Zhimeng Lv

miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo

In this study, we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus. Target screening of RNA-Seq data successfully identified interleukin-1 receptor-associated kinase (AjIRAK−1) as a putative target of miR-133. This result was further validated by negative expression profiles in Vibrio splendidus-challenged coelomocytes and lipopolysaccharide (LPS)-exposed cell cultures. HEK-293T cells transfected with a dual-luciferase reporter fused to the 3′UTR of wild-type or mutant AjIRAK-1 exhibited a 52.9% reduction in luciferase activity (p < 0.01) compared to controls. Co-infection with a miR-133 mimics or a specific siRNA targeting AjIRAK-1 significantly repressed the mRNA and protein expression levels of AjIRAK-1 and its downstream molecules, such as AjTRAF6 and Ajp105, in primary coelomocytes. In contrast, a miR-133 inhibitor significantly increased the expression of these TLR pathway members. The injection of miR-133 agomir or AjIRAK-1 siRNA into sea cucumbers not only decreased the expression of AjIRAK-1 and its downstream molecules but also significantly increased V. splendidus coelomocyte phagocytosis. All of the present data provide direct evidence that miR-133 is involved in TLR cascade modulation through AjIRAK-1 targeting to promote V. splendidus coelomocyte phagocytosis in these non-model invertebrates.

miR-200 modulates coelomocytes antibacterial activities and LPS priming via targeting Tollip in Apostichopus japonicus

In order to explore the potential roles of microRNAs (miRNAs) in regulating Toll-like receptor (TLR) pathways, we identified Toll interacting protein as a putative target of miR-200 in Apostichopus japonicus coelomocytes by RNA-seq screening (denoted as AjTollip). The positive expression profiles of miR-200 and AjTollip were detected in both LPS exposure primary coelomocytes and Vibrio splendidus challenge sea cucumber. Co-infection miR-200 mimics significantly elevated the expression of AjTollip and its down-stream molecules. In contrast, miR-200 inhibitor significantly repressed the expression of these TLR-pathway members. More importantly, miR-200 displayed not only to enhance coelomocytes antibacterial activities, but to suppress LPS priming in vitro. Overall, all these results will enhance our understanding on miR-200 regulatory roles in anti-bacterial process in sea cucumber.

A β-integrin from sea cucumber Apostichopus japonicus exhibits LPS binding activity and negatively regulates coelomocyte apoptosis.

Integrins are a family of membrane glycoproteins, which are the major receptors for extracellular matrix and cell-cell adhesion molecules. In this study, a 1038 bp sequence representing the full-length cDNA of a novel β-integrin subunit (designated as AjITGB) was cloned from Apostichopus japonicas by using combined transcriptome sequencing and RACE approaches. The deduced amino acid sequence of AjITGB shared a conserved tripeptide Arg-Gly-Asp (RGD) binding domain with an S-diglyceridecysteine or N-Palm cysteine residue (C(31)), a transmembrane domain, and a β-integrin cytoplasmic domain. Spatial distribution analysis showed that AjITGB was constitutively expressed in all tested tissues with dominant expression in the muscles and weak expression in the respiratory tree. The pathogen Vibrio splendidus challenge and LPS stimulation could both significantly down-regulate the mRNA expression of AjITGB. Functional investigation revealed that recombinant AjITGB displayed higher LPS binding activity but lower binding activity to PGN and MAN. More importantly, knockdown of AjITGB by specific siRNA resulted in the significant promotion of coelomocyte apoptosis in vitro. Results indicated that AjITGB may serve as an apoptosis inhibitor with LPS binding activity during host-pathogen interaction in sea cucumber.

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

ABSTRACT 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 &mgr;g &mgr;L−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. HIGHLIGHTSFull‐length cDNA of cathepsin B gene were firstly identified in Apostichopus japonicus.AjCTSB was ubiquitously expressed in all examined tissues with the larger magnitude in intestine.AjCTSB mRNA in coelomocytes could be significantly induced by Vibrio splendidus or LPS.Recombinant AjCTSB exhibited dose‐dependent CTSB activities.Coelomocytes apoptosis was significantly inhibited in vivo after AjCTSB knock‐down.

Molecular cloning and functional characterization of theta class glutathione S-transferase from Apostichopus japonicus

Abstract Glutathione S‐transferases (GSTs) are the superfamily of multifunctional detoxification isoenzymes and play crucial roles in innate immunity. In the present study, a theta class GST homology was identified from A. japonicus (designated as AjGST‐&THgr;) by RACE approaches. The full‐length cDNA of AjGST‐&THgr; was of 1013 bp encoded a cytosolic protein of 231 amino acids residues. Structural analysis revealed that AjGST‐&THgr; processed the characteristic N‐terminal GSH‐binding site (G‐site) and the C‐terminal hydrophobic substrate binding site (H‐site). Multiple sequence alignment and phylogenetic analysis together supported that AjGST‐&THgr; belonged to a new member of theta class GST protein subfamily. Spatial expression analysis revealed that AjGST‐&THgr; was ubiquitously expressed in all examined tissues with the larger magnitude in intestine. The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up‐regulate the mRNA expression of AjGST‐&THgr; when compared with control group. The recombinant protein was expressed in Escherichia coli and the purified AjGST‐&THgr; showed high activity with GST substrate. Meantime, disc diffusion assay showed that recombinant AjGST‐&THgr; protein could markedly improve bacterial growth under Cumene hydroperoxide exposure. More importantly, the recombinant AjGST‐&THgr; could effectively prevent primary coelomocytes apoptosis after LPS exposure. Our present findings suggested that AjGST‐&THgr; might play significantly roles in the modulation of immune response and protect cells from pathogens infection in A. japonicus. HighlightsThe full‐length cDNA of AjGST‐&THgr; was identified in Apostichopus japonicus.AjGST‐&THgr; was ubiquitously expressed in all examined tissues with the larger magnitude in intestine.The Vibrio splendidus challenge in vivo and LPS stimulation in vitro could both significantly up‐regulate AjGST‐&THgr; mRNA expression.The recombinant AjGST‐&THgr; could markedly improve bacterial growth under Cumene hydroperoxide exposure.The recombinant AjGST‐&THgr; could effectively prevent primary coelomocytes apoptosis in LPS exposed coelomocytes.

An invertebrate β-integrin mediates coelomocyte phagocytosis via activation of septin2 and 7 but not septin10

We have previously confirmed that β-integrin from Apostichopus japonicus (designated AjITGB) binds LPS and mediates the immune response. In this study, we found that AjITGB positively promoted the echinoderm immune cells located in the coelomic cavity (designated as coelomocyte) phagocytic activities. Flow cytometry assay indicated that the phagocytic percentage was significantly decreased, by a 37.3% and 41.36%, after AjITGB siRNA inference in vitro and in vivo, respectively, a result consistent with the decrease observed with an anti-AjITGB antibody blocking treatment. These decreased phagocytic activities were partially recovered by rAjITGB supplementation. To better understand the molecular mechanism underlying this phagocytosis, three phagocytic-related proteins, including Ajseptin2, Ajseptin7 and Ajseptin10, were cloned and further characterized. Completely consistent expression profiles were detected between AjITGB and Ajseptin2/7, at both the mRNA and protein levels, in V. splendidus-challenged sea cucumber. Furthermore, Ajseptin2/7 expression levels were significantly down-regulated in the AjITGB silencing group and could be partially recovered to its original level after rAjITGB administration. However, Ajseptin10 displayed no significant change in the same condition. A phagocytic assay further indicated that Ajseptin2/7, but not Ajseptin10, was crucial in mediating coelomocyte phagocytosis. The knockdown of the three genes by specific siRNAs indicated that Ajseptin2/7 significantly decreased coelomocyte phagocytosis, and this decrease was completely recovered after rAjITGB supplementation. There was no significant change in the phagocytosis rate in both the AjSEPT10 siRNA interference and rAjITGB supplementation groups. All our results confirmed that AjITGB modulates coelomocyte phagocytosis via the activation of Ajseptin2/7 but not AjSEPT10 and further supported the divergent roles of Ajseptins in AjITGB-mediated phagocytosis.

Microsomal glutathione transferase 1 attenuated ROS-induced lipid peroxidation in Apostichopus japonicus

&NA; Microsomal glutathione transferase (mGST) is a membrane bound glutathione transferase in multifunctional detoxification isoenzymes family and also plays crucial roles in innate immunity. In the present study, a novel microsomal GST homology was identified from Apostichopus japonicus (designated as AjmGST1) by RACE approaches. The full‐length cDNA of AjmGST1 was of 1296 bp encoded a protein of 169 amino acids residues. Multiple sequence alignment and phylogenetic analysis together supported that AjmGST1 belonged to a new member in invertebrates mGST family. Spatial expression analysis revealed that AjmGST1 was ubiquitously expressed in all examined tissues with the larger magnitude in tentacle. Time‐course expression of AjmGST1 mRNA in coelomocytes was up‐regulated after Vibrio splendidus challenge from 6 h until 72 h with the peak expression in 24 h, compared with that in the control group. Similarly, the induced expression of AjmGST1 expression was also detected in lipopolysaccharide (LPS) exposed primary coelomocytes. The purified recombinant protein of AjmGST1 showed high activity with GST substrate at pH of 7.0 and temperature of 35 °C. Meantime, the recombinant AjmGST1 depressed H2O2‐induced MDA production both in vivo and in vitro. All of these results indicated that AjmGST1 was an important regulator in elimination of lipid peroxidation under immune response. HighlightsFull‐length cDNA of microsomal glutathione transferase were identified in Apostichopus japonicus.AjmGST was ubiquitously expressed in all examined tissues with the larger magnitude in tentacle.AjmGST1 mRNA in coelomocytes was up‐regulated in vitro and in vivo.The recombinant AjmGST1 showed high activity with GST substrate.The recombinant AjmGST1 depressed H2O2‐induced MDA production in vitro and in vivo.

