Single Carbohydrate Recognition Domain Research Articles

A novel C-type lectin, perlucin, from the small abalone, Haliotis diversicolor involved in the innate immune defense against Vibrio harveyi infection

C-type lectins (CTLs), a member of pattern recognition receptors, play an important role in the innate immunity by recognizing invading microorganisms. In this study, a novel perlucin gene (designated as HdPer 3), a typical CTLs was cloned and characterized from the small abalone Haliotis diversicolor. The open reading frame of HdPer 3 was 471 bp, encoding a protein of 156 amino acids that included a single carbohydrate-recognition domain. HdPer 3 was widely expressed in all tested tissues and developmental stage. HdPer 3 expression was significantly up-regulated after Vibrio harveyi infection, suggesting that HdPer 3 was activated in response to bacterial infection. The encapsulation ability of hemocytes could be significantly enhanced by the recombinant protein HdPer 3 (rHdPer 3). To understand the regulation mechanism of the HdPer 3, HdPer 3 was silenced in vivo by RNAi. Knocking down HdPer 3 decreased the hemocytes phagocytosis. Meanwhile, knocking down HdPer 3 can reduce the expression of 2 phagocytosis-related genes (Rab and Dynamin), TNF-α, and 2 MAPK pathway-related genes (MAPK-X1 and Ras) after V. harveyi infection. Moreover, HdPer 3 interference could increase the bacterial load in the hemolymph and the mortality of abalones after V. harveyi infection. All these results suggested that HdPer 3 played a crucial role in the defense against V. harveyi infection by recognizing bacterial pathogens and activating the expression of immune-related genes.

The crystal structure of Nictaba reveals its carbohydrate-binding properties and a new lectin dimerization mode.

Nictaba is a (GlcNAc)n-binding, stress-inducible lectin from Nicotiana tabacum that serves as a representative for the Nictaba-related lectins, a group of proteins that play pivotal roles in plant defense mechanisms and stress response pathways. Despite extensive research into biological activities and physiological role(s) of the lectin, the three-dimensional structure of Nictaba remained largely unknown. Here, we report crystal structures for Nictaba in the apo form and bound to chitotriose. The structures reveal that the Nictaba protomer has a jelly-roll fold, similar to the cucumber lectin Cus17, but exhibit a unique and previously unseen mode of dimerization. The chitotriose binding mode, similar to Cus17, centers around the central GlcNAc residue, providing insights into the determinants of specificity of Nictaba towards carbohydrate structures. By integrating these structural insights with inputs from glycan arrays, molecular docking, and molecular dynamics simulations, we propose that Nictaba employs a single carbohydrate-recognition domain within each of the two subunits in the dimer to display pronounced specificity towards GlcNAc-containing carbohydrates. Furthermore, we identified amino acid residues involved in the extended binding site capable of accommodating structurally diverse high-mannose and complex N-glycans. Glycan array and in silico analyses revealed interactions centered around the conserved Man3GlcNAc2 core, explaining the broad recognition of N-glycan structures. Collectively, the structural and biochemical insights presented here fill a void into the atlas of lectin structure-function relationships and pave the way for future developments in plant stress biology and lectin-based applications.

A novel exoskeletal-derived C-type lectin facilitates phagocytosis of hemocytes in the oriental river prawn Macrobrachium nipponense

C-type lectins (CTLs) execute critical functions in multiple immune responses of crustaceans as a member of pattern recognition receptors (PRRs) family. In this study, a novel CTL was identified from the exoskeleton of the oriental river prawn Macrobrachium nipponense (MnLec3). The full-length cDNA of MnLec3 was 1150 bp with an open reading frame of 723 bp, encoding 240 amino acids. MnLec3 protein contained a signal peptide and one single carbohydrate-recognition domain (CRD). MnLec3 transcripts were widely distributed at the exoskeleton all over the body. Significant up-regulation of MnLec3 in exoskeleton after Aeromonas hydrophila challenged suggested the involvement of MnLec3 as well as the possible function of the exoskeleton in immune response. In vitro tests with recombinant MnLec3 protein (rMnLec3) manifested that it had polysaccharide binding activity, a wide spectrum of bacterial binding activity and agglutination activity only for tested Gram-negative bacteria (Escherichia coli, Vibrio anguillarum and A. hydrophila). Moreover, rMnLec3 significantly promoted phagocytic ability of hemocytes against A. hydrophila in vivo. What's more, MnLec3 interference remarkably impaired the survivability of the prawns when infected with A. hydrophila. Collectively, these results ascertained that MnLec3 derived from exoskeleton took an essential part in immune defense of the prawns against invading bacteria as a PRR.

Molecular characterization and functional analysis of galectin-1 from silver pomfret (Pampus argenteus)

Galectins, as members of lectin families, exhibit a high affinity for β-galactosides and play diverse roles in biological processes. They function as pattern recognition receptors (PRRs) with important roles in immune defense. In this study, galectin-1, designated as SpGal-1, was identified and characterized from silver pomfret (Pampus argenteus). The SpGal-1 comprises an open reading frame (ORF) spanning 396 base pairs (bp) and encodes a deduced amino acid (aa) sequence containing a single carbohydrate recognition domain (CRD). Sublocalization analysis revealed that SpGal-1 was mainly expressed in the cytoplasm. The mRNA transcripts of SpGal-1 were ubiquitously detected in various tissues, with a higher expression level in the intestine. In addition, when exposed to Photobacterium damselae subsp. damselae (PDD) infection, both the liver and head kidney exhibited significantly increased SpGal-1 mRNA expression. The recombinant protein of SpGal-1 (named as rSpGal-1) demonstrated hemagglutination against red blood cells (RBCs) from Larimichthys crocea and P. argenteus in a Ca2+ or β-Mercaptoethanol (β-ME)-independent manner. Notably, rSpGal-1 could bind with various pathogen-associated molecular patterns (PAMPs) including D-galactose, D-mannose, lipopolysaccharide (LPS), and peptidoglycan (PGN), with highest affinity to PGN. Moreover, rSpGal-1 effectively interacted with an array of bacterial types encompassing Gram-positive bacteria (Staphylococcus aureus and Nocardia seriolae) and Gram-negative bacteria (PDD and Escherichia coli, among others), with the most robust binding affinity towards PDD. Collectively, these findings highlight that SpGal-1 is a crucial PRR with involvement in the host immune defense of silver pomfret.

A C-type lectin with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs from Tegillarca granosa is involved in the innate immune defense

C-type lectins (CTLs), a superfamily of Ca2+-dependent carbohydrate-recognition proteins, serve as pattern recognition receptors (PRRs) in the immune response of many species. However, little is currently known about the CTLs of the commercially and ecologically important bivalve species, blood clam (Tegillarca granosa). In this study, a CTL (designated as TgCTL-1) with a single carbohydrate-recognition domain (CRD) containing unique QPN/WDD motifs was identified in the blood clam through transcriptome and whole-genome searching. Multiple alignment and phylogenetic analysis strongly suggested that TgCTL-1 was a new member of the CTL superfamily. Expression analysis demonstrated that TgCTL-1 was highly expressed in the hemocytes and visceral mass of the clam under normal condition. In addition, the expression of TgCTL-1 was shown to be significantly up-regulated upon pathogen challenge. Moreover, the recombinant TgCTL-1 (rTgCTL-1) displayed agglutinating and binding activities against both the gram-positive and gram-negative bacteria tested in a Ca2+-dependent manner. Furthermore, it was found that the in vitro phagocytic activity of hemocytes was significantly enhanced by rTgCTL-1. In general, our results showed that TgCTL-1 was an inducible acute-phase secretory protein, playing crucial roles in recognizing, agglutinating, and binding to pathogenic bacteria as well as modulating phagocytic activity of hemocytes in the innate immune defense of blood clam.

A C-type lectin in saliva of Aedes albopictus (Diptera: Culicidae) bind and agglutinate microorganisms with broad spectrum.

