An in vitro approach for simulating divergent Golgi O-glycosylation of tumor-associated MUC1 from normal MUC1
Year: 2026
Type: article
Abstract: Peptide O-glycosylation 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 relocation 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 processes that are i) extended GALNT reaction time and ii) prevention of inhibition by subsequent glycosylation enzymes such as 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 and more importantly that its preference is to sialyate the S9 and T13 sites in the SAPDTR motif. This is especially the case when the MUC1 concentration is high (i.e., high-level of expression), 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 modelling 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.
Source: Nature Communications
Institutions: University of Cape Town, University of Sri Jayewardenepura
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $6,990
Award: G4165398850
OpenAlex ID: W7155175031