The nuclear receptor coactivator 4 regulates ferritinophagy induced by vibrio splendidus in coelomocytes of Apostichopus japonicus.

Iron homeostasis is vital for the host's defense against pathogenic invasion and the ferritinophagy is a crucial mechanism in maintaining intracellular iron homeostasis by facilitating the degradation and recycling of stored iron. The nuclear receptor coactivator 4 (NCOA4) serves as a ferritinophagy receptor, facilitating the binding and delivery of ferritin to the autophagosome and lysosome. However, NCOA4 of the sea cucumber Apostichopus japonicus (AjNCOA4) has not been reported until now. In this study, we identified and characterized AjNCOA4 in A. japonicus. This gene encodes a polypeptide containing 597 amino acids with an open reading frame of 1794 bp. The inferred amino acid sequence of AjNCOA4 comprises an ARA70 domain. Furthermore, a multiple sequence alignment demonstrated varying degrees of sequence homology between AjNCOA4 from A. japonicus and other NCOA4 orthologs. The phylogenetic tree of NCOA4 correlates with the established timeline of metazoan evolution. Expression analysis revealed that AjNCOA4 is expressed in all tested tissues, including the body wall, muscle, intestine, respiratory tree, and coelomocytes. Following challenge with Vibrio splendidus, the coelomocytes exhibited a significant increase in AjNCOA4 mRNA levels, peaking at 24 h. We successfully obtained recombinant AjNCOA4 protein through prokaryotic expression and prepared a specific polyclonal antibody. Immunofluorescence and co-immunoprecipitation experiments demonstrated an interaction between AjNCOA4 and AjFerritin in coelomocytes. RNA interference-mediated knockdown of AjNCOA4 expression resulted in elevated iron ion levels in coelomocytes. Bacterial stimulation enhanced ferritinophagy in coelomocytes, while knockdown of AjNCOA4 reduced the occurrence of ferritinophagy. These findings suggest that AjNCOA4 modulates ferritinophagy induced by V. splendidus in coelomocytes of A. japonicus.

Transcriptome analysis reveals key transcription factors and pathways of polian vesicle associated with cell proliferation in Vibrio splendidus-challenged Apostichopus japonicus.

Polian vesicle is thought to produce coelomocytes and contribute to the sea cucumber's immune system. Our previous work has indicated that polian vesicle was responsible for cell proliferation at 72 h post pathogenic challenge. However, the transcription factors related to the activation of effector factors and the molecular process behind this remained unknown. In this study, to reveal the early functions of polian vesicle in response to the microbe, a comparative transcriptome sequencing of polian vesicle in V. splendidus-challenged Apostichopus japonicus, including normal group (PV 0 h), pathogen challenging for 6 h (PV 6 h) and 12 h (PV 12 h) was performed. Compared PV 0 h to PV 6 h, PV 0 h to PV 12 h, and PV 6 h to PV 12 h, we found 69, 211, and 175 differentially expressed genes (DEGs), respectively. KEGG enrichment analysis revealed the DEGs, including several transcription factors such as fos, FOS-FOX, ATF2, egr1, KLF2, and Notch3 between PV 6 h and PV 12 h were consistently enriched in MAPK, Apelin and Notch3 signaling pathways related to cell proliferation compared with that in PV 0 h. Important DEGs involved in cell growth were chosen, and their expression patterns were almost the same as the transcriptome profile analysis by qPCR. Protein interaction network analysis indicated that two DEGs of fos and egr1 were probably significant as key candidate genes controlling cell proliferation and differentiation in polian vesicle after pathogenic infection in A. japonicus. Overall, our analysis demonstrates that polian vesicles may play an essential role in regulating proliferation via transcription factors-mediated signaling pathway in A. japonicus and provide new insights into hematopoietic modulation of polian vesicles in response to pathogen infection.

TMT-based proteomics analysis of growth advantage of triploid Apostichopus japonicus.

