Search

Serine protease inhibitors (SPIs) play a crucial role in regulation of both host and bacterial serine protease. They are classified into several protein families, where Kazal-type inhibitors are one of families with multi-domain. In the present study, the polymorphism of AiSPI from Bay scallop Argopecten irradians was found to be associated with disease resistance of bay scallop against Listonella anguillarum. Nine single nucleotide polymorphisms (SNPs) were identified in the exon region of AiSPI, where five SNPs were non-synonymous mutation. Three of these mutations were located in "kazal-like 3"domain, two SNP loci positioned at +536, +1312 were selected for further association studies. For the locus +536, the genotype frequency of A/G in the resistant stock (12.8%) was significantly lower (p < 0.05) than that in the susceptible stock (35.1%), while, the genotype A/A in the resistant stock (87.2%) was significantly higher in comparison with susceptible stock (64.9%) (p < 0.05). The G allele frequencies were 6.4% and 17.6% in resistant stock and susceptible stock, respectively, and χ 2 -test revealed a significant difference in the frequency distribution between the two stocks (p < 0.05). But there was no significant association between the mutation C-T at locus +1312 with either resistant or susceptible group (p > 0.05). The genotype frequencies of T/T, T/C, C/C at locus +1312 were 94.6%, 2.7% and 2.7% respectively in the susceptible stock, while 100%, 0% and 0% respectively in the resistant stock. The amino acid change for the mutation at locus +536 A-G was from asparagine to serine, and the predicted homology model of this amino acid variation could affect its function as well as the structural integrity of the domain. In vitro elastase inhibition assay of the protein variants at locus +536 was conducted to explicate the effect of SNP. The increasing concentration of protein (0 mmol/L- 2.93 mmol/L) was incubated with 80 nmol/L elastase where the residual enzyme activity values for rAiSPI (N) with A variant and rAiSPI (S) with G variant were started to reduce from 0.40 to 0.215 and 0.435 to 0.356, respectively. The elastase inhibition ability of rAiSPI (N) variant was significantly higher than that of rAiSPI (S) (p < 0.01). The results suggested that the mutation at locus +536A/A significantly associated with disease resistance of bay scallop would shed light for selective breeding program., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins consisting of at least one carbohydrate-recognition domain (CRD), which participate in nonself-recognition and clearance of invaders. In invertebrate, some multidomain C-type lectins have been identified, but their relative functions and binding mechanism are still meager. In the present study, A C-type lectin (CfLec-4) with four CRDs from Chlamys farreri was selected to investigate its possible function in innate immunity. The mRNA expression of CfLec-4 in hemocytes was significantly up-regulated (P<0.01) after the stimulations of β-glucan, LPS or PGN, and reached the highest expression level at 3, 6, 12 h post-stimulation, which was 27.9-, 22.6- or 47.9-fold of that in blank group, respectively. Immunohistochemistry assay with polyclonal antibody specific for CfLec-4 revealed that the endogenous CfLec-4 was mainly located in the hepatopancreas, kidney and gonad of the scallops. The recombinant CfLec-4 (rCflec-4) could bind LPS, PGN, glucan and mannose in vitro, but could not bind LTA. Furthermore, rCflec-4 displayed a broader bacteria binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as Gram-negative bacteria Escherichia coli, Vibrio anguillarum and fungi Pichia pastoris. Meanwhile, rCfLec-4 could significantly (P<0.01) enhance the phagocytosis of hemocytes in vitro. The results clearly suggested that four-CRD containing CfLec-4 not only served as PRR with wider recognition spectrum, but also functioned as an opsonin participating in the clearance of invaders in scallops. It could be inferred that the diversity and complexity of CRDs in C-type lectins endowed these receptors with comprehensive recognition spectrum and multiple immune functions against complex living environment., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In the present study, the expression of some immune-related genes was examined as indicator