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.     

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.     

Primary structure characterization of Bothrops jararacussu snake venom lectin

The complete amino acid sequence of the lectin from Bothrops jararacussu snake venom (BJcuL) is reported. The sequence was determined by Edman degradation and amino acid analysis of the S-carboxymethylated BJcuL derivative (RC-BJcuL) and from its peptides originated from enzymatic digestion. The sequence of amino acid residues showed that this lectin displays the invariant amino acid residues characterized in C-type lectins. Amino acids analysis revealed a high content of acidic amino acids and leucine. These findings suggest that BJcuL, like other snake venom lectins, possesses structural similarities to the carbohydrate recognition domain (CRD) of calcium-dependent animal lectins belonging to the C-type -galactoside binding lectin family.     

Characterization of Multisugar-Binding C-Type Lectin (SpliLec) from a Bacterial-Challenged Cotton Leafworm, Spodoptera littoralis

Background

Various proteins that display carbohydrate-binding activity in a Ca²⁺-dependent manner are classified into the C-type lectin family. They have one or two C-type carbohydrate-recognition domains (CRDs) composed of 110–130 amino acid residues in common. C-type lectins mediate cell adhesion, non-self recognition, and immuno-protection processes in immune responses and thus play significant roles in clearance of invaders, either as cell surface receptors for microbial carbohydrates or as soluble proteins existing in tissue fluids. The lectin of Spodoptera littoralis is still uncharacterized.

Methodology

A single orf encoding a deduced polypeptide consisting of an 18-residue signal peptide and a 291-residue mature peptide, termed SpliLec, was isolated from the haemolymph of the cotton leafworm, S. littoralis, after bacterial challenge using RACE-PCR. Sequence analyses of the data revealed that SpliLec consists of two CRDs. Short-form CRD₁ and long-form CRD₂ are stabilized by two and three highly conserved disulfide bonds, respectively. SpliLec shares homology with some dipteran lectins suggesting possible common ancestor. The purified SpliLec exhibited a 140-kDa molecular mass with a subunit molecular mass of 35 kDa. The hemagglutination assays of the SpliLec confirmed a thermally stable, multisugar-binding C-type lectin that binds different erythrocytes. The purified SpliLec agglutinated microorganisms and exhibited comparable antimicrobial activity against gram (+) and gram (−) bacteria too.

Conclusions

Our results suggested an important role of the SpliLec gene in cell adhesion and non-self recognition. It may cooperate with other AMPs in clearance of invaders of Spodoptera littoralis.     

亚洲玉米螟免疫凝集素基因的克隆、序列分析及组织表达(英文)

C型凝集素作为模式识别分子可以识别部分脂多糖(LPS),进而参与昆虫细胞的防御反应。本文通过RT-PCR和3′/5′RACE技术从亚洲玉米螟Ostriniafurnacalis 5龄幼虫血细胞中克隆得到免疫凝集素基因(OfIML)。OfIMLmRNA全长为1241 bp,其中开放读码框(ORF)为924 bp,编码307个氨基酸(aa),分子量约为34.65 ku。与其它昆虫的C型凝集素比对分析结果显示,OfIML属于鳞翅目免疫凝集素,并且含有一个独特的结构特征,即一前一后2个糖识别域,氨基末端(CRD1,aa#1-135)和羧基末端(CRD2,aa#136-287)。RT-PCR检测OfIML在幼虫组织中的分布结果表明,其在血细胞、表皮、脂肪体、中肠、马氏管和气管中都有表达。OfIML GenBank登录号为ABZ81710。OfIML是一种昆虫免疫凝集素,含有2个糖识别域,根据其分子结构及在组织分布中的结果显示可能在亚洲玉米螟的免疫反应中起重要作用。     

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.     

Development of an Argopecten-Specific 18S rRNA Targeted Genetic Probe

Comparison of 18S ribosomal RNA gene sequences between diverse bivalve species, including eight scallop species, allowed the design of an 18S rRNA targeted oligonucleotide probe (BS-1364) that was specific for scallops belonging to the genus Argopecten (bay and calico scallops). The high sequence similarity of the 18S rRNA gene between Argopecten irradians and Argopecten gibbus (98.8%) prevented the design of an A. irradians species-specific probe. Hybridization studies using amplified 18S rDNA from a diverse collection of bivalve species demonstrated that the specificity of the digoxygenin-labeled probe was consistent with the predicted specificity indicated by sequence comparison. Hybridization studies using laboratory-spawned bay scallop veligers indicated that a single veliger could be detected by probe hybridization in a blot format, and that probe hybridization signal was proportional (r ²= .99) to the abundance of veligers. Methods for rRNA extraction and blotting were developed that allowed bay scallop veligers to be specifically and quantitatively identified in natural plankton samples. Preliminary studies conducted in Tampa Bay, Florida, suggest that introduced scallops can successfully spawn and produce veligers under in situ conditions. The Argopecten-specific probe and methods developed in this study provide the means to study the production and fate of bay scallop larvae in nature and provide evidence that scallops introduced into Tampa Bay have the potential for successful reproduction and enhancement of scallop stocks. Received January 25, 1999; accepted May 7, 1999.     

