Abstract
Dry eye syndrome is associated with tear film deficiency, owing to either insufficient supply or excessive loss, and with anomalous tear composition. To counteract the symptoms of discomfort caused by tear deficiency, the use of artificial tears is needed. Artificial tears are usually characterized by an high viscosity, which should increase their residence 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 a low viscosity polysaccharidic molecule 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. The purpose of the investigation was the assessment of the mucoadhesive properties of purified AG by evaluating its ability to interact with mucins. Several methodological approaches were envisaged to develop a suitable and friendly assay to study the interaction between mucin and AG; however, only the gel filtration approach allowed to evaluate the interaction. The ability of AG to interact with highly representative mucins (MUC1, MUC2 and MUC3) of the ocular surface was investigated. A quantitative measurement of the interaction between AG and MUC1 came from “frontal” gel filtration chromatography. Moreover, two components of purified AG (AG-I and AG-II) were identified by gel filtration chromatography and their interaction with MUC1 was studied. The results denoted a potential use of AG as possible component of artificial tears; being capable to interact with mucins, it may remain on the ocular surface, prolonging the hydratation of the eye.
Moreover, thanks to AG low viscosity, it may avoid the discomfort caused by the high viscosity
eye drops, widely used in artificial tears.
1. Introduction
1.1. The Ocular Surface System
The surface of the eye is an extraordinary and vital component of vision. The smooth, wet surface of the cornea is the major refractive surface of the visual system, which, along with corneal transparency, enables light to proceed through the lens and onto the retina for photoreceptor activation. The maintenance of correct properties of the ocular surface has, however, a cost.
Unlike all other wet surfaced epithelia of the body, the ocular surface is directly exposed to the outside world where it is especially subject to desiccation, injury, and pathogens. As a consequence, numerous protective mechanisms are provided by the Ocular Surface System (Fig.
1.1), to ensure vision. The Ocular Surface System is defined as the ocular surface, which includes the surface and glandular epithelia of the cornea, the conjunctiva, the lacrimal, accessory lacrimal, and meibomian glands, and their apical (tears) and basal (connective tissue) matrices; the eyelashes with their associated glands of Moll and Zeis; those components of the eyelids responsible for the blink; and the nasolacrimal duct. All components of the system are linked functionally by continuity of the epithelia, by innervation, and by the endocrine, vascular, and immune systems. A unit within the ocular surface system has been termed the “Lacrimal Functional Unit” (Stern et al., 1998). It is composed of the lacrimal glands (both main and accessory), the ocular surface, and the interconnecting innervation. This term emphasizes the interplay between the lacrimal gland, the ocular surface, narrowly defined as the corneal and conjunctival epithelia, and the nervous system. Since the functions of all regions of the Ocular Surface System are closely integrated, especially by innervation from the trigeminal nerves, signals from one region of the system influences the blink, goblet cell secretion, lacrimation, and/or lacrimal gland gene expression (Fang et al., 2005; International Dry Eye Workshop, 2007;
Gipson, 2007).
Figure 1.1. The Ocular Surface System. (a) Sagittal section showing that the ocular surface epithelium is continuous (pink) with regional specializations on and in the cornea, conjunctiva, lacrimal, and accessory lacrimal glands, and meibomian gland. Each specialized region of this ocular surface epithelium generates components of the tear film (blue). (B) Frontal view of the Ocular Surface System, which includes the surface and glandular epithelia of the cornea, conjunctiva, lacrimal gland, accessory lacrimal glands, and meibomian gland (note enlarged lower lid segment) and their apical (tears) and basal connective tissue matrices, the eye lashes, those components of the eyelids responsible for the blink, and the nasolacrimal duct. The functions of the system’s components are integrated or linked by innervation, and the endocrine, vascular, and immune systems (from Gipson, 2007).
1.2. The tear film
The preocular tear film is vital for the normal function of the ocular surface. It is maintained on the cornea and ocular surface by the blinking of the eyelid to replenish the tears over the cornea, through continuous constitutive secretion of tear components by all areas of the ocular surface epithelia and by specializations on the apical surfaces of the corneal and conjunctival epithelia.
This unique barrier on the ocular surface is adapted to defend against continuous assault from the
external environment. It functions to combat desiccation, microbial colonisation and the effects of
toxins, abrasive particles and a variety of environmental chemicals. It also maintains transparency
and ensures the smooth optical quality of the cornea as a refracting surface. To perform these
functions it must be continually removed and renewed (Corfield et al., 1997). The nasolacrimal
epithelial system adsorbs tear components and is believed to control and regulate tear outflow, helping to maintain the appropriate tear level, a fine balance between secretion and outflow (Paulsen et al., 2003). Tear film, in its natural or basal state is transparent, colourless and around only 7 µl in volume (Dilly, 1994). The classical description of the tear film (Wolff, 1954) encompassed three layers: an outer lipid layer, an intermediate aqueous layer and an inner mucus layer. More recent data has disputed the existence of boundaries between the layers, with the aqueous and mucus layer more likely comprising a single phase, increasing in mucus concentration towards the epithelium (Fig. 1.2) (Dilly, 1994). The lipid layer is secreted by meibomian glands and contains lipids such as fatty acids, phospholipids and cholesterol (Allen, 1983). The aqueous-mucus layer, also indicated with the term aqueous-mucin layer, derived from the lacrimal and other accessory glands, conjunctival and corneal cells, is composed primarily of water (95%), salts, but also a multitude of proteins which serve a defensive purpose such as lysozyme, immunoglobulins, defensins, growth factors, transferrin and trefoil factors. However, the main components that are responsible for its viscous and elastic gel-like properties are the glycoproteins mucins. In the aqueous layer, goblet cells probably secrete the most mucins, but shedding of membrane-bound mucins from conjunctival and corneal cells also contribute to the aqueous-mucin layer (Dilly, 1985; Greiner et al., 1985; Gipson et al., 1992; Dilly, 1994; Gipson et al., 1995; Gipson and Inatomi, 1997; Gipson, 2007). The tear-epithelial cell interface is critical for tear film maintenance on the corneal and conjunctival epithelia. As with all wet-surfaced epithelia of the body, maintenance of fluids on the cell surface is facilitated through membrane specialization on the apical surface membrane, where it abuts the luminal surface. The apical cell membrane adjacent to the tear film interface is thrown into short membrane folds, termed microplicae (Fig. 1.2). Membrane-associated mucins emanate from the tips of the microplicae and extend up to 500 nm from the membrane to form the glycocalyx, now considered to be an integral part of the tear film (Gipson and Argueso, 2003; Gipson et al., 2004; Gipson, 2004). The glycocalyx is heavily rich in carbohydrates, essentially hydrophilic and inherently wettable (Nichols et al., 1985; Tiffany, 1990; Tiffany 1994; Dilly, 1994). Laser interferometry and confocal microscopy were used to accurately measure the thickness of the tear film and its component layers. Earlier estimates of overall tear film thickness were around 7 µm (Dilly, 1994).
