An in vitro one-pot synthetic biology approach to simulating diverging Golgi O-glycosylation of tumor-associated MUC1 from normal tissue MUC1

Peptide O-glycosylation is a non-template-driven process that relies on the coordinated action of glycosyltransferases (GTs) within the endoplasmic reticulum (ER) and Golgi apparatus. An in vitro one-pot synthetic biology approach was developed to investigate the specificity and kinetics of GT O-GalNAc glycosylation that leads to tumor antigen glycoforms of mucin 1 (MUC1). The focus is to experimentally simulate the divergent glycosylation pathways that lead to the synthesis of cancer-associated antigens (Tn, T) and their sialylated derivatives. First, the biosynthetic details of the defining first step of GALNT re-localization from the ER to the Golgi was modeled using the one-pot method. Our findings reveal that an ER enriched with GALNTs results in complete Galnac (Tn) MUC1 site occupancy. This comes about as a function of two pro-cesses that are i) extended GALNT reaction time and ii) prevention of inhibition by subsequent glycosylation enzymes like C1GALT1. The modeling confirms that B3GNT6 has negligible specificity for MUC1 Tn, explaining the absence of core 3 and core 4 structures in MUC1 in both normal and cancerous breast cell lines. Moreover, ST6GALNAC1, and not ST6GALNAC2, is primarily responsible for α-2-6 sialylation of Tn and T antigens. Computer reaction dynamic simulations combined with kinetic experimental analysis show that ST6GALNAC1 prefers fully glycosylated MUC1 but moreover that its preference is the sialyation the S9 and T13 sites in the SAPDTR motif. This is especially the case when MUC1 concentra-tion is great (i.e., highly expressed), suggesting that sTn upregulation on MUC1 in cancer is linked to the occupancy status of S9 and T13 glycosylated sites, that were previously found to be cancer-associated. The results from the one-pot synthesis approach presented here demonstrate its ability to simulate cellular glycosylation within the Golgi-ER. This systems model-ling unpacks the molecular details of enzyme localization and substrate glycan occupancy that is fundamental to the regulatory mechanisms that gives rise to tumor-associated MUC1 antigens.