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Found 68 structures.
Displayed structures from 1 to 15
| -6)-b-D-Glcp-(1- | Show graphically |
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Structure type: polymer chemical repeating unit
Trivial name: pustulan, β-1,6-glucan, β-(1,6)-glucan
Compound class: CPS, cell wall polysaccharide, glucan
Contained glycoepitopes: IEDB_135614,IEDB_141806,IEDB_142488,IEDB_146664,IEDB_241101,IEDB_983931,SB_192
The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1→6)-β-D-glucan homopolymer, β-D-Glcp-(1-[→6)-β-D-Glcp-(1-]n →, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1→6)-β-D-glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1→6)-β-D-glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1→6)-β-D-glucan, but, instead, produced a different O-chain (designated serotype 02); these three strains expressed their own characteristic CPSs. (1→6)-β-D-Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1→6)-β-D-glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the β-D-Glcp-(1→6)-β-D-Glcp linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1→6)-β-D-glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1→6)-β-D-glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis. In addition, these (1→6)-β-D-glucan background antibodies may be protective against A. suis infection. The characterization herein of (1→6)-β-D-glucan is the foundation for the development of a serotyping system for A. suis.
Lipopolysaccharide, capsule, Actinobacillus suis, serotyping system, (1-6)-b-D-Glucan
NCBI PubMed ID: 11086692A cell surface antigen-typing system was devised for the swine pathogen Actinobacillus suis and used to examine the prevalence of different lipopolysaccharide (O) types in healthy and diseased pigs. The strains examined in this study were isolated from a variety of locations in Canada and from Kansas. Lipopolysaccharide preparations of 151 isolates of A. suis were characterized by immunoblotting using polyclonal antisera generated to strains SO4 (O1/K1), H89-1173 (O2/K3), and VSB 3714, a rough strain. Approximately 54% (62 of 114) of A. suis isolates from diseased pigs, all (11 of 11) isolates from healthy pigs, and all (4 of 4) reference strains reacted with O1/K1 antiserum. More than 80% (18 of 22) of A. suis strains used for bacterin production and approximately 41% (47 of 114) of isolates from diseased pigs bound O2/K3 antiserum. One isolate appeared to be rough, and five were untypeable. O1/K1- and O2/K3-reactive strains were equally prevalent in Kansas, whereas O2/K3-reactive strains were more common in Quebec and western Canada and O1/K1 strains were most common in Ontario. The fact that virtually all of the strains submitted for bacterin production were O2/K3-reactive strains is consistent with the notion that these strains may be more virulent than O1/K1 strains; alternatively, this may reflect geographic or other biases. In addition, we observed cross-reactivity between A. suis cell surface antigens and swine antisera to several other important pathogens. This finding may explain why previous attempts to develop a simple serodiagnostic test for A. suis have been unsuccessful.
serotype, Actinobacillus suis, Actinobacillus, Prevalence
NCBI PubMed ID: 11015398The cell wall of Aspergillus fumigatus is composed of a branched PI,3 glucan covalently bound to chitin, β-1,3, β-1,4 glucans, and galactomannan, that is embedded in an amorphous cement composed of α-1,3 glucan, galactomannan and polygalactosamin. The mycelial cell wall of A. fumigatus is very different from the yeast Saccharomyces cerevisiae cell wall, and in particular lacks 01,6 glucans and proteins covalently bound to cell wall polysaccharides. The differences in cell wall composition between the mould A. fumigatus and the yeast S. cerevisiae are also reflected at the genomic level where unique features have been identified in A. fumigatus. A single gene codes for the glucan synthase catalytic subumit; this finding has lead to the development of a RNAi methodology for the disruption of essential genes in A. fumigatus. In contrast to the glucan synthase, multiple genes have been found in the chitin synthase and the α-glucan synthase families; in spite of homologous sequences, each gene in each family have very different function. Similarly homologous mannosyltransferase genes are found in yeast and moulds but they lead to the synthesis of very different N-mannan structures. This chemo-genomic comparative analysis has also suggested that GPI-anchored proteins do not have a role of linker in the three dimensional organization of the fungal cell wall.
cell wall, mannan, glucan, GPI, chitin
Publication DOI: 10.1080/13693780400029155An endo-1,3-β-glucanase was purified from Tunicase®, a crude enzyme preparation from Cellulosimicrobium cellulans DK-1, and determined to be a 383-residue protein (Ala1-Leu383), comprising a catalytic domain of the glycoside hydrolase family 16 and a C-terminal carbohydrate-binding module family 13. The Escherichia coli expression system of the catalytic domain (Ala1-Thr256) was constructed, and the protein with N-terminal polyhistidine tag was purified using a Ni-nitrilotriacetic acid column. We analyzed enzymatic properties of the recombinant catalytic domain, its variants, and the Tunicase®-derived full-length endo-1,3-β-glucanase. Substitution of Glu119 with Ala and deletion of Met123, both of the residues are located in the catalytic motif, resulted in the loss of hydrolytic activity. In comparison between the full-length enzyme and isolated catalytic domain, their hydrolytic activities for soluble substrates such as laminarin and laminarioligosaccharides were similar. In contrast, the hydrolytic activity of the full-length enzyme for insoluble substrates such as curdlan and yeast-glucan was significantly higher than that of the catalytic domain. It should be noted that the acid stabilities for the hydrolysis of laminarin were clearly different. Secondary structure analysis using circular dichroism showed that the full-length enzyme was more acid stable than was the catalytic domain, possibly because of domain interactions between the catalytic domain and the carbohydrate-binding module.
Catalytic Domain, β-1, Carbohydrate-binding module, Enzymatic activity, 3 glucan, Acid tolerance
NCBI PubMed ID: 21979581The structure of cell wall of yeasts (genus Saccharomyces) is one of the factors that determine their health-promoting properties connected to the presence of b-glucans and mannoprotein. The aim of the study was to determine the influence of glycerol as a carbon source on structural polymers of cell wall (b-glucan and mannoprotein) of probiotic yeasts Saccharomyces cerevisiae var. boulardii and brewer’s yeasts S. cerevisiae R9. Significant increase of the percentage of polysaccharide content in the cell wall dry weight of S. cerevisiae R9 brewer’s yeasts was noted (in the range of 10–20 %) after cultivation in medium containing glycerol at a concentration of 2–5 % and pH 4.0. The highest content of carbohydrates in probiotic yeasts’ cell wall (58 %) was observed after cultivation in medium containing 3 % of glycerol and pH 5.0. The cell wall of probiotic yeasts was characterized by higher content of mannoprotein comparing with cell wall preparation of brewer’s yeasts S. cerevisiae R9 composed mainly of b-glucans. After cultivation in mediums with 2 and 3 % of glycerol, the cell of brewer’s yeasts contained the highest amount of b(1,3/1,6)-glucan in dry weight of the cell wall (about 36 %). Glycerol at a concentration of 3 and 5 % also intensified mannoprotein biosynthesis in cell wall of S. cerevisiae R9, approximating their content to those noted in the cells of probiotic yeasts (about 29 % (w/w) of dry weight of the cell wall) after cultivation in a medium of pH 5.0 containing 3 % of glycerol.
cell wall, β-glucan, Saccharomyces cerevisiae, mannoprotein, probiotic yeast
Publication DOI: 10.1007/s00217-013-2016-8In budding yeast, the neck that connects the mother and daughter cell is the site of essential functions such as organelle trafficking, septum formation and cytokinesis. Therefore, the morphology of this region, which depends on the surrounding cell wall, must be maintained throughout the cell cycle. Growth at the neck is prevented, redundantly, by a septin ring inside the cell membrane and a chitin ring in the cell wall. Here, we describe recent work supporting the hypothesis that attachment of the chitin ring, which forms at the mother–bud neck during budding, to β-1,3-glucan in the cell wall is necessary to stop growth at the neck. Thus, in this scenario, chemistry controls morphogenesis.
NCBI PubMed ID: 23949603Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.
NCBI PubMed ID: 23397570A biochemical pathway for the incorporation of cell wall protein into the cell wall of Neurospora crassa was recently proposed. In this pathway, the DFG-5 and DCW-1 endo-α-1,6-mannanases function to covalently cross-link cell wall protein-associated N-linked galactomannans, which are structurally related to the yeast outer chain mannans, into the cell wall glucan-chitin matrix. In this report, we demonstrate that the mannosyltransferase enzyme Och1p, which is needed for the synthesis of the N-linked outer chain mannan, is essential for the incorporation of cell wall glycoproteins into the Candida albicans cell wall. Using endoglycosidases, we show that C. albicans cell wall proteins are cross-linked into the cell wall via their N-linked outer chain mannans. We further demonstrate that the Dfg5p and Dcw1p α-1,6-mannanases are needed for the incorporation of cell wall glycoproteins into the C. albicans cell wall. Our results support the hypothesis that the Dfg5p and Dcw1p α-1,6-mannanases incorporate cell wall glycoproteins into the C. albicans cell wall by cross-linking outer chain mannans into the cell wall glucan-chitin matrix.
cell wall, mannan, N-glycans, endo-mannanases
NCBI PubMed ID: 26048011Covalent cross-links between chitin and glucan at the yeast cell wall are created by the transglycosylase activity of redundant proteins Crh1 and Crh2, with cleavage of β-1,4 linkages of the chitin backbone and transfer of the generated molecule containing newly created reducing end onto the glucan acceptor. A three-dimensional structure of Crh1 was generated by homology modeling based on the crystal structure of bacterial 1,3-1,4-β-D-glucanase, followed by site-directed mutagenesis to obtain molecular insights into how these enzymes achieve catalysis. The residues of both proteins that are involved in their catalytic and binding activities have been characterized by measuring the ability of yeast cells expressing different versions of these proteins to transglycosylate oligosaccharides derived from β-1,3-glucan, β-1,6-glucan and chitin to the chitin at the cell wall. Within the catalytic site, residues E134 and E138 of Crh1, as well as E166 and E170 of Crh2, corresponding to the nucleophile and general acid/base, and also the auxiliary D136 and D168 of Crh1 and Crh2, respectively, are shown to be essential for catalysis. Mutations of aromatic residues F152, Y160 and W219, located within the carbohydrate-binding cleft of the Crh1 model, also affect the transglycosylase activity. Unlike Crh1, Crh2 contains a putative carbohydrate-binding module (CBM18) of unknown function. Modeling and functional analysis of site-directed mutant residues of this CBM identified essential amino acids for protein folding and stability, as well as residues that tune the catalytic activity of Crh2.
cell wall, glucan, glycoside hydrolase, chitin, transglycosylation, computer modelling, carbohydrate binding module
NCBI PubMed ID: 25495733The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock.
cell wall, glycosyltransferases, Microscopy
NCBI PubMed ID: 26220968Plant innate immunity relies in first place on the detection of invading microbes. Thus, plants evolved receptors to sense unique molecules of the microbe, the so called microbe-associated molecular patterns or MAMPs. The best studied fungal MAMP is chitin, an important structural building block of the fungal cell wall. Over the past years several plant receptors for chitin have been characterized as well as different strategies adopted by fungi to evade chitin recognition. Despite its strong activity as an elicitor of plant defense chitin represents only a small percentage of the cell wall of most fungi compared to other complex sugars. β-glucan, the most abundant fungal cell wall polysaccharide, also serves as a MAMP, but the mechanisms of β-glucan perception and signaling in plants are largely unknown. In contrast to that the β-glucan recognition and signaling machineries are well characterized in mammals. The C-type lectin receptor Dectin-1 is a key component of these machineries. In this review we describe valuable knowledge about the existence of at least one β-glucan receptor in plants and about the hindrances in β-glucan research. Additionally we discuss possible future perspectives of glucan research and the possibility to transfer the gathered knowledge from mammalian systems to plants.
β-glucan, chitin, Dectin-1, fungal MAMPs, pattern recognition receptor, plant immune system
NCBI PubMed ID: 26688467Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present ubiquitously at the cell surface in all eukaryotes. They play a crucial role in the interaction of the cell with its external environment, allowing the cell to receive signals, respond to challenges, and mediate adhesion. In yeast and fungi, they also participate in the structural integrity of the cell wall and are often essential for survival. Roughly four decades after the discovery of the first GPI-APs, this review provides an overview of the insights gained from studies of the GPI biosynthetic pathway and the future challenges in the field. In particular, we focus on the biosynthetic pathway in Saccharomyces cerevisiae, which has for long been studied as a model organism. Where available, we also provide information about the GPI biosynthetic steps in other yeast/ fungi. Although the core structure of the GPI anchor is conserved across organisms, several variations are built into the biosynthetic pathway. The present Review specifically highlights these variations and their implications. There is growing evidence to suggest that several phenotypes are common to GPI deficiency and should be expected in GPI biosynthetic mutants. However, it appears that several phenotypes are unique to a specific step in the pathway and may even be species-specific. These could suggest the points at which the GPI biosynthetic pathway intersects with other important cellular pathways and could be points of regulation. They could be of particular significance in the study of pathogenic fungi and in identification of new and specific antifungal drugs/ drug targets.
cell wall, Candida albicans, Saccharomyces cerevisiae, GPI anchor biosynthesis, antifungal drug target
NCBI PubMed ID: 29679465| LIP-(1-6)-+ | LIP-(1-6)-a-D-Glcp-(1-1)-a-D-Glcp | Show graphically |
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Structure type: oligomer
Compound class: glycolipid, trehalolipid
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_742521,IEDB_983931,SB_192
Structural studies on the major glycolipid isolated from Rothia mucilaginosa were carried out utilising specific chemical degradation, NMR spectroscopy and matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI TOF-MS). The glycolipid was found to be a dimannosylacylmonoglyceride in which the carbohydrate part was the glycerol-linked dimannoside α-D-Manp-(1→3)-α-D-Manp-(1→3)-sn-Gro (Man A-Man B-Gro), of which Man B was esterified at O-6 by a fatty acid residue. A second fatty acid substituted the secondary methylene position of the glycerol residue, in contrast to the glycolipid previously found in R. dentocariosa and Saccharopolyspora strains, in which the second fatty acid esterified the primary methylene position of glycerol. Results of the ELISA experiment with rabbit specific antibacterial sera indicate that these two major glycolipids are antigenic, and the patterns of serological reactivity are similar but not identical.
NMR, structure, glycolipid, Rothia mucilaginosa, Stomatococcus, Opportunistic agent, Dimannosylacylmonoglyceride
NCBI PubMed ID: 15535967In this report we examined the structure and properties of surface-active lipids of Rhodococcus ruber. Most historical interest has been in the glycolipids of Rhodococcus erythropolis, which have been extensively characterised. R. erythropolis has been of interest due to its great metabolic diversity. Only recently has the metabolic potential of R. ruber begun to be explored. One major difference in the two species is that most R. ruber strains are able to oxidise the gaseous alkanes propane and butane. In preparation for investigation of the effects of gas metabolism on biosurfactant production, we set out to characterise the biosurfactants produced during growth on liquid n-alkanes and to compare these with R. erythropolis glycolipids.
lipid, biosurfactant, propane, alkane, butane, Rhodococcus ruber
NCBI PubMed ID: 12111164Biosurfactants are surface-active biomolecules that are produced by various micro-organisms. They show unique properties i.e. lower toxicity, higher biodegradability and environmental compatibility compared to their chemical counterparts. Glycolipids and lipopeptides have prompted application in biotechnology and cosmetics due to their multi-functional profile i.e. detergency, emulsifying, foaming and skin hydrating properties. Additionally, some of them can be served as antimicrobials. In this study the current status of research and development on rhamnolipids, sophorolipids, mannosylerythritol lipids, trehalipids, xylolipids and lipopeptides particularly their commercial application in cosmetics and biopharmaceuticals, is described
biotechnology, biopharmaceuticals, biosurfactants, antimicrobials, cosmetics
NCBI PubMed ID: 25970073Like other hydrocarbon-oxidising bacteria, rhodococci respond to the presence of alkanes by producing biosurfactant molecules to improve their ability to utilise these hydrophobic compounds as growth substrates. In the rhodococci these surfactants are predominantly glycolipids, the majority of which remain cell-bound during unrestricted growth. Most work has been done on the trehalose mycolates formed by Rhodococcus erythropolis, but nitrogen-limited conditions lead to the production of anionic trehalose tetraesters also. As surfactants, these compounds, whether purified or in crude form, are able to reduce the surface tension of water from 72 mN m-1 to a low of 26, thus making them among the most potent biosurfactants known. They are also able to reduce the interfacial tension between water and a hydrophobic phase (e.g. n-hexadecane) from 43 mN m-1 to values less than one (Table 1). Biosurfactants have about a ten- to 40-fold lower critical micelle concentration than synthetic surfactants. Such properties suggest a range of industrial applications, where a variety of surface-active characteristics are appropriate. Interest in biosurfactants as industrial chemicals results from the toxicity of many petrochemical-derived surfactants. Currently world-wide surfactant production is on a very large scale, and the demand for them is increasing. However, the drive towards less environmentally damaging chemicals makes biosurfactants attractive as they have lower toxicity. The reason they have not achieved a significant market share is the cost of production, which is considerably higher than for synthetic surfactants. This problem is being addressed using several strategies. An approach where there is great scope for improvement with the rhodococci is an understanding of the genetic basis of glycolipid production, which is largely unknown. They may find applications in the near future in the environmental remediation industries, where the requirement for purified molecules is of less importance. This review summarises knowledge of the chemistry, biochemistry and production of Rhodococcus surface-active lipids. Where they have been used, or there is potential for use, in industrial applications is discussed
glycolipids, biosurfactants, remediation, rhodococci
NCBI PubMed ID: 10068789Rhodococcus erythropolis DSM 43215 produced a surface-active trehalose lipid whose formation was induced by n-alkanes to a maximum of 2.1 g/l in a 50 l batch culture on 2% (w/v) n-alkanes of chain length C12 to C18. The glycolipid was extracted from the biomass with n-hexane and was purified by repeated chromatography on silica gel. It contained α,α-trehalose as the sole non-reducing sugar. The lipid moiety was characterized by 13C nuclear magnetic resonance spectroscopy and mass spectrometry and consisted predominantly of saturated long-chain α-branched β-hydroxy fatty acids (mycolic acids) ranging from C32H64O3 to C38H76O3, of which C34H68O3 and C35H70O3 predominated. The molar ratio of trehalose to mycolic acids was 1:2. 13C nuclear magnetic resonance analysis of the O-hexamethyltrehalose obtained by saponification of the permethylated trehalose dimycolates revealed, with the aid of deuterium exchange, that the ester linkages of mycolic acids are to both primary alcohol groups at the C-6 and C-6’ positions of the trehalose
glycolipids, Rhodococcus erythropolis, trehalose dimycolates
Publication DOI: 10.1099/00221287-115-2-491Biosurfactants are in demand by the global market as natural commodities that can be added to commercial products or use in environmental applications. These biomolecules reduce the surface/interfacial tension between fluid phases and exhibit superior stability to chemical surfactants under different physico-chemical conditions. Biotechnological production of biosurfactants is still emerging. Fungi are promising producers of these molecules with unique chemical structures, such as sophorolipids, mannosylerythritol lipids, cellobiose lipids, xylolipids, polyol lipids and hydrophobins. In this review, we aimed to contextualize concepts related to fungal biosurfactant production and its application in industry and the environment. Concepts related to the thermodynamic and physico-chemical properties of biosurfactants are presented, which allows detailed analysis of their structural and application. Promising niches for isolating biosurfactant-producing fungi are presented, as well as screening methodologies are discussed. Finally, strategies related to process parameters and variables, simultaneous production, process optimization through statistical and genetic tools, downstream processing and some aspects of commercial products formulations are presented.
biosurfactants, filamentous fungi, yeasts, screening, bioprocess, downstream
NCBI PubMed ID: 34131819| -3)-b-D-Glcp-(1- | Show graphically |
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Structure type: homopolymer
Trivial name: glucan, β-1,3-glucan, curdlan, curdlan-type polysaccharide 13140, paramylon, curdlan, laminarin, β-glucan, curdlan, β-(1,3)-glucan, β-(1,3)-glucan, curdlan, curdlan, β-1,3-glucan, paramylon, reserve polysaccharide, b-glucan, β-1,3-D-glucan, laminaran, botryosphaeran, laminaran type β-D-glucan, latiglucan I, pachymaran, Curdlan, zymosan A, β-glucan, curdlan, laminarin, zymosan, zymosan, glucan particles, zymosan, β-(1-3)-glucan, β-(1,3)-glucan, β-(1,3)glucan, pachymaran, D-glucan (DPn)540, pachyman, laminaran, curdlan, zymosan, zymosan, β-(1,3)-glucan, zymosan A, zymosan, β-1,3-glucan, curdlan, β-1,3-glucan, curdlan, β-1,3-glucan, curdlan, pachyman, β-(1,3)-glucan, curdlan, callose, a water-insoluble β-(1→3)-glucan, fermentum β-polysaccharide, water-insoluble glucan, alkali-soluble β-glucan (PeA3), alkali-soluble polysaccharide (PCAP), callose, laminarin
Compound class: EPS, O-polysaccharide, cell wall polysaccharide, lipophosphoglycan, glycoprotein, LPG, glucan, polysaccharide, glycoside, β-glucan, β3-glucan, cell wall glucan
Contained glycoepitopes: IEDB_1397514,IEDB_142488,IEDB_146664,IEDB_153543,IEDB_158555,IEDB_161166,IEDB_2278476,IEDB_2278477,IEDB_558869,IEDB_857743,IEDB_983931,SB_192
Extensive surveys for the effects of various β-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1→3)-β-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1→3)-β-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 β-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)-β-D-glucans, laminarans, mixed linkage (1→3), (1→4)-β-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1→3)-β-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1→3)-β-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.
NCBI PubMed ID: 8339297The four recombinant glucosyltransferases (GTFs), GtfJ, GtfK, GtfL and GtfM, that had previously been cloned from Streptococcus salivarius ATCC 25975, were individually expressed in Escherichia coli and their glucan products and kinetic properties were analysed. GtfJ was a primer-dependent GTF which synthesized an insoluble glucan composed mainly of α-(1→3)-linked glucosyl residues in the presence of dextran T-10. GtfK was primer-stimulated, and produced a linear soluble dextran without any detectable branch points both in the absence and in the presence of dextran T-10. GtfL was primer-independent and produced a mixed-linkage insoluble glucan composed of approximately equal proportions of α-(1→3)- and α-(1→6)-linked glucosyl residues. GtfL was inhibited by dextran T-10. GtfM was primer-independent and produced a soluble dextran with approximately 5% α-(1→3)-linked glucosyl residues. GtfM was essentially unaffected by the presence of dextran T-10. The results confirmed that each enzyme represented one of the four possible combinations of primer-dependency and product solubility and that each possessed unique biosynthetic properties. The soluble dextrans formed by GtfK and GtfM, as well as the mixed-linkage insoluble glucan formed by GtfL, were also capable of acting as primers for the primer-dependent GtfJ and the primer-stimulated GtfK. Unexpectedly, the linear dextran produced by GtfK was by far the least effective either at priming itself or at activating and priming the primer-dependent GtfJ.
13C-NMR, Streptococcus salivarius, Glucosyltransferases, kinetics, Glucans
NCBI PubMed ID: 7545511A patent culture of Alcaligenes faecalis var. myxogenes (ATCC 31749) synthesizes "curdlan" during the stationary phase of an aerobic batch fermentation following the depletion of assimilable nitrogen. Because this /3-1,3-homoglucan exopolymer is water insoluble, the fermentation broth is of a relatively low viscosity, and consequently offers little resistance to oxygen transfer from gas to the liquid. However, the layer of insoluble exopolymer surrounding the cell mass offers a resistance to oxygen transfer from the liquid to the cell, thereby necessitating an unexpectedly high dissolved oxygen concentration for maximal productivity. The shear sensitive nature of this fermentation restricts improving oxygen transfer by increasing agitation intensity. The requirement for high volumetric oxygen transfer can be met by low shear designs with axial-flow impellers, providing gas dispersion is assisted by the use of sparging devices consisting of microporous materials. The specific respiration rates for growing and curdlan-producing stationary-phase culture were determined to be and 2.7 mmol O2/g cell/h, respectively. At a cell density of 3 g cell/L, the maximal rate of curdlan biosynthesis was about 100 mg/g ceU/h, with a requirement for dissolved oxygen (DO) of 6.5 mg O2/L (86% air saturation at 30 ~ and I atm.). Whereas, at constant impeller speed, the volumetric oxygen transfer was improved both by increasing the air sparging rate and by using O2-enriched air (30% O2), productivity was not consistently improved by operating at a twofold higher cell density and at a DO> 6.5 mg O2/L. At higher cell densities, it would appear that shear must be increased to minimize culture clumping and assist in oxygen transfer to the cell.
exopolysaccharide, aeration, Curdlan, shear, oxygen demand, DOcrit, OTR
Publication DOI: 10.1007/BF02920581In order to elucidate the biosynthetic process of cellulose and curdlan, 13Clabeled polysaccharides were biosynthesized by Acetobacter xylinum (IFO 13693) and Agrobacterium sp. (ATCC 317491, from culture media containing D-(1-13C)glucose, D-(2-13C)glucose, D-(4-13C)glucose, or D-(6-13C)glucose as the carbon source, and their structures were determined by 13C NMR spectroscopy. The labeling was mainly found in the original position, indicating direct polymerization of introduced glucoses. In addition, the transfer of labeling from C-2 to C-I, C-3 and C-5, from C-4 to C-l, C-2 and C-3, and from C-6 to C-1 was found in celluloses. In curdlan, the transfer of labeling from C-1 to C-3, from C-2 to C-I and C-3, from C-4 to C-I, C-2 and C-3, and from C-6 to C-I and C-3 was observed. From analysis of this labeling, the biosynthetic process of cellulose and curdlan was explained as involving six routes. The percentages of each route via which cellulose or curdlan is biosynthesized were estimated for upper (C-t to C-3) and lower portions (C-4 to C-6) of glucosidic units in the polysaccharides. It is noted that very few polysaccharides are formed via the Embden-Meyerhof pathway. The lower half (C-4 to C-6) structure of introduced glucoses is well preserved in the polysaccharides.
Publication DOI: 10.1016/0144-8617(94)90184-8Curdlan was carboxymethylated in an aqueous alkaline medium using monochloroacetic acid as the etherifying agent. The structure of carboxymethylated curdlan (CMc) was analyzed by FT-IR and NMR spectroscopy, which revealed that the carboxymethyl group was introduced mainly at the C-6 position as well as at the C-2 and C-4 positions. Furthermore, CMc was compared with the native curdlan by using rheology and DSC methods. It was found that in water, both polysaccharides behaved as pseudoplastic fluids and fit the power law and Herschel-Bulkley rheological models well. Both the storage shear modulus G' and the loss shear modulus G'' of CMc aqueous solutions decreased and became more frequency dependent with decreasing concentration in comparison with the curdlan aqueous suspensions. The modulus-temperature curve also suggested that the gel characteristic of curdlan has been lost after chemical modification, which is consistent with the DSC results. AFM images revealed differences in the conformation of native and carboxymethylated curdlan, which changed from the aggregation of macromolecules to triple helices. All the experimental results suggest that the hydrogen bonds that bind curdlan with interstitial water to form the micelles have been destroyed completely and that the hydrophobic interactions related to the methylene groups at C-6 formed above 55 degrees C disappeared due to the introduction of the hydrophilic carboxymethyl group.
hydrogen bond, Rheology, Curdlan, carboxymethylation, physico-chemical property, gelation, hydrophobicity
NCBI PubMed ID: 16310757Alcaligenes faecalis var. myxogenes 10C3, which we isolated from soil, produces a water-soluble and an insoluble extracellular polysaccharide. The former (succinoglycan) is composed of glucose, galactose, pyruvic acid and succinic acid (molar proportions 7:1:1:1) with (β 1-3)-, (β 1-4)- and (β 1-6)-glucosidic linkages. The latter (curdlan) is composed entirely of (β 1-3)-linked D-glucose and forms a resilient firm gel when heated in suspension. The organism also produces extracellularly a repeating-unit octasaccharide of succinoglycan and cyclic (β 1-2)-D-glucan. These polymers or oligomers are also produced by many strains of Agrobacterium and Rhizobium. Spontaneous mutation in ability to produce these polysaccharides or oligosaccharides occurs in these strains. The structures of succinoglycan, and similar polymers containing riburonic acid or galactose as the end residue of the side chain, were elucidated by successive fragmentation with two special enzymes obtained from Cytophaga arvensicola followed by methylation analysis. It is interesting that the unit compound in the biosynthesis of succinoglycan is identical with the unit compound in the enzymic decomposition of the polymer. Studies on curdlan gel by X-ray, 13C n.m.r. and electron-microscopic analysis and other physicochemical methods showed that the molecular structure of curdlan changes from a single helix to a triple stranded helix on heat treatment at high temperature. Curdlan seems to be useful for making new types of jelly products and may also be useful in new procedures for food production. Interestingly, curdlan possesses marked antitumour activity. Extracellular isoamylase (EC 3.2.1.68; glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB 15, which we isolated from soil, is very useful for elucidation of the structure of amylopectin and glycogen, and also for the commercial production of amylose or maltose alone or in combination with beta-amylase. Maltose is useful as a sugar for injection, being better than glucose. Maltitol is easily produced from maltose by chemical reduction and is used as a low-calorie sweetener. The isoamylase is also effective for enhancing the production of glucose from starch by the action of glucoamylase.
bacterial polysaccharides, β-Glucans, Alcaligenes faecalis
NCBI PubMed ID: 6400487A 13C nuclear magnetic resonance (NMR) study of a resilient gel of a curdlan-type polysaccharide 13140, a (1 goes to 3)-β-D-glucan, from Alcaligenes faecalis var. myxogenes IFO 13140, was performed in an effort to understand the gel structure. It was found that very broad 13C resonance peaks of line widths ca. 150 (C-1-C-5) and 50 HZ (C-6) are able to be seen in the gel state. Because of a fivefold increase of the peak intensities with respect to those in an aqueous suspension by gelation, these peaks are unequivocally ascribed to certain regions relevant to the gel structure. Those 13 C NMR peaks, however, account for only 20-30 (as viewed from C-1-C-5) and 60% (from C-6) of the total gel, the peak areas of the rest being lost. With respect to those of the disordered low molecular weight acid degraded fraction, fraction II, and laminaran, downfield displacements of C-1, C-3, and C-4 signals are found to take place by amounts of 2.8, 3.2, and 0.9 ppm, respectively, while the remaining peaks (C-2, C-5, and C-6) are unchanged. In view of similar differences of chemical shifts between cyclodextrins and linear (1 goes to 4)-α-D-glucans (Colson, P., Jennings, H.J., and Smith, I.C.P. (1974), J. Am. Chem. Soc. 96, 8081-8087), the observed downfield 13C shifts of the glucosidic bonds in the gel are explained by the presence of the fixed conformation of the preferred dihedral angles, in which internal rotations around the glucosidic bonds are not allowed. Combined with the results of theoretical prediction and the downfield shift of C-4 signals, which is consistent with the presence of an O-4'...O-5 intramolecular hydrogen bond, the observed 13C peaks are ascribed to a region of single helical conformation, whereas the peak-loss portion of 13C NMR signals (70-80 and 40% from C-1 and C-6, respectively) is presumably ascribed to the multiple-helical junction zones for the gel structure and their vicinities. The variation of the line widths as well as the peak positions is also found to take place by stepwise addition of NaOH (greater than 0.22 M). The onset of the conformational transition, helix to random coil, is in good agreement with the change of viscosity, specific rotation, optical rotatory dispersion, and absorption maximum shift by complex formation with Congo Red reported by Ogawa et al. (Ogawa, K., Watanabe, T., Tsurugi, J., and Ono, S. (1972), Carbohydr. Res. 23, 399-405)
conformation, 13C NMR, Curdlan, Alcaligenes faecalis
NCBI PubMed ID: 843521The biosynthesis and export of bacterial cell-surface polysaccharides is known to occur through several distinct mechanisms. Recent advances in the biochemistry and structural biology of several proteins in synthase-dependent polysaccharide secretion systems have identified key conserved components of this pathway in Gram-negative bacteria. These components include an inner-membrane-embedded polysaccharide synthase, a periplasmic tetratricopeptide repeat (TPR)-containing scaffold protein, and an outer-membrane beta-barrel porin. There is also increasing evidence that many synthase-dependent systems are post-translationally regulated by the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Here, we compare these core proteins in the context of the alginate, cellulose, and poly-β-D-N-acetylglucosamine (PNAG) secretion systems.
exopolysaccharide, alginate, cellulose, synthase, poly-β-D-N-acetylglucosamine
NCBI PubMed ID: 23117123The β-glycan polysaccharide family includes cellulose, the abundant and ubiquitous plant cell wall structural polymer, invertebrate and fungal chitin, yeast cell wall glucans, hyaluronan and other extracellular matrix polymers of vertebrates, as well as numerous extracellular bacterial polysaccharides of commercial and agronomic significance. The biogenesis of these polymers is receiving increasing attention as genes encoding their synthases are discovered Though still rudimentary our basic understanding of the biochemistry of the synthetic processes and their control is growing rapidly. This review offers an overview, from a comparative viewpoint, of our current knowledge of enzymic events in β-glycan synthesis.
biosynthesis, classification, active sites, β-glycans, mechanisms, beta-glycans
Publication DOI: 10.4052/tigg.12.211Bacterial glucans have aroused increasing interest in commercial applications in the food and pharmaceutical sectors. A number of bacterial glucans have been reported over recent decades, and their structure, production, and functional properties have been extensively studied. In this paper, we review recent researches on bacterial glucans, with emphasis on the production, physical and chemical properties, and the new developments in food, biomedical, pharmaceutical, and other industrial applications.
food, property, production, pharmaceutical, Bacterial glucans, Biomedical
NCBI PubMed ID: 27678120The macrophage-stimulating properties of some structurally related polysaccharides were studied in vitro. When the polysaccharides were presented to the macrophages in a sterically fixed form, i.e. as microparticles, they induced the release of interleukin 1 (IL-1) from the macrophages. Microparticulate 1.3-β-glucan (curdlan) induced nonspecific macrophage mediated tumour cell killing while 1.4-α-glucan (starch), 1.6-α-glucan (dextran), and 1.6-α-mannan were without effect. The corresponding soluble polysaccharides did not stimulate the macrophages. Kinetic studies showed that although IL-1 was released immediately after stimulation, the macrophages needed a time lag of several days to develop tumour cytotoxicity. The development of cytotoxicity paralleled binding of tumour cells to the macrophages. Resident and inflammatory peritoneal macrophages showed differences in their responses to the polysaccharides. Stationary, resident peritoneal macrophages stimulated by macroparticles secreted high levels of IL-1 but expressed a low cytotoxic activity, while newly recruited inflammatory macrophages released lower levels of IL-1 but readily killed the tumour cells. The influence of cyclo-oxygenase products on the IL-1 release and macrophage cytotoxicity was also investigated. When cyclo-oxygenase was blocked with indomethacin, a significantly higher release of IL-1, and then an increased cytotoxicity, were obtained with 1.3-β-glucan stimulated macrophages. The results suggest that microparticulate polysaccharides may be useful for studies on the induction of macrophage differentiation and also for studies on nonspecific cellular immune responses in vitro and in vivo.
macrophage stimulation
NCBI PubMed ID: 3494301Fungal cell walls are predominantly composed of glucans, mannans, and chitin. Recognition of these glycans by the innate immune system is a critical component of host defenses against the mycoses. Complement, an important arm of innate immunity, plays a significant role in fungal pathogenesis, especially the alternative pathway (AP). Here we determine that the glycan monosaccharide composition and glycosidic linkages affect AP activation and C3 deposition. Furthermore, properdin, a positive regulator of the AP, contributes to these functions. AP activation by glycan particles that varied in composition and linkage was measured by C3a generation in serum treated with 10 mM EGTA and 10 mM Mg(2+) (Mg-EGTA-treated serum) (AP specific; properdin functional) or Mg-EGTA-treated serum that lacked functional properdin. Particles that contained either β1→3 or β1→6 glucans or both generated large and similar amounts of C3a when the AP was intact. Blocking properdin function resulted in 5- to 10-fold-less C3a production by particulate β1→3 glucans. However, particulate β1→6 glucans generated C3a via the AP only in the presence of intact properdin. Interestingly, zymosan and glucan-mannan particles (GMP), which contain both β-glucans and mannans, also required properdin to generate C3a. The β1→4 glycans chitin and chitosan minimally activated C3 even when properdin was functional. Finally, properdin binding to glucan particles (GP) and zymosan in serum required active C3. Properdin colocalized with bound C3, suggesting that in the presence of serum, properdin bound indirectly to glycans through C3 convertases. These findings provide a better understanding of how properdin facilitates AP activation by fungi through interaction with the cell wall components.
properdin
NCBI PubMed ID: 21878570This study examined the response of various forms and sources of glucans toward two different Limulus amebocyte lysate (LAL) methods, the modified LAL, and Glucatell. The glucans studied were curdlan, laminarin, yeast glucan, barley glucan, paramylon, pullulan, pustulan, mannan, and pachyman (as part of the Glucatell kit). Both methods provided largely similar results for each of the glucans; however, the Glucatell method yielded slightly higher responses to certain structures that may not necessarily be of fungal origin, leading to falsely greater positive results. The performance of each method to measure fungal glucan concentration specifically was then assessed.
glucan, Curdlan, dectin receptor, glucatell, LAL, Limulus amebocyte lysate
NCBI PubMed ID: 21830869Curdlan is a microbial polysaccharide composed exclusively of β-(1,3)-linked glucose residues. Until now only bacteria belonging to the Alcaligenes and Agrobacterium species have been reported to produce Curdlan. In this study, a bacterium capable of producing extracellular Curdlan, identified as Pseudomonas sp. on the basis of 16S rDNA gene sequencing, was isolated from soil samples. From the HPLC, permethylation linkage analysis, 13C NMR, and FT-IR analytical data, the polysaccharide consisted exclusively of glucose; the most prominent sugar was 1,3-linked glucose, and most glycosidic bonds joining these sugar residues were of the β-type. This also supported that the exopolysaccharide produced by Pseudomonas sp. was actually Curdlan. In addition, the Pseudomonas sp. was studied for the production of Curdlan by conventional "one-factor-at-a-time technique" and response surface methodology (RSM). It was observed that glucose and yeast extract were the most suitable carbon source and nitrogen source for Curdlan production, respectively. By using RSM, Curdlan production was increased significantly by 188%, from 1.25 to 2.35 g/L, when the strain was cultivated in the optimal condition developed by RSM, and the highest Curdlan production rate of 0.81 g/(L h) was obtained. To the best of the authors' knowledge, this is the first report on Curdlan production by Pseudomonas sp.
Pseudomonas, production, optimization, Curdlan, insoluble polysaccharide
NCBI PubMed ID: 22533491An antitumor β-1,3-glucan (PVG) was purified from PVA-S by sequential use of a-amylase digestion, ethanol precipitation, ion exchange chromatography on diethyl aminoethyl Sephadex A-25, ammonium sulfate fractionation, and GPC on Sepharose CL-2B. PVG showed [α]D + 28.4° (water). From the results of methylation analysis and carbon 13 nuclear magnetic resonance spectroscopy, PVG is a β-1,3-glucan branched at position 6 of every fifth 3-substituted β-glucosyl unit. PVG formed a complex with Congo Red in neutral and dilute alkali solution, but this complex was dissociated at more than 0.2N NaOH. PVG showed potent antitumor activity against the solid form of sarcoma 180 tumor in ICR mice.
polysaccharide, β-1, antitumor agent, Peziza vesiculosa, vesiculogen, 3-glucan, sarcoma 180
NCBI PubMed ID: 3830434Recently, a number of bioactive molecules, including antitumor agents, have been identified in various higher basidiomycetes mushrooms. Polysaccharides are the best known and most potent mushroom derived substances that display immuno-pharmacological properties. In this study, the water soluble crude polysaccharide HEB-P, which was obtained from the liquid culture broth of Hericium erinaceus by ethanol precipitation, was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This fractionation process resulted in two polysaccharide fractions that were termed HEB-NP Fr I and HEB-AP Fr I. Of the fractions, HEB-AP Fr I was able to upregulate the functional events mediated by activated macrophages, such as production of nitric oxide (NO) and expression of cytokines (IL-1β and TNF-α). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicate that HEB-AP Fr I was a low molecular mass polysaccharide with a laminarin-like triple helix conformation of the β-1,3-branched-β-1,2-mannan.
Hericium erinaceus, immunostimulating polysaccharide, laminarin-like triple helix, macrophage activation
Publication DOI: 10.1016/j.carbpol.2009.04.036MD-Fraction, a highly purified, soluble β-(1,3) (1,6)-glucan obtained from Grifola frondosa (an oriental edible mushroom), has been reported to inhibit tumor growth by modulating host immunity. β -Glucan, a major component of the fungal cell wall, is generally recognized by PRRs expressed on macrophages and DCs, such as Dectin-1, and the ability of β-glucans to modulate host immunity is influenced by their structure and purity. Most cellular studies have used particulate β-glucans, such as yeast zymosan (crude β-glucan) and curdlan (purified β-glucan). However, little is known about the cellular mechanism of soluble β-glucans, including MD-Fraction, despite significant therapeutic implications. In this study, we investigated the cellular mechanism of MD-Fraction in murine resident macrophages and compared it with two well-known β-glucan particles. MD-Fraction induced GM-CSF production rapidly through Dectin-1-independent ERK and p38 MAPK activation. Subsequently, MD-Fraction-induced GM-CSF enhanced proliferation and Dectin-1 expression, which permitted Dectin-1-mediated TNF-α induction through the Syk pathway. Curdlan induced not only the proliferation and activation of Dectin-1/Syk signaling in a manner similar to MD-Fraction but also the uncontrolled, pro inflammatory cytokine response. Contrastingly, zymosan reduced proliferation and Dectin-1 expression significantly, indicating that the mechanism of macrophage activation by MD-Fraction differs from that of zymosan. This is the first study to demonstrate that purified β-glucans, such as MD-Fraction and curdlan, induce GM-CSF production directly, resulting in Dec-tin-1/Syk activation in resident macrophages. In conclusion, we demonstrated that MD-Fraction induces cell proliferation and cytokine production without excessive inflammation in resident macrophages, supporting its immunotherapeutic potential.
Curdlan, β-glucan, MAPK, zymosan, Grifola frondosa
NCBI PubMed ID: 22028332The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.