to understand the development of immune defense system during the ontogenesis of scallop Chlamys farreri. The mRNA transcripts of pattern-recognition receptors (PRRs) including CfPGRP-S1, CfLGBP, CfLec-1 and CfLec-3 were observed at a low level or even undetected at early developmental stages from eggs to blastula, and then began to increase overwhelmingly in trochophore. For the genes of immune effector including CfLYZ, CfLBP/BPI, CfSOD and CfCAT, their mRNA transcripts were higher expressed in embryos, and increased significantly in D-hinged or early veliger larvae. The whole-mount immunofluorescence assay revealed two immunoreactive spots of CfPGRP-S1 were first observed in the mid-ventral region of prototroch in trochophore, and the immunopositive fluorescence of CfLGBP, CfLec-1 and CfLec-3 appeared at the same spots in early D-hinged larvae. Most of the PRRs were located in velum, mouth, esophagus and stomach region in early and mid-veliger larvae, and especially the strong immunopositive fluorescence of CfLec-3 was observed in velum. The immunoreactive areas of CfLYZ, CfLBP/BPI, CfSOD and CfCAT were observed in trochophore and early D-hinged larvae. After D-hinged larvae, they distributed in different tissues from the edge of velum, mouth, esophagus to the region around digestive gland. After bacterial challenge, the mRNA expression of CfLGBP, CfLec-1 and CfLec-3 did not change significantly in trochophore, while a down-regulation of CfPGRP-S1 was observed at 6 h (P < 0.05). The expression of CfPGRP-S1 and CfLGBP decreased or increased inversely in D-hinged and late veliger larvae respectively, whereas CfLec-1 and CfLec-3 increased significantly during 6-24 h after bacterial challenge in the two stages (P < 0.05). In contrast, the expressions of immune effectors in trochophore and late veliger larvae were significant up-regulated at 6 h, 12 h or 24 h after bacterial challenge (P < 0.05). However, in late D-hinged larvae, CfLYZ and CfSOD expressions were significantly down-regulated at 6 h, while CfLBP/BPI expression was up-regulated at 6 h and 24 h post challenge (P < 0.05). These results indicated that the immune defense system of scallop might appear firstly in the mid-ventral region of prototroch in trochophore, and developed maturely after late D-hinged larvae. The developing immune system in the D-hinged and late veliger larvae could respond to the immune stimulation in different manner., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition protein (PGRP) is a pattern recognition receptor, playing important roles in the innate immune response against invasive pathogens. The single nucleotide polymorphism (SNP) loci in scallop PGRP gene (CfPGRP) were screened from Chlamys farreri to investigate their association with disease resistance of scallop against Listonella anguillarum. Thirteen SNP sites were identified in PGRP domain of CfPGRP, and two of them at positions 4407 and 4408 which are located in the same codon resulted in a nonsynonymous substitution. The genotype frequency of CG/CG in the resistant stock was significantly lower than that in susceptible stock (0% vs 32.4%), while that of CG/TA in the resistant stock was significantly higher than that in susceptible stock (P < 0.01). The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfPGRP-S1 (R) with CG variant in 4407-4408 site, rCfPGRP-S1 (Y) with TA variant in 4407-4408 site, were elucidated by examining their P/N value at 405 nm with ELISA assay. The in vitro binding activities of the two rCfPGRP-S1 variants to both lipopolysaccharide (LPS) and peptidoglycan (PGN) varied (P < 0.05) in a dose-dependent manner, and rCfPRPP-S1(Y) exhibited significantly higher affinity to PGN and LPS than that of rCfPGRP-S1(R) (P < 0.05). The growth inhibition assay was conducted to find the antibacterial activities of the two variants. Both rCfPGRP-S1(R) and rCfPGRP-S1 (Y) displayed obvious activity to suppress the growth of Escherichia coli, but there was no significant difference in suppression activity of two variants (P > 0.05). The results suggested that the polymorphism at locus 4407-4408 of CfPGRP-S1 considerably affected its PAMP binding activity, and the SNP locus 4407-4408 CG/TA was associated with disease resistance of scallop against L. anguillarum infection, which could be used as a candidate marker for future selection in zhikong scallop breeding program., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F' as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) is a pattern recognition receptor (PRR) recognizing and binding both LPS and β-1, 3-glucan, playing important roles in innate immunity. In the present study, the single nucleotide polymorphisms (SNPs) were assessed in LGBP gene from scallop Chlamys farreri (designated CfLGBP), and eight SNPs were found in its potential LPS and glucanase binding motif. The locus +7679 with the transition of G-A, which produced an amino acid substitution at codon 360 from a non polar Glycine to polar Serine, was selected to inspect their association with disease resistance/susceptibility to Listonella anguillarum. Three genotypes G/G, G/A and A/A, were revealed at locus +7679, and their frequencies were 89.7%, 7.7% and 2.6% in the resistant stock, while 63.2%, 34.2% and 2.6% in the susceptible stock, respectively. The frequency of genotypes G/G and G/A were significantly different (P < 0.05) between the two stocks. The pathogen-associated molecular patterns (PAMP) binding activity of two recombinant proteins, rCfLGBP (G) with G variant at locus +7679 and rCfLGBP (S) with A variant at locus +7679, were elucidated by ELISA assay. The binding affinities of both LPS and β-glucan binding affinity were varied in a dose-dependent manner, where the binding affinity of rCfLGBP (G) was significantly higher than that of rCfLGBP (S) (P < 0.05). The results collectively suggested that the polymorphism of +7679 G/G in CfLGBP possibly enhances the binding activity of LPS and β-glucan, and was associated to disease resistance of scallop against L. anguillarum, which could be a potential marker applied in future selection of scallop with enhanced resistance to L. anguillarum., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins which play significant roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, a novel C-type lectin with four dissimilar carbohydrate-recognition domains (CRDs) was identified from Argopectenirradians (designated as AiCTL-9). The full-length cDNA of AiCTL-9 was of 2291 bp with an open reading frame of 1827 bp encoding a polypeptide of 608 amino acids with a signal sequence and four CRDs. The motifs determining carbohydrate binding specificity in each CRD of AiCTL-9 were different, and they were YPT in CRD1, EPD in CRD2, EPN in CRD3 and QPN in CRD4, respectively. All the four CRDs shared the similar potential tertiary structure of a typical double-loop structure with Ca(2+)-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The mRNA transcripts of AiCTL-9 were mainly detected in hepatopancreas as well as gonad, and also marginally detectable in mantle, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after the challenges of fungi PichiapastorisGS115 (P<0.05), Gram-positive bacteria Micrococcusluteus (P<0.05) and Gram-negative bacteria Vibrioanguillarum (P<0.01). The recombinant AiCTL-9 (rAiCTL-9) could bind various PAMPs, including LPS, PGN, mannan and glucan, and also displayed agglutinating activity to fungi P. pastorisGS115, Gram-positive bacteria Bacillussubtilis and Gram-negative bacteria EscherichiacoliTOP10F' as well as V. anguillarum in a Ca(2+) dependent manner. Moreover, rAiCTL-9 could initiate the cellular adhesion of hemocytes and enhance their encapsulation invitro. All these results implied that AiCTL-9 was a novel PRR involved in immune response of scallop against a large number of pathogens by recognizing different PAMPs and enhancing scallop hemocytes encapsulation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tumor necrosis factor (TNF) signaling pathway plays crucial roles in the regulation of various immune responses. In the present study, a TNF signaling pathway related regulatory factor, TRAF and TNF receptor-associated protein (TTRAP), was firstly identified from the mollusk Zhikong scallop Chlamys farreri (designated as CfTTRAP). The full-length cDNA of CfTTRAP was of 2326bp, containing an open reading frame (ORF) of 1008 bp encoding a polypeptide of 335 amino acids with the predicted molecular weight of 38.4 kDa. There was an Exo&#95;endo&#95;phos domain in CfTTRAP, and it was well conserved when compared with other TTRAPs, especially the endonuclease activity related motifs. The