POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN TRANSDUCED CELLS

Abstract: A polynucleotide (hpa) encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity.

Description: POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN TRANSDUCED CELLS FIELD AND BACKGROUND OF THE INVENTION The present invention relates to a polynucleotide, referred to hereinbelow as hpa, encoding a polypeptide having heparanase activity, vectors including same and transduced cells expressing heparanase. The invention further relates to a recombinant protein having heparanase activity. Heparan sulfate proteoglycans: Heparan sulfate proteoglycans (HSPG) are ubiquitous macromolecules associated with the cell surface and extra cellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (1-4). The basic HSPG structure includes a protein core to which several linear heparan sulfate chains are covalently attached. These polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N- linked acetyl groups (1-4). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPG in embryonic morphogenesis, angiogenesis, neurite outgrowth and tissue repair (1- 5). HSPG are prominent components of blood vessels (3). In large blood vessels they are concentrated mostly in the intima and inner media, whereas in capillaries they are found mainly in the subendothelial basement membrane where they support proliferating and migrating endothelial cells and stabilize the structure of the capillary wall. The ability of HSPG to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Cleavage of the heparan sulfate (HS) chains may therefore result in degradation ofthe subendothelial ECM and hence may play a decisive role in extravasation of blood-borne cells. HS catabolism is observed in inflammation, wound repair, diabetes, and cancer metastasis, suggesting that enzymes which degrade HS play important roles in pathologic processes. Heparanase activity has been described in activated immune system cells and highly metastatic cancer cells (6-8), but research has been handicapped by the lack of biologic tools to explore potential causative roles of heparanase in disease conditions. Involvement of Heparanase in Tumor Cell Invasion and Metastasis: Circulating tumor cells arrested in the capillary beds of different organs must invade the endothelial cell lining and degrade its underlying basement membrane (BM) in order to invade into the extravascular tissue(s) where they establish metastasis (9, 10). Metastatic tumor cells often attach at or near the intercellular junctions between adjacent endothelial cells. Such attachment of the metastatic cells is followed by rupture of the junctions, retraction of the endothelial cell borders and migration through the breach in the endothelium toward the exposed underlying BM (9). Once located between endothelial cells and the BM, the invading cells must degrade the subendothelial glycoproteins and proteoglycans of the BM in order to migrate out of the vascular compartment. Several cellular enzymes (e.g., collagenase IV, plasminogen activator, cathepsin B, elastase, etc.) are thought to be involved in degradation of BM (10). Among these enzymes is an endo-β-D-glucuronidase (heparanase) that cleaves HS at specific intrachain sites (6, 8, 11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma (11), fibrosarcoma and melanoma (8) cells. Moreover, elevated levels of heparanase were detected in sera from metastatic tumor bearing animals and melanoma patients (8) and in tumor biopsies of cancer patients (12). The control of cell proliferation and tumor progression by the local microenvironment, focusing on the interaction of cells with the extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells, was investigated previously by the present inventors. This cultured ECM closely resembles the subendothelium in vivo in its morphological appearance and molecular composition. It contains collagens (mostly type III and IV, with smaller amounts of types I and V), proteoglycans (mostly heparan sulfate- and dermatan sulfate- proteoglycans, with smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entactin and elastin (13, 14). The ability of cells to degrade HS in the cultured ECM was studied by allowing cells to interact with a metabolically sulfate labeled ECM, followed by gel filtration (Sepharose 6B) analysis of degradation products released into the culture medium (11). While intact HSPG are eluted next to the void volume of the column (Kav<0.2, Mr ~ 0.5x1 θ6), labeled degradation fragments of HS side chains are eluted more toward the V t ofthe column (0.5<kav<0.8, Mr =5-7xl0 3 ) (11). The heparanase inhibitory, effect of various non-anticoagulant species of heparin that might be of potential use in preventing extravasation of blood-borne cells was also investigated by the present inventors. Inhibition of heparanase was best achieved by heparin species containing 16 sugar units or more and having sulfate groups at both the N and O positions. While O-desulfation abolished the heparanase inhibiting effect of heparin, O-sulfated, N-acetylated heparin retained a high inhibitory activity, provided that the N-substituted molecules had a molecular size of about 4,000 daltons or more (7). Treatment of experimental animals with heparanase inhibitors (e.g., non-anticoagulant species of heparin) markedly reduced (>90%) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (7, 8, 16). Heparin fractions with high and low affinity to anti-thrombin III exhibited a comparable high anti-metastatic activity, indicating that the heparanase inhibiting activity of heparin, rather than its anticoagulant activity, plays a role in the anti- metastatic properties ofthe polysaccharide (7). Heparanase activity in the urine of cancer patients: In an attempt to further elucidate the involvement of heparanase in tumor progression and its relevance to human cancer, urine samples for heparanase activity were screened (16a). Heparanase activity was detected in the urine of some, but not all, cancer patients. High levels of heparanase activity were determined in the urine of patients with an aggressive metastatic disease and there was no detectable activity in the urine of healthy donors. Heparanase activity was also found in the urine of 20% of normal and microalbuminuric insulin dependent diabetes mellitus (IDDM) patients, most likely due to diabetic nephropathy, the most important single disorder leading to renal failure in adults. Possible involvement of heparanase in tumor angiogenesis: Fibroblast growth factors are a family of structurally related polypeptides characterized by high affinity to heparin (17). They are highly mitogenic for vascular endothelial cells and are among the most potent inducers of neovascularization (17, 18). Basic fibroblast growth factor (bFGF) has been extracted from the subendothelial ECM produced in vitro (19) and from basement membranes of the cornea (20), suggesting that ECM may serve as a reservoir for bFGF. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues and blood vessels (21). Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Studies on the interaction of bFGF with ECM revealed that bFGF binds to HSPG in the ECM and can be released in an active form by HS degrading enzymes (15, 20, 22). It was demonstrated that heparanase activity expressed by platelets, mast cells, neutrophils, and lymphoma cells is involved in release of active bFGF from ECM and basement membranes (23), suggesting that heparanase activity may not only function in cell migration and invasion, but may also elicit an indirect neovascular response. These results suggest that the ECM HSPG provides a natural storage depot for bFGF and possibly other heparin-binding growth promoting factors (24, 25). Displacement of bFGF from its storage within basement membranes and ECM may therefore provide a novel mechanism for induction of neovascularization in normal and pathological situations. Recent studies indicate that heparin and HS are involved in binding of bFGF to high affinity cell surface receptors and in bFGF cell signaling (26, 27). Moreover, the size of HS required for optimal effect was similar to that of HS fragments released by heparanase (28). Similar results were obtained with vascular endothelial cells growth factor (VEGF) (29), suggesting the operation of a dual receptor mechanism involving HS in cell interaction with heparin-binding growth factors. It is therefore proposed that restriction of endothelial cell growth factors in ECM prevents their systemic action on the vascular endothelium, thus maintaining a very low rate of endothelial cells turnover and vessel growth. On the other hand, release of bFGF from storage in ECM as a complex with HS fragment, may elicit localized endothelial cell proliferation and neovascularization in processes such as wound healing, inflammation and tumor development (24, 25). Expression of heparanase by cells of the immune system: Heparanase activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of HS by a specific heparanase activity (6). The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules, etc.) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens, etc.), suggesting its regulated involvement in inflammation and cellular immunity. Some of the observations regarding the heparanase enzyme were reviewed in reference No. 6 and are listed hereinbelow: First, a proteolytic activity (plasminogen activator) and heparanase participate synergistically in sequential degradation of the ECM HSPG by inflammatory leukocytes and malignant cells. Second, a large proportion of the platelet heparanase exists in a latent form, probably as a complex with chondroitin sulfate. The latent enzyme is activated by tumor cell-derived factor(s) and may then facilitate cell invasion through the vascular endothelium in the process of tumor metastasis. Third, release of the platelet heparanase from α-granules is induced by a strong stimulant (i.e., thrombin), but not in response to platelet activation on ECM. Fourth, the neutrophil heparanase is preferentially and readily released in response to a threshold activation and upon incubation ofthe cells on ECM. Fifth, contact of neutrophils with ECM inhibited release of noxious enzymes (proteases, lysozyme) and oxygen radicals, but not of enzymes (heparanase, gelatinase) which may enable diapedesis. This protective role of the subendothelial ECM was observed when the cells were stimulated with soluble factors but not with phagocytosable stimulants. Sixth, intracellular heparanase is secreted within minutes after exposure of T cell lines to specific antigens. Seventh, mitogens (Con A, LPS) induce synthesis and secretion of heparanase by normal T and B lymphocytes maintained in vitro. T lymphocyte heparanase is also induced by immunization with antigen in vivo. Eighth, heparanase activity is expressed by pre-B lymphomas and B- lymphomas, but not by plasmacytomas and resting normal B lymphocytes. Ninth, heparanase activity is expressed by activated macrophages during incubation with ECM, but there was little or no release of the enzyme into the incubation medium. Similar results were obtained with human myeloid leukemia cells induced to