Polyploid breeding can produce new species with a faster growth rate, higher disease resistance, and higher survival rate, and has achieved significant economic benefits. This study investigated the protein differences in the body wall of triploid Apostichopus japonicus and diploid A. japonicus using isotope-labeled relative and absolute quantitative Tandem Mass Tag technology. A total of 21,096 independent peptides and 4621 proteins were identified. Among them, there were 723 proteins with significant expression differences, including 413 up-regulated proteins and 310 down-regulated proteins. The differentially expressed proteins (DEPs) were enriched in 4519 Gene Ontology enrichment pathways and 320 Kyoto Encyclopedia of Genes and Genomes enrichment pathways. Twenty-two key DEPs related to important functions such as growth and immunity of triploid A. japonicus were screened from the results, among which 20 were up-regulated, such as cathepsin L2 cysteine protease and fibrinogen-like protein A. Arylsulfatase A and zonadhesin were down-regulated. The up-regulated proteins were mainly involved in oxidative stress response, innate immune response, and collagen synthesis in triploid A. japonicus, and the down-regulated proteins were mainly associated with the sterility of triploid A. japonicus. In addition, the transcriptome and proteome were analyzed jointly to support proteome data. In this study, the differences in protein composition between triploid and diploid A. japonicus were analyzed for the first time, and the results revealed the underlying reasons for the growth advantage of triploid A. japonicus.

Global N6-methyladenosine methylation analysis reveals the positive correlation between m6A modification and mRNA abundance during Apostichopus japonicus disease development.

N6-methyladenosine (m6A), the most abundant epitranscriptomic modification in eukaryotic messenger RNA (mRNA), plays important roles in regulation of gene expression for fundamental biological processes and diverse physiological functions, including combating with pathogen infection. Here, we were first profile transcriptome-wide m6A sequencing in four stages of skin ulceration syndrome-diseased Apostichopus japonicus following Vibrio splendidus infection, including Control (healthy), Early (small ulcer), Later (extensive ulcer), and Resistant (no ulcer) groups. Our results revealed that three experimental groups were all extensively methylated by m6A and the proportion of the m6A modified genes were also significantly increased to 28.90% (Early), 27.97% (Later), and 29.98% (Resistant) when compared with Control group (15.15%), indicating m6A modification could be induced by V. splendidus infection. Intriguingly, we discovered a positive correlation between the m6A methylation level and mRNA abundance, indicating a positive regulatory role of m6A in sea cucumber gene expression during V. splendidus infection. Moreover, genes with specific and differentially expressed m6A methylation in Later group were both enriched in cell adhesion, while Early and Resistant groups were both mainly involved in DNA conformation change and chromosome organization when compared with Control, suggesting the higher-methylated m6A might serve as "conformational marker" and associated to the initiation of related anti-disease genes transcription in order to improve disease resistance of sea cucumber. Subsequently, we selected the pivotal genes enriched in cell adhesion pathway and found that the IggFc-binding protein (FcGBP) and Fibrocystin-L both had higher levels of m6A methylation and higher level of mRNA expressions in Later group. Conversely, Fibrinogen C domain-containing protein 1 (F1BCD1) gene presented as an antibacterial role in sea cucumber and showed higher mRNA expression and higher m6A methylation in Resistant group and lower mRNA level in Later group. The levels of m6A methylation and mRNA abundance of FcGBP and F1BCD1 genes indicates disease occurrence or disease resistant were also verified by MeRIP-qPCR. Overall, our study presents the first comprehensive characterize of dynamic m6A methylation modification in the different stages of disease in sea cucumber. These data provide an invaluable resource for future studies of function and biological significance of m6A in mRNA in marine invertebrates.

ROS-mediated BNIP3-dependent mitophagy promotes coelomocyte survival in Apostichopus japonicus in response to Vibrio splendidus infection.

