Chondroitin Sulfate And Cancer

X, sulfated or unsubstituted. U, uronic acid of either C5 epimerization state.

X, sulfated or unsubstituted. U, uronic acid of either C5 epimerization state.

the uronic acid. Protonation of the anomeric oxygen completes the reaction with the breaking of the glycosidic bond. At its simplest, it can be visualized as a proton acceptance and donation mechanism (Jedrzejas, 2000). Hydro-lytic cleavage proceeds slightly differently, first with proton donation to the glycosidic bond. This breaks away the glycosidic oxygen, creating an O5 oxonium ion. Water addition neutralizes the oxonium ion and saturates all carbons (Linhardt et al., 1986). One should note that although lyases can only cleave linkages on the nonreducing side of uronic acid, the hydrolases have no such limitation and can cleave either bond.

B. GalAG Lyase-Mediated Degradation of GAGs

Chondroitinases (Table VI) are produced in soil and intestinal bacteria. Polysaccharides trafficked to the intestinal lumen with epithelial cells provide these bacteria with their nutrient source. Chondroitin lyases have been isolated and characterized from a variety genera, most notably Pedobacter (Fethiere et al., 1999; Huang et al., 1999; Michelacci et al., 1987; Pojasek et al., 2001, 2002; Yamagata et al., 1968) and Proteus (Hamai et al., 1997; Huang et al., 2003; Michel et al., 2004; Ryan et al., 1994; Sato et al., 1994; Yamagata et al., 1968). Chondroitinases have also been isolated from Arthrobacter (Lunin et al., 2004), Aeromonas (Kitamikado and Lee,

1975), Bacillus (Ernst et al., 1995), and Bacteroides (Guthrie et al., 1985; Hwa and Salyers, 1992a,b; Linn et al., 1983).

C. Chondroitinases from Pedobacter heparinus

Chondroitinase ABC from P. heparinus is a broad substrate specificity lyase that acts on a variety of GAGs, including C4S, C6S, DS, chondroitin, chondroitin D and E. The enzyme is unable to catalyze the depolymerization of hyaluronate, heparin and HS, or KS. It has a molecular weight in excess of 70 kDa and an optimal activity temperature of 30°C. The enzyme is fully active against DS, which is unusual for cABC lyases generally. Other cABC lyases have DS activities under 40% of their C4S activity (Ernst et al., 1995). Chondroitinase ABC causes the weight average molecular weight of a GalAG substrate to decrease slowly. The enzyme acts exclusively on the reducing end of the oligosaccharide chain, suggesting a wholly exolytic activity (Michelacci et al., 1987). The enzyme does not seem amenable to modeling by Michaelis-Menten kinetics, and thus some other mechanism may be in force. The reaction is inhibited by its product (UA-GalNAc4S), by excess substrate (Michelacci et al., 1987), Ca2+, PO^, Fe3+, and Mn2+.

Chondroitinase AC (EC 4.2.2.5) from P. heparinus is a 75-kDa enzyme that acts on C4S, C6S, chondroitin, and hyaluronate (Fig. 6) (Huang et al., 2001). It is completely incapable of DS cleavage. At the onset of reaction, the substrate's molecular weight drops dramatically, producing intermediate hexasaccharide and tetrasaccharide, with further degradation over time (Rye and Withers, 2002b). This suggests a predominantly endolytic mode of action. Heparin and DS are notable inhibitors of enzyme activity (Pojasek et al., 2001; Rye and Withers, 2002a,b,c).

Chondroitinase B from P. heparinus has been characterized extensively (Fig. 7) (Huang et al., 1999; Michel et al., 2004; Pojasek et al., 2001, 2002). It is the only known lyase that cleaves DS as its sole substrate. Chondro-itinase B can cleave DS b(1,4) bonds that contain the commonly occurring 4-O-sulfate but also linkages with sulfation at the 2-O-position, 6-O-position, or both. X-ray crystallography (Huang et al., 1999), site-directed mutagenesis (Michel et al., 2004; Pojasek et al., 2002), and modeling studies (Pojasek et al., 2002) have identified critical amino acids involved in substrate binding and catalytic activity. Chondroitinase B's activity is nonran-dom, nonprocessive, and endolytic, preferring longer substrate to shorter ones. It works primarily by cutting internal bonds proximal to the reducing end, although other cleavage sites are also susceptible. An Arg364Ala mutant has been shown to have an altered mode of action yielding an altered product profile. This phenomenon is most likely explained through the differentiated binding between this mutant and DS. Additionally, a complex between Ca2+, chondroitinase B, and DS is required for substrate cleavage. It has been proposed that the Ca2+ ion neutralizes the IdoA carboxylate in

