Control of N-linked oligosaccharide synthesis: cellular levels of dolichyl phosphate are not the only regulatory factor.
When MDCK cells were incubated in the presence of the protein synthesis inhibitor puromycin or cycloheximide, there was a rapid and concentration-dependent inhibition in the incorporation of [2-3H]mannose into lipid-linked oligosaccharide and into protein. However, mannose incorporation into dolichyl-P-mannose was not affected. Interestingly, these inhibitors did block [6-3H]glucosamine incorporation into dolichyl-PP-GlcNAc as well as into lipid-linked oligosaccharides. Similar results were obtained when other cell lines were used and also when inhibitors of protein glycosylation such as beta-hydroxynorvaline and beta-fluoroasparagine were used. Cells incubated in puromycin did not show any changes in the levels of sugar nucleotides, GDP-mannose or UDP-GlcNAc, or in the in vitro activities of the glycosyltransferases that add mannose to the lipid-linked oligosaccharides. The inhibition of mannose incorporation into lipid-linked oligosaccharides could not be overcome by addition of dolichyl-P to the inhibited cells, even though the addition of dolichyl-P to control cells stimulated mannose incorporation into dolichyl-P-mannose, lipid-linked oligosaccharides, and protein from 3- to 5-fold. Thus, limitations in the levels of dolichyl-P do not appear to be a major factor in this inhibition. On the other hand, addition of the tripeptide acceptor N-acyl-Asn-Try-Thr did overcome the puromycin inhibition to some extent, suggesting that accumulation of some intermediate such as lipid-linked oligosaccharides might be involved in the inhibition.
Pan
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Elbein
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D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D004288:Dolichol Phosphates / Q000378:metabolism* / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism; D006017:Glycolipids / Q000096:biosynthesis; D006023:Glycoproteins / Q000096:biosynthesis; D007668:Kidney; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D010455:Peptides / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
D004288:Dolichol Phosphates; C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D006017:Glycolipids; D006023:Glycoproteins; D009844:Oligosaccharides; D010455:Peptides; D011105:Polyisoprenyl Phosphate Sugars; D011500:Protein Synthesis Inhibitors; C028306:dolichol monophosphate; D004287:Dolichol Monophosphate Mannose; D005964:Glucosyltransferases; D008358:Mannose