Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas.

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host's immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.

Cloning and characterisation of NMDA receptors in the Pacific oyster, Crassostrea gigas (Thunberg, 1793) in relation to metamorphosis and catecholamine synthesis.

Bivalve metamorphosis is a developmental transition from a free-living larva to a benthic juvenile (spat), regulated by a complex interaction of neurotransmitters and neurohormones such as L-DOPA and epinephrine (catecholamine). We recently suggested an N-Methyl-D-aspartate (NMDA) receptor pathway as an additional and previously unknown regulator of bivalve metamorphosis. To explore this theory further, we successfully induced metamorphosis in the Pacific oyster, Crassostrea gigas, by exposing competent larvae to L-DOPA, epinephrine, MK-801 and ifenprodil. Subsequently, we cloned three NMDA receptor subunits CgNR1, CgNR2A and CgNR2B, with sequence analysis suggesting successful assembly of functional NMDA receptor complexes and binding to natural occurring agonists and the channel blocker MK-801. NMDA receptor subunits are expressed in competent larvae, during metamorphosis and in spat, but this expression is neither self-regulated nor regulated by catecholamines. In-situ hybridisation of CgNR1 in competent larvae identified NMDA receptor presence in the apical organ/cerebral ganglia area with a potential sensory function, and in the nervous network of the foot indicating an additional putative muscle regulatory function. Furthermore, phylogenetic analyses identified molluscan-specific gene expansions of key enzymes involved in catecholamine biosynthesis. However, exposure to MK-801 did not alter the expression of selected key enzymes, suggesting that NMDA receptors do not regulate the biosynthesis of catecholamines via gene expression.

Optimization of the UDP-Xyl biocatalytic synthesis from Crassostrea gigas by orthogonal design method.

UDP-Xyl, a nucleotide sugar involved in the biosynthesis of various glycoconjugates, is difficult to obtain and quite expensive. Biocatalysis using a one-pot multi-enzyme cascade is one of the most valuable biotransformation processes widely used in the industry. Herein, two enzymes, UDP-glucose (UDP-Glc) dehydrogenase (CGIUGD) and UDP-Xyl synthase (CGIUXS) from the Pacific oyster Crassostrea gigas, which are coupled together for the biotransformation of UDP-Xyl, were characterized. The optimum pH was determined to be pH 9.0 for CGIUGD and pH 7.5 for CGIUXS. Both enzymes showed the highest activity at 37 °C. Neither enzyme is metal ion-dependent. On this basis, a single factor and orthogonal test were applied to optimize the condition of biotransformation of UDP-Xyl from UDP-Glc. Orthogonal design L9 (33) was conducted to optimize processing variables of enzyme amount, pH, and temperature. The conversion of UDP-Xyl was selected as an analysis indicator. Optimum variables were the ratio of CGIUGD to CGIUXS of 2:5, enzymatic pH of 8.0, and temperature of 37 °C, which is confirmed by three repeated validation experiments. The UDP-Xyl conversion was 69.921% in a 1 mL reaction mixture by optimized condition for 1 h. This is the first report for the biosynthesis of UDP-Xyl from oyster enzymes.

CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes.

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ±â€¯4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ±â€¯1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.

The Dicer from oyster Crassostrea gigas functions as an intracellular recognition molecule and effector in anti-viral immunity.

