Purification and Characterization of Neuropeptides from Starfish, Asterias amurensis

Abstract

Two peptides (AaANP and AaPSYP) from the tube feet tissue and two peptides (AaSMP 1 and AaSMP 2) from pyloric caeca tissue were purified from Asterias amurensis and their characteristics were investigated. These peptides were purified in three to six steps of reverse-phase, gel-filtration and ion-exchange HPLC. The primary structure of the purified AaANP, AaPSYP, AaSMP 1 and AaSMP 2 were determined by automated amino acid sequencer and MALDI-TOF mass spectrometer.

AaANP: ANYHARGGKPRGGFRRYT (2064.28 Da) AaPSYP: PSYTGPVLGPKQSAEHKREFTQSQMPAGKHVHSLQMGSNNGA- (6137.2 Da) AaSMP 1: GGSRGAFDPLSAGFTD (1554.62 Da) AaSMP 2: FGGSGAFDPLSAGFTDR (1701.8 Da)

AaANP and AaPSYP were identified as a novel contractile peptide, and AaSMP 1 and AaSMP 2 were identified as peptides similar to starfish myorelxant peptide (SMP) derived from starfish Patiria pectinifera. AaPSYP was partially identified as transgelin protein of Asterias rubens. Also, the primary structure of AaPYSP is similar to that of human transgelin. In order to elucidate the complete primary structure of purified AaANP, AaANP-OH and AaANP-NH2 forms were synthesized, after then the retention time on HPLC. As a result, the native and the synthetic (AaANP-OH) peptides eluted with the same retention time. Secondly, in order to identify the complete primary structure of AaSMP 1 and AaSMP 2, peptides of -OH type and -NH2 type respectively were synthesized, and the retention time on HPLC and the relaxing activity were compared. As a result, the native and the synthetic (-OH) peptides eluted with the same retention time. In order to investigate the pharmacological activities of purified peptides, each peptide was introduced to various muscle systems of echinoderms. First of all, when AaANP was put into apical muscle and tube feet of starfish (P. pectinifera and A. amurensis), the response of the peptide appeared in only apical muscle of A. amurensis. Whereas, apical muscle of P. pectinifera and tube feet of each species did not react to AaANP. Also AaANP was treated to muscles of sea urchin and sea cucumber, but there were no activities. In the same manner as above, AaSMP 1 and AaSMP 2 was measured for relaxing activity in various muscle systems. In apical muscles and tube feet of starfish, the well-known relaxing peptides S1, S2 (derived from Asterias rubens) and SMP (derive from P. pectinifera) were used as comparative peptides. The order of the ED50 in the apical muscle was AaSMP 2 = SMP ≥ AaSMP 1 > S1 = S2. At the tube feet, the order of the ED50 was AaSMP 2 > AaSMP 1 = SMP > S1 > S2. Also in A. amurensis, AaSMP 1 and AaSMP 2 showed relaxing activity in apical muscle, but lower effects in tube feet, and it was confirmed that the activity was very similar to that of the previously found SMP of P. pectinifera. In sea urchin and sea cucumber, likely AaANP, AaSMP 1 and AaSMP 2 did not show any response. These results suggest that AaSMP 1 and AaSMP 2 function as broad relaxants in the starfish muscle, which mean that the contributions are different for each muscle system. Cloning and sequencing of a cDNA encoding the AaANP results show that it comprised 2569 bp including open reading frame (ORF) of 816 bp. The ORF was translated into 272 amino acids. The AaANP precursor protein was identified to AN peptide of A. rubens. But the activity of AN peptide is still unkonwn. Based on cDNA sequence of purified peptides, relative expression levels of starfish tissue were calculated. AaANP was highly expressed in radial nerve cord but gonad (generative tissue) was very low in gene expression. In AaSMP, it has 1959 bp including ORF of 1080 bp. The ORF of AaSMP was translated into 359 amino acids. The AaSMP precursor protein had homology with myorelaxant peptide of P. pectinifera and myorelaxant-type peptide of A. rubens. And AaSMP was relatively high expressed in radial nerve cord but it was expressed in other tissue (tube feet, apical muscle, stomach and pyloric caeca). The gene expression was very low only in gonad. Collectively, these data indicate that AaANP and AaPSYP have a general physiological role as a muscle contractant, and AaSMPs has role as a muscle relaxant in starfish. AaANP and AaPSYP are identified a novel contractile peptide. In case of AaANP, it was confirmed to have an amino acid sequence contrast to the previously reported results of the EST analysis. AaPYSP was highly conserved in various echinoderms and its function was confirmed for the first time in this study. AaSMP is the muscle relaxing peptides first identified in A. amurensis. AaANP, AaPSYP, and AaSMP were identified as neuropeptides exhibiting various physiological activities in vivo in echinoderms. Therefore, using the purified peptide in this study, it is shown the possibility of developing therapeutic agents for various diseases.