recombinant protein of CfTTRAP exhibited prominent endonuclease activity to digest the genome DNA from C. farreri in the presence of Mg(2+), but it could not digest genome DNA of Escherichia coli and Bacillus subtilis, indicating CfTTRAP was a new member of Mg(2+)/Mn(2+)-dependent phosphodiesterase enzymes (MDP) superfamily. The mRNA transcripts of CfTTRAP were detected in all tested tissues of scallop, including muscle, mantle, gonad, gill, kidney and hemocytes. The expression level of CfTTRAP mRNA in hemocytes varied greatly after the stimulation of LPS, PGN or β-glucan. LPS induced significant down-regulation (P<0.05) of CfTTRAP mRNA expression, while PGN or β-glucan up-regulated the expression significantly (P<0.01), indicating that this regulatory factor was involved in modulating immune responses towards different stimulus. The present results provided new evidences for the potential roles of such molecule in C. farreri, and further confirmed the existence of TTRAP modulated TNF signaling pathway in mollusk., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Dopa decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyzes the decarboxylation of L-Dopa to dopamine, and involved in complex neuroendocrine-immune regulatory network. The function for DDC in the immunomodulation remains unclear in invertebrate., Methodology: The full-length cDNA encoding DDC (designated CfDDC) was cloned from mollusc scallop Chlamys farreri. It contained an open reading frame encoding a polypeptide of 560 amino acids. The CfDDC mRNA transcripts could be detected in all the tested tissues, including the immune tissues haemocytes and hepatopancreas. After scallops were treated with LPS stimulation, the mRNA expression level of CfDDC in haemocytes increased significantly (5.5-fold, P<0.05) at 3 h and reached the peak at 12 h (9.8-fold, P<0.05), and then recovered to the baseline level. The recombinant protein of CfDDC (rCfDDC) was expressed in Escherichia coli BL21 (DE3)-Transetta, and 1 mg rCfDDC could catalyze the production of 1.651±0.22 ng dopamine within 1 h in vitro. When the haemocytes were incubated with rCfDDC-coated agarose beads, the haemocyte encapsulation to the beads was increased significantly from 70% at 6 h to 93% at 24 h in vitro in comparison with that in the control (23% at 6 h to 25% at 24 h), and the increased haemocyte encapsulation was repressed by the addition of rCfDDC antibody (which is acquired via immunization 6-week old rats with rCfDDC). After the injection of DDC inhibitor methyldopa, the ROS level in haemocytes of scallops was decreased significantly to 0.41-fold (P<0.05) of blank group at 12 h and 0.47-fold (P<0.05) at 24 h, respectively., Conclusions: These results collectively suggested that CfDDC, as a homologue of DDC in scallop, modulated the immune responses such as haemocytes encapsulation as well as the ROS level through its catalytic activity, functioning as an indispensable immunomodulating enzyme in the neuroendocrine-immune regulatory network of mollusc.

As a member of pattern-recognition receptors (PRRs), the Toll-like receptor (TLR) and its signaling pathway play pivotal roles in recognizing various pathogen-associated molecular patterns (PAMPs), and buildup the front-line against invading pathogens. In the present study, the sequence features and mRNA expression profiles of five key genes involved in TLR signal pathway were characterized, and their functions in the immune responses were also investigated in order to validate the TLR signaling pathway and its potential roles in the immune defense of Zhikong scallop Chlamys farreri. These five genes, including CfTLR, CfMyD88, CfTRAF6, CfIκB and CfNFκB, exhibited significant similarity with their homologues from other model organisms, and contained the typical motifs. A strong interaction between the TIR domain from CfTLR and CfMyD88 protein was revealed via ELISA assays. The mRNA transcripts of these five genes were all up-regulated after LPS stimulation, indicating that they were involved in the immune response against LPS. When CfTLR expression was inhibited by RNAi technology, the mRNA expression level of CfMyD88, CfTRAF6, CfIκB, CfNFκB and G-type lysozyme were all decreased, while those of superoxide dismutase and catalase were increased. After Listonella anguillara challenge, the apoptosis level