Chenghua Li

A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity.

The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a 5-terminal untranslated region (UTR) of 62 bp and a 3 UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21(DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LPS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk.

Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis

Anti-lipopolysaccharide factor (ALF) represents one kind of basic proteins, which binds and neutralizes LPS and exhibits strong antibacterial activity against Gram-negative R-type bacteria. The ALF gene of Chinese mitten crab Eriocheir sinensis (Milne Edwards, 1853) (denoted as EsALF) was identified from haemocytes by expressed sequence tag (EST) and PCR approaches. The full-length cDNA of EsALF consisted of 700 nucleotides with a canonical polyadenylation signal-sequence AATAAA, a polyA tail, and an open-reading frame of 363bp encoding 120 amino acids. The high similarity of EsALF-deduced amino acid sequence shared with the ALFs from other species indicated that EsALF should be a member of ALF family. The mRNA expression of EsALF in the tissues of heart, gonad, gill, haemocytes, eyestalk and muscle was examined by Northern blot analysis and mRNA transcripts of EsALF were mainly detected in haemocytes, heart and gonad. The temporal expression of EsALF in haemocytes after Vibrio anguillarum challenge was recorded by quantitative real-time RT-PCR. The relative expression level of EsALF was up-regulated rapidly at 2 h post-injection and reached 3-fold to that in blank group. After a drastic decrease to the original level from 4 to 8 h, the expression level increased again and reached 4-fold to that in the blank group at 12 h post-injection. The genomic DNA sequence of EsALF gene consists of 1174 bp containing three exons and two introns. The coding sequence of the EsALF mature peptide was cloned and expressed in Escherichia coli BL21(DE3)-pLysS to further elucidate its biological functions. The purified recombinant product showed bactericidal activity against both Gram-positive (G+) and Gram-negative (G-) bacteria, which demonstrated that the rEsALF was a broad-spectrum antibacterial peptide. All these results indicated that EsALF was an acute-phase protein involved in the immune responses of Chinese mitten crab, and provided a potential therapeutic agent for disease control in aquaculture.

Differential toxicological effects induced by mercury in gills from three pedigrees of Manila clam Ruditapes philippinarum by NMR-based metabolomics

Mercury is a hazardous pollutant in the Bohai marine environments due to its high toxicity to the marine organisms and subsequent ecological risk. Manila clam Ruditapes philippinarum is one of important sentinel organisms in ‘Mussel Watch Program’ launched in China and therefore used as a bioindicator in marine and coastal ecotoxicology. There are dominantly distributed three pedigrees of clam (White, Liangdao Red and Zebra) in Yantai population endowed with different tolerances to environmental stressors. In this study, gill tissues were collected from both untreated and mercury exposed White, Liangdao Red and Zebra clams, and the extracts were analyzed by NMR-based metabolomics to compare the original metabolomes and the toxicological effects induced by mercury exposure in three pedigrees. The major abundant metabolites in White clam sample were branched-chain amino acids, lactate, alanine, arginine, acetoacetate, glutamate, succinate, citrate, malonate and taurine, while the metabolite profile of Liangdao Red clam sample comprises relative high levels of alanine, arginine, glutamate, succinate and glycogen. For Zebra clam sample, the metabolite profile exhibited relatively high amount of aspartate, acetylcholine and homarine. After 48xa0h exposure of 20xa0μg l−1 Hg2+, the metabolic profiles from all the three pedigrees of clams commonly showed significant increases in alanine, arginine, glutamate, aspartate, α-ketoglutarate, glycine and ATP/ADP, and decreases in citrate, taurine and homarine. The unique metabolic differences between the metabolomes of gill tissues from Hg2+-exposed White, Liangdao Red and Zebra clams were found, including elevated acetylcholine and branched-chain amino acids in White clams, and the declined succinate in both White and Liangdao Red samples as well as the declined betaine in Zebra and White clams. Overall, our findings showed the differential toxicological responses to mercury exposure and that White clams could be a preferable bioindicator for the metal pollution monitoring based on the metabolic changes from gill compared with other two (Liangdao Red and Zebra) pedigrees of clams.

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5-terminal untranslated region (UTR) of 60 bp and a 3-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys(53), Cys(128), Cys(144), Cys(152)) involved in the formation of disulfides bridges and the potential Ca(2+)/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca(2+), and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.

A lectin (CfLec-2) aggregating Staphylococcus haemolyticus from scallop Chlamys farreri

Lectins are a family of carbohydrate-recognition proteins which play crucial roles in innate immunity. In this study, a new lectin (CfLec-2) gene was cloned from Chlamys farreri by EST and RACE approaches. The full-length cDNA of CfLec-2 was composed of 708bp, encoding a typical long form carbohydrate-recognition domain of 130 residues. The deduced amino acid sequence showed high similarity to Brevican in Homo sapiens, C-type lectin-1 and lectin-2 in Anguilla japonica. The cDNA fragment encoding the mature peptide of CfLec-2 was recombined into plasmid pET-32a (+) and expressed in Escherichia coli Rosseta-Gami (DE3). The recombinant CfLec-2 (rCfLec-2) protein exhibited aggregative activity toward Staphylococcus haemolyticus, and the agglutination could be inhibited by d-mannose but not EDTA or d-galactose, indicating that CfLec-2 was a Ca2+ independent lectin. Moreover, rCfLec-2 could suppress the growth of E. coli TOP10F. These results suggested that CfLec-2 was perhaps involved in the recognition and clearance of bacterial pathogens in scallop.

