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.

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.

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.

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.

Serpin-type serine protease inhibitor mediates coelomocyte apoptosis in Apostichopus japonicus.

Serine protease inhibitors (SPIs, serpins) are a protein superfamily involved in almost all physiological processes in all organisms. In this study, a novel serpin was identified from Apostichopus japonicus (Ajserpin) by using high-throughput sequencing and RACE approaches. The full-length cDNA of Ajserpin was 1893 bp with a 5'-untranslated region (UTR) of 130 bp, a 3'-UTR of 587 bp, and an open reading frame of 1176 bp encoding a polypeptide of 391 amino acids with a deduced molecular weight of 43.8 kDa. Ajserpin shares the standard structure of SPI, including three ß-sheets and eight α-helices. The deduced amino acid sequences of Ajserpin had no nuclear location signal and signal peptide structure. The phylogenetic tree and immunofluorescence showed that Ajserpin belonged to the clade B subfamily and was mainly located in the cytoplasm and nucleus. Sequence comparison and protein inhibition experiments showed that the active site (P1-P1' site) of Ajserpin was Arginine and Serine, which displayed inhibitory activity toward trypsin in a dose-dependent manner. Tissue distribution analysis showed that Ajserpin transcripts were constitutively expressed in all examined tissues with the peak in the body wall. Ajserpin mRNA transcripts could be induced in Vibrio splendidus-challenged sea cucumber or lipopolysaccharide-exposed coelomocytes. Furthermore, Ajserpin knockdown by small interfering RNAs could inhibit coelomocytes apoptosis. All our results revealed that Ajserpin might serve as an immune regulator in sea cucumber.

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.

Sedoheptulose kinase bridges the pentose phosphate pathway and immune responses in pathogen-challenged sea cucumber Apostichopus japonicus.

The sedoheptulose kinase carbohydrate kinase-like protein (CARKL) is critical for immune cell activation, reactive oxygen species (ROS) production, and cell polarization by restricting flux through the pentose phosphate pathway (PPP). To date, little is known about CARKL in regulating immune responses in marine invertebrates. In this study, we first cloned and characterized the CARKL gene from Apostichopus japonicus (designated as AjCARKL). Time-course analysis revealed that Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly downregulated AjCARKL mRNA expression. Furthermore, AjCARKL overexpression in cultured coelomocytes not only significantly inhibited the mRNA expression level of the rate-limiting enzyme glucose-6-phosphate dehydrogenase of the PPP but sharply decreased coelomocyte proliferation, ROS production, and phagocytic rate. Additionally, AjCARKL overexpression in mouse peritoneal macrophages (RAW264.7 cells) significantly attenuated the intracellular ROS production and sensitized the M2 phenotype macrophage polarization. These results revealed that AjCARKL serves as a rheostat for cellular metabolism and is required for proper immune response by negatively regulating PPP in pathogen-challenged A. japonicus.

Cyclophilin A mediates coelomocyte apoptosis via the NF-κB/Bcl-2 signaling pathway in Apostichopus japonicus.

As a multifunctional protein, cyclophilin A (CypA) plays an important role in cell apoptosis. In our previous work, we found that CypA from Apostichopus japonicus (AjCypA), as a cofactor, could modulate nuclear translocation of NF-κB. However, the immune function of AjCypA is largely unknown. In the present study, we found that siRNA-mediated AjCypA knockdown in vivo significantly increased the coelomocyte apoptosis rate. In addition, the expression of B-cell lymphoma-2 (AjBcl-2, an anti-apoptosis gene) was synchronously downregulated. To better understand the connection between AjCypA and AjBcl-2 expression, we cloned the promoter of AjBcl-2 via genomic walking, which spanned 1870 bp and contained four potential binding sites of NF-κB. Dual-luciferase reporter assay revealed that the full-length sequence and all truncated fragments exhibited high transcriptional activity. Moreover, 1 µg/mL LPS exposure significantly increased the luciferase activity of P1 (-1870/+57) by 2.31-fold and 3.15-fold at 12 and 24 h, respectively. Furthermore, the four potential NF-κB binding sites and pCMV-Flag2C-AjNF-κB co-transfection assay demonstrated that NF-κB could regulate the expression of AjBcl-2 via the NF-κB binding sites of AjBcl-2 promoter. All results supported that AjCypA mediates coelomocyte apoptosis via NF-κB/AjBcl-2 signaling pathway in A. japonicus.

