Carbohydrate-binding Motif Research Articles

Structure and Glycan-Binding Properties of the Vibrio Vulnificus Hemolysin B-Trefoil Lectin

Pore forming toxins (PFTs) are secreted by bacterial pathogens as water-soluble monomers that oligomerize on the cell surface to form transmembrane channels. To increase their affinity for the membrane, many PFTs utilize proteins, carbohydrates and/or lipids as receptors. Previous studies suggest that a PFT secreted by Vibrio cholerae, V. cholerae cytolysin (VCC), recognizes cell surface glycans in its mode of action. However, while VCC contains two domains whose structures resemble known carbohydrate binding motifs, a β-trefoil and β-prism fold, only the β-prism domain actively binds carbohydrate ligands. This presents the question of why an organism would make a protein with an inactive lectin domain. Sequence analysis identified that orthologs of VCC contain either both of the lectin-like domains, or only the β-trefoil domain. Here, we utilize functional and structural approaches to investigate the carbohydrate-binding activities of a VCC ortholog, Vibrio vulnificus hemolysin (VVH), which contains only a β-trefoil lectin-like domain. Our studies identify that the VVH β-trefoil lectin domain binds a broad range of branched sugars containing multiple N-acetyllactosamine (LacNAc) repeats, with micromolar affinity. This is in contrast to the β-prism domain of VCC, which binds a narrower range of complex N-glycans with nanomolar affinity. We solved the X-ray crystal structure of the VVH β-trefoil domain identifying two interesting traits. First, the β-trefoil lectin domain alone forms a heptameric ring characteristic of many PFTs. Second, VVH contains a unique loop and coordinating residues absent in VCC. We hypothesize an evolutionary relationship between VVH and VCC such that upon gaining the more specific β-prism domain, activity of the β-trefoil domain was lost.

Characterization of the Pseudomonas aeruginosametalloendopeptidase, Mep72, a member of the Vfr regulon

BackgroundPseudomonas aeruginosa Vfr (the virulence factor regulator) enhances P. aeruginosa virulence by positively regulating the expression of numerous virulence genes. A previous microarray analysis identified numerous genes positively regulated by Vfr in strain PAK, including the yet uncharacterized PA2782 and PA2783.ResultsIn this study, we report the detailed characterization of PA2783 in the P. aeruginosa strain PAO1. RT-PCR analysis confirmed that PA2782-PA2783 constitute an operon. A mutation in vfr significantly reduced the expression of both genes. The predicted protein encoded by PA2783 contains a typical leader peptide at its amino terminus end as well as metalloendopeptidase and carbohydrate binding motifs at its amino terminus and carboxy terminus regions, respectively. An in-frame PA2783::phoA fusion encoded a hybrid protein that was exported to the periplasmic space of Escherichia coli and P. aeruginosa. In PAO1, the proteolytic activity of the PA2783-encoded protein was masked by other P. aeruginosa extracellular proteases but an E. coli strain carrying a PA2783 recombinant plasmid produced considerable proteolytic activity. The outer membrane fraction of an E. coli strain in which PA2783 was overexpressed contained specific endopeptidase activity. In the presence of cAMP, purified recombinant Vfr (rVfr) bound to a 98-bp fragment within the PA2782-PA2783 upstream region that carries a putative Vfr consensus sequence. Through a series of electrophoretic mobility shift assays, we localized rVfr binding to a 33-bp fragment that contains part of the Vfr consensus sequence and a 5-bp imperfect (3/5) inverted repeat at its 3′ and 5′ ends (TGGCG-N22-CGCTG). Deletion of either repeat eliminated Vfr binding.ConclusionsPA2782 and PA2783 constitute an operon whose transcription is positively regulated by Vfr. The expression of PA2783 throughout the growth cycle of P. aeruginosa follows a unique pattern. PA2783 codes for a secreted metalloendopeptidase, which we named Mep72. Mep72, which has metalloendopeptidase and carbohydrate-binding domains, produced proteolytic and endopeptidase activities in E. coli. Vfr directly regulates the expression of the PA2782-mep72 operon by binding to its upstream region. However, unlike other Vfr-targeted genes, Vfr binding does not require an intact Vfr consensus binding sequence.

