Novel Schizosaccharomyces pombe N-linked GalMan₉GlcNAc isomers: role of the Golgi GMA12 galactosyltransferase in core glycan galactosylation

Schizosaccharomyces pombe synthesizes very large N-linked galactomannans, which are elongated from the Man₉GlcNAc₂ core that remains after the trimming of three Glc residues from the Glc₃Man₉GlcNAc₂ originally transferred from dolichyl pyrophosphate to nascent proteins in the endoplasmic reticulum. Prior to elongation of the galactomannan outer chain, the Man₉GlcNAc₂ core is modified into a family of Hex₁₀₋₁₅GlcNAc₂ structures by the addition of both Gal and Man residues (Ziegler et al. (1994) J. Biol. Chem., 269, 12527–12535). To understand the pathway of Man₉GlcNAc₂ modification, the Hex₁₀GlcNAc-sized pool was isolated by Bio-Gel P-4 gel filtration from the endo H-released N-glycans of S.pombe glycoproteins. This pool yielded four major fractions, a, b, c, and g, on preparative high pH, anion exchange chromatography, that represented 10, 29, 46, and 13% of the total Hex₁₀GlcNAc present, respectively. Structures of the glycan isomers present in each fraction were determined by one- and two-dimensional ¹H NMR spectroscopy techniques. Fraction a is principally (∼93%) a Man₁₀GlcNAc with a new α1,2-linked Man cap on the upperarm of Man₉GlcNAc. Fraction b contained two isomers of GalMan₉GlcNAc in which an α1,2-linked terminal Gal had been added either to the upper (b₁, 30%) or middle-arm (b₂, 70%) of Man₉GlcNAc. The gma12⁻α1,2-galactosyltransferase-negative S.pombe strain (Chappell et al. (1994) Mol. Biol. Cell., 5, 519–528) did not make fraction b implying that the gma12p galactosyltransferase is responsible for synthesis of both isomers b₁ and b₂. Isomer c is Man₁₀GlcNAc in which a new branching α1,6-linked Man had been added to the lowerarm α1,3-linked core residue as found earlier in Saccharomyces cerevisiae and Pichia pastoris. Fraction g had less than molar stoichiometry of both Gal and Glc. The major isomer (g₁, 85%) is the Man₉GlcNAc core with an α1,3-linked branching Gal on the penultimate 2-O-substituted Man of the lower arm. This residue is also found on a novel O-linked oligosaccharide recently described in S.pombe; Manα1,2 (Galα1,3)Manα1,2Mannitol (Gemmill and Trimble (1999)Glycobiology, 9, 507–515). The second isomer (g₂, 15%) is the partially processed Glc₂Man₉GlcNAc intermediate. Defining these Hex₁₀GlcNAc structures provides a starting point for understanding the enzymology of N-linked galactomannan core heterogeneity seen on S.pombe glycoproteins.