Toxicological responses to acute mercury exposure for three species of Manila clam Ruditapes philippinarum by NMR-based metabolomics

The Manila clam (Ruditapes philippinarum) has been considered a good sentinel species for metal pollution monitoring in estuarine tidal flats. Along the Bohai coast of China, there are dominantly distributed three species of clams (White, Liangdao Red and Zebra in Yantai population) endowed with distinct tolerances to environmental stressors. In this study, adductor muscle samples were collected from both control and acute mercury exposed White, Liangdao Red and Zebra clams, and the extracts were analyzed by NMR-based metabolomics to compare the metabolic profiles and responses to the acute mercury exposure to determine the most sensitive clam species capable of acting as abioindicator for heavy metal pollution monitoring. The major abundant metabolites in the White clam sample were branched-chain amino acids (leucine, isoleucine and valine), lactate, arginine, aspartate, acetylcholine, homarine and ATP/ADP, while the metabolite profile of Zebra clam sample comprised high levels of glutamine, acetoacetate, betaine, taurine and one unidentified metabolite. For the Liangdao Red clam sample, the metabolite profile relatively exhibited high amount of branched-chain amino acids, arginine, glutamate, succinate, acetylcholine, homarine and two unassigned metabolites. After 48h exposure of 20μgL(-1) Hg(2+), the metabolic profiles showed significant differences between three clam species, which included increased lactate, succinate, taurine, acetylcholine, betaine and homarine and decreased alanine, arginine, glutamine, glutamate, acetoacetate, glycine and ATP/ADP in White clam samples, and elevated succinate, taurine and acetylcholine, and declined glutamine, glycine, and aspartate in Liangdao Red clam samples, while the increased branched-chain amino acids, lactate, succinate, acetylcholine and homarine, and reduced alanine, acetoacetate, glycine and taurine were observed in the Zebra clam samples. Overall, our findings showed that White clams could be a preferable bioindicator for the metal pollution monitoring based on the more sensitive metabolic changes in the adductor muscle compared with other two (Liangdao Red and Zebra) clam species.

Molecular Characterization of a Novel Big Defensin from Clam Venerupis philippinarum

Antimicrobial peptides (AMPs) are important mediators of the primary defense mechanism against microbial invasion. In the present study, a big defensin was identified from Venerupis philippinarum haemocytes (denoted as VpBD) by RACE and EST approaches. The VpBD cDNA contained an open reading frame (ORF) of 285 bp encoding a polypeptide of 94 amino acids. The deduce amino acid sequence of VpBD shared the common features of big defensin including disulfide array organization and helix structure, indicating that VpBD should be a new member of the big defensin family. The mRNA transcript of VpBD was up-regulated significantly during the first 24 hr after Vibrio anguillarum challenge, which was 7.4-fold increase compared to that of the control group. Then the expression decreased gradually from 24 hr to 96 hr, and the lowest expression level was detected at 96 hr post-infection, which was still 3.9-fold higher than that of control. The mature peptide of VpBD was recombined in Escherichia coli and purified for minimum inhibitory concentration (MIC) determination. The rVpBD displayed broad-spectrum inhibitory activity towards all tested bacteria with the highest activity against Staphyloccocus aureus and Pseudomonas putida. These results indicated that VpBD was involved in the host immune response against bacterial infection and might contribute to the clearance of invading bacteria.

Molecular cloning and characterization of peroxiredoxin 6 from Chinese mitten crab Eriocheir sinensis.

Peroxiredoxin is a superfamily of antioxidative proteins that play important roles in protecting organisms against the toxicity of reactive oxygen species (ROS). In this study, the full-length cDNA encoding peroxiredoxin 6 (designated EsPrx6) was cloned from Chinese mitten crab Eriocheir sinensis by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EsPrx6 was of 1076 bp, containing a 5 untranslated region (UTR) of 69 bp, a 3 UTR of 347 bp with a poly (A) tail, and an open reading frame (ORF) of 660 bp encoding a polypeptide of 219 amino acids with the predicted molecular weight of 24 kDa. The conserved Prx domain, AhpC domain and the signature of peroxidase catalytic center identified in EsPrx6 strongly suggested that EsPrx6 belonged to the 1-Cys Prx subgroup. Quantitative real-time RT-PCR was employed to assess the mRNA expression of EsPrx6 in various tissues and its temporal expression in haemocytes of crabs challenged with Listonella anguillarum. The mRNA transcript of EsPrx6 could be detected in all the examined tissues with highest expression level in hepatopancreas. The expression level of EsPrx6 in haemocytes was down-regulated after bacterial challenge and significantly decreased compared to the control group at 12h. As time progressed, the expression level began to increase but did not recover to the original level during the experiment. The results suggested the involvement of EsPrx6 in responses against bacterial infection and further highlighted its functional importance in the immune system of E. sinensis.

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.