BIOSYNTHESIS OF LIPID-LINKED OLIGOSACCHARIDES .1. PREPARATION OF LIPID-LINKED OLIGOSACCHARIDE SUBSTRATES

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, Man9 (GlcNAc)2-pyrophosphoryl-dolichol was prepared by isolation and enzymatic synthesis using procine pancreatic microsomes, while Glc3Man9(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. © 1992.