Single Carbohydrate Recognition Domain Research Articles

HcCUB-Lec, a newly identified C-type lectin that contains a distinct CUB domain and participates in the immune defense of the triangle sail mussel Hyriopsis cumingii

As pattern recognition receptors (PRRs), C-type lectins (CTLs) play crucial roles in recognizing and eliminating pathogens in innate immunity. In this study, a novel CTL (HcCUB-Lec) was identified from the triangle sail mussel Hyriopsis cumingii. The full-length of HcCUB-Lec cDNA was 1558 bp with an open reading frame of 1281 bp that encodes a putative protein of 426 amino acid residues, including an N-terminal signal peptide, a complement Uegf Bmp1 (CUB) domain, a single carbohydrate recognition domain (CRD), and a transmembrane domain. Quantitative real-time PCR analysis revealed that HcCUB-Lec transcript was distributed in all examined tissues with the highest levels in hepatopancreas and was significantly upregulated in gills and hepatopancreas after immune challenge with Staphyloccocus aureus and Vibrio parahaemolyticus. When HcCUB-Lec was silenced by RNAi, the expression levels of three antimicrobial peptides, including whey acidic protein (HcWAP), defensin (HcDef), and lysozyme (HcLyso), were dramatically decreased in gills. The recombinant HcCUB-Lec and its individual CUB and CRD domains can bind with Gram-positive bacteria (S. aureus and Bacillus subtilis), Gram-negative bacteria (V. parahaemolyticus and Aeromonas hydrophila), and polysaccharides (lipopolysaccharide and peptidoglycan). Moreover, rHcCUB-Lec and its domains could also agglutinate S. aureus and V. parahaemolyticus in the presence of Ca2+ and can clear V. parahaemolyticus in H. cumingii. Results of this study suggest that HcCUB-Lec acts as an antimicrobial PRR that participates in the innate immune responses of H. cumingii.

Different angioregulatory activity of monovalent galectin-9 isoforms

Galectin-9 consists of two peptide-linked carbohydrate recognition domains (CRDs), but alternative splicing and proteolytic processing can give rise to multiple galectin-9 isoforms. Some of these consist of a single CRD and can exert different functions in cell biology. Here, we explored the role of these galectin-9 isoforms in endothelial cell function and angiogenesis. For this, we compared the effects of the two separate CRDs (Gal-9N and Gal-9C) with the tandem repeat galectin-9M on endothelial cell proliferation, migration, sprouting and tube formation in vitro as well as on angiogenesis in vivo using the chicken chorioallantoic membrane (CAM) assay. Galectin-9 isoforms significantly affected proliferation in quiescent endothelial cells and migration in activated endothelial cells. Interestingly, both monovalent gal-9 CRDs displayed opposite effects compared to gal-9M on proliferation and migration. Sprouting was significantly inhibited by gal-9C, while all isoforms appeared to stimulate tube formation. Angiogenesis in vivo was hampered by all three isoforms with predominant effects on vessel length. In general, the isoforms induced only subtle concentration-dependent effects in vitro as well as in vivo. Collectively, the effects of different galectin-9 isoforms in endothelial cell biology depend on the cellular activation status. While opposing effects can be observed on a cellular level in vitro, all galectin-9 isoforms hamper angiogenesis in vivo. This warrants further investigation of the regulatory mechanisms that underlie the diverging roles of galectin-9 isoforms in endothelial cell biology since this could provide therapeutic opportunities.

