Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae.
The periplasmic invertase was purified from Saccharomyces cerevisiae och1::LEU2 disruptant cells (delta och1), which have a defect in elongation of the outer chain attached to the N-linked core oligosaccharides (Nakayama, K., Nagasu, T., Shimma, Y., Kuromatsu, J., and Jigami, Y. (1992) EMBO J. 11, 2511-2519). Structural analysis of the pyridylaminated (PA) neutral oligosaccharides released by hydrazinolysis and N-acetylation confirmed that the och1 mutation causes a complete loss of the alpha-1,6-polymannose outer chain, although the PA oligosaccharides (Man9GlcNAc2-PA and Man10GlcNAc2-PA), in which one or two alpha-1,3-linked mannose(s) attached to the endoplasmic reticulumn (ER)-form core oligosaccharide (Man8GlcNAc2) were also detected. Analysis of the delta och1 mnn1 strain oligosaccharides released from total cell mannoprotein revealed that the delta och1 mnn1 mutant eliminates the alpha-1,3-mannose attached to the core and accumulates predominantly a single ER-form oligosaccharide species (Man8GlcNAc2), suggesting a potential use of this strain as a host cell to produce glycoproteins containing mammalian high mannose type oligosaccharides. The delta och1 mnn1 alg3 mutants accumulated Man5GlcNAc2 and Man8GlcNAc2 in total cell mannoprotein, confirming the lack of outer chain addition to the incomplete corelike oligosaccharide and the leaky phenotype of the alg3 mutation. All the results suggest that the OCH1 gene encodes an alpha-1,6-mannosyltransferase that is functional in the initiation of alpha-1,6-polymannose outer chain addition to the N-linked core oligosaccharide (Man5GlcNAc2 and Man8GlcNAc2) in yeast.
Nakanishi-Shindo
Y
Y
National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Nakayama
K
K
Tanaka
A
A
Toda
Y
Y
Jigami
Y
Y
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005656:Fungal Proteins / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry; D008358:Mannose / Q000737:chemistry*; D008364:Mannosyltransferases / Q000235:genetics; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D043324:beta-Fructofuranosidase
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C075636:OCH1 protein, S cerevisiae; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D008364:Mannosyltransferases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D008358:Mannose