Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

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

Mechanism by which orally administered beta-1,3-glucans enhance the tumoricidal activity of antitumor monoclonal antibodies in murine tumor models

Antitumor mAb bind to tumors and activate complement, coating tumors with iC3b. Intravenously administered yeast beta-1,3;1,6-glucan functions as an adjuvant for antitumor mAb by priming the inactivated C3b (iC3b) receptors (CR3; CD11b/CD18) of circulating granulocytes, enabling CR3 to trigger cytotoxicity of iC3b-coated tumors. Recent data indicated that barley beta-1,3;1,4-glucan given orally similarly potentiated the activity of antitumor mAb, leading to enhanced tumor regression and survival. This investigation showed that orally administered yeast beta-1,3;1,6-glucan functioned similarly to barley beta-1,3;1,4-glucan with antitumor mAb. With both oral beta-1,3-glucans, a requirement for iC3b on tumors and CR3 on granulocytes was confirmed by demonstrating therapeutic failures in mice deficient in C3 or CR3. Barley and yeast beta-1,3-glucan were labeled with fluorescein to track their oral uptake and processing in vivo. Orally administered beta-1,3-glucans were taken up by macrophages that transported them to spleen, lymph nodes, and bone marrow. Within the bone marrow, the macrophages degraded the large beta-1,3-glucans into smaller soluble beta-1,3-glucan fragments that were taken up by the CR3 of marginated granulocytes. These granulocytes with CR3-bound beta-1,3-glucan-fluorescein were shown to kill iC3b-opsonized tumor cells following their recruitment to a site of complement activation resembling a tumor coated with mAb.     

Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance

In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.     

Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.     

Covalent association of beta-1,3-glucan with beta-1,6-glucosylated mannoproteins in cell walls of Candida albicans.

Yeast and hyphal walls of Candida albicans were extracted with sodium dodecyl sulfate (SDS). Some of the extracted proteins reacted with a specific beta-1,6-glucan antiserum but not with a beta-1,3-glucan antiserum. They lost their beta-1,6-glucan epitope after treatment with ice-cold aqueous hydrofluoric acid, suggesting that beta-1,6-glucan was linked to the protein through a phosphodiester bridge. When yeast and hyphal walls extracted with SDS were subsequently extracted with a pure beta-1,3-glucanase, several mannoproteins that were recognized by both the beta-1,6-glucan antiserum and the beta-1,3-glucan antiserum were released. Both epitopes were sensitive to aqueous hydrofluoric acid treatment, suggesting that beta-1,3-glucan and beta-1,6-glucan are linked to proteins by phosphodiester linkages. The possible role of beta-glucans in the retention of cell wall proteins is discussed.     

Phagocytosis by human neutrophils is stimulated by a unique fungal cell wall component

Innate immunity depends upon recognition of surface features common to broad groups of pathogens. The glucose polymer beta-glucan has been implicated in fungal immune recognition. Fungal walls have two kinds of beta-glucan: beta-1,3-glucan and beta-1,6-glucan. Predominance of beta-1,3-glucan has led to the presumption that it is the key immunological determinant for neutrophils. Examining various beta-glucans for their ability to stimulate human neutrophils, we find that the minor cell wall component beta-1,6-glucan mediates neutrophil activity more efficiently than beta-1,3-glucan, as measured by engulfment, production of reactive oxygen species, and expression of heat shock proteins. Neutrophils rapidly ingest beads coated with beta-1,6-glucan while ignoring those coated with beta-1,3-glucan. Complement factors C3b/C3d are deposited on beta-1,6-glucan more readily than on beta-1,3-glucan. Beta-1,6-glucan is also important for efficient engulfment of the human pathogen Candida albicans. These unique stimulatory effects offer potential for directed stimulation of neutrophils in a therapeutic context.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells

Pattern recognition receptors, non-clonal immune proteins recognizing common microbial components, are critical for non-self recognition and the subsequent induction of Rel/NF-kappaB-controlled innate immune genes. However, the molecular identities of such receptors are still obscure. Here, we present data showing that Drosophila possesses at least three cDNAs encoding members of the Gram-negative bacteria-binding protein (DGNBP) family, one of which, DGNBP-1, has been characterized. Western blot, flow cytometric, and confocal laser microscopic analyses demonstrate that DGNBP-1 exists in both a soluble and a glycosylphosphatidylinositol-anchored membrane form in culture medium supernatant and on Drosophila immunocompetent cells, respectively. DGNBP-1 has a high affinity to microbial immune elicitors such as lipopolysaccharide (LPS) and beta-1,3-glucan whereas no binding affinity is detected with peptidoglycan, beta-1,4-glucan, or chitin. Importantly, the overexpression of DGNBP-1 in Drosophila immunocompetent cells enhances LPS- and beta-1,3-glucan-induced innate immune gene (NF-kappaB-dependent antimicrobial peptide gene) expression, which can be specifically blocked by pretreatment with anti-DGNBP-1 antibody. These results suggest that DGNBP-1 functions as a pattern recognition receptor for LPS from Gram-negative bacteria and beta-1, 3-glucan from fungi and plays an important role in non-self recognition and the subsequent immune signal transmission for the induction of antimicrobial peptide genes in the Drosophila innate immune system.     

Cell wall assembly by Pneumocystis carinii. Evidence for a unique gsc-1 subunit mediating beta -1,3-glucan deposition

Pneumocystis carinii remains a persistent cause of severe pneumonia in immune compromised patients. Recent studies indicate that P. carinii is a fungal species possessing a glucan-rich cyst wall. Pneumocandin antagonists of beta-1,3-glucan synthesis rapidly suppress infection in animal models of P. carinii pneumonia. We, therefore, sought to define the molecular mechanisms of beta-glucan cell wall assembly by P. carinii. Membrane extracts derived from freshly purified P. carinii incorporate uridine 5'-diphosphoglucose into insoluble carbohydrate, in a manner that was completely inhibited by the pneumocandin L733-560, an antagonist of Gsc-1-type beta-glucan synthetases. Using degenerative polymerase chain reaction and library screening, the P. carinii Gsc-1 catalytic subunit of beta-1,3-glucan synthetase was cloned and characterized. P. carinii gsc1 exhibited homology to phylogenetically related fungal beta-1,3-glucan synthetases, encoding a predicted 214-kDa integral membrane protein with 12 transmembrane domain structure. Immunoprecipitation of P. carinii extracts, with a synthetic peptide anti-Gsc-1 antibody, specifically yielded a protein of 219.4 kDa, which was also capable of incorporating 5'-diphosphoglucose into insoluble glucan carbohydrate. As opposed to other fungi, the expression of gsc-1 mRNA is uniquely regulated over P. carinii's life cycle, having minimal expression in trophic forms, but substantial expression in the thick-walled cystic form of the organism. These results indicate that P. carinii contains a unique catalytic subunit of beta-1,3-glucan synthetase utilized in cyst wall formation. Because synthesis of beta-1,3-glucan is absent in mammalian cells, inhibition of the P. carinii Gsc-1 represents an attractive molecular target for therapeutic exploitation.     

Isolation, purification and characterization of beta-1,3-glucan binding protein from the plasma of marine mussel Perna viridis

A beta-1,3-glucan binding protein (betaGBP) specific for laminarin (a beta-1,3-glucan) was detected for the first time in a mollusc, Perna viridis. betaGBP was isolated and purified from the plasma using laminarin precipitation and affinity chromatography on laminarin-Sepharose 6B, respectively. It agglutinated bakers yeast, bacteria, and erythrocytes and enhanced prophenoloxidase (proPO) activity of the plasma in a dose-dependent manner. The purified betaGBP appeared as a single band in native-PAGE and the purity was conformed by HPLC. The protein has a molecular weight estimate of 510kDa as determined by SDS-PAGE and in isoelectric focusing the purified betaGBP was focused as a single band at pI 5.3. betaGBP was found to possess inherent serine protease activity but lacked beta-1,3-glucanase activity and all these results suggest that plasma betaGBP of P. viridis functions as a recognition molecule for beta-1,3-glucan on the surface of microbial cell walls. This recognition and binding lead to the activation of the prophenoloxidase cascade mediated by the inherent serine protease activity of betaGBP. Presence of agglutinating activity and serine protease activity shows that betaGBP is a bifunctional protein. The findings are discussed in light of the importance of this protein in the innate immune response of P. viridis, and they implicate evolutionary link with similar proteins found in other invertebrates.     

