Abstract
Purpose:
Galectin-3 is a multimeric carbohydrate-binding protein that binds glycans on the transmembrane mucin MUC16 at the ocular surface to provide barrier function and prevent cellular damage. Tumor necrosis factor-α (TNF-α) is a central regulator of inflammation at the ocular surface, disrupting barrier function and promoting epithelial damage. Here, we investigated the effect of TNF-α on the interaction between galectin-3 and MUC16 in corneal epithelial cells.
Methods:
Immortalized human corneal-limbal epithelial (HCLE) cells were grown in conditions that promote differentiation and stratification. Tears from human subjects were collected by micropipette after instillation of 60 μL of sterile saline. MUC16 was detected by Western blot. The relative potency of the interaction between galectin-3 and MUC16 was evaluated using galectin-3 affinity chromatography with lactose fractionation. The contribution of N- and O-glycans on MUC16 towards galectin-3 binding was evaluated by enzymatic assays. HCLE cells were treated with or without 40 ng/mL TNF-α for 48 h at 37°C.
Results:
Affinity chromatography showed that MUC16 from HCLE cells bound to immobilized galectin-3 and could be eluted at different lactose concentrations, representing high (0.05-0.5 M) and low (0-0.05 M) affinity interactions. Similar to HCLE cells, MUC16 from tear samples also eluted at different lactose concentrations, consistent with the presence of multiple MUC16 glycoforms in the tear fluid. High-affinity binding of MUC16 to galectin-3 was dramatically abrogated by treatment of HCLE cell extracts with N-glycanase, indicating that MUC16 N-glycans are critical to promoting strong galectin-3 interactions. Interestingly, removal of O-glycans promoted high-affinity binding of MUC16 to galectin-3. Treatment of HCLE cells with TNF-α induced a 47% decrease in the amount of MUC16 with high-affinity towards galectin-3.
Conclusions:
Data from this study demonstrate an important role for mucin N-glycans in mediating high-affinity interactions between MUC16 and galectin-3. Potential alteration of surface N-glycosylation by the proinflammatory cytokine TNF-α may impair MUC16’s affinity towards galectin-3 and, therefore, promote barrier dysfunction at the ocular surface.
Keywords: 485 cornea: surface mucins •
486 cornea: tears/tear film/dry eye •
482 cornea: epithelium