Graduate School of Life Science and Faculty of Advanced Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo, 001-0021, Japan.
Chemistry. 2020 Sep 25;26(54):12363-12372. doi: 10.1002/chem.202002652. Epub 2020 Aug 28.
Human NOTCH1 receptor contains 36 epidermal growth factor (EGF)-like repeating domains, in which O-glycosylation status of EGF12 domain regulates the interaction with Notch ligands. Our interest is focused on the effect of specific O-glycosylation states on the structural behavior of EGF11 and EGF10, because they appeared to affect molecular mechanism in receptor-ligand interactions by inducing some conformational alterations in these domains and/or the regions connecting two domains. To understand the structural impact of various O-glycosylation patterns on the pivotal EGF-like repeats 10, 11, and 12, we performed chemical synthesis and NMR studies of site-specifically O-glycosylated EGF11 and EGF10. Our strategy enabled us to synthesize four EGF11 and five EGF10 modules. The specific O-glycosylation states affected in vitro folding of EGF10 more than EGF11, while calcium ion had a larger effect on EGF11 folding. Comprehensive NMR studies shed light on the new type "sugar bridges" crosslinking Thr-O-GlcNAc in the consensus sequence C5-X-X-G-X-(T/S)-G-X-X-C6 and an amino acid in the hinge region between the domains, 445Thr-O-GlcNAc-IIe451 in domain 11 and 405Thr-O-GlcNAc-Gln411 in domain 10, respectively.
人类 NOTCH1 受体包含 36 个表皮生长因子（EGF）样重复结构域，其中 EGF12 结构域的 O-糖基化状态调节与 Notch 配体的相互作用。我们的兴趣集中在特定的 O-糖基化状态对 EGF11 和 EGF10 结构行为的影响，因为它们似乎通过在这些结构域和/或连接两个结构域的区域中诱导某些构象改变来影响受体-配体相互作用的分子机制。为了了解各种 O-糖基化模式对关键 EGF 样重复 10、11 和 12 的结构影响，我们对 EGF11 和 EGF10 进行了化学合成和 NMR 研究。我们的策略使我们能够合成四个 EGF11 和五个 EGF10 模块。特定的 O-糖基化状态对 EGF10 的体外折叠影响大于 EGF11，而钙离子对 EGF11 折叠的影响更大。综合 NMR 研究揭示了新型“糖桥”交联 Thr-O-GlcNAc 在保守序列 C5-X-X-G-X-(T/S)-G-X-X-C6 中的作用，以及结构域间铰链区域的氨基酸作用，分别为 11 结构域中的 445Thr-O-GlcNAc-IIe451 和 10 结构域中的 405Thr-O-GlcNAc-Gln411。