Beta-1,3-glucan receptor and peptidoglycan receptor are present as separate entities within insect prophenoloxidase activating system

Silkworm plasma was passed over a peptidoglycan-Sepharose 4B column or a CPB column [CPB, curdlan type polysaccharide (beta-1,3-glucan) bead] in the absence of divalent cation and the effluents from the columns were named plasma-PG and plasma-CPB, respectively. Prophenoloxidase activating system in plasma-PG was triggered by beta-1,3-glucan but not by peptidoglycan and the system in plasma-CPB was triggered by peptidoglycan but not by beta-1,3-glucan, suggesting that the peptidoglycan-Sepharose 4B column and the CPB column remove peptidoglycan-receptor and beta-1,3-glucan-receptor, respectively, from plasma. This result indicates that both receptors exist as separate entities in silkworm plasma. It is suggested that plasma-PG and plasma-CPB may be used as specific reagents to detect minute amounts of beta-1,3-glucan and peptidoglycan.     

Isolation of extracellular 28- and 42-kilodalton beta-1,3-glucanases and comparison of three beta-1,3-glucanases produced by Bacillus circulans IAM1165.

Bacillus circulans IAM1165 produces three major extracellular beta-1,3-glucanases (molecular masses, 28, 42, and 91 kDa) during the stationary phase of growth. The 28- and 42-kDa enzymes were purified to homogeneity from the culture supernatant in this study. The properties of these two enzymes were examined, together with those of the 91-kDa enzyme previously isolated. The enzymatic properties of the 28- and 42-kDa beta-1,3-glucanases closely resemble each other. The enzymes belong to a category of endo type 1,3-beta-D-glucan glucanohydrolases. The enzymes were active at pH 4.0 to 7.0. The optimum temperature of the reactions was 60 degrees C when laminarin (a soluble beta-1,3-glucan) was used as the substrate at pH 7.0. The enzymes hydrolyzed barley glucan and lichenan (beta-1,3-1,4-glucans) more effectively than laminarin. Of the three enzymes, the 42-kDa enzyme lysed fungal cell walls the most effectively.     

Cloning of the RHO1 gene from Candida albicans and its regulation of beta-1,3-glucan synthesis.

The Saccharomyces cerevisiae RHO1 gene encodes a low-molecular-weight GTPase. One of its recently identified functions is the regulation of beta-1,3-glucan synthase, which synthesizes the main component of the fungal cell wall (J. Drgonova et al., Science 272:277-279, 1996; T. Mazur and W. Baginsky, J. Biol. Chem. 271:14604-14609, 1996; and H. Qadota et al., Science 272:279-281, 1996). From the opportunistic pathogenic fungus Candida albicans, we cloned the RHO1 gene by the PCR and cross-hybridization methods. Sequence analysis revealed that the Candida RHO1 gene has a 597-nucleotide region which encodes a putative 22.0-kDa peptide. The deduced amino acid sequence predicts that Candida albicans Rho1p is 82.9% identical to Saccharomyces Rho1p and contains all the domains conserved among Rho-type GTPases from other organisms. The Candida albicans RHO1 gene could rescue a S. cerevisiae strain containing a rho1 deletion. Furthermore, recombinant Candida albicans Rho1p could reactivate the beta-1,3-glucan synthesis activities of both C. albicans and S. cerevisiae membranes in which endogenous Rho1p had been depleted by Tergitol NP-40-NaCl treatment. Candida albicans Rho1p was copurified with the beta-1,3-glucan synthase putative catalytic subunit, Candida albicans Gsc1p, by product entrapment. Candida albicans Rho1p was shown to interact directly with Candida albicans Gsc1p in a ligand overlay assay and a cross-linking study. These results indicate that Candida albicans Rho1p acts in the same manner as Saccharomyces cerevisiae Rho1p to regulate beta-1,3-glucan synthesis.     

Antitumor effect of soluble beta-1,3-glucan from Agrobacterium sp. R259 KCTC 1019

Beta-1,3-glucans enhance immune reactions such as antitumor, antibacterial, antiviral, anticoagulatory, and wound healing activities. beta-1,3-Glucans have various functions depending on the molecular weight, degree of branching, conformation, water solubility, and intermolecular association. The molecular weight of the soluble glucan was about 15,000 as determined by a high-performance size exclusion chromatography. From the infrared (IR) and 13C NMR analytical data, the purified soluble glucan was found to exclusively consist of beta-D-glucopyranose with 1,3 linkage. We tested the immunestimulating activities of the soluble beta-1,3-glucan extracted from Agrobacterium sp. R259 KCTC 1019 and confirmed the following activities. IFN-gamma and each cytokines were induced in the spleens and thymus of mice treated with soluble beta-1,3-glucan. Adjuvant effect was observed on antibody production. Nitric oxide was synthesized in monocytic cell lines treated with beta-1,3-glucan. The cytotoxic and antitumor effects were observed on various cancer cell lines and ICR mice. These results strongly suggested that this soluble beta-1,3-glucan could be a good candidate for an immune-modulating agent.     

