Synthetic Human NOTCH1 EGF Modules Unraveled Molecular Mechanisms for the Structural and Functional Roles of Calcium Ions and O-Glycans in the Ligand-Binding Region
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- 作者：Shun Hayakawa ; Ryosuke Koide ; Hiroshi Hinou ; Shin-Ichiro Nishimura
- 刊名：Biochemistry
- 出版年：2016
- 出版时间：February 9, 2016
- 年：2016
- 卷：55
- 期：5
- 页码：776-787
- 全文大小：750K
- ISSN：1520-4995
文摘
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.