Amano Koh, Chiba Yasunori, Kasahara Yoshiko, Kato Yukinari, Kaneko Mika Kato, Kuno Atsushi, Ito Hiromi, Kobayashi Kazuo, Hirabayashi Jun, Jigami Yoshifumi, Narimatsu Hisashi
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.
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3232-7. doi: 10.1073/pnas.0710412105. Epub 2008 Feb 22.
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.
粘蛋白型O-聚糖是哺乳动物细胞中最典型的O-聚糖，具有多种不同的生物学功能。在此，我们报道了一种能够产生粘蛋白型糖链的基因工程酵母菌株。将编码枯草芽孢杆菌UDP-半乳糖/ N-乙酰半乳糖胺4-表异构酶、人UDP-半乳糖/ N-乙酰半乳糖胺转运蛋白、人ppGalNAc-T1和果蝇core1 β1-3半乳糖基转移酶的基因导入酿酒酵母。该工程酵母能够产生含有O-聚糖的MUC1a肽以及一种粘蛋白样糖蛋白——人血小板反应蛋白（hPod；也称为聚集素），hPod是一种血小板聚集因子，其活性需要唾液酸化-core1结构。体外唾液酸化后，酵母来源的hPod能够诱导血小板聚集。有趣的是，尽管在凝集素微阵列上两种hPod蛋白中均能检测到唾液酸化-core1，但用ppGalNAc-T3替代ppGalNAc-T1会导致血小板聚集诱导活性丧失。通过在培养基中添加罗丹宁-3-乙酸衍生物，可抑制酵母中常见的对MUC1a的O-甘露糖基化修饰。我们在此描述的酵母系统能够产生在不同糖基化位点修饰的糖蛋白，具有用于基础研究和药物应用的潜力。
