epiglucan and laminaran

Heparin has been the drug of choice in clinical pre-surgical and post-surgical prophylaxis of thrombotic events. However, because of its side-effects, such as bleeding and other disadvantages (i.e. chemical inhomogeneity and variability of its physiological activities), alternatives to heparin are an important field of research. A necessary procedure in the development of new drugs is the evaluation of structure-activity relationships. Genuine neutral polysaccharides were chemically modified and examined for their anticoagulant activities. The linear beta-1,3-glucan curdlan, an easily available bacterial polysaccharide, served as the basic polymer. It could be established that the anticoagulant activity was dependent on the degree of sulfation and the molecular weight. For heparin, the sulfation pattern, i.e. the actual location of the sulfate groups along the heparin chain, was of importance in addition to the degree of sulfation. Therefore, we investigated whether there was also a relationship between the substitution pattern of the curdlan sulfates and their anticoagulant activity. For determination of the substitution pattern of the sulfated polysaccharides, a method was developed that is based on synthesis of the partially alkylated alditol acetates of the polymer and examination of these derivatives using combined gas chromatography-mass spectrometry. In addition to the analytical data, the structure-activity relationship of anticoagulative curdlan sulfates is presented.

Topics: Animals; beta-Glucans; Blood Coagulation Tests; Dimethylformamide; Drug Evaluation; Fibrinolytic Agents; Gas Chromatography-Mass Spectrometry; Glucans; Glycosaminoglycans; Heparin; Heparinoids; Humans; Methylation; Molecular Weight; Neovascularization, Pathologic; Polysaccharides; Structure-Activity Relationship; Sulfates; Thrombosis

Bacterial β-glucans are exopolysaccharides (EPSs), which can protect bacteria or cooperate in biofilm formation or in bacterial cell adhesion.

Topics: Anti-Inflammatory Agents; beta-Glucans; Caco-2 Cells; Cell Adhesion; Coculture Techniques; Cytokines; Gene Expression Regulation; Glucans; Humans; Interleukin-10; Interleukin-8; Lactococcus lactis; Pediococcus; THP-1 Cells; Tumor Necrosis Factor-alpha

Alcoholic liver disease (ALD) has become a health issue globally. Laminarin, a low molecular weight marine-derived β-glucan, has been identified with multiple biological activities. In this study, the ameliorative effect on ALD of laminarin isolated from brown algae was investigated. Phenotypic, pathological alterations and biochemical characteristics indicated that laminarin administration (100 mg/kg/day) significantly alleviated liver injury and improved liver function in the alcohol-induced mice. Gene chip results indicated that laminarin treatment caused 52 up-regulated and 13 down-regulated genes in the hepatic tissues of alcohol-induced damage mice, and most of these genes are associated with regulation of oxidative stress (such as CYP450/glutathione-dependent antioxidation), Wnt signaling pathway, retinol metabolism, and cAMP pathway based on GO and KEGG analysis. PPI network analysis indicated that the downstream target genes lied in the hub of the net. Our experiments also confirmed the changed expressions of some target genes. Taken together, these results suggest that laminarin can ameliorate alcohol-induced damage in mice and its molecular mechanism lies in modulating anti-oxidation pathway, WNT pathway, and cAMP pathway, which regulate the expressions of downstream target genes and alleviate alcohol-induced damage. Our study provides new clue to prevent alcohol-induced damage and will be benefit to develop functional foods. PRACTICAL APPLICATIONS: This study verified the positive effect on alcoholic liver disease (ALD) of laminarin, a water-soluble brown algae-derived β-glucan, linked by β-(1,3) glycosidic bonds with β-(1,6) branches. Laminarin significantly alleviated liver injury and improved liver function of ALD mice. Moreover, transcriptomics and bioinformatics analysis further revealed the gene expression patterns, hub targets, and signalings including CYP450/glutathione, Wnt, retinol metabolism, cAMP pathways regulated by laminarin. This research is the first evidence for hepatoprotective effect of laminarin against ALD and its molecular mechanism, which will be advantage to develop functional foods or adjuvant therapy of ALD.

Topics: Animals; beta-Glucans; Ethanol; Glutathione; Liver Diseases, Alcoholic; Mice; Vitamin A

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, β-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of β-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Topics: Bacterial Proteins; beta-Glucans; Cellulose; Cloning, Molecular; Galactose; Glucans; Glycoside Hydrolases; Hot Temperature; Hydrogen-Ion Concentration; Mannans; Planctomycetales; Planctomycetes; Substrate Specificity; Talaromyces; Xylans

An endo-β-1,3(4)-glucanase AnENG16A from Aspergillus nidulans shows distinctive catalytic features for hydrolysis of β-glucans. AnENG16A hydrolyzed Eisenia bicyclis laminarin to mainly generate 3-O-β-gentiobiosyl-d-glucose and hydrolyzed barley β-glucan to mainly produce 3-O-β-cellobiosyl-d-glucose. Using molecular exclusion chromatography, we isolated and purified 3-O-β-cellobiosyl-d-glucose and 3-O-β-gentiobiosyl-d-glucose, respectively, from AnENG16A-hydrolysate of barley β-glucan and E. bicyclis laminarin. Further study reveals that 3-O-β-cellobiosyl-d-glucose had 8.99-fold higher antioxidant activity than barley β-glucan and 3-O-β-gentiobiosyl-d-glucose exhibited 43.0% higher antioxidant activity than E. bicyclis laminarin. Notably, 3-O-β-cellobiosyl-d-glucose and 3-O-β-gentiobiosyl-d-glucose exhibited 148.9% and 116.0% higher antioxidant activity than laminaritriose, respectively, indicating that β-1,4-linkage or -1,6-linkage at non-reducing end of β-glucotrioses had enhancing effect on antioxidant activity compared to β-1,3-linkage. Furthermore, 3-O-β-cellobiosyl-d-glucose showed 237.9% higher antioxidant activity than cellotriose, and laminarin showed 5.06-fold higher antioxidant activity than barley β-glucan, indicating that β-1,4-linkage at reducing end of β-glucans or oligosaccharides resulted in decrease of antioxidant activity compared to β-1,3-linkage.

Topics: Antioxidants; Aspergillus nidulans; beta-Glucans; Biphenyl Compounds; Catalysis; Cellobiose; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glucose; Hordeum; Hydrolysis; Molecular Structure; Picrates; Structure-Activity Relationship; Substrate Specificity

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe-derived or modified-self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying β-glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different β-glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) β-1,3-glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long β-1,3-glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short β-1,3-glucans. Hydrolysis of the β-1,6 side-branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long-chain β-glucans. Moreover, in contrast to the recognition of short β-1,3-glucans in A. thaliana, perception of long β-1,3-glucans in N. benthamiana and rice is independent of CERK1, indicating that β-glucan recognition may be mediated by multiple β-glucan receptor systems.

Topics: Arabidopsis; beta-Glucans; Brachypodium; Capsella; Glucans; Hordeum; Nicotiana; Oligosaccharides; Plant Immunity; Plant Leaves; Plant Proteins; Receptors, Immunologic; Species Specificity

We previously reported that chitinases reconstituted heat-inactivated stipe cell wall extension in a steady and continuous extension profile by cleaving chitins cross-linked to various polysaccahrides, whereas, endo-β-1,3-glucanases reconstituted heat-inactivated stipe wall extension in a profile of an initially fast extension and subsequent termination of extension due to its degradation of β-1,3-glucan but not other polysaccharides such as β-1,6-glucans cross-linked to chitins. Thus, a novel endo-β-1,6-glucanase, GH30A, from Coprinopsis cinerea was cloned and characterized to study cross-linking of β-1,6-glucan and wall extensibility in stipe walls. GH30A had higher activity and better thermophilicity than reported β-1,6-glucanases. GH30A hydrolyzed pustulan having β-1,6-linkages but not other polysaccharides without β-1,6-linkages; GH30A did not cleave gentiobiose and single β-1,6-linkage branches in laminarin from Laminaria digitata but cut consecutive β-1,6-linkage branches in laminarin from Eisenia bicyclis. GH30A reconstituted heated-inactivated stipe cell wall extension with release of glucose and gentiobiose, indicating that β-1,6-glucans were present and cross-linked to chitins in stipe walls, and cleaving β-1,6-glucans cross-linked to chitins by GH30A led to wall loosening for extension. However, GH30A individually or in combination with endo-β-1,3-glucanase reconstituted-stipe wall extension profile was similar to individual endo-β-1,3-glucanase's, exploring that chitins were also cross-linked to other polysaccharides besides β-1,3-glucans and β-1,6-glucans.

Topics: Agaricales; beta-Glucans; beta-Glucosidase; Cell Wall; Chitin; Chitinases; Fungal Proteins; Glucans; Hydrolysis

Adipocytes from white-adipose tissue are known to produce inflammatory cytokines, which play a major role in energy balance and metabolism. While they can respond to pathogen-associated molecular pattern (PAMPs) such as lipopolysaccharide (LPS) from bacteria, it is not known whether adipocytes can be stimulated by fungal cells. Previously, adipocytes were shown to produce toll-like receptor 2 (TLR2), a β-glucan receptor, suggesting that they could respond to β-glucan on the fungal cell wall. In this study, we show that heat-killed yeast induce an inflammatory response in adipocytes. Using fungal-like particles, namely laminarin-coated beads (LCB), we find that these particles trigger the expression of many key inflammatory genes in dose- and time-dependent fashions in adipocytes. These results suggest that β-glucan on the fungal cell wall is sufficient to elicit an inflammatory response in adipocytes. In addition, we show that both LCB and LCB-treated conditioned medium from RAW 264.7 murine macrophages (LCB-RM) induce the expression of those inflammatory genes through IKKβ-IκBα proteins. Together, we conclude that the fungal-like particles and the conditioned medium elicit an inflammatory response in adipocytes through the canonical or classical NF-κB pathway.

Topics: 3T3-L1 Cells; Adipocytes; Adipose Tissue, White; Animals; beta-Glucans; Culture Media, Conditioned; Cytokines; Glucans; Inflammation; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; NF-KappaB Inhibitor alpha; Signal Transduction; Tumor Necrosis Factor-alpha

Airway exposure to 1,3-β-D-glucan (β-glucan), an essential component of the cell wall of several pathogenic fungi, causes various adverse responses, such as pulmonary inflammation and airway hypersensitivity. The former response has been intensively investigated; however, the mechanism underlying β-glucan-induced airway hypersensitivity is unknown. Capsaicin-sensitive lung vagal (CSLV) afferents are very chemosensitive and stimulated by various insults to the lungs. Activation of CSLV afferents triggers several airway reflexes, such as cough. Furthermore, the sensitization of these afferents is known to contribute to the airway hypersensitivity during pulmonary inflammation. This study was carried out to determine whether β-glucan induces airway hypersensitivity and the role of the CSLV neurons in this hypersensitivity. Our results showed that the intratracheal instillation of β-glucan caused not only a distinctly irregular pattern in baseline breathing, but also induced a marked enhancement in the pulmonary chemoreflex responses to capsaicin in anesthetized, spontaneously breathing rats. The potentiating effect of β-glucan was found 45 min later and persisted at 90 min. However, β-glucan no longer caused the irregular baseline breathing and the potentiating of pulmonary chemoreflex responses after treatment with perineural capsaicin treatment that blocked the conduction of CSLV fibers. Besides, the potentiating effect of β-glucan on pulmonary chemoreflex responses was significantly attenuated by N-acetyl-L-cysteine (a ROS scavenger), HC-030031 (a TRPA1 antagonist), and Laminarin (a Dectin-1 antagonist). A combination of Laminarin and HC-030031 further reduced the β-glucan-induced effect. Indeed, our fiber activity results showed that the baseline fiber activity and the sensitivity of CSLV afferents were markedly elevated by β-glucan instillation, with a similar timeframe in anesthetized, artificially ventilated rats. Moreover, this effect was reduced by treatment with HC-030031. In isolated rat CSLV neurons, the β-glucan perfusion caused a similar pattern of potentiating effects on capsaicin-induced Ca

Topics: Acetanilides; Acetylcysteine; Animals; beta-Glucans; Capsaicin; Cells, Cultured; Glucans; Lectins, C-Type; Lung; Male; Neurons, Afferent; Purines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Reflex; Respiration; Respiratory Hypersensitivity; Sensory Receptor Cells; TRPA1 Cation Channel; Vagus Nerve

There is evidence that major components of the fungi cell wall not only define fungal properties and survival but also are responsible for their biological activities. Some data indicate that structural components of the fungal cell wall exert stimulatory/modulatory effects on immunocompetent cells acting as pathogen-associated molecular patterns (PAMPs). Fungal components can influence the activity of certain immune cell populations by affecting cell maturation and proliferation, promoting phagocytosis, cytotoxic activity, and cell migration, as well as production of various mediators. However, there is little information available concerning the impact of fungal-derived components on peripheral blood mononuclear cell (PBMC) activation. The aim of this study was to determine whether certain fungi-associated molecules, i.e., β-(1,3)-glucans (zymosan and curdlan) and mannan activate in vitro human PBMCs to synthesize cytokines, including chemokines. We documented that PBMCs, in response to stimulation with zymosan, curdlan, and mannan, express cytokines IFN-γ and GM-CSF, and chemokine CCL3, both at protein and transcript levels, as well as cytokine IL-1β and chemokine CXCL8, at mRNA level. Our observations support the idea that fungal-derived components can activate immune cells, including PBMCs, by stimulation of cytokine/chemokine production. A thorough understanding of this interaction is of prime importance since it influence both pathophysiological and immune processes as well as anti-fungal defense mechanisms.

