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

• Published in: Glycobiology (Oxford) 2008-04, Vol.18 (4), p.303-314
• Main Authors: Chigira, Yuko, Oka, Takuji, Okajima, Tetsuya, Jigami, Yoshifumi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Carbohydrate Sequence; Efficiency; EGF domain; Epidermal Growth Factor - chemistry; Epidermal Growth Factor - metabolism; Fringe; Fucosyltransferases - genetics; Fucosyltransferases - metabolism; Glycosylation; Hexosyltransferases - genetics; Hexosyltransferases - metabolism; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; Lectins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Models, Biological; Molecular Sequence Data; Monosaccharide Transport Proteins - genetics; O-fucose; Organisms, Genetically Modified; Protein Engineering; protein O-fucosyltransferase 1; Protein Processing, Post-Translational - genetics; Protein Structure, Tertiary; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Signal Transduction - genetics; yeast
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/cwn008

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.