X-ray crystal structure of a galactose-specific C-type lectin possessing a novel decameric quaternary structure

Rattlesnake venom lectin (RSL) from the western diamondback rattlesnake (Crotalus atrox) is an oligomeric galactose-specific C-type lectin. The X-ray crystal structure of RSL, in complex with lactose and thiodigalactoside, at 2.2 and 2.3 A resolution, respectively, reveals a decameric protein composed of two 5-fold symmetric pentamers arranged in a staggered, back-to-back orientation. Each monomer corresponds to a single canonical C-type lectin carbohydrate recognition domain devoid of accessory domains and is disulfide-bonded to a monomer in the other pentamer. The structure is the first example of that of a carbohydrate complex of a vertebrate galactose-specific C-type lectin. The 10 carbohydrate-binding sites, located on the rim of the decamer, suggest a role for multivalent interactions and a mechanism for RSL's ability to promote receptor cross-linking and cell aggregation.     

Human intelectin is a novel soluble lectin that recognizes galactofuranose in carbohydrate chains of bacterial cell wall

Galactofuranosyl residues are present in various microorganisms but not in mammals. In this study, we identified a human lectin binding to galactofuranosyl residues and named this protein human intelectin (hIntL). The mature hIntL was a secretory glycoprotein consisting of 295 amino acids and N-linked oligosaccharides, and its basic structural unit was a 120-kDa homotrimer in which 40-kDa polypeptides were bridged by disulfide bonds. The hIntL gene was split into 8 exons on chromosome 1q21.3, and hIntL mRNA was expressed in the heart, small intestine, colon, and thymus. hIntL showed high levels of homology with mouse intelectin, Xenopus laevis cortical granule lectin/oocyte lectin, lamprey serum lectin, and ascidian galactose-specific lectin. These homologues commonly contained no carbohydrate recognition domain, which is a characteristic of C-type lectins, although some of them have been reported as Ca(2+)-dependent lectins. Recombinant hIntL revealed affinities to d-pentoses and a d-galactofuranosyl residue in the presence of Ca(2+), and recognized the bacterial arabinogalactan of Nocardia containing d-galactofuranosyl residues. These results suggested that hIntL is a new type lectin recognizing galactofuranose, and that hIntL plays a role in the recognition of bacteria-specific components in the host.     

Expression of isolated C-type carbohydrate recognition domains

A galactose-binding lectin from the venom of the snake Trimeresurus stejnegeri consists of isolated carbohydrate recognition domains, belonging to group VII of the C-type animal lectins. As a first step toward determining the tertiary structure of the galactose-specific lectin, we produced the lectin in Escherichia coli. By in vitro refolding and affinity chromatography, modest amounts (8 mg/liter) of active recombinant proteins were obtained. The recombinant protein was homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. Its amino acid sequence without the initiated methionine at the N-terminus was also characterized by mass spectrometry. The data of hemagglutination and enzyme-linked lectin binding assays demonstrated that the recombinant lectin showed similar sugar-binding activity as the native protein. In addition, fluorescence spectroscopy and circular dichroism also showed obviously their structural similarity.     

Xerocomus chrysenteron lectin: identification of a new pesticidal protein

Xerocomus chrysenteron is an edible mushroom with insecticidal properties. In an earlier work, we found that proteins are responsible for this toxicity. Here we describe the purification of a approximately 15 kDa lectin, named XCL, from the mushroom. Its cDNA and gDNA were cloned by PCR strategies and a recombinant form was expressed in Escherichia coli. Sequence alignments and sugar specificity showed that this protein is the third member of a new saline-soluble lectin family present in fungi. This protein, either purified from mushroom or expressed in vitro in E. coli, was found to be toxic to some insects, such as the dipteran Drosophila melanogaster and the hemipteran, Acyrthosiphon pisum. The lectin possesses a high insecticidal activity compared to lectin isolated from leguminosae (Lathyrus ochrus) or from the snowdrop (Galanthus nivalis).     

Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures. The new C-type lectin-like molecule, designated liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), is a type II integral membrane protein of approximately 40 kDa in size with a single C-type lectin-like domain at the COOH terminus, closest in homology to DC-SIGNR, DC-SIGN, and CD23. LSECtin mRNA was only expressed in liver and lymph node among 15 human tissues tested, intriguingly neither expressed on hematopoietic cell lines nor on monocyte-derived dendritic cells (DCs). Moreover, LSECtin is expressed predominantly by sinusoidal endothelial cells of human liver and lymph node and co-expressed with DC-SIGNR. LSECtin binds to mannose, GlcNAc, and fucose in a Ca(2+)-dependent manner but not to galactose. Our results indicate that LSECtin is a novel member of a family of proteins comprising CD23, DC-SIGN, and DC-SIGNR and might function in vivo as a lectin receptor.     

Ecdysterone selectively stimulates the expression of a 23000-Da heat-shock protein-beta-galactosidase hybrid gene in cultured Drosophila cells

To study the regulated expression of cloned heat-shock genes in homologous cells, hybrid Drosophila heat-shock-Escherichia coli beta-galactosidase genes were constructed. Segments of the ecdysterone-inducible 23,000-Da heat-shock protein (hsp23) gene and of two other hsp genes (hsp84 and 70), which are not hormone regulated, were functionally linked to the bacterial coding sequence, and the resulting hybrid genes were introduced into cultured, hormone-responsive Drosophila cells by transfection. All hybrid genes directed the synthesis of E. coli-specific beta-galactosidase in heat-treated cells. hsp23 hybrid gene expression was stimulated strongly by ecdysterone, while the activities of the other hybrid genes were not affected at all by the hormone. A hybrid gene with only 147 bp of hsp23 promoter sequence could not be activated by either heat or ecdysterone treatment. Thus, far upstream sequences contain signals required for the regulated expression of the hsp23 gene in Drosophila cells.     

A lectin (CfLec-2) aggregating Staphylococcus haemolyticus from scallop Chlamys farreri

Lectins are a family of carbohydrate-recognition proteins which play crucial roles in innate immunity. In this study, a new lectin (CfLec-2) gene was cloned from Chlamys farreri by EST and RACE approaches. The full-length cDNA of CfLec-2 was composed of 708bp, encoding a typical long form carbohydrate-recognition domain of 130 residues. The deduced amino acid sequence showed high similarity to Brevican in Homo sapiens, C-type lectin-1 and lectin-2 in Anguilla japonica. The cDNA fragment encoding the mature peptide of CfLec-2 was recombined into plasmid pET-32a (+) and expressed in Escherichia coli Rosseta-Gami (DE3). The recombinant CfLec-2 (rCfLec-2) protein exhibited aggregative activity toward Staphylococcus haemolyticus, and the agglutination could be inhibited by d-mannose but not EDTA or d-galactose, indicating that CfLec-2 was a Ca2+ independent lectin. Moreover, rCfLec-2 could suppress the growth of E. coli TOP10F'. These results suggested that CfLec-2 was perhaps involved in the recognition and clearance of bacterial pathogens in scallop.     

Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10

Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.     

Gene expression and molecular characterization of a novel C-type lectin,encapsulation promoting lectin (EPL), in the rice armyworm,Mythimna separata

Insect cellular immune reactions differ depending on the target species. Phagocytosis is activated to scavenge microorganisms such as bacteria and fungi. On the other hand, larger invaders such as parasitoid wasps are eliminated by activation of encapsulation. In this study, we hypothesized that novel determinants regulate cellular immunities independent of surface molecular pattern recognition involving pattern recognition receptors (PRRs). Immune-related genes differentially expressed depending on the treated material size were screened in larval hemocytes of the rice armyworm, Mythimna separata. Consequently, we identified a novel C-type lectin gene up-regulated by injection of large beads but not small beads of identical material. Examination of in vitro effect of the recombinant protein on the immune reactions clarified that the protein activated encapsulation reaction, while it suppressed phagocytosis. These results suggest that this novel C-type lectin designated “encapsulation promoting lectin (EPL)” regulates cellular immunity by a novel immune target size-recognition mechanism.     

