Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactosedependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, ␤-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins.

These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).

The mucin-rich environment of the intact corneal epithelium is thought to contribute to the prevention of Staphylococcus aureus infection. This study examined whether O-glycans, which constitute the majority of the mucin mass of epithelial cell glycocalyces, prevented bacterial adhesion and growth. Abrogation of mucin O glycosylation using the chemical primer benzyl-␣-GalNAc resulted in increased adherence of parental strain RN6390 to apical human corneal-limbal epithelial (HCLE) cells and to biotinylated cell surface protein in static and liquid phase adhesion assays, consistent with a role of mucin O-glycans in preventing bacterial adhesion. Comparable results were found with ALC135, an isogenic mutant strain defective in the accessory gene regulators agr and sar, indicating that the agr-and/or sar-regulated virulence factors did not play a major role in mediating adhesion to the corneal cell surface after mucin O-glycan truncation. In exoglycosidase digestion studies, treatment with sialidase from Arthrobacter ureafaciens-which hydrolyzed mucin-associated O-acetyl sialic acid-but not from Clostridium perfringens resulted in an increase in RN6390 and ALC135 adhesion. Abrogation of mucin O glycosylation in HCLE cell cultures did not affect bacterial growth. Overall, these data indicate that mucin O-glycans contribute to the prevention of bacterial adherence to the apical surface of corneal epithelial cells and suggest that alteration of cell surface glycosylation from disease or trauma, including that stemming from contact lens wear, could contribute to a higher risk of infection.