Comprehensive Investigations of MUC1 O‑Glycosylation Process Reveal Initial Site Preference by the Polypeptide GalNAc Transferases

Tumor-associated MUC1 is coated with a high density of O-GalNAc glycans, which are initiated by a family of polypeptide N-acetyl-α-galactosaminyltransferases (GalNAc-Ts). However, the O-glycosylation process of MUC1 by each GalNAc-T isoform remains unclear. Here, we successfully obtained 14 human GalNAc-Ts with high catalytic activity based on a bacterial expression system. Employing MUC1-derived peptides as substrates, we systematically investigated the catalytic properties and site specificity of these GalNAc-Ts by chromatography and mass spectrometry, and found that they could be classified into two clusters. These two GalNAc-T clusters initially catalyze the threonine residue within GSTA or GVTS motifs, respectively, resulting in high O-glycosylation occupancy of both motifs. Moreover, molecular dynamics simulations and site-directed mutagenesis confirmed that the initial O-glycosite preference of GalNAc-Ts on MUC1 is controlled by two critical residues within the peptide-binding pocket. Swapping of the corresponding residues between two GalNAc-T clusters could exchange their initial O-glycosite preference. Quantum mechanics calculations further revealed the detailed catalytic mechanisms of GalNAc-Ts. Our work contributes to understanding the catalytic synthesis of multisite O-glycosylation of MUC1 by GalNAc-Ts, facilitating the development of O-glycosite-specific MUC1 vaccines.