Outer membrane protein OmpU is related to iron balance in Vibrio alginolyticus.

Outer membrane protein U (OmpU) is a major porin from Vibrio alginolyticus and has been considered a vaccine candidate against infection by V. alginolyticus. After pre-incubated with polyclonal antibody against rOmpU, V. alginolyticus showed a 78% decrease in extracellular iron level, suggesting that interruption of OmpU could increase intracellular iron level. The mRNA expression of ompU under iron-limited conditions was determined using real-time reverse transcriptase PCR. The mRNA level of ompU was downregulated to 0.27-, 0.036- and 0.019-fold after the addition of the iron chelator 2,2'-bipyridyl for 10, 30 and 60 min, respectively. In addition, the promoter of ompU contained a ferric uptake regulator (Fur) binding site, which revealed the potential regulation of ompU by Fur and iron. Fur from V. alginolyticus was purified and used for electrophoretic mobility shift assay. The result showed that in the absence of Fe2+, purified recombinant Fur could specifically bind to the promoter DNA of ompU, while in the presence of Fe2+, the binding of Fur and the promoter DNA was suppressed. Our study preliminarily explored the function of OmpU in iron balance in V. alginolyticus, and these findings were helpful in understanding iron metabolism in V. alginolyticus.

Bcl-2 mediates coelomocytes apoptosis by suppressing cytochrome c release in Vibrio splendidus challenged Apostichopus japonicus.

Apoptosis is an evolutionarily conserved immune response and plays a fundamental role in many physiological processes. In this study, the important apoptosis regulator of Bcl-2 homolog from economic marine animal Apostichopus japonicus (AjBcl-2) was cloned and its roles in V. splendidus infection explored. The AjBcl-2 gene contains 3263 nucleotides, with a 5' UTR of 519 bp, an ORF of 660 bp encoding 219 aa sequences, and a 3' UTR of 2084 bp. The AjBcl-2 protein shared a conserved Bcl domain and three Bcl-2 homology domains by SMART program. In healthy sea cucumbers, AjBcl-2 mRNA was expressed in all examined tissues with the peak expression in coelomocytes. The mRNA and protein levels of AjBcl-2 in coelomocytes were depressed at 12 h and 24 h, and induced at 48 h post V. splendidus challenge. In the same conditions, coelomocytes apoptosis rates were significantly increased at 24 h and decreased at 48 h. Moreover, siRNA-mediated AjBcl-2 knockdown significantly increased the coelomocytes apoptosis rates, which could be partially recovered by recombinant AjBcl-2 administration. Furthermore, there was an increase in the AjCyt c protein expression coupled with the downregulation expression of AjBcl-2 post AjBcl-2 silencing. Our results suggested that AjBcl-2 suppressed apoptosis by preventing the AjCyt c release in coelomocytes, and thus mediating V. splendidus infection in sea cucumbers.

Cloning and functional analysis the first NLRC4-like gene from the sea cucumber Apostichopus japonicus.

The NOD-like receptor family member 4 (NLRC4) plays a crucial role in regulating the innate immune responses and cell apoptosis pathways in vertebrates. However, the function of the NLRC4 counterpart in invertebrates remains elusive. In this study, the first NLRC4-like gene was cloned and characterized from Apostichopus japonicus (designated as AjNLRC4-like) with RACE technology. The full-length cDNA of the AjNLRC4-like gene was 4065 bp, which consisted of a 5'-untranslated region (UTR) of 387 bp, a 3'-UTR of 159 bp, and a complete open reading frame of 3519 bp encoding a polypeptide of 1172 amino acid residues. Structural analysis revealed that AjNLRC4-like protein contained two IG domains (31-132 and 251-353 amino acids), a common NACHT (600-757 amino acids), and no LRR and CARD domains compared with the vertebrate NLRC4. Spatial expression analysis revealed that the AjNLRC4-like was ubiquitously expressed in all the examined tissues with larger magnitude in the intestine. The mRNA expression of the AjNLRC4-like was significantly upregulated by 2.86- and 2.92-fold at 24 h after the Vibrio splendidus challenge in vivo and the lipopolysaccharide (LPS) treatment in vitro, respectively, compared with that of the control group. The purified recombinant AjNLRC4-NACHT protein displayed higher binding activities to various pathogen-associated molecular patterns (PAMPs), including LPS, peptidoglycan, and mannan. Further functional analysis indicated that the apoptosis of coelomocytes was significantly inhibited by 11.37% after specific AjNLRC4-like siRNA treatment, and the inflammatory caspase Ajcaspase-1 was synchronously decreased by 0.28-fold in the same condition. Collectively, these results supported that the uncanonical AjNLRC4-like protein may share similar functions to the vertebrate NLRC4 as the pattern recognition receptor and in mediating coelomocyte apoptosis in the pathogen-challenged sea cucumber.

