Engineering of mucin-type human glycoproteins in yeast cells
Engineering of mucin-type human glycoproteins in yeast cells
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DOI：
10.1073/pnas.0710412105
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发表时间：
2008-03-04
影响因子：
11.1
通讯作者：
Narimatsu, Hisashi
中科院分区：
文献类型：
--
作者：
Amano, Koh;Chiba, Yasunori;Narimatsu, Hisashi
粘蛋白型o -聚糖是哺乳动物细胞中发现的最典型的o -聚糖，具有许多不同的生物学作用。在这里，我们报告了一种能够产生粘蛋白型糖链的基因工程酵母菌株。将枯草芽孢杆菌（Bacillus subtilis） UDP-Gal/GaINAc 4- epimase、人UDP-Gal/GaINAc转运体、人ppGalNAc-T1和果蝇（Drosophila melanogaster） core1 β 1-3 GalT编码基因导入酿酒酵母（Saccharomyces cerevisiae）。这种工程酵母能够产生一种含有o -聚糖的MUC1a肽，以及一种类似粘蛋白的糖蛋白，人足蛋白（hPod，也称为aggrus），这是一种血小板聚集因子，需要唾液核结构才能发挥活性。体外唾液化后，酵母hPod可诱导血小板聚集。有趣的是，将ppGalNAc-T1替换为ppGalNAc-T3会导致血小板聚集诱导活性的丧失，尽管在凝集素微阵列上两种hPod蛋白中都检测到sialyl-core1。在培养基中添加罗丹宁-3-乙酸衍生物，可抑制大多数o -甘露糖基化（酵母中常见的一种修饰）对MUC1a的修饰。我们在这里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，并且在基础研究和制药应用中具有潜在的用途。
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/GaINAc 4-epimerase, human UDP-Gal/GaINAc transporter, human ppGalNAc-T1, and Drosophila melanogaster core1 beta 1-3 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.