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 C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.     

Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from mixed tissues of E. sinensis challenged with LPS. Eight genes involved in immune response were identified from 319 single colonies. Among them, two different C-type lectin-like domain (CTLD)-containing proteins were firstly identified in Chinese mitten crab. The full-length cDNA sequences of two C-type lectin-like domain (CTLD)-containing proteins named EsCTLDcp-1 and EsCTLDcp-2 were cloned by 5' RACE. The deduced amino acid sequences of EsCTLDcp-1 and EsCTLDcp-2 possessed several conserved features of C-type lectin subfamily. The tissue distribution of EsCTLDcp-1 and EsCTLDcp-2 was examined by Real-time PCR. In the normal Chinese mitten crab, the expression of EsCTLDcp-2 was detected in all tested tissues such as haemolymph, muscle, intestine, gill, heart, gonad and hepatopancreas, whereas in muscle, intestine, gill, heart and hepatopancreas for EsCTLDcp-1. The highest expressions of EsCTLDcp-1 and EsCTLDcp-2 were both observed in hepatopancreas. LPS significantly induced the expression of EsCTLDcp-1 and EsCTLDcp-2 in the hepatopancreas at the different time points. The induced fold change of EsCTLDcp-1 and EsCTLDcp-2 increased significantly from 2 h for EsCTLDcp-1 and 4 h for EsCTLDcp-2, and reached a maximum at 12 h, then dropped at 24 h. A differential pattern was found in Chinese mitten crab challenged with Chinese mitten crab pathogen Aeromonas hydrophila. The expression of EsCTLDcp-1 increased significantly at 2 h post-challenge crabs with A. hydrophila, then decreased at 4 h and 8 h, after that increased at 12 h and 24 h. The expression of EsCTLDcp-2 was decreased at the all time points. All these data suggest a differential role of EsCTLDcp-1 and EsCTLDcp-2 in the crab innate immune response to bacterial infection.     

Molecular cloning, characterization and expression analysis of macrophage migration inhibitory protein (MIF) in Chinese mitten crab, Eriocheir sinensis

Macrophage migration inhibitory factor (MIF) as a multi-functional cytokine mediating both innate and adaptive immune responses, however, their function within the innate immune system of invertebrates remains largely unknown. Therefore, we investigated the immune functionality of MIF in Chinese mitten crab (Eriocheir sinensis), a commercially important and disease vulnerable aquaculture species. The full-length MIF cDNA (704 bp) was cloned via PCR based upon an initial expressed sequence tag (EST) isolated from a E. sinensis cDNA library. The MIF cDNA contained a 363 bp open reading frame (ORF) that encoded a putative 120 amino acid (aa) protein. Comparisons with other reported invertebrate and vertebrate MIF sequences revealed conserved enzyme active sites. MIF mRNA expression in E. sinensis was (a) tissue-specific, with the highest expression observed in hepatotpancreas, and (b) responsive in hemocytes, hepatopancreas and gill to a Vibrio anguillarum challenge, with peak exposure observed 8 h, 12 h and 12 h post-injection, respectively. Collectively, data demonstrate the successful isolation of MIF from the Chinese mitten crab, and its involvement in the innate immune system of an invertebrate.     

