4. Discussion and Conclusions
Eye mucins are the main glycoproteins which protect the corneal surface. The interaction of mucins with ophthalmic preparations is important for their residence on the ocular surface, in particular for the amelioration of pathologies as the dry eye syndrome. Dry eye is associated with tear film deficiency, owing to either insufficient supply or excessive loss, and with anomalous tear composition. One of the essential functions of the tear film is to lubricate the ocular surface during blinking and eye movement. The eyes are constantly in motion, and the ocular surface is delicate; without adequate lubrication, ocular surface damage occurs and symptoms of discomfort develop or increase. Ocular lubrication is also essential for contact lens wearers, which utilize artificial tears to increase both lubrication and retention of the tear film. A general feature of artificial tears is their high viscosity which should increase the residence of the substance on the ocular surface. However, the high viscosity may lead up to inconveniences such as sticky feeling and solidification. In this regard, a strategy to antagonize the discomfort of dry eye is the use of low viscosity polysaccharidic molecules that may endure on the ocular surface without unpleasant disadvantages. Arabinogalactan (AG), a natural polysaccharide present in conifers of the genus Larix (Larch), is a no-viscous polymer which was recently shown to be a potential therapeutic product for dry eye protection and for the treatment of corneal wounds (Burgalassi et al., 2007).
The purpose of the investigation was the assessment of the mucoadhesive properties of purified AG by evaluating its ability to interact with mucins. The major mucin target used in this work was MUC1, one of the most abundant and studied membrane-associated mucins present on the ocular surface, which allowed to perform a consistent investigation on AG adhesion ability. Several methodological approaches were envisaged to develop a suitable and friendly assay to study the interaction between MUC1 and AG. However, most of them ended to be disappointing with the only exception, as I will discuss, of the most unfriendly and time consuming approach. Nevertheless, the bunch of information coming from those methodological attempts to optimize mucin-AG binding assay, revealed to be useful in terms of procedures to link mucins to insoluble matrixes. The main problem arose from the highly glycosylated surrounding of mucins, combined with the analogous polysaccaridic nature of AG. Moreover, the modest but still effective negative charge density of AG, due to a number of glucuronic acid residues, leads both molecules to similarly interact with several supports. This fact makes it difficult to discriminate the specific binding, if present, between mucin and AG. In this regard, the equilibrium dialysis approach using either classical cellulose or
hydrophobic membranes, resulted in a high undesirable interaction of arabinogalactan with the membrane, which affected the permeation of the ligand, impairing, in any event, the attainment of the equilibrium. Different supports were tested in order to perform the immobilization of mucins. Among ion exchanger resins, only anionic exchanger, such as polylysine-coated glass and DEAE-cellulose were suitable to effectively bind mucin. No conditions were found in the pH range from 3 to 8, useful to link MUC1 to cation exchangers such as Dowex resins (50WX2 and 88). Unfortunately, AG interacts with positive charged supports irrespectively of the presence of mucin. To circumvent the nonspecific interaction of AG on ionic supports, the study was carried on using non-ionic materials as polystyrene and polyvinyl chloride. Polyvinyl chloride gave better results concerning the adhesion of MUC1 (0,45 µg/cm2) than polystyrene (0,1 µg/cm2). An even better result in terms of MUC1 linkage to the support came from mucin covalent immobilization on glass fiber filters. This revealed to be the most promising method to immobilize mucin on support, allowing in the optimized conditions to reach up to 36 µg/cm2 of bound MUC1. However, both PVC layers and glass fiber filters were still a good target for AG binding, whose nonspecific adhesion on those supports compromised the usefulness of these materials to assemble an AG binding assay. On the contrary polystyrene have chances since the lower nonspecific AG adhesion. However, due to the very low level of mucin adhesion on the plate, also this approach failed in allowing the optimization of the binding assay mucin:AG. Despite so, this technique is very promising and certainly would deserve further studies before to be definitively ruled out.
