Structural characterization of the outer core and the O-chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5.

The point of attachment of the O-chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O-chain repeating unit, present in both the wild-type strain PAO1 and O-chain deficient mutant strains AK1401 and PAO-rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one-dimensional and two-dimensional NMR spectroscopy. Furthermore, the one-dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O-chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the beta-glucosyl residues and the translocation of the alpha-rhamnosyl residue, followed by the attachment of the first O-chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with alpha-1, 6-linkage specificity and another with alpha-1,3-linkage specificity. It is also plausible that an alpha-1,3 rhamnosyltransferase facilitates the addition of the 'new' alpha-rhamnosyl residue that will act as a receptor for the attachment of the single O-antigen repeating unit in the LPS of the semi-rough mutant. The 2-amino-2-deoxy-fucosyl residue of the first O-chain repeating unit directly attached to the core was found to have a beta-anomeric configuration instead of an alpha configuration, characteristic for this residue as a component of the O-chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O-chain containing LPS, differing from the O-chain deficient rough LPS.     

A human IgM M-protein in a patient with unknown bleeding disorder binds to beta-galactosyl and beta-glucosyl residues

In a patient with an unknown bleeding disorder and an IgM lambda paraproteinemia, we demonstrated by thin-layer chromatography immunostaining and enzyme-linked immunosorbent assay that this protein specifically bound to a number of glycolipids and glycoproteins which have terminal beta-galactosyl or beta-glucosyl residues. Binding to galactosylceramide or glucosylceramide was inhibited by both galactosylceramide and glucosylceramide. From these studies, it is apparent that the M-protein recognized both beta-galactosyl and beta-glucosyl residues. This M-protein was also shown to prolong the partial thromboplastin time of normal plasma. Thus, this case represents an example of anti-carbohydrate specificity of an IgM M-protein in association with a spontaneous bleeding disorder.     

Studies on lectins. XLIX. The use of glycosyl derivatives of Dextran T-500 for affinity electrophoresis of lectins

p-Aminophenyl glycosides and glycosylamines were coupled to periodate oxidized Dextran T-500 either directly or through an epsilon-aminocaproic acid spacer. The new glycosylated derivatives of dextran specifically precipitate lectins having the appropriate carbohydrate specificity, and thus were used in the preparation of affinity gels for affinity electrophoresis of lectins. The apparent strength of interaction of several lectins with carbohydrate residues immobilized in this way was less than with carbohydrates immobilized in O-glycosyl polyacrylamide copolymers. The presence of epsilon-aminocaproic spacer had no effect on the strength of interaction. The advantages of this type of macromolecular derivative of the ligand for affinity electrophoresis and some differences between the glycosylated dextrans and O-glycosyl polyacrylamide copolymers are discussed. Dextrans containing bound p-aminophenyl alpha-D-mannopyranoside and p-aminophenyl alpha-D-glucopyranoside were used to study the binding properties of concanavalin A and the lectin from Lathyrus sativus seeds. For the investigation of interaction of lectins from Ricinus communis and Glycine soja seeds, dextran derivatives containing bound p-aminophenyl alpha- and beta-D-galactopyranosides and alpha- and beta-D-galactopyranosylamines were used.     

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.     

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the beta-glucosyl Yariv reagent

An arabinogalactan-protein (AGP) from pressed juice of Echinacea purpurea herb was isolated from a high molecular weight fraction by precipitation with the beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of other AGPs: i.e., a high amount of polysaccharide (83%) with a ratio of galactose to arabinose of 1.8:1, some uronic acids (4-5%), and a low protein content (7%) with high levels of serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.2 x 10(6) Da. Linkage and 13C NMR analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and terminal units of Araf-(1-->5)-Araf-(1-->. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule. Complete loss of reactivity toward the beta-glucosyl Yariv antigen was then noticed.     

New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.     

Automated docking to explore subsite binding by glycoside hydrolase family 6 cellobiohydrolases and endoglucanases

Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.     

