The monoclonal antibody directed to difucosylated type 2 chain (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc; Y Determinant)

One of the monoclonal (AH-6) antibodies prepared by hybridoma technique against human gastric cancer cell line MKN74 was found to react with a series of glycolipids having the Y determinant (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc). The structure of one such glycolipid isolated from human colonic cancer and from dog intestine was identified as lactodifucohexaosyl-ceramide (Fuc alpha 1 leads to 2Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide; IV3,III3Fuc2nLc4Cer). The hapten glycolipid did not react with monoclonal antibodies directed to Lea, Leb, and X-hapten structures, and the AH-6 antibody did not react with the X-hapten ceramide pentasaccharide (Gal beta 1 leads to 4[Fuc alpha 1 leads to 3]GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), H1 glycolipid (Fuc alpha 1 leads to 2Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc beta 1 leads to 1-ceramide), nor with glycolipids having the Leb (Fuc alpha 1 leads to 2Gal beta 1 leads to 3[Fuc alpha 1 leads 4]GlcNAc beta 1 leads to R) determinant. The antibody reacted with blood group O erythrocytes, but not with A erythrocytes. Immunostaining of thin layer chromatography with the monoclonal antibody AH-6 indicated that a series of glycolipids with the Y determinant is present in tumors and in O erythrocytes.     

Wheat germ agglutinin chromatography of GlcNac beta 1-3(GlcNAc beta 1-6)Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, obtained by in vitro synthesis and by partial cleavage of teratocarcinoma poly-N-acetyllactosaminoglycans

GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.     

Enzymatic in vitro synthesis of radiolabeled pentasaccharides GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc and the isomeric Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc.

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).     

Escherichia coli beta-galactosidase unexpectedly cleaves the hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc without branch specificity

The branch specificity of Escherichia coli beta-galactosidase (EC 3.2.1.23) was studied by analyzing the cleavage of the branched hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc (1). This hexasaccharide was cleaved to pentasaccharides Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (3) and GlcNAc beta 1-3(Gal-beta 1-4GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (4) without any appreciable branch specificity. Even the further conversions of the pentasaccharides 3 and 4 into the tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc seemed to proceed at similar rates, without any appreciable branch specificity. In marked contrast to the hexasaccharide 1, the pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal (2), missing the reducing end GlcNAc, is known to be cleaved selectively at the 6-branch; this finding was confirmed in the present study. The different behaviour of hexasaccharide 1 and pentasaccharide 2 reflects differences in the reactivity of their 6-branches; the preferred conformations of these closely related molecules may be quite different.     

The binding specificity of the marker antibodies Tra-1-60 and Tra-1-81 reveals a novel pluripotency-associated type 1 lactosamine epitope

The expression of the epitopes recognized by the monoclonal antibodies Tra-1-60 and Tra-1-81 is routinely used to assess the pluripotency status of human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells. Although it is known that the epitopes recognized by Tra-1-60 and Tra-1-81 are carbohydrates, the exact molecular identity of these epitopes has been unclear. Glycan array analysis with more than 500 oligosaccharide structures revealed specific binding of Tra-1-60 and Tra-1-81 to two molecules containing terminal type 1 lactosamine: Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc and Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3GlcNAcβ1-3)Galβ1-4Glc. The type 1 disaccharide in itself was not sufficient for binding, indicating that the complete epitope requires an extended tetrasaccharide structure where the type 1 disaccharide is β1,3-linked to type 2 lactosamine. Our mass spectrometric analysis complemented with glycosidase digestions of hESC O-glycans indicated the presence of the extended tetrasaccharide epitope on an O-glycan with the likely structure Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAc. Thus, the present data indicate that the pluripotency marker antibodies Tra-1-60 and Tra-1-81 recognize the minimal epitope Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc, which is present in hESCs as a part of a mucin-type O-glycan structure. The exact molecular identity of Tra-1-60 and Tra-1-81 is important for the development of improved tools to characterize the pluripotent phenotype.     

Core tetrasaccharide liberated by endo-beta-D-N-acetylglucosaminidase D from lactosamine-type oligosaccharides of Semliki Forest virus membrane proteins.

