Mucins and Galectin-3 in Ocular Surface Health and Disease

Chapter 25

Mucins and Galectin-3 in Ocular Surface Health and Disease Downloaded by UNIV OF ARIZONA on December 19, 2012 | http://pubs.acs.org Publication Date (Web): December 18, 2012 | doi: 10.1021/bk-2012-1115.ch025

Jerome Mauris and Pablo Argüeso* Schepens Eye Research Institute and Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, Massachusetss 02114 *E-mail: [email protected]

The barrier function of ocular surface epithelia is multi-level, from the stratified structure of the corneal and conjunctival epithelia, to the thick glycocalyx present on the foremost apical cell membranes. Major glycoconjugates on the epithelial glycocalyx include membrane-associated mucins, a group of heavily O-glycosylated, high molecular weight glycoproteins involved in cell protection against damage and infection. In this mini review, we summarize recent evidence indicating that mucin O-glycans associate with the carbohydrate-binding protein galectin-3 to provide barrier function at the ocular surface, and discuss the relevance of this interaction to ocular surface disease.

Anatomy of the Ocular Surface The healthy ocular surface is composed of mucosa lining the cornea and conjunctiva, as well as the intervening transition area between them, known as the limbus (1). The cornea is a transparent, avascular tissue that is the primary refractive element of the visual system. The conjunctiva is a mucous membrane that covers the inner surface of the upper and lower eyelids and extends to the limbus. Both cornea and conjunctiva, as well as the limbus, are covered by a © 2012 American Chemical Society In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

stratified, squamous, nonkeratinizing epithelium, consisting of 5 to 7 cell layers within the cornea, and 3 to 10 cell layers within the conjunctiva. The conjunctiva is unique among the nonkeratinizing squamous tissues in that secretory goblet cells are intercalated between the epithelial cells. The role of the ocular surface epithelia is to maintain optical clarity by regulating the hydration of the cornea and conjunctiva, and to protect the globe from mechanical, toxic, and infectious trauma (2).

Downloaded by UNIV OF ARIZONA on December 19, 2012 | http://pubs.acs.org Publication Date (Web): December 18, 2012 | doi: 10.1021/bk-2012-1115.ch025

Mucins and the Epithelial Glycocalyx Traditionally, the cellular barrier function on mucosal surfaces has been ascribed to intercellular junctions that seal the paracellular space and connect individual epithelial cell membranes (3). It is now well accepted, however, that a second barrier is formed by dense microvilli and a complex glycocalyx containing high levels of membrane-associated mucins on the apical surface of mucosal epithelial cells (4). Based on molecular characterization, cell surface mucins are defined by their unique structural characteristics, including the presence on the extracellular domain of multiple tandem repeats of amino acids rich in serine, threonine and proline residues that are densely O-glycosylated (5). In addition, they contain a single transmembrane domain that anchors the large ectodomain to the plasma membrane, as well as a short cytoplasmic tail with intracellular signaling capabilities (6). The mucin ectodomain has a long filamentous structure that can extend up to 500 nm above the plasma membrane and, therefore, provide steric hindrance (7). Cell surface mucins produced in the ocular surface include MUC1, MUC4 and MUC16 (8). Early analyses of the chemical composition of purified ocular surface mucins revealed that approximately 55% of the mucin mass was carbohydrate, with galactose, N-acetylgalactosamine, N-acetylglucosamine and sialic acid as major constituents, and fucose, mannose, and glucose as minor components (9). Further structural analyses have shown that short core 1-based structures (Galβ1-3GalNAcα1-Ser/Thr, also known as the Thomsen Friedenreich or T-antigen) are prevalent at the ocular surface (10). This finding is supported by glycogene microarray analysis demonstrating the expression of core 1 β 1,3-galactosyltransferase (T-synthase or c1galt1), the enzyme responsible for biosynthesis of core 1 O-glycans, and Cosmc, a molecular chaperone specific for c1galt1, at the human ocular surface (11). Due to the presence of central tandem repeats in mucins, it is possible to speculate that hundreds of clustered core 1 O-glycans would be present on each mucin ectodomain, providing a high degree of multivalency for optimal interactions with carbohydrate-binding proteins (12).

