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P. Argueso, Z. Cao, J. Ricciuto, I. K. Gipson, N. Panjwani; MUC16 is a Cell Surface Ligand for Galectin-3 on Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2746.
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© ARVO (1962-2015); The Authors (2016-present)
Galectins are a family of ß-galactoside-binding proteins that may participate in the stabilization of the tear film by interacting with carbohydrates on the corneal epithelial cell surface glycocalyx. The purpose of this study was to determine whether the highly O-glycosylated, cell surface-associated mucin, MUC16, is a counter-receptor for galectin-3 at the ocular surface epithelia.
Assay for galectin-3 in human corneal (HCLE) and conjunctival (HCjE) epithelial cell cultures, as well as in normal tear fluid, was performed by Western blot analysis using a monoclonal rat anti-human galectin-3 antibody. For pull-down assays, HCLE protein extracts in RIPA buffer were incubated with Sepharose-conjugated galectin-3. Bound proteins were eluted with 100 mM ß-lactose, separated by agarose gel electrophoresis, and probed with the OC125 antibody against MUC16. To determine whether secreted galectin-3 is retained on the epithelial cell surface glycocalyx, cell surface proteins on apical cell membranes of HCLE cultures were biotinylated, isolated with a neutravidin-agarose column, and analyzed by Western blot.
By immunoblot, binding of the galectin-3 antibody to a ≈30kDa molecular weight band, consistent with the monomeric size of galectin-3, was detected in protein extracts from HCEC and HCjE cell cultures. Binding to human tears was weak. The cell surface-associated mucin, MUC16, from HCLE protein extracts bound to Sepharose-conjugated galectin-3 beads. ß-Lactose effectively eluted MUC16 from the galectin-3 affinity column, indicating that MUC16-galectin-3 interaction is galactose dependent. No MUC16 was detected in the unbound material. Cell surface biotinylation revealed that both MUC16 and galectin-3 were present on apical cell membranes in HCLE cells.
These results indicate that MUC16 is a counter-receptor for galectin-3 at the ocular surface epithelia. Retention of galectin-3 at the cell surface through its association with O-glycans on MUC16 may contribute to the formation of the glycocalyx barrier at the ocular surface epithelia.
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