Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of MissouriKansas 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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Abstract |
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Key words: heparanases/proteoglycan/pgsE-606/S-domain
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Introduction |
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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appears to be different from the previously identified extracellular heparanases (K.J.Bame, I.Venkatesan, and L.Heath, unpublished observations).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 56 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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Results |
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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 618 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 610 residues (10 of the 14 wild-type domains) and large domains consisting of 1218 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). 6061 HS chains have, on average, only one small S-domain per molecule, while the 6062 HS chains have approximately 4 S-domains per molecule, two-thirds of which are small. There are 8 S-domains on each 6063 HS glycosaminoglycan, which are distributed equally between small and large domains.
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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 1620 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
). 6061 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-typelike products when they act on 6062 and 6063 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 210 sugar residues. The second explanation is more likely, since the 6062 and 6063 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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Discussion |
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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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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 (500050,000 c.p.m.) were incubated with heparin-affinity purified CHO heparanase (11.5 µg protein) for 1624 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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Acknowledgments |
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Abbreviations |
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Footnotes |
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References |
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