of those CfTLR-suppressed scallops was significantly lower than that in control groups (p<0.05) at the beginning of bacteria challenge, while the cumulative mortality was significantly higher than that of control groups (p<0.05). These results collectively favored that a rather canonical MyD88-dependent TLR pathway existed in scallop and this pathway was involved in immune signaling to active the diverse downstream reaction including anti-oxidant, anti-bacteria and apoptosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of proteins that can bind pathogen-associated molecular patterns (PAMPs) and microorganisms through the recognition of carbohydrates, thus they are directly involved in innate defense mechanisms as part of the acute-phase response to infection. In this study, the cDNA of a novel C-type lectin (designated as AiCTL-7) was cloned from bay scallop Argopecten irradians by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of AiCTL-7 was of 651 bp containing a 525 bp open reading frame which encoded a signal peptide of 15 residues and a conserved carbohydrate-recognition domain (CRD) of 174 residues with the EPD and WSD motifs instead of the invariant EPN and WND motifs for determining the carbohydrate-binding specificity and constructing Ca(2+)-binding site 2 in vertebrates. The deduced amino acid sequence of AiCTL-7 CRD shared homology not only with the CRDs of C-type lectins in mollusks, but also with the fish lectin CRDs. The mRNA transcripts of AiCTL-7 were mainly detected in the tissue of hepatopancreas and also marginally detectable in kidney, gonad, hemocytes, heart and adductor of health scallop. After challenge with fungi Pichia pastoris GS115 and Gram-negative bacteria Listonella anguillarum, the relative expression level of AiCTL-7 was up-regulated significantly in hepatopancreas and hemocytes. The CRD of AiCTL-7 was recombined and expressed in Escherichia coli, and the recombinant protein (rAiCTL-7) aggregated P. pastoris remarkably in a Ca(2+)-dependent manner, and this agglutination could be inhibited by d-mannose, but not by d-galactose or β-1,3-glucan. However, rAiCTL-7 displayed no obvious agglutinating activity against L. anguillarum. These results collectively indicated that AiCTL-7 was involved in the primitive acute-phase response to microbial invasion as an important pattern recognition receptor (PRR) in the innate immune system of scallops., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: C-type lectins are a superfamily of Ca(2+) dependent carbohydrate-recognition proteins that play significant diverse roles in nonself-recognition and clearance of invaders. Though they are well characterized in vertebrates, the study of the potential function and mechanism of C-type lectins in invertebrate immunity is still in its infancy., Methodology: A C-type lectin (CfLec-1) from scallop Chlamys farreri, a dominant cultured mollusk species in China, was selected to investigate its mRNA expression, localization and the possible functions in innate immunity in the present study. After scallop was stimulated by three typical PAMPs, the mRNA expression of CfLec-1 in hemocytes was poles apart. It was significantly up-regulated (p<0.01) after scallops were stimulated by LPS or β-glucan, but significantly down-regulated (p<0.01) after PGN stimulation. The binding ability of recombinant CfLec-1 (designated as rCfLec-1) towards eight PAMPs was investigated subsequently by PAMPs microarray, which revealed rCfLec-1 could bind LPS, PGN and mannan in vitro, indicating CfLec-1 served as a PRR involved in the pathogen recognition. Immunofluorescence assay with polyclonal antibody specific for CfLec-1 revealed that CfLec-1 was mainly located in the mantle and gill of the scallop. CfLec-1 could bind to the surface of scallop hemocytes and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-1 antibody. Meanwhile, rCfLec-1 could also enhance the phagocytic activity of scallop hemocytes against Escherichia coli., Conclusions: The results clearly suggested that CfLec-1 in C. farreri not only served as a PRR involved in the PAMPs recognition, but also functioned as an opsonin participating in the clearance of invaders. It is therefore suspected that CfLec-1 could be an attachment-molecule to nonself-agents acting as an alternative to immunoglobulin in vertebrates.

