3-linked Glucose Research Articles

Exploiting diol reactivity for the access to unprecedented low molecular weight curdlan sulfate polysaccharides

Curdlan is a bacterial sourced polysaccharide, consisting of a linear backbone of β-1 → 3-linked glucose (Glc) units. The high interest in pharmaceutical applications of curdlan and derivatives thereof is fueling the study of multi-step sequences for regioselective modifications of its structure. Here we have developed semi-synthetic sequences based on a regioselective protection-sulfation-deprotection approach, allowing the access to some, new, low molecular weight curdlan polysaccharide derivatives with unprecedented sulfation patterns. Three different semi-synthetic schemes were investigated, all relying upon the installation of a cyclic benzylidene protecting group on Glc O-4,6-diols, followed by either direct sulfation and deprotection, or some additional steps – including a hydrolytic or oxidative cleavage of the benzylidene rings – prior to sulfation and deprotection. The six obtained polysaccharides were subjected to a detailed structural characterization by 2D-NMR analysis, revealing that some of them showed the majority of Glc units along the polymeric backbone decorated by unprecedented sulfation motifs.

Characterization and optimization of schizophyllan production from date syrup

This study demonstrates the efficient utilization of low-cost agricultural substrates, particularly date syrup, by Schizophyllum commune ATCC 38548 for schizophyllan production. Initially, one factor-at-a-time method was used to find the best carbon and nitrogen sources for schizophyllan production. Subsequently, response surface methodology was employed to optimize the level of culture medium components to maximize substrate conversion yield and schizophyllan production in submerged culture. Maximum product yield (0.12g schizophyllan/g date syrup) and schizophyllan production (8.5g/l) were obtained at concentrations of date syrup and corn steep liquor, inoculum size and agitation rate at 7.02%w/v, 0.10%w/v, 7.68%v/v and 181rpm, respectively. Sugar composition analysis, FTIR, NMR and molar mass determination revealed the purity and molecular properties of recovered schizophyllan produced from date syrup as glycosidic linkage analysis showed three main schizophyllan characteristic peaks arising from the 3-linked, 3,6-linked and terminal glucose residues. Finally, process economic analysis suggested that use of date syrup and corn steep liquor as nutrients would result in approximately 6-fold reduction in cost of raw materials for schizophyllan production as compared to conventional carbon and nitrogen sources such as sucrose and malt extract.

Isolation of cellotriosyl blocks from barley β-glucan with endo-1,4-β-glucanase from Trichoderma reesei

Mixed-linkage β-glucan from barley, with a cellotriosyl to cellotetraosyl ratio of 2.9, was hydrolysed with two endo-1,4-β-glucanases (cellulases) and one non-cellulolytic β-glucanase isolated from Trichoderma reesei. The hydrolysates were precipitated in 90% ethanol and the fragments obtained were further treated with lichenase, followed by analysis of the oligosaccharides released. One of the endo-1,4-β glucanases, Cel 5A (EG II), selectively degraded cellotetraosyl units in the polymer and left the cellotriosyl units unhydrolysed. Blocks of cellotriosyl units were isolated on a larger scale using this enzyme. The isolated blocks were fractionated into four fractions using gel permeation chromatography on Biogel P-6 and the structures of the blocks were analysed by 1H NMR spectroscopy. The fractions essentially contained cellotriosyl units of different sizes with a 3-linked glucose residue at the non-reducing end and a reducing end linked to two 4-linked glucose residues. The results thus indicated that the enzyme could hydrolyse a 4-linked glucose residue next to the 3-linked residue at the non-reducing terminal but left two 4-linked glucose residues at the reducing end. The isolated blocks of cellotriosyl units had a molecular weight distribution that fits a theoretical model based on random blocks of triosyl units in the mixed-linkage β-glucan.

The influence of mutanase and dextranase on the production and structure of glucans synthesized by streptococcal glucosyltransferases

Glucanohydrolases, especially mutanase [α-(1 → 3) glucanase; EC 3.2.1.59] and dextranase [α-(1 → 6) glucanase; EC 3.2.1.11], which are present in the biofilm known as dental plaque, may affect the synthesis and structure of glucans formed by glucosyltransferases (GTFs) from sucrose within dental plaque. We examined the production and the structure of glucans synthesized by GTFs B (synthesis of α-(1 → 3)-linked glucans) or C [synthesis of α-(1 → 6)- and α-(1 → 3)-linked glucans] in the presence of mutanase and dextranase, alone or in combination, in solution phase and on saliva-coated hydroxyapatite beads (surface phase). The ability of Streptococcus sobrinus 6715 to adhere to the glucan, which was formed in the presence of the glucanohydrolases was also explored. The presence of mutanase and/or dextranase during the synthesis of glucans by GTF B and C altered the proportions of soluble to insoluble glucan. The presence of either dextranase or mutanase alone had a modest effect on total amount of glucan formed, especially in the surface phase; the glucanohydrolases in combination reduced the total amount of glucan. The amount of (1 → 6)-linked glucan was reduced in presence of dextranase. In contrast, mutanase enhanced the formation of soluble glucan, and reduced the percentage of 3-linked glucose of GTF B and C glucans whereas dextranase was mostly without effect. Glucan formed in the presence of dextranase provided fewer binding sites for S. sobrinus; mutanase was devoid of any effect. We also noted that the GTFs bind to dextranase and mutanase. Glucanohydrolases, even in the presence of GTFs, influence glucan synthesis, linkage remodeling, and branching, which may have an impact on the formation, maturation, physical properties, and bacterial binding sites of the polysaccharide matrix in dental plaque. Our data have relevance for the formation of polysaccharide matrix of other biofilms.

Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R.

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillus rhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical (1)H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; D-glucose, 2; D-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional (1)H and (13)C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: [3Rha alpha-3Glc beta-3[Gal4,6(R)Py alpha-2]Rha alpha-3Rha alpha-3Rha alpha-2Glc alpha-](n) where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete (1)H and (13)C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.

Structure determination of the exopolysaccharide produced by Lactobacillus rhamnosus strains RW-9595M and R

Exopolysaccharides (EPSs) were isolated and purified from Lactobacillusrhamnosus strains RW-9595M, which has been shown to possess cytokine-stimulating activity, and R grown under various fermentation conditions (carbon source, incubation temperature and duration). Identical 1H NMR spectra were obtained in all cases. Molecular masses were determined by gel permeation chromatography. The primary structure was elucidated using chemical and spectroscopic techniques. Organic acid, monosaccharide and absolute configuration analyses gave the following composition: pyruvate, 1; d-glucose, 2; d-galactose, 1; and l-rhamnose, 4. Methylation analysis indicated the presence of three residues of 3-linked rhamnose, and one residue each of 2,3-linked rhamnose, 2-linked glucose, 3-linked glucose and 4,6-linked galactose. The EPS was submitted to periodate oxidation followed by borohydride reduction. Monosaccharide analysis of the resulting polysaccharide gave the new composition: rhamnose, 4; and glucose, 1. Methylation analysis confirmed the loss of the 2-linked glucose and 4,6-linked galactose residues. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the native EPS was consistent with the following heptasaccharide repeating unit: {3Rhaα-3Glcβ-3[Gal4,6(R)Pyα-2]Rhaα-3Rhaα-3Rhaα-2Glcα-}n where Rha corresponds to rhamnose (6-deoxymannose) and Py corresponds to pyruvate acetal. Complete 1H and 13C assignments are reported for the native and the corresponding pyruvate-hydrolysed polysaccharide. Electrospray MS and MS/MS data are given for the oligosaccharide produced by Smith degradation.

Structure of succinoglycan from an infectious strain of Agrobacterium radiobacter

The exopolysaccharide produced by a cystic fibrosis clinical isolate of Agrobacterium radiobacter was shown by monosaccharide and methylation analyses, degradation with succinoglycanase and NMR analysis to be a succinoglycan with the structure shown below. ( S)-pyruvic acid is found stoichiometrically as 4,6- O-ketal substituent of terminal glucose. Succinic acid is present in 40% of the repeating units and it is attached to O-6 of the 3-linked glucose next to the pyruvate carrying sugar. Some evidence is found that a small amount of succinic acid (ca. 6% of the total) is linked to O-6 of another undetermined glucose.

A soluble carbohydrate elicitor from Blumeria graminis f. sp. tritici is recognized by a broad range of cereals

Wheat plants rapidly recognize pathogenic and non-pathogenic conidia of the powdery mildew fungusBlumeria (syn. Erysiphe)graminis on their leaf surfaces. This suggests that a chemical signal emanates from conidia at the pre-penetration stage of infection. Conidia of B. graminis f. sp. tritici were found to contain an elicitor that was easily washed off their surface. The elicitor activity is heat stable and could not be removed by phenol extraction. By contrast, elicitor activity is sensitive to periodate oxidation and partial acid hydrolysis suggesting that the elicitor activity resides in a carbohydrate moiety. Analysis of carbohydrates revealed mostly glucose, with smaller amounts of xylose and mannose. The glucosyl residues of the B. graminis elicitor were found to be linked (1→2)-, (1→4), and (1→6)-, with (1→4, 1→6)- branch point residues, and no 3-linked glucose residues were detected. As treatment with β -mannanase significantly reduced elicitor activity, mixed-linkage (1→4), (1→6)-mannosyl residues appeared to be important for elicitor activity. The B. graminis elicitor induced the expression of all defence-related genes tested in wheat and also induced resistance to subsequent attack by B. graminis f. sp. tritici. In contrast, a hypersensitive response was not induced by the elicitor in the absence or the presence of a challenging inoculum of B. graminis f. sp. tritici. The elicitor also induced the accumulation of thaumatin-like proteins in barley, oat, rye, rice and maize, but did not induce necrosis in any of these species. This suggests that the B. graminis elicitor represents a host non-specific determinant of non-self recognition in cereals activating general defence responses other than the hypersensitive reaction.

Structural aspects of glucans formed in solution and on the surface of hydroxyapatite.

Streptococcus mutans glucosyltransferases (GtfB, -C, and -D) and their products formed from sucrose, glucans, play an essential role in the pathogenesis of dental caries. Enzymatically active Gtf is found in whole human saliva (solution), and incorporated into the salivary pellicle that is formed on teeth in vivo (surface). GtfB glucans are predominantly 1,3-linked; however, surface-formed glucans from GtfB contain greater amounts of 3-linked glucose than glucans formed in solution. In contrast, the major linkage of glucans formed on the surface by GtfB in the presence of sucrose and starch hydrolysates in 4-linked glucose. GtfC-derived glucans in solution have a major linkage of 6-linked glucose, while surface-formed glucans from the same enzyme have 3-linked glucose as the major linkage. GtfD glucans formed either in solution or on the surface are predominantly 1,6-linked; however, surface-formed glucans contain more 6-linked glucose than solution-formed glucans. Digestion with the glucanohydrolases mutanase and dextranase shows differences in susceptibility among glucans formed either in solution or on the surface by each of the Gtf enzymes, and differences are also seen in the soluble end products from these digestions. Our results show that the same Gtf enzyme can form structurally distinct glucans in solution and on a surface. These observations are important in the study of naturally occurring microbial films.

<I>Flavobacterium dormitator</I> var. <I>glucanolytioae</I>のβ-1, 3-グルカナーゼによる糖転移

Transglycosylation to stevioside by β-1, 3-glucanase from Flavobacterium dormitator var. glucanolyticae FA-5, in the presence of β-1, 3-glucans has been observed to give a series of glycosylated steviosides. One of the glycosylated steviosides derived from stevioside and laminaran by the β-1, 3-glucanase, ST-1, was purified by silicagel column chromatography. By chemical modification and 13CNMR spectroscopy, the ST-1 was identified as a β-1, 3-monoglycosyl stevioside which was glycosylated at the terminal glucose residue of C-13 sophorose side chain of stevioside molecule. Other products of the glycosylated steviosides were identified as a series of steviosides having different lengths of linear β-1, 3-linked glucose residues.

Biosynthesis of the mycobacterial O-methylglucose lipopolysaccharide. Characterization of putative intermediates in the initiation, elongation, and termination reactions.

From the 70% ethanol extract of Mycobacterium smegmatis cells, we isolated a mixture of weakly acidic oligosaccharides composed mainly of glucose and 6-O-methylglucose. The elution pattern from a Bio-Gel P-4 column suggested that the oligosaccharides were smaller than the O-methylglucose polysaccharide (MGP) and could be biosynthetic precursors. Analysis by fast-atom-bombardment mass spectrometry revealed that the oligosaccharides fit into a pattern for polysaccharide synthesis based on an alternate glucosylation-methylation mechanism until the chain reached the composition methylglucose11glucose5glyceric acid, at which time 2 glucose units are added to give glucose2methylglucose11glucose5glyceric acid. The addition of the last 2 glucoses and methylation of one of them to give mature MGP (methylglucose1glucose3methylglucose11glucose5glyceric acid) apparently occurs rapidly because the expected intermediates were not observed. Only 4 glucose units are present at the glyceric acid end of some molecules during all stages of the elongation process, and these represent precursors of a minor MGP homolog with an extra methyl group on the beta 1----3-linked glucose unit of MGP. alpha-D-Glucopyranosyl-(1----2)-D-glyceric acid and alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranosyl-(1----2)-D-glycer ic acid were also isolated from the extract and correspond in structure to the expected initial precursors.

The chemical structure of an antitumor polysaccharide in fruit bodies of Grifola frondosa (maitake).

