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Ashley M Woodward, Pablo Argueso; N-glycans Promote the Retention of Galectin-3 in the Corneal Epithelial Glycocalyx. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5634.
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© ARVO (1962-2015); The Authors (2016-present)
In the healthy corneal epithelium, galectin-3 binds glycans on the cell surface glycocalyx to provide barrier function and prevent cellular damage. Previous reports have demonstrated the importance of mucin O-glycans in galectin-3 binding and barrier function; however, no studies have examined the significance of mucin N-glycans. This study investigated the role of mucin N-glycans in promoting affinity and cell surface retention of galectin-3 in immortalized human corneal-limbal epithelial (HCLE) cells.
HCLE cell cultures were grown in a multilayered cell culture system. Affinity chromatography was used to determine the relative affinity of galectin-3 towards O- and N-glycans on the transmembrane mucin, MUC16. Mucin glycans were enzymatically digested using O-glycosidase and PNGase F, an enzyme that cleaves asparagine N-linked oligosaccharides. In addition, tunicamycin was used to inhibit N-glycan biosynthesis in cell culture. The presence of MUC16, galectin-3, and N-glycans was evaluated by immunoblot and lectin (PHA-L & ConA) blot analysis.
By affinity chromatography, enzymatic removal of N-glycans, but not O-glycans, reduced the relatively strong binding of MUC16 towards galectin-3. Treatment of HCLE cells with PNGase F to remove N-glycans from the apical cell surface led to increased levels of galectin-3 in cell culture supernatants. Treatment of HCLE cells with tunicamycin resulted in decreased cell surface protein expression of galectin-3. Interestingly, localization of MUC16 at the cell surface was also affected by tunicamycin.
Data from this study demonstrate that N-glycans promote high-affinity interactions between galectin-3 and MUC16 on human corneal epithelial cells, and suggests that alterations in N-glycosylation during ocular surface pathology may disrupt glycocalyx barrier function.
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