Engineering of mucin-type human glycoproteins in yeast cells.
Mucin-type O-glycans are the most typical O-glycans found in mammalian cells and assume many different biological roles. Here, we report a genetic engineered yeast strain capable of producing mucin-type sugar chains. Genes encoding Bacillus subtilis UDP-Gal/GalNAc 4-epimerase, human UDP-Gal/GalNAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 beta1-3 GalT were introduced into Saccharomyces cerevisiae. The engineered yeast was able to produce a MUC1a peptide containing O-glycan and also a mucin-like glycoprotein, human podoplanin (hPod; also known as aggrus), which is a platelet-aggregating factor that requires a sialyl-core1 structure for activity. After in vitro sialylation, hPod from yeast could induce platelet aggregation. Interestingly, substitution of ppGalNAc-T1 for ppGalNAc-T3 caused a loss of platelet aggregation-inducing activity, despite the fact that the sialyl-core1 was detectable in both hPod proteins on a lectin microarray. Most of O-mannosylation, a common modification in yeast, to MUC1a was suppressed by the addition of a rhodanine-3-acetic acid derivative in the culture medium. The yeast system we describe here is able to produce glycoproteins modified at different glycosylation sites and has the potential for use in basic research and pharmaceutical applications.
Amano
Koh
K
Research Center for Medical Glycoscience and Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Chiba
Yasunori
Y
Kasahara
Yoshiko
Y
Kato
Yukinari
Y
Kaneko
Mika Kato
MK
Kuno
Atsushi
A
Ito
Hiromi
H
Kobayashi
Kazuo
K
Hirabayashi
Jun
J
Jigami
Yoshifumi
Y
Narimatsu
Hisashi
H
D006023:Glycoproteins / Q000096:biosynthesis*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009077:Mucins; D015202:Protein Engineering / Q000379:methods*; D015003:Yeasts / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; D009077:Mucins; D016695:Glycosyltransferases