Mucin biosynthesis. Properties of a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase.

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-beta 1----3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1----3(Glc-NAc beta 1----6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.     

Biosynthesis of marsupial milk oligosaccharides. II: Characterization of a beta 6-N-acetylglucosaminyltransferase in lactating mammary glands of the tammar wallaby, Macropus eugenii.

Tammar wallaby (Macropus eugenii) mammary glands contain a UDP-GlcNAc:Gal beta 1----3Gal beta 1----4Glc beta 1----6-N-acetylglucosaminyltransferase (GlcNAcT) whose activity has been characterized with respect to the effect of pH, apparent Km for acceptor, effects of bivalent metal ions, acceptor specificity and identity of products. The enzyme did not show an absolute requirement for any bivalent metal ion but its activity was increased markedly by Mg2+, Ca2+ and Ba2+ and, to a lesser extent, by Mn2+. When Gal beta 1----3Gal beta 1----4Glc was used as acceptor, the product was Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----4Glc. With Gal beta 1----3Gal beta 1----3Gal beta 1----4Glc as acceptor, the product was shown, by 1H-NMR spectroscopy and exo-beta-galactosidase digestion, to be a novel pentasaccharide with the structure Gal beta 1----3[GlcNAc beta 1----6]Gal beta 1----3Gal beta 1----4Glc, suggesting that the enzyme recognises the non-reducing end of the acceptor substrate, rather than the reducing end.     

Biosynthesis of polylactosaminoglycans. Novikoff ascites tumor cells contain two UDP-GlcNAc:beta-galactoside beta 1----6-N-acetylglucosaminyltransferase activities

Novikoff ascites tumor cells contain a UDP-GlcNAc:beta-galactoside beta 1----6-N-acetylglucosaminyltransferase (beta 6-GlcNAc-transferase B) that acts on galactosides and N-acetylgalactosaminides in which the accepting sugar is beta 1----3 substituted by a Gal or GlcNAc residue. Characterization of enzyme products by 1H-NMR and methylation analysis indicates that an R beta 1----3(GlcNAc beta 1----6)Gal- branching point is formed such as occurs in blood-group-I-active substances. The enzyme does not show an absolute divalent cation requirement and 20 mM EDTA is not inhibitory. The activity is strongly inhibited by Triton X-100 at concentrations of greater than or equal to 0.2%. Competition studies suggest that a single enzyme acts on Gal beta 1----3Gal beta 1----4Glc, GlcNAc beta 1----3Gal beta 1----4GlcNAc and GlcNAc beta 1----3GalNAc alpha-O-benzyl (Km values 0.71, 0.83 and 0.53 mM, respectively). Gal beta----3Gal beta 1----4Glc as an acceptor substrate for beta 6-GlcNAc-transferase B does not inhibit the incorporation of GlcNAc in beta 1----6 linkage to the terminal Gal residues of asialo-alpha 1-acid glycoprotein catalyzed by a beta-galactoside beta 1----6-N-acetylglucosaminyltransferase (beta 6-GlcNAc-transferase A) previously described in Novikoff ascites tumor cells [D. H. Van den Eijnden, H. Winterwerp, P. Smeeman & W.E.C.M. Schiphorst (1983) J. Biol. Chem. 258, 3435-3437]. Neither is Triton X-100 at a concentration of 0.8% inhibitory for the activity of beta 6-GlcNAc-transferase A. This activity is absent from hog gastric mucosa microsomes, which has been described to contain high levels of beta 6-GlcNAc-transferase B. [F. Piller, J. P. Cartron, A. Maranduba, A. Veyrières, Y. Leroy & B. Fournet (1984) J. Biol. Chem. 259, 13,385-13,390]. Our results show that Novikoff tumor cells contain two beta-galactoside beta 6-GlcNAc-transferases, which differ in acceptor specificity and tolerance towards Triton X-100. A role for these enzymes in the synthesis of branched polylactosaminoglycans and of O-linked oligosaccharide core structures having blood-group I activity is proposed.     

Studies on glycosyltransferases in fusion-defective conA-resistant L6 rat myoblast cell lines

1. Sialyl- and galactosyl-transferase activities were determined in wild type and conA-resistant L6 rat myoblasts with substrates derived from fetuin, alpha 1-acid glycoprotein and bovine submaxillary mucin; fetuin was the best acceptor for both enzyme activities, whereas the mucin did not act as an acceptor. 2. The optimum pH for sialyltransferase was 6.6 in both cell lines. 3. The optimum pH for galactosyltransferase in the wild type cell line was 6.2 which was slightly higher than the value of 5.8 found for the conA-resistant cells. 4. Values for Km for both enzyme activities increased five to ten-fold in the variant cell line with both acceptors. 5. The main sialyltransferase activity was the Gal beta 1----4GlcNAc alpha 2----3sialyltransferase for N-linked chains. The galactosyltransferase was most likely the enzyme that is responsible for the synthesis of the Gal beta 1----4GlcNAc structure.     

