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Role of Mucins in the Function of the Corneal and Conjunctival Epithelia

The surface of the eye is covered by a tear film, which is held in place by a wet-surfaced, stratified, corneal and conjunctival epithelia. Both are vital for light refraction and protection of vision. Maintenance of tear film on the ocular surface, lubrication, and provision of a pathogen barrier on this wet surface is facilitated by a class of large, highly glycosylated, hydrophilic glycoproteins--the mucins. In the past 15 years, a number of mucin genes have been cloned, and based on protein sequence, categorized as either secreted or membrane associated. Both types of mucins are expressed by ocular surface epithelia. Goblet cells intercalated within the stratified epithelium of the conjunctiva secrete the large gel-forming mucin MUC5AC, and lacrimal gland epithelia secrete the small soluble mucin MUC7. Apical cells of the stratified epithelium of both corneal and conjunctival epithelium express at least three membrane-associated mucins (MUCs 1, 4, and 16), which extend from their apical surface to form the thick glycocalyx at the epithelium-tear film interface. The current hypothesis regarding mucin function and tear film structure is that the secreted mucins form a hydrophilic blanket that moves over the glycocalyx of the ocular surface to clear debris and pathogens. Mucins of the glycocalyx prevent cell-cell and cell-pathogen adherence. The expression and glycosylation of mucins are altered in drying, keratinizing ocular surface diseases.

Functions of ocular surface mucins in health and disease

Purpose of reviewThe purpose of the present review is to describe new concepts on the role of mucins in the protection of corneal and conjunctival epithelia and to identify alterations of mucins in ocular surface diseases. Recent findingsNew evidence indicates that gel-forming and cell surface-associated mucins contribute differently to the protection of the ocular surface against allergens, pathogens, extracellular molecules, abrasive stress, and drying. SummaryMucins are high-molecular weight glycoproteins characterized by their extensive O-glycosylation. Major mucins expressed by the ocular surface epithelia include cell surface-associated mucins MUC1, MUC4, MUC16, and the gel-forming mucin MUC5AC. Recent advances using functional assays have allowed the examination of their roles in the protection of corneal and conjunctival epithelia. Alterations in mucin and mucin O-glycan biosynthesis in ocular surface disorders, including allergy, nonautoimmune dry eye, autoimmune dry eye, and infection, are presented.

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types 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. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins.

The TFOS International Workshop on Contact Lens Discomfort: report of the contact lens interactions with the tear film subcommittee.

Department of Ophthalmology, New Zealand National Eye Centre, University of Auckland, Auckland, New Zealand School of Optometry and Vision Science, University of New South Wales, Sydney, New South Wales, Australia Schepens Eye Research Institute and Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts Optometric Technology Group Research & Consultancy, London, United Kingdom Centre for Contact Lens Research, School of Optometry and Vision Sciences, University of Waterloo, Waterloo, Ontario, Canada Glasgow Caledonian University, Glasgow, United Kingdom University of Miami, Miller School of Medicine Department of Ophthalmology, Bascom Palmer Eye Institute, Miami, Florida Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan

Mucin-type O-glycans in Tears of Normal Subjects and Patients with Non-Sjogren's Dry Eye

PURPOSE O-linked carbohydrates (O-glycans) contribute to the hydrophilic character of mucins in mucosal tissues. This study was conducted to identify the repertoire of O-glycans in the tear film and the glycosyltransferases associated with their biosynthesis, in normal subjects and patients with non-Sjögrens dry eye. METHODS Human tear fluid was collected from the inferior conjunctival fornix. O-glycans were released by hydrazinolysis, labeled with 2-aminobenzamide, and analyzed by fluorometric, high-performance liquid chromatography (HPLC) coupled with exoglycosidase digestions. O-glycan structures identified in tears were related to potential biosynthetic pathways in human conjunctival epithelium by using a glycogene microarray database. Lectin-binding analyses were performed with agglutinins from Arachis hypogaea, Maackia amurensis, and Sambucus nigra. RESULTS The O-glycan profile of human tears consisted primarily of core 1 (Gal beta 1-3GalNAc alpha 1-Ser/Thr)-based structures. Mono-sialyl O-glycans represented approximately 66% of the glycan pool, with alpha2-6-sialyl core 1 being the predominant O-glycan structure in human tears (48%). Four families of glycosyltransferases potentially related to the biosynthesis of these structures were identified in human conjunctiva. These included 13 polypeptide-GalNAc-transferases (GALNT), the core 1 beta-3-galactosyltransferase (T-synthase), three alpha2-6-sialyltransferases (ST6GalNAc), and two alpha2-3-sialyltransferases (ST3Gal). No significant differences in total amount of O-glycans were detected between tears of normal subjects and patients with dry eye, by HPLC and lectin blot. Likewise, no differences in glycosyltransferase expression were found by glycogene microarray. CONCLUSIONS This study identified the most common mucin-type O-glycans in human tears and their expected biosynthetic pathways in ocular surface epithelia. Patients with non-Sjögrens dry eye showed no alterations in composition and amount of O-glycans in the tear fluid.

Structure and Biological Roles of Mucin-type O-glycans at the Ocular Surface

Mucins are major components in mucus secretions and apical cell membranes on wet-surfaced epithelia. Structurally, they are characterized by the presence of tandem repeat domains containing heavily O-glycosylated serine and threonine residues. O-glycans contribute to maintaining the highly extended and rigid structure of mucins, conferring to them specific physical and biological properties essential for their protective functions. at the ocular surface epithelia, mucin-type O-glycan chains are short and predominantly sialylated, perhaps reflecting specific requirements of the ocular surface. Traditionally, secreted mucins and their O-glycans in the tear film have been involved in the clearance of debris and pathogens from the surface of the eye. New evidence, however, shows that O-glycans on the cell-surface glycocalyx have additional biological roles in the protection of corneal and conjunctival epithelia, such as preventing bacterial adhesion, promoting boundary lubrication, and maintaining the epithelial barrier function through their interaction with galectin-3. Abnormalities in mucin-type O-glycosylation have been identified in many disorders where the stability of the ocular surface is compromised. This review summarizes recent advances in understanding the structure, biosynthesis, and function of mucin-type O-glycans at the ocular surface and their alteration in ocular surface disease.

The role of calcium in mucin packaging within goblet cells

Recent reports hypothesize that calcium plays an important role in providing cationic shielding to keep negatively charged mucins condensed and tightly packed within mucus granules of goblet cells. Vitamin D controls mineral ion homeostasis and intestinal calcium absorption, which is mediated by the nuclear vitamin D receptor (VDR). Hypocalcemia is observed in mice in which the VDR has been ablated. The purpose of this study was to test the hypothesis that normal levels of calcium are required for the physiological packaging of mucins, by comparing the morphology and mucin extractability of conjunctival goblet cells of VDR-ablated to wild-type control mice. Whole eyes from C57/129/sv hybrid wild-type, VDR-ablated, and VDR-ablated mice fed a diet high in calcium to normalize serum ionized calcium levels were fixed in situ and processed for light and transmission electron microscopy (TEM). Mucin extractability from sections of mouse eyes was assessed by lectin-blot, using helix pomatia agglutinin (HPA), and mucin content within goblet cells was assessed by immunohistochemistry, using an antibody specific to the goblet cell mucin Muc5AC. Altered mucin packaging in the goblet cells of VDR-ablated mice as compared to control mice was observed by both light and electron microscopy. In the VDR-ablated mice, the mucin packets varied in size and staining. In contrast, in the controls, the secretory granules appeared regular and uniform. By TEM, mucin packets in the VDR-ablated mice showed dispersed fibrillar and less electron-dense material compared to the homogeneous and more electron-dense packets in wild type. The appearance of mucin packets in the VDR-ablated mice with restored calcium levels was comparable to those of the wild-type control mice. HPA binding to mucin extracted from sections of VDR mouse eyes was reduced when compared to that from wild type. By immunohistochemistry, there was markedly less binding of the antibody to the mucin Muc5AC to goblet cells of VDR-ablated mice compared to controls.VDR-ablated mice presented altered conjunctival mucin packaging. There were lower levels of extractable and immunohistochemically localizable mucin in VDR-ablated mouse conjunctivas than in the wild-type controls. Restoration of ionized calcium levels in the VDR-ablated mice prevented altered mucin packaging, supporting the hypothesis that calcium is required for the physiological packaging of mucins in goblet cells.

A Metalloproteinase Secreted by Streptococcus pneumoniae Removes Membrane Mucin MUC16 from the Epithelial Glycocalyx Barrier

The majority of bacterial infections occur across wet-surfaced mucosal epithelia, including those that cover the eye, respiratory tract, gastrointestinal tract and genitourinary tract. The apical surface of all these mucosal epithelia is covered by a heavily glycosylated glycocalyx, a major component of which are membrane-associated mucins (MAMs). MAMs form a barrier that serves as one of the first lines of defense against invading bacteria. While opportunistic bacteria rely on pre-existing defects or wounds to gain entry to epithelia, non opportunistic bacteria, especially the epidemic disease-causing ones, gain access to epithelial cells without evidence of predisposing injury. The molecular mechanisms employed by these non opportunistic pathogens to breach the MAM barrier remain unknown. To test the hypothesis that disease-causing non opportunistic bacteria gain access to the epithelium by removal of MAMs, corneal, conjunctival, and tracheobronchial epithelial cells, cultured to differentiate to express the MAMs, MUCs 1, 4, and 16, were exposed to a non encapsulated, non typeable strain of Streptococcus pneumoniae (SP168), which causes epidemic conjunctivitis. The ability of strain SP168 to induce MAM ectodomain release from epithelia was compared to that of other strains of S. pneumoniae, as well as the opportunistic pathogen Staphylococcus aureus. The experiments reported herein demonstrate that the epidemic disease-causing S. pneumoniae species secretes a metalloproteinase, ZmpC, which selectively induces ectodomain shedding of the MAM MUC16. Furthermore, ZmpC-induced removal of MUC16 from the epithelium leads to loss of the glycocalyx barrier function and enhanced internalization of the bacterium. These data suggest that removal of MAMs by bacterial enzymes may be an important virulence mechanism employed by disease-causing non opportunistic bacteria to gain access to epithelial cells to cause infection.

Molecular basis for MMP9 induction and disruption of epithelial cell–cell contacts by galectin-3

ABSTRACT Dynamic modulation of the physical contacts between neighboring cells is integral to epithelial processes such as tissue repair and cancer dissemination. Induction of matrix metalloproteinase (MMP) activity contributes to the disassembly of intercellular junctions and the degradation of the extracellular matrix, thus mitigating the physical constraint to cell movement. Using the cornea as a model, we show here that a carbohydrate-binding protein, galectin-3, promotes cell–cell detachment and redistribution of the tight junction protein occludin through its N-terminal polymerizing domain. Notably, we demonstrate that galectin-3 initiates cell–cell disassembly by inducing matrix metalloproteinase expression in a manner that is dependent on the interaction with and clustering of the matrix metalloproteinase inducer CD147 (also known as EMMPRIN and basigin) on the cell surface. Using galectin-3-knockout mice in an in vivo model of wound healing, we further show that increased synthesis of MMP9 at the leading edge of migrating epithelium is regulated by galectin-3. These findings establish a new galectin-3-mediated regulatory mechanism for induction of metalloproteinase expression and disruption of cell–cell contacts required for cell motility in migrating epithelia.

Glycogene Expression in Conjunctiva of Patients with Dry Eye: Downregulation of Notch Signaling

PURPOSE Glycoconjugates regulate a variety of biological events in mucosal surfaces, such as differentiation of postmitotic epithelial cells and maintenance of the wet-surfaced phenotype. This study aimed to identify the repertoire of genes (glycogenes) involved in biosynthesis of glycoconjugates in conjunctiva of normal subjects and patients with dry eye. METHODS RNA from conjunctival impression cytology samples was amplified and hybridized to a custom-designed glycogene microarray. Intensity data were converted to expression values and analyzed by ANOVA. Microarray data for selected Notch glycogenes were confirmed by quantitative real-time PCR. Notch receptors and ligands were immunolocalized on conjunctival biopsies by confocal microscopy. RESULTS By microarray, 424 glycogenes were identified in normal conjunctival epithelium; galectins, glycosyltransferases, mucins, Notch signaling molecules, and proteoglycans were among the most highly expressed. In dry eye, 46 glycogenes were significantly downregulated, including five members of the Notch signaling pathway (Notch1, Notch 2, Notch 3, Jagged1, Delta1), four Wnt signaling molecules (Wnt4, -5A, Frizzled6, -7), and three heparan sulfate glycotransferases (HS2ST1, HS3ST6, EXTL2). Only interferon-induced transmembrane protein 1 was upregulated. By real-time PCR, expression ratios of Notch1, Notch 3, and Jagged1 in dry eye were 0.43, 0.56, and 0.50, respectively, compared to controls (P < 0.05). Notch1, Notch3, and Jagged1 were immunolocalized throughout the conjunctival epithelium, whereas Notch2 and Delta1 were distributed apically. CONCLUSIONS This study revealed the differential glycogene expression profiles in normal subjects and patients with dry eye. Downregulation of Notch signaling in dry eye may result in abnormal differentiation of the conjunctival epithelium and have implications in the pathogenesis of the disease.