FIGURE 6 Structural comparision of cAC and cABC I. (A) Structure of chondroitinase AC from Pedobacter heparinus based on the crystal structure of Fethiere et al. (1999) and Huang et al. (2001). (B) Structure of chondroitinase ABC I from Proteus vulgaris based on the crystal structure by Huang et al. (2003). The similarities in the domains of both enzymes are evident. On a closer inspection, the middle domain of cABC I has very little sequence identity with the catalytic domain of cAC (and bacterial hyaluronidases). However, the residues that are implicated to play important roles in catalysis are conserved in both enzymes (Prabhakar et al., 2005). These catalytic residues are shown in full (purple). The more open cleft of cABC I is possibly suggestive of this enzyme's ability to accommodate a variety of oligosaccharide geometries and thus its wider substrate specificity.

FIGURE 6 Structural comparision of cAC and cABC I. (A) Structure of chondroitinase AC from Pedobacter heparinus based on the crystal structure of Fethiere et al. (1999) and Huang et al. (2001). (B) Structure of chondroitinase ABC I from Proteus vulgaris based on the crystal structure by Huang et al. (2003). The similarities in the domains of both enzymes are evident. On a closer inspection, the middle domain of cABC I has very little sequence identity with the catalytic domain of cAC (and bacterial hyaluronidases). However, the residues that are implicated to play important roles in catalysis are conserved in both enzymes (Prabhakar et al., 2005). These catalytic residues are shown in full (purple). The more open cleft of cABC I is possibly suggestive of this enzyme's ability to accommodate a variety of oligosaccharide geometries and thus its wider substrate specificity.

the catalytic groove, and two residues, Lys250 and Arg271, act as Bronsted base and acid, respectively (Michel et al., 2004). This strategy makes use of the flexibility of the IdoA moiety.

Very little is known about chondroitinase C from P. heparinus, an enzyme specific for 6-O-sulfated CS linkages. Cross-reactivity with C4S is

FIGURE 7 Chondroitinase B from Pedobacter heparinus. Structure of chondroitinase B based on the crystal structure of Huang etal. (1999) and Michel etal. (2004). Chondroitinase B is the only known enzyme that cleaves dermatan sulfate as its sole substrate. The structure shows the right-handed parallel b'helix fold representative in chondroitinase B and pectate lyases. The authors thank Dr Rahul Raman for support in preparing this schematic.

FIGURE 7 Chondroitinase B from Pedobacter heparinus. Structure of chondroitinase B based on the crystal structure of Huang etal. (1999) and Michel etal. (2004). Chondroitinase B is the only known enzyme that cleaves dermatan sulfate as its sole substrate. The structure shows the right-handed parallel b'helix fold representative in chondroitinase B and pectate lyases. The authors thank Dr Rahul Raman for support in preparing this schematic.

the result of the general heterogeneity of GAG polymers in which oligosaccharides that may contain predominantly C6S actually exist as copolymers with C4S.

D. Chondroitinases from Proteus vulgaris

The conventional broad substrate specificity enzyme from P. vulgaris (Ernst etal., 1995; Sato etal., 1994; Yamagata etal., 1968) has actually been found to comprise two distinct lyases, chondroitinases ABC I and II. Both enzymes cleave a wide variety of GalAGs and hyaluronan. Chondroitinase ABC I has been recombinantly expressed and characterized (Hamai et al., 1997; Michel et al., 2004), and its structure has been elucidated (Fig. 7) (Huang et al., 2003). It has a molecular weight of 105 kDa and optimally processes CS and DS substrates at 37°C. CS substrates are processed at greater rates than DS: 40% greater activity with C6S and more than a twofold increase with C4S. Activity is maximal for CS substrates at a pH of 7.9; for hyaluronan, the optimal pH is 6.1. Chondroitinase ABC I activity is inhibited by Zn2+ and heparin. The products of an exhaustive digestion of CS or DS comprise a mixture of unsaturated tetrasaccarides and disaccharides (Prabhakar et al., 2005). Putative catalytic residues of cABC I include His501, Tyr508, Arg560, and Glu653 (Prabhakar et al., 2005). Arginine-500 may also be involved in substrate catalysis (Huang et al., 2003). The lyase acts using the proton acceptance and donation mechanism described earlier. Chondroitinase ABC I is commercially available.

