Acceptor-substrate recognition by N-acetylglucosaminyltransferase-V: critical role of the 4"-hydroxyl group in beta-D-GlcpNAc-(1-->2)-alpha-D-Manp(1-->6)-beta-D-Glcp-OR.
The enzyme N-acetylglucosaminyltransferase-V (GlcNAcT-V) transfers GlcNAc from UDP-GlcNAc to the OH-6' group of oligosaccharides terminating in the sequence beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-beta-D-Glcp (or Manp)-OR (5, R = (CH2)7CH3) to yield the sequence beta-D-GlcpNAc-(1-->2)-[beta-D-GlcpNAc-(1-->6)]-alpha-D-Manp-(1--> 6)- beta-D-Glcp (or Manp)-OR. Biosynthetically, if beta-(1-->4)-galactosyltransferase acts first on 5, the product beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->6)-be ta-D-Glcp (or Manp)-OR (7) is no longer a substrate for GlcNAcT-V even though it retains the active OH-6' group. The reason for this loss in activity is examined in this paper. Six analogues of the acceptor trisaccharide 5, all with the reducing-end D-gluco configuration, were chemically synthesized. A key feature of the synthetic scheme is the use of 1,2-diaminoethane for the efficient removal of N-phthalimdo protecting groups. In these analogues OH-4 of the terminal sugar unit, the site of galactosylation by GalT in the normal GlcNAc-terminating trisaccharide 5, was systematically replaced by OMe, F, NH2, NHAc, and H, as well as inverted to the galacto configuration. The interactions of the resulting trisaccharide analogues with GlcNAcT-V from hamster kidney were then evaluated kinetically. All six compounds were found to be essentially inactive either as acceptors or as inhibitors of GlcNAcT-V. The conclusion is reached that galactosylation of natural acceptors for GlcNAcT-V destroys acceptor activity, not by introduction of the steric bulk of an added sugar residue, but by destroying an important hydrogen-bonding interaction of terminal OH-4 of the GlcNAc residues with the enzyme. This OH-4 group is therefore designated as a key polar group for GlcNAcT-V.
Kanie
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Department of Chemistry, University of Alberta, Edmonton, Canada.
Crawley
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SC
Palcic
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MM
Hindsgaul
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D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004187:Disaccharides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D007202:Indicators and Reagents; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008322:Mammals; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014312:Trisaccharides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D004187:Disaccharides; D007202:Indicators and Reagents; D009844:Oligosaccharides; D014312:Trisaccharides; D014537:Uridine Diphosphate N-Acetylglucosamine; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase