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Pablo Argueso, Ana Guzman-Aranguez, Ashley Woodward, Jesus J. Pintor; Inhibition of Mucin O-Glycosylation Promotes Endocytosis and Nanoparticle Uptake in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4394.
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Recent evidence has shown that O-glycans on cell surface-associated mucins contribute to maintaining barrier function by interacting with β-galactoside-binding lectins on the epithelial glycocalyx; however, the mechanisms involved have not been completely characterized. In this work, we have evaluated whether abrogation of O-glycosylation promotes endocytosis and particle uptake in human corneal epithelial (HCLE) cells.
Downregulation of mucin O-glycosylation in HCLE cells was carried out using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1, a critical galactosyltransferase required for the synthesis of core 1 O-glycans. Subcellular membrane vesicles were fractionated by ultracentrifugation in a 5-20% (w/v) continuous gradient of iodixanol. Fractions were analyzed by western blot using a mouse monoclonal MUC16 antibody. HCLE cells were incubated with 0.1 µm carboxylate-modified fluorescent nanospheres, and uptake analyzed by confocal microscopy and fluorometry before and after inhibition of endocytosis. Tight junction integrity was evaluated using transepithelial electrical resistance and ZO-1 staining.
Fractionation of membrane fragments revealed that trafficking of the cell surface mucin MUC16 was altered in cells transfected with c1galt1 shRNA. Moreover, in particle internalization studies, c1galt1 shRNA-transfected cells had a 1.63-fold increase in nanoparticle uptake as compared to scramble control. Nanoparticle internalization was dramatically reduced at 4°C, when active transport processes are blocked, and by sodium azide, a general inhibitor of endocytic processes. Dynasore and nocodazole significantly reduced nanoparticle uptake by 37% and 36%, respectively, suggesting a mechanism for coated pit budding and vesicular trafficking in nanoparticle uptake. The involvement of the clathrin-mediated pathway was supported by a significant decrease in uptake observed with chlorpromazine (44%) and hypertonic media (53%). Downregulation of c1galt1 expression did not decrease the transepithelial electrical resistance or ZO-1 staining, indicating that increased nanoparticle uptake occurs through the transcellular pathway.
These results indicate that mucin O-glycans hinder endocytosis and nanoparticle uptake, and suggest that transient manipulation of the glycocalyx barrier is an alternative approach to delivering therapeutic nanoparticles to the cornea.
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