Immunochemical studies on blood groups. Purification and characterization of radioactive 3H-reduced di- to hexasaccharides produced by alkaline beta-elimination-borohydride 3H reduction of Smith degraded blood group A active glycoproteins

Treatment of blood group A active glycoprotein from human ovarian cyst fluid by one stage of Smith degradation followed by alkaline beta-elimination in the presence of NaB[ 3H4 ] (Carlson degradation) liberated tritiated oligosaccharide alditols. The carbohydrate mixture was fractionated by gel filtration, elution from charcoal, paper chromatography, and high pressure liquid chromatography. Structures were established based on sugar composition, periodate oxidation, methylation analysis, and analysis of oligosaccharide alditols as permethylated and N-trifluoroacetylated derivatives by gas-liquid chromatography-mass spectrometry. The following structures have been deduced: Gal beta 1----3GalNAc-ol, GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1---- 3GlcNAc beta 1----6(3-deoxy)GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1---- 6GalNAc -ol, Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1----3[GlcNAc beta 1----6]GalNAc-ol, Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3GalNAc-ol, GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1---- 6GalNAc -ol, GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3[Gal beta 1----4GlcNAc beta 1----6]Gal beta 1----3GalNAc-ol, Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol. The smaller structures represent pieces of the larger structures. Together they provide direct evidence for the core structure of the carbohydrate side chains in the blood group substances as proposed by K. O. Lloyd and E. A. Kabat [1968) Proc. Natl. Acad. Sci. U.S.A. 61, 1470-1477). Oligosaccharides previously isolated after Carlson degradation of intact human ovarian cyst fluid HLeb , Lea, and B substances and from human and horse B substances contained various alpha-linked L- fucopyranose and alpha-linked Gal substitutions on the composite structure.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of the O-glycosidic oligosaccharide chains of the major Zajdela hepatoma ascites-cell-membrane glycoprotein

Glycoprotein MII2, the major cell surface glycoprotein (molecular mass 110 kDa) of Zajdela hepatoma ascites cells, contains about 25 O-glycosidic oligosaccharide chains per molecule. They were released as oligosaccharide-alditols by alkaline borohydride treatment of MII2, and purified by gel filtration on Bio-Gel P-6 followed by high-voltage paper electrophoresis. Four oligosaccharide-alditol fractions (A-D) were obtained in relative yields of 8:6:3:3. The structure of the components of fractions A-C was determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis, to be as follows. (A) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B1) NeuAc alpha(2----3)Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (B2) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol; (C) NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol. On the basis of sugar composition and characteristics on Bio-Gel P-6 filtration, paper electrophoresis and thin-layer chromatography, the structure of the carbohydrate component of fraction D is proposed to be as follows. (D) NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary structure determination of five sialylated oligosaccharides derived from bronchial mucus glycoproteins of patients suffering from cystic fibrosis. The occurrence of the NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)] GlcNAc beta(1----.) structural element revealed by 500-MHz 1H NMR spectroscopy

The structure of sialylated carbohydrate units of bronchial mucins obtained from cystic fibrosis patients was investigated by 500-MHz 1H NMR spectroscopy in conjunction with sugar analysis. After subjecting the mucins to alkaline borohydride degradation, sialylated oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high performance liquid chromatography. Five compounds could be obtained in a rather pure state; their structures were established as the following: A-1, NeuAc alpha(2----3)Gal beta(1----4) [Fuc alpha(1----3)]GlcNAc beta(1----3)Gal-NAc-ol; A-2, NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)-[GlcNAc beta (1----3)]GalNAc-o1; A-3, NeuAc alpha(2----3)Gal beta-(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----3)Gal beta(1----3) GalNAc-o1; A-4, NeuAc alpha(2----3)Gal beta(1----4)[Fuc alpha(1----3)]Glc-NAc NAc beta(1----6)[GlcNAc beta(1----3)]GalNAc-o1; A-6,NeuAc alpha-(2----3) Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----6)[Gal beta-(1----4) GlcNAc beta(1----3)]GalNAc-o1. The simultaneous presence of sialic acid in alpha(2----3)-linkage to Gal and fucose in alpha(1----3)-linkage to GlcNAc of the same N-acetyllactosamine unit could be adequately proved by high resolution 1H NMR spectroscopy. This sequence constitutes a novel structural element for mucins.     

