3 ENZYMES INVOLVED IN OLIGOSACCHARIDE-LIPID ASSEMBLY IN CHINESE-HAMSTER OVARY CELLS DIFFER IN LIPID SUBSTRATE PREFERENCE

Initial steps in N-linked glycosylation involve formation of a large o ligosaccharide structure on a lipid carrier, dolichyl phosphate. We ha ve previously characterized Chinese hamster ovary (CHO) glycosylation mutants (Lec9 cells) that utilize the polyisoprenoid lipid polyprenyl phosphate rather than dolichyl phosphate in these glycosylation reacti ons. Polyprenyl phosphate differs from dolichyl phosphate only in the degree of saturation of its terminal isoprenyl unit. Our goal was to d etermine whether the glycosylation defect of Lec9 cells could be expla ined simply by knowing lipid substrate preferences of the enzymes invo lved in the assembly of oligosaccharide-lipid (OSL) intermediates. In this study, we have used in vitro assay systems to compare the ability of dolichyl phosphate and polyprenyl phosphate to act as substrates f or three glycosyl transferase enzymes involved in OSL assembly. In ord er to insure that we were only examining lipid substrate preferences o f the enzymes and not other potential defects present in Lec9 cells, w e used membranes prepared from wild-type cells in these in vitro react ions. Our results indicate that one of the enzymes, mannosylphosphoryl dolichol (MPD) synthase, exhibited a significant preference for the do lichol substrate. Glucosylphosphoryldolichol (GPD) synthase, on the ot her hand, showed no binding specificity for the dolichol substrate, al though the enzyme used the dolichol substrate at a twofold higher rate . N,N'-diacetylchitobiosylpyrophosphoryldolichol (CPD) synthase was ab le to use either lipid substrate with equal efficiency. These results suggest that not all glycosyl transferases in this pathway show a pref erence for dolichol derivatives. Moreover, in conjunction with other s tudies from our laboratory, these results help explain the glycosylati on phenotype of Lec9 cells; that is, they synthesize less OSL and the structure of the major OSL is Man(5)GlcNAc(2)-P-P-lipid rather than Gl c(3)Man(9)GlcNAc(2)-P-P-lipid.