Topics: beta-Glucans; Cells, Cultured; Cytokines; Fungi; Glucans; Humans; Lectins, C-Type; Leukocytes, Mononuclear; Mannans; Oxazines; Protein Kinase Inhibitors; Pyridines; RNA, Messenger; Syk Kinase; Zymosan

Some β-glucans have attracted attention due to their functionality as an immunostimulant and have been used in processed foods. However, accurately measuring the β-glucan content of processed foods using existing methods is difficult. We demonstrate a new method, the Sodium hypochlorite Extracting and Enzymatic Digesting (SEED) assay, in which β-glucan is extracted using sodium hypochlorite, dimethyl sulfoxide, and 5 mol/L sodium hydroxide and then digested into β-glucan fragments using Westase which is an enzyme having β-1,6- and β-1,3 glucanase activity. The β-glucan fragments are further digested into glucose using exo-1,3-β-d-glucanase and β-glucosidase. We measured β-glucan comprising β-1,3-, -1,6-, and -1,(3),4- bonds in various polysaccharide reagents and processed foods using our novel method. The SEED assay was able to quantify β-glucan with good reproducibility, and the recovery rate was >90% for food containing β-glucan. Therefore, the SEED assay is capable of accurately measuring the β-glucan content of processed foods.

Topics: beta-Glucans; beta-Glucosidase; Food Analysis; Food Handling; Glucan 1,3-beta-Glucosidase; Glucans; Hordeum; Sodium Hypochlorite

C-type lectin receptors (CLRs) comprise a large family of immunoreceptors that recognize polysaccharide ligands exposed on pathogen surfaces and are conserved among mammals. However, interspecies differences in their ligand spectrums are not fully understood. Dectin-1 is a well-characterized CLR that recognizes β-glucan. We report here that seaweed-derived fucan activates cells expressing human Dectin-1 but not mouse Dectin-1. Low-valency β-glucan components within fucan appeared to be responsible for this activation, as the ligand activity was eliminated by β-glucanase treatment. The low-valency β-glucan laminarin also acted as an agonist for human Dectin-1 but not for mouse Dectin-1, whereas the high-valency β-glucan curdlan activated both human and mouse Dectin-1. Reciprocal mutagenesis analysis revealed that the ligand-binding domain of human Dectin-1 does not determine its unique sensitivity to low-valency β-glucan. Rather, we found that its intracellular domain renders human Dectin-1 reactive to low-valency β-glucan ligand. Substitution with two amino acids, Glu

Topics: Amino Acid Substitution; Animals; beta-Glucans; Cell Line; Glucans; Humans; Lectins, C-Type; Mice; Mutagenesis; Mutation, Missense; Polysaccharides; Protein Domains; Species Specificity; Substrate Specificity

A novel endo-β-1,3(4)-glucanase gene, cel16A, was cloned from the fungus Humicola insolens Y1. The 988-bp full-length gene encoded a 286-residue polypeptide consisting of a putative signal peptide of 20 residues and a catalytic domain belonging to glycosyl hydrolase family 16. It was successfully overexpressed in Pichia pastoris GS115. The purified recombinant Cel16A exhibited highest specific activity toward barley β-glucan, followed by lichenan and laminarin, but not toward CMC-Na, birchwood xylan, Avicel and filter paper, indicating that Cel16A is an endo-β-1,3(4)-glucanases. Recombinant Cel16A had a pH optimum at 5.5 and a temperature optimum at 55 °C with a specific activity of 693 U/mg toward barley β-glucan. It exhibited good stability over pH 5.0-9.0 and at temperatures up to 50 °C, retaining over 80% maximum activity. The K

Topics: Amino Acid Sequence; beta-Glucans; Catalytic Domain; Chromatography, Affinity; Cloning, Molecular; Endo-1,3(4)-beta-Glucanase; Enzyme Assays; Enzyme Stability; Fungal Proteins; Gene Expression; Genetic Vectors; Glucans; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Pichia; Protein Sorting Signals; Recombinant Proteins; Sequence Alignment; Sordariales; Substrate Specificity

Topics: Antigens, Fungal; beta-Glucans; False Positive Reactions; Food; Galactose; Glucans; Glucocorticoids; Graft vs Host Disease; Hematopoietic Stem Cell Transplantation; Humans; Invasive Fungal Infections; Kelp; Male; Mannans; Methylprednisolone; Middle Aged; Proteoglycans; Sezary Syndrome; Tomography, X-Ray Computed; Transplantation Conditioning

Ganoderma lucidum BCCM 31549 has a long established role for its therapeutic activities. In this context, much interest has focused on the possible functions of the (1,3)-β-D-glucan (G) produced by these cultures in a stirred-tank bioreactor and extracted from their underutilized mycelium. In the existing study, we report on the systematic production of G, and its sulfated derivative (GS). The aim of this study was to investigate G and its GS from G. lucidum in terms of their antibacterial properties and cytotoxicity spectrum against human prostate cells (PN2TA) and human caucasian histiocytic lymphoma cells (U937). (1)H NMR for both G and GS compounds showed β-glycosidic linkages and structural similarities when compared with two standards (laminarin and fucoidan). The existence of characteristic absorptions at 1,170 and 867 cm(-1) in the FTIR (Fourier Transform Infrared Spectroscopy) for GS demonstrated the successful sulfation of G. Only GS exhibited antimicrobial activity against a varied range of test bacteria of relevance to foodstuffs and human health. Moreover, both G and GS did not show any cytotoxic effects on PN2TA cells, thus helping demonstrate the safety of these polymers. Moreover, GS showed 40% antiproliferation against cancerous U937 cells at the low concentration (60 μg/ ml) applied in this study compared with G (10%). Together, this demonstrates that sulfation clearly improved the solubility and therapeutic activities of G. The water-soluble GS demonstrates the potential multifunctional effects of these materials in foodstuffs.

Topics: Anti-Bacterial Agents; Apoptosis; beta-Glucans; Bioreactors; Cell Line, Tumor; Glucans; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Molecular Weight; Mycelium; Polysaccharides; Proteoglycans; Reishi; Solubility

Infection and inflammation suppress the expression and activity of several drug transporters in liver. In the intestine, P-glycoprotein (P-gp/MDR1), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) are important barriers to the absorption of many clinically important drugs. The expression and activity of these proteins were examined under inflammation. Drug transport was determined in jejunum and ileum segments isolated from 1.0 mg/kg, 5.0 mg/kg, and 7.5 mg/kg indomethacin-treated or control rats in diffusion chambers. Transport of laminaran, used as a model compound of (1-3) β-D-glucan, was measured for 120 min in the presence or absence of inhibitors. Reverse transcription-polymerase chain reaction was used to measure mRNA levels. Compared with controls, levels of Mdr1a mRNA were significantly decreased in the jejunum and ileum of 7.5 mg/kg indomethacin-treated rats. Both reductions in the basolateral to apical efflux of laminaran and increases in the apical to basolateral influx of laminaran were observed, resulting in significant increases in the apical to basolateral absorption of laminaran in 7.5 mg/kg indomethacin-treated rats. The inhibitory effect of verapamil on laminaran transport was observed in control rats but not in indomethacin-treated rats. Fluorescein isothiocyanate dextran 40,000 permeability, membrane resistance, and claudin-4 mRNA level were not altered, indicating no change in the paracellular pathway. These results indicate that indomethacin-induced inflammation reduces the intestinal expression and activity of P-gp in rats, which elicits corresponding changes in the intestinal transport of laminaran. Hence, inflammatory diseases may impose variability in drug bioavailability through alterations in the intestinal expression and activity of drug transporters.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; beta-Glucans; Biological Transport; Disease Models, Animal; Down-Regulation; Glucans; Ileum; Indomethacin; Inflammation; Intestinal Absorption; Jejunum; Male; Proteoglycans; Rats, Wistar; RNA, Messenger; Time Factors

β-D-Glucans are known to present antitumor, anticancer, and anti-inflammatory activities that are influenced by their own antioxidant capacity. The antioxidant activity of botryosphaeran, an exopolysaccharide of the (1 → 3;1 → 6)-β-D-glucan type produced by the Botryosphaeria rhodina MAMB-05 was evaluated and compared to some other β-D-glucans (lasiodiplodan an exocellular (1 → 6)-β-D-glucan from Lasiodiplodia theobromae, laminarin and curdlan), and oligosaccharides, disaccharides, and monosaccharides in a study of scavenging activities of free radicals in-vitro. Botryosphaeran displayed high total antioxidant activity (80%) as well as good scavenging activity against hydroxyl radical (90.6%), superoxide anion (37%), hydrogen peroxide (38%), and nitric oxide radical (90%). No reducing power, metal-chelating capacity or inhibition of lipid peroxidation was observed for these β-D-glucans. The results demonstrated that botryosphaeran exhibited effective antioxidant activity as supported by many different assays, suggesting that this β-D-glucan may serve as a source of a new bioactive compound with effective antioxidant activity.

Topics: Antioxidants; Benzothiazoles; beta-Glucans; Free Radical Scavengers; Glucans; Hydrogen Peroxide; Hydroxyl Radical; Nitric Oxide; Sulfonic Acids; Superoxides

Laminarin is a β-1,3-D-glucan displaying occasional β-1,6 branches. This storage polysaccharide of brown algae constitutes an abundant source of carbon for marine bacteria such as Zobellia galactanivorans. This marine member of the Bacteroidetes possesses five putative β-1,3-glucanases [four belonging to glycosyl hydrolase family 16 (GH16) and one to GH64] with various modular architectures. Here, the characterization of the β-glucanase ZgLamC is reported. The catalytic GH16 module (ZgLamCGH16) was produced in Escherichia coli and purified. This recombinant enzyme has a preferential specificity for laminarin but also a significant activity on mixed-linked glucan (MLG). The structure of an inactive mutant of ZgLamCGH16 in complex with a thio-β-1,3-hexaglucan substrate unravelled a straight active-site cleft with three additional pockets flanking subsites -1, -2 and -3. These lateral pockets are occupied by a glycerol, an acetate ion and a chloride ion, respectively. The presence of these molecules in the vicinity of the O6 hydroxyl group of each glucose moiety suggests that ZgLamCGH16 accommodates branched laminarins as substrates. Altogether, ZgLamC is a secreted laminarinase that is likely to be involved in the initial step of degradation of branched laminarin, while the previously characterized ZgLamA efficiently degrades unbranched laminarin and oligo-laminarins.

