Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
Glycobiology. 2010 Mar;20(3):287-99. doi: 10.1093/glycob/cwp173. Epub 2009 Nov 11.
Notch is a key cell surface protein receptor that is a vital component of intercellular signaling occurring during development. The O-glucosylation of the extracellular Notch epidermal growth factor-like (EGF) repeats has recently been found to play an important role in the proper functioning of Notch in Drosophila. Previous efforts to identify the fine structure of the O-glucose-containing glycan of mammalian Notch have been hindered by limitations associated with approaches used to date. Here, we report the development of an alternative strategy that can be used to study this modification from a range of different tissues. To implement this approach, we have generated standards of the D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc trisaccharide, isomers of this structure, as well as the d-Xyl-alpha1-3-d-Glc disaccharide found previously on secreted EGF-containing proteins of the blood coagulation cascade. Following derivatization with 8-aminopyrene-1,3,6-trisulfonate (APTS), we use these standards in capillary electrophoretic analyses of O-glycans released from Notch1 EGF repeats in conjunction with exo-alpha-xylosidase digestion. These studies collectively reveal that the O-glucose-containing glycan decorating mammalian Notch is the D-Xyl-alpha1-3-D-Xyl-alpha1-3-D-Glc trisaccharide; an assignment in accord with previous predictions. Given the demonstrated importance of this modification in the function of Notch in Drosophila, we expect that the identification of this glycan decorating mammalian Notch1 should aid studies into the functional role of O-glycosylation of mammalian Notch isoforms. Wider application of this approach should facilitate identification of other EGF-containing proteins bearing this O-glycan and aid in their study.
Notch 是一种关键的细胞表面蛋白受体，是发育过程中细胞间信号传递的重要组成部分。最近发现，Notch 细胞外表皮生长因子样 (EGF) 重复物的 O-葡糖基化在果蝇 Notch 的正常功能中发挥着重要作用。以前，人们试图确定哺乳动物 Notch 中含有 O-葡萄糖的聚糖的精细结构，但由于迄今为止使用的方法存在局限性，这些努力受到了阻碍。在这里，我们报告了一种替代策略的开发，该策略可用于从多种不同组织中研究这种修饰。为了实施这种方法，我们生成了 D-Xyl-α1-3-D-Xyl-α1-3-D-Glc 三糖、这种结构的异构体以及先前在血液凝固级联的分泌性 EGF 含有蛋白质上发现的 d-Xyl-α1-3-d-Glc 二糖的标准。用 8-氨基芘-1,3,6-三磺酸 (APTS) 衍生化后，我们将这些标准与外切-α-木糖苷酶消化一起用于从 Notch1 EGF 重复物中释放的 O-聚糖的毛细管电泳分析。这些研究共同表明，修饰哺乳动物 Notch 的 O-葡萄糖聚糖是 D-Xyl-α1-3-D-Xyl-α1-3-D-Glc 三糖；这一分配与先前的预测一致。鉴于该修饰在果蝇 Notch 功能中的重要性，我们预计鉴定这种糖基化修饰的哺乳动物 Notch1 应该有助于研究哺乳动物 Notch 同工型的 O-糖基化的功能作用。这种方法的更广泛应用应该有助于鉴定其他含有这种 O-聚糖的 EGF 含有蛋白质，并有助于对其进行研究。
