Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

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.     

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 (Cys⁵³, Cys¹²⁸, Cys¹⁴⁴, Cys¹⁵²) 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.     

Molecular cloning,characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper,Epinephelus coioides

C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell–cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys⁷¹, Cys¹⁵², Cys¹⁶⁷, Cys¹⁷⁵) involved in the formation of disulphide bridges and the potential Ca²⁺/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca²⁺-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca²⁺-dependent manner.     

Expression of allograft inflammatory factor-1 (AIF-1) in response to bacterial challenge and tissue injury in the pearl oyster,Pinctada martensii

Allograft inflammatory factor-1 (AIF-1), an interferon (IFN)-γ-inducible calcium-binding cytokine, is associated with the inflammatory response and defense. We cloned and analyzed the expression pattern of the AIF-1 gene of the pearl oyster Pinctada martensii, hereafter designated PmAIF-1. The full-length PmAIF-1 cDNA is 946 bp in length and consists of a 5′-untranslated region (UTR) of 120 bp, a 3′-UTR of 376 bp, and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with an estimated molecular mass of 17 kDa. Sequence analysis reveals that PmAIF-1 contains two EF hand Ca⁺²-binding motifs like those in previously characterized AIF-1s while alignment with known AIF-1 protein sequences reveals higher similarity to invertebrate orthologs than to those of vertebrates.Quantitative PCR analysis reveals that PmAIF-1 is constitutively expressed, with the highest expression detected in hemocytes, and the expression level of PmAIF-1 mRNA was significantly up-regulated in hemocytes, gill, digestive gland under bacterial challenge and tissue injury. After challenged by gram-negative bacteria Vibrio alginolyticus and Vibrio parahaemolyticus, gram-positive bacteria Bacillus subtilis, the expression level of this gene in hemocytes were all up-regulated and reached the maximum point at 12 h (5.80 folds, P < 0.01), 6 h (5.02 folds, P < 0.01) and 12 h (5.49 folds, P < 0.01), respectively. Under shell damage and mantle injury, PmAIF-1 mRNA increased gradually in the first 3 h and reached a peak of expression at 6 h post-injury. These findings suggest that PmAIF-1 is an acute-response protein involved in the innate immune responses of pearl oysters, and provide general information about the mechanisms of innate immune defense against bacterial infection in pearl oysters.     

SpALF4: A newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand β-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.     

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.     

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.     

Mechanism for Recognition of an Unusual Mycobacterial Glycolipid by the Macrophage Receptor Mincle

Binding of the macrophage lectin mincle to trehalose dimycolate, a key glycolipid virulence factor on the surface of Mycobacterium tuberculosis and Mycobacterium bovis, initiates responses that can lead both to toxicity and to protection of these pathogens from destruction. Crystallographic structural analysis, site-directed mutagenesis, and binding studies with glycolipid mimics have been used to define an extended binding site in the C-type carbohydrate recognition domain (CRD) of bovine mincle that encompasses both the headgroup and a portion of the attached acyl chains. One glucose residue of the trehalose Glcα1–1Glcα headgroup is liganded to a Ca²⁺ in a manner common to many C-type CRDs, whereas the second glucose residue is accommodated in a novel secondary binding site. The additional contacts in the secondary site lead to a 36-fold higher affinity for trehalose compared with glucose. An adjacent hydrophobic groove, not seen in other C-type CRDs, provides a docking site for one of the acyl chains attached to the trehalose, which can be targeted with small molecule analogs of trehalose dimycolate that bind with 52-fold higher affinity than trehalose. The data demonstrate how mincle bridges between the surfaces of the macrophage and the mycobacterium and suggest the possibility of disrupting this interaction. In addition, the results may provide a basis for design of adjuvants that mimic the ability of mycobacteria to stimulate a response to immunization that can be employed in vaccine development.     

Molecular cloning and expression profiles of nitric oxide synthase (NOS) in mud crab Scylla paramamosain

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in vertebrates and invertebrates in recent years. However, it keeps unknown of nitric oxide (NO) system and NOS gene family in mud crab Scylla paramamosain, an important cultured commercial crustacean in China and Pacific area. In this report, the cDNA of NOS containing full-length ORF was cloned from mud crab, S. paramamosain. It was of 4424 bp, including a 5′-terminal untranslated region (UTR) of 239 bp, a 3′-terminal UTR of 540 bp, which contained two ATTTA motifs, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids. Structural analysis indicated that NOS contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time PCR analysis revealed S. paramamosain NOS (SpNOS) to be expressed in all tissues examined, with the highest expression in midintestine and the weakest level in heart and eyestalk. The expression profiles of SpNOS indicated that the NOS expression levels were significantly induced in midintestine, hepatopancrease and hemocytes after challenged with Vibrio Parahaemolyticus, the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). The NOS activity in hemocytes showed significant increase during at 24 h-48 h time period after immune challenges with V. Parahaemolyticus, poly I:C and LPS. Results here may suggest that the inducible NOS play an important role in mud crab’s defense against pathogenic infection.     

C-type lectin in Chlamys farreri (CfLec-1) mediating immune recognition and opsonization

Background

C-type lectins are a superfamily of Ca²⁺ 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.