C-type lectin B (SpCTL-B) regulates the expression of antimicrobial peptides and promotes phagocytosis in mud crab Scylla paramamosain

As pattern recognition receptors, C-type lectins (CTLs) play important roles in immune system of crustaceans through identifying and binding to the conservative pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. In this study, a new CTL, SpCTL-B, was identified from the hemocytes of mud crab Scylla paramamosain. The full-length of SpCTL-B cDNA was 1278 bp with an open reading frame (ORF) of 348 bp. The predicted SpCTL-B protein contains a single carbohydrate-recognition domain (CRD). SpCTL-B transcripts were distributed in all examined tissues with the highest levels in hepatopancreas. After challenged with Vibrio parahaemolyticus, LPS, polyI:C and white spot syndrome virus (WSSV), the mRNA levels of SpCTL-B in hemocytes and hepatopancreas were up-regulated. The recombinant SpCTL-B (rSpCTL-B) purified by Ni-affinity chromatography showed stronger binding activities with Staphylococcus aureus, β-hemolytic Streptococcus, Escherichia coli, Aeromonas hydrophila, Vibrio alginolyticus than those with V. parahaemolyticus and Saccharomyces cerevisiae. rSpCTL-B exhibited a broad spectrum of microorganism-agglutination activities against Gram-positive bacteria (S. aureus, β-hemolytic Streptococcus) and Gram-negative bacteria (E. coli, V. parahaemolyticus, A. hydrophila, V. alginolyticus) in a Ca2+-dependent manner. The agglutination activities of rSpCTL-B could be inhibited by D-mannose and LPS, but not by d-fructose and galactose. The antimicrobial assay showed that rSpCTL-B exhibited the growth inhibition against all examined gram-positive bacteria and gram-negative bacteria. When SpCTL-B was silenced by RNAi, the bacterial clearance ability in mud crab was decreased and the transcript levels of five antimicrobial peptides (AMPs) (SpCrustin, SpHistin, SpALF4 (anti-lipopolysaccharide factor), SpALF5 and SpALF6) were significantly decreased in hemocytes. In our study, knockdown of SpCTL-B could down-regulate the expression of SpSTAT at mRNA transcriptional level and protein translational level in mud crab. Meantime, the phagocytosis rate and the expression of three phagocytosis related genes were declined after RNAi of SpCTL-B in hemocytes in mud crab. Collectively, our results suggest that SpCTL-B might play its roles as a pattern recognition receptor (PRR) in immune response towards pathogens infection through influencing the expression of AMPs and the phagocytosis of hemocytes in mud crab S. paramamosain.

The functional characterization and comparison of two single CRD containing C-type lectins with novel and typical key motifs from Portunus trituberculatus

C-type lectins are a superfamily of Ca2+-dependent carbohydrate-recognition proteins, which play crucial roles in innate immunity including nonself-recognition and pathogen elimination. In the present study, two single-CRD containing C-type lectins were identified from swimming crab Portunus trituberculatus (designated as PtCTL-2 and PtCTL-3). The open reading frame (ORF) of PtCTL-2 encoded polypeptides of 485 amino acids with a signal peptide and a single carbohydrate-recognition domain (CRD), while PtCTL-3's ORF encoded polypeptides of 241 amino acids with a coiled-coil region and a single-CRD. The key motifs determining carbohydrate binding specificity in PtCTL-2 and PtCTL-3 were EPR (Glu-Pro-Arg) and QPD (Gln-Pro-Asp). EPR is a motif being identified for the first time, whereas QPD is a typical motif in C-type lectins. Different PAMPs binding features of the two recombinant proteins - PtCTL-2 (rPtCTL-2) and PtCTL-3 (rPtCTL-3) have been observed in our experiments. rPtCTL-2 could bind three pathogen-associated molecular patterns (PAMPs) with relatively high affinity, including glucan, lipopolysaccharide (LPS) and peptidoglycan (PGN), while rPtCTL-3 could barely bind any of them. However, rPtCTL-2 could bind seven kinds of microbes and rPtCTL-3 could bind six kinds in microbe binding assay. Moreover, rPtCTL-2 and rPtCTL-3 exhibited similar agglutination activity against Gram-positive bacteria, Gram-negative bacteria and fungi in agglutination assay. All these results illustrated that PtCTL-2 and PtCTL-3 could function as important pattern-recognition receptors (PRR) with broad nonself-recognition spectrum involved in immune defense against invaders. In addition, the results of carbohydrate binding specificity showed that PtCTL-2 with novel key motif had broad carbohydrate binding specificity, while PtCTL-3 with typical key motif possessed different carbohydrate binding specificity from the classical binding rule. Furthermore, PtCTL-2 and PtCTL-3 could also function as opsonin to enhance encapsulation of hemocytes against Ni-NTA beads.

