Glycobiology, 2000, Vol. 10, No. 7 715-726
© 2000 Oxford University Press
The spacing of S-domains on HS glycosaminoglycans determines whether the chain is a substrate for intracellular heparanases
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri–Kansas City, Kansas City, MO 64110, USA
Received on November 29, 1999; revised on January 28, 2000; accepted on January 28, 2000.
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Heparanases are mammalian endoglucuronidases that degrade heparan sulfate (HS) glycosaminoglycans to short 5–6 kDa pieces. In the Golgi, HS glycosaminoglycans are modified by a series of interdependent reactions which result in chains that have regions rich in N- and O-sulfate groups and iduronate residues (S-domains), separated by regions that are nearly devoid of sulfate. Structural analysis of the short HS chains produced by Chinese hamster ovary (CHO) cell heparanases indicate that the enzymes recognize differences in sulfate content between S-domains and unmodified sequences, and cleave the chain at junctions between these regions. To look more closely at whether the spacing of S-domains on the gly- cosaminoglycan influences its ability to be cleaved by heparanases, we examined the susceptibility of the HS chains synthesized by the proteoglycan synthesis mutant, pgsE-606. PgsE-606 cells are deficient in the modification enzyme N-deacetylase/N-sulfotransferase I, and synthesize HS chains that have fewer N- and O-sulfate groups and iduronate residues compared to wild-type (Bame et al., (1991), J. Biol. Chem., 266, 10287). HS glycosaminoglycans were isolated from wild-type and pgsE-606 cells and separated into populations based on sulfate content. Compared to wild-type HS, which has 14 S-domains, pgsE-606 cells synthesize three HS species, 606–1, 606–2, and 606–3, with 1, 4, and 8 S-domains, respectively. The spacing of the S-domains on the pgsE-606 HS chains is similar to the spacing the modified sequences on wild-type HS, indicating that each mutant glycosaminoglycan is composed of wild-type-like sequences and sequences devoid of S-domains. When incubated with partially purified CHO heparanases, only the portion of the mutant HS chains that had S-domains were degraded. Structural analysis of the heparanase-products confirmed that both the number and the arrangement of S-domains on the HS glycosaminoglycan are important for heparanase susceptibility. The structure of the different pgsE-606 HS chains also suggests mechanisms for the placement of S-domains when the gly- cosaminoglycan is synthesized.
Key words: heparanases/proteoglycan/pgsE-606/S-domain
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The fine structure of heparan sulfate (HS) glycosaminoglycans is crucial for the interaction of the proteoglycans with a variety of protein ligands, such as antithrombin III (Bourin and Lindahl, 1993
), basic fibroblast growth factor (bFGF) (Turnbull et al., 1992), and lipoprotein lipase (Parthasarathy et al., 1994). The arrangement of sulfate groups and uronic acid epimers necessary for these interactions results from a series of interdependent reactions that modify the glycosaminoglycan chains as the proteoglycan is synthesized in the Golgi (Lindahl et al., 1998). HS is initially created as a chain of alternating GlcUA and GlcNAc residues attached to a core protein. In some regions of the glycosaminoglycan, GlcN is deacetylated and N-sulfated, GlcUA is epimerized to IdoUA, and O-sulfate groups are added to GlcN and IdoUA residues at C6 and C2, respectively. Minor sulfation at C3 of GlcN and C2 of GlcUA may also occur. At the end of the modification process, the glycosaminoglycan is composed of highly sulfated, IdoUA-rich, flexible regions (S-domains), interspersed with more rigid regions of [GlcNAc-GlcUA] disaccharides that contain very little sulfate (Lyon and Gallagher, 1998). Bridging these two domains are "mixed sequences" where GlcNAc disaccharides and GlcNS disaccharides alternate (Lyon and Gallagher, 1998). In most HS species, S-domains range from 2–9 disaccharides and are separated by mixed and unmodified sequences that average 16–18 disaccharides (Lyon and Gallagher, 1998). Both the extent and the spacing of modified regions are regulated by the cell, and appear to be determined by the cell type or differentiation state, rather than by the protein core (Bernfield et al., 1992; David et al., 1992; Kato et al., 1994; Bame et al., 1994).