The cABC II lyase from P. vulgaris has been scrutinized far less thoroughly (Hamai et al., 1997; Ryan et al., 1994). Chondroitinase ABC II is 100 kDa in size and optimally processes GalAG substrates at pH 7.9 at 40°C. Although cABC II is just as broad as cABC I in terms of substrate profile, it is a far less efficient enzyme regarding catalytic activity. In fact, cABC II processes C4S 16-fold less effectively than cABC I and DS 8-fold less (Hamai et al., 1997; Ryan et al., 1994). The product profile for cABC II is similar to that of cABC I.

E. Chondroitinase AC from Arthrobacter aurescens

Chondroitinase AC from A. aurescens (Hiyama and Okada, 1976, 1977) is a 76-kDa enzyme that processes C4S, C6S, chondroitin, and hyaluronan. It is inhibited by DS. The enzyme processes these substrates exolytically, producing almost exclusively disaccharide products (Jandik et al., 1994). Chondroitinase AC will cleave CS/DS copolymers but only at linkages containing glucuronic acid (Gu et al., 1993; Linhardt et al., 1991).

F. Bacterial Hyaluronidases

Hyaluronidases have been purified from the bacterial genera Propionibacterium, Peptostreptococcus, Staphylococcus, Streptococcus, and Strep-tomyces (Linhardt et al., 1986). Specific activities of hyaluronidases vary widely. The lyase from Peptostreptococcus has a remarkably high turnover number, with an activity of 600,000 IU/mg (Tam and Chan, 1985). Hyaluronidase from Staphylococcus has a specific activity of 15 IU/mg (Rautela and Abramson, 1973; Vesterberg, 1968). Though hyaluronate is the major substrate, C4S and C6S can also be cleaved by many hyaluronidases, albeit at lower rates. Because of the great variation in catalytic rates among hyaluronidases and the relative inconsequentiality of pH for activity, it has been suggested that these enzymes may employ a mechanism fundamentally distinct from other GAG lyases.

VIII. Perspectives_

The limited availability and vast heterogeneity of tissue-derived GalAGs (and other GAGs) has hampered efforts to characterize their sequence-specific influences on proteins and other signaling molecules. Their complex nontemplate-driven biosynthesis precludes the possibility of amplification, unlike DNA and proteins, further complicating glycobiological study. Novel synthetic strategies for the fabrication of complex polysaccharides are still in their infancy. These limitations have forced investigators to cultivate sensitive analytical systems to examine GAG properties.

FIGURE 8 Techniques for GalAG sequencing analysis. Three major developments have propelled techniques to characterize the fine structural elements of complex polysaccharides: (1) the development of enzymatic tools to specifically degrade functional groups on an oligosaccharide chain, (2) analytical chemical approaches, including mass spectrometry, capillary electrophoresis, and nuclear magnetic resonance to establish complementary, orthogonal sets of structural data, and (3) a bioinformatics platform to integrate disparate data elements into a numerical strategy for sequencing. These techniques allow for the rapid and bias-free sequencing of complex polysaccharides by considering all possible compositions and converging at a single solution.

FIGURE 8 Techniques for GalAG sequencing analysis. Three major developments have propelled techniques to characterize the fine structural elements of complex polysaccharides: (1) the development of enzymatic tools to specifically degrade functional groups on an oligosaccharide chain, (2) analytical chemical approaches, including mass spectrometry, capillary electrophoresis, and nuclear magnetic resonance to establish complementary, orthogonal sets of structural data, and (3) a bioinformatics platform to integrate disparate data elements into a numerical strategy for sequencing. These techniques allow for the rapid and bias-free sequencing of complex polysaccharides by considering all possible compositions and converging at a single solution.

TABLE VII Biotechnological Applications of Chondroitinases

Enzyme

Application

Organization

cABC

Promoting neural plasticity in the central

Cambridge University; Massa

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