三疣梭子蟹HMGBa基因克隆及其应答不同病原入侵的表达特征

为研究三疣梭子蟹(Portunus trituberculatus)高迁移率族蛋白B (High-mobility group box protein, HMGB)在其先天免疫中发挥的功能, 利用RACE技术首次克隆得到了三疣梭子蟹HMGBa基因, 命名为PtHMGBa。其cDNA序列全长1030 bp, 其中5′端非编码区(UTR)为94 bp, 3′端非编码区(UTR)为255 bp, 开放阅读框(ORF)为681 bp, 编码一个含有227个氨基酸, 分子量25.82 kD, 理论等电点为5.94的蛋白质。PtHMGBa蛋白包含2个HMG盒结构域和一个酸性尾部结构域。分析表明, 三疣梭子蟹HMGBa氨基酸序列与凡纳滨对虾(Litopenaeus vannamei) HMGBa相似度最高。实时荧光定量PCR结果显示, PtHMGBa基因在血细胞和肝胰腺的表达量最高, 在眼柄中表达量最低。在副溶血弧菌和WSSV感染过程中, PtHMGBa基因在肝胰腺和血细胞中均出现了表达上调。其中, 经副溶血弧菌感染后, 该基因在上述2种组织中分别于48h和6h达到表达量的峰值; 经WSSV感染后, 该基因在2种组织中均在12h达到表达量的峰值。结果表明PtHMGBa基因参与了三疣梭子蟹抵御外来病原的免疫响应, 研究为深入开展三疣梭子蟹和其他甲壳动物的免疫调控机理提供了科学依据。     

Molecular cloning and immune responsive expression of a novel C-type lectin gene from bay scallop Argopecten irradians

C-type lectins are Ca(2+)-dependent carbohydrate-recognition proteins that play crucial roles in innate immunity. The cDNA of C-type lectin (AiCTL1) in the bay scallop Argopecten irradians was cloned by expressed sequence tag (EST) and RACE techniques. The full-length cDNA of AiCTL1 was 660 bp, consisting of a 5'-terminal untranslated region (UTR) of 30 bp and a 3' UTR of 132 bp with a polyadenylation signal sequence AATAAA and a poly(A) tail. The AiCTL1 cDNA encoded a polypeptide of 166 amino acids with a putative signal peptide of 20 amino acid residues and a mature protein of 146 amino acids. The deduced amino acid sequence of AiCTL1 was highly similar to those of the C-type lectins from other animals and contained a typical carbohydrate-recognition domain (CRD) of 121 residues, which has four conserved disulfide-bonded cysteine residues that define the CRD and two additional cysteine residues at the amino terminus. AiCTL1 mRNA was dominantly expressed in the hemocytes of the bay scallop. The temporal expression of AiCTL1 mRNA in hemocytes was increased by 5.7- and 4.9-fold at 6h after injury and 8h after injection of bacteria, respectively. The structural features, high similarity and expression pattern of AiCTL1 indicate that the gene may be involved in injury healing and the immune response in A. irradians.     

Immulectin, an inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase.

Immulectin, a C-type lectin from the tobacco hornworm, Manduca sexta, was cloned from a larval fat body cDNA library. The immulectin cDNA encodes a 309 residue polypeptide. Immulectin synthesis was induced by injection of killed gram-positive or gram-negative bacteria or yeast. After injection of bacteria, immulectin mRNA appeared in fat body and immulectin protein was detected in hemolymph. Immulectin contains two carbohydrate recognition domains. The carboxyl-terminal carbohydrate recognition domain is most similar (36% identity) to a lipopolysaccharide-binding protein from the American cockroach, Periplaneta americana. It also shares 26-35% identity to carbohydrate recognition domains of various mammalian C-type lectins. Two immulectin isoforms were identified in the hemolymph of bacteria-injected larvae. Recombinant immulectin agglutinated gram-positive and gram-negative bacteria and yeast. Addition of recombinant immulectin to M. sexta plasma stimulated activation of phenol oxidase. A combination of immulectin with lipopolysaccharide from E. coli activated phenol oxidase more rapidly and to a higher level than immulectin alone, whereas lipopolysaccharide by itself had little effect on phenol oxidase activation. Immulectin synthesized in response to bacterial or fungal infection may help to trigger protective responses in M. sexta in a manner similar to mannose-binding protein, a C-type lectin that functions in the mammalian innate immune system.     

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.     

Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition.     

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca²⁺ binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln¹²²-Pro¹²³-Asn¹²⁴) in LvLectin-1, but QPD (Gln¹²⁸-Pro¹²⁹-Asp¹³⁰) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P < 0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P < 0.01) and 12 h (P < 0.05), but the expression level of LvLectin-1 down-regulated significantly (P < 0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.