In addition to modulating interactions between proteoglycans and protein ligands, the versatile structure of HS may determine how the glycosaminoglycans are degraded by heparanases. These endo-ß-glucuronidases (Oldberg et al., 1980; Oosta et al., 1982; Bame and Robson, 1997; Pikas et al., 1998) are responsible for the normal turnover of HS inside cells (Yanagishita and Hascall, 1992; Bame et al., 1998), and are important for basement membrane and extracellular matrix remodeling during inflammation, angiogenesis or metastatic tumor growth (Hulett et al., 1999; Vlodavsky et al., 1999). At least two extracellular heparanases have been identified, a 9 kDa protein that is identical to CTAP-III (Rechter et al., 1999) and a 50 kDa enzyme which is primarily expressed in lymphoid tissue and placenta (Hulett et al., 1999; Kussie et al., 1999; Toyoshima and Nakajima, 1999; Vlodavsky et al., 1999). There may also be unique intracellular enzymes, since CHO cells contain three separable activities (Bame et al., 1998), and at least one of them appears to be different from the previously identified extracellular heparanases (K.J.Bame, I.Venkatesan, and L.Heath, unpublished observations). The role of different substrate modifications in heparanase specificity has been examined by measuring the inhibition of activity in the presence of chemically modified heparin chains (Irimura et al., 1986; LaPierre et al., 1996; Freeman and Parish, 1998), or the cleavage of chemically defined glycosaminoglycan substrates (Thunberg et al., 1982; Freeman and Parish, 1998; Pikas et al., 1998). They show that heparanases require a modified substrate, and suggest that 2-O-sulfate groups, found in S-domains, are essential. Experiments that showed heparanases are prevented from degrading the substrate when bFGF is bound to S-domains (Tumova and Bame, 1997) support the notion that the modified regions play a role in enzyme action. While these studies provided meaningful information about the role of the sulfated residues, they did not address the importance of the HS domain structure for heparanase action. Heparanases from a variety of sources do not cleave heparin as well as HS (Klein and von Figura, 1979; Freeman and Parish, 1998; Pikas et al., 1998). Since heparin, a highly modified glycosaminoglycan, lacks the unmodified sequences and domain structure of HS (Lyon and Gallagher, 1998), these findings indicate that the unmodified regions of the substrate are important in heparanase recognition and cleavage as well.
Structural analysis of the short HS glycosaminoglycans produced in Chinese hamster ovary (CHO) cells show they have an S-domain located near the end of the molecule (Bame and Robson, 1997). These findings led us to propose a model where heparanases recognize differences in sulfate content between S-domains and unmodified regions, and cleave the glycosaminoglycan at domain junctions, presumably within the mixed sequences. This model is consistent with the known substrate specificities of heparanases. We have extended these studies by examining the heparanase susceptibility of the HS synthesized by the CHO proteoglycan synthesis mutant pgsE-606. PgsE-606 cells are deficient in N-deacetylase/N-sulfotransferase I (NDST I) activity (Bame and Esko, 1989; Wei et al., 1993), which determines the extent of sulfation and uronic acid epimerization as the molecules transit through the Golgi (Lindahl et al., 1998). Because of this deficiency, pgsE-606 HS chains contain approximately half the amounts of N-sulfate, O-sulfate, and IdoUA residues as the wild-type CHO-K1 glycosaminoglycans (Bame et al., 1991a). Unlike the wild-type polysaccharide, pgsE-606 HS chains are not completely cleaved to short 5–6 kDa pieces by intracellular heparanases (Bame, 1993), suggesting that the sulfation pattern of the glycosaminoglycan influences its ability to be a substrate.
In this study, nascent HS chains were purified from wild-type CHO-K1 and pgsE-606 cells, and pooled into populations based on sulfate content. The susceptibility of each HS population to heparin-affinity purified CHO heparanases (Bame et al., 1998) was shown to be dependent on the extent of chain sulfation and number of S-domains. Structural analysis of the nascent HS chains and the heparanase-products showed that the regions of the mutant glycosaminoglycans that were degraded by heparanases had S-domains that were spaced along the HS like those found on wild-type chains. These results indicate that both the number and arrangement of S-domains along the glycosaminoglycan are important for determining whether the HS chain is susceptible to heparanases. The arrangement of S-domains on the undersulfated pgsE-606 HS chains also suggest possible mechanisms for the formation of these regions as the glycosaminoglycans are synthesized in the Golgi.
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PgsE-606 cells synthesize populations of nascent HS chains with different amounts of N-sulfate
Confluent wild-type CHO-K1 and mutant pgsE-606 cells were incubated with [3H]glucosamine or [35S]H2SO4 for 7 h, and the newly synthesized, nascent HS glycosaminoglycans were purified by DEAE-Sephacel and Sepharose CL-6B gel filtration chromatography. The long chains were then applied to an anion exchange HPLC column, since previous studies had suggested that a small proportion of the HS glycosaminoglycans synthesized by pgsE-606 cells were sulfated to a similar extent as wild-type molecules (Bame et al., 1994
). Unlike the wild-type chains, which elute from the anion exchange resin as a symmetrical peak, the pgsE-606 HS chains elute as three definable peaks, which we have designated as 1, 2, and 3 (Figure 1, Table I). Since all the pgsE-606 HS chains are the same size (Figure 4), the difference in the elution position on the anion exchange column should be due to differences in sulfate content. This hypothesis is supported by the observation that the chains that elute earlier from the anion exchange column have a smaller [35S]-c.p.m. to [3H]-c.p.m. ratio than glycosaminoglycans that elute at higher salt concentrations (Table I).
