Substrate Specificity of Heparanases from Human Hepatoma and Platelets*

Dagmar Sandbäck PikasDagger §, Jin-ping LiDagger , Israel Vlodavsky, and Ulf LindahlDagger

From the Dagger  Department of Medical Biochemistry and Microbiology, Uppsala University, The Biomedical Center, Box 575, S-751 23 Uppsala, Sweden and the  Department of Oncology, Hadassah University Hospital, P.O. Box 12000, Jerusalem 91120, Israel

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Heparan sulfate proteoglycans, attached to cell surfaces or in the extracellular matrix, interact with a multitude of proteins via their heparan sulfate side chains. Degradation of these chains by limited (endoglycosidic) heparanase cleavage is believed to affect a variety of biological processes. Although the occurrence of heparanase activity in mammalian tissues has been recognized for many years, the molecular characteristics and substrate recognition properties of the enzyme(s) have remained elusive.

In the present study, the substrate specificity and cleavage site of heparanase from human hepatoma and platelets were investigated. Both enzyme preparations were found to cleave the single beta -D-glucuronidic linkage of a heparin octasaccharide. A capsular polysaccharide from Escherichia coli K5, with the same (-GlcUAbeta 1,4-GlcNAcalpha 1,4-)n structure as the unmodified backbone of heparan sulfate, resisted heparanase degradation in its native state as well as after chemical N-deacetylation/N-sulfation or partial enzymatic C-5 epimerization of beta -D-GlcUA to alpha -L-IdceA. By contrast, a chemically O-sulfated (but still N-acetylated) K5 derivative was susceptible to heparanase cleavage. O-Sulfate groups, but not N-sulfate or IdceA residues, thus are essential for substrate recognition by the heparanase(s). In particular, selective O-desulfation of the heparin octasaccharide implicated a 2-O-sulfate group on a hexuronic acid residue located two monosaccharide units from the cleavage site, toward the reducing end.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Heparin is synthesized by connective tissue type mast cells, as part of serglycin proteoglycans, and is subsequently stored intracellularly in cytoplasmic granules. Heparan sulfate (HS),1 structurally related to heparin, is also elaborated in proteoglycan form; contrary to heparin, heparan sulfate chains may be associated with a variety of core proteins (1, 2). Heparan sulfate proteoglycans typically occur at the surface of most types of cells as well as in the extracellular matrix. They have been attributed a variety of biological effects that generally appear to be mediated through interactions between the constituent polysaccharide chains and various proteins (2-5).

The highly extended polysaccharide chains, Mr 60,000-100,000, of heparin proteoglycans may be cleaved shortly after completed biosynthesis, in the mast cell, into fragments similar in size to commercially available heparin (Mr 5000-25,000). The enzyme responsible for this conversion was identified as an endo-beta -D-glucuronidase (6). Endoglycosidases capable of partially depolymerizing HS chains have been demonstrated in a variety of cells and tissues, such as placenta (7), skin fibroblasts (8), platelets (9), melanoma (10), lymphoma (11), hepatocytes (12), CHO cells (13), and endothelial cells (14). Such enzymes, commonly referred to as "heparanases," contribute to the intracellular degradation of HS proteoglycans (15, 16) but are also released into the extracellular matrix, where they appear to modulate several processes of pathophysiological importance. Heparanases thus contribute to the remodeling of extracellular matrix and basement membranes prerequisite to angiogenesis (17) and to the egress of metastatic tumor cells (18) and other types of blood-borne cells (19) from the vasculature. They may regulate growth factor action by releasing heparan sulfate-bound growth factors such as basic fibroblast growth factor from cell surfaces or from the extracellular matrix (20, 21). Heparanases have also been implicated in the release of subendothelial bound lipoprotein lipase (22). Moreover, cleavage of HS chains on the vascular endothelium may affect the local concentration of HS with blood anticoagulant properties, thereby modulating the coagulation process (9).

Studies by Oldberg et al. (9) suggested that the heparanase obtained from human platelets cleaves endo-beta -D-glucuronidic linkages in HS, thus mimicking the action of the heparin-degrading enzyme in mast cells. Similar endoglucuronidase activity was reported in subsequent similar studies on heparanases from different sources, such as human placenta (7), human platelets (23), mouse melanoma (24), and CHO cells (25). In contrast, Hoogewerf et al. (26) attributed endo-alpha -D-glucosaminidase properties to a heparanase purified from human platelets and further proposed, based on amino acid sequencing, that this enzyme was identical to connective tissue-activating peptide-III/neutrophil-activating peptide-2. These components and beta -thromboglobulin are differently truncated forms of platelet basic protein, a member of the CXC-chemokine family.

Several attempts have been made to define the substrate recognition properties of heparanases from various sources (6, 9, 16, 25, 27-31). The approaches taken have involved structural comparison between polysaccharides susceptible or resistant to an enzyme, sequence analysis of fragments generated by enzymatic cleavage, and studies of inhibitory effects of chemically modified heparin derivatives. While most of these studies pointed to the importance of sulfate groups, no unified pattern emerged, and the minimal structural requirements for substrate recognition have remained to be defined.

The aim of the present study was to pinpoint the substrate recognition properties of two heparanases from different sources, human hepatoma and platelets. Both enzymes were identified as endo-beta -D-glucuronidases, based on their action on a well defined oligosaccharide substrate. Further model substrates were generated by systematic modification of the capsular polysaccharide generated by Escherichia coli K5, which has the same structure as the precursor polymer in heparin/HS biosynthesis. Testing various K5 polysaccharide derivatives as substrates for the heparanases revealed that whereas neither N-sulfate groups nor IdceA units are required for substrate recognition, the presence of O-sulfate groups, particularly on the hexuronic acid units, appears to be essential for enzyme action.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Enzymes-- Platelet heparanase was partially purified from 500 ml of human platelet-rich plasma (kindly given by Dr. C.-H. Heldin, The Ludwig Institute, Uppsala, Sweden) according to a modification of a previously described procedure (23). Briefly, the enzyme was recovered following fractionation on heparin-Sepharose, phenyl-Sepharose CL-4B and (passage through) DEAE-Sephacel. The partially purified enzyme contained <3 µg of protein/ml and was stored in portions at -20 °C. Hepatoma heparanase was partially purified from a human hepatoma cell line (Sk-hep-1), essentially as described for the placental heparanase (32), omitting the ammonium sulfate precipitation and adding a second cation exchange (CM-Sepharose) chromatography at pH 7.2. Briefly, the cells were grown in suspension in Dulbecco's modified Eagle's medium (4.5 g of glucose/liter) containing 10% calf serum to a concentration of 106 cells/ml in batches of 50 liters. Cell lysate was subjected to 1) cation exchange (CM-Sepharose) chromatography performed at pH 6.0; 2) cation exchange (CM-Sepharose) chromatography at pH 7.2; 3) heparin-Sepharose chromatography at pH 7.2; and 4) ConA-Sepharose chromatography at pH 6.0. Active fractions were eluted by NaCl gradients in 10 mM phosphate citrate buffer (steps 1-3) and by phosphate citrate buffer containing 1 M NaCl and 0.25 M alpha -methyl mannoside (step 4). The partially purified (~240,000-fold purification) preparation eluted from ConA-Sepharose contained 10-20 µg of protein/ml and consisted primarily of a ~46-kDa protein. Commercially available human heparanase was purchased as beta -thromboglobulin from Calbiochem.

K5-derived Saccharides-- Capsular polysaccharide from E. coli K5 was kindly given by Dr. V. Cavazzoni (Department of Industrial Microbiology, University of Milan, Italy).

A 35S-labeled heparin-like (or HS-like) polysaccharide was generated by incubating 25 µg of K5 polysaccharide with 4.8 mg of solubilized mouse mastocytoma microsomal enzymes and 3.5 MBq of 3'-phosphoadenosine-5'-phospho[35S]sulfate (PAPS) (specific activity, 6.6 TBq/mol), essentially as described by Kusche et al. (33). Anion exchange chromatography of the product on DEAE-Sephacel showed an elution position similar to that of standard chondroitin 4-sulfate from cartilage (data not shown).

Metabolically 3H-labeled K5 polysaccharide was prepared by growing E. coli K5 in the presence of D-[1-3H]glucose (34). The products were partially or exhaustively chemically N-deacetylated (by hydrazinolysis) (35) and were then N-sulfated (36). A portion of ~50% N-sulfated [3H]K5 polysaccharide was incubated with D-glucuronyl C-5 epimerase, purified from bovine liver, yielding a product in which ~30% of the D-glucuronyl residues located between adjacent N-sulfated GlcN units had been converted into IdceA (34).

Chemically O-sulfated K5 polysaccharide, obtained by treating the native product with pyridine-sulfur trioxide (according to route A as described by Casu et al. (37), except that no N-deacetylation/N-sulfation was performed) was kindly given by Dr. A. Naggi (Ronzoni Institute, Milan, Italy). NMR analysis of this material indicated a fully 6-O-sulfated polysaccharide with some sulfate groups also on GlcUA.2 The absence of N-sulfate groups was shown by the resistance of the polysaccharide to depolymerization by HNO2 at pH 1.5 (38) (data not shown). To radiolabel this material, 0.5 mg of the polysaccharide was first partially N-deacetylated by hydrazinolysis (in 0.55 ml of hydrazine hydrate (Fluka) containing 1% hydrazine sulfate) at 100 °C for 5 min (35). The sample was desalted on a PD-10 column (Amersham Pharmacia Biotech) in 10% aqueous ethanol and lyophilized. The polysaccharide was cleaved at the generated N-unsubstituted GlcN residues by nitrous acid treatment at pH 3.9 (38) and labeled by reduction of the resultant terminal anhydromannose units with 0.5 mCi of NaB3H4 (39). The 3H-labeled product (specific activity, ~100 × 103 cpm/µg of hexuronic acid) was recovered by gel filtration on Sephadex G-15 (1 × 150 cm) in 0.2 M NH4HCO3 and further separated on a Superose 12 gel filtration column in 0.5 M NaCl, 50 mM Tris-HCl, pH 7.4. Fractions containing material of Mr >5000 (specific activity, ~10 × 103 cpm/µg of hexuronic acid) were pooled, dialyzed against H2O, and lyophilized.

