N- and O-linked oligosaccharides completely lack galactose residues in the gms1och1 mutant of Schizosaccharomyces pombe

Abstract

Unlike their counterparts in budding yeast Saccharomyces cerevisiae, the glycoproteins of Schizosaccharomyces pombe contain, in addition to alpha-D-mannose (Man), a large number of alpha-D-galactose (Gal) residues. In both yeasts, large outer chains are attached to the oligosaccharide cores of glycoproteins during export via Golgi. Formation of the yeast-specific large outer chain is initiated by alpha-1,6-mannosylatransferase encoded by the och1+ gene, the disruption of which blocked outer chain elongation. We previously reported that N-linked oligosaccharide structures of S. pombe och1Delta mutant consisted of Gal(2-6)Man(9)GlcNAc(2) with alpha-linked Gal residues attached to the core oligosaccharide moiety. The disruption of gms1+, a gene encoding the UDP-galactose transporter required for the synthesis of galactomannan, abolished cell surface galactosylation in S. pombe. In this study, we constructed a gms1Deltaoch1Delta double mutant and determined the N- and O-linked oligosaccharide structures present on the cell surface. Oligosaccharides were liberated from glycoproteins by hydrazinolysis and labeled with the fluorophore, 2-aminopyridine. The pyridylaminated N-linked oligosaccharides were analyzed by high-performance liquid chromatography in combination with alpha1,2-mannosidase digestion and partial acetolysis. These analyses revealed that the N-linked oligosaccharides of gms1Deltaoch1Delta cells consisted of alpha1,2-linked Man-extended core oligosaccharides (Man(8-12)GlcNAc2) from which the fission yeast-specific alpha-linked Gal residues were completely absent.