A galectin from roughskin sculpin, Trachidermus fasciatus: Molecular cloning and characterization

Galectins are a family of β-galactoside-binding lectins, which have been proved to be involved in host–pathogen interactions by recognizing pathogen associated molecular patterns (PAMPs) on the surface of virus, bacteria, fungi and protozoa.In this study, a galactoside-binding lectin homolog was identified from roughskin sculpin Trachidermus fasciatus, named TfGal. The full-length of TfGal cDNA was 1016 bp with a 5′ untranslated region (UTR) of 134 bp and a 3′ UTR of 474 bp, and the open reading frame (ORF) is 408 bp. The deduced protein was composed of 135 amino acids, including a carbohydrate-recognition domain and a galactoside-type carbohydrate-binding motif H-NPR/W--E-R. The deduced amino acid sequence shared 58.52%–87.4% similarities with the galectins of the other fishes.Quantitative real-time PCR (qRT-PCR) analysis demonstrated that TfGal mRNA was abundantly expressed in the ovary, heart, stomach, skin, moderately expressed in the liver, brain, gills, spleen, and rarely expressed in the hemocytes, meat and intestine. The expression of TfGal mRNA in the hemocytes and the skin was dramatically up-regulated after challenged with LPS, reaching the highest level at 2 h post-challenge, and then dropped abruptly, while the expression of TfGal mRNA in the liver was up-regulated at 2–6 h post-challenge, and then returned to the normal level, with an increase at 96 h post-challenge again. However, no obvious change of the expression of TfGal mRNA was detected in the stomach. Recombinant TfGal purified from Escherichia coli (BL21) could agglutinate and/or bind microorganisms in Ca2+-independent manner. These results suggested that TfGal might be involved in the innate immune response of roughskin sculpin.

Alteration of the Carbohydrate-Binding Specificity of a C-type Lectin CEL-I Mutant with an EPN Carbohydrate-Binding Motif

CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins.

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 Ca2+-binding site 2 was EPN (Glu135-Pro136-Asn137). 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.

Arabidopsis F-box protein containing a Nictaba-related lectin domain interacts with N-acetyllactosamine structures

The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N- and O-glycans containing N-acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly-N-acetyllactosamine ([Galβ1-4GlcNAc]n) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides.

In silico Analysis of Molecular Mechanisms of Galanthus nivalis Agglutinin-Related Lectin-Induced Cancer Cell Death from Carbohydrate-Binding Motif Evolution Hypothesis

Galanthus nivalis agglutinin-related lectins, a superfamily of strictly mannose-binding-specific lectins widespread amongst monotyledonous plants, have drawn a rising attention for their remarkable anti-proliferative and apoptosis-inducing activities toward various types of cancer cells; however, the precise molecular mechanisms by which they induce tumor cell apoptosis are still only rudimentarily understood. Herein, we found that the three conserved motifs "QXDXNXVXY," the mannose-specific binding sites, could mutate at one or more amino acid sites, which might be a driving force for the sequential evolution and thus ultimately leading to the complete disappearance of the three conserved motifs. In addition, we found that the motif evolution could result in the diversification of sugar-binding types that G. nivalis agglutinin-related lectins could bind from specific mannose receptors to more types of sugar-containing receptors in cancer cells. Subsequently, we indicated that some sugar-containing receptors such as TNFR1, EGFR, Hsp90, and Hsp70 could block downstream anti-apoptotic or survival signaling pathways, which, in turn, resulted in tumor cell apoptosis. Taken together, our hypothesis that carbohydrate-binding motif evolution may impact the G. nivalis agglutinin-related lectin-induced survival or anti-apoptotic pathways would provide a new perspective for further elucidating the intricate relationships between the carbohydrate-binding specificities and complex molecular mechanisms by which G. nivalis agglutinin-related lectins induce cancer cell death.

Carbohydrate-binding motif in chitinase 3-like 1 (CHI3L1/YKL-40) specifically activates Akt signaling pathway in colonic epithelial cells

Host-microbial interactions play a key role during the development of colitis. We have previously shown that chinase 3-like 1 (CHI3L1) is an inducible molecule overexpressed in colonic epithelial cells (CECs) under inflammatory conditions. In this study, we found that chitin-binding motif (CBM) of CHI3L1 is specifically associated with the CHI3L1-mediated activation of the Akt-signaling in CEC by transfecting the CBM-mutant CHI3L1 vectors in SW480 CECs. Downstream, CHI3L1 enhanced the secretion of IL-8 and TNFα in a dose-dependent manner. We previously show that 325 through 339 amino-acids in CBM are crucial for the biological function of CHI3L1. Here we demonstrated that 325th-339th residues of CBM in CHI3L1 is a critical region for the activation of Akt, IL-8 production, and for a specific cellular localization of CHI3L1. In conclusion, CBM region of CHI3L1 is critical in activating Akt signaling in CECs, and the activation may be associated with the development of chronic colitis.

CDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata

Galectins can recognize and specifically bind to β-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters.

Deleted in Malignant Brain Tumors-1 Protein (DMBT1): A Pattern Recognition Receptor with Multiple Binding Sites

Deleted in Malignant Brain Tumors-1 protein (DMBT1), salivary agglutinin (DMBT1SAG), and lung glycoprotein-340 (DMBT1GP340) are three names for glycoproteins encoded by the same DMBT1 gene. All these proteins belong to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins: a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. In addition to SRCR domains, all DMBT1s contain two CUB domains and one zona pellucida domain. The SRCR domains play a role in the function of DMBT1s, which is the binding of a broad range of pathogens including cariogenic streptococci, Helicobacter pylori and HIV. Mucosal defense proteins like IgA, surfactant proteins and lactoferrin also bind to DMBT1s through their SRCR domains. The binding motif on the SRCR domains comprises an 11-mer peptide in which a few amino acids are essential for binding (GRVEVLYRGSW). Adjacent to each individual SRCR domain are glycosylation domains, where the attached carbohydrate chains play a role in the binding of influenza A virus and Helicobacter pylori. The composition of the carbohydrate chains is not only donor specific, but also varies between different organs. These data demonstrate a role for DMBT1s as pattern recognition molecules containing various peptide and carbohydrate binding motifs.

A multidomain galectin involved in innate immune response of pearl oyster Pinctada fucata

Galectins could specifically bind to β-galactoside residues and play crucial roles in innate immune responses of vertebrates and invertebrates. In this study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from pearl oyster Pinctada fucata (designated as PoGal). PoGal cDNA was 2138 bp long and consisted of a 5′-untranslated region (UTR) of 120 bp, a 3′-UTR of 350 bp with two cytokine RNA instability motifs (ATTTA), and an open reading frame (ORF) of 1668 bp encoding a polypeptide of 555 amino acids with an estimated molecular mass of 63.4 kDa and a theoretical isoelectric point of 4.8. PoGal contained four CRDs, each CRD of PoGal all had the conserved carbohydrate-binding motifs H-NPR and WG-ER. PoGal shared 43.7% and 62.9% identity to those of bay scallop and eastern oyster, respectively, which were only two galectins with four CRDs. The phylogenetic analysis revealed that all galectins with four CRDs formed a single clade. PoGal mRNA was constitutively expressed in all detected tissues, and the expression level of PoGal mRNA was significantly up-regulated in digestive gland, mantle, haemocyte, gonad and intestine after Vibrio alginolyticus stimulation. The expression profile analysis showed that the expression level of PoGal mRNA was significantly up-regulated at 4, 8 and 12 h after V. alginolyticus stimulation. These results suggested that PoGal was a constitutive and inducible acute-phase protein that perhaps involved in innate immune response of pearl oyster.

Identification and molecular analysis of a novel C-type lectin from Scophthalmus maximus

C-type lectins are calcium-dependent carbohydrate-binding proteins that play important roles in innate immunity. In this study, a C-type lectin homologue (SmLec1) was identified from turbot ( Scophthalmus maximus) and analyzed at expression and functional levels. The open reading frame of SmLec1 is 504 bp, with a 5′-untranslated region (UTR) of 101 bp and a 3′-UTR of 164 bp. The deduced amino acid sequence of SmLec1 shares 34%–38% overall identities with the C-type lectins of several fish species. In silico analysis identified in SmLec1 conserved C-type lectin features, including a carbohydrate-recognition domain, four disulfide bond-forming cysteine residues, and the mannose-type carbohydrate-binding motif. In addition, SmLec1 possesses a putative signal peptide sequence and is predicted to be localized in the extracellular. Expression of SmLec1 was highest in liver and responded positively to experimental challenges with fish pathogens. Recombinant SmLec1 (rSmLec1) purified from yeast was able to agglutinate the Gram-negative fish pathogen Listonella anguillarum but not the Gram-positive pathogen Streptococcus iniae. The agglutinating ability of rSmLec1 was abolished in the presence of mannose and ethylenediaminetetraacetic acid and by elevated temperature (65 °C). Further analysis showed that rSmLec1 could stimulate kidney lymphocyte proliferation and enhance the killing of bacterial pathogen by macrophages. Taken together, these results suggest that SmLec1 is a unique mannose-binding C-type lectin that possesses apparent immunomodulating property and is likely to be involved in host defense against bacterial infection.