Curdlan, Alcaligenes faecalis, arthritis
NCBI PubMed ID: 22328069Pullulan is a water-soluble polysaccharide produced by yeast-like fungus Aureobasidium pullulans. It is a regularly repeating copolymer with the chemical structure {→6)-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→4)-α-D-glucopyranosyl-(1→}n and viewed as a succession of α-(1→6)-linked (1→4)-α-D-triglucosides i.e. maltotriose (G3). Pullulan has a wide range of commercial applications in biomedical and food industries. Because of its strictly linear structure, pullulan is also a very valuable tool in basic research as well as a well-defined model substance. This chapter focuses on the current literature on pullulan mainly its microbial sources, structural geometry, fermentative production, biosynthesis aspects, peculiar characteristics and varied applications.
biosynthesis, structure, fermentative production, pullulan, Aureobasidium pullulans, food and biomedical applications
Publication DOI: 10.1007/978-94-007-2214-9_24In the first science review on the then nascent Metabolic Engineering field in 1991, Dr. James E. Bailey described how improving erythropoietin (EPO) glycosylation can be achieved via metabolic engineering of Chinese hamster ovary (CHO) cells. In the intervening decades, metabolic engineering has brought sweet successes in glycoprotein engineering, including antibodies, vaccines, and other human therapeutics. Today, not only eukaryotes (CHO, plant, insect, yeast) are being used for manufacturing protein therapeutics with human-like glycosylation, newly elucidated bacterial glycosylation systems are enthusiastically embraced as potential breakthrough to revolutionize the biopharmaceutical industry. Notwithstanding these excitement in glycoprotein, the sweet metabolic engineering reaches far beyond glycoproteins. Many different types of oligo- and poly-saccharides are synthesized with metabolically engineered cells. For example, several recombinant hyaluronan bioprocesses are now in commercial production, and the titer of 2'-fucosyllactose, the most abundant fucosylated trisaccharide in human milk, reaches over 20 g/L with engineered E. coli cells. These successes represent only the first low hanging fruits, which have been appreciated scientifically, medically and fortunately, commercially as well. As one of the four building blocks of life, sugar molecules permeate almost all aspects of life. They are also unique in being intimately associated with all major types of biopolymers (including DNA/RNA, proteins, lipids) meanwhile they stand alone as bioactive polysaccharides, or free soluble oligosaccharides. As such, all sugar moieties in biological components, small or big and free or bound, are important targets for metabolic engineering. Opportunities abound at the interface of glycosciences and metabolic engineering. Continued investment and successes in this branch of metabolic engineering will make vastly diverse sugar-containing molecules (a.k.a. glycoconjugates) available for biomedical applications, sustainable technology development, and as invaluable tools for basic scientific research. This short review focuses on the most recent development in the field, with emphasis on the synthesis technology for glycoprotein, polysaccharide, and oligosaccharide.
biosynthesis, glycosylation, Metabolic engineering
NCBI PubMed ID: 26655367Three structural classes of (1→3)-β-D-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (1→3)-β-glucans and side-chain-branched (1→3,1→2)-β-glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (1→3,1→6)-β-glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (1→3)-β-glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (1→3)-β-glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.
Agrobacterium, glucan, Alcaligenes faecalis, bacterial cellulose, Triple Helix
NCBI PubMed ID: 15818477Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications.
biosynthesis, gene clusters, Bacterial exopolysaccharides, polysaccharide engineering, tailor-made exopolysaccharides
NCBI PubMed ID: 26074894A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells
β-glucan, Aureobasidium pullulans, stimulation of macrophages, RNA expression
NCBI PubMed ID: 25875639ZG16p is a soluble mammalian lectin that interacts with mannose and heparan sulfate. Here we describe detailed analysis of the interaction of human ZG16p with mycobacterial phosphatidylinositol mannosides (PIMs) by glycan microarray and NMR. Pathogen-related glycan microarray analysis identified phosphatidylinositol mono- and di-mannosides (PIM1 and PIM2) as novel ligand candidates of ZG16p. Saturation transfer difference (STD) NMR and transferred NOE experiments with chemically synthesized PIM glycans indicate that PIMs preferentially interact with ZG16p by using the mannose residues. The binding site of PIM was identified by chemical-shift perturbation experiments with uniformly 15N-labeled ZG16p. NMR results with docking simulations suggest a binding mode of ZG16p and PIM glycan; this will help to elucidate the physiological role of ZG16p
NMR spectroscopy, chemical synthesis, lectins, carbohydrate microarrays, microarrays, phosphatidyl inositol mannoside
NCBI PubMed ID: 25919894Cervical cancer is the fourth-ranked cancer in the world and is associated with a large number of deaths annually. Chemotherapy and radiotherapy are known as the common therapeutic approaches in the treatment of cervical cancer, but because of their side effects and toxicity, researchers are trying to discovery alternative therapies. β-glucans, a group of glucose polymers that are derived from the cell wall of fungi, bacteria, and etc. it has been showed that β-glucans have some anti-cancer properties which due to their impacts on adaptive and innate immunity. Along to these impacts, these molecules could be used as drug carriers. In this regard, the application of β-glucans is a promising therapeutic option for the cancer prevention and treatment especially for cervical cancer. Herein, we have summarized the therapeutic potential of β-glucans alone or as adjuvant therapy in the treatment of cervical cancer. Moreover, we highlighted β-glucans as drug carriers for preventive and therapeutic purposes.
bacteria, β-Glucans, fungi, cervical cancer
NCBI PubMed ID: 32138756β-1,3-1,4-glucanases (or lichenases; EC 3.2.1.73) comprise one of the main enzymes used in industry during recent decades. These enzymes hydrolyze β-glucans containing β-1,3 and β-1,4 linkages, such as cereal β-glucans and lichenan. The β-1,3-1,4-glucanases are produced by a variety of bacteria, fungi, plants and animals. A large number of microbial β-1,3-1,4-glucanases have potential application in industrial processes, such as feed, food and detergent industries. The present review summarizes the available studies with respect to β-1,3-1,4-glucanases production conditions, enzyme biochemical properties and potential industrial application.
production, β-1, Applications, biochemical properties, 3-1, 4-glucanases
NCBI PubMed ID: 30430579The genome and natural habitat of Chitinophaga pinensis suggest it has the ability to degrade a wide variety of carbohydrate-based biomass. Complementing our earlier investigations into the hydrolysis of some plant polysaccharides, we now show that C. pinensis can grow directly on spruce wood and on the fungal fruiting body. Growth was stronger on fungal material, although secreted enzyme activity was high in both cases, and all biomass-induced secretomes showed a predominance of β-glucanase activities. We therefore conducted a screen for growth on and hydrolysis of β-glucans isolated from different sources. Most noncrystalline β-glucans supported good growth, with variable efficiencies of polysaccharide deconstruction and oligosaccharide uptake, depending on the polysaccharide backbone linkage. In all cases, β-glucan was the only type of polysaccharide that was effectively hydrolyzed by secreted enzymes. This contrasts with the secretion of enzymes with a broad range of activities observed during growth on complex heteroglycans. Our findings imply a role for C. pinensis in the turnover of multiple types of biomass and suggest that the species may have two metabolic modes: a "scavenging mode," where multiple different types of glycan may be degraded, and a more "focused mode" of β-glucan metabolism. The significant accumulation of some types of β-gluco-oligosaccharides in growth media may be due to the lack of an appropriate transport mechanism, and we propose that this is due to the specificity of expressed polysaccharide utilization loci. We present a hypothetical model for β-glucan metabolism by C. pinensis that suggests the potential for nutrient sharing among the microbial litter community. It is well known that the forest litter layer is inhabited by a complex microbial community of bacteria and fungi. However, while the importance of fungi in the turnover of natural biomass is well established, the role of their bacterial counterparts is less extensively studied. We show that Chitinophaga pinensis, a prominent member of an important bacterial genus, is capable of using both plant and fungal biomass as a nutrient source but is particularly effective at deconstructing dead fungal material. The turnover of dead fungus is key in natural elemental cycles in the forest. We show that C. pinensis can perform extensive degradation of this material to support its own growth while also releasing sugars that may serve as nutrients for other microbial species. Our work adds detail to an increasingly complex picture of life among the environmental microbiota.
bacteria, Bacteroidetes, biomass recycling, carbohydrate active enzymes, polysaccharide utilization loci, β-glucan polysaccharides
NCBI PubMed ID: 30413479The aim of the study was a comprehensive, comparative study evaluating the impact of the addition level (0.125%, 0.25%, 0.5%, 1%) of highly purified β-glucans isolated from bacteria (curdlan), fungi (scleroglucan) and oats on the stability, rheological and microstructural properties of milk gel during acidification with glucono-δ-lactone. Viscosity and susceptibility to shear thinning were lowest in aqueous solutions containing oat β-glucan (OBG) and highest in solutions containing curdlan. Regardless of their addition level, curdlan and scleroglucan produced pseudoplastic fluid. Coagulation was most rapidly induced by scleroglucan, and it was most delayed under the influence of OBG. The susceptibility of acidified milk gel to phase separation was significantly influenced by the hydration properties and source of β-glucan. Acidification of gels with the lowest concentration (0.125%) of scleroglucan and OBG promoted aggregation of the caseins and then phase separation. This behaviour was not observed in gels containing linear β-glucan (curdlan). The images acquired under a confocal microscope revealed that the all analysed preparations significantly affected the formation of protein complexes whose size and shape were closely linked with the type of added β-glucan. The morphology of samples containing curdlan most closely resembled the structure of the control gel. The addition (up to 0.5%) of scleroglucan and OBG resulted in a gel with low stability. Separate protein structures and clusters of β-glucan were found. The addition of higher levels of β-glucan resulted in a more homogeneous microstructure of the product, which was similar to the control acidified gels.
Curdlan, scleroglucan, oat β-glucan, confocal, phase separation, interactions
Publication DOI: 10.1016/j.foodhyd.2020.106486Natural polysaccharides derived from renewable biomass sources are regarded as environmentally friendly and sustainable polymers. As the third most abundant biomacromolecule in nature, after proteins and nucleic acids, polysaccharides are also closely related with many different life activities. In particular, β-glucans are one of the most widely reported bioactive polysaccharides and are usually considered as biological response modifiers. Among them, β-glucans with triple-helix conformation have been the hottest and most well-researched polysaccharides at present, especially lentinan and schizophyllan, which are clinically used as cancer therapies in some Asian countries. Thus, creation of these active triple-helix polysaccharides is beneficial to the research and development of sustainable "green" biopolymers in the fields of food and life sciences. Therefore, full fundamental research of triple-helix polysaccharides is essential to discover more applications for polysaccharides. In this Review, the recent research progress of chain conformations, bioactivities, and structure-function relationships of triple-helix β-glucans is summarized. The main contents include the characterization methods of the macromolecular conformation, proof of triple helices, bioactivities, and structure-function relationships. We believe that the governments, enterprises, universities, and institutes dealing with the survival and health of human beings can expect the development of natural bioproducts in the future. Hence, a deep understanding of β-glucans with triple-helix chain conformation is necessary for application of natural medicines and biologics for a sustainable world.
conformation, β-Glucans, schizophyllan, lentinan, triple-helix
NCBI PubMed ID: 31986015β-glucans are the dietary nutrients present in oats, barley, algae, and mushrooms. The macromolecules are well known for their immune-modulatory activity; however, how the human gut bacteria digest them is vaguely understood. In this study, Bacteroides uniformis JCM 13288 T was found to grow on laminarin, pustulan, and porphyran. We sequenced the genome of the strain, which was about 5.05 megabase pairs and contained 4868 protein-coding genes. On the basis of growth patterns of the bacterium, two putative polysaccharide utilization loci for β-glucans were identified from the genome, and associated four putative genes were cloned, expressed, purified, and characterized. Three glycoside hydrolases (GHs) that were endo-acting enzymes (BuGH16, BuGH30, and BuGH158), and one which was an exo-acting (BuGH3) enzyme. The BuGH3, BuGH16, and BuGH158 can cleave linear exo/endo-β-1-3 linkages while BuGH30 can digest endo-β-1-6 linkages. BuGH30 and BuGH158 were further explored for their roles in digesting β-glucans and generation of oligosaccharides, respectively. The BuGH30 predominately found to cleave long chain β-1-6-linked glucans, and obtained final product was gentiobiose. The BuGH158 used for producing oligosaccharides varying from degree of polymerization 2 to 7 from soluble curdlan. We demonstrated that these oligosaccharides can be utilized by gut bacteria, which either did not grow or poorly grew on laminarin. Thus, B. uniformis JCM 13288 T is not only capable of utilizing β-glucans but also shares these glycans with human gut bacteria for potentially maintaining the gut microbial homeostasis.
Enzymes, cross-feeding, glycan utilization, gut bacteria, macroalgae
NCBI PubMed ID: 33043794Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg-1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.
UDP-glucose, β-1, 3-glucan, carbon storage, Euglena gracilis, uridylyltranferase
NCBI PubMed ID: 30223004Euglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytEuglena gracilis is a eukaryotic single-celled and photosynthetic organism grouped under the kingdom Protista. This phytoflagellate can accumulate the carbon photoassimilate as a linear β-1,3-glucan chain called paramylon. This storage polysaccharide can undergo degradation to provide glucose units to obtain ATP and reducing power both in aerobic and anaerobic growth conditions. Our group has recently characterized an essential enzyme for accumulating the polysaccharide, the UDP-glucose pyrophosphorylase (Biochimie vol 154, 2018, 176-186), which catalyzes the synthesis of UDP-glucose (the substrate for paramylon synthase). Additionally, the identification of nucleotide sequences coding for putative UDP-sugar pyrophosphorylases suggests the occurrence of an alternative source of UDP-glucose. In this study, we demonstrate the active involvement of both pyrophosphorylases in paramylon accumulation. Using techniques of single and combined knockdown of transcripts coding for these proteins, we evidenced a substantial decrease in the polysaccharide synthesis from 39 ± 7 μg/106 cells determined in the control at day 21st of growth. Thus, the paramylon accumulation in Euglena gracilis cells decreased by 60% and 30% after a single knockdown of the expression of genes coding for UDP-glucose pyrophosphorylase and UDP-sugar pyrophosphorylase, respectively. Besides, the combined knockdown of both genes resulted in a ca. 65% reduction in the level of the storage polysaccharide. Our findings indicate the existence of a physiological dependence between paramylon accumulation and the partitioning of sugar nucleotides into other metabolic routes, including the Leloir pathway's functionality in Euglena gracilis.
sugar nucleotide, Euglena gracilis, knockdown, reserve polysaccharide, RNAi
NCBI PubMed ID: 33675853Euglena gracilis Z is a micro-algae that is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of Euglena gracilis Z has β-1, 3-glucan structure. Euglena gracilis Z and paramylon are reported to affect the immune system. In this study, we investigated the protective effects of Euglena gracilis Z and paramylon against influenza virus infection in mice. Euglena gracilis Z and paramylon were administered to mice as a 2% dietary mixture ad libitum. At 2 weeks after initiation of dietary administration, mice were infected intranasally with influenza virus A/PR/8/34 (H1N1). Survival rate was monitored 10 days after infection. In addition, we performed virus titer and cytokine profiles in the lung. High survival rates were observed for Euglena gracilis Z and paramylon-treated groups compared to the control group. Significantly lower virus titer in the lung was observed in the Euglena gracilis Z and paramylon-treated groups compared to the control group from day 1 after infection. Higher amount of IL-1β, IL-6, IL-12 (p70), IFN-γ, and IL-10 was observed in the paramylon groups compared to the control group. Our data therefore reveals a novel immunoregulatory role of the Euglena gracilis Z and paramylon which provides protection against influenza virus infection.
β-1, 3-D-glucan, Euglena gracilis Z, Influenza virus, Interferon-γ, Interleukin-12, paramylon
NCBI PubMed ID: 28974421β-Glucans are a class of polysaccharides consisting of D-glucose units that are polymerized primarily via the β-1,3 glycosidic bonds, in addition to the β-1,4 and/or β-1,6 bonds. They are present in various food products such as cereals, mushrooms, and seaweeds and are known for their numerous effects on the human body, depending on their structures, which are diverse. The major physicochemical properties of β-glucans include their antioxidant property, which is responsible for the scavenging of reactive oxygen species, and their role as dietary fiber for preventing the absorption of cholesterol, for promoting egestion, and for producing short-chain fatty acids in the intestine. Dietary β-glucans also exert immunostimulatory and antitumor effects by activation of cells of the mucosal immune system via β-glucan receptors, such as dectin-1. In this review, we elaborate upon the diversity of the structures and functions of β-glucans present in food, along with discussing their proposed mechanisms of action. In addition to the traditional β-glucan-containing foods, recent progress in the commercial mass cultivation and supply of an algal species, Euglena gracilis, as a food material is briefly described. Mass production has enabled consumption of paramylon, a Euglena-specific novel β-glucan source. The biological effects of paramylon are discussed and compared with those of other β-glucans.
β-glucan, Euglena gracilis, paramylon, food functionality, food material
NCBI PubMed ID: 29491277Progression to non-alcoholic steatohepatitis (NASH) manifests as hepatitis, fibrosis, and sometimes carcinoma, resulting in liver failure. Various clinical trials have indicated that several pharmacological agents, including angiotensin II receptor blockers (ARBs) or farnesoid X receptor (FXR) agonists, are effective in NASH treatment. In addition, functional foods are expected to be important alternatives for treating or preventing NASH. Recently, focus has been directed toward microalgae as dietary supplements, mainly for lifestyle-related diseases, because they contain various nutrients and functional ingredients. Specifically, a unicellular microalga Euglena gracilis stores a unique β-1,3-glucan particle called paramylon that stimulates the immune system. In this study, we evaluated the effects of Euglena and paramylon on NASH in Stelic Animal Model (STAM) mice using Sirius red staining and confirmed that oral administration of Euglena or paramylon inhibits the process of liver fibrosis. Moreover, compared with controls, paramylon decreased non-alcoholic fatty liver disease (NAFLD) activity scores related to inflammation. These results indicate that the oral administration of Euglena and paramylon inhibits fibrosis and ameliorates NASH.
Fibrosis, paramylon, Euglena, microalgae, NASH
NCBI PubMed ID: 30680167Mixed paramylon esters prepared from paramylon (a storage polysaccharide of Euglena), acetic acid, and a long-chain fatty acid by one-pot synthesis using trifluoroacetic anhydride as a promoter and solvent were shown to have thermoplasticity. Size exclusion chromatography indicated that the mixed paramylon esters had a weight average molecular weight of approximately 4.9-6.7×10(5). Thermal analysis showed that these esters were stable in terms of the glass transition temperature (>90°C) and 5% weight loss temperature (>320°C). The degree of substitution of the long alkyl chain group, a dominant factor determining thermoplasticity, was controlled by tuning the feed molar ratio of acetic acid and long-chain fatty acid to paramylon. These results implied that the one-pot synthesis is useful for preparing structurally-well defined thermoplastic mixed paramylon esters with high molecular weight.
esterification, paramylon, Euglena, One-pot synthesis, thermoplastic
NCBI PubMed ID: 25563938Acylation of paramylon, a storage polysaccharide of Euglena gracilis, using multiple acid anhydrides yielded thermoplastic paramylon mixed esters without significant depolymerization. DSC examination showed that the shorter the acyl chain, the higher both the melting and glass transition temperature of the ester. TG analyses revealed their higher thermostability with the 5% weight loss temperature of ∼330°C. Melt volume flow rate examination revealed that the longer the acyl chain, the higher the thermoplasticity of the ester and that the esters exhibited higher thermoplasticity than structurally analogous esters made from cellulose and curdlan. A notable feature of the thermoplastic paramylon mixed esters is the availability of hot-pressing as a means of molding them into a film. Light transmittance and XRD measurements revealed that these films were transparent and in the amorphous state. Tensile tests indicated that the films had adequate mechanical strength comparable to those of the cellulose and curdlan analogues.
acylation, β-1, 3-glucan, paramylon, Euglena, thermoplastic
NCBI PubMed ID: 27702530Paramylon is a β-1,3-D-glucan isolated from Euglena gracilis Z. This study was designed to evaluate the suppressive effects of the oral administration of paramylon on the development of atopic dermatitis (AD)-like skin lesions induced by repeated application of 2,4,6-trinitrochlorobenzene (TNCB) in sensitized NC/Nga mice. The effects of paramylon were assessed by measuring macroscopical and histopathological findings of skin, ear swelling, serum levels of total IgE, interleukin-4 (IL-4) and interferon-γ (IFN-γ) and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon inhibited the development of AD-like skin lesions as exemplified by a significant decrease in dermatitis scores for the back, ear swelling and hypertrophy of the skin, infiltration of inflammatory cells in the skin, and serum IgE levels. Oral administration of paramylon reduced serum levels of both IL-4 and IFN-γ and IL-18 and IL-12 contents in the skin lesions. Oral administration of paramylon did not cause weight loss, as was observed with prednisolone. These results suggest that paramylon inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing both the T-helper (Th) 1 and Th 2 cell responses. Our results indicate that paramylon treatment could provide an effective alternative therapy for the management of AD.
β-1, 3-D-glucan, Euglena gracilis Z, Interferon-γ, paramylon, atopic dermatitis, interleukin-4
NCBI PubMed ID: 20160419Euglena gracilis Z is a microorganism classified as a microalga and is used as a food or nutritional supplement. Paramylon, the carbohydrate storage substance of E. gracilis Z, is reported to affect the immunological system. This study evaluated the symptom-relieving effects of E. gracilis Z and paramylon in rheumatoid arthritis in a collagen-induced arthritis mouse model. The efficacy of both substances was assessed based on clinical arthritis signs, as well as cytokine (interleukin [IL]-17, IL-6, and interferon [IFN]-γ) levels in lymphoid tissues. Additionally, the knee joints were harvested and histopathologically examined. The results showed that both substances reduced the transitional changes in the visual assessment score of arthritis symptoms compared with those in the control group, indicating their symptom-relieving effects on rheumatoid arthritis. Furthermore, E. gracilis Z and paramylon significantly reduced the secretion of the cytokines, IL-17, IL-6, and IFN-γ. The histopathological examination of the control group revealed edema, inflammation, cell hyperplasia, granulation tissue formation, fibrosis, and exudate in the synovial membrane, as well as pannus formation and articular cartilage destruction in the femoral trochlear groove. These changes were suppressed in both treatment groups. Particularly, the E. gracilis Z group showed no edema, inflammation, and fibrosis of the synovial membrane, or pannus formation and destruction of articular cartilage in the femoral trochlear groove. Furthermore, E. gracilis Z and paramylon exhibited symptom-relieving effects on rheumatoid arthritis and suppressed the secretion of cytokines IL-17, IL-6, and IFN-γ. These effects were likely mediated by the regulatory activities of E. gracilis Z and paramylon on Th17 immunity. In addition, the symptom-relieving effects of both substances were comparable, which suggests that paramylon is the active component of Euglena gracilis Z.
Euglena gracilis Z, paramylon, rheumatoid arthritis
NCBI PubMed ID: 29389956Background/aim: β-Glucan has been shown to modulate immune system and potentially aid wound healing. A naturally-available β-glucan, paramylon, is available in the form of a film, which would be an ideal form to use in wound care. The aim of this study was to examine the therapeutic efficacy of paramylon film as a wound dressing. Materials and methods: An acute wound was created on the skin of the posterior aspect of mice and wound healing was observed for 5 days. Mice were treated with either paramylon film or conventional cellulose film. Results: The time course of changes in wound size revealed that paramylon film dressing application leads to significantly faster wound contraction than conventional cellulose film. The dressing suppressed elevation of the inflammatory cytokines interferon gamma, interleukin-6, and vascular endothelial growth factor. Conclusion: β-Glucan paramylon film can facilitate wound healing by inhibiting inflammatory aggression and has potential application as a novel wound dressing.
cytokine, β-glucan, paramylon, wound dressing
NCBI PubMed ID: 29936461The intestinal tract contains over half of all immune cells and peripheral nerves and manages the beneficial interactions between food compounds and the host. Paramylon is a β-1,3-glucan storage polysaccharide from Euglena gracilis (Euglena) that exerts immunostimulatory activities by affecting cytokine production. This study investigated the signaling mechanisms that regulate the beneficial interactions between food compounds and the intestinal tract using cell type-specific calcium (Ca2+) imaging in vivo and in vitro. We successfully visualized Euglena- and paramylon-mediated Ca2+ signaling in vivo in intestinal epithelial cells from mice ubiquitously expressing the Yellow Cameleon 3.60 (YC3.60) Ca2+ biosensor. Moreover, in vivo Ca2+ imaging demonstrated that the intraperitoneal injection of both Euglena and paramylon stimulated dendritic cells (DCs) in Peyer's patches, indicating that paramylon is an active component of Euglena that affects the immune system. In addition, in vitro Ca2+ imaging in dorsal root ganglia indicated that Euglena, but not paramylon, triggers Ca2+ signaling in the sensory nervous system innervating the intestine. Thus, this study is the first to successfully visualize the direct effect of β-1,3-glucan on DCs in vivo and will help elucidate the mechanisms via which Euglena and paramylon exert various effects in the intestinal tract.
immune system, β-1, 3-glucan, Euglena gracilis, Ca2+ signaling, intestinal epithelial cell, intravital imaging, small intestine
NCBI PubMed ID: 32751743A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBPMLG-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBPMLG-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBPMLG-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBPMLG-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBPMLG-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBPMLG-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.
X-ray crystallography, β-glucan, Bacteroides ovatus, carbohydrate microarrays, polysaccharide utilization loci, protein-carbohydrate interactions, SusD-like proteins
NCBI PubMed ID: 34817219In this study, curdlan sulphate - chitosan nanoparticles were prepared through polyelectrolyte complexing at a mass ratio of 2:1 respectively. The curdlan was produced by fermentation with Agrobacterium sp. ATCC 31750, which was then sulphated to form the polyanionic polymer. A first-line tuberculosis drug, Rifampicin and a phytochemical, DdPinitol, were encapsulated into Curdlan Sulphate (CS) - Chitosan Nanoparticles (C) (CSC NPs) of size 205.41 ± 7.24 nm. The drug release kinetics followed a Weibull model with initial burst release (48 % Rifampicin and 27 % d-Pinitol within 6 h), followed by a sustained release. The prepared CSC: d-PIN + RIF NPs was cytocompatible and entered the M.smegmatis infected macrophages through multiple endocytic pathways including clathrin, caveolae and macropinocytosis. They showed superior bactericidal activity (2.4-2.7 fold) within 4 h when compared to free drug Rifampicin (1.6 fold). The drug encapsulated CSC: RIF suppressed the pro-inflammatory gene (TNF-α by 3.66 ± 0.19 fold) and CSC: d-PIN + RIF increased expression of the anti-inflammatory gene (IL-10 by 13.09 ± 0.47 fold). Expression of TGF- β1 gene also increased when treated with CSC: d-PIN + RIF (13.00 ± 0.19 fold) which provided the immunomodulatory activity of the encapsulated CSC NPs. Thus, curdlan sulphate - chitosan polyelectrolyte complex can be a potential nanocarrier matrix for intracellular delivery of multiple drugs.
gene expression, Agrobacterium, macrophages, chitosan, cellular uptake, curdlan sulphate, endocytotic inhibitors, polyelectrolyte complex
NCBI PubMed ID: 33593559In recent decades, the composition, structure, biosynthesis, and function of bacterial extracellular polysaccharides (EPS) have been extensively studied. EPS are synthesized through different biosynthetic pathways. The genes responsible for EPS synthesis are usually clustered on the genome or large plasmids of bacteria. Generally, different EPS synthesis gene clusters direct the synthesis of EPS with different chemical structures and biological activities. A better understanding of the gene functions involved in EPS biosynthesis is critical for the production of EPS with special biological activities. Genetic engineering methods are usually used to study EPS synthesis related genes. This review organizes the available information on EPS, including their structures, synthesis of related genes, and highlights the research progress of modifying EPS gene clusters through gene-editing methods.
genetic engineering, gene clusters, bacterial extracellular polysaccharides
Publication DOI: 10.1016/j.ijbiomac.2021.01.139Euglena, a flagellated unicellular protist, has recently received widespread attention for various high-value metabolites, especially paramylon, which was only found in Euglenophyta. The limited species and low biomass of Euglena has impeded paramylon exploitation and utilization. This study established an optimal cultivation method of Euglena pisciformis AEW501 for paramylon production under mixotrophic cultivation. The results showed that the optimum mixotrophic conditions were 20 °C, pH 7.0, and 63 μmol photons m-2∙s-1, and the concentrations of sodium acetate and diammonium hydrogen phosphate were 0.98 g L-1 and 0.79 g L-1, respectively. The maximal biomass and paramylon content were 0.72 g L-1 and 71.39% of dry weight. The algal powder contained more than 16 amino acids, 6 vitamins, and 10 unsaturated fatty acids under the optimal cultivation. E. pisciformis paramylon was pure β-1,3-glucan-type polysaccharide (the purity was up to 99.13 ± 0.61%) composed of linear glucose chains linked together by β-1,3-glycosidic bonds. These findings present a valuable basis for the industrial exploitation of paramylon with E. pisciformis AEW501.
characterization, paramylon, Euglena pisciformis AEW501, mixotrophic cultivation, nutritional components analysis
NCBI PubMed ID: 36005522Paramylon is a linear β-1,3-glucan, similar to curdlan, produced as intracellular granules by the microalga Euglena gracilis, a highly versatile and robust strain, able to grow under various trophic conditions, with valorization of CO2, wastewaters, or food byproducts as nutrients. This review focuses in particular on the various processing routes leading to new potential paramylon based products. Due to its crystalline structure, involving triple helices stabilized by internal intermolecular hydrogen bonds, paramylon is neither water-soluble nor thermoplastic. The few solvents able to disrupt the triple helices, and to fully solubilize the polymer as random coils, allow non derivatizing shaping into films, fibers, and even nanofibers by a specific self-assembly mechanism. Chemical modification in homogeneous or heterogeneous conditions is also possible. The non-selective or regioselective substitution of the hydroxyl groups of glucosidic units leads to water-soluble ionic derivatives and thermoplastic paramylon esters with foreseen applications ranging from health to bioplastics.
chemical modification, β-1, 3-glucan, paramylon, crystallinity, processing, solvents
NCBI PubMed ID: 35450615Paramylon from Euglena gracilis (EGP) is a polymeric polysaccharide composed of linear β-1,3 glucan. EGP has been proved to have antibacterial activity, but its effect is weak due to its water insolubility and high crystallinity. In order to change this deficiency, this experiment carried out carboxymethylated modification of EGP. Three carboxymethylated derivatives, C-EGP1, C-EGP2, and C-EGP3, with a degree of substitution (DS) of 0.14, 0.55, and 0.78, respectively, were synthesized by varying reaction conditions, such as the mass of chloroacetic acid and temperature. Fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), and nuclear magnetic resonance (NMR) analysis confirmed the success of the carboxymethylated modification. The Congo red (CR) experiment, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetry (TG) were used to study the conformation, surface morphology, crystalline nature, and thermostability of the carboxymethylated EGP. The results showed that carboxymethylation did not change the triple helix structure of the EGP, but that the fundamental particles' surface morphology was destroyed, and the crystallization area and thermal stability decreased obviously. In addition, the water solubility test and antibacterial experiment showed that the water solubility and antibacterial activity of the EGP after carboxymethylation were obviously improved, and that the water solubility of C-EGP1, C-EGP2, and C-EGP3 increased by 53.31%, 75.52%, and 80.96% respectively. The antibacterial test indicated that C-EGP3 had the best effect on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), with minimum inhibitory concentration (MIC) values of 12.50 mg/mL and 6.25 mg/mL. The diameters of the inhibition zone of C-EGP3 on E. coli and S. aureus were 11.24 ± 0.15 mm and 12.05 ± 0.09 mm, and the antibacterial rate increased by 41.33% and 43.67%.
antibacterial activity, paramylon, carboxymethylated modification, water solubility
NCBI PubMed ID: 35893986Microbial exopolysaccharides (EPSs) are valuable extracellular macromolecules secreted as capsules or slime layers. Various microorganisms, including bacteria, yeasts, fungi, and algae have been studied for their ability to produce EPSs. Microbial EPSs exist as homopolysaccharides or heteropolysaccharides with various properties such as different monosaccharide compositions, structural conformation, molecular weight, and functional groups. They are cost-effective alternatives to plant and animal-derived polysaccharides because the microbial cells produced them in large quantities by biotechnological processes using low-cost substrates such as industrial wastes in a short time. Microbial EPSs are safe, biodegradable, and compatible polymers. They have extensive bioactivities, including antibacterial, antifungal, antiviral, antioxidant, antitumor, antidiabetic, antiulcer, anticoagulant, antiaging, immunomodulatory, wound healing, and cholesterol-lowering activities. Microbial EPSs owing to biological activities, special biochemical structures, and attractive physicochemical properties find plenty of potential applications in various industries. The enhancement of the production of EPSs and improving their properties can be provided by genetic engineering methods. The current review aims to provide a comprehensive examination of the therapeutic activities of microbial EPSs in infectious diseases and metabolic disorders, with a focus on the mechanisms involved. Also, the effect of the physicochemical characteristics of EPSs on these bioactivities was discussed to reveal the structure-activity relationship.
bacteria, exopolysaccharides, physicochemical properties, bioactivities
NCBI PubMed ID: 37256348Natural polysaccharides have an important role in pharmaceutical industries and are considered a huge source and importance. Polysaccharides are biopolymers characterized by complex secondary structures performing several roles in plants, animals, and different other microorganisms. As it has versatility such as hydrophilicity, good stability, safety, lack of toxicity, and biodegradability in nature, some of them are extensively used for food packaging, pharmaceutical formulations, and various kind of sustainable and renewable goods in biomedical industries. In this context, we focused on natural polysaccharides from different sources such as plants, animals, algae, and microbes. Here, we concentrate on the chemical structures along with their commercial importance in the pharmaceutical industry. Moreover, a summary of the formulations and their applications in different natural polysaccharides has been highlighted in this review. Therefore, this study might be helpful for the development of not only different pharmaceutical formulations but also advantageous in food and biomedical industries in the future.
microbial polysaccharides, algal polysaccharides, bioactive polymer, natural polysaccharides, pharmaceutical formulation, plant polysaccharides
Publication DOI: 10.1016/j.eurpolymj.2022.111801Paramylon is a storage polysaccharide produced by Euglena glacilis. Paramylon nanofibers form spontaneously through the self-assembly of single-strand paramylon. However, paramylon nanofibers are well-dispersed only in strongly alkaline solutions. Therefore, a method of fabricating a novel fiber network using paramylon was developed, without requiring the use of additives to prevent aggregation. Paramylon was dissolved in dimethyl sulfoxide at a high concentration, and this thick, viscous solution was added dropwise to ethanol. The resulting spheres (paramylon beads) were dispersed in water and homogenized. The aqueous dispersion contained a web-like structure comprising submicron fibers with widths of ~500 nm (paramylon web). Gradual removal of water from the aqueous dispersions produced centimeter-sized translucent paper-like films (paramylon papers). The translucency suggested that the submicron fibers did not excessively aggregate but still maintained small interstices within the paper owing to the rigid web-like structure.
paramylon, Euglena, submicron fiber, fiber network
Publication DOI: 10.1016/j.polymer.2023.125984Intraperitoneally or intravenously administered (1→3)-β-D-glucan remained in the liver and spleen, for a long time without major structural changes, but the priming activity to lipopolysaccharide (LPS)-triggered tumor necrosis factor alpha (TNF-α) production was reduced more quickly. The relationship between the deposited glucan contents and the antitumor activity was examined by comparing kinetics of the activity using solid form Sarcoma 180 tumor in ICR mice. We used three kinds of soluble glucans, sonifilan (SPG), grifolan (GRN), and SSG, and a particulate glucan, zymosan (ZYM). These were administered 5 weeks before (-5W) the tumor inoculation and the tumor weight was compared 5 weeks after the inoculation. Compared with the activity of those administered at the optimum timing, all of the glucans reduced the activity about 5 fold, although significant activity still remained, especially in the case of SPG. Five weeks after intraperitoneal (SPG, GRN, SSG) or intravenous (ZYM) administration of the glucans, all were found in the liver and spleen in significant quantities. These facts strongly suggested that the activity of the glucan was reduced not only because of chemical/physical degradation but also a certain physiological inactivation mechanism.
NCBI PubMed ID: 8787813Natural product complex carbohydrate polymers are currently being developed as prophylactic and/or therapeutic drugs. These water-soluble carbohydrate pharmaceuticals, which are primarily β-1, 3-D-glucan polymers, belong to the class of drugs known as biological response modifiers (BRMs). A major obstacle to the development, understanding and clinical utilization of carbohydrate BRMs has been the difficulty involved in accurately characterizing high molecular weight (MW) carbohydrate polymers. Recent advances in aqueous gel permeation chromatography (GPC), differential viscometry (DV) and multi- significance of these observations are readily apparent. As stated above, the characterization and quality control of natural product carbohydrate BRMs has, in the past, relied heavily on bioassays and conventional GPC. The data presented demonstrate the applicability of high performance GPC with multiple detectors in the analysis of complex carbohydrate BRMs. This technology when combined with other analytical techniques such as high-field 13C and 1H-NMR, elemental analysis, helix coil transition analysis and exhaustive methylation/gas chromatography/mass spectrometry (12) provide a much more complete description of this novel class of pharmaceuticals.
Publication DOI: 10.1080/10826079208016179The cell wall-less fz, sg, os-1 ("slime") triple mutant of Neurospora crassa lacks (1,3)-β-D-glucan synthase activity. fz, sg, os-1 segregants from slime x wild-type crosses initially germinate as a plasmodium (slime-like), but develop hyphae in a few hours and acquire a stable mycelial phenotype (mycelial intermediate). The cell wall-less phenotype (stable slime) can be reisolated from mycelial intermediates by filtration-enrichment selection in medium of high osmolarity. Pairs of mycelial intermediate and stable slime obtained from a single slime-like segregant were comparatively studied. Mycelial intermediate strains synthesize a cell wall with normal amounts of (1,3)-β-glucan, chitin, and other polysaccharides and possess (1,3)-β-glucan synthase activity with apparently normal properties (i.e., association with membranes, stability, Km app, Vmax, stimulation by GTP). The enzyme was dissociated by treatment with Tergitol NP-40 and NaCl into a membrane-bound catalytic center and a soluble factor which activates the enzyme in the presence of GTP. Heterologous reconstitution assays demonstrated that stable slime spheroplasts had normal activity of the soluble activating factor, but were severely deficient in membrane-bound activity. The genetic composition of the viable progeny of stable slime or mycelial intermediate x wild-type crosses failed to show differences between the two extreme phenotypes of slime. However, the analysis of heterokaryons demonstrated that the stable slime homokaryotic progeny of stable slime/wild-type heterokaryons were not viable. In contrast, the behavior of mycelial intermediate/wild-type heterokaryons was normal. Apparently, stable slime strains differed from the original mycelial intermediate in a mutation(s) which arose spontaneously during the filtration-enrichment selection applied to mycelial intermediates in order to obtain the cell wall-less phenotype. This new trait impaired conidial germination and might be the actual cause of the loss of (1,3)-β-glucan synthase activity and cell wall.
NCBI PubMed ID: 7614368A protease zymogen present in the plasma fraction of the hemolymph of silkworm, Bombyx mori, was purified to homogeneity as judged by SDS/PAGE and IEF/PAGE. An activating system for the zymogen was also isolated from the plasma fraction and was shown to be triggered by zymosan (yeast cell wall polysaccharide containing β-1,3-glucan) or peptidoglycan. Using this system, the purified zymogen was activated and the active enzyme was purified to homogeneity. The physiological function of the zymogen or its active form is not yet known, but the active form was shown to have narrower substrate specificity than trypsin. Among 33 peptide derivatives examined, Boc-Gln-Arg-Arg-NH-Mec and Boc-Val-Pro-Arg-NH-Mec (Boc = tert-butoxycarbonyl, NH-Mec = 4-methylcoumaryl-7-amide) were the best and the second best substrates, respectively. The purified zymogen was determined to be a 39-kDa protein consisting of a single polypeptide. The active form of the zymogen was labeled with [3H]diisopropylfluorophosphate and was completely inactivated by (p-amidinophenyl)methanesulfonyl fluoride. The molecular mass of the [3H]-labeled enzyme was determined to be 38 kDa in SDS/PAGE under reducing conditions. These results indicate that the 39-kDa protein purified in the present study is a zymogen of a serine-type protease and that the activation of the zymogen occurs by limited proteolysis.
NCBI PubMed ID: 7737188Crude membrane preparations of fungi contain the enzyme glucan synthase (EC 2.4.1.34) which produces a polymer of glucose linked through 1,3-β-glycosidic bonds. This polymer is a major structural element of the fungal cell wall. Preparations of glucan synthase are contaminated with the enzyme glycogen synthase (EC 2.4.1.11). Glycogen synthase forms the storage carbohydrate glycogen, a polymer of glucose consisting of mainly 1,4-α-glycosidic linkage. Both enzymes utilize uridine diphosphoglucose as substrate. Discrimination of glucan synthase from glycogen synthase activity has relied upon the inclusion of glycogen-degrading enzymes in the crude reactions. The polysaccharide reaction products of glucan synthase assays have been characterized by their susceptibility to enzymatic degradation by various glucanohydrolases. These degradative enzymes are impure and inclusion of appropriate control polysaccharides often leads to ambiguous results. A method for comparative qualitative analysis of polysaccharides formed in fungal glucan synthase reactions has been developed using high-performance anion-exchange chromatography. Using this method, polymers of glucose with 1,3-β-glycosidic linkage and 1,4-α linkage can be readily distinguished. This method has been applied to map oligosaccharides derived by partial acid hydrolysis from fungal glucan synthase reaction products from Candida albicans protoplasts prepared by two different methods.
NCBI PubMed ID: 8297006The relationship between the actin cytoskeleton and cell wall synthesis was studied by light and electron microscopy in protoplasts of Saccharomyces cerevisiae DBY 1693 containing the act1-1 allele. Since protoplasting also disturbs the actin cytoskeleton, these mutant protoplasts had a double error in their actin cytoskeletons. In the period between the onset of wall synthesis and completion of the wall, protoplasts grown at the permissive temperature showed an even distribution of actin patches all over the surface on which a new cell wall was being synthesized. After wall completion, actin patches partially disappeared, but then re-appeared, accumulated in growth regions at the start of polarized growth. This was compared with the pattern of actin patches observed in intact temperature-sensitive actin mutant cells cultivated at the permissive temperature. Electron microscopy of freeze-etched replicas revealed finger-like invaginations of the plasma membrane in both the actin mutant cells and their protoplasts. These structures showed a very similar distribution to the actin patches detected by rhodamine phalloidin staining in the fluorescence microscope. A hypothesis is presented, explaining the role of actin patches/finger-like invaginations of the plasma membrane in the synthesis of β-(1→3)-D-glucan wall microfibrils in yeast cells.
NCBI PubMed ID: 7670632Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1----3)-β-D-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1----3)-β-D-glucan elementary fibrils depicted by electron microscopy. The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.
NCBI PubMed ID: 1417414A yeast cell wall glycoprotein with a molecular weight of 40,000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene. The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI. This gene encoded a serine-rich protein of 239 amino acids with a molecular weight of 24,267. The presence of hydrophobic sequences in the N- and C-termini of the CWP1 protein suggests that it is secreted as a glycosylphosphatidylinositol-anchored protein and is subsequently integrated into the cell wall. Since a gene disruption experiment showed no growth defect, the CWP1 gene is not essential for growth. Mutant CWP1 protein deficient in the C-terminal hydrophobic sequence was secreted into the culture medium, not anchored to the cell wall, thereby indicating that this hydrophobic sequence plays a crucial role in anchoring to the cell wall. Homology between the CWP1 protein and TIP1 family of cold shock proteins suggests that they belong to a new family of cell wall proteins.
NCBI PubMed ID: 8543563The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1→3)-glucanase and endo-β-(1→6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1→3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1→3)- or endo-β-(1→6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1→3)-glucanase it can be concluded that this fibrillar component is composed of β-(1→3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.