Via complex salivary mixture, mosquitos can intervene immune response and be helpful to transmit several viruses causing deadly human diseases. Some C-type lectins (CTLs) of mosquito have been reported to be pattern recognition receptor to either resist or promote pathogen invading. Here, we investigated the expression profile and agglutination function of an Aedes albopictus CTL (Aalb_CTL2) carrying a single carbohydrate-recognition domain (CRD) and WND/KPD motifs. The results showed that Aalb_CTL2 was found to be specifically expressed in mosquito saliva gland and its expression was not induced by blood-feeding. The recombinant Aalb_CTL2 (rAalb_CTL2) could agglutinate mouse erythrocytes in the presence of calcium and the agglutinating activity could be inhibited by EDTA. rAalb_CTL2 also displayed the sugar binding ability to D-mannose, D-galactose, D-glucose, and maltose. Furthermore, it was demonstrated that rAalb_CTL2 could bind and agglutinate Gram positive bacteria Staphylococcus aureus and Bacillus subtilis, Gram negative bacteria Escherichia coli and Pseudomonas aeruginosa, as well as fungus Candida albicans in vitro in a calcium dependent manner. However, rAalb_CTL2 could not promote type 2 dengue virus (DENV-2) replication in THP-1 and BHK-21 cell lines. These findings uncover that Aalb_CTL2 might be involved in the innate immunity of mosquito to resist microorganism multiplication in sugar and blood meals to help mosquito survive in the varied natural environment.

Identification and functional characterization of C-type lectins and crustins provide new insights into the immune response of Portunus trituberculatus

A meticulous understanding of the immune characteristics of aquaculture animals is the basis for developing precise disease prevention and control strategies. In this study, four novel C-type lectins (PtCTL-5, PtCTL-6, PtCTL-7 and PtCTL-8) including a single carbohydrate-recognition domain (CRD), and four novel crustins (Ptcrustin-1, Ptcrustin-2, Ptcrustin-3 and Ptcrustin-4) with a single whey acidic protein (WAP) domain were identified from the swimming crab Portunus trituberculatus. Tissue distribution analysis indicated that most of the target genes were predominantly expressed in the hepatopancreas in all examined tissues, except for Ptcrustin-1 which were mainly expressed in the gills. Our results showed that the eight genes displayed various transcriptional profiles across different tissues. In hemocytes, the PtCTL-7 responded quickly to Vibrio alginolyticus and exhibited much more strongly up-regulation than other three PtCTLs. The Ptcrustin-1 rapidly responded to V. alginolyticus within 3 h in all the three tested tissues. Furthermore, recombinant proteins of PtCTL-5 and PtCTL-8 were successfully obtained, and both of them displayed bacterial binding activities toward V. alginolyticus, V. harveyi and Staphylococcus aureus, and only showed antibacterial activity against V. harveyi. These findings provided new insights into the diverse immune response of P. trituberculatus and laid theoretical foundations for the development of precise disease prevention and control strategies in P. trituberculatus farming. Moreover, the specific anti-V. harveyi activities exhibited by rPtCTL-5 and rPtCTL-8 suggested their promising application prospects for controlling diseases caused by V. harveyi.

Crucial roles of a novel exoskeletal-derived lectin in innate immunity of the oriental river prawn, Macrobrachium nipponense.

As important pattern recognition receptors (PRRs), C-type lectins play crucial roles in the crustacean innate immune system. In this study, a novel C-type lectin, designated as MnLec1, was obtained from the exoskeleton of the oriental river prawn Macrobrachium nipponense for the first time. The full-length cDNA of MnLec1 was 1329bp with an open reading frame of 774bp. The predicted MnLec1 protein contains a single carbohydrate-recognition domain with an EPN/LND motif and one Ca2+ binding site-2. MnLec1 transcripts were widely detected in the tested tissues of M.nipponense and significantly up-regulated after Aeromonas hydrophila challenge. The recombinant MnLec1 protein was found to have a wide spectrum of binding activities towards various microorganisms, agglutinate two kinds of Gram-negative bacteria (Escherichia coli and A.hydrophila) in a Ca2+ -independent manner. What's more, the survivability of prawns was significantly down-regulated after RNAi of MnLec1 when infected with A.hydrophila. Collectively, these findings suggest that MnLec1 from the exoskeleton might function as a PRR and play a crucial role in immune defense against invading pathogens in M.nipponense.

