Journal of Lipid Research, Vol 35, 1861-1868, Copyright © 1994 by Lipid Research, Inc.

ARTICLES

Three enzymes involved in oligosaccharide-lipid assembly in Chinese hamster ovary cells differ in lipid substrate preference

KR McLachlan and SS Krag
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, MD 21205.

Initial steps in N-linked glycosylation involve formation of a large oligosaccharide structure on a lipid carrier, dolichyl phosphate. We have previously characterized Chinese hamster ovary (CHO) glycosylation mutants (Lec9 cells) that utilize the polyisoprenoid lipid polyprenyl phosphate rather than dolichyl phosphate in these glycosylation reactions. Polyprenyl phosphate differs from dolichyl phosphate only in the degree of saturation of its terminal isoprenyl unit. Our goal was to determine whether the glycosylation defect of Lec9 cells could be explained simply by knowing lipid substrate preferences of the enzymes involved 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 for three glycosyl transferase enzymes involved in OSL assembly. In order to insure that we were only examining lipid substrate preferences of the enzymes and not other potential defects present in Lec9 cells, we used membranes prepared from wild-type cells in these in vitro reactions. Our results indicate that one of the enzymes, mannosylphosphoryldolichol (MPD) synthase, exhibited a significant preference for the dolichol substrate. Glucosylphosphoryldolichol (GPD) synthase, on the other hand, showed no binding specificity for the dolichol substrate, although the enzyme used the dolichol substrate at a twofold higher rate. N,N'-diacetyl- chitobiosylpyrophosphoryldolichol (CPD) synthase was able to use either lipid substrate with equal efficiency. These results suggest that not all glycosyl transferases in this pathway show a preference for dolichol derivatives.(ABSTRACT TRUNCATED AT 250 WORDS)
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