Appel-Lee synthesis of glycosyl inositols, substrates for inositol dehydrogenase from Bacillus subtilis

We recently reported that inositol dehydrogenase (EC 1.1.1.18) from Bacillus subtilis can catalyze the highly stereoselective oxidation of 1l-4-O-substituted myo-inositol derivatives, as well as disaccharides melibiose and isomaltose, but not gentiobiose or maltose, consistent with the requirement of an alpha-(1-->6) linkage. We believed that the enzyme might therefore catalyze efficient stereoselective oxidation of the appropriate alpha-linked glycosyl inositols. We have synthesized alpha-D-glucopyranosyl-(1-->4)-(DL)-myo-inositol and alpha-d-galactopyranosyl-(1-->4)-(DL)-myo-inositol using the Appel-Lee protocol to couple benzyl-protected glycosyl donors to protected inositols. This method failed in our hands using glycosyl donors derived from D-mannose and 2-azido-2-deoxy-D-glucose. When myo-inositol 1,3,5-monoorthoformate is used as the acceptor, the reaction is regiospecific for the 4/6-position. We report here the mildest conditions known for the removal of the orthoformate group. 2-Azido-2-deoxy-alpha-D-glucopyranosyl-(1-->4)-(DL)-myo-inositol was synthesized using the trichloroacetimidate derivative as the donor, and all three pseudo-disaccharides were substrates for inositol dehydrogenase. The glucopyranosyl and galactopyranosyl derivatives displayed apparent second-order rate constants comparable to that of myo-inositol.     

Purification and properties of Aspergillus niger beta-glucosidase.

Beta-glucosidase was purified from a crude cellulase preparation from Aspergillus niger by affinity chromatography on a methacrylamide-N-methylene-bis-methacrylamide copolymer bearing cellobiamine. The purified enzyme was a dimer with an isoelectric point of 4.0. The molecular mass of the enzyme was estimated to be 240 kDa by gel-permeation chromatography. The enzyme hydrolyzed specifically beta-glucosidic bonds and catalyzed transglucosylation of the beta-glucosyl group of cellobiose to yield 4-O-beta-gentiobiosylglucose in the presence of organic solvents or under neutral conditions.     

Sucrose 6-alpha-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex

A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-alpha-D-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 x 10(5) M-1 min-1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomeric form of D-glucose released on (i) alkaline hydrolysis of denatured glucosyl-enzyme and (ii) native enzyme hydrolysis of sucrose. The beta-anomer was identified in the former case and the alpha-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a beta-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzes at the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a beta-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Biosynthesis of Ethyl-β-glucoside in Extracts of Pea Seedlings

An enzyme, which lacks cellobiase activity, responsible for the synthesis of ethyl-beta-glucoside has been found in the extracts of pea hooks (1-centimeter length of the apical portion of epicotyl) and has been partially purified by ammonium sulfate fractionation. The enzyme can transfer the glucosyl moiety from a group of phenolic beta-glucosides to ethanol. A specific beta-glucosyl donor, isosuccinimide beta-glucoside, isolated from the extracts of pea seedlings shows the highest activity. The characteristics of the enzyme which synthesizes ethyl-beta-glucoside and the glucosyl donor, isosuccinimide beta-glucoside, have been studied. The significance of this system (enzyme and isosuccinimide beta-glucoside) has been discussed.     

Localization of lectins in legume cotyledons.

High-resolution techniques for the localization of lectins are described. Concanavalin A (Con A) and phytohaemagglutinin (PHA) are localized using a fluorescent method with (FITC)-labelled immunoglobulins which bind to the lectins in sections of jack and red kidney bean cotyledons. Specificity is defined by the use of specific sugar inhibitors. Both Con A and PHA are found in cytoplasmic sites. Lectins with beta-glycoside specificity are detected with red-coloured artificial carbohydrate antigens. The beta-galactosyl and beta-glucosyl antigens bind specifically to clusters of spherical bodies in the intercellular spaces, to cell wall sites, and to the periphery of the cytoplasm associated with the cell membrane.     