Organisms produce high levels of reactive oxygen species (ROS) to kill pathogens or act as signaling molecules to induce immune responses; however, excessive ROS can result in cell death. To maintain ROS balance and cell survival, mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates. In marine invertebrates, however, mitophagy and its functions remain largely unknown. In the current study, Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential (ΔΨm) and mitophagosome formation. The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide (LPS) treatment increased mitophagy flux. To explore the regulatory mechanism of mitophagy, we cloned Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a common mitophagy receptor, from sea cucumber Apostichopus japonicus (AjBNIP3) and confirmed that AjBNIP3 was significantly induced and accumulated in mitochondria after V. splendidus infection and LPS exposure. At the mitochondrial membrane, AjBNIP3 interacts with microtubule-associated protein 1 light chain 3 (LC3) on phagophore membranes to mediate mitophagy. After AjBNIP3 interference, mitophagy flux decreased significantly. Furthermore, AjBNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide (H2O2), ROS scavengers, and ROS inhibitors. Finally, inhibition of BNIP3-mediated mitophagy by AjBNIP3 small interfering RNA (siRNA) or high concentrations of lactate increased apoptosis and decreased coelomocyte survival. These findings highlight the essential role of AjBNIP3 in damaged mitochondrial degradation during mitophagy. This mitophagy activity is required for coelomocyte survival in A. japonicus against V. splendidus infection.

Transcriptomic and Metabolomic Analyses Provide Insights into the Growth and Development Advantages of Triploid Apostichopus japonicus.

Polyploid breeding is widely used in aquaculture as an important area of new research. We have previously grown Apostichopus japonicus triploids with a growth advantage. The body length, body weight, and aestivation time of triploid and diploid A. japonicus were measured in this study, and the transcriptome and metabolome were used to examine the growth advantage of triploids A. japonicus. The results showed that the proportion of triploid A. japonicus with a body length of 6-12 cm and 12-18 cm was significantly higher than that of diploid A. japonicus, and triploid A. japonicus had a shorter aestivation time (39 days) than diploid (63 days). We discovered 3296 differentially expressed genes (DEGs); 13 DEGs (for example, cyclin-dependent kinase 2) related to growth advantage, immune regulation, and energy storage were screened as potential candidates. According to Gene Ontology (GO) enrichment analysis, DEGs were significantly enriched in the cytoplasm (cellular component), ATP binding process (molecular function), oxidation-reduction process (biological process), and other pathways. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment data, DEGs were significantly enriched in ribosome production and other areas. We discovered 414 significant differential metabolites (SDMs), with 11 important SDMs (for example, nocodazole) linked to a growth advantage. SDMs are significantly enriched in metabolic pathways, as well as other pathways, according to the KEGG enrichment results. According to a combined transcriptome and metabolome analysis, 6 DEGs have regulatory relationships with 11 SDMs, which act on 11 metabolic pathways together. Our results further enrich the biological data of triploid A. japonicus and provide useful resources for genetic improvement of this species.

IL-17/IL-17 Receptor Pathway-Mediated Inflammatory Response in Apostichopus japonicus Supports the Conserved Functions of Cytokines in Invertebrates.

Inflammation participates in host defenses against infectious agents and contributes to the pathophysiology of many diseases. IL-17 is a well-known proinflammatory cytokine that contributes to various aspects of inflammation in vertebrates. However, the functional role of invertebrate IL-17 in inflammatory regulation is not well understood. In this study, we first established an inflammatory model in the Vibrio splendidus-challenged sea cucumber Apostichopus japonicus (Echinodermata). Typical inflammatory symptoms, such as increased coelomocyte infiltration, tissue vacuoles, and tissue fractures, were observed in the V. splendidus-infected and diseased tissue of the body wall. Interestingly, A. japonicus IL-17 (AjIL-17) expression in the body wall and coelomocytes was positively correlated with the development of inflammation. The administration of purified recombinant AjIL-17 protein also directly promoted inflammation in A. japonicus Through genome searches and ZDOCK prediction, a novel IL-17R counterpart containing FNIII and hypothetical TIR domains was identified in the sea cucumber genome. Coimmunoprecipitation, far-Western blotting, and laser confocal microscopy confirmed that AjIL-17R could bind AjIL-17. A subsequent cross-linking assay revealed that the AjIL-17 dimer mediates the inflammatory response by the specific binding of dimeric AjIL-17R upon pathogen infection. Moreover, silencing AjIL-17R significantly attenuated the LPS- or exogenous AjIL-17-mediated inflammatory response. Functional analysis revealed that AjIL-17/AjIL-17R modulated inflammatory responses by promoting A. japonicus TRAF6 ubiquitination and p65 nuclear translocation and evenly mediated coelomocyte proliferation and migration. Taken together, our results provide functional evidence that IL-17 is a conserved cytokine in invertebrates and vertebrates associated with inflammatory regulation via the IL-17-IL-17R-TRAF6 axis.

