Relative contributions of dectin-1 and complement to immune responses to particulate β-glucans

Glucan particles (GPs) are Saccharomyces cerevisiae cell walls chemically extracted so they are composed primarily of particulate β-1,3-D-glucans. GPs are recognized by Dectin-1 and are potent complement activators. Mice immunized with Ag-loaded GPs develop robust Ab and CD4(+) T cell responses. In this study, we examined the relative contributions of Dectin-1 and complement to GP phagocytosis and Ag-specific responses to immunization with OVA encapsulated in GPs. The in vitro phagocytosis of GPs by bone marrow-derived dendritic cells was facilitated by heat-labile serum component(s) independently of Dectin-1. This enhanced uptake was not seen with serum from complement component 3 knockout (C3(-/-)) mice and was also inhibited by blocking Abs directed against complement receptor 3. After i.p. injection, percent phagocytosis of GPs by peritoneal macrophages was comparable in wild-type and Dectin-1(-/-) mice and was not inhibited by the soluble β-glucan antagonist laminarin. In contrast, a much lower percentage of peritoneal macrophages from C3(-/-) mice phagocytosed GPs, and this percentage was further reduced in the presence of laminarin. Subcutaneous immunization of wild-type, Dectin-1(-/-), and C3(-/-) mice with GP-OVA resulted in similar Ag-specific IgG(1) and IgG(2c) type Ab and CD4(+) T cell lymphoproliferative responses. Moreover, while CD4(+) Th1 and Th2 responses measured by ELISPOT assay were similar in the three mouse strains, Th17 responses were reduced in C3(-/-) mice. Thus, although Dectin-1 is necessary for optimal phagocytosis of GPs in the absence of complement, complement dominates when both an intact complement system and Dectin-1 are present. In addition, Th-skewing after GP-based immunization was altered in C3(-/-) mice.     

Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.     

Double diastereoselection explains limitations in synthesizing mannose-containing β-(1,3)-glucans

It is known that 3-O-glycosylation of glucosidic acceptors bearing acyl groups in the 4 and 6 positions instead of a 4,6-O-benzylidene ring mainly affords α-glycosides. Described here is an unexpected stereochemical outcome for elongation at glucose O-3 of a β-d-Glcp-(1→3)-α-d-Manp disaccharide using peracetylated ethyl thioglucoside as a donor. This unexpected reaction was correlated with match-mismatch effects, as shown by efficient coupling of the same acceptor by a donor of l-configuration.     

Location of a cell-wall hydroxyproline-rich glycoprotein,cellulose and β-1,3-glucans in apical and differentiated regions of maize mycorrhizal roots

The 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 -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.Abbreviations CBH I cellobiohydrolase - DAPI 4,6-diamino-2-phenylindole - HRGP hydroxyproline-rich glycoprotein The research was supported by the Italian Murst (40%) grant and by an International Project between Spain and Italy (Azioni Integrate).     

Specific Fusion of β-1,4-Endoglucanase and β-1,4-Glucosidase Enhances Cellulolytic Activity and Helps in Channeling of Intermediates

Identification and design of new cellulolytic enzymes with higher catalytic efficiency are a key factor in reducing the production cost of lignocellulosic bioalcohol. We report here identification of a novel β-glucosidase (Gluc1C) from Paenibacillus sp. strain MTCC 5639 and construction of bifunctional chimeric proteins based on Gluc1C and Endo5A, a β-1,4-endoglucanase isolated from MTCC 5639 earlier. The 448-amino-acid-long Gluc1C contained a GH superfamily 1 domain and hydrolyzed cellodextrin up to a five-sugar chain length, with highest efficiency toward cellobiose. Addition of Gluc1C improved the ability of Endo5A to release the reducing sugars from carboxymethyl cellulose. We therefore constructed six bifunctional chimeric proteins based on Endo5A and Gluc1C varying in the positions and sizes of linkers. One of the constructs, EG5, consisting of Endo5A-(G₄S)₃-Gluc1C, demonstrated 3.2- and 2-fold higher molar specific activities for β-glucosidase and endoglucanase, respectively, than Gluc1C and Endo5A alone. EG5 also showed 2-fold higher catalytic efficiency than individual recombinant enzymes. The thermal denaturation monitored by circular dichroism (CD) spectroscopy demonstrated that the fusion of Gluc1C with Endo5A resulted in increased thermostability of both domains by 5°C and 9°C, respectively. Comparative hydrolysis experiments done on alkali-treated rice straw and CMC indicated 2-fold higher release of product by EG5 than that by the physical mixture of Endo5A and Gluc1C, providing a rationale for channeling of intermediates. Addition of EG5 to a commercial enzyme preparation significantly enhanced release of reducing sugars from pretreated biomass, indicating its commercial applicability.     

