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 14 bp indel polymorphism in the promoter region is associated with different responses to porcine circovirus type 2 infection by regulating MRC1 transcription.

Mannose receptor, C type 1 (MRC1) is a key factor in regulating the body's immune response to resist pathogen invasions. In this study, mRNA expressions of MRC1 gene in nine porcine organs/tissues were compared between Laiwu (LW) and Yorkshire × Landrace crossbred (YL) pigs prior to and post PCV2 infection. We found that, for pigs uninfected with PCV2, MRC1 mRNA expressions in the lung, spleen, large intestine, small intestine and mesenteric lymph node tissues of LW were significantly higher than those of YL pigs (P < 0.05). After PCV2 infection, MRC1 mRNA levels in the liver, kidney and mesenteric lymph node were significantly increased in LW pigs (P < 0.05); while, significantly decreased in the heart and lung tissues of YL pigs (P < 0.05). The transcriptional activity of porcine MRC1 promoter was further analyzed to investigate the molecular mechanism underlying these expressional differences in response to PCV2 infection. Luciferase assay indicated that a 14 bp indel polymorphism "GTTTTTTTTTTTTT" at the site -864 of MRC1 promoter contributed to the transcriptional activity. The frequency of 14 bp insertion in LW and Dapulian pigs, generally resistant to PCV2 infection, was higher than that in Duroc, Landrace and Yorkshire pigs, which were sensitive to PCV2 infection. The promoter with 14 bp insertion displayed higher MRC1 transcription level both prior to and post PCV2 infection compared with that carrying no insertion in PK15 cells (P < 0.01). The results suggest that this 14 bp indel polymorphism is associated with different responses to PCV2 infection by regulating MRC1 transcription.

Comprehensive Study of the Proteome and Transcriptome of the Venom of the Most Venomous European Viper: Discovery of a New Subclass of Ancestral Snake Venom Metalloproteinase Precursor-Derived Proteins.

The nose-horned viper, its nominotypical subspecies Vipera ammodytes ammodytes ( Vaa), in particular, is, medically, one of the most relevant snakes in Europe. The local and systemic clinical manifestations of poisoning by the venom of this snake are the result of the pathophysiological effects inflicted by enzymatic and nonenzymatic venom components acting, most prominently, on the blood, cardiovascular, and nerve systems. This venom is a very complex mixture of pharmacologically active proteins and peptides. To help improve the current antivenom therapy toward higher specificity and efficiency and to assist drug discovery, we have constructed, by combining transcriptomic and proteomic analyses, the most comprehensive library yet of the Vaa venom proteins and peptides. Sequence analysis of the venom gland cDNA library has revealed the presence of messages encoding 12 types of polypeptide precursors. The most abundant are those for metalloproteinase inhibitors (MPis), bradykinin-potentiating peptides (BPPs), and natriuretic peptides (NPs) (all three on a single precursor), snake C-type lectin-like proteins (snaclecs), serine proteases (SVSPs), P-II and P-III metalloproteinases (SVMPs), secreted phospholipases A2 (sPLA2s), and disintegrins (Dis). These constitute >88% of the venom transcriptome. At the protein level, 57 venom proteins belonging to 16 different protein families have been identified and, with SVSPs, sPLA2s, snaclecs, and SVMPs, comprise ∼80% of all venom proteins. Peptides detected in the venom include NPs, BPPs, and inhibitors of SVSPs and SVMPs. Of particular interest, a transcript coding for a protein similar to P-III SVMPs but lacking the MP domain was also found at the protein level in the venom. The existence of such proteins, also supported by finding similar venom gland transcripts in related snake species, has been demonstrated for the first time, justifying the proposal of a new P-IIIe subclass of ancestral SVMP precursor-derived proteins.

Studies Into ß-Glucan Recognition in Fish Suggests a Key Role for the C-Type Lectin Pathway.

