Trisaccharide GlcNAc Research Articles

Dimerization of the fungal defense lectin CCL2 is essential for its toxicity against nematodes.

Lectins are used as defense effector proteins against predators, parasites and pathogens by animal, plant and fungal innate defense systems. These proteins bind to specific glycoepitopes on the cell surfaces and thereby interfere with the proper cellular functions of the various antagonists. The exact cellular toxicity mechanism is in many cases unclear. Lectin CCL2 of the mushroom Coprinopsis cinerea was previously shown to be toxic for Caenorhabditis elegans and Drosophila melanogaster. This toxicity is dependent on a single, high-affinity binding site for the trisaccharide GlcNAc(Fucα1,3)β1,4GlcNAc, which is a hallmark of nematode and insect N-glycan cores. The carbohydrate-binding site is located at an unusual position on the protein surface when compared to other β-trefoil lectins. Here, we show that CCL2 forms a compact dimer in solution and in crystals. Substitution of two amino acid residues at the dimer interface, R18A and F133A, interfered with dimerization of CCL2 and reduced toxicity but left carbohydrate-binding unaffected. These results, together with the positioning of the two carbohydrate-binding sites on the surface of the protein dimer, suggest that crosslinking of N-glycoproteins on the surface of intestinal cells of invertebrates is a crucial step in the mechanism of CCL2-mediated toxicity. Comparisons of the number and positioning of carbohydrate-binding sites among different dimerizing fungal β-trefoil lectins revealed a considerable variability in the carbohydrate-binding patterns of these proteins, which are likely to correlate with their respective functions.

1-Methyl 1′-cyclopropylmethyl: an acid labile O-protecting group for polymer-supported oligosaccharide synthesis

R, S-1-Methyl 1′-cyclopropylmethanol can be converted to its trichloroacetimidate derivative. Lewis acid catalyzed etherification can then be used to prepare methyl cyclopropylmethyl (MCPM) ethers. Thus, ethyl 2,3,4-tri- O-benzyl-6- O-( R, S-1-methyl 1′-cyclopropylmethyl)-1-thio-α/β- d-glucopyranoside was prepared and the linker polymer combination (MPEG)(DOX)OH glycosylated. The MCPM group was cleaved with 10% trifluoroacetic acid in CH 2Cl 2 and the resulting alcohol glycosylated. After protecting group manipulations and cleavage the peracetylated disaccharide GlcNAc(β1→6)GlcOAc was isolated. Similarly the 4,6- O-phenylboronate diester of ethyl 1-thio-β- d-galactopyranoside was regioselectively etherified at O-3 and after boronate cleavage the sugar benzoylated to give ethyl 2,4,6-tri- O-benzoyl-3- O-( R, S-1-methyl 1′-cyclopropylmethyl)-1-thio-β- d-galactopyranoside. Through a similar sequence of glycosylation of (MPEG)(DOX)OH, MCPM deprotection, glycosylation, functional group manipulation and cleavage the peracylated disaccharide GlcNAc(β1→3)Gal(β1→)DOXOAc was prepared. Finally, the donor 2,6-di- O-benzoyl-4- O-levulinoyl-3- O-( R, S-1-methyl 1′-cyclopropylmethyl)-1-thio-β- d-galactopyranoside was prepared and elaborated into the trisaccharide GlcNAc(β1→3)[Glc(β1→4)]Gal(β1→)DOXOH. This trisaccharide was elaborated into the pentasaccharide Neu5Ac(α2→3)Gal(β1→4)GlcNAc(β1→3) [Glc(β1→4)]Gal(β1→)DOXOH using GalE-LgtB fusion and CMP-Neu5Ac synthetase/sialyltransferase fusion enzymes. This pentasaccharide is a single repeat unit of the capsular polysaccharide of Group B Streptococcus type 1A.

Synthesis of 3- O-sulfoglucuronyl lacto- N-neotetraose 2-aminoethyl glycoside and biotinylated neoglycoconjugates thereof

The 2-aminoethyl glycoside of pentasaccharide 3- O-sulfo-GlcA(β-1→3)Gal(β-1→4)GlcNAc(β-1→3)Gal(β-1→4)Glc(β ( 1) and its conjugates with biotin and biotinylated polyacrylic acid were synthesized as molecular probes to investigate the recognition of the HNK-1 epitope containing carbohydrates by proteins. Key steps in the first of two investigated schemes for the preparation of the target compound 1 were (a) assembling of the pentasaccharide backbone (compound 10) by glycosylation of selectively substituted allyl glycoside of the trisaccharide GlcNAc(β-1→3)Gal(β-1→4)Glc(β with glucuronyl-galactose glycosyl donor, (b) transformation of the allyl aglycon in 10 into 2-azidoethyl one (to give 11), (c) selective deprotection of the OH group at C-3 of the GlcA residue in 11 via saponification, intramolecular formation of 6,3-lacton ( 13) and its methanolysis, and (d) subsequent O-sulfation. The alternative scheme with the use of 2-azido-ethyl glycoside of the trisaccharide GlcNAc(β-1→3)Gal(β-1→4)Glc(β instead of the allyl glycoside 6 was less effective due to smaller yield at the step of pentasaccharide synthesis. Additionally to 1 the 2-aminoethyl glycosides of the oligosaccharides GlcA(β-1→3)Gal(β-1→4)GlcNAc(β-1→3)Gal(β-1→4)Glc(β, 3- O-sulfo-GlcA(β-1→3)Gal(β, and GlcA(β-1→3)Gal(β were also synthesized.

1H 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).

?1,3-Fucosylation of branched blood group I-type oligo-(N-acetyllactosamino)glycans by human milk transferases is restricted to distalN-acetyllactosamine units: The resulting isomers are separated by WGA-agarose chromatography

A partially purified preparation of alpha 1,3-fucosyltransferase(s) from human milk was used to [14C]fucosylate oligosaccharides containing Gal beta 1-4GlcNAc units. Substitution of N-acetyllactosamine at position 3' with a beta-linked N-acetyl-glucosamine enhanced the reactivity of the acceptor, whereas similar substitution at position 6' was inhibitory. Thus, the trisaccharide GlcNAc beta 1-6Gal beta 1-4GlcNAc (5), the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (11) and the triply branched decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (26) gave remarkably poor yields of alpha 1,3-fucosylated products in comparison to GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). beta 1,4-Galactosyl derivatives of 5 and 11, however, gave good yields of alpha 1,3-fucosylated products, but the fucosylation was restricted to the distal N-acetyllactosamine units of Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc (16), Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (18) and also in Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (22). Immobilized wheat germ agglutinin (WGA), possessing high affinity for 16 [1], revealed no affinity for the fucosylated derivative Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6Gal beta 1-4GlcNAc (17). The isomeric heptasaccharides Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (19) and Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4GlcNAc (20) were readily separated from each other on WGA agarose, and so were the isomeric nonasaccharides Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (23) and Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-6]Gal beta 1-4GlcNAc (24).

Single mid-chain GlcNAc?1-6Gal?1-4R sequences of linear oligosaccharides are resistant to endo-?-galactosidase ofBacteroides fragilis

Endo-beta-galactosidase (EC 3.2.1.103) of Bacteroides fragilis, at 250 mU ml-1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc beta 1-6Gal beta 1-4GlcNAc, or those of tetrasaccharides Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4Glc. The isomeric glycans which contained the GlcNAc beta 1-3Gal beta 1-4GlcNAc/Glc sequence were readily cleaved.

Syntheses of model oligosaccharides of biological significance. XI. A short synthesis of fucosylated chitobiosides, also bound to asparagine in a synthon (as in N-linked glycoproteins)

We describe a simple and efficient method for the preparation of the trisaccharide GlcNAc(β1-4)-[Fuc(α 1-6)-]GlcNAc(β 1-) (1) and of the protected form of GlcNAc(β 1-4)-[Fuc(α 1-6)-]GlcNAc(β1-Asn) (2). The key intermediate is benzyl 4,6-benzylidene chitobioside 5 giving the desired trisaccharide by insitu anomerization–glycosylation reaction with 2,3,4-tribenzylfucosyl bromide. The benzyl glycoside in the trisaccharide 6 has been replaced by acetate and then bromine; this glycosylating agent was used to prepare methyl and 8-methoxycarbonyloctyl glycosides as well as isothiocyanate 12, in a series of reactions. The latter compound gave, on reaction with 1-benzyl N-benzyloxycarbonyl-L-asparate, compound 13 (a protected derivative of 2), which should serve as a synthon for syntheses of glycopeptides. Keywords: glycopeptide, synthesis; oligosaccharide, synthesis; chitobiosides; fucosylated chitobiosides; N-linked oligosaccharides.