Scavenger receptors (SRs) are significant endocytic receptors contributing to constant internal environment. SR-cysteine-rich (SRCR) domain-containing SR is the most important class of SRs which has been so far reported exclusively in mammals and birds. In the present study, a novel SRCR domain-containing SR (CfSR) was firstly identified from scallop Chlamys farreri. The full-length cDNA of CfSR was of 2639 bp encoding a polypeptide of 804 amino acids with a signal peptide, six SRCR domains, a UPAR-like domain and a ShK toxin-like domain. All the SRCR domains contain highly conserved six cysteine residues to form three pairs of intradomain disulfide, among which SRCR-D5 was assumed to participate in ligand-binding. An attachment site of sequence CTTPLCN was found in UPAR-like domain, indicating CfSR was an anchor protein. This prediction was confirmed by its localization on the outer surface of hemocytes with immunofluorescence assay. The mRNA expression of CfSR was up-regulated significantly by the stimulations of lipopolysaccharides, peptidoglycan and β-glucan. A truncated CfSR (from V⁴⁵⁶ to T⁸⁰⁴) including SRCR-D5 was recombined and expressed in Escherichia coli, and the recombined protein displayed unique broad ligand-binding properties not only for acetylated low density lipoprotein (Ac-LDL) and dextran sulfate, but also for various pathogen associated molecular patterns, such as LPS, PGN, mannan and zymosan. All the results indicated that CfSR, the most primitive SR identified to date, was a versatile PRR involved in immune recognition, and the existence of functional SR might trace back to at least mollusk phylum., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient and highly conserved cytokine with multiple functions. In the present study, a MIF-like gene was cloned from Zhikong scallop Chlamys farreri (designated as CfMIF) based on expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CfMIF was of 2296bp, consisting of a 5' untranslated region (UTR) of 60bp, a 3' UTR of 1903bp with a poly(A) tail and an open reading frame (ORF) of 333bp encoded 111 amino acid residues with a calculated molecular mass of 12.6kDa and a theoretical isoelectric point of 5.63. The deduced amino acid sequence of CfMIF shared 27-50.5% similarity with those of other known MIFs. A conserved MIF domain was identified in the deduced amino acid sequence of CfMIF, and conserved proline(2) and lysine(33) were also found to be present in CfMIF. Phylogenetic analysis revealed that CfMIF is one of MIF members. The tissue distribution and temporal expression of CfMIF in hemocytes of scallop after lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan stimulation were detected by real-time RT-PCR. CfMIF gene was ubiquitously expressed in six selected tissues of healthy scallops, with the higher expression levels in hepatopancreas, mantle and gill. In comparison with the control group, the expression of CfMIF mRNA in hemocytes was up-regulated significantly at 6h, 24h and 48h after LPS treatment, and at all time points after PGN and glucan treatment. The cDNA fragment encoding mature peptide of CfMIF was recombined and expressed in Escherichia coli BL21 (DE3) pLysS. The recombinant protein of CfMIF (rCfMIF) promoted sheep fibroblast migration into scraped spaces in vitro. These results generated from the present study encourage us to suggest that CfMIF was a novel member of MIF family, and it was involved in immune response and wound healing by promoting fibroblast migration., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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 (Glu(269)-Pro(270)-Asp(271)) motif at the Ca(2+)-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., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

Dopamine beta hydroxylase (DBH) is a critical enzyme in the biosynthesis of catecholamines, and also plays an important role in complex neuroendocrine-immune regulatory network. In the present study, the cDNA encoding dopamine beta hydroxylase (designated CfDBH) was cloned from Chlamys farreri by using rapid amplification of cDNA ends (RACE) approaches and expression sequence tag (EST) analysis. The full-length cDNA of CfDBH was of 2302 bp, containing a 5' untranslated region (UTR) of 32 bp, a 3' UTR of 461 bp with a poly (A) tail, and an open reading frame (ORF) of 1809 bp encoding a polypeptide of 603 amino acids. The deduced amino acid sequence of CfDBH contained a signal peptide, a DOMON domain and a Cu2&#95;monooxygen domain, and it shared 39.4%-42.9% similarity with other reported DBHs. The conserved domains in CfDBH and the amino acid sequence similarity with other DBHs strongly suggested that it was a homologue of DBH