Β-glucan Binding Research Articles

Single-cell RNA-sequencing reveals Eriocheir sinensis hemocyte subpopulations and their molecular responses to Spiroplasma eriocheiris infection

The hemocytes is the central part of the Eriocheir sinensis innate immune system, however, their subclasses have not been completely defined and the single-cell gene transcriptional profiles and functions are still unknown. In the present study, the single-cell mRNA sequencing (scRNA-seq) was carried out to build a single-cell gene expression map of E. sinensis hemocytes and further analysis of the interaction molecular mechanism between the crab and novel aquatic pathogens Spiroplasma eriocheiris. By scRNA-seq, we successfully identified nine cell clusters (Hem1–9) and predicted their functions according to the specific marker genes. Pseudotime inference analysis revealed that all the identified cells undergo three stages, state 1 (the initial point), state 2 and state 3 (the other two branches). After being infected by S. eriocheiris, lots of different expression genes (DEGs) were identified in each cell cluster compared with the control group (with fold change ≥2 and p-value <0.05). The bioinformatics analysis found that the Hem1 and Hem5 were the main cell clusters synthesis and release of antimicrobial peptides (AMPs) for help the host against the pathogen infection. The Hem5 also could activate the proPO system to participate in the process of host eliminate the invading S. eriocheiris. Hem7, Hem8, and Hem9 were mainly responsible for killing the invading pathogen through phagocytosis. In particular, there were also many immune-related genes identified, including β-glucan binding protein (BGBP), Clip-domain serine proteases (CLIPs), phenoloxidase 1 (PO1), anti-lipopolysaccharide factors (ALFs), syndecan, and lysozyme C, which not only played critical roles in the crab immune system but also could serve as the marker genes for identifying cell subgroup. In conclusion, the results obtained in the present study could provide pivotal insights into the crab hemocytes cell-type-specific molecular mechanisms response S. eriocheiris infection at a single-cell level.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-β-1,3-glucanase on β1-3/1-6-glucan

The glycoside hydrolase family 55 (GH55) includes inverting exo-β-1,3-glucosidases and endo-β-1,3-glucanases, acting on laminarin, which is a β1-3/1-6-glucan consisting of a β1-3/1-6-linked main chain and β1-6-linked branches. Despite their different modes of action toward laminarin, endo-β-1,3-glucanases share with exo-β-1,3-glucosidases conserved residues that form the dead-end structure of subsite-1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-β-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-β-1,3-glucanases, degraded internal β-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. β1-3-Glucans lacking β1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal β1-3-linkage of the laminarioligosaccharide moiety at thereducing end side of the main chain β1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21s-1mM-1) were much lower than to laminarin (5920s-1mM-1). These results indicate that β-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites-1 and-2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-β-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-β-1,3-glucanases.

The secretory Candida effector Sce1 licenses fungal virulence by masking the immunogenic β-1,3-glucan and promoting apoptosis of the host cells.

Candida albicans deploys a variety of mechanisms such as morphological switch and elicitor release to promote virulence. However, the intricate interactions between the fungus and the host remain poorly understood, and a comprehensive inventory of fungal virulence factors has yet to be established. In this study, we identified a C. albicans secretory effector protein Sce1, whose induction and secretion are associated with vagina-simulative conditions and chlamydospore formation. Sequence alignment showed that Sce1 belongs to a Pir family in C. albicans, which is conserved across several fungi and primarily characterized as a β-glucan binding protein in the Saccharomycescerevisiae. Mechanically, Sce1 is primarily localized to the cell wall in a cleaved form as an alkali-labile β-1,3-glucan binding protein and plays a role in masking β-glucan in acidic environments and chlamydospores, a feature that might underline C. albicans' ability to evade host immunity. Further, a cleaved short form of Sce1 protein could be released into extracellular compartments and presented in bone marrow-derived macrophages infected with chlamydospores. This cleaved short form of Sce1 also demonstrated a unique ability to trigger the caspases-8/9-dependent apoptosis in various host cells. Correspondingly, genetic deletion of SCE1 led to dampened vaginal colonization of C. albicans and diminished fungal virulence during systemic infection. The discovery of Sce1 as a versatile virulence effector that executes at various compartments sheds light on the fungus-host interactions and C. albicans pathogenesis.

