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.     

Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core-type 1, two contained core-type 3 and two contained core-type 5. The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: A4b, GalNAc alpha(1----3) [NeuAc alpha(2----6)]GalNAcol; A4c, GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4d, Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4e, GalNAc alpha(1----3)-[NeuGc alpha(2----6)]GalNAcol; A4f, GlcNAc beta(1----3)[NeuGc alpha (2----6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core-type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H-NMR data for structure A4e, which is a novel structure, are presented for the first time.     

Biosynthesis of a disialylated sequence in N-linked oligosaccharides: identification of an N-acetylglucosaminide (alpha 2----6)-sialyltransferase in Golgi apparatus from rat liver

Rat liver Golgi apparatus are shown to have a CMP-N-acetylneuraminate: N-acetylglucosaminide (alpha 2----6)-sialyltransferase which catalyzes the conversion of the human milk oligosaccharide LS-tetrasaccharide-a (NeuAc alpha 2----3Gal beta 1---- 3GlcNAc beta 1----3Gal beta 1----4Glc) to disialyllacto -N- tetraose containing the terminal sequence: (formula: see text) found in N-linked oligosaccharides of glycoproteins. The N-acetylglucosaminide (alpha 2----6)-sialyltransferase has a marked preference for the sequence NeuAc alpha 2----3-Gal beta 1---- 3GlcNAc as an acceptor substrate. Thus, the order of addition of the two sialic acids in the disialylated structure shown above is proposed to be first the terminal sialic acid in the NeuAc alpha 2----3Gal linkage followed by the internal sialic acid in the NeuAc alpha 2---- 6GlcNAc linkage. Sialylation in vitro of the type 1 branches (Gal beta 1---- 3GlcNAc -) of the N-linked oligosaccharides of asialo prothrombin to produce the same disialylated sequence is also demonstrated.     

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.     

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.     

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.     

Sulfated N-linked carbohydrate chains in porcine thyroglobulin

N-linked carbohydrate chains of porcine thyroglobulin were released by the hydrazinolysis procedure. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis, the acidic fractions were further separated by high-performance liquid chromatography on Lichrosorb-NH2, and analyzed by 500-MHz 1H-NMR spectroscopy and, partially, by permethylation analysis. Of the acidic oligosaccharide-alditols, the following sulfated carbohydrate chains could be identified: NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3[(SO3Na----3)Gal beta 1----4GlcNAc beta1----2-Mana alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc-ol and NeuAc alpha 2----6Gal beta 1----4(SO3Na----)0-1 GlcNAc beta 1----2-Man alpha 1----3[NeuAc alpha 2----6Gal beta 1----4(SO3Na----6)1-0GlcNAc beta 1----2Man alpha 1----6]Man beta 1----4GlcNAc beta 1----4[Fuc alpha 1----6]GlcNAc- ol. The sulfated structural elements for porcine thyroglobulin form novel details of N-linked carbohydrate chains. They contribute to the fine structure of these oligosaccharides and are another type of expression of microheterogeneity.     

Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy

The analysis of the carbon-13 chemical shift data of NeuAc alpha (2----3)Gal beta (1----4)Glc and NeuAc alpha (2----3)Gla beta-(1----4)GlcNAc and their respective NeuAc alpha (2----6) isomers established distinct and different conformations of the sialic acid residue, depending on the type of anomeric linkage [alpha-(2----3) vs. alpha (2----6)]. Interactions between the NeuAc residue and the Glc or GlcNAc residue are particularly strong in the case of the alpha (2----6) isomers. Similar effects are observed for the larger oligosaccharides [II3(NeuAc)2Lac and IV6NeuAcLcOse4] and even in intact glycoproteins and polysaccharides. It is proposed that the NeuAc alpha (2----3) isomers assume an extended conformation with the sialic residue at the end (terminal) of the oligosaccharide chain or branch. The NeuAc alpha (2----6) isomers are assumed to be folded back toward the inner core sugar residues.     

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.     

The structures of the carbohydrate moieties of bovine blood coagulation factor X

Bovine blood coagulation factor X contains both asparagine-linked and threonine-linked oligosaccharides. The asparagine-linked chain is a mixture of a tridecasaccharide 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 and a dodecasaccharide NeuAc alpha 2 leads to 6 Gal 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 and their partial desialylation products. The threonine-linked chain is a mixture of NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)GalNAc, NeuAc alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, NeuGly alpha 2 leads to 3Gal beta 1 leads to 3 (NeuAc alpha 2 leads to 6)GalNAc, and NeuGly alpha 2 leads to 3Gal beta 1 leads to 3(NeuGly alpha 2 leads to 6)GalNAc, and their partial desialized forms. The carbohydrate moieties of the factor X subgroups, factors X1 and X2, are identical.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

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.     

