Ohashi Takao, Ikeda Yuka, Tanaka Naotaka, Nakakita Shin-ichi, Natsuka Shunji, Giga-Hama Yuko, Takegawa Kaoru
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Japan.
Biosci Biotechnol Biochem. 2009 Feb;73(2):407-14. doi: 10.1271/bbb.80712. Epub 2009 Feb 7.
Unlike the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe synthesizes large outer chains on the N-linked oligosaccharides that consist mainly of D-Gal and D-Man residues. The fission yeast och1(+) gene product has alpha1,6-mannosyltransferase activity, and Och1p is the key enzyme in the initiation of outer chain elongation. Although the in vitro substrate specificity of S. pombe Och1p has been reported (Yoko-o et al., FEBS Lett., 489, 75-80 (2001)), the structure of the N-linked oligosaccharides of och1Delta cells has not been investigated. In this study, we report a structural analysis of S. pombe N-linked oligosaccharides. Lectin blot analysis indicated that galactose residues were attached to the cell surface glycoproteins of the och1Delta cells. We conducted a structural analysis of pyridylaminated N-linked oligosaccharides prepared from galactomannoproteins by HPLC and (1)H NMR. These analyses revealed that the N-linked oligosaccharides of the och1Delta cells displayed heterogeneity in the glycan consisting of Hex(11-15)GlcNAc(2). The structural heterogeneity arose mainly from the addition of alpha1,2- and alpha1,3-Gal residues to the Man(9)GlcNAc(2) core structure.
与出芽酵母酿酒酵母不同，裂殖酵母粟酒裂殖酵母在N-连接寡糖上合成主要由D-半乳糖和D-甘露糖残基组成的大外链。裂殖酵母och1(+)基因产物具有α1,6-甘露糖基转移酶活性，Och1p是外链延伸起始的关键酶。尽管已报道了粟酒裂殖酵母Och1p的体外底物特异性（Yoko-o等人，《欧洲生物化学学会联合会快报》，489，75 - 80（2001）），但尚未研究och1Δ细胞N-连接寡糖的结构。在本研究中，我们报告了粟酒裂殖酵母N-连接寡糖的结构分析。凝集素印迹分析表明，半乳糖残基附着于och1Δ细胞的细胞表面糖蛋白。我们通过高效液相色谱和（1）H核磁共振对从半乳甘露糖蛋白制备的吡啶胺化N-连接寡糖进行了结构分析。这些分析表明，och1Δ细胞的N-连接寡糖在由己糖（11 - 15）GlcNAc（2）组成的聚糖中表现出异质性。结构异质性主要源于在Man(9)GlcNAc(2)核心结构上添加了α1,2-和α1,3-半乳糖残基。