Chemically N- and O-sulfated K5 polysaccharide was as described (40) (preparation Bb-2, fraction 4, recovered after chromatography on antithrombin-Sepharose). This material was subjected to partial depolymerization by limited deamination at pH 1.5 followed by reduction of the products with NaB3H4 (40). Labeled polysaccharide (Mr >5000; specific activity, ~15 × 103 cpm/µg of hexuronic acid) was isolated by gel chromatography on Bio-Gel P-10 (1 × 150-cm column; fine grade) in 0.5 M NH4HCO3.

Heparin/Heparan Sulfate-derived Saccharides-- N-[3H]Acetyl-labeled heparin (specific activity, 88 × 103 cpm/µg of hexuronic acid) was prepared as described by Höök et al. (41) and separated into fractions with high and low affinity for antithrombin by affinity chromatography (42).

An octasaccharide with high affinity for antithrombin was recovered following partial deaminative cleavage of heparin. Samples were labeled at the reducing end by reduction with NaB[3H]4 (specific activity of product, ~100 × 103 cpm/µg of hexuronic acid) or by treatment with [14C]acetic anhydride following N-deacetylation by hydrazinolysis (specific activity of product, ~130 × 103 cpm/µg of hexuronic acid) (28). A sample of 3H-labeled octasaccharide was degraded to labeled hexasaccharide by N-deacetylation followed by deamination (pH 3.9), and the hexasaccharide product was further converted to pentasaccharide by digestion with exo-beta -D-glucuronidase (42) (see also Fig. 2).

Metabolically 35S-labeled HS was prepared by growing NIH-3T3 cells in a cell culture dish (15 cm in diameter) in 15 ml of sulfate-free Dulbecco's modified Eagle's medium (with 1 mg/ml glucose, 13 mM L-glutamine, 100 units/ml penicillin, 50 µg/ml streptomycin, and 10% fetal calf serum) containing 3.7 MBq/ml 35SO42- overnight. [35S]HS was isolated essentially as described (43).

De-O-Sulfation of Saccharides-- Selective 2-O-desulfation was performed according to Jaseja et al. (Ref. 44; see also Ref. 45). An N-[14C]acetyl-labeled heparin octasaccharide (670 × 103 cpm, 5.3 µg of hexuronic acid) with high affinity for antithrombin was mixed with 4 mg of heparin tetrasaccharide (derived from bovine lung heparin by partial deamination at pH 1.5) in a total volume of 1 ml of 0.13 M NaOH, pH 12.5. The mixture was frozen at -70 °C and lyophilized. The radiolabeled material was separated from the tetrasaccharide carrier on a column (1 × 200 cm) of Sephadex G-25 (superfine grade) in 0.2 M NH4HCO3 and lyophilized.

6-O-Desulfation (accompanied by some 2-O-desulfation) of 3H-labeled antithrombin-binding heparin octasaccharide was performed as described (46). The pyridinium salt of the octasaccharide was treated with 1 ml of dimethyl sulfoxide/methanol (10:1) at 90 °C for 3.5 h. After the addition of 1 ml of H2O, the pH was raised to ~8 with NaOH. The sample was desalted on a PD-10 column in 0.2 M NH4HCO3, lyophilized, and finally re-N-sulfated by treatment with trimethylamine-sulfur trioxide (36).

Heparanase Incubation-- Samples of radiolabeled saccharide substrate, typically ~10,000 dpm, were mixed with heparanase (generally ~1 µg of hepatoma enzyme, ~0.5 µg of platelet enzyme, 5 µg of beta -thromboglobulin) in 10 mM citrate phosphate buffer, pH 5.8, 0.5 mM CaCl2, 100 mM NaCl, 0.5 mM dithiothreitol in a total volume of 200 µl. Occasionally, an internal standard of 35S-labeled heparan sulfate (10,000 dpm) was included as a positive control of enzyme activity. After incubation at 37 °C for 16 h, the samples were heated at 95 °C for 5 min. The digests were mixed with 200 µl of 2 M NaCl, 100 mM Tris-HCl, pH 7.4, 0.2% Triton X-100 and were then centrifuged for 10 min at 13,000 rpm before gel chromatography. Control samples were incubated in parallel without the addition of enzyme.

Analytical Procedures-- For compositional analysis of chemically O-sulfated K5 polysaccharide, the sample was N-deacetylated by hydrazinolysis for 5 h and was then subjected to deaminative cleavage by treatment with HNO2 at pH 3.9 (38). The resultant disaccharides were reduced with NaB3H4, isolated by gel chromatography, and finally analyzed by anion exchange HPLC on a Partisil-10 SAX column. For additional experimental details, see Ref. 47.

Structural analysis of N-sulfated saccharide was performed by deaminative cleavage (pH 1.5) at N-sulfated GlcN residues (38), followed by reduction with NaB3H4. The resultant 3H-labeled disaccharide was isolated by gel chromatography and analyzed by anion exchange HPLC.

Digestion of oligosaccharides with exo-beta -D-glucuronidase from bovine liver (type B-10; Sigma) was performed as described by Thunberg et al. (42).

Gel chromatography was performed as described in the figure legends. Fractions were analyzed by scintillation counting after the addition of OptiPhase "Hisafe" (Wallac).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Incubations of radiolabeled heparan sulfate (Fig. 1A) or heparin (Fig. 1B) with heparanase derived from hepatoma cells resulted in appreciable degradation of both polysaccharides, as shown by gel chromatography. Similar results were obtained with the platelet enzyme (data not shown). Heparin preparations with high or low affinity for antithrombin appeared equally susceptible to cleavage (data not shown). Experiments were undertaken to define the type of glycosidic bond cleaved by the enzymes and to elucidate their substrate recognition properties.


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Fig. 1.   Gel chromatography of heparan sulfate and heparin incubated with hepatoma heparanase. [35S]Heparan sulfate (A) or N-[3H]acetylated heparin (B) were incubated in the absence (open symbols) or presence (filled symbols) of hepatoma heparanase. The samples were analyzed by gel chromatography on a Superose 6 HR 10/30 column (A) or a Superose 12 HR 10/30 column (Pharmacia Biotech) (B) linked to an HPLC system. The columns were run in 1 M NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100 at 0.5 ml/min. Fractions of 0.5-1 ml were collected and analyzed for radioactivity. The positions of standard heparin oligomers are indicated by arrows.

Heparanases Cleave beta -D-Glucuronidic Linkages-- Previous experiments demonstrated that platelet heparanase cleaves a single glycosidic bond in a heparin octasaccharide having high affinity for antithrombin (28). Gel chromatography of the cleavage products along with oligosaccharide standards, obtained by partial deaminative cleavage of heparin, suggested that cleavage had occurred between monosaccharide units 3 and 4 from the nonreducing terminus (indicated by the open arrow in Fig. 2), in accord with the postulated beta -D-glucuronidase properties of the enzyme. By contrast, a platelet heparanase more recently isolated was claimed to be an alpha -D-glucosaminidase (26). The putative glucosaminidase activity was associated with purified connective tissue-activating peptide-III, which occurs in commercially available "beta -thromboglobulin" (a protein 4 amino acid residues shorter than connective tissue-activating peptide-III at the N terminus).


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Fig. 2.   Preparation of hexa- and pentasaccharides from an antithrombin-binding heparin octasaccharide. The open arrow indicates the beta -glucuronidic linkage cleaved by heparanases. The actual antithrombin-binding sequence corresponds to units 2-6 (within brackets). X represents hydrogen or SO3-.

Since our previous results (28) could conceivably have been misinterpreted due to inappropriate selection of oligosaccharide standards, we decided to repeat these experiments using standards obtained by degradation of the authentic octasaccharide (Fig. 2). This species, containing a reducing terminal anhydro-[3H]mannitol unit, was first N-deacetylated by hydrazinolysis and was then reacted with HNO2 at pH 3.9, yielding a labeled hexasaccharide. The hexasaccharide was further digested with exo-beta -D-glucuronidase to produce the pentasaccharide indicated (bottom structure in Fig. 2). The novel hexa- and pentasaccharide standards emerged at distinct elution positions upon gel chromatography (Sephadex G-25) and thus could be used as markers to assess the size of the labeled cleavage product obtained upon incubating the same octasaccharide with heparanase (Fig. 3). The results of such incubations indicate that the octasaccharide is susceptible to cleavage by enzyme from either hepatoma (Fig. 3A) or platelets (Fig. 3B). The labeled degradation product emerged on gel chromatography at the same elution position as the standard pentasaccharide derived from the octasaccharide substrate and was clearly separated from the related hexasaccharide species. The octasaccharide thus had been cleaved between residues 3 and 4 (see Fig. 2) This finding demonstrates that the endoglycosidase(s) of hepatoma and platelets attacks glucuronidic linkages. Similar results were obtained upon digestion of the octasaccharide with commercially available beta -thromboglobulin (Fig. 3C).


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Fig. 3.   Effect of heparanases on antithrombin-binding heparin octasaccharide. A 3H-labeled octasaccharide with high affinity for antithrombin was incubated with heparanases from different sources; hepatoma (A), platelets (B), and commercially available platelet-derived beta -thromboglobulin (C). The samples were run on a Sephadex G-25 column (1 × 200 cm, superfine grade) in 1 M NaCl, 50 mM Hepes-buffer, pH 7.4, 0.1% Triton X-100, in the presence of 0.5 mg of carrier heparin and N-[14C]acetyl-labeled octa- and tetrasaccharide standards to ensure the reproducibility of the elution positions. The tetrasaccharide standard was generated by exhaustive deaminative cleavage (pH 1.5) of the labeled octasaccharide, followed by reduction. Fractions of 1.2 ml were collected at a flow rate of 5 ml/h and analyzed by scintillation counting. Also, the 3H-labeled hexa- and pentasaccharides shown in Fig. 2 were analyzed on the same column. Their elution positions as well as the positions of the octa- and tetrasaccharide are indicated by arrows.

