Identification and transcriptional analysis of two types of lectins (SgCTL-1 and SgGal-1) from mollusk Solen grandis

C-type lectin and galectin are two types of animal carbohydrate-binding proteins which serve as pathogen recognition molecules and play crucial roles in the innate immunity of invertebrates. In the present study, a C-type lectin (designated as SgCTL-1) and galectin (designated as SgGal-1) were identified from mollusk Solen grandis, and their expression patterns, both in tissues and toward three pathogen-associated molecular patterns (PAMPs) stimulation were characterized. The full-length cDNA of SgCTL-1 and SgGal-1 was 1280 and 1466 bp, containing an open reading frame (ORF) of 519 and 1218 bp, respectively. Their deduced amino acid sequences showed high similarity to other members of C-type lectin and galectin superfamily, respectively. SgCTL-1 encoded a single carbohydrate-recognition domain (CRD), and the motif of Ca(2+)-binding site 2 was EPN (Glu(135)-Pro(136)-Asn(137)). While SgGal-1 encoded two CRDs, and the amino acid residues constituted the carbohydrate-binding motifs were well conserved in CRD1 but partially conserved in CRD2. Although SgCTL-1 and SgGal-1 exhibited different tissue expression pattern, they were both constitutively expressed in all tested tissues, including hemocytes, gonad, mantle, muscle, gill and hepatopancreas, and they were both highly expressed in hepatopancreas and gill. Furthermore, the mRNA expression of two lectins in hemocytes was significantly (P < 0.01) up-regulated with different levels after S. grandis were stimulated by lipopolysaccharide (LPS), peptidoglycan (PGN) or β-1,3-glucan. Our results suggested that SgCTL-1 and SgGal-1 from razor clam were two novel members of animal lectins, and they might function as pattern recognition receptors (PRRs) taking part in the process of pathogen recognition.     

Cloning and characterization of three novel WSSV recognizing lectins from shrimp Marsupenaeus japonicus

C-type lectins (CTLs) acting as pattern recognition receptors play essential roles in shrimp innate immune responses. Using WSSV envelope proteins (VP26, VP28, and VP281) to screen a phage display library of Marsupenaeus japonicus, three lectins (termed as MjLecA, MjLecB, and MjLecC) were found to interact with WSSV. Sequence analysis revealed that these MjLecs shared low similarities with each other. Phylogenetic analysis indicated MjLecA and MjLecB are likely to belong to the same lectin sub-family, while MjLecC belongs to another sub-family. These MjLecs showed broad, unique carbohydrate binding spectra. Also, the three MjLecs could interact with several envelope proteins of WSSV and could recognize a wide range of microorganisms. Moreover, binding of MjLecA or MjLecB to WSSV reduced the viral infection rate in vitro. These results suggest that various kinds of CTLs with structural and functional diversities may constitute a recognizing network against invading pathogens such as bacteria and virus, and play essential roles in the defence system of shrimp.     

拟穴青蟹两种新C-型凝集素基因的克隆与表达分析

为研究拟穴青蟹(Scylla paramamosain)中C-型凝集素(C-lectin)的功能, 从其肝胰腺中克隆获得全长858 bp和598 bp的两个C-型凝集素分子, 分别命名为Sp-lectin3和Sp-lectin4, 其推导的氨基酸序列含有信号肽和一个CRD, 其中Sp-lectin4还具有糖类结合的特征性基序QPD。Sp-lectin3和Sp-lectin4的开放阅读框分别由5个和4个外显子编码。在正常养殖青蟹的肝胰腺中该两个基因表达量最高, 其次是射精管(Sp-lectin3)或肠(Sp-lectin4); 除脑和储精囊外的所检测组织/器官中, Sp-lectin4表达量均高于Sp-lectin3。随着胚胎的发育, 该两个基因表达量逐渐升高, 峰期为溞状幼体, 而大眼幼体期的表达量急剧下降, 仔蟹期再回升; 在胚胎发育阶段除囊胚期外, Sp-lectin4表达量极显著高于Sp-lectin3(P0.01), 相反, 在胚后发育阶段除大眼幼体II期外, Sp-lectin3表达量却极显著高于Sp-lectin4(P0.01)。副溶血弧菌(Vibrio parahemolyticus) 人工感染, 可诱导Sp-lectin3在储精囊和射精管中的表达, 分别在感染后12h和6h显著上调(P0.05); 同时, 可诱导Sp-lectin4在肝胰腺中的表达, 并在感染后12h和18h显著上调(P0.05)。结果表明, Sp-lectin3和Sp-lectin4可能参与拟穴青蟹的抗细菌感染免疫反应, Sp-lectin3侧重于生殖系统如射精管和储精囊中发挥作用, 而Sp-lectin4侧重于在肝胰腺。       

Diversity in recognition of glycans by F-type lectins and galectins: molecular, structural, and biophysical aspects

