurensis. Each point represents the mean ± standard deviation determined from four separate experiments. Statistically significant differences between the effects of SMP and S1, SMP and S2, and S1 and S2 are represented by black, red and blue asterisks (*p < 0.05, ***p < 0.001 and ****p < 0.0001), respectively. The percentage relaxing activity was calculated by comparing each relaxation effect to the maximal contraction of apical muscle caused by 1 μM ACh. (b) Amino acid sequence of a 224‐residue SMP‐type precursor protein identified in A. rubens, which comprises a predicted 20‐residue signal peptide (blue) and eight copies of putative SMP‐like peptides (red) and putative dibasic cleavage sites (KR, green). The sequence of the cDNA encoding this protein is shown in Figure S2.
Figure 6. Multiple sequence alignment of the P. pectinifera starfish myorelaxant peptide (SMP) precursor with related neuropeptide precursors in other echinoderms. Highlighted red, green, blue, yellow and purple boxes represent multiple copies of neuropeptides separated by putative cleavage sites (KR or KK). All of the precursors contain multiple copies of related peptides: P. pectinifera
SMP precursor contains twelve copies of SMP and seven copies of SMP‐like peptides; A. rubens
SMP precursor contains eight copies of SMP‐like peptides; S. purpuratus neuropeptide precursor 6 (Spnp6) contains twenty‐one copies of nine SMP‐like peptides; Spnp7 precursor contains ten copies of nine SMP‐like peptides; A. japonicus neuropeptide precursor 7 (Ajnp7) contains six copies of five SMP‐like peptides. The sequences of Spnp6, Spnp7 and Ajnp7 are from (Rowe and Elphick 2012; Rowe et al. 2014).
Figure 7. Alignment of starfish myorelaxant peptide (SMP
a) with putative SMP‐like neuropeptides derived from echinoderm SMP‐type precursors: the starfish P. pectinifera and A. rubens; sea urchin S. purpuratus; sea cucumber A. japonicus. Conserved residues are highlighted in black and grey.
Figure 8. Alignment of echinoderm starfish myorelaxant peptide (SMP)‐type peptides with protostomian pedal peptide (PP)/orcokinin(OK)‐type peptides. The basic amino acids Lys, Arg and His are shown in the black with light grey highlighting, and the acidic residues Glu and Asp are shown in black with dark grey highlighting. All other amino acids are classified as hydrophobic (white with light grey highlighting) or hydrophilic (white with dark grey highlighting). Lower case ‘a’ denotes a C‐terminal amide group. Species abbreviations and references: Pp, P. pectinifera; Ar, A. rubens; Sp, S. purpuratus (Reich et al. 2015); Aj, A. japonicus (Du et al. 2012; Rowe and Elphick 2012); Ac, Aplysia californica (Moroz et al. 2006); Pd, Platynereis dumerilii (Conzelmann et al. 2011); Ce, Caenorhabditis elegans (Nathoo et al. 2001); Pc, Procambrus clarkii (Yasuda‐Kamatani and Yasuda 2000); Nv, Nasonia vitripennis (Hauser et al. 2010).
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