Depletion of manganese within the secretory pathway inhibits O-linked glycosylation in mammalian cells.
Proteins transiting the secretory pathway are posttranslationally modified by addition of oligosaccharides to asparagine N-linked and serine and threonine O-linked residues. The effects of divalent cation depletion on oligosaccharide processing of erythropoietin (EPO) and macrophage colony stimulating factor (M-CSF) were studied in Chinese hamster ovary cells. Treatment with A23187 did not inhibit M-CSF or EPO secretion but did inhibit addition of complex N-linked and O-linked oligosaccharides to both molecules. Similar results were obtained by treatment with thapsigargin, a potent inhibitor of the Ca(2+)-activated microsomal ATPase, indicating that the effect was due to depletion of divalent cations within the secretory pathway. Whereas addition of extracellular calcium chloride did not reverse the inhibition in complex N-linked and O-linked glycosylation, addition of manganese chloride partially reversed both defects. These results are consistent with a specific manganese requirement within the secretory pathway for the processing of complex N-linked oligosaccharides and the addition of O-linked oligosaccharides. Since there are no known specific inhibitors of O-linked glycosylation, the use of ionophores should significantly facilitate studies on the requirement and role of O-linked oligosaccharides in protein structure and function.
Kaufman
R J
RJ
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109.
Swaroop
M
M
Murtha-Riel
P
P
D000818:Animals; D016466:CHO Cells; D000001:Calcimycin / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002236:Carbohydrate Conformation; D002460:Cell Line; D002712:Chlorides / Q000494:pharmacology; D006224:Cricetinae; D004921:Erythropoietin / Q000378:metabolism*; D006031:Glycosylation; D016173:Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D008345:Manganese / Q000502:physiology*; D017895:Manganese Compounds / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002712:Chlorides; D017895:Manganese Compounds; D009844:Oligosaccharides; D013729:Terpenes; D004921:Erythropoietin; D000001:Calcimycin; D008345:Manganese; D019284:Thapsigargin; D016173:Macrophage Colony-Stimulating Factor; D000252:Calcium-Transporting ATPases; C025340:manganese chloride