INTRODUCTION
The ocular tear film is a complex mixture of ions, small molecules, glycoproteins and hundreds of proteins, many of which are bioactive [1,2]. The functions of the tear film consist of covering and wetting the surface of the cornea and the conjunctiva, maintaining their integrity, protecting against microbial challenge and preserving the visual acuity. A number of studies reported on differential proteomic analysis of tear fluids of healthy subjects and patients as an approach successfully employed for biomarker search and identification in dry eye disease [3,4], in climatic droplet keratopathy [5], in glaucoma [6] and in vernal keratoconjunctivitis (VKC) [7]. More recently, an increasingly growing interest in glycomic studies has emerged, since qualitative and quantitative glycan alterations are often crucial to afford key information on pathological mechanisms in a large variety of disorders and severe diseases such as rheumatoid arthritis [8,9], diabetes [10], inflammation [11], tumors [12] and Alzheimer’s disease [13-19].
Glycosylation is a complex post-translational modification of proteins, very sensitive to the biochemical environment, depending on the action of several glyco-enzymes. Even though it is not under direct genetic control, glycosylation affects several biological processes [20,21].
Glycosylation is an important factor in regulating the ocular surface homeostasis [22]. It was reported that inflammatory stress causesN -glycan processing deficiency in ocular autoimmune disease [23],while tear fluid N - glycosylation can provide specific and sensitive methods for biomarker discovery and disease diagnosis [24,10]. These pioneering works on tear glycomics suggested a reduced N- glycans fucosylation degree as potential biomarker for rosacea [24], and significant quantitative changes in some N- glycans in diabetes patients compared to diabetic retinopathy [10]. Both studies reported the identification of about 50 N- glycan species, pinpointing structures containing up to seven fucoses by Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF MS) [24], and complexN- glycans carrying up two sialic acids detected by Electrospray Mass Spectrometry (ESI-MS) [10]. Despite these achievements, a complete N- glycans profile of tear fluid is still missing because such investigations were performed on free (underivatized) glycans, undergoing sialic acids loss and fucose fragmentation when analyzed by MALDI MS and ESI MS, respectively. Nevertheless, such instances did not affect the aforesaid outcomes, as the analyses by MALDI MS dealt with the identification of N- glycan hyperfucosylated structures, while the research handled by ESI MS mainly pointed to extend the characterization of N- and O -linked sialylated species.
To acquire the human tear fluid N- glycoprofile without any loss of information, we carried out MALDI MS analysis on permethylatedN- glycans, a widely known glycomic approach allowing a high-sensitivity analysis of oligosaccharide species avoiding glycosidic linkage fragmentation. In the present study we moreover applied the MALDI MS and MS/MS approach to deeply characterize and compare the N- linked glycan profiles of tear fluid from normal subjects and from patients affected by VKC and atopic keratoconjunctivitis (AKC), aiming at identifying changes and, possibly, specific glycan features (glyco-biomarkers) associated to these ocular diseases.
VKC and AKC are two severe and persistent types of ocular allergy affecting the conjunctiva and the cornea, impairing the physiology and the function of the ocular surface and possibly causing significant complications leading to vision loss [25]. VKC is a chronic bilateral conjunctival inflammation typically occurring in areas with tropical and temperate climates and with a significant male preponderance, affecting mostly children and young adults [26]. AKC is a bilateral conjunctival and eyelid inflammation, which should be considered the ocular localization of atopic dermatitis [27]. Both VKC and AKC have a characteristic ropey, stringy mucous and/or serous discharge, and possible corneal complications, such as superficial punctate keratopathy (SPK), macroerosions and ulcers. If inappropriately treated, both VKC and AKC can lead to severe complications such as glaucoma, corneal scarring, and blindness. To date, no specific laboratory tests are suitable for VKC and AKC diagnosis and monitoring.