Structures of oligosaccharides cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein

Oligosaccharides were cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein and purified by Bio-Gel P-6 and paper chromatography. The structures of five oligosaccharides, determined by compositional analyses, quantitative periodate oxidation, chronic acid oxidation, methylation analyses and enzymatic degradations, were as follows: oligosaccharide I, beta DGal1----3DGalNAc-ol; II, beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; III, alpha LFuc1----2 beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; IV, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGal1NAcol; and V, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----4 beta DGlcNAc1----6[beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----3 beta DGal1----3]DGalNAc-ol. Of the oligosaccharides 60% had a molecular size of a decasaccharide or smaller, the tetra- and pentasaccharides II and III predominating. Oligosaccharides I through IV have been previously isolated from several glycoproteins by other laboratories; the decasaccharide, V, is a new structure.     

Structures of the O-linked oligosaccharides of the major cell surface sialoglycoprotein of MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma

Structures of the principal O-glycosides from the major cell surface sialoglycoprotein (ASGP-1) of the MAT-B1 and MAT-C1 ascites sublines of the 13762 rat mammary adenocarcinoma have been determined. Oligosaccharitols were released by alkaline borohydride treatments of ASGP-1 and purified by gel filtration, DEAE-Sephadex ion exchange chromatography, and high performance liquid chromatography. On the basis of carbohydrate composition, methylation analysis, periodate oxidation, and exoglycosidase digestion, the five major oligosaccharides released by mild alkaline borohydride were assigned the following structures: Component II-3: (NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)Ga 1 NAcOH(3----1 betaGa 1 3----2 alpha NeuAc) III-2a: (Ga 1 beta 1----4G1cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) III-2c: (Ga 1 alpha 1----3Ga 1 beta 1----4G1cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1 3----2 alpha NeuAc) IV-1a: (Ga 1 beta 1----4G 1 cNAc beta 1----6)Ga 1 NAcOH(3----1 beta Ga 1) IV-1c: (Ga 1 alpha 1----3Ga 1 beta 1----4G 1 cNAc beta 1----6) Ga 1 NAcOH(3----1 beta Ga 1) Fucosylated derivatives of III-2a, IV-1a, and IV-1c were found in smaller amounts with the fucose tentatively assigned to the 2-position of the lactosamine galactose. Components II-3, III-2a, and the fucosylated derivative of III-2A were found in both MAT-B1 and MAT-C1 sublines. The alpha-galactosides were found in detectable quantities only in subline MAT-B1. Oligosaccharides from MAT-C1 cells were enriched in sialic acid when compared to those from MAT-B1 cells. These results suggest that the 13762 ascites sublines, which bear different oligosaccharides, will provide models useful for the investigation of mechanisms regulating the expression of structures of the larger O-linked oligosaccharides.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.     

Chemical structure of neutral sugar chains isolated from human mature milk kappa-casein

The carbohydrate chains linked to human kappa-casein from mature milk were released by alkaline borohydride treatment as reduced oligosaccharides. The neutral oligosaccharides of lower molecular weight were fractionated and purified by gel filtration and preparative thin layer chromatographies. Seven neutral oligosaccharides (a di- (0.5%), two tetra- (30.5%), two penta- (5.4%) and two hexasaccharide alditols (10.9%] were obtained in homogeneity, and followed by methylation analysis with gas-liquid chromatography-mass spectrometry and by anomer analysis with 13C nuclear magnetic resonance. Their chemical structures were identified to be Gal beta 1----3GalNAc-ol (I), Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (II), Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (III), GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (IV), GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (V), Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Gal beta 1----4GlcNAc beta 1----6]GalNAc-ol (VI) and Fuc alpha 1----4GlcNAc beta 1----3/6Gal beta 1----3[Fuc alpha 1----4GlcNAc beta 1----6]GalNAc-ol (VII). Five oligosaccharide alditols (III-VII) were the novel carbohydrate chains of kappa-casein from mammalian milk.     

