Copper exposure does not alter the ability of intertidal sea cucumber Cucumaria miniata to tolerate emersion during low tide.

Intertidal animals experience cycles of tidal emersion from water and are vulnerable to copper (Cu) exposure due to anthropogenic toxicant input into marine waters. Both emersion and Cu toxicity can cause damage to physiological processes like aerobic metabolism, ammonia excretion, and osmoregulation, but the interactions of the combination of these two stressors on marine invertebrates are understudied. Mixed effects of 96 h of low and high Cu exposure (20 and 200 µg/L) followed by 6 h of tidal emersion were evaluated on the intertidal sea cucumber Cucumaria miniata. The respiratory tree accumulated the highest concentrations of Cu, followed by the introvert retractor muscle, body wall, and coelomic fluid. Emersion affected accumulation of Cu, perhaps by inhibiting excretion. 200 µg/L of Cu increased lactate production in the respiratory tree, indicative of damaged aerobic metabolism. Cu diminished ammonia excretion, but emersion increased oxygen uptake and ammonia excretion upon re-immersion. The combination of the two stressors did not have any interactive effects on metabolism or ammonia excretion. Neither Cu exposure nor emersion altered ion (sodium, potassium, calcium, magnesium) content of the coelomic fluid. Overall, results of this study suggest that Cu exposure does not alter C. miniata's high tolerance to emersion, and some potential strategies that this species uses to overcome environmental stress are illuminated.

Functional expression and mutant analysis of thioredoxin-fused CEL-III, a hemolytic lectin from the marine invertebrate Cucumaria echinata.

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. New expression system of CEL-III was constructed, and the recombinant thioredoxin-fused CEL-III (Trx-CEL-III) showed strong hemolytic and carbohydrate-binding activity as same as authentic CEL-III. Mutation analysis of Trx-CEL-III suggested that carbohydrate binding to subdomain 1α and 2ß of CEL-III might be important for the hemolytic activity.

Fucoidans from Cucumaria frondosa ameliorate renal interstitial fibrosis via inhibition of the PI3K/Akt/NF-κB signaling pathway.

The effects of Cucumaria frondosa polysaccharides (CFPs) on renal interstitial fibrosis by regulating the phosphatidylinositol-3-hydroxykinase/protein kinase-B/nuclear factor-κB (PI3K/AKT/NF-κB) signaling pathway were investigated in vivo and in vitro in this research. The common unilateral urethral obstruction (UUO) model was used to examine the renoprotective effect and its mechanism in vivo. Compared to the UUO group, CFP administration could ameliorate renal function, inhibit inflammation and fibrosis, and reduce the deposition of the extracellular matrix and epithelial-mesenchymal transition. Mechanistic results indicated that CFPs could inhibit the expression of the total protein of PI3K and the conversion of the AKT and NF-κB p65 phosphorylated proteins, thereby inhibiting the transduction of the PI3K/AKT/NF-κB pathway. In addition, CFP treatment could improve inflammation and fibrosis in HK-2 cells induced by TGF-ß1, and its in vitro mechanism was also verified to inhibit the PI3K/Akt/NF-κB signaling pathway. Overall, these results showed that CFP could alleviate renal interstitial fibrosis related to the PI3K/AKT/NF-κB signaling pathway.

Galactose-Specific, Hemolytic Lectin CEL-III from Cucumaria echinata.

CEL-III is a Ca2+-dependent and galactose-specific lectin purified from the sea cucumber, Cucumaria echinata; it exhibits hemolytic and hemagglutinating activities. CEL-III consists of the following three distinct domains: two N-terminal carbohydrate-binding domains (1 and 2), which adopt ß-trefoil folds such as the B-chain of ricin and are members of the (QXW)3 motif family, and domain 3, an oligomerization domain. After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores composed of CEL-III heptamers, leading to cell lysis or death. In this chapter, the purification and carbohydrate-coated microplate binding assay of CEL-III are described.

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.

BACKGROUND: CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. METHODS: Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. RESULTS: Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. CONCLUSIONS: Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. GENERAL SIGNIFICANCE: Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins.

Hemolytic lectin CEL-III heptamerizes via a large structural transition from α-helices to a ß-barrel during the transmembrane pore formation process.

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long ß-barrel as a transmembrane pore. This ß-barrel is composed of 14 ß-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a ß-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane ß-barrel in a position perpendicular to the plane of the lipid bilayer.

The protective effect of eicosapentaenoic acid-enriched phospholipids from sea cucumber Cucumaria frondosa on oxidative stress in PC12 cells and SAMP8 mice.

