Tear Fluid N-Glycan Profiling To Investigate Biomarkers In Vernal Keratoconjunctivitis

The tears covering the surface of the eye are an important component of the extracellular environment of the surface epithelial cell layer. Secretory and plasma contributions result in a fluid containing lipids, carbohydrates, proteins, and electrolytes which can affect the activity of the cells of the ocular surface. Different studies, based on proteomics and glycogene-chips, revealed several tear proteins are glycosylated, such as lacritin and proline rich protein 1. Mucins and carbohydrate-binding proteins were found to be among the most highly expressed glycogenes in human conjunctiva. These glycoproteins play a role in protection against pathogens and prevention of ocular surface desiccation,.In the present study, we analyze the N-linked glycome of tear fluid from patients suffering from vernal keratoconjunctivitis (VKC). VKC is a bilateral, chronic sight-threatening and severe inflammatory ocular disease mainly occurring in children and young male adults. Its predominant symptom is intense ocular itching, followed by tearing, mucous stringy discharge, severe photophobia, blepharospasm and foreign body sensation. Severe and inappropriately treated VKC can lead to severe ocular complications such as glaucoma, corneal scarring, and blindness. To date, no specific laboratory test is suitable for VKC diagnosis and monitoring. Tear samples of VKC patients and control subjects were treated with N-glycosidase F (PNGase F), an enzyme which specifically deglycosylates N-glycoprotein. Released N-glycans are purified and chemically derivatized by permethylation to improve the sensitivity of detection of molecular ions. Samples are analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). It is a valuable technique for biomarker detection and characterization of defective glycan structures. The eligibility of mass spectrometry for the study of glycosylation is due to its high sensibility and ability to analyze complex mixtures of glycans from biological samples. This approach allowed to distinguish tear fluid glycosylation pattern of VKC patients from control subjects: Spectra show tear fluid is rich in N-glycan structures. Data analysis evidences changes in terms of relative intensities for some structures. Preliminary study suggests the most significant peaks are related to mass to charge ratios 1906.7, 2592.3 and 2765.9. Each one of these three structures to 2966.5 ratios reveal meaningful changes between healthy and pathological conditions, as evidenced by the following histogram. These peaks could be used as potential biomarkers for VKC disease. Further structural characterization of the disease-associated N-glycans could confirm these preliminary results and allow to discover additional biomarkers.