Mucin biosynthesis. Enzymic properties of human-tracheal epithelial GDP-L-fucose:beta-D-galactoside alpha-(1----2)-L-fucosyltransferase

The human-tracheal, epithelial alpha-(1----2)-L-fucosyltransferase that transfers L-fucose from GDP-L-fucose to an acceptor containing a beta-D-galactopyranosyl group at the nonreducing terminal was characterized. Optimal enzyme activity was obtained at pH 6.5. 20-30mM MnCl2 (or CaCl2), and 0.05% Triton X-100 or 0.5% Tween 20. Mg2+ and Ba2+ ions moderately enhanced the enzyme activity, whereas Fe2+, Co2+, Zn2+, and Cd2+ ions were inhibitory. The enzyme activity was inhibited by N-ethylmaleimide and nucleotides of guanine, inosine, xanthine, and uridine. However, ATP and dithiothreitol did not affect the enzyme activity. The apparent Michaelis constant for GDP-L-fucose, freezing point-depressing glycoproteins (expressed as Gal----GalNAc----Thr), and phenyl beta-D-galactopyranoside was 0.29, 5.70, and 25.4mM, respectively. Under alkali-borohydride conditions (0.05M NaOH-M NaBH4, 45 degrees, 20 h), an L-[14C]fucosyltrisaccharide was released from the product obtained by use of freezing point-depressing glycoprotein as the acceptor. The alpha-L anomeric configuration of the fucoside was determined by the release of L-[14C]fucose from the purified trisaccharide by Turbo cornutus alpha-L-fucosidase. The (1----2) linkage of the L-fucosyl group to the D-galactosyl residue was established by methylation technique (m.s.-g.l.c.). The present enzyme has properties similar to those of the human milk alpha-(1----2)-L-fucosyltransferase which is encoded by a secretor gene.     

Mucin synthesis. UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase and UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase from pig and rat colon mucosa

Pig and rat colon mucosal membrane preparations catalyze the in vitro transfer of N-acetyl-D-glucosamine (GlcNAc) from UDP-GlcNAc to GalNAc-ovine submaxillary mucin to form GlcNAc beta 1-3GalNAc-mucin. Rat colon also catalyzes the in vitro transfer of GlcNAc from UDP-GlcNAc to GlcNAc beta 1-3GalNAc-mucin to form GlcNAc beta 1-3(GlcNAc beta 1-6) GalNAc-mucin. This is the first demonstration of in vitro synthesis of the GlcNAc beta 1-3GalNAc disaccharide and of the GlcNAc beta 1-3-(GlcNAc beta 1-6)GalNAc trisaccharide, two of the four major core types found in mammalian glycoproteins of the mucin type, i.e., those containing oligosaccharides with GalNAc-alpha-serine (threonine) linkages. The activity catalyzing synthesis of the disaccharide has been named UDP-GlcNAc:GalNAc-R beta 3-N-acetylglucosaminyltransferase (mucin core 3 beta 3-GlcNAc-transferase), while the activity responsible for synthesizing the trisaccharide has been named UDP-GlcNAc:GlcNAc beta 1-3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (mucin core 4 beta 6-GlcNAc-transferase). The beta 3-GlcNAc-transferase from pig colon is activated by Triton X-100, has an absolute requirement for Mn2+, and transfers GlcNAc to GalNAc-alpha-phenyl, GalNAc-alpha-benzyl, and GalNAc-ovine submaxillary mucin with apparent Km values of 5, 2, and 3 mM and Vmax values of 59, 62, and 37 nmol h-1 (mg of protein)-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Galactosyl transferases of baby hamster kidney (BHK) cells. Characterization of two oligosaccharide products synthesised using bovine asialo submaxillary-gland mucin as acceptor