NCBI PubMed ID: 4135002An indirect method to measure β-glucan, a major structural component of yeast cell walls, is available, but has the disadvantage of requiring the combined use of two assays. Recent reports describe the fungal index, which measures the difference between the conventional limulus test, in which factors C and G react with endotoxin and β-glucan, and a new endotoxin-specific test, in which only factor C reacts with endotoxin. The G test was developed as a direct method to measure β-glucan, and contains only factor G reacting with β-glucan alone. In this study, the G test was examined in sera of rabbits with experimental systemic candidiasis, and compared with the fungal index and mannan assay. The G test showed positive in all rabbits with systemic candidiasis faster and with higher titers than with the fungal index. Three rabbits with fulminant systemic candidiasis showed higher levels of reactivity with the G test and the fungal index than two rabbits with mild reactions. Mannan was positive by at least one serum in four of five rabbits by the latex agglutination test, and there was a good correlation between these assays. The G test is a good serodiagnostic method for the detection of candidiasis.
NCBI PubMed ID: 1403352During the past few years, cyto- and immunocytochemical techniques have been developed and widely used for locating and identifying various molecules in plant cell compartments. The last decade has witnessed tremendous improvements in molecular cytology, thus allowing an accurate in situ detection of various components thought to play important biological functions in the plant metabolism. The use of immunocytochemistry to investigate resistance mechanisms of plants upon pathogen attack has provided key information on the defense strategy that plants elaborate during a host-pathogen interaction. Of the various proteins induced in response to infection, chitinases and β-1,3-glucanases have been the focus of particular attention due to their believed antimicrobial activity through the hydrolysis of the main fungal wall components, chitin and β-1,3-glucans. Attention has also been paid to β-fructosidase, the enzyme that hydrolyzes sucrose into glucose and fructoside. The marked accumulation of this enzyme upon pathogen infection has led to the consideration that infection may greatly influence the metabolic activity of colonized tissues by creating alterations of source-sink relationships. Another facet of the plant's defense strategy that has been the focus of considerable interest is related to the accumulation of structural compounds, such as hydroxyproline-rich glycoproteins and callose, to reinforce the wall architecture, thus decreasing vulnerability to microbial enzymes. A number of alternatives designed to improve plant protection towards pathogen invasion have been suggested. Among these, the production of transgenic plants expressing constitutively a foreign resistance gene and the pretreatment of plants with elicitors of defense reactions have been the subject of intensive studies at the molecular, biochemical, and cytological levels. Results of such studies clearly demonstrate the important contribution that cyto- and immunocytochemical approaches can make to our knowledge of how plants defend themselves and how plant disease resistance can be directly enhanced. These approaches will undoubtedly be active areas for future research in the development of biological control alternatives in which the mode of action of the product used is of key importance.
NCBI PubMed ID: 7626800Glucans of fungal origin have been shown to inhibit the early stages of infection of Nicotiana by numerous viruses of different taxonomic groups. Several glucans were isolated from the cell walls of Phytophthora parasitica, Phytophthora megasperma f. sp. glycinea (Pmg) and Fusarium oxysporum, and their antiviral activity compared on tobacco leaves inoculated with tobacco mosaic virus. These polysaccharides consist of a mixture of (1→3)(1→6)-β-D-glucans with M(r) varying from 1.1 x 10(3) to 2 x 10(6). Requirements for a prominent antiviral activity of the fungal polysaccharides are a β-(1→3)(1→6)-D-glucan structure with mono-, di-, tri- or tetra-glucosidic side branches attached to a linear main chain of β-(1→3)-linked-D-glucose residues. Very high activity is correlated with a high degree of branching at position 6 and with the size and glycosidic nature of the side chains. The molecular masses and the organized structure of fungal β-D-glucans are not essential for their antiviral activity. The structural motif for antiviral activity in Nicotiana is distinct from that required for elicitation of phytoalexins in soybean cotyledons.
NCBI PubMed ID: 7786491The cell-wall components of the interface compartment in functioning mycorrhizal roots of maize (Zea mays L. cv. W64A) have been investigated with the use of immunocytochemistry and enzyme/lectin-gold techniques. The distribution of specific cell-wall probes was determined in the apical and differentiated regions of maize roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. Labelling experiments showed that a maize hydroxyproline-rich glycoprotein (HRGP), identified with a specific antibody, was particularly abundant in the apical dividing cells of the root meristem. Cellulose, located with a cellobiohydrolase-gold complex, showed a similar labelling pattern in the walls of both meristematic and differentiated parts of the roots. When the cortex was colonized by the mycorrhizal fungus, the HRGP and cellulose were expressed in two sites: the wall and the interface area created by invagination of the host membrane around the developing fungus. In contrast, in uninfected roots of the same age, they were only present in the inner part of the wall. A specific antibody against β-1,3-glucans demonstrated that these glucans were not laid down at the interface between the plant and fungus, while they appeared to be a skeletal component of the fungal wall, together with chitin.
cell wall, cellulose, β-1, arbuscular mycorrhizae, 3-glucans, hydroxyproline-rich glycoprotein, zea root meristem
Publication DOI: 10.1007/BF00199680The glucan synthase 1 gene (gs-1) is required for (1,3) β-glucan synthase activity [E.C. 2.4.1.34; UDP glucose:1,3-β-D-glucan 3-β-D-glucosyltransferase] and for cell wall formation. The gs-1 gene was cloned by functional complementation of the cell-wall-less defect of the (1,3) β-glucan synthase-deficient mutant, TM1, by using a genomic Neurospora crassa cosmid library. A 2568-nucleotide gs-1 cDNA sequence revealed a 532-amino acid open reading frame encoding a polypeptide of 59 kDa. The predicted gs-1 gene product has no obvious signal peptide cleavage sites or transmembrane domains. A gs-1 null mutant is defective for cell wall formation and (1,3) β-glucan synthase activity. The predicted GS-1 protein is weakly homologous to a putative Saccharomyces cerevisiae transcriptional regulatory protein.
NCBI PubMed ID: 7937796Pulse-chase experiments with [14C]glucose demonstrated that in the cell wall of wild-type Saccharomyces cerevisiae alkali-soluble (1-3)-β-glucan serves as a precursor for alkali-insoluble (1-3)-β-glucan. The following observations support the notion that the insolubilization of the glucan is caused by linkage to chitin: (i) degradation of chitin by chitinase completely dissolved the glucan, and (ii) disruption of the gene for chitin synthase 3 prevented the formation of alkali-insoluble glucan. These cells, unable to form a glucan-chitin complex, were highly vulnerable to hypo-osmotic shock indicating that the linkage of the two polymers significantly contributes to the mechanical strength of the cell wall. Conversion of alkali-soluble glucan into alkali-insoluble glucan occurred both early and late during budding and also in the ts-mutant cdc24-1 in the absence of bud formation.
NCBI PubMed ID: 7725794Glycosylphosphatidylinositol (GPI) membrane anchors are essential for the integration of yeast cell adhesion proteins into the cell wall, but mature cell-wall proteins are unlikely to be attached directly to the membrane. We thus propose that GPI-anchored glycoprotein forms are intermediates in a process that crosslinks the major components of the cell wall by transglycosylation. This mechanism may be critical for both the biosynthesis and overall architecture of the cell wall.
NCBI PubMed ID: 14731865We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1→3)-β-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a 'knr4 disrupted' tetrad demonstrated that mutant cell walls contained much less (1→3)-β-glucan and (1→6)-β-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.
NCBI PubMed ID: 7992508strain, structural, Pseudomonas, cell, determination, structural determination, cell wall, exopolysaccharide, Pseudomonas syringae, mannan, immunological activity, fungi, Cryphonectria parasitica
Journal NLM ID: 0043535A wovenable skin substitute (Sacchachitin) made from the residue of the fruiting body of Ganoderma tsugae was developed in this study. Chemical analysis revealed that the treated residue was a copolymer of β-1,3-glucan (ca 60%) and N-acetylglucosamine (ca 40%) with a filamental structure of mycelia form, as demonstrated by both optical and scanning electron microscopy. The pulp-like white residue was then woven into thin, porous sheets 7.0 cm in diameter and 0.1-0.2 mm in thickness by filtration and lyophilized for use as a skin substitute. The wound area produced by dissecting rat skin of full thickness was found to almost completely heal on the side covered with Sacchachitin, whereas the control side covered with cotton gauge was around 6.0 cm2 on the 28th day. Furthermore, the wound healing effects of the chitin sheet from crab shell (Beschitin) and Sacchachitin were not found to be significantly different.
3-glucan, wound healing, Ganoderma tsugae, fungal mycelia, skin substitute, poly(N-acetylglucosamine), b-1
NCBI PubMed ID: 9259514The BGL2 gene encodes a unique 1,3-β-glucosyltransferase (Bgl2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in Saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of Bgl2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked Bgl2p, as assessed by protein extraction, SDS-PACE and Western blot analysis, and enzyme assay; however, residual non-Bgl2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 degrees C; (2) an 8.7 +/- 1.6% slower growth rate at 37 degrees C for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-β- or 1,6-β-glucan. These data reveal that loss of Bgl2p does have a phenotype in C. albicans, and indicate that (1) loss of Bgl2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in walt structure), and (2) that additional 1,3-β-glucosyltransferases are present in the C. albicans BGL2 disruptant.
Candida albicans, BGL2, transglycosylation
NCBI PubMed ID: 9043114The polysaccharide β(1,3)-D-glucan is a component of the cell wall of many fungi. Synthesis of the linear polymer is catalysed by UDP-glucose β(1,3)-D-glucan β(3)-D-glucosyltransferase. Because this enzyme has a key role in fungal cell-wall synthesis, and because many organisms that are responsible for human mycoses, including Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, produce walls that are rich in β(1,3)-glucan, it has been and remains the focus of intensive study. From early characterization of the enzymatic activity in Saccharomyces cerevisiae, advances have been made in purification of the enzyme, identification of essential subunits and description of regulatory circuitry that controls expression and localization of different components of the multisubunit enzyme complex. Progress in each of these areas has been enhanced dramatically by the availability of specific inhibitors of the enzymatic reaction that produces β(1,3)-glucan. These natural product inhibitors have utility both as tools to dissect the biology of β(1,3)-glucan synthase and as sources for development of semisynthetic derivatives with clinical utility in treatment of human fungal disease. This review will focus on the biochemistry, genetics and regulation of the enzyme.
β(1, 3)-glucan, FKS genes, echinocandin
Publication DOI: 10.1080/mmy.39.1.55.66Mycelia of a wild strain Poria cocos were cultured in two media differing in one constituent: bran extract or corn steep liquor, and are designated as wb and wc, respectively. Six polysaccharide fractions were isolated sequentially from the two mycelia by 0.9% NaCl (PCM1), hot water (PCM2), 0.5 M NaOH (PCM3-I and -II) and 88% formic acid (PCM4-I and -II). Their chemical and physical characteristics were determined by infrared spectroscopy (IR), gas chromatography (GC), 13C NMR, light scattering (LS) and viscometry. The results indicated that wb-, wc-PCM1, and PCM2 were heteropolysaccharides mainly composed of α-d-glucose, mannose, and galactose, whereas wb-PCM3-I and wc-PCM3-I were mainly (1→3)-α-d-glucans, and wb- and wc-PCM3-II, PCM4-I and PCM4-II were (1→3)-β-D-glucans. Interestingly, (1→3) α- and (1→3)-β-D-glucans co-existed in the 0.5 M NaOH fraction and were separated individually into the two fractions (PCM3-I and PCM3-II) after neutralizing with acetic acid. The polysaccharides from wc-PCM cultured in media containing corn steep liquor contained relatively more protein. The polysaccharide fractions also existed in conformations including random coil (as in PCM0 and PCM1) and expanded chain (as in PCM3), and differed molecular mass. In addition, two exo-polysaccharides isolated from the two culture media by methanol precipitation (wb- and wc-PCM0) also differed in their monosaccharide composition.
conformation, polysaccharide, light scattering, composition, molecular mass, Poria cocos
NCBI PubMed ID: 12829396Six water-insoluble (1→3)-β-D-glucan fractions TM8-1 to TM8-6 with weight-average molecular mass Mw ranging from 5.76 to 77.4×104 obtained from the sclerotia of Pleurotus tuber-regium were sulfated to produce the water-soluble fractions S-TM8-1 to S-TM8-6 with Mw from 6.0 to 64.8×104. The degree of substitution (DS) of S-TM8 fractions was analyzed by elemental analysis (EA) to be 1.14–1.74. The 13C NMR results indicated that the C-6 was fully substituted, and C-2, C-4 were partially substituted by the sulfo-groups. The Mw and the intrinsic viscosity [η] of the S-TM8 fractions were measured, respectively, by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS and viscometry in phosphate buffer solution (PBS) at 37 °C. The dependences of [η] and radius of gyration 〈s2〉z1/2 on Mw for the S-TM8 samples were found to be [η]=1.89×10−2Mw0.70 (cm3/g) and 〈s2〉z1/2=1.12×10−4Mw0.81 (nm) in the Mw range tested. Based on current theories for a wormlike chain model, the molar mass per unit contour length ML and persistence length q of the S-TM8 were calculated to be 990 nm−1 and 8.5 nm, respectively. The relatively higher q value suggested a more expanded flexible chain of S-TM8 in PBS. The water-solubility and relatively expanded chain conformation of the STM8 fractions were considered to be significant to their antiviral activity. Water-insoluble (1→3)-β-D-glucans having different Mw values from the sclerotia of P. tuber-regium were sulfated to afford water-soluble derivatives. The modified β-glucans were shown by SEC-LLS and viscometry to have a more expanded flexible chain in aqueous solution than the native polysaccharides.
conformation, molecular mass, β-D-glucan, sulfonation, Pleurotus tuber-regium
NCBI PubMed ID: 14667707Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1→3)-α-d-glucan, a (1→3)-β-D-glucan, a (1→6)-β-D-glucan, and a α-galactomannan. To refine the structures of these polysaccharides, cell-wall glucans of S. pombe were extracted, fractionated, and analyzed by NMR spectroscopy. S. pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared. The alkali-insoluble fraction was treated with 0.5 M acetic acid or Zymolyase 100 T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction. 13C NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1→3)-α-d-glucan, a (1→3)-β-D-glucan, which may either be linear or slightly branched, and a highly branched (1→6)-β-D-glucan, in addition to α-galactomannan. The highly branched (1→6)-β-D-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1→3)-β-branched (1→6)-β-D-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by 13C NMR spectra in D2O. The data collectively indicate that (1→3)-α- and (1→3)-β-D-glucans are rigid and contribute to the cell shape, while the highly branched (1→6)-β-D-glucan and α-galactomannan are flexible.
NMR, cell-wall glucan, Schizosaccharomyces pombe
NCBI PubMed ID: 15337454A water-insoluble (1→3)-β-D-glucan isolated from fresh sclerotium of Poria cocos was, respectively, sulfated, carboxymethylated, methylated, hydroxyethylated, and hydroxypropylated, to afford five water-soluble derivatives. Their weight-average molecular masses (Mw) and intrinsic viscosities ([η]) were determined by size-exclusion chromatography combined with laser light scattering (SEC-LLS), LLS, and viscometry in phosphate buffer solution (PBS) at 37 °C. The antitumor activities, against Sarcoma 180 tumor cell (S-180) and gastric carcinoma cell strain (MKN-45 and SGC-7901) of the native β-glucan and the five derivatives, were tested in vitro and in vivo. The Mw values of the five derivatives in PBS were determined to be 3.8 × 104, 18.9 × 104, 16.0 × 104, 76.8 × 104, and 224.3 × 104, respectively. The high Mw values of the hydroxyethylated and hydroxypropylated derivatives in aqueous solution resulted from aggregation, and their true Mw values obtained in dimethyl sulfoxide were 20.1 × 104 and 19.1 × 104. The sulfated and carboxymethylated derivatives having DS of 1.0–1.3 show good water solubility, and exist as relatively expanded chains in aqueous solution. Interestingly, the native β-glucan did not show antitumor activity, whereas the sulfated and carboxymethylated derivatives exhibit significant antitumor activities against S-180 and gastric carcinoma tumor cells. This work showed that good water solubility, relatively high chain stiffness, and moderate molecular mass of the derivatives in aqueous solution contribute beneficial to enhancement of antitumor activity.
polysaccharide, methylation, light scattering, molecular mass, sulfation, carboxymethylation, antitumor activity, Poria cocos sclerotium, hydroxyethylation, hydroxypropylation
NCBI PubMed ID: 15476718Two botryosphaerans, exopolysaccharides (EPS) secreted by the ascomyceteous fungus Botryosphaeria rhodina, when grown on sucrose and fructose as sole carbon sources, were structurally compared after their isolation from the culture medium. Both EPS were submitted to trypsin digestion, and eluted as a single peak on gel filtration. Total acid hydrolysis yielded only glucose, and data from methylation analysis and Smith degradation indicated that both EPS constituted a main chain of glucopyranosyl β(1→3) linkages substituted at O-6. The products obtained after partial acid hydrolysis demonstrated side chains consisting of glucosyl- and gentiobiosyl- linked β(1→6) residues. C-13-NMR spectroscopy studies showed that all glucosidic linkages were of the β-configuration. The carbon source affected the side chain structures of botryosphaeran but not the main chain makeup. Sucrose produced less branching (21%) than fructose (31%). (c) 2005 Published by Elsevier Ltd.
exopolysaccharides, Botryosphaeria rhodina, beta(1->3;1->6)-D-glucans, carbon source, structural variation
Publication DOI: 10.1016/j.carbpol.2005.01.002The cell wall of Aspergillus fumigatus is composed of a branched PI,3 glucan covalently bound to chitin, β-1,3, β-1,4 glucans, and galactomannan, that is embedded in an amorphous cement composed of α-1,3 glucan, galactomannan and polygalactosamin. The mycelial cell wall of A. fumigatus is very different from the yeast Saccharomyces cerevisiae cell wall, and in particular lacks 01,6 glucans and proteins covalently bound to cell wall polysaccharides. The differences in cell wall composition between the mould A. fumigatus and the yeast S. cerevisiae are also reflected at the genomic level where unique features have been identified in A. fumigatus. A single gene codes for the glucan synthase catalytic subumit; this finding has lead to the development of a RNAi methodology for the disruption of essential genes in A. fumigatus. In contrast to the glucan synthase, multiple genes have been found in the chitin synthase and the α-glucan synthase families; in spite of homologous sequences, each gene in each family have very different function. Similarly homologous mannosyltransferase genes are found in yeast and moulds but they lead to the synthesis of very different N-mannan structures. This chemo-genomic comparative analysis has also suggested that GPI-anchored proteins do not have a role of linker in the three dimensional organization of the fungal cell wall.
cell wall, mannan, glucan, GPI, chitin
Publication DOI: 10.1080/13693780400029155A structural elucidation of polysaccharides extracted from the aposymbiotically cultured mycobiont of the lichen Ramalina peruviana was carried out in order to determine whether the polysaccharides found previously in the symbiotic thalli are produced by the mycobiont or photobiont or both. The mycobiont isolate was cultivated on a solid malt-yeast extract-medium and the freeze-dried colonies were detailed and the polysaccharides extracted successively with hot water and aq. 2% KOH, each at 100 degreesC. The alkaline extract was obtained in much higher yield (31.5%) and submitted to a freeze-thawing treatment, giving rise to a precipitate (PK2) of a mixture of (1→3),(1→4)-α-glucan (1.2:1 ratio, nigeran) and a (1→3)-β-glucan (laminaran). The mother liquor was treated with Fehling solution to give a precipitate (galactomannan). This had a (1→6)-linked α-D-mannopyranosyl main chain, substituted at O-4 and in small proportion at O-2,4 by β-Galp units. All three polysaccharides have previously been found in the symbiotic thalli of R. peruviana, showing that these are produced by the fungus, without the participation of the Trebouxia photobiont. Surprisingly, isolichenan, a cold-water soluble (1→3),(1→4)-α-linked-glucan (3:1 ratio) was not found in the isolated mycobiont, despite being the main polysaccharide found in the thalli.
polysaccharides, Galactomannan, lichen, Ramalina peruviana, cultured mycobiont, alpha- and beta-glucans, α- and β-Glucans
Publication DOI: 10.1111/j.1574-6968.2004.tb09740.xThe two main polysaccharides from the basidiomycetous fungus Laetiporus sulphureus were isolated, purified and characterized. The structural assignments were carried out using C-13, H-1, and 1H,(13) HSQC nuclear magnetic resonance spectroscopy, methylation analysis, and Smith degradation. One was a linear β-glucan having a (1→3)-linked main chain, namely laminaran. The other was a fucomannogalactan, which consisted of a main chain of (1→6)-linked α-D-galactopyranosyl residues, a part of them being substituted at O-2 by 3-O-D-mannopyranbsyl-L-fucopyranosyl, α-D-mannopyranosyl and in a minor proportion, α-L-fucopyranosyl groups. This heteropolysaccharide is related to those of other Basidiomycetes heterogalactans, although it differs distinctly in its side-chain structures. Whereas part of the single-unit L-fucopyranosyl and/or 3-O-α-mannopyranosyl-L-fucopyranosyl residues are present as side chains of the other heterogalactans, additional a-D-mannopyranosyl units are present in our fucomannogalactan of L. sulphureus.
beta-glucan, heterogalactan, fucomannogalactan, Laetiporus sulphureus
Publication DOI: 10.1016/S0378-1097(03)00853-XSeveral structurally different glucans (alpha- and beta-) and galactomannans were characterized as components of four species of the genus Ramalina, namely R. dendriscoides, R. fraxinea, R. gracilis and R. peruviana. Freeze-thawing treatment of hot aqueous extracts furnished as precipitates (PW) linear α-(D)-glucans of the nigeran type, with regularly distributed (1→3)- and (1→4)-linkages in a 1:1 ratio. The supernatants (SW) contained α-(D)-glucans with (1→3)- and (1→4)-linkages in a molar ratio of 3:1. The lichen residues were then extracted with 2% aq. KOH, and the resulting extracts Submitted to the freeze-thawing treatment, giving rise to precipitates (PK2) of a mixture of α-glucan (nigeran) and β-glucan, which were suspended in aqueous 0.5% NaOH at 50 degreesC, dissolving preferentially the β-glucan. These were linear with (1→3)-linkages (laminaran). The mother liquor of the KOH extractions (2% and 10% aq. KOH) was treated with Fehling's solution to give precipitates (galactomannans). The galactomannans are related. having (1→6)-linked α-(D)-mannopyranosyl main chains, substituted at O-4 and in a small proportion at O-2,4 by β-(D)-galactopyranosyl units. Despite the different habitats of these lichenized fungi, all species studied in this investigation have a similar pool of polysaccharides. (C) 2003 Elsevier Ltd. All rights reserved.
polysaccharides, lichenized fungi, galactomannans, Ramalina peruviana, alpha- and beta-glucans, lichens, Ramalina dendriscoides, Ramalina fraxinea, Ramalina gracilis
NCBI PubMed ID: 12895548The exopolysaccharide, Botryosphaeran, produced by the ligninolytic, ascomycetcous fungus Botryosphaeria sp., was isolated from the extracellular fluid by precipitation with ethanol, and purified by gel permeation chromatography to yield a carbohydrate-rich fraction (96%) composed mainly of glucose (98%). Infra-red and C-13 NMR spectroscopy showed that all the glucosidic linkages were in the β-configuration. Data from methylation analysis and Smith degradation indicated that Botryosphaeran was a (1→3)-β-(D)-glucan with approx 22% side branching at C-6. The products obtained from partial acid hydrolysis demonstrated that the side branches consisted of single (1→6)-β-linked glucosyl, and (1→6)-β-linked gentiobiosyl residues.
exopolysaccharide, (1 -> 3;1 -> 6)-beta-glucan, Botryosphaeran, Botyrosphaeria sp., Ascomyceteous fungus
Publication DOI: 10.1016/S0008-6215(03)00240-4A water-insoluble glucan, TEINS has been isolated from the hot alkaline extract of an edible mushroom Termitomyces eurhizus. The total carbohydrate content of the polysaccharide fraction was found to be 98.4%, and it was found to contain only glucose as the monosaccharide constituent. On the basis of total acid hydrolysis, a methylation experiment, periodate oxidation and (13)C NMR experiment, the repeating unit of the polysaccharide was established as: →3)-β-D-Glcp-(1→.
structure, NMR spectroscopy, Insoluble glucan, Termitomyces eurhizus, Edible mushroom
NCBI PubMed ID: 17064674The glucans of basidiomycetes are an important class of polysaccharides with potential biological activities. In this work, the β-glucans were isolated from the fruiting bodies of edible mushrooms, Pleurotus eryngii and Pleurotus ostreatoroseus, via extraction with hot water, and then fractionation by freeze-thawing. The insoluble glucans gave similar 13C NMR spectra, monosaccharide composition and methylation analyses, and P. eryngii was selected for further controlled Smith degradation, and DEPT and 1H (obs.), 13C HMQC spectroscopy. It was a branched β-glucan, with a main chain of (1→3)-linked-Glcp residues, substituted at O-6 by single-unit β-Glcp side-chains, on average to every third residue of the backbone, as in scleroglucan.
polysaccharides, β-Glucans, Edible mushrooms, Pleurotus spp.
Publication DOI: 10.1016/j.carbpol.2006.03.009Glucans of basidiomycetes are considered to be an important class of polysaccharides, as they can act as biological response modifiers. We now isolate a gel-forming, water-soluble β-glucan, with a molecular mass of 1200000 g/mol (HPSEC), from the fruit bodies of the edible mushroom Pleurotus florida, via alkaline extraction, followed by fractionation by freeze-thawing. Structural assignments were carried out using mono- and bi-dimensional nuclear magnetic resonance spectroscopy, monosaccharide composition, methylation analyses, and a controlled Smith degradation. It was a branched β-glucan, with a main chain of (1→3)-linked-Glcp residues, substituted at O-6 by single-unit Glcp side chains, on average to every fourth residue of the backbone.
polysaccharides, β-Glucans, Pleurotus florida, Edible mushrooms
NCBI PubMed ID: 18479676Two polysaccharides were isolated from the basidiomycete Flammulina velutipes, via successive hot extraction with water, 2% and 25% aq. KOH, and then submitted to freeze-drying. The precipitate formed by repeated freeze-thawing from the 2% aq. KOH extraction PK2 was analyzed by determination of its monosaccharide composition, as well as by methylation analyses using GC-MS, mono- ((13)C, (1)H NMR) and bidimensional ((1)H (obs.), (13)C HMQC) spectroscopy, and controlled Smith degradations. It was established to be a branched β-glucan, with a main chain of (1→3)-linked-Glcp residues, substituted at O-6 by single-unit β-Glcp side chains. The precipitate formed by repeated freeze-thawing from the 25% KOH extraction PK25 contained Xyl, Man, and Glc and was heterogeneous by HSPEC and extraction with DMSO gave a soluble xylomannan (XM). It was homogeneous with a molar mass 308000 g/mol (dn/dc=0.186). Using the above chemical analyses, it was a xylomannan with Man and Xyl in a 3:2 molar ratio. Its main chain consisted of (1→3)-linked α-Manp units, mainly substituted at O-4 by β-Xylp units or with some β-Xylp-(1→3)-β-Xylp groups.
Flammulina velutipes, Basidiomycete, Branched β-glucan, Xylomannan
NCBI PubMed ID: 16884744The three main polysaccharides from the lichenized fungus Umbilicaria mammulata were isolated and characterized. Their structures were determined using GC-MS of derived alditol acetates, GC-MS-methylation analysis and 13C NMR spectroscopy. Two of them were (1→3)-and (1→6)-linked β-glucans, namely laminaran and pustulan, respectively. The other was a galactofuranomannan, which was analyzed as well as a galactose-free polysaccharide formed on partial hydrolysis. The former consisted of a main chain of (1→6)-linked α-mannopyranosyl residues, part of them being substituted at O-2, O-4, and O-2,4 by complex, branched side chains containing α-mannopyranosyl and β-galactofuranosyl units. This heteropolysaccharide is related to those of other Umbilicariaceae, and its chemical characterization presents a useful tool for the systematics of lichenized fungi.
chemotaxonomy, lichen, Umbilicaria mammulata, Galactofuranomannan, Laminaran, Pustulan
Publication DOI: 10.1248/cpb.54.882Some polysaccharides isolated from natural sources show various important biological activities, such as antitumor, immunomodulatory, and anti-inflammatory effects, which are strongly affected by their chemical structures and chain conformations. This article attempts to review the current development on structural and conformational characterization of some importantly bioactive polysaccharides isolated from natural sources. The chemical structures were analyzed by FTIR, liquid-state NMR (one and two dimensions), solid-sate NMR, Raman spectroscopy, gas chromatography (GC), GC–Mass (GC–MS), and high-performance liquid chromatography (HPLC). The chain conformations of polysaccharides in solutions were investigated using static and dynamic light scattering, viscosity analysis based on the theory of dilute polymer solution, circular dichroism analysis, atomic force microscopy (AFM) including single molecular AFM and AFM-based single-molecule force spectroscopy, fluorescence correlation spectroscopy and NMR spectroscopy.
chemical structure, polysaccharides, biological activity, Chain conformation
Publication DOI: 10.1016/j.carbpol.2008.12.015Mushrooms have been valued as edible and medicinal resources, and antitumor substances have been identified in many mushroom species. Polysaccharides are the best known and most potent mushroom-derived substances with antitumor and immunomodulating properties. Although the isolation process, structural characterization and antitumor activity of mushroom polysaccharides have been extensively investigated in the past three decades, the relationship between the antitumor activity and the chemical composition as well as the high order structure of their active components is still not well established. These studies are still in progress in many laboratories, and the role of polysaccharides as antitumor agent is especially under intense debate. The purpose of the present review is to summarize the available information, and to reflect the current status of this research area with a view for future direction.
structure, biological activity, mushrooms, antitumor polysaccharides
Publication DOI: 10.1016/j.tifs.2006.07.013We have recorded high-resolution 13C-NMR spectra of linear (curdlan) and branched (lentinan, HA-β-glucan and its polyol and aldehyde derivatives) (1-3)-β-D-glucans in hydrate and gel states, in order to gain insight into their gelation mechanism. Network structure of curdlan turned out to be highly heterogeneous from its motional state, from liquid-like, through intermediate, to solid-like domains. They are studied by a variety of experiments, conventional high-resolution NMR by broad-band decoupling, high-power decoupling with magic angle spinning (MAS), and cross-polarization-magic-angle-spinning (CP-MAS). Nevertheless, we found that conformations of these distinct liquid-like and solid-like do- mains exhibit an identical single helix conformation with a small proportion of a triple helix form, supporting our previous view as to the gelation mechanism. In contrast, the network structure of branched (1-3)-β-D-glucans in the gel state arises mainly from the triple helix conformation. This means that gelation of branched (1-3)-β-D-glucan proceeds from partial association of the triple helical chains, previously proposed for gelation of a linear glucan. Furthermore, we found that conversion from the single chain to the single helix was not achieved readily by hydration of over 8h at 96% R. H. for branched glucan but the triple helix form is obtained when these samples are hydrated fully as in gel state.
conformation, 13C NMR, Curdlan, lentinan
NCBI PubMed ID: 2207281Linear (1→3)-β-D-glucans and side-chain-branched β-D-glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage; whereas branched (1→3),(1→6)-β-D-glucans constitute the most abundant components in the cell walls of fungi. In the present manuscript we describe the chemical characterization of a linear β-D-glucan secreted by mutants of the Basidiomycota biotrophic fungus Ustilago maydis affected in the Pal/Rim pathway involved in pH responses, that in contrast is not made by the wild type strain, at least in measurable amounts. The polysaccharide was obtained from the culture medium of a Δrim13::Cbx mutant of the fungus by ethanol precipitation, and analyzed using FT-IR spectroscopy, NMR spectroscopy in solid and liquid states, HPAEC-PAD chromatography, thin layer chromatography, enzymatic digestion and immunodetection. Our data revealed that, according to its characteristics, the polysaccharide is a (1→3)-β-D-glucan, and that the mutants used represent a relevant resource of this polysaccharide with wide applications.
polysaccharide, (1→3)-β-D-glucan, Ustilago maydis, Pal/Rim pathway
Publication DOI: 10.1016/j.carbpol.2011.06.077Host immune responses elicited by invading pathogens depend on recognition of the pathogen by specific receptors present on phagocytic cells. However, the reactions to viral, bacterial, parasitic and fungal pathogens vary according to the pathogen-associated molecular patterns (PAMPs) on the surface of the invader. Phagocytic cells are known to initiate a respiratory burst following an exposure to the pathogen, but the underlying and associated specific elements are poorly elucidated in fish. The present study describes the differential response of head kidney leukocytes from rainbow trout (Oncorhynchus mykiss) to different PAMPs mimicking viral (poly I:C), bacterial (flagellin and LPS) and fungal infections (zymosan and β-glucan). Transcript of cytokines related to inflammation (IL-1β, IL-6, IL-10 and TNF-α) was highly up-regulated following LPS exposure whereas flagellin or poly I:C induced merely moderate reactions. In contrast, IFN-γ expression was significantly higher in the poly I:C stimulated group compared to the LPS group. When head kidney cells were exposed to zymosan or β-glucan, genes encoding IL-1β, TNF-α, IL-6 and IL-10 became up-regulated. Their level of up-regulation was comparable to LPS but the kinetics differed. In particular, TNF-α induction was considerably slower when stimulated with zymosan or β-glucan. The gene encoding the COX-2 enzyme, a central element during initiation of inflammatory reactions, was significantly higher in stimulated cells although a depressing effect of high concentrations of LPS and zymosan became evident after 4. h exposure. This study suggests that rainbow trout leukocytes respond differently to viral, bacterial and fungal PAMPs, which may reflect activation of specific signaling cascades eventually leading to activation of different immune effector molecules.
inflammation, cytokines, immunostimulants, head kidney leukocytes, pathogen associated molecular patterns (PAMPs)
NCBI PubMed ID: 21147161The milk yeast Kluyveromyces lactis is an alternative model yeast to the well established Saccharomyces cerevisiae. The cell wall of these fungi consists of polysaccharides (i.e. long chains of β-1,3- and β-1,6-linked sugar chains and some chitin) and mannoproteins, both of which are continually adapted to environmental conditions in terms of their abundance and organization. This implies the need to perceive signals at the cell surface and to transform them into a proper cellular response. The signal transduction cascade involved in this process is generally referred to as the cell wall integrity (CWI) pathway. CWI signaling and cell wall composition have been extensively studied in the Baker's yeast S. cerevisiae and are also of interest in other yeast species with commercial potential, such as K. lactis. We here summarize the results obtained in the past years on CWI signaling in K. lactis and use a comparative approach to the findings obtained in S. cerevisiae to highlight special adaptations to their natural environments.
cell wall proteome, MAP kinase cascade, protein kinase C, transmission electron microscopy
NCBI PubMed ID: 216280801,3-β-Glucans are a class of natural polysaccharides with unique pharmacological properties and the ability to form single- and triple-helical structures that can be formed into resilient gels with the application of heat and humidity. The pharmacological capabilities of 1,3-β-glucans include the impartation of tumor inhibition, resistance to infectious disease, and improvements in wound healing. Curdlan is a linear 1,3-β-glucan that has been used extensively to study the nature of these helical structures and gels, and Curdlan sulfates have found ongoing application in the inhibition of HIV infection. 1,3-β-Glucan gels have been used in food science as stabilizers and encapsulating agents, in nanoscience as scaffolds to build nanofibers and nanowires, and in drug delivery to form nanoparticles and create helical micelles encapsulating polynucleotides. 1,3-β-Glucans are beginning to have enormous significance due to their dual nature as structure-forming agents and pharmacological substances, and research is especially focused on the application of these polymers in animal nutrition and drug delivery.
Glucans, drug delivery
NCBI PubMed ID: 21609131The role for adjuvants in human vaccines has been a matter of vigorous scientific debate, with the field hindered by the fact that for over 80 years, aluminum salts were the only adjuvants approved for human use. To this day, alum-based adjuvants, alone or combined with additional immune activators, remain the only adjuvants approved for use in the USA. This situation has not been helped by the fact that the mechanism of action of most adjuvants has been poorly understood. A relative lack of resources and funding for adjuvant development has only helped to maintain alum's relative monopoly. To seriously challenge alum's supremacy a new adjuvant has many major hurdles to overcome, not least being alum's simplicity, tolerability, safety record and minimal cost. Carbohydrate structures play critical roles in immune system function and carbohydrates also have the virtue of a strong safety and tolerability record. A number of carbohydrate compounds from plant, bacterial, yeast and synthetic sources have emerged as promising vaccine adjuvant candidates. Carbohydrates are readily biodegradable and therefore unlikely to cause problems of long-term tissue deposits seen with alum adjuvants. Above all, the Holy Grail of human adjuvant development is to identify a compound that combines potent vaccine enhancement with maximum tolerability and safety. This has proved to be a tough challenge for many adjuvant contenders. Nevertheless, carbohydrate-based compounds have many favorable properties that could place them in a unique position to challenge alum's monopoly over human vaccine usage.
carbohydrate, vaccines, immunity, glucan, inulin, Glycomics, adjuvants
NCBI PubMed ID: 21506649OBJECTIVE: Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis. METHODS: The roles of CR3, TLR2, MyD88, NOD1, NOD2, Dectin-1 and Dectin-2 were tested in vivo in arthritis elicited by intra-articular injections of zymosan, the fungal cell wall components curdlan, laminarin and mannan, and the bacterial cell wall peptidoglycan. RESULTS: Dectin-1, and to a lesser extent Dectin-2, contributed to arthritis. TLR2, MyD88 and CR3 played non-essential roles. Observations based on injection of curdlan, laminarin or mannan supported the dominant role of the Dectin-1 pathway in the joint. We demonstrated differential roles for NOD1 and NOD2 and identified NOD2 as a novel and essential mediator of zymosan-induced arthritis. CONCLUSIONS: Together, Dectin-1 and NOD2 are critical, sentinel receptors in the arthritogenic effects of zymosan. Our data identify a novel role for NOD2 during inflammatory responses within joint.
innate immunity, zymosan, Arthritis models, NOD2, In vivo inflammation
NCBI PubMed ID: 21424514The Candida albicans plasma membrane plays important roles in interfacing with the environment, morphogenesis, and cell wall synthesis. The role of the Sur7 protein in cell wall structure and function was analyzed, since previous studies showed that this plasma membrane protein is needed to prevent abnormal intracellular growth of the cell wall. Sur7 localizes to stable patches in the plasma membrane, known as MCC (membrane compartment occupied by Can1), that are associated with eisosome proteins. The sur7Δ mutant cells displayed increased sensitivity to factors that exacerbate cell wall defects, such as detergent (SDS) and the chitin-binding agents calcofluor white and Congo red. The sur7Δ cells were also slightly more sensitive to inhibitors that block the synthesis of cell wall chitin (nikkomycin Z) and β-1,3-glucan (caspofungin). In contrast, Fmp45, a paralog of Sur7 that also localizes to punctate plasma membrane patches, did not have a detectable role in cell wall synthesis. Chemical analysis of cell wall composition demonstrated that sur7Δ cells contain decreased levels of β-glucan, a glucose polymer that confers rigidity on the cell wall. Consistent with this, sur7Δ cells were more sensitive to lysis, which could be partially rescued by increasing the osmolarity of the medium. Interestingly, Sur7 is present in static patches, whereas β-1,3-glucan synthase is mobile in the plasma membrane and is often associated with actin patches. Thus, Sur7 may influence β-glucan synthesis indirectly, perhaps by altering the functions of the cell signaling components that localize to the MCC and eisosome domains.
cell wall, Cell Membrane
NCBI PubMed ID: 21115741Toxicological and immunomodulatory activities of botryosphaeran (BR), a newly emerged β-glucan that comprises a β-(1→3) backbone and β-(1→6) branched glucose residues were assessed. BR was 1820000 Da (M.W.) estimated by reversely-linear equation constructed by regression of logarithms of standard polysaccharides and their retention times of gel permeation chromatography. Sprague-Dawley rats were daily gavage-administered with BR at doses of 0, 1.25, 12.5, and 125 mg/kg body weight (BW) for 28 d. Serum hematological and biochemical analysis of all treatment were all within normal ranges. Mitogen-stimulated lymphoblastogenesis of spleno-lymphocytes was enhanced by BR at doses of 1.25 and 12.5 mg/kg BW. Through in vitro comparative assessments, RAW 264.7 macrophage (RAW) cells were treated with BR and two commercial β-glucans, zymosan (ZY) and barley β-glucan (GB), to characterize their relative immunomodulatory properties. All three β-glucans stimulated phagocytosis on fluorescence-labeled Escherichia coli. At dose levels from 5 to 200 μg/mL for 24h, nitric oxide produced by BR- and ZY-treated cells were higher than those produced by GB-treated and control groups. BR, ZY but GB also stimulated RAW cells in producing TNF-α. The results demonstrate that BR is toxicologically accepted and features as a potent immunomodulator.
gel permeation chromatography, β-glucan, immunomodulation, nitric oxide, Botryosphaeran, Sprague-Dawley rat
NCBI PubMed ID: 21185904A glucan was extracted with hotwater from the basidiomycete Pleurotus pulmonarius and shown to have a (1→3)-linked β-D-glucopyranosyl main-chain substituted at O-6 of every third unit by single β-D-glucopyranosyl nonreducing end units. This was shown by mono- and bidimensional nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and a controlled Smith degradation. The glucan was tested for its effects on the acetic acid-induced writhing reaction inmice, a typical model for quantifying inflammatory pain. It caused a marked and dose-dependent anti-inflammatory response, demonstrated by the inhibition of leukocytemigration to injured tissues (82±6%) with an ID50 of 1.19 (0.74–1.92) mg/kg. Furthermore, animals previously treated with the glucan (3 mg/kg i.p.), showed a reduction of 85±5% of writhes, after receiving the acetic acid injection. Furthermore, in the formalin test, the glucan (3–30 mg/kg, i.p.) also caused significant inhibition of both the early (neurogenic pain) and the late phases (inflammatory pain) of formalin-induced licking. However, it was more potent and effective in relation to the late phase of the formalin test, with mean ID50 values for the neurogenic and the inflammatory phases of >30 and 12.9 (6.7–24.6)mg/kg and the inhibitions observed were 43±5% and 96±4%, respectively. These data showed that the glucan had potent anti-inflammatory and analgesic (antinociceptive) activities, possibly by the inhibition of pro-inflammatory cytokines.
mushroom, β-glucan, chemical analysis, anti-inflammatory, Pleurotus pulmonarius, anti-inflammatory and analgesic effects, analgesic effects
NCBI PubMed ID: 18789924Antitumor activities of two (1→6)-branched (1→3)-β-D-glucans, isolated from the fruiting body of Auricularia auricula-judae ("kikurage", an edible mushroom), and other branched polysaccharides containing a backbone chain of (1→3)-α-d-glucosidic or (1→3)-α-d-mannosidic linkages [and their corresponding (1→3)-d-glycans, derived by mild, Smith degradation] were compared. Among these polysaccharides, a water-soluble, branched (1→3)-β-D-glucan (glucan I) of A. auricula-judae exhibited potent, inhibitory activity against implanted Sarcoma 180 solid tumor in mice. The alkali-insoluble, branched (1→3)-β-D-glucan (glucan II), a major constituent of the fruiting body, showed essentially no inhibitory activity. When the latter glucan, having numerous branches attached, was modified by controlled, periodate oxidation, borohydride reduction, and mild, acid hydrolysis, the resulting, water-soluble, degraded glucan, having covalently linked polyhydroxy groups attached at O-6 of the (1→3)-linked d-glucosyl residues, exhibited potent antitumor activity. Further investigations using the glucan-polyalcohol indicated that the attachment of the polyhydroxy groups to the (1→3)-β-D-glucan backbone may enhance the antitumor potency of the glucan. On the other hand, partial introduction of carboxymethyl groups into glucan II (d.s., 0.47-0.86), which altered the insolubility property, failed to enhance the antitumor activity. The interrelation between the antitumor activity and the structure of the branched (1→3)-β-D-glucan is discussed, on the basis of methylation and 13C-NMR studies of the periodate-modified glucans.
polysaccharide, β-glucan, antitumor activity, Auricularia auricula-judae
NCBI PubMed ID: 7196285Water-insoluble glucan was isolated from the baker's yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing.
polysaccharide, immunomodulator, yeast, tissue repair, water-insoluble glucan
NCBI PubMed ID: 22942695Cultures of the mycobiont Physcia kalbii were obtained from germinated ascospores and cultivated on Sabouraud-Sucrose-agar medium. Alkaline extraction of freeze-dried mycelia provided a branched (1→3),(1→6)-β-glucan and a glucomannan, whose chemical structure was determined by monosaccharide composition, methylation, controlled Smith degradation and NMR spectroscopic analysis. The β-glucan had a (1→3)-linked β-glucopyranosyl backbone, partially substituted (approx. 50% of the units) at O-6. The side chains were formed by 6-O- (~82%) and 2,6-O-linked-β- Glcp units, while the non-reducing ends were formed by β-glucopyranosyl residues. The glucomannan had (1→6)-linked α-Manp units in the main chain, almost all being substituted at O-2 by α-Manp and α-Glcp units. This glucomannan could be a typical polysaccharide of lichens from the family Physciaceae.
polysaccharides, lichen, Glucomannan, Branched β-glucan, mycobiont, Physcia
NCBI PubMed ID: 22981001The carbon-13 nmr spectra of two cyclodextrins and several linear glucans have been completely assigned. These assignments were made by comparison with the spectra of glucose, some of its specifically O-methylated derivatives, and a number of differently linked glucobioses and glucotrioses. This technique enables the composition, structure, and major sequence of a number of glucans to be determined. Conformational effects are also apparent in some of the spectra. The 1→4, 1→4, and 1→6 linkage sequence of the glucans from T. mesenterica and P. pullulans give, in addition to an anomeric signal for the 1→6 linkage, two separate anomeric signals for each of the 1→4 linkages. This multiplicity, apparent also in other carbon signals of the spectra, is due to the sensitivity of these carbons to the type of linkage on the neighboring glucose units. Some evidence that conformational effects are involved in this multiplicity is provided by a comparison of the spectra of the 1→4-linked cyclodextrins and linear glucans, where appreciable chemical shift differences are apparent for C1 and C4. These effects are influenced by pH modification and are attributed to differences in rotational isomers at the linked carbons.