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To look more closely at the sulfate content of the mutant glycosaminoglycans, an aliquot of each HS population was incubated with nitrous acid, pH 1.5, and the reaction mixtures were analyzed on a Superdex peptide FPLC column (Figure 2). At low pH, nitrous acid cleaves the HS chain at GlcNS residues, generating oligosaccharides whose length is dependent on the amount and spacing of the modified sugar. The distribution of the 3H-oligosaccharide species can be used to determine the percent of GlcN residues on the chain that are N-sulfated. The pattern of 3H-oligosaccharides generated by nitrous acid treatment of the long wild-type K1 chains is typical of what has been previously observed (Bame and Esko, 1989
). Nitrous acid converted all of the glycosaminoglycans to short oligosaccharides of up to 24–26 residues (Figure 2). The number of GlcNS residues in wild-type nascent HS is 33.2% (Table II), which is similar to the value originally reported (Bame and Esko, 1989). Nitrous acid treatment of the three pgsE-606 HS populations reflects the differences in sulfate content that were seen with elution from the anion exchange column. Most of the 606–1 3H-HS remains as long oligosaccharides of 20 or more residues (Figure 2), indicating that the sites of N-sulfation are widely spaced along the mutant glycosaminoglycan. The distribution of the nitrous acid oligosaccharides generated from 606–2 or 606–3 3H-HS is shifted to shorter species, showing increases in GlcNS residues for both glycosaminoglycan samples (Figure 2). The GlcNS content of 606–1, 606–2 and 606–3 HS is 12.9%, 18.2%, and 24.0%, respectively (Table II). Previous studies characterizing the structure of pgsE-606 HS chains showed the mutant glycosaminoglycans had about one-half the number of N-sulfate groups as the wild-type HS (Bame and Esko, 1989). If one takes into account the proportions of 606–1, 2, and 3 chains (Table I), the GlcNS content for the entire pgsE-606 HS population is 16.3%, which is still approximately one-half of the wild-type level.
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All four HS populations have the same proportion of free 35S-sulfate radioactivity, which represent the N-sulfate groups that were released by the nitrous acid treatment (Table III). This supports the previous analysis that showed the proportion of N-sulfated to O-sulfated residues on pgsE-606 HS was nearly identical to wild-type, even though the mutant glycosaminoglycans had fewer N-sulfate groups (Bame and Esko, 1989
). However, the three pgsE-606 HS chains have only half the amount of O-[35S]sulfate groups in the nitrous acid disaccharides. This is most likely a reflection of fewer 2-O-sulfate modifications on the mutant HS chains (Bame et al., 1991a). The defect seems to be specific for stretches of contiguous GlcNS disaccharides, since the percentage of O-[35S]sulfate groups in the nitrous acid tetrasaccharides and hexasaccharides is comparable for all the substrate glycosaminoglycans (Table III).
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S-Domain structure of wild-type and pgsE-606 HS
If our hypothesis that heparanases recognize differences in sulfate content between unmodified sequences and S-domains to bind to and cleave the HS glycosaminoglycan is correct, then the susceptibility of the pgsE-606 HS populations to CHO heparanases should be based on the number and distribution of these modified regions on the chain. The low pH nitrous acid analysis (Table II) shows that all three pgsE-606 HS populations have fewer S-sequences since each have fewer nitrous acid disaccharides than wild-type chains (Lyon and Gallagher, 1998
). However, they do not indicate the number of S-domains per chain, nor the location of these regions along the polysaccharide. These questions can be addressed by incubating the glycosaminoglycans with the bacterial polysaccharides lyases, heparitinase I and heparinase, which cleave the chain at specific sequences.