GH-16 Type β-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall.

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type β-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with β-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using β-glucans.

From Cancer Therapy to Winemaking: The Molecular Structure and Applications of β-Glucans and β-1, 3-Glucanases.

β-glucans are a diverse group of polysaccharides composed of β-1,3 or β-(1,3-1,4) linked glucose monomers. They are mainly synthesized by fungi, plants, seaweed and bacteria, where they carry out structural, protective and energy storage roles. Because of their unique physicochemical properties, they have important applications in several industrial, biomedical and biotechnological processes. β-glucans are also major bioactive molecules with marked immunomodulatory and metabolic properties. As such, they have been the focus of many studies attesting to their ability to, among other roles, fight cancer, reduce the risk of cardiovascular diseases and control diabetes. The physicochemical and functional profiles of β-glucans are deeply influenced by their molecular structure. This structure governs β-glucan interaction with multiple β-glucan binding proteins, triggering myriad biological responses. It is then imperative to understand the structural properties of β-glucans to fully reveal their biological roles and potential applications. The deconstruction of β-glucans is a result of β-glucanase activity. In addition to being invaluable tools for the study of β-glucans, these enzymes have applications in numerous biotechnological and industrial processes, both alone and in conjunction with their natural substrates. Here, we review potential applications for β-glucans and β-glucanases, and explore how their functionalities are dictated by their structure.

Development of a Highly Sensitive β-Glucan Detection System Using Scanning Single-Molecule Counting Method.

To overcome the limitations of the Limulus amebocyte lysate (LAL) assay method for the diagnosis of invasive fungal infection, we applied a reaction system combining recombinant β-glucan binding proteins and a scanning single-molecule counting (SSMC) method. A novel (1→3)-β-D-glucan recognition protein (S-BGRP) and a (1→6)-β-glucanase mutant protein were prepared and tested for the binding of (1→6)-branched (1→3)-β-D-glucan from fungi. S-BGRP and (1→6)-β-glucanase mutant proteins reacted with β-glucan from Candida and Aspergillus spp. Although LAL cross-reacted with plant-derived β-glucans, the new detection system using the SSMC method showed low sensitivity to plant (1→3)-β-D-glucan, which significantly improved the appearance of false positives, a recognized problem with the LAL method. Measurement of β-glucan levels by the SSMC method using recombinant β-glucan-binding proteins may be useful for the diagnosis of fungal infections. This study shows that this detection system could be a new alternative diagnostic method to the LAL method.

3D Structural Insights into β-Glucans and Their Binding Proteins.

β(1,3)-glucans are a component of fungal and plant cell walls. The β-glucan of pathogens is recognized as a non-self-component in the host defense system. Long β-glucan chains are capable of forming a triple helix structure, and the tertiary structure may profoundly affect the interaction with β-glucan-binding proteins. Although the atomic details of β-glucan binding and signaling of cognate receptors remain mostly unclear, X-ray crystallography and NMR analyses have revealed some aspects of β-glucan structure and interaction. Here, we will review three-dimensional (3D) structural characteristics of β-glucans and the modes of interaction with β-glucan-binding proteins.

Review: The structure and function of cellulase (endo-β-1,4-glucanase) and hemicellulase (β-1,3-glucanase and endo-β-1,4-mannase) enzymes in invertebrates that consume materials ranging from microbes, algae to leaf litter

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-β-1,4-glucanase and the hemicellulase enzymes, β-1,3-glucanase and endo-β-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-β-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like β-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-β-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest β-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. β-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses β-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as β-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their β-1,3-glucanase activity. The potential function of which is discussed. Endo-β-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.

Molecular recognition of the beta-glucans laminarin and pustulan by a SusD-like glycan-binding protein of a marine Bacteroidetes.