NMR, oligosaccharide, Bacterial polysaccharide, amylose
NCBI PubMed ID: 4464319β-glucan particles (GP) are polymeric carbohydrates, mainly found as components of cell wall fungi, yeast, bacteria and also in cereals such as barley and oat, and have been recently shown to have application in macrophage-targeted drug delivery. The aim of this study was to prepare and characterize GP containing a large payload of Rifabutin (RB), an anti-tuberculosis drug effective against MDR-TB at lower MIC than Rifampicin. GP were prepared from yeast cells by acidic and alkaline extraction were either spray dried or lyophilized, prior to RB loading and alginate sealing. The FTIR and 13C-NMR spectra of the GP confirmed a β-(1→3) linked glucan structure, with a triple-helical conformation. The spray dried GP exhibited better characteristics in terms of uniformity, size range (2.9 to 6.1 µm) and more than 75 % particles were below 3.5 μm. The RP-HPLC analysis of spray dried GP revealed drug entrapment and drug loading up to 81.46 ± 4.9 % and ~40.5 ± 1.9 %, respectively, as compared to those dried by lyophilization. Electron microscopy showed nearly spherical and porous nature of GP, and the presence of drug 'nanoprecipitates' filling the pore spaces. The formulation showed adequate thermal stability for pharmaceutical application. The particles were readily phagocytosed by macrophage(s) within 5 min of exposure. Drug release occurred in a sustained manner via diffusion, as the release kinetics best fit for drug release was obtained using Higuchi's equation. Thus, the spray dried GP-based-formulation technology holds promise for enhanced targeted delivery of anti-TB drug(s) to macrophage within a therapeutic window for the clearance of intracellular bacteria.
phagocytosis, macrophage, glucan particles, rifabutin, spray drying, targeted drug delivery
NCBI PubMed ID: 28507467More than 90% of the cell wall of the filamentous fungus Aspergillus fumigatus comprises polysaccharides. Biosynthesis of the cell wall polysaccharides is under the control of three types of enzymes: transmembrane synthases, which are anchored to the plasma membrane and use nucleotide sugars as substrates, and cell wall-associated transglycosidases and glycosyl hydrolases, which are responsible for remodeling the de novo synthesized polysaccharides and establishing the three-dimensional structure of the cell wall. For years, the cell wall was considered an inert exoskeleton of the fungal cell. The cell wall is now recognized as a living organelle, since the composition and cellular localization of the different constitutive cell wall components (especially of the outer layers) vary when the fungus senses changes in the external environment. The cell wall plays a major role during infection. The recognition of the fungal cell wall by the host is essential in the initiation of the immune response. The interactions between the different pattern-recognition receptors (PRRs) and cell wall pathogen-associated molecular patterns (PAMPs) orientate the host response toward either fungal death or growth, which would then lead to disease development. Understanding the molecular determinants of the interplay between the cell wall and host immunity is fundamental to combatting Aspergillus diseases.
polysaccharide, infection, interaction, fungi, mycelium, Conidia
NCBI PubMed ID: 28701066The Schizosaccharomyces pombe cell wall is a rigid exoskeletal structure mainly composed of interlinked glucose polysaccharides and galactomannoproteins. It is essential for survival of the fission yeast, as it prevents cells from bursting from internal turgor pressure and protects them from mechanical injuries. Additionally, the cell wall determines the cell shape and, therefore, a better knowledge of cell wall structure and composition could provide valuable data in S. pombe morphogenetic studies. Here, we provide information about this structure and the current reliable methods for rapid analysis of the cell wall polymers by specific enzymatic and chemical degradations of purified cell walls.
NCBI PubMed ID: 28733407The fungal wall is pivotal for cell shape and function, and in interfacial protection during host infection and environmental challenge. Here, we provide the first description of the carbohydrate composition and structure of the cell wall of the rice blast fungus Magnaporthe oryzae. We focus on the family of glucan elongation proteins (Gels) and characterize five putative β-1,3-glucan glucanosyltransferases that each carry the Glycoside Hydrolase 72 signature. We generated targeted deletion mutants of all Gel isoforms, that is, the GH72+ , which carry a putative carbohydrate-binding module, and the GH72- Gels, without this motif. We reveal that M. oryzae GH72+ GELs are expressed in spores and during both infective and vegetative growth, but each individual Gel enzymes are dispensable for pathogenicity. Further, we demonstrated that a Δgel1Δgel3Δgel4 null mutant has a modified cell wall in which 1,3-glucans have a higher degree of polymerization and are less branched than the wild-type strain. The mutant showed significant differences in global patterns of gene expression, a hyper-branching phenotype and no sporulation, and thus was unable to cause rice blast lesions (except via wounded tissues). We conclude that Gel proteins play significant roles in structural modification of the fungal cell wall during appressorium-mediated plant infection.
NCBI PubMed ID: 27568483The molecular composition of the cell wall is critical for the biology and ecology of each fungal species. Fungal walls are composed of matrix components that are embedded and linked to scaffolds of fibrous load-bearing polysaccharides. Most of the major cell wall components of fungal pathogens are not represented in humans, other mammals, or plants, and therefore the immune systems of animals and plants have evolved to recognize many of the conserved elements of fungal walls. For similar reasons the enzymes that assemble fungal cell wall components are excellent targets for antifungal chemotherapies and fungicides. However, for fungal pathogens, the cell wall is often disguised since key signature molecules for immune recognition are sometimes masked by immunologically inert molecules. Cell wall damage leads to the activation of sophisticated fail-safe mechanisms that shore up and repair walls to avoid catastrophic breaching of the integrity of the surface. The frontiers of research on fungal cell walls are moving from a descriptive phase defining the underlying genes and component parts of fungal walls to more dynamic analyses of how the various components are assembled, cross-linked, and modified in response to environmental signals. This review therefore discusses recent advances in research investigating the composition, synthesis, and regulation of cell walls and how the cell wall is targeted by immune recognition systems and the design of antifungal diagnostics and therapeutics.
NCBI PubMed ID: 285134151,3-β-D-glucan is a widespread polysaccharide in microorganisms, mushrooms and plants. Numerous benefits for the health of humans and animals have been described for more than 50 years. One readily available source for 1,3-β-D-glucan is the cell wall of the common baker’s or brewer’s yeast Saccharomyces cerevisiae. Conventional isolation processes involving treatments with hot alkali and acids, however, cause a certain degradation of the polymeric chains resulting not only in reduced yields but also a possible weakening of the beneficial effects. We have developed a new non-degrading process for the isolation of glucan from yeast cell walls comprising gentle extraction steps and enzymatic treatments without any extreme pH values. Depending on the quality of the utilized yeast cell walls, 1,3-β-D-glucan is obtained in a purity of up to 92% and a yield of 87%. During this process, the valuable byproduct mannoprotein - also beneficial for health - is easily obtained.
1, Saccharomyces cerevisiae, 3-β-D-glucan, mannoprotein, yeast cell walls
Publication DOI: 10.1016/S0144-8617(03)00162-0The structure of cell wall of yeasts (genus Saccharomyces) is one of the factors that determine their health-promoting properties connected to the presence of b-glucans and mannoprotein. The aim of the study was to determine the influence of glycerol as a carbon source on structural polymers of cell wall (b-glucan and mannoprotein) of probiotic yeasts Saccharomyces cerevisiae var. boulardii and brewer’s yeasts S. cerevisiae R9. Significant increase of the percentage of polysaccharide content in the cell wall dry weight of S. cerevisiae R9 brewer’s yeasts was noted (in the range of 10–20 %) after cultivation in medium containing glycerol at a concentration of 2–5 % and pH 4.0. The highest content of carbohydrates in probiotic yeasts’ cell wall (58 %) was observed after cultivation in medium containing 3 % of glycerol and pH 5.0. The cell wall of probiotic yeasts was characterized by higher content of mannoprotein comparing with cell wall preparation of brewer’s yeasts S. cerevisiae R9 composed mainly of b-glucans. After cultivation in mediums with 2 and 3 % of glycerol, the cell of brewer’s yeasts contained the highest amount of b(1,3/1,6)-glucan in dry weight of the cell wall (about 36 %). Glycerol at a concentration of 3 and 5 % also intensified mannoprotein biosynthesis in cell wall of S. cerevisiae R9, approximating their content to those noted in the cells of probiotic yeasts (about 29 % (w/w) of dry weight of the cell wall) after cultivation in a medium of pH 5.0 containing 3 % of glycerol.
cell wall, β-glucan, Saccharomyces cerevisiae, mannoprotein, probiotic yeast
Publication DOI: 10.1007/s00217-013-2016-8α-(1,3)-Glucan is a major component of the cell wall of Aspergillus fumigatus, an opportunistic human fungal pathogen. There are three genes (AGS1, AGS2 and AGS3) controlling the biosynthesis of α-(1,3)-glucan in this fungal species. Deletion of all the three AGS genes resulted in a triple mutant that was devoid of α-(1,3)-glucan in its cell wall; however, its growth and germination was identical to that of the parental strain in vitro. In the experimental murine aspergillosis model, this mutant was less pathogenic than the parental strain. The AGS deletion resulted in an extensive structural modification of the conidial cell wall, especially conidial surface where the rodlet layer was covered by an amorphous glycoprotein matrix. This surface modification was responsible for viability reduction of conidia in vivo, which explains decrease in the virulence of triple agsΔ mutant.
NCBI PubMed ID: 24244155Understanding the pathogenesis of an infectious disease is critical for developing new methods to prevent infection and diagnose or cure disease. Adherence of microorganisms to host tissue is a prerequisite for tissue invasion and infection. Fungal cell wall adhesins involved in adherence to host tissue or abiotic medical devices are critical for colonization leading to invasion and damage of host tissue. Here, with a main focus on pathogenic Candida species, we summarize recent progress made in the field of adhesins in human fungal pathogens and underscore the importance of these proteins in establishment of fungal diseases.
NCBI PubMed ID: 23397570Eight distinct polysaccharides (PS) of β-(1-3)-glucan type were tested for their capacity to render murine peritoneal exudate cells (PEC) cytotoxic. After intraperitoneal injection of lentinan, pachymaran and HE-pachyman 3 and 4 highly cytotoxic PEC were induced. Pachyman and HE-pachyman 1 and 2 were of moderate effect, whereas CM-pachymaran and HE-pachyman 3 and 4, highly cytotoxic PEC were induced. Pachyman and HE-pachymacrophages. The induction of PEC-dependent cytotoxicity exhibited a strict dose relationship. Optimal administration of PS resulted in the induction of cytotoxicity, which persisted for more than 25 days. Surprisingly, none of the PS tested was capable of rendering normal or thioglycollate-induced PEC cytoxic under in vitro conditions. It is suggested that the capacity of PS to render in vivo macrophages cytotoxic is related to the potency of these PS to activate the alternative pathway of complement system (APC) in so far as C3b may be the essential component required to render macrophages cytotoxic.
glucan, lentinan, T-cell immune adjuvants
NCBI PubMed ID: 6966608The mechanism of antitumour action of the polysaccharides, lentinan and carboxymethylpachymaran, was examined and compared with that of zymosan. It was revealed that (1) these polysaccharides did not show any direct cytocidal effect on tumour cells; (2) these polysaccharides had no antitumour action at all in neonatally thymectomized mice, indicating the important role of the thymus gland in tumour regression; and (3) in spite of these facts, these polysaccharides, contrary to zymosan, did not accelerate various conventional immune responses such as phagocytic activity, antibody production against heterologous antigen, increased number of peripheral lymphocytes, skin homograft rejection, and delayed hypersensitivity. This evidence suggests the presence of a complex and unknown mechanism for regression of transplanted tumour in the action of lentinan and carboxymethylpachymaran.
antitumor activity, neonatal thymectomy
NCBI PubMed ID: 4597986Despite a great deal of effort made by many researchers throughout the world, chemotherapeutic agents that attack cancer cells directly do not seem to have the expected effects except on some leukaemias. Besides, these agents show strong toxicity to the host, and reduce the host defense against infections, especially destroying lymphocytes and bone marrow cells. To find a new cancer drug that can activate or restore host defense mechanisms, we examined fungi, which had traditionally been said to be effective against cancer in Japan and other Asian countries, such as Ganoderma applanatum (Pers.) Pat. and Coriolus versicolor (Fr.) Quél., and several kinds of Japanese edible mushrooms. Test substances were administered intraperitoneally and screened for their ability to inhibit the growth of sarcoma 180 cells subcutaneously transplanted into swiss or ICR mice. This method, reported by Nakahara et al. [1], has been proven to be simple and suitable for screening of host-mediated anticancer drugs. Table 1 shows various antitumoral polysaccharides including lentinan that were isolated from fungi, basidiomysetes and yeast. Lentinan, a (1→3)-β-D-glucan with (1→6)-β-D-glucopyranoside branches isolated from an edible mushroom, Lentinus edodes (Berk.) Sing., exhibits a marked antitumor effect against sarcoma 180 cells transplanted subcutaneously at a dose of 1 mg/kg/day for ten days (Fig. 1) [2–4]. Its chemical and physical characteristics are listed in Table 2.
Francisella tularensis, Edible mushroom, delay type hypersensitivity, Lewis lung carcinoma, recurrent gastric cancer
Publication DOI: 10.1007/978-3-0348-8763-2_8Immunostimulants [1, 2] or immunopotentiators are drugs leading predominantly to a non-specific stimulation of immunological defence mechanisms. Most of them are not real antigens but antigenomimetics or so-called mitogens. Non-specific and non-antigen dependent stimulants do not affect immunological memory cells and, since their pharmacological efficacy fades comparatively quickly, they have to be administered either in intervals or continuously. Some immunostimulants may also stimulate T-suppressor cells and thereby reduce immune resistance, hence the term immunomodulation or immunoregulation, denoting any effect on, or change of, immune responsiveness is also very often used.
human immunodeficiency virus, aristolochic acid, viscum album, cichoric acid, phagocytosis index
Publication DOI: 10.1007/978-3-0348-8763-2_1We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from Kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 mug/ml and 10 mug/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from Saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cerevisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(-)6-linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403), mediates the phagocytosis of zymosan particles.
phagocytosis
NCBI PubMed ID: 6298248β-D-glucans of the laminaran type were prepared from 15 Cladonia spp., Cladonia bellidiflora, Cladonia boryi, Cladonia clathrata, Cladonia connexa, Cladonia crispatula, Cladonia furcata, Cladonia gracilis, Cladonia ibitipocae, Cladonia imperialis, Cladonia miniata, Cladonia penicillata, Cladonia salmonea, Cladonia signata, Cladonia substellata and Cladonia uncialis. They were extracted with 10% aqueous KOH at 100 degrees C, giving polysaccharides with varying yields and proportions of mannose, galactose and glucose. Their aqueous solutions were freeze-thawed giving precipitates of mixed α-glucan (nigeran) and β-glucans, which were isolated and suspended in aqueous 0.5% KOH at 50 degrees C, which preferentially dissolved the β-glucan. In the case of the C. uncialis product, it was subjected to methylation analysis, which gave rise to 2,4,6-tri-O-methylglucitol acetate only, corresponding to (1→3)-linkages. Its specific rotation (+4 degrees ) and one- and two-dimensional nuclear magnetic resonance (NMR) spectra were consistent with β-linkages. 13C and (1)H-1 signals were observed, respectively, at delta 102.8 (C-1), 86.0 (C-3), 76.2 (C-5), 72.6 (C-2), 68.3 (C-4) and 60.7 (C-6), and 4.55 (H-1), 3.31 (H-2), 3.49 (H-3), 3.27 (H-4), 3.27 (H-5), 3.48 (H-6) and 3.72 (H-6'). Similar (13)C-NMR spectra were obtained from the glucans from the other 14 Cladonia spp. The β-D-glucans of the laminaran type seems to be present in all Cladonia spp. being significant for chemotyping since it was observed in every species studied.
nuclear magnetic resonance, lichenized fungus, β-D-glucan, chemotyping, Laminaran, Cladonia spp.
NCBI PubMed ID: 11150667Non-prescriptional use of medicinal herbs among cancer patients is common around the world. The alleged anti-cancer effects of most herbal extracts are mainly based on studies derived from in vitro or in vivo animal experiments. The current information suggests that these herbal extracts exert their biological effect either through cytotoxic or immunomodulatory mechanisms. One of the active compounds responsible for the immune effects of herbal products is in the form of complex polysaccharides known as β-glucans. β-glucans are ubiquitously found in both bacterial or fungal cell walls and have been implicated in the initiation of anti-microbial immune response. Based on in vitro studies, β-glucans act on several immune receptors including Dectin-1, complement receptor (CR3) and TLR-2/6 and trigger a group of immune cells including macrophages, neutrophils, monocytes, natural killer cells and dendritic cells. As a consequence, both innate and adaptive response can be modulated by β-glucans and they can also enhance opsonic and non-opsonic phagocytosis. In animal studies, after oral administration, the specific backbone 1→3 linear β-glycosidic chain of β-glucans cannot be digested. Most β-glucans enter the proximal small intestine and some are captured by the macrophages. They are internalized and fragmented within the cells, then transported by the macrophages to the marrow and endothelial reticular system. The small β-glucans fragments are eventually released by the macrophages and taken up by other immune cells leading to various immune responses. However, β-glucans of different sizes and branching patterns may have significantly variable immune potency. Careful selection of appropriate β-glucans is essential if we wish to investigate the effects of β-glucans clinically. So far, no good quality clinical trial data is available on assessing the effectiveness of purified β-glucans among cancer patients. Future effort should direct at performing well-designed clinical trials to verify the actual clinical efficacy of β-glucans or β-glucans containing compounds.
antitumor activity
NCBI PubMed ID: 19515245Two water-soluble polysaccharide derivatives, carboxymethylated and quarternized glucans (CMGP and QGP) were synthesized for the first time from water-insoluble polysaccharides (GP) extracted from Ganoderma lucidum. Hollow microspheres were constructed using electrostatic layer-by-layer (LbL) deposition of the CMGP and QGP polyelectrolytes onto colloidal ZnO particles followed by the core decomposition with an acid solution. The structures of the multilayered CMGP/QGP microspheres were investigated by transmission electron microscopy (TEM), zeta potential and dynamic light scattering (DLS). The results revealed that the multilayer thickness increased regularly from 48 to 145 nm as the number of deposited CMGP/QGP layers was increased from two to seven, and the mean increment of thickness was ∼25 nm per layer, reflecting the high regularity of the layer-by-layer assembly. This work provided an easy method to construct hollow microcapsules with biocompatibility and controlled dimensions.
carboxymethylated polysaccharide, quaternized polysaccharide, ZnO microspheres, layer-by-layer assembly, hollow microcapsules
NCBI PubMed ID: 23768597The chemical structures of polysaccharides present in aposymbiotically cultured myco- and photobionts of the lichen Teloschistes flavicans were determined, in order to compare them with those previously found in the intact thallus. The mycobiont was cultured on a solid Lilly and Barnett medium and the resulting colonies were freeze dried, defatted, and their polysaccharides were extracted successively with 2%, 10% and 30% aq. KOH, each at 100 degrees C. The extracts were neutralized (HOAc) and fractionated, giving rise to three homogeneous fractions, PFSK2 from 2% KOH, which contained a (1→4),(1→6)-linked α-glucan (1:1 ratio, pullulan), fraction PK10 from 10% KOH extraction, which was a linear (1→3)-linked linear β-glucan (laminaran), and fraction PK30 from 30% KOH extraction, being a branched (1→3),(1→6)-linked β-glucan. The photobiont (Trebouxia sp. de Puymaly) was cultured in liquid nutrient medium, and after purification, a linear (1→5)-linked β-galactofuranan was characterized. The galactofuranan and the laminaran were not present in the symbiotic thallus, in contrast to the glucans, showing that the mycobiont alone produces them without participation of the photobiont.
polysaccharides, galactofuranan, Teloschistes flavicans, cultured mycobiont, α- and β-Glucans, cultured photobiont
NCBI PubMed ID: 18191406Lichens have been used for medicinal purposes throughout the ages, and beneficial claims have to some extent been correlated with their polysaccharide content. Of 13,500 lichen species growing worldwide, less than 100 species have been investigated for polysaccharide content. Lichen polysaccharides are mainly of three different structural types: β-glucans, α-glucans, and galactomannans. In addition, a few complex heteroglycans have recently been described, such as thamnolan, a water-soluble, immunologically active heteroglycan with a novel rhamnopyranosylgalactofuranan type of structure. A number of investigations have been carried out on biological effects of lichen polysaccharides, most notably antitumour, immunomodulating, antiviral, and memory-enhancing effects. The current review summarizes present knowledge on the structural characteristics and biological activity of lichen polysaccrides.
polysaccharides, biological activity, immunological activity, lichens, structural characteristics, antitumour activity
NCBI PubMed ID: 11345688For centuries, macrofungi have been used as food and medicine in different parts of the world. This is mainly attributed to their nutritional value as a potential source of carbohydrates, proteins, amino acids, and minerals. In addition, they also include many bioactive metabolites which make mushrooms and truffles common components in folk medicine, especially in Africa, the Middle East, China, and Japan. The reported medicinal effects of mushrooms include anti-inflammatory effects, with anti-inflammatory compounds of mushrooms comprising a highly diversified group in terms of their chemical structure. They include polysaccharides, terpenoids, phenolic compounds, and many other low molecular weight molecules. The aims of this review are to report the different types of bioactive metabolites and their relevant producers, as well as the different mechanisms of action of mushroom compounds as potent anti-inflammatory agents.
reishi, Ganoderma, ganoderic acid
NCBI PubMed ID: 25505823Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation.
cell wall, β-glucan, Candida albicans, b-glucan
NCBI PubMed ID: 25467440The cell wall of mushroom fruiting body is constituted of nondigestible macromolecules that are a rich source of dietary fiber with biological functions that are beneficial to human health. The cell wall components of an edible mushroom fruiting body from Pleurotus tuber-regium (PTR) were fractionated, and their chemical structures were investigated by chemical, physicochemical, and microscopic analyses. The present results suggest that the cell wall of the PTR mushroom fruiting body contains four main fractions: an outer fraction of polysaccharide and protein complex, which can be extracted using boiling water; a cold alkali-soluble fraction of heteropolysaccharides associated with a small amount of proteins; a hot alkali-soluble fraction of hyper-branched glucans; and an alkali-insoluble fraction of glucan-chitin complex with a normalized relative percentage of 3.6:21.9:55.7:18.8. The anomeric linkage of all the glucans was revealed by infrared spectroscopy to be β type. The structure of the major mushroom fruiting body cell wall polysaccharide (the hot alkali-soluble one, FHA-I) was elucidated by the methylation analysis to be composed of →1)-Glcp-(4→ linkages as the backbone with a 52% degree of branching consisting of →1)-Glcp-(6→ linkages in the side chains, whereas some →1)-Glcp-(3→ linkages might exist in the backbone or side chains. Size exclusion chromatography coupled with multiangle laser light scattering analysis revealed that FHA-I had a molecular weight of 4224000 g/mol and a root-mean-square radius of 30.4 nm. Both scanning electron and atomic force microscopy further showed the highly branched microstructure of FHA-I when dispersed in an aqueous sodium dodecyl sulfate solution.
cell wall polysaccharides, Pleurotus tuber-regium, dietary fiber, mushroom fruiting body
NCBI PubMed ID: 24625260Fungal cell walls contain several types of glycans, which play important roles in the pathogenesis of fungal infection and host immune response. Among them, glycosphingolipids have attracted much attention lately since they contribute actively to the fungi development and fungal-induced pathogenesis. Although glycosphingolipids are present in pathogenic and non-pathogenic fungi, pathogenic strains exhibit distinct glycan structures on their sphingolipids, which contribute to the regulatory processes engaged in inflammatory response. In Candida albicans, phospholipomannan (PLM) represents a prototype of these sphingolipids. Through its glycan and lipid moieties, PLM induces activation of host signaling pathways involved in the initial recognition of fungi, causing immune system disorder and persistent fungal disease. In this review, first we describe the general aspects of C. albicans sphingolipids synthesis with a special emphasize on PLM synthesis and its insertion into the cell wall. Then, we discuss the role of PLM glycosylation in regulating immune system activation and its contribution to the chronic persistent inflammation found in Candida infections and chronic inflammatory diseases.
regulation, glycosphingolipids, yeasts, inflammatory response
NCBI PubMed ID: 25394861Fungal glucans represent various structurally different d-glucose polymers with a large diversity of molecular mass and configuration. According to glucose anomeric structure, it is possible to distinguish α-D-glucans, β-D-glucans and mixed α,β-D-glucans. Further discrimination could be made on the basis of glycosidic bond position in a pyranoid ring, distribution of specific glycosidic bonds along a chain, branching and molecular mass. Fungal glucans can be chemically modified to obtain various derivatives of potential industrial or medicinal importance. NMR spectroscopy is a powerful tool in structural analysis of fungal glucans. Together with chemolytic methods like methylation analysis and periodate oxidation, NMR is able to determine exact structure of these polysaccharides. Fungal glucans or their derivatives exert various biological activities, which are usually linked to structure, molecular mass and substitution degree.
nuclear magnetic resonance, chemical modification, fungal glucans, structural diversity, structure–activity relationship
NCBI PubMed ID: 23218369LPS (lipopolysaccharide) induces platelet activation and is a well-known fundamental agent of septic shock and disseminated intravascular coagulation (DIC). Biological activity of (1→3)-β-D-glucan is related due to its anti-inflammatory, antioxidant, and antitumor properties. We focus our attention on the (1→3)-β-D-glucan (antiplatelet) properties. The main purpose of our study was to evaluate the influence of (1→3)-β-D-glucan from Saccharomyces cerevisiae on destructive activity of LPS (from Escherichia coli and Pseudomonas aeruginosa) on human blood platelets. We assess biochemically in vitro if (1→3)-β-D-glucan might combat the oxidative stress caused by LPS stroke associated with nitrative and oxidative damages of human platelet biomolecules. We also make an attempt by in silico molecular docking to determine the interactions between the molecules of (1→3)-β-D-glucan and LPS. Our conclusion is that protective mechanism of (1→3)-β-D-glucan against LPS action on blood platelets is due to as well: its antioxidant properties, as to its interaction with LPS-binding region of TLR4-MD-2 complex.
Lipopolysaccharide, endotoxin, TLR-4, (1-3)bDglucan, blood platelets
NCBI PubMed ID: 23911506β-Glucans from fungi exhibit a broad spectrum of biological activities including anti-tumor, immune-modulating and anti-inflammatory properties. The anti-inflammatory effect is mediated through the regulation of various inflammatory cytokines, such as nitric oxide (NO), interleukins (ILs), tumor necrosis factor alpha (TNF-α), interferon gamma (INF)-γ as well as non-cytokine mediator, prostaglandin E2 (PGE2). Up to now, the anti-inflammatory activity of β-glucans has received little attention. It is worthwhile to investigate the anti-inflammatory properties of fungal β-glucans in a separate review, discussing in vitro studies, animal studies and human studies on anti-inflammation effects of fungal β-glucans, as well as the structure-anti-inflammatory activity relationships.
cytokines, macrophages, anti-inflammatory, inflammatory responses
Publication DOI: 10.1016/j.tifs.2014.09.002Purified chitin-glucan complex (CGCpure) was extracted from Komagataella pastoris biomass using a hot alkaline treatment, followed by neutralization and repeated washing with deionized water. The co-polymer thus obtained had a β-glucan:chitin molar ratio of 75:25 and low protein and inorganic salts contents (3.0 and 0.9 wt%, respectively). CGCpure had an average molecular weight of 4.9 × 10(5)Da with a polydispersity index of 1.7, and a crystallinity index of 50%. Solid-state NMR provided structural insight at the co-polymer. X-ray diffraction suggests that CGCpure has α-chitin in its structure. CGCpure presented an endothermic decomposition peak at 315°C, assigned to the degradation of the saccharide structures. This study revealed that K. pastoris CGC has properties similar to other chitinous biopolymers and may represent an attractive alternative to crustacean chitin derived-products, being a reliable raw material for the development of new/improved pharmaceutical, cosmetic or food products.
structural analysis, molecular weight, chitin–glucan complex (CGC), Komagataella pastoris, thermal properties
NCBI PubMed ID: 26076647The major objective of the study was to analyse exopolysaccharide produced by a biofilm forming-clinical strain of Candida albicans. The biofilm-forming ability of C. albicans recovered from infected intrauterine devices (IUDs) was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay. The morphological characteristics of the biofilm were assessed using scanning electron microscopy (SEM), atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). Biochemical characterization of the exopolysaccharide was carried out by gel permeation chromatography, gas chromatography (GC), Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. Microscopic studies of C. albicans biofilm revealed complex, heterogeneous three-dimensional structure, in which yeast cells and hyphal elements were entrenched within exopolysaccharides matrix. Chromatographic analysis data indicated C. albicans exopolysaccharide (c. 300 kDa) to be made up of four major sugar units. The FTIR spectrum revealed specific absorbance of O-H, C-H, O=C=O, C=O, C-N and C-C ring stretching. (1) H and (13) C NMR data showed the presence of β (1→6) and β (1→3) linkages in the exopolysaccharide chain that were assigned to α-D-glucose and β-D-glucose, α-D-mannose, α-L-rhamnose and N-acetyl glucosamine (β-D-GlcNAc), respectively. Study suggested the production of a water soluble c. 300 kDa exopolysaccharide by C. albicans made up of glucose, mannose, rhamnose and N-acetyl glucosamine subunits.
exopolysaccharide, Biofilm, Candida albicans, Fourier transform infrared, infected intrauterine device, nuclear magenitc resonance
NCBI PubMed ID: 20002865Many species of higher basidiomycetes have traditionally been used because of their medicinal properties. The positive effects associated to the consumption of those fungi have been mainly attributed to cell wall polysaccharides, which have important structural roles and are present throughout the entire life cycles of fungi. One of the most consumed and studied species native of the Americas is Agaricus subrufescens, a mushroom prescribed in different countries for prophylaxis and noninvasive treatment of numerous health-related disorders. Prior to the process of extraction, purification, and application of these polysaccharides, one needs to be concerned with the preservation of the specimen and production of fungal biomass. Even though basidiomata (syn. fruiting body, mushrooms) generally yield larger volumes of biomass when compared to the mycelium, cultivation of mycelium allows a more efficient control of the process and, therefore, is the method of choice of polysaccharide production. Mycelial biomass can be produced by solid-state fermentation (SSF) or submersed fermentation (SmF). Further separation and concentration of bioactive polysaccharides can be done by means of porous membranes, such as tangential flow nanofiltration.
polysaccharide, biological activity, Agaricus subrufescens, fungal biomass, solid-state fermentation (SSF), submersed fermentation (SmF), nanofiltration
Publication DOI: 10.1007/978-3-319-16298-0_21In this study, we aim to investigate the physiochemical and biological properties of water-soluble phosphorylated polysaccharides (P-DIP) obtained from a water-insoluble polysaccharide (DIP) extracted from Dictyophora indusiata. A series of physiochemical properties were determined, including morphology, water-solubility, molecular weight, and degree of substitution (DS). To investigate the antioxidant activity of P-DIP, we determined the scavenging activity of hydroxyl radicals and DPPH, as well as the reducing power. MTT assay was performed to determine the cytotoxic effects of DIP and P-DIP on the cellular proliferation of MCF-7 and B16 cells. Compared with DIP, P-DIP showed a satisfactory water-solubility and significant increase in the antioxidant properties. Moreover, P-DIP also showed more significant inhibitory effects on the growth of MCF-7 and B16 tumor cells than the water-insoluble DIP. These results indicated that phosphorylation might contribute to the improvement of water solubility, as well as antioxidant and anti-tumor activities of natural DIP.
phosphorylation, Dictyophora indusiata, anti-oxidant and anti-tumor activity, dialyisis, centrifugation
NCBI PubMed ID: 25316421Biological activities of medicinal mushrooms have been attributed to β-(1→3),(1→6)-glucans that are present in the cell wall of fungi and some plants. Antitumor, immunomodulatory, antimicrobial, antinociception, antiinflammatory, prebiotic, antioxidant, and antidiabetic are some of different properties already described for β-(1→3),(1→6)-glucans. Immune activation systems, including specific β-glucan receptors like Dectin-1, complement (CR3), and Toll (TLR), have been identified to clarify these biological effects. The β-(1→3)-glucans are synthesized by β-(1→3)-glucan synthase (GLS), an enzyme belonging to the glucosyltransferase group, which has a catalytic unit (FKS) and another regulatory (RHO). The mechanisms for adding β-(1→6) branches to the non-reducing ends of the β-(1→3)-glucan chains are unclear until now. Due to the biological importance of β-(1→3),(1→6)-glucan, it is necessary to understand the biochemical and molecular mechanisms of its synthesis, both to optimize the production of bioactive compounds and to develop antifungal drugs that interrupt this process. Therefore, the aim of this review is to gather information about the potential of β-(1→3),(1→6)-glucans, their methods of isolation, purification, and chemical characterization, as well as how these biomolecules are synthesized by fungi and what studies involving biotechnology or molecular biology have contributed to this subject.
characterization, biotechnology, molecular biology, β-(1→3), (1→6)-glucans, β-(1→3)-glucan synthase, medicinal activities
NCBI PubMed ID: 26252967Consumption of edible mushrooms has been practiced since ages to promote human health and as traditional remedies for multiple human ailments. The excellent nutritional quality of these organisms collectively called as filamentous fungi for their filament like hyphal extension, owes to a high protein, low fat and cholesterol free profile. Over the last decade, a diverse repertoire of protein-glycan conjugates isolated from these organisms has been attributed with immunomodulatory, anticancer, and other therapeutic activities. An integrated use of conventional chemical analyses, improved separation technologies, and new generation high-throughput proteomic approaches has revealed features in these complex biomolecules unknown elsewhere amongst the eukaryotes. However, due to some serious technological bottlenecks, a comprehensive structure-function delineation of the fungal glycoproteome has eluded the scientists. If we can prevail over these constraints and dig deep into this unique niche of the fungal kingdom, the quest for new generation nutraceuticals and therapeutics will get headway.
glycoproteins, mushrooms, Glycoproteomics, filamentous fungi, polysaccharopeptide complexes, nutraceuticals, therapeutic enzymes
Publication DOI: 10.1002/9781118930458.ch20The Ascomycete Cordyceps militaris, an entomopathogenic fungus, is one of the most important traditional Chinese medicines. Studies related to its pharmacological properties suggest that this mushroom can exert interesting biological activities. Aqueous (CW and HW) and alkaline (K5) extracts containing polysaccharides were prepared from this mushroom, and a β-D-glucan was purified. This polymer was analysed by GC-MS and NMR spectrometry, showing a linear chain composed of β-D-Glc p (1R3)-linked. The six main signals in the 13C-NMR spectrum were assigned by comparison to reported data. The aqueous (CW, HW) extracts stimulated the expression of IL-1β, TNF-α, and COX-2 by THP-1 macrophages, while the alkaline (K5) extract did not show any effect. However, when the extracts were added to the cells in the presence of LPS, K5 showed the highest inhibition of the pro-inflammatory genes expression. This inhibitory effect was also observed for the purified β-(1→3)-D-glucan, that seems to be the most potent anti-inflammatory compound present in the polysaccharide extracts of C. militaris. In vivo, β-(1→3)-D-glucan also inhibited significantly the inflammatory phase of formalin-induced nociceptive response, and, in addition, it reduced the migration of total leukocytes but not the neutrophils induced by LPS. In conclusion, this study clearly demonstrates the anti-inflammatory effect of β-(1→3)-D-glucan.
Cordyceps militaris
NCBI PubMed ID: 25330371Glucans comprise an important class of polysaccharides present in basidiomycetes with potential biological activities. A (1→3)-β-D-glucan was isolated from Pleurotus sajor-caju via extraction with hot water followed by fractionation by freeze-thawing and finally by dimethyl sulfoxide extraction. The purified polysaccharide showed a 13C-NMR spectrum with six signals consisting of a linear glucan with a β-anomeric signal at 102.8 ppm and a signal at 86.1 ppm relative to O-3 substitution. The other signals at 76.2, 72.9, 68.3, and 60.8 ppm were attributed to C5, C2, C4, and C6, respectively. This structure was confirmed by methylation analysis, and HSQC studies. The β-D-glucan from P. sajor-caju presented an immunomodulatory activity on THP-1 macrophages, inhibited the inflammatory phase of nociception induced by formalin in mice, and reduced the number of total leukocytes and myeloperoxidase levels induced by LPS. Taken together, these results demonstrate that this β-D-glucan exhibits a significant anti-inflammatory activity.
β-D-glucan, fruiting bodies, Pleurotus sajor-caju, Edible mushroom, anti-inflammatory activity
NCBI PubMed ID: 25256522Roots of forest trees are associated with various ectomycorrhizal (ECM) fungal species that are involved in nutrient exchange between host plant and the soil compartment. The identification of ECM fungi in small environmental samples is difficult. The present study tested the feasibility of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy followed by hierarchical cluster analysis (HCA) to discriminate in situ collected ECM fungal species. Root tips colonized by distinct ECM fungal species, i.e., Amanita rubescens, Cenococcum geophilum, Lactarius subdulcis, Russula ochroleuca, and Xerocomus pruinatus were collected in mono-specific beech (Fagus sylvatica) and mixed deciduous forests in different geographic areas to investigate the environmental variability of the ECM FTIR signatures. A clear HCA discrimination was obtained for ECM fungal species independent of individual provenance. Environmental variability neither limited the discrimination between fungal species nor provided sufficient resolution to discern species sub-clusters for different sites. However, the de-convoluted FTIR spectra contained site-related spectral information for fungi with wide nutrient ranges, but not for Lactarius subdulcis, a fungus residing only in the litter layer. Specific markers for distinct ECM were identified in spectral regions associated with carbohydrates (i.e., mannans), lipids, and secondary protein structures. The present results support that FTIR spectroscopy coupled with multivariate analysis is a reliable and fast method to identify ECM fungal species in minute environmental samples. Moreover, our data suggest that the FTIR spectral signatures contain information on physiological and functional traits of ECM fungi.
