Novel Schizosaccharomyces pombe N-linked GalMan(9)GlcNAc isomers

Abstract

Schizosaccharomyces pombe synthesizes very large N-linked galactomannans, which are elongated from the Man(9)GlcNAc(2) core that remains after the trimming of three Glc residues from the Glc(3)Man(9)GlcNAc(2) originally transferred from dolichyl pyrophosphate to nascent proteins in the endoplasmic reticulum, Prior to elongation of the galactomannan outer chain, the Man(9)GlcNAc(2) core is modified into a family of Hex(10-15)GlcNAc(2) structures by the addition of both Gal and Man residues (Ziegler ef nl, (1994) J, Biol, Chem., 269, 12527-12535), To understand the pathway of Man(9)GlcNAc(2) modification, the Hex(10)GlcNAc-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 Hex(10)GlcNAc present, respectively. Structures of the glycan isomers present in each fraction were determined by one- and two-dimensional H-1 NMR spectroscopy techniques. Fraction a is principally (similar to 93%) a Man(10)GlcNAc with a new alpha 1,2-linked Man cap on the upper-arm of Man(9)GlcNAc, Fraction b contained two isomers of GalMan(9)GlcNAc in which an alpha 1,2-linked terminal Gal had been added either to the upper (b(1), 30%) or middle-arm (bz, 70%) of Man(9)GlcNAc, The gma12(-) alpha 1,2-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 b(1) and b(2), Isomer c is Man(10)GlcNAc in which a new branching alpha 1,6-linked Man had been added to the lower-arm alpha 1,3-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 (g(1), 85%) is the Man(9)GlcNAc core with an alpha 1,3-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; Mana alpha 1,2 (Gal alpha 1,3)Man alpha 1,2Mannitol (Gemmill and Trimble (1999) Glycobiology, 9, 507-515), The second isomer (g(2), 15%) is the partially processed Glc(2)Man(9)GlcNAc intermediate. Defining these Hex(10)GlcNAc structures provides a starting point for understanding the enzymology of N-linked galactomannan core heterogeneity seen on S.pombe glycoproteins.