GlcNAc Beta Research Articles

Ethnic differences in the expression of blood group antigens in the salivary gland secretory cells from German and Japanese non-secretor individuals

Type 1 ABO blood group antigens (peripheral core structure: Gal beta 1-3GlcNAc beta 1-R) are expressed mainly in endodermally-derived tissues, but are not synthesized in mesodermally-derived tissues. In the former tissues, H type 1 antigen is generated largely by alpha-2-L-fucosyltransferase encoded by secretor (Se) gene and acting on the terminal galactose of the type 1 precursor chain. This theory has been generally accepted, and it seems that the expression of ABO blood group antigens is absent, or expressed at a low level, in these tissues from non-secretor individuals. In this immunohistochemical study on the secretory cells of salivary glands, we found ethnic difference between German and Japanese non-secretor individuals in the expression of blood group antigens: i.e. the expression of the type 1 blood group antigens is present in these cells from Japanese non-secretor individuals but absent from German. A possible explanation is that another alpha-2-L-fucosyltransferase, independent of the secretor gene, is present in Japanese non-secretor individuals.

Expression of Gal beta 1-4GlcNAc sequences by human gastrointestinal neoplasms and their precursors as detected by Erythrina cristagalli and Erythrina corallodendron lectins

Lectins from Erythrina cristagalli (EGA) and Erythrina corallodendron (ECorA) are well-known to detect type 2 chain oligosaccharides (Gal beta 1-4GlcNAc). These carbohydrate moieties are the biosynthetic precursors of various ABH and Lewis blood group antigens and are therefore also related to tumor-associated carbohydrate antigens. For this reason, we investigated the expression of ECA and ECorA binding sites in a series of gastric, colorectal and pancreatic carcinomas as well as corresponding normal tissues. Additionally, a series of hyperplastic and adenomatous colorectal polyps was analyzed. According to our results, both lectins exhibited a strong reactivity with the great majority of gastrointestinal carcinomas. Regarding gastric carcinomas, a stronger reactivity with intestinal-type compared to the diffuse-type species could be observed. Some poorly differentiated tumors were not or only very faintly stained. In the case of colorectal carcinomas, liver metastases which were investigated comparatively, exhibited the same binding pattern as the primary tumors. Colorectal adenomas were stained in about half of the cases without significant relation to the grade of cellular atypia. Positivity observed in normal epithelia (i.e, gastric superficial epithelia, fundus neck cells and deep pyloric mucous glands, pancreatic acini and ductal structures) is in keeping with histogenetic relations between these normal histological structures and corresponding neoplasms. In areas exhibiting intestinal metaplasia, various portions showed cytoplasmic staining of columnar cells and/or of goblet cell vacuoles. On the other hand, columnar and goblet cells in normal colorectal tissue were only weakly stained in a number of specimens. Therefore, it can be concluded that Gal beta 1-4GlcNAc is overexpressed in neoplastic colorectal tissues. Summarized, ECA and ECorA are suitable tools to analyze the expression of Gal beta 1-4GlcNAc, the common precursor substance of various tumor-associated type 2 chain antigens in neoplastic tissues.

Identification of the reactive subunits of Aspergillus umbrosus involved in the antigenic response in farmer's lung

Farmer's lung (FL) is the most common form of extrinsic allergic alveolitis and the fungus Aspergillus umbrosus has frequently been associated with FL among Finnish farmers. IgG and IgA class antibodies against Aspergillus umbrosus are found in the serum of FL patients and healthy exposed farmers. So far the immunologically reactive subunits of Aspergillus umbrosus are unknown. The purpose of the study was to identify and localize the reactive subunits of Aspergillus umbrosus that are involved in the antigenic response in FL. The distribution of the primary antibody on the outer wall layers of 4- and 11 day-old fungi Aspergillus umbrosus was studied by pre-embedding immunolabelling with fluorescein isothiocyanate (FITC)- and gold-conjugated antibodies. The surface carbohydrates of the outer wall layers were studied using FITC-conjugated lectins. FITC-conjugated antibodies from FL patient sera reacted with spores (conidia), phialides and hyphae, whereas the fruiting body, cleistothecia, remained unstainable. Immunofluorescence labelling was confirmed by immunolabelling electron microscopy, and gold-conjugated IgG antibodies were found on the outer cell walls of spinulose conidia, and in a less degree on the outer cell walls of hyphae and phialides. Only single gold particles were found scattered on the surfaces of cleistothecia. The surface carbohydrates of Aspergillus umbrosus conidia, vesicles, phialides and hyphae had a high affinity to GlcNAc beta 1- and Man alpha 1-/Glc alpha 1-specific lectins and only GlcNAc beta 1-specific lectins also reacted with the outer cell walls of cleistothecia. Blocking of the antigenic sites of Aspergillus umbrosus by preincubating antigen-coated enzyme linked immunosorbent assay (ELISA) plates with lectins caused some inhibition, indicating that sites with the highest affinity for lectins were not always antigenic sites. Gold-and FITC -conjugated antibodies were distributed mostly on the outer wall layers of conidia, hyphae and phialides of Aspergillus umbrosus and GlcNAc beta 1-linked lectins had the strongest affinity to the outer wall layers.