Cluster Analysis, deciduous forests, field samples, infrared spectroscopy, mycorrhiza, soilborne fungi
NCBI PubMed ID: 24904624Virulence of Candida is linked with its ability to form biofilms. Once established, biofilm infections are nearly impossible to eradicate. Biofilm cells live immersed in a self-produced matrix, a blend of extracellular biopolymers, many of which are uncharacterized. In this study, we provide a comprehensive analysis of the matrix manufactured by Candida albicans both in vitro and in a clinical niche animal model. We further explore the function of matrix components, including the impact on drug resistance. We uncovered components from each of the macromolecular classes (55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid) in the C. albicans biofilm matrix. Three individual polysaccharides were identified and were suggested to interact physically. Surprisingly, a previously identified polysaccharide of functional importance, β-1,3-glucan, comprised only a small portion of the total matrix carbohydrate. Newly described, more abundant polysaccharides included α-1,2 branched α-1,6-mannans (87%) associated with unbranched β-1,6-glucans (13%) in an apparent mannan-glucan complex (MGCx). Functional matrix proteomic analysis revealed 458 distinct activities. The matrix lipids consisted of neutral glycerolipids (89.1%), polar glycerolipids (10.4%), and sphingolipids (0.5%). Examination of matrix nucleic acid identified DNA, primarily noncoding sequences. Several of the in vitro matrix components, including proteins and each of the polysaccharides, were also present in the matrix of a clinically relevant in vivo biofilm. Nuclear magnetic resonance (NMR) analysis demonstrated interaction of aggregate matrix with the antifungal fluconazole, consistent with a role in drug impedance and contribution of multiple matrix components. Importance: This report is the first to decipher the complex and unique macromolecular composition of the Candida biofilm matrix, demonstrate the clinical relevance of matrix components, and show that multiple matrix components are needed for protection from antifungal drugs. The availability of these biochemical analyses provides a unique resource for further functional investigation of the biofilm matrix, a defining trait of this lifestyle.
mannan, glucan
NCBI PubMed ID: 25096878D-Glucans have triggered increasing interest in commercial applications in the chemical and pharmaceutical sectors because of their technological properties and biological activities. The glucans are foremost among the polysaccharide groups produced by microorganisms with demonstrated activity in stimulating the immune system, and have potential in treating human disease conditions. Chemical alterations in the structure of D-glucans through derivatization (sulfonylation, carboxymethylation, phosphorylation, acetylation) contributes to their increased solubility that, in turn, can alter their biological activities such as antioxidation and anticoagulation. This review surveys and cites the latest advances on the biological and technological potential of D-glucans following chemical modifications through sulfonylation, carboxymethylation, phosphorylation or acetylation, and discusses the findings of their activities. Several studies suggest that chemically modified D-glucans have potentiated biological activity as anticoagulants, antitumors, antioxidants, and antivirals. This review shows that indepth future studies on chemically modified glucans with amplified biological effects will be relevant in the biotechnological field because of their potential to prevent and treat numerous human disease conditions and their clinical complications
exopolysaccharides, α- and β-Glucans, biomolecules
NCBI PubMed ID: 25239192Scleroglucan is a β-(1,3)-glucan which is highly branched at the 6-position with a single glucose residue. Acid hydrolysis of a high molecular weight scleroglucan gave a medium molecular weight, freely soluble material. Linkage analysis by the partially methylated alditol acetate method showed that the solubilized material had 30% branching. When the material was subjected to partial Smith degradations, the percent branching was reduced accordingly to 12% or 17%. After the percent branching was reduced, the average molecular weight of the samples increased considerably, indicating the assembly of higher ordered aggregate structures. An aggregate number distribution analysis was applied to confirm the higher aggregated structures. These aggregated structures gave the material significantly enhanced activity in an in vitro oxidative burst assay compared to the highly branched material
scleroglucan, de-branched, aggregate number distribution
NCBI PubMed ID: 26015027Health benefits of the polysaccharide (1→3)-β-D-glucan, reported to induce immunobiological, hypocholesterolemic, and hypoglycemic effects in humans and animals, have made the isolation, characterization, and assay of a viable glucan product critical. A new analytical method, based on internal standard proton NMR analysis, for the assay of solvent-wet samples containing (1→3)-β-D-glucan is presented. The method enables glucan identification, provides a solvent-free assay, and improves upon the previous multistep extraction and lyophilization procedure by reducing the 1-2 day analysis time to 1-2 h. NMR offers a rapid method for quantifying the glucan in commercial samples, such as nutraceuticals, as well as industrial samples enabling better evaluation of the efficacy of these carbohydrates in health-related applications
carbohydrate, nutraceutical, nutritional supplements
NCBI PubMed ID: 11559108Ciliary beating by respiratory epithelial cells continuously purges pathogens from the lower airways. Here we investigated the effect of the fungal cell wall polysaccharides Galactomannan (GM) and Zymosan (Zym) on the adrenergic activated particle transport velocity (PTV) of tracheal epithelium. Experiments were performed using tracheae isolated from male C57BL/6J mice. Transport velocity of the cilia bearing epithelial cells was measured by analysing recorded image sequences. Generation of reactive oxygen species (ROS) were determined using Amplex Red reagents. PCR experiments were performed on isolated tracheal epithelium to identify adrenergic receptor mRNA. The adrenergic receptors α1D, α2A, β1 and β2 have been identified in isolated tracheal epithelium. We found epinephrine responsible for an increase in PTV, which could only be reduced by selective β-receptor-inhibition. In addition, either GM or Zym prevented the epinephrine induced PTV increase. Furthermore, we observed a strong ROS generation evoked by GM or Zym. However, epinephrine induced increase in PTV recovered in the presence of GM and Zym after application of ROS scavengers. Both GM or Zym trigger reversible ROS generation in tracheal tissue leading to inhibition of the β-adrenergic increase in PTV
Galactomannan, zymozan, β-receptor-inhibition, reversible ROS generation
NCBI PubMed ID: 26571499β-glucans, a group of polysaccharides exist in many organism species such as mushrooms, yeasts, oats, barley, seaweed, but not mammalians, have a variety of biological activities and applications in drugs and other healthcare products. In recent years, β-glucans have been studied as adjuvants in anti-infection vaccines as well as immunomodulators in anti-cancer immunotherapy. β-glucans can regulate immune responses when administered alone and can connect innate and adaptive immunity to improve immunogenicity of vaccines. When β-glucans act as immunostimulants or adjuvants, a set of receptors have been revealed to recognize β-glucans, including dectin-1, complement receptor 3 (CR3), CD5, lactosylceramide, and so on. Therefore, this review is mainly focused on the application of β-glucans as immune adjuvants, the receptors of β-glucans, as well as their structure and activity relationship which will benefit future research of β-glucans.
vaccine, adjuvant, receptor, β-glucan, anti-tumor, anti-infection, structure and activity relationship
NCBI PubMed ID: 30049632Dietary fiber chemical and physical structures may be critical to the comprehension of how they may modulate gut bacterial composition. We purified insoluble polymers from Cookeina speciosa, and investigated its fermentation profile in an in vitro human fecal fermentation model. Two glucans, characterized as a (1→3),(1→6)-linked and a (1→3)-linked β-D-glucans were obtained. Both glucans were highly butyrogenic and propiogenic, with low gas production, during in vitro fecal fermentation and led to distinct bacterial shifts if compared to fructooligosaccharides. Specific increases in Bacteroides uniformis and genera from the Clostridium cluster XIVa, such as butyrogenic Anaerostipes and Roseburia were observed. The (1→3)-linked β-D-glucan presented a faster fermentation profile compared to the branched (1→3),(1→6)-linked β-D-glucan. Our findings support the view that depending on its fine chemical structure, and likely its insoluble nature, these dietary fibers can be utilized to direct a targeted promotion of the intestinal microbiota to butyrogenic Clostridium cluster XIVa bacteria.
Glucans, butyrate, Clostridium cluster XIVa, Cookeina speciose, in vitro fecal fermentation
NCBI PubMed ID: 293528781,3-β-glucan is considered a fungal biomarker and exposure to this agent induces lung inflammation. Previous studies have shown that 1,3-β-glucan affects Th1 and Th2 immune responses. Interleukin (IL)-10 and transforming growth factor (TGF)-β, as typical anti-inflammatory cytokines, suppress the Th1 immune response. To investigate the effects of 1,3-β-glucan on the secretion of cytokines in co-cultured mouse macrophages and lymphocytes in vitro, mice were exposed to 1,3-β-glucan or phosphate-buffered saline (PBS) by intratracheal instillation. Following extraction and co-culture of macrophages and lymphocytes, which were treated with or without 1,3-β-glucan in vitro, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of cytokines and real-time reverse transcription (RT)-polymerase chain reaction (PCR) was used to investigate the mRNA expression of forkhead box p3 (Foxp3) in the cells. We showed that 1,3-β-glucan exposure in vitro decreased the secretion of Th1 cytokines and increased the secretion of Th2 cytokines in the culture media. Furthermore, 1,3-β-glucan exposure in vitro increased the secretion of IL-10 and TGF-β in the culture media. According to these results, 1,3-β-glucan exposure in vitro is suggested to promote the secretion of anti-inflammatory cytokines, which may lead to a decrease in the levels of Th1 cytokines and an increase in the levels of Th2 cytokines. 1,3-β-glucan is suggested to induce regulatory lymphocytes, which partly contributes to an increased secretion of anti-inflammatory cytokines in co-cultured mouse macrophages and lymphocytes in vitro.
1, 3-beta-glucan, th1/th2 cytokine, anti-inflammatory cytokine
NCBI PubMed ID: 23799616In order to enhance the solubility of yeast β-D-glucan and enlarge the applicable area, we investigated dynamic high pressure microfluidization (DHPM) treatment with ionic liquids as solvent to prepare water-soluble yeast β-D-glucan (WSG). The solubility of β-D-glucan was greatly impacted by the pressure of DHPM, residence time, and ratio of β-D-glucan and EmimAc. The yield of WSG reached 79.25 % (w/w) with the water solubility of 72.50 % (w/w). The DHPM treatment did not destroy the ionic liquid structure therefore the ionic liquids could be recycled. FTIR and (13)C NMR spectrum results showed that WSG was the polysaccharides with (1→3)-β-D-glycosidic bonds, after modification, the hydrogen bonding was destroyed. The chain conformation of WSG in 0.1M NaNO3 aqueous solutions at 25 °C were studied by laser light scattering and viscometry. The Mark-Houwink equation was established to be [η]=2.09×10(-2) MW(0.63) (cm(3)g(-1)). According to the conformational parameters calculated from wormlike cylinder model, WSG existed as semi-stiff chain in aqueous solution.
structure, solubilization, yeast β-D-glucan
NCBI PubMed ID: 26691385Over the past several decades, research on the synthesis and organization of the cell wall polysaccharides of Aspergillus fumigatus has expanded our knowledge of this important fungal structure. Besides protecting the fungus from environmental stresses and maintaining structural integrity of the organism, the cell wall is also the primary site for interaction with host tissues during infection. Cell wall polysaccharides are important ligands for the recognition of fungi by the innate immune system and they can mediate potent immunomodulatory effects. The synthesis of cell wall polysaccharides is a complicated process that requires coordinated regulation of many biosynthetic and metabolic pathways. Continuous synthesis and remodeling of the polysaccharides of the cell wall is essential for the survival of the fungus during development, reproduction, colonization and invasion. As these polysaccharides are absent from the human host, these biosynthetic pathways are attractive targets for antifungal development. In this review, we present recent advances in our understanding of Aspergillus fumigatus cell wall polysaccharides, including the emerging role of cell wall polysaccharides in the host-pathogen interaction.
polysaccharide, cell wall, Biofilm, glycobiology, Aspergillus fumigatus
NCBI PubMed ID: 26920883Exopolysaccharides play an important structural and functional role in the development and maintenance of microbial biofilms. Although the majority of research to date has focused on the exopolysaccharide systems of biofilm-forming bacteria, recent studies have demonstrated that medically relevant fungi such as Candida albicans and Aspergillus fumigatus also form biofilms during infection. These fungal biofilms share many similarities with those of bacteria, including the presence of secreted exopolysaccharides as core components of the extracellular matrix. This review will highlight our current understanding of fungal biofilm exopolysaccharides, as well as the parallels that can be drawn with those of their bacterial counterparts.
polysaccharide, exopolysaccharide, Bacterial Adhesion, Biofilm, Extracellular matrix, carbohydrate biosynthesis
NCBI PubMed ID: 27129222Water-insoluble β-(1-3)-D-glucan isolated from the sclerotium of Poria cocos hardly exhibits biological activity. Therefore, it is advantageous to produce a value-added product from P. cocos. We extracted the β-(1-3)-D-glucan from the sclerotium of P. cocos and synthesized a carboxymethylated derivative. The structural and physiological properties of the derivative were investigated. The carboxymethylation of the polysaccharides was confirmed by Fourier transform infrared spectroscopy, and the degree of substitution (DS) and molecular weight were obtained by the potentiometric titration and gel permeation chromatography (GPC) analysis, respectively. The carboxymethylation caused the enhancement of in vitro bile acid binding capacity of the polysaccharides, which would be explained by the improved water solubility and structural changes caused by carboxymethylation. In addition, in vitro antiradical capacity of the derivative was observed by the method of 2,2-diphenyl-1-picrylhydrazyl (DPPH).
carboxymethylation, Poria cocos, DPPH, bile acid
NCBI PubMed ID: 19877636(1→3)-β-D-Glucans have received much attention with respect to their biological functions. A novel method to extract (1→3)-β-D-glucan from Saccharomyces cerevisiae cell wall is proposed in present work, which is based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL 1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal conditions were 52 °C, pH 5.5 and 1.5% (v/v) ethyl acetate. The kinetic behaviour of the yeast autolytic process under the optimized conditions was further studied. After 36 h of autolysis, 42.0% (w/w) cellular substances were released while the cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL 1 was treated by the novel method.
Saccharomyces cerevisiae, (1→3)-β-D-glucan, induced autolysis, sodium hypochlorite
Publication DOI: 10.1023/B:WIBI.0000007328.21451.78(1→3)-β-D-Glucan isolated from Poria cocos was phosphorylated to obtain a series of phosphorylated derivatives. Their structures, weight-average molecular weights (Mw), and chain conformation were studied by 13C NMR, 31P NMR, static laser light scattering and viscometry. The experimental results revealed that the phosphorylated glucan existed as relatively extended flexible chain in 0.15 M NaCl aqueous solution, and exhibited relatively strong inhibition against S-180 tumor cell in vitro and in vivo. In vivo, the fractions with relatively high molecular weight at low dosage exhibited stronger anti-tumor activities. The results revealed that the molecular weights and molecular conformation could influence the anti-tumor activities. The molecular weight ranging from 26000 to 268000 and the extended chain conformation were beneficial to enhance the anti-tumor activity, as a result of the increasing of the interaction between polysaccharide and immune system.
molecular weight, Chain conformation, (1→3)-β-D-glucan, anti-tumor activity, phosphorylated derivative
Publication DOI: 10.1016/j.carbpol.2009.05.019The fungal cell wall contains glycoproteins that interact with the host immune system. In the prominent pathogenic yeast Candida albicans, Pmr1 acts as a Golgi-resident ion pump that provides cofactors to mannosyltransferases, regulating the synthesis of mannans attached to glycoproteins. To gain insight into a putative conservation of such a crucial process within opportunistic yeasts, we were particularly interested in studying the role of the PMR1 homolog in a low-virulent species that rarely causes candidiasis, Candida guilliermondii. We disrupted C. guilliermondii PMR1 and found that loss of Pmr1 affected cell growth and morphology, biofilm formation, susceptibility to cell wall perturbing agents, mannan levels, and the wall composition and organization. Despite the significant increment in the amount of β1,3-glucan exposed at the wall surface, this positively influenced only the ability of the mutant to stimulate IL-10 production by human monocytes, suggesting that recognition of both mannan and β1,3-glucan, is required to stimulate strong levels of pro-inflammatory cytokines. Accordingly, our results indicate C. guilliermondii sensing by monocytes was critically dependent on the recognition of N-linked mannans and β1,3-glucan, as reported in other Candida species. In addition, chemical remotion of cell wall O-linked mannans was found to positively influence the recognition of C. guilliermondii by human monocytes, suggesting that O-linked mannans mask other cell wall components from immune cells. This observation contrasts with that reported in C. albicans. Finally, mice infected with C. guilliermondii pmr1Δ null mutant cells had significantly lower fungal burdens compared to animals challenged with the parental strain. Accordingly, the null mutant showed inability to kill larvae in the Galleria mellonella infection model. This study thus demonstrates that mannans are relevant for the C. guilliermondii-host interaction, with an atypical role for O-linked mannans.
virulence, cell wall, protein glycosylation, Candida guilliermondii, host-fungus interplay, mannosylation pathway
NCBI PubMed ID: 27994582Ganoderma 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). 1H 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.
cytotoxicity, antimicrobial activity, (1, Ganoderma lucidum, 3)-β-D-glucan sulfate
NCBI PubMed ID: 26907757Brewer's yeast is used in production of beer since millennia, and it is receiving increased attention because of its distinct fermentation ability and other biological properties. During fermentation, autolysis occurs naturally at the end of growth cycle of yeast. Yeast cell wall provides yeast with osmotic integrity and holds the cell shape upon the cell wall stresses. The cell wall of yeast consists of β-glucans, chitin, mannoproteins, and proteins that cross linked with glycans and a glycolipid anchor. The variation in composition and amount of cell wall polysaccharides during autolysis in response to cell wall stress, laying significant impacts on the autolysis ability of yeast, either benefiting or destroying the flavor of final products. On the other hand, polysaccharides from yeast cell wall show outstanding health effects and are recommended to be used in functional foods. This article reviews the influence of cell wall polysaccharides on yeast autolysis, covering cell wall structure changings during autolysis, and functions and possible applications of cell wall components derived from yeast autolysis.
cell wall, polysaccharides, β-glucan, MAPK, PKC, α-glucan, Bgs, GTPase
Publication DOI: 10.1016/j.tcsw.2018.10.001Nanoparticle-based delivery technologies have played a central role in a wide variety of applications, including cell therapy, gene transformation, and cellular delivery of molecular dyes. This work synthesized via ionic exchange a nanoparticle consisting of zinc-layered hydroxychloride coupled with yeast β-glucan (ZG), whose cellular immune response was evaluated using fish spleen leukocytes. Leukocytes from the marine Pacific red snapper (Lutjanus peru) were stimulated with zinc-layered hydroxychloride (ZHC) coupled with yeast β-glucan (GLU) and challenged with live Vibrio parahaemolyticus after 24 h. Structural characterization of this yeast glucan by proton nuclear magnetic resonance (NMR) indicated structures containing (1-6)-branched (1-3)-β-D-glucan. The ZHC and ZG were characterized with X-ray diffraction, infrared spectroscopy, scanning electron microscopy and thermogravimetric analysis. The results of the immunological study showed that ZHC, GLU or ZG were safe for leukocytes because cell viability was higher than 80% compared with DMSO or V. parahaemolyticus exposure. The ZG or GLU treatments enhanced nitric oxide production, superoxide dismutase, catalase and peroxidase activities. Induction of anti- and pro-inflammatory cytokine (IL-1β, IL-6, IL-8, IL-10, IL-12 and IL-17) genes was more pronounced in ZG or GLU treatments compared to the other groups. Based on the results, ZHC nanoparticles can be used as a delivery carrier of yeast β-glucan for enhancing immunity in fish and have great potential application in the aquaculture industry.
immune system, β-glucan, fish leukocytes, in vitro assay, layered hydroxide salt
NCBI PubMed ID: 30170109This review discusses the wealth of information available for the N. crassa cell wall. The basic organization and structure of the cell wall is presented and how the wall changes during the N. crassa life cycle is discussed. Over forty cell wall glycoproteins have been identified by proteomic analyses. Genetic and biochemical studies have identified many of the key enzymes needed for cell wall biogenesis, and the roles these enzymes play in cell wall biogenesis are discussed. The review includes a discussion of how the major cell wall components (chitin, β-1,3-glucan, mixed β-1,3-/β-1,4- glucans, glycoproteins, and melanin) are synthesized and incorporated into the cell wall. We present a four-step model for how cell wall glycoproteins are covalently incorporated into the cell wall. In N. crassa, the covalent incorporation of cell wall glycoproteins into the wall occurs through a glycosidic linkage between lichenin (a mixed β-1,3-/β-1,4- glucan) and a "processed" galactomannan that has been attached to the glycoprotein N-linked oligosaccharides. The first step is the addition of the galactomannan to the N-linked oligosaccharide. Mutants affected in galactomannan formation are unable to incorporate glycoproteins into their cell walls. The second step is carried out by the enzymes from the GH76 family of α-1,6-mannanases, which cleave the galactomannan to generate a processed galactomannan. The model suggests that the third and fourth steps are carried out by members of the GH72 family of glucanosyltransferases. In the third step the glucanosyltransferases cleave lichenin and generate enzyme/substrate intermediates in which the lichenin is covalently attached to the active site of the glucanosyltransferases. In the final step, the glucanosyltransferases attach the lichenin onto the processed galactomannans, which creates new glycosidic bonds and effectively incorporates the glycoproteins into the cross-linked cell wall glucan/chitin matrix.
cell wall, Galactomannan, glucan, filamentous fungi, melanin, Neurospora, glucanosyltransferase, mannanase
NCBI PubMed ID: 31649638Candida albicans is a commensal fungus that associates with human hosts. Under normal circumstances this interaction does not produce any severe life-threatening disease, as macrophages of the innate immune system will result in its clearance. However, disorders may arise in immunosuppressed individuals. To understand the bioactivity of Candida albicans cell wall polysaccharides, which represent an important component of its function, mannoprotein from this fungus was extracted, purified and analyzed. Mannoprotein with α-(1,2) and α-(1,6) linkages was investigated with use of HPLC and NMR. Co-incubation of mannoprotein with macrophages resulted in a mannoprotein with the potential to polarize macrophages to M1 and promote phagocytosis/microbial killing ability thus increasing the clearance of pathogens through Akt2. Moreover, mannoprotein within the cell wall promoted cell proliferation and inhibited apoptosis by activation of the Akt signaling pathway. Collectively, α-(1,6)(1,2)-mannoprotein, one of the five polysaccharides extracted from the cell wall of Candida albicans, demonstrates immune-enhancing effects by activation of the Akt signaling pathway. These findings provide important new insights into the biological effects of polysaccharides on macrophages. Such information can then serve as the foundation for the development of novel anti-fungal medications.
apoptosis, Candida albicans, Macrophage polarization, mannoprotein, cell cycle, Akt signal pathway
NCBI PubMed ID: 31005041The aim of this study was to investigate the effect of phosphorylation on the antioxidant activity of (1→3)-β-D-glucan from yeast cell wall. Alkali-insoluble (1→3)-β-D-glucan was extracted from yeast cell wall by an acid-base method. It was found that the purity of the sample was greatly improved after the precipitation was treated with alkali at 90 ℃ and then by acetic acid, which was about 96.5%. Phosphorylated (1→3)-β-D-glucan was prepared. Infrared (IR) spectra and nuclear magnetic resonance spectra (NMR) confirmed the successful introduction of phosphate into glucan. The substitution degree of phosphate was 0.18. The phosphorylated (1→3)-β-D-glucan could significantly increase SOD and CAT contents in serum, liver and brain of mice, and reduce MDA level in serum, liver and brain to a certain extent in vivo. This lays a solid foundation for the research and development of phosphorylated (1→3)-β-D-glucan antioxidant.
preparation, Structural characterization, yeast cell wall, antioxidant activities, phosphorylated (1→3)-β-D-glucan
NCBI PubMed ID: 31699554Zymosan (ZM), a naturally occurring insoluble macromolecule obtained from the cell wall of Saccharomyces cerevisiae, is used as a functional food (as dietary fiber), phagocytic stimulus, and immune potentiator. The present study aimed to increase its solubility and evaluate its immunological application. ZM was converted into soluble 6-amino-6-deoxy-β-(1-3)-glucan of a molecular weight of 296 kDa by reduction. Detailed structural characterization of aminated ZM was determined by Fourier transform infrared spectroscopy and two-dimensional NMR analysis (2D, COSY, TOCSY, ROSEY, NOSEY, and HSQC). Aminated ZM was biocompatible with Raw 264.7 macrophage cell lines up to a concentration of 100 μg/mL. Rhodamine tagging revealed that the aminated ZM microparticles were found localized within the nucleus of Raw 264.7 cells. Both native and aminated ZM showed a similar expression pattern of inflammatory genes in Raw 264.7.
amination, zymosan, Saccharomyces cerevisiae, immunological studies
NCBI PubMed ID: 32656418A rare Malaysian tiger milk mushroom Lignosus rhinocerus strain ABI (LRSA) was morphologically identified based on its pileus, stipe, and sclerotium. LRSA (515 bp) was sequenced and found to have 99% similar to L. rhinocerus strains CH31 and CH2. Phylogenetically, evolutionary distance (Knuc) and plasmid-matching software (ApE) for sequences of matching fungal species were used to verify that the isolate belonged to the L. rhinocerus species. The strain was cultured in a stirred-tank bioreactor and a mycelial β-glucan (G) was extracted for compound characterization. The structure of exopolysaccharide extract from mycelium of LRSA was studied using Fourier-transform infrared spectroscopy (FT-IR) and one-dimensional (1D) and two-dimensional (2D) Nuclear Magnetic Resonance (NMR). FT-IR spectroscopy showed that G exhibited a similar β-glycosidic structure to the standard (laminarin), and the presence of characteristic bands at 3277, 2919, 1638, 1545, 1400, 1078, and 896 1/cm confirmed the similarities. 1H and 13C NMR, as well as 2D NMR: homonuclear correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond coherence (HMBC) spectra, were used for structural elucidation of the β-glucan and confirmed the extracted material as (1,3)-β-D-glucan. In addition, the G compound exhibited antioxidant activities through total phenolic content (4.47 mg gallic acid equivalents/g), 2, 2-diphenyl-1-picrylhydrazyl (11.48 mg/mL), and ferric reducing antioxidant power (0.56 mg/mL) assays. These findings may facilitate the development of rare G production in a high-scale bioreactor using LRSA.
2D NMR, Antioxidant, (1, 3)-β-D-glucan, Lignosus rhinocerus, tiger milk mushroom, bioreactor fermentation
Publication DOI: 10.1016/j.bcab.2019.101455Beta-glucans as food hydrocolloids have demonstrated prebiotic potential recently. Novel hydrocolloid-based prebiotics could be developed by structural manipulation of existing carbohydrate candidates using different physical, chemical and/or biological approaches. However, there are no guidelines on choosing the most promising structural feature as the starting point(s) for modification. We performed a parallel in vitro fermentation of six native and carboxymethylated β-glucans from mushroom, bacteria, seaweed and cereal against five probiotic bacteria, including bifidobacteria and lactobacillus, and determined their growth parameters including the lag phase, maximum growth rate, and maximum population increase. An unsupervised analysis pipeline was developed to elucidate the structure-property relationship of β-glucans as prebiotic candidates and identified several chemical composition and structural features for modification to maximize their prebiotic potential. We adopted the random forest algorithm to quantify the relative importance of different composition and structural features in altering the three probiotic growth parameters. In terms of structural features, the desirable characteristics for prebiotic development are, in descending order of importance, low molecular weight, high water solubility, and no introduction of carboxymethyl functional group. Apart from structural features, we have identified the chemical composition as high purity, plays a significant importance in increasing the probiotic growth parameters, raising the relevance of including the purity in discussing the developmental cycle of prebiotic potential maximization. We suggest an integrated data alchemy approach including both machine learning and domain knowledge in making decision of choosing the promising starting points and direction for modification of existing hydrocolloids in prebiotic research.
chemical composition, Structural characterization, cyclic prebiotic potential maximization, structure-property relationship, machine learning, data alchemy approach
Publication DOI: 10.1016/j.foodhyd.2020.106001Polysaccharides from P. eryngii mushroom were selectively extracted using low-cost technologies (water at different conditions of temperature and pressure). Mannogalactan was the main polysaccharide in cold-water extracted fraction (CWEF), while a linear (1→6)-β-d-glucan was the main polymer in hot-water extracted fraction (HWEF). Autoclave-extracted fraction (AEF) contained a mixture of at least four different α- and β-glucans. The report of linear (1→6)-β-glucan and linear (1→3)-β-glucan is a new finding for P. eryngii fruiting bodies. The immunostimulatory properties of the fractions on THP-1 macrophages were studied. All fractions at 50, 250 and 500 μg/mL were not cytotoxic and produced different stimulus on NO, IL-1β and IL-10 secretion by the cells. Thus, our results showed that it is possible to concentrate different P. eryngii polysaccharides in selected fractions using a simple and low-cost procedure. Since biological effects depends on the polysaccharide structure, this technique allows the obtainment of fractions with distinct immunomodulatory activities.
polysaccharides, β-glucan, Grifola frondosa, mannogalactan, immunostimulatory properties, Pleurotus eryngii mushroom
NCBI PubMed ID: 33183624In the brewing process, the consumption of resources and the amount of waste generated are high and due to a lot of organic compounds in waste-water, the capacity of natural regeneration of the environment is exceeded. Residual yeast, the second by-product of brewing is considered to have an important chemical composition. An approach with nutritional potential refers to the extraction of bioactive compounds from the yeast cell wall, such as β-glucans. Concerning the potential food applications with better textural characteristics, spent brewer's yeast glucan has high emulsion stability and water-holding capacity fitting best as a fat replacer in different food matrices. Few studies demonstrate the importance and nutritional role of β-glucans from brewer's yeast, and even less for spent brewer's yeast, due to additional steps in the extraction process. This review focuses on describing the process of obtaining insoluble β-glucans (particulate) from spent brewer's yeast and provides an insight into how a by-product from brewing can be converted to potential food applications.
Saccharomyces cerevisiae, yeast, bioactive polysaccharides, particulate β-glucans, spent brewer's yeast
NCBI PubMed ID: 33467670The cell walls of fungi are critical for cellular structure and rigidity but also serve as a major communicator to alert the cell to the changing environment. In response to stresses encountered in human hosts, pathogenic fungi remodel their cell walls. Masking the β-1,3-glucan component of the cell wall is critical to escape detection by innate immune cells. We previously demonstrated that β-1,3-glucan is unmasked in response to host temperature stress when translatome reprogramming is defective in Cryptococcus neoformans Here, we used β-1,3-glucan unmasking as an output to identify signaling modules involved both in masking and in translatome reprogramming in response to host temperature stress. We reveal that the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway is involved in translatome reprogramming and that mutants in this pathway display moderate unmasking when grown at 37°C. Additionally, we show that mutants of the cell wall integrity (CWI)/Mpk1 MAPK pathway extensively unmask β-1,3-glucan. While the CWI pathway does not impact translatome reprogramming, our data suggest that it may play a role in the posttranslational regulation of transcription factors that govern masking.IMPORTANCECryptococcus neoformans is a fungal pathogen that causes devastating morbidity and mortality in immunocompromised individuals. It possesses several virulence factors that aid in its evasion from the host immune system, including a large polysaccharide capsule that cloaks the antigenic cell wall. Studies investigating how the cell wall is remodeled to keep this pathogen disguised in response to stress have been limited. We previously found that host temperature stress results in translatome reprogramming that is necessary for keeping the highly antigenic β-(1,3)-glucan component masked. Our data reveal signaling modules that trigger these responses and suggest the points of regulation at which these pathways act in achieving masking. Understanding these mechanisms may allow for therapeutic manipulation that may promote the immune recognition and clearance of this fungal pathogen.
cell wall, Glucans, Cryptococcus neoformans, stress response, cell signaling, host evasion
NCBI PubMed ID: 33568457Pentose-rich hydrolysate obtained from dilute acid pretreatment of oil palm empty fruit bunches was successfully consumed by pentose-consuming yeasts: Cyberlindnera jadinii (Cj) and Pichia jadinii (Pj). Nitrogen supplementation and no additional detoxification step were required. Pj produced 5.87 g/L of biomass using a C/N ratio of 14 after 120 h of fermentation, with xylose consumption of 71%. Cj produced 10.50 g/L of biomass after 96 h of fermentation with C/N ratio of 11.5, with maximum xylose consumption of 85%. β-Glucans, high value-added macromolecules, were further extracted from the yeast biomass, achieving yields of 3.1 and 3.0% from Pj and Cj, respectively. The isolated polysaccharides showed a chemical structure of β-(1,3)-glucan with residues of other molecules. Additionally, β-(1,6) branches seems to have been broken during isolation process. Further studies assessing β-glucans production at industrial scale should be carried out looking for nitrogen sources and optimizing the β-glucan isolation method.
β-glucan, Cyberlindnera jadinii, dilute acid pretreatment, oil palm empty fruit bunches, Pichia jadinii, bioprocess
NCBI PubMed ID: 33157450Background: The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital. Results: Here we examined the efficiency and reproducibility of 12 recombinant endo-β(1,3)-D-glucanases for specifically degrading the cell wall β(1,3)-D-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, β(1,3)-D-glucan and β(1,6)-D-glucan. We found that the GH16 endo-β(1,3)-D-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(1,3)-D-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-β(1,3)-D-glucanase no longer commercially available. Moreover, other recombinant β(1,3)-D-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides. Conclusions: The discovery of a reliable and efficient recombinant endo-β(1,3)-D-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall β(1,3)-D-glucan. This new commercial endo-β(1,3)-D-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis.
cell wall, fungi, β(1, fission yeast, 3)-D-glucan, recombinant endo-β(1, 3)-D-glucanase
NCBI PubMed ID: 34217291Vast efforts have been devoted to the development of antifungal drugs targeting the cell wall, but the supramolecular architecture of this carbohydrate-rich composite remains insufficiently understood. Here we compare the cell wall structure of a fungal pathogen Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. High-resolution solid-state NMR spectroscopy of intact cells reveals a rigid core formed by chitin, β-1,3-glucan, and α-1,3-glucan, with galactosaminogalactan and galactomannan present in the mobile phase. Gene deletion reshuffles the composition and spatial organization of polysaccharides, with significant changes in their dynamics and water accessibility. The distribution of α-1,3-glucan in chemically isolated and dynamically distinct domains supports its functional diversity. Identification of valines in the alkali-insoluble carbohydrate core suggests a putative function in stabilizing macromolecular complexes. We propose a revised model of cell wall architecture which will improve our understanding of the structural response of fungal pathogens to stresses.
polysaccharides, Aspergillus fumigatus, chitin, fungal cell wall, solid-state NMR
NCBI PubMed ID: 34732740A common edible mushroom Lentinula edodes, is an important source of numerous biologically active substances, including polysaccharides, with immunomodulatory and antitumor properties. In the present work, the biological activity of the crude, homogenous (Se)-enriched fraction (named Se-Le-30), which has been isolated from L. edodes mycelium by a modified Chihara method towards human peripheral blood mononuclear cells (PBMCs) and peripheral granulocytes, was investigated. The Se-Le-30 fraction, an analog of lentinan, significantly inhibited the proliferation of human PBMCs stimulated with anti-CD3 antibodies or allostimulated, and down-regulated the production of tumor necrosis factor (TNF)-? by CD3+ T cells. Moreover, it was found that Se-Le-30 significantly reduced the cytotoxic activity of human natural killer (NK) cells. The results suggested the selective immunosuppressive activity of this fraction, which is non-typical for mushroom derived polysaccharides.
polysaccharides, Lentinula edodes, selenium, immunosuppressant
NCBI PubMed ID: 34944419Although most fungi cause pathogenicity toward human beings, dynasties of the East Asian region have domesticated and utilized specific fungi for medical applications. The Japanese dynasty and nation have domesticated and utilized koji fermented with non-pathogenic fungus Aspergillus oryzae for more than 1300 years. Recent research has elucidated that koji contains medicinal substances such as Taka-diastase, acid protease, koji glycosylceramide, kojic acid, oligosaccharides, ethyl-α-d-glucoside, ferulic acid, ergothioneine, pyroglutamyl leucine, pyranonigrin A, resistant proteins, deferriferrichrysin, polyamines, Bifidobacterium-stimulating peptides, angiotensin I-converting enzyme inhibitor peptides, 14-dehydroergosterol, β-glucan, biotin, and citric acid. This review introduces potential medical applications of such medicinal substances to hyperlipidemia, diabetes, hypertension, cardiovascular and cognitive diseases, chronic inflammation, epidermal permeability barrier disruption, coronavirus disease 2019 (COVID-19), and anti-cancer therapy.
fermentation, Aspergillus, fungi, koji, medical application, medicinal substances, non-pathogenic fungus
NCBI PubMed ID: 33804991We previously described the biosynthesis, isolation, and immunosuppressive activity of the selenium-containing polysaccharide fraction isolated from the mycelial culture of Lentinula edodes. Structural studies have shown that the fraction was a protein-containing mixture of high molar mass polysaccharides α- and β-glucans. However, which of the components of the complex fraction is responsible for the immunosuppressive activity non-typical for polysaccharides of fungal origin has not been explained. In the current study, we defined four-polysaccharide components of the Se-containing polysaccharide fraction determined their primary structure and examined the effect on T- and B-cell proliferation. The isolated Se-polysaccharides, α-1,4-glucan (Mw 2250000 g/mol), unbranched β-1,6-D-glucan, unbranched β-1,3-D-glucan and β-1,3-branched β-1,6-D-glucan (Mw 110000 g/mol), are not typical as components of the cell wall of L. edodes. All are biologically active, but the inhibitory effect of the isolated polysaccharides on lymphocyte proliferation was weaker, though more selective than that of the crude fraction.
polysaccharides, T lymphocyte, Lentinula edodes, immunosuppressant, Se-containing polysaccharide
NCBI PubMed ID: 34500837Mushroom D-glucans are recognized as dietary fibers and as biologically active natural polysaccharides, with the advantages of being quite inexpensive for production, tolerable, and having a range of possible structures and physicochemical properties. The prebiotic potential of mushroom D-glucans has been explored in recent years, but the relationship between their various structural features and activity is poorly understood. This review focuses on comprehensively evaluating the prebiotic potential of mushroom D-glucans in face of their structural variations. Overall, mushroom D-glucans provide a unique set of different structures and physicochemical properties with prebiotic potential, where linkage type and solubility degree seem to be associated with prebiotic activity outcomes. The understanding of the effects of distinct structures and physicochemical properties in mushroom D-glucans on the gut microbiota contributes to the design and selection of new prebiotics in a more predictable way.
physicochemical properties, mushroom D-glucans, prebiotic effect
NCBI PubMed ID: 33838817The mushrooms have contributed to the development of active ingredients of fundamental importance in the field of pharmaceutical chemistry as well as of important tools in human and animal health, nutrition, and functional food. This review considers studies on the beneficial effects of medicinal mushrooms on the nutrition and health of humans and farm animals. An overview of the chemical structure and composition of mycochemicals is presented in this review with particular reference to phenolic compounds, triterpenoids and sterols, fatty acids and lipids, polysaccharides, proteins, peptides, and lectins. The nutritional value and chemical composition of wild and cultivated mushrooms in Italy is also the subject of this review which also deals with mushrooms as nutraceuticals and the use of mushrooms in functional foods. The nutraceutical benefits of UV irradiation of cultivated species of basidiomycetes to generate high amounts of vitamin D2 is also highlighted and the ability of the muhsrooms to inhibit glycation is analyzed. Finally, attention is paid to studies on bioactivities of some Italian wild and cultivated mushrooms with particular reference to species belonging to the genus Pleurotus. The review highlights the potential of medicinal mushrooms in the production of mycochemicals that represent a source of drugs, nutraceutical, and functional food. Graphic abstract: [Figure not available: see fulltext.]
mushroom, chemical structures, cultivation, nutrition, fungal diversity, mycochemicals
Publication DOI: 10.1007/s11101-021-09748-2Extracellular matrixes (ECMs), such as the cell walls and biofilms, are important for supporting cell integrity and function and regulating intercellular communication. These biomaterials are also of significant interest to the production of biofuels and the development of antimicrobial treatment. Solid-state nuclear magnetic resonance (ssNMR) and magic-angle spinning-dynamic nuclear polarization (MAS-DNP) are uniquely powerful for understanding the conformational structure, dynamical characteristics, and supramolecular assemblies of carbohydrates and other biomolecules in ECMs. This review highlights the recent high-resolution investigations of intact ECMs and native cells in many organisms spanning across plants, bacteria, fungi, and algae. We spotlight the structural principles identified in ECMs, discuss the current technical limitation and underexplored biochemical topics, and point out the promising opportunities enabled by the recent advances of the rapidly evolving ssNMR technology.
bacteria, algae, Plants, fungi, ssNMR
NCBI PubMed ID: 34878762As an endemic species,Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb. is widely distributed, such as in China, Korea, Japan, and North America, which have had a dual-purpose resource for medicines and food for over 2000 years. The applications of W. cocos were used to treat diseases including edema, insomnia, spleen deficiency, and vomiting. What's more, there have been wide uses of such edible fungi as a function food or dietary supplement recently. Up until now, 166 kinds of chemical components have been isolated and identified from W. cocos including triterpenes, polysaccharides, sterols, diterpenes, and others. Modern pharmacological studies showed that the components hold a wide range of pharmacological activities both in vitro and in vivo, such as antitumor, anti-inflammatory, antibacterial, anti-oxidant, and antidepressant activities. In addition, present results showed that the mechanisms of pharmacological activities were closely related to chemical structures, molecular signaling paths and the expression of relate proteins for polysaccharides and triterpenes. For further in-depth studies on this fungus based on the recent research status, this review provided some perspectives and systematic summaries of W. cocos in traditional uses, chemical components, pharmacological activities, separation and analysis technologies, and structure-activity relationships.
review, chemical components, pharmacological activities, structure-activity relationship (SAR), Wolfiporia cocos
NCBI PubMed ID: 35300566Fermentum is a common unicellular fungus with many biological activities attributed to β-polysaccharides. Different in vivo and in vivo experimental studies have long proven that fermentum β-polysaccharides have antioxidant, anti-tumor, and fungal toxin adsorption properties. However, there are many uncertainties regarding the relationship between the structure and biological activity of fermentum β-polysaccharides, and a systematic summary of fermentum β-polysaccharides is still lacking. Herein, we reviewed the research progress about the extraction, structure and modification, structure-activity relationship, activity and application of fermentum β-polysaccharides, compared the extraction methods of fermentum β-polysaccharide, and paid special attention to the structure-activity relationship and application of fermentum β-polysaccharide, which provided a strong basis for the development and application of fermentum β-polysaccharide.
activity, modification, structure-activity relationship, extraction, application, fermentum β-polysaccharides
NCBI PubMed ID: 36211774Termitomyces are well-known wild edible and medicinal basidiomycete mushrooms. The frequent consumption of Termitomyces stimulated studies on their health-promoting properties. Numerous health benefits of Termitomyces are associated with the main categories of components in Termitomyces, polysaccharides. Although the homopolysaccharides β-glucans are believed to be the major bioactive polysaccharides of Termitomyces, other heteropolysaccharides also possess biological activities. In this review, the extraction methods, chemical structures, and biological activities of polysaccharides from Termitomyces were thoroughly reviewed. The polysaccharides from different species of Termitomyces differ in molecular weight, monosaccharide composition, and linkages of constituent sugars. The health-promoting effects, including antioxidation, ulcer-healing and analgesic properties, immunomodulation, hypolipidemic and hepatoprotective effects, and antidiabetic properties of Termitomyces polysaccharides were summarized and discussed. Further studies were needed for a better understanding of the relationship between the fine chemical structure and health-promoting properties. This review provides a theoretical overview for future studies and utilization of Termitomyces polysaccharides.
polysaccharides, mushroom, Structures, bioactivities, Termitomyces
NCBI PubMed ID: 34936332Cordyceps militaris is a parasitic edible fungus and has been used as tonics for centuries. Polysaccharides are a major water-soluble component of C. militaris. Recently, C. militaris-derived polysaccharides have been given much attention due to their various actions including antioxidant, anti-inflammatory, anti-tumor, anti-hyperlipidemic, anti-diabetic, anti-atherosclerotic, and immunomodulatory effects. These bioactivities are determined by the various structural characteristics of polysaccharides including monosaccharide composition, molecular weight, and glycosidic linkage. The widespread use of advanced analytical analysis tools has greatly improved the elucidation of the structural characteristics of C. militaris-derived polysaccharides. However, the methods for polysaccharide structural characterization and the latest findings related to C. militaris-derived polysaccharides, especially the potential structure-activity relationship, have not been well-summarized in recent reviews of the literature. This review will discuss the methods used in the elucidation of the structure of polysaccharides and structural characteristics as well as the signaling pathways modulated by C. militaris-derived polysaccharides. This article provides information useful for the development of C. militaris-derived polysaccharides as well as for investigating other medicinal polysaccharides.
polysaccharide, structure-activity relationship, bioactivity, Cordyceps militaris, mechanisms of action
NCBI PubMed ID: 35711557Fungal β-glucans have received a lot of interest due to their proinflammatory activity towards cells of the innate immune system. Although commonly described as (1➔3)-β-glucans with varying degree of (1➔6)-branching, the fungal β-glucans constitute a diverse polysaccharide class. In this study, the alkali-soluble β-glucans from the edible mushroom Pleurotus eryngii were extracted and characterized by GC, GC-MS and 2D NMR analyses. The extracts contain several structurally different polysaccharides, including a (1➔3)-β-d-glucan with single glucose units attached at O-6, and a (1➔6)-β-d-glucan, possibly branched at O-3. The immunomodulatory activities of the P. eryngii extracts were assessed by investigating their ability to bind to the receptor dectin-1, and their ability to induce production of the proinflammatory cytokines TNF-α, IL-6 and IL-1β in LPS-differentiated THP-1 cells. Although the samples were able to bind to the dectin-1a receptor, they did not induce production of significant levels of cytokines in the THP-1 cells. Positive controls of yeast-derived (1➔3)-β-d-glucans with branches at O-6 induced cytokine production in the cells. Thus, it appears that the P. eryngii β-glucans are unable to induce production of proinflammatory cytokines in LPS-differentiated THP-1 cells, despite being able to activate the human dectin-1a receptor.