Topics: Amino Acid Sequence; beta-Glucans; Catalytic Domain; Cellulases; Crystallography, X-Ray; Flavobacteriaceae; Glucans; Models, Molecular; Molecular Sequence Data; Protein Conformation; Proteoglycans; Sequence Alignment; Substrate Specificity

Certain Saccharomyces cerevisiae strains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

Topics: beta-Glucans; Cell Membrane; Cell Wall; Chitin; Glucans; Killer Factors, Yeast; Polysaccharides; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spheroplasts

β(1-3)-Glucans, abundant in fungi, have the potential to activate the innate immune response against various pathogens. Although part of the action is exerted through the C-type lectin-like receptor Dectin-1, details of the interaction mechanism with respect to glucan chain-length remain unclear. In this study, we investigated a set of short β(1-3)-glucans with varying degree of polymerization (DP); 3, 6, 7, 16, and laminarin (average DP; 25), analyzing the relationship between the structure and interaction with the C-type lectin-like domain (CTLD) of Dectin-1. The interaction of short β(1-3)-glucans (DP6, DP16, and laminarin) with the CTLD of Dectin-1 was systematically analyzed by (1)H-NMR titration as well as by saturation transfer difference (STD)-NMR. The domain interacted weakly with DP6, moderately with DP16 and strongly with laminarin, the latter plausibly forming oligomeric protein-laminarin complexes. To obtain structural insights of short β(1-3)-glucans, the exchange rates of hydroxy protons were analyzed by deuterium induced (13)C-NMR isotope shifts. The hydroxy proton at C4 of laminarin has slower exchange with the solvent than those of DP7 and DP16, suggesting that laminarin has a secondary structure. Diffusion ordered spectroscopy revealed that none of the short β(1-3)-glucans including laminarin forms a double or triple helix in water. Insights into the interaction of the short β(1-3)-glucans with Dectin-1 CTLD provide a basis to understand the molecular mechanisms of β-glucan recognition and cellular activation by Dectin-1.

Topics: beta-Glucans; Carbohydrate Conformation; Carbohydrate Sequence; Deuterium; Glucans; Lectins, C-Type; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Protein Structure, Tertiary; Spectrum Analysis; Structure-Activity Relationship

Grapevine (Vitis vinifera) is susceptible to many pathogens which cause significant losses to viticulture worldwide. Chemical control is available, but agro-ecological concerns have raised interest in alternative methods, especially in triggering plant immunity by elicitor treatments. The β-glucan laminarin (Lam) and its sulfated derivative (PS3) have been previously demonstrated to induce resistance in grapevine against downy mildew (Plasmopara viticola). However, if Lam elicits classical grapevine defenses such as oxidative burst, pathogenesis-related (PR)-proteins and phytoalexin production, PS3 triggered grapevine resistance via a poorly understood priming phenomenon. The aim of this study was to identify the molecular mechanisms of the PS3-induced resistance. For this purpose we studied i) the signaling events and transcriptome reprogramming triggered by PS3 treatment on uninfected grapevine, ii) grapevine immune responses primed by PS3 during P. viticola infection. Our results showed that i) PS3 was unable to elicit reactive oxygen species (ROS) production, cytosolic Ca(2+) concentration variations, mitogen-activated protein kinase (MAPK) activation but triggered a long lasting plasma membrane depolarization in grapevine cells, ii) PS3 and Lam shared a common stress-responsive transcriptome profile that partly overlapped the salicylate- (SA) and jasmonate-(JA)-dependent ones. After P. viticola inoculation, PS3 specifically primed the SA- and ROS-dependent defense pathways leading to grapevine induced resistance against this biotroph. Interestingly pharmacological approaches suggested that the plasma membrane depolarization and the downstream ROS production are key events of the PS3-induced resistance.

Topics: beta-Glucans; Cell Death; Cell Membrane; Cyclopentanes; Disease Resistance; Gene Expression Regulation, Plant; Glucans; Oomycetes; Oxylipins; Plant Diseases; Plant Immunity; Reactive Oxygen Species; Salicylic Acid; Signal Transduction; Stress, Physiological; Transcriptome; Vitis

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a β-1,3-linked backbone and β-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, surprisingly little information is available on the biodegradation of schizophyllan. Enzymes that attack schizophyllan could be useful for controlled modifications of the polymer for novel applications. Enrichment cultures were used to isolate 20 novel fungal strains from soil samples, capable of growing on schizophyllan as a sole carbon source. Three additional strains were isolated as contaminants of stored schizophyllan solutions. Strains showing the highest levels of β-glucanase activity were identified as Penicillium simplicissimum, Penicillium crustosum, and Hypocrea nigricans. β-glucanases also showed activity against the similar β-glucans, laminarin and curdlan. By comparison, commercial β-glucanase from Trichoderma longibrachiatum and laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of the novel isolates. β-glucanases from P. simplicissimum and H. nigricans exhibited temperature optima of 60°C and 50°C against schizophyllan, respectively, with broad pH optima around pH 5.0. Partial purifications of β-glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple active endoglucanase species, including a 20-25 kD enzyme from P. simplicissimum.

Topics: Aspergillus; beta-Glucans; Fungal Proteins; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen-Ion Concentration; Hydrolysis; Hypocrea; Penicillium; Polysaccharides; Schizophyllum; Sizofiran; Soil Microbiology; Substrate Specificity; Temperature; Trichoderma

In a previous attempt to generate a protective vaccine against Candida albicans, a β-mannan tetanus toxoid conjugate showed poor immunogenicity in mice. To improve the specific activation toward the fungal pathogen, we aimed to target Dectin-1, a pattern-recognition receptor expressed on monocytes, macrophages, and dendritic cells. Laminarin, a β-glucan ligand of Dectin-1, was incorporated into the original β-mannan tetanus toxoid conjugate providing a tricomponent conjugate vaccine. A macrophage cell line expressing Dectin-1 was employed to show binding and activation of Dectin-1 signal transduction pathway by the β-glucan-containing vaccine. Ligand binding to Dectin-1 resulted in the following: 1) activation of Src family kinases and Syk revealed by their recruitment and phosphorylation in the vicinity of bound conjugate and 2) translocation of NF-κB to the nucleus. Treatment of immature bone marrow-derived dendritic cells (BMDCs) with tricomponent or control vaccine confirmed that the β-glucan-containing vaccine exerted its enhanced activity by virtue of dendritic cell targeting and uptake. Immature primary cells stimulated by the tricomponent vaccine, but not the β-mannan tetanus toxoid vaccine, showed activation of BMDCs. Moreover, treated BMDCs secreted increased levels of several cytokines, including TGF-β and IL-6, which are known activators of Th17 cells. Immunization of mice with the novel type of vaccine resulted in improved immune response manifested by high titers of Ab recognizing C. albicans β-mannan Ag. Vaccine containing laminarin also affected distribution of IgG subclasses, showing that vaccine targeting to Dectin-1 receptor can benefit from augmentation and immunomodulation of the immune response.

Topics: Animals; beta-Glucans; Binding Sites; Cell Line; Dendritic Cells; Drug Delivery Systems; Epitopes; Glucans; Lectins, C-Type; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Polysaccharides; Tetanus Toxoid; Trisaccharides; Vaccines, Conjugate

Streptomyces sp Mo endo-β-1,3-glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N-terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β-1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β-1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β-1,6-linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo-β-1,3-glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β-1,3 linkage between the 3rd and 4th glucosyl residue.

Topics: beta-Glucans; Disaccharides; DNA, Bacterial; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; Polysaccharides; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Streptomyces; Substrate Specificity; Temperature

Interleukin-23 (IL-23), a cytokine produced primarily by dendritic cells, is involved in host defense against gut pathogens and promotes innate immunity and inflammatory responses through the IL-23/interleukin-17 axis. We previously reported that extracts from edible mushrooms enhanced antimicrobial α-defensin production n HL60 cells. Because IL-23 is involved in defensin production, we hypothesized that edible mushrooms may modulate its secretion and gut inflammation. Eight-week-old C57BL/6 mice were fed the AIN76 diet or the same diet supplemented with 5% white button (WBM), portabella, or shiitake mushrooms. To assess in vivo and in vitro cytokine secretion, 7 to 8 mice per group received 3% dextran sodium sulfate (DSS) in drinking water during the last 5 days of the 6-week feeding period. To delineate the mechanisms by which mushrooms alter IL-23 secretion, J.744.1 cells were incubated with (100 μg/mL) WBM, portabella, and shiitake extracts without and with 100 μg/mL curdlan (a dectin-1 agonist) or 1 mg/mL laminarin (a dectin-1 antagonist). The dectin-1 receptor is a pattern-recognition receptor found in phagocytes, and its activation promotes antimicrobial innate immunity and inflammatory responses. In DSS-untreated mice, mushrooms significantly increased IL-23 plasma levels but decreased those of interleukin-6 (IL-6) (P < .05). In DSS-treated mice, mushroom-supplemented diets increased IL-6 and IL-23 levels (P < .05). Mushroom extracts potentiated curdlan-induced IL-23 secretion, and mushroom-induced IL-23 secretion was not blocked by laminarin in vitro, suggesting the involvement of both dectin-1-dependent and dectin-1-independent pathways. Although all mushrooms tended to increase IL-6 in the colon, only WBM and shiitake tended to increase IL-23 levels. These data suggest that edible mushrooms may enhance gut immunity through IL-23.

Topics: Animals; Anti-Infective Agents; beta-Glucans; Cell Line; Colitis; Dextran Sulfate; Dietary Supplements; Female; Glucans; Immunity, Innate; Inflammation; Interleukin-17; Interleukin-23; Interleukin-6; Lectins, C-Type; Macrophages; Mice; Mice, Inbred C57BL; Organ Size; Polysaccharides; Regression Analysis; Shiitake Mushrooms; Thymus Gland; Up-Regulation

Carnivory in plants evolved as an adaptation strategy to nutrient-poor environments. Thanks to specialized traps, carnivorous plants can gain nutrients from various heterotrophic sources such as small insects. Digestion in traps requires a coordinated action of several hydrolytic enzymes that break down complex substances into simple absorbable nutrients. Among these, several pathogenesis-related proteins including β-1,3-glucanases have previously been identified in digestive fluid of some carnivorous species. Here we show that a single acidic endo-β-1,3-glucanase of ~50 kDa is present in the digestive fluid of the flypaper-trapped sundew (Drosera rotundifolia L.). The enzyme is inducible with a complex plant β-glucan laminarin from which it releases simple saccharides when supplied to leaves as a substrate. Moreover, thin-layer chromatography of digestive exudates showed that the simplest degradation products (especially glucose) are taken up by the leaves. These results for the first time point on involvement of β-1,3-glucanases in digestion of carnivorous plants and demonstrate the uptake of saccharide-based compounds by traps. Such a strategy could enable the plant to utilize other types of nutritional sources e.g., pollen grains, fungal spores or detritus from environment. Possible multiple roles of β-1,3-glucanases in the digestive fluid of carnivorous sundew are also discussed.

Topics: Animals; beta-Glucans; Carnivory; Drosera; Glucan 1,3-beta-Glucosidase; Glucans; Hydrolysis; Plant Leaves; Polysaccharides; Proteolysis

Members of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to specifically bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. The tmCBP binding site is bipartite, comprising a canonical solvent-excluded region (subsite one), adjacent to a solvent-filled cavity (subsite two) where specific and semi-specific ligand recognition occur, respectively.. A molecular level understanding of binding pocket adaptation mechanisms that simultaneously allow both ligand specificity at subsite one and promiscuity at subsite two has potentially important implications in ligand binding and drug design studies. We sought to investigate the determinants of ligand binding selectivity in tmCBP through biophysical characterization of tmCBP in the presence of varying β-glucan oligosaccharides. Crystal structures show that whilst the amino acids that comprise both the tmCBP subsite one and subsite two binding sites remain fixed in conformation regardless of which ligands are present, the rich hydrogen bonding potential of water molecules may facilitate the ordering and the plasticity of this unique PBP binding site.. The identification of the roles these water molecules play in ligand recognition suggests potential mechanisms that can be utilized to adapt a single ligand binding site to recognize multiple distinct ligands.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Glucans; Binding Sites; Carrier Proteins; Cellulose; Circular Dichroism; Crystallography, X-Ray; Dextrins; Glucans; Hydrogen Bonding; Lectins; Ligands; Models, Molecular; Polysaccharides; Protein Conformation; Protein Denaturation; Protein Stability; Protein Structure, Secondary; Protein Structure, Tertiary; Substrate Specificity; Thermotoga maritima

β-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing β-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of β-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel β-1,3-glucanases with high catalytic efficiency and stability is of particular interest.. A β-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the β-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes β-1,3-glucan. Insoluble β-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains.. This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of β-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble β-1,3-glucan, and could be classified into CBM families.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; beta-Glucans; Catalytic Domain; Cloning, Molecular; Escherichia coli; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen-Ion Concentration; Hydrolysis; Molecular Sequence Data; Open Reading Frames; Paenibacillus; Polysaccharides; Recombinant Proteins; Sequence Analysis, DNA; Substrate Specificity; Temperature

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the nuclear magnetic resonance (NMR) solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone (15)N-(1)H groups of the protein, suggesting the formation of a large complex. Analytical ultracentrifugation (AUC) studies of formation of the N-βGRP-laminarin complex show that ligand binding induces self-association of the protein-carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to submicromolar concentrations. The structural model thus derived from this study for the N-βGRP-laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple-helical form of laminarin on the basis of an X-ray crystallographic structure of the N-βGRP-laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements conducted with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of the N-βGRP-laminarin macro complex and that a decreased stability is accompanied by a reduced level of activation of the proPO pathway. An increased level of β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of the βGRP-β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteas

Topics: Animals; beta-Glucans; Binding Sites; Carrier Proteins; Glucans; Immunity, Innate; Insect Proteins; Laminaria; Models, Molecular; Moths; Nuclear Magnetic Resonance, Biomolecular; Polysaccharides; Protein Structure, Tertiary

An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6-10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate.

Topics: Bacillus subtilis; beta-Glucans; Carboxymethylcellulose Sodium; Cellulose; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucans; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Polysaccharides; Substrate Specificity; Temperature

A carbohydrate-binding module from family 13 (CBM13), appended to the catalytic domain of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, was overexpressed in E. coli, and its interactions with β-glucans, laminarin and laminarioligosaccharides, were analyzed using surface plasmon resonance biosensor and isothermal titration calorimetry. The association constants for laminarin and laminarioligosaccharides were determined to be approximately 10(6) M(-1) and 10(4) M(-1), respectively, indicating that 2 or 3 binding sites in the α-, β-, and γ-repeats of CBM13 are involved in laminarin binding in a cooperative manner. The binding avidity is approximately 2-orders higher than the monovalent binding affinity. Mutational analysis of the conserved Asp residues in the respective repeats showed that the α-repeat primarily contributes to β-glucan binding. A Trp residue is predicted to be exposed to the solvent only in the α-repeat and would contribute to β-glucan binding. The α-repeat bound β-glucan with an affinity of approximately 10(4) M(-1), and the other repeats additionally bound laminarin, resulting in the increased binding avidity. This binding is unique compared to the recognition mode of another CBM13 from Streptomyces lividans xylanase.

Topics: Actinomycetales; Amino Acid Sequence; Aspartic Acid; beta-Glucans; Binding Sites; Calorimetry; Catalytic Domain; Circular Dichroism; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Lectins; Molecular Sequence Data; Polysaccharides; Protein Binding; Protein Structure, Tertiary; Repetitive Sequences, Amino Acid; Sequence Homology, Amino Acid; Streptomyces; Surface Plasmon Resonance; Tryptophan

A novel endo-1,3(4)-β-D-glucanase gene (bgl16C1) from Penicillium pinophilum C1 was cloned and sequenced. The 945-bp full-length gene encoded a 315-residue polypeptide consisting of a putative signal peptide of 18 residues and a catalytic domain belonging to glycosyl hydrolase family 16. The deduced amino acid sequence showed the highest identity (82%) with the putative endo-1,3(4)-β-glucanase from Talaromyces stipitatus ATCC 10500 and 60% identity with the characterized β-1,3(4)-glucanase from Paecilomyces sp. FLH30. The gene was successfully overexpressed in Pichia pastoris. Recombinant Bgl16C1 constituted 95% of total secreted proteins (2.61 g l⁻¹) with activity of 28,721 U ml⁻¹ in a 15-l fermentor. The purified recombinant Bgl16C1 had higher specific activity toward barley β-glucan (12,622 U mg⁻¹) than all known glucanases and also showed activity against lichenan and laminarin. The enzyme was optimally active at pH 5.0 and 55°C and exhibited good stability over a broad acid and alkaline pH range (>85% activity at pH 3.0-7.0 and even 30% at pH 11.0). All these favorable enzymatic properties make it attractive for potential applications in various industries.

Topics: Amino Acid Sequence; beta-Glucans; Bioengineering; Cloning, Molecular; Endo-1,3(4)-beta-Glucanase; Glucans; Molecular Sequence Data; Paecilomyces; Penicillium; Pichia; Polysaccharides; Recombinant Proteins; Sequence Alignment

A soluble homogeneous β-glucan, GFPBW1, with a molecular mass of 300 kDa was purified from the fraction of the fruit bodies of Grifola frondosa extracted with 5% NaOH. Using various methods, such as infrared spectroscopy, NMR, methylation and monosaccharide composition analysis, its structure was determined to be a β-D-(1-3)-linked glucan backbone with a single β-D-(1-6)-linked glucopyranosyl residue branched at C-6 on every third residue. It induced TNF-α and IL-6 production and the activation of Syk and NF-κB signaling in resident peritoneal macrophages from ICR mice, which could be significantly inhibited by the blocking reagent laminarin. A competitive phagocytosis assay with FITC-zymosan indicated that GFPBW1 could bind to DC-associated C-type lectin 1 (Dectin-1). The TNF-α secretion and activation of Syk/NF-κB signaling triggered by GFPBW1 were enhanced in RAW264.7 cells overexpressing wild but not mutant (Δ38 and Y15S) Dectin-1. Furthermore, GFPBW1 potentiated the Concanavalin A-induced proliferative response of splenocytes and inhibited Sarcoma-180 growth allografted in ICR mice but not in immunodeficient BALB/c nu/nu mice. These results suggested that the antitumor activity of GFPBW1 was partially associated with the activation of macrophages via the Dectin-1/Syk/NF-κB signaling pathway. This molecule could be a promising biological response modifier with clear application for antitumor therapies.

Topics: Animals; Antineoplastic Agents, Phytogenic; beta-Glucans; Carbohydrate Sequence; Cell Line, Tumor; Fruiting Bodies, Fungal; Gene Expression Regulation, Neoplastic; Glucans; Grifola; Injections, Subcutaneous; Interleukin-6; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Macrophages, Peritoneal; Mice; Mice, Inbred ICR; NF-kappa B; Phagocytosis; Polysaccharides; Protein-Tyrosine Kinases; Sarcoma; Signal Transduction; Syk Kinase; Tumor Necrosis Factor-alpha; Xenograft Model Antitumor Assays

Relatively little is known about how liposomal formulations modulate drug delivery to fungal pathogens. We compared patterns of hyphal cell wall binding for empty rhodmine-labeled liposomes and the clinically available amphotericin B-containing liposomal formulation (AmBisome) in Aspergillus fumigatus and Candida albicans. Following 0.5 h of coincubation with A. fumigatus , empty liposomes concentrated primarily in fungal septae along at the surface of the cell wall, suggesting that liposome uptake is concentrated in areas of the cell wall where linear glucan is exposed on the cell surface, which was confirmed by aniline blue staining. Consistent with this hypothesis, pretreatment of liposomes with soluble linear glucan (laminarin) decreased liposome binding in both Aspergillus and Candida fungal hyphae, while growth of Aspergillus hyphae in the presence of an agent that increases fungal cell wall surface exposure of linear β-glucans without cell death (caspofungin) increased liposome uptake throughout the Aspergillus fungal cell wall. Increasing the polyethylene glycol (PEG) concentration in liposomes from 0 to 30% significantly increased fungal uptake of liposomes that was only modestly attenuated when fungal cells were incubated in serum concentrations ranging from 10 to 100%. The presence of β-glucans on the fungal hyphae cell walls of Aspergillus fumigatus is one of the factors responsible for mediating the binding of liposome carriers to the hyphae and could explain possible synergy reported between liposomal amphotericin B and echinocanins.

Topics: Amphotericin B; Aniline Compounds; Antifungal Agents; Aspergillus fumigatus; beta-Glucans; Candida albicans; Caspofungin; Cell Wall; Chemistry, Pharmaceutical; Drug Carriers; Echinocandins; Glucans; Hyphae; Lipopeptides; Liposomes; Models, Molecular; Polyethylene Glycols; Polysaccharides

Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1 → 3)-elongation activity, (β1 → 4)- or (β1 → 6)-elongation, or (β1 → 6)-branching activities were also detected.

Topics: Azotobacter vinelandii; beta-Glucans; Enzyme Assays; Glucans; Glucosyltransferases; Models, Molecular; Molecular Structure; Polysaccharides; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas putida

The aim of the present study was to establish the optimum inclusion level of laminarin derived from Laminaria digitata on selected microbial populations, intestinal fermentation, cytokine and mucin gene expression in the porcine ileum and colon. A total of twenty-one pigs (mean body weight 17·9 kg) were randomly assigned to one of three dietary treatments: T1 - basal (control) diet, T2 and T3 - basal diets supplemented with laminarin included at 300 and 600 parts per million (ppm), respectively. Selected intestinal bacterial populations and volatile fatty acid (VFA) concentrations were measured in the ileum and colon. Relative gene expression levels for specific cytokine and mucin genes were investigated in ileal and colonic tissue in the absence and presence of a lipopolysaccharide (LPS) challenge. There was an up-regulation of MUC2 gene expression at the 300 ppm inclusion level in the ileum. In the colon, there was a significant reduction in the enterobacteriaceae population at the 300 ppm inclusion level (P = 0·0421). Dietary supplementation of 600 ppm laminarin led to a significant increase in MUC2 (P = 0·0365) and MUC4 (P = 0·0401) expression in the colon, and in the total VFA concentration in the caecum (P = 0·0489). A significant increase was also recorded in IL-6 (P = 0·0289) and IL-8 gene expression (P = 0·0245) in LPS-challenged colonic tissue at both laminarin inclusion levels. In conclusion, dietary inclusion of 300 ppm laminarin appears to be the optimum dose in the present study due to the reduction in the enterobacteriaceae populations and enhanced IL-6 and IL-8 cytokine expression in response to an ex vivo LPS challenge.

Topics: Animals; Anti-Bacterial Agents; beta-Glucans; Colon; Dietary Supplements; Enterobacteriaceae; Fatty Acids, Volatile; Fermentation; Gene Expression; Glucans; Ileum; Interleukin-6; Interleukin-8; Interleukins; Intestinal Mucosa; Intestines; Laminaria; Lipopolysaccharides; Mucin-2; Mucin-4; Mucins; Plant Extracts; Polysaccharides; Random Allocation; Swine; Up-Regulation

A full characterization of the high-resolution NMR spectrum of the laminarihexaose is described and used for the determination of the binding epitope of the more complex but structurally related laminarin. These biophysical data extend the current knowledge of β-glucans/Dectin-1 interactions and suggest different biological mechanisms in close relation with the size of the saccharidic chain.