Complementation of bacteriophage induction and recombination defects in Escherichia coli RecA(-) mutants by expression of the cloned T4 bacteriophage uvsX gene

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA(-) mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present.     

Escherichia coli ykfE ORFan gene encodes a potent inhibitor of C-type lysozyme

The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression of Escherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (K(i) approximately 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor of vertebrate lysozyme) at the E. coli genetic stock center.     

A scallop C-type lectin from Argopecten irradians (AiCTL5) with activities of lipopolysaccharide binding and Gram-negative bacteria agglutination

C-type lectins are a family of calcium-dependent carbohydrate-binding proteins. In the present study, a C-type lectin (designated as AiCTL5) was identified and characterized from Argopecten irradians. The full-length cDNA of AiCTL5 was of 673 bp, containing a 5' untranslated region (UTR) of 24 bp, a 3' UTR of 130 bp with a poly (A) tail, and an open reading frame (ORF) of 519 bp encoding a polypeptide of 172 amino acids with a putative signal peptide of 17 amino acids. A C-type lectin-like domain (CRD) containing 6 conserved cysteines and a putative glycosylation sites were identified in the deduced amino acid sequence of AiCTL5. AiCTL5 shared 11%-27.5% identity with the previous reported C-type lectin from A. irradians. The cDNA fragment encoding the mature peptide of AiCTL5 was recombined into pET-21a (+) with a C-terminal hexa-histidine tag fused in-frame, and expressed in Escherichia coli Origami (DE3). The recombinant AiCTL5 (rAiCTL5) agglutinated Gram-negative E. coli TOP10F' and Listonella anguillarum, but did not agglutinate Gram-positive bacteria Bacillus thuringiensis and Micrococcus luteus, and the agglutination could be inhibited by EDTA, indicating that AiCTL5 was a Ca(2+)-dependent lectin. rAiCTL5 exhibited a significantly strong activity to bind LPS from E. coli, which conformed to the agglutinating activity toward Gram-negative bacteria. Moreover, rAiCTL5 also agglutinated rabbit erythrocytes. These results indicated that AiCTL5 could function as a pattern recognition receptor to protect bay scallop from Gram-negative bacterial infection, and also provide evidence to understand the structural and functional diverse of lectin.     

Chicken CD69 and CD94/NKG2-like genes in a chromosomal region syntenic to mammalian natural killer gene complex

In mammals, natural killer (NK) cell C-type lectin receptors were encoded in a gene cluster called natural killer gene complex (NKC). The NKC is not reported in chicken yet. Instead, NK receptor genes were found in the major histocompatibility complex. In this study, two novel chicken C-type lectin-like receptor genes were identified in a region on chromosome 1 that is syntenic to mammalian NKC region. The chromosomal locations were validated with fluorescent in situ hybridization. Based on 3D structure modeling, sequence homology, chromosomal location, and phlylogenetic analysis, one receptor is the orthologue of mammalian cluster of differentiation 69 (CD69), and the other is highly homologous to CD94 and NKG2. Like CD94/NKG2 gene found in teleostean fishes, chicken CD94/NKG2 has the features of both human CD94 and NKG2A. Unlike mammalian NKC, these two chicken C-type lectin receptors are not closely linked but separated by 42 million base pairs according to the chicken draft genome sequence. The arrangement of several other genes that are located outside the mammalian NKC is conserved among chicken, human, and mouse. The chicken NK C-type lectin-like receptors in the NKC syntenic region indicate that this chromosomal region existed before the divergence between mammals and aves. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequences have been submitted to the GenBank nucleotide sequence database under the accession number chicken CD69 (DQ156495), CD94/NKG2 (DQ156496), and CD94/NKG2 variant (DQ241793).     

家蚕天蚕素cDNA原核表达及抗菌活性检测

采用RT-PCR方法从家蚕Bombyx mori新疆品种新蚕三号组织中扩增天蚕素cDNA片段,回收并克隆至Pmd18-T载体,进行序列分析。基因序列分析结果与已发表的天蚕素B的序列同源性为98%,表明所克隆的新疆家蚕天蚕素cDNA为独特的cDNA片段。将天蚕素基因与Pgex-4T-1融合表达载体中的谷胱甘肽转移酶基因融合,在大肠杆菌中表达, 结果表明经IPTG 诱导30 min后,pGEX-4T-1/天蚕素转化后的大肠杆菌生长明显受到抑制;当诱导210 min 后,大肠杆菌数量又开始增加,逐渐恢复至正常水平。说明天蚕素与谷胱甘肽转移酶基因融合表达后,在IPTG存在的短时间内仍然对原核细胞有较强的抗菌抑杀作用。     

The structure of a tunicate C-type lectin from Polyandrocarpa misakiensis complexed with D -galactose.

C-type lectins are calcium-dependent carbohydrate-recognising proteins. Isothermal titration calorimetry of the C-type Polyandrocarpa lectin (TC14) from the tunicate Polyandrocarpa misakiensis revealed the presence of a single calcium atom per monomer with a dissociation constant of 2.6 microM, and confirmed the specificity of TC14 for D -galactose and related monosaccharides. We have determined the 2.2 A X-ray crystal structure of Polyandrocarpa lectin complexed with D -galactose. Analytical ultracentrifugation revealed that TC14 behaves as a dimer in solution. This is reflected by the presence of two molecules in the asymmetric unit with the dimeric interface formed by antiparallel pairing of the two N-terminal beta-strands and hydrophobic interactions. TC14 adopts a typical C-type lectin fold with differences in structure from other C-type lectins mainly in the diverse loop regions and in the second alpha-helix, which is involved in the formation of the dimeric interface. The D -galactose is bound through coordination of the 3 and 4-hydroxyl oxygen atoms with a bound calcium atom. Additional hydrogen bonds are formed directly between serine, aspartate and glutamate side-chains of the protein and the sugar 3 and 4-hydroxyl groups. Comparison of the galactose binding by TC14 with the mannose binding by rat mannose-binding protein reveals how monosaccharide specificity is achieved in this lectin. A tryptophan side-chain close to the binding site and the distribution of hydrogen-bond acceptors and donors around the 3 and 4-hydroxyl groups of the sugar are essential determinants of specificity. These elements are, however, arranged in a very different way than in an engineered galactose-specific mutant of MBPA. Possible biological functions can more easily be understood from the fact that TC14 is a dimer under physiological conditions.     

Analysis of the Gal3 Signal Transduction Pathway Activating Gal4 Protein-Dependent Transcription in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GAL/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the GAL1 gene. Based on this result and the striking similarity between the GAL3 and GAL1 protein sequences we attempted to detect galactokinase activity that might be associated with the GAL3 protein. By both in vivo and in vitro tests the GAL3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by GAL1 is not likely due to galactokinase activity, but rather due to a distinct GAL3-like activity. Overall, the results indicate that GAL1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the GAL3-like activity of GAL1, functions beyond the galactose-specific GAL1, GAL7 and GAL10 enzymes are required for the LTA induction pathway.     

A molecular model of the carbohydrate recognition domain of a rat macrophage lectin and analysis of its binding site

A three-dimensional model of the carbohydrate recognition domain of a rat macrophage C-type lectin has been constructed by comparative modeling and assessed by inverse folding analysis. Comparative modeling in the presence of low sequence similarity was based on information provided by comparison of X-ray structures and sequence-structure alignments. The sequence-structure compatibility of the model was sound. Its binding site was analyzed in comparison to the X-ray structure of a galactose-specific mutant of the mannose-binding protein. The specificity of the macrophage lectin was discussed in light of mutagenesis data on asialoglycoprotein receptors.