Characterization of Heparanase from a Rat Parathyroid Cell Line*

Cell surface heparan sulfate proteoglycans undergo unique intracellular degradation pathways after they are endocytosed from the cell surface. Heparanase, an endo-β-glucuronidase capable of cleaving heparan sulfate, has been demonstrated to contribute to the physiological degradation of heparan sulfate proteoglycans and therefore regulation of their biological functions. A rat parathyroid cell line was found to produce heparanase with an optimal activity at neutral and slightly acidic conditions suggesting that the enzyme participates in heparan sulfate proteoglycan metabolism in extralysosomal compartments. To elucidate the detailed properties of the purified enzyme, the substrate specificity against naturally occurring heparan sulfates and chemically modified heparins was studied. Cleavage sites of rat heparanase were present in heparan sulfate chains obtained from a variety of animal organs, but their occurrence was infrequent (average, 1–2 sites per chain) requiring recognition of both undersulfated and sulfated regions of heparan sulfate. On the other hand intact and chemically modified heparins were not cleaved by heparanase. The carbohydrate structure of the newly generated reducing end region of heparan sulfate cleaved by the enzyme was determined, and it represented relatively undersulfated structures. O-Sulfation of heparan sulfate chains also played important roles in substrate recognition, implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of heparan sulfate proteoglycans. Further elucidation of the roles of heparanase in normal physiological processes would provide an important tool for analyzing the regulation of heparan sulfate-dependent cell functions.

Heparan sulfate proteoglycans (HSPGs) 1 are widely present in animal cells. They are one of the major constituents of basement membranes and plasma membranes. HSPGs present on the cell surface or in extracellular matrices have an ability to specifically interact with a variety of biologically active molecules including heparin-binding growth factors, cytokines, proteins involved in cell-cell interactions or cell-extracellular matrix interactions, and pathogens, such as viruses, prions, or plasmodia, thereby regulating biological activities of these molecules (1,2). Cell surface HSPGs are strategically located to be used for intercepting and regulating biological signals coming into cells. Thus, mechanisms involved in expressing HSPGs with proper carbohydrate modification, in maintaining them on the cell surface, in shedding them from the cell surface, and finally in controlling their endocytosis and intracellular degradation would all play important roles regulating biological functions of HSPGs.
Heparanase, an endo-␤-glucuronidase specifically cleaving HS, has drawn much attention for many years for its potential importance in HS metabolism. Heparanase activities have been detected in various tissues and cells, including placenta (3), platelets (4,5), liver (6), and Chinese hamster ovary cells (7). High levels of heparanase activities also have been attributed to some cancer cells, such as melanoma (8), hepatoma (9) and other carcinomas (10). Although a number of heparanase activities have been studied for the last 20 years, the first human (11)(12)(13)(14) and rat (this study; GenBank TM accession number AF184967) heparanases have been cloned only recently. Heparanases related to cancer cells appear to contribute to the disintegration of extracellular matrix and basement membrane by degrading the HSPGs present and therefore facilitating metastasis (11,12,14). In addition, heparanases are proposed to release growth factors bound to HSPG either at the cell surface or in the extracellular matrix and enhance cancer growth (15).
Heparanases during the normal cellular processes contribute to physiological degradation of HSPGs (16). Intracellular degradation processes of HS involving heparanases have been found in a variety of cells (17). It has been reported that cell surface HSPGs undergo unique intracellular degradation pathways after they are endocytosed from the cell surface (16). One of the degradation pathways involves a relatively slow and stepwise endoglycosidic degradation of HS by a heparanase, initially generating HS fragments of a specific length (ϳ10 kDa). This degradation process appears to occur within cellular compartments with neutral pH, suggesting the primary localization of heparanase in some extralysosomal compartments (16). HS fragments generated in the first step further undergo another heparanase cleavage in an acidic compartment that generates even shorter HS fragments with an average molecular mass of 5 kDa followed by the final degradation in the lysosome. This stepwise HSPG degradation suggests the presence of functionally distinct HS-degrading compartments, in which heparanase plays a pivotal role, and potential metabolic processes regulating biological functions of HS.
Detailed enzymatic properties of heparanase have not been fully elucidated because of the limited availability of the enzymatically active protein. Even information on its substrate specificity has been sparse. Enzyme cleavage sites appear to be present on most HS chains but are rather infrequent and consist of a range of structures but not a single type of structure. Thunberg et al. (18) reported susceptibility of a glucuronidic linkage in a defined heparin octasaccharide with antithrombin III binding property to a heparanase derived from platelet. Another report partially characterized a minor enzymatic activity among multiple heparanase activities found in Chinese hamster ovary cells, but the major heparanase activity in the system remained elusive (19). In the present study, using a heparanase derived from a rat parathyroid cell line, we have determined a range of heparanase substrate structures found in naturally occurring HS chains with structural diversity. The present study has provided information on the major substrate structure of heparanase and the occurrence of HS chains susceptible to the enzyme.  ), and chemically modified heparins (completely desulfated and N-acetylated heparin, completely desulfated and N-sulfated heparin, and N-desulfated and Nacetylated heparin) were obtained from Seikagaku Corp. (Tokyo, Japan). HS glycosaminoglycans from bovine kidney, intestine, lung, and aorta were a kind gift from Dr. K. Yoshida of Seikagaku Corp., and their characteristics have been reported previously (20). Total RNA isolation reagent (RNA STAT-60 TM ) was purchased from TEL-TEST, Inc. (Friendswood, TX). Other reagents used were of the highest grades commercially available.

Materials-Centricon
Preparation of Metabolically Radiolabeled HSPGs-35 S-Labeled HSPGs were prepared from metabolically radiolabeled rat osteoblastic cell (UMR 106) and rat parathyroid (PTr) cells as reported previously (21,22). Briefly, cell cultures at ϳ80% confluency were incubated for 16 -20 h at 37°C under 95% air, 5% CO 2 in Dulbecco's modified Eagle's medium/F-12 (1:1, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) in the presence of [ 35 S]sulfate at the concentration of 50 Ci/ml. After labeling for 24 h, cells were washed three times with Mg ϩ2 -and Ca ϩ2 -free phosphate-buffered saline and treated with 10 milliunits/ml chondroitinase ABC for 15 min at 37°C followed by incubation with trypsin (10 g/ml) for an additional 10 min. Materials released by trypsin containing cell surface HSPG were collected and centrifuged for 10 min at 3,000 rpm to remove cell debris. The supernatant made up to 4 M in guanidine HCl was applied to a Sephadex G-50 (Amersham Biosciences) column equilibrated with 8 M urea, 0.2 M NaCl, 0.05 M sodium acetate, pH 6.0, containing 0.5% Triton X-100. Excluded volume fractions were combined and applied onto Q-Sepharose (Amersham Biosciences) pre-equilibrated with the same 8 M urea buffer. After an extensive wash, bound macromolecules were eluted with 4 M guanidine HCl, 0.5% Triton X-100. The sample was further applied onto a Superose 6 (Amersham Biosciences) column in 4 M guanidine HCl, 0.5% Triton X-100 connected to an FPLC system (Amersham Biosciences) (23). The major 35 S-labeled peak was pooled, dialyzed against Mg 2ϩ -and Ca 2ϩ -free phosphate-buffered saline, and used as the substrate in heparanase assays. The final specific activity of 35 S in HS was ϳ8.7 ϫ 10 10 cpm/mmol.
Purification of Heparanase from PTr Cell Lysate-PTr cells were maintained in Minimum Essential Medium/F-12 (1:1) supplemented with 5% calf serum (Invitrogen) at 37°C under 95% air, 5% CO 2 . Confluent cells were washed three times with Mg 2ϩ -and Ca 2ϩ -free phosphate-buffered saline and extracted with 50 mM Tris-HCl, 25 mM KCl, pH 6.8, containing 0.5% Triton X-100 (ϳ1 ml of buffer was used per 10 6 cells) for 1 h at 4°C. The cell extract containing heparanase activity was then centrifuged (3,000 rpm, 15 min) and used immediately or stored at Ϫ20°C until further analysis. For the partial purification of the enzyme, PTr cell extract was applied on a heparin-Sepharose (Amersham Biosciences) column previously equilibrated with 50 mM Tris-HCl, 25 mM KCl, pH 6.8, containing 0.5% Triton X-100. The column was then washed extensively with the same buffer followed by the elution of bound proteins with a linear gradient of NaCl (0 -1.5 M). Fractions containing heparanase activity were pooled, dialyzed against 50 mM Tris-HCl, 25 mM KCl, pH 6.8, containing 0.5% Triton X-100, and used in further experiments.