Structural variations of O-linked oligosaccharides present in leukosialin isolated from erythroid, myeloid, and T-lymphoid cell lines

Structures of O-linked oligosaccharides of leukosialin isolated from K562 erythroid, HL-60 promyelocytic, and HSB-2 T-lymphoid cell lines were examined. Leukosialin was isolated by specific immunoprecipitation from cells which were metabolically labeled with [3H]glucosamine, and glycopeptides were isolated after Pronase digestion. O-Linked oligosaccharides were released by alkaline borohydride treatment, and the structures of purified oligosaccharides were elucidated by specific exoglycosidase digestion, Smith degradation, and methylation anaylsis. Oligosaccharides from K562 cells were found to be GalNAcOH, Gal beta 1----3GalNAcOH, NeuNAc alpha 2----6GalNAcOH, NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----6)GalNAcOH. On the other hand, oligosaccharides from HL-60 and HSB-2 cells were found to be NeuNAc alpha 2----3Gal beta 1----3GalNAcOH, NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAcOH, Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3)Gal beta 1----3)GalNAcOH, and NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6(NeuNAc alpha 2----3Gal beta 1----3)GalNAcOH. These results clearly indicate that leukosialin can be differently glycosylated with O-linked chains, and each erythroid or myeloid (and T-lymphoid) cell line expresses a characteristic set of O-linked oligosaccharides which differ in core structures as well as in sialylation.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

Biosynthesis of polylactosaminoglycans. Novikoff ascites tumor cells contain two UDP-GlcNAc:beta-galactoside beta 1----6-N-acetylglucosaminyltransferase activities

Novikoff ascites tumor cells contain a UDP-GlcNAc:beta-galactoside beta 1----6-N-acetylglucosaminyltransferase (beta 6-GlcNAc-transferase B) that acts on galactosides and N-acetylgalactosaminides in which the accepting sugar is beta 1----3 substituted by a Gal or GlcNAc residue. Characterization of enzyme products by 1H-NMR and methylation analysis indicates that an R beta 1----3(GlcNAc beta 1----6)Gal- branching point is formed such as occurs in blood-group-I-active substances. The enzyme does not show an absolute divalent cation requirement and 20 mM EDTA is not inhibitory. The activity is strongly inhibited by Triton X-100 at concentrations of greater than or equal to 0.2%. Competition studies suggest that a single enzyme acts on Gal beta 1----3Gal beta 1----4Glc, GlcNAc beta 1----3Gal beta 1----4GlcNAc and GlcNAc beta 1----3GalNAc alpha-O-benzyl (Km values 0.71, 0.83 and 0.53 mM, respectively). Gal beta----3Gal beta 1----4Glc as an acceptor substrate for beta 6-GlcNAc-transferase B does not inhibit the incorporation of GlcNAc in beta 1----6 linkage to the terminal Gal residues of asialo-alpha 1-acid glycoprotein catalyzed by a beta-galactoside beta 1----6-N-acetylglucosaminyltransferase (beta 6-GlcNAc-transferase A) previously described in Novikoff ascites tumor cells [D. H. Van den Eijnden, H. Winterwerp, P. Smeeman & W.E.C.M. Schiphorst (1983) J. Biol. Chem. 258, 3435-3437]. Neither is Triton X-100 at a concentration of 0.8% inhibitory for the activity of beta 6-GlcNAc-transferase A. This activity is absent from hog gastric mucosa microsomes, which has been described to contain high levels of beta 6-GlcNAc-transferase B. [F. Piller, J. P. Cartron, A. Maranduba, A. Veyrières, Y. Leroy & B. Fournet (1984) J. Biol. Chem. 259, 13,385-13,390]. Our results show that Novikoff tumor cells contain two beta-galactoside beta 6-GlcNAc-transferases, which differ in acceptor specificity and tolerance towards Triton X-100. A role for these enzymes in the synthesis of branched polylactosaminoglycans and of O-linked oligosaccharide core structures having blood-group I activity is proposed.     

