Antiadhesive character of mucin O-glycans at the apical surface of corneal epithelial cells

Abstract

Purpose: Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells.

Methods: Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber.

Results: Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface.

Conclusions: 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).

Figure 1

Alteration of mucin O-glycosylation in HCLE cells treated with benzyl-α-GalNAc. Addition of serum to HCLE cell cultures triggered the biosynthesis of cell surface-associated MUC16 and its H185 carbohydrate epitope, as determined by immunofluorescence (A) and Western blot (B), as well as the appearance of microplicae at the cell surface, as determined by scanning electron microscopy. In the presence of benzyl-α-GalNAc, HCLE cells did not synthesize the MUC16-associated H185 carbohydrate epitope. The inhibitor did not affect either the biosynthesis of MUC16 or the cell surface ultrastructural features of HCLE cells compared with serum treatment alone. Arrowheads: position of the 250-kDa molecular weight marker in the agarose gel. GAPDH was used as a loading control. Scale bars: 60 μm (immunofluorescence images), 5 μm (scanning electron microscopy images).

Figure 2

Effect of benzyl-α-GalNAc on biotinylated cell surface glycoproteins in HCLE cells. As determined by MAA binding, benzyl-α-GalNAc decreased O-linked sialylation on high molecular weight glycoproteins, which correlates with decreased binding of the H185 antibody to the cell surface–associated mucin MUC16 after benzyl-α-GalNAc treatment (Fig. 1). Binding of PNA to the mucin-associated T-antigen increased after benzyl-α-GalNAc treatment, most likely as a consequence of the reduced levels of terminal sialic acid on the cell surface mucins. Experiments were performed in duplicate.

Figure 3

Mucin O-glycans contribute to the prevention of epithelial cell surface adhesion under static conditions. Fluorescein-labeled HCLE cells and human fibroblasts in suspension showed reduced binding to HCLE cells grown under conditions that promote mucin O-glycan biosynthesis (Serum), compared with serum-free media (SFM) or to cells treated with the inhibitor benzyl-α-GalNAc (Serum+BG). In control experiments, fluorescein-labeled fibroblasts showed similar levels of adhesion to fibroblasts grown in the presence (FGM+BG) or absence (FGM) of benzyl-α-GalNAc, indicating that benzyl-α-GalNAc influences cell adhesion through the inhibition of O-glycosylation in mucin-expressing cells. Error bars, SEM.

Figure 4

Quantification of the number of transient adhering (rolling) cells under dynamic flow conditions in HCLE cells cultured under different conditions. (A) Inhibition of O-glycosylation with benzyl-α-GalNAc (Serum+BG) increased the number of rolling cells compared with the serum condition (Serum). Rolling cells were defined as cells moving less than 200 μm in five superimposed frames (t = 0.167 s). Error bars, SEM. (B) Representative images of different experimental growth conditions obtained after superimposing five consecutive frames taken from one field of view. In these experiments, the bright-field channel was open to allow visualization of HCLE cells grown on the culture slide. Movies corresponding to images are available online at http://www.iovs.org/cgi/content/full/49/1/197/DC1 (SFM, Movie 1; Serum, Movie 2; Serum+BG, Movie 3).

Figure 5

Cationized ferritin binding to HCLE cells after benzyl-α-GalNAc treatment. Apical surfaces of undifferentiated cells (SFM) and differentiated cells grown in the presence of benzyl-α-GalNAc (Serum+BG) have more negative charges than cells grown in the presence of serum (Serum). These results suggest that negative charges do not play a role in preventing cellular adhesion in HCLE cells. Error bars, SEM.