Rui Tada

Binding Capacity of a Barley β-d-Glucan to the β-Glucan Recognition Molecule Dectin-1

To clarify whether barley beta-glucans exhibit their biological effects via binding to dectin-1, a pivotal receptor for beta-1,3-glucan, the structure of barley beta-glucan E70-S (BBG-70) was unambiguously investigated by NMR spectroscopy and studied for its binding capacity and specificity to dectin-1 by ELISA. NMR spectroscopy confirmed that BBG-70 contains two different linkage glucans, namely, alpha-glucan and beta-glucan, which are not covalently attached to one another. Beta-glucan within BBG-70 is a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose residues but does not contain the continuous 1,3-linkage. Competitive ELISA revealed that highly purified barley beta-glucan E70-S (pBBG-70) inhibits the binding of soluble dectin-1 to sonifilan (SPG), a beta-1,3-glucan, although at a concentration higher than that of SPG and laminarin. It was found that barley beta-glucan can be recognized by dectin-1, implying that barley beta-glucan might, at least in part, exhibit its biological effects via the recognition by dectin-1 of the ligand sugar structure, which may be formed by 1,3-beta- and 1,4-beta-glucosyl linkage.

Barley-derived β-d-glucan induces immunostimulation via a dectin-1-mediated pathway

Barley-derived beta-glucan, a linear mixed-linkage beta-glucan composed of 1,3- and 1,4-beta-D-glucopyranose polymers, binds to dectin-1. However, whether it can trigger signal transduction via dectin-1 remains unclear. In this study, we used a reporter gene assay to determine whether barley-derived beta-d-glucan can activate NF-kappaB via dectin-1-mediated signaling when dectin-1 is cotransfected with Syk, CARD9, and Bcl10 in 293T cells. We found that barley-derived beta-D-glucan can activate NF-kappaB leading to cytokine production when dectin-1, Syk, CARD9, and Bcl10 are coexpressed in the cells. We also found that barley-derived beta-D-glucan can induce the phosphorylation of Syk and production of IL-6 in thioglycolate-elicited peritoneal macrophages. These results indicated that the immunostimulatory effects of barley-derived beta-d-glucan might be exerted, at least in part, via dectin-1.

An unambiguous structural elucidation of a 1,3-β-d-glucan obtained from liquid-cultured Grifola frondosa by solution NMR experiments

Grifolan LE (GRN-LE), a purified beta-D-glucan, which is obtained from liquid-cultured Grifola frondosa, exhibits various biological activities, including antitumor effects. Significant progress has been made in the study of these effects. However, an unambiguous structural characterization of GRN-LE using NMR spectroscopy has not been carried out as yet. It is well accepted that the biological effects of a beta-glucan depend on its primary structure, conformation, and molecular weight. In the present study, we unambiguously elucidate the primary structure of GRN-LE using NMR spectroscopy. The data presented here reveal that GRN-LE comprises a 1,3-beta-D-glucan backbone with a single 1,6-beta-D-glucosyl side branching unit on every third residue.

Structural characterisation and biological activities of a unique type β- d -glucan obtained from Aureobasidium pullulans

A β-d-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of β-d-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-β-d-glucan backbone with single 1-6-β-d-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this β-d-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-β-glucans like AP-FBG with such a high number of 1-6-β-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-β-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.

Undressing the fungal cell wall/cell membrane--the antifungal drug targets.

Being external, the fungal cell wall plays a crucial role in the fungal life. By covering the underneath cell, it offers mechanical strength and acts as a barrier, thus protecting the fungus from the hostile environment. Chemically, this cell wall is composed of different polysaccharides. Because of their specific composition, the fungal cell wall and its underlying plasma membrane are unique targets for the development of drugs against pathogenic fungal species. The objective of this review is to consolidate the current knowledge on the antifungal drugs targeting the cell wall and plasma membrane, mainly of Aspergillus and Candida species - the most prevalent fungal pathogens, and also to present challenges and questions conditioning the development of new antifungal drugs targeting the cell wall.

Agaricus brasiliensis-derived β-glucans exert immunoenhancing effects via a dectin-1-dependent pathway

Agaricus brasiliensis is a well-known medicinal mushroom. We have previously demonstrated that Agaricus-derived polysaccharides exhibit potent antitumor effects; however, the underlying mechanism(s) have not been elucidated yet. In this study, we examined the immunoenhancing activities of Agaricus extracts. Agaricus-derived polysaccharides were characterized as 1,6-β-glucan with a small amount of 1,3-β-glucan using anti-β-glucan antibody and nuclear magnetic resonance analysis. These polysaccharides strongly induced the production of various cytokines from both murine splenocytes and bone marrow-derived dendritic cells in the presence of exogenous granulocyte-macrophage colony-stimulating factor. Polysaccharide-induced cytokine production was significantly reduced in bone marrow-derived dendritic cells derived from dectin-1-deficient mice. Furthermore, a binding assay revealed that the Agaricus-derived polysaccharides can be recognized by dectin-1, a pivotal receptor for 1,3-β-glucan. Taken together, our results clearly indicate that the immunostimulation induced by Agaricus-derived polysaccharides is exerted, at least in part, via dectin-1 in combination with granulocyte-macrophage colony-stimulating factor.

