Effects of ocean acidification on immune responses of the Pacific oyster Crassostrea gigas.

Ocean acidification (OA), caused by anthropogenic CO2emissions, has been proposed as one of the greatest threats in marine ecosystems. A growing body of evidence shows that ocean acidification can impact development, survival, growth and physiology of marine calcifiers. In this study, the immune responses of the Pacific oyster Crassostrea gigas were investigated after elevated pCO2 exposure for 28 days. The results demonstrated that OA caused an increase of apoptosis and reactive oxygen species (ROS) production in hemocytes. Moreover, elevated pCO2 had an inhibitory effect on some antioxidant enzyme activities and decreased the GSH level in digestive gland. However, the mRNA expression pattern of several immune related genes varied depending on the exposure time and tissues. After exposure to pCO2 at ∼2000 ppm for 28 days, the mRNA expressions of almost all tested genes were significantly suppressed in gills and stimulated in hemocytes. Above all, our study demonstrated that elevated pCO2 have a significant impact on the immune systems of the Pacific oyster, which may constitute as a potential threat to increased susceptibility of bivalves to diseases.

Combined metabolome and proteome analysis of the mantle tissue from Pacific oyster Crassostrea gigas exposed to elevated pCO2.

Ocean acidification (OA) has been found to affect an array of normal physiological processes in mollusks, especially posing a significant threat to the fabrication process of mollusk shell. In the current study, the impact of exposure to elevated pCO2 condition was investigated in mantle tissue of Crassostrea gigas by an integrated metabolomic and proteomic approach. Analysis of metabolome and proteome revealed that elevated pCO2 could affect energy metabolism in oyster C. gigas, marked by differentially altered ATP, succinate, MDH, PEPCK and ALDH levels. Moreover, the up-regulated calponin-2, tropomyosins and myosin light chains indicated that elevated pCO2 probably caused disturbances in cytoskeleton structure in mantle tissue of oyster C. gigas. This work demonstrated that a combination of proteomics and metabolomics could provide important insights into the effects of OA at molecular levels.

Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum.

The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

The response profiles of HSPA12A and TCTP from Mytilus galloprovincialis to pathogen and cadmium challenge.

Heat shock 70 kDa protein 12A (HSPA12A) is an atypical member of HSP70 family, and the translationally controlled tumor protein (TCTP) is a novel HSP with chaperone-like activity. They are both involved in protecting organisms against various stressors. In the present study, the cDNAs of HSPA12A and TCTP (called MgHSPA12A and MgTCTP) were identified from Mytilus galloprovincialis by RACE approaches. The full-length cDNA of MgHSPA12A and MgTCTP encoded a peptide of 491 and 171 amino acids, respectively. Real-time PCR was employed to analyze the tissue distribution and temporal expression of these two genes after bacterial challenge and cadmium (Cd) exposure. It was found that the transcripts of MgHSPA12A and MgTCTP were dominantly expressed in gonad and muscle, respectively. The expression level of MgTCTP at 48 h post Vibrio anguillarum challenge was detected to be significantly up-regulated in hepatopancreas (P < 0.05). As concerned to Cd exposure, 2.0-fold increase of MgHSPA12A expression compared to that of the control was observed at 48 h in 5 µg/L Cd(2+)-treated group, while the expression levels of MgTCTP were significantly decreased after exposed to both 5 and 50 µg/L Cd(2+) for 24 h and 96 h. These results suggested the potential involvement of MgHSPA12A and MgTCTP in the mediation of the immune responses and environmental stress in mussels.

Responses of thioredoxin 1 and thioredoxin-related protein 14 mRNAs to cadmium and copper stresses in Venerupis philippinarum.

Thioredoxin (abbreviated as Trx) is an important ubiquitous disulfide reductase, which can protect organisms against various oxidative stresses. In the present study, thioredoxin 1 (named as VpTrx1) and thioredoxin-related protein (named as VpTrp14) were identified from Venerupis philippinarum, respectively. Similar to most Trx1s, VpTrx1 possessed all conserved features critical for the fundamental structure and function of Trx1s, such as the conserved catalytic residues (C-G-P-C), but lacked the other cysteine residues, while VpTrp14 contained the conserved motif (C-P-D-C). Quantitative Real-time PCR assay showed that VpTrx1 and VpTrp14 transcripts were distributed in a wide array of tissues most abundantly expressed in the hepatopancreas. The expression of VpTrp14 mRNA in the hepatopancreas was significantly up-regulated after exposure to 10 and 40µg/L Cd, while the VpTrx1 expression level was kept relatively constant. Both the expression levels of VpTrx1 and VpTrp14 in the hepatopancreas were induced after exposure to Cu, and increased to the peak value at 96h under the 40µg/L Cu exposure. These results showed that VpTrp14 transcripts responded to metal stress more acutely than VpTrx1, and both Trxs responded to Cu stress more sensitively than Cd. Together, it was suggested that VpTrx1 and VpTrp14 perhaps played important roles in the antioxidant responses against metal stress in V. philippinarum.