Dicer, as a member of ribonuclease III family, functions in RNA interference (RNAi) pathway to direct sequence-specific degradation of cognate mRNA. It plays important roles in antiviral immunity and production of microRNAs. In the present study, a Dicer gene was identified from oyster Crassostrea gigas, and its open reading frame (ORF) encoded a polypeptide (designed as CgDicer) of 1873 amino acids containing two conserved ribonuclease III domains (RIBOc) and a double-stranded RNA-binding motif (DSRM). The deduced amino acid sequence of CgDicer shared identities ranging from 18.5% to 46.6% with that of other identified Dicers. The mRNA transcripts of CgDicer were detectable in all the examined tissues of adult oysters, with the highest expression in hemocytes (11.21 ±â€¯1.64 fold of that in mantle, p < 0.05). The mRNA expression level of CgDicer in hemocytes was significantly up-regulated (36.70 ±â€¯11.10 fold, p < 0.01) after the oysters were treated with double-stranded RNA (dsRNA). In the primarily cultured oyster hemocytes, the mRNA transcripts of CgDicer were significantly induced at 12 h after the stimulation with poly(I:C), which were 2.04-fold (p < 0.05) higher than that in control group. Immunocytochemistry assay revealed that CgDicer proteins were mainly distributed in the cytoplasm of hemocytes. The two most important functional domains of CgDicer, DSRM and RIBOc, were recombinant expressed in Escherichia coli transetta (DE3), and the recombinant DSRM protein displayed significantly binding activity to dsRNA and poly(I:C) in vitro, while the recombinant RIBOc protein exhibited significantly dsRNase activity to cleave dsRNA in vitro. These results collectively suggested that CgDicer functioned as either an intracellular recognition molecule to bind dsRNA or an effector with ribonuclease activity, which might play a crucial role in anti-viral immunity of oyster.

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, Crassostrea virginica.

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2-3.3. This enzyme prefers to hydrolyze the acidic GSLs, II3SO3LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4-4.5. Since oyster ceramidase greatly prefers ceramides with C6 to C8 fatty acids, C6-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C16 and C18 fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.

A novel caspase-associated recruitment domain (CARD) containing protein (CgCARDCP-1) involved in LPS recognition and NF-κB activation in oyster (Crassostrea gigas).

Caspase-associated recruitment domain (CARD) containing proteins play critical roles in molecular interaction and regulation of various signaling pathways, such as the activation of caspase and NF-κB singling pathway in the process of apoptosis or inflammation. In the present study, a novel CARD containing protein (designed CgCARDCP-1) was identified and characterized from oyster Crassostrea gigas. Molecular feature analysis revealed that, the open reading frame (ORF) of CgCARDCP-1 gene was 759 bp encoding a polypeptide of 253 amino acids with a conserved N-terminal CARD domain and two transcriptional coactivator p15 (PC4) domains in C-terminus. Homologous alignment showed that the amino acid sequence of CgCARDCP-1 shared 30%-46% identity with that of caspase-2. By RT-PCR detection, the mRNA transcripts of CgCARDCP-1 were found to be widely distributed in various tissues of oyster with the highest expression level in hemocytes and mantle. And CgCARDCP-1 protein was mostly distributed in the cytoplasm of oyster hemocytes as shown by immunohistochemistry. Moreover, the CgCARDCP-1 mRNA expression level in hemocytes was significantly up-regulated after lipopolysaccharide (LPS) and Vibrio splendidus stimulations. The recombinant CgCARDCP-1 displayed strong binding activity with LPS in vitro. In addition, after transfected into the HEK-293T cell with luciferase reporter system, CgCARDCP-1 could significantly promote the NF-κB activation (1.29-fold, p < 0.05) compared to that in the control group. These results collectively demonstrated that the CgCARDCP-1 might serve as a recognition molecule for LPS and a regulator of NF-κB activation in the immune response of oyster.

Triple serine loop region regulates the aspartate racemase activity of the serine/aspartate racemase family.

Recently, we cloned and characterized eleven serine and aspartate racemases (SerR and AspR, respectively) from animals. These SerRs and AspRs are not separated by their racemase functions and form a serine/aspartate racemase family cluster based on phylogenetic analysis. Moreover, we have proposed that the AspR-specific triple serine loop region at amino acid positions 150-152 may be responsible for the large AspR activity. In the present study, to test this hypothesis, we prepared and characterized fourteen mutants in this region of animal SerRs and AspRs. The large AspR activity in Acropora and Crassostrea AspR was reduced to <0.04% of wild-type after substitution of the triple serine loop region. Conversely, introducing the triple serine loop region into Acropora, Crassostrea, and Penaeus SerR drastically increased the AspR activity. Those mutants showed similar or higher substrate affinity for aspartate than serine and showed 11-683-fold higher k cat and 28-351-fold higher k cat/K m values for aspartate than serine racemization. Furthermore, we introduced serine residues in all combinations at position 150-152 in mouse SerR. These mutants revealed that a change in the enzyme function from SerR to AspR can be caused by introduction of Ser151 and Ser152, and addition of the third serine residue at position 150 further enhances the enzyme specificity for aspartate due to a decrease in the serine racemase and serine dehydratase activity. Here, we provide convincing evidence that the AspR gene has evolved from the SerR gene by acquisition of the triple serine loop region.

Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO2.

Ocean acidification (OA) has deleterious impacts on immune response and energy homeostasis status of Mollusca. In the present study, the apoptosis ratio of hemocytes and the adenosine triphosphate (ATP) allocation in gill tissues were determined after Pacific oysters Crassostrea gigas were exposed to elevated CO2 environment (pH = 7.50) for 16 days.The apoptosis ratio in CO2 exposure group (35.2%) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO2 could be significantly inhibited (p < 0.05) by KH7, a specific inhibitor of a bicarbonate sensor soluble adenylyl cyclase (sAC). After CO2 exposure, sAC in oyster (CgsAC) was found to be clustered with mitochondria in the cytoplasm, and the pro-caspase-3 was cleaved into two small fragments. Moreover, the activities of caspase-3 and caspase-9 also increased post CO2 exposure and these increases could be inhibited by KH7. However, the activities of caspase-8 did not change significantly compared with that in the control group. After CO2 exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could also be inhibited by KH7. The ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO2 exposure, which was also inhibited by KH7. These results implied that the elevated CO2 could activate the mitochondria-CgsAC pathway of apoptosis and ATP metabolism in oyster, and this pathway played essential roles in maintaining the homeostasis and the balance of energy metabolism in response to OA.

The modulation role of serotonin in Pacific oyster Crassostrea gigas in response to air exposure.

Serotonin, also known as 5-hydroxytryptamine (5-HT), is a critical neurotransmitter in the neuroendocrine-immune regulatory network and involved in regulation of the stress response in vertebrates and invertebrates. In the present study, serotonin was found to be widely distributed in the tissues of Pacific oyster Crassostrea gigas, including haemolymph, gonad, visceral ganglion, mantle, gill, labial palps and hepatopancreas, and its concentration increased significantly in haemolymph and mantle after the oysters were exposed to air for 1 d. The apoptosis rate of haemocytes was significantly declined after the oysters received an injection of extra serotonin, while the activity of superoxide dismutase (SOD) in haemolymph increased significantly. After the stimulation of serotonin during air exposure, the apoptosis rate of oyster haemocytes and the concentration of H2O2 in haemolymph were significantly decreased, while the SOD activity was significantly elevated. Furthermore, the survival rate of oysters from 4th to 6th d after injection of serotonin was higher than that of FSSW group and air exposure group. The results clearly indicated that serotonin could modulate apoptotic effect and redox during air exposure to protect oysters from stress.

High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis.

HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl2 stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression.

Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.

Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.

CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca2+ increased significantly (p < 0.05). But, this increasing of Ca2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response.

Pacific oyster polyamine oxidase: a protein missing link in invertebrate evolution.

Polyamine oxidases catalyse the oxidation of polyamines and acetylpolyamines and are responsible for the polyamine interconversion metabolism in animal cells. Polyamine oxidases from yeast can oxidize spermine, N(1)-acetylspermine, and N(1)-acetylspermidine, while in vertebrates two different enzymes, namely spermine oxidase and acetylpolyamine oxidase, specifically catalyse the oxidation of spermine, and N(1)-acetylspermine/N(1)-acetylspermidine, respectively. In this work we proved that the specialized vertebrate spermine and acetylpolyamine oxidases have arisen from an ancestor invertebrate polyamine oxidase with lower specificity for polyamine substrates, as demonstrated by the enzymatic activity of the mollusc polyamine oxidase characterized here. This is the first report of an invertebrate polyamine oxidase, the Pacific oyster Crassostrea gigas (CgiPAO), overexpressed as a recombinant protein. This enzyme was biochemically characterized and demonstrated to be able to oxidase both N(1)-acetylspermine and spermine, albeit with different efficiency. Circular dichroism analysis gave an estimation of the secondary structure content and modelling of the three-dimensional structure of this protein and docking studies highlighted active site features. The availability of this pluripotent enzyme can have applications in crystallographic studies and pharmaceutical biotechnologies, including anticancer therapy as a source of hydrogen peroxide able to induce cancer cell death.

