Stichopus Japonicus Research Articles

Purification and partial characterization of an acid phosphatase from the body wall of sea cucumber Stichopus japonicus

A high molecular weight (HMW) acid phosphatase from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of anion exchange chromatography, gel filtration chromatography and high performance liquid chromatography (HPLC). The enzyme was purified 19.3-fold with a total yield of 1.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of MW 147.9 kDa. The enzyme displayed maximum activity at pH 4.0 and 50 °C with p-nitrophenyl phosphate as substrate. The enzyme activity appeared to be stable over pH 2.0–5.0 and up to 40 °C. The enzyme activity was enhanced slightly by Mg 2+, whereas inhibited strongly by Cu 2+ and Zn 2+. The enzyme hydrolyzes several phosphate esters, suggesting a probable non-specific nature. The amino acid sequences of three segments of the purified enzyme were analyzed by mass spectroscopy, which did not have any homology with previously described acid phosphatase.

The Research Progress of Antitumorous Effectiveness of Stichopus japonicus Acid Mucopolysaccharide in North of China

The sea cucumbers growing in the estuary of the Pohai of northern China are called Stichopus japonicus and are the orthodox holothurians in traditional Chinese medicine. There are multiple biological active ingredients in S. japonicus, and S. japonicus acid mucopolysaccharide (SJAMP) is one of the important ingredients. SJAMP has multiple pharmacologic actions, such as antitumor, immunologic regulation, anticoagulated blood, and antivirus. The research on antitumor has been carried out by way of animal experiments aiming at studying internal tumor-inhibiting effect of SJAMP, and the route of administration is usually peritoneal or intragastric. Additionally, sea cucumbers have been widely recognized and applied as medicated food or therapeutic prescriptions during and after the treatment of some tumors.

Purification and Characterization of a Cathepsin L-Like Enzyme from the Body Wall of the Sea CucumberStichopus japonicus

Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS-PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine7-amido-4-methylcoumarin with K(m) (69.92 microM) and k(cat) (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 degrees C. It showed thermal stability below 40 degrees C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.

Purification and partial characterization of a novel β-1,3-glucanase from the gut of sea cucumber Stichopus japonicus

For the first time, a β-1,3-glucanase was isolated from the gut of sea cucumber. The enzyme was purified to homogeneity by ammonium sulfate precipitation, ion exchange chromatography with diethylaminoethyl (DEAE)-Sepharose CL-6B, hydrophobic interaction chromatography with Butyl-Sepharose and preparative electrophoresis with polyacrylamide gel electrophoresis (PAGE). The molecular weight of the purified enzyme was estimated to be 37.5 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited optimum activity at pH 5.5 and 40 °C. The enzyme showed high pH stability within the range of pH 5.0–8.0 and thermostability up to 40 °C. The enzyme activity was markedly activated by Mn 2+, whereas strongly inactivated by Cu 2+ and Ag +. The K m and V max of the β-1,3-glucanase were 19.8 μg ml −1 and 2000 μg min −1 mg −1, respectively. The amino acid sequences of three segments of the purified enzyme were acquired by mass spectrometer method, which did not have any homology with previously described echinoderm β-1,3-glucanases, suggesting that it may be a novel member of echinoderm β-1,3-glucanase.

Determination of Triterpene Glycosides in Sea Cucumber (Stichopus japonicus) and Its Related Products by High-Performance Liquid Chromatography

A creative and sensitive method has been developed for the determination of triterpene glycosides concentrations in sea cucumber ( Stichopus japonicus) and related products by using d-quinovose (6-deoxyglucose) as the measurement standard by reverse-phase high-performance liquid chromatography (HPLC) and variable-wavelength detection. d-quinovose, which is a unique monosaccharide in holostane triterpene glycosides, was liberated by acid hydrolysis and precolumn derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP). PMP-quinovose was analyzed by HPLC with 22% acetonitrile in 0.05 M KH2PO4 aquatic solution (pH 5.2) as mobile phase. The calibration curves of d-quinovose were linear within the range of 6.56-164 mg/L (r(2) > 0.995). The contents of triterpene glycosides in various S. japonicus products were determined after appropriate pretreatment methods. The concentration of triterpene glycosides was calculated by the formula C = C(qui) x alpha (alpha = 8.5). The result showed that this method was a simple, rapid, and stable method for the determination of triterpene glycosides in S. japonicus products.

