Isolation and characterization of neuropeptides from the pyloric caeca of starfish, Patiria pectinifera

Abstract

The two fractions (A and B) from the pyloric caeca extract of starfish (Patiria pectinifera) were used to purify neuropeptides, and the relaxing and contractile activities of the isolated peptides were investigated. Pedal peptides/Orcokinin-like peptides (PP/OK) were purified from fraction A, and a myoactive neuropeptide which belongs to NG family peptides was purified from fraction B. To purify these peptides, the acidic extract of pyloric caeca was applied to a series of HPLC steps that comprise reversed-phase, ion exchange, and size exclusion HPLC. To examine the relaxing effect of the obtained fractions an in vitro bioassay on the apical muscle of starfish was performed. The molecular weights and the primary structures of the four purified peptides determined by LC-MS and Edman degradation, respectively, were as follows: the PP/OK peptide SMPa (FGKGGAYDLSAGFTD, 1602.72 Da), SMPb (FGMGGAYDLSAGFTD, 1605.66 Da), SMPc (FGMGGAYDLSAGFTE, 1619.68 Da), and NGFFYamide with a molecular weight of 646.29 Da. SMPa disegnaited as PpSMPa in this study, is a starfish myorelaxant peptide (SMP) with a free carboxyl terminus (-COOH) from the PP/OK family peptides, which was previously purified from the whole-body extract of starfish. In addition, SMPb and SMPc, which were putatively predicted to be produced as isotypes of SMPa, in the previous study, were purified from the pyloric caeca extract of P. pectinifera. SMPb is [Met3]-SMPa in which Lys, the third residue of SMPa, is substituted with Met and was designated as PpSMP in this study. PpSMPc is [Met3, Glu16]-SMPa in which Lys at the 3rd position and Asp at the 16th position of SMPa are substituted with Met and Glu, respectively. Meanwhile, NGFFYamine peptide was isolated from fraction B, which belongs to NG peptides, and was designated as PpNG in this study. The four purified peptides were synthesized using the solid-phase method. In addition, to investigate and compare the biological activities of free C-terminus PpSMPa-c and amidated forms were also synthesized. To compare the identity of native PpSMPb-c with the synthetic ones, the retention times on RP-HPLC were compared. As a result, the retention times of native PpSMPb-c were the same as those of the synthetic peptides with the free-carboxy terminals. To investigate the smooth muscle relaxing activity of PpSMPa-c, apical muscles and tube feet of P. pectinifera were used. PpSMPa-c showed relaxing activity that increased in a concentration-dependent manner for apical muscle and tube feet. The relaxing activity of PpSMPa-c was stronger on apical muscle than those on tube feet. PpSMPa-c with C-terminal amidation showed a relaxing activity similar to that of the natural peptides on apical muscle and tube feet preparations. However, on the apical muscle preparation, PpSMPc-NH2 had lower threshold concentration compared to native PpSMPc. To investigate the tissue specificity of PpSMPa-c’s smooth muscle relaxing activity, the retractor muscle of the sea urchin Mesocentrotus nudus was used. All PpSMPa-c showed weak relaxing response at 10-5 M on the retractor muscle of sea urchin. Although Sp1-Sp9 peptides that have sequence similarities with PpSMP was reported from sea urchin, their myoactivity on the tissues of sea urchins and other echinoderms has not yet been studied. The relaxing activity exerted by PpSMPa-c suggest that their homologs in sea urchin (Sp1-Sp9) may also possess relaxing activity on the retractor muscle of sea urchin. In addition, the tube feet preparation from starfishes P. pectinifera, Asterias amurensis, and Certonardoa semiregularis were used to compare the relaxing activity of PpSMPa. The relaxing activity of PpSMPa at a concentration of 10-5 M decreased in the following order: P. pectinifera > C. semiregularis > A. amurensis. Therefore, since PpSMPa-c showed relaxing responses on the apical muscle and tube feet of the starfishes, PpSMP neuropeptides may act as bioregulators that are involved in movement and feeding behavior of starfishes. The sequence of PpNG precursor was obtained by cDNA cloning. The cDNA cloning revealed the nucleotide sequence of 1978 bases encoding the PpNG precursor protein containing 240 amino acid residues. The open reading frame contains a predicted signal peptide, two copies of NGFFYamide with dibasic cleavage sites (KR), and the C-terminal neurophysin domain comprising 14 conserved cysteine residues. The RT-qPCR revealed an expression of PpNG in all tissue except gonads and a relatively high expression level in the radial nerve cord. This result means that PpNG is widely distributed in tissues of starfish P. pectinifera. To investigate PpNG’s activity, relaxing and contractile activities were measured using apical muscle, tube feet, and cardiac stomach of P. pectinifera. The relaxing response of PpNG showed relatively higher activity on the apical muscle of A. amurensis than on that of P. pectinifera. The dose-dependent relaxing activity of PpNG on the apical muscle of P. pectinifera was observed up to 10-6 M, but the potency of relaxing activity did not change at high concentrations’ 10-5 M and 10-4 M. On the other hand, the relaxing activity of PpNG on A. amurensis apical muscle increased in a dose-depend manner. This result suggests the different actions of PpNG on the apical muscles of P. pectinifera and A. amurensis. Meanwhile, PpNG showed a contractile activity on tube feet and cardiac muscle of P. pectinifera, and a potent response on the cardiac stomach was observed. Therefore, PpNG is suggest to play a role in feeding behavior and in locomotion in starfish. Finally, NG peptides derived from starfish (PpNG), sea urchin Strongylocentrotus purpuratus (SpNG), and sea cucumber Apostichopus japonicus (AjNG) were used to investigate their activity on the cardiac stomach of starfish. The results show that the conserved N-terminal sequence does not affect the bioactivity of the NG pentapeptides, but changes in the hydrophobic amino acid residues located at the variable C-terminus may affect the bioactivity of the NG peptides. In this study, for the first time, four neuropeptides, PpSMPa-c and PpNG, were purified from the pyloric caeca of starfish P. pectinifera. Also, their smooth muscle relaxing and contractile activities, and the cDNA cloning of PpNG were studied.