O-Sulfate Groups Are Essential for Heparanase Activity-- The capsular polysaccharide generated by E. coli K5 has the same (-GlcUAbeta 1,4-GlcNAcalpha 1,4-)n structure as the precursor polymer in heparin/HS biosynthesis. Completed HS chains contain major regions of such sequence that have escaped biosynthetic polymer modification (47). Incubation of a 3H-labeled K5 polysaccharide preparation, obtained by growing the E. coli K5 strain in the presence of [1-3H]glucose, with hepatoma heparanase did not lead to any appreciable degradation of the polymer (Fig. 4, A and B). By contrast, a sample of 35S-labeled heparan sulfate that was included as an internal control showed a drastic reduction in size (indicated by the arrows in Fig. 4, A and B). The glucuronidic linkages in the native K5 polysaccharide thus were resistant to enzymatic cleavage.


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Fig. 4.   Lack of effect of hepatoma heparanase on native K5 polysaccharide. Metabolically 3H-labeled K5 polysaccharide was incubated in the absence (A) or presence (B) of hepatoma heparanase as described under "Experimental Procedures." The samples were analyzed on the same Superose 6 column as was used in Fig. 1A. A sample of 35S-labeled HS was included as a positive control of enzyme activity; the peak elution positions of this material are indicated by arrows.

Previous studies demonstrated that incubation of K5 polysaccharide with solubilized microsomal enzymes from a heparin-producing mouse mastocytoma, in the presence of the 35S-labeled sulfate donor, PAPS, yielded a labeled polymer that contained the modified disaccharide units typically found in heparin and heparan sulfate (33). Incubation of such a polysaccharide, Mr >40,000, with hepatoma heparanase resulted in appreciable degradation into polydisperse products that ranged from Mr ~40,000 to <6000 (Fig. 5). Repeated exhaustive incubation of subfractions of these products (designated 1-3 in Fig. 5A) with heparanase did not induce any further degradation, as shown by gel chromatography (Fig. 5, B-D). The enzymatic modification of the K5 polysaccharide thus introduced heparanase cleavage sites that were relatively sparsely distributed.


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Fig. 5.   Effect of hepatoma heparanase on K5 polysaccharide modified by incubation with mouse mastocytoma microsomal enzymes. K5 polysaccharide was incubated with solubilized microsomal enzymes from mouse mastocytoma in the presence of [35S]PAPS (see "Experimental Procedures"). A sample of the 35S-labeled polysaccharide (66 × 103 cpm) was separated on a Sephacryl S-300 (Amersham Pharmacia Biotech) gel filtration column (1 × 100 cm) in 0.2 M NaCl before (open circle) and after (filled circle) incubation with hepatoma heparanase (A). The material degraded by the heparanase was divided in three pools, as indicated, dialyzed against water, and lyophilized prior to reincubation with hepatoma heparanase. The elution patterns of these products are shown in B-D, before (open circles) and after (filled circles) reincubation with heparanase. The elution positions of heparin standards (40) of different sizes (in kDa) are indicated in D.

The biosynthetic polymer modification process includes N-deacetylation and N-sulfation of GlcNAc residues, C-5 epimerization of GlcUA to IdceA units, and finally O-sulfation at various positions (3). To determine which of these various reactions contribute to substrate recognition by the heparanase, we undertook systematic chemical/enzymatic modification of the K5 polysaccharide, followed by analytical heparanase digestion of the various products (summarized in Table I). Metabolically 3H-labeled K5 polysaccharide was subjected to partial (~50%) or essentially complete chemical N-deacetylation/N-sulfation. Both products remained intact after heparanase digestion, even after 3 days of incubation with the repeated addition of fresh enzyme (data not shown). The presence of N-sulfate groups therefore is not sufficient for substrate recognition by the enzyme. The partially N-sulfated material was further modified by incubation with D-glucuronyl C-5 epimerase, resulting in conversion of a fraction of the GlcUA residues into IdceA units (see "Experimental Procedures"). Again, the product was resistant to cleavage by either hepatoma or platelet heparanase (data not shown).

                              
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Table I
Degradation of polysaccharides by heparanases
Degradation of radiolabeled polysaccharides by heparanases was indicated by a shift in elution position on gel chromatography (+). In all incubations of heparanase-resistent polysaccharides (-) the presence of active enzyme was ascertained by the inclusion of an internal 35S-labeled HS standard.

The main difference between the K5 polysaccharide modified through incubation with microsomal enzymes (in the presence of PAPS) and that obtained by chemical N-deacetylation/N-sulfation followed by enzymatic C-5 epimerization is the occurrence of O-sulfate groups in the former product. The difference in susceptibility to heparanase therefore would seem to implicate such groups in substrate recognition by this enzyme, possibly in conjunction with one or more of the other modifications (N-sulfate groups, IdceA units) introduced during heparin/heparan sulfate biosynthesis. The role of O-sulfate groups in heparanase action was directly assessed by means of a sample of K5 polysaccharide that had been chemically O-sulfated, under conditions expected to yield essentially complete 6-O-sulfation of GlcN residues, along with some O-sulfation of GlcUA units (37). The sample was prepared for heparanase incubation by limited N-deacetylation, deaminative cleavage at N-unsubstituted GlcN units, and reduction of cleavage products with NaB3H4, yielding a 3H-labeled polysaccharide with a molecular size larger than that (Mr ~15,000) of heparin (Fig. 6A). Remarkably, this model substrate was degraded by the heparanase to products ranging in Mr from ~1,500 to ~15,000 (Fig. 6B). Similar results were obtained with hepatoma and platelet heparanase preparations, as well as with beta -thromboglobulin (results not shown). Samples of K5 polysaccharide containing N-sulfate in addition to O-sulfate groups (see "Experimental Procedures") were also susceptible to heparanase degradation (not shown). Based on these findings, we conclude that O-sulfate groups are indeed essential for substrate recognition by heparanases, whereas N-sulfate groups are compatible with but not per se required for enzyme action.


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Fig. 6.   Effect of hepatoma heparanase on O-sulfated K5 polysaccharide. Chemically O-sulfated 3H-labeled K5 polysaccharide was incubated in the absence (A) or presence (B) of hepatoma heparanase and analyzed on the same Superose 12 column as was used in Fig. 1B.

Specification of O-Sulfate Requirement-- The O-sulfated K5 polysaccharide was subjected to compositional analysis, involving complete N-deacetylation, deaminative cleavage, reduction/3H-labeling of the resultant disaccharides, and separation of the products by anion exchange HPLC (Fig. 7). A major peak (67% of the total labeled disaccharides) appeared at the elution position (~45 min) of GlcUA-aManR(6-OSO3) that would correspond to a -GlcUA-GlcNAc(6-OSO3)- sequence in the intact polymer. In addition, a smaller (25% of total disaccharides), more retarded peak was observed after 90 min, presumably representing a di-O-sulfated disaccharide species. Treatment of the disaccharide mixture with exo-beta -D-glucuronidase eliminated the mono-O-sulfated species, as expected, but did not affect the di-O-sulfated component (data not shown). This result indicates that the latter compound contains a sulfate group on the GlcUA residue that confers resistance toward exoenzymatic cleavage of the corresponding glucuronidic linkage.


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Fig. 7.   Compositional analysis of O-sulfated K5 polysaccharide. Chemically O-sulfated K5 polysaccharide was degraded to 3H-labeled disaccharides as described under "Experimental Procedures." The products were applied to a Partisil-10 SAX column and eluted with a stepwise gradient of KH2PO4 as indicated (dashed line). The arrows indicate the elution positions of disaccharide standards: GlcUA-aManR(6-OSO3) (1); IdceA(2-OSO3)-aManR (2); IdceA(2-OSO3)-aManR(6-OSO3) (3); IdceA(2-OSO3)-aManR(3- and 6-OSO3) (4). The minor peaks emerging after ~5 and ~88 min represents GlcUA-aManR and tetrasaccharide, respectively.

The O-sulfate requirements for substrate recognition were further determined through selective desulfation experiments. For this purpose, we used a better defined, naturally occurring substrate known to contain both GlcN 6-O-sulfate and IdceA 2-O-sulfate groups, i.e. the antithrombin-binding heparin octasaccharide. This species was radiolabeled, either by reduction with NaB3H4 or by replacing the N-acetyl group adjacent to the nonreducing terminus with a [14C]acetyl substituent (see "Experimental Procedures" and Fig. 2). Incubation of a mixture of the two differentially labeled octasaccharide substrates with hepatoma heparanase yielded the expected [3H]pentasaccharide and [14C]trisaccharide products (Fig. 8, A and B). A sample of the [14C]acetyl-labeled octasaccharide was treated with alkali, under conditions known to effect 2-O-desulfation of IdceA units (44), but not 6-O-desulfation of GlcN residues (see "Addendum"). The 3H-labeled octasaccharide (not further modified) was mixed with the 2-O-desulfated 14C-labeled octasaccharide, and the mixture was digested with heparanase (Fig. 8, C and D). The 14C-labeled component (apparently somewhat reduced in size compared with the fully O-sulfated octasaccharide) was completely resistant to digestion, as shown by the lack of formation of any labeled trisaccharide. Moreover, the 3H-labeled, fully sulfated, octasaccharide was only marginally affected by the enzyme, indicated by the appearance of a minor labeled pentasaccharide peak. This observation suggests that the 2-O-desulfated component was not only resistant to heparanase digestion but also caused inhibition of the enzyme. By contrast, preferential 6-O-desulfation of the 3H-labeled octasaccharide had no apparent effect on its susceptibility toward heparanase, judging from the generation of 3H-labeled pentasaccharide upon incubation with either hepatoma or platelet enzyme (data not shown).


