Sialyltransferases: expression and application for chemo-enzymatic syntheses

Glycosylation is a complex yet common form of post-translational protein/lipid modification in the eukaryotic cells. It is processed by glycosyltransferases (GTs), a large group of enzyme, that are involved in the biosynthesis of glycoprotein and glycolipid sugar chains. Moreover, carbohydrates represent major components of the outer surface of mammalian cells and there is now abundant evidence that terminal glycosylation sequences are involved in adhesion, immune response, and neuronal outgrowth events. Sialylated oligosaccharide sequences have long been predicted to be information – containing molecules and critical determinants, e.g. in cell-cell recognition processes, cell-matrix interactions and maintenance of serum glycoproteins in the circulation. Enzymes responsible for the terminal sialylation are sialyltransferases (STs), a subset of the GT family that use CMP-NeuNAc as the activated sugar donor to catalyze the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. The Myelin-Associated Glycoprotein (MAG), expressed in myelin of the central and peripheral nervous system, has been identified as one of the neurite outgrowth-inhibitory proteins, together with Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp). Among all MAG physiological ligands, i.e. brain gangliosides, the GQ1bα is the most potent natural ligand identified so far. Moreover, only the sialic acid containing part of the whole GQ1bα molecule was shown to be important for MAG binding. Therefore, we decided to use a chemo-enzymatic approach for syntheses of the GQ1bα mimetics. For that purpose we expressed recombinant eukaryotic rST3Gal III (EC 2.4.99.6) and hST6Gal I (EC 2.4.99.1), and recombinant prokaryotic Campylobacter jejuni α-2,3/2,8 bifunctional sialyltransferase (Cst-II) using different expression systems. The enzymes were purified and biochemical characterized towards several natural and non-natural acceptor substrates, and used for the preparative chemo-enzymatic synthesis of different carbohydrate structures, e.g. [NeuNAcα(2,8)]NeuNAcα(2,3)Galβ(1,3)GlcNAc-β-OLem; NeuNAcα(2,3)Galβ(1,4)GlcNAc-β-OLem; NeuNAcα(2,3)Galβ(1,4)Glc-β-OLem;).