Purification and characterization of a salt-tolerant cellulase from the mangrove oyster, Crassostrea rivularis.

A cellulase with wide range of pH resistance and high salt tolerance was isolated from the digestive gland of the oyster Crassostrea rivularis living in mangrove forests. The 27 kDa cellulase named as CrCel was purified 40.6 folds by anion exchange chromatography and extraction from the gel after non-reducing sodium dodecylsufate-polyacrylamide gel electrophoresis. The specific activity of the purified cellulase was 23.4 U/mg against carboxymethyl cellulose (CMC). The N-terminal amino acid sequence of CrCel was determined to be NQKCQANSRV. CrCel preferably hydrolyzes ß-1,4-glucosidic bonds in the amorphous parts of cellulose materials and displays degradation activity toward xylan. The Km and Vmax values of CrCel for CMC were determined to be 2.1% ± 0.4% and 73.5 ± 3.3 U mg(-1), respectively. The optimal pH value and temperature of CrCel were 5.5 and 40°C, respectively. The enzyme was stable in a wide range of pH, retaining over 60% activity after incubation for 80 min in the pH range of 3.0-9.0. In addition, CrCel showed remarkable tolerance to salt and remained active at high NaCl concentrations, but also retained over 70% activity after incubation in 0.5-2 M NaCl for up to 24 h. On the basis of the N-terminal sequence alignment and its similar properties to other animal cellulases, CrCel was regarded as a member of glycosyl hydrolase family 45 ß-1,4-glucanases. CrCel is the first reported cellulase isolated from mangrove invertebrates, which suggests that it may participate in the assimilation of cellulolytic materials derived from the food sources of the oyster and contribute to the consumption of mangrove primary production. The unique properties of this enzyme make it a potential candidate for further industrial application.

Evaluation of impacted Brazilian estuaries using the native oyster Crassostrea rhizophorae: Branchial carbonic anhydrase as a biomarker.

In this study, we investigated the use of branchial carbonic anhydrase activity in a sessile filter feeding species, the oyster Crassostrea rhizophorae, as a biomarker. The oysters were collected in three human impacted Brazilian estuaries, following a crescent latitudinal gradient: in Pernambuco state (Itamaracá), in Espírito Santo state (Piraquê), and in Paraná state (Paranaguá), in August/2003 (Winter in the southern hemisphere) and February/2004 (Summer). Three sites were chosen in each estuary for oyster sampling: Reference (R), Contaminated 1 (C1, close to industrial/harbor contamination), and Contaminated 2 (C2, near to sewage discharges). Comparing to values in oysters sampled in reference sites, there was apparent inhibition in carbonic anhydrase activity (CAA) in gills of oysters from C1 of Itamaracá and from C2 of Piraquê, both cases in Summer. On the other hand, increased CAA was noted in C2 oysters of Itamaracá in winter, and of Paranaguá, in both seasons. Branchial CAA in C. rhizophorae was thus very responsive to coastal contamination. Data are consistent with its usefulness as a supporting biomarker for inexpensive and rapid analysis in the assessment of estuaries using a sessile osmoconformer species, but preferably allied to other biomarkers and with knowledge on the suite of contaminants present.

Cloning and characterization of an apoptosis-related DNA fragmentation factor (DFF) from oyster, Crassostrea hongkongensis.

