Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains.
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.
Chigira
Yuko
Y
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki.
Oka
Takuji
T
Okajima
Tetsuya
T
Jigami
Yoshifumi
Y
D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D002240:Carbohydrate Sequence; D004526:Efficiency; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases; D006031:Glycosylation; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D037102:Lectins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D030781:Organisms, Genetically Modified; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics*
D029681:Arabidopsis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D037102:Lectins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; C426650:SLC35C1 protein, human; C476110:mur1 protein, Arabidopsis; D004815:Epidermal Growth Factor; D005647:Fucosyltransferases; D005964:Glucosyltransferases; D006602:Hexosyltransferases; C106516:MFNG protein, human