differentiate to mature macrophages. Tenth, T-cell mediated delayed type hypersensitivity and experimental autoimmunity are suppressed by low doses of heparanase inhibiting non- anticoagulant species of heparin (30). Eleventh, heparanase activity expressed by platelets, neutrophils and metastatic tumor cells releases active bFGF from ECM and basement membranes. Release of bFGF from storage in ECM may elicit a localized neovascular response in processes such as wound healing, inflammation and tumor development. Twelfth, among the breakdown products of the ECM generated by heparanase is a tri-sulfated disaccharide that can inhibit T-cell mediated inflammation in vivo (31). This inhibition was associated with an inhibitory effect of the disaccharide on the production of biologically active TNFα by activated T cells in vitro (31). Other potential therapeutic applications'. Apart from its involvement in tumor cell metastasis, inflammation and autoimmunity, mammalian heparanase may be applied to modulate: bioavailability of heparin-binding growth factors (15); cellular responses to heparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8) (31a, 29); cell interaction with plasma lipoproteins (32); cellular susceptibility to certain viral and some bacterial and protozoa infections (33, 33a, 33b); and disintegration of amyloid plaques (34). Heparanase may thus prove useful for conditions such as wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases and viral infections. Mammalian heparanase can be used to neutralize plasma heparin, as a potential replacement of protamine. Anti-heparanase antibodies may be applied for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids. Common use in basic research is expected. The identification of the hpa gene encoding for heparanase enzyme will enable the production of a recombinant enzyme in heterologous expression systems. Availability of the recombinant protein will pave the way for solving the protein structure function relationship and will provide a tool for developing new inhibitors. Viral Infection: The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (33) and Dengue (33a) viruses to cells and for subsequent infection of the cells. Removal of the cell surface heparan sulfate by heparanase may therefore abolish virus infection. In fact, treatment of cells with bacterial heparitinase (degrading heparan sulfate) or heparinase (degrading heparan) reduced the binding of two related animal herpes viruses to cells and rendered the cells at least partially resistant to virus infection (33). There are some indications that the cell surface heparan sulfate is also involved in HIV infection (33b). Neurodegenerative diseases: Heparan sulfate proteoglycans were identified in the prion protein amyloid plaques of Genstmann-Straussler Syndrome, Creutzfeldt- Jakob disease and Scrape (34). Heparanase may disintegrate these amyloid plaques which are also thought to play a role in the pathogenesis of Alzheimer's disease. Restenosis and Atherosclerosis: Proliferation of arterial smooth muscle cells (SMCs) in response to endothelial injury and accumulation of cholesterol rich lipoproteins are basic events in the pathogenesis of atherosclerosis and restenosis (35). Apart from its involvement in SMC proliferation (i.e., low affinity receptors for heparin-binding growth factors), HS is also involved in lipoprotein binding, retention and uptake (36). It was demonstrated that HSPG and lipoprotein lipase participate in a novel catabolic pathway that may allow substantial cellular and interstitial accumulation of cholesterol rich lipoproteins (32). The latter pathway is expected to be highly atherogenic by promoting accumulation of apoB and apoE rich lipoproteins (i.e. LDL, VLDL, chylomicrons), independent of feed back inhibition by the cellular sterol content. Removal of SMC HS by heparanase is therefore expected to inhibit both SMC proliferation and lipid accumulation and thus may halt the progression of restenosis and atherosclerosis. There is thus a widely recognized need for, and it would be highly advantageous to have a polynucleotide encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity. SUMMARY OF THE INVENTION According to the present invention there is provided a polynucleotide, referred to hereinbelow as hpa, hpa cDNA or hpa gene, encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity. Cloning ofthe human hpa gene which encodes heparanase, and expression of recombinant heparanase by transfected host cells is reported. A purified preparation of heparanase isolated from human hepatoma cells was subjected to tryptic digestion and microsequencing. The YGPDVGQPR (SEQ ID NO: 8) sequence revealed was used to screen EST databases for homology to the corresponding back translated DNA sequence. Two closely related EST sequences were identified and were thereafter found to be identical. Both clones contained an insert of 1020 bp which included an open reading frame of 973 bp followed by a 27 bp of 3' untranslated region and a Poly A tail. Translation start site was not identified. Cloning of the missing 5' end o hpa was performed by PCR amplification of DNA from placenta Marathon RACE cDNA composite using primers selected according to the EST clones sequence and the linkers ofthe composite. A 900 bp PCR fragment, partially overlapping with the identified 3' encoding EST clones was obtained. The joined cDNA fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an open reading frame which encodes a polypeptide of 543 amino acids (SEQ ID NO: 10) with a calculated molecular weight of 61,192 daltons. Cloning an extended 5' sequence was enabled from the human SK-hepl cell line by PCR amplification using the Marathon RACE. The 5' extended sequence ofthe SK-hepl hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta (SEQ ID NO: 9). The assembled sequence contained an open reading frame, SEQ ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15, a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The ability of the hpa gene product to catalyze degradation of heparan sulfate in an in vitro assay was examined by expressing the entire open reading frame of hpa in insect cells, using the Baculovirus expression system. Extracts and conditioned media of cells infected with virus containing the hpa gene, demonstrated a high level of heparan sulfate degradation activity both towards soluble ECM-derived HSPG and intact ECM. This degradation activity was inhibited by heparin, which is another substrate of heparanase. Cells infected with a similar construct containing no hpa gene had no such activity, nor did non- infected cells. The ability of heparanase expressed from the extended 5' clone towards heparin was demonstrated in a mammalian expression system. The expression pattern of hpa RNA in various tissues and cell lines was investigated using RT-PCR. It was found to be expressed only in tissues and cells previously known to have heparanase activity. A panel of monochromosomal human/CHO and human/mouse somatic cell hybrids was used to localize the human heparanase gene to human chromosome 4. The newly isolated heparanase sequence can be used to identify a chromosome region harboring a human heparanase gene in a chromosome spread. According to further features in preferred embodiments of the invention described below, there is provided a polynucleotide fragment which includes a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. According to still further features in the described preferred embodiments the polynucleotide fragment includes nucleotides 63-1691 of SEQ ID NO:9 or nucleotides 139-1869 of SEQ ID NO: 13, which encode the entire human heparanase enzyme. According to still further features in the described preferred embodiments there is provided a polynucleotide fragment which includes a polynucleotide sequence capable of hybridizing with hpa cDNA, especially with nucleotides 1- 721 of SEQ ID NO:9. According to still further features in the described preferred embodiments the polynucleotide sequence which encodes the polypeptide having heparanase activity shares at least 60 % homology, preferably at least 70 % homology, more preferably at least 80 % homology, most preferably at least 90 % homology with SEQ ID NOs:9 or l3. According to still further features in the described preferred embodiments the polynucleotide fragment according to the present invention includes a portion (fragment) of SEQ ID NOs:9, or 13. For example, such fragments could include nucleotides 63-721 of SEQ ID NO:9 and/or a segment of SEQ ID NO:9 which encodes a polypeptide having the heparanase catalytic activity. According to still further features in the described preferred embodiments the polypeptide encoded by the polynucleotide fragment includes an amino acid sequence as set forth in SEQ ID NOs:10 or 14 or a functional part thereof. According to still further features in the described preferred embodiments the polynucleotide sequence encodes a polypeptide having heparanase activity, which shares at least 60 % homology, preferably at least 70 % homology, more preferably at least 80 % homology, most preferably at least 90 % homology with SEQ ID NOs: 10 or l4. According to still further features in the described preferred embodiments the polynucleotide fragment encodes a polypeptide having heparanase activity, which may therefore be allelic, species and/or induced variant of the amino acid sequence set forth in SEQ ID NOs:10 or 14. It is understood that any such variant may also be considered a homolog. According to still further features in the described preferred embodiments there is provided a single stranded polynucleotide fragment which includes a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity as described above. According to still further features in the described preferred embodiments there is provided a vector including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The vector may be of any suitable type including but not limited to a phage, virus, plasmid, phagemid, cosmid, bacmid or even an artificial chromosome. The polynucleotide sequence encoding a polypeptide having heparanase catalytic activity may include any of the above described polynucleotide fragments. According to still further features in the described preferred embodiments there is provided a host cell which includes an exogenous polynucleotide fragment including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The exogenous polynucleotide fragment may be any of the above described fragments. The host cell may be of any type such as prokaryotic cell, eukaryotic cell, a cell line, or a cell as a portion of a multicellular organism (e.g., cells of a transgenic organism). According to still further features in the described preferred embodiments there is provided a recombinant protein including a polypeptide having heparanase catalytic activity. According to still further features in the described preferred embodiments there is provided a pharmaceutical composition comprising as an active ingredient a recombinant protein having heparanase catalytic activity. According to still further features in the described preferred embodiments there is provided a medical equipment