Publications

• A mouse lymphoma cell line resistant to the leukoagglutinating lectin from Phaseolus vulgaris is deficient in UDP-GlcNAc: alpha-D-mannoside beta 1,6 N-acetylglucosaminyltransferase.

  Cummings R.D., Trowbridge I.S., Kornfeld S.

  J Biol Chem 257:13421-13427(1982) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

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

  Shoreibah M.G., Hindsgaul O., Pierce M.

  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 ra ... >> More

  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. << Less

  J Biol Chem 267:2920-2927(1992) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A trisaccharide acceptor analog for N-acetylglucosaminyltransferase V which binds to the enzyme but sterically precludes the transfer reaction.

  Khan S.H., Crawley S.C., Kanie O., Hindsgaul O.

  Development of inhibitors specific for the glycosyltransferases involved in the biosynthesis of asparagine-linked sugar chains has been undertaken in the hopes that these compounds may serve as tools to elucidate the roles of complex carbohydrates in biological recognition events. We report here t ... >> More

  Development of inhibitors specific for the glycosyltransferases involved in the biosynthesis of asparagine-linked sugar chains has been undertaken in the hopes that these compounds may serve as tools to elucidate the roles of complex carbohydrates in biological recognition events. We report here the first example of a glycosyltransferase acceptor analog in which strategic replacement of a nonreacting hydroxyl group with a larger substituent produces a molecule which is recognized by the enzyme but does not react because of a steric block to the glycosyl transfer reaction. N-Acetylglucosaminyltransferase V catalyzes the transfer of GlcNAc from the sugar nucleotide donor UDP-GlcNAc to the 6-OH group of mannose in the synthetic trisaccharide acceptor beta GlcNAc(1-->2)alpha Man(1-->6)beta Glc-O(CH2)7CH3 (Km = 23 +/- 2 microM; Vmax = 116 +/-3 pmol/h) to form the tetrasaccharide beta GlcNAc(1-->2)(beta GlcNAc(1-->6))alpha Man(1-->6)beta Glc-O(CH2)7CH3. The acceptor analog produced by replacement of the adjacent nonreacting 4-OH group of the mannose residue with an O-methyl group was not a substrate for the enzyme but was found to be a good competitive inhibitor of GlcNAc transferase V with Ki = 14 +/-2 microM. To test the theory that it was the presence of the large methyl group which prevented the glycosyl transfer reaction the 4'-deoxygenated analog was synthesized. It was found to be a good substrate with Km = 74 +/-6 microM and an almost 5-fold higher kcat (Vmax = 535 +/-13 pmol/h). NMR data show no evidence of important conformational differences between the trisaccharide analogs, and kinetic experiments detected no differences for the binding of UDP-GlcNAc in their presence. The conclusion was therefore reached that the large methyl group introduced on O-4' sterically prevented the formation of product even though both potential substrates were bound by the enzyme. This "steric exclusion" strategy offers potential for the design of inhibitors for that class of glycosyltransferases in which the reactive hydroxyl group is also an essential recognition element. << Less

  J Biol Chem 268:2468-2473(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A novel beta(1,6)-N-acetylglucosaminyltransferase V (GnT-VB).

  Kaneko M., Alvarez-Manilla G., Kamar M., Lee I., Lee J.-K., Troupe K., Zhang W., Osawa M., Pierce M.

  UDP-N-acetylglucosamine:alpha(1,6)-D-mannoside beta(1,6)-N-acetylglucosaminyltransferase (GnT-V, Mgat5) functions in the biosynthesis of N-linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct ... >> More

  UDP-N-acetylglucosamine:alpha(1,6)-D-mannoside beta(1,6)-N-acetylglucosaminyltransferase (GnT-V, Mgat5) functions in the biosynthesis of N-linked glycans and is transcriptionally upregulated by oncogene signaling. We report here the cloning and characterization of a human cDNA encoding a distinct enzyme with related substrate specificity, termed GnT-VB, which is predicted to have 53% similarity to the original amino acid sequence of GnT-V(A). Transient expression of GnT-VB cDNA in COS7 cells yielded significant increases of activity toward GnT-VA acceptors, including synthetic saccharides and N-linked glycopeptides, with some differences in specificity. Unlike GnT-VA, GnT-VB required divalent cation for full activity. EST databases showed expression of a 6 bp (+) splice isoform of GnT-VB; when expressed, this enzyme showed significantly reduced activity. CHO Lec4 cells, which do not express GnT-VA or B activity, lack synthesis of the N-linked beta(1,6) branch, and do not bind L-phytohemagglutinin (L-PHA), were transfected with GnT-VB or GnT-VA; both then bound significant amounts of L-PHA, demonstrating that both enzymes synthesized N-linked beta(1,6) branched glycans in vivo. Real-time polymerase chain reaction results showed that GnT-VB mRNA was highly expressed in brain and testis, with lesser levels in other tissues, while human GnT-VA showed a more general expression, but with low levels in brain and no expression in skeletal muscle. << Less

  FEBS Lett. 554:515-519(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The trisaccharide beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----6)-beta-D-Manp, as its 8-methoxycarbonyloctyl glycoside, is an acceptor selective for N-acetylglucosaminyltransferase V.

  Hindsgaul O., Tahir S.H., Srivastava O.P., Pierce M.

  Incubation of the trisaccharide acceptor, beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----6)-beta-D-Manp-O( CH2)8CO2Me with sonicates of Rous sarcoma-transformed baby-hamster kidney cells, which contain N-acetylglucosaminyltransferase V activity, resulted in the production of beta-D-GlcpNAc-(1----2)-[b ... >> More

  Incubation of the trisaccharide acceptor, beta-D-GlcpNAc-(1----2)-alpha-D-Manp-(1----6)-beta-D-Manp-O( CH2)8CO2Me with sonicates of Rous sarcoma-transformed baby-hamster kidney cells, which contain N-acetylglucosaminyltransferase V activity, resulted in the production of beta-D-GlcpNAc-(1----2)-[beta-D-GlcpNAc-(1----6)]-alpha-D-Manp-(1- ---6)-beta-D-Manp-O(CH2)8CO2Me (4). The product of the enzymic reaction was identified by comparison of its 1H-n.m.r. spectrum with that of authentic 4 whose chemical synthesis is also described. << Less

  Carbohydr Res 173:263-272(1988) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A secreted type of beta 1,6-N-acetylglucosaminyltransferase V (GnT-V) induces tumor angiogenesis without mediation of glycosylation: a novel function of GnT-V distinct from the original glycosyltransferase activity.

  Saito T., Miyoshi E., Sasai K., Nakano N., Eguchi H., Honke K., Taniguchi N.

  Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that beta1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltrans ... >> More

  Angiogenesis is the first regulatory step of tumor progression. Herein, we report on some findings that show that beta1,6-N-acetylglucosaminyltransferase V (GnT-V) functions as an inducer of angiogenesis that has a novel and completely different function from the original function of glycosyltransferase. A secreted type of GnT-V protein itself promoted angiogenesis in vitro and in vivo at physiological concentrations. The highly basic domain of GnT-V induced the release of fibroblast growth factor-2 from heparan sulfate proteoglycan on the cell surface and/or extracellular matrix, leading to angiogenesis. These findings provide some novel information on the relationship between GnT-V and tumor metastasis. The inhibition of GnT-V secretion or its expression represents a novel potential strategy for the inhibition of tumor angiogenesis. << Less

  J. Biol. Chem. 277:17002-17008(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Purification and characterization of UDP-N-acetylglucosamine: alpha-6-D-mannoside beta 1-6N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase V) from a human lung cancer cell line.

  Gu J., Nishikawa A., Tsuruoka N., Ohno M., Yamaguchi N., Kangawa K., Taniguchi N.

  A beta 1-6N-acetylglucosaminyltransferase (GnT-V) [EC 2.4.1.155] which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-D-6-mannoside has been purified up to 20,000-fold from the cultured supernatant of the QG small lung cancer cell line with a 37% yield. The iso ... >> More

  A beta 1-6N-acetylglucosaminyltransferase (GnT-V) [EC 2.4.1.155] which catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to alpha-D-6-mannoside has been purified up to 20,000-fold from the cultured supernatant of the QG small lung cancer cell line with a 37% yield. The isolation procedure included chromatography on phenyl-Sepharose, hydroxylapatite, UDP-hexanolamine Sepharose, and a biantennary sugar substrate (GnGn-bi-Asn) coupled to activated CH-Sepharose 4B. Sodium dodecyl sulfate gel electrophoresis under non-reducing conditions showed a single band of 73 kDa. Under reducing conditions, however, an additional component of 60 kDa was seen. Peptide mapping analysis indicated that both of these proteins were essentially identical, indicating that the 60-kDa component is probably a proteolytically cleaved form of the 73-kDa protein. Studies on the activity of the enzyme toward a variety of pyridylaminated sugars showed that the enzyme is most active toward triantennary (GnGnGn-tri-PA) and biantennary (GnGn-bi-PA) sugars. The Km values for GnGn-bi-PA and UDP-GlcNAc were 133 microM and 3.5 mM, respectively. These studies represent the first report of the enzymatic properties of a highly purified human GnT-V. << Less

  J Biochem 113:614-619(1993) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Molecular cloning and characterization of human GnT-IX, a novel beta1,6-N-acetylglucosaminyltransferase that is specifically expressed in the brain.

  Inamori K., Endo T., Ide Y., Fujii S., Gu J., Honke K., Taniguchi N.

  A novel beta1,6-N-acetylglucosaminyltransferase (beta1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5'-rapid amplification of cDNA end analysis and a further data base search. The o ... >> More

  A novel beta1,6-N-acetylglucosaminyltransferase (beta1, 6GnT) cDNA was identified by a BLAST search using the amino acid sequence of human GnT-V as a query. The full-length sequence was determined by a combination of 5'-rapid amplification of cDNA end analysis and a further data base search. The open reading frame encodes a 792 amino acid protein with a type II membrane protein structure typical of glycosyltransferases. The entire sequence identity to human GnT-V is 42%. When pyridylaminated (PA) agalacto biantennary N-linked oligosaccharide was used as an acceptor substrate, the recombinant enzyme generated a novel product other than the expected GnT-V product, (GlcNAcbeta1,2-Manalpha1,3-)[GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA. This new product was identified as [GlcNAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,3-][Glc-NAcbeta1,2-(GlcNAcbeta1,6-)Manalpha1,6-]Manbeta1,4-GlcNAcbeta1,4-GlcNAc-PA by mass spectrometry and 1H NMR. Namely, the new GnT (designated as GnT-IX) has beta1,6GnT activity not only to the alpha1,6-linked mannose arm but also to the alpha1,3-linked mannose arm of N-glycan, forming a unique structure that has not been reported to date. Northern blot analysis showed that the GnT-IX gene is exclusively expressed in the brain, whereas the GnT-V gene is expressed ubiquitously. These results suggest that GnT-IX is responsible for the synthesis of a unique oligosaccharide structure in the brain. << Less

  J. Biol. Chem. 278:43102-43109(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.