Topics: beta-Glucans; Binding Sites; Carbohydrate Sequence; Epitope Mapping; Glucans; Humans; Immunologic Factors; Lectins, C-Type; Macrophage-1 Antigen; Magnetic Resonance Spectroscopy; Membrane Proteins; Models, Molecular; Molecular Sequence Data; Nerve Tissue Proteins; Oligosaccharides; Polysaccharides; Protein Binding; Recombinant Proteins; Structure-Activity Relationship

β-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, β-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of β-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 μg/ml) of five commercial β-glucan preparations, differing in structure and source, on porcine monocyte-derived dendritic cells (MoDCs). Although all β-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another β-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlan-stimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1β (IL-1β), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when β-glucans are used as dietary supplement.

Topics: Animals; beta-Glucans; Cell Differentiation; Cytokines; Dendritic Cells; Glucans; Humans; Interleukins; Lymphocyte Activation; Mice; Monocytes; Polysaccharides; Swine; T-Lymphocytes; Zymosan

Phagocytosis is a fundamental aspect of innate resistance against microbes, including fungi. In this study we investigated the significance of beta-glucan on the surfaces of zymosan particles, derived from Saccharomyces cerevisiae, during phagocytosis by RAW 264.7 macrophages. Phagocytosis was assessed in vitro by macrophage exposure to zymosan particles followed by cell staining and light microscopy. Macrophage ingestion of zymosan was dependent on cellular recognition of the particles' beta-glucans since laminarin, a soluble beta-glucan, inhibited phagocytosis in a concentration dependent manner when added to cell cultures. In contrast, the presence of another carbohydrate, mannan, had no effect on zymosan phagocytosis by cells. In addition we showed that LPS and dexamethasone had opposing effects on phagocytosis of zymosan. LPS significantly augmented ingestion while in contrast dexamethasone, like laminarin, suppressed it. The LPS-enhanced ingestion of zymosan was insensitive to the presence of laminarin in cell cultures, however dexamethasone partially ameliorated the effects of LPS on phagocytosis. Our findings confirm beta-glucan as an important ligand identified by macrophages and required for zymosan phagocytosis in naïve cells, but not in cells previously exposed to LPS.

Topics: Animals; beta-Glucans; Cell Line; Dose-Response Relationship, Immunologic; Glucans; Lipopolysaccharides; Macrophages; Mice; Phagocytosis; Polysaccharides; Solubility; Zymosan

It has immense potential for immunotherapy and vaccination to target antigens to antigen-presenting cells (APCs). Here we described a method for delivering whole protein antigens to APCs via carbohydrate-mediated targeting of Dectin-1, which is a C-type lectin and mainly expresses on subpopulations of dendritic cells and macrophages. Laminarin, which is a beta-1-3 glucan and a typical ligand for Dectin-1, was chemically coupled to ovalbumin (OVA). Compared to OVA alone, the conjugate was effectively recognized and ingested by CHO cells stably expressing Dectin-1 and bound to bone marrow dendritic cells (BMDCs) via Dectin-1. Laminarin modification led to significant enhancement of OVA-specific CD4(+) T-cell response. Moreover, when used to immunize mice, the conjugate enhanced the primary IgG antibody response to OVA. Taken together, our data suggest that APCs targeting based on glucan-Dectin-1 interaction is a promising approach to improve vaccines.

Topics: Animals; Antibody Formation; Antigen-Presenting Cells; Antigens; beta-Glucans; CD4-Positive T-Lymphocytes; CHO Cells; Cricetinae; Cricetulus; Dendritic Cells; Glucans; Lectins, C-Type; Membrane Proteins; Mice; Mice, Inbred C57BL; Nerve Tissue Proteins; Ovalbumin; Polysaccharides; Vaccines

A critical component of host innate immunity is recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Dectin-1 is the primary PRR for exogenous beta-glucan, a component of fungal and bacterial cell walls. A previous study conducted in our laboratory demonstrated that administration of beta-glucan as a feed additive resulted in increased innate immune function of neonatal chickens, suggesting that chickens possess a Dectin-1-like beta-glucan receptor. In the present study, we demonstrated that heterophils and peripheral blood mononuclear cells (PBMCs) from day-old chicks had a significant increase in the generation of reactive oxygen species (ROS) following stimulation with the Dectin-1 specific agonist, curdlan. Pretreatment of heterophils and PBMCs with laminarin, a beta-glucan receptor blocking agent and specific inhibitor of Dectin-1 activity, significantly reduced the curdlan-induced ROS production. Together these data provide evidence for the first time of the presence of a functional Dectin-1-like beta-glucan receptor in chicken heterophils and PBMCs.

Topics: Animals; Animals, Newborn; beta-Glucans; Chickens; Glucans; Granulocytes; Immunity, Innate; In Vitro Techniques; Lectins, C-Type; Leukocytes, Mononuclear; Membrane Proteins; Nerve Tissue Proteins; Polysaccharides; Reactive Oxygen Species; Receptors, Immunologic; Respiratory Burst

The mechanism of the nonlinear concentration dependence of intestinal absorption of (1-->3)-beta-D-glucan was studied using in situ rat intestinal perfusion, as well as the in vitro Ussing-type chamber method mounted with rat intestinal tissue. The intestinal absorption rate constant of a (1-->3)-beta-D-glucan, laminaran, evaluated by the loop method increased significantly with increasing concentration of laminaran up to 0.5 muM in a nonlinear fashion and tended to decrease at higher concentrations. Mucosal-to-serosal directed permeation of the laminaran across rat ileal sheets evaluated by the in vitro Ussing-type chamber method also decreased in a dose-dependent fashion. Serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. In addition, the serosal-to-mucosal flux was reduced in the presence of metabolic inhibitor, 2,4-di-nitrophenol. These results suggest that laminaran is secreted into the intestinal lumen predominantly by the efflux transporting system. We conclude that intestinal transport of (1-->3)-beta-D-glucan involves specialized transporter or something similar in both absorptive and secretory directions, and complex nonlinear intestinal absorption characteristics can be ascribed to the participation of multiple transport mechanism.

Topics: 2,4-Dinitrophenol; Animals; beta-Glucans; Biological Transport; Dose-Response Relationship, Drug; Glucans; Ileum; In Vitro Techniques; Intestinal Absorption; Intestinal Mucosa; Male; Nonlinear Dynamics; Polysaccharides; Proteoglycans; Rats; Rats, Wistar; Serous Membrane

YCP, a novel (1,4)-alpha-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation.. Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production.. YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation.. We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.Acta Pharmacologica Sinica (2009) 30: 1008-1014; doi: 10.1038/aps.2009.93.

Topics: Animals; Ascomycota; beta-Glucans; CD11b Antigen; Cell Line; Enzyme Activation; Glucans; Humans; Lipopolysaccharides; Macrophage Activation; Macrophages; Mannose; Mice; Nitrites; p38 Mitogen-Activated Protein Kinases; Polysaccharides; Receptors, Cell Surface; Seawater; Signal Transduction; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 6

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

Topics: Animals; beta-Glucans; Binding Sites; Cellulase; Cellvibrio; Conserved Sequence; Endo-1,4-beta Xylanases; Evolution, Molecular; Glucans; Horseshoe Crabs; Lectins; Polysaccharides; Proteoglycans

The main cytokine induced by the interaction of oral epithelial cells with C. glabrata is granulocyte monocyte colony-stimulating factor (GM-CSF); however, the mechanisms regulating this response are unknown. Based on previously published information on the interactions of C. albicans with oral epithelial cells, we hypothesized that interaction with viable C. glabrata triggers GM-CSF synthesis via NF-kappaB activation. We found that C. glabrata-induced GM-CSF synthesis was adhesion-dependent, enhanced by endocytosis, and required fungal viability. NF-kappaB activation was noted during interaction of epithelial cells with C. glabrata, and pre-treatment with an NF-kappaB inhibitor partly inhibited GM-CSF synthesis. Blocking TLR4 with anti-TLR4 antibody did not inhibit GM-CSF production. In contrast, an anti-CDw17 antibody triggered significant inhibition of NF-kappaB activation and GM-CSF synthesis. beta-glucans did not stimulate GM-CSF synthesis, suggesting that the CDw17/NF-kappaB/GM-CSF pathway may be beta-glucan-independent. This study provides new insights into the mechanism of GM-CSF induction by C. glabrata.

Topics: Antibodies; Antigens, CD; beta-Glucans; Candida glabrata; Cell Line; Cell Line, Tumor; Cytochalasin D; Endocytosis; Epithelial Cells; Glucans; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lactosylceramides; Mouth Mucosa; NF-kappa B; Nucleic Acid Synthesis Inhibitors; Polysaccharides; Polysaccharides, Bacterial; Toll-Like Receptor 4; Zymosan

(1-->3)-beta-D-glucan is found in cell walls of some fungi, bacteria and plants. It plays a crucial role in bioaerosol-induced inflammatory reactions. To estimate the level of airborne (1-->3)-beta-D-glucan exposure, a monoclonal antibody-based two-site enzyme immunoassay (mAb-EIA) was developed. The results obtained with the mAb-EIA were compared with the results of a Limulus amoebocyte lysate-based assay for (1-->3)-beta-D-glucan. Three mAbs produced by mouse immunization with bovine serum albumin-conjugated laminarin were enriched by in vitro production in a modular mini-fermenter and affinity purified. Two mAbs were selected for the development of a two-site EIA specific for (1-->3)-beta-D-glucan. Different polysaccharides, fungal and plant seed extracts, and airborne inhalable dust from workplaces (poultry farms, pig stables, grain storage houses, and a laboratory animal facility) were sampled with portable pumps and measured with both the mAb-EIA and Glucatell assay. Using carboxymethylated curdlan as a standard, the mAb-EIA gave a steep dose-response curve for concentrations between 0.36-15 ng/ml. The mAb-EIA was specific for (1-->3)-beta-D-glucan and was sufficiently sensitive to detect (1-->3)-beta-D-glucan in airborne dust samples. In comparing the EIA results to the values obtained with the Glucatell assay, the correlation was found to be high (coefficient of correlation r(2)=0.91), and the mean ratio of the values was 1.7. Depending on the dust source, either the Glucatell assay or the mAb-EIA gave higher results. The mAb-EIA is sensitive enough to detect (1-->3)-beta-D-glucan in airborne dust samples collected with portable pumps. Thus, the assay is suited for the investigation of the health effects induced by exposure to this class of biologically active molecules.

Topics: Animals; Antibodies, Monoclonal; beta-Glucans; Dose-Response Relationship, Immunologic; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Glucans; Humans; Hybridomas; Inhalation Exposure; Limulus Test; Mice; Mice, Inbred BALB C; Occupational Exposure; Particulate Matter; Polysaccharides; Proteoglycans; Reproducibility of Results

The murine molecule dectin-1 (known as the beta-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal beta-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 angstroms resolution structure in which a short soaked natural beta-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and beta-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.

Topics: Animals; beta-Glucans; Cations, Divalent; Crystallography, X-Ray; Fungal Proteins; Glucans; Lectins, C-Type; Membrane Proteins; Mice; Models, Molecular; Nerve Tissue Proteins; Polysaccharides; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Immunologic; Structure-Activity Relationship

In this study we tested the in vitro and in vivo anti-Cryptococcus neoformans activity of an antilaminarin (anti-beta-glucan) monoclonal antibody (MAb 2G8) (immunoglobulin G2b) which was previously shown to inhibit the growth of beta-glucan-exposing Candida albicans cells. Here we show that MAb 2G8 binds to the cell wall of C. neoformans and inhibits its growth to an extent comparable to that observed for C. albicans. Binding and growth inhibition were detected almost equally for encapsulated and acapsular C. neoformans strains. In addition, at subinhibitory concentrations, MAb 2G8 reduced the capsule thickness without affecting protease or phospholipase production. Acapsular fungal cells, but not encapsulated fungal cells, were opsonized by the antibody and more efficiently phagocytosed and killed by human monocytes and by murine peritoneal macrophages. A single administration of MAb 2G8 resulted in a reduction in the fungal burden in the brains and livers of mice systemically infected with a highly virulent, encapsulated C. neoformans strain. This protective effect was also detected in neutropenic mice. Overall, these findings demonstrate that cell wall beta-glucan of encapsulated C. neoformans is accessible to antibodies which can exert remarkable anticryptococcal activities in vitro and in vivo.

