Detection of the lipid‐linked precursor oligosaccharide of N‐linked protein glycosylation in Drosophila melanogaster

Abstract

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...

Drosophila UDP-glucose:glycoprotein glucosyltransferase: sequence and characterization of an enzyme that distinguishes between denatured and native proteins.

“…The Drosophila extracellular matrix proteins that acted as excellent substrates for DUGT in vitro, had probably encountered DUGT during their assembly within the ER of the Kc 7E10 cells. Glucosylated high mannose dolichollinked oligosaccharide precursors have been identified in Drosophila, though the N-linked glycosylation pathway has not been studied extensively (Parker et al, 1991). Most, or all, of the N-linked oligosaccharides of the secreted proteins that we used are of the high mannose type, as digestion with either endoglycosidase H or F cleaved the individual proteins to a similar size.…”

Dynamic Developmental Elaboration of N-Linked Glycan Complexity in the Drosophila melanogaster Embryo

“…Previous studies of Drosophila N-linked glycans have documented the abundance of the high mannose oligosaccharides, M3N2-M9N2, and a single fucosylated paucimannose glycan, M3N2F, in adult and larval tissues (15,17,25,27,55). Minor paucimannose glycans with additional core fucosylation have also been characterized (16,21).…”

Protein N-Glycosylation in the Baculovirus-Insect Cell System

“…It is generally accepted that the insect N-glycosylation pathway begins like the mammalian pathway, with en bloc transfer of the oligosaccharide precursor from Glc 3 Man 9 GlcNAc 2 -PP-Dol to an appropriate asparagine residue in a newly synthesized polypeptide [20]. This conclusion is strongly supported by studies documenting the presence of the lipid-linked Nglycan in insect cell systems [50][51][52]. Several lines of biochemical evidence suggest that removal of the terminal glucose residues from Glc 3 Man 9 GlcNAc 2 is also accomplished in the same way in insect and mammalian cells.…”