caa a22 4500 469115254 CHVBK 20180323133122.0 cr unu---uuuuu 170328e19920801xx s 000 0 eng 10.1007/BF00731163 doi (NATIONALLICENCE)springer-10.1007/BF00731163 Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2- N -acetylglucosaminyl-transferase I using synthetic substrate analogues [Elektronische Daten] [Gabriele Möller, Folkert Reck, Hans Paulsen, Kanwal Kaur, Mohan Sarkar, Harry Schachter, Inka Brockhausen] UDP-GlcNAc: Manα3R β2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M r 42,000). TheV max for the pure enzyme with [Manα6(Manα3)Manα6](Manα3)Manβ4GlcNAcβ4GlcNAcβ-Asn as substrate was 4.6 µmol min−1 mg−1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in β1-2 linkage to the Manα3Manβ-terminus of the substrate. Several derivatives of Manα6(Manα3)Manβ-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the β-linked mannose of Manα6(Manα3)Manβ-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the α3-linked mannose (Man) of the substrate increases theK M 20-fold. Modifications on the α6-linked mannose or on the core structure affect mainly theK M and to a lesser degree theV max, e.g., substitutions of the Manα6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK M, whereas various other substitutions at the 3-position increase theK M slightly. Manα6(Manα3)4-O-methyl-Manβ4GlcNAc was found to be a weak inhibitor of GlcNAc-T I. Chapman & Hall, 1992 GlcNAc-transferase I nationallicence substrate specificity nationallicence glycoprotein biosynthesis nationallicence N-linked glycans nationallicence BSA : Bovine serum albumin nationallicence Bn : benzyl nationallicence Fuc, F : l-fucose nationallicence Gal, G : d-galactose nationallicence GalNAc, GA : N-acetyl-d-galactosamine nationallicence Glc : d-glucose nationallicence GlcNAc, Gn : N-acetyl-d-glucosamine nationallicence HPLC : high performance liquid chromatography nationallicence Man, M : d-mannose nationallicence mco : 8-methoxycarbonyl-octyl, (CH2)8 COOOCH3 nationallicence Me : methyl nationallicence MES : 2-(N-morpholino)ethanesulfonate nationallicence NMR : nuclear magnetic resonance nationallicence PMSF : phenylmethylsulfonylfluoride nationallicence pnp : p-nitrophenyl nationallicence SDS : sodium dodecyl sulfate nationallicence T : transferase nationallicence Tal : d-talose nationallicence Xyl : d-xylose nationallicence {0, 2 + F} : Manα6 (GlcNAcβ2Manα3) Manβ4GlcNAcβ4 (Fucα6) GlcNAc nationallicence {2, 2} : GlcNAcβ2Manα6 (GlcNAcβ2Manα3) Manβ4GlcNAcβ4GlcNAc nationallicence M5-glycopeptide, Manα6 (Manα3) Manα6 (Manα3) Manβ4 GlcNAcβ4GlcNAcβ-Asn nationallicence Möller Gabriele Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut Reck Folkert Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany aut Paulsen Hans Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany aut Kaur Kanwal Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada aut Sarkar Mohan Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut Schachter Harry Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut Brockhausen Inka Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut Glycoconjugate Journal Kluwer Academic Publishers 9/4(1992-08-01), 180-190 0282-0080 9:4<180 1992 9 10719 https://doi.org/10.1007/BF00731163 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00731163 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Möller Gabriele Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut NATIONALLICENCE 700 1- Reck Folkert Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany aut NATIONALLICENCE 700 1- Paulsen Hans Institut für Organische Chemie, Universität Hamburg, Hamburg, Germany aut NATIONALLICENCE 700 1- Kaur Kanwal Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada aut NATIONALLICENCE 700 1- Sarkar Mohan Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut NATIONALLICENCE 700 1- Schachter Harry Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut NATIONALLICENCE 700 1- Brockhausen Inka Research Institute, The Hospital for Sick Children, M5G 1X8, Toronto, Ontario, Canada aut NATIONALLICENCE 773 0- Glycoconjugate Journal Kluwer Academic Publishers 9/4(1992-08-01), 180-190 0282-0080 9:4<180 1992 9 10719 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer