N-acetylglucosamine Groups Research Articles

Identification of an N-Acetylglucosaminyltransferase in Dictyostelium discoideum That Transfers an “Intersecting” N-Acetylglucosamine Residue to High Mannose Oligosaccharides

Glycoproteins synthesized by the cellular slime mold Dictyostelium discoideum have been shown to contain asparagine-linked high-mannose oligosaccharides which have an N-acetylglucosamine group in a novel intersecting position (attached beta 1-4 to the mannose linked alpha 1-6 to the core mannose). We have used crude membrane preparations from vegetative D. discoideum (strain M4) to characterize the enzyme activity responsible for catalyzing the transfer of GlcNAc to the intersecting position of high-mannose oligosaccharides. UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase activity in these preparations attaches GlcNAc to the mannose residue-linked alpha 1-6 to the beta-linked core mannose of the following Man9GlcNAc oligosaccharide as shown by the arrow. (formula; see text) It will also attach GlcNAc to the same intersecting position and/or to the bisecting position (beta-linked core mannose) of the following Man5GlcNAc oligosaccharide. (formula; see text) An analysis of the pH profiles, effects of heat denaturation, and substrate inhibitions on the addition of GlcNAc to either the intersecting or bisecting position of this Man5GlcNAc oligosaccharide indicates that a single enzyme activity is responsible for transferring GlcNAc to both positions. Various oligosaccharides were assayed to determine the substrate specificity of the transferase activity. These data indicate that both the mannose-attached alpha 1-3 and the mannose-attached alpha 1-6 to the mannose receiving the GlcNAc play a critical role in substrate suitability; absence of the alpha 1-6 mannose results in at least a 90% decrease in activity, while absence of the alpha 1-3 mannose results in a completely inactive substrate. This suggests that the minimal substrate is the disaccharide Man alpha 1-3Man.

Hyaluronan affects extravascular water in lungs of unanesthetized rabbits

We have determined whether changes in lung hyaluronan content affect extravascular water in lungs of unanesthetized rabbits. Three groups of experiments were performed. In group 1 (n = 12), no infusions were given; in group 2, nine pairs of rabbits received either intravenous hyaluronidase (750 U.kg-1.min-1) or an equivalent volume of saline; in group 3, nine pairs of rabbits received either hyaluronidase or saline, followed by intravenous saline infusion amounting to 24% of body weight. At the end of each experiment, one lung was analyzed for extravascular lung water by the wet-dry method. Except for group 3, in all animals the other lung was analyzed for hyaluronan content by a method that involved hydrolyzing lung hyaluronan with fungal hyaluronidase to release reducing N-acetyl glucosamine groups, which were quantified. In group 1, lung hyaluronan, which varied from 50 to 159 micrograms/g dry wt (mean 106 +/- 35 micrograms/g dry wt), significantly correlated with variation in extravascular lung water (mean 4.2 +/- 0.3 g/g dry wt). In group 2 rabbits given hyaluronidase, lung hyaluronan was 40% lower and extravascular lung water was 14.6% lower than in paired controls (P less than 0.01). In group 3, volume expansion did not affect lung water, except after hyaluronidase when lung water was 47% higher than paired controls. We conclude that in the lung the content of hyaluronan is one of the determinants of extravascular water content.

Structural characterization of the somatostatin receptor in rat anterior pituitary membranes.

To structurally characterize the somatostatin receptor in the anterior pituitary, the chemical cross-linking reagent N-5-azido-nitrobenzoyloxysuccinimide was used to attach covalently [125I-Tyr11]somatostatin-14 to its receptor in pituitary membranes. Rat anterior pituitary membranes were incubated with [125I-Tyr11]somatostatin-14, washed, and then treated with 100 microM cross-linker, which was activated by exposure to UV light. Gel electrophoresis followed by autoradiography revealed a broad band centered at 88,000 mol wt. The appearance of this band was unaffected by dithiothreitol. Competitive inhibition of binding by unlabeled somatostatin resulted in a parallel inhibition of labeling of the 88,000 mol wt protein. The addition of guanine nucleotides in concentrations that inhibit binding similarly inhibited cross-linking. The cross-linked membranes were solubilized in Zwittergent 3-12, a nondenaturing detergent, and the glycosylation pattern of the labeled protein was investigated by incubation with various lectins coupled to agarose. The cross-linked protein was selectively adsorbed by wheat germ agglutinin, and this interaction was blocked by the addition of N,N',N"-triacetylchitotriose, indicating that the rat anterior pituitary somatostatin receptor is a glycoprotein containing polymeric beta-1-4 linked N-acetylglucosamine groups. The results of this study show that the rat anterior pituitary somatostatin receptor is a glycoprotein of 88,000 mol wt containing no disulfide-linked subunits.

Receptors for fucose-binding proteins of Lotus tetragonolobus isolated from mouse embryonal carcinoma cells. Structural characteristics of the poly(N-acetyllactosamine)-type glycan.

Receptors for fucose-binding proteins of Lotus tetragonolobus were isolated from N4-1 and F9 embryonal carcinoma cells. They were glycoproteins, whose major components had apparent relative molecular masses of more than 100,000. Carbohydrates released from the receptors of N4-1 cells by hydrazinolysis were separated into three fractions by gel filtration. Binding activity to the lectin was detected in the high-molecular-mass fraction. The composition of the large glycan was characteristic of the poly (N-acetyllactosamine)-type, and glucosamine was identified as the sugar involved in the protein-carbohydrate linkage. The glycan has one fucosyl residue per four N-acetyllactosamine units. Most of the fucose was linked to the C-3 hydroxyl group of N-acetylglucosamine. No Fuc alpha 1----2Gal or Fuc alpha 1----4GlcNAc linkage was detected. The glycan had a relative molecular mass of 9000 or more and the poly(N-acetyllactosamine) units were branched. N-Acetylgalactosamine residues were detected in non-reducing ends of at least a part of the glycan. Therefore, the glycan has a more complex structure than the related one from human granulocytes, although both of them have Fuc alpha 1----3GlcNAc termini.

Novel fucolipids of human adenocarcinoma: monoclonal antibody specific for trifucosyl Ley (III3FucV3FucVI2FucnLc6) and a possible three-dimensional epitope structure.

Immunization of mice with Ley-active trifucosylnonaosylceramide (III3FucV3FucVI2FucnLc6) isolated from human colonic adenocarcinoma (Nudelman, E., Levery, S. B., Kaizu, T., and Hakomori, S. (1986) J. Biol. Chem. 261, 11247-11253) followed by selection of hybridoma by positive reaction with this antigen and negative reaction with two other Ley antigens (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) resulted in successful isolation of the hybridoma producing IgM antibody, termed KH1, specific to Ley-active trifucosylnonaosylceramide, which does not cross-react with Ley-active hexaosyl- or octaosylceramides (III3FucIV2FucnLc4 and V3FucVI2FucnLc6) without internal fucosyl substitution. The three-dimensional structure of the trifucosylnonaosylceramide was simulated based on previously published glycosidic torsion angles for fucosyl type 2 chain (Lex and Ley) and for GlcNAc beta 1----3Gal beta as predicted by hard sphere exo-anomeric calculations (Thøgersen, H., Lemieux, R. U., Bock, K., and Meyer, B. (1982) Can. J. Chem. 60, 44-57). The picture thus constructed showed a broad nonpolar area consisting of the hydrophobic surface of the pyranosyl ring and acetamido group of N-acetylglucosamine and three CH3 groups of L-fucose; this hydrophobic area is adjacent to a hydrophilic area. In analogy to the detailed structure of Leb or Ley involved in their interactions with antibodies and lectins (Spohr, U., Hindsgaul, O., and Lemieux, R. U. (1985) Can. J. Chem. 63, 2644-2652), such a wide hydrophobic area adjacent to a hydrophilic region could be recognized by the antibody KH1, as shown in the model illustrated in the text. Since the axis of ceramide, which is inserted in the lipid bilayer, is perpendicular to the plane of type 2 chain, the epitope recognized by the antibody KH1 is located at the external nonpolar surface of the carbohydrate chain that is overlaid on the lipid bilayer.

Fertilization-induced modification of chorion N-acetylglucosamine groups blocks polyspermy in ascidian eggs