Identification and Deletion of Tft1, a Predicted Glycosyltransferase Necessary for Cell Wall β-1,3;1,4-Glucan Synthesis in Aspergillus fumigatus

Aspergillus fumigatus is an environmental mold that causes severe, often fatal invasive infections in immunocompromised patients. The search for new antifungal drug targets is critical, and the synthesis of the cell wall represents a potential area to find such a target. Embedded within the main β-1,3-glucan core of the A. fumigatus cell wall is a mixed linkage, β-D-(1,3;1,4)-glucan. The role of this molecule or how it is synthesized is unknown, though it comprises 10% of the glucans within the wall. While this is not a well-studied molecule in fungi, it has been studied in plants. Using the sequences of two plant mixed linkage glucan synthases, a single ortholog was identified in A. fumigatus (Tft1). A strain lacking this enzyme (tft1Δ) was generated along with revertant strains containing the native gene under the control of either the native or a strongly expressing promoter. Immunofluorescence staining with an antibody against β-(1,3;1,4)-glucan and biochemical quantification of this polysaccharide in the tft1Δ strain demonstrated complete loss of this molecule. Reintroduction of the gene into the knockout strain yielded reappearance in amounts that correlated with expected expression of the gene. The loss of Tft1 and mixed linkage glucan yielded no in vitro growth phenotype. However, there was a modest increase in virulence for the tft1Δ strain in a wax worm model. While the precise roles for β-(1,3;1,4)-glucan within A. fumigatus cell wall are still uncertain, it is clear that Tft1 plays a pivotal role in the biosynthesis of this cell wall polysaccharide.     

Construction and characterization of a fusion β-1,3-1,4-glucanase to improve hydrolytic activity and thermostability

A new fusion gene (Bgl-licMB), encoding β-1,3-1,4-glucanase both from Bacillus amyloliquefaciens (Bgl) and Clostridium thermocellum (licMB), was constructed via end-to-end fusion and expressed in Escherichia coli to improve hydrolytic activity and thermostability of β-1,3-1,4-glucanase. The results of enzymatic properties showed that the catalytic efficiency (K_cat/K_m) of the fusion enzyme for oat β-glucan was 2.7 and 20-fold higher than that of the parental Bgl and licMB, respectively, and that the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bgl and licMB were both less than 30%. These properties make this particular β-1,3-1,4-glucanase a good candidate for application in brewing and animal-feed industries.     

Structural and catalytic roles of residues located in β13 strand and the following β-turn loop in Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

Background

Fibrobacter succinogenes 1,3-1,4-β-d-glucanase (Fsβ-glucanase) is the only naturally occurring circularly permuted β-glucanase among bacterial glucanases with reverse protein domains. We characterized the functional and structural significance of residues 200–209 located in the domain B of Fsβ-glucanase, corresponding to the major surface loop in the domain A region of Bacillus licheniformis glucanase.

Methods

Rational design approaches including site-directed mutagenesis, initial-rate kinetics, and structural modeling analysis were used in this study.