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Fig. 8.   Lack of effect of hepatoma heparanase on 2-O-desulfated antithrombin-binding heparin octasaccharide. A mixture of N-[14C]acetyl- (~10,000 dpm) and [3H]aManR-labeled (~10,000 dpm) octasaccharide was incubated in the absence (A) or presence (B) of hepatoma heparanase and analyzed on a prototype gel chromatography column (1.6 × 60 cm) (properties similar to those of Superdex 30; Amersham Pharmacia Biotech) in 1 M NaCl, 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100. Fractions of 1 ml were collected at a flow rate of 1 ml/min. The same experiment was performed but with a 2-O-desulfated instead of fully O-sulfated 14C-labeled octasaccharide (C and D). The elution positions of heparin deca-, tetra-, and dimers are indicated by arrows.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The existence of heparanase type endoglycosidases has been recognized for over 2 decades, yet several basic features of the enzymes remain poorly understood. Although early reports pointed to the occurrence of distinct enzyme species with different substrate specificities (28, 48), neither the number of enzymes nor their substrate recognition properties have been defined. A recent comparison of the enzyme characteristics such as molecular size, pI, and pH optimum of heparanases from mouse melanoma cells, mouse macrophages, and human platelets suggested that the enzymes were highly similar, although the human platelet enzyme was found to be smaller than the murine homologs (49). On the other hand, Bame et al. (25) concluded, in a study of heparanases from CHO cells, that a single type of cells may contain several heparanases with different substrate specificities. While the present investigation does not provide any information as to the number of different enzymes, it is noted that the heparanase preparations recovered from human platelets and from hepatoma cells could not be distinguished on the basis of substrate specificity.

Identifying the target glycosidic bond for heparanase action has been a matter of controversy. Early studies of endoglycosidases from mouse mastocytoma (6) and from human platelets (9, 23) implicated beta -D-glucuronidic linkages. More recently, however, it was proposed that a heparanase from platelets has endo-alpha -D-glucosaminidase activity (26). The results of the present study, based on identification of fragments generated by heparanase cleavage of a well defined heparin octasaccharide, clearly point to glucuronidic linkages as the site of cleavage. These results applied also to the commercially available beta -thromboglobulin preparations that were claimed to express endoglucosaminidase activity and thus contradict the conclusion of Hoogewerf et al. (26).

The simplest polysaccharide available that is basically related to heparin/HS is the (-GlcUAbeta 1,4-GlcNAcalpha 1,4-)n species, which has a structure identical to that of the initial polymer formed in heparin/HS biosynthesis (50). This polysaccharide may be isolated in large quantities from cultures of E. coli K5 and thus provides a well defined starting point for attempts to elucidate the substrate specificity of heparanase. The resistance of this polymer to heparanase digestion indicates that substrate recognition depends on one or more of the structural features introduced during biosynthetic polymer modification. Indeed, exposing the K5 polysaccharide to the action of solubilized mastocytoma microsomal enzymes led to the generation of a product that was susceptible to heparanase-induced cleavage (Fig. 5). Notably, the relatively sparse distribution of susceptible bonds pointed to a composite recognition site involving a combination of different structural features. More specific information was obtained by screening a series of variously modified K5 polysaccharide samples for heparanase susceptibility (Table I). These experiments revealed that neither N-sulfate groups (see also Ref. 51) nor IdceA units were required, whereas O-sulfate groups were found to be essential for enzyme action.

