Glycobiology Advance Access originally published online on February 5, 2008
Glycobiology 2008 18(4):303-314; doi:10.1093/glycob/cwn008
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Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains
2 Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566
3 Department of Applied Molecular Biosciences, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
1 To whom correspondence should be addressed: Tel: +81-29-861-6160; Fax: +81-29-861-6161; e-mail: jigami.yoshi{at}aist.go.jp
Received on June 6, 2007; revised on January 29, 2008; accepted on January 31, 2008
Development of a heterologous system for the 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 factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the 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 / Fringe / O-fucose / protein O-fucosyltransferase 1 / yeast