Stichopus Japonicus Research Articles

Stichopus japonicus arginine kinase: gene structure and unique substrate recognition system

Arginine kinase from the sea cucumber Stichopus japonicus underwent a unique molecular evolution. Unlike the monomeric 40 kDa arginine kinases from molluscs and arthropods, Stichopus arginine kinase is dimeric, the same as cytoplasmic isoenzymes of the vertebrate creatine kinases. Its entire amino acid sequence is more similar to creatine kinases than to other arginine kinases, but the guanidino specificity region (GS region) is of the arginine kinase type. To elucidate its unusual evolution, the structure of the Stichopus arginine kinase gene was determined. It consisted of seven exons and six introns, and a part of the exon 2 of the Stichopus gene corresponds to the GS region. Compared with the structure of the human muscle creatine kinase gene (seven exons, six introns), the splice junctions of five introns were conserved exactly between the two genes, suggesting that these introns had been conserved for at least 500 million years. The entire sequence of Stichopus arginine kinase is distinctly included in the creatine kinase cluster in all tree construction methods examined. On the other hand, if the tree is constructed only from sequences corresponding to Stichopus exon 2, it is placed in the arginine kinase cluster. Thus we conclude that Stichopus arginine kinase evolved not from the arginine kinase gene but from the creatine kinase gene, and suggest that its GS region, determining substrate specificity, has been replaced by an arginine kinase type via exon shuffling. In typical arginine kinases four residues, Ser(63), Gly(64), Val(65) and Tyr(68) (numbering from the Limulus polyphemus sequence), in the GS region are highly conserved and are associated with substrate binding. Among them, Tyr(68) appears to play a crucial role by forming a hydrogen bond with the substrate, and is conserved exactly in all arginine kinases. However, in Stichopus arginine kinase, none of these four conserved residues were present. Nevertheless, the enzyme displays an affinity for the substrate arginine (K(m)=0.8 mM) comparable with other arginine kinases. This implies that a completely different substrate-binding system has been developed in Stichopus arginine kinase. We propose that the His(64) in Stichopus arginine kinase acts as a substitute for the Tyr(68) in other arginine kinases, and that the imidazole ring of His(64) is hydrogen bonded with the substrate arginine, thus stabilizing it.

Acclimation of the Sea Cucumber Apostichopus japonicus to Decreased Salinity at the Blastula and Gastrula Stages: Its Effect on the Desalination Resistance of Larvae at Subsequent Stages of Development

Apostichopus (= Stichopus) japonicus blastulae and gastrulae were acclimated for 18 h to salinities of 32‰ (control), 24 and 22‰ (the lower limit of the range of tolerance), and 20‰ (below the range of tolerance). Acclimation to 20‰ resulted in the appearance of teratic larvae, most of which subsequently died. Acclimation to 24, 22, and 20‰ led to a shift in the range of tolerance of the larvae at further stages of development. With a decrease in salinity, acclimated larvae developed more successfully than unacclimated larvae. Acclimated larvae attained the pentactula stage and settled at a salinity range of 32–20‰; unacclimated larvae, at 32–22‰. At different stages of development, acclimated larvae survived greater decreases in salinity than unacclimated larvae. The acclimation effects could be traced up to metamorphosis and settling, i.e., two weeks after the end of the acclimation process.

Expression of the otx gene in the ciliary bands during sea cucumber embryogenesis.

The Otx gene encodes a homeodomain transcription factor that has a highly conserved role in brain formation of both flies and vertebrates. To deduce evolutionary relationship of the chordate central nervous system to the larval or adult nervous system of nonchordate deuterostomes, we characterized the expression of the Otx gene (Sj-Otx) throughout the entire embryonic and larval development of the sea cucumber Stichopus japonicus. Sj-Otx transcripts were detected in fertilized eggs and in the posterior part of the archenteron of gastrulae. However, the expression was downregulated as embryos developed into auricularia larvae. Sj-Otx was expressed again in the ciliary bands of late auricularia larvae, just before metamorphosis to doliolaria larvae. The expression domain corresponded to the domains moving to the mouth during metamorphosis and sinking into the buccal cavity, but not to the five transverse ciliary bands of the doliolaria. The expression gradually disappeared during further development and was not detected in juveniles. These results indicate that the gene responsible for chordate brain formation is expressed in the ciliary bands of auricularia larvae.