Heparanase Assay-The heparanase assay was performed using the following methods. For the gel filtration method, radiolabeled HSPG (ϳ7,000 cpm) from PTr or UMR cells prepared as described above was incubated with PTr cell extract at 37°C. The reaction mixture (100 l) consisted of 50 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , pH 6.8, containing 0.5% Triton X-100. After defined incubation times, the reaction was stopped by the addition of 4 M guanidine-HCl (400 l), and samples were analyzed on a Superose 6 column (1.0 ϫ 30 cm) with an FPLC system. Chromatography was performed in 4 M guanidine-HCl, 0.05 M sodium acetate, pH 6.0, containing 0.5% Triton X-100 at a flow rate of 24 ml/h (23). Radioactivity in every 1-min fraction (400 l) was measured with OptiPhase "HighSafe" 3 (Wallac, Turku, Finland) using a Beckman liquid scintillation counter. For the ultrafiltration method, 35 S-labeled HSPG from UMR cells (approximately 3,000 cpm) was incubated with partially purified PTr heparanase at 37°C in 50 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , pH 6.8, containing 0.5% Triton X-100 in a total volume of 100 l. The reaction was stopped with the addition of 4 M guanidine-HCl (400 l), and the total solution was applied to a Centricon 30 ultrafiltration membrane unit followed by centrifugation at 3,000 rpm for 30 min. Filtrate containing depolymerized HS was then counted for radioactivity as described above.
Analysis of Carbohydrate Structure at the Heparanase Cleavage Site-HS preparations from bovine kidney, intestine, lung, and aorta and chemically modified heparin were reduced at their reducing terminal with 1 M NaBH 4 , 50 mM NaOH at 45°C for 24 h and desalted using Sephadex G-50 after neutralization. The reduced HS (250 g each) was incubated in 50 mM Tris-HCl, 25 mM KCl, 5 mM MgCl 2 , pH 6.8, for 1 h at 37°C with PTr heparanase purified by heparin-Sepharose, then precipitated with 80% ethanol, and dried after removing the supernatant. Heparanase-digested HS with newly generated reducing ends at the cleavage site was labeled with [ 3 H]NaBH 4 . Briefly, 5 mCi of [ 3 H]NaBH 4 (PerkinElmer Life Sciences) was dissolved in 250 l of 0.25 M non-radioactive NaBH 4 , 25 mM NaOH to give a final specific activity of 7.4 Ci/mol and mixed with 250 g of HS. The labeling was done for 24 h at 45°C followed by neutralization with 1 M acetic acid and removal of unreacted material using Sephadex G-50 chromatography (bed volume, 4 ml). Then the 3 H-labeled sample was subjected to various enzymatic digestions or chemical treatments as described below and was analyzed on a Superdex Peptide (1 ϫ 30 cm) gel filtration column in 0.65 M NaCl, 50 mM phosphate, pH 7.4 buffer connected to an FPLC system. Heparinase or heparitinase treatment of 3 H-labeled HS (both at 10 milliunits/sample) was carried out in 0.1 M Tris acetate buffer, pH 7.3 at 37°C for 1 h. Nitrous acid treatment (24) and periodate oxidation were done as described previously (25,26). Completeness of the heparanase digestion of non-radioactive or 3 H-labeled HS chains was monitored by co-incubation of 35 S-labeled HS in all samples (see examples in Figs. 3C and 5E). In some experiments, the intact HS chains in the preparations were directly labeled with [ 3 H]NaBH 4 as above.