Apoptosis plays an important pathophysiological role in the homeostasis of immune systems. DNA fragmentation factors (DFFs) have been shown to be essential for DNA fragmentation, and the resultant DNA fragments follow a laddering pattern during apoptosis in vertebrates. In invertebrates, the functions of the DFF orthologs are not well characterized; therefore, we cloned and characterized a bivalve DFFA ortholog from the Hong Kong oyster Crassostrea hongkongensis (designated ChDFFA). The full-length cDNA of ChDFFA is 1186 bp in length and encodes a putative protein of 200 amino acids that contains an N-terminal CAD domain and a DFF-C domain at its C-terminus. Real-time RT-PCR results showed that ChDFFA is ubiquitously expressed in several tissues, and its highest expression is in gill. Following a 3- to 48-h challenge by microbial infection, the expression of ChDFFA increased in hemocytes. Using fluorescence microscopy, ChDFFA was localized in nuclei when exogenously expressed in HeLa cells. In addition, over-expression of ChDFFA inhibited the transcriptional activities of p53/p21-Luc reporter genes in HEK293T cells. These results suggest that ChDFFA may be involved in immune response reactions in the Hong Kong oyster C. hongkongensis.

The first invertebrate RIG-I-like receptor (RLR) homolog gene in the pacific oyster Crassostrea gigas.

Retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) is a pivotal receptor that detects numerous RNA and DNA viruses and mediates the innate induction of interferons and pro-inflammatory cytokines upon viral infection. In the present study, we cloned and characterized the first RIG-I gene in a marine mollusk, Crassostrea gigas, and designated it as CgRIG-I. The full-length CgRIG-I cDNA is 3436 bp, including 5'- and 3'-untranslated regions (UTRs) of 93 bp and 286 bp, respectively, and an open reading frame (ORF) of 3057 bp. The gene encodes a 1018 amino acid polypeptide with an estimated molecular mass of 116.5 kDa. SMART analysis showed that the CgRIG-I protein had the typical conserved domains, including the caspase activation and recruitment domains (CARDs), the RNA helicase domain and the C-terminal regulatory domain (RD). Phylogenetic analysis revealed that CgRIG-I was grouped into the clade of its vertebrate homologs. Moreover, CgRIG-I expression could be specifically increased after stimulation by poly(I:C) rather than by other PAMPs such as lipopolysaccharide (LPS), peptidoglycan (PGN), heat-killed Listeria monocytogenes (HKLM) and heat-killed Vibrio alginolyticus (HKVA). Meanwhile, six IRF, three STAT and one NF-κB predicted sites were identified in the CgRIG-I promoter, which was consistent with its high responsiveness to poly(I:C). In summary, this report provides the first CgRIG-I sequence of a mollusk, but its function in the antiviral immune response requires further investigation.

Molecular cloning and differential expression in tissues of a tyrosinase gene in the Pacific oyster Crassostrea gigas.

Tyrosinases are a group of type-3 copper proteins that catalyze the first two reactions in the melanin biosynthesis in organisms ranging from bacteria, fungi, plants to animals. Tyrosinases are not only involved in pigmentation, but also play an important role in the innate immunity in invertebrates. Additionally, tyrosinases are also known to be involved in the biogenesis and pigmentation of shells. The recently published Crassostrea gigas genome sequences revealed that the Pacific oyster possesses at least 26 tyrosinase isoforms. However, their molecular features are largely understudied. In fact, the full-length mRNA sequence was determined for one of the tyrosinase genes (i.e., CgTry1; aka cgi-tyr1). Here we report the full-length transcript of a second C. gigas tyrosinase (CgTyr2) sequence and the determination of its sequence features characteristic to the tyrosinase family proteins. We also showed that CgTyr2 gene was differentially expressed with the highest level of expression in mantle edges, suggesting its potential role in the formation of periostracum/pigmentation. Our comprehensive phylogenetic reconstructions supported that hemocyanins possibly evolved from a tyrosinase by an ancient gene duplication followed by functional differentiation, and the current large number of tyrosinase isoforms in C. gigas and other mollusks were originated from multiple gene duplication events that took places before and after mollusk species were established.

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas.

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.