p44/42MAPK and p90RSK modulate thermal stressed physiology response in Apostichopus japonicus

Reversible protein phosphorylation (RPP) plays a key role in signal transduction, enzyme activity and metabolic maintenance in response to extreme changes in the environment. In this study, we identified 55 phosphorylated proteins in Apostichopus japonicus coelomocytes by iTRAQ analysis, and sixteen proteins displayed differently expressed profiles between thermal stressed and control groups. Mitogen-activated protein kinase (MAPK) signaling cascade was further characterized by Western blot. Spatial expression analysis revealed that phospho-p44/42MAPK levels were mainly detected in coelomocytes and muscle, and no signal in intestine and respiratory trees, although the protein were constitutively expressed in all examined tissues. Time-course expression analysis shown that the amount of phospho-p44/42MAPK decreased by 57-60% in coelomocytes and 29-40% in muscle in acute exposure stage of 20°C and 25°C, respectively. In higher temperature remaining stage, phospho-p44/42MAPK was both induced with 1.7-fold increase at 20°C and 5.2-fold at 25°C at the first 12h in coelomocytes, which was consistent with the change of p44/42MAPK. Similarly, 2.5-fold and 2.7-fold increases were detected in muscle at corresponding exposure temperature. In contrast, the p44/42MAPK in muscle showed depressed expression profiles. As time progressed, phospho-p44/42MAPK levels were all decreased significantly both in coelomocytes and in muscles. A reduction in phosphorylation levels was detected at 84h in 20°C exposed coelomocytes with 0.42-fold decrease, and at 36h in 25°C challenged muscle by 0.80-fold decrease. Correlated expression profiles between phospho-p90RSK and phospho-p44/42MAPK suggest that p44/42MAPK may be involved in temperature-induced metabolic suppression through targeting p90RSK in A. japonicus.

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

HighlightsFull‐length cDNA of GST2 were identified in Apostichopus japonicus.AjmGST2 belonged to a new member in invertebrate mGST family and close to mammalian LTC4S.AjmGST2 mRNA in coelomocytes was up‐regulated after Vibrio splendidus challenge or LPS exposure.LTC4 contents showed consistently increase to that of AjGST2 in LPS exposed coelomocytes.The increased LTC4 contents and depressed ROS production in coelomocytes were detected after AjmGST2‐silencing. &NA; 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 1917 bp 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 6 h challenge of pathogenic Vibrio splendidus and reached the peak expression at 48 h. 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 24 h. 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.

Characterization of NLRP3-like gene from Apostichopus japonicus provides new evidence on inflammation response in invertebrates

Abstract Inflammatory/defensive response after pathogen invasion is considered a local defense reaction in vertebrates. Inflammation response in Apostichopus japonicus was hardly determined due to scarce information available for nucleotide binding domain‐like receptor family, pyrin domain‐containing (NLRP) family. In the present study, invertebrate NLRP homologue was identified from A. japonicus (designated as AjNLRP3‐like) by rapid amplification of cDNA ends. Full‐length cDNA of AjNLRP3‐like measured 2970 bp with 2265 bp open reading frame encoding a 754‐amino acid (aa) residue protein. Structural analysis revealed that AjNLRP3‐like processed characteristic domains of pyrin (32–102aa) and NACHT (183–339aa). Multiple sequence alignment and phylogenetic analysis supported that AjNLRP3‐like belongs to a new member of NLRP3 protein subfamily. Spatial expression analysis revealed that AjNLRP3‐like was ubiquitously expressed in all examined tissues with larger magnitude in coelomocytes. Both Vibrio splendidus challenge in vivo and lipopolysaccharide stimulation in vitro significantly upregulated mRNA expression of AjNLRP3‐like when compared with the control group. NLRP3‐mediated inflammation response depended on release of lysosomal cathepsin B (CTSB) and subsequent activation of high‐mobility group box (HMGB) in vertebrates. We investigated expression profiles of AjNLRP3‐like and AjHMGB after AjCTSB knock‐down and discovered that AjNLRP3‐like was depressed by 0.66‐fold and 0.47‐fold, whereas AjHMGB was depressed by 0.70‐fold and 0.50‐fold at 24 and 48 h in AjCTSB‐silenced group, respectively. Similarly, down‐regulation of AjHMGB was also observed after AjNLRP3‐like knock‐down. This study therefore suggests that A. japonicus feature similar inflammatory events as those in vertebrates, and activation of AjNLRP3‐like depends on AjCTSB expression and release of AjHMGB. HighlightsFull‐length cDNA of NLRP3‐like were identified in Apostichopus japonicus.AjmNLRP3‐like was ubiquitously expressed in all examined tissues with the larger magnitude in coelomocytes.AjmNLRP3‐like mRNA in coelomocytes was up‐regulated in vitro and in vivo.AjmNLRP3‐like and AjHMGB were depressed after AjCTSB knock‐down treatment in vitro.AjmNLRP3‐like could activate the expression of AjHMGB, but not AjCTSB in vitro.