Identification and molecular profiling of DC-SIGN-like from big belly seahorse (Hippocampus abdominalis) inferring its potential relevancy in host immunity

Dendritic-cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) is a C-type lectin that functions as a pattern recognition receptor by recognizing pathogen-associated molecular patterns (PAMPs). It is also involved in various events of the dendritic cell (DC) life cycle, such as DC migration, antigen capture and presentation, and T cell priming. In this study, a DC-SIGN-like gene from the big belly seahorse Hippocampus abdominalis (designated as ShDCS-like) was identified and molecularly characterized. The putative, complete ORF was found to be 1368 bp in length, encoding a protein of 462 amino acids with a molecular mass of 52.6 kDa and a theoretical isoelectric point of 8.26. The deduced amino acid sequence contains a single carbohydrate recognition domain (CRD), in which six conserved cysteine residues and two Ca2+-binding site motifs (QPN, WND) were identified. Based on pairwise sequence analysis, ShDCS-like exhibits the highest amino acid identity (94.6%) and similarity (97.4%) with DC-SIGN-like counterpart from tiger tail seahorse Hippocampus comes. Quantitative real-time PCR revealed that ShDCS-like mRNA is transcribed universally in all tissues examined, but with abundance in kidney and gill tissues. The basal mRNA expression of ShDCS-like was modulated in blood cell, kidney, gill and liver tissues in response to the stimulation of healthy fish with lipopolysaccharides (LPS), Edwardsiella tarda, or Streptococcus iniae. Moreover, recombinant ShDCS-like-CRD domain exhibited detectable agglutination activity against different bacteria. Collectively, these results suggest that ShDCS-like may potentially involve in immune function in big belly seahorses.

A C-type lectin, Nattectin-like protein (CaNTC) in Qihe crucian carp Carassius auratus: Binding ability with LPS, PGN and various bacteria, and agglutinating activity against bacteria

C-type lectins (CTLs), as the members of pattern-recognition receptors (PRRs), play the significant roles in innate immunity through binding with pathogen-associated molecular patterns (PAMPs) on the surface of microbe. In the present study, a novel CTL, Nattectin-like protein (named as CaNTC), was investigated in Qihe crucian carp Carassius auratus. The full-length cDNA of CaNTC was composed of 776 bp, with a 152 bp 5′-untranslated region (UTR), a 492 bp ORF encoding a 163-aa protein, and a 132 bp 3′-UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of CaNTC contained a signal peptide, a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys57-Cys150, Cys126-Cys142), and an EPN/WND motif required for carbohydrate-binding specificity. With regard to the mRNA transcript of CaNTC, it was predominately expressed in liver. The temporal expressions of CaNTC were obviously up-regulated in liver, spleen and head-kidney after challenged by Aeromonas hydrophila and poly I: C, respectively, and the change pattern was in the time-depended manner. The recombinant CaNTC (rCaNTC) purified from Escherichia coli BL21 (DE3), exhibited strong binding ability with LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. With regard to the agglutinating activity of rCaNTC, rCaNTC was able to agglutinate rabbit erythrocytes and three kinds of bacteria (Gram-negative bacteria, Escherichia coli and A. hydrophila, and Gram-positive bacteria Staphylococcus aureus) in a Ca2+-dependent manner. These findings collectively demonstrated that CaNTC, as a PRR, could be involved in the innate immunity and play an important role in immune defense of C. auratus.

