Chemical Synthesis Demonstrates That Dynamic O‑Glycosylation Regulates the Folding and Functional Conformation of a Pivotal EGF12 Domain of the Human NOTCH1 Receptor

The interaction of the human NOTCH1 receptor and its ligands is a crucial step in initiating the intracellular signal transductions, in which O-glycosylation of the extracellular EGF-like domain strongly affects multiple aspects of cell differentiation, development, and cancer biology. However, consequences of biosynthetic O-glycosylation processes in the endoplasmic reticulum (ER) and Golgi on the folding of EGF domains remain unclear. Synthetic human NOTCH1 EGF12 modules allow for new insight into the crucial roles of O-glycosylation in the folding and conformation of this pivotal domain. Here, we show for the first time that predominant O-glucosylation at Ser458 facilitates proper folding of the EGF12 domain in the presence of calcium ion, while the nonglycosylated linear EGF12 peptide affords large amounts of misfolded products (>50%) during <i>in vitro</i> oxidative folding. Strikingly, O-fucosylation at Thr466 prior to O-glucosylation at Ser458 totally impedes folding of EGF12 independent of calcium ion, whereas modification of the Fucα1→ moiety with β-linked GlcNAc dramatically enhances folding efficiency. In addition, we elicit that extension of the Glcβ1→ moiety with xyloses is a negative-regulation mechanism in the folding of EGF12 when synthesis of a trisaccharide (Xylα1→3­Xylα1→3­Glcβ1→) dominates over the posttranslational modification at Thr466. Comprehensive nuclear magnetic resonance studies of correctly folded EGF12 modules demonstrate that noncovalently bonded bridges between sugars and peptide moieties, namely sugar bridges, contribute independently to the stabilization of the antiparallel β-sheet in the ligand-binding region. Our results provide evidence that the dynamic O-glycosylation status of the EGF12 domain elaborated in the ER and Golgi strongly affects folding and trafficking of the human NOTCH1 receptor

Convergent Solid-Phase Synthesis of Macromolecular MUC1 Models Truly Mimicking Serum Glycoprotein Biomarkers of Interstitial Lung Diseases

Synthetic macromolecular MUC1 glycopeptides have been used to unravel molecular mechanisms in antibody recognition of disease-specific epitopes. We have established a novel synthetic strategy for MUC1 tandem repeats having complex O-glycosylation states at each repeating unit based on convergent solid-phase fragment condensation under microwave irradiation. We have accomplished the synthesis of 77 amino acid MUC1 glycopeptides (MW = 12 759) having three major antigenic O-glycoforms [Tn, core 1 (T), and core 2 structures] at 10 designated positions out of 19 potential O-glycosylation sites. We demonstrate that the macromolecular MUC1 glycopeptide displaying the essential glycopeptidic neoepitope Pro-Asp-Thr­(sialyl-T)-Arg-Pro-Ala-Pro at two different tandem repeats is an excellent serum MUC1 model showing ideal stoichiometric binding with anti-KL6/MUC1 antibody in the sandwich ELISA to quantify human serum KL6/MUC1 levels as a critical biomarker of interstitial lung diseases