Interaction of arabinogalactan with mucins
Graphical abstract
Introduction
Natural polysaccharides have been widely used for many industrial and biomedical applications [1]. The mucoadhesivity of polysaccharides is generally considered advantageous for several of these applications. This is the case, for instance, in the development of ophthalmic preparations in which the mucoadhesivity of polysaccharides allows them to adhere to ocular surfaces [2], [3].
The aim of this work was to assess the mucoadhesive properties of western larch (Larex occidentalis) arabinogalactan by evaluating its ability to interact with mucins.
Arabinogalactan is a long-chain, densely branched polysaccharide abundantly present in plants as part of cell walls [4]. A significant presence of arabinogalactan has been detected in many herbs with assessed immuno-stimulant properties [5], [6]. In addition, arabinogalactan has been approved by the U.S. Food and Drug Administration as a source of dietary fiber [7]. The structure of the arabinogalactan backbone allows the identification of two types of the polysaccharide; the type I has β-1,4-galactan backbones, while the type II has β-1,3-galactan backbones [4]. The type II is often present also as the carbohydrate moiety of a class of highly glycosylated hydroxyproline-rich glycoproteins, known as arabinogalactan proteins, which are involved in several cell functions [8].
The woody tissues of western larch are especially rich in arabinogalactan type II (AG) and represent the main source of the commercially available arabinogalactan. It has been reported to be mainly composed of galactose and arabinose units in a molar ratio of approximately 6:1 [9]. The presence of glucuronic acid residues has also been detected and it has been demonstrated that the presence of uronic acids affect the behavior of AG on size exclusion chromatography [10].
One third of the AG molecule is composed of (1→3)-β-d-galactopyranose units, which constitute the main chain, while the rest consists of side groups (whose size varies from monosaccharides to oligosaccharides) which are (1→6)-linked to each galactose unit [11], [12]. AG exists naturally as an ordered assembly of molecules in which the (1→3)-β-D-galactan main chain is organized in triple helices [13], [14]. Side groups do not interfere with the stability of the chain triple helices, but generate a wide flexible branching surface with many exposed hydroxyl groups, which are able to interact via hydrogen bonds with both neighboring helices and several polysaccaridic targets. These features have been proposed to be responsible for the hydrocolloid nature of AG [13], [15].
The values reported of AG molecular weight (MW) vary considerably, ranging from 3 to 100 kDa. Evidence has been presented of the existence of two AG components characterized by different MW [9], [16], [17]. At the same time other authors have described a preparation of AG which was homogeneous in terms of MW [18], [19].
Mucins are extracellular proteins with a high MW ranging from 5 × 102 to 2 × 104 kDa and are among the largest known glycoproteins (for review see Ref. [20]). They are usually divided in two classes: membrane associated and secreted. The protein core of mucins of both classes displays a molecular mass ranging from 200 to 600 kDa, representing only approximately 20% of the mass of the mature glycoprotein. A common characteristic of all mucins is the high level of glycosylation, which occurs through glycans O-linked to serine and threonine residues. The hydrophilicity of mucins is associated with the heavy glycosylation and this helps to hold fluids onto epithelial surfaces and to clean the surfaces and/or to block surface microbial binding [21]. In the present study, through different gel filtration chromatographic approaches, it was possible to evidence and quantify an interaction between AG and mucins.
Section snippets
Materials
Arabinogalactan (AG), pharmaceutical grade, was supplied by Opocrin S.p.A. Modena, Italy. The product, a white dry powder, was 96.3% in AG with a galactose to galactose plus arabinose molar fraction of 0.86 and with a protein content less than 0.004%. Mucin from bovine submaxillary glands (BSM), mucin from porcine stomach (PSM), ferritin, bovine serum albumin (BSA), anthrone and fluorescein isothiocyanate were purchased from Sigma–Aldrich, Italy. Alpha-crystallin was isolated from bovine lens
Evidence of AG-mucin interaction
To define the mucoadhesivity of AG, different methodological approaches have been adopted [24], [25], [26]. In the present study evidence of the interaction of AG with mucins came from the chromatographic approach of molecular sieving on Sephacryl S300. The molecular sieving properties of this support, combined with its ability to interact with polysaccharidic molecules [27], resulted to be quite useful to monitor the AG-mucin interaction.
When BSM and AG were individually chromatographed on a
Conclusions
In the present study evidence of the interaction of AG with mucins came from a chromatographic approach on Sephacryl S300. Western Larch AG is able of a multiple binding interaction with mucins, with a dissociation constant for the AG-mucin adduct in the μM range. These features, in addition to the lower viscosity of AG compared to other polysaccharides, make AG an interesting tool for biomedical applications.
Acknowledgements
This work was supported in part by Opocrin S.p.A., Modena, Italy and in part by Pisa University, Pisa, Italy.
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Both authors contributed equally to the work.