A polysaccharide was extracted from fruit bodies of Grifola frondosa (maitake), and the chemical structure and antitumor activity were studied. The extracted polysaccharide could be hydrolyzed by ?A-glucanase into glucose, indicating it to be a β-glucan. The sample gave methyl 2, 3, 4, 6- tetra-Ο-, methyl 2, 4, 646-Ο-, methyl 2, 3, 4-tri -Ο- and methyl 2, 4-di-Ο-methylglucoside in the molar ratio of 4 : 2 : 1 : 4 on methylation. In the carbon-13 nuclear magnetic resonance spectrum, the signals of C-6' (related to (1-6) bonding) and C-3' (related to (1-3) bonding) were observed in addition to those of free C-6 and C-3. These results indicate that the major chain is made up of β-1, 6-linked glucose residues with branches of β-1, 3-linked glucose. This glucan inhibited the growth of Sarcoma 180 tumor in ICR mice.

Antitumor activity and structural characterization of polysaccharide fractions extracted with cold alkali from a fungus, Peziza vesiculosa.

Polysaccharides in the fruit body of Peziza vesiculosa were extracted with cold aqueous sodium hydroxide. The antitumor effect and structural features of the extracted fractions were examined. The above extraction gave a water-soluble heteroglycan fraction and a water-insoluble glucan fraction. The structural characteristics of these polysaccharides were estimated from the results of methylation analysis and 13C-nuclear magnetic resonance spectroscopy. The heteroglycan fraction was composed of glucose and mannose (20 : 1). The fraction contained a large amount of β-1, 3-glucosidic linkages (about 64%) and a small amount of α-1, 4-glucosidic linkages (about 30%). Some of the β-1, 3-linked glucose residues included branching points. The water-insoluble fraction contained a large amount of β-1, 3-linkages (about 90%). Both fractions showed potent antitumor activity against the solid form of sarcoma 180 tumor in ICR mice.

The mod A mutant of Dictyostelium discoideum is missing the alpha 1,3-glucosidase involved in asparagine-linked oligosaccharide processing.

The recessive mutation, mod A, in the Dictyostelium discoideum strain M31 results in an alteration in the post-translational modification of lysosomal enzymes. We now report studies which indicate that mod A is deficient in glucosidase II, an enzyme which is involved in the processing of asparagine-linked oligosaccharides. [2-3H]Mannose-labeled glycopeptides were prepared from three purified mod A lysosomal enzymes and compared to the equivalent glycopeptides from parental enzymes. The mod A glycopeptides were deficient in high mannose oligosaccharides containing two phosphomannosyl residues and accumulated oligosaccharides with one phosphomannosyl residue. The phosphate was present in the form of an acid-stable phosphodiester in both instances. There was also an increase in the amount of nonphosphorylated high mannose oligosaccharides mod A and these were larger than the corresponding material from the parental enzymes. In addition, the nonphosphorylated oligosaccharides were only partially degraded by alpha-mannosidase, indicating the presence of a blocking moiety. In vitro enzyme assays demonstrated that the mod A cells cannot remove the inner 1 leads to 3-linked glucose from a glucosylated high mannose oligosaccharide. The cells are also deficient in membrane-bound neutral p-nitrophenyl-alpha-D-glucosidase activity. This activity has been attributed to glucosidase II in other systems. Removal of the outer 1 leads to 2-linked glucose from Glc3Man9Glc-NAc2 is normal, demonstrating the presence of glucosidase I activity. We conclude from these data that M31 cells are deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the glucosylated oligosaccharides of newly glycosylated proteins. This defect can explain the mod A phenotype and is proposed to be the primary genetic defect in these cells.

A lectin-resistant mouse lymphoma cell line is deficient in glucosidase II, a glycoprotein-processing enzyme.

