Exploring the substrate specificities of α-2,6- and α-2,3-sialyltransferases using synthetic acceptor analogues

Abstract

The acceptor specificities of rat liver Gal(β1-4)GlcNAc α-2,6-sialyltransferase, recombinant fulllength human liver Gal(β1-4)GlcNAc α-2,6-sialyltransferase, and a soluble form of recombinant rat liver Gal(β1-3/4)GlcNAc α2,3-sialyltransferase were studied with a panel of analogues of the trisaccharide Gal(β1-4)GlcNAc(βl-2)Man(α1-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 α-2,6-sialyltransferases tolerated most of the modifications at the galactose residue to some extent, whereas the α-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 α-2,6-sialyltransferase were shown to be inhibitory towards the enzyme; likewise, the α-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.