NMR, THP-1, Dectin-1, dispersion, mushroom glucans
NCBI PubMed ID: 37839837Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, β-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications.
cell wall, solid-state NMR, Aspergillus sydowii, halophilic fungi
NCBI PubMed ID: 37925437Mushrooms have progressively transitioned from a rich man's diet to a popular universally accepted nutrient-rich food. Packed with multiple elixirs for health and human welfare, mushrooms have gained paramount importance. Mushroom polysaccharides have now become recognized as the icons of mushroom bioactivity. This being the case, detection, identification, characterization, structural elucidation of these polysaccharides becomes ideally unequivocal. This review briefly highlights the biological importance of mushroom polysaccharides. The analytical techniques currently applied for analysis of mushroom polysaccharides have been consolidated and summarized. The current scenario while using MALDI TOF MS for polysaccharide analysis and further its application for mushroom polysaccharide analysis has been comprehensively gathered and presented for the first time. This pioneering work, based on the accumulated information, addresses the gaps in this application area, points out to the challenges and limitations and proposes future recommendations for widening the prospects for fully utilizing this analytical tool (MALDI-TOF MS).
mass spectrometry, MALDI-TOF-MS, mushroom polysaccharides, medicinal properties
Publication DOI: 10.1016/j.trac.2023.117095Poria cocos alkali-soluble polysaccharide (PCAP), a water-insoluble β-glucan, is the main component of the total dried sclerotia of Poria cocos. However, its gelation behaviour and properties have yet to be comprehensively studied. In this study, an acid-induced physical hydrogel based on natural PCAP is fabricated. The acid-induced gelation in PCAP is explored with respect to the pH and polysaccharide concentration. PCAP hydrogels are formed in the pH range of 0.3-10.5, and the lowest gelation concentration is 0.4 wt%. Furthermore, dynamic rheological, fluorescence, and cyclic voltammetry measurements are performed to elucidate the gelation mechanism. The results reveal that hydrogen bonds and hydrophobic interactions play a dominant role in gel formation. Subsequently, the properties of the PCAP hydrogels are investigated using rheological measurements, scanning electron microscopy, gravimetric analysis, free radical scavenging, MTT assays, and enzyme-linked immunosorbent assays. The PCAP hydrogels exhibit a porous network structure and cytocompatibility, in addition to good viscoelastic, thixotropic, water-holding, swelling, antioxidant, and anti-inflammatory activities. Furthermore, using rhein as a model drug for encapsulation, it is demonstrated that its cumulative release behaviour from the PCAP hydrogel is pH dependent. These results indicate the potential of PCAP hydrogels for application in biological medicine and drug delivery.
Poria cocos, alkali-soluble polysaccharide, acid-induced hydrogel, drug release, hydrogel characteristics
NCBI PubMed ID: 37030457Ganoderma lucidum, widely used in traditional medicine, has several biological properties. Polysaccharides, mainly glucans, are known as one of its main bioactive compounds. Consequently, the achievement and chemical investigation of such molecules are of pharmaceutical interest. Herein, we obtained water-insoluble and water-soluble polysaccharides from G. lucidum by alkaline extraction. Fractionation process yielded three fractions (GLC-1, GLC-2, and GLC-3). All samples showed to be composed mainly of glucans. GLC-1 is a linear (1→3)-linked β-glucan; GLC-2 is a mixture of three different linear polysaccharides: (1→3)-β-glucan, (1→3)-α-glucan, and (1→4)-α-mannan; while GLC-3 is a branched β-glucan with a (1→4)-linked main chain, which is branched at O-3 or O-6 by (1→3)- or (1→6)-linked side chains. This research reports the variability of glucans in Ganoderma lucidum fruiting bodies and applicable methodologies to obtain such molecules. These polysaccharides can be further applied in biological studies aiming to investigate how their chemical differences may affect their biological properties
branched, polysaccharides, linear, mannan, Glucans, Ganoderma lucidum
NCBI PubMed ID: 38574411Glucans are the most abundant class of macromolecule polymers in fungi, which are commonly found in Ascomycota and Basidiomycota. Fungal glucans are not only essential for cell integrity and function but also crucial for the immense industrial interest in high value applications. They present a variety of structural characteristics at the nanoscale due to the high regulation of genes and the involvement of stochastic processes in synthesis. However, although recent findings have demonstrated the genes of glucans synthesis are relatively conserved across diverse fungi, the formation and organization of diverse glucan structures is still unclear in fungi. Here, we summarize the structural features of fungal glucans and the recent developments in the mechanisms of glucans biosynthesis. Furthermore, we propose the engineering strategies of targeted glucan synthesis and point out the remaining challenges in the synthetic process. Understanding the synthesis process of diverse glucans is necessary for tailoring high value glucan towards specific applications. This engineering strategy contributes to enable the sustainable and efficient production of glucan diversity
biosynthesis, gene, diversity, glucan, fungi, target glucan
NCBI PubMed ID: 38286552The fungal cell wall plays a critical role in regulating cellular integrity and communication, and serves as a frontline defense against stress. It is also a prime target for the development of antifungal agents. The cell wall is comprised of diverse polysaccharides and proteins and poses a challenging target for high-resolution structural characterization. Recently, the solid-state nuclear magnetic resonance (ssNMR) analysis of intact Aspergillus fumigatus cells has provided atomic-level insights into the structural polymorphism and functional assembly principles of carbohydrate components within the cell wall. This physical perspective, alongside structural information from biochemical assays, offers a renewed understanding of the cell wall as a highly complex and dynamic organelle. Here, we summarize key conceptual advancements in the structural elucidation of A. fumigatus mycelial and conidial cell walls and their responses to stressors. We also highlight underexplored areas and discuss the opportunities facilitated by technical advancements in ssNMR spectroscopy
polysaccharide, carbohydrate, cell wall, glucan, Aspergillus, fungi, antifungal, chitin, solid-state NMR
Publication DOI: 10.3390/jof10030219The interaction between the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) and the resistant cowpea (Vigna unguiculata (L.) Walp.) cv. Queen Anne is characterized by the deposition of callose around intracellular fungal structures. Ultrastructural examination of the early stages of infection by basidiospores of the fungus revealed two types of deposits induced by intracellular hyphae: a non-callose collar at the penetration site and a callose encasement around the invasion hypha. The callose encasement was developed from the site where the fungus encountered the inside of the plant cell wall and was separated from the fungus by the extrahyphal membrane and an extension of plant plasma membrane. The incidence of encasements was reduced in plants treated with inhibitors of transcription (actinomycin D), protein synthesis (cycloheximide), protein glycosylation (tunicamycin) and microfilament polymerization (cytochalasin E). Inhibitors of Golgi-associated vesicle transfer (monensin, brefeldin A) and anti-microtubule agents (colchicine, oryzalin) had no effect. When the fungus was killed by heat treatment in either the resistant or the susceptible cultivar, callose was deposited at various locations along the fungus, mostly in the extrahyphal matrix. The data suggest that unlike the extrahaustorial membrane surrounding D-haustoria of this fungus, the extrahyphal membrane is capable of generating callose. Since this process does not normally occur in the susceptible or the resistant cv. even when callose is deposited in the latter by regions of the plasma membrane not associated with the fungus, we conclude that the deposition of callose by the extrahyphal membrane is inhibited by the living fungus.
resistance, Biotroph, callose, nhibitors, monokaryon
Publication DOI: 10.1046/j.1469-8137.1997.00760.xLaminarin, a linear β-1,3 glucan (mean degree of polymerization of 33) was extracted and purified from the brown alga Laminaria digitata. Its elicitor activity on tobacco (Nicotiana tabacum) was compared to that of oligogalacturonides with a mean degree of polymerization of 10. The two oligosaccharides were perceived by suspension-cultured cells as distinct chemical stimuli but triggered a similar and broad spectrum of defense responses. A dose of 200 μg mL−1 laminarin or oligogalacturonides induced within a few minutes a 1.9-pH-units alkalinization of the extracellular medium and a transient release of H2O2. After a few hours, a strong stimulation of Phe ammonia-lyase, caffeic acid O-methyltransferase, and lipoxygenase activities occurred, as well as accumulation of salicylic acid. Neither of the two oligosaccharides induced tissue damage or cell death nor did they induce accumulation of the typical tobacco phytoalexin capsidiol, in contrast with the effects of the proteinaceous elicitor β-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight β-1,3–1,6 glucans from fungal cell walls, and the β-1,6–1,3 heptaglucan showed that the elicitor effects observed in tobacco with β-glucans are specific to linear β-1,3 linkages, with laminaripentaose being the smallest elicitor-active structure. In accordance with its strong stimulating effect on defense responses in tobacco cells, infiltration of 200 μg mL−1 laminarin in tobacco leaves triggered accumulation within 48 h of the four families of antimicrobial pathogenesis-related proteins investigated. Challenge of the laminarin-infiltrated leaves 5 d after treatment with the soft rot pathogen Erwinia carotovora subsp. carotovora resulted in a strong reduction of the infection when compared with water-treated leaves.
Journal NLM ID: 0401224Susceptible and resistant cotton lines were cytologically and histochemically investigated for their defense reactions to a highly aggressive and defoliating strain of Verticillium dahliae, a fungus responsible for vascular wilt. Cytochemistry showed that early responses consisted of reinforcement in structural barriers with polysaccharides, including callose and cellulose. Ultrastructural modifications of parenchyma cells of the vascular tissues were associated with strong production of terpenoids and phenolics. These defense reactions were detected early in roots of the resistant line, one to four days after inoculation, while they were seen later in roots of the susceptible line.
ultrastructure, terpenoids, phenolics, callose, cytochemistry, vascular wilt
Publication DOI: 10.1023/A:1018558225454In plants, cell walls are one of the first lines of defence for protecting cells from successful invasion by fungal pathogens and are a major factor in basal host resistance. For the plant cell to block penetration attempts, it must adapt its cell wall to withstand the physical and chemical forces applied by the fungus. Papillae that have been effective in preventing penetration by pathogens are traditionally believed to contain callose as the main polysaccharide component. Here, we have re-examined the composition of papillae of barley (Hordeum vulgare) attacked by the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) using a range of antibodies and carbohydrate-binding modules that are targeted to cell wall polysaccharides. The data show that barley papillae induced during infection with Bgh contain, in addition to callose, significant concentrations of cellulose and arabinoxylan. Higher concentrations of callose, arabinoxylan and cellulose are found in effective papillae, compared with ineffective papillae. The papillae have a layered structure, with the inner core consisting of callose and arabinoxylan and the outer layer containing arabinoxylan and cellulose. The association of arabinoxylan and cellulose with penetration resistance suggests new targets for the improvement of papilla composition and enhanced disease resistance.
cellulose, callose, Blumeria graminis, arabinoxylan, papillae, penetration, powdery mildew
NCBI PubMed ID: 25138067The antibiotic fungal toxin brefeldin A (BFA) causes synthesis of additional cell wall material in adult differentiated onion inner epidermal cells at concentrations of 5–30 μg/ml. This tertiary wall contains callose and is layered on the secondary cellulosic wall in a time- and dose-dependent manner. Initially, callose is found in pit fields in the form of small vesicular patches. With time and dose, depositions grow in size and form large plugs invaginating into the cell, where the adjacent cytoplasm forms bulky accumulations and contains many organelles including endomembranes. Within the cytoplasm, BFA exerts the characteristic morphological effects on the secretory system including changes of the Golgi stacks, formation of large vesicles, and proliferation of dilated cisternae of the endoplasmic reticulum. Higher concentrations of BFA (60 μg/ml) lead to disintegration of the Golgi apparatus; they have no effects on the cell wall, no callose synthesis occurs. We conclude from these observations that BFA has two independent targets in onion cells. BFA acts on the plasma membrane, hence operating as an elicitor of plant defense reactions and thus activates callose synthesis. BFA acts also on the membranes of the secretory system and influences budding and fusion of vesicles at the endoplasmic reticulum and at the dictyosomes. These two mechanisms occur in parallel, suggesting that the secretory system still can play its presumed role in callose synthesis. Only when dictyosomes are completely disintegrated, no more callose is formed.
cell wall, signal transduction, Golgi apparatus, callose, brefeldin A, onion inner epidermis
Publication DOI: 10.1007/BF01282925Symplastic intercellular transport in plants is achieved by plasmodesmata (PD). These cytoplasmic channels are well known to interconnect plant cells to facilitate intercellular movement of water, nutrients, and signaling molecules including hormones. However, it is not known whether Al may affect this cell-to-cell transport process, which is a critical feature for roots as organs of nutrient/water uptake. We have microinjected the dye lucifer yellow carbohydrazide into peripheral root cells of an Al-sensitive wheat (Triticum aestivum cv Scout 66) either before or after Al treatment and followed the cell-to-cell dye-coupling through PD. Here we show that the Al-induced root growth inhibition is closely associated with the Al-induced blockage of cell-to-cell dye coupling. Immunofluorescence combined with immuno-electron microscopic techniques using monoclonal antibodies against 1→3-β-D-glucan (callose) revealed circumstantial evidence that Al-induced callose deposition at PD may responsible for this blockage of symplastic transport. Use of 2-deoxy-D-glucose, a callose synthesis inhibitor, allowed us to demonstrate that a reduction in callose particles correlated well with the improved dye-coupling and reduced root growth inhibition. While assessing the tissue specificity of this Al effect, comparable responses were obtained from the dye-coupling pattern in tobacco (Nicotiana tabacum) mesophyll cells. Analyses of the Al-induced expression of PD-associated proteins, such as calreticulin and unconventional myosin VIII, showed enhanced fluorescence and co-localizations with callose deposits. These results suggest that Al-signal mediated localized alterations to calcium homeostasis may drive callose formation and PD closure. Our data demonstrate that extracellular Al-induced callose deposition at PD could effectively block symplastic transport and communication in higher plants.
callose, aluminium, plasmodesmata, cell-to-cell trafficking inhibition
NCBI PubMed ID: 11080277| a-D-Glcp-(1-1)-a-D-Glcp | Show graphically |
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Show legend Show as text |
Structure type: oligomer
C12H22O11
Trivial name: trehalose, trehalose dihydrate
Compound class: glucan
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_742521,IEDB_983931,SB_192
SUMMARY: The dynamic response of cellular carbohydrates to a NaCl shock in Rhizobium leguminosarum biovar trifolii TA-1 (0.25 M-NaCl) and Rhizobium meliloti SU-47 (0.4 M-NaCl) grown in NaCl-free medium was investigated in non-growing cell cultures and in cell suspensions, using in vivo NMR. After transferring cells grown in a NaCl-free medium to a glutamic-acid-free medium containing mannitol and NaCl, both strains immediately responded to the increased osmotic pressure by augmenting the cellular trehalose content of the cell. Without mannitol in the medium trehalose synthesis was slower, but clearly detectable. Its synthesis paralleled the breakdown of the reserve materials glycogen and poly-β-hydroxybutyric acid (PHB). NMR experiments with 25-fold-concentrated cell suspensions using 13C1-mannitol as substrate revealed that 15-20% of the trehalose synthesized was derived from mannitol, but 80-85% was from other sources. Trehalose was mainly formed from the internal pool of glycogen and/or PHB, whether mannitol was present or not, and reached 135 and 280 μg (mg cell protein)-1 in the strains TA-1 and SU-47, respectively. At low osmolarity, intracellular trehalose was metabolized by strains TA-1 and SU-47. Intracellularly accumulated phosphoglycerol-substituted and neutral cyclic (1,2)-β-glucans of SU-47 cells grown in the absence of NaCl were neither degraded nor excreted after exposure to NaCl. Strain TA-1, which only makes neutral cyclic (1,2)-β-glucans, continued to synthesize and excrete cyclic (1,2)-β-glucans after exposure to NaCl. By using in vivo 31P-NMR, a sharp peak at 1.34 p.p.m. was present in cell suspensions of strain SU-47. This peak, representing glycerol-1-phosphate-substituted cyclic glucans, was absent in strain TA-1.
Journal NLM ID: 0375371Three aldoses, six alditols, eight oligosaccharides, three polysaccharides and a glycopeptide were found in a Sticta sp. The most interesting of these were gentiobiose, -triose and -tetraose, O-α-D-Galp-(1→6)-O-β-D-Galp-(1→1)-D-glyceritol released on alkaline treatment, and a polysaccharide with predominant (1→4)-linked β-D-Xylp units. Present were a galactoglucomannan, a polysaccharide, possibly a mixture, containing β-D-Galf, and α-L-Araf units and a glycopeptide with α- and β-D-Galf, Galp, Glcp and Manp units, having a core with successive (1→2)-linked α-D-Manp residues substituted at O-3 with those of α-D-Galf. After four years storage of the lichen, the contents of the principal polyols, ribitol (0.86%), arabinitol (0.99%) and mannitol (2.50%) fell to practically zero, with the appearance of threitol (0.03%), xylitol (0.01%) and myo-inositol (0.03%). Also, the protein content fell from 34 to 18%. The most abundant amino acids were valine (10.5% of total) in fresh lichen and threonine (14.6%) in stored lichen.
carbohydrates, protein, glycopeptide, Sticta sp., Stictacea, compositional variation on storage
NCBI PubMed ID: 7763796Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.
biosynthesis, Mycobacterium tuberculosis, sulfolipid, SL-1
NCBI PubMed ID: 22194604Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.
Molecular mechanics, Rhizobium meliloti, lectinology, lectins as targets, computer assisted, NMR spectoscopic, crystallographic elucidation, sugar code, chemioal tailoring, phosphodiester backbone, microheterogeneity of glycan, monomer variability, N acetylneuramicinic, transgenic pollen, nitrogen enriched nutrients, non agglutinating ricin, hydrophobic molecules, phytopathogenic fungus, phosphomannose mutase, B bearing individuals, anti adhesion therapy, NMR spectrum, parenchymal host cells
NCBI PubMed ID: 10702616Two novel glycolipids, emmyguyacin A (1a) and emmyguyacin B (1b), were isolated at concentrations of 1.51 g/L from a potato dextrose agar fermentation of a sterile fungus species. The compounds inhibit replication of influenza A virus (A/X31) in MDCK cells by inhibiting the pH-dependent conformational change of hemagglutinin A (IC50 9 μM). The structures were deduced using one- and two-dimensional NMR techniques and mass spectrometric analyses on both the parent compounds and a host of degradation products and derivatives. A novel and unusual oxalic acid ester of a monohydroxylated fatty acid (5, 17-oxalyloxydocosanoic acid) is reported. The first isolation and characterization of the fatty acid 17-hydroxydocosanoic acid (3) itself is also reported as a saponification product of 1.
Publication DOI: 10.1021/np010345aThe title compound, C12H22O11·2H2O {systematic name: 6,6'-oxybis[2-(hydroxymethyl)-3,4,5,6-tetra-hydro-2H-pyran-3,4,5-triol] dihydrate}, is a disaccharide, which was isolated from Tremella fuciformis. The molecule contains two six-membered rings, both of which adopt a chair conformation. Extensive O-H∙∙∙O hydrogen bonds occur in the crystal structure
Trehalose, Tremella fuciformis, single-crystal X-ray study
NCBI PubMed ID: 22904949lichen
Publication DOI: 10.1016/0008-6215(88)85057-2Up-to-date information concerning the chemical structure and properties of trehalose, its natural occurrence and biological functions in plants, fungi, and prokaryotes, as well as its practical application, mainly in medicine and biotechnology, are reviewed. A special section deals with the role of trehalose and other protective polyols in stress processes in fungi.
bacteria, microorganisms, Trehalose, mycelial fungi, protective carbohydrates
NCBI PubMed ID: 25844437Fly agaric (Amanita muscaria) was investigated using a 1H NMR-based metabolomics approach. The caps and stems were studied separately, revealing different metabolic compositions. Additionally, multivariate data analyses of the fungal basidiomata and the type of soil were performed. Compared to the stems, A. muscaria caps exhibited higher concentrations of isoleucine, leucine, valine, alanine, aspartate, asparagine, threonine, lipids (mainly free fatty acids), choline, glycerophosphocholine (GPC), acetate, adenosine, uridine, 4-aminobutyrate, 6-hydroxynicotinate, quinolinate, UDP-carbohydrate and glycerol. Conversely, they exhibited lower concentrations of formate, fumarate, trehalose, α- and β-glucose. Six metabolites, malate, succinate, gluconate, N-acetylated compounds (NAC), tyrosine and phenylalanine, were detected in whole A. muscaria fruiting bodies but did not show significant differences in their levels between caps and stems (P value>0.05 and/or OPLS-DA loading correlation coefficient <0.4). This methodology allowed for the differentiation between the fruiting bodies of A. muscaria from mineral and mineral-organic topsoil. Moreover, the metabolomic approach and multivariate tools enabled to ascribe the basidiomata of fly agaric to the type of topsoil. Obtained results revealed that stems metabolome is more dependent on the topsoil type than caps. The correlation between metabolites and topsoil contents together with its properties exhibited mutual dependences.
NCBI PubMed ID: 25437454Trehalose is a disaccharide constituted by two molecules of D-glucose, whose unique structural and physicochemical properties are responsible for its high stability. This carbohydrate accumulates inside the cytosol of yeast during abiotic stress due to its protective effect against desiccation, high temperatures, freezing, high salinity and oxidation. For this reason, trehalose has important applications in food, cosmetic, pharmaceutical industries and research. In this study, it was performed a comparative analysis of trehalose production in non-conventional yeasts Saccharomyces kluyveri, Candida shehatae and Candida guilliermondii evaluating the effect of two growth temperatures (28 and 34 °C) and two methods of cell breakage (mechanical or thermal disruption by bead beating or boiling, respectively). The growth rate (cells/mL) of the cells cultured at 34 °C was lower in comparison with the growth at 28 °C. S. kluyveri produced the higher amount of biomass in comparison with the yeasts from the Candida, C. shehatae was the only yeast in which no trehalose production was detected at any temperature. Based on Tukey-Kramer test, it was determined that C. guilliermondii yields the higher amount of trehalose in contrast to the two other species of yeast tested.
temperature, stress, Candida shehatae, Candida guilliermondii, Saccharomyces kluyveri
Journal NLM ID: 101722971Fungal propagules survive stresses better than vegetative cells. Neosartorya fischeri, an Aspergillus teleomorph, forms ascospores that survive high temperatures or drying followed by heat. Not much is known about maturation and development of extreme stress resistance in fungal cells. This study provides a novel two-step model for the acquisition of extreme stress resistance and entry into dormancy. Ascospores of 11- and 15-day-old cultures exhibited heat resistance, physiological activity, accumulation of compatible solutes and a steep increase in cytoplasmic viscosity. Electron spin resonance spectroscopy indicated that this stage is associated with the removal of bulk water and an increase of chemical stability. Older ascospores from 15- to 50-day-old cultures showed no changes in compatible solute content and cytoplasmic viscosity, but did exhibit a further increase of heat resistance and redox stability with age. This stage was also characterized by changes in the composition of the mixture of compatible solutes. Mannitol levels decreased and the relative quantities of trehalose and trehalose-based oligosaccharides increased. Dormant ascospores of N. fischeri survive in low-water habitats. After activation of the germination process, the stress resistance decreases, compatible solutes are degraded and the cellular viscosity drops. After 5 h, the hydrated cells enter the vegetative stage and redox stability has decreased notably
Oligosaccharides, mannitol, Trehalose, Neosartorya fischeri, stress resistance
NCBI PubMed ID: 25040022BACKGROUND: Pullulan and glycogen have many applications and physiological functions. However, to date, it has been unknown where and how the pullulan is synthesized in the yeast cells and if cell wall structure of the producer can affect pullulan and glycogen biosynthesis. METHODS: The genes related to cell wall integrity were cloned, characterized, deleted and complemented. The cell wall integrity, pullulan biosynthesis, glycogen accumulation and gene expression were examined. RESULTS: In this study, the GT6 and GT7 genes encoding different α(1,2) mannosyltransferases in Aureobasidium melanogenum P16 were cloned and characterized. The proteins deduced from both the GT6 and GT7 genes contained the conserved sequences YNMCHFWSNFEI and YSTCHFWSNFEI of a Ktr mannosyltransferase family. The removal of each gene and both the two genes caused the changes in colony and cell morphology and enhanced glycogen accumulation, leading to a reduced pullulan biosynthesis and the declined expression of many genes related to pullulan biosynthesis. The swollen cells of the disruptants were due to increased accumulation of glycogen, suggesting that uridine diphosphate glucose (UDP-glucose) was channeled to glycogen biosynthesis in the disruptants, rather than pullulan biosynthesis. Complementation of the GT6 and GT7 genes in the corresponding disruptants and growth of the disruptants in the presence of 0.6 M KCl made pullulan biosynthesis, glycogen accumulation, colony and cell morphology be restored. GENERAL SIGNIFICANCE: This is the first report that the two α1,2 mannosyltransferases were required for colony and cell morphology, glycogen accumulation and pullulan biosynthesis in the pullulan producing yeast.
α1, A. melanogenum, glycogen accumulation, pullulan biosynthesis, 2 mannosyltransferases
NCBI PubMed ID: 29550432Aspergillus fumigatus mitogen-activated protein kinases (MAPKs) are involved in maintaining the normal morphology of the cell wall and providing resistance against cell wall-damaging agents. Upon cell wall stress, cell wall-related sugars need to be synthesized from carbohydrate storage compounds. Here we show that this process is dependent on cAMP-dependent protein kinase A (PKA) activity and regulated by the high-osmolarity glycerol response (HOG) MAPKs SakA and MpkC. These protein kinases are necessary for normal accumulation/degradation of trehalose and glycogen, and the lack of these genes reduces glucose uptake and glycogen synthesis. Alterations in glycogen synthesis were observed for the sakA and mpkC deletion mutants, which also displayed alterations in carbohydrate exposure on the cell wall. Carbohydrate mobilization is controlled by SakA interaction with PkaC1 and PkaR, suggesting a putative mechanism where the PkaR regulatory subunit leaves the complex and releases the SakA-PkaC1 complex for activation of enzymes involved in carbohydrate mobilization. This work reveals the communication between the HOG and PKA pathways for carbohydrate mobilization for cell wall construction.IMPORTANCE Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections such as invasive pulmonary aspergillosis, especially in immunocompromised patients. The fungal cell wall is the main component responsible for recognition by the immune system, due to the specific composition of polysaccharide carbohydrates exposed on the surface of the fungal cell wall called pathogen-associated molecular patterns (PAMPs). Key enzymes in the fungal cell wall biosynthesis are a good target for fungal drug development. This report elucidates the cooperation between the HOG and PKA pathways in the mobilization of carbohydrates for fungal cell wall biosynthesis. We suggest that the reduced mobilization of simple sugars causes defects in the structure of the fungal cell wall. In summary, we propose that SakA is important for PKA activity, therefore regulating the availability and mobilization of monosaccharides for fungal cell wall biosynthesis during cell wall damage and the osmotic stress response.
cell wall, glycogen, Trehalose, Aspergillus fumigatus, MpkC, SakA, high-osmotic glycerol pathway, protein kinase A
NCBI PubMed ID: 30538182Trehalose is a nonreducing disaccharide with exceptional physical and chemical properties that explain its careful preservation throughout evolution and its wide presence in the biosphere, comprising both prokaryotic and eukaryotic organisms. Indeed, bacteria contain an elaborate system for trehalose biosynthesis, which includes three different and well-characterized pathways (OtsAB, TreS and TreYZ) that provide them with ample physiological roles; for example, as carbon source, structural component or compatible solute. In contrast, in eukaryotes only the OtsAB orthologous pathway has been conserved (termed the TPS/TPP route). Surprisingly, however, trehalose synthesis does not occur in vertebrates, although they possess hydrolytic mechanisms. The trehalose metabolism has been carefully elucidated in fungi, where the sugar plays crucial physiological roles as reserve carbohydrate, stress protector or virulence factor. Recent research has also provided new evidence on whether trehalose serves as antifungal target, metabolic regulator or signaling molecule, paving new ways for biotechnological and clinical applications. All these topics are covered in this article.
synthesis, virulence, carbohydrate, signal, hydrolysis, stress, regulator, Trehalose, fungi, antifungal, reserve, trehalase
Publication DOI: 10.1016/B978-0-12-809633-8.12084-9Trehalose is a nonreducing disaccharide, and it plays an intracellular protective role in organisms under various stress conditions. In this study, the trehalose synthesis and its protective role in Pleurotus ostreatus were investigated. As a signal in metabolic regulation, reactive oxygen species (ROS) accumulated in the mycelia of P. ostreatus under heat stress (HS). Furthermore, mycelial growth was significantly inhibited, and the malondialdehyde (MDA) level significantly increased under HS. First, exogenous addition of H2O2 inhibited mycelial growth and elevated the MDA level, while N-acetyl cysteine (NAC) and vitamin C (VC) reduced the MDA level and recovered mycelial growth under HS by scavenging ROS. These results indicated that the mycelial radial growth defect under HS might be partly caused by ROS accumulation. Second, adding NAC and VC to the media resulted in rescued trehalose accumulation, which indicated that ROS has an effect on inducing trehalose synthesis. Third, the mycelial growth was recovered by addition of trehalose to the media after HS, and the MDA level was reduced. This effect was further verified by the overexpression of genes for trehalose-6-phosphate synthase (TPS) and neutral trehalase (NTH), which led to increased and reduced trehalose content, respectively. In addition, adding validamycin A (NTH inhibitor) to the media promoted trehalose accumulation and the recovered mycelial growth after HS. In conclusion, trehalose production was partly induced by ROS accumulation in the mycelia under HS, and the accumulated trehalose could promote the recovery of growth after HS, partly by reducing the MDA level in the mycelia.
Trehalose, ROS, growth inhibition rate, heat stress, MDA
NCBI PubMed ID: 31069486Trehalose, mannitol and arabitol are the main saccharides of extant fungal metabolism, but their occurrence and distribution in geological materials have rarely been considered. Here, we identify these sugars in Miocene lignites and for the first time in Late Cretaceous mudstones and coals. The co-occurrence of trehalose, mannitol and arabitol in the sedimentary rocks investigated suggests their fungal origin, because these three saccharides are major compounds present in most modern fungi, including the very common mycorrhizal and wood-rotting groups. Therefore, we conclude that these sugars should be treated as new fungal biomarkers (biomolecules) present in geological rocks. Trehalose and mannitol are major compounds in total extracts of the samples and a sum of their concentration reaches 4.6 μg/g of sample. The arabitol concentrations do not exceed 0.5 μg/g, but in contrast to trehalose, the concentration correlates well with mannitol (R^2 = 0.94), suggesting that they have the same, translocatory role in fungi. Based on the trehalose vs. mannitol and arabitol distributions in Cretaceous samples and their comparison with data for modern fungi, we preliminarily conclude that the coal seams from the Rakowice Małe (SW Poland) section were formed during warmer climatic periods than the overlying sediments. Furthermore, no DNA could be isolated from the samples of lignites and overlying sediments, whereas it was abundant in the control samples of maple, birch and oak wood degraded by fungi. This indicates an absence of recent fungi responsible for decay in lignites and implies that the saccharide origin is connected with ancient fungi. Other sugar alcohols and acids like D-pinitol, quinic acid and shikimic acid, were found for the first time in sedimentary rocks, and their source is inferred to be from higher plants, most likely conifers. The preservation of mono- and disaccharides of fungal origins in pre-Palaeogene strata implies that compounds previously thought as unstable can survive for tens to hundreds of millions of years without structural changes in immature rocks unaffected by secondary processes.
saccharides, biomarkers, fungi, lignites, Miocene, Cretaceous
Publication DOI: 10.1016/j.coal.2018.11.003Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C–60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C–60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.
oligosaccharide, sophorose, koji, amazake, saccharification
NCBI PubMed ID: 30414826The study of metabolomics in natural products using the diverse analytical instruments including GC-MS, LC-MS, and NMR is useful for the exploration of physiological and biological effects and the investigation of drug discovery and health functional foods. Cordyceps militaris has been very attractive to natural medicine as a traditional Chinese medicine, due to its various bioactive properties including anti-cancer and anti-oxidant effects. In this study, we analyzed the metabolite profile in 50% ethanol extracts of C. militaris fruit bodies from three development periods (growth period, matured period, and aging period) using 1H-NMR, and identified 44 metabolites, which are classified as 16 amino acids, 10 organic acids, 5 carbohydrates, 3 nucleotide derivatives, and 10 other compounds. Among the three development periods of the C. militaris fruit body, the aging period showed significantly higher levels of metabolites including cordycepin, mannitol (cordycepic acid), and β-glucan. Interestingly, these bioactive metabolites are positively correlated with antitumor growth effect; the extract of the aging period showed significant inhibition of HepG2 hepatic cancer cell proliferation. These results showed that the aging period during the development of C. militaris fruit bodies was more highly enriched with bioactive metabolites that are associated with cancer cell growth inhibition.
nuclear magnetic resonance, β-glucan, metabolomics, Cordyceps militaris, cordycepin, anti-cancer effect
NCBI PubMed ID: 31336431Cultured Cordyceps militaris is very popular. To gain dynamic insight into activity markers in fruiting body of Cordyceps militaris (C. militaris) in Bombyx mori (B. mori), also named silkworm. The development stages of samples at 3, 9, 12, 19, 27, and 33 days after inoculation (DAI) were collected. HPLC coupled with diode array detection and evaporative light-scattering detection method (HPLC-DAD-ELSD) was used to determine eight makers, including six nucleosides and two carbohydrates from the samples. C. militaris cultured 33 DAI with fifth star silkworm larva could accumulate higher levels of cordycepin (13.43 mg/g) than the highest reported cordycepin (8.57 g/L). The contents of cordycepin, adenosine, and trehalose were gradually increased with the formation of C. militaris fruiting bodies on silkworm larva, while mannitol was decreased. The change of guanosine was similar to uracil. Results suggested that mannitol could be accumulated in a short period during mycelium growth and could metabolize and transform into energy store and trehalose during fruit body formation. The inosine in the insect was completely utilized and transformed. The synergistic formation of cordycepin and adenosine or differences in metabolized pathways are a great possibility according to the same trend. Highlights: This research offered some reference to further find a certain regularity or metabolic mechanism.
Cordyceps militaris, cordycepin, adenosine, HPLC-DAD-ELSD, Bombyx mori
NCBI PubMed ID: 30442223Infectious diseases are serious public health problems, affecting a large portion of the world's population. A molecule that plays a key role in pathogenic organisms is trehalose and recently has been an interest in the metabolism of this molecule for drug development. The trehalose-6-phosphate synthase (TPS1) is an enzyme responsible for the biosynthesis of trehalose-6-phosphate (T6P) in the TPS1/TPS2 pathway, which results in the formation of trehalose. Studies carried out by our group demonstrated the inhibitory capacity of T6P in the TPS1 enzyme from Saccharomyces cerevisiae, preventing the synthesis of trehalose. By in silico techniques, we compiled sequences and experimentally determined structures of TPS1. Sequence alignments and molecular modeling were performed. The generated structures were submitted in validation of algorithms, aligned structurally and analyzed evolutionarily. Molecular docking methodology was applied to analyze the interaction between T6P and TPS1 and ADMET properties of T6P were analyzed. The results demonstrated the models created presented sequence and structural similarities with experimentally determined structures. With the molecular docking, a cavity in the protein surface was identified and the molecule T6P was interacting with the residues TYR-40, ALA-41, MET-42, and PHE-372, indicating the possible uncompetitive inhibition mechanism provided by this ligand, which can be useful in directing the molecular design of inhibitors. In ADMET analyses, T6P had acceptable risk values compared with other compounds from World Drug Index. Therefore, these results may present a promising strategy to explore to develop a broad-spectrum antibiotic of this specific target with selectivity, potency, and reduced side effects, leading to a new way to treat infectious diseases like tuberculosis and candidiasis.
Drug design, Trehalose, molecular docking, Saccharomyces cerevisiae, ADMET properties, trehalose-6-phosphate synthase
NCBI PubMed ID: 31478225Several factors affect the vegetative growth of fungi, such as temperature, pH, and culture medium. In addition to mycelial growth, these factors affect metabolite production. There are limited studies that have identified the metabolites produced by the fungus Bjerkandera adusta, which have potential biotechnological applications. Here, we evaluated the effects of temperature, culture medium, and incubation time on the production of mycelial mass and metabolites of B. adusta isolated from Pinus taeda. The highest mycelial mass was obtained at 24 °C, in the potato dextrose and malt extract media, upon incubation for 28 and 35 days. The disaccharide α-α-trehalose was for the first time isolated and identified by X-ray diffraction in this fungal genus.
sugar, Crystallography, Bjerkandera adusta, Pinus spp, in vitro culture
NCBI PubMed ID: 32370630In Saccharomyces cerevisiae, α-glucosidase (maltase) is a key enzyme in maltose metabolism. In addition, the overexpression of the α-glucosidase-encoding gene MAL62 has been shown to increase the freezing tolerance of yeast in lean dough. However, its cryoprotection mechanism is still not clear. RNA sequencing (RNA-seq) revealed that MAL62 overexpression increased uridine diphosphoglucose (UDPG)-dependent trehalose synthesis. The changes in transcript abundance were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and enzyme activity assays. When the UDPG-dependent trehalose synthase activity was abolished, MAL62 overexpression failed to promote the synthesis of intracellular trehalose. Moreover, in strains lacking trehalose synthesis, the cell viability in the late phase of prefermentation freezing coupled with MAL62 overexpression was slightly reduced, which can be explained by the increase in the intracellular glycerol concentration. This result was consistent with the elevated transcription of glycerol synthesis pathway members. The increased freezing tolerance by MAL62 overexpression is mainly achieved by the increased trehalose content via the UDPG-dependent pathway, and glycerol also plays an important role. These findings shed new light on the mechanism of yeast response to freezing in lean bread dough and can help to improve industrial yeast strains.
glycerol, Saccharomyces cerevisiae, RNA-seq, α-glucosidase, freezing tolerant, lean dough, maltose, trehalose synthesis pathway
NCBI PubMed ID: 33076920The effect of eight cold-resistant yeast strains (J3, J7, J8, J9, J12, J15, J18, and J25) of Wickerhamomyces anomalus on the lipid oxidation of cold stored fish mince (4 °C) were investigated. And the metabolites of these yeast were determined with gas chromatography-mass spectrometry. These strains could effectively inhibit the increase of hydroperoxides value (p < 0.05), and the inhibiting rate was positively correlated with the content of isolongifolene, xylitol, turanose, thymol-glucoside, and uridine. Especially, the J3, J7, J8, J9, J12, and J18 could eliminate a large part of thiobarbituric acid reactive substances (TBARS) (p < 0.05), the eliminating rate was proportionate to the aldehyde dehydrogenase activity. Several bacteriostatic metabolites were detected: thymol-glucoside, 2-phenylethanol, cedro, and 2,4-bis (1,1-dimethylethyl) phenol. In addition, W. anomalus produced many metabolites with fruit and floral notes. In conclusion, cold-resistant W. anomalus strains own antioxidant activity were potential new bio-preservatives in the cold storage of muscle products.
metabolite, Antioxidant, Wickerhamomyces anomalus, cold-resistant, lipid oxidation, bio-preservative
NCBI PubMed ID: 31445174| b-D-Glcp-(1-3)-b-D-Glcp | Show graphically |
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Show legend Show as text |
Structure type: oligomer
Trivial name: laminaribiose
Compound class: lipophosphoglycan
Contained glycoepitopes: IEDB_142488,IEDB_146664,IEDB_153543,IEDB_983931,SB_192
Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.
Molecular mechanics, Rhizobium meliloti, lectinology, lectins as targets, computer assisted, NMR spectoscopic, crystallographic elucidation, sugar code, chemioal tailoring, phosphodiester backbone, microheterogeneity of glycan, monomer variability, N acetylneuramicinic, transgenic pollen, nitrogen enriched nutrients, non agglutinating ricin, hydrophobic molecules, phytopathogenic fungus, phosphomannose mutase, B bearing individuals, anti adhesion therapy, NMR spectrum, parenchymal host cells
NCBI PubMed ID: 10702616| -3)-b-D-Glcp-(1- | Show graphically |
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Structure type: structural motif or average structure
Trivial name: β-1,3-glucan
Compound class: EPS, O-polysaccharide, cell wall polysaccharide, glucan
Contained glycoepitopes: IEDB_1397514,IEDB_142488,IEDB_146664,IEDB_153543,IEDB_158555,IEDB_161166,IEDB_2278476,IEDB_2278477,IEDB_558869,IEDB_857743,IEDB_983931,SB_192
The walls of infectious pathogens, which are essential for transmission, pathogenesis, and diagnosis, contain sugar polymers that are defining structural features, e.g., β-1,3-glucan and chitin in fungi, chitin in Entamoeba cysts, β-1,3-GalNAc in Giardia cysts, and peptidoglycans in bacteria. The goal here was to determine in which of three walled forms of Toxoplasma gondii (oocyst, sporocyst, or tissue cyst) is β-1,3-glucan, the product of glucan synthases and glucan hydrolases predicted by whole-genome sequences of the parasite. The three most important discoveries were as follows. (i) β-1,3-glucan is present in oocyst walls of Toxoplasma and Eimeria (a chicken parasite that is a model for intestinal stages of Toxoplasma) but is absent from sporocyst and tissue cyst walls. (ii) Fibrils of β-1,3-glucan are part of a trabecular scaffold in the inner layer of the oocyst wall, which also includes a glucan hydrolase that has a novel glucan-binding domain. (iii) Echinocandins, which target the glucan synthase and kill fungi, arrest development of the Eimeria oocyst wall and prevent release of the parasites into the intestinal lumen. In summary, β-1,3-glucan, which can be targeted by drugs, is an important component of oocyst walls of Toxoplasma but is not a component of sporocyst and tissue cyst walls. Importance: We show here that walls of Toxoplasma oocysts, the infectious stage shed by cats, contain β-1,3-glucan, a sugar polymer that is a major component of fungal walls. In contrast to fungi, β-1,3-glucan is part of a trabecular scaffold in the inner layer of the oocyst wall that is independent of the permeability barrier formed by the outer layer of the wall. While glucan synthase inhibitors kill fungi, these inhibitors arrest the development of the oocyst walls of Eimeria (an important chicken pathogen that is a surrogate for Toxoplasma) and block release of oocysts into the intestinal lumen. The absence of β-1,3-glucan in tissue cysts of Toxoplasma suggests that drugs targeted at the glucan synthase might be used to treat Eimeria in chickens but not to treat Toxoplasma in people.
mass spectrometry, parasites, β-1, 3-glucan, Toxoplasma, Toxoplasma gondii, Cyst wall, Eimeria
NCBI PubMed ID: 23015739The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a β-(1→3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. β-(1→3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The β-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of β-(1→3)- and β-(1→6)-D-glucosidic linkages. The specific activity of the β-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.
cell wall, MALDIMS, β-glucan, Candida spp., limulus test
Publication DOI: 10.1016/S0008-6215(99)00049-XPolysaccharides of foliose thalli of Pseudocyphellaria clathrata (De Not.) Malme, a fungus lichenized with a green photobiont, were extracted and chemically characterized. The thalli were successively extracted with water and 2% aqueous KOH, at 100 degrees C. After neutralization, the solution was subjected to freeze-thawing and Fehling treatment of the supernatant precipitated a structurally complex galactoglucomannan (GGM) that was identical to a similar fraction obtained from Pseudocyphellaria aurata (Ach.) Vainio, a fungus lichenized by the green alga Dictyochloropsis symbiotica. Isolated from the supernatant of the Fehling precipitation, was an arabinogalactomannoglucan (AGMG) that contained arabinose, galactose, mannose, and glucose in a 12:34:40:14 molar ratio. Methylation analysis showed 18 different structures, the principal being nonreducing galactofuranosyl end-units (25%) with the main chain composed by (1→3)-linked β-glucopyranosyl units, as seen by controlled Smith degradation.
polysaccharides, lichen, Pseudocyphellaria clathrata
Publication DOI: 10.1639/0007-2745(2005)108[118:PPBTCP]2.0.CO;2This report describes a method for the solubilization of a micro-particulate β-D-glucan. Insoluble glucan is dissolved in methyl sulfoxide and urea (8m) and partially phiosphorylated at 100°. The resulting water-soluble product is called glucan phosphate. The conversion rate is 70%, and the preparation is endotoxin free as determined by the Limulus lysate procedure. Glucan phosphate is composed of 34.66% C, 6.29% H, 42.83% O, and 2.23% P and has a repeating-unit empirical formula of (C6H10O5)7·PO3H2, indicating a phosphate group substitution on every seventh glucose subunit. Molecular-weight averages, polydispersity, and intrinsic viscosity were determined by aqueous high-performance size-exclusion chromatography (s.e.c.) with on-line, multi-angle laser light scattering (m.a.1.1.s) photometry and differential viscometry (d.v.). Two polymer peaks were resolved. Peak 1 (Mw = 3570000 daltons), represents ~2% of the total polymers, while peak 2 (Mw = 110000 daltons) comprises ~98% of polymers. 13C- and 31P-n.m.r. spectroscopy confirmed the β-1,3 interchain linkage and the presence of a phosphate group. In solution, glucan phosphate polymers self-associate in a triple-helical arrangement. The ability to prepare a immunologically active, non-toxic, water-soluble β-D-glucan preparation will greatly enhance the clinical utility of this class of compounds.
phosphate, glucan, Saccharomyces cerevisiae, solubilization
NCBI PubMed ID: 1804535The cell wall polysaccharides of brewers spent yeast Saccharomyces pastorianus (BSY) and the inoculum yeast (IY) were studied in order to understand the changes induced by the brewing process. The hot water and alkali extractions performed solubilized mainly mannoproteins, more branched for BSY than those of IY. Also, (31)P solid state NMR showed that the BSY mannoproteins were 3 times more phosphorylated. By electron microscopy it was observed that the final residues of alkali sequential extraction until 4M KOH preserved the yeast three-dimensional structure. The final residues, composed mainly by glucans (92%), showed that the BSY, when compared with IY, contained higher amount of (1→4)-linked Glc (43% for BSY and 16% for IY) and lower (1→3)-linked Glc (17% for BSY and 42% for IY). The enzymatic treatment of final residue showed that both BSY and IY had (α1→4)-linked Glc and (β1→4)-linked Glc, in a 2:1 ratio, showing that S. pastorianus increases their cellulose-like linkages with the brewing process.
glucan, mannoprotein, alcoholic fermentation stress, brewers spent yeast, sequential extraction
NCBI PubMed ID: 25839826Changes in cell wall structure of four strains of Sacccharomyces cerevisiae species (brewer's, baker's and probiotic yeast) after culturing on deproteinated potato juice water (DPJW) with diverse addition of glycerol and different pH were investigated. It allowed to select conditions intensifying biosynthesis of β(1,3)/(1,6)-glucan and mannoproteins of cell walls of tested strains. Yeast cell wall structural polysaccharides show biological activity and technological usability in food industry but also decide about therapeutic properties of yeast biomass. The highest increase in the thickness of walls (by about 100%) and β-glucan layer (by about 120%) was stated after cultivation of S. cerevisiae R9 brewer's yeast in DPJW supplemented with 5 and 10% (w/v) of glycerol and pH 7.0 while S. cerevisiae var. boulardi PAN yeast synthesized by ab. 70% thicker β-glucan layer when the pH of growth medium was equal to 5.0. The cells of brewer's yeast (S. cerevisiae R9), probiotic (S. cerevisiae CNCM 1-745) and baker's (S. cerevisiae 102) intensified the ratio of mannoproteins in the structure of cell walls cultivated in mediums supplemented with above 15% of glycerol what point out the protective action of glycoprotein's under osmotic stress conditions. The study confirms at the first time the possibility of using agro-industrial waste in biosynthesis of functional polysaccharides of S. cerevisiae cell wall. It could be an new advantage in production of yeast biomass with therapeutic properties or β-glucan preparation as a novel food ingredient.
glycerol, Saccharomyces cerevisiae, mannoproteins, β(1, waste potato juice water, 3)/(1, 6)-glucan
NCBI PubMed ID: 29885339Zymoseptoria tritici is a hemibiotrophic pathogen which causes Septoria leaf blotch in wheat. The pathogenesis of the disease consists of a biotrophic phase and a necrotrophic phase. The pathogen infects the host plant by suppressing its immune response in the first stage of infection. Hemibiotrophic pathogens of the genus Fusarium cause Fusarium head blight, and the necrotrophic Parastagonosporanodorum is responsible for Septoria nodorum blotch in wheat. Cell wall-degrading enzymes in plants promote infections by necrotrophic and hemibiotrophic pathogens, and trichothecenes, secondary fungal metabolites, facilitate infections caused by fungi of the genus Fusarium. There are no sources of complete resistance to the above pathogens in wheat. Defense mechanisms in wheat are controlled by many genes encoding resistance traits. In the wheat genome, the characteristic features of loci responsible for resistance to pathogenic infections indicate that at least several dozen genes encode resistance to pathogens. The molecular interactions between wheat and Z. tritici, P. nodorum and Fusarium spp. pathogens have been insufficiently investigated. Most studies focus on the mechanisms by which the hemibiotrophic Z. tritici suppresses immune responses in plants and the role of mycotoxins and effector proteins in infections caused by P. nodorum and Fusarium spp. fungi. Trichothecene glycosylation and effector proteins, which are involved in defense responses in wheat, have been described at the molecular level. Recent advances in molecular biology have produced interesting findings which should be further elucidated in studies of molecular interactions between wheat and fungal pathogens. The Clustered Regularly-Interspaced Short Palindromic Repeats/ CRISPR associated (CRISPR/Cas) system can be used to introduce targeted mutations into the wheat genome and confer resistance to selected fungal diseases. Host-induced gene silencing and spray-induced gene silencing are also useful tools for analyzing wheat-pathogens interactions which can be used to develop new strategies for controlling fungal diseases.
Fusarium, Parastagonospora nodorum, Zymoseptoria tritici, anatomical barriers, pattern-triggered immunity
NCBI PubMed ID: 29642627Fungal β-1,3-glucan glucanosyltransferases are glucan-remodeling enzymes that play important roles in cell wall integrity, and are essential for the viability of pathogenic fungi and yeasts. As such, they are considered possible drug targets, although inhibitors of this class of enzymes have not yet been reported. Herein we report a multidisciplinary approach based on a structure-guided design using a highly conserved transglycosylase from Sacharomyces cerevisiae, that leads to carbohydrate derivatives with high affinity for Aspergillus fumigatus Gel4. We demonstrate by X-ray crystallography that the compounds bind in the active site of Gas2/Gel4 and interact with the catalytic machinery. The topological analysis of noncovalent interactions demonstrates that the combination of a triazole with positively charged aromatic moieties are important for optimal interactions with Gas2/Gel4 through unusual pyridinium cation-π and face-to-face π-π interactions. The lead compound is capable of inhibiting AfGel4 with an IC50 value of 42 μm.
carbohydrates, Oligosaccharides, Glycomimetics, Aspergillus fumigatus, transglycosylases
NCBI PubMed ID: 29164827Candida biofilms resist the effects of available antifungal therapies. Prior studies with Candida albicans biofilms show that an extracellular matrix mannan-glucan complex (MGCx) contributes to antifungal sequestration, leading to drug resistance. Here we implement biochemical, pharmacological, and genetic approaches to explore a similar mechanism of resistance for the three most common clinically encountered non-albicansCandida species (NAC). Our findings reveal that each Candida species biofilm synthesizes a mannan-glucan complex and that the antifungal-protective function of this complex is conserved. Structural similarities extended primarily to the polysaccharide backbone (α-1,6-mannan and β-1,6-glucan). Surprisingly, biochemical analysis uncovered stark differences in the branching side chains of the MGCx among the species. Consistent with the structural analysis, similarities in the genetic control of MGCx production for each Candida species also appeared limited to the synthesis of the polysaccharide backbone. Each species appears to employ a unique subset of modification enzymes for MGCx synthesis, likely accounting for the observed side chain diversity. Our results argue for the conservation of matrix function among Candida spp. While biogenesis is preserved at the level of the mannan-glucan complex backbone, divergence emerges for construction of branching side chains. Thus, the MGCx backbone represents an ideal drug target for effective pan-Candida species biofilm therapy.IMPORTANCECandida species, the most common fungal pathogens, frequently grow as a biofilm. These adherent communities tolerate extremely high concentrations of antifungal agents, due in large part, to a protective extracellular matrix. The present studies define the structural, functional, and genetic similarities and differences in the biofilm matrix from the four most common Candida species. Each species synthesizes an extracellular mannan-glucan complex (MGCx) which contributes to sequestration of antifungal drug, shielding the fungus from this external assault. Synthesis of a common polysaccharide backbone appears conserved. However, subtle structural differences in the branching side chains likely rely upon unique modification enzymes, which are species specific. Our findings identify MGCx backbone synthesis as a potential pan-Candida biofilm therapeutic target.
Biofilm, Extracellular matrix, Candida, antifungal resistance, non-albicans
NCBI PubMed ID: 29615504There are about 40 species in the Pleurotus genus, including those with high economic significance, i.e. P. ostreatus and P. pulmonarius. The fruiting bodies of oyster mushrooms are of high nutritional and health-promoting value. In addition, many species belonging to the Pleurotus genus have been used as sources of substances with documented medicinal properties, such as high-molecular weight bioactive compounds (polysaccharides, peptides and proteins) and low-molecular weight compounds (terpenoids, fatty acid esters and polyphenols). The bioactive substances contained in the mycelium and fruiting bodies of Pleurotus species exhibit immunostimulatory, anti-neoplastic, anti-diabetic, anti-atherosclerotic, anti-inflammatory, antibacterial and anti-oxidative properties. Their multidirectional positive influence on the human organism is the result of interaction of bioactive substances. Extracts from individual Pleurotus species can be used for the production of dietary supplements increasing the organism’s immunity. They are also used for the production of cosmetics. They can be added to functional foods as probiotics, or used as natural preservatives or ingredients of special foodstuffs for patients with specific diseases.
Functional food, fruiting bodies, health-promoting activities, oyster mushrooms
Publication DOI: 10.2478/fhort-2018-0012Azole antifungals inhibit the fungal ergosterol biosynthesis pathway, resulting in either growth inhibition or killing of the pathogen, depending on the species. Here we report that azoles have an initial growth-inhibitory (fungistatic) activity against the pathogen Aspergillus fumigatus that can be separated from the succeeding fungicidal effects. At a later stage, the cell wall salvage system is induced. This correlates with successive cell integrity loss and death of hyphal compartments. Time-lapse fluorescence microscopy reveals excessive synthesis of cell wall carbohydrates at defined spots along the hyphae, leading to formation of membrane invaginations and eventually rupture of the plasma membrane. Inhibition of β-1,3-glucan synthesis reduces the formation of cell wall carbohydrate patches and delays cell integrity failure and fungal death. We propose that azole antifungals exert their fungicidal activity by triggering synthesis of cell wall carbohydrate patches that penetrate the plasma membrane, thereby killing the fungus. The elucidated mechanism may be potentially exploited as a novel approach for azole susceptibility testing.
cell wall, Microscopy, antifungal activity, azoles
NCBI PubMed ID: 30082817Deadly infections from opportunistic fungi have risen in frequency, largely because of the at-risk immunocompromised population created by advances in modern medicine and the HIV/AIDS pandemic. This review focuses on dynamics of the fungal polysaccharide cell wall, which plays an outsized role in fungal pathogenesis and therapy because it acts as both an environmental barrier and as the major interface with the host immune system. Human fungal pathogens use architectural strategies to mask epitopes from the host and prevent immune surveillance, and recent work elucidates how biotic and abiotic stresses present during infection can either block or enhance masking. The signaling components implicated in regulating fungal immune recognition can teach us how cell wall dynamics are controlled, and represent potential targets for interventions designed to boost or dampen immunity.
cell wall, glucan, innate immunity, fungi, evasion
NCBI PubMed ID: 29452950Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and β-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential β-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of β-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast β-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.
Genes, sequencing, Proteomics, Sprassis crispa
NCBI PubMed ID: 30375506The high mortality of invasive fungal infections, and the limited number and inefficacy of antifungals necessitate the development of new agents with novel mechanisms and targets. The fungal cell wall is a promising target as it contains polysaccharides absent in humans, however, its molecular structure remains elusive. Here we report the architecture of the cell walls in the pathogenic fungus Aspergillus fumigatus. Solid-state NMR spectroscopy, assisted by dynamic nuclear polarization and glycosyl linkage analysis, reveals that chitin and α-1,3-glucan build a hydrophobic scaffold that is surrounded by a hydrated matrix of diversely linked β-glucans and capped by a dynamic layer of glycoproteins and α-1,3-glucan. The two-domain distribution of α-1,3-glucans signifies the dual functions of this molecule: contributing to cell wall rigidity and fungal virulence. This study provides a high-resolution model of fungal cell walls and serves as the basis for assessing drug response to promote the development of wall-targeted antifungals
cell wall, cell wall polymers, fungi, solid-state NMR
NCBI PubMed ID: 30013106Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus Beauveria bassiana displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (Bbgas3). Bbgas3 expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of Bbgas3 resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of ΔBbgas3 aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The ΔBbgas3 mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the ΔBbgas3 mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT50, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9).IMPORTANCE Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene (Bbgas3) regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus Beauveria bassiana to grow at extreme pH. The loss of Bbgas3 resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the ΔBbgas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.
β-1, filamentous fungi, Beauveria bassiana, cell wall integrity, alkaline tolerance, high pH, 3-glycanosyltransferase
NCBI PubMed ID: 29802184Glycoengineered yeast cells, which express human-compatible glycan structures, are particularly attractive host cells to produce therapeutic glycoproteins. Disruption of OCH1 gene, which encodes an α-1,6-mannosyltransferase required for mannan-type N-glycan formation, is essential for the elimination of yeast-specific N-glycan structures. However, the gene disruption causes cell wall defects leading to growth defects. Here, we tried to identify factors to rescue the growth defects of och1Δ cells by in vivo mutagenesis using piggyBac (PB)-based transposon. We isolated a mutant strain, named 121, which could grow faster than parental och1Δ cells. The PB element was introduced into the promoter region of BEM4 gene and upregulated the BEM4 expression. Overexpression of BEM4 suppressed growth defects in och1Δ cells. The slow grow phenotypes were partially rescued by expression of Rho1p, whose function is regulated by Bem4p. Our results indicate that BEM4 would be useful to produce therapeutic proteins in glycoengineered yeast without the growth defects.
glycosylation, glycoengineering, budding yeast, piggyBac transposon
NCBI PubMed ID: 29882469| -4)-b-D-GlcpNAc-(1- | Show graphically |
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Show legend Show as text |
Structure type: homopolymer
Trivial name: chitin
Compound class: O-polysaccharide, cell wall polysaccharide, glucan, polysaccharide, chitin
Contained glycoepitopes: IEDB_135813,IEDB_137340,IEDB_141807,IEDB_151531,IEDB_153212,IEDB_241099,IEDB_423114,IEDB_423150,SB_74,SB_85
This chapter describes the range of glycan structures and pathways that are found in different parasitic protozoa. All parasitic protists express a range of glycoconjugates that form protective protein-rich or carbohydrate-rich surface coats. Protein-rich coats are typically found on developmental stages that inhabit nonhydrolytic niches, such as the bloodstream and nonacidified intracellular vacuoles. These coats are commonly dominated by a limited repertoire of antigenically diverse proteins that are commonly, but not always, glycosylphosphatidylinositol- (GPI-) anchored and modified with N- or O-glycans. Carbohydrate-rich coats are commonly found on developmental stages that dwell within hydrolytic environments, such as vertebrate and arthropod digestive tracts and lysosomal vacuoles. These coats are dominated by GPI-anchored glycoproteins that are heavily modified with N-glycans, O-glycans, or phosphoglycans. Free GPI glycolipids (not attached to protein) can also be abundant or dominant components of these coats. Some parasitic protists can also form highly resistant cyst stages encased within polysaccharide-rich cell walls. Considerable progress has been made in defining the structures of the surface and intracellular glycans of the parasitic protists, their biosynthesis and the role that individual components play in parasite infectivity.
O-glycosylation, Glycosylphosphatidylinositol, N-glycosylation, protozoan parasites, Phosphoglycosylation
Publication DOI: 10.1016/B978-0-12-374546-0.00012-2The impact of fungal infections on humans is a serious public health issue that has received much less attention than bacterial infection and treatment, despite ever-increasing incidence exacerbated by an increased incidence of immunocompromised individuals in the population. Candida species, in particular, cause some of the most prevalent hospital-related fungal infections. Fungal infections are also detrimental to the well-being of grazing livestock, with milk production in dairy cows, and body and coat condition adversely affected by fungal infections. Fungal cell walls are essential for viability, morphogenesis and pathogenesis: numerous anti-fungal drugs rely on targeting either the cell wall or cell membrane, but the pipeline of available bioactives is limited. There is a clear and unmet need to identify novel targets and develop new classes of anti-fungal agents. This mini review focuses on fungal cell wall structure, composition and biosynthesis in Candida spp., including C. auris. In addition, an overview of current advances in the development of cell wall targeted therapies is considered.
vaccines, glycoproteins, inhibitors, chitin, fungal cell wall
NCBI PubMed ID: 34746525The cell wall of Trichosporon cutaneum consists of 11% protein, 63% neutral carbohydrate, 9% glucosamine and 13% glucuronic acid. The sugars include glucose (32%), mannose (6%) and traces of xylose and galactose. The cell wall was fractionated with alkali to yield a mixture of alkali-soluble matrix components, and an alkali-insoluble glucan associated with chitin. The alkali-insoluble glucan contained a mixture of (1-3) and (1-6) glycosidic linkages. It was only partly susceptible to digestion by the β(1-3) glucanase, Zymolyase. The alkali-soluble fraction contained glucan, mannan and acidic polymers. The glucan was (1-3)-linked with no (1-6) linkages and only trace amounts of (1-3-6)-linked glucose. It was resistant to digestion by Zymolyase. Extensive hydrolysis of this fraction with trifluoroacetic acid released a high-molecular-mass glucuronan which had 1H- and 13C-NMR profiles matching those of the β(1-4) glucuronan, mucoric acid. Xylomannan was purified from isolated cell walls and from whole cells. It contained glucose, mannose, xylose, and D-glucuronic acid. It was very similar in composition and structure to the capsular polysaccharides of Cryptococcus neoformans, and to an extracellular polysaccharide produced by another yeast described as T. cutaneum. Electron microscopy showed that the cell wall of T. cutaneum has a lamellar structure characteristic of a basidiomycetous yeast rather than the electron-dense 'fuzzy coat' seen in Candida albicans.
NCBI PubMed ID: 8245838Deproteinated A. niger biomass contains several covalently bound amino acids. The most abundant are arginine, serine, and proline in molar ratio of 3: 2: 2. One order of magnitude less is the amount of valine, phenylalanine, leucine and glycine. On deacetylation and separation of chitosan from glucan, the main three amino acids remain bound predominantly to chitosan, whereas the hydrophobic amino acids accompany mainly glucan. The presence of arginine could be the cause of stronger basicity of fungal chitosan compared to polyglucosamine.
Publication DOI: 10.1007/BF00805812During the past few years, cyto- and immunocytochemical techniques have been developed and widely used for locating and identifying various molecules in plant cell compartments. The last decade has witnessed tremendous improvements in molecular cytology, thus allowing an accurate in situ detection of various components thought to play important biological functions in the plant metabolism. The use of immunocytochemistry to investigate resistance mechanisms of plants upon pathogen attack has provided key information on the defense strategy that plants elaborate during a host-pathogen interaction. Of the various proteins induced in response to infection, chitinases and β-1,3-glucanases have been the focus of particular attention due to their believed antimicrobial activity through the hydrolysis of the main fungal wall components, chitin and β-1,3-glucans. Attention has also been paid to β-fructosidase, the enzyme that hydrolyzes sucrose into glucose and fructoside. The marked accumulation of this enzyme upon pathogen infection has led to the consideration that infection may greatly influence the metabolic activity of colonized tissues by creating alterations of source-sink relationships. Another facet of the plant's defense strategy that has been the focus of considerable interest is related to the accumulation of structural compounds, such as hydroxyproline-rich glycoproteins and callose, to reinforce the wall architecture, thus decreasing vulnerability to microbial enzymes. A number of alternatives designed to improve plant protection towards pathogen invasion have been suggested. Among these, the production of transgenic plants expressing constitutively a foreign resistance gene and the pretreatment of plants with elicitors of defense reactions have been the subject of intensive studies at the molecular, biochemical, and cytological levels. Results of such studies clearly demonstrate the important contribution that cyto- and immunocytochemical approaches can make to our knowledge of how plants defend themselves and how plant disease resistance can be directly enhanced. These approaches will undoubtedly be active areas for future research in the development of biological control alternatives in which the mode of action of the product used is of key importance.
NCBI PubMed ID: 7626800The cell-wall components of the interface compartment in functioning mycorrhizal roots of maize (Zea mays L. cv. W64A) have been investigated with the use of immunocytochemistry and enzyme/lectin-gold techniques. The distribution of specific cell-wall probes was determined in the apical and differentiated regions of maize roots in the presence and in the absence of the mycorrhizal fungus, Glomus versiforme. Labelling experiments showed that a maize hydroxyproline-rich glycoprotein (HRGP), identified with a specific antibody, was particularly abundant in the apical dividing cells of the root meristem. Cellulose, located with a cellobiohydrolase-gold complex, showed a similar labelling pattern in the walls of both meristematic and differentiated parts of the roots. When the cortex was colonized by the mycorrhizal fungus, the HRGP and cellulose were expressed in two sites: the wall and the interface area created by invagination of the host membrane around the developing fungus. In contrast, in uninfected roots of the same age, they were only present in the inner part of the wall. A specific antibody against β-1,3-glucans demonstrated that these glucans were not laid down at the interface between the plant and fungus, while they appeared to be a skeletal component of the fungal wall, together with chitin.
cell wall, cellulose, β-1, arbuscular mycorrhizae, 3-glucans, hydroxyproline-rich glycoprotein, zea root meristem
Publication DOI: 10.1007/BF00199680Glycosylphosphatidylinositol (GPI) membrane anchors are essential for the integration of yeast cell adhesion proteins into the cell wall, but mature cell-wall proteins are unlikely to be attached directly to the membrane. We thus propose that GPI-anchored glycoprotein forms are intermediates in a process that crosslinks the major components of the cell wall by transglycosylation. This mechanism may be critical for both the biosynthesis and overall architecture of the cell wall.
NCBI PubMed ID: 14731865We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1→3)-β-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a 'knr4 disrupted' tetrad demonstrated that mutant cell walls contained much less (1→3)-β-glucan and (1→6)-β-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.
NCBI PubMed ID: 7992508In recent years it has become evident that the structural polysaccharide chitin is synthesized from a family of enzymes encoded by multiple CHS chitin synthase genes, and regulated by an array of ancillary gene products that influence CHS activation and localization. Considerable attention has therefore been given to elucidating the function of specific CHS gene products in individual fungi. In those fungi in which individual CHS genes have been deleted systematically, there is little evidence for redundancy of function in family members. Chs enzymes are now known that participate in lateral wall biosynthesis, septum synthesis and spore formation but the phenotype of some CHS gene mutations is subtle, and so the role of the corresponding isoenzymes remains obscure. Nonetheless, it has become clear that certain members of the CHS gene families of fungi are more important for growth, integrity and viability than others, and this knowledge has already led to the design of new classes of antifungal agents that are targeted against key enzyme activities. Future work in this area will help define how individual Chs enzymes are targeted to specific regions of the cell wall and at specific times of the cell cycle, and should facilitate the rational development of novel and highly specific antifungal agents.
cell wall, mutagenesis, morphogenesis, antifungal drugs, chitin synthesis
Publication DOI: 10.1080/mmy.39.1.41.53N-Acetylchitooligosaccharides (oligochitin, chitin oligosaccharides) of a specific size can act as potent elicitor signals for suspension-cultured rice cells as well as various plant cells which include many monocots and some dicots. We recently isolated and characterized a highly elicitor-active glucopentaose from the cell wall P-Glucan from rice blast disease fungus. The results indicated that rice and soybean cells recognize different structural units of fugal glucans as elicitor signals. Because this elicitor treatment can induce many defense reactions, it has been serving as an excellent model system for the study of the signal transduction cascade leading to the activation of defense-related genes. It is critically important to identify and characterize the receptor molecules which perceive the elicitor signal to clarify the whole signal transduction cascade. A 75 kDa chitin oligosaccharide binding protein in the plasma membrane of suspension-cultured rice cells was identified as a putative receptor for the elicitor and purified. Recent studies on the structure and function of the binding proteins for these oligosaccharide elicitors will provide a clue to understanding how these elicitors are perceived and transduced in rice and other plant cells and also how such recognition systems have evolved.
receptor, signal transduction, elicitor
Publication DOI: 10.4052/tigg.12.113Background: To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction. Findings. N-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25% [14C]-GlcNAc incorporation into [14C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to N-acetylchitooses as it was not observed with oligosaccharides in other series, such as β-(1,4), β-(1,3) or α-(1,6) glucooligosaccharides. Conclusions: The effect induced by the N-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity in vitro. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size.
Oligosaccharides, glycosyltransferase, chitin synthase, polymerization activation, polysaccharide synthase
NCBI PubMed ID: 22032207Chitin (β-(1-4)-poly-N-acetyl-D-glucosamine) is widely distributed in nature and is the second most abundant polysaccharide after cellulose. Chitin occurs in nature as ordered macrofibrils. It is the major structural component in the exoskeleton of crab and shrimp shells and the cell wall of fungi and yeast. As chitin is not readily dissolved in common solvents, it is often converted to its more deacetylated derivative, chitosan. Chitin, chitosan, and its derivatives are widely used in tissue engineering, wound healing, and as functional foods. Recently, easy methods for the preparation of chitin and chitosan nanofibers have been developed, and studies on biomedical applications of chitin and chitosan nanofibers are ongoing. Chitin and chitosan nanofibers are considered to have great potential for various biomedical applications, because they have several useful properties such as high specific surface area and high porosity. This review summarizes methods for the preparation of chitin and chitosan nanofibers. Further, biomedical applications of chitin and chitosan nanofibers in (i) tissue engineering, (ii) wound dressing, (iii) cosmetic and skin health, (iv) stem cell technology, (v) anti-cancer treatments and drug delivery, (vi) anti-inflammatory treatments, and (vii) obesity treatment are summarized. Many studies indicate that chitin and chitosan nanofibers are suitable materials for various biomedical applications.
drug delivery, cosmetics, chitosan, chitin, obesity, nanofibers, biomedical applications, tissue engineering, stem cells
NCBI PubMed ID: 25992423Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target.
cell wall, chitin, chitin synthase
NCBI PubMed ID: 24942743Chitin and β-glucan are conserved throughout evolution in the fungal cell wall and are the most common polysaccharides in fungal species. Together, these two polysaccharides form a structural scaffold that is essential for the survival of the fungus. In the present study, we demonstrated that Aspergillus fumigatus alkali-insoluble cell wall fragments (AIF), composed of chitin linked covalently to β-glucan, induced enhanced immune responses when compared with individual cell wall polysaccharides. Intranasal administration of AIF induced eosinophil and neutrophil recruitment, chitinase activity, TNF-α and TSLP production in mice lungs. Selective destruction of chitin or β-glucan from AIF significantly reduced eosinophil and neutrophil recruitment as well as chitinase activity and cytokine expression by macrophages, indicating the synergistic effect of the cell wall polysaccharides when presented together as a composite PAMP. We also showed that these cell wall polysaccharides induced chitin-specific IgM in mouse serum. Our in vivo and in vitro data indicate that chitin and β-glucan play important roles in activating innate immunity when presented as composite cell wall PAMPs.
inflammation, glucan, innate immunity, lung, chitin, Fungal cell wall polysaccharides
NCBI PubMed ID: 24286790Polysaccharides such as α- and β-glucans, chitin, and glycoproteins extensively modified with both N- and O-linked carbohydrates are the major components of fungal surfaces. The fungal cell wall is an excellent target for the action of antifungal agents, since most of its components are absent from mammalian cells. Recognition of these carbohydrate-containing molecules by the innate immune system triggers inflammatory responses and activation of microbicidal mechanisms by leukocytes. This review will discuss the structure of surface fungal glycoconjugates and polysaccharides and their recognition by innate immune receptors.
glycoconjugates, polysaccharides, innate immunity, fungal pathogens, pattern recognition receptors
NCBI PubMed ID: 25353009Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of β-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation.
cell wall, β-glucan, Candida albicans, b-glucan
NCBI PubMed ID: 25467440GPI-anchoring is a universal and critical post-translational protein modification in eukaryotes. In fungi, many cell wall proteins are GPI-anchored, and disruption of GPI-anchored proteins impairs cell wall integrity. After being synthesized and attached to target proteins, GPI anchors undergo modification on lipid moieties. In spite of its importance for GPI-anchored protein functions, our current knowledge of GPI lipid remodelling in pathogenic fungi is limited. In this study, we characterized the role of a putative GPI lipid remodelling protein, designated PerA, in the human pathogenic fungus Aspergillus fumigatus. PerA localizes to the endoplasmic reticulum and loss of PerA leads to striking defects in cell wall integrity. A perA null mutant has decreased conidia production, increased susceptibility to triazole antifungal drugs, and is avirulent in a murine model of invasive pulmonary aspergillosis. Interestingly, loss of PerA increases exposure of β-glucan and chitin content on the hyphal cell surface, but diminished TNF production by bone marrow-derived macrophages relative to wild type. Given the structural specificity of fungal GPI-anchors, which is different from humans, understanding GPI lipid remodelling and PerA function in A. fumigatus is a promising research direction to uncover a new fungal specific antifungal drug target.
Aspergillus fumigatus, GPI
NCBI PubMed ID: 24779420The cell wall structure of mushroom sclerotium was investigated by a fractionation of its soluble cell wall polysaccharides followed by chemical, physico-chemical and microscopic analyses. The present results suggest that cell wall structure of Pleurotus tuber regium sclerotium contains three main layers: an outer layer of glycoproteins, a middle layer of hyper-branched glucans and an inner layer of complex between hyper-branched glucan and chitin. The structure of the hyper-branched glucans were elucidated by the methylation analysis to be composed of →1)-Glcp-(4→ linkages as the backbone with some →1)-Glcp(6→ linkages existed in the side chains, while some →1)-Glcp-(3→ linkages might exist in the backbone or side chains. SEC-MALLS analysis revealed that hyper-branched β-glucans had a M-w ranging from 1400000 to 5200000 g/mol and R.M.S. radius ranging from 26 nm to 38.5 nm. SEM and AFM further showed that hyper-branched β-glucans were spherical in shape when dispersed in water.
Publication DOI: 10.1016/j.foodhyd.2013.09.023Within the domain of Eukarya, the fungi form a seperate kingdom. The typical formation of branched mycelia from single hyphae is based on cell wall production at the growing hyphal tip. There, excretory vesicle fuse with the membrane releasing cell wall synthesis enzymes like chitin synthase forming the polymer of N-acetyl glucosamin, the backbone of fungal cell walls. In addition, glucan synthases form the structural component β-1,3-glucan. Via β-1,6-glucan, cell wall proteins can be linked to the maturing cell wall, and α-1,3-glucan can form a matrix within the cell wall, but also a slimy matrix secreted into the medium. A layer of hydrophobins allows for growth into the air, but also facilitates formation of macroscopic structures like mushrooms.
cell wall, glucan, fungi, chitin
Publication DOI: 10.1002/biuz.201610599This study was devoted to polysaccharides extraction (chitin and chitosan) from Penicillium camembertii cell wall. A culture on solid medium was adopted under carefully selected conditions, appropriate to mycelium growth: duration 6 days, medium YPGA and pH 5. The temperature was adjusted (20 °C to 28 °C) in order to study the effect of temperature on chitin/chitosan production. Biomass decreased with increasing temperatures: 13 g/L at 20 °C and 11.6 g/L at 28 °C. For all tested temperatures, the yields of insoluble alkaline fractions (AIM) were almost identical (200 mg/g). The solubility of fractions in 2% acetic acid allowed obtaining two fractions: an insoluble fraction (AcIM) with 18% of maximum yield and soluble fraction (AcSM) with 1% yield. The SEM micrographs of AcIM fractions were similar to AIM fractions. These showed a compact structure different from commercial chitin. The presence of chitin in P. Camembertii cultured in YPGA medium was also confirmed by ATR spectroscopy.
chitin, Biomass, free chitosan, Penicillium camembertii, YPGA
NCBI PubMed ID: 31004649In 1799, Hatchett decalcified shells of crabs, lobsters, prawns and crayfish with mineral acids, observing that they produced a moderate effervescence and in a short time were found to be soft and plastic of a yellowish color and like a cartilage, which retained the original figure. Although this is the first mention of calcified chitin in invertebrates, the discovery of chitin is usually attributed both to Braconnot in 1811 who discovered chitin from fungi, and to Odier in 1823 who obtained a hornlike material after treatment of cockchafer elytra with potassium hydroxide. Chitin was first named fongine by Braconnot and then chitine by Odier. Children revealed the nitrogenous nature of chitin in 1824. The history of chitosan, the main derivative of chitin, dates back to 1859 with the work of Rouget. The name of chitosan was, however, introduced in 1894 by Hoppe-Seyler. In 1876, Ledderhose hydrolyzed arthropod chitin and discovered glykosamin, the first derivative of chitin. This review describes the 220 years of the development of chitin. I have roughly divided the story into five periods: discovery from 1799 to 1894, a period of confusion and controversy from 1894 to 1930, exploration in 1930–1950, a period of doubt from 1950 to 1970, and finally the period of application from 1970. The different periods are illustrated by examples of published studies, in particular from outstanding scholars who have left their mark on the history of this polysaccharide. Although this historic review is not exhaustive, it highlights the work of researchers who have contributed to the development of our knowledge of chitin throughout the 220 years of its history.
History, chitosan, chitin, discovery, Braconnot, controversy, exploration, period of doubt, period of application
Publication DOI: 10.1007/s10311-019-00901-0Chitin is a linear polysaccharide of the amino sugar N-acetyl glucosamine. It is present in the extracellular matrix of a variety of invertebrates including sponges, molluscs, nematodes and arthropods and fungi. Generally, it is an important component of protective or supportive extracellular matrices that cover the tissue that produces it or the whole body of the organism. Chitin fibres associate with each other adopting one of three possible crystalline organisations, i.e. α-, β- or γ-chitin. Usually, chitin fibre bundles interact with chitin-binding proteins forming higher order structures. Chitin laminae, which are two-dimensional sheets of α-chitin crystals with antiparallel running chitin fibres in association with β-folded proteins, are primary constituents of the arthropod cuticle and the fibrous extracellular matrix in sponges. A tri-dimensional composite material of proteins coacervates and β-chitin constitute hard biomaterials such as the squid beak. The molecular composition of γ-chitin-based structures that contribute to the physical barrier found in insect cocoons is less well studied. In principle, chitin is a versatile extracellular polysaccharide that in association with proteins defines the mechanical properties of tissues and organisms.
evolution, Extracellular matrix, barrier, Cuticle, body shape
NCBI PubMed ID: 31102240Chitin is an important structural polysaccharide, which supports and organizes extracellular matrices in a variety of taxonomic groups including bacteria, fungi, protists, and animals. Additionally, chitin has been recognized as a molecule that is required for Rhizobia-legume symbiosis and involved in arbuscular mycorrhizal signaling in the symbiotic interaction between terrestrial plants and fungi. Moreover, it serves as a unique molecular pattern in the plant defense system against pathogenic fungi and parasites, and in the innate and adaptive immune response of mammals and humans. In this review, we will focus on the prevalence and structural function of chitin in bacteria, fungi, and protists, with a particular focus on the evolution of chitin synthases and the function of chitin oligosaccharides as a signaling molecule in symbiosis and immunity.
Rhizobia, cell wall, fungi, protists, skeleton
NCBI PubMed ID: 31102241Chitin is one of the most important carbohydrates of the fungal cell wall, and is synthesized by chitin synthases. Chitin can be degraded by chitinases, which are important virulence factors in pathogenic fungi. Knowledge about the biosynthesis and degradation of chitin, and the enzymes responsible, has accumulated in recent years. In this review, we analyze the amino acid sequences of chitin synthases from several typical fungi. These enzymes can be divided into seven groups. While the different chitin synthases from a single fungus share a low degree of similarity, the same type of chitin synthase from different fungi shows high similarity. The number of chitinase genes in fungi display wide variation, from a single gene in Schizosaccharomyces pombe, to 36 genes in Trichoderma virens. Chitinases from different fungi can be divided into four groups. The functions of chitin synthases and chitinases in several typical fungi are summarized, and the crystal structures of chitinases and chitinase modification are also discussed.
crystal structure, modification, chitin, chitin synthase, chitinase
NCBI PubMed ID: 31102246The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.
α-1, conditional mutant, Saccharomyces cerevisiae, 6-mannosyltransferase, chitin synthase III, MET3 promoter
NCBI PubMed ID: 30706115This review discusses the wealth of information available for the N. crassa cell wall. The basic organization and structure of the cell wall is presented and how the wall changes during the N. crassa life cycle is discussed. Over forty cell wall glycoproteins have been identified by proteomic analyses. Genetic and biochemical studies have identified many of the key enzymes needed for cell wall biogenesis, and the roles these enzymes play in cell wall biogenesis are discussed. The review includes a discussion of how the major cell wall components (chitin, β-1,3-glucan, mixed β-1,3-/β-1,4- glucans, glycoproteins, and melanin) are synthesized and incorporated into the cell wall. We present a four-step model for how cell wall glycoproteins are covalently incorporated into the cell wall. In N. crassa, the covalent incorporation of cell wall glycoproteins into the wall occurs through a glycosidic linkage between lichenin (a mixed β-1,3-/β-1,4- glucan) and a "processed" galactomannan that has been attached to the glycoprotein N-linked oligosaccharides. The first step is the addition of the galactomannan to the N-linked oligosaccharide. Mutants affected in galactomannan formation are unable to incorporate glycoproteins into their cell walls. The second step is carried out by the enzymes from the GH76 family of α-1,6-mannanases, which cleave the galactomannan to generate a processed galactomannan. The model suggests that the third and fourth steps are carried out by members of the GH72 family of glucanosyltransferases. In the third step the glucanosyltransferases cleave lichenin and generate enzyme/substrate intermediates in which the lichenin is covalently attached to the active site of the glucanosyltransferases. In the final step, the glucanosyltransferases attach the lichenin onto the processed galactomannans, which creates new glycosidic bonds and effectively incorporates the glycoproteins into the cross-linked cell wall glucan/chitin matrix.
cell wall, Galactomannan, glucan, filamentous fungi, melanin, Neurospora, glucanosyltransferase, mannanase
NCBI PubMed ID: 31649638Invasive fungal infections are some of the most life-threatening infectious diseases in the hospital setting. In industrialized countries, the most common fungal species isolated from immunocompromised patients are Candida and Aspergillus spp. However, the number of infections due to Mucorales spp. is constantly increasing and little is known about the virulence factors of these fungi. The fungal cell wall is an important structure protecting fungi from the environment. A better knowledge of its composition should improve our understanding of host-pathogen interactions. Cell wall molecules are involved in tissue adherence, immune escape strategies, and stimulation of host defenses including phagocytosis and mediators of humoral immunity. The fungal cell wall is also a target of choice for the development of diagnostic or therapeutic tools. The present review discusses our current knowledge on the cell wall structure of Mucorales in terms of the polysaccharides and glyco-enzymes involved in its biosynthesis and degradation, with an emphasis on the missing gaps in our knowledge.
cell wall, polysaccharides, glucuronic acid, Mucorales, glyco-enzymes
NCBI PubMed ID: 30941108Chitin, chitosan and their complexes with β-glucan (chitin–glucan complex, CGC, and chitosan–glucan complex, ChGC) are value-added polysaccharides extracted from the cell-walls of many fungi. Commercial chitin and its deacetylated form, chitosan, are currently obtained from marine waste material, mostly animal sources (crustaceans and marine invertebrates), through harsh chemical procedures that have low reproducibility due to the variability of the composition of the sources and their seasonal character. These disadvantages are overcome by using fungi as sources of chitinous polymers. The extraction of chitin/chitosan from fungi cell-walls has the great advantage of yielding products with stable composition and properties, using simpler procedures, with the added benefit of also generating CGC and ChGC, two copolymers that combine the proven properties of chitin/chitosan with those of β-glucans. Over the last decades, fungal chitinous polymers have been the focus of extensive research that included optimization of the cultivation conditions of a wide range of species and the development of optimized extraction, purification and characterization techniques, as well as the demonstration of the biopolymers' biological properties, which include immunomodulatory, anticancer, antioxidant and antimicrobial activity. Given these properties, several attempts were made to develop applications for them in areas ranging from biomedicine and pharmaceuticals to food and agriculture. Despite their wide range of proven functional properties that include the ability to form different polymeric structures, as well as biological activity, fungal chitinous biopolymers are still underexplored. Nevertheless, these biopolymers hold great potential for development into valuable products or applications that are surely worth further investigation.
fungi, chitosan, chitin, cell-wall polysaccharides, chitin–glucan complex (CGC), chitosan–glucan complex (ChGC)
Publication DOI: 10.1002/jctb.6325Chitin is a unique structural exopolysaccharide abundantly found in nature. This exopolysaccharide has a unique chemical structure that acts as a protective outermost covering for most of the crustaceans in aquatic ecosystem. This fortification is because of the insoluble nature of this exopolysaccharide which consist of a linear chain of β-(1→4)-linked-N-acetylglucosamine units. Chitin is hydrolyzed with the help of a hydrolase known as chitinase. Variety of microbial species have been explored for chitinase production. Chitinolytic microbial species can be alternatively used for degradation of chitin instead of chemical treatment in agricultural sector. This biological approach has lesser environmental impact because of its apparently safe nature. In the current study, bioprospecting of chitinase producing species was conducted and different chitinolytic bacterial strains were screened for chitinase production which could have anti-fungal potential. Bacterial isolates were identified based on polyphasic approach and the enzyme production was optimized using one-variable-at-a-time technique. Hyphal extension method was used for determination of anti-fungal potential of chitinase.Glutamicibacter uratoxydans was indigenously isolated and identified for chitinase production. G. uratoxydans is a novel bacterial species which has not been previously explored to produce chitinase or other hydrolases. G. uratoxydans biosynthesized chitinase utilizing colloidal chitin as a sole source of carbon. The chitinase biosynthesized by G. uratoxydans is effectively potent against Aspergillus fumigatus thus, suggesting that this extracellular enzyme could be used for the treatment of fungal infection caused by filamentous fungi.
cell wall, antifungal activity, chitin, chitinase, Glutamicibacter uratoxydans
Publication DOI: 10.1007/s12649-019-00746-2Chitin and chitosan are two related polysaccharides that provide important structural stability to fungal cell walls. Often embedded deeply within the cell wall structure, these molecules anchor other components at the cell surface. Chitin-directed organization of the cell wall layers allows the fungal cell to effectively monitor and interact with the external environment. For fungal pathogens, this interaction includes maintaining cellular strategies to avoid excessive detection by the host innate immune system. In turn, mammalian and plant hosts have developed their own strategies to process fungal chitin, resulting in chitin fragments of varying molecular size. The size-dependent differences in the immune activation behaviors of variably sized chitin molecules help to explain how chitin and related chitooligomers can both inhibit and activate host immunity. Moreover, chitin and chitosan have recently been exploited for many biomedical applications, including targeted drug delivery and vaccine development.
