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-richO-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.