Structural characterization of a blood group A heptaglycosylceramide with globo-series structure. The major glycolipid based blood group A antigen of human kidney

A blood group A glycosphingolipid with the globo-series structure has been isolated from human kidney and structurally characterized. The structure was shown by mass spectrometry and proton NMR spectroscopy of the intact permethylated and permethylated-reduced derivatives together with degradation studies to be, GalNAc alpha 1----3Gal(2----1 alpha Fuc)beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1 Ceramide. This glycolipid reacts with both polyclonal and monoclonal anti-A blood group typing antisera and it is the major glycolipid based blood group A antigen present in the human kidney.     

Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

New evidence of the substrate specificity of endo-beta-N-acetylglucosaminidase D

The susceptibility of a variety of oligosaccharides to endo-beta-N-acetylglucosaminidase D was investigated. The oligosaccharides having the structures of Man alpha 1----6 (GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, derived from complex type triantennary sugar chains, released +/- Fuc alpha 1----6GlcNAcOT upon incubation with the enzyme at almost the same rate as Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. When the reaction products were reduced with NaB3H4 and analyzed by Bio-Gel P-4 column chromatography, a new radioactive peak was detected in both cases. This new radioactive oligosaccharide was confirmed to be Man alpha 1----6(GlcNAc beta 1----4Man alpha 1----3)Man beta 1----4GlcNAcOT in the former case and Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAcOT in the latter. These results indicated that endo-beta-N-acetylglucosaminidase D does not require the presence of a free hydroxyl group at the C-4 position of the alpha-mannosyl residue of the trisaccharide glycon: Man alpha 1----3Man beta 1----4GlcNAc beta 1----.     

Oligosaccharides at individual glycosylation sites in glycoprotein 71 of Friend murine leukemia virus

Glycoprotein 71 from Friend murine leukemia virus was digested with proteases and the glycopeptides obtained were isolated and assigned, by amino acid sequencing, to the eight N-glycosylated asparagines in the molecule; only Asn334 and Asn341 could not be separated. The oligosaccharides liberated from each glycopeptide by endo-beta-N-acetylglucosaminidase H, or by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, were fractionated and subjected to structural analysis by one- and two-dimensional 1H NMR, as well as by methylation/gas-liquid-chromatography/mass-fragmentography. At each glycosylation site, the substituents were found to be heterogeneous including, at Asn334/341 and Asn410, substitution by different classes of N-glycans: oligomannosidic oligosaccharides, mainly Man alpha 1----6(Man alpha 1----3)Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were detected at Asn168, Asn334/341 and Asn410. Hybrid species, partially sialylated, intersected and (proximally) funcosylated Man alpha 1----6(Man alpha 1----3)Man alpha 1----6 and Man alpha 1----3Man alpha 1----6 and Man alpha 1----3Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc beta 1----, were found at Asn12, as previously published [Schlüter, M., Linder, D., Geyer, R., Hunsmann, H., Schneider, J. & Stirm, S. (1984) FEBS Lett. 169, 194-198] and at Asn334/341. N-Acetyllactosaminic glycans, mainly partially intersected and fucosylated NeuAc alpha 2----3 or Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(NeuAc alpha 2----6 or NeuAc alpha 2----3Gal-beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNac beta 1----4GlcNAc beta 1---- with some bifurcation at ----6Man alpha 1----6, were obtained from Asn266, Asn302, Asn334/341, Asn374 and Asn410. In addition, Thr268, Thr277, Thr279, Thr304/309, as well as Ser273 and Ser275, were found to be O-glycosidically substituted by Gal beta 1----3GalNAc alpha 1----, monosialylated or desialylated at position 3 of Gal or/and position 6 of GalNAc.     

