Takeuchi Hideyuki, Haltiwanger Robert S
Department of Biochemistry and Cell Biology, Institute of Cell and Developmental Biology, Stony Brook University, Stony Brook, NY, USA.
Methods Mol Biol. 2013;1022:119-28. doi: 10.1007/978-1-62703-465-4_10.
Epidermal growth factor-like (EGF) repeats are found in numerous extracellular or transmembrane proteins including Notch. EGF repeats containing the appropriate consensus sequences can be modified with two unusual types of glycans: O-fucosylation and O-glucosylation. We have identified the glycosyltransferases that catalyze the addition of the first sugar to these consensus sites: protein O-fucosyltransferase 1 (Pofut1) and protein O-glucosyltransferase (Rumi/Poglut1). Recently, we have demonstrated that Rumi/Poglut1 shows protein O-xylosyltransferase activity as well. Here, we describe how we characterize the enzymatic activity of these enzymes, including preparation of the acceptor substrates, using bacterially expressed EGF repeats.
表皮生长因子样（EGF）重复序列存在于包括Notch在内的众多细胞外或跨膜蛋白中。含有适当共有序列的EGF重复序列可被两种不同寻常的聚糖修饰：O-岩藻糖基化和O-葡萄糖基化。我们已经鉴定出催化第一个糖添加到这些共有位点的糖基转移酶：蛋白O-岩藻糖基转移酶1（Pofut1）和蛋白O-葡萄糖基转移酶（Rumi/Poglut1）。最近，我们还证明Rumi/Poglut1也具有蛋白O-木糖基转移酶活性。在此，我们描述了我们如何表征这些酶的酶活性，包括使用细菌表达的EGF重复序列制备受体底物。
