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.     

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).     

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].     

Homonuclear three-dimensional NMR methods for the complete assignment of proton NMR spectra of oligosaccharides--application to Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc.

Two new homonuclear three-dimensional NMR techniques are described for the simplification of proton resonance assignment in oligosaccharides, namely HOHAHA-COSY and ROESY-COSY. The former technique is of value in the resonance assignment of gluco-configuration monosaccharide residues, whereas the latter is more suited to resonance assignment of galacto-configuration monosaccharide residues. The value of these techniques is illustrated by application to the proton resonance assignment of the pentasaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal beta 1-4Glc, a compound which exhibits a variety of assignment problems due to severe cross-peak overlap in conventional COSY or HOHAHA spectra.     

Toxin A from Clostridium difficile binds to rabbit erythrocyte glycolipids with terminal Gal alpha 1-3Gal beta 1-4GlcNAc sequences

The binding of Toxin A isolated from Clostridium difficile to rabbit erythrocyte glycolipids has been studied. Total lipid extracts from rabbit erythrocytes were subjected to thin-layer chromatography and toxin-binding glycolipids detected by using 125I-labeled Toxin A in a direct binding overlay technique. Two major and several minor toxin-binding glycolipids were detected in rabbit erythrocytes by this method. The results of structural analyses of the major toxin-binding glycolipids were consistent with a pentasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) and a branched decasaccharide-ceramide (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) previously identified as the two most abundant glycolipids in rabbit erythrocytes. 125I-Toxin A binding to these glycolipids could be inhibited by bovine thyroglobulin, monospecific antiserum to the toxin, or by treatment of the glycolipids with alpha-galactosidase. The absence of toxin interaction with isoglobotriaosylceramide (Gal alpha 1-3Gal beta 1-4Glc-Cer) isolated from canine intestine suggested that the GlcNAc residue present in the terminal Gal alpha 1-3Gal beta 1-4GLcNAc sequence common to all known toxin binding glycoconjugates is required for carbohydrate-specific recognition by Toxin A. These observations are consistent with the proposed carbohydrate binding specificity of Toxin A for the nonreducing terminal sequence, Gal alpha 1-3Gal beta 1-4GlcNAc.     

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.     

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.     

Three-dimensional structure of a glycosphingolipid having a novel carbohydrate linkage, Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta, determined by theoretical calculations

The novel glycosphingolipid, SEGLx (Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta Cer), which was identified by us (Kawakami Y, et al. (1993) J Biochem 114: 677-83), shows a characteristic spectrum on 1H-NMR analysis, in which the anomeric proton resonances of a reducing end galactose and a glucose are split. To elucidate the structural characteristics of SEGLx, we determined its three-dimensional (3D) structure by means of computer simulation, involving such techniques as molecular mechanics (MM2), the semiempirical molecular orbital method (AM1), molecular dynamics (Amber), and computer 3D modelling. With the hypothesis that all OH group(s) of a ceramide participate in intramolecular hydrogen bonds, two kinds of stable conformers, horizontal and right-angled ones, were formed, depending on the ceramide species. The present findings suggest that the chemical species of both the long chain base and fatty acid moieties, mainly the occurrence of OH group(s), affect the chemical shifts of the anomeric proton resonances not only of the reducing terminal galactose but also the penultimate glucose through the formation of intramolecular hydrogen bonds. Computer simulation through theoretical calculation and 3D modelling was shown to be the best means of confirming the results obtained by experimental analysis.     

Human urokinase contains GalNAc beta (1-4)[Fuc alpha (1-3)]GlcNAc beta (1-2) as a novel terminal element in N-linked carbohydrate chains.

Structural analysis of enzymically released N-linked carbohydrate chains of human urokinase (urinary-type plasminogen activator) by 1H NMR spectroscopy and FAB-MS demonstrated that the N-linked oligosaccharides on the only N-glycosylation site contain diantennary structures with the novel GalNAc beta (1-4) [Fuc alpha (1-3)]GlcNAc beta (1-2) element in the upper or the lower branch.     

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.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

Identification of rat {alpha}1 macroglobulin as an inhibitor of rat Gal{beta}1-4GlcNAc {alpha}2-6 sialyltransferase

Rat serum was found to contain an inhibitor of pure     

Distribution of Gal alpha 1----3Gal beta 1----4GlcNAc residues on secreted mammalian glycoproteins (thyroglobulin, fibrinogen, and immunoglobulin G) as measured by a sensitive solid-phase radioimmunoassay