Alternation of Venerupis philippinarum Hsp40 gene expression in response to pathogen challenge and heavy metal exposure.

HSP40 was an understudied protein family with co-chaperone activity. In the present study, a HSP40 homology was cloned from Venerupis philippinarum haemocytes (designated as VpHSP40) by EST analysis and RACE approaches. The expression profiles of VpHSP40 under Vibrio anguillarum challenge and heavy metal exposure were investigated by quantitative real-time RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression, and the highest expression level was detected at 24 h post-infection with 6.0-fold increase compared with that in the control group. During heavy metal exposure experiment, the expression of VpHSP40 could also be induced by Cu(2+) and Cd(2+) at different concentration, where Cu(2+) displayed more toxic effect on clam than that of Cd(2+). Concerning the same heavy metal, varied effect on VpHSP40 expression was detected at different concentration of heavy metal. Taking together, these results suggested that VpHSP40 was perhaps involved in mediating the immune responses and environmental stresses in V. philippinarum.

The first molluscan TCTP in Venerupis philippinarum: molecular cloning and expression analysis.

Translationally controlled tumor protein (TCTP) is one of the abundant and ubiquitously expressed proteins in metazoans. In the present study, the first molluscan TCTP (denoted as VpTCTP) was identified from Venerupis philippinarum haemocytes by EST and RACE approaches. The full-length cDNA of VpTCTP consisted of 1148 nucleotides with an open-reading frame of 555 bp encoding 184 amino acids. The deduced amino acid sequence of VpTCTP shared high similarity with TCTPs from other species, indicating that VpTCTP should be a new member of TCTP family. Several highly conserved motifs, including 5'terminal ologopyrimidine (5'TOP) starting sequence and rich AU and AUUT elements in 3'UTR, were also identified in VpTCTP. The tissue and temporal expression of VpTCTP after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. VpTCTP transcript could be detected in all examined tissues with the highest expression level in haemocytes and the lowest in hepatopancreas. Concerning the time-course expression in haemocytes, the relative expression of VpTCTP mRNA was down-regulated sharply from 6 h to 12 h post-infection. Then, the expression level was obviously up-regulated and reached 3.4-fold to that in the control group at 48 h post challenge. As time progressed, the expression of VpTCTP recovered to the original level at 96 h. All these results indicated that VpTCTP was an acute-phase protein involved in the immune response of V. philippinarum.

Identification of two small heat shock proteins with different response profile to cadmium and pathogen stresses in Venerupis philippinarum.

Small heat shock proteins (sHSPs) encompass a widespread and diverse class of proteins with molecular chaperone activity. In the present study, two sHSP isoforms (VpsHSP-1 and VpsHSP-2) were cloned from Venerupis philippinarum haemocytes by Rapid Amplification of cDNA Ends (RACE) approaches. The expression profiles of these two genes under Vibrio anguillarum challenge and cadmium exposure were investigated by quantitative real-time reverse transcriptase polymerase chain reaction. The bacterial challenge could significantly up-regulate the mRNA expression of both VpsHSP-1 and VpsHSP-2, with the increase of VpsHSP-2 expression occurred earlier than that of VpsHSP-1. During the cadmium exposure experiment, the expression level of both VpsHSP-1 and VpsHSP-2 decreased significantly with larger amplitude in VpsHSP-2. As time progressed, the expression levels of both genes were up-regulated with more increment in the low-chemical exposure groups. The differences in the response to pathogen stimulation and cadmium exposure indicated that there were functional diversity between the two structurally different molecules, VpsHSP-1 and VpsHSP-2, and they probably played distinct roles in mediating the environmental stress and immune responses in calm.

Cloning and characterization of an invertebrate type lysozyme from Venerupis philippinarum.

