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.