Abstract
BackgroundInteraction of transmembrane mucins with the multivalent carbohydrate-binding protein galectin-3 is critical to maintaining the integrity of the ocular surface epithelial glycocalyx. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells.Methodology/Principal FindingsAbrogation of galectin-3 biosynthesis in multilayered cultures of human corneal epithelial cells using siRNA, and in galectin-3 null mice, resulted in significant loss of corneal barrier function, as indicated by increased permeability to the rose bengal diagnostic dye. Addition of β-lactose, a competitive carbohydrate inhibitor of galectin-3 binding activity, to the cell culture system, transiently disrupted barrier function. In these experiments, treatment with a dominant negative inhibitor of galectin-3 polymerization lacking the N-terminal domain, but not full-length galectin-3, prevented the recovery of barrier function to basal levels. As determined by fluorescence microscopy, both cellobiose- and lactose-containing glycopolymers incorporated into apical membranes of corneal epithelial cells, independently of the chain length distribution of the densely glycosylated, polymeric backbones. Membrane incorporation of cellobiose glycopolymers impaired barrier function in corneal epithelial cells, contrary to their lactose-containing counterparts, which bound to galectin-3 in pull-down assays.Conclusions/SignificanceThese results indicate that galectin-3 multimerization and surface recognition of lactosyl residues is required to maintain glycocalyx barrier function at the ocular surface. Transient modification of galectin-3 binding could be therapeutically used to enhance the efficiency of topical drug delivery.
Highlights
The thick coat of carbohydrates in the glycocalyx that emerges from apical membranes of epithelial cells is critical to maintaining barrier function on mucosal surfaces
Abrogation of galectin-3 did not alter the biosynthesis of either galectin-8 or -9, two additional galectins expressed by the human ocular surface epithelia [20] (Figure S1)
Through studies performed during the last decade, it has become apparent that transmembrane mucins bind galectins in a carbohydrate-dependent manner to elicit a variety of biological functions under both physiological and pathological conditions [10,29,30]
Summary
The thick coat of carbohydrates in the glycocalyx that emerges from apical membranes of epithelial cells is critical to maintaining barrier function on mucosal surfaces This glycocalyx is important in preventing access of microbes to plasma membranes, and significantly restricts drug and vaccine targeting of epithelial cells [1]. Stratified human corneal and conjunctival epithelia express at least three membrane-associated mucins: MUCs 1, 4, and 16 [5]. These large molecules are characterized by the presence of heavily Oglycosylated, central tandem repeats of amino acids, with their carbohydrate component providing 50–90% of the mature glycoprotein’s molecular mass [6]. This study aimed to determine whether disruption of galectin-3 multimerization and insertion of synthetic glycopolymers in the plasma membrane could be used to modulate glycocalyx barrier function in corneal epithelial cells
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