DISCUSSION
MALDI-MS profiling and MALDI-MS/MS characterization of permethylatedN- glycans permitted to extend the repertoire of the identifiedN- glycans in the normal control tear fluid up to ≃ 150, as reported in Table S2. Many of the glycans shown in this table were detected for the first time because, as already mentioned in the introduction, MALDI analysis of not-derivatized glycans underestimates the amount of sialylated glycans, and ESI-MS underestimates the occurrence of strucures decorated with fucose units, whereas MALDI MS of permethylated glycans is an effective method to detect both theseN- glycan series. Although this approach does not allow to trace the proteins from which the identified glycans originated, it is important to point out that two glycoproteins are much more abundant in the normal tear fluid than others [34]: lactoferrin (1.56 g/L) and secretory IgA (2.07 g/L). Another major glycoprotein certainly contributing to the overall glycosylation profile is Immunoglobulin G (IgG), which is present, however, in a much lower concentration (0.004 g /L) [34]. The N- glycosylation of lactoferrin and IgA has been extensively studied [35,36] and it can be supposed that oligomannose and asymmetrically branched N- glycans originate largely from lactoferrin, complex-type glycans (mainly biantennary and bisected) derive predominantly by IgA, multi-fucosylated glycans come from the Secretory Component of IgA, and monoantennary and hybrid-type glycans arise from the joining chain of secretory IgA.
All the obtained tear MS profiles were rich in specific glycoforms, particularly those with a high fucosylation degree, indicating both core and peripheral decoration, these last essential component of antigens belonging to the Lewis family on cell membrane surface. Increased levels of fucosylation have been implicated in several diseases: a) inflammatory diseases, as N- glycans fucosylation plays a regulatory role for selectin-dependent leukocyte adhesion [37]; b) immunological response, owing to the regulatory role of fucosylated glycoforms in immune response and to the autoimmunity development [38,39] and c) cancer, promoting TNF-α activity in M1 inflammatory macrophages [40].
Large differences emerged from the comparison of N- glycome profiles from control, VKC and AKC tear fluids. In control samples, the base peak was the bisected species at m/z 1907.0. In VKC, dominant ions were assigned to the biantennary and core-fucosylated glycans at m/z 2605.3 and m/z 2966.5. The AKC MS profiles showed a huge increase of the ion at m/z 2792.4, (biantennary, disialylated unfucosylated glycoform), with respect to and VKC tears. These glycomic data were in good agreement with our previous results on quantitative proteomics of VKC tear fluid, showing increase of serotransferrin and lactotransferrin with a concomitant reduction of immunoglobulins [7]. Altoghether, these results led to assume that the peaks with increased intensities refer to glycans from lactotransferrin (m/z 2966.5 and 2605.3) [36,41,42] and from serotransferrin (m/z 2792.4) [29,43], whereas peaks showing decreased intensity are associated to immunoglobulins.
Serotransferrin is an iron-binding plasma glycoprotein, primarily expressed in the liver but also in corneal epithelial cells [44]. It has been shown that patients with allergy and allergic keratoconjunctivitis suffer an increased oxidative stress condition [45,46]. The capacity of binding free iron to protect against toxic radical oxygen species, gives to the serotransferrin an antioxidant effects inhibiting iron-mediated oxidation [45]. Serotransferrin is usually present in tears in very low concentrations, belongs to the ocular innate immune system and contributes, with lactoferrin, to the iron-sequestration mechanisms active against pathogens.
Serotransferrin and other serum proteins like albumin and IgG are not typically present in the ocular surface at high levels and, presumably, they may derive from plasma through a passive filtration from vessels. Their presence in the tear film could be due to a serum leakage, in response to ocular inflammation, to a stimulation of the conjunctiva or to a mild ocular trauma [47]. However, the dramatic increase of species at m/z 2792.4 in VKC and AKC MS profiles, the high serotransferrin tear levels in AKC and, in lesser extend, in VKC and the relative lower peaks related to immunoglobulins, may suggest a local production of serotransferrin in addition of serum leakage due to the ocular inflammation. A reduction in serotransferrin tear levels has been shown in a rabbit model of Sjögren syndrome-associated dry eye [48] and in saporin toxin denervated lacrimal glands rat-model despite normal tear production [49]. Differently from dry eye, ocular allergy is characterized by increased tearing and corneal sensitivity and by corneal nerve fiber abnormalities [50,51]. The antimicrobial, antiviral, antiparasitic and anti-inflammatory activity of serotransferrin and of other over-expressed tear fluid proteins, may explain the low rate of infection in ocular allergy patients and may function as an up-regulating mechanism to down-regulate the effects of pro-inflammatory cytokine overexpressed by the allergic reaction [52].
In conclusion, we identified in normal control tears multiple specific glycoforms, many of whom with a high fucosylation degree. TearN- glycome analysis highlighted profound and specific changes of tear proteome in allergic keratoconjunctivitis compared to control tears.