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.     

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.     

Primary structure of the glycan chains of normal C 1 esterase inhibitor (C 1-INH) after NMR analysis at 400 MHz

The primary structural analysis of O- and N-linked carbohydrate chains of the C-1-esterase inhibitor purified from normal serum was carried out by 400-MHz 1H-NMR spectroscopy. C-1-esterase inhibitor protein of a molecular weight of 116,000 daltons contains 24 O-glycans: NeuAc (alpha 2-3) Gal (beta 1-3) GalNAc, 4 N-glycans: NeuAc (alpha 2-6) Gal (beta 1-4) (GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-6) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc and 2 N-glycans: NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc. 30% of the N-glycans are fucosylated.     

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 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.     

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.     

Structure elucidation of sulphated oligosaccharides from recombinant human tissue plasminogen activator expressed in mouse epithelial cells.

Sulphated N-linked carbohydrate chains isolated from recombinant human tissue plasminogen activator expressed in mouse epithelial (C127) cells were analysed as oligosaccharide alditols by methylation analysis, liquid secondary ion mass spectrometry, and one- and two-dimensional 1H-NMR spectroscopy. The results demonstrate that the major component has the following novel structure: NeuAc-alpha 2-6Gal beta 1-4GlcNAc beta 1-2[NeuAc alpha 2-3Gal beta 1- 4GlcNAc beta 1-4]-Man alpha 1-3[NeuAc alpha 2-3(SO4-6)Gal beta 1- 4-GlcNAc beta 1-2Man alpha 1-6]-Man beta 1-4GlcNAc beta 1- 4[Fuc alpha 1-6]GlcNAc-o1.     

Assignment of the1H- and13C-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series

The assignment of the 13C- and 1H-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series was obtained from homonuclear and heteronuclear correlation spectroscopy. These analyses were performed on the following compounds: 1. Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 2. NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 3. Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 4. NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 5. NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc; 6. Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 7. Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; 8. NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc.     

Structural studies of the sugar chains of human parotid alpha-amylase

Human parotid amylase can be separated into three families of isoenzymes (A', A, and B) by Sephadex G-75 column chromatography. Isoenzymes in family B were free from carbohydrate, while those in family A were all glycoproteins. The carbohydrate moieties of family A isoenzymes were released from their polypeptide portions by hydrazinolysis and labeled by reduction with NaB[3H]4. The yield of total radioactive oligosaccharides indicated that family A isoenzymes all contain single asparagine-linked sugar chains in one molecule. The radioactive oligosaccharides were fractionated into one acidic and two neutral oligosaccharide fractions by paper electrophoresis and paper chromatography. By sequential exoglycosidase digestion in combination with a methylation study, their structures were determined to be: Gal beta 1 leads to 4 (Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3)GlcNAc beta 1 leads to 2Man alpha 1 leads to 6 and 3[Gal beta 1 leads to 4 GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc Gal beta 1 leads to 4(Fuc alpha 1 leads to 3) GlcNAc 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 4(Fuc alpha 1 leads to 6)GlcNAc.     

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.     

Structures of the asparagine-linked sugar chains of human chorionic gonadotropin.

The asparagine-linked sugar chains of human chorionic gonadotropin were released from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. More than 90% of the released radioactive oligosaccharides contained N-acetylneuraminic acid residues. After removal of N-acetylneuraminic acid residues by sialidase treatment, two neutral oligosaccharide fractions were obtained by paper chromatography. Sequential exoglycosidase digestion revealed that one of them was a mixture of two neutral oligosaccharides. The complete structures of the three oligosaccharides were elucidated by methylation analysis. It was confirmed that all the N-acetylneuraminic acid residues of the asparagine-linked sugar chains of human chorionic gonadotropin occur as NeuAc alpha 2 leads to 3Gal groupings by comparing the methylation analysis data for the acidic oligosaccharide mixture before and after sialidase treatment. Based on these results, the structures of the asparagine-linked sugar chains of human chorionic gonadotropin were confirmed to be +/- NeuAc alpha 2 leads to 3Gal 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 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc beta 1 leads to 4(+/- Fuc alpha 1 leads to 6)GlcNAc and Man alpha 1 leads to 6(NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4 GlcNAc beta 1 leads to Man alpha 1 leads to 3)Man beta 1 leads to 4 GlcNAc beta 1 leads to 4GlcNAc.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

Structural study of the sugar chains of alpha-amylases produced ectopically in tumors

About thirty percent of two alpha-amylases produced from a serous papillary cystadenocarcinoma of the ovarium (case 1) and a bronchioloalveolar adenocarcinoma of the lung (case 2) was glycoproteins containing 1 mol of asparagine-linked sugar chain, respectively. The structures of the sugar moieties were found by sequential enzymatic degradation and methylation analysis to be as follows: [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(3)][GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(6)]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc and [(Gal beta 1 leads to 4)0 or 1GlcNAc 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 4(Fuc alpha 1 leads to 6)GlcNAc. Structures of asparagine-linked sugar chains were the same in the tumors of cases 1 and 2 and were incomplete in comparison with those of the parotid amylase.     

Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.     

Isolation and NMR spectroscopic characterization of sialic acid compounds and hemofiltrate of chronic uremia patients

Eight sialyloligosaccharides have been isolated from the hemofiltrate of a patient with end stage renal disease using reverse osmosis, gel filtration, ion-exchange and high-performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H- and 13C-NMR spectroscopy: 1 NeuAc alpha 2-3Gal beta 1-4Glc; 2 NeuAc alpha 2-6Gal beta 1-4Glc; 3 NeuAc alpha 2-3Gal beta 1-4GlcNAc; 4 NeuAc-alpha 2-6Gal beta 1-4GlcNAc; 5 NeuAc alpha 2-3Gal beta 1-4-GlcNAc alpha 1-P; 6 NeuAc alpha 2-6Gal beta 1-4GlcNAc alpha 1-P; 7 NeuAc alpha 2-3Gal beta 1-3GalNAc alpha 1-P; 8 NeuAc alpha 2-8NeuAc. While compounds 1-7 are also components of normal human urine, di-N-acetyl-D-neuraminic acid (8) could be isolated for the first time from biological material. The origin and possible clinical relevance of these compounds have to be proved in further investigations.     

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.     

A new ganglioside in human meconium detected by antiserum against the human milk sialyloligosaccharide, LS-tetrasaccharide b

Antibodies directed against human milk sialyloligosaccharides [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59] are used to identify human meconium gangliosides by radioimmuneoverlay-thin-layer chromatography or by direct binding on nitrocellulose filters of sialyl[3H]oligosaccharide alditols obtained from gangliosides after ozonolysis and alkali-fragmentation. Thin-layer chromatograms of meconium monosialylgangliosides immunostained with rabbit antisera specific for LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) or LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) reveal their corresponding gangliosides, 6'-LM1 and a previously undescribed ceramide derivative of LS-tetrasaccharide b, respectively. The sialyl[3H]oligosaccharides derived from the monosialylganglioside fraction of meconium are separated by paper chromatography and assayed for binding to specific anti-sialyloligosaccharide sera. Antisera specific for LS-tetrasaccharide c and 3'-sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) identify their corresponding 3H-labeled haptens released from the major meconium gangliosides 6'-LM1 and GM3, respectively. Binding of a ganglioside-derived sialyl[3H]oligosaccharide by anti-LS-tetrasaccharide b serum is consistent with the presence in meconium of a monosialylganglioside with the following proposed structure: (formula; see text)     

Structural basis of the heterogeneity of asparagine-linked oligosaccharides of a Waldenstrom's macroglobulinemia immunoglobulin M

The extent of glycans heterogeneity in a pathological human immunoglobulin M ZAJ has been studied on oligosaccharides released by hydrazinolysis from the purified glycoprotein. After reduction with NaB3H4, asparagine-linked carbohydrate chains were separated by affinity chromatography on concanavalin A-Sepharose into oligomannosidic and N-acetyllactosaminic types. Glycans of the oligomannosidic type were further fractionated by HPLC and those of the N-acetyllactosamine type by preparative high-voltage electrophoresis. The primary structure of the main oligosaccharides was investigated on the basis of micro-methylation analysis, mass spectrometry and sequential exo-glycosidase digestion. Glycans of the oligomannosidic type varied in size from Man5GlcNAc2 to Man9GlcNAc2. N-Acetyllactosaminic glycans were found of the biantennary, bisected-biantennary and triantennary types. They presented a higher degree of heterogeneity due to the presence of a variable number of NeuAc and fucose residues. The new structures we report here were in addition to the major biantennary one we previously described on the basis of methylation analysis and 500 MHz 1H-NMR spectroscopy (Cahour, A., Debeire, P., Hartmann, L., Montreuil, J., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) FEBS Lett. 170, 343-349): NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)Glc-NAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)]Glc-NAc(beta 1-4) [Fuc(alpha 1-6)]GlcNAc.     

A novel sialylfucopentaose in human milk. Presence of this oligosaccharide is not dependent on expression of the secretor or Lewis fucosyltransferases

We have identified a novel oligosaccharide in human milk that is a fucosyl derivative of sialyltetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc). This oligosaccharide was purified by affinity chromatography on a column of immobilized Ricinus communis I lectin. Structural analyses of radiolabeled oligosaccharides by exoglycosidase digestions, binding by specific anti-carbohydrate antibodies, and analysis of the 3H-labeled glucitol derivative obtained after permethylation and hydrolysis are consistent with the following proposed structure. (formula; see text) The analyses of human milk sialylpentasaccharides from donors typed as Le(a-,b+), Le(a+,b-), and Le(a-,b-) secretor confirmed the secretor gene-dependent expression of the sialylated lacto-N-fucopentaose I (Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and the Lewis gene-dependent expression of the sialylated lacto-N-fucopentaose II (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-4Glc). However, the presence of this novel oligosaccharide in human milk is not dependent on the expression of either the secretor gene or the Lewis gene-specified fucosyltransferases.     

Structural study on the carbohydrate moiety of human placental alkaline phosphatase

Alkaline phosphatase purified from human placenta contains a single asparagine-linked sugar chain in one molecule. The sugar chain was quantitatively liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction, and separated by paper electrophoresis into one neutral and two acidic fractions. By a combination of sequential exoglycosidase digestion and methylation analysis, the structures of oligosaccharides in the neutral fraction were confirmed to be as follows: Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The acidic oligosaccharide fractions were mixtures of mono- and disialyl derivatives of the neutral fraction. All the sialic acid residues of the sugar chains occur as the NeuAc alpha 2----3Gal group. In the case of monosialyl derivatives, the N-acetylneuraminic acid was exclusively linked to the Man alpha 1----3 arm.