Multifunctionality of Campylobacter jejuni sialyltransferase CstII: Characterization of GD3/GT3 oligosaccharide synthase, GD3 oligosaccharide sialidase, and trans-sialidase activities

Abstract

CstII from bacterium Campylobacter jejuni strain OH4384 has been previously characterized as a bifunctional sialyltransferase having both α2,3-sialyltransferase (GM3 oligosaccharide synthase) and α2,8-sialyltransferase (GD3 oligosaccharide synthase) activities which catalyze the transfer of N-acetylneuraminic acid (Neu5Ac) from cytidine 5′-monophosphate (CMP)-Neu5Ac to C-3′ of the galactose in lactose and to C-8 of the Neu5Ac in 3′-sialyllactose, respectively (Gilbert M, Karwaski MF, Bernatchez S, Young NM, Taboada E, Michniewicz J, Cunningham AM, Wakarchuk WW. 2002. The genetic bases for the variation in the lipo-oligosaccharide of the mucosal pathogen, Campylobacter jejuni. Biosynthesis of sialylated ganglioside mimics in the core oligosaccharide. J Biol Chem. 277:327-337). We report here the characterization of a truncated CstII mutant (CstIIΔ32I53S) cloned from a synthetic gene whose codons are optimized for an Escherichia coli expression system. In addition to the α2,3- and α2,8-sialyltransferase activities reported before for the synthesis of GM3- and GD3-type oligosaccharides, respectively, the CstIIΔ32I53S has α2,8-sialyltransferase (GT3 oligosaccharide synthase) activity for the synthesis of GT3 oligosaccharide. It also has α2,8-sialidase (GD3 oligosaccharide sialidase) activity that catalyzes the specific cleavage of the α2,8-sialyl linkage of GD3-type oligosaccharides and α2,8-trans-sialidase (GD3 oligosaccharide trans-sialidase) activity that catalyzes the transfer of a sialic acid from a GD3 oligosaccharide to a different GM3 oligosaccharide (3′-sialyllactoside). The donor substrate specificity study of the CstIIΔ32I53S GD3 oligosaccharide synthase activity indicates that the enzyme is flexible in using different CMP-activated sialic acids and their analogs for the synthesis of GD3 oligosaccharides containing natural and nonnatural modifications at the terminal sialic acid. © The Author 2008. Published by Oxford University Press. All rights reserved.