title: Identification of novel α1,3-galactosyltransferase and elimination of α-galactose-containing glycans by disruption of multiple α-galactosyltransferase genes in Schizosaccharomyces pombe.
abstract: The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of D-galactose (Gal) in addition to D-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1(+)-otg3(+) (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2(+) gene product had Gal transfer activity toward a pyridylaminated Man(9)GlcNAc(2) oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3(+) gene product exhibited Gal transfer activity toward the pyridylaminated Man(9)GlcNAc(2) oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, (1)H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides.
authors.affiliation: Department of Bioscience and Biotechnology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Ohashi
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.forename: Kaoru
authors.initials: K
authors.lastname: Takegawa
mesh_terms: D000595:Amino Acid Sequence; D005690:Galactose / Q000737:chemistry*; D005700:Galactosyltransferases / Q000096:biosynthesis* / Q000737:chemistry*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D016681:Genome, Fungal; D037102:Lectins / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry*; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D037102:Lectins; D009844:Oligosaccharides; D011134:Polysaccharides; D005700:Galactosyltransferases; C047006:N-acetyllactosaminide alpha-1,3-galactosyltransferase; D005690:Galactose