Characterizations and the Mechanism Underlying Osteogenic Activity of Peptides from Enzymatic Hydrolysates of Stichopus japonicus.

Sea cucumber (Stichopus japonicus) is a kind of fishery product with high nutritional value. It exhibits a wide range of biological activity and has potential application in the food, pharmaceutical, and biomedical industries. However, there are no reports available on the effects of S. japonicus peptides (SJP) on bone mineral density regulations. The purpose of this work was to analyze the composition and osteogenic activity of SJP and explore its underlying mechanism. The results showed that SJP stimulated cell proliferation, differentiation, and mineralization in a dose-dependent manner. In addition, SJP could promote the proliferation of MC3T3-E1 cells by altering the cell cycle progression and regulating the expression of Cyclins. Besides, SJP activated the WNT/ß-catenin pathway and increased the nuclear level of the active form ß-catenin. Furthermore, SJP also induced the expression of bone morphogenetic protein (BMP-2) and increase the phosphorylation levels of p38, JNK, and ERK, suggesting that the osteogenic activity of SJP may be achieved through the activation of WNT/ß-catenin and BMP/MAPK signal pathways. In vivo, SJP significantly inhibited the serum levels of RANKL, ALP, and TRAP, whereas it increased the levels of osteocalcin and osteoprotegerin in OVX-mice. These results indicate that SJP may have the potential to stimulate bone formation and regeneration, and may be used as a functional food or nutritional supplement to prevent osteoporosis.

Transcriptome analysis reveals roles of polian vesicle in sea cucumber Apostichopus japonicus response to Vibrio splendidus infection.

Polian vesicle is originally regarded as a hematopoietic and inflammatory response organ in sea cucumber by the operations of cell depletion and heterogeneous cells injection, respectively. In the present study, to reveal the role and immune mechanisms of polian vesicle in response to pathogen, Vibrio splendidus, we first performed a comparative transcriptome analysis for the cells from polian vesicle wall in V. splendidus-challenged Apostichopus japonicus through RNA high-throughput sequencing technology. Briefly, 465,356,848 clean reads were obtained after cleaning up low-quality reads in total. Approximately 73% of the sequenced reads could be aligned to the reference genome of A. japonicus. The DEGs of CG (control group) vs TG 24 h (24 h post-infection group), CG vs TG 72 h (72 h post-infection group) and TG 24 h vs TG 72 h were 3762, 1391 and 3258, respectively. Gene Ontology (GO) annotation assay revealed that those genes associated with the processes such as cell process, cell, binding and catalytic activity were significantly induced in all three groups post V. splendidus infection. KEGG enrichment analysis suggested the DEGs in TG 24 h were enriched in Toll-like receptor (TLR) signaling pathway, complement and coagulation cascades, antigen processing and presentation and IL-17 signaling pathway compared with that in CG, while the pathways including ribosome biogenesis in eukaryotes, DNA replication, and cell cycle related with cell proliferation were mainly enriched in TG 72 h than that of CG. Furthermore, six important DEGs were chosen and showed the consistent expression patterns with the results of RNA-seq by qPCR. Overall, our analysis towards the current data demonstrates that polian vesicle may play an essential role in the regulation of immune response in A. japonicus and provide new insights into hematopoietic function of polian vesicle in response to pathogen infection.

Effects of Supplementary Selenium and Vitamin E on the Growth Performance, Antioxidant Enzyme Activity, and Gene Expression of Sea Cucumber Apostichopus japonicus.

