Publications

• The human UDP-N-acetylglucosamine: alpha-6-D-mannoside-beta-1,2-N-acetylglucosaminyltransferase II gene (MGAT2). Cloning of genomic DNA, localization to chromosome 14q21, expression in insect cells and purification of the recombinant protein.

  Tan J., D'Agostaro A.F., Bendiak B., Reck F., Sarkar M., Squire J.A., Leong P., Schachter H.

  UDP-GlcNAc:alpha-6-D-mannoside [GlcNAc to Man alpha 1-6] beta-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligomannose to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used ... >> More

  UDP-GlcNAc:alpha-6-D-mannoside [GlcNAc to Man alpha 1-6] beta-1,2-N-acetylglucosaminyltransferase II (GlcNAc-T II, EC 2.4.1.143) is a Golgi enzyme catalyzing an essential step in the conversion of oligomannose to complex N-glycans. A 1.2-kb probe from a rat liver cDNA encoding GlcNAc-T II was used to screen a human genomic DNA library in lambda EMBL3. Southern analysis of restriction endonuclease digests of positive phage clones identified two hybridizing fragments (3.0 and 3.5 kb) which were subcloned into pBlueScript. The inserts of the resulting plasmids (pHG30 and pHG36) are over-lapping clones containing 5.5 kb of genomic DNA. The pHG30 insert (3.0 kb) contains a 1341-bp open reading frame encoding a 447-amino-acid protein, 250 bp of G + C-rich 5'-upstream sequence and 1.4 kb of 3'-downstream sequence. The pHG36 insert (3.5 kb) contains 2.75 kb of 5'-upstream sequence and 750 bp of the 5'-end of the open reading frame. The protein sequence showed the domain structure typical of all previously cloned glycosyltransferases, i.e. a short 9-residue putative cytoplasmic N-terminal domain, a 20-residue hydrophobic non-cleavable putative signal-anchor domain and a 418-residue C-terminal catalytic domain. Northern analysis of human tissues showed a major message at 3 kb and minor signals at 2 and 4.5 kb. There is no sequence similarity to any previously cloned glycosyltransferases including human UDP-GlcNAc:alpha-3-D-mannoside [GlcNAc to Man alpha 1-3] beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-T I) which has 445 amino acids with a 418-residue C-terminal catalytic domain. The human GlcNAc-T I and II genes (MGAT1 and MGAT2) map to chromosome bands 5q35 and 14q21, respectively, by fluorescence in situ hybridization. The entire coding regions of human GlcNAc-T I and II are each on a single exon. There is 92% identity between the amino acid sequences of the catalytic domains of human and rat GlcNAc-T II. Southern analysis of restriction enzyme digests of human genomic DNA indicates that there is only a single copy of the MGAT2 gene. The full-length coding region of GlcNAc-T II has been expressed in the baculovirus/Sf9 insect cell system, the recombinant enzyme has been purified to near homogeneity with a specific activity of about 20 mumol.min-1.mg-1 and the product synthesized by the recombinant enzyme has been identified by high-resolution 1H-NMR spectroscopy and mass spectrometry. << Less

  Eur. J. Biochem. 231:317-328(1995) [PubMed] [EuropePMC]

• Control of glycoprotein synthesis. Bovine colostrum UDP-N-acetylglucosamine:alpha-D-mannoside beta 2-N-acetylglucosaminyltransferase I. Separation from UDP-N-acetylglucosamine:alpha-D-mannoside beta 2-N-acetylglucosaminyltransferase II, partial purification, and substrate specificity.

  Harpaz N., Schachter H.

  J Biol Chem 255:4885-4893(1980) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Control of glycoprotein synthesis. Purification of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver.

  Bendiak B., Schachter H.

  A new affinity chromatography adsorbant, in which UDP-GlcNAc has been linked to thiopropyl-Sepharose at the 5 position of the uracil via a 5-mercuri mercaptide bond, was utilized to purify UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II 60,000-fold from rat liver. After ex ... >> More

  A new affinity chromatography adsorbant, in which UDP-GlcNAc has been linked to thiopropyl-Sepharose at the 5 position of the uracil via a 5-mercuri mercaptide bond, was utilized to purify UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II 60,000-fold from rat liver. After extraction of rat liver membranes with Triton X-100, the enzyme was found to exist in two molecular weight forms of markedly differing size, separable on Sephadex G-200. The low Mr form was separated from the high Mr form on columns of CM-Sephadex and hydroxylapatite, and was further purified by sequential elutions with NaCl, UDP-GlcNAc, and EDTA from the 5-mercuri-UDP-GlcNAc affinity adsorbant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified low Mr form under reducing conditions revealed two protein bands of Mr 48,000 and 43,000. The purified enzyme catalyzes the transfer of N-acetylglucosamine from UDP-GlcNAc to the compound: (Formula: see text) The high Mr form of the enzyme, which eluted in the void volume of Sephadex G-200, was resistant to a number of treatments in attempts to reduce its molecular weight. These results suggest that the high Mr form of the enzyme may represent either a complex which normally exists in Golgi membranes as a result of strong protein-protein interactions or a protein with one or more "anchor" segments. << Less

  J Biol Chem 262:5775-5783(1987) [PubMed] [EuropePMC]

• The nonidentity of porcine N-acetylglucosaminyltransferases I and II.