in C. farreri. The mRNA expression of CfDBH in various tissues and its temporal expression in haemocytes of scallops stimulated with LPS were ascertained by Quantitative real-time RT-PCR. The mRNA transcripts of CfDBH were detected in all the examined tissues with the highest expression level in hepatopancreas. The expression level of CfDBH in haemocytes was up-regulated after LPS stimulation and increased to hundreds fold higher than that of the control group at 12 h, and then decrease significantly to 0.36-fold and 0.31-fold at 24 h and 48 h respectively. The results suggested pathogen infections significantly induced the expression level of CfDBH, and the activation of DBH could influence the immune response of scallop C. farreri through changing the concentration of catecholamines., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Peptidoglycan recognition protein (PGRP) is an essential molecule in innate immunity for both invertebrates and vertebrates, owing to its prominent ability in detecting and eliminating the invading bacteria. Several PGRPs have been identified from mollusk, but their functions and the underlined mechanism are still unclear. In the present study, the mRNA expression profiles, location, and possible functions of PGRP-S1 from Zhikong scallop Chlamys farreri (CfPGRP-S1) were analyzed. The CfPGRP-S1 protein located in the mantle, gill, kidney and gonad of the scallops. Its mRNA expression in hemocytes was up-regulated extremely after PGN stimulation (P<0.01), while moderately after the stimulations of LPS (P<0.01) and β-glucan (P<0.05). The recombinant protein of CfPGRP-S1 (designated as rCfPGRP-S1) exhibited high affinity to PGN and moderate affinity to LPS, but it did not bind β-glucan. Meanwhile, rCfPGRP-S1 also exhibited strong agglutination activity to Gram-positive bacteria Micrococcus luteus and Bacillus subtilis and weak activity to Gram-negative bacteria Escherichia coli. More importantly, rCfPGRP-S1 functioned as a bactericidal amidase to degrade PGN and strongly inhibit the growth of E. coli and Staphyloccocus aureus in the presence of Zn(2+). These results indicated that CfPGRP-S1 could not only serve as a pattern recognition receptor recognizing bacterial PGN and LPS, but also function as a scavenger involved in eliminating response against the invaders., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of Ca(2+) dependent carbohydrate-recognition proteins which play significant diverse roles in nonself-recognition and clearance of invaders. In the present study, a C-type lectin (CfLec-2) from Zhikong scallop Chlamys farreri was selected to investigate its functions in innate immunity. The mRNA expression of CfLec-2 in hemocytes was significantly up-regulated (P<0.01) after scallops were stimulated by LPS, PGN or β-glucan, and reached the highest expression level at 12h post-stimulation, which was 72.5-, 23.6- or 43.8-fold compared with blank group, respectively. The recombinant CfLec-2 (designated as rCfLec-2) could bind LPS, PGN, mannan and zymosan in vitro, but it could not bind β-glucan. Immunofluorescence assay with polyclonal antibody specific for CfLec-2 revealed that CfLec-2 was mainly located in the mantle, kidney and gonad. Furthermore, rCfLec-2 could bind to the surface of scallop hemocytes, and then initiated cellular adhesion and recruited hemocytes to enhance their encapsulation in vitro, and this process could be specifically blocked by anti-rCfLec-2 serum. These results collectively suggested that CfLec-2 from the primitive deuterostome C. farreri could perform two distinct immune functions, pathogen recognition and cellular adhesion synchronously, while these functions were performed by collectins and selectins in vertebrates, respectively. The synchronous functions of pathogen recognition and cellular adhesion performed by CfLec-2 tempted us to suspect that CfLec-2 was an ancient form of C-type lectin, and apparently the differentiation of these two functions mediated by C-type lectins occurred after mollusk in phylogeny., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a kind of pattern recognition receptor, which can recognize and bind LPS and beta-1, 3-glucan, and plays curial roles in the innate immune defense against Gram-negative bacteria and fungi. In this study, the functions of LGBP from Zhikong scallop Chlamys farreri performed in innate immunity were analyzed. Firstly, the mRNA expression of CfLGBP in hemocytes toward three typical PAMPS stimulation was examined by realtime PCR. It was up-regulated extremely (P < 0.01) post stimulation of LPS and beta-glucan, and also exhibited a moderate up-regulation (P < 0.01) after PGN injection. Further PAMPs binding assay with