Catalytic versatility of Bacillus pumilus beta-xylosidase: glycosyl transfer and hydrolysis promoted with alpha- and beta-D-xylosyl fluoride

Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose. The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of [14C]xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one [14C]xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flavonol glycosides of Warburgia ugandensis leaves

Four new flavonol gycosides: kaempferide 3-O-beta-xylosyl (1-->2)-beta-glucoside, kaempferol 3-O-alpha-rhamnoside-7,4'-di-O-beta-galactoside, kaempferol 3,7,4'-tri-O-beta-glucoside and quercetin 3-O-[alpha-rhamnosyl (1-->6)] [beta-glucosyl (1-->2)]-beta-glucoside-7-O-alpha-rhamnoside, were characterized from a methanolic leaf extract of Warburgia ugandensis. The known flavonols: kaempferol, kaempferol 3-rhamnoside, kaempferol 3-rutinoside, myricetin, quercetin 3-rhamnoside, kaempferol 3-arabinoside, quercetin 3-glucoside, quercetin, kaempferol 3-rhamnoside-4'-galactoside, myricetin 3-galactoside and kaempferol 3-glucoside were also isolated. Structures were established by spectroscopic and chemical methods and by comparison with authentic samples.     

Synthesis of glycosyl diglycerides

Synthesis of mono- and diglycosyl digly cerides with natural structure from 1,2-di-O-acyl-sn-glycerols, 1,2-O-isopropylidene-sn-glycerol, 2,5-methylene-D-mannitol by the orthoester method of glycosylation is reported.     

Synthesis of glycosyl phosphoramidates: novel isosteric analogues of glycosyl phosphates.

Several newer isosteric analogues of glycosyl phosphates, namely of glycosyl phosphoramidates, were synthesized in good yields using Staudinger reaction of their corresponding azides with trimethyl phosphite followed by de-O-acetylation. The structure and conformation of the fully protected analogue synthesized, namely 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl bismethoxyphosphoramidate, was established by X-ray crystallography.     

Transglycosylation of cellobiose by partially purified Trichoderma viride cellulase.

A commercial cellulase from Trichoderma viride was fractionated into three fractions, F1, F2, and F3, in order to investigate transglycosylation activities. Among these fractions, F3, which demonstrated highly hydrolytic activity toward p-nitrophenyl beta-D-glucopyranoside and Avicel, most effectively catalyzed the transglycosylation of cellobiose and converted cellobiose into beta-Glc-(1-->6)-beta-glc-(1-->4)-Glc and beta-Glc-(1-->6)-beta-Glc-(1-->6)-beta-Glc(1-->4)-Glc. The F3 fraction contained the enzyme to catalyze beta-glucosyl transfer toward only the C-6 position of the sugar acceptor, and thus it is expected to be of use for syntheses of functional oligosaccharides.     

Significant rate accelerated synthesis of glycosyl azides and glycosyl 1,2,3-triazole conjugates

An efficient and significantly rapid access of a series of glycosyl azides and glycosyl 1,2,3-triazole conjugates is reported using modified one-pot reaction conditions. In both cases yields were excellent and single diastereomers were obtained. Figure Rapid preparation of 4-substituted glycosyl 1,2,3-triazole conjugates from glycosyl bromides. CDRI communication no. 7340.     

Polysaccharide composition of the fruit juice of Morinda citrifolia (Noni)

An ethanol-insoluble, high molecular weight fraction was collected from the juice of Morinda citrifolia fruit grown in Viet Nam. The fraction is composed primarily of carbohydrate (67% (w/w)). The polysaccharide fraction consists predominantly of GalAp (53.6mol%), Araf (13.6mol%), Galp (17.9mol%) and Rhap (9.5mol%). Glycosyl linkage analysis suggests the polysaccharide fraction contains mostly the pectic polysaccharides, homogalacturonan (4-GalAp), rhamnogalacturonan I (4-GalAp, 2-Rhap, 2,4-Rhap), arabinan (5-Araf, 3,5-Araf, t-Araf), type I arabinogalactan (4-Galp, 3,4-Galp, t-Araf) and beta-glucosyl Yariv-binding type II arabinogalactan (3,6-Galp, t-Araf). Low levels of xyloglucan (4-Glcp, 4,6-Glcp, t-Xylp, t-Fucp), heteroxylan (4-Xylp) and heteromannan (4-Manp) are also present.