Later, data indicated an extent between 35 and 40 µm, the majority (~30 µm) contributed by a mucous gel situated adjacent to the epithelial surface (Prydal et al., 1992; Prydal et al., 1993;
Dilly, 1994) (Fig. 1.2). The physical state of the mucous gel and the changes of its
physicochemical properties as a consequence of modification of environmental factors, such as ionic strength and pH, play an important role in many diseases (Bansil and Turner, 2006). Since the existence of such an extended mucous layer, attention has focused on the contribution of mucins to the physiology of the tear film. Specialized defensive, surfactant and rheological properties of the tear film may be reflected in the structure of its mucins (Holly and Lemp, 1971;
Holly, 1973; Holly and Lemp, 1977; Dilly, 1994).
Figure 1.2. Schematic composition of the tear film and its interface with the ocular surface epithelium (from Gipson, 2007).
1.3. Mucins
Mucins, extracellular proteins with high molecular weights ranging from 0,5 to 20 MDa, are
among the largest known glycoproteins. A common characteristic of all mucins is the high level
of glycosylation (Dekker et al., 2002), consisting up to 80% of their mass as O-linked glycan
chains (Royle et al., 2008). All mucin polypeptide chains have domains rich in serine, threonine and proline (PTS repeats), whose hydroxyl groups are in O-glycosidic linkage with oligosaccharides. These domains are composed of tandemly repeated sequences that vary in number, length, and amino acid sequence from one mucin to another (Gendler and Spicer, 1995).
These large regions, up to 6000 amino acids in length, are usually devoid of cysteine residues and composed of short (8–169 amino acids) tandemly repeated peptides. PTS regions are found in all MUC-type mucins but also in many other glycoproteins, although the PTS regions in mucins are exceptionally long and usually comprise more than half the polypeptide. Non-PTS regions are the N- and C-terminal parts of the mucin polypeptides beyond the PTS regions. They usually contain all the cysteine residues of the mucins. Non-PTS regions are not excessively repeated, unlike PTS regions (Fig. 1.3) (Dekker et al., 2002).
The picture that emerges is that of a macromolecule with a complex organization into domains with different structures. From the polymeric viewpoint mucin can be considered as a multiblock copolymer with alternating polyelectrolytic domains having a grafted sugar brush, connected by flexible regions with less glycosylation and the tendency to form multimers (Bansil and Turner, 2006). The high carbohydrate content of mucins contributes to the physico-chemical properties required for protective roles at mucosal surfaces (Corfield et al., 1997), and is highly adaptable.
Mucins are capable of blocking microbial binding to the ocular surface (Fleiszig et al., 2002).
Thus, their carbohydrate content represents a bank of glycan structural sequences with the potential to create specific ligands (Hang and Bertozzi, 2005).
1.3.1. Mucin classification
Mucins have been divided in two categories, secreted mucins and membrane associated mucins
(Fig. 1.3) (Gendler and Spicer, 1995; Hollingsworth and Swanson, 2004).
Figure 1.3. Mucin composition and classification. Two examples of well-characterized mucins, MUC1 and MUC2, membrane associated and secreted mucin respectively. On the right: Mucin classification scheme (Modified from Dekker et al., 2002).
1.3.1.1. Secreted mucins
Of the seven secreted ocular mucins identified to date, five are the so-called gel-forming mucins
(MUC2, 5AC, 5B, 6 and 19; Fig. 1.3) (Moniaux et al., 2001; Gipson, 2007). Beside the central
glycosylated region (PTS regions), there are cysteine-rich regions (non-PTS regions), located at
the amino and carboxy terminals, and sometimes interspersed between the PTS-repeats. These
regions contain an amino acid composition more representative of globular proteins, relatively
little O-glycosylation and a few N-glycosylation sites (Bell et al., 2003) and a high proportion of
cysteine (>10%). These cysteine rich regions contain domains that possess sequence similarity to
domains of von Willebrand factor (Perez-Vilar and Hill, 1999; Turner et al., 1999; Bell et al.,
2001), and have been shown to be involved in dimerization via disulfide bond formation, and
subsequent polymerization of the dimers to form multimers (Sheehan et al., 2004). The gel-
forming mucins are secreted by goblet or glandular cells in all wet-surfaced epithelia of the body.
They are moved about on epithelial surfaces by various mechanisms, including tracheal cilia movement, peristalsis, or, in the case of the ocular surface, by eyelid movement, to clean the surfaces of particulate matter. Secreted mucins’ hydrophilic character, which results from its heavy glycosylation, helps to hold fluids on epithelial surfaces.
Beside the gel forming mucins, two small soluble mucins have been identified, MUC7 and MUC9 (Fig. 1.3), both of which lack cysteine-rich domains. MUC7 has been shown to have antimicrobial activity (Situ et al., 2003).
The aqueous-mucus layer of the ocular surface is mainly composed of MUC5AC (Gipson and Inatomi, 1998; Bansil and Turner, 2006), and there is an high similarity with MUC2. These mucins have an high homology in their non-PTS regions (Dekker et al., 2002; Schultz et al., 2000).
1.3.1.2. Membrane-associated mucins
Membrane-associated mucins (MAMs) have a single transmembrane domain, a short cytoplasmic tail, and a large, heavily glycosylated extracellular domain. These proteins are found in the glycocalyx of apical membranes of wet-surfaced epithelia (Hollingsworth and Swanson, 2004;
Blalock et al., 2008). They may extend as much as 500 nm from the apical epithelial surface (Bramwell et al., 1986; Hilkens et al., 1992; Gipson, 2004). To date, 10 MAMs have been identified (MUC1, 3A, 3B, 4, 12, 13, 15, 16, 17, and 20). (Fig. 1.3) (Hollingsworth and Swanson, 2004; Higuchi et al., 2004).
MUC1 is highly represented on ocular surface cell membrane and is the most studied (Inatomi et al., 1995; Inatomi et al., 1996; Pflugfelder et al., 2000; Argueso et al., 2003). MUC1 contains as well as an uninterrupted PTS repeat (Dekker et al., 2002), a transmembrane domain and a SEA domain (sea-urchin sperm protein enterokinase agrin) (Fig. 1.6). Most of the wet-surfaced epithelia express several MAMs, but each may have different functions due to differences in cytoplasmic tail sequence, intracellular signaling capability, and/or presence of binding domains.
For example, MUC1 cytoplasmic tail is capable of interacting with molecules involved in different signalling pathway, such as beta-catenin (Yamamoto et al., 1997).
Soluble forms of MUC1, MUC4 and MUC16 are constitutively released from the apical surfaces
of epithelial cells into luminal fluids in vivo, but little is known about the mechanisms of shedding
(Yin and Lloyd, 2001; Komatsu et al., 2002). At least three possible mechanisms for release are
possible. First, constitutive steady release may be brought about by an endogenous protease present normally in either the cell membrane or in the extracellular fluids (Hollingsworth and Swanson, 2004). Secondly, splice variants of the mucins lacking the transmembrane and cytoplasmic domain may be secreted from the apical surfaces (Moniaux et al., 2001). Although splice variants have been reported for MUC1 and 4, such variants have not been reported for MUC16. Thirdly, proteases or other inflammatory agents present in body fluids as a result of disease may induce aberrant “release” of MAMs from surfaces of affected epithelium (Blalock et al., 2008) However, soluble forms of MUC1, MUC4, and MUC16 have been detected in samples of normal human tear fluid, thus indicating the occurrence of their shedding in physiological conditions from the ocular surface epithelium (Spurr-Michaud et al., 2007).
