Peptide O-glycosylation relies on the coordinated action of glycosyltransferases across the endoplasmic reticulum (ER) and Golgi apparatus. However, the molecular mechanisms driving aberrant glycosylation in cancer remain poorly understood. Here we show an in vitro one-pot synthetic biology approach that simulates divergent glycosylation pathways to map the synthesis of mucin 1 (MUC1) tumor-associated antigens. By modeling the cancer-associated relocation of initiation enzymes (GALNTs) to the ER, we demonstrate that this spatial shift leads to complete GalNAc (Tn antigen) occupancy. This occurs because ER localization extends reaction times and prevents inhibition by downstream Golgi enzymes. Furthermore, combined kinetic and computer reaction dynamic simulations reveal that ST6GALNAC1 exclusively drives α−2-6 sialylation, with a strict preference for the T13 site on fully glycosylated MUC1. This suggests that cancer-associated sTn upregulation is directly linked to T13 occupancy. Ultimately, this systems modeling approach decodes the enzyme localization and substrate specificities fundamental to tumourigenesis. Here, an in vitro model shows cancer-linked relocation of GALNTs to the ER drives complete mucin 1 Tn antigen occupancy. ST6GALNAC1 then selectively sialylates the T13 site, driving tumourigenesis.
Nashed et al. (Wed,) studied this question.