A novel C-type lectin (Cflec-3) from Chlamys farreri with three carbohydrate-recognition domains

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-type lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by d-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop.     

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.     

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system.     

AiCTL-6, a novel C-type lectin from bay scallop Argopecten irradians with a long C-type lectin-like domain

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel C-type lectin gene from scallop Argopecten irradians (designated as AiCTL-6) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of AiCTL-6 was 1080 bp. The open reading frame encoded a polypeptide of 307 amino acids, including a signal sequence and a C-type lectin-like domain (CTLD) of 150 amino acid residues longer than any usual CTLD. It contained six conserved cysteine residues involved in the formation of three internal disulfide bridges and an EPD (Glu₂₆₉-Pro₂₇₀-Asp₂₇₁) motif at the Ca²⁺-binding site 2. The deduced amino acid sequence of AiCTL-6 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, AiCTL-6 mRNA was found mainly in hepatopancreas and gill, and marginally expressed in other tissues. After the scallops were challenged by Listonella anguillarum for 6 h, the mRNA expression of AiCTL-6 was up-regulated significantly to 7.2-fold compared to the blank group. While at 9 h post Micrococcus luteus challenge, its expression level was 60.1 times higher than that of the blank group. The functional activity of AiCTL-6 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiCTL-6 could agglutinate Gram-negative bacteria E. coli TOP10F′, Gram-positive bacteria M. luteus and Staphylococcus aureus. These results collectively suggested that AiCTL-6, as a novel member of C-type lectin family, contributed to the host defense mechanisms against invading microorganism in A. irradians.     

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 (H⁴⁶, H⁴⁸, H⁶³, H¹²⁰ for Cu binding, and H⁶³, H⁷¹, H⁸⁰, D⁸³ 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.     

Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria

The effect of live bacteria (Micrococcus lysodeikticus and Vibrio anguillarum), and PAMPs (poly I:C, zymosan, LPS, LTA and CpG) on the production of intermediate toxic radicals (respiratory burst activity and production of nitric oxide) and mytilin B, myticin C and lysozyme gene expression was studied in vivo and in vitro. In vitro, bacteria were able to modulate the haemocytes' respiratory burst activity, being significantly increased after 6 h of incubation. The effect of pathogen-associated molecular patterns (PAMPs) was also studied. Zymosan produced an increase of the PMA-mediated response but an inhibition of the zymosan-mediated response. A significant increase of nitric oxide production was found at all the sampled time points (1, 3 and 6 h) in comparison with controls on both, the Gram-positive and Gram-negative bacteria. The in vivo responses measured on haemocytes after M. lysodeikticus injection were faster than those induced by V. anguillarum. However, V. anguillarum induced stronger in vitro effects. Mytilin B, myticin C and lysozyme in vitro gene expression, occurred at short times after infection. The maximum in vitro expression was detected 3 h post-infection. The differences between M. lysodeikticus and V. anguillarum in different measured parameters may suggest that different signalling pathways might be involved. Moreover, among all assayed PAMPs, LPS elicited the highest response.     

Two classes of glutathione S-transferase genes with different response profiles to bacterial challenge in Venerupis philippinarum

Glutathione S-transferase (GST) is major cytosolic detoxification enzymes involved in many pathological and physiological processes. In the present study, two classes of GSTs (VpGST-1 and VpGST-2) were cloned from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. Sequence alignments and phylogenetic analysis together supported that they belonged to a new member of sigma and pi classes GSTs protein family, respectively. The expression profiles of these two genes under Vibrio anguillarum challenge were investigated by quantitative RT-PCR. The bacterial challenge could significantly up-regulate the mRNA expression of both VpGST-1 and VpGST-2 with larger amplitude in VpGST-2, and the feedback speed for VpGST-2 was more rapid than that of VpGST-1. The differences in the response to bacterial challenge indicated that they were functional diversity and probably played cooperative roles in mediating the Vibrio challenge in clam.     

A tandem-repeat galectin from blood clam Tegillarca granosa and its induced mRNA expression response against bacterial challenge

Galectins are a family of β-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, we have cloned and characterized the first galectin to be identified in Tegillarca granosa (designated Tg-GAL). The full-length cDNA of Tg-GAL was of 2,394 bp nucleotides, encoding a polypeptide of 354 amino acids. The amino acid sequence of T. granosa galectin (Tg-GAL) showed striking sequence similarity to invertebrate and vertebrate galectins in carbohydrate recognition domains (CRD) and contained amino acids that are crucial for binding β-galactoside sugars. Structurally, the Tg-GAL was a tandem repeat galectin containing two CRD connected by a unique peptide link. Quantitative real-time PCR was employed to investigate the tissue distribution of Tg-GAL mRNA and temporal expression in haemocytes of clams challenged with Vibrio parahaemolyticus, lipopolysaccharide (LPS) and peptidogylcan (PGN). The Tg-GAL mRAN expression was concentrated in hepatopancreas and mantle. The up-regulation of Tg-GAL after bacteria V. parahaemolyticus, LPS and PGN challenge showed that Tg-GAL might play a pivotal role in anti-bacterial immunity. Further study should investigate the effects of Tg-GAL absence by siRNA knockout.