Topics: Animals; Antibodies, Fungal; Antibodies, Monoclonal; beta-Glucans; Brain; Cell Wall; Cells, Cultured; Colony Count, Microbial; Cryptococcosis; Cryptococcus neoformans; Female; Glucans; Humans; Immunotherapy; Liver; Macrophages; Mice; Mice, Inbred BALB C; Monocytes; Phagocytosis; Polysaccharides; Protein Binding

(1-->3)-beta-D-Glucans, a cell wall component in most microfungi, are suggested to play a role in the development of respiratory and general symptoms in organic dust-related diseases. The mechanisms by which they induce these effects are, however, not clear. In the present study, mediator release and its potentiation by the (1-->3)-beta-D-glucan as well as by the (1-->6)-beta-D-glucan found in yeast and other fungi were therefore examined. Blood leucocytes from healthy volunteers and from patients allergic to house dust mite were incubated with (1-->3)-beta-D-glucans with increasing 1,6-branchings: curdlan [a linear (1-->3)-beta-D-glucan], laminarin and scleroglucan, and furthermore with pustulan, a linear (1-->6)-beta-D-glucan. Histamine release was not observed on exposure to the glucans only, but in the presence of anti-immunoglobulin E (IgE) antibody or specific antigens, all the glucans investigated led to an enhancement of the IgE-mediated histamine release. The glucans induced a significant potentiation of the mediator release when present at concentrations in the range of 2-5 x 10(-5) M. These results suggest that (1-->3)-beta-D-glucan as well as (1-->6)-beta-D-glucan aggravates IgE-mediated histamine release. Knowledge concerning the effects of glucans on immune responses may be of importance for understanding and treating inflammatory and allergic diseases.

Topics: Adult; Air Pollution, Indoor; Allergens; beta-Glucans; Cell Wall; Dust; Environmental Exposure; Fungi; Glucans; Histamine; Humans; Immunoglobulin E; In Vitro Techniques; Leukocytes; Middle Aged; Polysaccharides; Respiratory Hypersensitivity

To evaluate the in vitro microbicidal activity against Acanthamoeba castellanii of a murine monoclonal anti-idiotypic antibody (KTmAb) and a synthetic killer mimotope (KP), which mimic a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity through interaction with specific cell wall receptors, mainly constituted by beta-glucans.. Amoebicidal activity was investigated after incubation of trophozoites under different experimental conditions with laminarinase, KTmAb, KP and a scrambled decapeptide (SP). To confirm the specific interaction of KP with beta-glucans, the experiments were also carried out in the presence of laminarin (beta1-3-glucan) or pustulan (beta1-6-glucan); both glucan molecules were co-incubated with KP or SP.. KTmAb and KP exhibited a time-dependent killing activity, in comparison with SP or heat-inactivated KTmAb; this activity was completely abolished by pre-incubation with laminarin, but not by pustulan. Notably, in vitro amoebicidal activity was observed in the presence of laminarinase, an enzyme that specifically hydrolyses beta-glucans. Furthermore, KP specifically inhibited the growth of Acanthamoeba on infected contact lenses and the remaining adherent KP-treated trophozoites appeared strongly damaged.. The results indicate that the expression of beta1-3-glucan receptors in the cell membrane is probably modulated during cell growth of A. castellanii and is critical for the killing activity of KT-like molecules. Our data confirm the broad antimicrobial spectra of KTmAb and KP, emphasize the crucial role of beta1-3-glucan in microbial physiology and suggest the potential use of KTmAb and KP in the prevention and therapy of Acanthamoeba infections or in preventing Acanthamoeba contamination during storage of contact lenses.

Topics: Acanthamoeba castellanii; Amebicides; Animals; Antibodies, Anti-Idiotypic; Antibodies, Monoclonal; beta-Glucans; Cell Wall; Contact Lenses; Glucans; Killer Factors, Yeast; Mice; Mycotoxins; Oligopeptides; Polysaccharides

Haemocytes from the gastropod snail Lymnaea stagnalis (Linnaeus) were used as a model to characterize protein kinase C (PKC) signalling events in molluscan defence cells. Challenge of freshly collected haemocytes with the beta-1, 3-glucan laminarin resulted in a transient increase in the phosphorylation of haemocyte PKC, with maximal phosphorylation (represented by a 3.5-fold increase) occurring at 10 min; this effect was blocked by the PKC inhibitor, GF109203X. Moreover, extracellular signal-regulated kinase (ERK) was found to be a downstream target of molluscan PKC, operating via a MAPK/ERK kinase (MEK)-dependent mechanism. Pharmacological inhibition of PKC phosphorylation by U-73122 and ET-18-OCH(3) suggested that laminarin-dependent PKC signalling was modulated via phospholipase C (PLC); however, a role for phosphatidylinositol-3-kinase (PI-3-K) is unlikely since the PI-3-K inhibitor LY294002 was without effect. Generation of H(2)O(2) by haemocytes in response to laminarin was also investigated. H(2)O(2) output increased in a dose- and time-dependent manner, with 10 mg ml(-1) laminarin eliciting a 9.5-fold increase in H(2)O(2) production after 30 min. H(2)O(2) production was significantly attenuated by the PKC inhibitors, GF109203X and Gö 6976, and by the NADPH-oxidase inhibitor, apocynin. In conclusion, these data further our understanding of PKC signalling events in molluscan haemocytes and for the first time define a role for PKC in H(2)O(2) production by these defence cells. Given that H(2)O(2) is an important anti-pathogen molecule, and that haemocytes play a crucial role in the elimination of invading organisms, PKC signalling in these cells is likely to be crucial to the molluscan innate defence response.

Topics: Animals; beta-Glucans; Carbazoles; Estrenes; Extracellular Signal-Regulated MAP Kinases; Glucans; Hemocytes; Hydrogen Peroxide; Indoles; Lymnaea; Maleimides; Phosphatidylinositol 3-Kinases; Phospholipid Ethers; Phosphorylation; Polysaccharides; Protein Kinase C; Protein Kinase Inhibitors; Pyrrolidinones; Reactive Oxygen Species; Signal Transduction

The ability of Candida albicans to rapidly and reversibly switch between yeast and filamentous morphologies is crucial to pathogenicity, and it is thought that the filamentous morphology provides some advantage during interaction with the mammalian immune system. Dectin-1 is a receptor that binds beta-glucans and is important for macrophage phagocytosis of fungi. The receptor also collaborates with Toll-like receptors for inflammatory activation of phagocytes by fungi. We show that yeast cell wall beta-glucan is largely shielded from Dectin-1 by outer wall components. However, the normal mechanisms of yeast budding and cell separation create permanent scars which expose sufficient beta-glucan to trigger antimicrobial responses through Dectin-1, including phagocytosis and activation of reactive oxygen production. During filamentous growth, no cell separation or subsequent beta-glucan exposure occurs, and the pathogen fails to activate Dectin-1. The data demonstrate a mechanism by which C. albicans shape alone directly contributes to the method by which phagocytes recognize the fungus.

Topics: Animals; beta-Glucans; Candida albicans; Cell Line; Glucans; Hyphae; Lectins, C-Type; Macrophage Activation; Macrophages; Membrane Proteins; Mice; Nerve Tissue Proteins; Phagocytosis; Polysaccharides; Reactive Oxygen Species; Zymosan

We analyzed the human monocyte-stimulating ability of laminarin from Eisenia bicyclis, lichenan from Cetraria islandica, and their oligomers depolymerized with endo-1,3-beta-glucanase from Arthrobacter sp. The respective beta-glucan oligomers with different degrees of polymerization (DP) were fractionated from hydrolytic products of laminarin and lichenan using gel-filtration chromatography. The monocyte-conditioned medium pre-cultured in the presence of a fraction of beta-glucan oligomer (DP>/=8) from laminarin exhibited inhibitory activity against the proliferation of human myeloid leukemia U937 cells, while those pre-cultured with other beta-glucan oligomers and the original laminarin and lichenan showed little or no activity. NMR analysis indicated that the beta-glucan oligomer (DP>/=8) has an average DP value of 13, and its ratio of beta-1,3- to beta-1,6-linkages in glucopyranose units was estimated to be 1.3:1. These results indicate that the beta-1,3-glucan oligomer with a higher content of beta-1,6-linkage stimulates monocytes to inhibit the proliferation of U937 cells.

Topics: beta-Glucans; Biopolymers; Cell Division; Glucans; Humans; Molecular Structure; Monocytes; Nuclear Magnetic Resonance, Biomolecular; Polysaccharides; U937 Cells

Two endoglucanases (EGs), EG A and EG B, were purified to homogeneity from Penicillium occitanis mutant Pol 6 culture medium. The molecular weights of EG A and EG B were 31,000 and 28,000 kDa, respectively. The pI was about 3 for EG A and 7.5 for EG B. Optimal activity was obtained at pH 3.5 for both endoglucanases. Optimal temperature for enzyme activity was 60 degrees C for EG A and 50 degrees C for EG B. EG A was thermostable at 60 degrees C and remained active after 1 h at 70 degrees C. EGs hydrolyzed carboxymethylcellulose, phosphoric acid swollen cellulose, and beta-glucan efficiently, whereas microcrystalline cellulose (Avicel) and laminarin were poorly hydrolyzed. Only EG B showed xylanase activity. Furthermore, these EGs were insensitive to the action of glucose and cellobiose but were inhibited by the divalent cations Hg2+, Co2+, and Mn2+.

Topics: beta-Glucans; Carboxymethylcellulose Sodium; Cations, Divalent; Cellulase; Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Glucans; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Molecular Weight; Mutation; Penicillium; Polysaccharides; Substrate Specificity; Temperature; Xylans

To generate a vaccine to protect against a variety of human pathogenic fungi, we conjugated laminarin (Lam), a well-characterized but poorly immunogenic beta-glucan preparation from the brown alga Laminaria digitata, with the diphtheria toxoid CRM197, a carrier protein used in some glyco-conjugate bacterial vaccines. This Lam-CRM conjugate proved to be immunogenic and protective as immunoprophylactic vaccine against both systemic and mucosal (vaginal) infections by Candida albicans. Protection probably was mediated by anti-beta-glucan antibodies as demonstrated by passive transfer of protection to naive mice by the whole immune serum, the immune vaginal fluid, and the affinity-purified anti-beta-glucan IgG fractions, as well as by administration of a beta-glucan-directed IgG2b mAb. Passive protection was prevented by adsorption of antibodies on Candida cells or beta-glucan particles before transfer. Anti-beta-glucan antibodies bound to C. albicans hyphae and inhibited their growth in vitro in the absence of immune-effector cells. Remarkably, Lam-CRM-vaccinated mice also were protected from a lethal challenge with conidia of Aspergillus fumigatus, and their serum also bound to and markedly inhibited the growth of A. fumigatus hyphae. Thus, this novel conjugate vaccine can efficiently immunize and protect against two major fungal pathogens by mechanisms that may include direct antifungal properties of anti-beta-glucan antibodies.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Aspergillosis; Aspergillus fumigatus; Bacterial Proteins; beta-Glucans; Candida albicans; Candidiasis, Vulvovaginal; Female; Fungal Vaccines; Glucans; Magnetic Resonance Spectroscopy; Mice; Mice, Mutant Strains; Polysaccharides; Rats; Rats, Wistar; Serologic Tests; Vaccines, Conjugate

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.