Structure of Neuritogenic Active Ganglioside from the Sea CucumberStichopus japonicus

A ganglioside molecular species SJG-1 has been obtained from the n-hexane soluble lipid fraction of the chloroform/methanol extract of the sea cucumber Stichopus japonicus. On the basis of chemical and spectroscopic evidence, the structure of SJG-1 has been determined. SJG-1 possesses an N-glycolylneuraminic acid (sialic acid), nonhydroxy fatty acids and phytosphingosine-type long-chain bases as major ceramide components. The ganglioside SJG-1 exhibits neuritogenic activity toward the rat pheochromocytoma cell line PC12 cells in the presence of NGF.

Molecular Phylogenetic Analysis of Diverse Forms of Echinoderm Guanylyl Cyclases

We found many isoforms of membrane-bound guanylyl cyclase (membrane GC) in the gonads of several echinoid species including Hemicentrotus pulcherrimus (six isoforms), Glyptocidaris crenularis (four isoforms), Clypeaster japonicus (three isoforms), Diadema setosum (three isoforms), and Brissus agassizii (three isoforms). In addition to these membrane GC isoforms, several putative soluble GC isoforms were also found, one in H. pulcherrimus, two in G. crenularis, and three in D. setosum. Other echinoderm species also possess many GC isoforms in their gonads: the starfish, Asterina pectinifera, has five membrane GC isoforms; the brittle star, Ophioplocus japonicus, possesses six membrane and two putative soluble GC isoforms; and the sea cucumber, Stichopus japonicus, contains five membrane and two putative soluble GC isoforms. We also obtained the full-length cDNA sequence of some of these isoforms, one for sperm-activating peptide (SAP) receptor-type GC, and the other for a homolog of the vertebrate natriuretic peptide receptors/membrane GCs, and analyzed their phylogenetic relationship among various invertebrate and vertebrate GC isoforms.

Invertebrate compounds acting on the hemostatic mechanism.

Physiological secretions from some invertebrates have toxic effects on mammalian blood coagulation and fibrinolytic systems. Some of these effects occur because the substances contained in the secretions resemble the components of the hemostatic system. Some of the substances have been characterized, and have been found to have similar molecular weights or sequences, which may indicate a common ancestry. The components can be divided into five groups: antithrombic agents (group I); inhibitors and activators of the prothrombinase complex (group II); substances that affect platelet function (group III); substances that affect the fibrinolytic mechanism (group IV); and a group of miscellaneous agents whose activities are difficult to group together (group V). In group I special mention of the antithrombin agents in Hirudo medicinalis should be made. In group II, the agents affecting the prothrombinase complex are antistasin from Haementeria officinalis, ghilanten from Haementeria Ghiliani and the tick anticoagulant protein from Ornithodoros moubata, a factor V activator/inhibitor from Lonomia achelous and factor II and factor X activators from L. achelous and Lonomia obliqua. Examples of factors which affect platelet function (group III) are glossina from the black fly Glossina morsitans, calin from H. medicinalis, decorsin (a desintegrin) from Macrobdella decorsa, and FAGA from Stichopus japonicus selenka. The first three of these are inhibitors of platelet aggregation, and the last is an inducer. The plasminogen activators (group IV) from the L. achelous caterpillar and Eutriatoma maculata trigger the fibrinolytic system, whereas hementin from H. officinalis and hementerin from Haementeria depressa are directly fibrinolytic. The last group of substances (group V) include those with factor-XIIa-like activity from D. farinae, kallikrein-like activity and a factor XIII degrading enzyme from L. achelous, destabilase from H. medicinalis and prolixin S (nitroforin 2, or anti-factor-IXa) from Rhodnius prolixus. Some of these components have been well characterized, cloned and prepared in recombinant form, and seem to be very promising from the therapeutic point of view.

