Mucin O–Glycans Prevent Apical Cell–Cell Adhesion in Corneal Epithelial Cells Under Dynamic Flow Conditions

Abstract

Purpose: : We have hypothesized that O–glycans on membrane–associated mucins confer disadhesive properties to the apical surfaces of human corneal epithelial cells, preventing the tarsal conjunctival epithelial surfaces of the eye from adhering to the cornea while blinking or sleeping. The purpose of this study was to evaluate the role of membrane–associated mucins and their O–glycans on cell–cell adhesion, using a transient adhesion assay on telomerase–immortalized human corneal–limbal epithelial (HCLE) cells.

Methods: : The production of mucins and O–glycans in HCLE cells was evaluated by immunofluorescence microscopy and Western blot analysis using monoclonal antibodies to MUC1 (HMFG–2), MUC16 (OC125), and a terminal O–acetylated sialic acid epitope on MUC16 (H185). HCLE cultures were grown on cell culture slides and fitted onto a parallel plate laminar flow chamber. Trypsinized HCLE cells grown without serum and labeled with a fluorescent dye (6–CFDA) were perfused over the HCLE culture through the flow chamber at a shear stress of 0.8 dyn/cm². The number of transiently adherent cells was monitored by fluorescence video microscopy. HCLE cell surface features were assessed by field emission scanning electron microscopy.

Results: : HCLE cultures grown without serum on cell culture slides produced MUC1 on their apical surfaces but no MUC16. HCLE cells grown in serum for seven days produced MUC1, MUC16 and its carbohydrate epitope H185. Addition of 2 mM benzyl–α–GalNAc, an inhibitor of mucin O–glycosylation, to HCLE cells cultured in serum resulted in lack of binding of H185, without affecting either apomucin production or the ultrastructural morphology of the apical cell surfaces. The number of transiently adherent cells on HCLE cells producing both MUC1 and MUC16 was significantly lower (3.8 ±1.9) than those in which MUC16 was not produced (71.2 ±23.6, p<0.005). The number of transiently adherent cells on benzyl–α–GalNAc treated cultures (46.0 ±12.9) was higher than in control cultures grown with serum (p<0.05).

Conclusions: : These data indicate, that under dynamic flow conditions, mucin O–glycans prevent cell–cell adhesion in HCLE cells and suggest that glycosylated membrane–associated mucins may contribute to the disadhesive properties of the apical epithelial surfaces on the eye.

Keywords: cornea: surface mucins • cornea: epithelium • cell membrane/membrane specializations