A C-type lectin with a single CRD from Onychostoma macrolepis mediates immune recognition against bacterial challenge

C-type lectins (CTL) are a large group of pattern-recognition proteins and to play important roles in glycoprotein metabolism, multicellular integration, and immunity. Based on their overall domain structure, they can be classified as different groups that possess different physiological functions. A typical C-type lectin (named as OmLec1) was identified from the fish, Onychostoma macrolepis, an important cultured fish in China. Open reading frame of OmLec1 contains a 570 bp, encoding a protein of 189 amino acids that includes a signal peptide and a single carbohydrate-recognition domain. The phylogenetic analysis showed that OmLec1 could be grouped with C-type lectin from other fish. OmLec1 was expressed in all the tissues in our study, and the expression level was highest in liver. And its relative expression levels were significantly upregulated following infection with Aeromonas hydrophila. The recombinant OmLec1 protein (rOmLec1) could agglutinate some Gram-negative bacteria and Gram-positive bacteria in vitro in the presence of Ca2+, showing a typical Ca2+-dependent carbohydrate-binding protein. Furthermore, rOmLec1 purified from E. coli BL21 (DE3), strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-dependent manner. These results indicate that OmLec1 plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

A C-Type Lectin Highly Expressed in Portunus trituberculatus Intestine Functions in AMP Regulation and Prophenoloxidase Activation.

A C-type lectin (PtCLec2) from Portunus trituberculatus was identified for characterization of its role in defense and innate immunity. PtCLec2 contains a single carbohydrate-recognition domain (CRD) with a conserved QPD motif, which was predicted to have galactose specificity. The mRNA expression of PtCLec2 was predominantly detected in intestine and increased rapidly and significantly upon pathogen challenge. The recombinant PtCLec2 (rPtCLec2) could bind various microorganisms and PAMPs with weak binding ability to yeast and PGN. It agglutinated the tested Gram-negative bacteria (Vibrio alginolyticus and Pseudomonas aeruginosa), Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus), and rabbit erythrocytes in the presence of exogenous Ca2+, and these agglutination activities were suppressed by LPS, d-galactose, and d-mannose. Further, rPtCLec2 enhanced phagocytosis and clearance of V. alginolyticus, and displayed inhibitory activities against the tested bacteria. Knockdown of PtCLec2 decreased the transcription of two phagocytosis genes (PtArp and PtMyosin), three prophenoloxidase (proPO) system-related genes (PtPPAF, PtcSP1, and PtproPO), six antimicrobial peptides (AMPs) (PtALF4-7, PtCrustin1, and PtCrustin3), and PtRelish but upregulated the expression levels of PtJNK, PtPelle, and PtTLR. These results collectively indicate that PtCLec2 might perform its immune recognition function via binding and agglutination, and mediate pathogen elimination via regulating hemocyte phagocytosis, AMP synthesis, and proPO activation.

In silico characterization and expression analysis of eight C-type lectins in obscure puffer Takifugu obscurus

C-type lectins (CTLs) are a group of carbohydrate-binding proteins that play crucial roles in innate immune defense against invading pathogens. CTLs have been extensively studied in lower vertebrates, such as fish, for their roles in eliminating pathogens; however, their homologs in pufferfish are not well known. In the present study, eight CTLs from obscure puffer Takifugu obscurus (designated as ToCTL3–10 according to the order they were discovered) were obtained. All predicted ToCTL proteins contained a single carbohydrate recognition domain (CRD). ToCTL7 also contained one calcium-binding epidermal growth factor (EGF)-like domain (EGF_CA) and a transmembrane region. ToCTL9 also contained an SCP domain, an EGF domain, and an EGF-like domain. Bioinformatics analysis revealed that ToCTL3–10 mainly clustered with the corresponding CTL homologs of other pufferfish species. Tissue distribution analysis detected ToCTL3–10 in all tissues examined, including kidneys, liver, gills, spleen, intestines, and heart. Moreover, the expressions of ToCTL3–10 were significantly induced in the kidneys of obscure puffer following challenges with three Gram-negative bacterial pathogens, namely, Vibrio harveyi, Aeromonas hydrophila, and Edwardsiella tarda, and a synthetic analog of double-stranded RNA poly(I:C). The expression patterns of ToCTL3–10 in response to different immune stimulants were different. Our results indicated that the eight ToCTLs obtained herein might be involved in host defense against bacterial and poly(I:C) infections in T. obscurus.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

Functions of a C-type lectin with a single carbohydrate-recognition domain in the innate immunity and movement of the red flour beetle, Tribolium castaneum.