Structure and properties of hyaline spheres in holothuroid larvae

Summary The structure of the hyaline spheres (HS) in the larvae of the holothuroids Parastichopus californicus and Stichopus japonicus was examined by electron microscopy, and their sensitivity to a range of hydrolytic enzymes was tested. The HS are attached to epithelium and extend into the blastocoel. They consist primary of extracellular material but scattered cells surround them, and a few are found within matrix. Ultrastructurally, the matrix has a gel-like appearance and contains fine granules and fibrils. HS are dissolved very specifically by hyaluronidase (10 mg/ml, pH 5.4). Pronase, trypsin and papain at the same concentration damage both HS and the larval body to varying degrees. Surprisingly, HS sink when isolated from the larval body, so it is unlikely they serve as flotation devices. It seems more likely they serve as storage of structural elements utilized after metamorphosis.

Effect of Depolymerized Holothurian Glycosaminoglycan (DHG) on Tissue Factor Pathway Inhibitor: In Vitro and In Vivo Studies

Depolymerized holothurian glycosaminoglycan (DHG) is a glycosaminoglycan extracted from the sea cucumber Stichopus japonicus Selenka. In previous studies, we demonstrated that DHG has antithrombotic and anticoagulant activities that are distinguishable from those of heparin and dermatan sulfate. In the present study, we examined the effect of DHG on the tissue factor pathway inhibitor (TFPI), which inhibits the initial reaction of the tissue factor (TF)-mediated coagulation pathway. We first examined the effect of DHG on factor Xa inhibition by TFPI and the inhibition of TF-factor VIIa by TFPI-factor Xa in in vitro experiments using human purified proteins. DHG increased the rate of factor Xa inhibition by TFPI, which was abolished either with a synthetic C-terminal peptide or with a synthetic K3 domain peptide of TFPI. In contrast, DHG reduced the rate of TF-factor VIIa inhibition by TFPI-factor Xa. Therefore, the effect of DHG on in vitro activity of TFPI appears to be contradictory. We then examined the effect of DHG on TFPI in cynomolgus monkeys and compared it with that of unfractionated heparin. DHG induced an increase in the circulating level of free-form TFPI in plasma about 20-fold when administered i.v. at 1 mg/kg. The prothrombin time (PT) in monkey plasma after DHG administration was longer than that estimated from the plasma concentrations of DHG. Therefore, free-form TFPI released by DHG seems to play an additive role in the anticoagulant mechanisms of DHG through the extrinsic pathway in vivo. From the results shown in the present work and in previous studies, we conclude that DHG shows anticoagulant activity at various stages of coagulation reactions, i.e., by inhibiting the initial reaction of the extrinsic pathway, by inhibiting the intrinsic Xase, and by inhibiting thrombin.

Structure of fucose branches in the glycosaminoglycan from the body wall of the sea cucumber Stichopus japonicus

Fucose-branched chondroitin sulfate E was prepared from the body wall of sea cucumber Stichopus japonicus. The purified glycosaminoglycan (GAG) was chemically desulfated, followed by carboxyl reduction. Intact, desulfated, and desulfated/carboxyl-reduced GAG fractions were subjected to per- O-methylation. GC-MS analyses of the resultant partially methylated alditol acetates demonstrated that the fucose branch is formed by two fucopyranosyl residues linked glycosidically through position (1 → 3), and that the fucose branch and glucuronic acid are almost equimolar. In addition, it was elucidated that about 20% of the branches stretch from O-3 position of a glucuronic acid moiety of the core chondroitin sulfate polymer, while remaining fucose branches are postulated to protrude from O-4 and/or O-6 position(s) of a N-acetylgalactosamine moiety. This fucose branch was also confirmed to be highly sulfated according to six kinds of substitution pattern in methylation analysis.

Muscle regeneration in the holothurian Stichopus japonicus.