Identification of a Macrobrachium nipponense C-type lectin with a close evolutionary relationship to vertebrate lectins

C-type lectins (CTLs) are involved in the innate immune defense of vertebrates and invertebrates against invading pathogens. This study cloned and characterized a novel C-type lectin (MnCTL) of the oriental river prawn, Macrobrachium nipponense. The cloned MnCTL cDNA encompasses an open reading frame of 774 nucleotides and encodes polypeptides of 257 residues. The deduced MnCTL protein contains a single carbohydrate recognition domain (CRD) with an EPN (Glu-Pro-Asn) motif in calcium-binding site 2. Phylogenetic analysis indicated that MnCTL has a closer evolutionary relationship with vertebrate lectins than with invertebrate lectins. Tissue expression analysis showed that high levels of MnCTL are ubiquitously distributed in the gills and stomach of M. nipponense. Quantitative real-time RT-PCR (qRT-PCR) analysis showed that MnCTL expression was up-regulated by bacteria or white spot syndrome virus (WSSV) challenge. Knock-down of the MnCTL gene in WSSV-challenged prawns significantly decreased MnALF1 and MnALF2 transcript levels. The recombinant MnCRD (rMnCRD) agglutinated both Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Vibrio parahaemolyticus) in the presence of calcium. Furthermore, rMnCRD could bind to all the tested bacteria with different activities. The sugar-binding assay showed that rMnCRD was able to bind lipopolysaccharide and peptidoglycan in a concentration-dependent manner. In addition, rMnCRD could accelerate bacterial clearance. On the contrary, MnCTL silencing by dsRNA interference could weaken the bacterial clearance ability. All these findings implicated MnCTL were involved in the antiviral and antibacterial innate immunity of M. nipponense.

Silencing of galectin-1 inhibits retinal neovascularization and ameliorates retinal hypoxia in a murine model of oxygen-induced ischemic retinopathy

Aberrant neovascularization is a consequence of inappropriate angiogenic signaling and contributes to several diseases. Although many regulators of pathogenic angiogenesis have been identified, the understanding of this process remains incomplete. Galectin-1 (Gal-1), as a homodimeric protein with a single carbohydrate-recognition domain, is implicated in several pathologic processes, including angiogenesis; however, its involvement in retinal neovascularization (RNV) remains unknown. Here, we investigated the anti-angiogenic effect of silencing Gal-1 through intravitreal injection in a mouse model of oxygen-induced retinopathy (OIR). Our results revealed that Gal-1 was overexpressed and closely related to retinal neo-vessels in OIR retinas. After silencing Gal-1 via intravitreal injection of adenoviral-Gal-1-RNA interference (Ad-Gal-1-RNAi), RNV and retinal hypoxia were significantly attenuated, indicating the anti-angiogenic effect of Gal-1 inhibition. Western blot analysis and real-time polymerase chain reaction indicated that the expression of both neuropilin-1 (Nrp-1) and B cell lymphoma-2 (Bcl-2) decreased after intravitreal injection of Ad-Gal-1-RNAi, implying the possible involvement of Nrp-1 and Bcl-2 in Gal-1-related angiogenic processes. Additionally, whole-mount fluorescence and hematoxylin and eosin staining showed that intravitreal injection of Ad-Gal-1-RNAi did not significantly disrupt the retinal vasculature and neuronal structure of room air mice. Moreover, Ad-Gal-1-RNAi transfer promoted retinal vascular sprouting and increased retinal vascular perfusion, likely through decreased phosphorylation of myosin phosphatase target protein-1. Collectively, our results demonstrated that Gal-1 functions as an important regulator in RNV and offers a promising strategy for the treatment of RNV diseases, such as proliferative diabetic retinopathy and retinopathy of prematurity.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

A C-type lectin (MrLec) with high expression in intestine is involved in innate immune response of Macrobrachium rosenbergii

C-type lectins (CTLs) are pattern-recognition proteins that play an important role in innate immunity of vertebrates and invertebrates. In this study, a lectin cDNA named MrLec was cloned and characterized from giant freshwater prawns (Macrobrachiun rosenbergii). The full-length cDNA of MrLec was 1431 bp, which contained an open reading frame of 1041 bp that encoded a protein with 346 amino acids. MrLec was found to contain a typical signal peptide of 18 amino acids and a single carbohydrate-recognition domain with 121 amino acids. The phylogenetic analysis showed that MrLec was grouped with vertebrates and had 57% identity with C-type lectin 3 from Marsupenaeus japonicas. Tissue expression analysis showed that MrLec was ubiquitously distributed at a high level in the intestine, with lower expression levels in the hemocytes, heart, hepatopancreas, gill and stomach. Vibrio parahaemolyticus infection induced the upregulation of MrLec in the gills and intestine. For the white spot syndrome virus (WSSV) challenge, MrLec in gills was upregulated at 24, 36 and 48 h. In intestine, MrLec also went up at 36 and 48 h WSSV challenge. Recombinant MrLec can agglutinate (Ca2+-dependent) and bind both Gram-negative and Gram-positive bacteria. rMrLec could attach to lipopolysaccharide and peptidoglycan in a dose-dependent manner. These results indicated possible MrLec involvement in the immune response of giant freshwater prawns.

C-type lectin (MrCTL) from the giant freshwater prawn Macrobrachium rosenbergii participates in innate immunity

C-type lectins (CTLs) play important roles in the innate immunity of invertebrates. In this study, a novel CTL with a single carbohydrate recognition domain (CRD) containing an EPN (Glu-Pro-Asn) motif was identified from the giant freshwater prawn Macrobrachium rosenbergii. This CTL was designated as MrCTL. The cDNA of MrCTL is 1788 bp with a 657 bp open reading frame that encodes a protein of 218 amino acids. The cDNA and genome sequences of MrCTL show a polymorphism that leads to MrCTL isoforms. MrCTL was highly expressed in the gills and intestine of normal prawn, and its transcription increased after Vibrio parahaemolyticus or white spot syndrome virus (WSSV) challenge. Recombinant mature MrCTL and its single CRD could agglutinate (Ca2+-dependent) and bind both Gram-positive and Gram-negative bacteria. The recombinant proteins could attach to lipopolysaccharide and peptidoglycan in a dose-dependent manner. Recombinant MrCTL could accelerate bacterial clearance. Thus, MrCTL could serve as a pattern recognition receptor involved in the innate immunity of M. rosenbergii.

Molecular cloning and characterization of a C-type lectin in yellow catfish Tachysurus fulvidraco

This study represents the first report of a C-type lectin (ctl) in yellow catfish Tachysurus fulvidraco. The complete sequence of ctl complementary (c)DNA consisted of 685 nucleotides. The open reading frame potentially encoded a protein of 177 amino acids with a calculated molecular mass of c.y 20.204 kDa. The deduced amino-acid sequence contained a signal peptide and a single carbohydrate recognition domain with four cysteine residues and GlnProAsp (QPD) and TrpAsnAsp (WND) motifs. Ctl showed the highest identity (56.0%) to the predicted lactose binding lectin from channel catfish Ictalurus punctatus. Quantitative real-time (qrt)-PCR analysis showed that ctl messenger (m)RNA was constitutively expressed in all examined tissues in normal fish, with high expression in trunk kidney and head kidney, which was increased following Aeromonas hydrophila challenge in a duration-dependent manner. Purified recombinant Ctl (rCtl) from Escherichia coli BL21 was able to bind and agglutinate Gram-positive and Gram-negative bacteria in a calcium-dependent manner. These results suggested that Ctl might be a C-type lectin of T. fulvidraco involved in innate immune responses as receptors (PRR).

Antimicrobial functions of EsLecH, a C-type lectin, via JNK pathway in the Chinese mitten crab, Eriocheir sinensis

C-type lectins (CTLs) are pattern recognition proteins that play significant roles in the innate immune system by identifying and eliminating pathogens. Here, we have reported a CTL (EsLecH) from the Chinese mitten crab that can bind to microorganisms and regulate antimicrobial peptide (AMP) expression via the c-Jun N-terminal kinase (JNK) pathway. EsLecH was found to have an N-terminal signal peptide and a single carbohydrate recognition domain. The EsLecH transcript was detected abundantly in various tissues, and it was significantly upregulated in hemocytes after challenging with lipopolysaccharides and bacteria. Recombinant (r)EsLecH could bind to microorganisms, but at different levels. Ca2+ significantly increased rEsLecH binding affinity to microorganisms. Furthermore, growth inhibition by rEsLecH increased with increasing rEsLecH levels. Knockdown of EsLecH was accompanied by a significant reduction in AMP expression and JNK phosphorylation; AMP expression was reduced with JNK silencing and can not rescued by rEsLecH when absence of JNK. These results indicate that EsLecH could regulate AMPs via JNK signaling.

Single CRD containing lectin from Macrobrachium rosenbergii (MrLec) participates in innate immunity against pathogen infections.

As a type of pattern-recognition proteins, lectins perform important functions in the innate immunity of crustaceans, including prawns. Although several reports showed that C-type lectin domain family (CLEC) importantly functions in host-pathogen interactions, limited research has focused on CLEC in Macrobrachium rosenbergii. In the present study, a new single CRD containing CLEC (designated as MrLec) was reported in freshwater prawns, M.rosenbergii. The full-length cDNA of MrLec consisted of 1027bp with an open reading frame of 801bp, which encoded a peptide of 266 amino acid residues. Genomic sequence for MrLec was also obtained from the M.rosenbergii, which contain 4 exons and 3 introns. MrLec was found to contain a single carbohydrate-recognition domain with an EPN motif. MrLec was ubiquitously distributed in various tissues of a normal prawn, particularly in the hepatopancreas and gills. MrLec expression in the gills was significantly upregulated after a challenge with Vibrio parahaemolyticus and downregulated at 24h after MrLec RNA interference (MrLec-RNAi). The expression levels of some AMPs, including antilipopolysaccharide factor 1 (Alf1) and lysozyme 2 (Lyso2), also markedly decreased after MrLec-RNAi. Recombinant MrLec can agglutinate (Ca(2+)-dependent) and bind both Gram-negative and Gram-positive bacteria. Results suggested that MrLec participates in the recognition of invading pathogens and functions in the immune response of prawn against pathogen infections.

Antimicrobial response of galectin-1 from rock bream Oplegnathus fasciatus: Molecular, transcriptional, and biological characterization.

In this study, we describe the identification and characterization of a proto type galectin, galectin-1, from rock bream Oplegnathus fasciatus (OfGal-1). Galectins are evolutionarily conserved carbohydrate binding lectins that show a wide range of functions related to development and immune physiology. They have been identified as pattern recognition receptors of innate immune system that recognize a broad range of microbes. OfGal-1 cDNA comprised of 993 bp with an open reading frame of 408 bp that encodes 135 amino acids. A single carbohydrate recognition domain was present in the OfGal-1 amino acid sequence. The sequence comparison by multiple and pairwise alignments and the phylogenetic tree emphasized the strong evolutionary conservation of Gal-1. The typical β-sandwich structure was identified from the predicted tertiary structure. The constitutive expression of mRNA transcripts was detected in a wide range of tissues examined, with the highest expression in the heart. Immune challenges with live bacteria (Edwardsiella tarda and Streptococcus iniae), rock bream irido virus, and mitogens (lipopolysaccharide and poly I:C) modulated the expression of OfGal-1 mRNAs in the gills, head kidney, and liver. The recombinant OfGal-1 (rOfGal-1) strongly agglutinatinated the human erythrocytes, and this hemagglutination was inhibited by lactose and D-galactose. A wide range of bacteria (S. iniae, S. parauberis, Escherichia coli, Edwardsiella tarda, Vibrio anguillarum, Vibrio harveyi, and Vibrio tapetis) and a ciliate (Miamiensis avidus) were also effectively recognized by rOfGal-1. Significant antiviral activity against rock bream irido virus was also demonstrated by rOfGal-1. Collectively, results from the present study indicate that OfGal-1 can recognize a wide range of microbes and is a vital pattern recognition receptor in the innate immune system of rock bream.