Marine bacteria catabolize carbohydrate polymers of algae, which synthesize these structurally diverse molecules in ocean surface waters. Although algal glycans are an abundant carbon and energy source in the ocean, the molecular details that enable specific recognition between algal glycans and bacterial degraders remain largely unknown. Here we characterized a surface protein, GMSusD from the planktonic Bacteroidetes-Gramella sp. MAR_2010_102 that thrives during algal blooms. Our biochemical and structural analyses show that GMSusD binds glucose polysaccharides such as branched laminarin and linear pustulan. The 1.8 Å crystal structure of GMSusD indicates that three tryptophan residues form the putative glycan-binding site. Mutagenesis studies confirmed that these residues are crucial for laminarin recognition. We queried metagenomes of global surface water datasets for the occurrence of SusD-like proteins and found sequences with the three structurally conserved residues in different locations in the ocean. The molecular selectivity of GMSusD underscores that specific interactions are required for laminarin recognition. In conclusion, our findings provide insight into the molecular details of β-glucan binding by GMSusD and our bioinformatic analysis reveals that this molecular interaction may contribute to glucan cycling in the surface ocean.

β-Glucan-induced cooperative oligomerization of Dectin-1 C-type lectin-like domain.

Dectin-1 is a C-type lectin-like pattern recognition receptor that recognizes β(1-3)-glucans present on non-self pathogens. It is of great importance in innate immunity to understand the mechanism whereby Dectin-1 senses β(1-3)-glucans and induces intracellular signaling. In this study, we characterize the ligand binding and ligand-induced oligomerization of murine Dectin-1 using its C-type lectin-like domain (CTLD). Interaction of CTLD with laminarin, a β-glucan ligand, induced a tetramer of CTLD, as evidenced by size exclusion chromatography and multi-angle light scattering. Component analysis suggested a stoichiometry of four CTLD molecules bound to four laminarin molecules. Dimers and trimers of CTLD were not detected suggesting cooperative oligomerization. In order to map the amino acid residues of CTLD involved in β-glucan binding and domain oligomerization, we performed site-directed mutagenesis on surface-exposed and most conserved amino acid residues. Among the mutants examined, W221A, H223A and Y228A abolished oligomer formation. Since these residues are spatially arranged to form a hydrophobic groove, it is likely that W221, H223 and Y228 are directly involved in β-glucan binding. Interestingly, mutation of the residues on the other side of the hydrophobic groove, including Y141, R145 and E243, also exhibited reduced oligomer formation, suggesting involvement in protein-protein interactions guided by laminarin. Ligand titration using intrinsic tryptophan fluorescence revealed that wild-type CTLD binds laminarin cooperatively with a Hill coefficient of ~3, while the oligomer-reducing mutations, inside and outside the putative binding site abolish or decrease cooperativity. We suggest that the ligand-induced cooperative oligomer formation of Dectin-1 is physiologically relevant in sensing exogenous β-glucan and triggering intracellular signaling.

Phenoloxidase activation, antimicrobial, and antibiofilm properties of β-glucan binding protein from Scylla serrata crab hemolymph

In this study, we purified β-GBP from hemolymph of Scylla serrata crabs using affinity chromatography. The purified S. serrata β-GBP (Ss-β-GBP) had 100kDa molecular mass in the SDS-PAGE. MALDI-TOF/TOF analysis was conducted, revealing that the purified 100kDa protein had 96% similarity with β-GBP of Astacus leptodactylus. Ss-β-GBP was characterized using high-performance liquid chromatography (HPLC), X-ray diffraction (XRD) analysis, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy, which confirmed the structure of the Ss-β-GBP. The purified Ss-β-GBP was functionally analyzed by yeast agglutination and phagocytic reaction assays. Moreover, the PO enhancing ability of Ss-β-GBP was evidenced through PO activity. Specifically, the antibacterial activity of the Ss-β-GBP against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria was evaluated by determining its minimum inhibitory concentration (MIC)<60μg/ml for all tested species. Furthermore, the antibiofilm efficacy of Ss-β-GBP at 50 and 100μg/ml was outlined using light microscopy and confocal laser scanning microscopy (CLSM). Bacterial viability assays also outlined the dose-dependent activity of Ss-β-GBP based on the ratio of live/dead bacterial cells. The results of this study revealed that crab-borne Ss-β-GBP might be widely used to suppress the growth of pathogenic bacteria.