[3H]Mannose- and [3H]glucosamine-labeled lactosamine-type glycopeptides of Semliki Forest virus membrane proteins were stripped of their fucose, sialic acid, galactose and distal N-acetylglucosamine residues and subsequently digested with endo-beta-D-N-acetylglucosaminidase D from Diplococcus pneumoniae. Two products were obtained, a neutral tetrasaccharide and a residual glycopeptide fraction. The tetrasaccharide appeared to consist of two alpha-mannose residues, one beta-mannose residue and one N-acetylglucosamine residue located at the reducing terminus of the molecule. Results of Smith degradation, beta-elimination and acetolysis were compatible with four structures; (1) Man alpha-1-3[Man alpha 1-6]Man beta 1-4GlcNAc; (2) Man alpha 1-3Man beta 1-4[Man alpha 1-6] GlcNAc; (3) Man alpha 1-3Man alpha 1-4[Man beta 1-6]GlcNAc, or (4) Man alpha 1-6Man alpha 1-3Man beta-1-4GlcNAc. The reactivity of the viral glycopeptides with endo-beta-D-N-acetylglucosaminidase D and the chromatographic properties of the liberated core tetrasaccharide suggest that its most likely structure was Man alpha 1-3[Man alpha-1-6]Man beta 1-4GlcNAc. The core tetrasaccharide of glycans of membrane protein E3, one of the viral membrane proteins obtained from infected cell, was similar to that of the virion glycans.     

The carbohydrate of bovine prothrombin. Occurrence of Gal beta 1 leads to 3GlcNAc grouping in asparagine-linked sugar chains.

Bovine prothrombin contains three asparagine-linked sugar chains in 1 molecule. The sugar chains were quantitatively released from the polypeptide backbone by hydrazinolysis. All of the oligosaccharides thus obtained contain N-acetylneuraminic acid. Sialidase treatment of these acidic oligosaccharides released three isomeric oligosaccharides, N-1, N-2 and N-3. N-3 was a typical complex type asparagine-linked sugar chain widely found in other glycoprotein, while N-1 and N-2 were unique, because they contain Gal beta 1 leads to 3GlcNAc grouping in the outer chain moiety. By comparing the data of methylation analysis of the acidic oligosaccharides before and after sialidase treatment, the structures of the sugar chains of bovine prothrombin were confirmed as a mixture of NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc leads to Asn and their partially desialized forms.     

Control of glycoprotein synthesis. Detection and characterization of a novel branching enzyme from hen oviduct, UDP-N-acetylglucosamine:GlcNAc beta 1-6 (GlcNAc beta 1-2)Man alpha-R (GlcNAc to Man) beta-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.     

Mucin synthesis. Conversion of R1-beta 1-3Gal-R2 to R1-beta 1-3(GlcNAc beta 1-6)Gal-R2 and of R1-beta 1-3GalNAc-R2 to R1-beta 1-3(GlcNAc beta 1-6)GalNAc-R2 by a beta 6-N-acetylglucosaminyltransferase in pig gastric mucosa

A UDP-GlcNAc:R1-beta 1-3Gal(NAc)-R2 [GlcNAc to Gal(NAc)] beta 6-N-acetylglucosaminyltransferase activity from pig gastric mucosa microsomes catalyzes the formation of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal-R from GlcNAc beta 1-3Gal-R where -R is -beta 1-3GalNAc-alpha-benzyl or -beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-benzyl. This enzyme is therefore involved in the synthesis of the I antigenic determinant in mucin-type oligosaccharides. The enzyme also converts Gal beta 1-3Gal beta 1-4Glc to Gal beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc. The enzyme was stimulated by Triton X-100 at concentrations between 0 and 0.2% and was inhibited by Triton X-100 at 0.5%. There is no requirement for Mn2+ and the enzyme activity is reduced to 65% in the presence of 10 mM EDTA. Enzyme products were purified and identified by proton NMR, methylation analysis and beta-galactosidase digestion. Competition studies suggest that this pig gastric mucosal beta 6-GlcNAc-transferase activity is due to the same enzyme that converts Gal beta 1-3GalNAc-R to mucin core 2, Gal beta 1-3(GlcNAc beta 1-6)GalNAc-R, and GlcNAc beta 1-3GalNAc-R to mucin core 4, GlcNAc beta 1-3(GlcNAc beta 1-6)GalNAc-R. Substrate specificity studies indicate that the enzyme attaches GlcNAc to either Gal or GalNAc in beta (1-6) linkage, provided these residues are substituted in beta (1-3) linkage by either GlcNAc or Gal. The insertion of a GlcNAc beta 1-3 residue into Gal beta 1-3GalNAc-R to form GlcNAc beta 1-3Gal beta 1-3GalNAc-R prevents insertion of GlcNAc into GalNAc. These studies establish several novel pathways in mucin-type oligosaccharide biosynthesis.     

Human serum contains a novel beta 1,6-N-acetylglucosaminyltransferase activity that is involved in midchain branching of oligo (N-acetyllactosaminoglycans).