Topics: beta-Glucans; Cells, Cultured; Gene Expression Regulation, Plant; Genes, Plant; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glucuronidase; Methyltransferases; Nicotiana; Plant Diseases; Plant Leaves; Plant Proteins; Polysaccharides; Promoter Regions, Genetic; Protein Binding; Respiratory Burst; Reverse Transcriptase Polymerase Chain Reaction; Tobacco Mosaic Virus

Glucans are microbial cell wall carbohydrates that are shed into the circulation of patients with infections. Glucans are immunomodulatory and have structures that are influenced by bacterial or fungal species and growth conditions. We developed a method to covalently label carbohydrates with a fluorophore on the reducing terminus, and used the method to study the pharmacokinetics following intravenous administration of three highly purified and characterized glucans (glucan phosphate, laminarin and scleroglucan) that varied according to molecular size, branching frequency and solution conformation. Elimination half-life was longer (3.8+/-0.8 vs. 2.6+/-0.2 and 3.1+/-0.6 h) and volume of distribution lower (350+/-88 ml/kg vs. 540+/-146 and 612+/-154 ml/kg) for glucan phosphate than for laminarin and scleroglucan. Clearance was lower for glucan phosphate (42+/-6 ml/kg h) than for laminarin (103+/-17 ml/kg h) and scleroglucan (117+/-19 ml/kg h). Since plasma levels at steady state are inversely related to clearance, these differences suggest that pharmacokinetics could favor higher blood levels of glucans with certain physicochemical properties.

Topics: Animals; Area Under Curve; beta-Glucans; Glucans; Half-Life; Injections, Intravenous; Limulus Test; Linear Models; Male; Polysaccharides; Rats; Rats, Sprague-Dawley

An engineered, killer decapeptide (KP) has been synthesized based on the sequence of a recombinant, single-chain anti-idiotypic antibody (KT-scFv) acting as a functional internal image of a yeast killer toxin. Killer decapeptide exerted a strong fungicidal activity against Candida albicans, which was attributed to peptide interaction with beta-glucan. As this polysaccharide is also a critical component of the cryptococcal cell wall, we wondered whether KP was also active against Cryptococcus neoformans, a human pathogen of increasing medical importance. We found that KP was able to kill both capsular and acapsular C. neoformans cells in vitro. Furthermore, KP impaired the production of specific C. neoformans virulence factors including protease and urease activity and capsule formation, rendering the fungus more susceptible to natural effector cells. In vivo treatment with KP significantly reduced fungal burden in mice with cryptococcosis and, importantly, protected the majority of immunosuppressed animals from an otherwise lethal infection. Given the relevance of cryptococcosis in immunocompromised individuals and the inability of conventional drugs to completely resolve the infection, the results of the present study indicate KP as an ideal candidate for further studies on novel anticryptococcal agents.

Topics: Animals; Antifungal Agents; beta-Glucans; Candida albicans; Cryptococcosis; Cryptococcus neoformans; Dose-Response Relationship, Drug; Female; Glucans; Humans; Macrophages; Melanins; Mice; Mice, Inbred BALB C; Neutrophils; Peptides; Polysaccharides; Survival Rate; Virulence Factors

Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity via interaction with membrane receptors on macrophages, neutrophils, and natural killer cells. We investigated the binding of water-soluble glucans in primary cultures of normal human coronary or dermal vascular endothelial cells (VECs). Membranes from VECs exhibited saturable binding. Competition studies demonstrated the presence of at least two glucan binding sites on VECs. Glucan phosphate competed for all binding sites with a KD of 3.7 microM for coronary VECs and 11 microM for dermal VECs, respectively. Laminarin, a low molecular weight glucan, competed for 47 to 51 per cent of binding (KD = 2.8-2.9 microM), indicating the presence of at least two binding sites. Glucan (1 microg/mL) stimulated VEC nuclear factor kappaB nuclear binding activity and Interleukin 8 expression--but not that of vascular endothelial growth factor--in a time-dependent manner. This is the first report of pattern recognition receptors for glucan on human VECs. It also provides the first evidence that glucans can directly modulate the functional activity of VECs by stimulating cytokine gene. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal cell wall products and suggests that the response to glucans may not be confined to leukocytes.

Topics: Antigens, Fungal; Awards and Prizes; beta-Glucans; Binding Sites; Binding, Competitive; Cell Membrane; Cells, Cultured; Endothelium, Vascular; Glucans; Humans; Interleukin-8; NF-kappa B; Polysaccharides; Receptors, Immunologic; Saccharomyces cerevisiae

Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.

Topics: beta-Glucans; Binding Sites; Binding, Competitive; Carboxymethylcellulose Sodium; Cell Adhesion Molecules; Cell Membrane; Glucans; Humans; Lipoproteins, LDL; Monocytes; Poly C; Poly I; Polysaccharides; Receptors, Immunologic; Receptors, LDL; Receptors, Scavenger; Sizofiran; U937 Cells

Fungal cell wall glucans nonspecifically stimulate various aspects of innate immunity. Glucans are thought to mediate their effects via interaction with membrane receptors on macrophages, neutrophils, and NK cells. There have been no reports of glucan receptors on nonimmune cells. We investigated the binding of a water-soluble glucan in primary cultures of normal human dermal fibroblasts (NHDF). Membranes from NHDF exhibited saturable binding with an apparent dissociation constant (K(D)) of 8.9 +/- 1.9 microg of protein per ml and a maximum binding of 100 +/- 8 resonance units. Competition studies demonstrated the presence of at least two glucan binding sites on NHDF. Glucan phosphate competed for all binding sites, with a K(D) of 5.6 microM (95% confidence interval [CI], 3.0 to 11 microM), while laminarin competed for 69% +/- 6% of binding sites, with a K(D) of 3.7 microM (95% CI, 1.9 to 7.3 microM). Glucan (1 microg/ml) stimulated fibroblast NF-kappaB nuclear binding activity and interleukin 6 (IL-6) gene expression in a time-dependent manner. NF-kappaB was activated at 4, 8, and 12 h, while IL-6 mRNA levels were increased by 48% at 8 h. This is the first report of pattern recognition receptors for glucan on human fibroblasts and the first demonstration of glucan binding sites on cells other than leukocytes. It also provides the first evidence that glucans can directly modulate the functional activity of NHDF. These results provide new insights into the mechanisms by which the host recognizes and responds to fungal (1-->3)-beta-D-glucans and suggests that the response to glucans may not be confined to cells of the immune system.

Topics: beta-Glucans; Binding Sites; Binding, Competitive; Biosensing Techniques; Cell Line; Cell Membrane; Fibroblasts; Glucans; Humans; Interleukin-6; NF-kappa B; Polysaccharides; Saccharomyces cerevisiae; Skin

The laminaripentaose-producing beta-1,3-glucanase of Streptomyces matensis is a member of the glycoside hydrolase family GH-64. We have constructed and purified a recombinant hexahistidine-tagged form of the enzyme for characterisation. The enzyme, which exists as a monomer in solution, hydrolyses beta-1,3-glucan by a mechanism leading to overall inversion of the anomeric configuration. This is the first determination of the mechanism prevailing in glycoside hydrolase family GH-64 and this is the first characterisation of an 'inverting' beta-1,3-glucanase.

Topics: beta-Glucans; beta-Glucosidase; Biopolymers; Chromatography, Gel; Escherichia coli; Glucan 1,3-beta-Glucosidase; Glucans; Histidine; Hydrolysis; Magnetic Resonance Spectroscopy; Peptides; Polysaccharides; Protein Binding; Protein Structure, Quaternary; Recombinant Fusion Proteins; Stereoisomerism; Streptomyces

The C-terminal 155 amino acids of the putative laminarinase, Lam16A, from T. maritima comprise a highly thermostable family 4 CBM that binds beta-1,3- and beta-(1,3)(1,4)-glucans. Laminarin, a beta-1,3-glucan, presented two classes of binding sites for TmCBM4-2, one with a very high affinity (3.5 x 10(7) M(-1)) and one with a 100-fold lower affinity (2.4 x 10(5) M(-1)). The affinities for laminarioligosaccharides and beta-(1,3)(1,4)-glucans ranged from approximately 2 x 10(5) to approximately 2.5 x 10(6) M(-1). Cellooligosaccharides and laminariobiose were bound only very weakly (K(a)s approximately 5 x 10(3) M(-1)). Spectroscopic and mutagenic studies implicated the involvement of three tryptophan residues (W28, W58, and W99) and one tyrosine residue (Y23) in ligand binding. Binding was enthalpically driven and associated with large negative changes in heat capacity. Temperature and osmotic conditions profoundly influenced binding. For the first time in solution, the direct uptake and release of water in CBM binding are demonstrated.

Topics: Amino Acid Motifs; beta-Glucans; Binding, Competitive; Chromatography, Affinity; DNA Primers; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Hot Temperature; Mutagenesis, Site-Directed; Osmosis; Plasmids; Polymerase Chain Reaction; Polysaccharides; Protein Binding; Substrate Specificity; Thermotoga maritima

The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.

Topics: beta-Glucans; Carbohydrate Conformation; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycosylation; Gram-Negative Aerobic Bacteria; Hordeum; Hydrolysis; Magnetic Resonance Spectroscopy; Polysaccharides; Substrate Specificity

Pattern recognition proteins function in innate immune responses by binding to molecules on the surface of invading pathogens and initiating host defense reactions. We report the purification and molecular cloning of a cDNA for a 53-kDa beta1,3-glucan-recognition protein from the tobacco hornworm, Manduca sexta. This protein is constitutively expressed in fat body and secreted into hemolymph. The protein contains a region with sequence similarity to several glucanases, but it lacks glucanase activity. It binds to the surface of and agglutinates yeast, as well as gram-negative and gram-positive bacteria. Beta1,3-glucan-recognition protein in the presence of laminarin, a soluble glucan, stimulated activation of prophenoloxidase in plasma, whereas laminarin alone did not. These results suggest that beta1,3-glucan-recognition protein serves as a pattern recognition molecule for beta1,3-glucan on the surface of fungal cell walls. After binding to beta1,3-glucan, the protein may interact with a serine protease, leading to the activation of the prophenoloxidase cascade, a pathway in insects for defense against microbial infection.

Topics: Amino Acid Sequence; Animals; Base Sequence; beta-Glucans; Carrier Proteins; Cell Wall; Cloning, Molecular; Conserved Sequence; DNA, Complementary; Enzyme Activation; Escherichia coli; Fungi; Glucans; Glycoside Hydrolases; Hemolymph; Insect Proteins; Manduca; Micrococcus; Molecular Sequence Data; Monophenol Monooxygenase; Polysaccharides; Protein Binding; RNA, Messenger; Sequence Homology, Amino Acid

The transglycosylation reaction catalyzed by endo-1,3-beta-D-glucanase LIV from the marine mollusk Spisula sachalinensis was studied with the goal of preparing p-nitrophenyl (Np) 1,3- and 1,3; 1,6-D-glucooligosides. As donors we used the 1,3;1,6-beta-D-glucans with various content of beta-1, 6-glucoside bonds: laminarians [from Laminaria cichorioides (10%), L. gurjanovae (2%), and Fucus evanescens (35%)] and translam (25%); as acceptor we used the p-nitrophenyl beta-D-glucoside (GNp). The maximal yield of the transglycosylation products was found when using laminaran from Laminaria cichorioides; donors with a lower or higher content of beta-1,6-glucoside bonds were less efficient. The laminaran from F. evanescens and translam gave no Np-laminaribioside. At optimal conditions (10 mg/ml of laminaran from L. cichorioides and 5 mg/ml of GNp), maximal yields of Np-laminaribioside, Np-trioside, Np-tetraoside, and Np-pentaoside were 19, 8, 3, and 1%, respectively. The first two compounds were isolated by chromatography on silica gel, their physicochemical characteristics were obtained, and their structures were established by 13C NMR spectroscopy.