A 60-day feeding experiment was conducted to evaluate the effects of single selenomethionine (Se) and its mixture with vitamin E (VE) on the growth, antioxidant enzyme activities, and gene expression of juvenile sea cucumber Apostichopus japonicus. The design of the experiment contained two factors and 5 × 2 levels by means of adding various levels of Se and VE in the feed, i.e., combination of 0, 0.3, 0.6, 0.9, or 1.2 mg Se kg-1 and 0 or 200 mg VE kg-1. The results revealed that the specific growth rate and weight gain rate were the highest in the group with 0.3 mg Se kg-1 and 200 mg VE kg-1, followed by the group with 0.6 mg Se kg-1 without VE. Se significantly improved the activities of amylase and protease with VE also imposed positive effect on the amylase activity. Glutathione peroxidase (GPX) activity was highest in the group with 1.2 mg Se kg-1 and lowest with the basal diet. The activity of catalase (CAT) was increased while glutathione reductase (GR) activity was decreased in response to the addition of Se. No significant interactive effects of Se and VE on the enzyme activities were found except superoxide dismutase (SOD) activity. While relative expressions of GPX, CAT, and SOD genes were significantly responsive to the addition of dietary Se, VE significantly promoted the gene expression of SOD. The results suggested that Se and VE might have beneficial effects on the growth and antioxidant responses of A. japonicus.

Analysis of the gene transcription patterns and DNA methylation characteristics of triploid sea cucumbers (Apostichopus japonicus).

Breeding of polyploid aquatic animals is still an important approach and research hotspot for realizing the economic benefits afforded by the improvement of aquatic animal germplasm. To better understand the molecular mechanisms of the growth of triploid sea cucumbers, we performed gene expression and genome-wide comparisons of DNA methylation using the body wall tissue of triploid sea cucumbers using RNA-seq and MethylRAD-seq technologies. We clarified the expression pattern of triploid sea cucumbers and found no dosage effect. DEGs were significantly enriched in the pathways of nucleic acid and protein synthesis, cell growth, cell division, and other pathways. Moreover, we characterized the methylation pattern changes and found 615 differentially methylated genes at CCGG sites and 447 differentially methylated genes at CCWGG sites. Integrative analysis identified 23 genes (such as Guf1, SGT, Col5a1, HAL, HPS1, etc.) that exhibited correlations between promoter methylation and expression. Altered DNA methylation and expression of various genes suggested their roles and potential functional interactions in the growth of triploid sea cucumbers. Our data provide new insights into the epigenetic and transcriptomic alterations of the body wall tissue of triploid sea cucumbers and preliminarily elucidate the molecular mechanism of their growth, which is of great significance for the breeding of fine varieties of sea cucumbers.

Isolation of probiotics and their effects on growth, antioxidant and non-specific immunity of sea cucumber Apostichopus japonicus.

Probiotics play vital roles in controlling diseases, enhancing specific and non-specific immunity and stimulating growth in the aquaculture industry. However, the effect of fermentation of feed by probiotics on the immune ability of sea cucumber has not been reported to date. Here, three candidate probiotic strains (Bacillus species) were isolated from the culture seawater and sediment of sea cucumber, and fishmeal and scallop mantle fermented by the candidate probiotic strains were used to feed sea cucumber. The results showed that the free amino acid and small peptide contents of the fishmeal and scallop mantle were significantly increased after fermentation for 72 h. However, the weight gain (WG) and specific growth rate (SGR) of sea cucumber showed no significant differences among the fermented fishmeal, fermented scallop mantle and control groups. Scallop mantle fermented by the three candidate probiotics could increase the coelomocyte number and respiratory burst activity. The immune-related enzymatic activity was increased after consuming the fermented fishmeal and scallop mantle, while the activity of antioxidant enzymes was reduced. The expression levels of immune- and antioxidant-related genes were changed after consuming the fermented fishmeal and scallop mantle. Taken together, our results suggest that probiotics could increase the immunocompetence of sea cucumber, and fermented scallop mantle might be a potential substitute for fishmeal during feed preparation. Our results lay a foundation for further understanding the relationship between probiotics and the non-specific immunity of sea cucumber.

MiR-210 regulates coelomocyte proliferation through targeting E2F3 in Apostichopus japonicus.