Population genetic structure of sea cucumber, Stichopus japonicus in Korea using microsatellite markers

Sea cucumber (Stichopus japonicus) is a commercially valuable species in Korea. We examined the genetic characteristics of sea cucumber populations in Korea using microsatellite markers. A total of 144 sea cucumbers from five populations were typed for nine polymorphic microsatellite loci. A total of 139 different alleles were found over all loci and many alleles were unique. The average number of allele per locus ranged from 6 to 18.4. The average observed and expected heterozygosities ranged from 0.532 to 0.626 and from 0.719 to 0.789 respectively. All populations showed significant departure from Hardy–Weinberg equilibrium at almost all loci except one (Psj2409). This deviation was in the direction of heterozygote deficit. A phylogenetic tree revealed two distinct clusters. One cluster was formed by the eastern sea population. A second cluster consisted of the subpopulations of the western and southern sea populations. The eastern sea population showed genetic differences such as a larger number of alleles per locus, a larger number of unique alleles and a smaller number of the most common alleles, suggesting a higher genetic diversity in this population. These results provide basic information on natural population genetic structure of S. japonicus in Korea.

Autophagy plays a potential role in the process of sea cucumber body wall “melting” induced by UV irradiation

The changes of tissue appearances and structures in the process of UV-induced “melting” for sea cucumber (Stichopus japonicus) body wall were studied. And the localization and determination of acid phosphatase (ACP), Cathepsin B and Cathepsin L activities were also investigated. The results show that the connective tissue was damaged with many hollows emerging and the regular collagen bundles were broken apart into irregular fragments. Margination of condensed chromatin at the nuclear membrane was observed. Both Golgi’s body and endoplasmic reticulum swelled, curled, and eventually double-or multi-lamellar vesicles were formed. A number of autophagic vesicles distributed in all through the whole cytoplasm. ACP becomes more active after UV irradiation. The activities of cathepsin B and cathepsin L increased in UV-treated sea cucumbers and both achieved their maximum under certain conditions. It indicates that autophagy plays a potential role in the “melting” process for sea cucumber body wall induced by UV irradiation.

Effects of N-terminal deletion mutation on arginine kinase from the sea cucumber Stichopus japonicus

Deletion mutants of arginine kinase (AK) were constructed: AKND4, AKND6, AKND8, AKND10 (the first 4, 6, 8 and 10 amino acids of the N-terminal were deleted), to investigate the structural and functional roles of the N-terminal. Results showed that the deletion mutants assume less compact conformations compared to the wild-type, whereas no significant changes of the secondary or the quaternary structures were observed, implying that the deletions cause a perturbation in the tertiary structure or the hydrodynamic properties of the enzyme. The enzymatic and denaturing measurements showed that removal of the N-terminal residues decreased the activity and stability of the enzyme markedly. The instability increased in accord with the increased number of amino acid residues removed from the N-terminal of AK. It can be concluded that the N-terminal of AK plays an important role in maintaining the conformational stability and catalytic function of the enzyme.

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I 1 (existing in 1.8–1.4 M urea) and I 2 (existing in 0.8–0.4 M urea). I 1 was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I 2 was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates ( I B). Comparison of the properties of these intermediates suggested that I B in the kinetic process corresponded to I 1 in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.

Biologically active gangliosides from echinoderms

This review will summarize the authors’ studies on the structure and the biological activities of the gangliosides from echinoderms. Over 40 gangliosides have been obtained and characterized from 13 kinds of echinoderms, namely, Acanthaster planci, Asterina pectinifera, Asterias amurensis versicolor, Astropecten latespinosus, Luidia maculata, Linckia laevigata (starfish), Cucumaria echinata, Holothuria pervicax, H. leucospilota, Stichopus japonicus, S. chloronotus (sea cucumber), Comanthus japonica (feather star) and Ophiocoma scolopendrina (brittle star). The structural feature of the echinoderms gangliosides was their unique carbohydrate moieties. The biological property of the gangliosides from echinoderms, taking up in this review, was mainly neuritogenic activity.

