Effect of Site‐Specific O ‐Glycosylation on the Structural Behavior of NOTCH1 Receptor Extracellular EGF‐like Domains 11 and 10

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.