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.     

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)     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

The structures of the serine-linked sugar chains on human chorionic gonadotropin

The human chorionic gonadotropin beta-subunit tryptic COOH-terminal peptide (residues 123-145) which contains 3 serine-linked sugar chains was isolated. The sugar chains were cleaved by beta-elimination and then separated by gel filtration. The peaks were pooled and their compositions determined. The products of serial glycosidase digestion and periodate oxidation of the intact glycopeptide were also characterized. Of the serine-linked sugar chains, 13% were the hexasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,3 Gal beta 1,4 GlcNAc beta 1,6) GalNAc, 34% the tetrasaccharide NeuAc alpha 2,3 Gal beta 1,3 (NeuAc alpha 2,6) GalNAc, 43% the trisaccharide NeuAc alpha 2,3 Gal beta 1,3 GalNAc and 10% the disaccharide NeuAc alpha 2,6 GalNAc.     

A carbohydrate structural variant of MM glycoprotein (glycophorin A)

A variant of the MM glycoprotein (glycophorin A) was isolated from erythrocyte membranes of two individual donors, a mother (L.G.) and daughter (V.W.). This glycoprotein was found to be a carbohydrate variant in which, for both donors, certain O-glycosidically linked saccharides retained the core structure consisting of NeuAc(alpha 2,3)Gal(beta 1,3)GalNAc that is common to all O-linked saccharides of the MN glycoproteins, and, in addition, contained substituents, of varying chain lengths, on the primary carbinol of GalNAc. These saccharides were released from the polypeptide by beta-elimination in the presence of sodium borohydride, and aspects of their structure were investigated by glycosidase digestion and periodate oxidation. Thus, the smallest variant structure was deduced to be NeuAc(alpha 2,3)Gal(beta 1,3)[GlcNAc(beta 1,6)]H2GalNAc. The 6-O-linked GlcNAc appears to serve as the focus of further chain elongation reactions, involving alternate additions of Gal and GlcNAc residues and leading to the formation of several homologous structures. Two such structures, NeuAc(alpha 2,3)Gal(beta 1,3)[GlcNAc(beta 1,?) Gal(beta 1,3/4)GlcNAc(beta 1,6)]H2GalNAc and NeuAc(alpha 2,3) Gal(beta 1,3)[Gal(beta 1,3/4)GlcNAc(beta 1,6)]H2GalNAc were the predominant species present. A larger saccharide was also isolated and its partial sequence was determined to be Gal(beta 1,3/4)GlcNAc(beta 1,?)[Gal(beta 1,3/4)Glc-NAc(beta 1,?)] Gal(beta 1,3/4)GlcNAc(beta 1,6)[NeuAc(alpha 2,3)Gal-(beta 1,3)]H2GalNAc. Because the peptide portion of these glycoproteins contains two methionine residues, it was possible to isolate two CNBr glycopeptides from separate regions of the molecule, and to assess the distribution of these variant structures in the polypeptide. The saccharides were linked to about 2-3 Ser and/or Thr residues in the donor LG glycoprotein and one of the attachment sites was located within the CNBr glycooctapeptide representing the NH2 terminus. Considerable heterogeneity in saccharide structure was documented for this site, and it is likely that such heterogeneity occurs also at other sites. The variant saccharides bear structural similarities to the core region of O-linked saccharides of certain blood group-active mucins and ovarian cyst secretions, and to the outer sequences of N-linked carbohydrate units (I-, i-active) of the major glycoprotein of human erythrocytes, band 3. The structures of the variant saccharides suggest that they may be potential precursors of H blood group-active carbohydrates, present in varying degrees of maturity, and attached to an integral protein of erythrocytes.     

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)     

Kinetic parameters of a beta-D-galactoside alpha 2 leads to 6 sialyltransferase from embryonic chicken liver

A beta-D-galactoside alpha 2 leads to 6 sialyltransferase was purified 500-fold in 14% yield from 14-day embryonic chicken liver. Characterization of the product of the sialyltransferase catalysis was accomplished by separation and permethylation of double-labelled ([14C]NeuAc, [3H]Gal) oligosaccharides following their release from the glycoprotein fetuin by hydrazinolysis. The enzyme transfers NeuAc to Gal(beta 1 leads to 4)GlcNAc(beta 1 leads to)R-terminated oligosaccharides; no activity was found towards Gal(beta 1 leads to 3)GalNAc(alpha 1 leads to)R structures. The trisaccharide. NeuAc(alpha 2 leads to 6)Gal(beta 1 leads to 4)Glc, was shown to be a good inhibitor of the sialyltransferase. Kinetic investigations of the enzyme indicate it to have a sequential, random bi-bi mechanism.