Immune-modulatory effects of ß-glucans are generally considered beneficial to fish health. Despite the frequent application of ß-glucans in aquaculture practice, the exact receptors and downstream signalling remains to be described for fish. In mammals, Dectin-1 is a member of the C-type lectin receptor (CLR) family and the best-described receptor for ß-glucans. In fish genomes, no clear homologue of Dectin-1 could be identified so far. Yet, in previous studies we could activate carp macrophages with curdlan, considered a Dectin-1-specific ß-(1,3)-glucan ligand in mammals. It was therefore proposed that immune-modulatory effects of ß-glucan in carp macrophages could be triggered by a member of the CLR family activating the classical CLR signalling pathway, different from Dectin-1. In the current study, we used primary macrophages of common carp to examine immune modulation by ß-glucans using transcriptome analysis of RNA isolated 6 h after stimulation with two different ß-glucan preparations. Pathway analysis of differentially expressed genes (DEGs) showed that both ß-glucans regulate a comparable signalling pathway typical of CLR activation. Carp genome analysis identified 239 genes encoding for proteins with at least one C-type Lectin Domains (CTLD). Narrowing the search for candidate ß-glucan receptors, based on the presence of a conserved glucan-binding motif, identified 13 genes encoding a WxH sugar-binding motif in their CTLD. These genes, however, were not expressed in macrophages. Instead, among the ß-glucan-stimulated DEGs, a total of six CTLD-encoding genes were significantly regulated, all of which were down-regulated in carp macrophages. Several candidates had a protein architecture similar to Dectin-1, therefore potential conservation of synteny of the mammalian Dectin-1 region was investigated by mining the zebrafish genome. Partial conservation of synteny with a region on the zebrafish chromosome 16 highlighted two genes as candidate ß-glucan receptor. Altogether, the regulation of a gene expression profile typical of a signalling pathway associated with CLR activation and, the identification of several candidate ß-glucan receptors, suggest that immune-modulatory effects of ß-glucan in carp macrophages could be a result of signalling mediated by a member of the CLR family.

Identification and preliminary characterization of a putative C-type lectin receptor-like protein in the T. cruzi tomato lectin endocytic-enriched proteome.

Trypanosoma cruzi, the etiological agent of the Chagas' disease in Latin America undergoes a complex life cycle involving two hosts, a mammalian host and a reduviid insect vector (triatomine). In the insect midgut the parasite multiplies as epimastigote forms, which rely on endocytosis for their energy requirement. We recently showed that posttranslational modification of endocytic N-glycoproteins by tomato lectin (TL) binding-N-glycans is crucial for receptor-mediated endocytosis (RME) in epimastigote forms. In an attempt to characterize the endocytic proteome we used a TL affinity chromatography, which significantly enriched glycoproteins of the trypanosomal endocytic pathway. In addition to various lysosomal hydrolases, we found an endosomal C-type lectin-like protein, which displays some structural and topological characteristics of the mammalian lectin receptor superfamily. This lectin encoding a large transmembrane protein of around 375kDa contained three putative extracellular N-terminal C-type lectin domains (CTLD) and located inside the flagellar pocket (FP)/cytostome and endosomal compartments of the insect stage of the parasite and on the surface of the plasma membrane of intracellular amastigote parasites. Noteworthy, this endogenous lectin displayed similar sugar-binding specificity to that of TL and therefore could be important in either the N-glycan mediated endocytosis or parasite adhesion to host cells. We postulated that during the evolution of trypanosomatids, genes encoding lectin harboring 3 CTDLs represent an old acquisition present in free-living, monoxenic and heteroxenic trypanosomatids, which would have been secondarily lost in extracellular parasites from the T. brucei clade.

Recent insights into structures and functions of C-type lectins in the immune system.

The majority of the C-type lectin-like domains in the human genome likely to bind sugars have been investigated structurally, although novel mechanisms of sugar binding are still being discovered. In the immune system, adhesion and endocytic receptors that bind endogenous mammalian glycans are often conserved, while pathogen-binding C-type lectins on cells of the innate immune system are more divergent. Lack of orthology between some human and mouse receptors, as well as overlapping specificities of many receptors and formation of receptor hetero-oligomers, can make it difficult to define the roles of individual receptors. There is good evidence that C-type lectins initiate signalling pathways in several different ways, but this function remains the least well understood from a mechanistic perspective.

Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio) during Infection with Aeromonas sobria.

Mannose receptor (MR) is a member of pattern-recognition receptors (PRRs), which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR) was cloned from zebra fish (Danio rerio), which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II) domain, eight C-type lectin-like domains (CTLDs), a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1ß and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

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.

Function of a novel C-type lectin with two CRD domains from Macrobrachium rosenbergii in innate immunity.

C-type lectins play crucial roles in innate immunity. In the present study, a novel C-type lectin gene, designated as MrCTL, was identified from Macrobrachium rosenbergii. MrCTL contains 2 carbohydrate-recognition domains (CRDs), namely MrCRD1 and MrCRD2. The MrCRD1 contains a QEP motif and MrCRD2 contains a motif of EPD. MrCTL was mainly expressed in the hepatopancreas. The expression level of MrCTL in hepatopancreas was significantly upregulated after a challenge with Vibrio parahaemolyticus or White spot syndrome virus (WSSV). The recombinant MrCTL, MrCRD1 and MrCRD2 have an ability to agglutinate both Gram-negative (V. parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) in a calcium dependent manner. The recombinant MrCTL, MrCRD1 and MrCRD2 bind directly to all tested microorganisms. All these results suggested that MrCTL may have important roles in immune defense against invading pathogens in prawns.

Chicken NK cell receptors.

Natural killer cells are innate immune cells that destroy virally infected or transformed cells. They recognize these altered cells by a plethora of diverse receptors and thereby differ from other lymphocytes that use clonally distributed antigen receptors. To date, several receptor families that play a role in either activating or inhibiting NK cells have been identified in mammals. In the chicken, NK cells have been functionally and morphologically defined, however, a conclusive analysis of receptors involved in NK cell mediated functions has not been available. This is partly due to the low frequencies of NK cells in blood or spleen that has hampered their intensive characterization. Here we will review recent progress regarding the diverse NK cell receptor families, with special emphasis on novel families identified in the chicken genome with potential as chicken NK cell receptors.

Analysis of the mouse 129-strain Nkrp1-Clr gene cluster reveals conservation of genomic organization and functional receptor-ligand interactions despite significant allelic polymorphism.

The Nkrp1 (Klrb) family of NK cell receptors and their genetically linked Clr (Clec2) ligands are conserved between rodents and humans. Nonetheless, certain mouse and rat Nkrp1 genes exhibit significant allelic polymorphism between inbred strains. We previously demonstrated that the Nkrp1-Clr recognition system is genetically and functionally conserved between the B6 and BALB/c strains, with focused sequence divergence evident in certain genes (e.g., Nkrp1b,c). Here, we extend this finding by mapping the 129-strain Nkrp1-Clr gene cluster, which is structurally conserved yet displays significant sequence divergence relative to the B6 haplotype. In addition, we show that 129-strain NK cells possess comparable Nkrp1 and Clr transcript expression, and characterize several NKR-P1:Clr interactions that are functionally conserved between the B6 and 129 strains, including documented and novel receptor-ligand pairs. Thus, despite significant allelic polymorphism observed in the Nkrp1-Clr region, the overall genetic organization and functional repertoire appear to be conserved among mouse strains, in contrast to the striking variation observed in the corresponding Ly49 region. These data extend our knowledge of the complex genetically linked Nkrp1-Clr NK recognition system in mice.

A novel C-type lectin (FcLec4) facilitates the clearance of Vibrio anguillarum in vivo in Chinese white shrimp.

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.

Identification and comparative analysis of three novel C-type lectins from the silkworm with functional implications in pathogen recognition.