Extracts of BHK (baby hamster kidney) cells catalyse incorporation of galactose from UDP-galactose into asialo bovine submaxillary gland mucin. The galactosylated oligosaccharide products were released by alkaline-borohydride treatment and purified by Bio-Gel P2 chromatography and high-performance liquid chromatography. The structures of the oligosaccharide sequences synthesised have been identified unequivocally by high resolution 500 MHz 1H-NMR as galactosyl-(beta 1----3) N-acetylgalactosamine and galactosyl (beta 1----4) N-acetylglucosaminyl (beta 1----3)-N-acetylgalactosamine. Characterization of the latter sequence shows the presence in bovine mucin of the type III core sequence N-acetylglucosamine-(beta 1----3) N-acetylgalactosamine. Fractionation of BHK cell extracts on alpha-lactalbumin-Agarose has shown that the (beta 1----4)-galactosyl transferase responsible for synthesis of the trisaccharide binds to alpha-lactalbumin, a modulator of the (beta 1----4)-galactosyl transferase involved in N-glycan assembly. The evidence that the same transferase activity may be responsible for galactose transfer to both O-glycans and N-glycans is discussed.     

Biosynthesis of the blood group P antigen-like GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure: a novel N-acetylgalactosaminyltransferase in human blood plasma.

Human blood group O plasma was found to contain an N-acetylgalactosaminyltransferase which catalyzes the transfer of N-acetylgalactosamine from UDP-GalNAc to Gal beta 1-->4Glc, Gal beta 1-->4GlcNAc, asialo-alpha 1-acid glycoprotein, and Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc-ceramide, but not to Gal beta 1-->3GlcNAc. The enzyme required Mn2+ for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by beta-N-acetylhexosaminidase and released N-acetylgalactosamine. Apparent Km values for UDP-GalNAc, Mn2+, lactose, N-acetyllactosamine, and terminal N-acetyllactosaminyl residues of asialo-alpha 1-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5 mM, respectively. Studies on acceptor substrate competition indicated that all the acceptor substrates mentioned above compete for one enzyme, whereas the enzyme can be distinguished from an NeuAc alpha 2-->3Gal beta-1,4-N-acetylgalactosaminyltransferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of N-acetylgalactosamine to lactose revealed that N-acetylgalactosamine had been transferred to the carbon-3 position of the beta-galactosyl residue. Although the GalNAc beta 1-->3Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Gal alpha 1-->4Gal beta-1,3-N-acetylgalactosaminyltransferase, which is involved in the synthesis of the major P antigen-active glycolipid, GalNAc beta 1-->3Gal alpha 1-->4Gal beta 1-->4Glc-ceramide. Hence, the GalNAc beta 1-->3Gal beta 1-->4GlcNAc/Glc structure is synthesized by the novel Gal beta 1-->4GlcNAc/Glc beta-1,3-N-acetylgalactosaminyltransferase.     

Mucin biosynthesis: purification and characterization of a mucin beta 6N-acetylglucosaminyltransferase.

We have purified, to apparent homogeneity, a mucin beta 6N-acetylglucosaminyltransferase (beta 6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Gal beta 1-3GalNAc alpha benzyl (Bzl) to stabilize the beta 6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Superflow affinity column equilibrated with 1 mM Gal beta 1-3GalNAc alpha Bzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 mumol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the beta 6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The Km values for UDP-GlcNAc and Gal beta 1-3GalNAc alpha Bzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified beta 6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAc beta 1-3Gal beta R as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a beta 1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all beta 6GlcNAc structures found in mucin-type oligosaccharides.     

Biosynthesis of blood group i-active polylactosaminoglycans. Partial purification and properties of an UDP-GlcNAc:N-acetyllactosaminide beta 1----3-N-acetylglucosaminyltransferase from Novikoff tumor cell ascites fluid

An N-acetylglucosaminyltransferase has been partially purified from Novikoff tumor cell ascites fluid by affinity chromatography on concanavalin A-Sepharose. The enzyme was obtained in a highly concentrated form after lyophilization. The enzyme appeared to be highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The enzyme therefore could be described as an UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with oligosaccharides that form part of N-glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The enzyme also showed activity toward oligosaccharides related to blood group I- and i-active polylactosaminoglycans. In addition the enzyme together with calf thymus UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single enzyme species. It is concluded that the N-acetylglucosaminyltransferase functions in both the initiation and the elongation of polylactosaminoglycan chains of N-glycoproteins and possibly other glycoconjugates.     

Characterization and specific assay for a galactoside beta-3-galactosyltransferase of human kidney

Two galactosyltransferases identified as UDP-galactose:lactose (lactosylceramide) alpha-4- and beta-3-galactosyltransferases [Bailly P. et al. (1986) Biochem. Biophys. Res. Commun. 141, 84-91] have been characterized in human kidney microsomes. Using methyl beta-D-galactoside as acceptor substrate, we have determined the experimental conditions (pH 5.0, 4 mM Cd2+) in which only the beta-3-galactosyltransferase activity is detectable. The reaction product has been characterized by chemical methods and glycosidase studies. Under these experimental conditions, some of the enzyme properties have been further investigated. Apparent Km values are for UDP-galactose, 0.170 mM; for lactose, 242 mM; and for lactosylceramide, 2.5 mM. Acceptor specificity studies suggest that the beta-3-galactosyltransferase is specific for terminal Gal beta 1-4Glc(NAc) residues and responsible for elongation of oligosaccharide chains in glycolipids. Competition studies with lactose and N-acetylgalactosamine as acceptor substrates indicate that the transferase described here can be distinguished from the UDP-galactose:2-acetamide-2-deoxy-D-galactose beta-3-galactosyltransferase and therefore represents a novel enzyme capable of synthesizing unusual carbohydrate structures similar to those which accumulate in certain neurological diseases.     

Purification and enzymatic characterization of CMP-sialic acid: beta-galactosyl1----3-N-acetylgalactosaminide alpha 2----3-sialyltransferase from human placenta

A CMP-NeuAc:Gal beta 1----3GalNAc-R alpha 2----3-sialyltransferase has been purified over 20,000-fold from a Triton X-100 extract of human placenta by affinity chromatography on concanavalin A-Sepharose and CDP-hexanolamine-Sepharose in a yield of 10%. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions revealed that the enzyme consists of a major polypeptide species with a molecular weight of 41,000 and some minor forms with molecular weights of 40,000, 43,000, and 65,000, respectively, which can be resolved partially by gel filtration on Sephadex G-100. Isoelectric focusing revealed that the enzyme occurs in a major and a minor charged form with pI values of 5.0-5.5 and 6.0, respectively. Acceptor specificity studies indicated that the enzyme catalyzes the incorporation of sialic acid from CMP-NeuAc into glycoproteins, glycolipids, and oligosaccharides which possess a terminal Gal beta----3GalNAc unit. Analysis of the structure of the product chain by high-pressure liquid chromatography and thin layer chromatography as well as methylation analysis revealed that a NeuAc alpha 2----3Gal beta 1----3GalNAc sequence is elaborated. The best glycoprotein acceptors are antifreeze glycoprotein and porcine submaxillary asialo/afucomucin. The disaccharide Gal beta 1----3GalNAc-Thr shows values for Km and V which are close to those of the latter glycoprotein. Lactose as well as oligosaccharides in which galactose is linked beta 1----3 or beta 1----4 to N-acetylglucosamine are less efficient acceptors. Of the glycolipids tested only gangliosides GM1 and GD1b served as an acceptor. The enzyme does not show an absolute aglycon specificity, and attaches sialic acid regardless the anomeric configuration of the N-acetylgalactosaminyl residue in the accepting Gal beta 1----3GalNAc unit. By use of specific acceptor substrates it could be demonstrated that the purified enzyme is free from other known sialyltransferase activities. Studies with rabbit antibodies raised against a partially purified sialyltransferase preparation indicated that the enzyme is immunologically unrelated to a Gal beta 1----4GlcNAc-R alpha 2----3-sialyltransferase, which previously had been identified in human placenta (Van den Eijnden, D.H., and Schiphorst, W. E. C. M. (1981) J. Biol. Chem. 256, 3159-3162). Initial-rate kinetic studies suggest that the sialyltransferase operates through a mechanism involving a ternary complex of enzyme, sugar donor, and acceptor. This is the first report on the extensive purification and characterization of a sialyltransferase from a human tissue.     

Two glycosphingolipid sialyltransferases are localized in different sub-Golgi compartments in rat liver

A highly purified Golgi preparation from rat liver was fractionated on a sucrose density gradient and the activity of two sialyltransferases, CMP-NeuAc: Gal beta 1----4Glc-Cer (lactosylceramide) alpha-2----3sialyltransferase; Sat-1), and CMP-NeuAc:Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3) Gal beta 1----4Glc-Cer (GM1 ganglioside) alpha 2----3sialyltransferase; SAT-4), involved in the biosynthesis of gangliosides were assayed in the collected fractions. These two activities were recovered in different regions of the gradient; SAT-1 was found in a more dense region than SAT-4. This distribution coincided with that of two N-Asn linked oligosaccharide processing enzymes (UDP-GlcNAc:lysosomal enzyme precursor GlcNAc-1-phosphotransferase and UDP-Gal:ovalbumin galactosyltransferase), assumed as putative markers of cis- and trans-Golgi cisternae, respectively. These findings are consistent with the assembly of ganglioside oligosaccharide chains occurring in different sub-Golgi compartments.     