MiR-210 plays a crucial role in cell survival, migration, and regeneration in vertebrates. In our previous work, the expression of miR-210 was considerably induced in diseased Apostichopus japonicus with skin ulcer syndrome (SUS). To further explore the mechanism of miR-210 in regulating the SUS, this study identified E2F transcription factor 3 (E2F3), a candidate target of miR-210, from the sea cucumber A. japonicus via RNA-seq and RACE (designated as AjE2F3). A 1992 bp fragment representing the full-length cDNA of AjE2F3 was obtained, which includes an ORF of 1194 bp encoding a polypeptide of 398 amino acids with a molecular weight of 44.43 kDa. Expression profiling analysis suggested that the expression of AjE2F3 decreased while that of miR-210 increased in Vibrio splendidus-challenged sea cucumber coelomocytes. Dual-luciferase reporter assay revealed that miR-210 targeted AjE2F3 via binding to the 3'UTR region from 108 nt to 128 nt. MiR-210 overexpression in cultured coelomocytes repressed AjE2F3 at the mRNA level and reduced cell proliferation in vitro. Consistently, AjE2F3 overexpression significantly promoted coelomocyte proliferation, as assessed by MTT in vitro. Overall, our results indicated that miR-210 can suppress coelomocyte proliferation by targeting AjE2F3 in pathogen-challenged sea cucumbers.

Existence and functions of a kisspeptin neuropeptide signaling system in a non-chordate deuterostome species.

The kisspeptin system is a central modulator of the hypothalamic-pituitary-gonadal axis in vertebrates. Its existence outside the vertebrate lineage remains largely unknown. Here, we report the identification and characterization of the kisspeptin system in the sea cucumber Apostichopus japonicus. The gene encoding the kisspeptin precursor generates two mature neuropeptides, AjKiss1a and AjKiss1b. The receptors for these neuropeptides, AjKissR1 and AjKissR2, are strongly activated by synthetic A. japonicus and vertebrate kisspeptins, triggering a rapid intracellular mobilization of Ca2+, followed by receptor internalization. AjKissR1 and AjKissR2 share similar intracellular signaling pathways via Gαq/PLC/PKC/MAPK cascade, when activated by C-terminal decapeptide. The A. japonicus kisspeptin system functions in multiple tissues that are closely related to seasonal reproduction and metabolism. Overall, our findings uncover for the first time the existence and function of the kisspeptin system in a non-chordate species and provide new evidence to support the ancient origin of intracellular signaling and physiological functions that are mediated by this molecular system.

The iron-sulfur protein subunit of succinate dehydrogenase is critical in driving mitochondrial reactive oxygen species generation in Apostichopus japonicus.

Succinate dehydrogenase (SDH) is a mitochondrial enzyme with the unique ability to participate in both the tricarboxylic acid cycle and the electron transport chain to produce reactive oxygen species (ROS). The B subunit of SDH is required for succinate oxidation, which is critical for pro-inflammatory response. In this study, we cloned the iron-sulfur protein subunit of SDH from Apostichopus japonicus (denoted as AjSDHB) via RACE technology and explored its role in the immune system as a response to pathogen infection. The full-length cDNA of AjSDHB was 1442 bp with a complete open reading frame of 858 bp encoding 286 amino acids. Simple modular architecture research tool analysis revealed that AjSDHB contained two conserved domains, including a 2Fe-2S iron-sulfur cluster binding domain and a 4Fe-4S dicluster domain, without a signal peptide. Multiple sequence alignment demonstrated that AjSDHB shared a high degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates. Phylogenetic analysis supported the finding that AjSDHB is a new member of the SDHB protein subfamily. Tissue distribution analysis revealed that AjSDHB was expressed in all examined tissues and particularly highly expressed in the muscles. AjSDHB transcripts were markedly induced in coelomocytes both by Vibrio splendidus challenge in vivo and lipopolysaccharide exposure in vitro. Function analysis showed that siRNA-mediated AjSDHB knockdown could substantially reduce the mitochondrial membrane potential (ΔΨm) and further decrease mitochondrial ROS production in A. japonicus coelomocytes. By contrast, AjSDHB overexpression considerably increased ΔΨm and mitochondrial ROS production of A. japonicus coelomocytes. These results supported the idea that AjSDHB is involved in the innate immunity of A. japonicus through its participation in mitochondrial ROS generation.

4-Hydroxyphenylpyruvate dioxygenase from sea cucumber Apostichopus japonicus negatively regulates reactive oxygen species production.