Results

Our kinetic data showed that D202N and D206N exhibited a 1.8- and 1.5-fold increase but G207N, G207−, F205L, N208G and T204F showed a 7.0- to 2.2-fold decrease, in catalytic efficiency (k_cat/K_M) compared to the wild-type enzyme. The comparative energy ΔΔG_b value in individual mutant enzymes was well correlated to their catalytic efficiency. D206R mutant enzyme exhibited the highest relative activity at 50 °C over 10 min, whereas K200F was the most heat-sensitive enzyme.

Conclusions

This study demonstrates that Phe205, Gly207, and Asn208 in the Type II turn of the connecting loop may play a role in the catalytic function of Fsβ-glucanase.

General significance

Residues 200–209 in Fsβ-glucanase resided at the similar structural topology to that of Bacillus enzyme were found to play some similar catalytic function in glucanase.     

Genes encoding acidic and basic class III β-1,3-glucanases are expressed in tomato plants upon viroid infection

-1,3-glucanases are hydrolytic enzymes considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants. We have isolated and characterized two complementary DNA clones, corresponding to new -1,3-glucanases from tomato plants (Lycopersicon esculentum) which are expressed upon challenge with citrus exocortis viroid. Amino acid sequence comparison revealed that they are most similar to -1,3-glucanases from tobacco, particularly to PR-Q, the unique component of the class III -1,3-glucanase. The deduced amino acid sequences of the two tomato -1,3-glucanases indicate that, although being highly similar in amino acid sequence, they have different isoelectric points: pI 10.5 for the basic isoform (Tom PR-Q b) and pI 5.2 for the acidic one (Tom PR-Q a). The expression of these two -1,3-glucanase messenger RNAs (mRNAs) in response to viroid infection and ethephon treatments was examined. mRNAs for these two isoforms are coordinately expressed and induced similarly to mRNAs for other PR proteins, indicating that they are part of a general and coordinate mechanism of response of tomato plants susceptible to viroid infection.     

Developmental and hormonal regulation of β-1,3-glucanase in tobacco

A highly sensitive and specific rocket immunoassay was used to measure the content of an endo-type -1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of -1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, -naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l^-1 NAA and 0.3 mg·l^-1 kinetin are transferred onto medium without hormones or with either hormone added separately, the -1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is -1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This -1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.Abbreviation NAA -naphthaleneacetic acid     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

Regulation of calcium in pancreatic α- and β-cells in health and disease

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.     

β-1,4-endoglucanases from Talaromyces amestolkiae: Production of glucooligosaccharides from different β-glucans

Abstract

This article presents the purification and characterization of two β-1,4-endoglucanases from Talaromyces amestolkiae. The cellulase activities secreted by this fungus were studied in the presence of different carbon sources, attaining the maximal levels in the presence of Avicel as carbon source. In these conditions, two glycosylated β-1,4-endoglucanases with molecular masses of 25,573?kDa (EG1) and 51,825?kDa (EG2), were purified. Both isoenzymes have acidic isoelectric points, 5.4 and 4.6, respectively. Their optimum pH and temperature, either in crudes or after purification, were in the range normally used for the simultaneous saccharification and fermentation in bioethanol production. In addition, the enzymatic hydrolysis of different β-glucans by both enzymes was studied. In the assayed conditions, both enzymes hydrolysed carboxymethylcellulose, a typical substrate for endoglucanases, although EG2 was much more efficient. However, EG1 was also able to hydrolyse lichenan and laminarin. These findings suggest the potential interest of EG2 for specific hydrolysis of cellulose, present in plant cell walls, to produce bioethanol, while the more promiscuous enzyme EG1 could be used for production of glucooligosaccharides.     