Topics: Animals; Chromatography, Gel; Chromatography, High Pressure Liquid; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycosylation; Laminaria; Magnetic Resonance Spectroscopy; Mollusca; Nitrophenols; Oligosaccharides; Polysaccharides; Substrate Specificity

Incubation of plasma of the locust Locusta migratoria, with laminarin induced the precipitation of two major proteins with molecular masses of about 260,000 (P260) and 85,000 Da (P85). This precipitation was not observed when other polysaccharides, such as curdlan, dextran, chitin, cellulose or mannan were used. P260 and P85 were purified to homogeneity by a single step on heparin-sepharose chromatography. Since all attempts to separate P260 from P85, other than the use of sodium dodecyl sulfate, were unsuccessful, it is likely that these two molecules form a complex non-covalently associated. Treatment of P260-P85 complex with N-glycosidase F showed that P260 did not appear to be glycosylated whereas 6% of P85 molecular mass was due to N-linked carbohydrates. On the other hand, no change in molecular masses of P260 or P85 was observed once the complex had been treated with lipase. SDS-PAGE and Western blots of plasma and serum stained with blue Coomassie for proteins or with highly specific polysera to P260 or P85, respectively, showed that P260 was only present in plasma and P85 remained in both samples. This indicates that P260 is likely to be one of the most abundant plasma proteins directly involved in the coagulation process in Locusta migratoria. The addition of plasma or P260-P85 complex to a hemocyte lysate supernatant prior to its activation by laminarin induced a lower protease as well as phenoloxidase activity compared with the control. This reduction of activities was not observed in the presence of serum or when P260-P85 complex was added to a fully activated proPO system.

Topics: Amino Acid Sequence; Animals; beta-Glucans; Chemical Precipitation; Glucans; Glycoside Hydrolases; Grasshoppers; Insect Proteins; Molecular Sequence Data; Polysaccharides

Low-level (subanomole) determination of amino acids in samples of naturally derived polymeric carbohydrates has been demonstrated using vapor-phase acid hydrolysis and subsequent precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Application has been demonstrated for neutral polysaccharide polymers such as laminarin (beta-1,3; branched), curdland (beta-1,3; unbranched), pullulan (alpha-1, 6-maltotriose), glycogen (alpha-1,4-glucan), and inulin (polyfructose). Successful determination (acceptable recovery and lack of interferences) was possible in samples which also contained up to roughly 50 micrograms amino sugars (e.g., chitosan or glucans with copurified glucosamine oligomers), although optimum utility is for samples containing up to ca. 10 micrograms total amines. The limit of quantification was roughly 20 ng protein/ mg sample, based on analysis of reagent blanks, although the limit of detection was much lower (ca. 0.1 ng protein/mg sample). Incorporation of a relatively rapid hydrolysis (150 degrees C for 1.5 h) gave similar results to use of 110 degrees C for 24 h and allowed for relatively rapid processing. The method has shown good sensitivity, linearity, ruggedness, and ease. Recovery has been optimized, although yield varied somewhat depending on polymer composition.

Topics: Amino Acids; Aminoquinolines; beta-Glucans; Carbamates; Carbohydrates; Chromatography, High Pressure Liquid; Glucans; Glucosamine; Inulin; Laminaria; Polymers; Polysaccharides

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.

Topics: Aspergillus fumigatus; beta-Glucans; Cell Wall; Fungal Proteins; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycosylation; Hydrogen-Ion Concentration; Membrane Glycoproteins; Molecular Weight; Polysaccharides; Substrate Specificity; Temperature

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Topics: Actinomycetales; Amino Acid Sequence; Bacillus subtilis; Base Sequence; beta-Glucans; beta-Glucosidase; Cloning, Molecular; DNA Primers; DNA, Bacterial; Genes, Bacterial; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen-Ion Concentration; Isoelectric Point; Isoenzymes; Molecular Sequence Data; Polysaccharides; Recombinant Proteins; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Substrate Specificity

A 1,3-beta-glucanase purified from rice grain is a 33 kDa monomer with a pI of > or = 10.4. The enzyme was determined to be an endo-1,3-beta-glucanase (EC 3.2.1.39) by end product analysis using Laminaria digitata laminarin as substrate. Its amino-terminal amino acid sequence revealed strong homology to an endo-1,3-beta-glucanase from barley.

Topics: Amino Acid Sequence; beta-Glucans; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Molecular Sequence Data; Oryza; Polysaccharides; Substrate Specificity

The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley (1-->3, 1-->4)-beta-glucan by barley (1-->3)-beta-glucanase (E.C. 3.2.1.39) isoenzyme GII and (1-->3, 1-->4)-beta-glucanase (EC 3.2.1.73) isoenzyme EII, respectively, has been determined by 1H-NMR. Both enzymes catalyse hydrolysis with retention of anomeric configuration (e-->e) and may therefore operate via a double displacement mechanism. We predict that all other members of Family 17 of beta-glycosyl hydrolases also follow this stereochemical course of hydrolysis.

Topics: beta-Glucans; Carbohydrate Conformation; Carrier Proteins; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycoside Hydrolases; Hordeum; Magnetic Resonance Spectroscopy; Maltose-Binding Proteins; Models, Chemical; Polysaccharides; Recombinant Fusion Proteins

The use of a novel monoclonal antibody (mAb) that reacts with (1,6)-beta-glucan has permitted the study of the different covalent linkages between glucan and mannoproteins in the cell wall of Candida albicans. The mAb JRR1 was originally raised by immunization with Zymolyase extracts from C. albicans cell walls, but it soon became apparent that it reacted with a (1,6)-beta-glucan epitope. By using this antibody, we show the existence of glucan-mannoprotein complexes between the (1,6)-beta-glucan epitope recognized by the antibody and cell wall mannoproteins. The topology of the (1,6)-beta-glucan in the cell wall of C. albicans has also been studied.

Topics: Antibodies, Monoclonal; beta-Glucans; Binding, Competitive; Candida albicans; Cell Wall; Chromatography, Affinity; Epitopes; Fluorescent Antibody Technique; Fungal Proteins; Glucans; Mannans; Membrane Glycoproteins; Polysaccharides; Tunicamycin

A new 1-->3;1-->6-beta-D-glucan (translam) with immunostimulating activity was isolated from products of the laminaran (L. cichorioides) transformation with endo-1-->3-beta-D-glucanase L0 (Chlamys albidus). As compared with laminaran, translam has a higher molecular weight, contains a 2.5-fold number of 1-->6-bound glucose residues mainly about the nonreducing end of the molecule, but no mannitol. Some of the 1-->6-linked glucose residues are included, in contrast to laminaran in the main chain of 1-->3-beta-D-glucan. It was shown that the translam formation is due to the ability of endo-1-->3-beta-D-glucanase L0 to catalyze the synthesis not only of 1-->3-but also of 1-->6-glucosidic linkages.

Topics: Adjuvants, Immunologic; beta-Glucans; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycosylation; Magnetic Resonance Spectroscopy; Polysaccharides

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.

Topics: Animals; Antibodies, Monoclonal; Arthropod Proteins; beta-Glucans; Blood Cells; Cell Extracts; Dimethyl Sulfoxide; Endotoxins; Enzyme Activation; Enzyme Precursors; Enzyme-Linked Immunosorbent Assay; False Positive Reactions; Glucans; Hemolymph; Horseshoe Crabs; Limulus Test; Lipopolysaccharides; Mice; Polymyxin B; Polysaccharides; Sensitivity and Specificity; Serine Endopeptidases

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

Topics: Animals; Anticoagulants; beta-Glucans; Blood Proteins; Glucans; Horseshoe Crabs; Kinetics; Lipopolysaccharides; Oligosaccharides; Polysaccharides; Polysaccharides, Bacterial; Structure-Activity Relationship

By separating Limulus amebocyte lysate by cation-exchange chromatography with an SP-Toyopearl 650C column, a fraction insensitive to endotoxin, yet specifically sensitive to beta-glucan, was successfully obtained in the unadsorbed portion. This fraction showed beta-glucan dose-dependent clotting enzyme activity, although no sensitivity to endotoxin. This beta-glucan-dependent reaction showed no interference in the presence of endotoxin, with the fraction also showing sensitivity towards various kinds of beta-glucan, i.e. curdlan, pachyman, laminaran and lichenan. The sensitivity towards curdlan was approximately 10(-10) g/ml.

Topics: Animals; Bacterial Toxins; beta-Glucans; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Glucans; Horseshoe Crabs; Polysaccharides; Polysaccharides, Bacterial; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.

Topics: Amino Acid Sequence; Amino Acids; Animals; Astacoidea; beta-Glucans; Carrier Proteins; Chemical Precipitation; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Glucans; Immunoassay; Isoelectric Point; Molecular Sequence Data; Molecular Weight; Monophenol Monooxygenase; Peptide Hydrolases; Polysaccharides

The glycans used in an earlier study to define the ligand specificity of the human monocyte phagocytic receptor for unopsonized particulate activators were assessed for their capacities to activate the proteins of the human alternative complement pathway. Normal human serum was preincubated with glycans under conditions of chelation to prevent activation of the classical complement pathway, and the activation-depletion of the alternative complement pathway was determined by the subsequent capacity of the serum to lyse rabbit erythrocytes (Er). When serum was preincubated at a 1/2 dilution in 8 mM EGTA/2 mM Mg with increasing numbers of yeast glucan or zymosan particles, and was evaluated at final serum dilutions of 1/8, its capacity to lyse Er was found to be reduced by 50% with 1.9 X 10(6)/ml yeast glucan particles and 1.4 X 10(6)/ml zymosan particles. At 2 mg/ml of serum diluted 1/2 in 8 mM EGTA/2 mM Mg, nonturbid preparations of mannan, laminarin, or pyrogen-free inulin and turbid suspensions of cellulose, Sephadex, agarose, or purified inulin failed to activate the alternative complement pathway. In contrast, activation-depletion of the alternative pathway was induced by turbid preparations of crude inulin, nigeran, pachyman, barley beta-glucan, and pustulan, which at 700 micrograms/ml, 500 micrograms/ml, 350 micrograms/ml, 60 micrograms/ml, and 27 micrograms/ml, respectively, effected 50% reductions in the subsequent lysis of Er. After centrifugation of 2 mg/ml suspensions of barley beta-glucan at 1100 X G for 5 min and at 15,000 X G for 15 min, the supernatants contained 90 to 92% and 65% of the barley beta-glucan, respectively, as determined by the anthrone method. On a weight basis, the 1100 X G supernatant exhibited the same capacity to activate the alternative pathway as the corresponding original suspension, whereas the 15,000 X G supernatants had less than 3% of the original anti-complementary activity. Preincubation of adherent human monocytes with increasing concentrations of barley beta-glucan suspensions, 100,000 X G supernatants containing 64% of the original beta-glucan, and laminarin all decreased subsequent ingestion of 1.25 X 10(6) zymosan particles in a dose-related fashion. The numbers of monocytes from three different donors phagocytosing zymosan were reduced by 50% after pretreatment with 30 to 65 micrograms/ml, 25 to 48 micrograms/ml, and 12 to 15 micrograms/ml of barley beta-glucan suspensions, 100,000 X G supernatants of barley

Topics: beta-Glucans; Complement Activation; Complement Pathway, Alternative; Glucans; Hordeum; Humans; Mannans; Monocytes; Phagocytosis; Polysaccharides; Receptors, Complement; Zymosan

Mouse macrophages were cultured on chemically modified plastic dishes. On dishes covered with immobilized glycans, the macrophages were stimulated as judged by increased 14C-glucosamine incorporation, increased cytostatic and cytolytic capacities and by morphology as seen by scanning electron microscopy. The corresponding soluble glycans did not have the capacity to stimulate macrophages as measured by these criteria. Plastic surfaces covered with polyethylenimine showed stimulation of the macrophages with regard to some of the parameters measured. These results may indicate that the stimulation is a multistep process and that, contrary to earlier findings, it is not a prerequisite for stimulation that the glycan be intracellular. The results support the idea that a fixed steric arrangement of glycans is necessary for the stimulation of macrophages in vitro.

Topics: Amylose; Animals; beta-Glucans; Cell Line; Cytotoxicity, Immunologic; Glucans; Macrophage Activation; Macrophages; Mice; Polysaccharides; Stereoisomerism; Structure-Activity Relationship