C-type lectins (CTLs) are a superfamily of proteins found in almost all vertebrates and invertebrates. They play an important role in innate immune defences, development and epidermal structure. Here, a CTL with one carbohydrate-recognition domain containing a highly conserved Gln-Pro-Asp (QPD) motif was identified in Tribolium castaneum and given the name TcCTL5. Spatiotemporal analyses showed that Tcctl5 was highly expressed in the late pupa stage and mainly existed in the central nervous system and haemolymph. The transcript level of Tcctl5 was prominently induced after bacterial infection. Recombinant TcCTL5 proteins (rTcCTL5) were found to bind to lipopolysaccharide, peptidoglycan and tested bacteria and induce microbial agglutination in the presence of Ca2+ . Interestingly, when Tcctl5 was knocked down, the transcript level of antimicrobial peptides (AMPs) (attacin1, defensins3, coleoptericin1 and cecropins3) was prominently downregulated after induction with Gram-negative Escherichia coli. More interestingly, Tcctl5 was knocked down, leading to increased mortality and loss of locomotor activity, which exhibited less travel distances among early adults. These results demonstrate that Tcctl5 plays an important role in the innate immune reaction and the movement of T. castaneum. Thus, it may represent an alternative molecular target for pest control and thus reduce the use of pesticides in agricultural production.

A typical C-type lectin, perlucin-like protein, is involved in the innate immune defense of whiteleg shrimp Litopenaeus vannamei

C-type lectins are a large group of the pattern-recognition proteins, and have been reported to be involved in invertebrate innate immunity, such as cell adhesion, bacterial clearance, phagocytosis, prophenoloxidase activation and encapsulation. Here, a perlucin-like protein (PLP), a typical C-type lectin, was identified from the cDNA library of the shrimp, Litopenaeus vannamei. LvPLP contains a 540 bp open reading frame, encoding a protein of 179 amino acids that includes a single carbohydrate-recognition domain. Phylogenetic analysis showed that LvPLP was clustered into a single group together with other perlucins from molluscs. Quantitative real-time PCR revealed that LvPLP was expressed mainly in the hemocytes, hemolymph, heart and gills. The transcription of LvPLP was significantly induced at 9 h by both Gram− bacteria Vibrio parahaemolyticus and Vibrio anguillarum. Meanwhile, recombinant LvPLP (rLvPLP) bound directly to lipopolysaccharide and peptidoglycan with different affinity. rLvPLP showed a strong ability to bind to Gram+ (Staphylococcus aureus and Bacillus subtilis) and Gram− bacteria (V. parahaemolyticus and V. anguillarum), and could induce agglutination of V. parahaemolyticus and V. anguillarum, but not S. aureus and B. subtilis in the presence Ca2+. Further study showed that when LvPLP was knocked down by RNAi, three phagocytosis-related genes (peroxinectin, mas-like protein and dynamin) and four antimicrobial peptide (AMP) genes (crustin, ALF1, ALF2 and ALF3) were significantly decreased. Altogether, these results demonstrated that LvPLP played a vital role in L. vannamei immune response towards bacterial challenge by binding and agglutinating bacteria and influencing phagocytosis and AMP expression.

A stomach-specific lectin plays a crucial role in the innate immunity of red swamp crayfish, Procambarus clarkii

C-type lectins (CTLs) play important roles in innate immune system of crustaceans as pattern recognition receptors (PRRs). In this study, a novel CTL gene was obtained from the red swamp crayfish, Procambarus clarkii, designated as PcLec. PcLec encodes a peptide with 175 amino acids, with a signal peptide and a single carbohydrate recognition domain (CRD). The PcLec transcripts were specifically expressed in crayfish stomach and were induced by bacterial challenge. In vitro assays with recombinant PcLec protein revealed that it had bacterial binding activity, polysaccharide binding activity, bacterial agglutination activity, and antimicrobial activity. Most importantly, PcLec knockdown significantly impaired the survivability of crayfish upon oral infection with its pathogen A. hydrophila. According to these results, we infer that the PcLec plays a crucial role in antibacterial defense of crayfish.