Although lectins are "hard-wired" in the germline, the presence of tandemly arrayed carbohydrate recognition domains (CRDs), of chimeric structures displaying distinct CRDs, of polymorphic genes resulting in multiple isoforms, and in some cases, of a considerable recognition plasticity of their carbohydrate binding sites, significantly expand the lectin ligand-recognition spectrum and lectin functional diversification. Analysis of structural/functional aspects of galectins and F-lectins-the most recently identified lectin family characterized by a unique CRD sequence motif (a distinctive structural fold) and nominal specificity for l-Fuc-has led to a greater understanding of self/nonself recognition by proteins with tandemly arrayed CRDs. For lectins with a single CRD, however, recognition of self and nonself glycans can only be rationalized in terms of protein oligomerization and ligand clustering and presentation. Spatial and temporal changes in lectin expression, secretion, and local concentrations in extracellular microenvironments, as well as structural diversity and spatial display of their carbohydrate ligands on the host or microbial cell surface, are suggestive of a dynamic interplay of their recognition and effector functions in development and immunity.     

Molecular cloning and expression of a C-type lectin gene from Venerupis philippinarum

C-type lectins have been demonstrated to play important roles in invertebrate innate immunity by mediating the recognition of pathogens and clearing the micro-invaders. In the present study, a C-type lectin gene (denoted as VpCTL) was identified from Venerupis philippinarum by expressed sequence tag and rapid amplification of cDNA ends approaches. The full-length cDNA of VpCTL consists of 904 nucleotides with an open-reading frame of 456 bp encoding a peptide of 151 amino acids. The deduced amino acid sequence of VpCTL shared high similarity with C-type lectins from other species. The C-type lectin domain and the characteristic EPN and WND motifs were found in VpCTL. The VpCTL mRNA was dominantly expressed in the haemocytes of the V. philippinarum. After Listonella anguillarum challenge, the temporal expression of VpCTL mRNA in haemocytes was increased by 97- and 84-fold at 48 and 96 h, respectively. With high expression level in haemocytes and hepatopancreas, and the up-regulated expression in haemocytes indicted that VpCTL was perhaps involved in the immune responses to L. anguillarum challenge.     

A novel C-type lectin (Cflec-3) from Chlamys farreri with three carbohydrate-recognition domains

C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-type lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by d-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop.     

How C-type lectins detect pathogens

Glycosylation of proteins has proven extremely important in a variety of cellular processes, including enzyme trafficking, tissue homing and immune functions. In the past decade, increasing interest in carbohydrate-mediated mechanisms has led to the identification of novel carbohydrate-recognizing receptors expressed on cells of the immune system. These non-enzymatic lectins contain one or more carbohydrate recognition domains (CRDs) that determine their specificity. In addition to their cell adhesion functions, lectins now also appear to play a major role in pathogen recognition. Depending on their structure and mode of action, lectins are subdivided in several groups. In this review, we focus on the calcium (Ca(2+))-dependent lectin group, known as C-type lectins, with the dendritic cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) as a prototype type II C-type lectin organized in microdomains, and their role as pathogen recognition receptors in sensing microbes. Moreover, the cross-talk of C-type lectins with other receptors, such as Toll-like receptors, will be discussed, highlighting the emerging model that microbial recognition is based on a complex network of interacting receptors.     

Two C-type lectins from shrimp Litopenaeus vannamei that might be involved in immune response against bacteria and virus

C-type lectins play crucial roles in innate immunity to recognize and eliminate pathogens efficiently. In the present study, two C-type lectins from shrimp Litopenaeus vannamei (designated as LvLectin-1 and LvLectin-2) were identified, and their expression patterns, both in tissues and toward pathogen stimulation, were then characterized. The full-length cDNA of LvLectin-1 and LvLectin-2 was 567 and 625 bp, containing an open reading frame (ORF) of 471 and 489 bp, respectively, and deduced amino acid sequences showed high similarity to other members of C-type lectin superfamily. Both two C-type lectins encoded a single carbohydrate-recognition domain (CRD). The motif of Ca²⁺ binding site 2 in CRD, which determined carbohydrate-binding specificity, was QPN (Gln¹²²-Pro¹²³-Asn¹²⁴) in LvLectin-1, but QPD (Gln¹²⁸-Pro¹²⁹-Asp¹³⁰) in LvLectin-2. Two C-type lectins exhibited similar tissue expression pattern, for their mRNA were both constitutively expressed in all tested tissues, including hepatopancreas, muscle, gill, hemocytes, gonad and heart, furthermore they were both mostly expressed in hepatopancreas, though the expression level of LvLectin-2 was much higher than LvLectin-1. The expression level of two C-type lectins mRNA in hemocytes varied greatly after the challenge of Listonella anguillarum or WSSV. After L. anguillarum challenge, the expression of both C-type lectins were significantly (P < 0.01) up-regulated compared with blank group, and LvLectin-1 exhibited higher level than LvLectin-2; while after the stimulation of WSSV, the expression of LvLectin-2 was significantly up-regulated at 6 h (P < 0.01) and 12 h (P < 0.05), but the expression level of LvLectin-1 down-regulated significantly (P < 0.01) to 0.4-fold at 6 and 12 h post-stimulation. The results indicated that the two C-type lectins might be involved in immune response toward pathogen infection, and they might perform different recognition specificity toward bacteria or virus.     

Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.     

Crystal structure of the trimeric alpha-helical coiled-coil and the three lectin domains of human lung surfactant protein D.

BACKGROUND: Human lung surfactant protein D (hSP-D) belongs to the collectin family of C-type lectins and participates in the innate immune surveillance against microorganisms in the lung through recognition of carbohydrate ligands present on the surface of pathogens. The involvement of this protein in innate immunity and the allergic response make it the subject of much interest. RESULTS: We have determined the crystal structure of a trimeric fragment of hSP-D at 2.3 A resolution. The structure comprises an alpha-helical coiled-coil and three carbohydrate-recognition domains (CRDs). An interesting deviation from symmetry was found in the projection of a single tyrosine sidechain into the centre of the coiled-coil; the asymmetry of this residue influences the orientation of one of the adjacent CRDs. The cleft between the three CRDs presents a large positively charged surface. CONCLUSIONS: The fold of the CRD of hSP-D is similar to that of the mannan-binding protein (MBP), but its orientation relative to the alpha-helical coiled-coil region differs somewhat to that seen in the MBP structure. The novel central packing of the tyrosine sidechain within the coiled-coil and the resulting asymmetric orientation of the CRDs has unexpected functional implications. The positively charged surface might facilitate binding to negatively charged structures, such as lipopolysaccharides.     

Structure of a C-type carbohydrate recognition domain from the macrophage mannose receptor

The mannose receptor of macrophages and liver endothelium mediates clearance of pathogenic organisms and potentially harmful glycoconjugates. The extracellular portion of the receptor includes eight C-type carbohydrate recognition domains (CRDs), of which one, CRD-4, shows detectable binding to monosaccharide ligands. We have determined the crystal structure of CRD-4. Although the basic C-type lectin fold is preserved, a loop extends away from the core of the domain to form a domain-swapped dimer in the crystal. Of the two Ca(2+) sites, only the principal site known to mediate carbohydrate binding in other C-type lectins is occupied. This site is altered in a way that makes sugar binding impossible in the mode observed in other C-type lectins. The structure is likely to represent an endosomal form of the domain formed when Ca(2+) is lost from the auxiliary calcium site. The structure suggests a mechanism for endosomal ligand release in which the auxiliary calcium site serves as a pH sensor. Acid pH-induced removal of this Ca(2+) results in conformational rearrangements of the receptor, rendering it unable to bind carbohydrate ligands.     

DC-SIGN and L-SIGN are high affinity binding receptors for hepatitis C virus glycoprotein E2

The hepatitis C virus (HCV) genome codes for highly mannosylated envelope proteins, which are naturally retained in the endoplasmic reticulum. We found that the HCV envelope glycoprotein E2 binds the dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and the related liver endothelial cell lectin L-SIGN through high-mannose N-glycans. Competing ligands such as mannan and an antibody directed against the carbohydrate recognition domains (CRD) abrogated binding. While no E2 interaction with distant monomeric CRDs on biosensor chips could be detected, binding is observed if CRDs are closely seeded (Kd = 48 nm) and if the CRD is part of the oligomeric-soluble extracellular domain of DC-SIGN (Kd = 30 nm). The highest affinity is seen for plasma membrane-expressed DC-SIGN and L-SIGN (Kd = 3 and 6 nm, respectively). These results indicate that several high-mannose N-glycans in a structurally defined cluster on E2 bind to several subunits of the oligomeric lectin CRD. High affinity interaction of viral glycoproteins with oligomeric lectins might represent a strategy by which HCV targets to and concentrates in the liver and infects dendritic cells.     

A tandem-repeat galectin from blood clam Tegillarca granosa and its induced mRNA expression response against bacterial challenge

Galectins are a family of β-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, we have cloned and characterized the first galectin to be identified in Tegillarca granosa (designated Tg-GAL). The full-length cDNA of Tg-GAL was of 2,394 bp nucleotides, encoding a polypeptide of 354 amino acids. The amino acid sequence of T. granosa galectin (Tg-GAL) showed striking sequence similarity to invertebrate and vertebrate galectins in carbohydrate recognition domains (CRD) and contained amino acids that are crucial for binding β-galactoside sugars. Structurally, the Tg-GAL was a tandem repeat galectin containing two CRD connected by a unique peptide link. Quantitative real-time PCR was employed to investigate the tissue distribution of Tg-GAL mRNA and temporal expression in haemocytes of clams challenged with Vibrio parahaemolyticus, lipopolysaccharide (LPS) and peptidogylcan (PGN). The Tg-GAL mRAN expression was concentrated in hepatopancreas and mantle. The up-regulation of Tg-GAL after bacteria V. parahaemolyticus, LPS and PGN challenge showed that Tg-GAL might play a pivotal role in anti-bacterial immunity. Further study should investigate the effects of Tg-GAL absence by siRNA knockout.