Abstract
We have examined the synthesis and processing of asparagine-linked oligosaccharides from Aedes albopictus C6/36 mosquito cells. These cells synthesized a glucose-containing lipid-linked oligosaccharide with properties identical to that of Glc3Man9GlcNAc2-PP-dolichol. Results of brief pulse label experiments with [3H]mannose were consistent with the transfer of Glc3Man9GlcNAc2 to protein followed by the rapid removal of glucose residues. Pulse-chase experiments established that further processing of oligosaccharides in C6/36 cells resulted in the removal of up to six alpha-linked mannose residues yielding Man3GlcNAc2 whose structure is identical to that of the trimannosyl "core" of N-linked oligosaccharides of vertebrate cells and yeast. Complex-type oligosaccharides were not observed in C6/36 cells. When Sindbis virus was grown in mosquito cells, Man3GlcNAc2 glycans were preferentially located at the two glycosylation sites which were previously shown to have complex glycans in virus grown in vertebrate cells. These Man3GlcNAc2 structures are the most extensively processed oligosaccharides in A. albopictus, and as such, are analogous to the complex glycans of vertebrate cells. We suggest that determinants of oligosaccharide processing which reside in the polypeptide are universally recognized despite evolutionary divergence of the oligosaccharide-processing pathway between insects and vertebrates.
Highlights
We have examined the synthesis and processing of viewed in Ref. 2)
These same siteisn virus grown in BHK, CEF, and CHceOlls? A study of oligosaccharide processing inc&6 cells reveals that MansGlcNAcpstructures,which arise from highmannose oligosaccharides, constitutethe mostextensively processed oligosaccharides in these cells
When Sindbis virus is grown in mosquito cells, MansGlcNAcn is preferentially located a t two glycosylation sites which have complex-typeoligosaccharides in Sindbis virus grown in vertebrate cells [10]
Summary
We have examined the synthesis and processing of viewed in Ref. 2) These observations suggest that oligosacasparagine-linked oligosaccharides from Aedes albop- charide processing is regulated in somefashion ictus Cs/36 mosquito cells. If oligosaccharide strucshown to havecomplex glycans in virus grownin ver- tures are differenitn insect and vertebratceells, do extentsof tebrate cells These MansGlcNAcz structures are the oligosaccharide processing a t individual glycosylation sites of most extensively processed oligosaccharides in A. al- Cs/36-grown Sindbis virus parallel extents of processing at bopictus, and as such, are analogous to the complex glycans of vertebrate cells. When Sindbis virus is grown in mosquito cells, MansGlcNAcn is preferentially located a t two glycosylation sites which have complex-typeoligosaccharides in Sindbis virus grown in vertebrate cells [10] These results suggest that extentsof oligosaccharide processing are. The abbreviations used are: endo H, endo-8-N-acetylglucosaminidase H; endo D, endo 8-N-acetylglucosaminidase D; BHK, baby hamster kidney; CEF, chicken embryo fibroblasts; CHO, Chinese hamster ovary; EDTA, (ethylenedinitri1o)tetraacetic acid HPLC, high performance liquid chromatography; L-15, Leibovitz-15;MEM, minimal essential medium; MeOH, methanol; Dol-PP, dolichol pyrophosphate
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