Biosynthesis in vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core.

The galactosyltransferase, GalT-4, which catalyses the biosynthesis in vitro of neolactotetraosylceramide, nLcOse4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (Glc NAc beta 1-3Gal beta 1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc beta 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn2+). The K(m) values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 microM, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-alpha 1-acid glycoprotein or N-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc beta 1-1-3Gal beta 1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for an N-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH2 beta 1-3Gal beta 1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while the K(m) and Vmax of the reacetylated substrate (GlcNAc beta 1-3Gal beta 1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.

Occurrence and Structural Analysis of Highly Sulfated Multiantennary N‐linked Glycan Chains Derived from a Fertilization‐Associated Carbohydrate‐Rich Glycoprotein in Unfertilized Eggs of Tribolodon hakonensis

This study represents the first detailed investigation of the nature of highly sulfated (keratan-sulfate-like) complex-type asparagine-linked glycans having a tetraantennary core structure and shows the effectiveness of fast-atom-bombardment mass spectrometric (FAB-MS) methods incorporating derivatization and mild methanolysis for analyzing such complex types of sulfated glycans. The structure of the N-glycan chains was unambiguously established by a combination of compositional analysis, methylation analysis, mild methanolysis for desulfation, hydrazinolysis/nitrous acid deamination, enzymatic (endo-beta-galactosidase and peptide:N-glycosidase F) digestions, and instrumental analyses (1H-NMR spectroscopy and FAB-MS) which revealed the novel repeating sulfated carbohydrate sequences, +/- Gal beta 1-->4Gal beta 1[-->(HSO3-->6)GlcNAc beta 1-->3(+/- Gal beta 1-->4)Gal beta 1]n--> (see Structure I; p + q + r + s approximately 14). This sequence is unique in: (a) the skeletal structure is similar to that of keratan sulfate but is completely devoid of 6-O-sulfated Gal residues and (b) the presence of branched Gal residues in the sequence -->4GlcNAc beta 1-->3(Gal beta 1-->4)Gal beta 1-->. [formula: see text]

A human antibody binds to alpha-galactose receptors and mimics the effects of Clostridium difficile toxin A in rat colon

BACKGROUND & AIMS: Nearly all human sera contain an immunoglobulin G antibody (antigalactose) that binds the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc expressed on cells from most mammals but not humans. Because the Clostridium difficile toxin A receptor in rodents contains this trisaccharide, the aim of this study was to examine whether antigalactose could mimic the enterotoxic effects of toxin A and bind to receptors containing this trisaccharide. METHODS: Fluid secretion, [3H]-mannitol permeability, and release of rat mast cell protease II and prostaglandin E2 were measured after luminal exposure of rat colon to either purified human anti-galactose, control immunoglobulin G, toxin A, or buffer. RESULTS: Toxin A (5 micrograms) and antigalactose (250 micrograms) but not control immunoglobulin (250 micrograms) stimulated colonic fluid secretion and caused increased mannitol permeability and rat mast cell protease II release. Antigalactose and toxin A and, to a lesser degree, control immunoglobulin G also stimulated release of prostaglandin E2, but only toxin A produced acute inflammation of rat colonic mucosa. Antigalactose and toxin A bound specifically to a single class of colonic brush border receptors with dissociation constants of 10(-6) mol/L and 5.4 x 10(-8) mol/L, respectively. CONCLUSIONS: Fluid secretion, increased permeability, and mast cell activation occur in rat colon when toxin A or human antigalactose immunoglobulin G bind to receptors bearing the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc. (Gastroenterology 1996 Jun;110(6):1704-12)

Characterization of Porcine Platelet Glycoproteins Recognized by Human Natural “Anti-Gal” Antibodies

Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain.

