Notch signaling pathway plays key roles on cell developing process. Human NOTCH protein contains 36 EGF-like repeats on extra cellular domain. Each EGF domains have conserved three disulfide bonds. Many EGF domains are glycosylated by α-O-fucose [mature form is Neu5Acα(2→3)Galβ(1→4)GlcNAcβ(1→3)Fucα1→Thr], β-O-glucose [Xylα(1→3)Xylα(1→3)Glcβ1→Ser or Glcβ1→Ser] and β-O-GlcNAc [Neu5Acα(2→3)Galβ(1→4)GlcNAcβ1→Thr]. However, the effects of O-glycosylation on protein structure, especially on its folding process are still unclear. Considering that some variants carrying different and immature glycoforms can be produced during the biosynthetic processes and O-glycosylation site reside between two cysteine residues which participate in disulfide bonds, oxidative formation of three disulfide cross-linkages may be affected significantly by dynamic O-glycosylation alteration. We hypothesized that the efficacy of the accurate folding of the NOTCH1 EGF domain depends on the interaction of individual glycoforms with the proximal peptide regions. To examine this hypothesis, focusing on pivotal EGF domains involving ligand binding site, we synthesized model glycopeptides chemically and in vitro folding reactions from their unstructured status were assessed by monitoring HPLC profiles of the products. In addition, to check the interaction of O-glycans with peptide backbone, we also performed NMR structural analysis of each EGF domain. Our results indicate characteristics of folding processes as well as conformational stabilizations of each EGF domain, which are related to glycosylation states, are quite different. Structural analysis of synthetic EGF domains unveiled underlying novel regulation mechanism in the protein folding.

Dynamic O-glycosylation states regulate the folding and conformational characteristics of individual human NOTCH1 EGF domains

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.