The failure to find suitable assay conditions making use of immobilized mucin was determinant for the optimization of an alternative method to firstly assess and then quantitate the mucin:AG interaction. The first clear evidence of the AG ability to interact with mucins in solution came through a gel filtration approach. This kind of approach, although required much time, is so far the only method that allowed to obtain positive and confident results. The rationale of the experimental approach was the predictable change in the elution volume of a ligand targeting an high molecular weight molecule, when subjected to a molecular sieving chromatography. Such an approach, which should be obviously successful for thigh binding interactions, may fail when the interactive process is weaker and therefore sensitive to the sieving properties of the chromatographic support. In these conditions, a fine setting of the elution conditions is required, in order to disclose the ligand:target interaction. The chromatographic analysis of MUC1 and AG, individually run on Sephacryl S300, revealed well distinct retention times and elution profiles compatibles with a complete resolution of the two molecules (Fig. 3.9, A) However, when a mixture of MUC1 and AG were
chromatographed in the same conditions, a significant change in the elution profiles of both the molecular species was observed. In particular (Fig. 3.9, B), a significant shift of the elution peak of AG toward higher molecular weight and a considerable “tailing” of the mucin peak were observed. Despite this result was consistent with an interaction between AG and MUC1, the possibility of an alteration of the column efficiency due to the presence of MUC1 (high molecular weight, highly glycosylated protein) was considered. Such a possibility has been ruled out, since the elution profiles of both cytochrome C and chymotrypsinogen, proteins showing a molecular weight similar to AG, were unaffected by the presence of mucin (Fig. 3.12). Moreover, a sign of specificity of the interaction between AG and MUC1 was indicated by the lack of any change of elution features of AG when chromatographed with different high molecular weight proteins and glycoproteins, such as alpha crystallin, ferritin and thyroglobulin (Fig. 3.13).
As already mentioned, a critical aspect in order to evaluate the interaction between MUC1 and AG by the gel filtration approach was the definition of the analysis conditions. In fact, as shown in Fig. 3.10 and Fig. 3.11, the relative concentrations of mucin and AG, as well as their absolute values at a defined ratios affected the elution profiles. This fact makes it clear that, even though very useful to underline the occurrence of a MUC1:AG interaction, such a chromatographic approach cannot be successfully used for quantitative measurements. Above all that, a relevant result was the estimation of the effectiveness of the interaction between AG and MUC1 by “frontal” gel filtration chromatography. By this methodological approach a series of chromatographic analysis of the target molecule using different ligand concentrations in the elution buffer were performed, and the level of the trapped ligand under the target elution peak was measured. With such a technique, by using concentrations of AG ranging from 0,06 to 0,23 mg/ml a dissociation constant for the MUC1-AG complex of 5,2 ± 0,55 x 10-6 M was estimated by Schatchard plot (Fig. 3.17).
AG from Western larch is known to contain different components, possibly characterized by a different content in glucuronic acid, that can be resolved on size exclusion chromatography, lowering the ionic strength of the eluant (see Introduction section, paragraph 1.6.2.). The presence of more than one component was verified for the AG preparation used in this work. As already mentioned, when AG was subjected to chromatography in standard buffer, a single peak was observed; however, the analysis performed at reduced ionic strength revealed the occurrence of two components, AG-I and AG-II, associated to the two resolved peaks (I and II) appearing in the elution profile of Fig. 3.1, B. To rule out the possibility of an equilibrium between these two forms of AG, the eluted fractions of peak I and II were collected into two
distinct pools, concentrated and individually subjected to chromatography. Indeed, as shown in Fig.3.1, C, the two AG components appeared to be independent. In fact, for the pool of peak I, the elution profile revealed, together with AG-I, only a very modest presence of AG-II, likely present in the peak I pool as a contaminant. Furthermore, the elution profile of the peak II pool, revealed the presence of a unique component at the expected retention time for AG-II. Once assessed the occurrence of AG-I and AG-II components in the AG sample, their ability to interact with MUC1 was verified from a qualitative point of view. As shown in Fig. 3.18, an identical shift of the elution peaks of AG-I and AG-II was observed when the two AG components were subjected to gel filtration chromatography in the presence of MUC1, suggesting that both AG forms are able to interact with mucin.