C-type lectins can act as pattern recognition receptors (PRRs) in innate immunity. Previously, we identified two C-type lectins from silkworm (Bombyx mori), BmLBP and BmMBP, as PRRs. In the present study, we identified three homologs of these lectins by searching the silkworm genome database. These novel B. mori low-expression lectins were designated BmLEL-1, BmLEL-2, and BmLEL-3. Although Western-blot analysis failed to detect BmLEL-1, -2, or -3 in plasma, affinity precipitation of larval plasma with various microorganisms revealed that BmLEL-1 and -2 bind to rough and smooth strains of Gram-negative bacteria, respectively. BmLEL-1, -2, and -3 were found to be expressed in testis and ovary, where BmLEL-2 expression was up-regulated after bacteria infection. These results indicate that the novel C-type lectins might play a role in the innate immunity in these tissues as PRRs. Here, we discuss the roles and members of the C-type lectins as primary PRRs in B. mori cellular immunity.

Cflec-4, a multidomain C-type lectin involved in immune defense of Zhikong scallop Chlamys farreri.

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, a novel multidomain C-type lectin gene from scallop Chlamys farreri (designated as Cflec-4) was cloned by RACE approach based on EST analysis. The full-length cDNA of Cflec-4 was of 2086 bp. The open reading frame was of 1830bp and encoded a polypeptide of 609 amino acids, including a signal sequence and four dissimilar carbohydrate-recognition domains (CRDs). The deduced amino acid sequence of Cflec-4 shared high similarities to other C-type lectin family members. The phylogenetic analysis revealed the divergence between the three N-terminal CRDs and the C-terminal one, suggesting that the four CRDs in Cflec-4 originated by repeated duplication of different primordial CRD. The potential tertiary structure of each CRD in Cflec-4 was typical double-loop structure with Ca2+-binding site 2 in the long loop region and two conserved disulfide bridges at the bases of the loops. The tissue distribution of Cflec-4 mRNA was examined by fluorescent quantitative real-time PCR. In the healthy scallops, the Cflec-4 transcripts could be only detected in gonad and hepatopancreas, whereas in the Listonella anguillarum challenged scallops, it could be also detected in hemocytes. These results collectively suggested that Cflec-4 was involved in the immune defense of scallop against pathogen infection and provided new insight into the evolution of C-type lectin superfamily.

Porcine DC-SIGN: molecular cloning, gene structure, tissue distribution and binding characteristics.

DC-SIGN, a human C-type lectin, is involved in the transmission of many enveloped viruses. Here we report the cloning and characterization of the cDNA and gene encoding porcine DC-SIGN (pDC-SIGN). The full-length pDC-SIGN cDNA encodes a type II transmembrane protein of 240 amino acids. Phylogenetic analysis revealed that pDC-SIGN, together with bovine, canis and equine DC-SIGN, are more closely related to mouse SIGNR7 and SIGNR8 than to human DC-SIGN. pDC-SIGN has the same gene structure as bovine, canis DC-SIGN and mouse SIGNR8 with eight exons. pDC-SIGN mRNA expression was detected in pig spleen, thymus, lymph node, lung, bone marrow and muscles. pDC-SIGN protein was found to express on the surface of monocyte-derived macrophages and dendritic cells, alveolar macrophages, lymph node sinusoidal macrophage-like, dendritic-like and endothelial cells but not of monocytes, peripheral blood lymphocytes or lymph node lymphocytes. A BHK cell line stably expressing pDC-SIGN binds to human ICAM-3 and ICAM-2 immunoadhesins in a calcium-dependent manner, and enhances the transmission of porcine reproductive and respiratory syndrome virus (PRRSV) to target cells in trans. The results will help better understand the biological role(s) of DC-SIGN family in innate immunity during the evolutionary process.

The IRBIT domain adds new functions to the AHCY family.

