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Glycobiology Advance Access published online on February 5, 2008
Glycobiology, doi:10.1093/glycob/cwn008
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains

Yuko Chigira1, Takuji Oka1, Tetsuya Okajima2 and Yoshifumi Jigami1

1 Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan
2 Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Address correspondence to: Yoshifumi Jigami, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan, Tel: +81 29 861 6160; Fax: +81 29 861 6161; E-mail: jigami.yoshi{at}aist.go.jp

Received on June 6, 2007; accepted on January 31, 2008

Development of a heterologous system for production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factor VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

Key words: EGF domain / protein O-fucosyltransferase 1 / O-fucose / yeast / Fringe


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