comprising a medical device containing, as an active ingredient a recombinant protein having heparanase catalytic activity. According to still further features in the described preferred embodiments there is provided a heparanase overexpression system comprising a cell overexpressing heparanase catalytic activity. According to still further features in the described preferred embodiments there is provided a method of identifying a chromosome region harboring a human heparanase gene in a chromosome spread comprising the steps of (a) hybridizing the chromosome spread with a tagged polynucleotyde probe encoding heparanase; (b) washing the chromosome spread, thereby removing excess of non-hybridized probe; and (c) searching for signals associated with said hybridized tagged polynucleotyde probe, wherein detected signals being indicative of a chromosome region harboring a human heparanase gene. The present invention can be used to develop new drugs to inhibit tumor cell metastasis, inflammation and autoimmunity. The identification of the hpa gene encoding for heparanase enzyme enables the production of a recombinant enzyme in heterologous expression systems. BRIEF DESCRIPTION OF THE DRAWINGS The invention herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1 presents nucleotide sequence and deduced amino acid sequence of hpa cDNA. A single nucleotide difference at position 799 (A to T) between the EST (Expressed Sequence Tag) and the PCR amplified cDNA (reverse transcribed RNA) and the resulting amino acid substitution (Tyr to Phe) are indicated above and below the substituted unit, respectively. Cysteine residues and the poly adenylation consensus sequence are underlined. The asterisk denotes the stop codon TGA. FIG. 2 demonstrates degradation of soluble sulfate labeled HSPG substrate by lysates of High Five cells infected with p¥hpa2 virus. Lysates of High Five cells that were infected with p¥hpa2 virus (•) or control pF2 virus (□) were incubated (18 h, 37 °C) with sulfate labeled ECM-derived soluble HSPG (peak I). The incubation medium was then subjected to gel filtration on Sepharose 6B. Low molecular weight HS degradation fragments (peak II) were produced only during incubation with the pFhpal infected cells, but there was no degradation of the HSPG substrate ( ■ * ■ ) by lysates of pF2 infected cells. FIGs. 3a-b demonstrate degradation of soluble sulfate labeled HSPG substrate by the culture medium of pFhpa2 and pFhpa4 infected cells. Culture media of High Five cells infected with p¥hpa2 (3a) or p¥hpa4 (3b) viruses (•), or with control viruses (D) were incubated (18 h, 37 °C) with sulfate labeled ECM- derived soluble HSPG (peak I, V). The incubation media were then subjected to gel filtration on Sepharose 6B. Low molecular weight HS degradation fragments (peak II) were produced only during incubation with the hpa gene containing viruses. There was no degradation of the HSPG substrate by the culture medium of cells infected with control viruses. FIG. 4 presents size fractionation of heparanase activity expressed by p¥hpa2 infected cells. Culture medium of pFhpa2 infected High Five cells was applied onto a 50 kDa cut-off membrane. Heparanase activity (conversion of the peak I substrate, (V) into peak II HS degradation fragments) was found in the high (> 50 kDa) (•), but not low (< 50 kDa) (o) molecular weight compartment. FIGs. 5a-b demonstrate the effect of heparin on heparanase activity expressed by τpFhpa2 and pFhpa4 infected High Five cells. Culture media of p¥hpa2 (5a) and pFhpa4 (5b) infected High Five cells were incubated (18 h, 37 °C) with sulfate labeled ECM-derived soluble HSPG (peak I, ^) in the absence (• ) or presence (Δ) of 10 μg/ml heparin. Production of low molecular weight HS degradation fragments was completely abolished in the presence of heparin, a potent inhibitor of heparanase activity (6, 7). FIGs. 6a-b demonstrate degradation of sulfate labeled intact ECM by virus infected High Five and S£21 cells. High Five (6a) and Sf21 (6b) cells were plated on sulfate labeled ECM and infected (48 h, 28 °C) with pFhpa4 (•) or control pFl (o) viruses. Control non-infected Sf21 cells (R) were plated on the labeled ECM as well. The pH of the cultured medium was adjusted to 6.0 - 6.2 followed by 24 h incubation at 37 °C. Sulfate labeled material released into the incubation medium was analyzed by gel filtration on Sepharose 6B. HS degradation fragments were produced only by cells infected with the hpa containing virus. FIG. 7a-b demonstrate degradation of sulfate labeled intact ECM by virus infected cells. High Five (7a) and Sf21 (7b) cells were plated on sulfate labeled ECM and infected (48 h, 28 °C) with pFhpa (•) or control pFl (α) viruses. Control non-infected Sf21 cells (R) were plate on labeled ECM as well. The pH of the cultured medium was adjusted to 6.0 - 6.2, followed by 48 h incubation at 28 °C. Sulfate labeled degradation fragments released into the incubation medium was analyzed by gel filtration on Sepharose 6B. HS degradation fragments were produced only by cells infected with the hpa containing virus. FIGs. 8a-b demonstrate degradation of sulfate labeled intact ECM by the culture medium of p¥hpa4 infected cells. Culture media of High Five (8a) and Sf21 (8b) cells that were infected with pFhpa4 (•) or control pFl (D) viruses were incubated (48 h, 37 °C, pH 6.0) with intact sulfate labeled ECM. The ECM was also incubated with the culture medium of control non-infected Sf21 cells (R). Sulfate labeled material released into the reaction mixture was subjected to gel filtration analysis. Heparanase activity was detected only in the culture medium of p¥hpa4 infected cells. FIGs. 9a-b demonstrate the effect of heparin on heparanase activity in the culture medium of p¥hpa4 infected cells. Sulfate labeled ECM was incubated (24 h, 37 °C, pH 6.0) with culture medium of p¥hpa4 infected High Five (9a) and Sf21 (9b) cells in the absence (•) or presence (V) of 10 μg/ml heparin. Sulfate labeled material released into the incubation medium was subjected to gel filtration on Sepharose 6B. Heparanase activity (production of peak II HS degradation fragments) was completely inhibited in the presence of heparin. FIGs. lOa-b demonstrate purification of recombinant heparanase on heparin-Sepharose. Culture medium of Sf21 cells infected with pYhpa4 virus was subjected to heparin-Sepharose chromatography. Elution of fractions was performed with 0.35 - 2 M NaCl gradient iy). Heparanase activity in the eluted fractions is demonstrated in Figure 10a (•). Fractions 15-28 were subjected to 15% SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining. A correlation is demonstrated between a major protein band (MW ~ 63,000) in fractions 19 - 24 and heparanase activity. FIGs. l la-b demonstrate purification of recombinant heparanase on a Superdex 75 gel filtration column. Active fractions eluted from heparin- Sepharose (Figure 10a) were pooled, concentrated and applied onto Superdex 75 FPLC column. Fractions were collected and aliquots of each fraction were tested for heparanase activity (C, Figure 11a) and analyzed by SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining (Figure l ib). A correlation is seen between the appearance of a major protein band (MW ~ 63,000) in fractions 4 - 7 and heparanase activity. FIGs. 12a-e demonstrate expression of the hpa gene by RT-PCR with total RNA from human embryonal tissues (12a), human extra-embryonal tissues (12b) and cell lines from different origins (12c-e). RT-PCR products using hpa specific primers (I), primers for GAPDH housekeeping gene (II), and control reactions without reverse transcriptase demonstrating absence of genomic DNA or other contamination in RNA samples (III). M- DNA molecular weight marker VI (Boehringer Mannheim). For 12a: lane 1 - neutrophil cells (adult), lane 2 - muscle, lane 3 - thymus, lane 4 - heart, lane 5 - adrenal. For 12b: lane 1 - kidney, lane 2 - placenta (8 weeks), lane 3 - placenta (11 weeks), lanes 4-7 - mole (complete hydatidiform mole), lane 8 - cytotrophoblast cells (freshly isolated), lane 9 - cytotrophoblast cells (1.5 h in vitro), lane 10 - cytotrophoblast cells (6 h in vitro), lane 11 - cytotrophoblast cells (18 h in vitro), lane 12 - cytotrophoblast cells (48 h in vitro). For 12c: lane 1 - JAR bladder cell line, lane 2 - NCITT testicular tumor cell line, lane 3 - SW-480 human hepatoma cell line, lane 4 - HTR (cytotrophoblasts transformed by SV40), lane 5 - HPTLP-I hepatocellular carcinoma cell line, lane 6 - EJ-28 bladder carcinoma cell line. For 12d: lane 1 - SK-hep-1 human hepatoma cell line, lane 2 - DAMI human megakaryocytic cell line, lane 3 - DAMI cell line + PMA, lane 4 - CHRF cell line + PMA, lane 5 - CHRF cell line. For 12e: lane 1 - ABAE bovine aortic endothelial cells, lane 2 - 1063 human ovarian cell line, lane 3 - human breast carcinoma MDA435 cell line, lane 4 - human breast carcinoma MDA231 cell line. FIG. 13 presents a comparison between nucleotide sequences of the human hpa and a mouse EST cDNA fragment (SEQ ID NO: 12) which is 80 % homologous to the 3' end (starting at nucleotide 1066 of SEQ ID NO:9) of the human hpa. The aligned termination codons are underlined. FIG. 14 demonstrates the chromosomal localization of the hpa gene. PCR products of DNA derived from somatic cell hybrids and of genomic DNA of hamster, mouse and human of were separated on 0.7 % agarose gel following amplification with hpa specific primers. Lane 1 - Lambda DNA digested with BstEU, lane 2 - no DNA control, lanes 3 - 29, PCR amplification products. Lanes 3-5 - human, mouse and hamster genomic DNA, respectively. Lanes 6- 29, human monochromosomal somatic cell hybrids representing chromosomes 1- 22 and X and Y, respectively. Lane 30 - Lambda DNA digested with _5_ tEII. An amplification product of approximately 2.8 Kb is observed only in lanes 5 and 9, representing human genomic DNA and DNA derived from cell hybrid carrying human chromosome 4, respectively. These results demonstrate that the hpa gene is localized in human chromosome 4. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a polynucleotide, referred to hereinbelow interchangeably as hpa, hpa cDNA or hpa gene, encoding a polypeptide having heparanase activity, vectors including same, transduced cells expressing heparanase and a recombinant protein having heparanase activity. Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting. The present invention can be used to develop treatments for various diseases, to develop diagnostic assays for these diseases and to provide new tools for basic research especially in the fields of medicine and biology. Specifically, the present invention can be used to develop new drugs to inhibit tumor cell metastasis, inflammation and autoimmunity. The identification of the hpa gene encoding for the heparanase enzyme enables the production of a recombinant enzyme in heterologous expression systems. Furthermore, the present invention can be used to modulate bioavailability of heparin-binding growth factors, cellular responses to heparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8), cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and some bacterial infections, and disintegration of neurodegenerative plaques. Recombinant heparanase is thus a potential treatment for wound healing, angiogenesis, restenosis, atherosclerosis, inflammation, neurodegenerative diseases (such as, for example, Genstmann-Straussler Syndrome, Creutzfeldt- Jakob disease, Scrape and Alzheimer's disease) and certain viral and some bacterial and protozoa infections. Recombinant heparanase can be used to neutralize plasma heparin, as a potential replacement of protamine. As used herein, the term "modulate" includes substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or condition, or substantially preventing the appearance of clinical symptoms of a disease or condition. A "modulator" therefore includes an agent which may modulate a disease or condition. Modulation of viral, protozoa and bacterial infections includes any effect which substantially interrupts, prevents or reduces any viral, bacterial or protozoa activity and/or stage of the virus, bacterium or protozoon life cycle, or which reduces or prevents infection by the virus, bacterium or protozoon in a subject, such as a human or lower animal. As used herein, the term "wound" includes any injury to any portion of the body of a subject including, but not limited to, acute conditions such as thermal burns, chemical burns, radiation burns, burns caused by excess exposure to ultraviolet radiation such as sunburn, damage to bodily tissues such as the perineum as a result of labor and childbirth, including injuries sustained during medical procedures such as episiotomies, trauma-induced injuries including cuts, those injuries sustained in automobile and other mechanical accidents, and those caused by bullets, knives and other weapons, and post-surgical injuries, as well as chronic conditions such as pressure sores, bedsores, conditions related to diabetes and poor circulation, and all types of acne, etc. Anti-heparanase antibodies, raised against the recombinant enzyme, would be useful for immunodetection and diagnosis of micrometastases, autoimmune lesions and renal failure in biopsy specimens, plasma samples, and body fluids. Such antibodies may also serve as neutralizing agents for heparanase activity. Cloning of the human hpa gene encoding heparanase and expressing recombinant heparanase by transfected cells is herein reported. This is the first mammalian heparanase gene to be cloned. A purified preparation of heparanase isolated from human hepatoma cells was subjected to tryptic digestion and microsequencing. The YGPDVGQPR (SEQ ID NO:8) sequence revealed was used to screen EST databases for homology to the corresponding back translated DNA sequences. Two closely related EST sequences were identified and were thereafter found to be identical. Both clones contained an insert of 1020 bp which includes an open reading frame of 973 bp followed by a 3' untranslated region of 27 bp and a Poly A tail, whereas a translation start site was not identified. Cloning of the missing 5' end was performed by PCR amplification of DNA from placenta Marathon RACE cDNA composite using primers selected according to the EST clones sequence and the linkers ofthe composite. A 900 bp PCR fragment, partially overlapping with the identified 3' encoding EST clones was obtained. The joined cDNA fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an open reading frame which encodes, as shown in Figure 1 and SEQ ID NO:l l, a polypeptide of 543 amino acids (SEQ ID NO: 10) with a calculated molecular weight of 61,192 daltons. A single nucleotide difference at position 799 (A to T) between the EST clones and the PCR amplified cDNA was observed. This difference results in a single amino acid substitution (Tyr to Phe) (Figure 1). Furthermore, the published EST sequences contained an unidentified nucleotide, which following DNA sequencing of both the EST clones was resolved into two nucleotides (G and C at positions 1630 and 1631 in SEQ ID NO:9, respectively). The ability of the hpa gene product to catalyze degradation of heparan sulfate in an in vitro assay was examined by expressing the entire open reading frame in insect cells, using the Baculovirus expression system. Extracts and conditioned media of cells infected with virus containing the hpa gene, demonstrated a high level of heparan sulfate degradation activity both towards soluble ECM-derived HSPG and intact ECM, which was inhibited by heparin, while cells infected with a similar construct containing no hpa gene had no such activity, nor did non- infected cells. The expression pattern of hpa RNA in various tissues and cell lines was investigated using RT-PCR. It was found to be expressed only in tissues and cells previously known to have heparanase activity. Cloning an extended 5' sequence was enabled from the human SK-hepl cell line by PCR amplification using the Marathon RACE. The 5' extended sequence ofthe SK-hepl hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta (SEQ ID NO:9). The assembled sequence contained an open reading frame, SEQ ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs: 14 and 15, a polypeptide of 592 amino acids, with a calculated molecular weight of 66,407 daltons. This open reading frame was shown to direct the expression of catalitically active heparanase in a mammalian cell expression system. The expressed heparanase was detectable by anti heparanase antibodies in Western blot analysis. A panel of monochromosomal human/CHO and human/mouse somatic cell hybrids was used to localize the human heparanase gene to human chromosome 4. The newly isolated heparanase sequence can therefore be used to identify a chromosome region harboring a human heparanase gene in a chromosome spread. Thus, according to the present invention there is provided a polynucleotide fragment (either DNA or RNA, either single stranded or double stranded) which includes a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The term "heparanase catalytic activity" or its equivalent term "heparanase activity" both refer to a mammalian endoglycosidase hydrolyzing activity which is specific for heparan or heparan sulfate proteoglycan substrates, as opposed to the activity of bacterial enzymes (heparinase I, II and III) which degrade heparin or heparan sulfate by means of β-elimination (37). In a preferred embodiment of the invention the polynucleotide fragment includes nucleotides 63-1691 of SEQ ID NO:9, or nucleotides 139-1869 of SEQ ID NO: 13, which encode the entire human heparanase enzyme. However, the scope of the present invention is not limited to human heparanase since this is the first disclosure of an open reading frame (ORF) encoding any mammalian heparanase. Using the hpa cDNA, parts thereof or synthetic oligonucleotides designed according to its sequence will enable one ordinarily skilled in the art to identify genomic and/or cDNA clones including homologous sequences from other mammalian species. The present invention is therefore further directed at a polynucleotide fragment which includes a polynucleotide sequence capable of hybridizing (base pairing under either stringent or permissive hybridization conditions, as for example described in Sambrook, J., Fritsch, E.F., Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.) with hpa cDNA, especially with nucleotides 1-721 of SEQ ID NO:9. In fact, any polynucleotide sequence which encodes a polypeptide having heparanase activity and which shares at least 60 % homology, preferably at least 70 % homology, more preferably at least 80 % homology, most preferably at least 90 % homology with SEQ ID NOs: 9 or 13 is within the scope of the present invention. The polynucleotide fragment according to the present- invention may include any part of SEQ ID NOs: 9 or 13. For example, it may include nucleotides 63-721 of SEQ ID NO:9, which is a novel sequence. However, it may include any segment of SEQ ID NOs: 9 or 13 which encodes a polypeptide having the heparanase catalytic activity. When the phrase "encodes a polypeptide having heparanase catalytic activity" is used herein and in the claims section below it refers to the ability of directing the synthesis of a polypeptide which, if so required for its activity, following post translational modifications, such as but not limited to, proteolysis (e.g., removal of a signal peptide and of a pro- or preprotein sequence), methionine modification, glycosylation, alkylation (e.g., methylation), acetylation, etc., is catalytically active in degradation of, for example, ECM and cell surface associated HS. In a preferred embodiment ofthe invention the polypeptide encoded by the polynucleotide fragment includes an amino acid sequence as set forth in SEQ ID NOs: 10 or 14 or a functional part thereof, i.e., a portion harboring heparanase catalytic activity. However, any polynucleotide fragment which encodes a polypeptide having heparanase activity is within the scope of the present invention. Therefore, the polypeptide may be allelic, species and/or induced variant of the amino acid sequence set forth in SEQ ID NOs: 10 or 14 or functional part thereof. In fact, any polynucleotide sequence which encodes a polypeptide having heparanase activity, which shares at least 60 % homology, preferably at least 70 % homology, more preferably at least 80 % homology, most preferably at least 90 % homology with SEQ ID NOs: 10 or 14 is within the scope of the present invention. The invention is also directed at providing a single stranded polynucleotide fragment which includes a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity as described above. The term "complementary" as used herein refers to the ability of base pairing. The single stranded polynucleotide fragment may be DNA or RNA or even include nucleotide analogs (e.g., thioated nucleotides), it may be a synthetic oligonucleotide or manufactured by transduced host cells, it may be of any desired length which still provides specific base pairing (e.g., 8 or 10, preferably more, nucleotides long) and it may include mismatches that do not hamper base pairing. The invention is further directed at providing a vector which includes a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The vector may be of any type. It may be a phage which infects bacteria or a virus which infects eukaryotic cells. It may also be a plasmid, phagemid, cosmid, bacmid or an artificial chromosome. The polynucleotide sequence encoding a polypeptide having heparanase catalytic activity may include any of the above described polynucleotide fragments. The invention is further directed at providing a host cell which includes an exogenous polynucleotide fragment encoding a polypeptide having heparanase catalytic activity. The exogenous polynucleotide fragment may be any of the above described fragments. The host cell may be of any type. It may be a prokaryotic cell, an eukaryotic cell, a cell line, or a cell as a portion of an organism. The exogenous polynucleotide fragment may be permanently or transiently present in the cell. In other words, transduced cells obtained following stable or transient transfection, transformation or transduction are all within the scope of the present invention. The term "exogenous" as used herein refers to the fact that the polynucleotide fragment is