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.

Molecular cloning and functional characterization of MYC transcription factor in pathogen-challenged Apostichopus japonicus.

Myelocytomatosis viral oncogene (MYC), a transcription factor in the MYC family, plays vital roles in vertebrate innate immunity by regulating related immune gene expressions. In this study, we cloned and characterized an MYC gene from sea cucumber Apostichopus japonicus via RNA-seq and RACE approaches (designated as AjMYC). A 2074 bp fragment representing the full-length cDNA of AjMYC was obtained. This gene includes an open reading frame (ORF) of 1296 bp encoding a polypeptide of 432 amino acid residues with the molecular weight of 48.85 kDa and theoretical pI of 7.22. SMART analysis indicated that AjMYC shares an MYC common HLH motif (354-406 aa) at the C-terminal. Spatial expression analysis revealed that AjMYC is constitutively expressed in all detected tissues with peak expression in the tentacle. Vibrio splendidus-challenged sea cucumber could significantly boost the expression of AjMYC transcripts by a 5.58-fold increase in the first stage. Similarly, 2.75- and 3.23-fold increases were detected in LPS-exposed coelomocytes at 1 and 24 h, respectively. In this condition, coelomocyte apoptotic rate increased from 11.98% to 56.23% at 1 h and to 59.08% at 24 h. MYC inhibitor treatment could not only inhibit the expression of AjMYC and Ajcaspase3, but also depress the coelomocyte apoptosis. Furthermore, AjMYC overexpression in EPC cells for 24 h also promoted the cell apoptosis rate from 21.31% to 45.85%. Collectively, all these results suggested that AjMYC is an important immune factor in coelomocyte apoptosis toward pathogen-challenged sea cucumber.

Divergent proteomics response of Apostichopus japonicus suffering from skin ulceration syndrome and pathogen infection.

Skin ulceration syndrome (SUS) of sea cucumber is a common and serious disease that affects the stable development of Apostichopus japonicus in the culture industry. The part of sea cucumber that suffers from major injury and is directly observed is the body wall, in which protein variations should be the most direct evidence of the disease. To understand the response mechanisms of A. japonicus in SUS progression, we investigated protein changes in the body wall of diseased A. japonicus induced by Vibrio splendidus and individuals with natural diseases by isobaric tags for relative and absolute quantification (iTRAQ). About 119 proteins were identified in the two iTRAQ groups. A comparison of the protein expression profiles among two SUS conditions revealed that the mode of action induced by V. splendidus (Vs-SUS) was completely different from those in individuals with natural disease (ND-SUS). Most of the differentially expressed proteins (DEPs) (33 in 37 DEPs) were significantly depressed in the Vs-SUS group. Only 13 proteins in 27 DEPs showed similar trend to those in the ND-SUS group. Many important proteins involved in major intercellular signaling pathways associated with SUS disease were identified based on the KEGG and GO database search. Many proteins were located in the mitochondria and mainly involved in the oxidative stress pathway. Glutathione metabolism pathway was associated with reactive oxygen (ROS) production in the ND-SUS group. In the Vs-group, most of the proteins were concentrated in the cytoplasm and were related to immunity and extracellular matrix stability. In the ND-SUS group, the activity of key enzymes (CAT, GPx) that eliminate mitochondrial ROS production and structural stable protein (HSP60, HSP10) decreased, whereas those of complement proteins (C3, C3-2) that promoted ROS production was upregulated. This finding supported that oxidative damage caused by ROS might be the main effector for SUS in the ND-SUS group. The challenge with V. splendidus led to the breakdown of the defense capability of sea cucumber and suppressed the expression of immune-related proteins, such as C-type lectin, caspase, STAT, and cystatin. The downregulation of TIMP led to MMP1 overexpression. Members of the MMP family could directly degrade the extracellular matrix, which may be the main reason for the cell matrix degradation and induced SUS disease in the Vs-SUS group. Hence, ROS and extracellular matrix degradation enzymes could play important roles in the formation of SUS in sea cucumber. Results provide insights into the complex molecular mechanism of SUS in sea cucumber.