The number of S-domains was examined by incubating each of the 3H-HS glycosaminoglycans with the bacterial polysaccharide lyase, heparitinase I (EC 4.2.2.8) for 24 h, and analyzing the reaction mixture on a Superdex FPLC column (Figure 3). Heparitinase I primarily cleaves the glycosidic bond between GlcN and GlcUA residues, without any regard to the substituent at the amino group (Linhardt, 1994). The enzyme may also act at some GlcNAc and IdoUA sequences, although this reaction not as efficient (Desai et al., 1993). The unmodified and mixed sequences on the glycosaminoglycan should be cleaved by heparitinase I to disaccharides and tetrasaccharides, respectively, while the S-domains should be resistant to the enzyme (Lyon and Gallagher, 1998). Therefore, the distribution of the resistant oligosaccharides will indicate the number and size of S-domains for each HS population. As expected, the major heparitinase I-products for all four HS glycosaminoglycan samples are disaccharides, with the amount of this product inversely proportional to the GlcNS content (Figure 3, Table IV). Interestingly, none of the HS populations had many heparitinase I-tetrasaccharide products, which represent sequences with alternating glucuronate and iduronate disaccharides. The remaining 3H-radioactivity, representing the S-domains, eluted as oligosaccharides of 6–18 residues. Using the distribution of the heparitinase-resistant fragments, the number of S-domains was calculated for each HS population (see Materials and methods). Wild-type K1 HS glycosaminoglycans have approximately 14 S-domains per chain (Table IV), consistent with our previous findings (Tumova and Bame, 1997). The S-domains fall into two size classes: small domains consisting of 6–10 residues (10 of the 14 wild-type domains) and large domains consisting of 12–18 residues (4 of the 14 wild-type domains). All three pgsE-606 HS populations have fewer S-domains than wild-type HS, with a different distribution of small and large domains (Table IV). 606–1 HS chains have, on average, only one small S-domain per molecule, while the 606–2 HS chains have approximately 4 S-domains per molecule, two-thirds of which are small. There are 8 S-domains on each 606–3 HS glycosaminoglycan, which are distributed equally between small and large domains.
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To look at the distribution of S-domains on the different 3H-HS glycosaminoglycans, they were incubated with the bacterial polysaccharide lyase, heparinase (EC 4.2.2.7), and the products analyzed on the TSK 4000 gel filtration column (Figure 4). Heparinase cleaves the glycosidic bond between GlcNS and IdoUA(2S) residues (Linhardt, 1994
). Because this sequence is only found in modified regions, the sizes of the cleaved products can give an idea of the frequency of S-domains along the glycosaminoglycan (Lyon and Gallagher, 1998). However, this procedure cannot quantitate the number of S-domains, since the enzyme will not cleave any S-domain that lacks an IdoUA(2S) residue. This specificity is important, since the mutant HS chains have fewer 2-O-sulfate groups than expected from the defect in N-sulfation (Bame et al., 1991a). The bacterial lyase cleaves most of the wild-type 3H-HS to short glycosaminoglycans of ~10 kDa (Kav = 0.70–0.74), as well as longer molecules (Kav < 0.6) which are portions of the chain that either lack S-domains or IdoUA(2S) residues in the S-domain, and oligosaccharides (Kav = 0.91) which arise from the lyase cleaving within the modified region. The three nascent pgsE-606 3H-HS chains are degraded to different extents by heparinase (Figure 4). As expected, 606–1, which has the fewest S-domains, is only slightly susceptible to the bacterial enzyme, while 606–2 and 606–3 are cleaved more extensively. Similar size short heparinase-products are generated from wild-type, 606–2 and 606–3 3H-HS (Figure 4), indicating that in the regions where the mutant glycosaminoglycans are modified, the spacing of S-domains is analogous to the wild-type polysaccharide.
Heparanases cleave the pgsE-606 HS chains to differ extents, depending on the number of S-domains
The heparanase susceptibility of each HS sample was examined by incubating the four 3H-HS populations with heparin-affinity purified CHO enzymes (Bame et al., 1998). This preparation contains at least three activities that appear to have different cleavage sites (K.J.Bame, I.Venkatesan, and H.D.Stelling, unpublished observations); however, since the separate activities cleave the pgsE-606 HS chains in a similar manner as the combined enzymes (data not shown), we are reporting our findings with the combined activities. The 3H-HS substrates were incubated with CHO heparanases for 16–20 h at 37°C to insure that the chains were cleaved as completely as possible, and then the reaction mixture was analyzed on a TSK 4000 gel filtration column (Figure 4). As shown previously, CHO heparanases convert nascent, 81 kDa wild-type chains to short, 6 kDa fragments (Tumova and Bame, 1997). 606–1 HS is only slightly cleaved by CHO heparanases, generating large intermediate-size fragments and short oligosaccharides that are comparable in size to the wild-type product (Figure 4). CHO heparanases generate more of these short, wild-type–like products when they act on 606–2 and 606–3 3H-HS chains, indicating that increasing the number of S-domains makes the glycosaminoglycans more susceptible to the enzymes. The elution positions of the intermediate-size and short chain products from the three mutant HS populations suggest that the heparanase cleavage sites are clustered along each chain (Figure 4). The sizes of the CHO heparanase products are similar to the sizes of the 3H-chains generated by bacterial heparinase, suggesting that the cleavage sites for the bacterial and mammalian enzymes are located close to each other on the glycosaminoglycan.