Pathway of glycine betaine biosynthesis in Aspergillus fumigatus

ABSTRACT The choline oxidase (CHOA) and betaine aldehyde dehydrogenase (BADH) genes identified in Aspergillus fumigatus are present as a cluster specific for fungal genomes. Biochemical and molecular analyses of this cluster showed that it has very specific biochemical and functional features that make it unique and different from its plant and bacterial homologs. A. fumigatus ChoAp catalyzed the oxidation of choline to glycine betaine with betaine aldehyde as an intermediate and reduced molecular oxygen to hydrogen peroxide using FAD as a cofactor. A. fumigatus Badhp oxidized betaine aldehyde to glycine betaine with reduction of NAD+ to NADH. Analysis of the AfchoAΔ::HPH and AfbadAΔ::HPH single mutants and the AfchoAΔAfbadAΔ::HPH double mutant showed that AfChoAp is essential for the use of choline as the sole nitrogen, carbon, or carbon and nitrogen source during the germination process. AfChoAp and AfBadAp were localized in the cytosol of germinating conidia and mycelia but were absent from resting conidia. Characterization of the mutant phenotypes showed that glycine betaine in A. fumigatus functions exclusively as a metabolic intermediate in the catabolism of choline and not as a stress protectant. This study in A. fumigatus is the first molecular, cellular, and biochemical characterization of the glycine betaine biosynthetic pathway in the fungal kingdom.

A highly branched 1,3-β-D-glucan extracted from Aureobasidium pullulans induces cytokine production in DBA/2 mouse-derived splenocytes.

We recently elucidated the structure of a highly branched 1,3-beta-D-glucan with 6-monoglucopyranosyl side chains, extracted from Aureobasidium pullulans (AP-FBG). Although the biological effects of beta-D-glucans are known to depend on their structures, the effects of a highly branched 1,3-beta-D-glucan on the production of cytokines by leukocytes in mice have not yet been elucidated. In this study, we found that AP-FBG strongly induced the production of various cytokines, especially Th1 cytokines (e.g., IFN-gamma and IL-12p70) and Th17 cytokines (e.g., IL-17A), but did not induce the production of IL-4, IL-10, and TNF-alpha in DBA/2 mouse-derived splenocytes in vitro.

Induction of IFN-γ by a highly branched 1,3-β-d-glucan from Aureobasidium pullulans in mouse-derived splenocytes via dectin-1-independent pathways

We have previously elucidated the precise structure of a unique type of 1,3-β-D-glucan, AP-FBG (Aureobasidium pullulans-fermented β-D-glucan), from the fungus A. pullulans and found that AP-FBG strongly induced the production of various cytokines in DBA/2 mouse-derived splenocytes in vitro. However, the mechanism(s) of action of AP-FBG on in vitro mouse primary cells have not been characterized in detail. Herein, we report that the production of IFN-γ in DBA/2 mouse-derived splenocytes by AP-FBG was not inhibited following treatment with an anti-dectin-1 neutralizing antibody. In addition, AP-FBG not only failed to activate dectin-1-mediated signaling pathways, examined by a reporter gene assay but also failed to bind to dectin-1, a pivotal receptor for 1,3-β-D-glucan. Taken together, AP-FBG induced cell activation via dectin-1-independent pathways.

Attachment of class B CpG ODN onto DOTAP/DC-chol liposome in nasal vaccine formulations augments antigen-specific immune responses in mice

BackgroundTo overcome infectious diseases, the development of mucosal vaccines would be an effective strategy, since mucosal surfaces are the entry site for most pathogens. In general, protein antigens show inherently poor immunogenicity when administered by the mucosal route. Therefore, co-administration of an appropriate mucosal adjuvant is required to exert immune responses toward pathogen-derived antigens effectively. However, the development of a safe and effective mucosal adjuvant system is still challenging. Although, recent studies reported that oligodeoxynucleotides (ODNs) containing immunostimulatory CpG motifs (CpG ODNs) act as potent mucosal adjuvants and are useful in the formulation of nasal vaccines, there are some disadvantages. For instance, the administration of phosphorothioate (PS)-modified CpG ODNs can induce adverse systemic effects, such as splenomegaly, in a dose-dependent manner. Therefore, a reduced dose of CpG ODN might be crucial when used as vaccine adjuvant for clinical purposes. Therefore, we prepared a CpG ODN-loaded cationic liposome, and evaluated its mucosal adjuvant activity.ResultsWe prepared a CpG ODN-loaded DOTAP/DC-chol liposome that was stable during our experiments, by mixing CpG ODNs and liposomes at an N/P ratio of 4. Further, we demonstrated that the attachment of class B CpG ODN to the DOTAP/DC-chol liposomes synergistically enhanced antigen-specific IgA production in the nasal area than that induced by CpG ODN and DOTAP/DC-chol liposomes alone. The endpoint titers were more than tenfolds higher than that induced by either single CpG ODN or single DOTAP/DC-chol liposomes. Additionally, although serum IgG1 responses (indicated as a Th2 response) remained unchanged for DOTAP/DC-chol liposomes and CpG ODN-loaded DOTAP/DC-chol liposomes, the CpG ODN-loaded DOTAP/DC-chol liposomes synergistically induced the production of serum IgG2a (indicated as a Th1 response) than that by the individual liposomes.ConclusionsWe conclude that the advantage of using DOTAP/DC-chol liposome harboring CpG ODN is it induces both antigen-specific mucosal IgA responses and balanced Th1/Th2 responses. Therefore, such a combination enables us to resolve the adverse effects of using CpG ODNs (as a mucosal adjuvant) by reducing the overall dose of CpG ODNs. Further, the biodegradable and essentially non-antigenic nature of the liposomes makes it superior than the other existing mucosal adjuvants.