A study on β-glucan binding protein (β-GBP) and its involvement in phenoloxidase cascade in Indian white shrimp Fenneropenaeus indicus

The present study reports the purification of novel immune molecule β-1, 3 glucan binding protein from the heamolymph of the Indian white shrimp, Fenneropenaeus indicus (Fiβ-GBP). The purified Fiβ-GBP had 95kDa molecular weight in SDS-PAGE analysis. MALDI-TOF/TOF analysis revealed that the purified Fiβ-GBP showed similarity to various crustacean proteins; 48 and 46% similarity was observed for β-1, 3 glucan binding protein of Chinese white shrimp Fenneropenaeus chinensis and banana shrimp Fenneropenaeus merguiensis, with MOWSE score of 3.11e+12 and 2.05e+8, respectively. The phenoloxidase activity (PO) of Fiβ-GBP was evaluated and, in the presence of laminarin, PO activity increased significantly. Substrate specificity assay demonstrated that Fiβ-GBP had the specific binding site for soluble or insoluble β-glucan (laminarin), since the PO activity increased in the presence of laminarin when compared to other sugars. Enzymatic activities revealed that the optimum temperature and pH for Fiβ-GBP activating PO were 40°C and pH 7–8. Moreover, even at 100°C Fiβ-GBP enhanced PO activity highlighting that Fiβ-GBP was thermostable and thermophilic in nature. Among various divalent metallic ions, Fiβ-GBP significantly promoted the PO activity in presence of Mg2+ and Ca2+. The breakdown of para nitroanilide from Nα−Benzoyl-l-Arginine 4-Nitroanilide hydrochloride showed that serine protease activity was induced by Fiβ-GBP and also increased concentration of Fiβ-GBP evoked the activity. Furthermore, hemolytic activity tests revealed that PO reaction product induced RBC membrane damage and cell shrinkage. Lastly, Baclight bacterial viability assays showed maximum killing effect of PO reaction product on both Gram positive and Gram negative bacteria.

Identification and functionality prediction of pathogenesis-related protein 1 from legume family.

The production and accumulation of pathogenesis-related (PR) proteins in plants is one of the important responses to biotic and abiotic stress. Large number of identified PR proteins has been categorized into 17 functional families based on their structure, phylogenetics, and biological activities. However, they are not widely studied in legume crops. Using 29 PR1 proteins from Arabidopsis thaliana, as query, here we have predicted 92 candidate PR1 proteins through the PSI-BLAST and HMMER programs. These candidate proteins were comprehensively analyzed with, multiple sequence alignment, domain architecture studies, signal peptide, and motif extraction followed by phylogenetic analysis. Further, response of two candidate PR1 proteins from chickpea against Fusarium oxysporum f.sp.ciceri attack was validated using qRT-PCR followed by their 3D structure prediction. To decipher mode of action for PR1s, docking of pathogen extracellular matrix components along with fungal elicitors was performed with two chickpea PR1 proteins. Based on these findings, we propose carbohydrate to be the unique pathogen-recognition feature for PR1 proteins and β-glucanase activity via β-glucan binding or modification.

Tryptophan introduction can change β-glucan binding ability of the carbohydrate-binding module of endo-1,3-β-glucanase.

Endo-1,3-β-glucanase from Cellulosimicrobium cellulans DK-1 has a carbohydrate-binding module (CBM-DK) at the C-terminal side of a catalytic domain. Out of the imperfect tandem α-, β-, and γ-repeats in CBM-DK, the α-repeat primarily contributes to β-glucan binding. This unique feature is derived from Trp273 in α-repeat, whose corresponding residues in β- and γ-repeats are Asp314 and Gly358, respectively. In this study, we generated Trp-switched mutants, W273A/D314W, D270A/W273A/D314W, W273A/G358W, and D270A/W273A/G358W, and analyzed their binding abilities toward laminarioligosaccharides and laminarin. While the binding affinities of D270A/W273A and W273A mutants were either lost or much lower than that of the wild-type, those of Trp-switched mutants recovered, indicating that a Trp introduction in β- or γ-repeat can substitute the α-repeat by primarily contributing to β-glucan binding. Thus, we have successfully engineered a CBM-DK that binds to laminarin by a mechanism different from that of the wild-type, but with similar affinity.

