Enzymatic introduction of a fluorescent sialic acid into oligosaccharide chains of glycoproteins.
Aiming at the introduction of a fluorescent sialic acid into glycoconjugates, 5-acetamido-9-(3-fluoresceinylthio-ureido)-3,5,9-trideoxy-2-non ulosonic acid (9-fluoresceinyl-NeuAc) was synthesized which has an intact carbon chain. a) Despite the space-filling substituent at C-9, the fluorescent NeuAc analogue was activated to the corresponding CMP-glycoside by CMP sialic acid synthase from bovine brain. Whereas the Km value of the synthase was little affected by the modification (Km = 2.1 mM, for NeuAc Km = 1.4 mM), the V value decreased to 7.5%. b) CMP-9-fluoresceinyl-NeuAc was synthesized on a preparative scale (17% overall yield), and characterized by analytical HPLC, absorption and fluorescence spectra. c) 9-Fluoresceinyl-NeuAc was transferred onto asialo-alpha 1-acid glycoprotein by both Gal beta 1, 4GlcNAc alpha 2, 6sialyltransferase and Gal beta 1,4(3)GlcNAc alpha 2,3sialyltransferase (rat liver), and onto antifreeze glycoprotein by GalNAc alpha 2,6-sialyltransferase (porcine submaxillary glands). Using analytical HPLC, transfer was confirmed after release of the fluorescent sialic acid by Vibrio cholerae sialidase. d) Initial rate studies indicated a low Km value of Gal beta-1,4GlcNAc alpha 2,6sialyltransferase, and GalNAc alpha 2,6sialyltransferase (specific for O-linked oligosaccharide chains) for CMP-9-fluoresceinyl-NeuAc.
Gross
H J
HJ
Institut für Biochemie II, Universität Heidelberg, Federal Republic of Germany.
Brossmer
R
R
D000818:Animals; D005452:Fluoresceins / Q000378:metabolism*; D006023:Glycoproteins / Q000096:biosynthesis*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D000216:N-Acylneuraminate Cytidylyltransferase / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis*; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism*
D005452:Fluoresceins; D006023:Glycoproteins; D009844:Oligosaccharides; D012794:Sialic Acids; C057965:5-acetamido-9-(3-fluoresceinylthioureido)-3,5,9-trideoxy-2-nonulosonsonic acid; D012799:Sialyltransferases; D009713:Nucleotidyltransferases; D000216:N-Acylneuraminate Cytidylyltransferase