N- and O-glycosylation and phosphorylation of the bar secretion leader derived from the barrier protease of Saccharomyces cerevisiae.
A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha 1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure.
Jars
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MU
ZymoGenetics, Inc., Seattle, Washington 98102, USA.
Osborn
S
S
Forstrom
J
J
MacKay
V L
VL
D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000378:metabolism*; D005285:Fermentation; D005656:Fungal Proteins / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005656:Fungal Proteins; D021382:Protein Sorting Signals; D010450:Endopeptidases