Glycosyltransferase Activity Can Be Modulated by Small Conformational Changes of Acceptor Substrates

Abstract

A range of N-acetyllactosamine derivatives (compounds 4-7) that have restricted mobilities around their glycosidic linkages have been employed to determine how small changes in conformational properties of an oligosaccharide acceptor affect catalytic efficiencies of glycosylations by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI. Restriction of conformational mobility was achieved by introducing tethers of different length and chemical composition between the C-6 and C-2' hydroxyl of LacNAc. Compound 4 is a 2',6-anhydro derivative which is highly constrained and can adopt only two unusual conformations at the LacNAc glycosidic linkage. Compound 5 is modified by a methylene acetal tether and can exist in a larger range of conformations; however, the Phi dihedral angle is restricted to values smaller than 30 degrees, which are not entirely similar to minimum energy conformations of LacNAc. The ethylene-tethered 6 can attain conformations in the relatively large energy plateau of LacNAc that include syn conformations A and B, whereas compound 7, which is modified by a methylamide tether, can only reside in the B-conformer. 2',6-Dimethoxy derivative 2 was employed to determine the effect of alkylation of the C-6 and C-2' hydroxyls of 5 and 6 whereas 3 was used to reveal the effects of the C-6 amide and C-2' alkylation of 7. The apparent kinetic parameters of transfer to the conformationally constrained 4-7 and reference compounds 1-3 catalyzed by alpha-2,6- and alpha-2,3-sialyltransferases and alpha-1,3-fucosyltransferases IV and VI were determined, and the results correlated with their conformational properties. The data for 4-6 showed that each enzyme recognizes N-acetyllactosamine in a low minimum energy conformation. A small change in conformational properties such as in compound 5 resulted in a significant loss of catalytic activity. Larger conformational changes such as in compound 4 abolished all activity of the sialyltransferases whereas the fucosyltransferases showed some activity, albeit very low. The kinetic data for compounds 4 and 5 demonstrate clearly that different glycosyltransferases respond differently to conformational changes, and the fucosyltransferases lost less activity than the sialyltransferases. Correlating apparent kinetic parameters of conformationally constrained 6 and 7 and their reference compounds 2 and 3 further supports the fact that different enzymes respond differently and indicates that sialyltransferases and fucosyltransferases recognize N-acetyllactosamine in a different conformation. Collectively, the data presented here indicate that small conformational changes of an oligosaccharide acceptor induced by, for example, the protein structure can be employed to modulate the patterns of protein glycosylation.

In Vitro Evidence for the Dual Function of Alg2 and Alg11: Essential Mannosyltransferases in N-Linked Glycoprotein Biosynthesis

“…Plasticity of the active site and the flexibility between acceptor-and donor-binding sites may help to explain how enzymes with the same overall structure are used for the formation of more than one type of glycosidic linkage and for the transfer of more than one monosaccharide. It has been shown that constraining glycosyl acceptors into a variety of conformations can have a significant impact on the kinetic parameters of the glycosyltransferases that recognize them (62). Much more work is needed to begin to understand how Alg2 can carry out an R1,3-mannosylation when the trisaccharide intermediate is bound, while binding of the tetrasaccharide induces an R1,6-mannosylation, thus changing the linkage specificity by changing the substrate.…”

Sialyltransferase inhibition and recent advances

“…Considerable effort has also been dedicated to the design and synthesis of inhibitors of sialylation processing enzymes focusing on the receptor specificity by using modified lactose and lactosamine [37][38][39]. Through the studies of structure-effective relationship, Wlasichuk and co-workers reported that the C-6′ hydroxyl and acetamido group of LacNAc are essential for sialylation by rat liver α2,6-sialyltransferase [37].…”

“…Through the studies of structure-effective relationship, Wlasichuk and co-workers reported that the C-6′ hydroxyl and acetamido group of LacNAc are essential for sialylation by rat liver α2,6-sialyltransferase [37]. Methylation of the C-6 and C-2′ hydroxyls of LacNAc had only a minimal effect on α2,3-sialyltransferase [39], but there was no inhibition of α2,3-sialyltransferase when the C-2′ hydroxyl of LacNAc was epimerized [38]. In addition, fluorine-containing mucin core 2 oligosaccharide analogs in which the fluorine atom located at different positions of galactose residue were synthesized in a convergent way for probing carbohydrate-enzyme interaction and carbohydrate-selectin interaction.…”

Tailoring recombinant protein quality by rational media design

“…Fucosyltransferases and sialyltransferases may compete for the same acceptor substrates, which was demonstrated with N-acetyllactosamine (LacNAc) derivatives 120 . As a result, selective inhibition of sialic acid addition by adding a fluorinated sialic acid analog increases fucosylation 112 .…”