Molecular characterization, inducible expression and functional analysis of an IKKß from the tropical sea cucumber Holothuria leucospilota.

The inhibitory kappa B kinase (IKK) is a critical regulator for the nuclear factor-κB (NF-κB) pathway. In this study, an IKKß named as HLIKKß was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLIKKß is 4246 bp in size, containing a 132 bp 5'-untranslated region (UTR), a 1783 bp 3'-UTR and a 2331 bp open reading frame (ORF) encoding a protein of 776 amino acids with a deduced molecular weight of 89.66 kDa. HLIKKß contains a kinase domain (KD) at its N-terminal, a leucine zipper (LZ) and a helix-loop-helix (HLH) motif at its C-terminal. In the KD, a conserved active loop (SXXXS) were identified. The results of luciferase reporter assay and ELISA assay showed that over-expressed HLIKKß in HEK293T cells could activate the nuclear factor-κB (NF-κB) and induce the secretion of proinflammatory cytokines TNF-α and IL-1ß. When HLIKKß was silenced by siRNA, the apoptosis rate of sea cucumber coelomocytes was increased significantly, indicating the anti-apoptotic function of HLIKKß. Moreover, the up-regulation of HLIKKß mRNA was observed in the sea cucumber coelomocytes after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge, suggesting that the HLIKKß might play important roles in the innate immune defense of sea cucumber against the viral and bacterial infections.

The first cloned echinoderm tumor necrosis factor receptor from Holothuria leucospilota: Molecular characterization and functional analysis.

In this study, an echinoderm tumor necrosis factor receptor named HLTNFR-16 was first cloned from the tropical sea cucumber Holothuria leucospilota. The full-length cDNA of HLTNFR-16 is 3675 bp in size, containing a 415 bp 5'-untranslated region (UTR), a 2024 bp 3'-UTR and a 1236 bp open reading frame (ORF) encoding a protein of 411 amino acids with a deduced molecular weight of 45.63 kDa. The HLTNFR-16 protein contains a signal peptide, four TNFR domains (the last three were identified as extracellular cysteine-rich domains), a transmembrane region and a death domain. Phylogenetic analysis showed that HLTNFR-16 was clustered into a clade with TNFR-16s in other species, indicating that this echinoderm TNFR may be a new member of the TNFR-16 subfamily. The results of TUNEL assay showed that the over expression of HLTNFR-16 could induce apoptosis in HEK293T cells. When HLTNFR-16 was silenced by siRNA, the apoptosis of sea cucumber coelomocytes induced by inactivated Vibrio harveyi was suppressed significantly, indicating that HLTNFR-16 is important for apoptosis induction. Additionally, luciferase reporter assay exhibited that the over-expressed HLTNFR-16 in HEK293T cells could activate the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Moreover, the secretion of proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-18 in HEK293T cells was increased by the over-expression of HLTNFR-16. This study provides evidences for the potential roles of sea cucumber TNFR in the innate immunity.

The first cloned sea cucumber FADD from Holothuria leucospilota: Molecular characterization, inducible expression and involvement of apoptosis.

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5'-untranslated region (UTR), a 1334-bp 3'-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.

Cytotoxic activity of Holothuria tubulosa (Echinodermata) coelomocytes.

The immune system of marine invertebrates, in particular that of holothurians, still requires further study. Our research showed that coelomocyte cells contained in the coelomic fluid of the sea cucumber, Holothuria tubulosa, are able to lyse, in vitro, red blood cells in rabbits and sheep. A plaque-forming assay showed spherule cells to be the effector cells, able to release cytotoxic molecules after xenogenic cell contact. The coelomocyte lysate supernatant, analysed by polyacrylamide gel electrophoresis overlay technique, using rabbit and sheep erythrocytes, showed two different haemolytic protein patterns: one calcium dependent and the other calcium independent. The fractions of each pattern were resolved on a polyacrylamide gel and calcium-dependent and independent coelomocyte lysate patterns were compared.

The first tropical sea cucumber caspase-8 from Holothuria leucospilota: Molecular characterization, involvement of apoptosis and inducible expression by immune challenge.

In this study, the first tropical sea cucumber caspase-8 named HLcaspase-8 was identified from Holothuria leucospilota. The full-length cDNA of HLcaspase-8 is 2293 bp in size, containing a 245 bp 5'-untranslated region (UTR), a 521 bp 3'-UTR and a 1527 bp open reading frame (ORF) encoding a protein of 508 amino acids with a deduced molecular weight of 57.47 kDa. Besides the common signatures of caspase family including conserved cysteine active site pentapeptide motif QACQG, P20 domain and P10 domain, HLcaspase-8 also contains a characteristic DED domain. The over-expression of HLcaspase-8 in HEK293T cells showed that HLcaspase-8 protein could induce apoptosis and the apoptosis could be promoted by TNF-α, indicating that the apoptosis induced by HLcaspase-8 might also be triggered via a receptor-mediated pathway. Moreover, the expression of HLcaspase-8 in in vitro experiments performed in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic Acid [poly (I:C)] challenge, suggesting that the sea cucumber caspase-8 might play some important roles in the innate immune defense against bacterial and viral infections.

Changes in holothurian coelomocyte populations following immune stimulation with different molecular patterns.

Echinoderms possess a variety of cells populating the coelomic fluid; these cells are responsible for mounting defense against foreign agents. In the sea cucumber Holothuria glaberrima, four different coelomocyte types were readily distinguished using morphological, histochemical and physiological (phagocytic activity) parameters: lymphocytes, phagocytes, spherulocytes and "giant" cells (listed in order of abundance). Monoclonal antibodies generated against sea cucumber tissues and one polyclonal against sea urchin mayor yolk protein (MYP) were also used to characterize these cell populations. The effects of several pathogen-associated molecular patterns (PAMPs): Lipopolysaccharides from Escherichia. coli (LPS), heat-killed Staphylococcus aureus (SA) and a synthetic dsRNA were studied on coelomocyte cell populations. PAMPs increased the phagocytic activity of the holothurian coelomocytes, and were able to induce selective immune responses in several of these populations, demonstrating the ability of the sea cucumber to respond to a different variety of immune challenges. Overall, these results show the variety of cells that populate the coelomic fluid of the holothurian and demonstrate their involvement in immune reactions. These animals represent an untapped resource for new findings into the evolution and development of the immune response not only in invertebrates but also in phylogenetically shared reactions with vertebrates.

T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): possible involvement in differential recognition of bacteria.

In invertebrates, cellular and humoral components are evolved to maintain their body immunity and integrity. Both these factors respond to different antigens such as microorganisms, vertebrate erythrocytes and foreign proteins. In this article, we report a study of a lectin (HSL) involved in immune response in the echinoderm, sea cucumber (Holothuria scabra). Correlative studies indicate that the expression of this defensive lectin is induced by bacterial challenge, wherein cell wall glycoconjugates of bacteria are involved in lectin induction. HSL showed strong broad spectrum antibacterial activity against both gram-positive and gram-negative bacteria. Under in vitro conditions, purified HSL mediate agglutination of the test bacteria, there by indicating a possible mode of action in physiological situation.

Identification of nerve plexi in connective tissues of the sea cucumber Holothuria glaberrima by using a novel nerve-specific antibody.

The echinoderm nervous system is one of the least studied among invertebrates, partly because the tools available to study the neurobiology of this phylum are limited. We have now produced a monoclonal antibody (RN1) that labels a nervous system component of the sea cucumber Holothuria glaberrima. Western blots show that our antibody recognizes a major band of 66 kDa and a minor band of 53 kDa. Immunohistological experiments show that, in H. glaberrima, the antibody distinctly labels most of the known nervous system structures and some components that were previously unknown or little studied. A surprising finding was the labeling of nervous plexi within the connective tissue compartments of all organs studied. Double labeling with holothurian neuropeptides and an echinoderm synaptotagmin showed that RN1 labeled most, if not all, of the fibers labeled by these neuronal markers, but also a larger component of cells and fibers. The presence of a distinct connective tissue plexus in holothurians is highly significant since these organisms possess mutable connective tissues that change viscosity under the control of the nervous system. Therefore, the cells and fibers recognized by our monoclonal antibodies may be involved in controlling tensility changes in echinoderm connective tissue.

Effects of thermal stress on the immune and oxidative stress responses of juvenile sea cucumber Holothuria scabra.

Holothuria scabra is the most valued and cultured tropical sea cucumber, given the great demand of this species for human consumption. However, despite its ecological and economic relevance, little is known regarding its immune responses under thermal stress. Here, the main goal was to study the response of sea cucumbers to temperature stress, assessing sub-organismal alterations and acclimation capacities of juveniles to temperature changes. After changing temperature (1 °C/day) for 6 days, organisms were exposed to temperature conditions of 21 °C (cold), 27 °C (control), and 33 °C (warm) over a 30 day period. At each 15-day interval (T0, T15, and T30), six replicates per condition were killed for biochemical analysis. Immune responses were addressed by studying the activity of phenoloxidase (PO) and prophenoloxidase (ProPO) in the coelomic fluid. Antioxidant defence responses-catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR) enzymatic activities-were measured in the muscle and respiratory tree tissues, whereas oxidative damage was evaluated by measuring levels of superoxide radicals (ROS), DNA-strand breaks and lipid peroxidation (LPO). Juvenile H. scabra increased SOD and PO activities when temperature was elevated, and revealed low levels of ROS and damage in both cold and warm treatments throughout the experiment, confirming the organism's moderate thermal stress. After the short acclimation period, the immune and antioxidant responses prevented damage and maintained homeostasis. This multi-biomarker approach highlights its usefulness to monitor the health of H. scabra and to gain insight concerning the use of this high-valued species in global-scale aquaculture from different temperature regions.

Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra.

The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra.

Long Non-Coding RNAs (lncRNAs) of Sea Cucumber: Large-Scale Prediction, Expression Profiling, Non-Coding Network Construction, and lncRNA-microRNA-Gene Interaction Analysis of lncRNAs in Apostichopus japonicus and Holothuria glaberrima During LPS Challenge and Radial Organ Complex Regeneration.

Long non-coding RNA (lncRNA) structurally resembles mRNA but cannot be translated into protein. Although the systematic identification and characterization of lncRNAs have been increasingly reported in model species, information concerning non-model species is still lacking. Here, we report the first systematic identification and characterization of lncRNAs in two sea cucumber species: (1) Apostichopus japonicus during lipopolysaccharide (LPS) challenge and in heathy tissues and (2) Holothuria glaberrima during radial organ complex regeneration, using RNA-seq datasets and bioinformatics analysis. We identified A. japonicus and H. glaberrima lncRNAs that were differentially expressed during LPS challenge and radial organ complex regeneration, respectively. Notably, the predicted lncRNA-microRNA-gene trinities revealed that, in addition to targeting protein-coding transcripts, miRNAs might also target lncRNAs, thereby participating in a potential novel layer of regulatory interactions among non-coding RNA classes in echinoderms. Furthermore, the constructed coding-non-coding network implied the potential involvement of lncRNA-gene interactions during the regulation of several important genes (e.g., Toll-like receptor 1 [TLR1] and transglutaminase-1 [TGM1]) in response to LPS challenge and radial organ complex regeneration in sea cucumbers. Overall, this pioneer systematic identification, annotation, and characterization of lncRNAs in echinoderm pave the way for similar studies and future genetic, genomic, and evolutionary research in non-model species.

Immune-related genes associated with intestinal tissue in the sea cucumber Holothuria glaberrima.

We have analyzed 5,173 expressed sequence tags (ESTs) from three cDNA libraries of normal and regenerating intestinal tissue of the sea cucumber Holothuria glaberrima and found 22 putative immune-related genes. These sequences showed similarities (e-value approx 10(-8)) to genes involved in immune processes or expressed by immune cells. Sequences were analyzed using bioinformatic tools to determine a putative identity. In addition, phylogenetic analyses were performed to find relationships with similar proteins in other organisms. The mRNAs for ten sequences were detected in coelomocytes by using reverse transcription-polymerase chain reaction (RT-PCR). Moreover, five of them showed a significant increase in expression after an LPS challenge, while the other five showed no significant changes. These results show the variety of immune molecules that may be found in holothurians and support the idea that the invertebrate immune system is more than a collection of simple innate responses. Our study also provides new data of importance in deciphering the evolution and development of the immune system.

Purification and characterization of a T-antigen specific lectin from the coelomic fluid of a marine invertebrate, sea cucumber (Holothuria scabra).

A novel lectin was purified from the coelomic fluid of the sea cucumber Holothuria scabra (HSL), subjected to bacterial challenge. HSL is a monomeric glycoprotein of molecular mass 182 kDa. The lectin is highly thermostable as it retains full activity for 1 h at 80 degrees C. Further, the hemagglutination activity of HSL is unaffected by pH in the range 2-11. Unlike other lectins purified from marine invertebrates, the hemagglutination activity of HSL does not require any divalent metal ions. The affinity profile of HSL was studied by a combination of hemagglutination inhibition and fluorescence spectroscopy. HSL binds to desialylated glycoproteins, MealphaGal, T-antigen and T (alpha-ser)-antigen with a distinction between beta1-4 and beta1-3 linkages. Mealpha-T-antigen was a potent ligand having highest affinity (Ka 8.32 x 10(7)M(-1)). Monosaccharide binding is enthalphically driven while disaccharide binding involves both entropic and enthalpic contributions.