A novel C-type lectin with a YPD motif from Portunus trituberculatus (PtCLec1) mediating pathogen recognition and opsonization

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins that function as pattern recognition receptors (PRRs) in innate immune system. In this study, a new C-type lectin was identified from the swimming crab Portunus trituberculatus (PtCLec1). The full-length cDNA of PtCLec1 was 873 bp encoding 176 amino acids. The predicted PtCLec1 protein contained a signal peptide and a single carbohydrate-recognition domain with a special YPD motif. The PtCLec1 transcripts were mainly detected in hepatopancreas and its relative expression levels were significantly up-regulated after the challenges of Vibrio alginolyticus, Micrococcus luteus and Pichia pastoris. The recombinant PtCLec1 (rPtCLec1) could bind all the tested pathogen-associated molecular patterns (PAMPs), including lipopolysaccharides (LPS), peptidoglycan (PGN) and glucan (GLU), and microorganisms, including V. alginolyticus, V. parahaemolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, M. luteus and P. pastoris. It also exhibited strong activity to agglutinate bacteria and yeast in a Ca2+-dependent manner, and such agglutinating activity could be inhibited by d-galactose and LPS. Moreover, rPtCLec1 revealed antimicrobial activity against the tested Gram-negative (V. alginolyticus, V. parahaemolyticus and P. aeruginosa) and Gram-positive bacteria (S. aureus and M. luteus), and promoted the clearance of V. alginolyticus in vivo and hemocyte phagocytosis in vitro. Knockdown of PtCLec1 could down-regulate the expression of phagocytosis-related genes, but enhance the expression levels of prophenoloxidase (proPO) system-related genes, mannose-binding lectin (MBL), antimicrobial peptides (AMPs), MyD88 and Relish. All these results indicate that PtCLec1 might act as a PRR in immune recognition and an opsonin in pathogen elimination.

Two C-type lectins (ReCTL-1, ReCTL-2) from Rimicaris exoculata display broad nonself recognition spectrum with novel carbohydrate binding specificity

C-type lectins are Ca2+-dependent carbohydrate-binding proteins containing one or more carbohydrate-recognition domains (CRDs). C-type lectins play crucial roles in innate immunity, including nonself-recognition and pathogen elimination. In the present study, two C-type lectins (designated ReCTL-1 and ReCTL-2) were identified from the shrimp Rimicaris exoculata which dwells in deep-sea hydrothermal vents. The open reading frames of ReCTL-1 and ReCTL-2 encoded polypeptides of 171 and 166 amino acids respectively, which were both composed of a signal peptide and a single CRD. The key motifs determining the carbohydrate binding specificity of ReCTL-1 and ReCTL-2 were respectively Glu–Pro–Ala (EPA) and Gln–Pro–Asn (QPN), which were firstly discovered in R. exoculata. ReCTL-1 and ReCTL-2 displayed similar pathogen-associated molecular pattern (PAMP) binding features and they bound three PAMPs—β-glucan, lipopolysaccharide and peptidoglycan—with relatively high affinity. In addition, both could efficiently recognize and bind Gram-positive bacteria, Gram-negative bacteria and fungi. However, ReCTL-1 and ReCTL-2 exhibited different microbial agglutination activities: ReCTL-1 agglutinated Staphylococcus aureus and Saccharomyces cerevisiae, while ReCTL-2 agglutinated Micrococcus luteus, Vibrio parahaemolyticus and V. fluvialis. Both ReCTL-1 and ReCTL-2 inhibited the growth of V. fluvialis. All these results illustrated that ReCTL-1 and ReCTL-2 could function as important pattern-recognition receptors with broad nonself-recognition spectra and be involved in immune defense against invaders, but their specificities are not the same. In addition, the two ReCTLs possessed different carbohydrate binding specificities from each other and from the classical pattern: ReCTL-1 with an EPA motif bound d-galactose and l-mannose, while ReCTL-2 with a QPN motif bound d-fucose and N-acetylglucosamine.