The regeneration of longitudinal muscle bands (LMBs) in the sea cucumber Stichopus japonicus was studied using light and electron microscopic and immunocytochemical methods. Previous investigations of holothurian organs showed the presence of some cytoskeletal proteins which were specific for LMBs only. One of them, the 98 KDa protein, was isolated by means of SDS-electrophoresis and used as an antigen to obtain polyclonal antibodies. When tested on paraffin sections of sea cucumber organs, the antibodies were shown to interact only with coelomic epithelial cells covering the LMBs. The antibodies were used to study LMB regeneration after transverse cutting. During regeneration no signs of myocyte dedifferentiation or mitotic division were observed. In the wound region, damaged myocytes degenerated and muscle bundles desintegrated. However, the coelomic epithelial cells dedifferentiated and began to invade the LMB. Just beneath the surface these cells formed clusters (muscle bundle rudiments). The number and size of the clusters gradually increased, the cells lengthened and developed contractile filaments. These observations suggest that new muscle bundles arise from coelomic epithelial cells covering the LMBs. The migration of coelomic epithelial cells into the damaged LMBs and their myogenic transformation are the basic mechanism of holothurian muscle regeneration.

Comparative biochemical studies of carotenoids in sea cucumbers

The results of an investigation of the carotenoids in the seven species of sea cucumber ( Stichopus japonicus, Holothuria leucospilota, H. moebi and H. pervicax of the order Aspidochirotida, Cucumaria japonica, C. echinata and Pentacta australis of the order Dendrochirotida), from the comparative biochemical point of view, are reported. β-Carotene, β-echinenone, canthaxanthin, phoenicoxanthin and astaxanthin were common in all the sea cucumbers examined. A series of novel marine carotenoids (cucumariaxanthin A, B and C) was obtained from the sea cucumbers of the order Dendrochirotida, while they could not be found from those of the order Aspidochirotida. Significant differences in the carotenoid patterns of the two orders were also observed. The structures of cucumariaxanthin A, B and C have been determined, by chemical and spectroscopic investigations, to be (9Z,9′Z)-5,6,5′,6′-tetrahydro-β,β-carotene-4,4′-dione, (9Z,9′Z)-4′-hydroxy-5,6,5′,6′-tetrahydro-β,β-caroten-4-one, and (9Z,9′Z)-5,6,5′,6′-tetrahydro-β,β-carotene-4,4'-diol, respectively. From the experimental results of carotenoids in the sea cucumbers examined, an oxidative metabolic pathway for β-carotene to astaxanthin, and a new reductive and isomeric metabolic pathway for canthaxanthin to cucumariaxanthin C (via cucumariaxanthin A and B) are proposed.

Effect of compounds elevating cyclic nucleotide levels on dithiothreitol-induced oocyte maturation in the holothurian Stichopus japonicus

We investigated the effect of compounds elevating the intracellular cyclic nucleotide levels (cAMP and cGMP) on oocyte maturation induced by dithiothreitol (DTT) in sea cucumber Stichopus japonicus. Forskolin, theophylline, 3-isobutyl-1-methylxanthine and sodium nitroprusside inhibited the DTT-induced oocyte maturation in Stichopus japonicus. The results suggest that, in holothurians, cyclic nucleotides are negative secondary messengers of oocyte maturation, i.e. a decrease in the level of cAMP and cGMP is a necessary condition for the reinitiation of meiosis in the oocytes of these animals.

Hyaline spheres in auricularia ofStichopus japonicus

Summary The hyaline spheres (HS) in auricularia of Stichopus japonicus consist of extracellular material in which solitary cells are present. This extracellular material can be stained by dyes for lipids. There are no membranes or other structural elements in HS. The cells within HS are most probably responsible for secreting the extracellular material. HS are not embryonic inductors of the ciliary rings which appear during metamorphosis. They probably store nutrients which permit larvae to complete metamorphosis successfully, but this remains to be established.

Amino acid sequence of a C-type lectin CEL-IV from the marine invertebrate Cucumaria echinata.