The mechanism and site of proteolytic cleavage in the extracellular domain of MUC1 have been studied in uterine epithelium in vitro, where the constituitive shedding of the protein is induced by agents such as tumor necrosis factor-alpha, phorbol-12-myristate-13-acetate and membrane type matrix metalloproteinase 1 (Thathiah et al., 2003; Thathiah et al., 2004). Agents that have been suggested to induce aberrant MAMs release include neutrophil elastase (Kim et al., 1987) and N-acetylcysteine (Berry et al., 2004). Release of the extracellular domains of MAMs appears to be independent of the intracellular cleavage and re-association that occurs in MAMs after protein synthesis in the endoplasmic reticulum during processing and assembly of the full-size protein (Levitin et al., 2005; Soto et al., 2006). However, there is a lack of information on the specific mechanisms of constitutive extracellular domain shedding, or induced release of MAMs at the ocular surface.
Other MAMs present on the ocular surface are MUC3A and MUC3B (Gipson, 2007). MUC3 was one of the first MUC protein, found in 1990 (Taylor-Papadimitriou, 1991), but it has been discovered that there are in fact two products of closely related and adjacent genes, MUC3A and 3B, with 98% homology (Pratt et al., 2000). These mucins characteristically have a transmembrane domain, a SEA domain and one or two epidermal-growth-factor (EGF)-like domains (Fig. 1.6) (Dekker et al., 2002).
1.3.2. Mucin glycosylation
Mucins from many sources have been examined for carbohydrate content and oligosaccharide patterns (Moore and Tiffany, 1981; Roussel et al., 1988; Schachter and Brockhausen, 1992;
Lamblin et al., 1992; Montreuil et al., 1996; Corfield et al., 2001; Rose and Voynow, 2006).
Mucin-type oligosaccharide chains show a considerable variety in length, branching and terminal substitution. Chains consist of three regions: core, backbone and periphery (Fig. 1.4). Variation exists in each region. Thus, for any mucin molecule, hundreds of O-linked oligosaccharides may occur.
Figure. 1.4. Hypothetical composite oligosaccharide structure found in mucins. Principal features include: O- glycosidic linkage of N-acetyl-D-galactosamine to serine and threonine residues on the polypeptide chain; CORE region, with core 2 as an example; BACKBONE of poly-N-acetyllactosamine type; PERIPHERAL groups such as sialic acid, sulphate and a blood group antigen. Gal, galacotse; Glc, glucose; Fuc, fucose; GlcNAc, N-acetyl glucosamine; GalNAc, N-acetyl galactosamine (Corfield et al. 1997).
The glycan chains comprise galactose, N-acetylgalactosamine, N-acetylglucosamine, fucose, sialic acids and traces of mannose and sulphate. Typically, the oligosaccharide chains, consisting of 5–15 monomers, exhibit moderate branching and are attached to the protein through N-acetyl- galactosamine by O-glycosidic bonds to the hydroxyl side chains of serine and threonine and arranged in a ‘‘bottle brush’’ configuration (Bansil and Turner, 2006). The small proportion of N- linked oligosaccharides present in mucins seems to be relevant for the correct folding and subcellular transport (Hollingsworth and Swanson, 2004; Bell et al, 2003).
Mucin-type O-linked glycosylation is initiated by a family of enzymes known as N-
acetylgalactosaminyltransferases (GALNT) that transfer α-N-acetylgalactosamine to Ser/Thr
residues in the protein backbone to form the Tn-antigen, an immediate precursor in biosynthesis of Thomsen-Friedenreich (T) antigen (galactose-N-acetylgalactosamine). It has been estimated that in about 90% of all cancers and some leukemias, T and Tn antigens are expressed and uncovered (Springer, 1995) (Fig. 1.5). The Tn-antigen can be elongated by other glycosyltransferases to generate a series of O-linked glycans (Fig. 1.5) (Guzman-Aranguez et al., 2009).
Figure 1.5. Pathways of O-glycosylation on the ocular surface epithelia. Glycosyltransferases potentially involved in the biosynthesis of mucin-type O-glycans include thirteen polypeptide GalNAc-transferases (GALNT) involved in the initiation of mucin-type O-glycosylation, the core 1 β-3-galactosyltransferase (T-synthase) involved in the addition of galactose to the Tn antigen, three α2-6-sialyltransferases (ST6GalNAc), and two α2-3-sialyltransferases (ST3Gal). Core 2 (dotted line) in conjunctival epithelial cells is synthesized by core 2 β1,6 N- acetylglucosaminyltransferase (C2GnT-M). The relative levels of expression of individual glycosyltransferase isoforms are indicated in parenthesis. Symbols represent galactose (●), N-acetylgalactosamine (□), N- acetylglucosamine (■), and N-acetylneuraminic acid (♦) (from Guzman-Aranguez et al., 2009).
To date, eight core structures have been identified, of which core 1 and core 2 (Fig 1.5) are the
most common. The core structures are then extended, by the sequential action of a series of
glycosyltransferases organised along established pathways, to generate the wide variety of O- glycan chains (Hanisch, 2001; Gipson and Argueso, 2003; Guzman-Aranguez et al., 2009).
Chains vary in charges ranging from neutral to highly negative, depending on the presence of sialic acid and sulphate (Roussel et al., 1988; Montreuil et al., 1996).
Core 1-based structures are the major mucin-type O-glycans at the ocular surface; there are three major differences between tissue-associated O-glycans in conjunctiva and those found in tears (Guzman-Aranguez et al., 2009). First, the conjunctival epithelium contains core 2-based structures, including galactosyl core 2 and di α2-3 sialyl galactosyl core 2. Second, α2-3 sialyl core 1 is the predominant O-glycan in human conjunctival tissue (47.4%), whereas in tears the prominent O-glycan is α2-6 sialyl core 1 (48%). And third, disialyl core 1 is present in conjunctival mucin but not in tears. These discrepancies could be due to several factors; it is possible that specific mucin-type O-glycans in conjunctival tissue are associated to intracellular or cell surface glycoproteins and, therefore, not secreted into the tear film. It is also possible that the difference in O-glycan composition between conjunctival epithelium and tear film is due to degradation by glycosidase activity in tears (Matthews et al., 2001; Guzman-Aranguez et al., 2009).
Bacteria have been shown to adapt to the library of glycan structures produced by the mucosal barriers of their hosts by evolving families of glycan binding lectins that facilitate colonization (Sharon and Lis, 1997), suggesting the importance of individual glycan structures in host defence and bacterial recognition. Analyses of canine ocular mucin O-glycans (Carrington et al., 1998) and of human ocular mucins (Berry et al., 1996; Argueso et al., 1998; Chao et al., 1988) provided compositional information, and showed that canine ocular mucin glycans are smaller compared with those found at other mucosal surface. Royle et al. (Royle et al., 2008) identified and compared the ocular glycan profile for ocular mucins in man, dog, and rabbit, to facilitate future studies into host microbial interactions in ocular infectious disease (Royle et al., 2008).
