Hayakawa Shun, Koide Ryosuke, Hinou Hiroshi, Nishimura Shin-Ichiro
Graduate School of Life Science, Hokkaido University , N21, W11, Kita-ku, Sapporo 001-0021, Japan.
Medicinal Chemistry Pharmaceuticals, Company Ltd. , N21, W12, Kita-ku, Sapporo 001-0021, Japan.
Biochemistry. 2016 Feb 9;55(5):776-87. doi: 10.1021/acs.biochem.5b01284. Epub 2016 Jan 27.
The Notch signaling pathway is an evolutionarily highly conserved mechanism that operates across multicellular organisms and is critical for cell-fate decisions during development and homeostasis in most tissues. Notch signaling is modified by posttranslational glycosylations of the Notch extracellular EGF-like domain. To evaluate the structural and functional roles of various glycoforms at multiple EGF domains in the human Notch transmembrane receptor, we established a universal method for the construction of NOTCH1 EGF modules displaying the desired O-glycans at the designated glycosylation sites. The versatility of this strategy was demonstrated by the rapid and highly efficient synthesis of NOTCH1 EGF12 concurrently having a β-D-glucopyranose-initiated glycan (Xylα1 → 3Xylα1 → 3Glcβ1 →) at Ser458 and α-L-fucopyranose-initiated glycan (Neu5Acα2 → 3Galβ1 → 4GlcNAcβ1 → 3Fucα1 →) at Thr466. The efficiency of the proper folding of the glycosylated EGF12 was markedly enhanced in the presence of 5 mM CaCl2. A nuclear magnetic resonance study revealed the existence of strong nuclear Overhauser effects between key sugar moieties and neighboring amino acid residues, indicating that both O-glycans contribute independently to the intramolecular stabilization of the antiparallel β-sheet structure in the ligand-binding region of EGF12. A preliminary test using synthetic human NOTCH1 EGF modules showed significant inhibitory effects on the proliferation and adhesiveness of human breast cancer cell line MCF-7 and lung adenocarcinoma epithelial cell line A549, demonstrating for the first time evidence that exogenously applied synthetic EGF modules have the ability to interact with intrinsic Notch ligands on the surface of cancer cells.
Notch信号通路是一种在进化上高度保守的机制，在多细胞生物中发挥作用，对大多数组织发育和体内平衡过程中的细胞命运决定至关重要。Notch信号通过Notch细胞外EGF样结构域的翻译后糖基化修饰。为了评估人Notch跨膜受体多个EGF结构域中各种糖型的结构和功能作用，我们建立了一种通用方法，用于构建在指定糖基化位点展示所需O-聚糖的NOTCH1 EGF模块。该策略的通用性通过快速高效合成同时在Ser458处具有β-D-吡喃葡萄糖起始聚糖（Xylα1→3Xylα1→3Glcβ1→）和在Thr466处具有α-L-吡喃岩藻糖起始聚糖（Neu5Acα2→3Galβ1→4GlcNAcβ1→3Fucα1→）的NOTCH1 EGF12得以证明。在5 mM CaCl2存在下，糖基化EGF12正确折叠的效率显著提高。核磁共振研究揭示了关键糖部分与相邻氨基酸残基之间存在强烈的核Overhauser效应，表明两种O-聚糖都独立地有助于EGF12配体结合区域中反平行β-折叠结构的分子内稳定。使用合成人NOTCH1 EGF模块的初步测试显示对人乳腺癌细胞系MCF-7和肺腺癌上皮细胞系A549的增殖和黏附具有显著抑制作用，首次证明外源性应用的合成EGF模块有能力与癌细胞表面的内源性Notch配体相互作用。
