Product-identification and substrate-specificity studies of the GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (fuc→asn-linked GlcNAc) 6-α-l-fucosyltransferase in a golgi-rich fraction from porcine liver

Golgi-rich membranes from porcine liver have been shown to contain an enzyme that transfers l-fucose in α-(1→6) linkage from GDP-l-fucose to the asparagine-linked 2-acetamido-2-deoxy-d-glucose r residue of a glycopeptide derived from human α₁-acid glycoprotein. Product identification was performed by high-resolution, ¹H-n.m.r. spectroscopy at 360 MHz and by permethylation analysis. The enzyme has been named GDP-l-fucose: 2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, because the substrate requires a terminal β-(1→2)-linked GlcNAc residue on the α-Man (1→3) arm of the core. Glycopeptides with this residue were shown to be acceptors whether they contained 3 or 5 Man residues. Substrate-specificity studies have shown that diantennary glycopeptides with two terminal β-(1→2)-linked GlcNAc residues and glycopeptides with more than two terminal GlcNAc residues are also excellent acceptors for the fucosyltransferase. An examination of four pairs of glycopeptides differing only by the absence or presence of a bisecting GlcNAc residue in β-(1→4) linkage to the β-linked Man residue of the core showed that the bisecting GlcNAc prevented 6-α-l-fucosyltransferase action. These findings probably explain why the oligosaccharides with a high content of mannose and the hybrid oligosaccharides with a bisecting GlcNAc residue that have been isolated to date do not contain a core l-fucosyl residue.     

Capsular and extracellular polysaccharides from Rhizobium microsymbionts of Acacia decurrens

The capsular polysaccharide produced by a Rhizobium isolated from a root nodule of Acacia decurrens is composed of 3-O-methyl- -rhamnose: -rhamnose: - mannose: -glucose: -galacturonic acid in the molar ratios of 1:2:2:4:1. The extracellular polysaccharide is similarly constituted. Structural analyses indicate a decasaccharide repeating-unit in which the -rhamnosyl groups occur as single-unit side-chains. The 3-O-methyl- -rhamnosyl and one of the α- -rhamnosyl groups are (1→6)-linked to two of the -glucosyl residues. The other α- -rhamnosyl group is (1→4)-linked to the -galacturonic acid residue. The main-chain residues are all (1→3)-linked, and are partially identified as -(1→3)-α- -GalpA-(1→3)-α- -Manp- (1→3)-α- -Glcp-(1→3)-.     

Enzymatic redox isomerization of 1,6-disaccharides by pyranose oxidase and NADH-dependent aldose reductase

Pyranose 2-oxidase, a homotetrameric FAD-flavoprotein from the basidiomycete Trametes multicolor, catalyzes regioselectively the oxidation of the 1→6 disaccharides allolactose [β- -Galp-(1→6)- -Glc], gentiobiose [β- -Glcp-(1→6)- -Glc], melibiose [α- -Galp-(1→6)- -Glc], and isomaltose [α- -Glcp-(1→6)- -Glc] at position C-2 of their reducing moiety. The resulting glycosyl -arabino-hexos-2-uloses can be reduced specifically at C-1 by NAD(P)H-dependent aldose reductase from the yeast Candida tenuis. By this novel, two-step redox isomerization process the four disaccharide substrates could be converted to the corresponding keto-disaccharides allolactulose [β- -Galp-(1→6)- -Fru], gentiobiulose [β- -Glcp-(1→6)- -Fru], melibiulose [α- -Galp-(1→6)- -Fru], and isomaltulose (palatinose, [α- -Glcp-(1→6)- -Fru]) in high yields. These products could find application in food technology as alternative sweeteners.     

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rha-(1→, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rha-(1→, which was identified in a previous study.     

β-N-Acetylhexosaminidase-catalysed synthesis of non-reducing oligosaccharides

A large panel of fungal β-N-acetylhexosaminidases was tested for the regioselectivity of the β-GlcNAc transfer onto galacto-type acceptors ( -galactose, lactose, 2-acetamido-2-deoxy- -galactopyranose). A unique, non-reducing disaccharide β- -GlcpNAc-(1→1)-β- -Galp and trisaccharides β- -GlcpNAc-(1→4)-β- -GlcpNAc-(1→1)-β- -Galp, β- -Galp-(1→4)-β- -Glcp-(1→1)-β- -GlcpNAc and β- -Galp-(1→4)-α- -Glcp-(1→1)-β- -GlcpNAc were synthesised under the catalysis of the β-N-acetylhexosaminidase from the Aspergillus flavofurcatis CCF 3061 with -galactose and lactose as acceptors. The use of 2-acetamido-2-deoxy- -galactopyranose as an acceptor with the β-N-acetylhexosaminidases from A. flavofurcatis CCF 3061, A. oryzae CCF 1066 and A. tamarii CCF 1665 afforded only β- -GlcpNAc-(1→6)- -GalpNAc.     