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.     

Mannan-Binding Lectin of the Sea Urchin Strongylocentrotus nudus

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/β-protein with eight β-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.     

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.     

C-Type lectin-like domains in Caenorhabditis elegans: predictions from the complete genome sequence

Protein modules related to the C-type carbohydrate-recognition domains of animal lectins are found in at least 125 proteins encoded in the Caenorhabditis elegans genome. Within these proteins, 183 C-type lectin-like domains (CTLDs) have been identified. The proteins have been classified based on the overall arrangement of modules within the polypeptides and based on sequence similarity between the CTLDs. The C.elegans proteins generally have different domain organization from known mammalian proteins containing CTLDs. Most of the CTLDs are divergent in sequence from those in mammalian proteins. However, 19 show conservation of most of the amino acid residues that ligate Ca(2+)to form a carbohydrate-binding site in vertebrate C-type carbohydrate-recognition domains. Seven of these domains are particularly similar in sequence to mannose- and N-acetylglucosamine-binding domains in the vicinity of this Ca(2+)site.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Comparative physiology of young and old cohorts of bay scallop Argopecten irradians irradians (Lamarck): mortality,growth, and oxygen consumption

The bay scallop Argopecten irradians (Lamarck) undergoes rapid population decline in its second year of life. Pre-(1st-yr) and postreproductive (2nd-yr) bay scallops were held in cages at two sites on Long Island, New York, U.S. Survival, growth, and metabolic rates of the two cohorts were compared monthly throughout the autumn and winter. Second-year scallops, the harvestable crop of the year, maintained a positive energy balance until late November–December. Both age classes experienced similar relative tissue weight losses during overwintering (9–11% at the site where milder environmental conditions prevailed, and 24–25% at the more stressful site). Ambient water temperature explained a significant proportion (93%) of the seasonal variation in the rate of oxygen consumption. Thus the northern bay scallop A.i. irradians shows a limited ability to acclimatise oxygen consumption to seasonal temperature changes over the range of 1–23°C. A significant increase in oxygen uptake was associated with increased gametogenic activity of young scallops in May. Metabolic rate at this time was 50% higher than that predicted based on temperature, providing an estimate of the metabolic cost of reproduction in this species. The weight-normalized oxygen uptake rate of senescent scallops was significantly lower than that of young scallops. Mass natural mortality of the older cohort occurred during the winter, before the onset of a second gametogenesis; only 50% of the population survived beyond late January in 1985. Mortality was delayed by 2.5 months during a similar experiment conducted in 1986. Results of this study suggest that senescent mortality of New York bay scallop populations is not directly linked to the energy drain of a second reproductive event following overwintering stress.     

Development of Expressed Sequence Tags from the Bay Scallop, Argopecten irradians irradians

The bay scallop, Argopecten irradians irradians, introduced from North America, has become one of the most important aquaculture species in China. Inan effort to identify scallop genes involved in host defense, a high-quality cDNA library was constructed from whole body tissues of the bay scallop. A total of 5828 successful sequencing reactions yielded 4995 expressed sequence tags (ESTs) longer than 100 bp. Cluster and assembly analyses of the ESTs identified 637 contigs (consisting of 2853 sequences) and 2142 singletons, totaling 2779 unique sequences. Basic Local Alignment Search Tool (BLAST) analysis showed that the majority (73%) of the unique sequences had no significant homology (E-value ≤ 0.005) to sequences in GenBank. Among the 748 sequences with significant GenBank matches, 160 (21.4%) were for genes related to metabolism, 131 (17.5%) for cell/organism defense, 124 (16.6%) for gene/protein expression, 83 (11.1%) for cell structure/motility, 70 (9.4%) for cell signaling/communication, 17 (2.3%) for cell division, and 163 (21.8%) matched to genes of unknown functions. The list of host-defense genes included many genes with known and important roles in innate defense such as lectins, defensins, proteases, protease inhibitors, heat shock proteins, antioxidants, and Toll-like receptors. The study provides a significant number of ESTs for gene discovery and candidate genes for studying host defense in scallops and other molluscs.     

An immune responsive multidomain galectin from bay scallop Argopectens irradians

Galectins are a family of β-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from bay scallop Argopectens irradians (designated AiGal1) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of AiGal1 was of 2235 nucleotides, encoding a polypeptide of 549 amino acids. SMART program analysis revealed that AiGal1 contained four galectin CRDs, and all the CRDs contained the two consensus motifs essential for ligand-binding. Quantitative real-time PCR was employed to investigate the tissue distribution of AiGal1 mRNA and temporal expression in haemocytes of scallops challenged with Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The AiGal1 mRNA could be detected in all tested tissues with the highest expression level in hepatopancreas. After challenged by V. anguillarum and M. luteus, the expression level of AiGal1 mRNA was both up-regulated and reached the maximum level at 9 h (1.52 fold, P < 0.05) and 18 h (2.89 fold, P < 0.01) post challenge, respectively. However, there was no significant difference in the mRNA expression of AiGal1 in haemocytes after P. pastoris challenge (P > 0.05). These results collectively indicated that AiGal1 was a new member of the galectin family and involved in the immune responses against bacterial infection.