1.3.3. Mucin genes
Currently, approximately 20 mucin genes (designated muc) have been identified, cloned and
partially sequenced in the human, and homologs to many of them have been identified in the
mouse and rat (Perez-Vilar and Hill, 1992). These are abundantly expressed in epithelial and
glandular tissues. Seven of these genes have been identified at the human ocular surface and in
the lacrimal glands (Gipson, 2004; Paulsen and Berry, 2006). Only three muc genes (muc1, muc2
and muc5B) have been totally sequenced due to the large size of the central tandem repeats, which are difficult to be accurately assembled. For some authors, many of the smaller membrane bound mucins are not considered ‘‘true’’ mucins since they only share the PTS repeats and glycosylation with other mucins (Dekker et al., 2002). The genes for secreted mucins MUC2, MUC5AC, MUC5B and MUC6 are located in a cluster within 500 kb on the short arm of chromosome 11 (11p15) (Pigny et al., 1996). The 11p15 mucins share considerable overall homology in their non- PTS regions (21–33%) and have probably evolved through gene duplication of one ancestral gene (Desseyn et al., 1998).
Figure 1.6. Deducted polypeptides from muc genes. The chromosomal location of each mucin gene is indicated on the right. All mucin sequences were aligned with their C terminus to the right. Each type of peptide domain is depicted in a separate colour, indicated in the legend. MUC16, MUC7 and MUC8 show no homology to any other known mucin. MUC11 cannot be aligned because it is so far only characterized by its PTS region; its gene has the same chromosomal location as MUC3A, MUC3B and MUC12, and the possibility cannot be excluded that the MUC11 cDNA sequence is, in fact, continuous with the MUC12 cDNA, of which only the C terminus has been identified. The representations are based on the full-length cDNA sequences. Any splice variants (e.g. MUC1, MUC3A, MUC3B and MUC4) are not accounted for, and allelic variation (known to exist in at least some MUCs as variations in the number of tandemly repeated PTS sequences) was also not taken into account. Dashed lines indicate unknown sequences. The brackets around the N terminus of MUC6 indicate that this sequence is not publicly available. The scale of the deduced polypeptides is indicated by the bar representing 500 amino acids (aa) (from Dekker et al., 2002).
Several membrane-bound mucin genes are probably related to each other, especially these
localized to chromosomal locus 7q22: muc3A, muc3B and muc12 (Fox et al., 1992; Gum et al.,
1997; Van Klinken et al., 1997; Williams et al., 1999a; Williams et al., 1999b; Pratt et al., 2000).
Moreover, the membrane associated MUC1 is also related to the 7q22 mucins (Gendler et al., 1988; Middleton-Price et al., 1988; Ligtenberg et al., 1991) (Fig 1.6). The PTS-repeat sequences for each muc gene are unique to each species, whereas the cys-rich regions share a large degree of similarity (Perez-Vilar and Hill, 1999). Different collections of muc genes are expressed in different tissues (Dekker et al., 2002).
1.3.4. Physical and colloidal properties of mucins in dilute solution
Mucins have both hydrophobic and hydrophilic regions with the ability to form hydrogen bonds, and electrostatic interactions. This wide range of interactions causes them to aggregate forming gels and mucoadhesive interactions with other substances. These physical properties are of direct relevance to the physiological functions of mucus in normal and disease states (Bansil and Turner, 2006).
Mucins have been difficult to characterize, owing to their large molecular weight, polydispersity and high degree of glycosylation (Bansil and Turner, 2006). Earlier biophysical studies (Harding, 1989; Bansil et al., 1995), primarily using light scattering methods, showed that mucins were a somewhat stiffened random coil with a radius of gyration around 100 nm. NMR studies of MUC1 (Fontenot et al., 1993; Otvos and Cudic, 2003) revealed very little alpha helix, a small amount of beta and mostly random coil. The large size of mucins has, on the other hand, turned out to be of great advantage in imaging the molecule directly. Earlier transmission electron micrographic studies (Fiebrig et al., 1995) revealed long fibers approximately 400 nm long in pig gastric mucin (PGM). More recent Atomic Force Microscopy (AFM) studies of ocular mucins (Round et al., 2002) showed individual fibers with a broad distribution of contour lengths. While most of the fibers were between 200–600 nm long, the tail of the distribution extended to 1500 nm. They also estimated a persistence length of about 36 nm from these images, which confirmed the extended nature of the polymer. Another AFM study of ocular mucin (Brayshaw et al., 2004) demonstrated the multimeric nature of mucin, by observing in situ depolymerisation on treatment with dithiothreitol. Longer fibers, up to 2 µm in length, were observed in PGM (Deacon et al., 2000);
AFM images of PGM which was about 50% deglycosylated showed that the deglycosylated
portions re-folded forming compact globular structures (Hong et al., 2005). Thus the sugars seem
to be important for maintaining the extended conformation of mucin. The conformation of mucin
also depends on factors such as pH and ionic strength (Cao et al., 1999). As described by Lee et
al. (Lee et al., 2005), the conformation of PGM in dilute solution examined by viscosity and
circular dichroism measurements revealed similar changes as function of pH (Bansil and Turner, 2006).
Taylor et al. (Taylor et al., 2003) used rheological techniques to investigate the structure and formation of the pig gastric mucus gel and showed that both transient and non-transient interactions are responsible in maintaining the gel matrix. Purified mucins also has been shown to form gels. The gelation of mucin is a complex problem, involving the interplay of electrostatic and hydrophobic interactions, somewhat reminiscent of the association of multiblock copolymers in solvents that have differential solubility with respect to the component blocks. The entanglement of the sugar side chains further contributes to the high viscosity of mucin solutions.
Mucin glycoproteins has also been shown to exhibit liquid crystalline order. Viney et al. (Viney et al., 1993) had shown that slug mucin forms nematic liquid crystals (the molecules in the liquid align themselves into a threadlike shape). A detailed study of the temperature and concentration dependence of the liquid crystalline behaviour of commercially available mucin suggested that the interactions of interdigitated sugar side chains were responsible for the nematic behaviour (Davies and Viney, 1998). These observations were confirmed by neutron scattering (Waigh et al., 2002). Purified PGM, on the other hand, did not show this liquid crystalline orientation, suggesting that commercially available mucin, which consists of a single mucin monomer, is more rigid and easier to orient than the multimeric form of native PGM in which the non- glycosylated portions serve as flexible links between the rigid, glycosylated, monomeric domains.
The well known tendency of other substances to adhere to mucin, known as mucoadhesivity, is not surprising given that this glycoprotein exhibits electrostatic, hydrophobic, and hydrogen bonding interactions (Harding et al., 1999). By the same token the molecule can be anti-adhesive, for example to negatively charged species (Berry et al., 2001). Bovine submaxillary mucin could be adsorbed to hydrophobic polystyrene surface, rendering the surface hydrophilic (Shi and Caldwell, 2000).
In view of the protective function of mucus, studying the diffusion of other molecules through it
is of considerable physiological interest. It is also an important factor in the design of drugs which
have to diffuse through the mucus layer, or kept from entering it. While many small molecules
diffuse readily through mucus, the diffusion of larger particles depends both on size and muco-
adhesive interactions. While many small viruses (20–200 nm) could diffuse through cervical
mucus, others, such as the Herpes Simplex virus, got stuck to the mucus (Olmsted et al., 2001).
Due to its high viscosity, water and other low viscosity fluids injected under pressure gradient across an unstirred mucin solution do not simply diffuse, but are transported by a viscous fingering mechanism (Bhaskar et al., 1992; Fujita et al., 2000).
Mucus is the first barrier with which nutrients and enteric drugs must interact and diffuse through, in order to be absorbed and gain access to the circulatory system and their target end organs.
There is great interest in methods to optimize these so-called muco-adhesive interactions for improved drug delivery. Various molecular interactions have been exploited to enhance mucoadhesion, including, polyelectrolytic interactions, hydrogen bonds (Harding et al., 1999) and disulfide binding (Leitner et al., 2003). Mucin can be used as a high molecular weight additive to improve the adherence of artificial tear drops in treating dry eye syndrome (Khanvilkar et al., 2001). Efforts to develop nanoparticles for mucosal DNA vaccines and gene therapy are also being considered (Dawson et al., 2004). The abnormality of the sugars in cell membrane bound mucins from cancerous cells has also been targeted as a potential for cancer vaccine development (Rathborne, 1993).
1.4. Alterations of ocular surface in pathologies: Dry eye syndrome
Dry eye is a disorder of the tear film due to tear deficiency or excessive evaporation, which causes damage to the ocular surface and is associated with symptoms of ocular discomfort, as defined by the Dry Eye Workshop in 1995 (Lemp, 1995). This definition was improved in the light of new knowledge about the roles of tear hyperosmolarity and ocular surface inflammation in dry eye and the effects of dry eye on visual function. According to the final definition, which came in 2007, dry eye is a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort (Begley et al., 2003; Adatia et al., 2004; Vitale et al., 2004), visual disturbance (Rieger, 1992; Liu et al., 1999; Goto et al., 2002) and tear film instability (Holly et al., 1973; Bron, 2001; Goto et al., 2003) with potential damage to the ocular surface. It is accompanied by increased hosmolarity of the tear film (Farris et al., 1986; Gilbard, 1994;
Murube, 2006; Tomlinson et al; 2006) and inflammation of the ocular surface (Pflungfelder et al.,
1999; Tsubota et al., 1999; Dry Eye Workshop, 2007). A healthy tear film nourishes, lubricates
and protects the ocular surface. Any dysfunction of the main or accessory lacrimal glands, the
meibomian glands, eyelids, cornea, conjunctiva or the connecting neural reflex arcs (the
components which together form the lacrimal functional unit) causes tear film instability,
symptoms of grittiness and irritation, ocular surface inflammation and ultimately signs of ocular surface damage and visual impairment (Stern et al., 1998).
The prevalence of dry eye has been reported as 9% of patients over 40 years of age, increasing to 15% of those over 65 (McCarty et al., 1998; Schein et al., 1997). Given the trend towards an aging population, the impact of dry eye on clinical eye care services seems likely to increase in years to come. In optometric practice, dry eye remains the primary reason for reduced wearing times and for contact lens failure with studies reporting around 50% prevalence of self-reported dry eye in contact lens wearers compared with 20% in non-contact lens wearers (Nichols et al., 2005). Women were found to report dry eye more frequently than men, with 40% of the men and 62% of the women classified as having dry eye (Nichols et al., 2006). The reasons for this were not explored, but potential contributing factors were considered to be hormone fluctuations during the menstrual cycle or after menopause and use of oral contraceptives or hormone replacement therapy. It was also noted that symptom reporting by women, in general, tends to be higher than that by men (Ladwig et al., 2000).
1.4.1. Dry eye aetiology
Although dry eye patients report similar symptoms of dryness, grittiness, irritation and burning, the causes can be diverse. Pathogenic events that disturb homeostasis, and are not promptly neutralized by appropriate reaction of the Ocular Surface System, create a deleterious cycle of events that result in the appearance of disease (Johnson and Murphy, 2004). The core mechanisms of dry eye are driven by tear hyperosmolarity and tear film instability. Tear hyperosmolarity causes damage to the surface epithelium by activating a cascade of inflammatory events at the ocular surface and a release of inflammatory mediators into the tears. Epithelial damage involves cell death by apoptosis, a loss of goblet cells, and disturbance of mucin expression, leading to tear film instability. This instability exacerbates ocular surface hyperosmolarity and completes the vicious circle. Tear film instability can be initiated, without the prior occurrence of tear hyperosmolarity, by several aetiologies, including xerophthalmia, ocular allergy, topical preservative use, and contact lens wear (Dry Eye Workshop, 2007) (Fig.
1.7).
Figure 1.7. Events and pathogenicity of ocular surface diseases. (from Johnson and Murphy, 2004).
Dry eye can be classified into two main aetiological groups: aqueous deficient and evaporative (Lemp, 1995; Dry Eye Workshop, 2007). Aqueous deficient dry eye encompasses both Sjögren’s Syndrome and non-Sjögren’s causes of lacrimal gland dysfunction. Sjögren’s Syndrome is a systemic autoimmune condition characterized by a combination of keratoconjunctivitis sicca and dry mouth
(Sjögren and Block, 1971
). Evaporative dry eye is divided into intrinsic and extrinsic causes, where intrinsic factors include meibomian gland dysfunction and lid abnormalities, and extrinsic factors include contact lens wear and ocular surface disease such as allergy. Aqueous deficient dry eye occurs when the main or accessory lacrimal glands are compromised.
Evaporative dry eye, on the other hand, occurs with defective meibomian glands, ocular surface irregularities, anomalies of lid structure or by the wearing of contact lenses (Dry Eye Workshop, 2007). Contact lenses induce dry eye through lipid layer disruption, tear film thinning, corneal desiccation as a consequence of lens dehydration, loss of lid conformity and/or blink alteration.
All contact lenses disrupt tear film structure to some extent. While patients with healthy ocular
surfaces and tear film prior to lens fitting may be able to withstand this disruption, those with a fragile tear system are more inclined to report dry eye symptoms with contact lens wear. Aqueous deficient and evaporative dry eye may co-exist but it is important to establish the likeliest cause by thorough assessment, in order to manage the dry eye most effectively (Fig. 1.8).
Figure 1.8. Major aetiological causes of dry eye (from DryEyeWorkshop, 2007).
The basis for symptoms in dry eye is not truly known (Johnson and Murphy, 2004). There is still debate about fundamental issues, such as the processes involved in tear rupture, the presence of inflammatory mediators at the surface of the eye and, in particular, the importance and the quantity of ocular mucins at the ocular surface (Johnson and Murphy, 2004).
1.4.2. Dry eye and mucin alterations
Alterations in mucin expression and/or mucin glycosylation could be implicated in the pathophysiology of dry eye (Caffery et al., 2008), but only a limited number of studies addressing these issues have been conducted.
The first possibility is an alteration in mucin glycosylation. Altered mucin O-glycosylation has
been frequently observed with several pathologies, including IgA nephropathy, cystic fibrosis,
inflammatory bowel disease, and cancer (Berger, 1999; Brockhausen, 1999; Barratt et al., 2004;
Xia et al., 2005; Bodger et al., 2006). In the eye, several reports have described alterations in the carbohydrate composition of MAMs at the apical glycocalyx of conjunctival epithelial cells in patients with dry eye. These include a reduction in lectin and antibody binding to cell-surface carbohydrate epitopes such as sialic acid and core 1, and alteration in the distribution of glycosyltransferases involved in mucin-type O-glycosylation (Versura et al., 1986; Watanabe et al., 1997; Danjo et al., 1998; Argueso et al., 2003; Argueso et al., 2006). Since MAMs are involved in resisting rose bengal dye penetration, Danjo et al. (Danjo et al., 1998) observed significant differences in dye patterns to conjunctival cells in normal eyes compared with those of patients with dry eye symptoms, showing alterations in cell surface mucin O-glycosylation in aqueous-deficient dry eye correlates with epithelial damage at the ocular surface.
As regards secreted mucins, Guzman-Aranguez et al. (Guzman-Aranguez et al., 2009) evaluated the expression of carbohydrate epitopes in dry eye tear fluid, detecting no changes in the total amount of mucin O-glycans in tears of dry eye patients, using HPLC. Similarly, no differences were detected in lectin binding to tear glycoproteins in dry eye, indicating lack of alterations in core 1 O-glycosylation and glycoprotein sialylation in the disease. Hence, no changes in O-glycan composition and amount of secreted mucins occur in the tears of these patients, in contrast with MAMs alterations in carbohydrate distribution on the epithelial glycocalyx in dry eye.
The second possibility is an alteration in the expression of muc genes. Guzman-Aranguez et al.
(Guzman-Aranguez et al., 2009) tried to identify genes with altered expression in dry eye patients
by analyzing microarray data from conjunctival samples collected by impression cytology. The
results revealed no differences in mRNA expression of mucin-type glycosyltransferases between
normal subjects and dry eye patients. These results are in agreement with those obtained by
Imbert et al. (Imbert et al., 2006) using real-time RT-PCR, showing no alterations in GALNT
expression in conjunctival epithelium in patients with aqueous-deficient dry eye. In these studies,
however, the conjunctiva was isolated and homogenized previous to analysis, while Argueso et
al. (Argueso et al., 2003) using immunofluorescence in situ, reported changes in the cell-type and
cell-layer distribution of GALNT in pathologically keratinized conjunctival epithelia. Although
no localization studies have been performed with other mucin-type glycosyltransferases,
alteration in the local distribution of these enzymes, not in their overall expression, can be
hypothesize and might correlate with altered cell surface O-glycosylation and epithelial damage
(Guzman-Aranguez et al., 2009).
Finally, there is the question of the correlation between the expression of muc genes and their protein products. Although several reports have shown alterations of mucin glycosylation in dry eye (Garcer et al., 1998; Jones et al., 1998; Danjo et al., 2000; Pisella et al., 2000), to date, very little is known about correlation of the expression of specific mucin genes and their protein products. Using an antibody against the cysteine rich domain of the goblet cell-specific mucin MUC5AC, reduced secreted MUC5AC protein levels have been observed in the tears of Sjögren’s syndrome patients, together with reduced mRNA transcripts in their conjunctival cells (Argueso et al., 2002) compared to normal subjects. On the other hand, a study by Zhao et al.
(Zhao et al., 2001) found no difference in MUC5AC levels in the tears of dry eye subjects versus normals. Using histochemical staining of surface conjunctival epithelial cells gathered from impression cytology, reduced expression of MUC16 in non-Sjögren syndrome dry eye was reported and observed that this was associated with rose bengal staining (Blalock et al., 2007;
Danjo et al., 1998). These observations stand in contrast to a recent study by Caffery et al.
(Caffery et al., 2008), that demonstrated an increased expression of membrane associated MUC16 in the conjunctiva of patients with Sjögren syndrome and in aqueous deficient dry eye patients, compared to normal subjects.
Contrasting results have been obtained also for MUC1. Jones et al. (Jones et al., 1998) reported a
decrease in MUC1 protein levels in patients with Sjögren syndrome, detected by
immunofluorescent staining of conjunctival epithelium, using an antibody raised against the PTS
region of MUC1. However, interpretation of these results is of concern, because the number of
tandem repeats per mucin molecule varies due to genetic polymorphism, and probes against
regions within the PTS regions of mucins are not useful as quantitative tools. At the genetic level,
a specific splice-variant of the muc1 gene was found to be slightly reduced in dry eye subjects
(Imbert et al., 2006). Argueso et al. (Argueso et al., 2002), have failed to find differences in muc1
or muc4 gene expression between controls and Sjögren subjects (Argueso et al., 2002). On the
contrary, Caffery et al. (Caffery et al., 2008) showed that Sjögren subjects express greater
quantities of MUC1 protein and mRNA compared to control subjects. These results, combined
with the previous MUC16 results (Caffery et al., 2008), suggest an excess of both MUC1 and
MUC16 proteins in the tear film of Sjögren subjects and increased levels of mRNA for these
mucins in conjunctival cells.
The finding of increased tear mucins in Sjögren syndrome is of particular interest as the observation of excess mucus in the tears of Sjögren patients is a long standing clinical finding.
(Caffery et al., 2010). Mechanistically, albeit simplistically, it appears that increased expression of mRNAs coding for MUC1 and MUC16 are followed by excess shedding of these species into the tear film, or vice versa. The excess expression of mucins in disease states is a well studied phenomenon. In fact, excess mucin production in humans is an ancient defense mechanism (Basbaum et al., 1999) and non-ocular mucous membranes demonstrate excess mucus production under adverse conditions in dogs, rats, and humans (Marom et al., 1983; Johnson et al., 1985;
Yanni et al., 1989). Excess mucin of the ocular surface occurs most commonly in ocular allergy and here there is excess of transmembrane MUC1, MUC4, and MUC16 (Dogru et al., 2005). In vitro, inflammatory mediators that have been found in the tear film of dry eye patients have been
shown to increase MUC1 expression in a human limbal corneal epithelial cell line (Albertsmeyer et al., 2010). Also, ocular cicatricial pemphigoid patients demonstrate increased mucin production (Argueso et al., 2003). This increased mucin levels in the early stages of the keratinisation process, or during inflammation, suggests that ocular surface cells can participate in compensatory attempts to synthesize more mucin to maintain a wet surface phenotype. The shedding of the extra cellular portion of MUC1 has been studied in infection and is considered to be a reaction of the molecule to changes in its environment that then signals the cell to which it is attached (McCauley et al., 2007). It is suggested that the release of the extracellular portion produces activation, proliferation or apoptotic response of the epithelium (McCauley et al., 2007).
Perhaps this exposed portion of MUC1 is shed because of physiologic changes associated with severe dry eye disease and this activity then signals the epithelial cells to produce more MUC1.
Caffery et al. findings would suggest that this compensatory or protective mechanism is most prevalent in Sjögren dry eye disease, but may be related to disease severity (Caffery et al., 2010).
As described in Paragraph 1.3.1.2, extracellular domains of MUC1, 4 and 16 can be released from the ocular surface by agents present in tears. Neutrophil elastase and tumor necrosis factor present in higher amounts in dry eye patients’ tears may cause MAMs release (Blalock et al., 2008).
Since membrane associated mucins are proposed to function in lubrication, hydration, and
protection of the ocular surface, understanding the mechanism of their ectodomain release may
help to better clarify the aetiology of dry eye syndrome.
1.5. Artificial Tears
Ocular lubrication is essential for contact lens wearers, which utilize artificial tears to increase both lubrication and retention of the tear film. Human tears are composed of water, electrolytes, small molecules such as carbohydrates and lipids, and a variety of proteins. The use of artificial tears has obvious limitations. Artificial tears cannot completely substitute complex composition of natural tears. The integrity of the three-layered lipid, aqueous, and mucin structure, vital to the effective functioning of the tear film is not reproduced. Artificial tears act by adding volume to the tear film, but they can only do this while they remain in contact with the surface of the eye. A simple saline eye-drop will remain in contact with the eye surface for only a few seconds.
Therefore, to increase the permanence time on the corneal surface and also have a good tolerability, these preparations are generally made viscous by adding agents of high molecular weight normally hydrosoluble polymers of synthetic, semi synthetic or natural origin. The preference has been given to compositions based on macromolecular compounds of natural origin such as cellulose derivatives (in particular, cellulose esters like carboxy- methylcellulose, and alcohol derivatives of cellulose ethers like hydroxylpropylmethylcellulose), glycosaminoglycans (in particular, hyaluronic acid, a polysaccharide present in many human and animal tissues and fluids, and widely used in ophthalmic preparations) and polysaccharides having suitable rheological properties (such as polysaccharide extracted from tamarind seed, TSP). Synthetic or semi synthetic compounds include polyvinyl alcohol, polyvinyl pyrrolidone, polyethylene glycol and dextran. All these high molecular weight polymers are called hydrogels, and their mucous adhesive properties help in prolonging the time tears stay on the eye. Hydrogels are actually polymers that are endowed with the property of swelling up in water and retaining the moisture.
There is no significant difference in the clinical efficacy of different hydrogels that are used in different artificial tears. In addition, there have been no large scale, masked, comparative clinical trials to evaluate the wide variety of ocular lubricants (Dry Eye Workshop, 2007).
An important feature in the selection of artificial tears is the type of preservative used to increase
their shelf life. The more commonly used preservatives are benzalkonium chloride and
chlorobutanol. In particular, benzalkoniun chloride can increase the eye surface irritation and
disease. Newer preservatives such as sodium perborate, sodium chloride and polyquaternium-1 (a
polycationic polymer) are less damaging to the eye surface than benzalkoniun chloride (Gobbels
and Spitznas, 1989; Lopez Bernal and Ubels, 1991).
To increase the retention time of artificial tears, a recent development is the introduction of ingredients that have some bioadhesive properties. For example, Hydroxypropyl (HP)-Guar (a nonionic polymeric thickener) forms a cross linked viscoelastic gel. It has been suggested that HP-guar preferentially binds to the more dry or damaged areas of the surface epithelial cells, providing protection for these cells. Once exposed to the eye surface, HP-Guar forms a gel with increased viscosity and bioadhesive properties that promote the retention of the demulcents present in the preparation (polyethylene glycol 400 & polypropylene glycol) (Christensen et al., 2004). These viscous tears have a longer retention time but are not easily drained out of the eye through the lacrimal outflow system. Therefore, the high viscosity may lead up to inconveniences such as sticky feeling and crystallization on the edge of eyelids and lashes, causing an unacceptable blurry vision in some individuals (Ridder et al., 2005).
1.6. Arabinogalactans
A strategy to antagonize the discomfort of dry eye is the use of arabinogalactan (AG), a low
viscosity polysaccharidic molecule that may endure on the ocular surface without unpleasant
disadvantages. Especially in the case of problems related to contact lens use, it is instead
extremely important for a possible product used as a tear fluid supplement, to have a low
viscosity, besides being well-tolerated and having no irritant effects on the eye. Arabinogalactans
are a class of long-chain, densely branched polysaccharides with a molecular weight ranging
between 10 and 120 kDa and a central structure consisting of a chain of galactopyranose units. In
nature they are found in various microbe systems, especially Mycobacteria, where they are
complexed with peptidoglycan and mycolic acid to form the cell wall. Many edible and non-
edible plants are rich sources of arabinogalactans, mainly in a glycoproteic form. Many herbs
with acknowledged immuno-stimulant properties, such as Echinacea purpurea, Baptisia tintoria
and Thuja occidentalis, contain significant quantities of arabinogalactans. The woody tissues of
plants of the Larex genus are particularly rich in arabinogalactans, especially Larex occidentalis,
also known as Western larch, Mountain larch or Western tamarack, native of the Pacific and
Inland Northwest United States and Canada, which represents the main source of
arabinogalactans for industrial use (Odonmazing et al., 1994) and also Larex dahurica (original
from central Asia), Larex dicidua (European) and Larex leptolepis (Japanese). AG is approved by
the U.S. Food and Drug Administration (FDA) as a source of dietary fiber (Hauer and Aderer,
1993; Kelly et al., 1999).
Arabinogalactans from Western larch are composed of two fractions, the more abundant high weight fraction AG-A with a maximum molecular weight of about 37 kDa, and the low weight fraction AG-B of about 7-10 kDa, AG-B has been variously reported to occur in a wide range of proportions relative to AG-A, and it is unclear from the literature whether a typical A/B ratio exists (Ponder and Richards, 1997a). The purest form of AG-A is the principal material investigated in literature and is commercially available. (Ponder and Richards, 1997a). Hereafter, the term arabinogalactan (AG) is referred to the high weight component AG-A of Western larch.
1.6.1. Arabinogalactan composition
All the arabinogalactans isolated from larch are nitrogen-less polysaccharides. They are
composed of galactose and arabinose units in a molar ratio of approx. 6:1. A trace amount of
glucuronic acid is generally also found (Ponder and Richards, 1997a). One third of the molecule
is composed of (1→3)-β-D-galactopyranose units, which constitute the main chain, while the rest
consists of lateral groups bonded in position (1→6) to each galactose unit, whose size varies from
monosaccharides to olygosaccharides (Stephen, 1983; Ponder and Richards, 1997c). The AG
composition is illustrated in Fig. 1.9, where ten main-chain residues are shown and seven of
which carry side chains at C-6. The structure of the lateral groups is not uniform. Often, the
lateral group is the disaccharide β-D-galactopyranose-(1→6)-β-D-galactopyranose or β-L-
arabinopyranose-(1→3)-α-L-arabinofuranose. Less frequently, there is the monomer β-D-
galactopyranose or the monomer α-L-arabinofuranose (Jones and Reid, 1963; Fu and Timell,
1972; Ponder and Richards, 1997c). Four types of less represented side chains with more than two
residues (indicated from I to IV, in Fig. 1.9) are highly variable, with numerous possible linkage
environments for the arabinose residues. Arabinose residues occur mostly on the periphery of the
AG molecule at the ends of relatively long side chains (Ponder and Richards, 1997c; Ponder,
1998). The high variability of the side chains, makes it difficult to envisage a regular repeating
unit in AG. It is therefore unlikely that all types of even the major large side chains are
represented in any single AG molecule (Ponder, 1998).
Figure 1.9. Schematic representation of arabinogalactan composition. The figure illustrate only major features of an average or typical AG molecule. Percentages above the figure are estimates of the percentage of main-chain residues carrying the type of attached side chain (if any) shown beneath. Arap, arabinopyranose; Araf, arabinofuranose; Galp, galactopyranose. (from Ponder and Richards, 1997c).
Another possible linked main chain residues (e.g. branched at C-4), though probable, are not indicated in Fig. 1.9 and neither is the known small amount (0.2%) of glucuronic acid residues (Ponder and Richards, 1997a).