Carbohydrate binding specificities of several poly-N-acetyllactosamine-binding lectins

The structural requirements for the interaction of the Asn-linked poly-N-acetyllactosamine-type oligosaccharide moieties of glycoproteins with various N-acetylglucosamine-binding lectins were investigated by means of affinity chromatography on immobilized lectin-Sepharose columns. High molecular weight glycopeptides containing poly-N-acetyllactosamine-type oligosaccharides obtained by Pronase digestion of human erythrocyte ghosts were treated with 0.1 M trifluoroacetic acid at 100 degrees C for 40 min and then several oligosaccharide fragments were purified with an amino-bonded silica column. Among these oligosaccharide fragments, trisaccharide Gal beta 1-4GlcNAc beta 1-6Galol bound to the wheat germ agglutinin (WGA)- and pokeweed mitogen (PWM)-Sepharose columns, and also showed affinity to the Datura stramonium agglutinin (DSA)-, Lycopersicon esculentum (tomato) agglutinin- and Solanum tuberosum (potato) agglutinin-Sepharose columns. Pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Galol showed weaker affinity to the WGA- and PWM-Sepharose columns, compared to the trisaccharide. Trisaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Galol showed weak affinity to the WGA-Sepharose column and did not show any affinity to the other lectin-Sepharose columns. Hexasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAcol bound only to the DSA-Sepharose column, indicating that only DSA does not require a GlcNAc beta(1-6)- linkage for interaction.

Control of Glycoprotein Synthesis: Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine: GlcNAcβ-6(GlcNAcβ1–2)Manα-R (GlcNAc to Man) β-4-N-acetylglucosaminyltransferase VI

Hen oviduct membranes were shown to contain high activity of a novel enzyme, UDP-GlcNac:GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-R (GlcNAc to Man) beta 4-GlcNAc-transferase VI. The enzyme was shown to transfer GlcNAc in beta 1-4 linkage to the D-mannose residue of GlcNAc beta 1-6 (GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or methyl. Radioactive enzyme products were purified by several chromatographic steps, including high performance liquid chromatography, and structures were determined by proton nmr, fast atom bombardment-mass spectrometry, and methylation analysis to be GlcNAc beta 1-6 ([14C]GlcNAc beta 1-4) (GlcNAc beta 1-2) Man alpha-R. The enzyme is stimulated by Triton X-100 and has optimum activity at a relatively high MnCl2 concentration of about 100 mM; Co2+, Mg2+, and Ca2+ could partially substitute for Mn2+. A tissue survey demonstrated high GlcNAc-transferase VI activity in hen oviduct and lower activity in chicken liver and colon, duck colon, and turkey intestine. No activity was found in mammalian tissues. Hen oviduct membranes cannot act on GlcNAc beta 1-6Man alpha-R but have a beta 4-GlcNAc-transferase activity that converts GlcNAc beta 1-2Man alpha-R to GlcNAc beta 1-4(GlcNAc beta 1-2) Man alpha-R where R is either 1-6Man beta-(CH2)8COOCH3 or 1-6Man beta methyl. The latter activity is probably due to GlcNAc-transferase IV which preferentially adds GlcNAc in beta 1-4 linkage to the Man alpha 1-3 arm of the GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn core structure of asparagine-linked glycans. The minimum structural requirement for a substrate of beta 4-GlcNAc-transferase VI is therefore the trisaccharide GlcNAc beta 1-6(GlcNAc beta 1-2) Man alpha-; this trisaccharide is found on the Man alpha 6 arm of many branched complex asparagine-linked oligosaccharides. The data suggest that GlcNAc-transferase VI acts after the synthesis of the GlcNAc beta 1-2Man alpha 1-3-, GlcNAc beta 1-2Man alpha 1-6-, and GlcNAc beta 1-6 Man alpha 1-6-branches by GlcNAc-transferases I, II, and V, respectively, and is responsible for the synthesis of branched oligosaccharides containing the GlcNAc beta 1-6(GlcNAc beta 1-4)(GlcNAc beta 1-2)Man alpha 1-6Man beta moiety.

Sulfation of the tumor cell surface sialomucin of the 13762 rat mammary adenocarcinoma.