Distribution of S-domains on intermediate-size heparanase products
If CHO heparanases require S-domains to cleave the HS substrate, we would predict that the intermediate-size products (Figure 4) should lack these modified sequences. To test this, we incubated nascent pgsE-606 substrates exhaustively with CHO heparanases, purified the intermediate-size chains by gel filtration, and examined the structure of the product glycosaminoglycans. To insure that the intermediate-size glycosaminoglycans were no longer substrates for the mammalian enzymes, an aliquot was reincubated with fresh heparanases and analyzed by gel filtration to confirm that the reaction had gone to completion (data not shown). When treated with pH 1.5 nitrous acid, all three intermediate-size chains are cleaved primarily to oligosaccharides of 14 or more residues (Figure 5, Table II), indicating that, for the most part, the GlcNS residues are spaced very far apart on the HS chain. The presence of 35S-nitrous acid disaccharides, tetrasaccharides and hexasaccharides (Figure 5, Table III) indicates that each intermediate-size product has at least one S-domain on the molecule. Since the elution position of the intermediate-size products does not shift significantly on the TSK 4000 column upon treatment with bacterial heparinase (Figure 6), it suggests that either the S-domain does not have a heparinase-susceptible sequence or that it is at the end of the polysaccharide, and the gel filtration column cannot distinguish between molecules that differ in 2–10 sugar residues. The second explanation is more likely, since the 606–2 and 606–3 intermediate products each have a small peak of 3H-radioactivity that elutes with a Kav of 0.91 (Figure 6, inset), which should be the oligosaccharides that are generated when the bacterial enzyme cleaves within an S-domain.
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Distribution of S-domains on short heparanase products
The three short pgsE-606 CHO heparanase-products were also examined for the presence of S-domains. While none of the mutant products are as N-sulfated as the short wild-type chains (Table II), they all have an S-domain, since disaccharides and tetrasaccharides are generated when the molecules are treated with nitrous acid (Figure 7). Approximately 18% of the 3H-radioactivity in the 606–1 short HS pool is due to intermediate chains that were pooled along with the short products (see Figure 8). The nitrous acid profile of the 606–1 short HS chains reflects this contamination, since there are oligosaccharides that still elute at the void volume of the Superdex column. Therefore, the percentage of GlcNS residues is probably higher than what we are reporting. Further evidence that each short HS product has an S-domain is the appearance of the 3H-oligosaccharide peak when the glycosaminoglycans are incubated with bacterial heparinase (Figure 8). The incubation of the short heparanase-products with the bacterial enzyme also shows that the S-domains are at the end of the glycosaminoglycan, since the shifts in the elution positions of the major 3H-peaks correlate with a loss of only 10% of the molecules. If the S-domain were in the middle of the chain, a larger shift in elution position would be observed.
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After CHO heparanases degrade the HS chain, internal S-domains will now be located near an end of the short glycosaminoglycan product (Bame and Robson, 1997
). This means that when the short HS products are treated with nitrous acid, new 35S-oligosaccharides will be generated, which reflect the distance between the S-domain and the cleaved bond. Because heparanases cleave the substrate so that the product’s reducing sugar is GlcUA, the new oligosaccharides generated by nitrous acid will actually be mono-, tri-, or pentasaccharides, which elute on the Superdex column in the disaccharide and tetrasaccharide peaks (data not shown). Therefore, by comparing the differences in the distribution of nitrous acid 35S-oligosaccharides between the substrate and short product HS, we can get an idea of the distance of the S-domain from the heparanase cleavage site. All the short chains have a higher percentage of nitrous acid 35S-tetrasaccharides than the parent substrate, and with the exception of 606–3, a higher percentage of nitrous acid 35S-disaccharides as well (Table III). In every case the increase in tetrasaccharides is greater than the increase in disaccharides, suggesting that the modified sequences of the S-domain begin, on average, two to three residues from the bond cleaved by heparanases. |
Discussion |
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In this study, we used HS chain substrates, synthesized by wild-type CHO-K1 and pgsE-606 cells, to show that the placement of S-domains along the glycosaminoglycan is critical for heparanase action. PgsE-606 cells synthesize three different forms of HS, that because of the deficiency in NDST I, have progressively fewer S-domains than the wild-type chain. Structural analysis indicates that the S-domains are clustered on the mutant glycosaminoglycan so that a portion of the molecule is modified like wild-type HS, while the rest of the chain lacks modified sequences. Our studies show that only the portion of the glycosaminoglycan that has regularly spaced S-domains is susceptible to heparanases. Thus, wild-type HS is completely cleaved by the enzymes to short, 6 kDa chains, while only a portion of the pgsE-606 HS chain is converted to the 6 kDa product (Figure 9). The amount of short pgsE-606 6 kDa chains produced by CHO heparanases is proportional to the number of S-domains on the glycosaminoglycan. The other heparanase product from each pgsE-606 HS is an intermediate-size chain that comes from the region of the mutant molecule that is devoid of S-domains. Structural studies of all the heparanase products indicate that they have a single, terminal S-domain, yet they are no longer susceptible to the enzymes. This result suggests that the intracellular heparanases in CHO cells may need to recognize two S-domains in order to align properly on the HS substrate.
