A quantitative and site-specific chemoenzymatic glycosylation approach for PEGylated MUC1 peptides
A quantitative and site-specific chemoenzymatic glycosylation approach for PEGylated MUC1 peptides
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DOI：
10.1039/c3sc52641k
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发表时间：
2014-01-01
期刊：
影响因子：
8.4
通讯作者：
Schwientek, Tilo
中科院分区：
文献类型：
--
作者：
Bello, Claudia;Farbiarz, Karine;Schwientek, Tilo
完全控制复杂的翻译后修饰(PTM)，如O-糖基化，是测试和了解这些修饰在蛋白质功能中的生物学作用的先决条件。尽管在过去的几年里取得了长足的进步，但高通量和易于使用的合成复杂糖基化多肽的方法仍然缺乏。我们在这里提出了一种有效的方法来制备均一的位点特异性O-糖基化多肽。通过将单分散聚乙二醇(PEG)聚合物临时连接到这些多肽的N-末端，实现了顺序的化学酶糖基化和从反应组分中分离。随后通过蛋白水解法去除部分聚乙二醇组分，可以定量回收均一的O-糖肽，适合作为糖蛋白合成的基础。在这里，我们演示了葡萄糖醛酸化的MUC1变体的制备，MUC1是人类粘蛋白家族中的一个著名成员。合成了均一的O-糖基化的变异体，并将用于研究O-连接的葡萄糖醛酸表位在人类MUC1串联重复序列的功能环境中的作用。
Full control over complex post-translational modifications (PTMs), such as O-glycosylation, is a prerequisite for testing and understanding the biological role of these modifications in protein function. Despite considerable progress over the last years, high throughput and easy-to-use methods for the synthesis of complex glycosylated peptides are still missing. We present here an efficient methodology to produce homogeneous site-specifically O-glycosylated peptides. Sequential chemoenzymatic glycosylation and separation from the reaction components are achieved via the temporary attachment of a monodisperse polyethylene glycol (PEG) polymer to the N-terminus of these peptides. Subsequent proteolytic removal of the PEG moiety allows quantitative recovery of homogeneous O-glycopeptides, suitable as building blocks for glycoprotein synthesis. Here, we demonstrate the preparation of glucuronylated variants of MUC1, a well-known member of the human mucin family. Homogeneously O-glycosylated variants were synthesized and will be used to study the role of O-linked glucuronic acid epitopes within the functional environment of the human MUC1 tandem repeat.