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections and providing nutrition as digestion enzymes. In the present study, an invertebrate type lysozyme (denoted as VpLYZ) was identified from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. The full-length cDNA of VpLYZ consisted of 805 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open-reading frame of 558bp encoding a polypeptide of 185 amino acids with a calculated molecular mass of 20.87kD and theoretical pI of 8.44. The high similarity of VpLYZ with other i-type lysozymes from mollusk indicated that VpLYZ should be a new member of i-type lysozyme family. Similar to most i-type lysozymes, VpLYZ possessed all conserved features critical for the fundamental structure and function of i-type lysozymes, such as three catalytic residues (Glu19, Asn72 and Ser75) and i-type specific motif CL(E/L/R/H)C(I/M)C. By semi-quantitative RT-PCR analysis, mRNA transcript of VpLYZ was found to be most abundantly expressed in the tissues of gills, hepatopancreas and haemocytes, weakly expressed in the tissues of muscle, foot and mantle. After clams were challenged by Vibrio anguillarum, the mRNA level of VpLYZ in overall haemocyte population was recorded by quantitative real-time RT-PCR. VpLYZ mRNA was down-regulated sharply from 6h to 12h post-infection. Then, the expression level increased to the peak at 72h and recovered to the original level at 96h. All these results indicated that VpLYZ was involved in the immune response against microbe infection and contributed to the clearance of bacterial pathogens.

Identification and characterization of an intracellular Cu, Zn-superoxide dismutase (icCu/Zn-SOD) gene from clam Venerupis philippinarum.

Superoxide dismutase (SOD, EC 1.15.1.1) represents one kind of enzyme involved in scavenging the high level of reactive oxygen species (ROS) into molecular oxygen and hydrogen peroxide. In the present study, the intracellular Cu/Zn-SOD gene (icCu/Zn-SOD) of Venerupis philippinarum (denoted as VpSOD) was identified from haemocytes by homology cloning and RACR approaches. The full-length cDNA of VpSOD consisted of 910 nucleotides with a canonical polyadenylation signal sequence AATAAA, a polyA tail, and an open-reading frame of 465 bp encoding 154 amino acids. The deduced amino acid of VpSOD shared high similarity with the icCu/Zn-SODs from other species, indicating that VpSOD should be a new member of icCu/Zn-SOD family. Several highly conserved motifs including Cu, Zn binding sites (H(46), H(48), H(63), H(120) for Cu binding, and H(63), H(71), H(80), D(83) for Zn binding), intracellular disulfide bond and two Cu, Zn SOD signatures were also identified in VpSOD. The temporal expression of VpSOD in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of VpSOD mRNA was up-regulated rapidly at 6 h post-infection and reached 18-fold of the control group. After a drastic decrease at 12 h, the expression level increased again and reached 22-fold to that in the control group at 96 h post-infection. All these results indicated that VpSOD was an acute-phase protein involved in the immune responses of V. philippinarum.

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization.

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anti-cancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.

Two thymosin-repeated molecules with structural and functional diversity coexist in Chinese mitten crab Eriocheir sinensis.

Recently, beta-thymosin-like proteins with multiple thymosin domains (defined as thymosin-repeated proteins) have been identified from invertebrate. In the present study, the cDNAs of two thymosin-repeated proteins (designated EsTRP1 and EsTRP2) were cloned from Chinese mitten crab by expressed sequence tags (EST) techniques. BLAST analysis presented three and two thymosin domains in EsTRP1 and EsTRP2, respectively, with the identities amongst the five domains varying from 47% to 100%. Both EsTRP1 and EsTRP2 shared high similarities with previously identified vertebrate beta-thymosins and invertebrate thymosin-repeated proteins (TRPs) with the identities ranging from 43% to 78%, indicating that EsTRPs were new members of the beta-thymosin family. Real-time RT-PCR assay was adopted to determine the tissue distribution of EsTRPs and their temporal expression profile in hemocytes after pathogen stimulation and injury challenge. The expression of EsTRP1 transcript was predominantly detectable in the tissues of hemocytes, hepatopancreas and gonad with the highest expression in hemocytes, while the highest expression level of EsTRP2 was found in heart. EsTRP1 mRNA expression in hemocytes significantly increased at 3 and 48h after Listonella anguillarum challenge, but there was no significant variation in EsTRP2 temporal expression profile. The injury challenge reduced the mRNA expression of EsTRPs, with the down-regulation of EsTRP2 expression occurred earlier than that of EsTRP1. The cDNA fragments encoding their mature peptides of EsTRP1 and EsTRP2 were recombined and expressed in Escherichia coli. The activities of recombinant proteins (rEsTRP1 and rEsTRP2) were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and lysoplate assay. rEsTRP2 could significantly accelerate the growth of human hepatocellular carcinoma cell line, but there was no significant effect of rEsTRP1 on the tumor cell proliferation. Both rEsTRP1 and rEsTRP2 did not possess the ability of killing Micrococcus luteus and L. anguillarum. The differences in the tissue distribution of mRNA transcripts, the response to pathogen stimulation and injury challenge, and the effect of recombinant proteins on human cell proliferation, indicated that there were functional diversity between the two structurally different molecules, EsTRP1 and EsTRP2.