Macrophage migration inhibitory factor is involved in inflammation response in pathogen challenged Apostichopus japonicus.

Macrophage migration inhibitory factor (MIF) is a cytokine and plays critical roles in inflammatory and immune responses in vertebrates. However, its functional role in inflammation has not been well studied in invertebrates. In the present study, we cloned and characterized MIF gene from Apostichopus japonicus by RNA-seq and RACE approaches (designated as AjMIF). A 1047 bp fragment representing the full-length cDNA of AjMIF was obtained, including a 5' UTR of 100 bp, an open reading frame (ORF) of 366 bp encoding a polypeptide of 121 amino acids residues with the molecular weight of 13.43 kDa and theoretical isoelectric point of 5.63 and a 3' UTR of 580 bp. SMART analysis showed that AjMIF has conserved MIF domain (2-117aa) similar to its mammalian counterparts. The amino terminal proline residue (P2) and invariant lysine residue (K33) which are critical active sites of tautomerase activity in mammalian MIF were also detected. Phylogenic analysis and multiple alignments have shown that AjMIF shared higher degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates. For Vibrio splendidus challenged sea cucumber, the peak expression of AjMIF mRNAs in coelomocytes were detected at 6 h (23.5-fold) and remained at high levels until 24 h (4.01-fold), and returned to normal level at 48 h in comparison with that of the control group. Similarly, a significant increase in the relative mRNA levels of AjMIF was also found in 10 µg mL-1 LPS-exposed primary cultured coelomocytes. Functional analysis indicated that recombinant AjMIF incubation could promote inflammatory response related genes of Ajp105, AjVEGF, AjMMP1 and AjHMGB3 expression by 1.35-fold, 1.36-fold, 1.83-fold and 1.27-fold increase, respectively, which was consistent with the findings in vertebrate MIFs. All these results collectively suggested that AjMIF had a similar function to MIFs in higher animals and might serve as a candidate cytokine in inflammatory regulation in sea cucumber.

VEGF-like protein from Apostichopus japonicus promotes cell proliferation and migration.

Vascular endothelial growth factor (VEGF) is a key conservative regulator of inflammation response by promoting cell proliferation, migration, and vascular permeability. It also induces the release of inflammatory factors in vertebrates. We previously characterized NLR family pyrin domain containing 3 and HMGB3 homology in Apostichopus japonicus, providing the occurrence of inflammation in this species. However, to our knowledge, other inflammation-related molecules, such as VEGF, have rarely been investigated. In the present study, a novel VEGF homolog was identified from A. japonicus (designated as AjVEGF) by rapid amplification of cDNA ends. Full-length cDNA of AjVEGF was 3181 bp with a putative open reading frame of 1752 bp encoding 583 amino acid (aa) residue protein. Structural analysis revealed that AjVEGF processed characteristic VEGF domains of platelet-derived growth factor domain (132-232 aa) and CXC domain (223-270 aa). Multiple sequence alignment and phylogenetic analysis both supported that AjVEGF belongs to a new member of VEGF protein subfamily. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro could significantly upregulate mRNA expression of AjVEGF compared with the control group. Functional analysis indicated that recombinant AjVEGF promoted coelomocyte proliferation and migration not only in sea cucumber but also in human colorectal adenocarcinoma cells (HT29). This consistent function was also detected for human VEGFs. Taken together, these findings suggest that AjVEGF has a similar function of VEGF in higher animals and might serve as a candidate cytokine in sea cucumber inflammation.

Microsomal glutathione transferase 2 modulates LTC4 synthesis and ROS production in Apostichopus japonicus.