Since gel filtration approach was the only successful method to demonstrate an interaction between MUC1 and AG, the behaviour on gel filtration of other mucin types, MUC2 and MUC3, was analyzed. As for MUC1, MUC3 is another membrane associated mucin present on the ocular surface. MUC2, a secreted mucin of the ocular surface is, among the commercially available mucins, the most similar to MUC5AC the main secreted mucin in the ocular district. Differently from what observed for MUC1, which was apparently homogeneous on Sephacryl S300 chromatography, the elution profiles of both MUC2 and MUC3 commercial preparations, displayed several peaks with different distribution of sugar content (Fig. 3.19). An attempt to elucidate the composition of MUC2 and MUC3 was made estimating the reactivity of the resolved peaks toward specific antibodies against human MUC2 and MUC3. Unfortunately, no-cross reactivity was detected between these antibodies and any fraction of porcine MUC2 and MUC3 under investigation. For both MUC2 and MUC3, the lowest retention time peak, similar to that of MUC1, was the only one apparently carrying glucidic moieties (Fig. 3.19). Thus, for both mucins, they were selected to verify the interaction with AG. Indeed, the glycosylated peak of both MUC2 and MUC3, being the only one well resolved from the AG elution peak, was the only component, suitable for testing the ability to interact with the ligand by a chromatographic approach.
At this point, a brief comment on the fluorescent labelled derivative of AG, FITC-AG, we adopted to increase the sensibility of detection of AG in the studies of interaction with MUC1 immobilized on different supports, as well as to monitor AG in some of the chromatographic analyses with MUC2 and MUC3. When FITC-AG was subjected to chromatographic analysis in standard conditions on Sephacryl S300, it eluted as a single symmetrical peak, exactly as the unmodified AG. This allowed to use it to monitor the interacting event with the target mucins. Indeed, its use appeared unavoidable in the MUC2 and MUC3 analyses, since the
higher degree of glycosylation of these targets, as compared with MUC1, would make very difficult the evaluation of the occurred overlapping with the ligand by the anthrone assay method. It is worth to note that the attempt to separate two components from FITC-AG failed. In fact the chromatographic analysis of FITC-AG at low ionic strength revealed only one component, possibly related to AG-I. Actually the insertion of negative charges following FITC labelling of AG (O'Driscoll et al., 1991) may well explain such a behaviour.
When the glycosylated component of MUC3 and FITC-AG were subjected together to a chromatographic analysis, a change in the elution profile of the ligand, overlapping the mucin peak, was observed (Fig. 3.20). As observed for MUC1, this result is consistent with an interaction between AG and MUC3. When the secreted mucin MUC2, after isolation, as for MUC3, of the glucidic moiety carrying component, was subjected to chromatography together with FITC-AG, only a very modest increase of the peak width of the ligand was observed (Fig. 3.22). The presented results would suggest that AG may interact preferentially with membrane associated mucins with respect to secreted mucins. More examples of both membrane bound mucins and secreted mucins require to be analysed in order to validate this conclusion.
The specific interaction, at least so far, between AG and membrane associated mucins, as MUC1 and MUC3, would suggest that AG may hold on the ocular surface, favouring the hydration and possibly the thickness of the corneal hydrate layer. Such a link between mucins and water may end to re-establish a normal moisturizing of the ocular surface of dry eye patients, as depicted in the simplistic model drown below.
Figure 4.1. Simplistic model of normal and dry eye patients. The figure would represent a normal ocular surface highly hydrated (A), dry eye ocular surface (B) and dry eye ocular surface in the presence of AG (C).
Taken together, these results denote a potential use of AG as possible component of artificial tears; being capable to interact with membrane-associated mucins, it may remain on the ocular surface, and thanks to its low viscosity, it may avoid the discomfort caused by the high viscosity eye drops, widely used in artificial tears. The use of AG in artificial tear formulations could be useful for treatment of dry eye and for contact lens wearers. Since alterations of mucin glycosylation pattern was observed in dry eye patients, the study could be developed evaluating the interactive capability of AG associated to different mucin glycosylation state. In conclusion, AG is a natural low viscosity polysaccharide with a wide potentiality in the treatment of ocular surface pathologies; this thesis work underlines its capability to interact with different ocular surface proteins, and define a confident method to test the interaction AG-mucins. The future perspective could be oriented toward in vitro and in
vivo tests of AG ophthalmic solutions, to obtain a formulation with the most promising