Identification and characterization of microsatellite markers derived from expressed sequence tags of the sea cucumber Stichopus japonicus

AbstractEleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Stichopus japonicus cDNA libraries. The numbers of alleles ranged from three to 10, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and from 0.077 to 0.690, respectively. Significant deviations from Hardy–Weinberg expectations were observed at eight loci due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of S. japonicus.

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber Stichopus japonicus

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.

Distribution and seasonal activity variation of proteases in digestive tract of sea cucumber Stichopus japonicus

Distribution and seasonal activity variation of proteases in digestive tract of sea cucumber Stichopus japonicus

Feeding and growth on bivalve biodeposits by the deposit feeder Stichopus japonicus Selenka (Echinodermata: Holothuroidea) co-cultured in lantern nets

Suspension aquaculture of filter-feeding bivalves has been developing rapidly in coastal waters in the world, especially in China. Previous studies have demonstrated that dense populations of filter-feeding bivalves in shallow water can produce a large amount of faeces and pseudofaeces (biodeposits) that may lead to negative impacts on the benthic environment. To determine whether the deposit feeder Stichopus ( Apostichopus) japonicus Selenka can feed on bivalve biodeposits and whether the sea cucumber can be co-cultured with bivalves in suspended lantern nets, three experiments were conducted, two in tanks in the laboratory and one in the field. In a 3-month flow-through experiment, results showed that sea cucumbers grew well with specific growth rate (SGR) reaching 1.38% d − 1 , when cultured in the bottom of tanks (10 m 3 water volume) where scallops were cultured in suspension in lantern nets. Moreover, results of another laboratory experiment demonstrated that sea cucumbers could survive well on bivalve biodeposits, with a feeding rate of 1.82 ± 0.13 g dry biodeposits ind − 1 d − 1 , absorption efficiency of organic matter in biodeposits of 17.2% ± 5.5%, and average SGR of 1.60% d − 1 . Our longer-term field experiments in two coastal bays (Sishili Bay and Jiaozhou Bay, northern China) showed that S. japonicus co-cultured with bivalves also grew well at growth rates (0.09–0.31 g wet weight ind − 1 d − 1 ) depending on individual size. The results suggest that bivalve lantern nets can provide a good habitat for sea cucumbers; and the co-culture of bivalve molluscs with sea cucumbers may provide an additional valuable crop with no additional inputs.

The roles of C-terminal loop residues of dimeric arginine kinase from sea cucumber Stichopus japonicus in catalysis, specificity and structure

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by MgATP to form a high-energy compound phosphoarginine (Parg) and MgADP in forward reaction in invertebrates. To detect the different catalytical mechanisms among Stichopus-AK (dimer) and Limulus-AK (monomer) and Torpedo creatine kinase (dimeric CK) and to reveal the structural role of the C-terminal domain loop (C-loop) of dimeric AK, six single-site mutants, E314D, E314Q, E314V, F315A, F315H and F315Y were constructed as well as two multi-site variants, S312R/F315H/V319E (formed by substituting the C-loop of monomeric AK for that of dimeric AK, termed the AAloop) and S312G/E314V/F315D/E317A/S318A/G321S (formed by substituting the C-loop of dimeric CK for that of dimeric AK, termed the ACloop). The AK activity of the three mutants at Glu 314 decreased significantly, from 60- to 500-fold. The ACloop showed only slight AK activity, unlike the same construction in Limulus-AK. In addition, all Phe 315 mutants including the AAloop which retained Glu 314 had modest AK activity (5–84% of the wild type). All the results above suggested that Glu 314 played a more significant role in catalysis in dimeric AK than in the monomer. In addition, ANS profiles indicated that the tolerance of the three Glu 314 mutants to denaturant decreased slightly compared with wild type AK. Though monomeric AK has a His residue at site 315, mutants F315H and the AAloop could not resist any perturbation of denaturant, and the mutants showed a Gibbs free energy of about 2.7 kJ/mol lower than wild type AK. Therefore Phe 315 in dimeric AK has a different role from His 315 in monomeric AK. This might contribute to the stabilization of the native conformation, while His 315 in Limulus AK directly binded to the carboxylate of arginine. Taking all the results above together, we suggested a unique mechanism in dimeric AK, different from both monomeric AK and dimeric CK.