SusG: A Unique Cell-Membrane-Associated α-Amylase from a Prominent Human Gut Symbiont Targets Complex Starch Molecules

SusG is an alpha-amylase and part of a large protein complex on the outer surface of the bacterial cell and plays a major role in carbohydrate acquisition by the animal gut microbiota. Presented here, the atomic structure of SusG has an unusual extended, bilobed structure composed of amylase at one end and an unprecedented internal carbohydrate-binding motif at the other. Structural studies further demonstrate that the carbohydrate-binding motif binds maltooligosaccharide distal to, and on the opposite side of, the amylase catalytic site. SusG has an additional starch-binding site on the amylase domain immediately adjacent to the active cleft. Mutagenesis analysis demonstrates that these two additional starch-binding sites appear to play a role in catabolism of insoluble starch. However, elimination of these sites has only a limited effect, suggesting that they may have a more important role in product exchange with other Sus components.

Expression Profiling of Galectin-3-Depleted Melanoma Cells Reveals its Major Role in Melanoma Cell Plasticity and Vasculogenic Mimicry

Galectin-3 (Gal-3) is a beta-galactoside-binding protein that is involved in cancer progression and metastasis. Using a progressive human melanoma tissue microarray, we previously demonstrated that melanocytes accumulate Gal-3 during the progression from benign to dysplastic nevi to melanoma and further to metastatic melanoma. Herein, we show that silencing of Gal-3 expression with small hairpin RNA results in a loss of tumorigenic and metastatic potential of melanoma cells. In vitro, Gal-3 silencing resulted in loss of tumor cell invasiveness and capacity to form tube-like structures on collagen ("vasculogenic mimicry"). cDNA microarray analysis after Gal-3 silencing revealed that Gal-3 regulates the expression of multiple genes, including endothelial cell markers that appear to be aberrantly expressed in highly aggressive melanoma cells, causing melanoma cell plasticity. These genes included vascular endothelial-cadherin, which plays a pivotal role in vasculogenic mimicry, as well as interleukin-8, fibronectin-1, endothelial differentiation sphingolipid G-protein receptor-1, and matrix metalloproteinase-2. Chromatin immunoprecipitation assays and promoter analyses revealed that Gal-3 silencing resulted in a decrease of vascular endothelial-cadherin and interleukin-8 promoter activities due to enhanced recruitment of transcription factor early growth response-1. Moreover, transient overexpression of early growth response-1 in C8161-c9 cells resulted in a loss of vascular endothelial-cadherin and interleukin-8 promoter activities and protein expression. Thus, Gal-3 plays an essential role during the acquisition of vasculogenic mimicry and angiogenic properties associated with melanoma progression.

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.

Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevisATCC 8287

BackgroundLactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group.ResultsIn this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179–435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.ConclusionThis study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

Multiplicity of carbohydrate-binding sites in β-prism fold lectins: occurrence and possible evolutionary implications

The beta-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit/domain. Until recently, beta-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit/domain. However, the recently determined structure of the beta-prism fold I lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for two-lectin folds and which carry one or more relevant carbohydrate-binding motifs. The very recent observation of a beta-prism I fold lectin, griffthsin, with three binding sites in each domain further confirmed the need for such an analysis. The analysis demonstrates substantial diversity in the number of binding sites unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of beta-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion.It would seem that the preponderance of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the beta-prism II fold, is related to the role of plant lectins in defence.

Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins

The larvicidal activity of Bacillus thuringiensis subsp. israelensis against dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a beta-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene ( Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3′ and 5′ rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey ( Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have 11 amino acids residues, which are relatively invariant in animals’ C-type lectin CRDs. Five residues at Ca 2+ binding site 1 are conserved in Fclectin. The potential Ca 2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control.

Regioselectivity in acylation of oligosaccharides catalyzed by the metalloprotease thermolysin

Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized on Celite as biocatalyst has been carried out. The reactions were performed in DMSO, a good solvent for carbohydrates, where the enzyme has previously shown its activity in transesterifications of sucrose, maltose and maltose-containing oligosaccharides. Surprisingly, no reaction was observed for glucose or the glucose-containing disaccharides, trehalose and lactose. In contrast, laurate monoesters of several sucrose-containing tri- and tetrasaccharides were synthetized through a one step transesterification using vinyl laurate as the acylating agent. Enzyme regioselectivity was accurately determined by HPLC/MS and the structure of the main regioisomers was established by a combination of NMR experiments. The preferred position of acylation in all cases was the 2-OH of the α- d-glucopyranose moiety linked 1→2 to the β- d-fructofuranose unit. These results correlate with the regioselectivity observed in the case of the disaccharide sucrose. A general carbohydrate binding motif for catalysis by thermolysin is proposed.