3-O-methylation of mannose residues. A novel reaction in the processing of N-linked oligosaccharides occurring in Mucor rouxii.

Yeast- and mycelial-form cells of the dimorphic fungus Mucor rouxii incubated with [U-14C]glucose were found to synthesize Man-P-dolichol, Glc-P-dolichol, and Glc3Man9GlcNAc2-P-P-dolichol. The structure of the oligosaccharide moiety of the latter was similar to that of the same compound isolated from other eucaryotic cells. Oligosaccharides that migrated on paper chromatography as Man6-30GlcNAc standards were obtained upon treatment of delipidated proteins with a protease and endo-beta-N-acetylglucosaminidase H. The oligosaccharides that migrated apparently as single substances on paper chromatography could be separated into three different populations by paper electrophoresis in sodium borate buffer. The fastest migrating substances contained only mannose and N-acetylglucosamine residues, whereas the other two contained, in addition, different proportions of 3-O-methylmannose units. The oligosaccharides with the highest content of 3-O-methylmannose residues appeared to be completely resistant to alpha-mannosidase degradation; they were, however, cleaved by endo-beta-N-acetylglucosaminidase H. Mycelial cells synthesized a much higher proportion of 3-O-methylmannose-containing oligosaccharides than yeast cells. Cells incubated with [methyl-14C]methionine were found to label only the N-linked oligosaccharides containing 3-O-methylmannose residues. It is concluded that transfer of Glc3Man9GlcNAc2 to protein is followed by excision of glucose and probably one or two mannose residues, followed by further mannosylation and in some cases also methylation of oligosaccharides. This represents a novel reaction in the processing of N-linked oligosaccharides.