The complete amino acid sequence of a Ca(2+)-dependent lectin, CEL-IV, from the marine invertebrate Cucumaria echinata was analyzed. The established sequence showed that CEL-IV comprises 157 amino acid residues with a molecular mass of 17,098 Da (without disulfide bonds). From comparison with other proteins, CEL-IV was apparently homologous with the C-type lectin family. The identity was relatively high with a sea cucumber (Stichopus japonicus) lectin SJL-I (40.0%) and a sea urchin (Anthocidaris crassispina) lectin echinoidin (32.6%). In CEL-IV, one interchain and two intrachain disulfide bonds were identified. Interestingly, one of the two intrachain disulfide bonds that were highly conserved among the other C-type lectins was missing, suggesting that this might be a characteristic feature of C-type lectins in the Holothuroidea.

Preliminary study of management of red tide water by the filter feeder Mytilus edulis galloprovincialis

An examination was made to determine whether red tide water masses could be controlled by the filter-feeding bivalve, Mytilus edulis galloprovincialis L., cultured on rope collectors. The mussels effectively retained food particles larger than about 4 μm, and their retention rate increased with increase in plankton density between 10 5 and 5×10 6 cells ml −1, this being equal to the density of plankton in red tide water masses. Thus, plankton in red tide water masses should be removed rapidly from seawater by mussels through their retention and excretion, since the faeces excreted by mussels that had been fed on plankton were deposited speedily. Furthermore, the faeces excreted by mussels which contain a large amount of organic matter unabsorbed by the mussels, were eaten by an edible deposit-feeding sea cucumber, Stichopus japonicus. Thus, certain species of plankton concentrating in red tide water masses may serve not only as food sources for filter feeders and deposit feeders, but can also be removed from the seawater.

Purification and characterization of two Ca(2+)-dependent lectins from coelomic plasma of sea cucumber, Stichopus japonicus.

Two structurally distinct lectins were purified from the coelomic plasma of holothurian, Stichopus japonicus, by affinity chromatography on a porcine stomach mucin-conjugated agarose column, gel filtration on a Superose 6 column, and ion-exchange chromatography on a HiTrap Q-FPLC. The two lectins showed apparent molecular masses of about 400 kDa (SPL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PAGE under reducing conditions. Both lectins showed hemagglutination activity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-terminal amino acid sequences were highly homologous to but distinct from those of a Ca(2+)-dependent (C-type) lectin named SJL-I purified from the same species. In addition to porcine stomach mucin, the hemagglutination activity of SPL-1 was strongly inhibited by uronic acids such as galacturonic acid, and glucuronic acid, while the activity of SPL-2 was inhibited by GalNAc and galactosides. Both lectins were adsorbed on clotted coelomocytes in the presence of Ca2+ but not in the presence of inhibitory sugars or EGTA, suggesting the presence of an endogenous carbohydrate ligand(s) for plasma C-type lectins in the clot. However, coelomocyte clotting occurred normally even in the presence of inhibitory sugars, but was strongly inhibited by synthetic GRGDSP peptide or EGTA, suggesting the participation of integrin but not the lectin-carbohydrate interaction in the clotting events.

EFFECT OF MECHANICAL VIBRATION ON ACTIVE TENSION IN THE LONGITUDINAL RETRACTOR MUSCLE OF A SEA CUCUMBER STICHOPUS JAPONICUS

1. The effect of mechanical vibration on active tension in an echinoderm somatic smooth muscle was studied using the longitudinal retractor muscle (LRM) of a sea cucumber Stichopus japonicus. 2. The steady contracture tension in LRM fibres maximally activated with 10(-3) mol l-1 acetylcholine (ACh) was reduced by vibrations (peak-to-peak amplitude, 0.5­2.5 % of l0, where l0 is the slack length of the muscle; frequency, 5­100 Hz). The extent of reduction of active contracture tension increased with increasing amplitude of vibration, but it did not change appreciably with increasing frequency of vibration. 3. The steady contracture tension in LRM fibres submaximally activated with 10(-5) mol l-1 ACh was more markedly reduced by vibrations than was that in maximally activated fibres. 4. The vibration-induced reduction of active contracture tension disappeared when temperature was lowered from 20­23 to 0 °C. 5. The development of contracture tension in LRM fibres activated with ACh was not affected by mechanical vibration. 6. These results are discussed in connection with the vibration-induced decrease in the rate of breakage of the actin­myosin linkages responsible for isometric force generation.

Amino Acid Sequence of a Lectin from the Sea Cucumber, Stichopus japonicus, and Its Structural Relationship to the C-Type Animal Lectin Family1

The complete amino acid sequence of SJL-I, a lectin from the sea cucumber, Stichopus japonicus, was determined by sequence analysis of peptides derived on enzymatic and chemical fragmentation of the protein. SJL-I consists of 143 amino acid residues and its molecular mass was calculated to be 15,837 Da. Comparison of the sequence of SJL-I with a database revealed that SJL-I exhibits apparent homology with C-type lectins, especially with those of marine invertebrates. The highest homology (identity 28.6%) was found with echinoidin, a lectin from the sea urchin, Anthocidaris crassispina. Comparison of the sequence of SJL-I with those of other C-type lectins indicated that the conserved amino acids are relatively abundant in the C-terminal half of their carbohydrate-recognition domains (CRDs), that can be considered to be involved in binding with Ca2+ as well as carbohydrates.

Purification and characterization of two lectins from the sea cucumber Stichopus japonicus.

Two Ca(2+)-dependent lectins were purified from the sea cucumber Stichopus japonicus by affinity chromatography on lactosyl-Sepharose 4B and ion-exchange chromatography on Q-Sepharose. Their molecular masses were estimated to be 13kDa (SJL-I) and 15 kDa (SJL-II) on SDS-PAGE. SJL-I agglutinated rabbit erythrocytes as well as human A, B, and O-type erythrocytes, but SJL-II agglutinated only rabbit erythrocytes. Hemagglutination by SJL-I was competitively inhibited by N-acetyl-D-galactosamine and galactose-containing carbohydrates. On the other hand, only lactose, melibiose, and raffinose gave weak inhibition of hemagglutination by SJL-II, suggesting that SJL-II may have high specificity for particular complex carbohydrate(s) on the surface of rabbit erythrocytes. SJL-II was activated at ten times lower Ca2+ concentration than SJL-I. Both lectins lost activity in acidic pH, while SJL-I appeared more stable down to pH 4.5.

Fibrinolysis relating substances in marine creatures

1. 1. Extracts with physiological saline solution were obtained from about 20 species of invertebrates and seaweed. Tosyl- l-Arg-MeOH hydrolysing and fibrin plate lytic activity were detected in the invertebrates Stichopus japonicus, Crassost gigas, Tapes japonica, and Kintai-gai as well as the seaweed Codiales codium. 2. 2. These activities were all labile against heat (at 65°C for 1 hr). Except for the extract from Stichopus japonicus, lytic activities against fibrin plates with and without plasminogen were similar. 3. 3. The extract from S. japonicus showed plasminogen activating potency as well as the existence of urokinase (UK) activity enhancing factor. 4. 4. On the other hand, the extract of the seaweed Hizikia fusiformis showed a strong UK inhibiting activity. 5. 5. A fraction of fibrinolytic enzyme was obtained from the extract of S. japonicus by absorption to the celite affinity chromatography. It was orally administered to rabbits at a dosage of 40 mg/kg/day. 6. 6. Fibrinolytic activity was determined periodically on the eugloblin fraction of plasma samples collected from these animals. 7. 7. As compared with the pretreatment value, the activity increased about 2 times ( P < 0.01) and 3 times ( P < 0.005) after 4 and 8 weeks, respectively, of the treatment. 8. 8. After 8 weeks of treatment, the kidney of treated rabbits was extracted with 2 M KCl. The activity of tissue plasminogen activator (free-type TPA) was revealed to be enhanced significantly ( P < 0.001) in the extracts. 9. 9. The fibrinolytic enzyme increased in the blood was recognized by zymography to be mainly the UK type plasminogen activator with mol. wt of 53,000.