Structural studies of the antigen III cell wall polysaccharide of Trichosporon domesticum

Cell wall and soluble polysaccharides that reacted with Trichosporon domesticum factor III serum were isolated from the type strain of T. domesticum. The fractions contained O-acetyl groups, which contributed to the serological reactivity. The antigenic structure was characterized by chromatographic and spectroscopic methods. The polysaccharide has an α-(1→3)- -mannan backbone with hetero-oligosaccharide side chains consisting of a 2-O-substituted β- -glucuronic acid residue bound to O-2 of the mannose residue, β- -xylopyranosyl residues located in the middle of the side chain, and a nonreducing terminal α- -arabinopyranosyl residue bound to O-4 of xylose. The mannan backbone is O-acetylated at O-6 of the mannose residues.     

Morphological and structural characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its anti-exercise fatigue activity

The crude polysaccharide was obtained from Gynostemma pentaphyllum Makino by water extraction followed by ethanol precipitation. The polysaccharide was successively purified by chromatography on DEAE-52 and SephadexG-150 column, and three polysaccharide fractions were obtained and termed GPP1-a, GPP2-b, and GPP3-a, respectively. The administration with GPP1-a markedly prolonged exhaustive exercise time of the mice. Structural features of GPP1-a were investigated by a combination of instrumental and chemical analyses, including atomic force microscope (AFM), scanning electron microscope (SEM), partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, gas chromatography–mass spectrometry (GC–MS) analysis and NMR spectroscopy. The results indicate that GPP1-a has a backbone of (1 → 4)-linked α-d-Glucose residues, which occasionally branches at O-6. The branches are mainly composed of (1 → 6)-linked α-d-Glucose, (1 → 3)-linked β-d-Galactose and (1 → 6)-linked α-d-Galactose residues, and terminated with β-d-Galactose residues and β-l-Arabinose residues.     

Saponins from Albizzia lucida

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-α- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[α- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers

Oligomeric procyanidins were isolated from the leaves and flowers of hawthorn (Crataegus laevigata). A trimer, epicatechin-(4β→8)-epicatechin-(4β→6)-epicatechin, and a pentamer consisting of (−)-epicatechin units linked through C-4β/C-8 bonds have been isolated from hawthorn for the first time, in addition to known procyanidins including dimers B-2, B-4 and B-5, trimers C-1 and epicatechin-(4β→6)-epicatechin-(4β→8)-epicatechin, and tetramer D-1. A fraction containing a hexamer was also found.     

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-α- -mannosidase from Aspergillus phoenicis

A novel 1,6-α- -mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only α- -Manp-(1→6)- -Manp and did not act on α- -Manp-(1→2)- -Manp, or α- -Manp-(1→3)- -Manp. The 1,6-α- -mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were 21% mannobiose and 5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 °C. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1–6 linkages were found in both of them.     

Enzymatic synthesis of α- -fucosyl-N-acetyllactosamines and 3′-O-α- -fucosyllactose utilizing α- -fucosidases

An α- -fucosidase from porcine liver produced α- -Fuc-(1→2)-β- -Gal-(1→4)- -GlcNAc (2′-O-α- -fucosyl-N-acetyllactosamine, 1) together with its isomers α- -Fuc-(1→3)-β- -Gal-(1→4)- -GlcNAc (2) and α- -Fuc-(1→6)-β- -Gal-(1→4)- -GlcNAc (3) through a transglycosylation reaction from p-nitrophenyl α- -fucopyranoside and β- -Gal-(1→4)- -GlcNAc. The enzyme formed the trisaccharides 1–3 in 13% overall yield based on the donor, and in the ratio of 40:37:23. In contrast, transglycosylation by Alcaligenes sp. α- -fucosidase led to the regioselective synthesis of trisaccharides containing a (1→3)-linked α- -fucosyl residue. When β- -Gal-(1→4)- -GlcNAc and lactose were acceptors, the enzyme formed regioselectively compound 2 and α- -Fuc-(1→3)-β- -Gal-(1→4)- -Glc (3′-O-α- -fucosyllactose, 4), respectively, in 54 and 34% yields, based on the donor.     

Some structural features of the -glucan from the seed of Mirabilis jalapa

The cotyledon of the seed of Mirabilis jalapa was found to contain a -glucan. Methylation, periodate oxidation, and graded and enzymic hydrolysis studies were conducted to elucidate its structure. For every 38 -glucosyl residues therein, 34 are (1→4)- and 3 are (1→3)-linked; the -glucosyl unit at the branch point is linked through O-1, O-2, and O-4. In some places in the chain, there are at least three (1→3)-linked -glucosyl residues in a sequence. Both α- and β- -glucosidic linkages are present in the polysaccharide, the former preponderating. The -glucan gave with iodine a faint blue color that had λ_max 420 nm.     

Enzymatic synthesis of three pNP-α-galactobiopyranosides: application of the library of fungal α-galactosidases

The regioselectivity of the transglycosylation reaction catalyzed by extracellular α-galactosidases from filamentous fungi was studied using p-nitrophenyl α- -galactopyranoside. Regioisomers of p-nitrophenyl α- -galactobiopyranoside α(1→2), α(1→3) and α(1→6) were isolated and characterized. α-Galactosidases with pronounced regioselectivity towards α-Gal-O-R acceptor were identified.     

High-Performance Capillary Electrophoretic Characterization of Different Types of Oligo- and Polysialic Acid Chains

We have carried out comparative structural analysis of novel oligo- and polysialic acid chains from diverse sources. Controlled acid hydrolysates of (a) colominic acid, α2→8-linked homopolymer ofN-acetylneuraminic acid (Neu5Ac), (b) α2→8-linked oligo/polyNeu5Gc chains present in rainbow trout egg polysialoglycoprotein, and (c) α2→8-linked oligomers of deaminoneuraminic acid (KDN) residues of KDN-rich glycoprotein derived from rainbow trout vitelline envelope were analyzed by high-performance capillary electrophoresis (HPCE). The results showed that three different types of α2→8-linked oligosialic acids having same degree of polymerization can be separated by HPCE. A partial hydrolysate of colominic acid with mild acid was shown by CE to form intramolecular esters during the controlled hydrolysis and the subsequent workup procedure. In contrast, lactonization of (→5-O_glycolyl-Neu5Gcα2→)n, α2→5-O_glycolyl-linked homopolymer ofN-glycolylneuraminic acid (Neu5Gc) present in the egg jelly coat of sea urchin, did not take place as readily as in (→8Neu5Acα2→)n.     

Large-scale production of the carbohydrate portion of the sialyl–Tn epitope, α-Neup5Ac-(2→6)- -GalpNAc, through bacterial coupling

α-Neup5Ac-(2→6)- -GalpNAc, the carbohydrate portion of sialyl–Tn epitope of the tumor-associated carbohydrate antigen, was prepared by a whole-cell reaction through the combination of recombinant Escherichia coli strains and Corynebacterium ammoniagenes. Two recombinant E. coli strains overexpressed the CMP-Neup5Ac biosynthetic genes and the α-(2→6)-sialyltransferase gene of Photobacterium damsela. C. ammoniagenes contributed to the production of UTP from orotic acid. α-Neup5Ac-(2→6)- -GalpNAc was accumulated at 87 mM (45 g/L) after a 25-h reaction starting from orotic acid, N-acetylneuraminic acid, and 2-acetamide-2-deoxy- -galactose.     

A polysaccharide from Lichina pygmaea and L. confinis supports the recognition of Lichinomycetes

The lichen-forming order Lichinales, generally characterized by prototunicate asci and the development of thalli with cyanobacteria, has recently been recognized as a separate class of ascomycetes, Lichinomycetes, as a result of molecular phylogenetic studies. As alkali and water-soluble (F1SS) polysaccharides reflect phylogeny in other ascomycetes, a polysaccharide from Lichina pygmaea and L. confinis was purified and characterized to investigate whether these F1SS compounds in the Lichinomycetes were distinctive. Nuclear magnetic resonance (NMR) spectroscopy and chemical analyses revealed this as a galactomannan comprising a repeating unit consisting of an α-(1→6)-mannan backbone, mainly substituted by single α-galactofuranose residues at the O-2- or the O-2,4- positions linked to a small mannan core. With the exception of the trisubstituted mannopyranose residues previously described in polysaccharides from other lichens belonging to orders now placed in Lecanoromycetes, the structure of this galactomannan most closely resembles those found in several members of the Onygenales in Eurotiomycetes. Our polysaccharide data support molecular studies showing that Lichina species are remote from Lecanoromycetes as the galactofuranose residues are in the α-configuration. That the Lichinomycetes were part of an ancestral lichenized group can not be established from the present data because the extracted polysaccharide does not have the galactofuranose residue in the β configuration; however, the data does suggest that an ancestor of the Lichinomycetes contained a mannan and was part of an early radiation in the ascomycetes.     