externally introduced into the cell. Therein it may be present in a single of any number of copies, it may be integrated into one or more chromosomes at any location or be present as an extrachromosomal material. The invention is further directed at providing a heparanase overexpression system which includes a cell overexpressing heparanase catalytic activity. The cell may be a host cell transiently or stably transfected or transformed with any suitable vector which includes a polynucleotide sequence encoding a polypeptide having heparanase activity and a suitable promoter and enhancer sequences to direct overexpression of heparanase. However, the overexpressing cell may also be a product of an insertion (e.g., via homologous recombination) of a promoter and/or enhancer sequence downstream to the endogenous heparanase gene of the expressing cell, which will direct overexpression from the endogenous gene. The term "overexpression" as used herein in the specification and claims below refers to a level of expression which is higher than a basal level of expression typically characterizing a given cell under otherwise identical conditions. The invention is further directed at providing a recombinant protein including a polypeptide having heparanase catalytic activity. The recombinant protein may be purified by any conventional protein purification procedure close to homogeneity and/or be mixed with additives. The recombinant protein may be manufactured using any ofthe cells described above. The recombinant protein may be in any form. It may be in a crystallized form, a dehydrated powder form or in solution. The recombinant protein may be useful in obtaining pure heparanase, which in turn may be useful in eliciting anti- heparanase antibodies, either poly or monoclonal antibodies, and as a screening active ingredient in an anti-heparanase inhibitors or drugs screening assay or system. The invention is further directed at providing a pharmaceutical composition which include as an active ingredient a recombinant protein having heparanase catalytic activity. Formulations for topical administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, stents, active pads, and other medical devices may also be useful. In fact the scope of the present invention includes any medical equipment such as a medical device containing, as an active ingredient, a recombinant protein having heparanase catalytic activity. Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable. Formulations for parenteral administration may include, but are not limited to, sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons ordinarily skilled in the art can easily determine optimum dosages, dosing methodologies and repetition rates. Further according to the present invention there is provided a method of identifying a chromosome region harboring a human heparanase gene in a chromosome spread, the method is executed implementing the following method steps, in which in a first step the chromosome spread (either interphase or metaphase spread) is hybridized with a tagged polynucleotyde probe encoding heparanase. The tag is preferably a fluorescent tag. In a second step according to the method the chromosome spread is washed, thereby excess of non-hybridized probe is removed. Finally, signals associated with the hybridized tagged polynucleotyde probe are searched for, wherein detected signals being indicative of a chromosome region harboring the human heparanase gene. One ordinarily skilled in the art would know how to use the sequences disclosed herein in suitable labeling reactions and how to use the tagged probes to detect, using in situ hybridization, a chromosome region harboring a human heparanase gene. Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non-limiting fashion. EXAMPLES The following protocols and experimental details are referenced in the Examples that follow: Purification and characterization of heparanase from a human hepatoma cell line and human placenta: A human hepatoma cell line (Sk-hep- 1) was chosen as a source for purification of a human tumor-derived heparanase. Purification was essentially as described in U.S. Pat. No. 5,362,641 to Fuks, which is incorporated by reference as if fully set forth herein. Briefly, 500 liter, 5x10 1 1 cells were grown in suspension and the heparanase enzyme was purified about 240,000 fold by applying the following steps: (i) cation exchange (CM- Sephadex) chromatography performed at pH 6.0, 0.3-1.4 M NaCl gradient; (ii) cation exchange (CM-Sephadex) chromatography performed at pH 7.4 in the presence of 0.1% CHAPS, 0.3-1.1 M NaCl gradient; (iii) heparin-Sepharose chromatography performed at pH 7.4 in the presence of 0.1% CHAPS, 0.35-1.1 M NaCl gradient; (iv) ConA- Sepharose chromatography performed at pH 6.0 in buffer containing 0.1 % CHAPS and 1 M NaCl, elution with 0.25 M α-methyl mannoside; and (v) HPLC cation exchange (Mono-S) chromatography performed at pH 7.4 in the presence of 0.1 % CHAPS, 0.25-1 M NaCl gradient. Active fractions were pooled, precipitated with TCA and the precipitate subjected to SDS polyacrylamide gel electrophoresis and/or tryptic digestion and reverse phase HPLC. Tryptic peptides of the purified protein were separated by reverse phase HPLC (C8 column) and homogeneous peaks were subjected to amino acid sequence analysis. The purified enzyme was applied to reverse phase HPLC and subjected to N-terminal amino acid sequencing using the amino acid sequencer (Applied Biosystems). Cells: Cultures of bovine corneal endothelial cells (BCECs) were established from steer eyes as previously described (19, 38). Stock cultures were maintained in DMEM (1 g glucose/liter) supplemented with 10 % newborn calf serum and 5 % FCS. bFGF (1 ng/ml) was added every other day during the phase of active cell growth (13, 14). Preparation of dishes coated with ECM: BCECs (second to fifth passage) were plated into 4- well plates at an initial density of 2 x 10 5 cells/ml, and cultured in sulfate-free Fisher medium plus 5 % dextran T-40 for 12 days. Na 2 35 SO 4 (25 μCi/ml) was added on day 1 and 5 after seeding and the cultures were incubated with the label without medium change. The subendothelial ECM was exposed by dissolving (5 min., room temperature) the cell layer with PBS containing 0.5 % Triton X-100 and 20 mM NH 4 OH, followed by four washes with PBS. The ECM remained intact, free of cellular debris and firmly attached to the entire area of the tissue culture dish (19, 22). To prepare soluble sulfate labeled proteoglycans (peak I material), the ECM was digested with trypsin (25 μg/ml, 6 h, 37 °C ), the digest was concentrated by reverse dialysis and the concentrated material was applied onto a Sepharose 6B gel filtration column. The resulting high molecular weight material (Kav< 0.2, peak I) was collected. More than 80 % of the labeled material was shown to be composed of heparan sulfate proteoglycans (11, 39). Heparanase activity: Cells (1 x 10 6 /35-mm dish), cell lysates or conditioned media were incubated on top of 35 S-labeled ECM (18 h, 37 °C) in the presence of 20 mM phosphate buffer (pH 6.2). Cell lysates and conditioned media were also incubated with sulfate labeled peak I material (10-20 μl). The incubation medium was collected, centrifuged (18,000 x g, 4 °C, 3 min.), and sulfate labeled material analyzed by gel filtration on a Sepharose CL-6B column (0.9 x 30 cm). Fractions (0.2 ml) were eluted with PBS at a flow rate of 5 ml/h and counted for radioactivity using Bio-fluor scintillation fluid. The excluded volume (V 0 ) was marked by blue dextran and the total included volume (V£) by phenol red. The latter was shown to comigrate with free sulfate (7, 11, 23). Degradation fragments of HS side chains were eluted from Sepharose 6B at 0.5 < Kav < 0.8 (peak II) (7, 11, 23). A nearly intact HSPG released from ECM by trypsm - and, to a lower extent, during incubation with PBS alone - was eluted next to V 0 (Kav < 0.2, peak I). Recoveries of labeled material applied on the columns ranged from 85 to 95 % in different experiments (11). Each experiment was performed at least three times and the variation of elution positions (Kav values) did not exceed +/- 15 %. Cloning of hpa cDNA: cDNA clones 257548 and 260138 were obtained from the I.M.A.G.E Consortium (2130 Memorial Parkway SW, Hunstville, AL 35801). The cDNAs were originally cloned in EcoRl and Notl cloning sites in the plasmid vector pT3T7D-Pac. Although these clones are reported to be somewhat different, DNA sequencing demonstrated that these clones are identical to one another. Marathon RACE (rapid amplification of cDNA ends) human placenta (poly- A) cDNA composite was a gift of Prof. Yossi Shiloh of Tel Aviv University. This composite is vector free, as it includes reverse transcribed cDNA fragments to which double, partially single stranded adapters are attached on both sides. The construction of the specific composite employed is described in reference 39a. Amplification of hp3 PCR fragment was performed according to the protocol provided by Clontech laboratories. The template used for amplification was a sample taken from the above composite. The primers used for amplification were: First step: 5'-primer: API : 5'-CCATCCTAATACGACTCACTATAGGG C-3', SEQ ID NO:l; 3*-primer: HPL229: 5'-GTAGTGATGCCATGTAACTGA ATC-3', SEQ ID NO:2. Second step: nested 5'-primer: AP2: 5'- ACTCACTATAGGGCTCGAGCG GC-3', SEQ ID NO:3; nested 3'- primer: HPL171 : 5'-GCATCTTAGCCGTCT TTCTTCG-3', SEQ ID NO:4. The HPL229 and HPL171 were selected according to the sequence of the EST clones. They include nucleotides 933-956 and 876-897 of SEQ ID NO:9, respectively. PCR program was 94 °C - 4 min., followed by 30 cycles of 94 °C - 40 sec, 62 °C - 1 min., 72 °C - 2.5 min. Amplification was performed with Expand High Fidelity (Boehringer Mannheim). The resulting ca. 900 bp hp3 PCR product was digested with Bfrl and Pv_.II. Clone 257548 (phpal) was digested with EcoRI, followed by end filling and was then further digested with Bfrl. Thereafter the Pvull - Bfrl fragment of the hp3 PCR product was cloned into the blunt end - Bfrl end of clone phpal which resulted in having the entire cDNA cloned in pT3T7-pac vector, designated phpal. DNA Sequencing: Sequence determinations were performed with vector specific and gene specific primers, using an automated DNA sequencer (Applied Biosystems, model 373A). Each nucleotide was read from at least two independent primers. Computer analysis of sequences: Database searches for sequence similarities were performed using the Blast network service. Sequence analysis and alignment of DNA and protein sequences were done using the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the University of Wisconsin. RT-PCR: RNA was prepared using TRI-Reagent (Molecular research center Inc.) according to the manufacturer instructions. 