| Abstrakt: |
Schizosaccharomyces pombe whole‐cell glycoproteins, previously depleted of N‐linked glycans by sequential treatment with endo‐ß‐N‐acetylglucosaminidase H and peptide‐N4‐asparagine amidohydrolase F, were ß‐eliminated with 0.1 M NaOH/1 M NaBH4 to release the O‐linked oligosaccharides. The saccharide‐alditols were separated by gel‐exclusion chromatography into pools from Hexitol to Hex4Hexitol in size. Analysis of the Hexitol pool indicated Man to be the only sugar linked to Ser or Thr residues. The Hex1Hexitol pool contained two components, Galα1,2Man‐ol (2A) and Manα1,2Man‐ol (2B). The Hex2Hexitol pool contained two components, Galα1,2Manα1,2Man‐ol (3A) and Manα1,2Manα1,2Man‐ol (3B). The two Hex3Hexitol components were Galα1,2(Galα1,3)Manα1,2Man‐ol (4A) and Manα1,2(Galα1,3)Manα1,2Man‐ol (4B). The Hex4Hexitol component was found to be a single isomer with the composition of Galα1,2(Galα1,3)Manα1,2Manα1,2Man‐ol (5AB). Surprisingly, galactobiose was not detected in any of these oligosaccharides. The gma12 (T. G. Chappell and G. Warren (1989) J. Cell Biol., 109, 2693–2707) and gth1 (T. G. Chappell personal communication) α1,2‐galactosyltransferase‐deficient mutants and the gma12/gth1 double mutant S.pombe strains were similarly examined. The results indicated that gma12p is solely responsible for the addition of terminal α1,2‐linked Gal in compound 2A, while one or both of gma12p and gth1p are required for the α1,2‐linked Gal in 4A. Both transferases are largely responsible for terminal Gal in isomer 5AB. Neither gma12 nor gth1 had any discernible effect on the structure of the large N‐linked galactomannans as determined by 1H NMR spectroscopy. Thus, while gth1p and gma12p appear responsible for adding α1,2‐linked Gal to terminal Man, neither adds galactose side chains to the N‐linked poly α1,6‐Man outerchain, nor the O‐linked branch‐forming α1,3‐linked Gal. Furthermore, the presence of Hexα1,2(Galα1,3)Manα1,2‐ structures in the O‐linked glycans implies the presence of a novel branch‐forming α1,3‐galactosyltransferase in S.pombe. [ABSTRACT FROM PUBLISHER] |