Evaluation of deoxygenated oligosaccharide acceptor analogs as specific inhibitors of glycosyltransferases

The glycosyltransferases controlling the biosynthesis of cell-surface complex carbohydrates transfer glycosyl residues from sugar nucleotides to specific hydroxyl groups of acceptor oligosaccharides. These enzymes represent prime targets for the design of glycosylation inhibitors with the potential to specifically alter the structures of cell-surface glycoconjugates. With the aim of producing such inhibitors, synthetic oligosaccharide substrates were prepared for eight different glycosyltransferases. The enzymes investigated were: A, alpha(1----2, porcine submaxillary gland); B, alpha(1----3/4, Lewis); C, alpha(1----4, mung bean); D, alpha(1----3, Lex)-fucosyltransferases; E, beta(1----4)-galactosyltransferase; F, beta(1----6)-N-acetylglucosaminyltransferase V; G, beta(1----6)-mucin-N-acetylglucosaminyltransferase ("core-2" transferase); and H, alpha(2----3)-sialyltransferase from rat liver. These enzymes all transfer sugar residues from their respective sugar nucleotides (GDP-Fuc, UDP-Gal, UDP-GlcNAc, and CMP-sialic acid) with inversion of configuration at their anomeric centers. The Km values for their synthetic oligosaccharide acceptors were in the range of 0.036-1.3 mM. For each of these eight enzymes, acceptor analogs were next prepared where the hydroxyl group undergoing glycosylation was chemically removed and replaced by hydrogen. The resulting deoxygenated acceptor analogs can no longer be substrates for the corresponding glycosyltransferases and, if still bound by the enzymes, should act as competitive inhibitors. In only four of the eight cases examined (enzymes A, C, F, and G) did the deoxygenated acceptor analogs inhibit their target enzymes, and their Ki values (all competitive) remained in the general range of the corresponding acceptor Km values. No inhibition was observed for the remaining four enzymes even at high concentrations of deoxygenated acceptor analog. For these latter enzymes it is suggested that the reactive acceptor hydroxyl groups are involved in a critical hydrogen bond donor interaction with a basic group on the enzyme which removes the developing proton during the glycosyl transfer reaction. Such groups are proposed to represent logical targets for irreversible covalent inactivation of this class of enzyme.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

The presence of N-acetyllactosamine and lactose: beta (1-3)N-acetylglucosaminyltransferase activity in human urine

Normal human urine was found to contain beta (1-3)N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to N-acetyllactosamine and lactose. Lacto-N-tetraose which carries the terminal Gal beta (1-3)GlcNAc structure was a poor acceptor. The product of the transferase reaction with N-acetyllactosamine as acceptor was identified by methylation analysis as GlcNAc beta (1-3)Gal beta (1-4)GlcNAc. The beta-linkage of the GlcNAc in the synthesized trisaccharide was confirmed by the action of the specific beta-N-acetylhexosaminidase. The enzyme requires Mn2+ ions for its activity, shows a broad pH optimum from 7 to 9, and appears to have a molecular weight of about 200,000 as estimated by Sephadex gel filtration.     

Purification and characterization of rat kidney UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase.

In order to investigate the molecular mechanism of the specific increase of UDP-N-acetylglucosamine:alpha-6-D-mannoside beta-1,6-N-acetylglucosaminyltransferase (GlcNAcT-V, EC 2.4.1.155) activity after viral or oncogenic transformation, we have purified the enzyme from a Triton X-100 extract of rat kidney acetone powder. GlcNAcT-V was purified by sequential affinity chromatography using first UDP-hexanolamine-agarose and then a synthetic oligosaccharide inhibitor-agarose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed two major bands at apparent molecular masses of 69 and 75 kDa. The enzyme was recovered in a 26% final yield with a 450,000-fold increase in specific activity to a Vmax of 18.8 mumols/(mg.min). Enzyme activity was stabilized and enhanced by the addition of 20% glycerol, 0.5 mg/ml IgG, and 0.2 M NaCl. The optimal ranges of pH and Triton X-100 concentrations for enzyme activity were 6.5-7.0 and 1.0-1.5%, respectively. The divalent cations, Mn2+, Ca2+, and Mg2+, were each found to have a negligible (less than 10%) effect on activity; moreover, the enzyme was fully active in the presence of 20 mM EDTA. The Km value of the purified enzyme toward a synthetic trisaccharide acceptor was 90 microM, and the Ki value toward a synthetic active site inhibitor was 140 microM.     

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

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

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

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

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.