the polyclonal antibody specific for CfLGBP proved that the recombinant CfLGBP (designated as rCfLGBP) could bind not only LPS and beta-glucan, but also PGN in vitro. More importantly, rCfLGBP exhibited obvious agglutination activity towards Gram-negative bacteria Escherichia coli, Gram-positive bacteria Bacillus subtilis and fungi Pichia pastoris. Taking the results of immunofluorescence assay into account, which displayed CfLGBP was expressed specifically in the immune cells (hemocytes) and vulnerable organ (gill and mantle), we believed that LGBP in C. farreri, serving as a multi-functional PRR, not only involved in the immune response against Gram-negative and fungi as LGBP in other invertebrates, but also played significant role in the event of anti-Gram-positive bacteria infection. As the first functional research of LGBP in mollusks, our study provided new implication into the innate immune defense mechanisms of C. farreri and mollusks., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The globular C1q-domain-containing (C1qDC) proteins are a family of versatile pattern recognition receptors via their globular C1q (gC1q) domain to bind various ligands including several PAMPs on pathogens. In this study, a new gC1q-domain-containing protein (AiC1qDC-1) gene was cloned from Argopecten irradians by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNA of AiC1qDC-1 was composed of 733bp, encoding a signal peptide of 19 residues and a typical gC1q domain of 137 residues containing all eight invariant amino acids in human C1qDC proteins and seven aromatic residues essential for effective packing of the hydrophobic core of AiC1qDC-1. The gC1q domain of AiC1qDC-1, which possessed the typical 10-stranded beta-sandwich fold with a jelly-roll topology common to all C1q family members, showed high homology not only to those of C1qDC proteins in mollusk but also to those of C1qDC proteins in human. The AiC1qDC-1 transcripts were mainly detected in the tissue of hepatopancreas and also marginally detectable in adductor, heart, mantle, gill and hemocytes by fluorescent quantitative real-time PCR. In the microbial challenge experiment, there was a significant up-regulation in the relative expression level of AiC1qDC-1 in hepatopancreas and hemocytes of the scallops challenged by fungi Pichia pastoris GS115, Gram-positive bacteria Micrococcus luteus and Gram-negative bacteria Listonella anguillarum. The recombinant AiC1qDC-1 (rAiC1qDC-1) protein displayed no obvious agglutination against M. luteus and L. anguillarum, but it aggregated P. pastoris remarkably. This agglutination could be inhibited by d-mannose and PGN but not by LPS, glucan or d-galactose. These results indicated that AiC1qDC-1 functioned as a pattern recognition receptor in the immune defense of scallops against pathogens and provided clues for illuminating the evolution of the complement classical pathway., (Copyright 2010 Elsevier Ltd. All rights reserved.)

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles as pattern recognition receptors (PRRs) in the innate immunity. In this study, the full-length cDNA of a C-type lectin was cloned from scallop Chlamys farreri (designated as Cflec-5) by expression sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of Cflec-5 was of 1412 bp. The open reading frame encoded a polypeptide of 153 amino acids, including a signal sequence and a conserved carbohydrate-recognition domain with the EPN motif determining the mannose-binding specificity. The deduced amino acid sequence of Cflec-5 showed high similarity to members of C-type lectin superfamily. The quantitative real-time PCR was performed to investigate the tissue distribution of Cflec-5 mRNA and its temporal expression profiles in hemocytes post pathogen-associated molecular patterns (PAMPs) stimulation. In healthy scallops, the Cflec-5 mRNA was mainly detected in gill and mantle, and marginally in other tissues. The mRNA expression of Cflec-5 could be significantly induced by lipopolysaccharide (LPS) and glucan stimulation and reached the maximum level at 6 h and 12 h, respectively. But its expression level did not change significantly during peptidoglycan (PGN) stimulation. The function of Cflec-5 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta Gami (DE3). The recombinant Cflec-5 agglutinated Pichia pastoris in a calcium-independent way. The agglutinating activity could be inhibited by d-mannose, LPS and glucan, but not by d-galactose or PGN. These results collectively suggested that Cflec-5 was involved in the innate immune response of scallops and might contribute to nonself-recognition through its interaction with various PAMPs., (2010 Elsevier Ltd. All rights reserved.)