First molluscan TNFR homologue in Zhikong scallop: Molecular characterization and expression analysis

Tumor necrosis factor receptors (TNFRs) are a superfamily of proteins characterized by the unique cysteine-rich domain (CRD) and their important roles in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The first member of the molluscan TNFR family, designated as CfTNFR, was identified from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTNFR was of 1334 bp, consisting of a 5' UTR of 17 bp, a 3'UTR of 69 by with a poly (A) tail, and an open reading frame (ORE) of 1248 by encoding a polypeptide of 415 amino acids with a theoretical isoelectric point of 8.33 and predicted molecular weight of 47.07 kDa. There were a signal peptide, a CRD, a transmembrane region and a death domain in the deduced amino acid sequence of CfTNFR, suggesting that it was a typical type 1 membrane protein. The high identities (22-40%) of CfTNFR with other TNFR superfamily members indicated that CfTNFR should be a member of TNFR superfamily, and moreover, it should be the first death domain-containing TNFR found in invertebrates. Phylogenetic analysis revealed that CfTNFR was closely related to TNFR-like proteins from Strongylocentrotus purpuratus, Drosophila melanogaster and Ciona intestinalis, and they formed a separate branch apart from vertebrate TNFRs. The spatial expression of CfTNFR transcripts in healthy and bacteria challenged scallops was examined by quantitative real-time PCR. CfTNFR transcripts could be detected in all tested tissues, including haemocytes, gonad, gill, mantle and hepatopancreas, and significantly up-regulated in the tissues of gonad, gill, mantle and hepatopancreas after Listonella anguillarum challenge, indicating that CfTNFR was constitutive and inducible acute-phase protein involved in immune defence. The present results suggested the existence of the TNFR-like molecules and TNF-TNFR system in low invertebrates, and provided new insights into the role of CfTNFR in scallop innate immune responses to invading microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.

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. (C) 2008 Elsevier Ltd. All rights reserved.

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 Ca2+/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 Ca2+, 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. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of a C-type lectin from the bay scallop Argopecten irradians

C-type lectins are Ca2+ dependent carbohydrate-recognition proteins that play crucial roles in the invertebrate innate immunity, such as nonself recognition, activation of proPO system, antibacterial activity, promotion of phagocytosis and nodule formation. In this study, a novel C-type lectin of bay scallops Argopecten irradians (Ai Lec) was identified using expressed sequence tag (EST) and RACE techniques. The Ai Lec cDNA encoded a polypeptide of 171 amino acids with a putative signal peptide of 21 amino acid residues and a mature protein of 150 amino acids. The deduced amino acid sequence of Ai Lec was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 131 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. The expression of Ai Lec transcript was dominantly detected in the hepatopancreas and slightly detected in the haemocytes of normal scallops. 6 h after Vibrio anguillarum-challenge and 8 h after Micrococcus luteus-challenge, the temporal expression of Ai Lec mRNA in hemocytes was increased by 4.4- and 3.6-folds, respectively. The results suggested that Ai Lec was a constitutive and inducible acute-phase protein and might be involved in immune response to Gram-negative and Gram-positive microbial infection in bay scallop A. irradians.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: Gene cloning, expression and activity analysis

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549 bp full-length cDNA of EsproPO contained a 2040 bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68 bp, and a long 3'-UTR of 1441 bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2 h, 12 h and 48 h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab. (c) 2007 Elsevier Ltd. All rights reserved.

cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone contributing to the folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. The full-length cDNA of Zhikong scallop Chlamysfarreri HSP90 (designated CfHSP90) was cloned by EST and rapid RACE techniques. It was of 2710 bp, including an open reading frame (ORF) of 2181 bp encoding a polypeptide of 726 amino acids with all the five HSP90 family signatures. BLAST analysis revealed that the CfHSP90 gene shared high similarity with other known HSP90 genes. Fluorescent real-time quantitative RT-PCR was used to examine the expression pattern of CfHSP90 mRNA in haemocytes of scallops exposed to Cd2+, Pb2+ and Cu2+ for 10 and 20 days, respectively. All the three heavy metals could induce CfHSP90 expression. There was a clear dose-dependent expression pattern of CfHSP90 after heavy metals exposure for 10 days or 20 days. Different concentrations of the same metal resulted in different effects on CfHSP90 expression. The results indicated that CfHSP90 responded to various heavy metal stresses with a dose-dependent expression pattern as well as exposure time effect, and could be used as a molecular biomarker in a heavy metal polluted environment. (c) 2007 Elsevier Inc. All rights reserved.

Molecular cloning and characterization of a catalase gene from Zhikong scallop Chlamys farreri

Catalase is one of the central enzymes involved in scavenging the high level of reactive oxygen species (ROS) by degradation of hydrogen peroxide to oxygen and water. The full-length catalase cDNA of Zhikong scallop Chlamys farreri (denoted as CfCAT) was identified from hemocytes by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The nucleotide sequence of CfCAT cDNA consisted of 3146 bp with a 5' UTR of 103 bp, an unusually long 31 UTR of 1519 bp with a canonical polyadenylation signal sequence AATAAA and a potyA tail, and an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acids with predicted molecular weight of 57.5 kDa. The deduced amino acid sequence of CfCAT has significant homology to catalases from animals, plants and bacteria. Several highly conserved motifs including the proximal heme-ligand signature sequence RLFSYNDTH, the proximal active site signature FNRERIPERVVHAKGGGA, and the three catalytic amino acid residues of His(72), Asn(145) and Tyr(355) were identified in the deduced amino acid sequence of CfCAT. The CfCAT was demonstrated to be a peroxisomal glycoprotein with two potential glycosylation sites and a peroxisome targeting signal of ANL that was consistent with human, mouse and rat catalases. The time-course expression of CfCAT in hemocytes was measured by quantitative real-time PCR. The expression of CfCAT increased gradually and reached the highest point at 12 h post-Vibrio infection, then recovered to the original level at 24 h. All these results indicate that CfCAT, a constitutive and inducible protein, is a member of the catalase family and is involved in the process against ROS in scallop. (c) 2007 Published by Elsevier Ltd.

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 363 bp 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 8h, 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 1174bp 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. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning and mRNA expression of peptidoglycan recognition protein (PGRP) gene in bay scallop (Argopecten irradians, Lamarck 1819)

Peptidoglycan recognition proteins (PGRPs) are a type of pattern recognition molecules (PRM) that recognize the unique cell wall component peptidoglycan (PGN) of bacteria and are involved in innate immunity. The first bivalve PGRP cDNA sequence was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and PCR technique. The full-length cDNA of bay scallop PGRP (designated AiPGRP) gene contained 10 18 bp with a 615-bp open reading frame that encoded a polypeptide of 205 amino acids. The predicted amino acid sequence of AiPGRP shared high identity with PGRP in other organisms, such as PGRP precursor in Trichoplusia ni and PGRP SC2 in Drosophila melanogaster. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of AiPGRP in different tissues and the temporal expression of AiPGRP in the mixed primary cultured hemocytes challenged by microbial components lipopolyssacharide (LPS) from Escherichia coli and PGN from Micrococcus luteus. Higher-level mRNA expression of AiPGRP was detected in the tissues of hemocytes, gonad and kidney. The expression of AiPGRP in the mixed primary cultured hemocytes was up regulated after stimulated by PGN, while LPS from E. coli did not induce AiPGRP expression. The results indicated that AiPGRP was a constitutive and inducible expressed protein that was mainly induced by PGN and could be involved in scallop immune response against Gram-positive bacteria infection. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri

Toll-like receptors (TLRs) are an ancient family of pattern recognition receptors, which show homology with the Drosophila Toll protein and play key roles in detecting various non-self substances and then initiating and activating immune system. In this report, the full length of the first bivalve TLR (named as CfToll-1) is presented. CfToll-1 was originally identified as an EST (expressed sequence tag) fragment from a cDNA library of Zhikong scallop (Chlamys farreri). Its complete sequence was obtained by the construction of Genome Walker library and 5' RACE (rapid amplification of cDNA end) techniques. The full length cDNA of CfToll-1 consisted of 4308 nucleotides with a polyA tail, encoding a putative protein of 1198 amino acids with a 5' UTR (untranslated region) of 211 bp and a 3'UTR of 500 bp. The predicted amino acid sequence comprised an extracellular domain with a potential signal peptide, nineteen leucine-rich repeats (LRR), two LRR-C-terminal (LRRCT) motifs, and a LRR-N-terminal (LRRNT), followed by a transmembrane segment of 20 amino acids, and a cytoplasmic region of 138 amino acids containing the Toll/IL-1R domain (TIR). The deduced amino acid sequence of CfToll-1 was homologous to Drosophila melanogaster Tolls (DmTolls) with 23-35% similarity in the full length amino acids sequence and 30-54% in the TIR domain. Phylogenetic analysis of CfToll-1 with other known TLRs revealed that CfToll-1 was closely related to DmTolls. An analysis of the tissue-specific expression of the CfToll-1 gene by Real-time PCR showed that the transcripts were constitutively expressed in tissues of haemocyte, muscle, mantle, heart, gonad and gill. The temporal expressions of CfToll-1 in the mixed primary cultured haemocytes were observed after the haemocytes were treated with 1 mu g ml(-1) and 100 ng ml(-1) lipopolysaccharide (LPS), respectively. The expression of CfToll-1 was up-regulated and increased about 2-fold at 6 h with the treatment of 1 mu g ml(-1) LPS. The expression of CfToll-1 was down-regulated with the treatment of 100 ng ml(-1) LPS. The results indicated that the expression of CfToll-1 could be regulated by LPS, and this regulation was dose-dependent. (c) 2006 Elsevier Ltd. All rights reserved.

Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri

C-type lectin is a family of Ca2+ dependent carbohydrate-recognition proteins which play crucial roles in the innate immunity of invertebrates by mediating the recognition of host cells to pathogens and clearing microinvaders as a pattern recognition protein (PRP). The cDNA of Zhikong scallop Chlamys farreri C-type lectin (designated CFLec-1) was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of CFLec-1 was 1785 bp, consisting of a 5'-terminal untranslated region (UTR) of 66 bp and an unusually long 3' UTR of 1040 bp with seven polyadenylation signal sequences AATAAA and a poly(A) tail. The CFLec-1 cDNA encoded a polypeptide of 221 amino acids with a putative signal peptide of 15 amino acid residues and a mature protein of 206 amino acids. Analysis of the protein domain features indicated a typical long-form carbohydrate-recognition domain (CRD) of 130 residues in the CFLec-1 deduced amino acid sequence. The expression pattern of CFLec-1 transcripts in healthy and bacterial challenged scallops was studied by semi-quantitative RT-PCR. mRNA transcripts of CFLec-1 could be mainly detected in the tissues of haemocytes, gill, gonad and mantle of unchallenged scallops, whereas the expression of CFLec-1 transcripts was increased in all the tested tissues after heat-killed Vibrio anguillarum challenge. The temporal expression of CFLec-1 mRNA in haemolymph challenged by Micrococcus luteus and V anguillarum was both up-regulated and reached the maximum level at 8 and 16 It post stimulation, respectively, and then dropped back to the original level. In order to investigate its immune functions, CFLec- I was recombined and expressed in Escherichia coli BL21(DE3)-pLysS as a fusion protein with thioredoxin. The recombinant CFLec-1 agglutinated bacteria E. coli JM109 in vitro, and the agglutination was Ca2+ dependent which could be inhibited by EDTA. But it did not agglutinate M. luteus, Candida lipolytica and animal erythrocytes including rabbit, rat, mouse, chicken, human group A, human group B, human group O. Meanwhile, the recombinant CFLec-1 could inhibit the growth of both E. coli JM 109 and M. luteus, but no inhibition activity against V anguillarum. These result indicated that CFLec-1 was a constitutive and inducible PRP which was involved in the reorganization and clearance of invaders in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and expression of a novel Kazal-type serine proteinase inhibitor gene from Zhikong scallop Chlamys farreri, and the inhibitory activity of its recombinant domain

