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17 1.5 Glycosaminoglycans isolated from shellfish

Many researchers, including Dietrich and co-workers, reported the isolation of GAGs similar to heparin with comparable anti-thrombin activities from mollusc’s invertebrates. Similarly, heparin-like substance with relative ability to bind antithrombin III (ATIII) has been isolated from the marine clams, anomalocardia brasiliana (Pejler et al, 1987: Dietrich et al, 1985). Another GAG similar to heparin GAG, with an AT-binding region comparable to that of mammalian heparin has also been purified from another clam species, Mercenaria mercenaria (Jordan and Marcum, 1986). Additionally, heparin, (a highly sulfated polysaccharide, commonly isolated from mast cell or mucosa and also used as an anticoagulant in the clinic) (Bjork and Lindahl, 1982) has been reported to have some other biological activities, such as; anti-viral activity, bind to a variety of growth factors, inhibit complement activation, and regulate angiogenic activity (Weiler et al, 1992; Jackson et al, 1991; Casu 1985; Folkman et al, 1983).

1.6 Biosynthesis of GAGs

HS and CS are synthesised in the Golgi, whereby the individual GAG chains are O- linked to a core protein, forming a large polysaccharide-protein conjugate known as proteoglycan (PG) (Sasisekharan et al, 2006: Silbert and Sugumaran, 2002: Sugahara and Kitagawa, 2002). However, KS can be either N-linked or O-linked to the core protein of the PG (Funderburgh 2002). HA is not synthesised in the Golgi from the core protein but rather by an integral plasma membrane synthase, which secretes the nascent chain immediately (Itano and Kimata, 2002). All GAGs, with the exception of HA, are biosynthesised as proteoglycans and the linkage region is the same in all, except for keratan sulfate. This

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linkage region consists of the tetrasaccharide; glucuronic acid (GlcA), galactose (Gal), Gal, and xylose (Xyl)-linked to the hydroxyl group of serine in the polypeptide core (Sugahara and Kitagawa, 2000). Biosynthesis of all GAGs, except HA is initiated from a core protein, rich in serine-glycine repeats, which create the platform for the attachment of one or more GAG chains via the linkage region (Kimura et al, 1984). Contrarily, HA GAGs lack a covalently bound protein core and is synthesised at the intracellular surface of the plasma membrane (Afratis et al., 2012)

Biosynthesis of GAG is a complex non-template-driven process that involves many enzymes that assemble the GAG polymer and then sulfate them at specific positions. GAGs are synthesised as homopolymers, which are modified subsequently by N-deacetylation and N-sulfation. This is followed by numerous modifications, including sulfation, epimerisation and desulfation; all of which are performed in a spatiotemporal manner, producing mature and functional GAG chains that exert biological functions dependent on their specific structure. This is common to both types of GAG chain and is formed through the stepwise addition of each monosaccharide residue by the respective specific glycosyltransferase (Grimshaw,1997).

After the attachment of the linker, the first modification to the chain determines whether the chain matures into a CS or HS. This modification involves the transfer of a GlcNAc (HS) or a GalNAc (CS) monosaccharide. The enzyme GlcNAcT-I transfers GlcNAc to the tetrasaccharide linker, initiating HS biosynthesis (Rohrmann et al, 1985). This enzyme is different from the glycosyltransferase GlcNAcT-II that is involved in the elongation of the HS GAG chains. In the case of CS, the initiating GalNAc residue is transferred to the linker region by the enzyme GalNAcT. It is not very clear whether the activity of this enzyme differs from the GalNAc transferase activity of chondroitin synthase, which is involved in the

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chain-elongation process. In all cases real chain-building ensues after the transfer of GlcNAc (HS) or GalNAc (CS) to the linker.

In the case of HS chains, a multidomain glycosyl-transferase successively adds GlcNAc and GlcA; thus, initiating the chain-elongation process. This multidomain enzyme is encoded by EXT1 and EXT2 of the EXT family of genes. Although EXT1 and EXT2 possess the ability to transfer both GlcNAc and GlcA, it has been established that these enzymes form a single oligomeric unit, which is required for complete in vivo chain-elongation activity. Similarly, in the case of CS, chondroitin synthase, also a known multidomain enzyme, transfers GalNAc and GlcA successively for chain elongation (Sashisekhare et al, 2006). Molecular cloning has been achieved for four of the glycosyl transferases responsible for the biosynthesis of the linkage region tetrasaccharide (Uyama et al, 2007: Kitagawa et al, 1998). Firstly, xylosyl transferase (XylT) transfers a Xyl residue from UDP-Xyl to specific serine residues in core proteins in the endoplasmic reticulum and the cis-Golgi compartments (Figure 1. 3) (Uyama et al, 2007). Secondly, Two XylTs, XylT-1 and XylT-2, were cloned, and their amino acid sequences found to be significantly homologous. In contrast enzymatic activities were only shown by XylT-1, not by XylT-2. Following the transfer of a Xyl residue, two Gal residues are transferred to the Xyl residue by two kinds of galactosyl transferases (GalTs), GalT-I and GalT-II, in the cis- 1-O-Ser, in the medial- and trans-Golgi compartments (Figure 1.3) (Uyama et al, 2007).

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Figure 1.3; Schema of the biosynthetic assembly of the GAG backbones by various glycosyltransferases.

A number of glycosyltransferases are required for the synthesis of the backbones of GAG’s.e.g XylT, Xyl transferase; GalT-I, Gal transferase-I; GalT-II, Gal transferase-II, GlcAT-I, GlcA transferase-I; GalNAcT-I, GalNAc transferase-I; CS GlcAT-II, CS GlcA transferase-II;GalNAcT-II, GalNAc transferase-II; CS polymerase, GlcA/GalNAc transferase; GlcNAcT-I, GlcNAc transferase-I; GlcNAcT-II,GlcNAc transferase-II; HS polymerase, GlcA/GlcNAc transferase; ChSy-1, chondroitin synthase-1; ChSy-2, chondroitin synthase-2; and ChPF, chondroitin polymerising factor.

The biosynthesis of the linkage region is completed by the actions of GlcAT-I; transferring a GlcA residue to the linkage trisaccharide, Galβ1→3Galβ1→4Xylβ. Moreover, one study highlights that the expression level of GlcAT-I correlates well with the amount of