Detection of Heparanase in Rat Parathyroid
Cell Line-Metabolically radiolabeled HSPG was incubated with PTr cell extract as described under "Experimental Procedures" for different times at 4°C. A low temperature of 4°C was used to slow down the enzymatic activity to clarify reaction kinetics. The degradation of intact radiolabeled proteoglycans to smaller fragments (average molecular mass, ϳ10 kDa) was already visible after 5 min of incubation and increased in a time-dependent manner reaching a plateau by 90 min of incubation (Fig. 1). This process was not inhibited by a mixture of protease inhibitors, suggesting the presence of HS-specific enzyme (data not shown).

Determination of Optimum pH for Enzyme Activity and
Inhibitory Effect of Heparin-Most of the heparanase activities reported so far appear to act at acidic pH (14,27). To determine the optimal degradation conditions for PTr heparanase, the ultrafiltration heparanase assay was carried out at different pH values as described under "Experimental Procedures." Degradation of 35 S-labeled HSPG was extensive at both neutral and slightly acidic pH (Fig. 2). This discrepancy from the previous reports suggested that either the enzyme differs in the biochemical properties from the one reported previously or specific electrolyte compositions at neutral pH used in the present study allowed its optimal activity. Especially it was noted that the presence of divalent cations such as Mg ϩ2 and Ca ϩ2 enhanced the enzyme activity, while the addition of EDTA inactivated it (data not shown). Freeman et al. (28) have reported that heparanases derived from rat liver, B16 melanoma cells, and human umbilical vein endothelial cells as well as rat and human carcinoma cell lines were capable of cleaving heparin, while heparanase from PTr cells did not cleave hepa-rin (Fig. 3A). In fact, heparin strongly inhibited the enzyme activity with an IC 50 ϭ 0.048 g/ml (Fig. 4A). Similarly, all chemically modified heparins tested were not degraded by heparanase (Fig. 3, B and C) but showed variable degrees of enzyme inhibition. Thus, inhibitory activity of intact and chemically modified heparins on the enzyme activity was analyzed to evaluate the relative contribution of specific sulfate groups (Fig. 4B). Analysis using Lineweaver-Burk plots indicated that inhibition of heparanase activity on HS as the substrate by the intact heparin was competitive with K i ϭ 0.032 g/ml. The effect of N-desulfation was minimal on the inhibitory activity of heparin (filled triangle) and resulted in a K i value of 0.046 g/ml, while the O-desulfation (filled square) significantly reduced its inhibitory activity of heparin resulting in K i ϭ 1.0 g/ml (values represent the average based on multiple experiments). As expected, totally desulfated heparin and hyaluronic acid did not show any inhibitory activity (data not shown). Interestingly both chondroitin 4-sulfate and 6-sulfate showed a significant inhibition with K i ϭ 1.2 g/ml (data not shown). Pretreatment of chondroitin sulfate with nitrous acid at pH 1.5 did not alter the K i value, indicating that this inhibition was genuine to chondroitin sulfate and not due to contaminating heparin in the chondroitin sulfate preparation.
Analysis of Substrate Structures of Heparanase-HS glycosaminoglycans prepared from bovine lung, kidney, intestine, and aorta were 3 H-labeled at their original reducing ends, digested with partially purified heparanase, and analyzed by Superose 6 chromatography (Fig. 5). Except the HS preparation from aorta, all HS preparations were susceptible to the enzyme and generated HS fragments with a limit size ranging from 7 to 9 kDa estimated against glycosaminoglycan molecular mass standards. Then, in the next experiment, the common carbohydrate structure (or range of structures) susceptible to heparanase cleavage was determined. HS chains from bovine lung, kidney, and intestine were first reduced at their natural reducing ends with non-radioactive borohydride and then incubated with partially purified PTr heparanase. Newly generated reducing ends by the enzyme cleavage were labeled with [ 3 H]NaBH 4 as described under "Experimental Procedures." 