Innate immunity in a pyralid moth: functional evaluation of domains from a beta-1,3-glucan recognition protein

Invertebrates, like vertebrates, utilize pattern recognition proteins for detection of microbes and subsequent activation of innate immune responses. We report structural and functional properties of two domains from a beta-1,3-glucan recognition protein present in the hemolymph of a pyralid moth, Plodia interpunctella. A recombinant protein corresponding to the first 181 amino-terminal residues bound to beta-1,3-glucan, lipopolysaccharide, and lipoteichoic acid, polysaccharides found on cell surfaces of microorganisms, and also activated the prophenoloxidase-activating system, an immune response pathway in insects. The amino-terminal domain consists primarily of an alpha-helical secondary structure with a minor beta-structure. This domain was thermally stable and resisted proteolytic degradation. The 290 residue carboxyl-terminal domain, which is similar in sequence to glucanases, had less affinity for the polysaccharides, did not activate the prophenoloxidase cascade, had a more complicated CD spectrum, and was heat-labile and susceptible to proteinase digestion. The carboxyl-terminal domain bound to laminarin, a beta-1,3-glucan with beta-1,6 branches, but not to curdlan, a beta-1,3-glucan that lacks branching. These results indicate that the two domains of Plodia beta-1,3-glucan recognition protein, separated by a putative linker region, bind microbial polysaccharides with differing specificities and that the amino-terminal domain, which is unique to this class of pattern recognition receptors from invertebrates, is responsible for stimulating prophenoloxidase activation.     

Purification and partial characterization of a beta-1,3-glucan-binding-protein membrane receptor from blood cells of the crayfish Pacifastacus leniusculus.

A receptor for the 100 kDa beta-1,3-glucan-binding protein [Duvic, B. and S?derh?ll, K. (1990) J. Biol. Chem. 265, 9327-9332] has been purified from hemocyte membranes of the crayfish Pacifastacus leniusculus. The purification was achieved by DEAE-cellulose chromatography of detergent-solubilized membranes. The receptor had an apparent molecular mass of 350 kDa when subjected to native polyacrylamide-gel electrophoresis and was composed of two non-covalently associated subunits of about 230 kDa and 90 kDa, as judged by SDS/polyacrylamide-gel electrophoresis or two-dimensional electrophoresis. The receptor could only bind the beta-1,3-glucan-binding protein if this protein had previously reacted with a beta-1,3-glucan, laminarin, and the binding site was located on the 230 kDa subunit. The binding of laminarin-treated beta-1,3-glucan-binding protein to its receptor was a saturable process and binding data indicated a single high-affinity-binding site with a Kd of 0.35 +/- 0.15 microM as determined by Scatchard analysis. The receptor had a requirement for divalent cations and a pH optimum of 6.5 for binding the laminarin-treated beta-1,3-glucan-binding protein. Laminarin, as well as oligosaccharides such as D-glucose, sialic acid, N-acetyl glucosamine or methyl-alpha-D-mannoside, could not affect the binding of the beta-1,3-glucan-binding protein to its receptor.     

Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae.

A major protein of Saccharomyces cerevisiae cell walls is a 29-kilodalton glycoprotein which shows lectinlike binding to beta-1,3-glucan and chitin. It was solubilized by heating isolated cell walls at 90 degrees C and purified to homogeneity by running two high-pressure liquid chromatography columns. With the sequence information of the N terminus and seven peptides, two oligonucleotides were synthesized and the gene was cloned. Its sequence is similar to those of two plant beta-glucanases, and the protein was shown to possess beta-1,3-exoglucanase activity with laminarin as substrate. Haploid yeast cells contained one copy of the gene (BGL2). Gene disruption did not result in a phenotype.     

Yeast glucan in the cyst wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of beta-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains beta-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is beta-1,3-glucan laminaripentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is beta-1,3-glucan.