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Because we have not determined the placement of O-sulfate groups in the nitrous acid disaccharides and tetrasaccharides derived from the short HS products, we cannot address whether a specific arrangement of 6-O and 2-O sulfate groups within S-domains is required for heparanase recognition and cleavage. Our analysis is also limited by the heparanase preparation we used, which contains at least three activities that appear to have different cleavage sites (K.J.Bame, I.Venkatesan, and H.D.Stelling, unpublished observations). However, we can make some general conclusions about the modification state of the chain near the cleavage site, since most of the sulfate groups in the pgsE-606 glycosaminoglycans will be in S-domains. Our data suggest that heparanase activities in CHO cells cleave a sequence that is two to four residues away from the start of an S-domain. The differences in the lengths between the susceptible bond and the S-domain arise because CHO cell heparanases generate short glycosaminoglycans that have the S-domain at either the nonreducing end (class I) or reducing end (class II) of the molecule (Bame and Robson, 1997
). Bai et al. (1997) and Pikas et al. (1998) have proposed that in order to recognize the substrate, heparanases require a single 2-O-sulfated uronic acid very close to the cleaved bond. Our work does not directly test this hypothesis. However, since the pgsE-606 HS glycosaminoglycans have fewer 2-O-sulfated iduronate residues than expected for the N-sulfation defect (Bame et al., 1991a), it may mean that CHO heparanases only require a 2-O-sulfate group in the S-domain, and its location within the modified region is not important.
Alternatively, it may be that CHO heparanases recognize a three-dimensional structure rather than a particular arrangement of O-sulfate groups. Structural studies with heparin oligosaccharides show that the conformation of glycosidic linkages in IdoUA-rich sequences causes the O-sulfate groups to be clustered on one side of the polysaccharide chain (Mulloy et al., 1993). This conformation may emphasize the differences between N-sulfated and N-acetylated sequences. Thus, it is possible that the only requirement for the mammalian enzymes is that the S-domains are O-sulfated. This would explain why all four HS glycosaminoglycan substrates are degraded more extensively by CHO heparanases than by the bacterial heparinase, which requires a 2-O-sulfate group to cleave the chain. A structural recognition model would also explain why pgsF-17 HS chains that lack 2-O-sulfate groups in S-domains are still cleaved by CHO heparanases in vitro, although not as quickly or to the same extent as wild-type substrate (Bai et al., 1997). If the enzymes required a 2-O-sulfate group to recognize the HS chain, the pgsF-17 glycosaminoglycan should not be degraded at all. Our data also suggest that the size of the S-domain may not be a critical factor for heparanase recognition and cleavage either. CHO heparanases completely degrade nascent wild-type HS chains to 6-kDa pieces, even though the chains have S-domains that range from 6 to 18 residues. This result suggests that as long as there is a minimum sequence that can form an S-domain, it will be recognized by heparanases.
A structural recognition model is attractive when one considers functions of intracellular heparanases. Proposed roles for these enzymes include catalyzing the initial catabolic steps necessary for the complete degradation of HS glycosaminoglycans in lysosomes (Kresse and Glössl, 1987), and the creation of the variety of short HS fragments required to regulate proteoglycan interactions (Yayon et al., 1991) or protect ligands from inactivation and degradation (Pillalraisetti et al., 1997; Sperinde and Nugent, 1998; Tumova et al., 1999). The structure of HS chains has been shown to change with developmental state or upon aging, which alters the ability of proteins to bind to the glycosaminoglycan (Brickman et al., 1998; Feyzi et al., 1998). If a specific sequence required by heparanases was changed by development, aging or other differentiation processes, the intracellular degradation of the HS glycosaminoglycans might be inhibited. Instead, if the enzymes recognize a secondary structure formed by iduronate-rich sequences, which should not change significantly even if the placement of O-sulfate groups within the domain is altered (Mulloy et al., 1994), they would still be able to degrade the glycosaminoglycan at every developmental stage or differentiated state, and generate the short HS fragments that may be essential for other cellular functions.
