title: Exploring the substrate specificities of alpha-2,6- and alpha-2,3-sialyltransferases using synthetic acceptor analogues.
abstract: The acceptor specificities of rat liver Gal(beta 1-4)GlcNAc alpha-2,6-sialyltransferase, recombinant full-length human liver Gal(beta 1-4)GlcNAc alpha-2,6-sialyltransferase, and a soluble form of recombinant rat liver Gal(beta 1-3/4)GlcNAc alpha-2,3-sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-O)(CH2)7CH3. These analogues contain structural variants of D-galactose, modified at either C3, C4 or C5 by deoxygenation, fluorination, O-methylation, epimerization, or by the introduction of an amino group. In addition, the enantiomer of D-galactose is included. The alpha-2,6-sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the alpha-2,3-sialyltransferase displayed a narrower specificity. Molecular dynamics simulations were performed in order to correlate enzymatic activity to three-dimensional structure. Ineffective acceptors for rat liver alpha-2,6-sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the alpha-2,3-sialyltransferase was found to be inhibited by all non-substrates. Modified sialyloligosaccharides were obtained on a milligram scale by incubation of effective acceptors with one of each of the three enzymes, and characterized by 500-MHz 1H-NMR spectroscopy.
authors.affiliation: Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
authors.forename: J A
authors.initials: JA
authors.lastname: Van Dorst
authors.forename: J M
authors.initials: JM
authors.lastname: Tikkanen
authors.forename: C H
authors.initials: CH
authors.lastname: Krezdorn
authors.forename: M B
authors.initials: MB
authors.lastname: Streiff
authors.forename: E G
authors.initials: EG
authors.lastname: Berger
authors.forename: J A
authors.initials: JA
authors.lastname: Van Kuik
authors.forename: J P
authors.initials: JP
authors.lastname: Kamerling
authors.forename: J F
authors.initials: JF
authors.lastname: Vliegenthart
mesh_terms: D000818:Animals; D003198:Computer Simulation; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D051381:Rats; D011994:Recombinant Proteins; D012799:Sialyltransferases / Q000378:metabolism*; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase
chemical_list: D011994:Recombinant Proteins; D014312:Trisaccharides; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase; D000094663:beta-Galactoside alpha-2,3-Sialyltransferase