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L-Type lectins (LTLs) contain a luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These type I membrane proteins are involved in the early secretory pathway of animals, and have functions in glycoprotein sorting, trafficking and targeting. Recent studies suggest that LTLs may be involved in immune responses in vertebrates, but no functional studies have been reported. This study reports an LTL, designated as MjLTL1, from the kuruma shrimp Marsupenaeus japonicus. MjLTL consists of a signal peptide, leguminous lectin domain, and transmembrane region. It was upregulated following challenge of shrimp with Vibrio anguillarum. MjLTL1 could agglutinate several bacteria with the presence of calcium, and bind to several Gram-positive and Gram-negative bacteria through lipopolysaccharide and peptidoglycan binding. MjLTL1 could enhance the clearance of V. anguillarum in vivo. MjLTL1 silencing by RNA interference could impair bacterial clearance ability. Further study suggested that MjLTL1 promoted hemocyte phagocytosis. To analyze the possible mechanism, a disintegrin and metalloprotease-like protein (MjADAM) mediating the proteolytic release of extracellular domains from the membrane-bound precursors was also studied in the shrimp. MjADAM exhibited similar tissue location and expression profiles to MjLTL1. After knockdown of MjADAM, the hemocyte phagocytosis rate also declined significantly. ADAM was reported to have an ectodomain shedding function to LTL and release the ectodomain of the lectin from cell membrane. Therefore, our results suggest that the extracellular domain of MjLTL1 might be released from the cell surface as a soluble protein by MjADAM, and function as an opsonin involved in the antibacterial immune responses in shrimp., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lectins play important roles in many biological processes, including protein trafficking, cell signaling, pathogen recognition, as effector molecules, and so on, because of their capacity to bind carbohydrates. Presently, seven groups of lectins have been identified in shrimp: C-type, L-type, P-type, M-type, fibrinogen-like domain lectins, galectins, and calnexin/calreticulin. These lectins have different structures, diverse expression patterns, and multiple functions in the shrimp immune response. This review summarizes the research progress and analyzes the diversity of shrimp lectins, focusing mainly on the C-type lectin family. Shrimp C-type lectins show considerable diversity in their domain architectures, sugar substrates, tissue distributions, expression patterns responding to pathogen challenge and functions in shrimp immunity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

C-type lectins (CTLs) are characterized by the presence of a C-type carbohydrate recognition domain (CTLD) that by recognizing microbial glycans, is responsible for their roles as pattern recognition receptors in the immune response to bacterial infection. In addition to the CTLD, however, some CTLs display additional domains that can carry out effector functions, such as the collagenous domain of the mannose-binding lectin. While in vertebrates, the mechanisms involved in these effector functions have been characterized in considerable detail, in invertebrates they remain poorly understood. In this study, we identified in the kuruma shrimp (Marsupenaeus japonicus) a structurally novel CTL (MjCC-CL) that in addition to the canonical CTLD, contains a coiled-coil domain (CCD) responsible for the effector functions that are key to the shrimp’s antibacterial response mediated by antimicrobial peptides (AMPs). By the use of in vitro and in vivo experimental approaches we elucidated the mechanism by which the recognition of bacterial glycans by the CTLD of MjCC-CL leads to activation of the JAK/STAT pathway via interaction of the CCD with the surface receptor Domeless, and upregulation of AMP expression. Thus, our study of the shrimp MjCC-CL revealed a striking functional difference with vertebrates, in which the JAK/STAT pathway is indirectly activated by cell death and stress signals through cytokines or growth factors. Instead, by cross-linking microbial pathogens with the cell surface receptor Domeless, a lectin directly activates the JAK/STAT pathway, which plays a central role in the shrimp antibacterial immune responses by upregulating expression of selected AMPs. [ABSTRACT FROM AUTHOR]

Increasing number of evidence suggests that the hemolymph of numerous apparently healthy invertebrates is unsterile. Investigation of hemolymph microbiota properties and the homeostasis between host and bacteria is helpful to reveal bacteria pathogenesis, host immunity, and possible utilization in disease control. Crustaceans represent a large family of aquatic animals. Therefore, crustacean fishery is of important economic value worldwide. Research related to crustacean hemolymph microbiota has been performed over the years. In the present study, we conclude currently available information and present a comprehensive analysis regarding homeostasis between host and bacteria. In general, the presence of microbiota in crustacean hemolymph is an endemic event and can be influenced by internal and external factors. Opportunistic bacteria may have generated some changes or mutations under hemolymph stress. Meanwhile, hosts suppress hemolymph microbiota proliferation with the help of some critical antimicrobial peptides and lectins. The hemolymph microbiota may be beneficial for hosts as resistance against external damages. In addition, the hemolymph microbiota may be utilized in aquaculture. [ABSTRACT FROM AUTHOR]

Highlights: [•] Both rEsLecA and rEsLecG could bind towards wide range of microorganisms. [•] Both rEsLecA and rEsLecG induced the aggregation against microbial pathogens. [•] Both rEsLecA and rEsLecG exhibited microbial inhibition and even bactericidal activity. [•] Both rEsLecA and rEsLecG could stimulate the cellular encapsulation in vitro. [Copyright &y& Elsevier]

Abstract: As pattern recognition receptors (PRRs), C-type lectins (CTLs) play significant roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (EsLecD) was identified from the crustacean Eriocheir sinensis. The cloning of full-length EsLecD cDNA was based on the initial expressed sequence tags (ESTs) isolated from a hepatopancreatic cDNA library. The full-length EsLecD cDNA of 686 bp with an open reading frame of 468 bp encodes a putative protein of 155 aa residues, including an N-terminal signal peptide and a single carbohydrate-recognition domain (CRD). By quantitative RT-PCR analysis, the EsLecD transcript was mainly detected in the hepatopancreas but rarely in other tissues, and it was significantly upregulated in the hepatopancreas after immune challenge with lipopolysaccharides. The recombinant EsLecD protein (rEsLecD) exhibited the ability to bind to all tested microorganisms, including bacteria and yeast. Meanwhile, calcium significantly increased the binding affinity of rEsLecD toward microorganisms, but it was not essential. The binding of rEsLecD induced the aggregation of microbial pathogens. Moreover, rEsLecD was capable of inhibiting the growth of microorganisms and even directly killing bacteria. Interestingly, rEsLecD could stimulate cellular encapsulation in vitro. In conclusion, results of this study suggest that EsLecD acts as an antibacterial PRR participating in the innate immunity of invertebrates. [Copyright &y& Elsevier]

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate. [ABSTRACT FROM AUTHOR]

Pattern recognition receptors (PPRs) are part of the initial step of a host defense against pathogens in detecting pathogen-associated molecular patterns. However, determinants of the specificity of this recognition by innate immune molecules of invertebrates remain largely unknown. In this study, we investigated the potential involvement of an invertebrate PRR C-type lectin in the antimicrobial response of the crustacean Eriocheir sinensis. Based on the initial expressed sequence tags (EST) of a hepatopancreatic cDNA library, the full-length EsLecF cDNA was cloned and determined to contain a 477-bp open reading frame encoding a putative 158-amino-acid protein. A comparison with other reported invertebrate and vertebrate C-type lectin superfamily sequences revealed the presence of a common carbohydrate recognition domain (CRD). EsLecF transcripts in E. sinensis were mainly detected in the hepatopancreas and were inducible by a lipopolysaccharide (LPS) injection. The recombinant EsLecF (rEsLecF) protein produced via a prokaryotic expression system and affinity chromatography was found to have a wide spectrum of binding activities towards various microorganisms, and its microbial-binding activity was calcium-independent. Moreover, the binding of rEsLecF induced the aggregation of microbial pathogens. Results of the microorganism growth inhibitory assay and antibacterial assay revealed capabilities of rEsLecF in suppressing microorganism growth and directly killing bacteria, respectively. Furthermore, rEsLecF could enhance cellular encapsulation in vitro. Collectively, the findings presented here demonstrated the successful isolation of a novel C-type lectin in a crustacean and highlighted its critical role in the innate immunity of an invertebrate. [ABSTRACT FROM AUTHOR]

Abstract: The basic mechanism of host fighting against pathogens is pattern recognition receptors recognized pathogen-associated molecular patterns. However, the specificity of recognition within the innate immune molecular of invertebrates remains largely unknown. For this reason, we investigated the immune functionality of two pattern recognition receptors, C-type lectin EsLecA and EsLecG, post lipopolysaccharides (LPS) challenge in Chinese mitten crab (Eriocheir sinensis), which is a commercially important and disease vulnerable aquaculture species. The cloning of full-length EsLecA and EsLecG cDNA were based on the initial expressed sequence tags (EST) isolated from a hepatopancreatic cDNA library via PCR. The EsLecA cDNA contained a 480-bp open reading frame that encoded a putative 159-amino-acid protein, while EsLecG cDNA contained a 465-bp open reading frame that encoded a putative 154-amino-acid protein. Comparison, with other reported invertebrate and vertebrate sequences, revealed the presence of carbohydrate recognition domains that were common among C-type lectin superfamilies. EsLecA and EsLecG mRNA expression in E. sinensis were (a) both detected in all tissues, including the hepatopancreas, gills, hemocytes, testis, accessory gland, ovary, muscle, stomach, intestine, heart, thoracic ganglia and brain, and (b) responsive in hepatopancreas, gill, hemocytes post-LPS immuno-challenge all appeared dramatically variation. Collectively, the data presented here demonstrate the successful isolation of two novel C-type lectins from the Chinese mitten crab, and their role in the innate immune system of an invertebrate. [Copyright &y& Elsevier]

C-type lectins are one family of pattern recognition receptors (PRRs) that play important roles in innate immunity. In this work, cDNA and genomic sequences for a new C-type lectin ( FcLec5) were obtained from the Chinese white shrimp Fenneropenaeus chinensis. FcLec5 cDNA contains an open reading frame of 1,008 bp and its genomic sequence is 1,137 bp with 4 exons and 3 introns. The predicted FcLec5 protein contains a signal peptide and two carbohydrate recognition domains (CRDs). The N-terminal CRD of FcLec5 has a predicted carbohydrate recognition motif of Gln-Pro-Asp (QPD), while the C-terminal CRD contains a motif of Glu-Pro-Gln (EPQ). Northern blot analysis showed that FcLec5 mRNA was specifically expressed in hepatopancreas. FcLec5 protein was expressed in hepatopancreas and secreted into hemolymph. Real-time PCR showed that FcLec5 transcript exhibited different expression profiles after immune-challenged with Vibrio anguillarum or White Spot Syndrome Virus (WSSV). Recombinant FcLec5 and its two individual CRDs could agglutinate most bacteria tested, and the agglutinating activity was Ca-dependent. Besides, the agglutinating activity to gram-negative bacteria is higher than that to gram-positive bacteria. Direct binding assay showed that recombinant FcLec5 could bind to all microorganisms tested (five gram-positive and four gram-negative bacteria, as well as yeast) in a Ca-independent manner. Recombinant FcLec5 also directly bound to bacterial peptidoglycan, lipopolysaccharide and lipoteichoic acids. These results suggest that FcLec5 may act as a PRR for bacteria via binding to bacterial cell wall polysaccharides in Chinese white shrimp. [ABSTRACT FROM AUTHOR]

Phagocytosis is a conserved cellular response among metazoans. Opsonins are some molecules that label targets to increase their susceptibility to phagocytosis. Opsonins are usually captured by receptors on the surface of phagocytes. Our previous study found the C-type lectin FcLec4 from Chinese white shrimp Fenneropenaeus chinensis might function as an opsonin to facilitate bacterial clearance. In the present study we purified the native FcLec4 protein and confirmed its opsonic activity in the near relation, kuruma shrimp Marsupenaeus japonicus. The possible receptor of FcLec4 was identified as β-integrin by panning a T7 phage display library of shrimp hemocytes and then confirmed by co-immunoprecipitation assay. We further proved that the interaction between FcLec4 and β -integrin did not rely on the carbohydrate recognition domain but on the N terminus of FcLec4. In addition, inhibition of FcLec4 expression using RNAi delayed bacterial clearance, and β -integrin knockdown suppressed the opsonic activity of FcLec4. This study is the first to show the direct interaction between an opsonin and its receptor in crustaceans. Our study provides new insights into invertebrate phagocytosis and the functions of C-type lectins. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are key recognition proteins in shrimp immunity. A few years ago we reviewed sequence information, ligand specificity, expression profiles and specific functions of the shrimp CTLs. Since then, multiple integrated studies that implemented biochemical approaches using both the native and recombinant proteins, functional genetic approaches using RNA interference, and mechanistic studies by analyzing protein-protein interactions were carried out. Results from these rigorous studies revealed the functions and mechanisms of action of selected members of the shrimp CTL family. This review focuses on this new knowledge, that includes unique structural aspects, functions, and mechanisms in host-pathogen interactions, the functional relevance of regions other than the C-type lectin domain, and the regulation of transcription of shrimp CTLs. Thus, this review aims to provide a detailed update of recent studies that have contributed to our better understanding of the shrimp immune events that involve CTL functions. • C-type lectins play important roles in shrimp-pathogen interactions. • Additional regions are essential for the function of shrimp C-type lectins. • This review highlights the in vivo immune-related events of C-type lectins. [ABSTRACT FROM AUTHOR]