  Oppenheimer C.L., Eckhardt A.E., Hill R.L.

  J Biol Chem 256:11477-11482(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Control of glycoprotein synthesis. Kinetic mechanism, substrate specificity, and inhibition characteristics of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver.

  Bendiak B., Schachter H.

  Purified rat liver UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II (Bendiak, B., and Schachter, H. (1987) J. Biol. Chem. 262, 5775-5783) has been characterized kinetically, and its substrate specificity and inhibition characteristics have been determined. Kinetic data indi ... >> More

  Purified rat liver UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II (Bendiak, B., and Schachter, H. (1987) J. Biol. Chem. 262, 5775-5783) has been characterized kinetically, and its substrate specificity and inhibition characteristics have been determined. Kinetic data indicate an ordered, or largely ordered sequential mechanism, with UDP-GlcNAc binding prior to the acceptor. The minimal acceptor structure required for full activity is: (Formula: see text) The acceptor molecule must have a terminal Man alpha 1-6 residue, and a terminal GlcNAc beta 1-2Man alpha 1-3 branch to display any activity, but does not require the reducing GlcNAc residue, as the enzyme was about 50% as active after reduction of this residue to N-acetylglucosaminitol. Additional residues (Gal beta 1-4 on the GlcNAc beta 1-2Man alpha 1-3 arm, or a bisecting GlcNAc beta 1-4 on the beta-Man residue) abolish catalytic activity. These results suggest a rigid order in the biosynthesis of all N-linked complex oligosaccharides (bisected and nonbisected bi-, tri-, and tetraantennary), since the enzyme must act to completion prior to the action of either UDP-Gal:GlcNAc beta 1-4 galactosyltransferase or N-acetylglucosaminyltransferase III to make such structures. Inhibition studies with nucleotides, sugars, nucleotide-sugars, and their respective analogues revealed that analogues of UDP and UTP, in which the hydrogen at the 5 position of the uracil was substituted with -CH3, bromine, or mercury (as the mercaptide) were good reversible inhibitors of the enzyme, whereas substitution at other sites lessened the inhibitory potency, usually to a large degree. << Less

  J Biol Chem 262:5784-5790(1987) [PubMed] [EuropePMC]

• Isolation and properties of alpha-D-mannose:beta-1,2-N-acetylglucosaminyltransferase from trachea mucosa.

  Mendicino J., Chandrasekaran E.V., Anumula K.R., Davila M.

  A beta-1,2-N-acetylglucosaminyltransferase which transfers N-acetylglucosamine from UDP-N-acetylglucosamine to the branched terminal mannosyl residues found in the complex type oligosaccharide units of N-linked glycoproteins was isolated from swine trachea mucosa. The enzyme was purified from micr ... >> More

  A beta-1,2-N-acetylglucosaminyltransferase which transfers N-acetylglucosamine from UDP-N-acetylglucosamine to the branched terminal mannosyl residues found in the complex type oligosaccharide units of N-linked glycoproteins was isolated from swine trachea mucosa. The enzyme was purified from microsomes after solubilization with 1% Triton X-100 and 1% Nonidet P-40, and it was also isolated from a soluble extract of this tissue. The enzyme was purified by chromatography on DEAE-cellulose, cellulose phosphate, and Sephacryl S300 columns and by affinity chromatography on a Sepharose 4B column containing covalently bound ovomucoid. The purified enzyme forms beta-1,2 bonds between N-acetylglucosamine and terminal-branched mannosyl residues of glycoproteins and glycopeptides. The enzyme has an absolute specificity for terminal branched mannosyl residues, and solute specificity for terminal branched mannosyl residues, and no activity is observed with mannose, p-nitrophenyl alpha-D-mannoside, p-nitrophenyl beta-O-mannoside, and glycopeptides which contain unbranched oligosaccharide chains terminating in free mannose. Evidence obtained by kinetic and structural analysis shows that the purified enzyme catalyzes the reactions shown in Scheme I. << Less

  Biochemistry 20:967-976(1981) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Glycosyltransferases involved in elongation of N-glycosidically linked oligosaccharides of the complex or N-acetyllactosamine type.

  Schachter H., Narasimhan S., Gleeson P., Vella G.

  Methods Enzymol 98:98-134(1983) [PubMed] [EuropePMC]

  This publication is cited by 9 other entries.