title: Cell-free N-glycosylation in Dictyostelium discoideum: analysis of wild-type and mutants defective in lipid-linked oligosaccharide biosynthesis.
abstract: N-glycosylation was measured in wild-type cell lysates of Dictyostelium discoideum and in two mutant strains that synthesize a truncated lipid-linked oligosaccharide, Man6GlcNAc2 lacking terminal mannose and glucose residues. Endogenous lipid-linked oligosaccharide (LLO) was transferred to octanoyl-Asn-[125I]Tyr-ThrNH2 by membrane fractions. About 50% of the glycopeptide product remained associated with membranes. Taurocholate and saponin promoted and preserved glycosylation, but NP-40 and Triton X-100 did not. Using this artificial assay, the rate and extent of transfer of the truncated lipid-linked oligosaccharide in extracts of the two mutant strains, HL241 and HL243, was reduced 5-10-fold relative to that of wild-type. The low activity found in the mutant strains appears to result from either reduced affinity of the truncated LLO for the transferase or from its improper topological localization in the membrane. When protein N-glycosylation is measured in living cells it is nearly normal in HL241, but it is 3-4-fold decreased in HL243. Although the results of the in vitro and in vivo assays differ, they are not in conflict. Rather, they suggest that the static in vitro assay may be capable of revealing subtleties in the productive positioning of LLO and the oligosaccharyl transferase. The decrease in glycosylation seen in intact HL243 cells may be a consequence of the pleiotropic effects of the primary mutation rather than a direct result of the altered LLO structure. Genetic analysis showed that the mutation in HL241 is recessive, while the mutation in HL243 is dominant and prevents normal development. Thus, the two mutants share a lesion in lipid-linked oligosaccharide biosynthesis and in cell-free glycosylation, but differ in their in vivo glycosylation. Their primary defects are probably different.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla 92093.
authors.forename: H H
authors.initials: HH
authors.lastname: Freeze
authors.forename: P
authors.initials: P
authors.lastname: Koza-Taylor
authors.forename: J A
authors.initials: JA
authors.lastname: Jones
authors.forename: W F
authors.initials: WF
authors.lastname: Loomis
mesh_terms: D002474:Cell-Free System; D003902:Detergents; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D004171:Diploidy; D006031:Glycosylation; D006238:Haploidy; D006602:Hexosyltransferases; D007700:Kinetics; D050356:Lipid Metabolism; D008351:Mannans; D008565:Membrane Proteins; D009154:Mutation; D009844:Oligosaccharides / Q000096:biosynthesis*; D013696:Temperature; D014166:Transferases / Q000378:metabolism
chemical_list: D003902:Detergents; D008351:Mannans; D008565:Membrane Proteins; D009844:Oligosaccharides; C064465:mannosyl(6)-N-acetylglucosamine(2); D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase