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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Engineering of mucin-type human glycoproteins in yeast cells
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Engineering of mucin-type human glycoproteins in yeast cells

机译：酵母细胞中粘蛋白型人糖蛋白的工程改造

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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 β1-3 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 ppGal-NAc-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.
机译：粘蛋白型O-聚糖是在哺乳动物细胞中发现的最典型的O-聚糖，并承担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。将编码枯草芽孢杆菌UDP-Gal / GalNAc 4-表异构酶，人UDP-Gal / GalNAc转运蛋白，人ppGalNAc-T1和果蝇果蝇core1β1-3GalT的基因引入酿酒酵母中。经过改造的酵母能够产生含有O-聚糖和粘蛋白样糖蛋白的人Podoplanin（hPod；也称为aggrus）的MUC1a肽，这是一种血小板聚集因子，需要唾液酸-core1结构才能发挥活性。体外唾液酸化后，酵母中的hPod可以诱导血小板聚集。有趣的是，用ppGalNAc-T3替代ppGal-NAc-T1会导致血小板聚集诱导活性的丧失，尽管事实是在凝集素微阵列上的两种hPod蛋白中均可检测到唾液酸-core1。通过在培养基中添加若丹宁-3-乙酸衍生物，可抑制大多数O-甘露糖基化（一种在酵母中常见的修饰）对MUC1a的作用。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，并具有在基础研究和药物应用中的潜力。

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