Conclusion
Our findings highlight the crucial role of bisecting GlcNAc in chemoresistance and suggest the possibility of reversing chemoresistance by modulating the specific glycosylation in breast cancer therapy.
Methods
Levels and regulatory roles of bisecting N-acetylglucosamine (GlcNAc) in chemoresistant breast cancer cells were determined in vitro and in vivo. Glycoproteomics guided identification of site-specific bisecting GlcNAc on P-glycoprotein (P-gp). Co-immunoprecipitation coupled mass spectrometry (Co-IP-MS) and proximity labelling MS identified the interactome of P-gp, and the biological function of site-specific bisecting GlcNAc was investigated by site/truncation mutation and structural simulations.
Results
Bisecting GlcNAc levels were reduced in chemoresistant breast cancer cells, accompanied by an enhanced expression of P-gp. Enhanced bisecting GlcNAc effectively reversed chemoresistance. Mechanical study revealed that bisecting GlcNAc impaired the association between Ezrin and P-gp, leading to a decreased expression of membrane P-gp. Bisecting GlcNAc suppressed VPS4A-mediated P-gp recruitment into microvesicles, and chemoresistance transmission. Structural dynamics analysis suggested that bisecting GlcNAc at Asn494 introduced structural constraints that rigidified the conformation and suppressed the activity of P-gp.