The recognition of pathogen-associated molecular patterns is accomplished by the recognition modules of pattern recognition receptors (PRRs). Leucine-rich repeats (LRRs) and C-type lectin-like domain (CTLD) represent the two most universal categories of recognition modules. In the current study, we identified a novel soluble and bacteria-inducible PRR comprising LRRs and a CTLD from the hepatopancreas of kuruma shrimp Marsupenaeus japonicus and named it Leulectin. The module arrangement of Leulectin is unique among all organisms. Both modules, together with the whole molecule, protected shrimp against Vibrio infection. By screening the pathogen-associated molecular patterns that shrimp might encounter, Leulectin was found to sense Vibrio flagellin through the LRRs and to recognize LPS through CTLD. The LRR-flagellin interaction was confirmed by pull-down and far-Western assays and was found to rely on the fourth LRR of Leulectin and the N terminus of flagellin. The recognition of LPS was determined by the long loop region of CTLD in a calcium-independent manner. By sensing the flagellin, LRRs could prevent its attachment to shrimp cells, thereby inhibiting Vibrio colonization. With the ability to recognize LPS, CTLD could agglutinate the bacteria and promote hemocytic phagocytosis. Our study clearly showed the division of labor and the synergy between different recognition modules and provided new insights into the concept of pattern recognition and the function of soluble PRRs in the antibacterial response. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus-associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to viral protein 28, the most abundant and functionally relevant envelope protein of WSSV. Furthermore, cell-surface calreticulin was identified as a receptor of MjsvCL, and the interaction between these proteins was a determinant for the viral infection-promoting activity of MjsvCL. The MjsvCL-calreticulin pathway facilitated virus entry likely in a cholesterol-dependent manner. This study provides insights into a mechanism by which soluble CLs capture and present virions to the cell-surface receptor to facilitate viral infection. [ABSTRACT FROM AUTHOR]

Some aquatic invertebrates such as shrimp contain low albeit stable numbers of bacteria in the circulating hemolymph. The proliferation of this hemolymph microbiota in such a nutrient-rich environment is tightly controlled in healthy animals, but the mechanisms responsible had remained elusive. In the present study, we report a C-type lectin (MjHeCL) from the kuruma shrimp (Marsupenaeus japonicus) that participates in restraining the hemolymph microbiota. Although the expression of MjHeCL did not seem to be modulated by bacterial challenge, the down-regulation of its expression by RNA interference led to proliferation of the hemolymph microbiota, ultimately resulting in shrimp death. This phenotype was rescued by the injection of recombinant MjHeCL, which restored the healthy status of the knockdown shrimp. A mechanistic analysis revealed that MjHeCL inhibited bacterial proliferation by modulating the expression of antimicrobial peptides. The key function of MjHeCL in the shrimp immune homeostasis might be related to its broader recognition spectrum of the hemolymph microbiota components than other lectins. Our study demonstrates the role of MjHeCL in maintaining the healthy status of shrimp and provides new insight into the biological significance of C-type lectins, a diversified and abundant lectin family in invertebrate species. [ABSTRACT FROM AUTHOR]

Highlights: [•] LdlrLec1 and LdlrLec2 expression were obviously upregulated upon WSSV challenge. [•] Injection of rLdlrLec1 and rLdlrLec2 into shrimp inhibited WSSV replication. [•] Knocking down the expression of LdlrLec1 and 2 increased WSSV replication in vivo. [•] LdlrLec1 and LdlrLec2 interact with VP28 of WSSV and inhibit the viral infection. [Copyright &y& Elsevier]

Abstract: C-type lectins function as pattern recognition receptors and play important roles in the innate immune system of crustaceans. In this study, we reported a new CTL gene (designated as PcLec5) from red swamp crayfish, Procambarus clarkii. PcLec5 was mainly distributed in hepatopancreas, gills and intestine, and the PcLec5 transcripts were up-regulated in all the three tissues after challenge with bacteria Vibrio anguillarum. For further functional analyses, PcLec5 was recombinantly expressed in Escherichia coli and anti-PcLec5 polyclonal antiserum was prepared. The results of bacteria binding assay revealed that PcLec5 could selectively bind to 5 of 9 kinds of bacteria we used and had a tendency to bind to Gram-negative bacteria. Sugar binding assay showed that PcLec5 could bind to peptidoglycan, lipoteichoic acid and lipopolysaccharide, with the highest affinity to LPS. Furthermore, bacteria-clearance experiment showed PcLec5 could selectively facilitate the clearance of injected bacteria in crayfish, and the bacteria-clearance facilitating spectrum of PcLec5 was totally in agreement with its bacteria binding spectrum. These results suggested PcLec5 function as a pattern recognition receptor in crayfish immune system and had certain selectivity on bacteria pathogens. [Copyright &y& Elsevier]

Abstract: C-type lectins play important roles in the innate immune system of crustaceans. In this study, a novel C-type lectin gene, designated as PcLec4, was obtained from the red swamp crayfish (Procambarus clarkii). Quantitative real-time polymerase chain reaction revealed that PcLec4 is mainly expressed in the crayfish hepatopancreas and intestine, and the PcLec4 mRNA expression is upregulated after challenged with the bacteria Vibrio anguillarum. PcLec4 was recombinantly expressed in Escherichia coli and anti-PcLec4 polyclonal antiserum was prepared. Binding experiments revealed that the recombinant PcLec4 binds to various bacteria and polysaccharides on the bacterial surface, which suggests that PcLec4 recognizes bacterial pathogens. Overexpression of PcLec4 in crayfish using the pIeLec4 vector was performed. The results show that the crayfish overexpressing PcLec4 eliminate injected V. anguillarum more quickly than the control, which suggests that PcLec4 elicits further immune response for removing invading bacteria. The results of the survival experiment confirmed the function of PcLec4 in resisting V. anguillarum because PcLec4 overexpression in crayfish significantly increased the crayfish survival rate. These results reveal that PcLec4 has an important role in the antibacterial immunity of crayfish, and in vivo PcLec4 overexpression might be used as a disease control strategy in aquiculture. [Copyright &y& Elsevier]

Abstract: C-type lectins are abundantly found in crustaceans. They function in the immune system by recognizing, opsonizing, or agglutinating. Some of them also feature anti-microbial activity. In this study, we identify a hepatopancreas-specific C-type lectin (PcLec2) that responds significantly to immune challenges in red swamp crayfish (Procambarus clarkii). Recombinant PcLec2 shows no agglutinating or anti-microbial activity. It can bind to lipopolysaccharides and bacterial cells in the absence of calcium, and its binding to Gram-negative bacteria is stronger than that to Gram-positive ones. Moreover, the protein can stimulate the activation of prophenoloxidase both in vitro and in vivo. We conclude that this C-type lectin may be an upstream detector of the prophenoloxidase activating system in crayfish. [ABSTRACT FROM AUTHOR]

Abstract: C-type lectins may function as pattern-recognition receptors (PRRs) and play important roles in immune responses. In this work, a cDNA for a new C-type lectin, FcLec3, was obtained from Chinese white shrimp Fenneropenaeus chinensis using expressed sequence tag analysis and rapid amplification of the cDNA ends. FcLec3 contains an N-terminal signal peptide and a carbohydrate recognition domain (CRD). RT-PCR analysis showed that FcLec3 was mainly expressed in hepatopancreas and that the expression of FcLec3 was obviously up-regulated by Vibrio anguillarum or white spot syndrome virus (WSSV) challenge. Recombinant FcLec3 could agglutinate Gram-negative and -positive bacteria with the presence of calcium. A following agglutination inhibitory test indicated that FcLec3 could recognize muramic acid and peptidoglycan. Besides, pull-down assay showed that the recombinant protein could interact with VP28, one major envelope protein of WSSV. These results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp. [Copyright &y& Elsevier]

Abstract: C-type lectins play important roles in innate immunity of invertebrates. In the present study, we report a novel C-type lectin, named FcLec4, from the Chinese white shrimp Fenneropenaeus chinensis. FcLec4 contains a single carbohydrate recognition domain (CRD) with a putative signal peptide. Phylogenetic analysis indicated that FcLec4 was distant from most reported C-type lectins from shrimps. The expression of FcLec4 increased at both mRNA and protein level after stimulation of Vibrio anguillarum. Recombinant FcLec4 could agglutinate both Gram-positive and -negative bacteria in the presence of calcium. The recombinant protein could bind to peptidoglycan and selectively bind to microorganisms. Interestingly, the tight binding of recombinant FcLec4 to V. anguillarum might facilitate the subsequent clearance of the bacteria in vivo. To the best of our knowledge, this might be the first report that a C-type lectin was found to be directly involved in the anti-V. anguillarum response in shrimps. [Copyright &y& Elsevier]

