Biosynthesis of lipid-linked oligosaccharides: I. Preparation of lipid-linked oligosaccharide substrates

Abstract

In order to purify the glycosyltransferases involved in the assembly of lipid-linked oligosaccharides and to be able to study the acceptor substrate specificity of these enzymes, methods were developed to prepare and purify a variety of lipid-linked oligosaccharides, differing in the structure of the oligosaccharide moiety. Thus, Man 9 (GlcNAc) 2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using procine pancreatic microsomes, while Glc 3Man 9(GlcNAc) 2-PP-dolichol was isolated from Madin-Darby canine kidney cells. Treatment of these oligosaccharide lipids with a series of selected glycosidases led to the preparation of Manα1,2Manα1,2Manα1,3[Manα1,6(Manα1,3)Manα1,6]Manβ1,4GlcNAcβ1,4GlcNAc-PP-dolichol; Manα1,2Manα1,2Manα1,3[Manα1,6]Manβ1,4GlcNAcβ1,4GlcNac-PP-dolich; and Manα1,6(Manα1,3)Manα1,6[Manα1,3]Manβ1,4GlcNAc-β1,4GlcNAc-PP-dolichol. The preparation, isolation, and characterization of each of these lipid-linked oligosaccharide substrates are described.