As a wide distribution molecule, 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) catalyzes the second step in the tyrosine catabolism pathway. This process commonly occurs in all aerobic life forms. The broad distribution of these metabolites suggests that they have an important role in many organisms. A portion of the 4-HPPD homology sequence was also identified in Apostichopus japonicus transcriptome. However, the functional roles of A. japonicus 4-HPPD remain unclear. In the current study, a 4-HPPD homolog was cloned from A. japonicus (designated as AjHPPD). The nucleotide sequence analysis showed that the open reading frame of AjHPPD was 1149 bp and encoded a 382-amino-acid residue polyprotein with glyoxalase_4 (residues 20-133) and glyoxalase (residues 180-335) domains. The spatial expression analysis revealed that AjHPPD was ubiquitously expressed in all examined tissues with large-magnitude in the respiratory tree and was minimally expressed in coelomocytes. Compared with a control group, the significant increase in transcription of AjHPPD mRNA in the Vibrio splendidus-challenged sea cucumber was 2.10-fold (p < 0.01) at 48 h and returned to the normal level at 72 and 96 h. Similarly, compared with a control group, the significant increase in the transcription of AjHPPD mRNA was 3.36-fold (p < 0.01) at 24 h after stimulation with 10 mg mL-1 of LPS. On the one hand, silencing AjHPPD in vitro could inhibit the expression of pentose phosphate pathway (PPP) flux enzyme glucose-6-phosphate dehydrogenase (G6PD) at the mRNA level and prevent the clearance of reactive oxygen species (ROS) in sea cucumbers. On the other hand, interference of AjHPPD by using specific siRNA can result in the significant promotion of coelomocyte apoptosis with a 1.61-fold increase in vitro. AjHPPD negatively regulated ROS levels by modulating tyrosine catabolism on AjG6PD expression and coelomocyte apoptosis in response to pathogen infection.

A tandem-repeat galectin-1 from Apostichopus japonicus with broad PAMP recognition pattern and antibacterial activity.

Galectins belong to the family of carbohydrate-binding proteins and play major roles in the immune and inflammatory responses of both vertebrates and invertebrates. In the present study, one novel galectin-1 protein named AjGal-1 was identified from Apostichopus japonicas with an open reading frame of 1179 bp encoding a polypeptide of 392 amino acids. The deduced amino acids sequence of AjGal-1 contained three carbohydrate recognition domains (CRDs) which shared 34-37% identity with that of other galectin proteins from echinodermata, fishes, and birds. In the phylogenetic tree, AjGal-1 was closely clustered with galectins from Mesocentrotus nudus and Paracentrotus lividus. The mRNA transcripts of AjGal-1 were ubiquitously expressed in all the detected tissues, including gut, longitudinal muscle, gonad, coelomocytes, respiratory tree, tentacle and body wall, with the highest expression level in coelomocytes. After Vibrio splendidus stimulation, the mRNA expression levels of AjGal-1 in coelomocytes were significantly increased at 6 and 12 h (P < 0.01) compared with that in control group, and went back to normal level at 72 h. The recombinant protein of AjGal-1 (rAjGal-1) could bind various PAMPs including d-galactose, lipopolysaccharide (LPS), peptidoglycan (PGN) and mannose (Man), and exhibited the highest affinity to d-galactose. Meanwhile, rAjGal-1 could also bind and agglutinate different kinds of microorganisms, including gram-negative bacteria (V. splendidus and Escherichia coli), gram-positive bacteria (Micrococus leteus), and fungi (Pichia pastoris). rAjGal-1 also exhibited anti-microbial activity against V. splendidus and E. coli. All these results suggested that AjGal-1 could function as an important PRR with broad spectrum of microbial recognition and anti-microbial activity against the invading pathogen in A. japonicas.

The potential role of Krüppel-like factor 13 (Aj-klf13) in the intestine regeneration of sea cucumber Apostichopus japonicus.