Serine proteinase inhibitors (SPIs) play important roles in host physiological and immunological processes in all multicellular organisms. A novel Kazal-type SPI gene was cloned from the Zhikong scallop Chlamys farreri (designated as CfKZSPI) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfKZSPI was of 1788 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) encoding a polypeptide of 509 amino acids with a putative signal peptide of 22 amino acids. The deduced amino acid sequence of CfKZSPI contained 12 tandem Kazal domains with high similarity to other Kazal-type SPIs. The temporal expression of CfKZSPI in hemocytes after Vibrio anguillorum challenge was recorded by quantitative real-time RT-PCR. The relative mRNA expression level of CfKZSPI was up-regulated and reached 43.6-fold at 3 h post-challenge. After a decrease at 6 h, the expression Level increased again and reached 207.8-fold at 12 h post-challenge. The 12th Kazal domain of CfKZSPI was recombined into pET-32a(+) and expressed in Escherichia coli Rosetta-gami (DE3) to investigate its inhibitory activity. The purified recombinant protein (rCf KZSPI-1 2) showed significant inhibitory activity against trypsin but no activity against thrombin. When the molar ratio of inhibitor to trypsin reached 1:1, almost 90% of the enzyme activity could be inhibited, which suggested that one molecule of rCfKZSPI-12 was able to inhibit one molecule of trypsin. Kinetics analysis with Dixon plot showed that the inhibition constant (K-i) of rCfKZSPI-12 to trypsin was 173 nmol L-1. These results indicated that CfKZSPI was a novel Kazal-type SPI with significant inhibitory activity against trypsin, and was suspected to be involved in scallop immune response. (c) 2008 Elsevier Ltd. All rights reserved.

Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri

LPS-induced TNF-alpha factor (LITAF) is a novel transcriptional factor that was first discovered in LPS-stimulated human macrophage cell line THP-1. LITAF can bind to TNF-a promoter to regulate its expression. The first scallop LITAF (named as CfLITAF) was cloned from Zhikong scallop Chlamys farreri by Expressed Sequence Tag (EST) and Polymerase Chain Reaction (PCR) techniques. The cDNA of CfLITAF was of 1240 bp and consisted of a 5' untranslated region (UTR) of 112 bp, a 3' UTR of 678 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 16.08 kDa and theoretical isoelectric point of 6.77. A typical conserved LITAF-domain was identified in CfLITAF by SMART analysis. Homology analysis of the deduced amino acid sequence of CfLITAF with other known sequences by using the BLAST program revealed that CfLITAF was homologous to the LITAF from human and rat (Identity = 46%), cattle, horse, mouse and chicken (Identity = 48%), western clawed frog (Identity = 42%), and zebrafish (Identity = 50%). The mRNA expression of CfLITAF in different tissues including haemocytes, muscle, mantle, heart, gill and gonad, and the temporal expression in haemocytes challenged by LPS or peptidoglycan (PGN) were measured by Real-time RT-PCR. CfLITAF mRNA transcripts could be detected in all tissues examined and be up-regulated in haemocytes after LPS challenge. No significant changes were observed after PGN stimulation. All these data indicated the existence of LITAF in scallop and also provided clue on the presence of TNF-alpha-like molecules in invertebrates. (C) 2007 Elsevier Ltd. All rights reserved.

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 T-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 6 h 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 6 h 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 LIDS 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. (c) 2008 Elsevier Ltd. All rights reserved.