1.6.2. Arabinogalactan and size exclusion chromatography behaviour
The small proportion of glucuronic acid units of AG, has been considered to affect the behaviour
of AG on size exclusion chromatography (SEC) (Ponder and Richards, 1997a). The elution
profile of AG changes related to the ionic strength of the eluent, as shown in Fig. 1.10. The
authors evidenced, when using water as eluent, three peaks referred to as A2, A1, A0, going from
the lower to the higher retention time. Using 50 mM NaNO
3as eluent, the three peaks co-elute,
while at least a 100-fold dilution of the 50 mM NaNO
3eluent was needed to achieve a significant
separation of the three peaks. With all of the eluents used (Fig. 1.10), the A0 peak eluted at about
the same retention time. Therefore the retention time for this form of AG (AG-A0) was controlled
essentially by its size in all cases, while the retention times of the other forms (AG-A1 and AG-
A2) is affected by the ionic strength of the eluent. This effect was a consequence of the partial
exclusion of negatively charged species, due to the presence of glucuronic acid residues, from the
stationary phase when low ionic strength eluent was used (Ponder and Richards, 1997a). The
source of the charges on the stationary phase (Shodex, polyhydroxymethylmethacrylate) is
unclear, but their presence is acknowledged by the manufacturer who recommends use of ionic
eluents precisely for the purpose of eliminating their non-SEC effects (Kato, 1995). Thus AG seems to be constituted by different forms characterized by different levels of glucuronic acid.
Figure 1.10. Elution profile of AG-A on size exclusion chromatography Shodex KB-804. Effect of eluent concentrations (from Ponder and Richards 1997a).
1.6.3. Arabinogalactan structure
Ordered tertiary structures occur frequently in natural polysaccharides, often as multiple helices maintained by hydrogen bonding and susceptible to disruption by alkali or heat (Rees et al., 1983). Of particular interest are certain (1→6)-branched (1→3)-β-D-glucopyranans such as scleroglucan, which exists in a triple elical conformation, both in the solid state and in aqueous solution, and which is susceptible to an alkali-induced order-disorder transition from triple helix to single chain random coil (Brigand, 1993). Ponder and Richards shown that AG exist naturally as ordered assemblies of molecules (multiplexes) that can be disrupted by alkali to form individual, unassociated molecules, i.e. disordered arabinogalactan (DAG) (Ponder and Richards, 1997b; Ponder, 1998). Compositional analysis of AG and DAG revealed no significant differences between them; each were found to exhibit a molar Galactose/Arabinose ratio of 5,7.
Analysis by SEC shown that smaller AG molecules undergo the transition in preference to larger ones, probably because, longer helices are more stable than shorter ones. Compelling evidence that the transition does not involve scission of covalent bonds consists of its reversibility by simple evaporation or freezing of an aqueous solution of DAG. (Ponder and Richards, 1997b).
Ponder and Richards (Ponder and Richards, 1997b) used SEC approach to define a possible
structure for AG, through the SEC analysis of DAG structures. Their hypothesis of the existence of triplex structures of AG was finally confirmed by more recent X-ray diffraction study (Chandrasekaran and Janaswamy, 2002), which has demonstrated that (1→3)-β-galactan, the arabinogalactan main chain, can assume the same triple-helical structure (Fig. 1.11) of (1→3)-β- glucan first reported for curdlan (Deslandes et al., 1980) and later adopted for scleroglucan (Bluhm et al., 1982).
Figure 1.11. Representation of the (1→3)-β-D-galactan triple helix. In the side view (left) the vertical line is the helix axis. The axial view (right) clearly shows the formation of O-2H···O-2 hydrogen bonds (dashed lines) between neighboring 3-fold related chains (from Chandrasekaran and Janaswamy, 2002).
Chandrasekaran and Janaswamy demonstrated that the galactan triple helix can also
accommodate a disaccharide galactose-(1→6)-galactose substituted at the 6th position in every
galactose unit in the main chain. The side group attachment is not unique as it can be done in
several ways while preserving the helix symmetry. The variety of molecular models that could be
generated, but not discriminated on steric grounds is a clear testimony that the arabinogalactan
molecule is extremely polymorphic. Such polymorphism is the major structural reason for the
lack of crystallinity leading to a diffraction pattern that give rise three possible types of triple
helix models (Fig. 1.12). The side groups appear to perform dual roles in the arabinogalactan
molecule that resembles a bottle brush. Firstly, like the bristles of the brush, they shield the
galactan triple helix from external turbulences so that the central core stays strong. Secondly, like
flexible bristles, they can interact with neighbouring helices via hydrogen bonds. These features are in agreement with Ponder and Richards data (Ponder and Richards, 1997a; Ponder and Richards, 1997b; Ponder and Richards, 1997c; Ponder, 1998) and fully consistent with the expected solution behaviour of arabinogalactan (Whistler, 1997) as a hydrocolloid and not a gelling agent that would require some lateral organization or partial crystallinity.
Figure 1.12. Possible triple helix models of AG. Two mutually perpendicular views of two turns of the arabinogalactan triple helix in models a, b and c. Their respective fingerprints, namely pinwheel, hexagon and snowflake shapes are discernible in the bottom panels (from Chandrasekaran and Janaswamy, 2002).
In conclusion, morphological studies on AG have shown that this polymer has great
conformational freedom and may take on many distinct shapes, but the main chain generally has a
rigid triple spiral helix structure while the lateral groups form flexible branches with many
exposed hydroxyl groups (Chandrasekaran and Janaswamy, 2002). This is considered to be the
reason underlying the mucoadhesive characteristics of the polymer.
1.6.4. Arabinogalactan physical properties
AG dispersions from 0.2 to 10% in water showed a Newtonian rheological behaviour and viscosity values ranging from 1.00 to 1.58 mPa·s (Burgalassi et al., 2007), which allow to classify it as a non-viscous polymer. The viscosity of tear fluid is reported to be 1.02-1.93 mPa·s (Lemp et al., 1970), and viscosizers are commonly added to ophthalmic formulations to lengthen their per- ocular residence time. The maximum allowable viscosity of ophthalmic solutions has been indicated to be in the range 15-30 mPa·s (Chrai and Robinson, 1974) but in the case of mucoadhesive polymers, lower values may produce the same results (Saettone et al., 1994;
Oechsner and Keipert, 1999). Moreover, the application of highly viscous formulations renders administration more difficult and may cause ocular discomfort, usually resulting in a reduced patient compliance (Winfield et al., 1990). The results of rheologic measurement of the adhesive capacity of polysaccharides as hyaluronic acid (HA), tamarind seed polysaccharide (TSP) and AG toward mucin-polymers are summarized in Table 1.1. The polymeric solutions showed a wide viscosity range, from 1.38 mPa·s of AG to 24.40 mPa·s of HA, but an inspection of the Adhesion Index (AI) values (Table 1.1) shows that AG interact with mucin in vitro more than other tested polymers (Burgalassi et al., 2007).
Table 1.1. Rheologic assessment of mucin-polymers adhesive bond strength. ηm, ηp, ηt, and ηb represent viscosity coefficients of mucin, polymer, the mucin-polymer system and bioadhesive component respectively. AI, adhesion index (from Burgalassi et al., 2007).