The study of the expression of Gal alpha 1----3Gal beta 1----4GlcNAc residues on mammalian glycoconjugates is of particular interest since as many as 1% of circulating IgG antibodies in man (the natural anti-Gal antibody) interact specifically with this carbohydrate residue. In recent studies, we have found that Gal alpha 1----3Gal beta 1----4GlcNAc residues are abundant on red cells and nucleated cells of nonprimate mammals, prosimians, and New World monkeys, but their expression is diminished in Old World monkeys, apes, and humans. In the present work, we have analyzed the expression of these residues on secreted mammalian glycoproteins. For this purpose, we have developed a radioimmunoassay (RIA) which enables the quantification of Gal alpha 1----3Gal beta 1----4GlcNAc residues on the secreted glycoproteins. Purified biotinylated anti-Gal was used as the antibody in the RIA, and bovine thyroglobulin enriched for Gal alpha 1----3Gal beta 1----4GlcNAc residues served as a solid-phase antigen. In this study, it is reported for the first time that the evolutionary pattern of Gal alpha 1----3Gal beta 1----4GlcNAc residue distribution in in vivo secreted glycoproteins is similar to that observed in membranes of cell lines and of red cells. Thyroglobulin, fibrinogen, or IgG molecules from nonprimate mammals and from New World monkeys express varying amounts of Gal alpha 1----3Gal beta 1----4GlcNAc residues ranging between 0.01 and 11 residues per molecule, whereas no such residues are present on any of these glycoproteins of human or Old World monkey origin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of DNA complementary to human UDP-GalNAc: Fuc alpha 1----2Gal alpha 1----3GalNAc transferase (histo-blood group A transferase) mRNA

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.     

Fibulin-5 binds human smooth-muscle cells through alpha5beta1 and alpha4beta1 integrins, but does not support receptor activation

Fibulin-5, an extracellular matrix glycoprotein expressed in elastin-rich tissues, regulates vascular cell behaviour and elastic fibre deposition. Recombinant full-length human fibulin-5 supported primary human aortic SMC (smooth-muscle cell) attachment through alpha5beta1 and alpha4beta1 integrins. Cells on fibulin-5 spread poorly and displayed prominent membrane ruffles but no stress fibres or focal adhesions, unlike cells on fibronectin that also binds these integrins. Cell migration and proliferation were significantly lower on fibulin-5 than on fibronectin. Treatment of cells on fibulin-5 with a beta1 integrin-activating antibody induced stress fibres, increased attachment, migration and proliferation, and stimulated signalling of epidermal growth factor receptor and platelet-derived growth factor receptors alpha and beta. Fibulin-5 also modulated fibronectin-mediated cell spreading and morphology. We have thus identified the beta1 integrins on primary SMCs that fibulin-5 interacts with, and have shown that failure of fibulin-5 to activate these receptors limits cell spreading, migration and proliferation.     

Structures of neutral O-linked polylactosaminoglycans on human skim milk mucins. A novel type of linearly extended poly-N-acetyllactosamine backbones with Gal beta(1-4)GlcNAc beta(1-6) repeating units

O-Linked oligosaccharides were isolated from human skim milk mucins and from mucin-derived glycopeptides by reductive beta-elimination. The released alditols were fractionated by DEAE-Sephadex chromatography and purified by high performance liquid chromatography on primary amine bonded phase. The structures of the major neutral oligosaccharide alditols could be established by fast atom bombardment and electron impact mass spectrometry, combined with methylation analysis, 500-MHz 1H nuclear magnetic resonance spectroscopy, and endo-beta-galactosidase (from Bacteroides fragilis, EC 3.2.1.103) digestion (where n = 0-3): (formula; see text) Major O-glycosidically linked oligosaccharides on skim milk mucins are of the Gal beta(1-3)[GlcNAc beta(1-6)] GalNAc core type 2 and exhibit linearly extended backbone chains of the poly N-acetyllactosamine type comprizing up to at least four repeating units, which are linked by the hitherto unknown sequence GlcNAc-beta(1-6) Gal rather than GlcNAc beta(1-3)Gal. A considerable portion of neutral alditols is represented by branched isomers of the linear species, which are distinguished by their content of 3,6-disubstituted galactose and their partial resistance to endo-beta-galactosidase digestion.     

Stimulation of release of Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase from the FAZA hepatoma cell line by dexamethasone and phorbol ester

1. Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase was assayed in FAZA hepatoma cells and the cell culture medium following growth of cells in presence of dexamethasone and phorbol ester. 2. There was about a seven-fold increase in sialyltransferase activities in cells and medium in presence of dexamethasone with the maximum effect occurring at 10(-6)-10(-7) M dexamethasone. 3. The presence of 10(-6) M phorbol ester in the culture medium increased sialyltransferase activities in cells and medium by ca 40% over the values found with dexamethasone alone. 4. The use of the FAZA hepatoma cell line for studies on sialyltransferase is compared with the primary hepatocyte system reported on earlier (Woloski et al., 1986).     

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.