Biosynthesis of a blood group H1 antigen by alpha 1, 2-fucosyltransferase in PC12 cells.

We have examined the expression of GDP-fucose: glycosphingolipid fucosyltransferase activity in PC12 cells and PC12 sublines in relation to the neuronal differentiation induced by nerve growth factor (NGF) or dexamethasone. Transfer of fucose to paragloboside (nLc4Cer) yielded a product which was determined to be a blood group H1 antigen (Fuc alpha 1-2Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc-Cer) by gas chromatography/mass spectrometry analysis and enzymatic hydrolysis, suggesting that PC12 cells have an alpha 1,2-fucosyltransferase. Lactosylceramide was also fucosylated at a reduced rate. When the differentiation of PC12 cells and PC12 subline cells, PC12D and MR31, was induced by exposure to either NGF or dexamethasone, the fucosyltransferase activity for nLc4Cer was found to decrease in both cell lines, suggesting the association with cell differentiation. This is the first report of the presence of an alpha 1,2-fucosyltransferase in cultured neuronal cell lines which catalyses the in vitro biosynthesis from nLc4Cer of a type-2 chain glycosphingolipid having the blood group H1 determinant. The disaccharides, beta-lactose and N-acetyllactosamine, were also fucosylated by PC12 cell enzyme, although the specificity for the carbohydrate structure was different from that for glycosphingolipids.

Substrate specificity of bovine liver cytosolic neutral alpha-mannosidase activated by Co2+.

A cytosolic neutral alpha-mannosidase was purified from bovine liver. Its molecular weight was found to be 500,000 on gel filtration. The activity of the enzyme toward Man alpha 1-6-(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc-PA was increased 26-fold by preincubation with 1 mM Co2+. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc was hydrolyzed by the enzyme to Man alpha 1-3Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc, which was further hydrolyzed to Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc. The rate of hydrolysis was 15-fold greater than that of Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. This substrate specificity suggested that the enzyme could be involved in the degradation of oligomannose-type sugar chains with one GlcNAc residue released from glycoproteins by endo-beta-N-acetylglucosaminidase, and supported a pathway for glycoprotein catabolism via oligomannosyl glycans with one GlcNAc residue proposed on the basis of an earlier study on a cytosolic neutral alpha-mannosidase from Japanese quail oviduct [Oku, H. and Hase, S. (1991) J. Biochem. 110, 982-989].

Purification and characterization of hen oviduct alpha 1,2-mannosidase.

An alpha-mannosidase capable of hydrolyzing three Man alpha 1,2-residues from pyridylamine-(PA-) labeled Man9GlcNAc2 was purified from hen oviduct. The purity of the preparation was analyzed by PAGE; its molecular weight was 42,000 by SDS-PAGE or 50,000 by gel filtration. The pH optimum was 6.5. The enzyme was inactivated with EDTA; enzyme activity was restored by the addition of Ca2+. The enzyme activity was inhibited by 1-deoxymannojirimycin, but not by swainsonine. The substrate specificity of the purified enzyme was analyzed using PA-oligomannose-type sugar chains. When Man9GlcNAc2-PA was digested, Man alpha 1-6(Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4Glc-NAc-PA was obtained as an end product, and the enzyme was incapable of hydrolyzing p-nitrophenyl alpha-D-mannoside and Man alpha 1,3- or Man alpha 1,6-residues. Judging from these characteristics, the enzyme was classified as a Man9-mannosidase or Golgi mannosidase I and speculated to participate in the processing or catabolism of glycoproteins.

α2→3Sialyltransferase associated with the synthesis of CA 19-9 in colorectal tumors