Sperm bind to the chorion (vitelline envelope) of Ascidia nigra, which has many exposed N-acetylglucosamine (Glc Nac) groups. The Glc Nac-binding lectin wheat germ agglutinin (WGA), and p-nitrophenyl N-acetylglucosaminide block sperm binding as does N-acetylglucosaminidase digestion. Thus chorionic Glc Nac is an important component of the sperm receptor. Sperm bind to the chorion in increasing numbers during the first minute after fertilization, then bound sperm fall off to reach a low level by 2 min. Sperm unbinding is blocked by Glc Nac but not other sugars. Chorion binding of fluorescent WGA is also depressed after fertilization. Supernumerary sperm which have unbound from chorions gain a WGA binding site at the tip of the head, demonstrating the transfer of a Glc Nac group from chorion to sperm upon release. Glycerol-extracted eggs bind sperm during the first minute but fail to show the decline seen with living eggs. Supernatant obtained after fertilization of a dense egg suspension contains a trypsin-sensitive factor which modifies glycerinated eggs so that sperm binding is greatly inhibited. The block to polyspermy forms with the same kinetics as sperm unbinding, and is also sensitive to Glc Nac. This suggests that release of a chorion modifying factor removes Glc Nac residues from the chorion, releasing extra sperm and preventing polyspermy.

Characterization of the parasitic interface between Erysiphe pisi and Pisum sativum using fluorescent probes

The constituents of the interface between a strain of Erysiphe pisiand two susceptible and five resistant lines of Pisum sativum were investigated by fluorescence microscopy after treatment with specific reagents. Fragments of epidermes were detached so that the haustoria and extrahaustorial membranes were exposed directly to the reagents. The specificities of the 23 reagents employed included α- and β-linkages in polysaccharides; constituent sugars; aldehyde, amino, sulphydryl and disulphide groups; proteins, lysine, arginine, lipid, calcium and anions; and anion transport sites. The main conclusions are that in the susceptible cultivars the extrahaustorial membrane includes polysaccharides (β1–4 linked) with small amounts of α-glucose, α-mannose and galactose, β-linked N-acetylglucosamine (haustorial face only), protein, arginine, amino and sulphydryl groups and calcium. The extrahaustorial matrix includes varying quantities of β-linked polysaccharides. Haustorial walls contain β-linked polysaccharides including α-glucose, α-mannose, galactose and N-acetyl glucosamine (β-linked). The haustorial cytoplasm contains protein and releases fluorescein from fluorescein diacetate and 3- o-methyl fluorescein phosphate. The main differences between the susceptible lines and the resistant lines is that the resistant line, show enhanced reactivity of terminal glucose, mannose and N-acetylglucosamine groups in the extrahaustorial membranes of some lines. The last is probably due to greater accessibility of lectin effected by rupture of extrahaustorial membranes. Constituents of extrahaustorial membranes fluorescing with 4-acetamido-4′- iso-thiocyanato-stilbene-2,2′-disulphonic acid developed ore slowly and included less cross-linked protein. Calcium was rare in the extrahaustorial membranes and host cytoplasm in resistant lines. The results are discussed in relation to the molecular and functional properties of the interface.

Mechanism of glycosylation in the Golgi apparatus.

A major role of the Golgi apparatus in liver is the terminal glycosylation of secreted serum proteins and of plasma membrane glycoproteins. Galactosyltransferase is a membrane-bound Golgi enzyme that transfers galactose directly from uridine diphosphogalactose (UDP-Gal) to terminal N-acetylglucosamine groups of N-asparagine-linked glycoproteins during secretion. Sialytransferase then transfers sialic acid from cytidine monophosphosialic acid (CMP-NAN) to the newly added terminal galactose of the glycoprotein. In the cell, the transfer reaction must occur on the lumen side of the Golgi membrane. UDP-Gal is synthesized mainly in the cytoplasm and CMP-NAN is synthesized in the nucleus in liver. An important question for understanding the mechanism is, how do these nucleotide sugars gain access to the transferases? A second question involves uridine diphosphate (UDP), a highly inhibitory product of galactosyltransferase. How is UDP removed from the lumen of the Golgi fast enough to prevent product inhibition of the galactosyltransferase? We have shown that isolated Golgi, although vesiculated, retains its original orientation. The vesicles are oriented with greater than 90% of both galactosyltransferase and sialyl-transferase on the luminal side of the vesicles. Using intact vesicles, we can show that UDP-Gal is taken up via a saturable carrier system present in the Golgi membrane. During galactosylation in vitro, UDP formed in the lumen of Golgi vesicles is rapidly converted to UMP by a nucleoside diphosphatase in the lumen. Uridine monophosphate, which is much less inhibitory to the galactosyltransferase than UDP, is then transported out of the lumen by a second carrier and is broken down further to uridine by 5'-nucleotidase on the cytoplasmic side of the Golgi vesicles. The transport of nucleotides appears unique to the Golgi membranes, since neither rough endoplasmic reticulum nor plasma membrane vesicles from rat liver accumulate these nucleotides.