Transcriptional expression of immune system genes inLitopenaeus vannameiduring ontogenetic development

The gene expression level for immune system genes was evaluated at different lifecycle stages in the white shrimp (Litopenaeus vannamei). The gene-coding pattern recognition protein, β-glucan binding protein–high density lipoprotein (BGBP), from haemocytes, the effector proteins peroxinectin and clotting protein (CP), the antibacterial proteins Lysozyme, Penaeidin 3 and prophenoloxidase (proPO), and the small heat-shock protein 10 were studied. The expression of BGBP was detected from the Nauplii 3 substage and beyond (except for PL1). Peroxinectin and CP were constitutively transcribed throughout the different substages. Lysozyme, Penaeidin and proPO were also detected throughout the experimental stages (except eggs and nauplii). Lysozyme was transcribed during most of the shrimp substages, from Zoea 2 and beyond. Penaeidin 3 exhibited the lowest expression level of the genes evaluated; however, its transcription was similar to those of adults from post-larvae 11 and forward. proPO was higher transcribed during last post-larval substages. Heat-shock protein-10 was detected in all shrimp substages and exhibited higher expression at the PL substages compared to the first larval substages. Evidence suggests that the shrimp immune system is partially activated from the early larval substages, but is not completed until the shrimp have undergone the metamorphic substages.

Molecular cloning, relative expression, and structural analysis of pattern recognition molecule β-glucan binding protein from mangrove crab Episesarma tetragonum.

A full-length cDNA of a β-glucan binding protein (β-GBP) gene was identified from the mangrove crab Episesarma tetragonum. The open reading frame of the E. tetragonum β-GBP (Epte β-GBP) is 1,167 bp long, encoding a polypeptide of 389 amino acids. The deduced amino acid sequence of Epte β-GBP gene has conserved a potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites. Phylogenetic analysis of the Epte β-GBP gene showed the similarity with β-GBPs of other crustaceans and arthropods. Quantitative RT-PCR results showed the upregulation of Epte β-GBP gene expression in E. tetragonum hemocytes following a 12-H challenge in response to β-glucan (β-G). Epte β-GBP was involved in the regulation and activation of the prophenoloxidase cascade. A three-dimensional structure of active Epte β-GBP was modeled by homology modeling and refined with molecular dynamics simulations. A structural aspect of the protein is discussed based on experimental and theoretical results obtained.

Gene expression responses of white shrimp (Litopenaeus vannamei) infected with necrotizing hepatopancreatitis bacterium

Abstract Necrotizing hepatopancreatitis bacterium (NHPB) affects the production of shrimp farms along Latin America. NHPB is one of the main threats to shrimp culture; therefore the defense mechanisms and molecular mediators that act against this bacterium ought to be well understood. The aim of this study was to study the changes in the immune response and the expression of genes coding for proteins of the immune system in the white shrimp, Litopenaeus vannamei, infected with NHPB. Shrimp infected with NHPB and healthy shrimp were maintained under laboratory conditions during 24 d. Haemolymph and hepatopancreas were collected from shrimp every six days. Total hemocyte count (THC), activities of α-2-Macroglobulin (A-2-M), phenoloxidase and total phenoloxidase from haemolymph were monitored. The expression of clottable protein (CP) and lipopolysaccharide & β-glucan binding protein (LGBP) were determined from hepatopancreas, whereas the expression of A-2-M, prophenoloxidase (proPO) and transglutaminase (TGase) was determined from hemocytes. THC showed no significant differences among infected and healthy shrimp at any of the four times. The specific activity of A-2-M significantly decreased on days 18th and 24th post-infection. In the case of the specific activity of total phenoloxidase and phenoloxidase a significant decrease was detected only in day 18 post-infection. CP gene expression decreased at 6th day post-infection, while at 18 days showed an increase of about 2.5-fold. LGBP expression increased in the first six days and at the 24th day post-infection had a drastic decline. The expression of A-2-M showed no significant changes in any of the four times. proPO expression increased more than 4-fold at day 12th post-infection but decreased at 18th day post-infection. TGase expression increased 3.5-fold at day 12th post-infection. The results found in this study suggest an active participation of the proPO system and the clotting reaction against NHP infection, mostly on days 12th and 18th post-infection.

The Effects of &amp;#946; -Glucans on Dendritic Cells and Implications for Cancer Therapy

β-Glucans are polysaccharides of β-D-glucose extracted from the cell walls of different species of mushrooms, yeast, oat, barley, seaweeds, algae and bacteria. Modern biomedical research has identified β-glucans as biological response modifiers (BRM) with anti-tumor properties that elicit potent immune responses through their recognition by a variety of pattern recognition receptors (PRRs) on dendritic cells (DCs), macrophages and neutrophils. Complement receptor-3 (CR3), lactosylceramides, scavenger receptors and dectin-1 are involved in β-glucan recognition, triggering a series of signaling events that modulate innate and subsequently adaptive immune responses. β-Glucan binding to specific receptors in DCs and macrophages triggers their activation and maturation, increases their antigen-presentation ability and enhances the production of proinflammatory cytokines that stimulate the polarization of TH1 or TH17 responses, and induces the activation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Moreover, large β-glucans can be degraded by macrophages into smaller moieties, when released, prime CR3 receptor on neutrophils and natural killer (NK) cells mediating CR3-dependent cellular cytotoxicity (CR3-DCC) of iC3b opsonized tumor cells. Elucidating the molecular mechanisms of β- glucan-induced signaling in immune cells is essential for the design of new therapeutic strategies against cancer. Future studies should be done to translate β-glucan research to the clinic.

Purification and characterization of a β-glucan binding protein from the haemolymph of freshwater prawnMacrobrachium rosenbergii

β-glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β-glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β-glucan increased the specific PO activity compared with that of β-glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS-PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS-PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non-covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti-βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL−1. Furthermore, the applicability of the developed ELISA is discussed.

β-Glucan modulates the lipopolysaccharide-induced innate immune response in rat mammary epithelial cells

Mastitis, caused by mammary pathogenic bacteria which are frequent implications of Escherichia coli, is an important disease affecting women and dairy animals worldwide. The β-glucan binding of dectin-1 can induce its own intracellular signaling and can mediate a variety of cellular responses. This work was to investigate the effect of β-glucan on the lipopolysaccharide (LPS)-induced inflammatory response and related innate immune signaling in primary rat mammary epithelial cells. Cells were treated with serum-free medium added with a DMSO solution containing β-glucans at concentrations of 0, 1, 5, 25μmol/L for 12h, and then exposed to 10μg/mL LPS for 40min. Moreover, cells were pretreated with BAY 11-7082 to inhibit NF-κB and then successively exposed to 5μmol/L β-glucan, 10μg/mL LPS, 5μmol/L β-glucan and 10μg/mL LPS, according to the specific experimental design. Normal control cultures contained an equal volume of DMSO, which was collected at the same time. After incubating rat mammary epithelial cells for40 min with 10μg/mL LPS, TLR4, MyD88 and NF-κB expression all increased (P<0.05), as did the secretion of TNF-α and IL-1β (P<0.05), but IκB and β-casein expression both decreased (P<0.05). Treatment with different concentrations of β-glucan for 12h activated Dectin1/Syk, which subsequently suppressed TLR4, MyD88 and NF-κB expression and TNF-α and IL-1β secretion. However, it restored the IκB and β-casein expression that had been induced by the 40min incubation with 10μg/mL LPS. Pretreatment with BAY 11-7082 at 10µmol/L for 2h partially prevented NF-κB induction by LPS, but the presence of β-glucan prevented this inactivation. BAY 11-7082 could not simultaneously inhibit LPS induction of TLR4, MyD88 and β-glucan activation of Dectin1/Syk in rat mammary epithelial cells. These findings demonstrated that β-glucan activation of Dectin1/Syk attenuated LPS induction of TLR4/MyD88/NF-κB and inhibited the LPS-induced inflammation factors in mammary epithelial cells, thereby providing a possibly protective effect of β-glucan in the prevention of LPS-induced dysfunction in mammary epithelial cells.