Incubation of UDP-GlcNAc and radiolabeled GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc beta 1-3Gal (4) and the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc beta 1-6Gal (6), together with the trisaccharides GlcNAc beta 1-6Gal beta 1-4GlcNAc (7) and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta 1,4-galactosyltransferase and UDP-[6-3H]Gal gave the octasaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4GlcNAc beta 1-3([6-3H]-Gal beta 1-4GlcNAc beta 1-6)[U-14C] Gal beta 1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3Gal (18) and the branched pentasaccharide GlcNAc beta 1-3-([6-3H]Gal beta 1-4GlcNAc beta 1-6) [U-14C]Gal beta 1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (20). The serum beta 1,6-GlcNAc transferase catalyzed also the formation of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (11) from UDP-GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (10). The pentasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (16), too, served as an acceptor for the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

The synthesis of linear trilactosamine

TrilactosamineGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-sp, where sp = O(CH₂)₃NH₂ is a spacer, was synthesized. The tetrasaccharide fragment Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-sp was obtained by successive glycosylation using elongation by one monosaccharide residue at a time; and the tetrasaccharide was then transformed into a hexasaccharide with a disaccharide glycosyl donor. A 2,2,2-trichloroethoxycarbonyl group was used for the protection of the glucosamine amino group.     

GDP-fucose: beta-N-acetylglucosamine (Fuc to (Fuc alpha 1----6GlcNAc)-Asn-peptide)alpha 1----3-fucosyltransferase activity in honeybee (Apis mellifica) venom glands. The difucosylation of asparagine-bound N-acetylglucosamine

Incubation of honeybee (Apis mellifica) venom-gland extracts with GDP-[14C]fucose and GlcNAc beta 1----2Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----6)GlcNAc beta 1----N-Asn-peptide(NAc) gave a labeled product in 40% yield. Analysis by 500-MHz 1H-NMR spectroscopy indicated the transferred fucose-(Fuc) residue to be alpha 1----3-linked to the Asn-bound GlcNAc. Further proof was provided by one-dimensional and two-dimensional 1H-NMR analysis of the incubation mixture, after incubation with beta-N-acetylhexosaminidase. The established carbohydrate structure (formula; see text) proves the existence of a novel alpha 1----3-fucosyltransferase with the ability to effect difucosylation of the Asn-bound GlcNAc in N-glycans.     

The tumor suppressor EXT-like gene EXTL2 encodes an alpha1, 4-N-acetylhexosaminyltransferase that transfers N-acetylgalactosamine and N-acetylglucosamine to the common glycosaminoglycan-protein linkage region. The key enzyme for the chain initiation of heparan sulfate.

We previously demonstrated a unique alpha-N-acetylgalactosaminyltransferase that transferred N-acetylgalactosamine (GalNAc) to the tetrasaccharide-serine, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser (GlcA represents glucuronic acid), derived from the common glycosaminoglycan-protein linkage region, through an alpha1,4-linkage. In this study, we purified the enzyme from the serum-free culture medium of a human sarcoma cell line. Peptide sequence analysis of the purified enzyme revealed 100% identity to the multiple exostoses-like gene EXTL2/EXTR2, a member of the hereditary multiple exostoses (EXT) gene family of tumor suppressors. The expression of a soluble recombinant form of the protein produced an active enzyme, which transferred alpha-GalNAc from UDP-[3H]GalNAc to various acceptor substrates including GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Interestingly, the enzyme also catalyzed the transfer of N-acetylglucosamine (GlcNAc) from UDP-[3H]GlcNAc to GlcAbeta1-3Galbeta1-O-naphthalenemethanol, which was the acceptor substrate for the previously described GlcNAc transferase I involved in the biosynthetic initiation of heparan sulfate. The GlcNAc transferase reaction product was sensitive to the action of heparitinase I, establishing the identity of the enzyme to be alpha1, 4-GlcNAc transferase. These results altogether indicate that EXTL2/EXTR2 encodes the alpha1,4-N-acetylhexosaminyltransferase that transfers GalNAc/GlcNAc to the tetrasaccharide representing the common glycosaminoglycan-protein linkage region and that is most likely the critical enzyme that determines and initiates the heparin/heparan sulfate synthesis, separating it from the chondroitin sulfate/dermatan sulfate synthesis.     