Structure of novel glyceroglycolipids in Adzuki bean (Vigna angularis) seeds

In addition to monoglycosyl diacylglycerol, three glyceroglycolipids with two to four hexose units were isolated from Adzuki bean seeds. Their structures were determined using enzymatic hydrolysis, permethylation analysis, nuclear magnetic resonance spectroscopy, and reversed-phase high pressure liquid chromatography, as folows: Gal(beta 1----3')- for the monoglycosyl diacyglycerol, Gal(alpha 1----6)-Gal(beta 1----3')- and Gal(beta 1----6)-Gal(beta1----3')- for the diglycosyl diacylglycerol, [Gal(beta 1----6]2-Gal(beta 1----3')- and Gal(beta 1----6)-Gal(alpha 1----6)-Gal(beta 1----3')- for the triglycosyl diacylglycerol, and [Gal(beta 1----6)]3-Gal(beta 1----3')- and [Gal(beta 1----6)]2-Gal(alpha 1----6)-Gal(beta 1----3')- for the tetraglycosyl diacylglycerol. Except for Gal(alpha 1----6)-Gal(beta 1----3')-diacylglycerol, all of the glycolipids with oligosaccharide moieties had novel glycosidic linkage patterns differing from those in the well-known glyceroglycolipids in nature. The predominant diacylglycerol species component was characterized as dilinolenin. However, the molecular species composition of the diacylglycerol moieties of the two diglycosyl isomers with different glycosidic linkage patterns were found to be qualitatively the same but different quantitatively, as follows: in the well-known isomer Gal(alpha 1----6)-Gal(beta 1----3')-, 39% dilinolenin, 15% palmitoyl linolenin, and 14% linoleoyl linolenin; and in the novel isomer Gal(beta 1----6)-Gal(beta 1----3')-, 35% dilinolenin, 24% dilinolein, and 21% linoleoyl linolenin.     

Biosynthesis of marsupial milk oligosaccharides. II: Characterization of a beta 6-N-acetylglucosaminyltransferase in lactating mammary glands of the tammar wallaby, Macropus eugenii.

Tammar wallaby (Macropus eugenii) mammary glands contain a UDP-GlcNAc:Gal beta 1----3Gal beta 1----4Glc beta 1----6-N-acetylglucosaminyltransferase (GlcNAcT) whose activity has been characterized with respect to the effect of pH, apparent Km for acceptor, effects of bivalent metal ions, acceptor specificity and identity of products. The enzyme did not show an absolute requirement for any bivalent metal ion but its activity was increased markedly by Mg2+, Ca2+ and Ba2+ and, to a lesser extent, by Mn2+. When Gal beta 1----3Gal beta 1----4Glc was used as acceptor, the product was Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----4Glc. With Gal beta 1----3Gal beta 1----3Gal beta 1----4Glc as acceptor, the product was shown, by 1H-NMR spectroscopy and exo-beta-galactosidase digestion, to be a novel pentasaccharide with the structure Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----3Gal beta 1----4Glc, suggesting that the enzyme recognises the non-reducing end of the acceptor substrate, rather than the reducing end.     

Asparagine-linked sugar chains of fetuin: occurrence of tetrasialyl triantennary sugar chains containing the Gal beta 1----3GlcNAc sequence

Asparagine-linked sugar chains were quantitatively released from fetuin by hydrazinolysis. Structural analysis of the sugar chains by sequential exoglycosidase digestion in combination with methylation analysis and Smith degradation revealed that most of them have typical biantennary (8%) and triantennary (74%) structures containing different amounts of N-acetylneuraminic acid residues. In addition, an unusual tetrasialyl triantennary sugar chain (17%) containing the Gal beta 1----3GlcNAc sequence in the outer chain moiety was detected, and its structure was elucidated as NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)-GlcNAc beta 1----4(NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2)Man alpha 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAc.     

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.     

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.