NCBI PubMed ID: 31807896The fungal cell wall serves as the interface between the cell and the environment. Fungal cell walls are composed largely of polysaccharides, primarily glucans and chitin, though in many fungi stress-resistant cell types elaborate additional cell wall structures. Here, we use solid-state nuclear magnetic resonance spectroscopy to compare the architecture of cell wall fractions isolated from Saccharomyces cerevisiae spores and Cryptococcus neoformans melanized cells. The specialized cell walls of these two divergent fungi are highly similar in composition. Both use chitosan, the deacetylated derivative of chitin, as a scaffold on which a polyaromatic polymer, dityrosine and melanin, respectively, is assembled. Additionally, we demonstrate that a previously identified but uncharacterized component of the S. cerevisiae spore wall is composed of triglycerides, which are also present in the C. neoformans melanized cell wall. Moreover, we identify a tyrosine-derived constituent in the C. neoformans wall that, although it is not dityrosine, is a non-pigment constituent of the cell wall. The similar composition of the walls of these two phylogenetically distant species suggests that triglycerides, polyaromatics, and chitosan are basic building blocks used to assemble highly stress-resistant cell walls and the use of these constituents may be broadly conserved in other fungal species.
chitosan, Saccharomyces cerevisiae, chitin, fungal cell wall, solid-state NMR, Cryptococcus neoformans, melanin, macromolecular assembly, dityrosine, triglycerides
NCBI PubMed ID: 33271921Cryptococcus neoformans and Cryptococcus gattii are two species complexes in the large fungal genus Cryptococcus and are responsible for potentially lethal disseminated infections. These two complexes share several phenotypic traits, such as production of the protective compound melanin. In C. neoformans, the pigment associates with key cellular constituents that are essential for melanin deposition within the cell wall. Consequently, melanization is modulated by changes in cell-wall composition or ultrastructure. However, whether similar factors influence melanization in C. gattii is unknown. Herein, we used transmission EM, biochemical assays, and solid-state NMR spectroscopy of representative isolates and "leaky melanin" mutant strains from each species complex to examine the compositional and structural factors governing cell-wall pigment deposition in C. neoformans and C. gattii. The principal findings were the following. 1) C. gattii R265 had an exceptionally high chitosan content compared with C. neoformans H99; a rich chitosan composition promoted homogeneous melanin distribution throughout the cell wall but did not increase the propensity of pigment deposition. 2) Strains from both species manifesting the leaky melanin phenotype had reduced chitosan content, which was compensated for by the production of lipids and other nonpolysaccharide constituents that depended on the species or mutation. 3) Changes in the relative rigidity of cell-wall chitin were associated with aberrant pigment retention, implicating cell-wall flexibility as an independent variable in cryptococcal melanin assembly. Overall, our results indicate that cell-wall composition and molecular architecture are critical factors for the anchoring and arrangement of melanin pigments in both C. neoformans and C. gattii species complexes.
Molecular Structure, cell wall, polysaccharides, virulence factor, opportunistic pathogen, nuclear magnetic resonance (NMR), fungi, Cryptococcus, solid-state NMR, melanin
NCBI PubMed ID: 31896575The human gut microbiota has been revealed in recent years as a factor that plays a decisive role in the maintenance of human health, as well as in the development of many non-communicable diseases. This microbiota can be modulated by various dietary factors, among which complex carbohydrates have a great influence. Although most complex carbohydrates included in the human diet come from vegetables, there are also options to include complex carbohydrates from non-vegetable sources, such as chitin and its derivatives. Chitin, and its derivatives such as chitosan can be obtained from non-vegetable sources, the best being insects, crustacean exoskeletons and fungi. The present review offers a broad perspective of the current knowledge surrounding the impacts of chitin and its derived polysaccharides on the human gut microbiota and the profound need for more in-depth investigations into this topic. Overall, the effects of whole insects or meal on the gut microbiota have contradictory results, possibly due to their high protein content. Better results are obtained for the case of chitin derivatives, regarding both metabolic effects and effects on the gut microbiota composition.
polysaccharides, insect, gut microbiota, prebiotic, chitosan, chitin, crustacean
NCBI PubMed ID: 32545663Two chitosan extracts were prepared by chemical and enzymatic treatment of Ganoderma lucidum mushroom, as an alternative source to crustacean shells. The molecular weight of the enzymatic extract was lower than that of the chemical one and of shrimp chitosan, as determined by viscosity measurements. Characteristic signals were identified in the 1 H-NMR spectra and high deacetylation degree indicated good physico-chemical properties for both mushroom chitosan extracts. The scavenging capacity of mushroom chitosan extracts was moderate against the synthetic radicals of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH), but higher values were observed for the enzymatic extract, compared to the chemical extract and shrimp chitosan. In vitro cytotoxicity was evaluated in L929 mouse fibroblast cell lines and the results of MTT assay showed good cytocompatibility in the tested range of concentrations. The growth of Gram-positive bacteria was inhibited more than Gram-negative bacteria in the presence of mushroom chitosan extracts, in particular by the chemical one, indicating their efficiency as antimicrobial agents. All these results strengthen the evidence of mushroom polysaccharide preparations availability for biomedical applications.
extraction, deacetylation, fungi, radical scavenging activity, α-amylase
NCBI PubMed ID: 32333466To develop more ecologically sustainable agricultural practices requires that we reduce our reliance on synthetic chemical pesticides for crop protection. This will likely involve optimized biocontrol approaches - the use of beneficial soil microbes to attack potential plant pathogens to protect plants from diseases. Many bacterial species, including strains of Bacillus subtilis, have been explored for their biocontrol properties, as they can control the growth of harmful fungi, often by disrupting the fungal cell wall. A strain that is not often considered for this particular application is Bacillus subtilis natto, primarily known for fermenting soybeans via cell wall degradation in the Japanese probiotic dish "natto." Because deconstruction of the fungal cell wall is considered an important biocontrol trait, we were motivated to explore the possible anti-fungal properties of the B. subtilis natto strain. We show that B. subtilis natto can use complex fungal material as a carbon source for growth, and can effectively deconstruct fungal cell walls. We found degradation of fungal cell wall proteins, and showed that growth on a mix of peptides was very strong. We also found that intact fungal cell walls can induce the secretion of chitinases and proteases. Surprisingly, we could show that chitin, the bulk component of the fungal cell wall, does not permit successful growth of the natto strain or induce the secretion of chitinolytic enzymes, although these were produced during exposure to proteins or to complex fungal material. We have further shown that protease secretion is likely a constitutively enabled mechanism for nutrient scavenging by B. subtilis natto, as well as a potent tool for the degradation of fungal cell walls. Overall, our data highlight B. subtilis natto as a promising candidate for biocontrol products, with relevant behaviors that can be optimized by altering growth conditions. Whereas it is common for bacterial biocontrol products to be supplied with chitin or chitosan as a priming polysaccharide, our data indicate that this is not a useful approach with this particular bacterium, which should instead be supplied with either glucose or attenuated fungal material.
protease, secretome, fungal cell wall, chitinase, biocontrol, Bacillus subtilis natto
NCBI PubMed ID: 32296406Vast efforts have been devoted to the development of antifungal drugs targeting the cell wall, but the supramolecular architecture of this carbohydrate-rich composite remains insufficiently understood. Here we compare the cell wall structure of a fungal pathogen Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. High-resolution solid-state NMR spectroscopy of intact cells reveals a rigid core formed by chitin, β-1,3-glucan, and α-1,3-glucan, with galactosaminogalactan and galactomannan present in the mobile phase. Gene deletion reshuffles the composition and spatial organization of polysaccharides, with significant changes in their dynamics and water accessibility. The distribution of α-1,3-glucan in chemically isolated and dynamically distinct domains supports its functional diversity. Identification of valines in the alkali-insoluble carbohydrate core suggests a putative function in stabilizing macromolecular complexes. We propose a revised model of cell wall architecture which will improve our understanding of the structural response of fungal pathogens to stresses.
polysaccharides, Aspergillus fumigatus, chitin, fungal cell wall, solid-state NMR
NCBI PubMed ID: 34732740Background: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as β(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of β(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. Purpose: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. Methods: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms 'natural antifungals' and 'plant extracts' with 'fungal cell wall'. Results: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. Conclusion: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.
Plant Extracts, chitin, fungal cell wall, antifungal drugs, bioactive natural products, cell wall synthases, systemic fungal infection
NCBI PubMed ID: 33958276Extracellular matrixes (ECMs), such as the cell walls and biofilms, are important for supporting cell integrity and function and regulating intercellular communication. These biomaterials are also of significant interest to the production of biofuels and the development of antimicrobial treatment. Solid-state nuclear magnetic resonance (ssNMR) and magic-angle spinning-dynamic nuclear polarization (MAS-DNP) are uniquely powerful for understanding the conformational structure, dynamical characteristics, and supramolecular assemblies of carbohydrates and other biomolecules in ECMs. This review highlights the recent high-resolution investigations of intact ECMs and native cells in many organisms spanning across plants, bacteria, fungi, and algae. We spotlight the structural principles identified in ECMs, discuss the current technical limitation and underexplored biochemical topics, and point out the promising opportunities enabled by the recent advances of the rapidly evolving ssNMR technology.
bacteria, algae, Plants, fungi, ssNMR
NCBI PubMed ID: 34878762Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, β-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications.
cell wall, solid-state NMR, Aspergillus sydowii, halophilic fungi
NCBI PubMed ID: 37925437Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field 1 H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.
NMR, NMR spectroscopy, cell wall, peptide, Schizophyllum commune, proton detection
NCBI PubMed ID: 36181715Pleurotus ostreatus, also known as the oyster mushroom, is a popular edible mushroom cultivated worldwide. This review aims to survey recent progress in the molecular genetics of this fungus and demonstrate its potential as a model mushroom for future research. The development of modern molecular genetic techniques and genome sequencing technologies has resulted in breakthroughs in mushroom science. With efficient transformation protocols and multiple selection markers, a powerful toolbox, including techniques such as gene knockout and genome editing, has been developed, and numerous new findings are accumulating in P. ostreatus. These include molecular mechanisms of wood component degradation, sexual development, protein secretion systems, and cell wall structure. Furthermore, these techniques enable the identification of new horizons in enzymology, biochemistry, cell biology, and material science through protein engineering, fluorescence microscopy, and molecular breeding
cell wall, genome editing, agaricomycete, breeding, mycelial materials, wood degradation
NCBI PubMed ID: 38372792Integral and membrane-associated proteins extracted from neuron-enriched perirhopalial tissue of the jellyfish Cyanea capillata were probed with a panel of lectins that recognize sugar epitopes of varying complexity. Of the 13 lectins tested, only concanavalin A, jacalin lectin and tomato lectin stained distinct bands on Western blots, indicating the presence of repeating α-1,6-mannoses, terminal Gal-α-1,6-GalNAc and repeating β-1,4-linked GlcNAc, respectively. In whole-mounted perirhopalial tissue, jacalin lectin stained several cell types, including neurons, muscle, cilia and mucus strands. Tomato lectin stained secretory cells intensely, and neurons in a punctate fashion. Concanavalin A stained cytoplasmic epitopes in both ecto-and endodermal cells, and ectodermal secretory cells and the mucus strands emanating from them. With the exception of tomato lectin's sugar epitope, the other sugar epitopes identified in this study are “non-complex.” This study suggests that while glycosylation of integral and membrane-associated proteins occurs in Cyanea, the sugars post-translationally linked to these proteins tend to be simple.
carbohydrate, linkage, sugar, Scyphozoa, nerve, Cyanea capillata (Cnidaria)
Publication DOI: 10.1007/BF00318487| a-D-Glcp-(1-3)-a-D-Glcp | Show graphically |
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Structure type: oligomer
Trivial name: nigerose
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_983931,SB_192
Theoretical calculations reveal that oligosaccharides are second to no other class of biochemical oligomery in terms of coding capacity. As integral part of cellular glycoconjugates they can serve as recognitive units for receptors (lectins). Having first been detected in plants, lectins are present ubiquitously. Remarkably for this field, they serve as bacterial and viral adhesins. Following a description of these branches of lectinology to illustrate history, current status and potential for medicinal chemistry, we document that lectins are involved in a wide variety of biochemical processes including intra- and intercellular glycoconjugate trafficking, initiation of signal transduction affecting e. g. growth regulation and cell adhesion in animals. It is thus justified to compare crucial carbohydrate epitopes with the postal code ensuring correct mail routing and delivery. In view of the functional relevance of lectins the design of high-affinity reagents to occupy their carbohydrate recognition domains offers the perspective for an attractive source of new drugs. Their applications can be supposed to encompass the use as cell-type-selective determinant for targeted drug delivery and as blocking devices in anti-adhesion therapy during infections and inflammatory disease. To master the task of devising custom-made glycans/glycomimetics for this purpose, the individual enthalpic and entropic contributions in the molecular rendezvous between the sugar receptor under scrutiny and its ligand in the presence of solvent molecules undergoing positional rearrangements need to be understood and rationally exploited. As remunerative means to this end, cleverly orchestrated deployment of a panel of methods is essential. Concerning the carbohydrate ligand, its topological parameters and flexibility are assessed by the combination of computer-assisted molecular-mechanics and molecular-dynamics calculations and NMR-spectroscopic measurements. In the presence of the receptor, the latter technique will provide insights into conformational aspects of the bound ligand and into spatial vicinity of the ligand to distinct side chains of amino acids establishing the binding site in solution. Also in solution, the hydrogen-bonding pattern in the complex can be mapped with monodeoxy and monofluoro derivatives of the oligosaccharide. Together with X-ray crystallographic and microcalorimetric studies the limits of a feasible affinity enhancement can be systematically probed. With galactoside-binding lectins as instructive mo del, recent progress in this area of drug design will be documented, emphasizing the general applicability of the outlined interdisciplinary approach.
Molecular mechanics, Rhizobium meliloti, lectinology, lectins as targets, computer assisted, NMR spectoscopic, crystallographic elucidation, sugar code, chemioal tailoring, phosphodiester backbone, microheterogeneity of glycan, monomer variability, N acetylneuramicinic, transgenic pollen, nitrogen enriched nutrients, non agglutinating ricin, hydrophobic molecules, phytopathogenic fungus, phosphomannose mutase, B bearing individuals, anti adhesion therapy, NMR spectrum, parenchymal host cells
NCBI PubMed ID: 10702616Several lactic acid bacteria are able to produce water-soluble and water-insoluble homoexopolysaccharides (HoEPS) from sucrose. In this study, structures of all HoEPS which were fermentatively produced by Leuconostoc mesenteroides subsp. dextranicum NRRL B-1121 and B-1144, Leuconostoc mesenteroides subsp. mesenteroides NRRL B-1149, B-1438 and B-1118, Leuconostoc suionicum DSM 20241, and Liquorilactobacillus satsumensis DSM 16230 were systematically analyzed. Monosaccharide analysis, methylation analysis, NMR spectroscopy, size-exclusion chromatography, and different enzymatic fingerprinting methods were used to obtain detailed structural information. All strains produced water-soluble dextrans and/or levans as well as water-insoluble glucans. Levans showed different degrees of branching and high molecular weights, whereas dextrans had comparable structures and broader size distributions. Fine structures of water-soluble HoEPS were analyzed after endo-dextranase and endo-levanase hydrolysis. Water-insoluble glucans were composed of different portions of 1,3-linkages (5 to 40 %). Hydrolysis with endo-dextranase and endo-mutanase yielded further information on block sizes and varying fine structures. Overall, clear differences between HoEPS yields and structures were observed
enzymatic hydrolysis, Dextran, levan, mutan, HPAEC-PAD/MS, HPSEC-RI
NCBI PubMed ID: 37940249Koji amazake, prepared from rice koji, is a traditional Japanese sweet beverage. The main source of sweetness is glucose derived from rice starch following digestion by enzymes of Aspergillus oryzae during saccharification. The temperature of this process was empirically determined as 45°C–60°C, but no studies have systematically investigated the effect of temperature on saccharification efficiency. We addressed this in the present study by evaluating saccharification efficiency at various temperatures. We found that glucose content was the highest at 50°C (100%) and was reduced at temperatures of 40°C (66.4%), 60°C (91.9%), and 70°C (76.6%). We previously reported that 12 types of oligosaccharides are present in koji amazake; the levels of eight of these, namely nigerose, kojibiose, trehalose, isomaltose, gentiobiose, raffinose, panose, and isomaltotriose, were the highest at 50°C–60°C, whereas sophorose production was maximal at 70°C. Based on these findings, we initially performed saccharification at 50°C and then switched the temperature to 70°C. The maximum amount of each saccharide including sophorose that was produced was close to the values obtained at these two temperatures. Thus, oligosaccharide composition of koji amazake is dependent on saccharification temperature. These findings provide useful information for improving the consumer appeal of koji amazake by enhancing oligosaccharide content.
oligosaccharide, sophorose, koji, amazake, saccharification
NCBI PubMed ID: 30414826Increasing knowledge of the role of the intestinal microbiome in human health and well-being has resulted in increased interest in prebiotics, mainly oligosaccharides of various origins. To date, there are no reports in the literature on the prebiotic properties of oligosaccharides produced by the hydrolysis of pure fungal α-(1→3)-glucan. The aim of this study was to prepare α-(1→3)-glucooligosaccharides (α-(1→3)-GOS) and to perform initial evaluation of their prebiotic potential. The oligosaccharides were obtained by acid hydrolysis of α-(1→3)-glucan isolated from the fruiting bodies of Laetiporus sulphureus and then, characterized by HPLC. Fermentation of α-(1→3)-GOS and reference prebiotics was compared in in vitro pure cultures of Lactobacillus, Bifidobacterium, and enteric bacterial strains. A mixture of α-(1→3)-GOS, notably with a degree of polymerization of 2 to 9, was obtained. The hydrolysate was utilized for growth by most of the Lactobacillus strains tested and showed a strong bifidogenic effect, but did not promote the growth of Escherichia coli and Enterococcus faecalis. α-(1→3)-GOS proved to be effective in the selective stimulation of beneficial bacteria and can be further tested to determine their prebiotic functionality.
prebiotic, fruiting bodies, Laetiporus sulphureus, α-(1→3)-glucan, α-(1→3)-glucooligosaccharides
NCBI PubMed ID: 33255915| /Variants 1/-+ | /Variants 0/-b-Manp6Ac-(1-1)-Ery-ol /Variants 0/ is: C18={6}-(1-3)- OR (exclusively) C17={6}-(1-3)- OR (exclusively) C16={6}-(1-3)- OR (exclusively) C15={6}-(1-3)- OR (exclusively) C14={6}-(1-3)- OR (exclusively) C13={6}-(1-3)- OR (exclusively) C12={6}-(1-3)- /Variants 1/ is: Vl-(1-2)- OR (exclusively) Oco-(1-2)- OR (exclusively) Hxo-(1-2)- OR (exclusively) Dco-(1-2)- OR (exclusively) But-(1-2)- | Show graphically |
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Structure type: oligomer
Compound class: glycolipid
Contained glycoepitopes: IEDB_114707,IEDB_137485,IEDB_1394182,IEDB_144983,IEDB_152206,IEDB_983930,SB_44,SB_72
When grown on vegetable oils and their derivatives, the smut fungus Ustilago maydis (DSM 4500 and ATCC 14826) produces several glycolipids under nitrogen-limiting conditions. With 45 g l(-1) sunflower oil fatty acids (technical grade) a yield of 30 g l(-1) glycolipid was achieved. The resulting mixture contained predominantly mannosylerythritol lipids together with smaller amounts of cellobiose lipids. The production of the more polar cellobiose lipids was enhanced when glucose was used as carbon source. The molecular structure of the main components of the glycolipid mixture were elucidated by a combination of NMR spectroscopic and mass-spectrometric techniques.
NMR, mannosylerythritol lipids, derivatives, mannosylerythritol lipids candida-antarctica identification biosurfactants alkanes
Publication DOI: 10.1007/s002530051359Biosurfactants are in demand by the global market as natural commodities that can be added to commercial products or use in environmental applications. These biomolecules reduce the surface/interfacial tension between fluid phases and exhibit superior stability to chemical surfactants under different physico-chemical conditions. Biotechnological production of biosurfactants is still emerging. Fungi are promising producers of these molecules with unique chemical structures, such as sophorolipids, mannosylerythritol lipids, cellobiose lipids, xylolipids, polyol lipids and hydrophobins. In this review, we aimed to contextualize concepts related to fungal biosurfactant production and its application in industry and the environment. Concepts related to the thermodynamic and physico-chemical properties of biosurfactants are presented, which allows detailed analysis of their structural and application. Promising niches for isolating biosurfactant-producing fungi are presented, as well as screening methodologies are discussed. Finally, strategies related to process parameters and variables, simultaneous production, process optimization through statistical and genetic tools, downstream processing and some aspects of commercial products formulations are presented.
biosurfactants, filamentous fungi, yeasts, screening, bioprocess, downstream
NCBI PubMed ID: 34131819| a-D-Galp-(1-1)-+ | Subst-(1-17)-17HOBeh-(1-3)-a-D-Glcp l?17HOBeh = 17-hydroxybehenic acid; Subst = oxalic acid = SMILES O=C(O){1}C(=O)O | Show graphically |
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Structure type: oligomer
; 775.40918 [M+Na]+
C36H65O16
Trivial name: emmyguyacin A
Contained glycoepitopes: IEDB_136906,IEDB_137472,IEDB_141794,IEDB_142488,IEDB_144998,IEDB_146664,IEDB_151528,IEDB_190606,IEDB_983931,SB_192,SB_7
Two novel glycolipids, emmyguyacin A (1a) and emmyguyacin B (1b), were isolated at concentrations of 1.51 g/L from a potato dextrose agar fermentation of a sterile fungus species. The compounds inhibit replication of influenza A virus (A/X31) in MDCK cells by inhibiting the pH-dependent conformational change of hemagglutinin A (IC50 9 μM). The structures were deduced using one- and two-dimensional NMR techniques and mass spectrometric analyses on both the parent compounds and a host of degradation products and derivatives. A novel and unusual oxalic acid ester of a monohydroxylated fatty acid (5, 17-oxalyloxydocosanoic acid) is reported. The first isolation and characterization of the fatty acid 17-hydroxydocosanoic acid (3) itself is also reported as a saponification product of 1.
Publication DOI: 10.1021/np010345a| a-D-Glcp-(1-1)-+ | Subst-(1-17)-17HOBeh-(1-3)-a-D-Glcp l?17HOBeh = 17-hydroxybehenic acid; Subst = oxalic acid = SMILES O=C(O){1}C(=O)O | Show graphically |
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Structure type: oligomer
Trivial name: emmyguyacin B
Compound class: glycoside
Contained glycoepitopes: IEDB_142488,IEDB_144998,IEDB_146664,IEDB_742521,IEDB_983931,SB_192
Background: Natural products acquire vast and intriguing structural diversity and have been recognized as a tremendously diverse source of new lead compounds. Numerous bioactive secondary metabolites are present in the form of glycosylated molecules in which the sugar parts are normally associated with the interaction along with molecular recognition of the cellular target. Scope and approach: The presence of sugar entities are crucial as well as in some cases necessary, for therapeutic effects. Establishing novel and potent glycosylated secondary metabolites has formed a main goal in the natural product field from fungi and bacteria. These compounds possess a diverse range of sugar units. Key findings and conclusions: Fungi is considered one of the important sources for approved drugs with a diverse range of mode of action. The sugar part in numerous pharmacologically active natural products enhances bioavailability, biological potential, reduce toxicity, and improve stability. The vast majority of glyocosides showed antimicrobial effects, cytotoxic, antiviral and antiinflammatory effects. Notably, numerous fungal glycosides presented in this review illustrate significant antimicrobial effects towards various microorganisms especially against plant pathogens. The antimicrobial effects of these fungal glycosides indicate that these metabolites could be employed as natural preservatives in food in order to abolish or control the growth of pathogenic and spoilage microorganisms.
glycoside, antimicrobial, fungi, food preservative, secondary metabolites
Publication DOI: 10.1016/j.tifs.2021.02.029Two novel glycolipids, emmyguyacin A (1a) and emmyguyacin B (1b), were isolated at concentrations of 1.51 g/L from a potato dextrose agar fermentation of a sterile fungus species. The compounds inhibit replication of influenza A virus (A/X31) in MDCK cells by inhibiting the pH-dependent conformational change of hemagglutinin A (IC50 9 μM). The structures were deduced using one- and two-dimensional NMR techniques and mass spectrometric analyses on both the parent compounds and a host of degradation products and derivatives. A novel and unusual oxalic acid ester of a monohydroxylated fatty acid (5, 17-oxalyloxydocosanoic acid) is reported. The first isolation and characterization of the fatty acid 17-hydroxydocosanoic acid (3) itself is also reported as a saponification product of 1.
Publication DOI: 10.1021/np010345a| 17HOBeh-(1-3)-+ | a-D-Galp-(1-1)-a-D-Glcp l?17HOBeh = 17-hydroxybehenic acid | Show graphically |
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Structure type: oligomer
Contained glycoepitopes: IEDB_136906,IEDB_137472,IEDB_141794,IEDB_142488,IEDB_144998,IEDB_146664,IEDB_151528,IEDB_190606,IEDB_983931,SB_192,SB_7
Two novel glycolipids, emmyguyacin A (1a) and emmyguyacin B (1b), were isolated at concentrations of 1.51 g/L from a potato dextrose agar fermentation of a sterile fungus species. The compounds inhibit replication of influenza A virus (A/X31) in MDCK cells by inhibiting the pH-dependent conformational change of hemagglutinin A (IC50 9 μM). The structures were deduced using one- and two-dimensional NMR techniques and mass spectrometric analyses on both the parent compounds and a host of degradation products and derivatives. A novel and unusual oxalic acid ester of a monohydroxylated fatty acid (5, 17-oxalyloxydocosanoic acid) is reported. The first isolation and characterization of the fatty acid 17-hydroxydocosanoic acid (3) itself is also reported as a saponification product of 1.
Publication DOI: 10.1021/np010345a| a-D-Galp-(1-1)-a-D-Glcp | Show graphically |
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Structure type: oligomer
Contained glycoepitopes: IEDB_136906,IEDB_137472,IEDB_141794,IEDB_142488,IEDB_144998,IEDB_146664,IEDB_151528,IEDB_190606,IEDB_983931,SB_192,SB_7
Two novel glycolipids, emmyguyacin A (1a) and emmyguyacin B (1b), were isolated at concentrations of 1.51 g/L from a potato dextrose agar fermentation of a sterile fungus species. The compounds inhibit replication of influenza A virus (A/X31) in MDCK cells by inhibiting the pH-dependent conformational change of hemagglutinin A (IC50 9 μM). The structures were deduced using one- and two-dimensional NMR techniques and mass spectrometric analyses on both the parent compounds and a host of degradation products and derivatives. A novel and unusual oxalic acid ester of a monohydroxylated fatty acid (5, 17-oxalyloxydocosanoic acid) is reported. The first isolation and characterization of the fatty acid 17-hydroxydocosanoic acid (3) itself is also reported as a saponification product of 1.
Publication DOI: 10.1021/np010345a| b-D-Glcp-(1-3)-Subst Subst = ergosterol = SMILES O{3}[C@@H]4C/C3=C/C=C1\[C@H](CC[C@]2([C@H]1CC[C@@H]2[C@@H](/C=C/[C@H](C)C(C)C)C)C)[C@@]3(C)CC4 | Show graphically |
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Structure type: monomer
C34H56O10
Trivial name: ergosterol-β-D-glucopyranoside, ergosterol β-D-glucopyranoside
Compound class: saponin glycoside, glycoside, steroid glycoside, sterylglycoside
Contained glycoepitopes: IEDB_142488,IEDB_146664,IEDB_983931,SB_192
To defend themselves against fungal pathogens, plants produce numerous antifungal proteins and peptides, including defensins, some of which have been proposed to interact with fungal cell surface glycosphingolipid components. Although not known as a phytopathogen, the filamentous fungus Neurospora crassa possesses numerous genes similar to those required for plant pathogenesis identified in fungal pathogens (Galagan, J. E., et al. 2003. Nature 422: 859-868), and it has been used as a model for studying plant-phytopathogen interactions targeting fungal membrane components (Thevissen, K., et al. 2003. Peptides. 24: 1705 1712). For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa. The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as β-glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N-2'-hydroxy-(E)-Delta(3)-octadecenoate as the fatty-N-acyl component attached to the long-chain base (4E,8E)-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N-2'-hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-β-glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.
mass spectrometry, nuclear magnetic resonance spectroscopy, ceramide, tandem mass spectrometry, Electrospray Ionization, collision-induced dissociation, glucoside, Aspergillus fumigatus, sphingolipid, cerebroside, ergosterol, sterol
NCBI PubMed ID: 15654124Activity guided fractionations led to the isolation of two antitumor compounds 5a,8a-epidioxy-24(R)-methylcholesta-6,22-dien-3β-D-glucopyranoside and 5,6-epoxy-24(R)-methylcholesta-7,22-dien-3β-ol from the methanol extract of Cordyceps sinensis. Two previously known compounds, ergosteryl-3-O-β-D-glucopyranoside and 22-dihydroergosteryl-3-O-β-D-glucopyranoside were also isolated. The structures of hitherto unknown sterols were established by 1D and 2D NMR spectroscopic techniques with the former synthesized in order to confirm the identity of the sugar moiety by chemical correlation. The glycosylated form of ergosterol peroxide was found to be a greater inhibitor to the proliferation of K562, Jurkat, WM-1341, HL-60 and RPMI-8226 tumor cell lines by 10 to 40% at 10 mg/ml than its previously identified aglycone, 5a,8a-epidioxy-24(R)-methylcholesta-6,22-dien-3β-ol
antitumor activity, Cordyceps sinensis, steroidal glycosides, Cordyceps sinenis
NCBI PubMed ID: 10423860Cryptococcosis is an infectious disease caused by pathogenic fungi, such as Cryptococcus neoformans and Cryptococcus gattii. The ceramide structure (methyl-d18:2/h18:0) of C. neoformans glucosylceramide (GlcCer) is characteristic and strongly related to its pathogenicity. We recently identified endoglycoceramidase-related protein 1 (EGCrP1) as a glucocerebrosidase in C. neoformans and showed that it was involved in the quality control of GlcCer by eliminating immature GlcCer during the synthesis of GlcCer (Ishibashi, Y., Ikeda, K., Sakaguchi, K., Okino, N., Taguchi, R., and Ito, M. (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1). J. Biol. Chem. 287, 368-381). We herein identified and characterized EGCrP2, a homologue of EGCrP1, as the enzyme responsible for sterylglucoside catabolism in C. neoformans. In contrast to EGCrP1, which is specific to GlcCer, EGCrP2 hydrolyzed various β-glucosides, including GlcCer, cholesteryl-β-glucoside, ergosteryl-β-glucoside, sitosteryl-β-glucoside, and para-nitrophenyl-β-glucoside, but not α-glucosides or β-galactosides, under acidic conditions. Disruption of the EGCrP2 gene (egcrp2) resulted in the accumulation of a glycolipid, the structure of which was determined following purification to ergosteryl-3β-glucoside, a major sterylglucoside in fungi, by mass spectrometric and two-dimensional nuclear magnetic resonance analyses. This glycolipid accumulated in vacuoles and EGCrP2 was detected in vacuole-enriched fraction. These results indicated that EGCrP2 was involved in the catabolism of ergosteryl-β-glucoside in the vacuoles of C. neoformans. Distinct growth arrest, a dysfunction in cell budding, and an abnormal vacuole morphology were detected in the egcrp2-disrupted mutants, suggesting that EGCrP2 may be a promising target for anti-cryptococcal drugs. EGCrP2, classified into glycohydrolase family 5, is the first steryl-β-glucosidase identified as well as a missing link in sterylglucoside metabolism in fungi
metabolism, glycolipid, fungi, glycosidase, sterol
NCBI PubMed ID: 25361768The pathogenic fungus Cryptococcus neoformans causes cryptococcosis, an opportunistic infectious disease resulting in 600,000 deaths per year. The two major glycolipids in C. neoformans are glucosylceramide (GlcCer) with a fungus-specific ceramide (methyl d18 : 2/h18 : 0) and ergosteryl β-glucoside (EG); however, the catabolic pathway of these glycolipids has not yet been uncovered. We found two homologues of endoglycoceramidase (EGCase, EC 3.2.1.123) in C. neoformans, designated Endoglycoceramidase-related Protein 1 and 2 (EGCrP1 and EGCrP2). EGCase hydrolyzes the O-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids. However, EGCrP1 and EGCrP2 show completely different specificities; that is, EGCrP1 is a neutral glucocerebrosidase specific to GlcCer, whereas EGCrP2 is an acid β-glucosidase capable of hydrolyzing not only GlcCer but also various β-glucosides, including pNP β-glucoside and EG. Using each disruption mutant of egcrp1 and egcrp2, we elucidated that EGCrP1 plays an integral role in quality control of the fungus-specific GlcCer by eliminating immature GlcCer, which are byproducts of the GlcCer synthesis pathway, whereas EGCrP2 is involved in the catabolism of EG in the vacuoles of C. neoformans. The analysis of egcrp1-disrupted mutants also revealed that the quality control of fungus-specific GlcCer is strongly linked to the formation of the polysaccharide capsule, an important virulence factor. On the other hand, the disruption of EG catabolism resulted in growth arrest, dysfunction in cell budding, and abnormal vacuole morphology. These results indicate that catabolism of two different glycolipids plays different physiological roles in C. neoformans and strongly suggest EGCrP1 and EGCrP2 as targets for anti-cryptococcal drugs with a new mechanism of action
capsule, Cryptococcus neoformans, sterylglucoside, glycolipid catabolism, glucosylceramide quality control, vacuole
Publication DOI: 10.4052/tigg.1504.1ECordyceps sinensis, also called DongChongXiaCao (winter worm, summer grass) in Chinese, is becoming increasingly popular and important in the public and scientific communities. This study summarizes the chemical constituents and their corresponding pharmacological actions of Cordyceps sinensis. Many bioactive components of Cordyceps sinensis have been extracted including nucleoside, polysaccharide, sterol, protein, amino acid, and polypeptide. In addition, these constituents’ corresponding pharmacological actions were also shown in the study such as anti-inflammatory, antioxidant, antitumour, antiapoptosis, and immunomodulatory actions. Therefore can use different effects of C. sinensis against different diseases and provide reference for the study of Cordyceps sinensis in the future
polysaccharides, Cordyceps sinensis, nucleosides, sterol compounds
NCBI PubMed ID: 25960753Six new ergostane-type steroids; (22E)-3β,5α,6α,11-tetrahydroxy-9(11)-seco-ergosta-7,22-dien-9-one (1), (22E)-8,14-epoxyergosta-6,22-diene-3β,5α,9α-triol (2), (22E)-4α,5α-epoxyergosta-7,22-diene-3β,6β-diol (3), (22E)-3β,4β,5α-trihydroxyergosta-7,22-dien-6-one (4), (22E)-ergosta-7,22-diene-3β,5β,6α-triol (5), and (22E)-6β-methoxyergosta-7,22-diene-3β,5α-diol 3-O-β-D-glucopyranoside (6) were isolated from the fruiting bodies of king trumpet mushroom (Pleurotus eryngii), along with fourteen known compounds (7-20). All isolated compounds were evaluated for their inhibitory effects on macrophage activation using a nitric oxide production inhibition assay.
Pleurotus eryngii, steroid, ergostane, inhibitory effect, nitric oxide production
NCBI PubMed ID: 27423395A new β-tetralonyl glucoside, methylberchemiaside (1), along with five known compounds (2-6) were isolated from a fungus Colletotrichum sp . GDMU-1 derived from the leaves of Santalum album. Their structures were determined by detailed analysis of spectroscopic data. All compounds were tested for the inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-treated RAW264.7 cells. Among them, compounds 4 and 5 showed moderate anti-inflammatory activity with IC50 value of 30.4 and 8.9 μM, respectively.
anti-inflammatory activity, β-tetralonyl glucoside
NCBI PubMed ID: 29553810Cryptococcus spp. are responsible for life-threatening infections in humans causing mortality rates of 70% in developing countries. Antifungal therapy to combat cryptococcosis is based on the combination of amphotericin B, azoles, and 5-flucytosine. However, treatment failure is frequently triggered by antifungal resistance, drug-drug interactions, and toxicity. New alternatives to prevent cryptococcosis are imperative. Here, we discuss the roles of lipids in the immunological control of the disease caused by Cryptococcus spp. Recently, remarkable advances on immunology of fungal infections have been made and a number of studies indicated the potential of vaccine formulations to combat cryptococcosis. New formulations exploiting virulence regulators and genetically modified attenuated strains have been tested. In this context, lipids have emerged as virulence regulators and immunogens to be explored. Glucosylceramide (GlcCer), sterylglycosides (SGs), and lipid-containing extracellular vesicles have been recently tested in vaccine formulations and their anticryptococcal efficacy was confirmed in vivo. Together, the data discussed here encourage the use of fungal lipids in anticryptococcal vaccinal strategies.
glucosylceramide, cryptococcosis, antifungal vaccine, immunogenic lipids, sterylglycosides, extracellular vesicles
Publication DOI: 10.1007/s40475-019-00178-xInvasive fungal infections pose an increasing threat to human hosts, especially in immunocompromised individuals. In response to the increasing morbidity and mortality of fungal infections, numerous groups have shown great strides in uncovering novel treatment options and potential efficacious vaccine candidates for this increasing threat due to the increase in current antifungal resistance. Steryl glycosides are lipid compounds produced by a wide range of organisms, and are largely understudied in the field of pathogenicity, especially to fungal infections. Published works over the years have shown these compounds positively modulating the host immune response. Recent advances, most notably from our lab, have strongly indicated that steryl glycosides have high efficacy in protecting the host against lethal Cryptococcal infection through acting as an immunoadjuvant. This review will summarize the keystone studies on the role of steryl glycosides in the host immune response, as well as elucidate the remaining unknown characteristics and future perspectives of these compounds for the host-fungal interactions.
immunomodulatory, adjuvants, fungal pathogenesis, lipid metabolism, host immunity, sterols, steryl glycosides, vaccine candidates
NCBI PubMed ID: 32102324Previously, we reported that the polar lipid fraction from the golden oyster mushroom, Pleurotus citrinopileatus, suppresses colon injuries which result from apoptosis induced by inflammatory stresses in vivo and in vitro (Yamashita et al., J. Oleo Sci., 69, 751-757 (2020)). Here, we investigated the use of lipid classes in mushroom polar lipid fraction in alleviating colon injury using differentiated Caco-2 cells as an intestinal tract model. The mushroom polar lipid fraction was separated into four fractions using silica thin layer chromatography. Each mushroom polar lipid fraction suppressed lipopolysaccharide (LPS)-induced decreases in the viability of intestinal cells, and the effects of sphingolipid fractions were significantly stronger than those of fraction that did not contain sphingolipids. Addition of sphingolipid fractions suppressed the expression of apoptosis-related proteins (e.g., death receptors and caspases) in the LPS-treated cells. Mushroom polar lipids, especially sphingolipids suppress intestinal apoptosis induced by inflammatory stress, and highly polar sphingolipids may exert stronger suppressive effects.
inflammation, mushroom, apoptosis, sphingolipid, sphingoid base
NCBI PubMed ID: 32788522The structure, properties, distribution in nature, and biological activity of sterol glycosides and acylgylcosides are reviewed.
biological activity, sterol glycoside, acylgylcoside
Publication DOI: 10.1007/BF02323277| LIP-(1-2)-+ | b-D-Glcp-(1-1)-S,R-9b1SphdC19 | Show graphically |
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Structure type: monomer
Compound class: glycolipid, glycosphingolipid, cerebroside
Contained glycoepitopes: IEDB_137339,IEDB_142488,IEDB_146664,IEDB_983931,SB_192,SB_5
Cerebroside fraction was obtained from fresh fruiting bodies of Lentinus edodes and separated into ten molecular species by reverse-phase high-performance liquid chromatography. The species were identified by GLC, GC-MS and NMR. Their component glycosides and sphingoids were uniformly glucose and (4E,8E)-9-methyl-4,8-sphingadienine, respectively. The component fatty acids were 2-hydroxy acids with the carbon chain length of 16, 15, 14, 18, 24, 17, 25, 26, 22 and 23 (from major to minor). The cerebrosides with the C14-18 fatty acids showed strong fruiting-inducing activity in Schizophyllum commune. Those with the C22 and C23 ones had one-eighth and one-sixteenth of the activity, respectively, and those with C24-26 had no detectable activity. 22 and 23 must be the carbon chain lengths of the component fatty acid of the sphingolipids critical for expression of biological activity.
structure-activity relationship, Lentinus edodes, cerebroside, fruiting inducing activity
NCBI PubMed ID: 2917142Fungal infections are becoming more prevalent and problematic due to the continual rise of immune deficient patients as well as the progressive development of drug resistance towards currently available antifungal drugs. There has been a significant increase in the development of antifungal compounds with a similar mechanism of action of current drugs. In contrast, there has been very little progress in developing compounds inhibiting totally new fungal targets or/and fungal pathways. This review focuses on novel compounds recently discovered to target the fungal sphingolipids and their metabolizing enzymes.
sphingolipids, fungal infections, aspergillosis, cryptococcosis, candidiasis, drug development, antifungals
NCBI PubMed ID: 32825250| New query | Export IDs | Home | Help |
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