A novel assay method specific for alpha 2-->3sialyltransferase that seems to be responsible for the synthesis of CA 19-9 antigen was developed and the levels of the enzyme in colorectal tumor tissues were measured and compared with the levels of alpha 1-->4fucosyltransferase and the CA 19-9 antigen. Lacto-N-biose I (Gal beta 1-->3GlcNAc beta, Lewis(c) [Le(c)]) and 6-O-methyl-Le(c) (Gal beta 1-->3[6OMe]GlcNAc beta) were synthesized and covalently attached to bovine serum albumin (BSA). These two substrates were incubated with extracts from colorectal tissues in the presence of cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) and their resulting products were detected by a sequential use of anti-BSA monoclonal antibody-coated beads and 125I-labelled anti-sialylated Le(c) antibody. Levels of alpha 2-->3sialyltransferase and alpha 1-->4fucosyltranferase activities and CA 19-9 antigen were measured in the extracts from colorectal tumors and their adjacent normal tissues. 6-O-Methyl-Le(c)-BSA showed a strong acceptor activity compared with Le(c)-BSA and was used as a specific acceptor for alpha 2-->3sialyltransferase. Similar elevation patterns alpha 2-->3sialyltransferase activities and CA 19-9 antigen levels were observed in tumor extracts but no clear correlation was present between the level of alpha 1-->4fucosyltransferase activities and CA 19-9 antigen levels were observed in tumor extracts but no clear correlation was present between the level of alpha 1-->4fucosyltransferase activities and CA 19-9 antigen levels in the same extracts. The accumulation of CA 19-9 antigen in colorectal tumors might be caused mainly by the activation of alpha 2-->3sialyltransferase but not by that of alpha 1-->fucosyltransferase.

Glycosylation of Recombinant Ancrod from Agkistrodon rhodostoma after Expression in Mouse Epithelial Cells

The thrombin-like serine protease ancrod from the Malayan pit viper Agkistrodon rhodostoma was expressed in mouse epithelial cells (C127). Oligosaccharide constituents were liberated from tryptic glycopeptides by treatment with peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase F. Neutral oligosaccharide alditols obtained after reduction and enzymic desialylation were separated by two-dimensional HPLC and characterized by methylation analysis, liquid secondary-ion mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and sequential degradation with exoglycosidases. In contrast to natural ancrod, the recombinant glycoprotein carries exclusively diantennary, triantennary and tetraantennary N-glycans with Gal beta 4 GlcNAc beta (type-2) antennae which were, in part, further substituted by host-cell-specific structural elements such as Gal alpha 3 residues or N-acetyllactosamine repeats. As a characteristic feature, a substantial proportion of the oligosaccharides bears a GalNAc beta 4Glc-NAc antenna. Studies at the level of individual N-glycosylation sites demonstrated that glycans with N, N'-diacetyllactosediamine units are not specifically attached but occur at all sites in varying amounts. Hence, the putative recognition signal (Pro70-Lys-Lys) for glycoprotein hormone N-acetylgalactosaminyltransferase, present in this glycoprotein in close proximity to Asn79, does not convey site-specific transfer of GalNAc residues in these cells.

Use of diethyl squarate for the coupling of oligosaccharide amines to carrier proteins and characterization of the resulting neoglycoproteins by MALDI-TOF mass spectrometry.

The 8-methoxycarbonyloctyl glycosides of GlcNAc beta, Gal beta 1-4Glc beta, Fuc alpha 1-2Fuc alpha 1-3GalNAc beta and Fuc alpha 1-2Gal beta 1-3[Fuc alpha 1-4]GlcNac beta were converted to primary amines by reaction with neat ethylenediamine and then coupled to bovine serum albumin (BSA) using diethyl squarate as the connector. The average degree of incorporation of the sugar onto the protein, as well as the molecular weight distribution, could be conveniently determined using matrix assisted laser desorption ionization/time of flight (MALDI-TOF) mass spectrometry thus avoiding cumbersome structure-dependent colour-tests or analysis of cleaved ligand. The present coupling method has the advantages of proceeding under very mild conditions, yielding controlled incorporation values and can reliably be used for the coupling of very small amounts (mg) of oligosaccharide.

Localization of the Putative Sialic Acid-binding Site on the Immunoglobulin Superfamily Cell-surface Molecule CD22

B-lymphocyte antigen CD22 is a member of the recently described sialoadhesin family of immunoglobulin-like cell-surface glycoproteins that bind glycoconjugates terminating in sialic acid. One prominent ligand for CD22 is the highly glycosylated leukocyte surface protein CD45. Using surface plasmon resonance spectroscopy, we characterized the interaction of recombinant mouse CD22 with native CD45 purified from rat thymus (CD45-thy). By in situ desialylation and resialylation of immobilized CD45-thy, we show that mouse CD22 binds to the sialoglycoconjugate NeuGc alpha 2-6Gal beta 1-4GlcNAc carried on CD45-thy N-glycans. Previous studies have shown that the sialic acid-binding site lies within the two membrane-distal domains of CD22 (domains 1 and 2), which are V-set and C2-set immunoglobulin superfamily domains, respectively. To further localize the binding site, we have made 42 single amino acid substitutions throughout both domains. All 12 mutations that abrogated binding to CD45-thy without disrupting antibody binding were of residues within the GFCC'C" beta-sheet of domain 1. These residues are predicted to form a contiguous binding site centered around an arginine residue in the F strand that is conserved in all members of the sialoadhesin family. Our results provide further evidence that immunoglobulin superfamily cell adhesion molecules use the GFCC'C" beta-sheet of membrane-distal V-set domains to bind structurally diverse ligands, suggesting that this surface is favored for cell-cell recognition.

The reducing end of alpha Gal oligosaccharides contributes to their efficiency in blocking natural antibodies of human and baboon sera.

Synthetic galactosyl oligosaccharides were tested for their ability to inhibit the cytotoxic reaction of human and baboon natural antibodies on PK15 cells in culture. Methyl-alpha-Gal gave weak inhibition, Gal alpha 1-3Gal substantially inhibited the reaction (400muM), and Gal alpha 1-3Gal beta 1-4GLcNAc was ten times more efficient (30 muM). The modification from alpha to beta anomeric configuration of the nonreducing end resulted in a complete loss of activity, while substitutions at the reducing end induced only a partial loss of activity. These observations suggest that natural anti-alphaGal antibodies recognize the epitope from its nonreducing end, but that substitutions at the reducing terminus can modify the antibody-binding capacity. Modified tri- and tetrasaccharides are better inhibitors than the disaccharide but not as good as Gal alpha 1-3Gal beta 1-4GlcNAc. The reducing terminus therefore contributes some energy to the reaction, indicating that certain oligosaccharides will be of more potential clinical use than others.

Identification of a GDP-Fuc:Gal beta 1-3GalNAc-R (Fuc to Gal) alpha 1-2 fucosyltransferase and a GDP-Fuc:Gal beta 1-4GlcNAc (Fuc to GlcNAc) alpha 1-3 fucosyltransferase in connective tissue of the snail Lymnaea stagnalis.

Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in alpha 1 -2 linkage to terminal Gal of type 3 (Gal beta 1-3GalNAc) acceptors, and in alpha 1-3 linkage to GlcNAc ot type 2 (Gal beta 1-4GlcNAc) acceptors. The alpha 1-2 fucosyltransferase was active with Gal beta 1-3GalNAc beta 1-OCH2CH=CH2 (Km = 12mM, V(max) = 1.3 mUml-1) and Gal beta 1-3GalNAc (km =20 mM, V(max) = 2.1 mUml-1), whereas the alpha 1-3 fucosyltransferase was active with Gal beta 1-4GlcNAc (Km = 23 mM, V(max) = 1.1 mUml-1). The products formed from from Gal beta 1-3GalNAc beta 1-OCH2CH=CH2 and Gal beta 1-4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz 1H-NMR spectroscopy and methylation analysis to be Fucalpha1-2Gal beta 1-3GalNAc beta 1-OCH2CH=CH2 and Gal beta 1-4(Fucalpha1-3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.

Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-beta-glucosides using UDP gal:GlcNAc beta 1,4 galactosyltransferase.

We previously showed that human melanoma, CHO and other cells can convert beta-xylosides into structural analogs of ganglioside GM3. We have investigated several potential acceptors including a series of n-alkyl-beta-D-glucosides (n = 6-9). All were labeled with 3H-galactose when incubated with human melanoma cells. Octyl-beta-D-glucoside (Glc beta Octyl) was the best acceptor, whereas neither octyl-alpha-D-glucoside nor N-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis of the products by a combination of chromatographic methods and specific enzyme digestions showed that the acceptors first received a single Gal beta 1,4 residue followed by an alpha 2,3 linked sialic acid. Synthesis of these products did not affect cell viability, adherence, protein biosynthesis, or incorporation of radiolabeled precursors into glycoprotein, glycolipid or proteoglycans. To determine which beta 1,4 galactosyl transferase synthesized Gal beta 1,4Glc beta Octyl, we analyzed similar incubations using CHO cells and a mutant CHO line (CHO 761) which lacks GAG-core specific beta 1,4 galactosyltransferase. The mutant cells showed the same level of incorporation as the control, eliminating this enzyme as a candidate. Thermal inactivation kinetics using melanoma cell microsomes and rat liver Golgi to galactosylate Glc beta Octyl showed the same half-life as UDP-Gal:GlcNAc beta 1,4 galactosyltransferase, whereas LacCer synthase was inactivated at a much faster rate. We show that Glc beta Octyl is a substrate for purified bovine milk UDP-Gal:GlcNAc beta 1,4 galactosyltransferase. Furthermore, the galactosylation of Glc beta Octyl by CHO cell microsomes can be competitively inhibited by GlcNAc or GlcNAc beta MU. These results indicate that UDP-Gal:GlcNAc beta 1,4 galactosyltransferase is the enzyme used for the synthesis of the alkyl lactosides when cells or rat liver Golgi are incubated with alkyl beta glucosides.

Molecular mimicry in the recognition of glycosphingolipids by Gal alpha 3 Gal beta 4 GlcNAc beta-binding Clostridium difficile toxin A, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13: characterization of a binding-active human glycosphingolipid, non-identical with the animal receptor.

Glycoconjugates with terminal Gal alpha 3Gal beta 4GlcNAc beta sequences have been shown to be recognized by three carbohydrate-binding proteins; toxin A of Clostridium difficile, human natural anti alpha-galactosyl IgG and the monoclonal antibody Gal-13. However, the biological significance of this binding specificity in humans is unclear, since unsubstituted Gal alpha 3Gal beta 4GlcNAc beta sequences are not found in human tissues, due to suppression of the gene coding for the enzyme Gal beta 3-transferase. To explore this inconsistency, the binding of toxin A, human natural anti alpha-galactosyl IgG, and the Gal-13 monoclonal antibody to various glycosphingolipids was examined using the thin-layer chromatogram binding assay. The binding to Gal alpha 3Gal beta 4GlcNAc beta-terminated glycosphingolipids of rabbit erythrocytes was confirmed. A minor binding-active compound was also detected in the non-acid glycosphingolipid fraction of human erythrocytes. This glycosphingolipid was isolated and characterized by EI mass spectrometry, gas chromatography-EI mass spectrometry after degradation, and proton NMR spectroscopy, as GalNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, corresponding to the x2 glycosphingolipid isolated before form this source. Two additional binding-active glycosphingolipids were found. One was GalNAc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4 Glc beta 1 Cer, produced from blood group A-active GalNAc alpha 3 (Fuc alpha 2) Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1 Cer by acid-induced defucosylation. The other was GlcNAc beta 3 Gal beta 4 GlcNAc beta 3 Gal beta 4 Glc beta 1 Cer, generated from NeuGc alpha 3Gal beta 4GlcNAc beta 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer by enzymatic hydrolysis. A number of other glycosphingolipid sequences, including the human Le(x), Le(y), and I blood group determinants, suggested to act as receptors for toxin A, were not recognized by the three ligands. Despite the different terminal substituents and anomerity of the binding-active glycosphingolipids, calculated minimum energy conformations demonstrated topographical similarities in the spatial orientation of the terminal trisaccharides, possibly accounting for the cross-reactivity.

Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection.

Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta-N-acetylhexosaminidase at different enzyme concentrations.

Structural Analysis ofN-Linked Oligosaccharide of Mitogenic Lectin-B from the Roots of Pokeweed (Phytolacca Americana)

The structure of an asparagine (N-) linked oligosaccharide of pokeweed lectin-B (PL-B) and the amino acid sequences around two glycosylation sites were identified. The pyridylamino (PA) oligosaccharide prepared from PL-B was eluted as a single peak on both reversed-phase (RP-) HPLC and size-fractionation (SF-) HPLC, and its structure was estimated to be Man alpha 1-->6(Man alpha 1-->3)(Xyl beta 1-->2) Man beta 1-->4GlcNAc beta 1-->4(Fuc alpha 1-->3)GlcNAc by a combination of component analysis, successive exoglycosidase digestions, IS-MS analysis, and 500 MHz 1H-NMR. Two tryptic glycopeptides were isolated from the reduced and S-pyridylethylated PL-B after gel filtration followed by RP-HPLC, indicating the presence of two glycosylation sites in PL-B. The amino acid sequences around the two glycosylation sites were determined to be Cys-Gly-Val-Asp-Phe-Gly-Asn(CHO)-Arg.