1.25 μg were taken for reverse transcription reaction using MuMLV Reverse transcriptase (Gibco BRL) and Oligo (dT)i5 primer, SEQ ID NO:5, (Promega). Amplification of the resultant first strand cDNA was performed with Taq polymerase (Promega). The following primers were used: HPU-355: 5'-TTCGATCCCAAGAAGGAATCAAC-3', SEQ ID NO:6, nucleotides 372-394 in SEQ ID NO:9 or 11. HPL-229: 5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID NO:7, nucleotides 933-956 in SEQ ID NO:9 or 11. PCR program: 94 °C - 4 min., followed by 30 cycles of 94 °C - 40 sec, 62 °C - 1 min., 72 °C - 1 min. Expression of recombinant heparanase in insect cells: Cells, High Five and Sf21 insect cell lines were maintained as monolayer cultures in SF900II-SFM medium (GibcoBRL). Recombinant Baculovirus: Recombinant virus containing the hpa gene was constructed using the Bac to Bac system (GibcoBRL). The transfer vector pFastBac was digested with Sail and NotI and ligated with a 1.7 kb fragment of phpal digested with Xhol and Nøtl. The resulting plasmid was designated pVa.shpal. An identical plasmid designated pVasihpa4 was prepared as a duplicate and both independently served for further experimentations. Recombinant bacmid was generated according to the instructions of the manufacturer with pFasXhpal, p¥asϋιpa4 and with pFastBac The latter served as a negative control. Recombinant bacmid DΝAs were transfected into Sf21 insect cells. Five days after transfection recombinant viruses were harvested and used to infect High Five insect cells, 3 x 10 6 cells in T-25 flasks. Cells were harvested 2 - 3 days after infection. 4 x 10 6 cells were centrifiiged and resuspended in a reaction buffer containing 20 mM phosphate citrate buffer, 50 mM ΝaCl. Cells underwent three cycles of freeze and thaw and lysates were stored at -80 °C. Conditioned medium was stored at 4 °C. Partial purification of recombinant heparanase: Partial purification of recombinant heparanase was performed by heparin-Sepharose column chromatography followed by Superdex 75 column gel filtration. Culture medium (150 ml) of Sf21 cells infected with pFhpa4 virus was subjected to heparin- Sepharose chromatography. Elution of 1 ml fractions was performed with 0.35 - 2 M ΝaCl gradient in presence of 0.1 % CHAPS and 1 mM DTT in 10 mM sodium acetate buffer, pH 5.0. A 25 μl sample of each fraction was tested for heparanase activity. Heparanase activity was eluted at the range of 0.65 - 1.1 M ΝaCl (fractions 18-26, Figure 10a). 5 μl of each fraction was subjected to 15 % SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining. Active fractions eluted from heparin-Sepharose (Figure 10a) were pooled and concentrated (x 6) on YM3 cut-off membrane. 0.5 ml of the concentrated material was applied onto 30 ml Superdex 75 FPLC column equilibrated with 10 mM sodium acetate buffer, pH 5.0, containing 0.8 M ΝaCl, 1 mM DTT and 0.1 % CHAPS. Fractions (0.56 ml) were collected at a flow rate of 0.75 ml/min. Aliquots of each fraction were tested for heparanase activity and were subjected to SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining (Figure l ib). EXAMPLE 1 Cloning of the hpa gene Purified fraction of heparanase isolated from human hepatoma cells (SK- hep-1) was subjected to tryptic digestion and microsequencing. EST (Expressed Sequence Tag) databases were screened for homology to the back translated DNA sequences corresponding to the obtained peptides. Two EST sequences (accession Nos. N41349 and N45367) contained a DNA sequence encoding the peptide YGPDVGQPR (SEQ ID NO:8). These two sequences were derived from clones 257548 and 260138 (I.MA.G.E Consortium) prepared from 8 to 9 weeks placenta cDNA library (Soares). Both clones which were found to be identical contained an insert of 1020 bp which included an open reading frame (ORF) of 973 bp followed by a 3' untranslated region of 27 bp and a Poly A tail. No translation start site (AUG) was identified at the 5' end of these clones. Cloning of the missing 5' end was performed by PCR amplification of DNA from a placenta Marathon RACE cDNA composite. A 900 bp fragment (designated hp3), partially overlapping with the identified 3' encoding EST clones was obtained. The joined cDNA fragment, 1721 bp long (SEQ ID NO:9), contained an open reading frame which encodes, as shown in Figure 1 and SEQ ID NO: 11, a polypeptide of 543 amino acids (SEQ ID NO: 10) with a calculated molecular weight of 61,192 daltons. The 3' end of the partial cDNA inserts contained in clones 257548 and 260138 started at nucleotide G 721 of SEQ ID NO:9 and Figure 1. As further shown in Figure 1, there was a single sequence discrepancy between the EST clones and the PCR amplified sequence, which led to an amino acid substitution from Tyr 246 in the EST to Phe 246 in the amplified cDNA. The nucleotide sequence ofthe PCR amplified cDNA fragment was verified from two independent amplification products. The new gene was designated hpa. As stated above, the 3' end of the partial cDNA inserts contained in EST clones 257548 and 260138 started at nucleotide 721 of hpa (SEQ ID NO:9). The ability of the hpa cDNA to form stable secondary structures, such as stem and loop structures involving nucleotide stretches in the vicinity of position 721 was investigated using computer modeling. It was found that stable stem and loop structures are likely to be formed involving nucleotides 698-724 (SEQ ID NO:9). In addition, a high GC content, up to 70 %, characterizes the 5' end region of the hpa gene, as compared to about only 40 % in the 3' region. These findings may explain the immature termination and therefore lack of 5' ends in the EST clones. To examine the ability of the hpa gene product to catalyze degradation of heparan sulfate in an in vitro assay the entire open reading frame was expressed in insect cells, using the Baculovirus expression system. Extracts of cells, infected with virus containing the hpa gene, demonstrated a high level of heparan 5 sulfate degradation activity, while cells infected with a similar construct containing no hpa gene had no such activity, nor did non- infected cells. These results are further demonstrated in the following Examples. EXAMPLE 2 l o Degradation of soluble ECM-derived HSPG Monday er cultures of High Five cells were infected (72 h, 28 °C) with recombinant Bacoluvirus containing the pFast/zpα plasmid or with control virus containing an insert free plasmid. The cells were harvested and lysed in heparanase reaction buffer by three cycles of freezing and thawing. The cell 15 lysates were then incubated (18 h, 37 °C) with sulfate labeled, ECM-derived HSPG (peak I), followed by gel filtration analysis (Sepharose 6B) of the reaction mixture. As shown in Figure 2, the substrate alone included almost entirely high molecular weight (Mr) material eluted next to V 0 (peak I, fractions 5-20, Kav < 20 0.35). A similar elution pattern was obtained when the HSPG substrate was incubated with lysates of cells that were infected with control virus. In contrast, incubation of the HSPG substrate with lysates of cells infected with the hpa containing virus resulted in a complete conversion of the high Mr substrate into low Mr labeled degradation fragments (peak II, fractions 22-35, 0.5 < Kav < 25 0.75). Fragments eluted in peak II were shown to be degradation products of heparan sulfate, as they were (i) 5- to 6-fold smaller than intact heparan sulfate side chains (Kav approx. 0.33) released from ECM by treatment with either alkaline borohydride or papain; and (ii) resistant to further digestion with papain 30 or chondroitinase ABC, and susceptible to deamination by nitrous acid (6, 11). Similar results (not shown) were obtained with Sf21 cells. Again, heparanase activity was detected in cells infected with the hpa containing virus (pF hpa), but not with control virus (pF). This result was obtained with two independently generated recombinant viruses. Lysates of control not infected 35 High Five cells failed to degrade the HSPG substrate. In subsequent experiments, the labeled HSPG substrate was incubated with medium conditioned by infected High Five or Sf21 cells. As shown in Figures 3a-b, heparanase activity, reflected by the conversion of the high Mr peak I substrate into the low Mr peak II which represents HS degradation fragments, was found in the culture medium of cells infected with the p¥hpa2 or pFhpa4 viruses, but not with the control pFl or pF2 viruses. No heparanase activity was detected in the culture medium of control non-infected High Five or Sf 1 cells. The medium of cells infected with the pFhpa4 virus was passed through a 50 kDa cut off membrane to obtain a crude estimation of the molecular weight of the recombinant heparanase enzyme. As demonstrated in Figure 4, all the enzymatic activity was retained in the upper compartment and there was no activity in the flow through (<50 kDa) material. This result is consistent with the expected molecular weight ofthe hpa gene product. In order to further characterize the hpa product the inhibitory effect of heparin, a potent inhibitor of heparanase mediated HS degradation (40) was examined. As demonstrated in Figures 5a-b, conversion of the peak I substrate into peak II HS degradation fragments was completely abolished in the presence of heparin. Altogether, these results indicate that the heparanase enzyme is " expressed in an active form by insect cells infected with Baculovirus containing the newly identified human hpa gene. EXAMPLE 3 Degradation of HSPG in intact ECM Next, the ability of intact infected insect cells to degrade HS in intact, naturally produced ECM was investigated. For this purpose, High Five or Sf21 cells were seeded on metabolically sulfate labeled ECM followed by infection (48 h, 28 °C) with either the pFhpa4 or control ρF2 viruses. The pH of the medium was then adjusted to pH 6.2-6.4 and the cells further incubated with the labeled ECM for another 48 h at 28 °C or 24 h at 37 °C. Sulfate labeled material released into the incubation medium was analyzed by gel filtration on Sepharose 6B. As shown in Figures 6a-b and 7a-b, incubation of the ECM with cells infected with the control pF2 virus resulted in a constant release of labeled material that consisted almost entirely (>90%) of high Mr fragments (peak I) eluted with or next to V 0 . It was previously shown that a proteolytic activity residing in the ECM itself and/or expressed by cells is responsible for release of the high Mr material (6). This nearly intact HSPG provides a soluble substrate for subsequent degradation by heparanase, as also indicated by the relatively large amount of peak I material accumulating when the heparanase enzyme is inhibited by heparin (6, 7, 12, Figure 9). On the other hand, incubation of the labeled ECM with cells infected with the pFhpa4 virus resulted in release of 60- 70% of the ECM-associated radioactivity in the form of low Mr sulfate-labeled fragments (peak II, 0.5 <Kav< 0.75), regardless of whether the infected cells were incubated with the ECM at 28 °C or 37 °C. Control intact non-infected Sf21 or High Five cells failed to degrade the ECM HS side chains. In subsequent experiments, as demonstrated in Figures 8a-b, High Five and Sf21 cells were infected (96 h, 28 °C) with pFhpa4 or control pFl viruses and the culture medium incubated with sulfate-labeled ECM. Low Mr HS degradation fragments were released from the ECM only upon incubation with medium conditioned by pFhpa4 infected cells. As shown in Figure 9, production of these fragments was abolished in the presence of heparin. No heparanase activity