Although they act on essentially the same substrate, it is possible that intracellular heparanases have different substrate requirements than the enzymes secreted from cells. The substrate studies that suggested a 2-O-sulfate group was required for heparanases to cleave the HS chain were done with a partially purified preparation of the 50 kDa platelet heparanase (Pikas et al., 1998). It may be that this secreted enzyme needs a 2-O-sulfate group to recognize and cleave the HS molecule, while the intracellular CHO activities only require a 3-dimensional structure.
Our study also raises some intriguing questions about how the biosynthetic enzymes modify HS chains as they are synthesized in the Golgi. In the regions where the mutant pgsE-606 glycosaminoglycans are modified, the spacing between S-domains is like wild-type, which suggests that the initial modification steps are regulated to occur at specific intervals. One possible mechanism may be that as the NDST enzyme moves along the growing HS chain, some type of secondary structure causes it to pause and modify a stretch of GlcN residues. The secondary structure could be the iduronate-rich sequences formed when the NDST previously paused, so that one S-domain is responsible for forming the next one. This model provides a positive feedback mechanism, since the formation of the first S-domain promotes the formation of subsequent domains, and would result in regular spacing between modified regions. However, it does not address the question of how the first modification steps are initiated. Nor does this model address why pgsE-606 cells synthesize HS glycosaminoglycans with different numbers of S-domains. There are probably several factors that play a role in determining the extent of modification of the HS glycosaminoglycan, such as the concentration and distribution of modification enzymes in the Golgi compartments (Bame et al., 1994), and the concentration of the sulfate donor, PAPS, in the organelle (Abeijon et al., 1997). Since pgsE-606 cells synthesize less NDST enzyme (Bame and Esko, 1989) and have a 2-fold decrease in the intracellular PAPS concentration compared to wild-type cells (Bame et al., 1991b), it is possible that both factors are involved in the distribution of S-domains observed on the mutant HS glycosaminoglycans. At least two NDST enzymes have been cloned (Lindahl et al., 1998), so it should be possible to examine the intracellular distribution of these activities in both pgsE-606 and wild-type CHO cells.
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Materials and methods |
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Cell culture
Chinese hamster ovary cells (CHO-K1) were obtained from the American Culture Collection (CCL-61, Rockville MD) and cultured in Ham’s F12 medium (Gibco-BRL, Grand Island NY) as described previously (Bame and Esko, 1989
). CHO mutant cell line pgsE-606 was obtained from Dr. J.D.Esko (University of California, San Diego) and cultured in the same manner as the parental line. All radioactive labeling experiments were performed with sulfate-free defined medium with 2 or 10 mM glucose (Bame and Esko, 1989).
HS glycosaminoglycan isolation
Confluent cells were incubated with 50 µCi/ml [35S]H2SO4 or [3H]-glucosamine (NEN Life Sciences) in defined F-12 media to incorporate label into long, nascent HS chains. Cells were released from the culture dish with 0.125% trypsin, and the long glycosaminoglycans in the trypsinate were isolated (Bame, 1993). Labeled glycosaminoglycans were purified on DEAE-Sephacel columns (Pharmacia LKB Biotechnology, Inc., Uppsala Sweden) and precipitated with 80% (v/v) ethanol in the presence of 0.5 mg/ml chondroitin sulfate carrier (Bame, 1993). Residual peptides were removed by alkaline borohydride treatment. Chondroitin sulfate chains were exhaustively degraded by chondroitin-ABC lyase (EC 4.2.2.4., ICN Biochemicals, Costa Mesa, CA) and separated from labeled HS chains by gel filtration chromatography on a Sepharose CL-6B column (102 x 1 cm, Pharmacia), equilibrated and run in 0.2 M NH4HCO3 (Bame, 1993). Fractions containing the nascent HS species were pooled and concentrated on DEAE Sephacel columns, desalted by PD10 columns (Pharmacia) and lyophilized to dryness. The lyophilized long chains were resuspended in water and applied to an anion exchange HPLC column to separate the HS into populations based on sulfate content. The column was run in 0.05 M KH2PO4, pH 6.0, 0.2% Zwittergent, and labeled HS was eluted with a NaCl gradient from 0.05 to 0.7M (Bame, 1993). Intermediate and short HS chains were generated from each population of long pgsE-606 glycosaminoglycans by incubation with heparin-affinity purified CHO heparanase activities as described below. Short HS chains from wild-type K1 were purified from the cells remaining on the dish after trypsinization (Tumova and Bame, 1997).
Partial purification of heparanase activities from CHO cells
The CHO heparanase preparation used in these experiments was the heparin affinity-purified fraction from CHO cells (Bame et al., 1998). Heparanase activities were isolated from CHO cell homogenates by low speed centrifugation, followed by ammonium sulfate precipitation, cation exchange chromatography, and heparin affinity chromatography. Further purification of the heparanase activities from CHO cells suggests there may be at least four separate enzymes in the heparin-affinity purified material (Bame et al., 1998). Activities in CHO cells have been shown to be endoglucuronidases and there is no exoglycosidic activity associated with the partially purified heparanase preparation (Bame and Robson, 1997).
HS glycosaminoglycan degradation assay
Labeled HS chains (5000–50,000 c.p.m.) were incubated with heparin-affinity purified CHO heparanase (1–1.5 µg protein) for 16–24 h at 37°C (Tumova and Bame, 1997). The reaction volume was 75 µl, and 21 mM citrate, 57 mM phosphate buffer was used to maintain pH 5.5. The reaction was stopped with 0.1 M Tris, pH 8.0, 0.5 M NaCl, and the reaction products were analyzed on a TSK 4000 gel filtration column (7.5 x 30 mm, TosoHaas, Montgomeryville, PA). The column was equilibrated in 0.1 M KH2PO4, pH 6.0, 0.5 M NaCl, 0.2% Zwittergent, and run at a flow rate of 0.5 ml/min (Tumova and Bame, 1997). The column was standardized with heparin, HS and chondroitin sulfate molecules of known molecular weight (Tumova and Bame, 1997).
Chain treatments
Labeled HS was incubated with low pH nitrous acid (Shively and Conrad, 1976) and the size and proportion of 3H- or 35S-oligosaccharides generated was examined by chromatography on a Superdex peptide FPLC column (1.3 x 31 cm, Pharmacia) equilibrated in 0.5 M pyridinium acetate, pH 5.0. The column was run at a flow rate of 0.5 ml/minute and 0.4 min. fractions were collected. The column was standardized using Biogel P2-column purified 3H-oligosaccharides generated by low pH nitrous acid treatment of [3H]-glucosamine labeled CHO-K1 HS or 3H-reduced CHO-K1 HS (Bame and Robson, 1997). The percent of the GlcN residues that are N-sulfated is determined as N = 
[([[3H]GlcNRn/n) x 100/total c.p.m.], where [3H]GlcNRn is the number of counts in a peak for a particular oligosaccharide of n disaccharides.
The number and size of the S-domains on the glycosaminoglycan were examined by incubating the 3H-HS chains with the bacterial polysaccharide lyase, heparitinase I (EC 4.2.2.8, Seikagaku, Ijamsville MD). Nascent 3H-HS chains were mixed with 100 µg of bovine kidney HS (Sigma) and incubated with 40 mU/ml heparitinase I (EC 4.2.2.8) in 50 mM sodium phosphate, pH 7.6, for 16 h at 37°C (Linhardt, 1994). To insure that the reaction went to completion, another aliquot of enzyme was added and the mixture was incubated an additional 8 h. The reaction was stopped by heating the mixture at 100°C for 2 min, and the size and proportions of 3H-oligosaccharides were examined by chromatography on the Superdex peptide FPLC column. Since the nascent CHO HS glycosaminoglycans are approximately 370 residues (Tumova and Bame, 1997), the number of S-domains (N) on each chain is determined as N = [([3H]GlcNRn/total c.p.m.) x (370/n)] where [3H]GlcNRn is the number of counts in a peak for a particular oligosaccharide of n residues, and n is equal to or greater than 6.
The distribution of S-domains along the glycosaminoglycan was analyzed by incubating the 3H-HS chains with the bacterial polysaccharide lyase, heparinase (EC 4.2.2.7, Seikagaku). Nascent, intermediate-size and short 3H-HS chains were mixed with 10 µg of heparin (Sigma) and incubated with 100 mU/ml heparinase (EC 4.2.2.7) in 20 mM sodium acetate, 1 mM calcium acetate, pH 7.0, for 12 h at 37°C (Linhardt, 1994). To insure that the reaction went to completion, another aliquot of enzyme was added, and the mixture incubated an additional 12 h. The reaction was stopped by the addition of 0.1 M Tris, pH 8.0, 0.5M NaCl, and the reaction products were analyzed by TSK 4000 gel filtration chromatography.
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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This work was supported by Grant MCB-9418859 from the National Science Foundation.
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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HS, heparan sulfate; CHO, Chinese hamster ovary; bFGF, basic fibroblast growth factor; NDST, N-deacetylase/N-sulfotransferase; GlcNAc, N-acetylglucosamine; GlcNS, N-sulfoglucosamine; GlcN, glucosamine; GlcUA, glucuronic acid; IdoUA, iduronic acid.
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1 To whom correspondence should be addressed
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