A galectin related protein from Oplegnathus fasciatus: Genomic, molecular, transcriptional features and biological responses against microbial pathogens

Galectins, a family of β-galactoside-binding lectins, are pattern recognition receptors that recognize pathogen-associated molecular patterns and are subsequently involved in the opsonization, phagocytosis, complement activation, and killing of microbes. Here, we report a novel galectin related protein (GRP) identified from rock bream (Oplegnathus fasciatus), designated OfGal like B. The cDNA of OfGal like B is 517 bp with an open reading frame (ORF) of 438 bp, encoding 145 amino acids, with a single carbohydrate recognition domain (CRD). However, only two of the seven critical residues responsible for carbohydrate recognition were identified in the CRD. There was no signal peptide identified in the OfGal like B protein. The genomic structure of OfGal like B, determined using a bacterial artificial chromosome (BAC) genomic library, consists of four exons and three introns. Homology assessment, multiple sequence alignment, and phylogenetic analysis indicated that OfGal like B is an evolutionarily conserved lectin that is closely related to the proto-type galectins. OfGal like B mRNA was constitutively expressed in a wide range of tissues in healthy rock breams. When challenged with bacterial or viral stimulants, OfGal like B was up-regulated in the gills and spleen of rock breams, indicating that it likely plays an important role during bacterial and viral infections. Furthermore, recombinant OfGal like B (rOfGal like B) lacked carbohydrate-binding activity but was able to recognize and agglutinate bacteria, including Streptococcus iniae, Listeria monocytogenes, Vibrio tapetis, Escherichia coli, and Edwardsiella tarda, and a ciliate parasite, Miamiensis avidus. These results collectively suggest that OfGal like B is involved in pathogen recognition and plays a significant role(s) in the innate defense mechanism of rock bream.

Cloning and the mRNA expression of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis in response to pathogenic inoculation

Crustaceans are deficient in an adaptive immune system and depend solely on their innate immunity. One kind of pattern recognition proteins which plays an important role in the shrimp immunity is lectin. A new C-type lectin called FmLC2 was cloned from the stomach of the banana shrimp Fenneropenaeus merguiensis by means of RT-PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE). Its full-length cDNA contains 1098 bp with a single open reading frame of 738 bp, encoding a peptide of 245 amino acids. The deduced amino acid sequence of FmLC2 consists of a signal peptide of 17 amino acids with a molecular mass of 28,115 Da and an isoelectric point of 6.94. The primary structure of FmLC2 comprises a single carbohydrate recognition domain (CRD) with a QPD (Gln-Pro-Asp) motif and one Ca2+ binding site. Like other C-type lectins, its CRD structure contains a double-loop characteristic being stabilized by two conserved disulfide linkages. The mRNA expression of FmLC2 was detected specifically in the stomach and gills, less was found in the hepatopancreas. Upon inoculation of shrimp with Vibrio harveyi or white spot syndrome virus (WSSV), the FmLC2 expression either in stomach or gills was higher than in the hepatopancreas. Besides, its expression in these tissues was up-regulated to reach the highest levels at 12 or 18 h for V. harveyi or WSSV stimulation, respectively. RNAi-based silencing of FmLC2 resulted in suppression of its expression, increases in mortality when the shrimp were challenged with V. harveyi or WSSV, and the median lethal time was reduced compared with controls. These results suggest that FmLC2 may serve as receptor molecules which recognize invading bacterial and viral pathogens and thus contribute a role in the shrimp immune response.