A mechanistic role for protein oligomerization in the phosphagen kinase family

Creatine and arginine kinases catalyze reversible phosphoryl transfer between ATP and creatine or arginine, respectively. Although these two kinases share approximately 80% sequence identity, the quaternary structure of arginine kinase (AK) is typically monomeric, whereas that of creatine kinase (CK) is dimeric or octomeric. Previous structural and functional studies of dimeric CK show that the binding of a transition state analogue complex [Cr-NO3-MgADP] by the two active sites displays negative cooperativity. The goal of this project is to determine whether protein oligomerization, specifically negative cooperativity, plays a functional role in the mechanism of these enzymes. Viscosity (trehalose) variation studies and pre-steady state kinetic analyses by rapid quench of the reactions catalyzed by wild-type dimeric CK, the R147A/R151A monomeric CK and an unusual dimeric AK from Stichopus japonicus, were conducted to examine the rate-determining step of each reaction. Collectively, the results show that the rate of the reaction catalyzed by the monomeric enzymes is limited by the rate of product release, whereas that of the dimeric enzymes is limited by phosphoryl transfer. Thus, negative cooperativity within the phosphagen kinase family enhances catalytic efficiency by decreasing the free energy barrier for product release. This work was supported by NSF grant #0344432.

Nervous system development of the sea cucumber Stichopus japonicus

The nervous system development of the sea cucumber Stichopus japonicus was investigated to explore the development of the bilateral larval nervous system into the pentaradial adult form typical of echinoderms . The first nerve cells were detected in the apical region of epidermis in the late gastrula. In the auricularia larvae, nerve tracts were seen along the ciliary band. There was a pair of bilateral apical ganglia consisted of serotonergic nerve cells lined along the ciliary bands. During the transition to the doliolaria larvae, the nerve tracts rearranged together with the ciliary bands, but they were not segmented and remained continuous. The doliolaria larvae possessed nerves along the ciliary rings but strongly retained the features of auricularia larvae nerve pattern. The adult nervous system began to develop inside the doliolaria larvae before the larval nervous system disappears. None of the larval nervous system was observed to be incorporated into the adult nervous system with immunohistochemistry. Since S. japonicus are known to possess an ancestral mode of development for echinoderms, these results suggest that the larval nervous system and the adult nervous system were probably formed independently in the last common ancestor of echinoderms.

Characterization and subunit composition of collagen from the body wall of sea cucumber Stichopus japonicus

Pepsin-solubilized collagen (PSC) without telopeptides was prepared from the body wall of the sea cucumber Stichopus japonicus and isolated by selective precipitation with NaCl. The PSC exhibited a maximum absorbance at 220 nm. The subunit of PSC was isolated by Sephacryl S-300 HR. The results of SDS-PAGE suggested that purified collagen from S. japonicus was a 1 α trimer (about 135 kDa) while 1 α chain resembling α 1 chain of type I collagen of vertebrate. The thermal stability temperature ( T s) was 57.0 °C as measured by DSC, about 5.0 °C lower than that of type I collagen of calf. Peptide mapping and amino acid analysis of PSC also revealed the difference between invertebrate and vertebrate. However, the presence of ( α 1) 3 trimers was evident.

Depolymerized Holothurian Glycosaminoglycan and Heparin Inhibit the Intrinsic Tenase Complex by a Common Antithrombin-Independent Mechanism.

