The catalytic mechanism of UDP-GlcNAc: Mannoside N-acetylglucosamintransferases I and II

Research project P 14343 Mechanism of GlcNAc-transferases I and II Josef GLÖSSL 08.05.2000 The covalent modification of proteins with oligosaccharides at selected asparagine residues is termed N- glycosylation. The formation of N-glycans is often crucial for the proper synthesis and function of the respective glycoprotein. Most N-linked oligosaccharides on mammalian glycoproteins are either of the oligomannosidic or the complex type. The key enzymes for the conversion of oligomannosidic N-glycans into complex-type N-linked oligosaccharides are N-acetylglucosaminyltransferases I (GnT-I) and II (GnT-II). In spite of the physiological significance of GnT-I and GnT-II, the catalytic mechanism of these enzymes is only poorly understood. This project aims at a detailed molecular characterisation of the enzymatic properties of GnT-I and GnT-II. Our main approach is based on site-directed mutagenesis of recently cloned GnT-I and GnT-II DNAs. We will mutate amino acid residues of the enzymes that are strictly conserved from plants to man. The mutant cDNAs will then be expressed in a heterologous system. Finally, the catalytic properties of the corresponding proteins will be bona fide characterised. This project will substantially improve the current knowledge of glycosyltransferase action and be a first step towards the rational design of novel inhibitors which may prove valuable tools to modify the N-glycosylation machinery of eukaryotic cells in vivo.