Selective O-desulfation of the antithrombin-binding heparin octasaccharide, previously found to be cleaved by the enzyme, provided more detailed insight into the substrate recognition properties of the heparanase(s) studied. While 6-O-desulfation of this molecule did not prevent the predicted degradation upon incubation with heparanase, the 2-O-desulfated analog not only resisted cleavage but also appeared to inhibit the enzyme (Fig. 8). The single 2-O-sulfated hexuronic acid residue in position 5 of the octasaccharide substrate (Fig. 2) would therefore seem to be essential for substrate recognition and subsequent cleavage of the linkage between units 3 and 4. While we cannot exclude the possibility of a promoting effect on heparanase action, an obligatory requirement for the 3-O-sulfate group on unit 4 appears unlikely for several reasons. First, structural analysis of the chemically O-sulfated K5 polysaccharide, found to be a substrate for the heparanase preparations, revealed no evidence of 3-O-sulfated GlcN units. Moreover, heparin with low affinity for antithrombin was degraded by heparanase to about the same extent as high affinity heparin, despite an ~10-fold lower content of GlcN 3-O-sulfate groups (39). Finally, Bai et al. (16) recently found aberrant turnover of HS in CHO cells defective in a sulfotransferase required for 2-O-sulfation of hexuronic acid residues and concluded that 2-O-sulfated IdceA units are important for cleavage of HS by intracellular heparanase.3 The results of the present study suggest that the single 2-O-sulfated hexuronic acid unit required for substrate recognition by the heparanase may be either GlcUA or IdceA. Furthermore, the N-sulfate groups that are located close to the target glucuronidic linkage for the heparanase in the antithrombin-binding region (Fig. 2) and in HS (16) are prerequisite to the incorporation of the essential 2-O-sulfate groups during polysaccharide biosynthesis (3, 4) but apparently are not per se required for heparanase action. The structural characteristics of a minimally O-sulfated HS sequence required for substrate recognition by the heparanase(s) (cleavage at the arrow) thus could be summarized as follows (see also Bai et al. (16)).
<AR><R><C><UP> </UP></C></R><R><C><UP>↓     </UP></C></R><R><C><UP>-HexA-GlcN-GlcUA-GlcN-</UP></C></R><R><C><UP>‖       ‖ </UP></C></R><R><C><UP>Ac/SO<SUB>3</SUB> Ac/SO</UP><SUB><UP>3</UP></SUB></C></R></AR><FENCE><AR><R><C><UP>IdceA</UP><FENCE><UP>2-OSO</UP><SUB><UP>3</UP></SUB></FENCE></C></R><R><C><UP>GlcUA</UP><FENCE><UP>2-OSO</UP><SUB><UP>3</UP></SUB></FENCE></C></R></AR></FENCE><AR><R><C><UP> </UP></C></R><R><C><UP> </UP></C></R><R><C><UP>-GlcN-</UP></C></R><R><C><UP>   ‖</UP></C></R><R><C><UP>Ac/SO</UP><SUB><UP>3</UP></SUB></C></R></AR>
<UP><SC>Structure</SC> 1</UP>
The number of distinct heparanase type enzymes occurring in the mammalian organism is unknown. However, all three enzyme preparations investigated in the present study (two of which were derived from platelets and might contain the same enzyme protein) showed similar substrate recognition properties. Preliminary analysis of heparanase from human placenta also indicated similar specificity (data not shown). Still, the information available is not sufficient to fully define the minimal recognition sequence for any of the enzymes. The future development of this area will depend on the molecular cloning of the various enzymes followed by characterization of the recombinant proteins and their catalytic properties, using well defined, preferably synthetic, oligosaccharides as substrates.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Annamaria Naggi and Dr. Benito Casu (Ronzoni Institute, Milan, Italy) for the gift of O-sulfated K5 polysaccharide and for constructive criticism of the manuscript.

    Addendum

The predicted selective 2-O-desulfation was ascertained by structural analysis of alkali-treated octasaccharide. Deaminative cleavage of (reduced) octasaccharide (treatment with nitrous acid at pH 1.5), followed by reduction of the products with NaB3H4, yielded a labeled disaccharide that corresponded to units 5 and 6 of the structure shown in Fig. 2. While this disaccharide was identified (by anion exchange HPLC; see "Experimental Procedures" and the legend to Fig. 7) exclusively as the disulfated species, IdceA(2-OSO3)-aManR(6-OSO3), the corresponding component from the alkali-treated octasaccharide was IdceA-aManR(6-OSO3) (data not shown). The 2-O-sulfate group thus had been removed, whereas the 6-O-sulfate group remained bound to the GlcN residue.

The interpretation of the results of heparanase incubations are potentially complicated by alkali-induced modifications other than 2-O-desulfation of the octasaccharide. These include N-deacetylation of GlcNAc residues as well as conversion of 3-O-sulfated GlcNSO3 units to an aziridine derivative that resists cleavage by nitrous acid (Ref. 45; A. Naggi and B. Casu, personal communication). In accord with these observations ~30% of the initial N-[14C]acetyl label was released from the octasaccharide due to the alkali treatment (data not shown). However, the yield of 3H-labeled disaccharide derived from units 5 and 6, as compared with that of the hexasaccharide 1-6 (Fig. 2), following HNO2/NaB3H4 treatment (see above) indicated that the GlcNSO3 unit 4 remained largely susceptible to deamination (data not shown). We therefore conclude that more than half of the total 2-O-desulfated octasaccharide molecules would have escaped the side reactions, hence that the observed resistance of the modified octasaccharide to heparanase cleavage should be ascribed to the lack of the 2-O-sulfate group.

    FOOTNOTES

* This work was supported by Swedish Medical Research Council Grant 2309, European Commission Grant BIO4-CT95-0026, and Polysackaridforskning AB (Uppsala, Sweden).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 46-18-4714242; Fax: 46-18-4714209; E-mail: Dagmar.Pikas{at}medkem.uu.se.

1 The abbreviations used are: HS, heparan sulfate; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; aManR, 2,5-anhydro-D-mannitol; CHO, Chinese hamster ovary; HPLC, high pressure liquid chromatography.

2 B. Casu, personal communication.

3 While this conclusion is probably correct, it is recalled that a 2-O-sulfated IdceA unit was identified as part of the substrate recognition site for the 3-O-sulfotransferase involved in the biosynthesis of the antithrombin-binding region (52); a polysaccharide devoid of HexA 2-O-sulfate groups thus will probably lack GlcN 3-O-sulfate substituents.

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Abstract
Introduction
Procedures
Results
Discussion
References

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