Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis
Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis
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DOI：
10.1093/glycob/cwu089
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发表时间：
2015-01-01
期刊：
影响因子：
4.3
通讯作者：
Clausen, Henrik
中科院分区：
文献类型：
--
作者：
Kong, Yun;Joshi, Hiren J.;Clausen, Henrik
N-乙酰氨基半乳糖转移酶（GalNAc）型（粘蛋白型）O-糖基化是一种丰富且高度多样的蛋白质修饰。这种类型的0-糖基化在高尔基体中由多达20种同源多肽GalNAc-T同工酶的大家族起始，所述同工酶将GalNAc转移至Ser、Thr和可能的Tyr残基。然后，这些GalNAc残基被大量的糖基转移酶进一步延长，以构建各种复杂的O-聚糖结构。确定O-聚糖位点占有率的因素仍然知之甚少，尽管很明显，细胞中单个同工酶的底物特异性和GalNAc-Ts的库是关键参数。GalNAc-T同工酶在细胞和组织中差异表达，原则上允许细胞产生依赖于存在的同工型的特定子集的独特0-糖蛋白组。使用重组表达的GalNAc-Ts对受体肽底物特异性进行体外分析一直是探测单个同种型活性的首选方法，但这些研究受到蛋白质中实际O-糖基化位点的生物学验证和可测试底物数量的阻碍。在这里，我们提出了一个系统的分析10人GalNAc-T同工酶的活性与195肽底物覆盖已知的O-糖基化位点，并提供了一个全面的数据集，用于评估亚型特异性贡献的O-糖蛋白质组。
N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.