Microsomal glutathione transferase 2 (mGST2) is an integral membrane protein involved in detoxication of xenobiotics, and has also been suggested to catalyze the biosynthesis of pro-inflammatory mediator leukotriene C4 (LTC4) as homologous to LTC4 synthase (LTC4S) in mammals. In the present study, a novel mGST2 homology was identified from Apostichopus japonicus (designated as AjmGST2) by RACE approaches. The full-length cDNA of AjmGST2 was of 1917bp encoding a polypeptide of 161 amino acids residues. Multiple sequences alignment and phylogenetic analysis together supported that AjmGST2 belonged to a new member in invertebrate mGSTs family and close to mammalian LTC4S. Spatial expression analysis revealed that AjmGST2 was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. AjmGST2 transcripts in coelomocytes were slightly induced post 6h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48h. The increased expression profiles of AjmGST2 were also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. Consistently, LTC4 contents were also induced by a 1.56-fold increase in the same condition. Functional assay further revealed that AjmGST2 might be functioned as LTC4S to promote LTC4 synthesis. AjmGST2 knock-down by specific siRNA significantly depressed LTC4 contents with 27.0% decrease at 24h. Meantime, ROS levels were elevated by 40.1% in vitro. All of these results indicated that AjmGST2 performed dual functions roles as LTC4S and ROS eliminator in sea cucumber immune response.

Molecular cloning and functional characterization of cathepsin B from the sea cucumber Apostichopus japonicus.

Cathepsin B (CTSB), a member of lysosomal cysteine protease, is involved in multiple levels of physiological and biological processes, and also plays crucial roles in host immune defense against pathogen infection in vertebrates. However, the function of CTSB within the innate immune system of invertebrates, particularly in marine echinoderms, has been poorly documented. In this study, the immune function of CTSB in Apostichopus japonicus (designated as AjCTSB), a commercially important and disease vulnerable aquaculture specie, was investigated by integrated molecular and protein approaches. A 2153 bp cDNA representing the full-length of AjCTSB was cloned via overlapping ESTs and RACE fragments. AjCTSB contained an open reading frame of 999 bp encoding a secreted protein of 332 amino acid residues with a predicted molecular mass of 36.8 kDa. The deduced amino acid of AjCTSB shared a typical activity center containing three conserved amino acid residues (Cys108, His277 and Asn297). Phylogenetic tree analysis also supported that AjCTSB was a new member of CTSB family with clustering firstly with invertebrate CTSBs. Quantitative real time PCR analysis revealed that AjCTSB was ubiquitously expressed in all examined tissues with the highest levels in intestine. The Vibrio splendidus challenged sea cucumber and LPS-exposed coelomocytes could both significantly boost the expression of AjCTSB. Moreover, the purified recombinant AjCTSB exhibited dose-dependent CTSB activities at the concentration ranged from 0 to 0.24 µg µL-1. Further functional analysis indicated that coelomocytes apoptosis was significantly inhibited by 0.16-fold in vivo and the apoptosis execution Ajcaspase 3 was extremely reduced in Apostichopus japonicus coelomocytes treated with specific AjCTSB siRNA. Collectively, all these results suggested that AjCTSB was an important immune factor and might be served as apoptosis enhancers in pathogen challenged sea cucumber.

NF-κB/Rel, not STAT5, regulates nitric oxide synthase transcription in Apostichopus japonicus.

Nitric oxide (NO) is an important signaling molecular in the immune system of all vertebrates and invertebrates for pathologic and physiologic process, and it is largely produced by inducible nitric oxide synthase (iNOS). To uncover key mechanisms regulating NOS expression in sea cucumber Apostichopus japonicus, we amplified a fragment of the NOS promoter by genome walking approach and characterized putative transcription factor binding motifs using luciferase assay. Transient transfection of EPC cells using 5'-deletion constructs linked to luciferase reporter revealed that the region -614/+39 contributed importantly to expression of the AjNOS gene, and the -614 bp of the 5'-flanking region of the AjNOS gene responded well to LPS. Analysis of the functional promoter region revealed the presence of two potential NF-κB (-375 bp to -366 bp, -76 bp to -67 bp) and three STAT binding sites (-284 bp to -276 bp, -95 bp to 87 bp, -81 bp to -73 bp). When luciferase reporter vector and expression vector co-transfected revealed that NF-κB/Rel, but not STAT5, activate the AjNOS promoter fragment. Furthermore, two truncated reporter vectors co-transfected with vector expressing NF-κB/Rel revealed that the first NF-κB binding site (-375 bp to -366 bp) was essential for the ability of this promoter to induce AjNOS transcription. In addition, blocking the AjRel by SN50 (NF-κB inhibitory peptide) depressed the AjNOS expression and NO production both in vivo and in vitro, respectively, revealing that AjRel might directly modulate AjNOS. All our findings confirmed that NF-κB dependent mechanisms regulating expression of AjNOS and suggested a means of linking NO production to the immune response.