Krüppel-like factor13 (klf13), a member of the Krüppel-like factor family, plays a vital role in cell proliferation and differentiation. When sea cucumber Apostichopus japonicus is attacted by predators, it can spit viscera in order to escape attack, and then complete the intestine regeneration process within 15 days. However, the potential role of klf13 from A. japonicus (Aj-klf13) in the intestine regeneration of sea cucumber A. japonicus still remains unknown. In present paper, the full-length cDNA of klf13 gene from A. japonicus was cloned by RACE techniques, and it was composed of 2496 bp, including a 245 bp 5' UTR, a 1396 bp 3' UTR and a 855 bp open reading frame, which encoded a polypeptide of 284 amino acids and C2H2 zinc finger domains. The expression level of Aj-klf13 showed an increasing trend in intestine regeneration process of sea cucumber, and it reached the highest at 6 days, returning to the normal at 15 days. By western blot, the expression level of Aj-KLF13 protein was basically consistent with that of Aj-klf13 gene. The expression locations of protein by immunofluorescence indicated that Aj-KLF13 was widely expressed in the normal physiological state and intestine regeneration process of sea cucumbers, which was in the nucleus. There was tissue specificity of the protein, which was mainly distributed in luminal epithelium and coelomic epithelium. These results indicate that Aj-klf13 plays a crucial role in the intestine regeneration process of sea cucumber A. japonicus.

MYC regulates coelomocytes apoptosis by targeting Bax expression in sea cucumber Apostichopus japonicus.

Myelocytomatosis viral oncogene (MYC), a multifunctional transcription factor, (TF) exerts various physiological and pathological effects on animals. AjMYC could induce coelomocyte apoptosis in Apostichopus japonicus, but the underlying molecular mechanism remains poorly understood. In this study, the promoter sequence of apoptosis regulator Bcl-2-associated X (Bax) was cloned by genomic walking. The AjBax promoter region spaning 1189 bp, containing several transcription factor binding sites, included four potential E-boxes (-1030 bp to -1019 bp, -785 bp to -774 bp, -570 bp to -559 bp, -100 bp to -89 bp), two P53 binding sites (-439 bp to -430 bp, -845 bp to -836 bp), and one NF-κB site (-191 bp to -182 bp). Transient transfection of EPC cells with 5'-deletion constructs linked to luciferase reporter revealed that the region -1189/+454 contributed importantly to the expression of the AjBax. In addition, the AjBax promoter was induced by LPS, PGN or MAN. The four potential MYC binding sites were cotransfected with AjMYC in EPC cell whether AjMYC could activate AjBax expression as a transcriptional factor. Only P1 (-1189/+454) fragment containing the first MYC binding site transfection increased the luciferase activity by 2.08-fold (p < 0.01) compared with the control. The first MYC binding site -1030/-1019 was essential to induce AjBax transcription. Further functional assay indicated that AjBax was significantly induced by 3.54-fold increase (p < 0.01) after AjMYC overexpression in sea cucumber coelomocytes. All our findings supported that AjMYC could regulate coelomocyte apoptosis by directly targeting AjBax expression in A. japonicus.

Isolation and identification of zinc-chelating peptides from sea cucumber (Stichopus japonicus) protein hydrolysate.

BACKGROUND: Zinc is known to play an essential role in the biological activities in the human body. In this study, a zinc-chelating peptide (ZCP) produced by Alcalase-assisted hydrolysis of the body wall of sea cucumber was isolated and identified. The ZCP was purified stepwise by ultrafiltration, anion-exchange chromatography, and gel filtration chromatography, in conjunction with ultraviolet-visual (UV-visual) spectrophotometry, which was used to analyze each purified fraction. RESULTS: Analysis of the purified ZCP revealed that its zinc-chelating ability was 33.31%. Analysis of isothermal titration calorimetry suggested that the binding of ZCP and zinc (N ≈ 2) was endothermic, with weak binding affinity. Fourier transform infrared spectroscopy spectra (FTIR) indicated that carboxylic and amide groups in ZCP were the primary binding sites of Zn. Sequencing the result by ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry (UPLC-Q-TOF-MS/MS) showed that a representative ZCP had the sequence WLTPTYPE with a molecular weight of 1005.5 Da. CONCLUSION: These results provide a promising foundation for the production of zinc supplements from sea-cucumber-derived ZCPs. © 2019 Society of Chemical Industry.