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.     

Cell-associated oligosaccharides of Bradyrhizobium spp.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.     

An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium

An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.     

Activation of serum prophenoloxidase in arthropod immunity. The specificity of cell wall glucan activation and activation by purified fungal glycoproteins of crayfish phenoloxidase.

This study shows that the activation of crayfish serum prophenoloxidase by carbohydrates was specific for beta-1,3-glucans. Fractionation of the beta-1,3-glucan laminaran into laminaran M and laminaran G showed that both activated the proenzyme, but the G-chain had somewhat higher affinity for the proenzyme. Methylation analysis of these two fractions revealed that there were no 1,6-linkages present. Laminaripentaose, a linear pentasaccharide composed of (1 leads to 3)-linked beta-D-glucopyranosyl residues was also active but had a lower affinity for the proenzyme than laminaran G. Laminaran completely inhibited the activation of prophenoloxidase by the pentaose. In the concentrations tested, laminaran was not inhibitory to the phenoloxidase activity. Purified extracellular glycoproteins of the parasitic fungus Aphanomyces astaci also strongly activated crayfish serum prophenoloxidase. Only high molecular weight glycoproteins were effective. Exo-beta-1,3-glucanase treatment decreased the activating capacity, suggesting that at least part of the glycoproteins consisted of beta-1,3-glucans. The significance of these results in the defence against parasitic fungi in crayfish is discussed.     

Linear beta-1,3 glucans are elicitors of defense responses in tobacco

Laminarin, a linear beta-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 microg 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 H(2)O(2). 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 beta-megaspermin. Structure activity studies with laminarin, laminarin oligomers, high molecular weight beta-1, 3-1,6 glucans from fungal cell walls, and the beta-1,6-1,3 heptaglucan showed that the elicitor effects observed in tobacco with beta-glucans are specific to linear beta-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 microg 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.     

Enzymatic synthesis of cello-oligosaccharides by rice BGlu1 {beta}-glucosidase glycosynthase mutants

Rice BGlu1 beta-glucosidase is a glycosyl hydrolase family 1 enzyme that acts as an exoglucanase on beta-(1,4)- and short beta-(1,3)-linked gluco-oligosaccharides. Mutations of BGlu1 beta-glucosidase at glutamate residue 414 of its natural precursor destroyed the enzyme's catalytic activity, but the enzyme could be rescued in the presence of the anionic nucleophiles such as formate and azide, which verifies that this residue is the catalytic nucleophile. The catalytic activities of three candidate mutants, E414G, E414S, and E414A, in the presence of the nucleophiles were compared. The E414G mutant had approximately 25- and 1400-fold higher catalytic efficiency than E414A and E414S, respectively. All three mutants could catalyze the synthesis of mixed length oligosaccharides by transglucosylation, when alpha-glucosyl fluoride was used as donor and pNP-cellobioside as acceptor. The E414G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than that of E414S and E414A, respectively, and gave yields of up to 70-80% insoluble products with a donor-acceptor ratio of 5:1. (13)C-NMR, methylation analysis, and electrospray ionization-mass spectrometry showed that the insoluble products were beta-(1,4)-linked oligomers with a degree of polymerization of 5 to at least 11. The BGlu1 E414G glycosynthase was found to prefer longer chain length oligosaccharides that occupy at least three sugar residue-binding subsites as acceptors for productive transglucosylation. This is the first report of a beta-glucansynthase derived from an exoglycosidase that can produce long-chain cello-oligosaccharides, which likely reflects the extended oligosaccharide-binding site of rice BGlu1 beta-glucosidase.     

Preparation and NMR characterization of glucosamine oligomers bearing an azide function using chitosan

In this study, a procedure to produce glucosamine oligomers with the amino functions transformed into azido groups was optimized, and HPLC purification afforded to the isolation of nine different oligosaccharides derivatives, with the reducing end transformed in alditol. These oligomers differed for the degree of polymerization and for the type of alditol at the reducing end. The first group comprehended species from di- to hexasaccharide, with all the amino functions converted into an azido group. The second and the third groups were isolated in minor yields, and were both constituted from tri- and tetrasaccharides; the difference between the two groups regarded exclusively the type of alditol found at the reducing end, which was a glucosaminitol in the first case, or a N-acetylglucosaminitol in the other. Products were fully characterized by 2D NMR spectroscopy. The azido moieties installed on these oligosaccharides can be further exploited in Cu(I) catalyzed azido-alkyne cycloaddition reactions.     

New oligo-β-(1,3)-glucan derivatives as immunostimulating agents

Oligo-β-(1,3)-glucans were chemically modified in order to introduce a structural variation specifically on the reducing end of the oligomers. The impact of well defined structural modulations was further studied on cancer cells and murin models to evaluate their cytotoxicity and immunostimulating potential.     

Distinct carbohydrate recognition domains of an invertebrate defense molecule recognize Gram-negative and Gram-positive bacteria

Coelomic fluid of Eisenia foetida earthworms (Oligochaeta, Annelida) contains a 42-kDa defense molecule named CCF for coelomic cytolytic factor. By binding microbial antigens, namely the O-antigen of lipopolysaccharide (LPS), beta-1,3-glucans, or N,N'-diacetylchitobiose present, respectively, on Gram-negative bacteria or yeast cell walls, CCF triggers the prophenoloxidase activating pathway. We report that CCF recognizes lysozyme-predigested Gram-positive bacteria or the peptidoglycan constituent muramyl dipeptide as well as muramic acid. To identify the pattern recognition domains of CCF, deletion mutants were tested for their ability to reconstitute the prophenoloxidase cascade in E. foetida coelomic fluid depleted of endogenous CCF in the presence of LPS, beta-1,3-glucans, N,N'-diacetylchitobiose, and muramic acid. In addition, affinity chromatography of CCF peptides was performed on immobilized beta-1,3-glucans or N,N'-diacetylchitobiose. We found that the broad specificity of CCF for pathogen-associated molecular patterns results from the presence of two distinct pattern recognition domains. One domain, which shows homology with the polysaccharide and glucanase motifs of beta-1,3-glucanases and invertebrate defense molecules located in the central part of the CCF polypeptide chain, interacts with LPS and beta-1,3-glucans. The C-terminal tryptophan-rich domain mediates interactions of CCF with N,N'-diacetylchitobiose and muramic acid. These data provide evidence for the presence of spatially distinct carbohydrate recognition domains within this invertebrate defense molecule.     

Some thaumatin-like proteins hydrolyse polymeric β-1,3-glucans

Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.     

Carbohydrate binding properties of banana (Musa acuminata) lectin II. Binding of laminaribiose oligosaccharides and beta-glucans containing beta1,6-glucosyl end groups.

This paper extends our knowledge of the rather bizarre carbohydrate binding poperties of the banana lectin (Musa acuminata). Although a glucose/mannose binding protein which recognizes alpha-linked gluco-and manno-pyranosyl groups of polysaccharide chain ends, the banana lectin was shown to bind to internal 3-O-alpha-D-glucopyranosyl units. Now we report that this lectin also binds to the reducing glucosyl groups of beta-1,3-linked glucosyl oligosaccharides (e.g. laminaribiose oligomers). Additionally, banana lectin also recognizes beta1,6-linked glucosyl end groups (gentiobiosyl groups) as occur in many fungal beta1,3/1,6-linked polysaccharides. This behavior clearly distinguishes the banana lectin from other mannose/glucose binding lectins, such as concanavalin A and the pea, lentil and Calystegia sepium lectins.     

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.     

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.     

The beta-1,3-glucanosyltransferase gas4p is essential for ascospore wall maturation and spore viability in Schizosaccharomyces pombe

Meiosis is the developmental programme by which sexually reproducing diploid organisms generate haploid gametes. In yeast, meiosis is followed by spore morphogenesis. The formation of the Schizosaccharomyces pombe ascospore wall requires the co-ordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans. During sporogenesis, the beta-1,3-glucan synthase bgs2p synthesizes linear beta-1,3-glucans, which remain unorganized and alkali-soluble until covalent linkages are set up between beta-1,3-glucans and other cell wall components. Several proteins belonging to the glycoside hydrolase family 72 (GH72) with beta-1,3-glucanosyltransferase activity have been described in other organisms, such as the Saccharomyces cerevisiae Gas1p or the Aspergillus fumigatus Gel1p. Here we describe the characterization of gas4(+), a new gene that encodes a protein of the GH72 family. Deletion of this gene does not lead to any apparent defect during vegetative growth, but homozygous gas4Delta diploids show a sporulation defect. Although meiosis occurs normally, ascospores are unable to mature or to germinate. The expression of gas4(+) is strongly induced during sporulation and a yellow fluorescent protein (YFP)-gas4p fusion protein localizes to the ascospore periphery during sporulation. We conclude that gas4p is required for ascospore maturation in S. pombe.     

PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans

PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and beta-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between beta-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in beta-1,3-glucan-associated beta-1, 6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between beta-1,6- and beta-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process beta-1,3-glucans and make available acceptor sites for the attachment of beta-1,6-glucans.     

Phosphoglycerol substituents present on the cyclic beta-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol.

The synthesis of periplasmic cyclic beta-1,2-glucans is a property unique to species of the family Rhizobiaceae. For this reason, it is generally believed that these molecules may play an important role in the plant infection process. In the present study, we determined that the cyclic beta-1,2-glucans produced by Rhizobium meliloti 1021 were predominantly anionic in character and contained both phosphoglycerol and succinic acid substituents. In addition, we demonstrated that phosphatidylglycerol was the source of the phosphoglycerol substituents present on these oligosaccharides and that greater than 60% of the total phospholipid turnover in this organism involved this substitution reaction.     

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.     

Elicitor and resistance-inducing activities of beta-1,4 cellodextrins in grapevine, comparison with beta-1,3 glucans and alpha-1,4 oligogalacturonides

Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response.     

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.     

Characterization of an exo-beta-1,3-galactanase from Clostridium thermocellum

A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.