Effect of β-1/3,1/6-glucan upon immune responses and bacteria in the gut of healthy common carp (Cyprinus carpio)

β-glucans are frequently included in the diet of healthy common carp Cyprinus carpio as a pre-emptive measure for combatting disease. In order to study the effect this has on the relationship between the gut bacteria and host immune response, carp were maintained on either a β-glucan free diet or feed containing 0.1% MacroGard®, a β-1/3, 1/6-glucan, for up to 7 weeks and analysis of innate immune gene expression and molecular analysis of the gut bacteria was performed. The data reveals feeding of MacroGard® to healthy carp does not induce bactericidal innate immune gene expression in the gut but does appear to alter bacterial species richness that did not have a negative effect on overall health. Analysis of innate immune gene expression within the upper midgut revealed that there were significant changes over time in the expression of Interleukin (il)-1β, inducible nitric oxide synthase (inos), mucin (muc2) and C-reactive protein (crp2). Diet did not affect the number of copies of the bacterial 16s rDNA gene in the gut, used as a as a measure of total bacteria population size. However, PCR-denaturing gradient gel electrophoresis (DGGE) analysis revealed a shift in bacterial species richness with MacroGard feeding. Bactericidal immune gene expression of crp2, muc2 and il-1β was weakly correlated with gut bacteria population size indicating a potentially limited role of these genes in interacting with the gut bacteria in healthy carp in order to maintain gut homeostatic conditions. These findings highlight the importance of considering both host immunity and the microbiome together in order to fully elucidate the effeect of immunomodulants, such as β-glucans, upon gut health.     

Differences in N-acetyllactosamine synthesis between β-1,4-galactosyltransferases I and V

Unlike classical -1,4-galactosyltransferase (-1,4-GalT I), -1,4-GalT V (formerly IV*) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95: 472-477]. The human -1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that -1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 µM and 41 µM towards UDPGal, respectively. No significant inhibition of N-acetyllactosamine synthesis with -lactalbumin was observed for -1,4-GalT V but the significant inhibition with -lactalbumin was observed for -1,4-GalT I.     

The expression and function of β-1,4-galactosyltransferase-I in dendritic cells

β-1,4-Galactosyltransferase-I (GalTI) is unusual among the galactosyltransferase family, which has two isoforms that differ only in the length of their cytoplasmic domains [1]. In this study, we found that both the long and short isoforms of GalTI were expressed in human monocyte-derived dendritic cells (MoDCs), and localized in the cytoplasm near nucleus and cytomembrane. The expression level of GalTI and cellular adhesion ability was increased when DCs continued to mature. We also demonstrated that the cellular adhesion ability of DCs was inhibited by α-lactalbumin (α-LA) via interference with cell surface GalTI function, suggesting that the adhesion ability was positively correlated with the expression of cell surface long GalTI. α-LA also could inhibit DC-T clustering and CD4⁺ T cell proliferation. Collectively, the data suggests that GalTI might act as a key adhesion molecular participating in T cells-DCs contacts.     

Quantification and characterization of β-lactam resistance genes in 15 sewage treatment plants from East Asia and North America

The emerging antibiotic resistance genes in the aquatic environment have aroused public concern. As β-lactam is the most widely used group of antibiotics, β-lactam resistance genes were selected to investigate their distribution and diversity in the activated sludge from 15 geographically different sewage treatment plants (STPs) of China, Singapore, USA, and Canada. Specific PCR and quantitative real-time PCR (q-PCR) were used to investigate the occurrence and abundance of nine β-lactam resistance genes. Five genes (OXA-1, OXA-2, OXA-10, ampC, and TEM-1) were detected in most of the sludge collected, while three genes (mecA, CTX-M-1, and SME) were not found in any sludge sample. The total abundances of the six detected β-lactam resistance genes in the 15 STPs varied from 5.34?×?10(1) copies/ng DNA (ampC) to 5.49?×?10(4) copies/ng DNA (OXA-1). Overall, OXA-1 had the highest total concentration, followed by IMP and OXA-10. Noticeably, the abundances of TEM-1 in Chinese STPs were generally higher than those in the STPs of other countries, while the abundances of OXA-2 and IMP in the STPs of North America were much greater than those of East Asia. A total of 78 clones carrying β-lactam resistance genes were randomly selected from six clone libraries for phylogenetic diversity analysis; the similarity of these cloned genes to known β-lactam resistance genes with sequence identities ranged from 96% to 100%. Furthermore, OXA-1, ampC, and IMP were found to be more diverse than the other β-lactam resistance genes.