ASGP-1, the major cell surface sialomucin of the 13762 ascites rat mammary adenocarcinoma, is at least 0.5% of the total ascites cell protein and has sulfate on 20% of its O-linked oligosaccharide chains. We have used this system to investigate the O-glycosylation pathway in these cells and to determine the temporal relationship between sulfation and sialylation. The two major sulfated oligosaccharides (S-1 and S-2) were isolated as their oligosaccharitols by alkaline borohydride elimination, anion exchange HPLC, and ion-suppression HPLC. From structural analyses S-1 is proposed to be a branched, sulfated trisaccharide -O4S-GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and S-2 its sialylated derivative -O4S-GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNac. Pulse labeling with sulfate indicated that sulfation occurred primarily on a form of ASGP-1 intermediate in size between immature and mature sialomucin. Pulse-chase analyses showed that the intermediate could be chased into mature ASGP-1. The concomitant conversion of S-1 into S-2 had a half-time of less than 5 min. Monensin treatment of the tumor cells led to a 95% inhibition of sulfation with the accumulation of unsulfated trisaccharide GlcNAc beta 1,6-(Gal beta 1,3)-GalNAc and sialylated derivative GlcNAc beta 1,6-(NeuAc alpha 2,3-Gal beta 1,3)-GalNAc. These data suggest that sulfation of ASGP-1 is an intermediate synthetic step, which competes with beta-1,4-galactosylation for the trisaccharide intermediate and thus occurs in the same compartment as beta-1,4-galactosylation. Moreover, sulfation precedes sialylation, but the two are rapidly successive kinetic events in the oligosaccharide assembly of ASGP-1.

Biosynthesis of a tumor cell surface sialomucin. Maturation and effects of monensin.

The major cell surface glycoprotein (ascites sialoglycoprotein-1 (ASGP-1] of ascites 13762 rat mammary tumor cells is a large (Mr greater than 500,000), highly glycosylated sialomucin which is present in great abundance (greater than 0.5% of total cell protein). Thus, these tumors provide a useful system for investigating the biosynthesis of O-glycosylated glycoproteins. Previous studies in this system have demonstrated that initiation of O-linked oligosaccharides occurs throughout most of the transit period of ASGP-1 from the endoplasmic reticulum to the cell surface. By pulse-chase threonine labeling and precipitation with peanut agglutinin, ASGP-1 is first observed as an immature lightly glycosylated form (Mr approximately 200,000) which is converted to a more mature, more heavily glycosylated form (designated the premature or P form) with a half-time of about 30 min. The P form is then more gradually converted into the mature ASGP-1. Analysis of glucosamine-labeled oligosaccharitols obtained from the immature form showed primarily unsialylated derivatives consisting of the structures of the size of the tetrasaccharide Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc and smaller, whereas the mature form showed a mixture of sialylated and unsialylated structures. Desialylation of glucosamine-labeled mature form resulted in a glycoprotein intermediate in size between the immature and mature forms, indicating that the size change with maturation is not solely due to sialylation. Treatment of the cells with 10(-6) M monensin significantly reduced the conversion of immature to mature form without inhibiting initiation of O-linked oligosaccharides and without preventing sialylation. Analysis of oligosaccharitols obtained from ASGP-1 of monensin-treated cells showed that the major oligosaccharides are trisaccharide GlcNAc beta 1,6(Gal beta 1,3)GalNAc and sialylated trisaccharide GlcNAc beta 1,6(NeuAc alpha 2,3-Gal-beta 1,3) GalNAc. These results suggest that monensin specifically disrupts the compartment of the biosynthetic pathway which adds most of the beta 1,4-Gal to the oligosaccharides of ASGP-1 and that this compartment is separate from the primary site of sialylation.

Biosynthesis of blood group I antigens. Identification of a UDP-GlcNAc:GlcNAc beta 1-3Gal(-R) beta 1-6(GlcNAc to Gal) N-acetylglucosaminyltransferase in hog gastric mucosa.

A beta 1-6N-acetylglucosaminyltransferase has been identified in microsomal preparations from hog gastric mucosa which is able to synthesize branch points in branched lactosaminoglycans (blood group I antigenic structures). The enzyme can be assayed specifically using the synthetic trisaccharide GlcNAc beta 1-3Gal beta 1-4Glc beta-OMe as acceptor. The product of the transferase reaction was isolated and identified by methylation analysis as, (Formula: see text) Into this tetrasaccharide two galactose residues were incorporated by the specific beta-N-acetylglucosaminide beta 1-4-galactosyltransferase from bovine milk. Thus a hexasaccharide was formed which was shown to inhibit strongly a murine monoclonal and a human anti-I antibody. Using a variety of oligosaccharides and glycolipids, which correspond to structures found in linear lactosaminoglycan chains, the acceptor substrate specificity of the branching enzyme was determined. From these results it is concluded that branching occurs only during the elongation process at the nonreducing end and follows a well-defined order. N-Acetylglucosamine is first transferred to position 3 of a terminal galactose followed immediately by the addition of a second N-acetylglucosamine to position 6; only then the 1-3 and the 1-6 branches are further elongated by galactose residues.