The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc-GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galβ1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galβ1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and β-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups.

Carbohydrate–protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6±1.1 pN and 7.1±1.1 pN were determined for the MUC1(Tn)—MGL and MUC1(STn)—MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27.1±4.4 and 36.9±3.6 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.

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