During the past few years, the IRBIT domain has emerged as an important add-on of S-adenosyl-L-homocystein hydrolase (AHCY), thereby creating the new family of AHCY-like proteins. In this review, we discuss the currently available data on this new family of proteins. We describe the IRBIT domain as a unique part of these proteins and give an overview of its regulation via (de)phosphorylation and proteolysis. The second part of this review is focused on the potential functions of the AHCY-like proteins. We propose that the IRBIT domain serves as an anchor for targeting AHCY-like proteins towards cytoplasmic targets. This leads to regulation of (i) intracellular Ca2+ via the inositol 1,4,5-trisphosphate receptor (IP3R), (ii) intracellular pH via the Na+/HCO3 - cotransporters (NBCs); whereas inactivation of the IRBIT domain induces (iii) nuclear translocation and regulation of AHCY activity. Dysfunction of AHCY-like proteins will disturb these three important functions, with various biological implications.

Extended and bent conformations of the mannose receptor family.

In mammals, the mannose receptor family consists of four members, Endo180, DEC-205, phospholipase A2 receptor and the mannose receptor. The extracellular domains of all these receptors contain a similar arrangement of domains in which an N-terminal cysteine-rich domain is followed by a single fibronectin type II domain and eight or ten C-type lectin-like domains. This review focuses on the three-dimensional structure of the receptors in the mannose receptor family and its functional implication. Recent research has revealed that several members of this family can exist in at least two configurations: an extended conformation with the N-terminal cysteine-rich domain pointing outwards from the cell membrane and a bent conformation where the N-terminal domains fold back to interact with C-type lectin-like domains at the middle of the structure. Conformational transitions between these two states seem to regulate the interaction of these receptors with ligands and their oligomerization.

Cloning, expression and characterization of two C-type lectins from the venom gland of Bungarus multicinctus.

C-type lectins found in many animals are non-enzymatic proteins and able to bind with mono- and oligosaccharides in a Ca(2+)-dependent fashion. Here, we report the cloning of two C-type lectins named BML-1 and BML-2 from the venom gland of Bungarus multicinctus, and expression of their mature peptides with 135 and 137 amino acids as inclusion bodies. Recombinant BML-1 and BML-2 proteins with 135 amino acids formed monomers, and those with 137 amino acids formed homodimers and monomers and both of them displayed certain hemagglutinating activity to rabbit erythrocytes. The results of Western blotting and immuno-affinity chromatography demonstrated that C-type lectins in B. multicinctus formed dimers in physiological conditions, and their molecular weight is lower than previous predictions. This is the first report of the cloning of the BML-2 gene from the venom gland of B. multicinctus, as well as an investigation of its confirmation and biological functions.

Evolutionary analysis reveals collective properties and specificity in the C-type lectin and lectin-like domain superfamily.

Members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily share a common fold and are involved in a variety of functions, such as generalized defense mechanisms against foreign agents, discrimination between healthy and pathogen-infected cells, and endocytosis and blood coagulation. In this work we used ConSurf, a computer program recently developed in our lab, to perform an evolutionary analysis of this superfamily in order to further identify characteristics of all or part of its members. Given a set of homologous proteins in the form of multiple sequence alignment (MSA) and an inferred phylogenetic tree, ConSurf calculates the conservation score in every alignment position, taking into account the relationships between the sequences and the physicochemical similarity between the amino acids. The scores are then color-coded onto the three-dimensional structure of one of the homologous proteins. We provide here and at http://ashtoret.tau.ac.il/ approximately sharon a detailed analysis of the conservation pattern obtained for the entire superfamily and for two subgroups of proteins: (a) 21 CTLs and (b) 11 heterodimeric CTLD toxins. We show that, in general, proteins of the superfamily have one face that is constructed mostly of conserved residues and another that is not, and we suggest that the former face is involved in binding to other proteins or domains. In the CTLs examined we detected a region of highly conserved residues, corresponding to the known calcium- and carbohydrate-binding site of the family, which is not conserved throughout the entire superfamily, and in the CTLD toxins we found a patch of highly conserved residues, corresponding to the known dimerization region of these proteins. Our analysis also detected patches of conserved residues with yet unknown function(s).