Role of Galectin-3 in Barrier Function Despite extensive molecular characterization of mucins at the ocular surface during the last 15 years, their organization at the plasma membrane and the mechanism by which they promote barrier function is not well understood. Using 410 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


glycogene microarray analysis, we demonstrated that galectin-3 was one of the most highly expressed glycogenes in human conjunctival epithelium (11). This finding led us to evaluate the role of galectins in maintaining barrier function at the ocular surface through interaction with mucin O-glycans. Galectins are a family of animal lectins defined by their affinity towards β-galactosides and the presence of at least one evolutionary conserved carbohydrate-binding domain (13). In recent years, several studies have revealed that galectin-3 exists as a monomer in solution, but that it can self-associate through intermolecular interactions involving the N-terminal domain when bound to a multivalent ligand (14) and, therefore, mediate crosslinking of counter-receptors (15–18). It has also been shown that the resulting galectin-ligand lattices on the cell surface are robust and resistant to lateral movement of membrane components (19). Data from our laboratory demonstrated that galectin-3 localizes to apical membranes of apical epithelial cells in human conjunctival and corneal tissue (20). Moreover, galectin-3 colocalized with the membrane-associated mucins MUC1 and MUC16 on the apical surface of epithelial cells, and both mucins bound to galectin-3 affinity columns in a galactose-dependent manner, indicating that transmembrane mucins are counter-receptors for galectin-3 at the ocular surface. The relevance of this interaction to ocular surface barrier function was evaluated using the rose bengal uptake assay. Rose bengal, a derivative of fluorescein, is an organic anionic dye that has been clinically used for many decades to assess damage to the ocular surface epithelium in ocular surface disease (21). Stratification and differentiation of cultured immortalized corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and core 1 O-glycans on their apical surfaces, provide protection against rose bengal penetrance (22). Abrogation of the mucin-galectin interaction in this in vitro system, using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye (20). Similarly, downregulation of mucin O-glycosylation in corneal epithelial cells using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 also reduced cell surface galectin-3 and increased epithelial permeability. These findings indicate that galectin-3 through interactions with mucin O-glycans at the apical membrane of corneal epithelial cells forms a cell surface lattice important to barrier function. Using cell surface biotinylation and subcellular fractionation, as well as confocal laser scanning microscopy, we recently showed that knockdown of c1galt1 in human corneal epithelial cells also stimulates the endocytosis of plasma membrane proteins and enhances the internalization of nanoparticles in a clathrin-dependent manner (23). Therefore, it is possible to speculate that, when bound to cell surface O-glycans, galectin-3 effectively promotes lattice formation and prevents the endocytosis of plasma membrane proteins and extracellular material.

411 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Barrier Function in Ocular Surface Disease Ocular surface pathology is commonly associated with loss of epithelial barrier function (21). Several studies have reported alterations in the biosynthesis of mucins and mucin O-glycans at the ocular surface under pathological conditions, including dry eye, allergy, pterygium, ocular rosacea, and infection [reviewed by Mantelli (24) and Guzman-Aranguez (10)]. Within these ocular surface diseases, dry eye has been extensively studied, as it affects between 6 and 43 million people in the United States alone (25), and the options for effective pharmacological treatment are limited. Dry eye is a multifactorial disease of the ocular surface that is prevalent in women and that results in symptoms of discomfort, visual disturbance, and tear film instability, with potential damage to ocular surface epithelia. A number of reports have described alterations in the carbohydrate composition of the apical glycocalyx in patients with dry eye. These include a reduction in lectin and antibody binding to cell-surface carbohydrate epitopes such as sialic acid as well as core 1, and alteration in the distribution of glycosyltransferases involved in mucin-type O-glycosylation (26–30). Unlike with mucins, limited research has been conducted to date on the role of galectins in ocular surface epithelial pathology. To our knowledge, only one manuscript has been published describing alterations in galectin-3 in ocular surface disease (31). Although the study included only a small number of patients, the authors found increased galectin-3 levels in tears of patients with ocular inflammatory disease, such as sarcoidosis and adenoviral conjunctivitis, as compared to normal individuals. This result would suggest that barrier disruption in ocular surface disease results in lack of interaction of galectin-3 with mucin O-glycans on the apical epithelial glycocalyx, followed by release of the lectin into the tear fluid. Increased understanding of the ocular surface barrier under physiological and pathological conditions has led to interest in developing pharmaceutical agents that modulate mucin biosynthesis. Two therapeutic agents targeting the production of mucins at the ocular surface have been recently developed for dry eye and marketed in Japan as Mucosta® ophthalmic suspension (rebamipide) and DIQUAS™ ophthalmic solution (diquafosol tetrasodium). So far, however, it is not clear whether the mechanism of action of these drugs is to promote ocular surface health by restoring the mucin O-glycan and galectin-3 interaction on superficial cells at the ocular surface. Studies aimed at determining whether carbohydrate-lectin interactions on the epithelial glycocalyx can be modulated to treat patients with ocular surface barrier dysfunction are likely to prove to be rewarding.

Acknowledgments Supported by the National Eye Institute Grant R01 EY014847 (PA).

412 In Galectins and Disease Implications for Targeted Therapeutics; Klyosov, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

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Mucins and Galectin-3 in Ocular Surface Health and Disease

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