Abstract: Lectins are regarded as potential immune recognition proteins. In this study, a novel C-type lectin (Fc-Lec2) was cloned from the hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-Lec2 is 1219bp with an open reading frame (ORF) of 1002bp that encodes a protein of 333 amino acids. Fc-Lec2 contains a signal peptide and two different carbohydrate recognition domains (CRDs) arranged in tandem. The first CRD contains a QPD (Gln-Pro-Asp) motif that has a predicted binding specificity for galactose and the second CRD contains a EPN (Glu-Pro-Asn) motif for mannose. Fc-Lec2 was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated in the hepatopancreas of shrimp challenged with bacteria or viruses. Recombinant mature Fc-Lec2 and its two individual CRDs (CRD1 and 2) did not have hemagglutinating activity against animal red blood cells, but agglutinated some Gram-positive and Gram-negative bacteria in a calcium-dependent manner. The three recombinant proteins also bound to bacteria in the absence of calcium. Fc-Lec2 seems to have broader specificity and higher affinity for bacteria and polysaccharides (peptidoglycan, lipoteichoic acid and lipopolysaccharide) than each of the two individual CRDs. These data suggest that the two CRDs have synergistic effect, and the intact lectin may be more effective in response to bacterial infection, the Fc-Lec2 performs its pattern recognition function by binding to polysaccharides of pathogen cells. [Copyright &y& Elsevier]

Abstract: Lectins play important roles in animal innate immune responses by serving as pattern recognition receptors, opsonins, or effector molecules. Here, we report a novel hepatopancreas-specific C-type lectin, designated Fc-hsL, from the hepatopancreas of the Chinese shrimp, Fenneropenaeus chinensis. The cDNA of Fc-hsL is 571bp long with a 480bp open reading frame that encodes a 159-residue protein. Fc-hsL contains a signal peptide and a single C-type lectin-like domain (CTLD) or carbohydrate recognition domain (CRD). It has an EPN(Glu-Pro-Asn) motif with a predicted ligand-binding site specific for mannose. Fc-hsL was constitutively expressed in the hepatopancreas of normal shrimp, and its expression was up-regulated following challenge of shrimp with bacteria or virus. Fc-hsL was not detected in other tissues but was induced in the stomach of immune-challenged shrimp. Fc-hsL protein was detected in both hemolymph and the hepatopancreas of bacteria- and virus-challenged shrimp. Recombinant mature Fc-hsL has no hemagglutinating activity, but calcium-dependent agglutinating activity against some Gram-positive and Gram-negative bacteria was detected. The rFc-hsL also has binding activity to some Gram-positive and Gram-negative bacteria and high antimicrobial activity against some bacteria and fungi. These in vitro functions of recombinant Fc-hsL were calcium-independent. Fc-hsL may act as a pattern recognition receptor in antibacterial defense and as an effector in innate immunity of Chinese shrimp. [Copyright &y& Elsevier]

Abstract: C-type lectins participate in pathogen recognition and other defense responses in innate immunity as well as in cell–cell interactions. A new cDNA encoding a 335-residue polypeptide containing two tandem C-type lectin domains was cloned from the haemocytes of Helicoverpa armigera (Ha-lectin). Northern hybridizations revealed that the mRNA of Ha-lectin was expressed constitutively in haemocytes, and was up-regulated following injections of bacteria, yeast, or virus. Ha-lectin expression was also induced in the fat body when larvae were injected with bacteria, yeast or 20-hydroxyecdysone and a non-steroidal ecdysone agonist, RH-2485. The Ha-lectin was detected in granular haemocytes. The recombinant protein (rHa-lectin) expressed in Escherichia coli had hemagglutinating and sugar-binding activities. The native Ha-lectin protein was identified in haemocytes and plasma using a polyclonal antiserum raised against rHa-lectin by immunoblotting techniques. All together, our results suggest that the Ha-lectin gene is involved in innate immunity, and may also participate in the molting process. [Copyright &y& Elsevier]