Alzheimer's disease (AD) is a common neurodegenerative disorders, in which oxidative stress plays an important role. The present study investigated the effect of eicosapentaenoic acid-enriched phospholipids (EPA-enriched PL) from the sea cucumber Cucumaria frondosa on oxidative injury in PC12 cells induced by hydrogen peroxide (H2O2) and tert-butylhydroperoxide (t-BHP). We also studied the effect of EPA-enriched PL on learning and memory functions in senescence-accelerated prone mouse strain 8 (SAMP8) in vivo. Pretreatment with EPA-enriched PL resulted in an enhancement of survival in a dose-dependent manner in H2O2 or t-BHP damaged PC12 cells. EPA-enriched PL pretreatment could also reduce the leakage of lactate dehydrogenase (LDH), and increase the intracellular total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity compared with the H2O2 or t-BHP group. The down-regulated Bcl-2 mRNA level and up-regulated Bax, Caspase-9, and Caspase-3 mRNA expression induced by H2O2 or t-BHP could be restored by EPA-enriched PL pretreatment. These results demonstrated that EPA-enriched PL exhibited its neuroprotective effects by virtue of its antioxidant activity, which might be achieved by inhibiting the mitochondria-dependent apoptotic pathway. The neuroprotective effect of EPA-enriched PL was also verified in vivo test: the EPA-enriched PL administration prevented the development of learning and memory impairments in SAMP8 mice. Our results indicated that EPA-enriched PL could offer an efficient and novel strategy to explore novel drugs or functional food for neuronprotection and cognitive improvement.

High-value components and bioactives from sea cucumbers for functional foods--a review.

Sea cucumbers, belonging to the class Holothuroidea, are marine invertebrates, habitually found in the benthic areas and deep seas across the world. They have high commercial value coupled with increasing global production and trade. Sea cucumbers, informally named as bêche-de-mer, or gamat, have long been used for food and folk medicine in the communities of Asia and Middle East. Nutritionally, sea cucumbers have an impressive profile of valuable nutrients such as Vitamin A, Vitamin B1 (thiamine), Vitamin B2 (riboflavin), Vitamin B3 (niacin), and minerals, especially calcium, magnesium, iron and zinc. A number of unique biological and pharmacological activities including anti-angiogenic, anticancer, anticoagulant, anti-hypertension, anti-inflammatory, antimicrobial, antioxidant, antithrombotic, antitumor and wound healing have been ascribed to various species of sea cucumbers. Therapeutic properties and medicinal benefits of sea cucumbers can be linked to the presence of a wide array of bioactives especially triterpene glycosides (saponins), chondroitin sulfates, glycosaminoglycan (GAGs), sulfated polysaccharides, sterols (glycosides and sulfates), phenolics, cerberosides, lectins, peptides, glycoprotein, glycosphingolipids and essential fatty acids. This review is mainly designed to cover the high-value components and bioactives as well as the multiple biological and therapeutic properties of sea cucumbers with regard to exploring their potential uses for functional foods and nutraceuticals.

Hemolytic C-type lectin CEL-III from sea cucumber expressed in transgenic mosquitoes impairs malaria parasite development.

The midgut environment of anopheline mosquitoes plays an important role in the development of the malaria parasite. Using genetic manipulation of anopheline mosquitoes to change the environment in the mosquito midgut may inhibit development of the malaria parasite, thus blocking malaria transmission. Here we generate transgenic Anopheles stephensi mosquitoes that express the C-type lectin CEL-III from the sea cucumber, Cucumaria echinata, in a midgut-specific manner. CEL-III has strong and rapid hemolytic activity toward human and rat erythrocytes in the presence of serum. Importantly, CEL-III binds to ookinetes, leading to strong inhibition of ookinete formation in vitro with an IC(50) of 15 nM. Thus, CEL-III exhibits not only hemolytic activity but also cytotoxicity toward ookinetes. In these transgenic mosquitoes, sporogonic development of Plasmodium berghei is severely impaired. Moderate, but significant inhibition was found against Plasmodium falciparum. To our knowledge, this is the first demonstration of stably engineered anophelines that affect the Plasmodium transmission dynamics of human malaria. Although our laboratory-based research does not have immediate applications to block natural malaria transmission, these findings have significant implications for the generation of refractory mosquitoes to all species of human Plasmodium and elucidation of mosquito-parasite interactions.

C-type lectin-like carbohydrate recognition of the hemolytic lectin CEL-III containing ricin-type -trefoil folds.

CEL-III is a Ca(2+)-dependent hemolytic lectin, isolated from the marine invertebrate Cucumaria echinata. The three-dimensional structure of CEL-III/GalNAc and CEL-III/methyl alpha-galactoside complexes was solved by x-ray crystallographic analysis. In these complexes, five carbohydrate molecules were found to be bound to two carbohydrate-binding domains (domains 1 and 2) located in the N-terminal 2/3 portion of the polypeptide and that contained beta-trefoil folds similar to ricin B-chain. The 3-OH and 4-OH of bound carbohydrate molecules were coordinated with Ca(2+) located at the subdomains 1alpha, 1gamma, 2alpha, 2beta, and 2gamma, simultaneously forming hydrogen bond networks with nearby amino acid side chains, which is similar to carbohydrate binding in C-type lectins. The binding of carbohydrates was further stabilized by aromatic amino acid residues, such as tyrosine and tryptophan, through a stacking interaction with the hydrophobic face of carbohydrates. The importance of amino acid residues in the carbohydrate-binding sites was confirmed by the mutational analyses. The orientation of bound GalNAc and methyl alpha-galactoside was similar to the galactose moiety of lactose bound to the carbohydrate-binding site of the ricin B-chain, although the ricin B-chain does not require Ca(2+) ions for carbohydrate binding. The binding of the carbohydrates induced local structural changes in carbohydrate-binding sites in subdomains 2alpha and 2beta. Binding of GalNAc also induced a slight change in the main chain structure of domain 3, which could be related to the conformational change upon binding of specific carbohydrates to induce oligomerization of the protein.