3 H-Labeled samples were further submitted to digestion by HSdegrading enzymes or chemical treatments followed by gel filtration chromatography on a Superdex Peptide column. Representative chromatographic analyses for the bovine kidney HS are shown in Fig. 6. Nitrous acid treatment at high pH (pH 4.5) resulted in little change in the chromatogram, indicating that GlcNH 2 was not present in the majority of 3 H-labeled HS fragments (Fig. 6B). Low pH nitrous acid treatment generated extensively degraded HS oligosaccharides (Fig. 6C): ϳ50% of 3 H activity in trisaccharide, 8% in pentasaccharide, and the rest in hepta-and larger oligosaccharide positions, indicating that the first GlcNSO 3 was present on 50% of HS oligosaccharides on the fourth sugar from the cleavage site, 8% at the sixth sugar, and so on. Heparinase treatment resulted in little digestion, indicating the lack of highly sulfated regions susceptible FIG. 6. Analysis of heparanase cleavage site. HS fragments released upon heparanase degradation were reduced with [ 3 H]borohydride, and they were submitted to analysis by gel-permeation chromatography on a Superdex Peptide column after chemical treatment or enzyme digestion. A, intact HS fragments; B, after high pH (pH 4.5) nitrous acid treatment; C, after low pH (pH 1.5) nitrous acid treatment; D, after heparinase digestion; E, after heparitinase digestion; F, after periodate oxidation. Arrows in C and E correspond to the elution positions of octa-, hexa-, tetra-, and disaccharides, respectively. to the enzyme on the non-reducing end side near the cleavage site (Fig. 6D). Periodate oxidation generated a peak that contained virtually all the radioactivity at the position corresponding to smaller than monosaccharide size, indicating the absence of 2-O-sulfation on the first GlcUA (Fig. 6F). Heparitinase digested almost all radioactive HS oligosaccharides into two closely eluting products (roughly 50% of radioactivity in each peak) of trisaccharide size (Fig. 6E). Analysis of each peak using an ion-exchange column in high performance liquid chromatography suggested that the early eluting peak was a monosulfated trisaccharide, while the later eluting peak was a non-sulfated trisaccharide (data not shown). This suggested that the third sugar from the cleavage site to the direction toward the non-reducing end was GlcUA. Results of the same set of analyses for other HS preparations from other organs were essentially the same except that the proportions of peaks after heparitinase digestion and low pH nitrous acid treatment differed slightly (see Table I). These results were summarized to illustrate the carbohydrate structure of the non-reducing end side of the heparanase cleavage site (Fig. 7). The structural features near the cleavage site were similar among all HS preparations analyzed and represented a range of structures with a relatively undersulfated region of HS chain. The data were consistent with the cleavage with an endo-␤-glucuronidase with none of the reducing end GlcUA sulfated. The second residue downstream from the cleavage site was GlcNAc of which ϳ50% was O-sulfated, thus it was not an obligated sulfation. The third residue was GlcUA. The fourth residue was GlcN of which ϳ50% was N-sulfated. Since glucuronidic linkages between the third and the second and between the fifth and the fourth residue were not susceptible to the heparanase, the GlcN on the reducing end side of the cleavage site may have a different sulfation pattern from those present on the second and fourth GlcN, e.g. containing two or more sulfate residues.

DISCUSSION
Most recent studies on heparanase have emphasized its roles in pathological processes such as cancer metastasis (8,11,12,14) or inflammation (29). In the present study, however, we have focused on the function of heparanase in the normal cellular catabolism of HSPG, which is likely to be the main physiological function of the enzyme. The exclusive localization of heparanase in the cell and its absence in the conditioned medium demonstrated in this study indicated its primary function as the enzyme responsible for the intracellular degradation of HSPG as observed in a number of metabolic studies of cell surface HSPGs. Heparanase activities found in blood vessels and tumor masses as reported in other papers (11,29), on the other hand, were present mostly in the extracellular spaces and therefore appeared to reflect distinct or nonphysiological usages of the enzyme. The optimum pH for the parathyroid heparanase ranged from neutral to slightly acidic, suggesting the enzyme to be functional in prelysosomal compartments in the cell. This was consistent with results from previous experiments demonstrating that endoglycosidic degradation of HSPG in rat parathyroid cells was insensitive to lysosomotropic agents such as chloroquine (17). Based on these results we postulate that putative localization of rat parathyroid heparanase is in prelysosomal compartments that function at near neutral pH, e.g. endosomes, where HSPGs undergo specific degradation with slow turnover rates (16).
