The Disrupted Glycocalyx as a Source of Ocular Surface Biomarkers

Abstract

The glycocalyx is a dense and diverse coat of glycans and glycoconjugates responsible for maintaining cell surface integrity and regulating the interaction of cells with the external environment. Transmembrane mucins such as MUC1 and MUC16 comprise a major component of the epithelial glycocalyx and are currently used to monitor disease progression in cancer. At the ocular surface, multiple lines of evidence indicate that abnormal expression of the enzymes responsible for glycan biosynthesis during pathological conditions impairs the glycosylation of transmembrane mucins. It is now becoming clear that these changes contribute to modify the interaction of mucins with galectin-3, a multimeric lectin crucial for preserving the ocular surface epithelial barrier. This review highlights the potential of using the epithelial glycocalyx as a reliable source for the generation of biomarkers to diagnose and monitor ocular surface disease.

The surface of the eye is faced with the critical function of forming a protective barrier that prevents cellular damage and systemic infection while allowing the exchange of molecules with the external environment. It is formed by a highly specialized layer of corneal and conjunctival epithelial cells and it is covered by a tear film that contributes to moisturize and maintain a smooth refractive surface.¹ A number of pathological conditions are known to impair the integrity of the ocular surface epithelia. Among them, dry eye is the most prevalent form of ocular surface disease affecting millions of adults worldwide.² For many years the lack of biological measures of ocular surface disease has slowed down progress in clinical research and patient care, particularly in dry eye. However, recent technological advances have led to a resurgence of studies directed at finding minimally invasive biomarkers to assist in the diagnosis of ocular surface disease and to determine progression or responsiveness to therapy.³ This review provides an overview of the structure of the healthy ocular surface epithelial glycocalyx and its potential to serve as a source of biomarkers in pathological conditions.

THE GLYCOCALYX AS A BIOMARKER

The glycocalyx is a dense and diverse coat of glycans and glycoconjugates present in all animal cells. One of the more important characteristics of the glycocalyx is that it is dynamic, therefore, its composition changes to ensure an adequate response to physiological and pathological conditions. This feature has been extensively exploited in the field of cancer for the development of biomarkers with proven clinical value. Here, abnormal expression of transmembrane mucins has led to current diagnostic assays for the detection of a variety of tumors.^{4, 5} The cancer antigen 15–3 (CA15–3), the soluble product of the MUC1 gene, is the most commonly used marker in breast cancer and is used to monitor response to treatment and disease recurrence.^{5, 6} Similarly, the CA125 test is commonly used to detect elevated levels of MUC16 in blood, an early sign of ovarian cancer in patients at high risk of the disease.⁷ An additional FDA-approved test evaluates CA19–9, a sialylated Lewis carbohydrate antigen widely used for the diagnosis of pancreatic cancer.^{8, 9} Not surprisingly, the use of the glycocalyx as a source of biomarkers is not restricted exclusively to tumors. The vascular endothelial surface is covered with an abundant amount of glycoproteins, proteoglycans and associated plasma proteins. Increased serum levels of syndecan-1 and glycosaminoglycans resulting from a disrupted endothelial glycocalyx have been associated with deteriorating cardiac and renal function,^{10, 11} and organ failure in sepsis,^{12, 13} and have been consequently proposed as biomarkers to monitor disease progression.

THE OCULAR SURFACE EPITHELIAL GLYCOCALYX

Research during the last decade has redefined and highlighted the contribution of apical plasma membranes on epithelial cells to the protection of the ocular surface. It is this area adjacent to the tear film interface where short membrane folds form to produce microplicae (Fig. 1). Transmembrane mucins emanate from the tips of these structures, extending up to 500 nm above the plasma membrane, far above other glycoconjugates, to form a distinct glycocalyx.^14–16 These mucins have single membrane-spanning regions with large extracellular glycosylated domains containing tandem repeats of amino acids rich in serine and threonine residues. The most apical portion of the human ocular surface epithelia produces primarily three mucin genes named MUC1, MUC4 and MUC16.¹⁷ Approximately 55% of the mucin products are carbohydrate chains containing galactose, N-acetylgalactosamine, N-acetylglucosamine and terminal sialic acid derivatives.^{18, 19} Under physiological conditions, galactosyl residues on mucin glycans are cross-linked by the multimeric lectin galectin-3 on the apical cell glycocalyx. This interaction is mediated by two major classes of glycans, N-glycans and O-glycans, and is critical to maintain the epithelial barrier and to prevent cellular damage.^{20, 21} In addition to transmembrane mucins, the ocular surface glycocalyx is rich in glycoconjugates such as proteoglycans (i.e., heparan sulfate and chondroitin sulfate) and gangliosides.^{22, 23}

FIG. 1. The ocular surface epithelial glycocalyx.

Transmembrane mucins emanating up to 500 nm above the plasma membrane interact with multimeric galectin-3 to promote epithelial integrity. Major classes of glycans at the ocular surface include O-glycans, commonly attached to serine (Ser) or threonine (Thr) amino acids, and N-glycans, attached to asparagine (Asn). Mucins, glycoproteins and glycolipids are embedded in the plasma membrane.

