Schizosaccharomyces pombe produces novel Gal(0-2)Man(1-3) O-linked oligosaccharides

Schizosaccharomyces pombe whole-cell glycoproteins, previously depleted of N-linked glycans by sequential treatment with endo-beta-N-acetylglucosamini dase H and peptide-N-4-asparagine amidohydrolase F, were beta-eliminated wi th 0.1 M NaOH/1 M NaBH4 to release the O-linked oligosaccharides. The sacch aride-alditols were separated by gel-exclusion chromatography into pools fr om Hexitol to Hex(4)Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex(1)Hexitol pool contained two components, Gal alpha 1,2Man-ol (2A) and Man alpha 1,2Ma n-ol (2B), The Hex(2)Hexitol pool contained two components, Gal alpha 1,2Ma n alpha 1,2Man-ol (3A) and Man alpha 1,2Man alpha 1,2Man-ol (3B), The two H ex(3)Hexitol components were Gal alpha 1,2(Gal alpha 1,3)Man alpha 1,2Man-o l (4A) and Man alpha 1,2(Gal alpha 1,3)Man alpha 1,2Man-ol (4B), The Hex(4) Hexitol component was found to be a single isomer with the composition of G al alpha 1,2(Gal alpha 1,3)Man alpha 1,2Man alpha 1,2Man-ol (5AB), Surprisi ngly, galactobiose was not detected in any of these oligosaccharides. The g ma12 (T. G, Chappell and G, Warren (1989) J, Cell Biol,, 109, 2693-2707) an d gth1 (T. G, Chappell personal communication) alpha 1,2-galactosyltransfer ase-deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsibl e for the addition of terminal alpha 1,2-linked Gal in compound 2A, while o ne or both of gma12p and gth1p are required for the alpha 1,2-linked Gal in 4A, Both transferases are largely responsible for terminal Gal in isomer 5 AB, Neither gma12 nor gth1 had any discernible effect on the structure of t he large N-linked galactomannans as determined by H-1 NMR spectroscopy. Thu s, while gth1p and gma12p appear responsible for adding ctl,2-linked Gal to terminal Man, neither adds galactose side chains to the N-linked poly alph a 1,6-Man outerchain, nor the O-linked branch-forming alpha 1,3-linked Gal. Furthermore, the presence of Hex alpha 1,2(Gal alpha 1,3)Man alpha 1,2- st ructures in the O-linked glycans implies the presence of a novel branch-for ming alpha 1,3-galactosyltransferase in S,pombe.