Immunologically active (1-->3)-(1-->4)-alpha-D-glucan from Cetraria islandica.

A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean M(r) of Ci-3 was determined to be 2000 kD, compared to 6-8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1-->3)- and (1-->4)-alpha-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.     

Rhamnopyranosylgalactofuranan, a new immunologically active polysaccharide from Thamnolia subuliformis.

A complex polysaccharide, Ths-3, consisting mainly of rhamnopyranosyl and galactofuranosyl units, has been isolated from the water extract of the lichen Thamnolia subuliformis using ethanol fractionation, dialysis, ion-exchange chromatography, gel filtration and preparative GP-HPLC. The mean M(r) of Ths-3 was determined to be 1450 kD, and the monosaccharide composition is gal/rha/glc/xyl/man in the ratio of 40:31:13:10:6. The structure of Ths-3 was further elucidated by methylation analysis by GC-MS and NMR spectroscopy and found to be basically composed of (1-->3)-linked beta-D-galactofuranosyl units with branches on C6, and rhamnosyl units being predominantly (1-->2)-linked with branches on C3 and C4, while some units are (1-->3)-linked. Glucose, mannose and galactofuranose are found as terminal units and glucose and mannose are also (1-->4)-linked, while xylose is only present as terminal units. The trisaccharide xylglcglc was detected after partial hydrolysis of the polysaccharide. The immunomodulating activity of Ths-3 was tested in an in vitro phagocytosis assay and the classical anticomplementary assay, and proved to be active in both tests. The authors suggest the trivial name thamnolan for Ths-3.     

Reversible accumulation of (1-->3,1-->4)-beta-glucan endohydrolase in wheat leaves under sugar depletion.

A (1-->3,1-->4)-beta-D-glucan endohydrolase [(1-->3,1-->4)-beta-glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1-->3,1-->4)-beta-glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1-->3,1-->4)-beta-glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1-->3,1-->4)-beta-glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1-->3,1-->4)-beta-glucanase accumulation under sugar depletion remains to be elucidated.     

Evolution and differential expression of the (1-->3)-beta-glucan endohydrolase-encoding gene family in barley, Hordeum vulgare.

The (1-->3)-beta-D-glucan glucanohydrolases [(1-->3)-GGH; EC 3.2.1.39] of barley (Hordeum vulgare L., cv Clipper) are encoded by a small gene family. Amino acid sequences deduced from cDNA and genomic clones for six members of the family exhibit overall positional identities ranging from 44% to 78%. Specific DNA and oligodeoxyribonucleotide (oligo) probes have been used to demonstrate that the (1-->3)-GGH-encoding genes are differentially transcribed in young roots, young leaves and the aleurone of germinated grain. The high degree of sequence homology, coupled with characteristic patterns of codon usage and insertion of a single intron at a highly conserved position in the signal peptide region, indicate that the genes have shared a common evolutionary history. Similar structural features in genes encoding barley (1-->3,1-->4)-beta-glucan 4-glucanohydrolases [(1-->3,1-->4)-GGH; EC 3.2.1.73] further indicate that the (1-->3)-GGHs and (1-->3,1-->4)-GGHs are derived from a single 'super' gene family, in which genes encoding enzymes with related yet quite distinct substrate specificities have evolved, with an associated specialization of function. The (1-->3,1-->4)-GGHs mediate in plant cell wall metabolism through their ability to hydrolyse the (1-->3,1-->4)-beta-glucans that are the major constituents in barley walls, while the (1-->3)-GGHs, which are unable to degrade the plant (1-->3,1-->4)-beta-glucans, can hydrolyse the (1-->3)- and (1-->3,1-->6)-beta-glucans of fungal cell walls.     

Synthesis of the alpha-L-Araf-(1-->2)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->2)]-beta-D-Galp-(1-->6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.

For the characterization of the supposed epitope of an arabinogalactan, isolated from the extract of the cell-cultured Echinacea purpurea, the title hexasaccharide was synthesized. The whole synthetic route was based on the 6-O-(methoxydimethyl)methyl ether (MIP) protecting group strategy. 2-O-Benzyl-3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose was used to prepare the desired glycosyl donor and glycosyl acceptor both carrying a persistent O-benzyl group at position 2'. Reaction of the digalactose donor and the digalactose acceptor resulted in a beta-(1-->6)-linked galactose-containing tetrasaccharide in which OH-2' and OH-2"' were substituted with benzyl groups. Hydrogenolytic removal of the benzyl groups of the tetragalactose compound gave the diol aglycon which was diarabinosylated in one step to furnish the protected target compound, whose deprotection led to the title hexasaccharide. All of the synthesized compounds were characterized by 1H and 13C NMR spectra, as well as by MALDI-TOF mass-spectrometry measurements.     

Synthesis of divalent beta-(1-->6)-branched (1-->3)-glucohexaose and trivalent beta-(1-->6)-branched (1-->3)-glucotriose

Hexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based dimers were synthesized by twofold glycosidation of the hexaosyl trichloroacetimidate with hexylene 1,6-diol, diethylene glycol and triethylene glycol, respectively. Meanwhile, a triose, beta-1D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based trimer was obtained by glycosidation of the triosyl trichloroacetimidate with a glycerol-derived triol scaffold.     

Enzyme-linked immunosorbent assay specific for (1-->6) branched, (1-->3)-beta-D-glucan detection in environmental samples.

(1-->3)-beta-D-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1-->6) branched, (1-->3)-beta-D-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1-->3)-beta-D-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall beta-D-glucans as a detector reagent. The assay was highly specific for (1-->6) branched, (1-->3)-beta-D-glucans (such as that from Saccharomyces cerevisiae) and did not show any response at 200 ng/ml to curdlan, laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and endotoxins. The detection level was 0.8 ng/ml for baker's yeast glucan and Betafectin. A coefficient of variation of 7.8% was obtained for (1-->3)-beta-D-glucans in house dust samples. Metal working fluids spiked with (1-->3)-beta-D-glucans inhibited the glucan assay. Because the assay is specific for (1-->6) branched, (1-->3)-beta-D-glucans and is sensitive and reproducible, it will be useful for the investigation of health effects from exposure to this class of biologically active molecules.     

Synthesis of mannose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose

Coupling of the trisaccharide acceptor 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-5-O-acetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (2) with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-alpha-D-annopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (1) gave an alpha-linked hexasaccharide 3, while coupling of 2 with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (7) produced alpha- 8 and beta-linked 12 hexasaccharides in a ratio of 3:2. Deprotection of 3, 8, and 12 afforded the analogues of the immunomodulator beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-D-Glcp (A).     

Synthesis of mannose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose (I)

alpha-D-Manp-(1-->3)-[alpha-D-Manp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[alpha-D-Manp-(1-->6)]-D-Glcp and alpha-D-Manp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-alpha-D-Manp-(1-->6)]-D-Glcp were synthesized in a regio- and stereoselective way as the mannose-containing analogues of the immunomodulating beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-D-Glcp.     

Insight into multi-site mechanisms of glycosyl transfer in (1-->4)beta-D-glycans provided by the cereal mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.

Synthases of cellulose, chitin, hyaluronan, and all other polymers containing (1-->4)beta-linked glucosyl, mannosyl and xylosyl units have overcome a substrate orientation problem in catalysis because the (1-->4)beta-linkage requires that each of these sugar units be inverted nearly 180 degrees with respect to its neighbors. We and others have proposed that this problem is solved by two modes of glycosyl transfer within a single catalytic subunit to generate disaccharide units, which, when linked processively, maintain the proper orientation without rotation or re-orientation of the synthetic machinery in 3-dimensional space. A variant of the strict (1-->4)beta-D-linkage structure is the mixed-linkage (1-->3),(1-->4)beta-D-glucan, a growth-specific cell wall polysaccharide found in grasses and cereals. beta-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1-->3)beta-D-linkages. In reactions in vitro at high substrate concentration, a polymer composed of almost entirely cellotriosyl and cellopentosyl units is made. These results support a model in which three modes of glycosyl transfer occur within the synthase complex instead of just two. The generation of odd numbered units demands that they are connected by (1-->3)beta-linkages and not (1-->4)beta-. In this short review of beta-glucan synthesis in maize, we show how such a model not only provides simple mechanisms of synthesis for all (1-->4)beta-D-glycans but also explains how the synthesis of callose, or strictly (1-->3)beta-D-glucans, occurs upon loss of the multiple modes of glycosyl transfer to a single one.     

An efficient and stereoselective synthesis of beta-D-Arap-(1-->2)-beta-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-alpha-D-Manp, a tetrasaccharide fragment of Leishmania major lipophosphoglycan.

A tetrasaccharide fragment of Leishmania major lipophosphoglycan (which seems to be involved in a biological mechanism for the parasite transmission) has been synthesised using the thioglycoside, trichloroacetimidate and halide-exchange glycosylation procedures and step-wise chain elongation strategy.     

Physicochemical properties of (1 → 6)-branched (1 → 3)-β-D-glucans. 1. Physical dimensions estimated from hydrodynamic and electron microscopic data

The physical dimensions of several (1 → 6) branched (1 → 3) -β-D -glucan samples obtained from different organisms and their derivatives have been studied by electron microscopy, light scattering measurements, viscometry, and gel permeation chromatography. The electron micrographs indicate that in most samples these biopolymers are adequately described as linear worm-like coils. A sample reconstituted from alkaline media appeared as a blend of the linear, circular, and aggregated polymer morphologies. The average mass per unit length, M_L = M_w/L_w for the macroscopically linear samples, was estimated to be 2100 ± 200 g mol^?1 nm^?1. The parameter m_L was determined from the contour lengths obtained by electron microscopy and the molecular weight by light scattering measurements. The observed M_L was consistent with the triple-helical structure reported from x-ray diffraction studies and observed degree of side-chain substitution. From the molecular snapshots shown in the electron micrographs, the persistence lengths of these β-D -glucans were determined to be 140 ± 30 nm. The experimentally determined intrinsic viscosities were consistent with these estimates of M_L and persistence length. Comparison of the molecular weight distributions obtained from gel permeation chromatography and those deduced from the electron micrographs indicates that number and weight average contour lengths are more reliable than z and z + 1 averages. © 1993 John Wiley & Sons, Inc.     

Synthesis of galactose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose and its lauryl glycoside

Coupling of the trisaccharide acceptor either 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-5-O-acetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (13) or lauryl 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,5-di-O-acetyl-alpha-D-glucopyranoside (15) with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate (12) gave alpha-linked hexasaccharides 14 and 16, respectively, while coupling of either 13 or 15 with trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate 17 did not afford any hexasaccarides. The analogues of the immunomodulator beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-beta-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp (1) was obtained by deprotection of 14 and 16.     

Studies on (beta,1-->5) and (beta,1-->6) linked octyl Gal(f) disaccharides as substrates for mycobacterial galactosyltransferase activity.

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.     

Chemical analysis of a new (1-->3)-, (1-->6)-branched glucan from an edible mushroom, Pleurotus florida

A glucan that was soluble in aqueous sodium chloride was isolated from the aqueous extract of the fruiting bodies of Pleurotus florida. On the basis of total hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR studies (1H, 13C, TOCSY, DQF-COSY, NOESY, and HSQC), the structure of the repeating unit of the polysaccharide is established as: This glucan stimulates the phagocytic activity of macrophages.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

beta-Furfuryl-beta-glucoside. An endogenous activator of higher plant UDP-glucose: (1----3)-beta-glucan synthase.

We have recently established the existence of endogenous activators of higher plant UDP-glucose: (1----3)-beta-glucan synthase (Callaghan, T., Ross, P., Weinberger-Ohana, P., and Benziman, M. (1988) Plant Physiol. 86, 1099-1103). Here we report the purification and chemical analysis of the most abundant and specific compound, termed Activator I, isolated from Vigna radiata. This compound was extensively purified by a multistep procedure which yielded 0.1 mg of purified activator/g of fresh tissue. Enzyme digestion, neutral sugar analysis, GC/MS of permethylated derivatives, and NMR analysis of native Activator I indicated that the compound contains a single beta-linked glucosyl residue. High resolution FAB-MS indicated an elemental composition of C11H16O7 (Mr = 260), with a calculated Mr of 98 for the aglycone. 13C, DEPT, and COSY NMR spectra showed that the aglycone molecule is an oxygen heterocycle of 5 carbons, consistent with a structure of beta-furfuryl alcohol. Comparison of IR and GC/EI-MS spectra of authentic beta-furfuryl alcohol with native aglycone confirmed the conclusion that Activator I is beta-furfuryl-beta-glucoside. Chemically synthesized beta-furfuryl-beta-glucoside has identical chemical properties and biological activity when compared with the purified endogenous activator (Ka = 50 microM).     

Synthesis of beta-(1-->6)-branched (1-->3)-glucododecaose and -glucopentadecaose with alternate beta- and alpha-bonds in the backbone

Beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)](2-3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp were synthesized as their methoxyphenyl glycosides in a concise way with a trisaccharide as the building block.     

Biosynthesis of (1-->3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana.

The aim of this work was to develop a biochemical approach to study (1-->3)-beta-D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH 6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive homotropic cooperativity of (1-->3)-beta-D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta-D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta-D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3)-beta-D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of the in vitro products are influenced by the detergent used for protein extraction. The reaction products synthesized by different detergent extracts were characterized by infrared spectroscopy, methylation analysis, 13C-NMR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1-->3)-beta-D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.     

Structure of epiglucan, a highly side-chain/branched (1 --> 3;1 --> 6)-beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht

The extracellular fungal polysaccharide, epiglucan, synthesised by Epicoccum nigrum is a side-chain/branched (1 --> 3;1 --> 6)-D-beta-glucan. Methylation analysis, 13C DEPT NMR and specific enzymic digestion data show slight variation in branching frequency among the epiglucans from the three strains examined. The (1 --> 3)-beta-linked backbone has (1 --> 6)-beta-linked branches at frequencies greater than the homologous glucans, scleroglucan and schizophyllan, from Sclerotium spp. and Schizophyllum commune, respectively. The structural analyses do not allow a distinction to be made between structures I and II. [structures: see text] Epiglucan displays non-Newtonian shear thinning rheological properties, typical of these glucans.