The presence of a glycan of the same molecular size as the lipid linked precursor oligosaccharide (Glc,Man,GlcNAc,) of the N-linked protein glycosylation pathway in mammalian cells has been detected in a glycolipid fraction of cultured Drosophila ntefanogaster cells. Oligosaccharide sequencing studies were consistent with the existence of a glucosylated high mannose containing structure, which may be the common precursor for N-linked protein glycosylation in insect cells.N-Linked protein glycosylation; Glycolipid; Lipid-linked precursor oligosaccharide; Metabolic labelling: Drosophila n~ekogonasrer
1, INTRODUCTIONA consensus for the major pathway of N-linked glycosylation in vertebrates has been established /l]. This pathway involves the synthesis of the precursor oligosaccharide, GlucoseJvIannoseJV-Acetylglucosamine, (Glc,Man,GlcNAc2), attached to dolichol, a long chain isoprenoid alcohol. The glpan is transferred en-Hoc to nascent polypeptide chains in the endoplasmic reticulum. The precursor oligosaccharide is processed to give the diversity of final carbohydrate structures seen in mature glycoproteins. It is apparent that the initial synthesis and transfer reaction are the same in all eukaryotes analysed except for Trypanosoma cruzi, Crithidia fasciculata and Leishmania mexicana [2]. The precursor oligosaccharide transferred onto proteins in these organisms lacks the three glucoses. Non-vertebrate eukaryotes also differ in the processing of the precursor oligosaccharides 12-41.In vertebrates processing is begun by the removal of the three glucoses from the precursor oligosaccharide by two enzymes. These are cl-glucosidase 1 (EC. 32.106) which removes the terminal a-l-2 linked glucose, and ol-glucosidase II (EC. 3.2.106, mannosyl-oligosaccharide glucosidase) which then removes the two remaining a-1-3 linked glucoses [1,4]. Our objective is to identify similar processing enzymes in Drosophila melanogaster. However, in the light of observations such as those on the three organisms lacking three terminal glucoses in their precursor, it is necessary to proCorrespondence address: G,F. Parker, Genetics Laboratory, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
58vide biochemical evidence for the existence of a glucosylated precursor oligosaccharide in this organism.
MATERIALS AND METHODSBio-Gel P-4, Dowex AGSOXl2-100, Dowex AG3-X4A and Chelex 100 were obtained from Rio-Rad laboratories, radioactive mannose ([2-'I-I]mannose 16.3 Ci/mmol) was purchased from Amersham Ltd, amino acids and organic buffers rep ccl1 culture were purchased from Sigma. QAE-Sephadex A-25 was obtained from Pharmacia Ltd. (Milton Keynes, UK). Foetal calf serum was supplied by Globepharm (Surrey, UK). Glutamine and trypsin were purchased from Gibco (Gibco/BRL, UK). Yeast extract powder was from Lab M, chloroform and methanol were 'analar' grade supplied by BDH.Porcine liver ce-glucosidase I was prepared from detergent extracts of isolated microsomes and purified using carboxypentyl deoxynojirimicyn-aga...