Specific detection of N-acetylglucosamine-containing oligosaccharide chains on ovine submaxillary asialomucin

Human milk beta-N-acetylglucosaminide beta 1 leads to 4-galactosyltransferase (EC 2.4.1.38) was used to galactosylate ovine submaxillary asialomucin to saturation. The major [14C]galactosylated product chain was obtained as a reduced oligosaccharide by beta-elimination under reducing conditions. Analysis by Bio-Gel filtration and gas-liquid chromatography indicated that this compound was a tetrasaccharide composed of galactose, N-acetylglucosamine and reduced N-acetylgalactosamine in a molar ratio of 2:0.9:0.8. Periodate oxidation studies before and after mild acid hydrolysis in addition to thin-layer chromatography revealed that the most probable structure of the tetrasaccharide is Gal beta 1 leads to 3([14C]Gal beta 1 leads to 4GlcNAc beta 1 leads to 6)GalNAcol. Thus it appears that Gal beta 1 leads to 3(GlcNAc beta 1 leads to 6)GalNAc units occur as minor chains on the asialomucin. The potential interference of these chains in the assay of alpha-N-acetylgalactosaminylprotein beta 1 leads to 3-galactosyltransferase activity using ovine submaxillary asialomucin as an acceptor can be counteracted by the addition of N-acetylglucosamine.     

Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures.

The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.     

The sugar chains of cold-insoluble globulin. A protein related to fibronectin.

Cold-insoluble globulin isolated from bovine plasma contains six asparagine-linked sugar chains in 1 molecule (a dimeric form). These sugar chains were released from the polypeptide backbone by hydrazinolysis and labeled by reduction with NaB[3H]4. Most of these sugar chains contain N-acetylneuraminic acid and can be separated by paper electrophoresis. By combination of sequential exoglycosidase digestion and methylation study, their structures were elucidated as Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6 or 4Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(NeuAc alpha 2 leads to 4 or 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc, NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]-Man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc and NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[NeuAc alpha 2 leads to 4Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]man beta 1 leads to 4GlcNAc beta 1 leads to 4GlcNAc.     

Synthesis of O-alpha-D-glucopyranosyl-(1----4)-O-alpha -D-xylopyranosyl-(1----4)-O-alpha -D-xylopyranosyl-(1----4)-D-glucopyranose as a substrate analogue of alpha amylase

The tetrasaccharide a-D-Glcp-(1----4)-a-D-Xylp-(1----4)-a-D-Xylp-(1----4)-D- Glcp (1) has been synthesized, as a substrate analogue of alpha amylase, by silver perchlorate-catalyzed glycosylation of benzyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-a-D-xylopyranosyl)-beta-D- glucopyranoside (30) with 2,3-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-a-D- glucopyranosyl)-a-D-xylopyranosyl chloride or by methyl triflate-promoted condensation of 30 with methyl 2,3-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)-1-thio- beta-D-xylopyranoside, followed by removal of protecting groups of the resulting tetrasaccharide derivative 40.     

Human alpha3-fucosyltransferases convert chitin oligosaccharides to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus

Human alpha3-fucosyltransferases (Fuc-Ts) are known to convert N-acetyllactosamine to Galbeta1-4(Fucalpha1-3)GlcNAc (Lewis x antigen); some of them transfer fucose also to GalNAcbeta1-4GlcNAc, generating GalNAcbeta1-4(Fucalpha1-3)GlcNAc determinants. Here, we report that recombinant forms of Fuc-TV and Fuc-TVI as well as Fuc-Ts of human milk converted chitin oligosaccharides of 2-4 GlcNAc units efficiently to products containing a GlcNAcbeta1-4(Fucalpha1-3)GlcNAcbeta1-4R determinant at the nonreducing terminus. The product structures were identified by mass spectrometry and nuclear magnetic resonance experiments; rotating frame nuclear Overhauser spectroscopy data suggested that the fucose and the distal N-acetylglucosamine are stacked in the same way as the fucose and the distal galactose of the Lewis x determinant. The products closely resembled a nodulation factor of Mesorhizobium loti but were distinct from nodulation signals generated by NodZ-enzyme.     

Synthesis of a repeating unit and the dimer of the repeating unit of the major chain of Shigella flexneri O-antigen polysaccharide

Methyl glycoside of the tetrasaccharide GlcNAc(beta 1-2)Rha(alpha 1-2)Rha(alpha 1-3)Rha, which represents a repeating unit of the basic chain of Shigella flexneri O-antigenic polysaccharides, was synthesized using acylated monosaccharide synthons. A dimer of the repeating unit, octasaccharide [GlcNAc(beta 1-2)Rha(alpha 1-2) Rha(alpha 1-3)Rha(alpha 1-3)]2-OMe was obtained by TrClO4-catalyzed condensation of two tetrasaccharide blocks.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.