was detected in the culture medium of control, non-infected cells. These results indicate that the heparanase enzyme expressed by cells infected with the pFhpa4 virus is capable of degrading HS when complexed to other macromolecular constituents (i.e. fibronectin, laminin, collagen) of a naturally produced intact ECM, in a manner similar to that reported for highly metastatic tumor cells or activated cells ofthe immune system (6, 7). EXAMPLE 4 Purification of recombinant heparanase The recombinant heparanase was partially purified from medium of pFhpa4 infected Sf21 cells by Heparin-Sepharose chromatography (Figure 10a) followed by gel filtration of the pooled active fractions over an FPLC Superdex 75 column (Figure 11a). A ~ 63 kDa protein was observed, whose quantity, as was detected by silver stained SDS-polyacrylamide gel electrophoresis, correlated with heparanase activity in the relevant column fractions (Figures 10b and l ib, respectively). This protein was not detected in the culture medium of cells infected with the control pFl virus and was subjected to a similar fractionation on heparin-Sepharose (not shown). EXAMPLE 5 Expression of the hpa gene in various cell types, organs and tissues Referring now to Figures 12a-e, RT-PCR was applied to evaluate the expression of the hpa gene by various cell types and tissues. For this purpose, total RNA was reverse transcribed and amplified. The expected 585 bp long cDNA was clearly demonstrated in human kidney, placenta (8 and 11 weeks) and mole tissues, as well as in freshly isolated and short termed (1.5-48 h) cultured human placental cytotrophoblastic cells (Figure 12a), all known to express a high heparanase activity (41). The hpa transcript was also expressed by normal human neutrophils (Figure 12b). In contrast, there was no detectable expression of the hpa mRNA in embryonic human muscle tissue, thymus, heart and adrenal (Figure 12b). The hpa gene was expressed by several, but not all, human bladder carcinoma cell lines (Figure 12c), SK hepatoma (SK-hep-1), ovarian carcinoma (OV 1063), breast carcinoma (435, 231), melanoma and megakaryocytic (DAMI, CHRF) human cell lines (Figures 12d-e). The above described expression pattern of the hpa transcript was determined to be in a very good correlation with heparanase activity levels determined in various tissues and cell types (not shown). EXAMPLE 6 hpa homologous genes EST databases were screened for sequences homologous to the hpa gene. Three mouse ESTs were identified (accession No. Aal 77901, from mouse spleen, Aa067997 from mouse skin, Aa47943 from mouse embryo), assembled into a 824 bp cDNA fragment which contains a partial open reading frame (lacking a 5' end) of 629 bp and a 3' untranslated region of 195 bp (SEQ ID NO: 12). As shown in Figure 13, the coding region is 80% similar to the 3' end of the hpa cDNA sequence. These ESTs are probably cDNA fragments of the mouse hpa homolog that encodes for the mouse heparanase. Searching for consensus protein domains revealed an amino terminal homology between the heparanase and several precursor proteins such as Procollagen Alpha 1 precursor, Tyrosine-protein kinase-RYK, Fibulin-1, Insulinlike growth factor binding protein and several others. The amino terminus is highly hydrophobic and contains a potential trans-membrane domain. The homology to known signal peptide sequences suggests that it could function as a signal peptide for protein localization. EXAMPLE 7 Isolation of an extended 5 ' end of hpa cDNA from human SK-hepl cell line The 5' end of hpa cDNA was isolated from human SK-hepl cell line by PCR amplification using the Marathon RACE (rapid amplification of cDNA ends) kit (Clontech). Total RNA was prepared from SK-hepl cells using the TRI-Reagent (Molecular research center Inc.) according to the manufacturer instructions. Poly A+ RNA was isolated using the mRNA separator kit (Clonetech). The Marahton RACE SK-hepl cDNA composite was constructed according to the manufacturer recommendations. First round of amplification was performed using an adaptor specific primer API: 5'-CCATCCTAATACG ACTCACTATAGGGC-3', SEQ ID NO: l, and a hpa specific antisense primer hpl-629: 5'-CCCCAGGAGCAGCAGCATCAG-3', SEQ ID NO: 17, corresponding to nucleotides 119-99 of SEQ ID NO:9. The resulting PCR product was subjected to a second round of amplification using an adaptor specific nested primer AP2: 5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:3, and a hpa specific antisense nested primer hpl-666 5'- AGGCTTCGAGCGCAGCAGCAT-3', SEQ ID NO: 18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The PCR program was as follows: a hot start of 94 °C for 1 minute, followed by 30 cycles of 90 °C - 30 seconds, 68 °C - 4 minutes. The resulting 300 bp DNA fragment was extracted from an agarose gel and cloned into the vector pGEM-T Easy (Promega). The resulting recombinant plasmid was designated pHPSKl . The nucleotide sequence of the pHPSKl insert was determined and it was found to contain 62 nucleotides of the 5' end of the placenta hpa cDNA (SEQ ID NO:9) and additional 178 nucleotides upstream, the first 178 nucleotides of SEQ ID NOs:13 and l5. A single nucleotide discrepancy was identified between the SK-hepl cDNA and the placenta cDNA. The "T" derivative at position 9 of the placenta cDNA (SEQ ID NO:9), is replaced by a "C" derivative at the corresponding position 187 ofthe SK-hepl cDNA (SEQ ID NO: 13). The discrepancy is likely to be due to a mutation at the 5' end of the placenta cDNA clone as confirmed by sequence analysis of sevsral additional cDNA clones isolated from placenta, which like the SK-hepl cDNA contained C at position 9 of SEQ ID NO:9. The 5' extended sequence of the SK-hepl hpa cDNA was assembled with the sequence of the hpa cDNA isolated from human placenta (SEQ ID NO: 9). The assembled sequence contained an open reading frame which encodes, as shown in SEQ ID NOs: 14 and 15, a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons. The open reading frame is flanked by 93 bp 5' untranslated region (UTR). EXAMPLE 8 Isolation ofthe upstream genomic region ofthe hpa gene The upstream region of the hpa gene was isolated using the Genome Walker kit (Clontech) according to the manufacturer recommendations. The kit includes five human genomic DNA samples each digested with a different restriction endonuclease creating blunt ends: EcoRV, Seal, Oral, Pvull and Sspl. The blunt ended DNA fragments are ligated to partially single stranded adaptors. The Genomic DNA samples were subjected to PCR amplification using the adaptor specific primer and a gene specific primer. Amplification was performed with Expand High Fidelity (Boehringer Mannheim). A first round of amplification was performed using the apl primer: 5'-G TAATACGACTCACTATAGGGC-3', SEQ ID NO: 19, and the hpa specific antisense primer hpl-666: 5'-AGGCTTCGAGCGCAGCAGCAT-3', SEQ ID NO: 18, corresponding to nucleotides 83 - 63 of SEQ ID NO:9. The PCR program was as follows: a hot start of 94 °C - 3 minutes, followed by 36 cycles of 94 °C - 40 seconds, 67 °C - 4 minutes. The PCR products of the first amplification were diluted 1 :50. One μl of the diluted sample was used as a template for a second amplification using a nested adaptor specific primer ap2: 5'-ACTATAGGGCACGCGTGGT-3', SEQ ID NO:20, and a hpa specific antisense primer hpl-690, 5'-CTTGGGCTCACC TGGCTGCTC-3', SEQ ID NO:21, corresponding to nucleotides 62-42 of SEQ ID NO: 9. The resulting amplification products were analyzed using agarose gel electrophoresis. Five different PCR products were obtained from the five amplification reactions. A DNA fragment of approximately 750 bp which was obtained from the Sspl digested DNA sample was gel extracted. The purified fragment was ligated into the plasmid vector pGEM-T Easy (Promega). The resulting recombinant plasmid was designated pGHP6905 and the nucleotide sequence ofthe hpa insert was determined. A partial sequence of 594 nucleotides is shown in SEQ ID NO: 16. The last nucleotide in SEQ ID NO: 13 corresponds to nucleotide 93 in SEQ ID: 13. The DNA sequence in SEQ ID NO: 16 contains the 5' region of the hpa cDNA and 501 nucleotides of the genomic upstream region which are predicted to contain the promoter region ofthe hpa gene. EXAMPLE 9 Expression ofthe 592 amino acids HPA polypeptide in a human 293 cell line The 592 amino acids open reading frame (SEQ ID NOs: 13 and 15) was constructed by ligation of the 110 bp corresponding to the 5' end of the SK-hepl hpa cDNA with the placenta cDNA. More specifically the Marathon RACE - PCR amplification product of the placenta hpa DNA was digested with Sacl and an approximately 1 kb fragment was ligated into a Sαcl-digested pGHP6905 plasmid. The resulting plasmid was digested with Earl and Aatll. The Earl sticky ends were blunted and an approximately 280 bp EarVb mt-Aatϊl fragment was isolated. This fragment was ligated with pVasthpa digested with EcoRI which was blunt ended using Klenow fragment and further digested with Aatll. The resulting plasmid contained a 1827 bp insert which includes an open reading frame of 1776 bp, 31 bp of 3' UTR and 21 bp of 5' UTR. This plasmid was designated pFastLΛ ?α. A mammalian expression vector was constructed to drive the expression of the 592 amino acids heparanase polypeptide in human cells. The hpa cDNA was excised prom pFastLhpa with _5_._.HII and Notl. The resulting 1850 bp BssHϊl-Notl fragment was ligated to a mammalian expression vector pSI (Promega) digested with Mlul and Notl. The resulting recombinant plasmid, pSI zp Met2 was transfected into a human 293 embryonic kidney cell line. Transient expression of the 592 amino-acids heparanase was examined by western blot analysis and the enzymatic activity was tested using the gel shift assay. Both these procedures are described in length in U.S. Pat. application No. 09/071,739, filed May 1, 1998, which is incorporated by reference as if fully set forth herein. Cells were harvested 3 days following transfection. Harvested cells were re-suspended in lysis buffer containing 150 mM NaCl, 50 mM Tris pH 7.5, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail (Boehringer Mannheim). 40 μg protein extract samples were used for separation on a SDS- PAGE. Proteins were transferred onto a PVDF Hybond-P membrane (Amersham). The membrane was incubated with an affinity purified polyclonal anti heparanase antibody, as described in U.S. Pat. application No. 09/071,739. A major band of approximately 50 kDa was observed in the transfected cells as well as a minor band of approximately 65 kDa. A similar pattern was observed in extracts of cells transfected with the pShpa as demonstrated in U.S. Pat. application No. 09/071,739. These two bands probably represent two forms of the recombinant heparanase protein produced by the transfected cells. The 65 kDa protein probably represents a heparanase precursor, while the 50 kDa protein is suggested herein to be the processed or mature form. The catalytic activity of the recombinant protein expressed in the pShpaMet2 transfected cells was tested by gel shift assay. Cell extracts of transfected and of mock transfected cells were incubated overnight with heparin (6 μg in each reaction) at 37 °C, in the presence of 20 mM phosphate citrate buffer pH 5.4, 1 mM CaCl2, 1 mM DTT and 50 mM NaCl. Reaction mixtures were then separated on a 10 % polyacrylamide gel. The catalytic activity of the recombinant heparanase was clearly demonstrated by a faster migration of the heparin molecules incubated with the transfected cell extract as compared to the control. Faster migration indicates the disappearance of high molecular weight heparin molecules and the generation of low molecular weight degradation products. EXAMPLE 10 Chromosomal localization ofthe hpa gene Chromosomal mapping of the hpa gene was performed utilizing a panel of monochromosomal human/CHO and human/mouse somatic cell hybrids, obtained from the UK HGMP Resource Center (Cambridge, England). 