A single-CRD C-type lectin from oyster Crassostrea gigas mediates immune recognition and pathogen elimination with a potential role in the activation of complement system

C-type lectins (CTLs), serving as pattern recognition receptors (PRRs), are a superfamily of Ca2+-dependent carbohydrate-recognition proteins that participate in nonself-recognition and pathogen elimination. In the present study, a single carbohydrate-recognition domain (CRD) CTL was identified from oyster Crassostrea gigas (designated as CgCLec-2). There was only one CRD within the deduced amino acid sequence of CgCLec-2 consisting of 129 amino acid residues. A conserved EPN (Glu246-Pro247-Asn248) motif was found in Ca2+-binding site 2 of CgCLec-2. The CgCLec-2 mRNA could be detected in all the examined tissues at different expression levels in oysters. The mRNA expression of CgCLec-2 in hemocytes was up-regulated significantly at 6 h post Vibrio splendidus challenge. The recombinant CgCLec-2 (rCgCLec-2) could bind various Pathogen-Associated Molecular Patterns (PAMPs), including lipopolysaccharide, mannan and peptidoglycan, and displayed strong binding abilities to Vibrio anguillarum, V. splendidus and Yarrowiali polytica and week binding ability to Staphylococcus aureus. It could also enhance the phagocytic activity of oyster hemocytes to V. splendidus and exhibited growth suppression activity against gram-positive bacteria S. aureus but no effect on gram-negative bacteria V. splendidus. Furthermore, the interaction between rCgCLec-2 and rCgMASPL-1 was confirmed by GST Pull down. The results suggested that CgCLec-2 served as not only a PRR in immune recognition but also a regulatory factor in pathogen elimination, and played a potential role in the activation of complement system.

A C-type lectin fold gene from Japanese scallop Mizuhopecten yessoensis, involved with immunity and metamorphosis.

C-type lectins are a superfamily of Ca(2+)-dependent carbohydrate-recognition proteins that are well known for their participation in pathogen recognition and clearance. In this study, a putative C-type lectin fold (MyCLF) gene was identified from the Japanese scallop Mizuhopecten yessoensis. The full-length of MyCLF was 645 bp, encoding a polypeptide of 167 amino acids. MyCLF carried a signal peptide of 20 amino acid residues, and a single carbohydrate recognition domain, having relatively high amino acid sequence conservation with C-type lectins reported for other bivalves. The expression of MyCLF mRNA transcripts in adult tissues, after bacterial challenge and during different developmental stages was determined using real-time quantitative RT-PCR. MyCLF was mainly distributed in the mantle, gill, and kidney. The expression of MyCLF clearly increased 3 h after Vibrio anguillarum challenge, and dropped to a minimum level after 9 h compared to the control group. During embryonic development, the expression level increased in the gastrulae, trochophore and early D-shaped larvae, decreased in D-shaped larvae, and then increased hundreds of times in metamorphosing larvae. The results suggested that MyCLF was involved in an immune response and it may play important roles during the metamorphosis phase of M. yessoensis.

Molecular cloning of a C-type lectin with one carbohydrate recognition domain from Fenneropenaeus merguiensis and its expression upon challenging by pathogenic bacterium or virus

Lectins, one type of pattern recognition proteins, play important roles in an innate immunity of crustaceans including shrimp. A new C-type lectin designated FmLC1 was cloned from the hepatopancreas of banana shrimp Fenneropenaeus merguiensis by procedures of PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE). The full-length cDNA is composed of 706bp with a single open reading frame of 477bp, encoding a peptide of 158 amino acid residues. Its deduced amino acid sequence comprises a putative signal peptide of 17 amino acids and has an estimated molecular mass of 17,934Da with a theoretical pI of 4.46. The primary sequence of FmLC1 contains a single carbohydrate recognition domain (CRD) with an EPS (Glu-Pro-Ser) motif and one Ca2+ binding site, stabilized by two disulfide bonds. FmLC1 mRNA was detected to express specifically in the hepatopancreas, a master organ in shrimp. Its expression in the hepatopancreas was up-regulated to reach the maximum at 12 or 48h following challenge of shrimp with Vibrio harveyi or white spot syndrome virus, respectively. These results suggest that FmLC1 may participate in recognition of invading pathogens such as bacteria and viruses, and play roles in the immune response of shrimp even at different stages of the clearance of pathogens.