Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin.
A cultured cell line of the mosquito, Aedes aegypti, is sensitive to tunicamycin as expected from the ability of crude membrane preparations to catalyse the formation of N-acetylglucosamine-linked dolichyl pyrophosphate. Formation of dolichylphosphomannose was also detected and this reaction was totally insensitive to tunicamycin. Incorporation of radioactive mannose into total acid-precipitable glycoproteins was inhibited greater than 90% in whole cells by tunicamycin, while the incorporation of leucine and glucosamine was less affected. Separation of the radioactive hexosamines from acid hydrolysates of cells incubated with [14C]glucosamine and tunicamycin showed predominant labelling of galactosamine, whereas in control cells not treated with the drug both glucosamine and galactosamine were labelled equally. Evidently, mosquito cells synthesise N-glycosidically linked carbohydrate chains assembled through tunicamycin-sensitive steps involving dolichyl pyrophospho-oligosaccharides, and O-glycosidically linked chains rich in N-acetylgalactosamine, the assembly of which is unaffected by tunicamycin. These results support structural evidence (Butters, T.D. and Hughes, R.C. (1981) Biochim. Biophys. Acta 640, 655-671) for the presence of high mannose N-glycans and N-acetylgalactosamine-rich O-glycans in mosquito cell glycoproteins. The absence of complex N-glycans was confirmed by the demonstration of negligible activities of N-acetylglucosaminyl-, galactosyl- and sialyltransferases responsible for assembly of the terminal sequences of N-glycans of mature mammalian glycoproteins.
D000330:Aedes / Q000378:metabolism*; D000818:Animals; D050260:Carbohydrate Metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005944:Glucosamine / Q000031:analogs & derivatives*; D006023:Glycoproteins / Q000096:biosynthesis*; D007700:Kinetics; D037102:Lectins; D008565:Membrane Proteins / Q000096:biosynthesis*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology*
D006023:Glycoproteins; D037102:Lectins; D008565:Membrane Proteins; D014415:Tunicamycin; D011092:Polyethylene Glycols; D017830:Octoxynol; D005944:Glucosamine