A DNA Aptamer Targeting Galectin-1 as a Novel Immunotherapeutic Strategy for Lung Cancer

Galectin-1 (Gal-1) is a pleiotropic homodimeric β-galactoside-binding protein with a single carbohydrate recognition domain. It has been implicated in several biological processes that are important during tumor progression. Several lines of evidence have indicated that Gal-1 is involved in cancer immune escape and induces T cell apoptosis. These observations all emphasized Gal-1 as a novel target for cancer immunotherapy. Here, we developed a novel Gal-1-targeting DNA aptamer (AP-74 M-545) and demonstrated its antitumor effect by restoring immune function. AP-74 M-545 binds to Gal-1 with high affinity. AP-74 M-545 targets tumors in murine tumor models but suppresses tumor growth only in immunocompetent C57BL/6 mice, not in immunocompromised non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. Immunohistochemistry revealed increased CD4+ and CD8+ T cells in AP-74 M-545-treated tumor tissues. AP-74 M-545 suppresses T cell apoptosis by blocking the binding of Gal-1 to CD45, the main receptor and apoptosis mediator of Gal-1 on T cells. Collectively, our data suggest that the Gal-1 aptamer suppresses tumor growth by blocking the interaction between Gal-1 and CD45 to rescue T cells from apoptosis and restores T cell-mediated immunity. These results indicate that AP-74 M-545 may be a potential strategy for cancer immunotherapy.

Identification and Characterization of a Novel Quadruple-Domain Galectin from the Hong Kong Oyster Crassostrea hongkongensis

Galectins, i.e. β-galactoside-binding animal lectins, play essential roles in the innate immunity systems of vertebrates and invertebrates. In the present study, a full-length cDNA coding for a galectin from the Hong Kong oyster Crassostrea hongkongensis (designated ChGal) was cloned and characterized. The complete cDNA of ChGal included an open reading frame (ORF) of 1668 bp, as well as 5′- and 3′-untranslated regions (UTRs) of 75 bp and 302 bp, respectively. The ORF coded for a putative protein of 555 amino acids with an estimated molecular mass of 63.4 kDa and a theoretical isoelectric point of 5.0. Sequence analysis revealed that ChGal contains four carbohydrate recognition domains (CRDs), each containing the conserved carbohydrate-binding motifs H-NPR and WG-ER. Phylogenetic analysis produced an unrooted tree with four clades: single CRDs, C-terminal CRDs with tandem-repeat galectin clade, N-terminal CRDs with tandem-repeat galectin clade, and quadruple-CRD clusters. ChGal clustered in the quadruple-domain galectins, which are divided into four subgroups on the basis of the position of each individual CRD. Recombinant ChGal exhibited strong agglutinating activity against Escherichia coli, Vibrio alginolyticus, and Bacillus thuringiensis. ChGal mRNA was constitutively expressed in all tissues tested, with the highest level detected in the digestive gland. The expression of ChGal significantly increased in haemocytes in response to V. alginolyticus challenge. These results suggest that ChGal play an important role in the innate immune responses of the Hong Kong oyster.

2-Transmembrane C-type lectin from oriental river prawn Macrobrachium nipponense participates in antibacterial immune response

As a type of pattern-recognition proteins (PRRs), C-type lectins (CTLs) perform important functions in non-self recognition and clearance of pathogens in innate immunity. In this study, a unique 2-transmembrane CTL (designated as Mn-2TM-cLec) with a single carbohydrate recognition domain (CRD) was isolated from Macrobrachium nipponense. The full-length cDNA of Mn-2TM-cLec consisted of 3265 bp with an 837 bp open reading frame encoding a protein with 278 amino acids. Mn-2TM-cLec was ubiquitously distributed in various tissues of normal prawn, particularly in the hemocytes, hepatopancreas, and gills. The expression of Mn-2TM-cLec was significantly up-regulated in the gills and hepatopancreas after the prawns were challenged with Staphylococcus aureus and Vibrio parahaemolyticus. RNA interference knock-down of Mn-2TM-cLec gene decreased the transcription levels of three antimicrobial peptides (anti-lipopolysaccharide factor (ALF) 1, ALF2, and Crustin (Crus) 1) after V. parahaemolyticus infection. The recombinant CRD of Mn-2TM-cLec could bind lipopolysaccharide, peptidoglycans, and diverse bacterial strains and agglutinate S. aureus and V. parahaemolyticus in a Ca2+-dependent manner. In addition, the rCRD enhanced the clearance of V. parahaemolyticus injected in prawns. In summary, Mn-2TM-cLec might act as a PRR to participate in the prawn immune defense against pathogens through its antimicrobial activity.