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.