DISRUPTION OF THE GLYCOCALYX IN OCULAR SURFACE DISEASE

A number of studies have evaluated the content of transmembrane mucins in ocular surface disease, focusing primarily on their (i) cellular expression, (ii) protein levels in tear fluid, and (iii) glycosylation status. While changes in mucin glycosylation in dry eye appear to be well documented, there is no consensus on whether alteration of mucin transcription or protein biosynthesis is a good indicator of disease. An early study of the ocular surface of postmenopausal women with dry eye noted increased expression and biosynthesis in MUC1 and MUC16 that positively correlated with diagnostic tests.²⁴ Subsequently, other investigators found no evidence that would support a correlation between MUC1 or MUC16 protein concentration, or MUC1 mRNA expression, with a range of symptomatic data in postmenopausal women.²⁵ To complicate things further, an additional study reported decreased expression of MUC1 and MUC4 in patients with non-Sjögren’s dry eye.²⁶ In Sjögren’s patients, our own studies failed to find differences in the levels of MUC1 or MUC4 expression compared to healthy individuals,²⁷ although the amounts of both soluble MUC16 and MUC16 mRNA appear to increase.²⁸ In addition to drying diseases, potential alteration of transmembrane mucins at the ocular surface has been reported in atopic keratoconjunctivitis,²⁹ infection,³⁰ pseudophakic bullous keratopathy,³¹ complete androgen insensitivity syndrome,³² and pterygium.³³

It is now well established that changes in cell surface glycosylation occur during pathological processes at the ocular surface. These alterations arise from an abnormal expression of the enzymes responsible for glycan biosynthesis. During homeostatic conditions, the distribution of glycosyltransferases involved in the initiation of mucin-type O-glycosylation is exquisitely regulated in a cell-layer- and cell-type-specific manner.³⁴ Likewise, the proper expression of N-glycosylation processing enzymes assures stability and barrier function of the MUC16 mucin.²⁰ In ocular cicatricial pemphigoid, however, the expression of these enzymes is significantly altered as the ocular surface becomes keratinized.^{34, 35} This impairment in glycosyltransferase expression in ocular surface drying conditions has led to propose that mucin glycosylation could be used in the diagnosis, monitoring and management of epithelial dysfunction. Certainly, antibodies to mucin carbohydrates whose distribution is altered in dry eye disease are available. An example is the H185 monoclonal antibody. This antibody, developed by Ilene Gipson in the nineties, binds to apical membranes of apical cells of human corneal, conjunctival, laryngeal, and vaginal epithelium, particularly at the tips of microplicae.³⁶ Subsequent structural characterization has demonstrated that the H185 epitope is an O-acetyl sialic acid derivative on MUC16.^{18, 37} Altered binding of the antibody to conjunctival epithelium correlates with severity in patients with dry eye symptoms,³⁸ and the antigen can be detected at elevated concentrations in pathological tears.²⁴ In addition, the H185 antibody has been used to monitor the progression of keratinization following the treatment of superior limbic keratoconjunctivitis.³⁹ Other antibodies against carbohydrate epitopes that bind to the ocular surface epithelia include KL-6 and CA19–9. KL-6 recognizes a sialylated carbohydrate epitope on MUC1 and produces a mosaic pattern in impression cytology samples of patients with dry eye disease⁴⁰ whereas CA19–9, used in the diagnosis of pancreatic cancer, diminishes in their tear fluid.⁴¹

GALECTIN-3 AS A DISEASE BIOMARKER

Galectin-3 is a β-galactoside-binding lectin highly expressed by the ocular surface epithelia.⁴² Despite being a soluble protein without a transmembrane domain, it localizes to cell membranes, predominantly on the apical portion of the stratified corneal and conjunctival epithelia.²¹ Altered expression levels of galectin-3 are observed in heart disease, kidney disease and cancer, and because the protein can be found on biological fluids including serum and urine, it has been proposed as a diagnostic biomarker. Of interest is that galectin-3 does not exhibit circadian variation and its expression is not associated with age, body mass index or sex.⁴³ In the eye, early studies suggested that galectin-3 was present only in tears from patients with ocular surface disorders.⁴⁴ Further investigation demonstrated that the concentration of galectin-3 in the normal tear fluid is 0.12 ng/μg total protein but significantly increases to 0.38 ng/μg total protein in patients with dry eye.⁴⁵ Importantly, this study also showed an association between elevated expression of MMP9 in dry eye subjects and the presence of cleaved galectin-3 in the tear fluid, as no degradation of galectin-3 was observed in tears of healthy subjects. Several cell types could contribute to the elevated concentration of galectin-3 in tears of patients with ocular surface disease, including immune and epithelial cells. We have hypothesized that disruption of mucin glycosylation on the epithelial glycocalyx could result in decreased affinity towards galectin-3 and its subsequent release into the tear fluid.⁴⁶ Certainly, the individual contribution of these and other cell types to galectin-3 content in biological fluids merits further investigation.

CONCLUSION

Key events during disease development and progression are changes in tissue glycosylation and the release, in many instances, of glycans and glycoconjugates into extracellular fluids. These compounds have constituted a valuable resource for the development of biomarkers.⁴⁷ In this context, attempts have been made to use tear fluid glycans as a diagnostic indicator of skin conditions and diabetes.^{48, 49} The epithelial glycocalyx is a reliable source for the generation of biomarkers. Research during the past decade has established that the ocular surface epithelial glycocalyx is sensitive to disruption during pathological states. The data generated indicate that the glycosylation of transmembrane mucins is impaired in ocular surface disease and supports the use of mucin glycan antibodies as novel tools for biomarker discovery in the ophthalmic field. Further, new advances indicate that disruption of the epithelial glycocalyx negatively affects the mucin-galectin interaction. Monitoring the integrity of galectin-3, and its release into the tear film, could be used to determine the severity and progression of dry eye, and in the prediction of treatment response. Many other components of the ocular surface epithelial glycocalyx remain understudied, and include proteoglycans, glycosaminoglycans, and glycosphingolipids.⁵⁰ Further exploration efforts should have important clinical implications for the diagnosis and treatment of ocular surface disease.