Depolymerized holothurian glycosaminoglycan (DHG) is a low molecular weight form (M.W. 12,500) of fucosylated chondroitin sulfate isolated from the sea cucumber Stichopus japonicus . DHG demonstrates antithrombotic efficacy in models of thrombin-induced pulmonary thromboembolism in the mouse, venous thrombosis in the rat, and dialysis during renal failure in the dog. The in vitro anticoagulant activities and antithrombotic efficacy of DHG are antithrombin-independent, and associated with lower bleeding tendency compared to unfractionated or low molecular weight heparin (LMWH). DHG has several potential mechanisms of action including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). DHG demonstrates significant affinity for both factor VIIIa and factor IXa, but the specific mechanism for inhibition of the intrinsic tenase complex (ITC) is undefined. We recently established the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of the ITC by LMWH (Yuan et al. Biochemistry 44:3615–3625, 2005). The mechanism and molecular target for ITC inhibition by DHG was likewise determined, and compared to inhibition by LMWH. DHG completely inhibited factor X activation with a 50-fold higher apparent affinity (KI ~2 nM) than observed for partial inhibition by LMWH (KI ~111 nM). DHG reduced the Vmax(app) for factor X activation, without a significant effect on the KM(app), consistent with non-competitive inhibition. DHG did not affect the in vitro half-life of factor VIIIa activity, or inhibit chromogenic substrate cleavage by factor IXa-phospholipid. However, DHG reduced the affinity (KD(app)) of factor IXa for factor VIIIa in a dose dependent fashion, suggesting that the decreased Vmax(app) for factor X resulted from reduced complex assembly. DHG competed the binding of factor IXa to immobilized LMWH with an EC50 ~ 35-fold lower than soluble LWMH, suggesting that the binding sites for DHG and LMWH overlap on the protease. Likewise, the relative affinity of DHG for factor IXa compared to LMWH correlated with inhibitor potency. Kinetic analysis of ITC inhibition employing factor IXa with mutations in the heparin-binding exosite demonstrated that relative affinity for DHG (KI) was: wild type>K241A>H92A>R170A>>R233A; with partial rather than complete inhibition of the mutants. This rank order for DHG potency correlated with the effect of these mutations on factor IXa-LMWH affinity, and the potency of LMWH for the ITC. Submaximal inhibitory concentrations of DHG also accelerated decay of the ITC, under condition where the half-life is primarily dependent on dissociation of the factor VIIIa A2 domain. Thus, DHG binds to an exosite on factor IXa that overlaps with the binding sites for LMWH and factor VIIIa, disrupting critical factor IXa-factor VIIIa interaction(s). These structurally diverse glycosaminoglycans share a common mechanism for inhibition of factor X activation by the ITC. This inhibition occurs at DHG concentrations that are significantly lower (KI ~ 2 nM) than required for optimal acceleration of thrombin inhibition by HCII (~2.4 μM), or inhibition of factor VIII activation by thrombin (> 80 nM). Accordingly, DHG represents a lead compound for analysis of this novel antithrombotic mechanism in the absence of confounding antithrombin-dependent activities.

The kinetic study of arginine kinase from the sea cucumber Stichopus japonicus with 5,5′-dithiobis-(2-nitrobenzoic acid)

The Stichopus japonicus arginine kinase (AK) is a significant dimeric enzyme. Its modification and inactivation course with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and the reactivation course of DTNB-modified AK by dithiothreitol were investigated on the basis of the kinetic theory of the substrate reaction during the modification of enzyme activity. The results show that the modification is a biphasic course while the inactivation is monophasic, with one essential reactive cysteine per subunit. The Cys 274 (numbering from the Stichopus sequence) is exposed to DTNB and is near the ATP binding site. The modified AK can be reactivated by an excess concentration of dithiothreitol in a monophasic kinetic course. The presence of ATP or the transition-state analog markedly slows the apparent reactivation rate constant. The analog components, arginine-ADP-Mg 2+ can induce conformational changes of the modified enzyme, but adding NO 3 − cannot induce further changes that occur with the native enzyme. The reactive cysteines’ location and its role in the catalysis of AK are discussed. The results suggest that the cysteine may be located in the hinge area of the two domains of AK. The reactive cysteine of AK, which was proposed to be Cys 274, may play an important role not in the binding of the transition-state analog but in the conformational changes caused by the transition-state analog.