H NMR analysis of novel sialylated and fucosylated lactose-based oligosaccharides having linear GlcNAc(β1–6)Gal and Neu5Ac(α2–6)GlcNAc sequences

Three novel oligosaccharides of human infant faeces have been fully characterised by methylation analysis and 500/600 MHz ¹H NMR spectroscopy including DQF-COSY, TQF-COSY, TOCSY and ROESY experiments. The oligosaccharides were shown to be lactose-based structures two of which were substituted at C-6 of Gal with either the Le^x trisaccharide, Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1-, or Neu5Ac(α2–6)Gal(β1–4)GlcNAc-(β1-. They differ from other free oligosaccharides previously isolated from the human by having the (1 → 6) linkage to Gal in the absence of a (1 → 3) branch. The third oligosaccharide has Neu5Ac(α2–6) linked to GlcNAc of the trisaccharide GlcNAc(β1–3)Gal(β1–4)Glc. This is a linear fragment of the disialylated tetrasaccharide sequence Neu5Ac(α2–3)Gal(β1–3)[Neu5Ac(α2–6)]GlcNAc(β1- found in the milk oligosaccharide disialyl LNT (the GlcNAc residue of the tetrasaccharide linked to lactose) and also of N-linked chains (GlcNAc linked to Man).     

Lupane-triterpene glycosides from leaves of Acanthopanax koreanum

Two new lupane-triterpene glycosides named acankoreosides C and D, were isolated from the leaves of Acanthopanax koreanum. Based on spectroscopic data, the chemical structures were determined as 3-O-β- -glucopyranosyl 3α,11α-dihydroxylup-20(29)-en-28-oic acid 28-O-α- -rhamnopyranosyl-(1→4)-β- -glucopyranosyl-(1→6)-β- -glucopyranosyl ester and 3α,11α-dihydroxylup-23-al-20(29)-en-28-oic acid 28-O-α- -rhamnopyranosyl-(1→4)-β- -glucopyranosyl-(1→6)-β- -glucopyranosyl ester, respectively.     

Structural characterization of an active polysaccharide from Phellinus ribis

A water-soluble polysaccharide named as PRP was isolated from the fruiting bodies of Phellinus ribis by hot water extraction, DEAE-cellulose and Superdex 30 column chromatography. Its structural characteristics were investigated by FT-IR, NMR spectroscopy, GLC-MS, methylation analysis, periodate oxidation and Smith degradation. Based on the data obtained, PRP was found to be a β-d-glucan containing a (1 → 4), (1 → 6)-linked backbone, with a single β-d-glucose at the C-3 position of (1 → 6)-linked glucosyl residue every eight residues, along the main chain. The glucan has a weight-average molecular weight of about 8.59 kDa by HPGPC determination using dextran samples as the standards. Preliminary activity tests in vitro revealed that PRP could stimulate the proliferation of spleen lymphocyte.     

Water-soluble (1→3), (1→4)-β-d-glucans from barley (Hordeum vulgare) endosperm. III. Distribution of cellotriosyl and cellotetraosyl residues

Cellotriosyl and cellotetraosyl residues, linked by single (1→3)-β-linkages, account for more than 90% of the 40°C water-soluble (1→3), (1→4)-β-d-glucan from barley flour. We have analysed their sequence dependence by treating the polymer as a two-state Markov chain with stationary distribution. Quantitation of the penultimate oligosaccharides released during hydrolysis of the (1→3), (1→4)-β-d-glucan with (1→3), (1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73) by analytical gel filtration chromatography enabled the relative abundance of two adjacent cellotriosyl, two adjacent cellotetraosyl and adjacent cellotetraosyl/cellotriosyl residues to be estimated and the sequence dependence to be evaluated.Within the theoretical and practical constraints of the method it is concluded that the cellotriosyl and cellotetraosyl residues are arranged in an essentially independent (random) fashion. Thus, any mechanism proposed for the biosynthesis of the molecule should explain this apparently random distribution of cellotriosyl and cellotetraosyl residues as well as the presence, in relatively low frequency, of blocks of up to 10 or more adjacent (1→4)-linkages.