In vitro galactosylation and sialylation of human immunoglobulin G

Abstract

Glycosylation is a common co- and post-translational modification of proteins which may have profound effects on protein structure and function. Glycoproteins consist of an ensemble of glycosylated variants, or glycoforms, and the glycosylation pattern of a glycoprotein is not random but dependent on the cell type, physiological conditions, and in some carse disease states. For further studies to elucidate the structures and biological functions of the glycoproteins, the large amount of glycoproteins is needed, however it has been hard to produce recombinant glycoproteins with minimizing the heterogeneity as well as increasing the level of selective glycosylation. In this study, therefore, we have developed a versatile tool for in vitro synthesis of oligosaccharides and glycoconjugates using human immunoglobulin G as a model of therapeutic glycoprotein. Human IgG contains truncated glycans such as terminal galactose(Gal) and N-acetylglucosamine (GlcNAc) residues which may cause rapid clearance of IgG from circulation as a consequence of binding to the aglycosylprotein receptor. To increase the level of terminal sialylation, we regalactosylated and/or resialylated IgG using β1,4-galactosyltransferase and/or α 2,3-sialyltransferase by using UDP-Gal and CMP-NeuAc, respectively. And to overcome the difficulty associated with the preparation of sugar-nucleotides, efficient enzymatic systems for the production of UDP-Gal and CMP-NeuAc were introduced.