Synthesis of pentasaccharide analogues of the N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V using tetrasaccharide precursors and recombinant beta-(1-->2)-N-acetylglucosaminyltransferase II.
Recombinant human UDP-GlcNAc: alpha-Man-(1-->6)R beta-(1-->2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143, GlcNAc-T II) was produced in the Sf9 insect cell/baculovirus expression system as a fusion protein with a (His)6 tag and partially purified by affinity chromatography on a metal chelating column. The partially purified enzyme was used to catalyze the transfer of GlcNAc from UDP-GlcNAc to R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3))beta-Man-O-octyl to form beta-GlcNAc(1-->2)R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha- Man(1-->3))beta-Man-O-octyl where there is either no modification of the alpha-Man(1-->6) residue (7), or where R is 3-deoxy (8), 4-deoxy (9) or 6-deoxy (10). The yields ranged from 64-80%. Products were characterized by 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 7-10 are pentasaccharide analogues of the biantennary N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V.
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
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Reck
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Meinjohanns
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Tan
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Grey
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Paulsen
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Schachter
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006031:Glycosylation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D017351:N-Acetylglucosaminyltransferases; C063312:beta-1,3-galactosyl-0-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase