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Noda Institute for Scientiﬁc Research GRANT
2011 Young Investigator Research Grant

Functional analysis of novel glycosyltransferases toward
the remodeling of the ﬁssion yeast glycans
Takao OHASHIInternational Center for Biotechnology, Osaka University

Research aims
The glycans from the ﬁssion yeast Schizosaccharomyces
pombe contain large amounts of a -linked D-galactose (Gal)in addition to a -linked D-mannose (Man) unlike other wellstudied yeasts or human. Alpha galactosylation, which isone of the signiﬁcant characteristics of S. pombe, can be aprimary hindrance to the development of therapeutic glycoprotein production system in this yeast, because a -galactosylation can cause the immune response in human body. To
overcome this problem, the complete elimination of a galactosylation is required. Previously we succeeded ineliminating a -linked Gal residues by disruption of a gms1⫹
gene, which encodes Golgi localized UDP-Gal transporter.
However UDP-Gal transporter activity is required for b 1,4galactosylation processes that produce the biantennarycomplex oligosaccharides in a subsequent humanization
step in S. pombe. As an alternative approach, we constructed a septuple a -galactosyltransferase-related genedisruptant (7GalTD) and then analyzed its glycan structures. The glycan structural analysis of the 7GalTD mutant
revealed that it still had a 1,3-linked Gal residues, indicating the presence of at least one more additional unidentiﬁed
a 1,3-galactosyltransferase1). In this study, we aimed toidentify the novel a 1,3-galactosyltransferase(s) and to construct the S. pombe a -galactosylation null mutant by multiple a -galactosyltransferase gene disruptions.

transferase genes, three genes (designated otg1⫹, otg2⫹ andotg3⫹ for alpha one, three-galactosyltransferase, respectively) with unknown functions were identiﬁed. We disrupted the three genes from the 7GalTD mutant to constructa strain lacking 10 presumptive a -galactosyltransferase
genes (10GalTD). The PA-glycans were prepared from glycoproteins from wild-type, gms1D, 7GalTD and 10GalTD
strains, and were analyzed by size-fractionation HPLC. The
chromatogram of the 10GalTD strain was quite similar tothat of the gms1D strain. In the otg1Dotg2Dotg3D triple disruption strain, the peaks corresponding to tetra-saccharides
containing an a 1,3-Gal residue were absent, but peaks corresponding to di- and tri-saccharides containing an a 1,2Gal residue were still present (Fig. 1).
To assess enzymatic activity of Otg proteins, they wereexpressed in 10GalTD cells. Solubilized membrane preparations from 10GalTD cells in which Otg proteins expressed
were used as the enzyme source (Fig. 2). Assays carried out
with UDP-Gal as donor substrate revealed that the Otg2
protein had Gal transfer activity toward a Man9GlcNAc2-PAand Mana 1,2-Mana 1,2-Man-PA. In addition, the Otg3
protein exhibited Gal transfer activity toward the Man9GlcNAc2-PA. Generation of an a 1,3-linkage was conﬁrmed byHPLC analysis, a -galactosidase digestion analysis, 1HMethods
The ﬁssion yeast strains were cultivated in YES medium
at 30°C and harvested in early stationary phase. Cell surface glycoproteins were extracted by autoclaving at 121°C
for 90 min in citrate buffer followed by precipitation withmethanol. The precipitates were dissolved in hot water, dialyzed and lyophilized. Oligosaccharides were liberated
from glycoproteins by hydrazinolysis, followed by N-acetylation. The reducing ends of the liberated oligosaccharides
were pyridylaminated (PA) as described2). The PA-oligosaccharides were analyzed by size-fractionation HPLC.
Results
In order to identify undiscovered a 1,3-galactosyltransferase(s), we searched for glycosyltransferase genes in theS. pombe genome sequence. Among the predicted glycosyl-Fig. 1. Size-fractionation HPLC of O-linked glycans from 10GalTD
strain.
The chromatograms show O-linked oligosaccharides from each ﬁssion
yeast strain. The vertical bars indicate the elution positions of the standard
Man-PA and previously identiﬁed peaks in wild-type strain. In theschematic structures shown, the vertical and the diagonal bars between theletters indicate a 1,2- and a 1,3-linkages, respectively. Abbreviations: G,galactose; M, mannose; PA, pyridylamino.

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glycosyltransferases with the ability to form a 1,3-linkages
between Gal and a -Man residues. Complete elimination ofa -galactosylation in S. pombe was achieved by multiple
deletions of newly found otg⫹ genes. Moreover, the10GalTD mutant constructed in the present study is likely
to be valuable with respect to subsequent b -galactosylation,
because a -galactosylation was absent, and simultaneously
the Golgi-localized UDP-Gal transporter was still present.
References
Ohashi, T. et al. (2011) Structural analysis of a 1,3-linked galactosecontaining oligosaccharides in Schizosaccharomyces pombe mutants
harboring single and multiple a -galactosyltransferase genes disruptions. Glycobiology. 21: 340–351.
2) Ohashi, T. et al. (2009) The och1 mutant of Schizosaccharomyces
pombe produces galactosylated core structures of N-linked oligosaccharides. Biosci. Biotechnol. Biochem. 73: 407–414.
3) Ohashi, T., Fujiyama, K., and Takegawa, K. (2012) Identiﬁcation ofnovel a 1,3-galactosyltransferase and elimination of a -galactosecontaining glycans by disruption of multiple a -galactosyltransferase
genes in Schizosaccharomyces pombe. J. Biol. Chem. 287: 38866–38875.
1)

Fig. 2. In vitro enzymatic assay of Otg proteins.
The size-fractionation HPLC proﬁles of the reaction mixtures using theacceptor substrate Man9GlcNAc2-PA (A), Mana 1,2Mana 1,2Man-PA (B)and solubilized enzyme extracts from 10GalTD expressing Otg proteins
are shown.

NMR spectroscopy and LC-MS/MS analysis. These results
indicate that Otg2p and Otg3p are involved in a 1,3-galactosylation of S. pombe oligosaccharides.
Conclusion
We have shown for the ﬁrst time that the Otg proteins are