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.
Highlights
We searched for unidentified ␣1,3-galactosyltransferases in Schizosaccharomyces pombe and identified three novel genes
The proteins encoded by the otg genes belong to glycosyltransferase family 8 (GT8) of the Carbohydrate-Active EnZymes (CAZy) database [35] and possess a DXD motif that is involved in divalent cation binding necessary for sugar-nucleotide binding
Otg Proteins Are Involved in ␣1,3-Galactosylation of O- and N-Linked Glycans—To examine the effects of deleting the otg1ϩ–otg3ϩ genes, the O-linked glycans were released from glycoproteins from wild-type, gms1⌬, 7GalT⌬, and 10GalT⌬ cells, fluorescently labeled by 2-aminopyridine, and were analyzed by size-fractionation HPLC (Fig. 4)
Summary
We searched for unidentified ␣1,3-galactosyltransferases in Schizosaccharomyces pombe and identified three novel genes (otg1ϩ–otg3ϩ). Such yeast-specific outer chain structures and the fission yeast-specific ␣-linked Gal residues may cause rapid removal of the corresponding glycoproteins from the blood stream or may provoke an immune response in humans [14] and, must be eliminated To this end we have been analyzing the precise glycan structures in S. pombe glycosylation mutants to allow use of this organism as an alternative glycoprotein-producing host [15,16,17,18,19,20,21,22,23]. By searching for putative glycosyltransferases in the S. pombe genome, we found three novel uncharacterized genes, otg1ϩ–otg3ϩ (␣ one, threegalactosyltransferase) Disruption of these three genes in the 7GalT⌬2 mutant resulted in a strain in which 10 ␣-galactosyltransferase genes had been deleted (10GalT⌬) and caused a complete loss of ␣-galactosylation in its glycan. In vitro enzymatic assay revealed that Otg2p had ␣-Gal transfer
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