Substrate specificity for the cleavage by heparanase was unique, especially with its infrequent occurrence and the range of carbohydrate structures involved. The presence of infrequent enzyme cleavage sites may be due to either the recognition of a single, unusual modification in HS chains such as GlcNH 2 or sulfated GlcUA by the enzyme or the requirement of extended carbohydrate sequences for the cleavage. Results of the present study indicated that the latter appears to be the case. Involvement of carbohydrate sequences with substantial size is characteristic to protein-carbohydrate binding shared by many heparin-or HS-binding proteins. Despite the presence of heparanase cleavage sites in low frequency, most HS chains with structural diversity prepared from various organs showed susceptibility, except ones from aorta, which may have been already exposed to the enzyme in the tissue.
O-Sulfation of HS seems to be important in both substrate recognition and in the degradation process ( Fig. 2A). This was supported by a significant reduction of heparanase-inhibitory activity by O-desulfation of heparin as well as by the inhibition of the enzyme by chondroitin sulfate. On the other hand, lack of highly sulfated residues susceptible to heparinase digestion (Fig. 5D) as well as periodate oxidation (Fig. 5E) on the nonreducing end side of the heparanase cleavage site suggested carbohydrate structures near the reducing end generated upon the cleavage seem to be composed mainly of unsulfated saccharide residues (Fig. 5), implying that rat parathyroid heparanase acts near the boundary of highly sulfated and undersulfated domains of HSPGs. Heparanase cleavage site structure proposed by the present study significantly differs from the one speculated by Bame and colleagues (19). Bame et al. (19) suggested that Chinese hamster ovary heparanase, as well as heparanase derived from placenta and liver, generates two classes of cleavage site structures. The first group (class I), representing the major products, which were not fully characterized, has relatively unmodified structures near the reducing end. It appeared to consist mostly of GlcUA and GlcNAc resi-  dues. On the contrary, the second group (class II), representing the minor product, was suggested to have a structure (GlcNSO 3 -IdoUA2S-GlcNSO 3 -HexUA-GlcNAc-GlcUA). The presence of distinct, multiple activities of heparanase could have been due to activities of an enzyme as suggested by authors (19), the action of another enzyme such as a hexosaminidase, or even the presence of multiple heparanases. There is the potential that the class I activity may resemble that of the parathyroid heparanase reported in the present study.
Whether parathyroid heparanase recognizes the structure similar to those proposed by Pikas et al. (9) is still uncertain. Pikas and her colleagues used a heparin octasaccharide with a defined structure that has antithrombin binding property as the substrate and suggested that the reducing end formed upon the heparanase cleavage can be summarized as the following sequence: HexUA-GlcNAc/SO 3 -GlcUA. Although the structure presented by Pikas et al. (9) is compatible with those of our present study, PTr heparanase was found to be totally inhibited by heparin and could not cleave it. Since the only substrate used in the study by Pikas et al. (9) was a short oligosaccharide, the effect of the carbohydrate truncation on the enzyme activity could not be evaluated.
Biological roles of heparanase have been postulated in diverse pathological conditions in addition to those in cancer metastasis. It is widely recognized that the shedding of HSPG from the endothelium by heparanase causes the loss of the endothelial cell barrier and enables extravasation of blood elements (30). HS fragments generated by heparanase may also stimulate the release of factors responsible for immune cell response. The exact roles of heparanase in normal physiological processes and cell function have been largely unknown. The enzyme participating in the metabolism of cell surface HSPGs is likely to induce the changes in cell functions and structure. The fragments of HS generated during the stepwise degradation by heparanase are likely to possess some biological activities depending on their structure and molecular size. It is possible that HS fragments generated in the stepwise degradation may slow down the degradation of basic fibroblast growth factor and prolong its intracellular life, which consequently could modulate some biological function of basic fibroblast growth factor (31). Tumova et al. (32) showed that a portion of internalized basic fibroblast growth factor was localized to the nucleus together with short HS chains, suggesting that HS fragments may function as a carrier that not only directs fibroblast growth factor to nucleus but also protects it from a rapid degradation. Moreover, HS fragments generated upon heparanase treatment can be released from the cell (29) to the extracellular space where they can function as a competitive inhibitor of HSPGs (33). Further elucidation of the biological roles of heparanase would provide pivotal information on these biological processes.