Glycosylation of asparagine residues of glycoproteins occurs by the transfer of a glucose3mannose9N-acetylglucosamine2 (Glc3Man9GlcNAc2) oligosaccharide from a lipid carrier to the nascent protein. Normally, this transfer is quickly followed by the stepwise removal of the glucose residues which are arranged in the sequence: Glc1 leads to 2Glc1 leads to 3Glc1 leads to 3Man. We now report studies which demonstrate that a lectin-resistant mutant of the BW5147 mouse lymphoma cell line is deficient in the enzyme which removes the two inner glucose residues. This cell line (PHAR2.7) was selected for resistance to the cytotoxic effects of Phaseolus vulgaris leukoagglutinating lectin (Trowbridge, I. S., Hyman, R., Ferson, T., and Mazauskas, C. (1978) Eur. J. Immunol. 8, 716-723). Glycopeptides prepared from cells equilibrium-labeled with either [2-3H]mannose or [6-3H]galactose were characterized using lectin affinity chromatography, treatment with specific endo- and exoglycosidases, sizing by paper chromatography, and methylation analysis. Approximately 50% of the radioactivity in [3H]mannose-labeled glycopeptides from the mutant cells is present as glucosylated high mannose-type oligosaccharides whereas parent cell glycopeptides labeled under similar conditions lack detectable amounts of these species. Using [3H]galactose labeling, the major glucosylated oligosaccharides were identified as Glc2Man9GlcNAc2 and Glc2Man8GlcNAc2. In vitro enzyme assays demonstrated that the mutant cells cannot remove either of the two inner 1 leads to 3-linked glucose residues. Removal of the outer 1 leads to 3-linked glucose is normal. We conclude from these data that the PHAR2.7 cell line is deficient in glucosidase II, the enzyme which removes the two inner glucose residues from the oligosaccharides of newly glycosylated proteins.

The effects of N-hexyl-O-glucosyl sphingosine on normal cultured human fibroblasts: a chemical model for Gaucher's disease

Normal human skin fibroblasts were grown in the presence of N-hexyl-O-glucosyl sphingosine (HGS), an inhibitor of aryl glucosidase and glucocerebrosidase. Tests of the cells with aryl glycosides showed that beta-glucosidase activity in the cells was drastically reduced while other enzyme activities (alpha-glucosidase, beta-galactosidase, and N-acetyl-beta-hexosaminidase) were normal or elevated. Exposure of cells to HGS for 28 days resulted in increased values for cell weight per plate, glucocerebroside concentration, and galactosyl-galactosylglucosyl ceramide concentration. The concentrations of total lipid, cholesterol, and protein were unchanged, as was the fatty acid distribution within the glycolipids. Chemically, the inhibitor-treated cells exhibited a model form of Gaucher's disease. Although many membranous cytoplasmic inclusions were induced by HGS, they were unlike the characteristic inclusions seen in individuals with the genetic disorder. Skin fibroblasts from a Gaucher patient showed no abnormalities in composition or appearance.

Carbohydrates of the fresh water algaTribonema aequale. I. Low molecular weight and polysaccharides

Glycerol, erythritol, xylitol, glucose and mannitol have been separated and characterised as low molecular weight carbohydrates of Tribonema aequale Pascher. Complex mixtures of water-soluble polysaccharides comprising glucose, galactose, mannose, rhamnose, fucose and traces of arabinose and xylose were also identified. The major alkali-soluble polysaccharides were characterised as a 1, 6-linked glucan and 1, 4- and 1, 3-linked xylans. These polysaccharides defied fractionation and it is possible that the various structural features are present in a single heteropolysaccharide. Evidence for the presence of a small proportion of 1, 3-linked glucose units was also obtained but enzymic studies failed to confirm the presence of a β-1, 3-linked glucan. Proof was obtained that the residual cell-wall comprises cellulose.

XIII.—The Polysaccharides Synthesized byMonodus subterraneuswhen Grown on Artificial Media under Bacteria-free Conditions.

SynopsisThis paper describes the isolation and characterization of the polysaccharides synthesized by the unicellular organism,Monodus subterraneus. Complete extraction of polysaccharide material proved difficult and the method finally adopted, after removal of colouring matter by immersion in dimethyl sulphoxide, was successive extractions with (i) cold water, (ii) hot water, (iii) aqueous ethanol, (iv) chlorite solution, and (v) dilute alkali. The major polysaccharide, isolated from (v), is a glucan. It was shown by periodate oxidation, methylation and enzymic studies to consist mainly ofβ-l, 4-linked glucose units. The presence of a small proportion ofβ-1, 3-linked glucose units, either incorporated with theβ-1, 4-linked units in a single polymer of the lichenin-type or in a separate polymer of the laminarin-type, was also established. Further small quantities of this mixed glucan were also deposited from (ii) and from (iii) on keeping these extracts in the cold. Heterogeneous crude polysaccharide material was isolated from (i), (ii), (iii) and (iv) by alcohol precipitation. The sugars present in these various precipitates and their respective oxopolysaccharides were identified chromatographically, and, in the case of the major constituents, separated and characterized as crystalline derivatives. The polysaccharide extracted by cold water was found to contain glucose, mannose, galactose, xylose and 12 per cent of sulphate; that extracted by hot water comprised glucose (major), galactose (major), and possibly fucose; and that extracted by chlorite solution consisted of glucose (major), xylose (major), ribose and a trace of galactose.