40 ng of each of the somatic cell hybrid DNA samples were subjected to PCR amplification using the hpa primers: hpu565 5'-AGCTCTGTAGATGTGC TATACAC-3', SEQ ID NO:22, corresponding to nucleotides 564-586 of SEQ ID NO:9 and an antisense primer hpll71 5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:23, corresponding to nucleotides 897-876 of SEQ ID NO:9. The PCR program was as follows: a hot start of 94 °C - 3 minutes, followed by 7 cycles of 94 °C - 45 seconds, 66 °C - 1 minute, 68 °C - 5 minutes, followed by 30 cycles of 94 °C - 45 seconds, 62 °C - 1 minute, 68 °C - 5 minutes, and a 10 minutes final extension at 72 °C. The reactions were performed with Expand long PCR (Boehringer Mannheim). The resulting amplification products were analyzed using agarose gel electrophoresis. As demonstrated in Figure 14, a single band of approximately 2.8 Kb was obtained from chromosome 4, as well as from the control human genomic DNA. A 2.8 kb amplification product is expected based on amplification of the genomic hpa clone (data not shown). No amplification products were obtained neither in the control DNA samples of hamster and mouse nor in somatic hybrids of other human chromosome. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope ofthe appended claims. LIST OF REFERENCES CITED HEREIN ABOVE BY NUMBERS 1. Wight, T.N., Kinsella, M.G., and Qwarnstromn, E.E. (1992). The role of proteoglycans in cell adhesion, migration and proliferation. Curr. Opin. Cell Biol, 4, 793-801. 2. Jackson, R.L., Busch, S.J., and Cardin, A.L. (1991). Gly cosaminogly cans: Molecular properties, protein interactions and role in physiological processes. Physiol. Rev., 71, 481-539. 3. Wight, T.N. (1989). Cell biology of arterial proteoglycans. Arteriosclerosis, 9, 1-20. 4. . Kjellen, L., and Lindahl, U. (1991). Proteoglycans: structures and interactions. Annu. Rev. Biochem., 60, 443-475. 5. 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Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn,- CM., Folkman, J., and Vlodavsky, I. (1989). Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules. Biochemistry, 28, 1737-1743. 16. Parish, C.R., Coombe, D.R., Jakobsen, K.B., and Underwood, PA. (1987). Evidence that sulphated polysaccharides inhibit tumor metastasis by blocking tumor cell-derived heparanase. Int. J. Cancer, 40, 511-517. 16a. Vlodavsky, I., Hua-Quan Miao., Benezra, M., Lider, O., Bar- Shavit, R., Schmidt, A., and Peretz, T. (1997). Involvement of the extracellular matrix, heparan sulfate proteoglycans and heparan sulfate degrading enzymes in angiogenesis and metastasis. In: Tumor Angiogenesis. Eds. C.E. Lewis, R. Bicknell & N. Ferrara. Oxford University Press, Oxford UK, pp. 125-140. 17. Burgess, W.H., and Maciag, T. (1989). The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem., 58, 575-606. 18. 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" , Naparstek, Y., Vlodavsky, I., and Cohen, I.R. (1989). Suppression of experimental autoimmune diseases and prolongation of allograft survival by treatment of animals with heparinoid inhibitors of T lymphocyte heparanase. J. Clin. Invest, 83, 752-756. 31. Lider, O., Cahalon, L., Gilat, D., Hershkovitz, R., Siegel, D., Margalit, R., Shoseyov, O., and Cohn, I.R. (1995). A disaccharide that inhibits tumor necrosis factor α is formed from the extracellular matrix by the enzyme heparanase. Proc. Natl. Acad. Sci. USA., 92, 5037-5041. 31a. Rapraeger, A., Krufka, A., and Olwin, B.R. (1991). Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science, 252, 1705-1708. 32. Eisenberg, S., Sehayek, E., Olivecrona, T., and Vlodavsky, I. (1992). Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix. J. Clin. Invest., 90, 2013-2021. 33. Shieh, M-T., Wundunn, D., Montgomery, R.I., Esko, J.D., and Spear, P.G. J. (1992). Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol., 116, 1273-1281. 33a. Chen, Y., Maguire, T., Hileman, R.E., Fromm, J.R., Esko, J.D., Linhardt, R.J., and Marks, R.M. (1997). Dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate. Nature Medicine 3, 866- 871. 33b. Putnak, J.R., Kanesa-Thasan, N., and Innis, B.L. (1997). A putative cellular receptor for dengue viruses. Nature Medicine 3, 828-829. 34. Narindrasorasak, S., Lowery, D., Gonzalez-DeWhitt, P., Poorman, R.A., Greenberg, B., Kisilevsky, R. (1991). High affinity interactions between the Alzheimer's beta-amyloid precursor protein and the basement membrane form of theparan sulfate proteoglycan. J. Biol. Chem., 266, 12878-83. 35. Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (Lond.)., 362:801-809. 36. Zhong-Sheng, J., Walter, J., Brecht, R., Miranda, D., Mahmood Hussain, M., Innerarity, T.L. and Mahley, W.R. (1993). Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells. J. Biol. Chem., 268, 10160-10167. 37. Ernst, S., Langer, R., Cooney, Ch.L., and Sasisekharan, R. (1995). Enzymatic degradation of gly cosaminogly cans. Critical Reviews in Biochemistry and Molecular Biology, 30(5), 387-444. 38. Gospodarowicz, D., Mescher, AL., Birdwell, CR. (1977). Stimulation of corneal endothelial cell proliferation in vitro by fibroblast and epidermal growth factors. Exp Eye Res 25, 75-89. 39. Haimovitz-Friedman, A., Falcone, D.J., Eldor, A., Schirrmacher, v., Vlodavsky, I., and Fuks, Z. (1991) Activation of platelet heparitinase by tumor cell-derived factors. Blood, 78, 789-796. 39a. Savitsky, K., Platzer, M., Uziel, T., Gilad, S., Sartiel, A., Rosental, A., Elroy-Stein, O., Siloh, Y. and Rotman, G. (1997). 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Claims: WHAT IS CLAIMED IS: 1. A polynucleotide fragment comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. 2. The polynucleotide fragment of claim 1 , wherein said polynucleotide sequence includes nucleotides 63-1691 of SEQ ID NO:9, or nucleotides 139-1869 of SEQ ID NO: 13. 3. The polynucleotide fragment of claim 1 , wherein said polynucleotide sequence includes nucleotides 63-721 of SEQ ID NO:9. 4. The polynucleotide fragment of claim 1 , wherein said polynucleotide is as set forth in SEQ ID NOs:9 or 13. 5. The polynucleotide fragment of claim 1 , wherein said polynucleotide sequence includes a segment of SEQ ID NOs:9 or 13, said segment encodes said polypeptide having said heparanase catalytic activity. 6. The polynucleotide fragment of claim 1 , wherein said polypeptide includes an amino acid sequence as set forth in SEQ ID NOs: 10 or 14. 7. The polynucleotide fragment of claim 1 , wherein said polypeptide includes a segment of SEQ ID NOs: 10 or 14, said segment harbors said heparanase catalytic activity. 8. The polynucleotide fragment of claim 1 , wherein said polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. 9. A single stranded polynucleotide fragment comprising a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity. 10. The polynucleotide fragment of claim 9, wherein said polynucleotide sequence includes at least a portion of SEQ ID NOs:9 or 13. 1 1. A vector comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. 12. The vector of claim 1 1 , wherein said polynucleotide sequence includes nucleotides 63-1691 of SEQ ID NO:9, or nucleotides 139-1869 of SEQ ID NO: 13. 13. The vector of claim 1 1 , wherein said polynucleotide sequence includes nucleotides 63-721 of SEQ ID NO:9. 14. The vector of claim 1 1 , wherein said polynucleotide sequence is as set forth in SEQ ID NOs:9 or 13. 15. The vector of claim 1 1 , wherein said polynucleotide sequence includes a segment of SEQ ID NOs:9 or 13, said segment encodes said polypeptide having said heparanase catalytic activity. 16. The vector of claim 1 1 , wherein said polypeptide includes an amino acid sequence as set forth in SEQ ID NOs: 10 or 14. 17. The vector of claim 11 , wherein said polypeptide includes a segment of SEQ ID NOs: 10 or 14, said segment harbors said heparanase catalytic activity. 18. The vector of claim 11, wherein said polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. 19. A host cell comprising an exogenous polynucleotide fragment including a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. 20. The host cell of claim 19, wherein said polynucleotide sequence includes nucleotides 63- 1691 of SEQ ID NO:9, or nucleotides 139- 1869 of SEQ ID NO: 13. 21. The host cell of claim 19, wherein said polynucleotide sequence includes nucleotides 63-721 of SEQ ID NO:9. 22. The host cell of claim 19, wherein said polynucleotide sequence is as set forth in SEQ ID NOs:9 or 13. 23. The host cell of claim 19, wherein said polynucleotide sequence includes a segment of SEQ ID NOs:9 or 13, said segment encodes said polypeptide having said heparanase catalytic activity. 24. The host cell of claim 19, wherein said polypeptide includes an amino acid sequence as set forth in SEQ ID NOs: 10 or 14. 25. The host cell of claim 19, wherein said polypeptide includes a segment of SEQ ID NOs: 10 or 14, said segment harbors said heparanase catalytic activity. 26. The host cell of claim 19, wherein said polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. 27. A host cell expressing a recombinant heparanase. 28. A recombinant protein comprising a polypeptide having heparanase catalytic activity. 29. The recombinant protein of claim 28, wherein said polypeptide includes a segment of SEQ ID NOs: 10 or 14. 30. A polynucleotide fragment comprising a polynucleotide sequence capable of hybridizing with nucleotides 1-721 of SEQ ID NO:9. 31. A polynucleotide sequence as set forth in SEQ ID NOs: 9 or 13. 32. A polynucleotide sequence homologous to SEQ ID NOs:9 or 13. 33. An amino acid sequence as set forth in SEQ ID NOs: 10 or 14. 34. An amino acid sequence homologous to SEQ ID NOs: 10 or 14. 35. A pharmaceutical composition comprising as an active ingredient a recombinant protein having heparanase catalytic activity. 36. A heparanase overexpression system comprising a cell overexpressing heparanase catalytic activity. 37. A modulator of heparin-binding growth factors, cellular responses to heparin-binding growth factors and cytokines, cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and bacterial infections or disintegration of neurodegenerative plaques comprising as an active ingredient a recombinant protein having heparanase catalytic activity. 38. A medical equipment comprising a medical device containing, as an active ingredient, a recombinant protein having heparanase catalytic activity. 39. The vector of claim 1 1 , wherein said vector is a baculovirus vector. 40. The host cell of claim 19, wherein said cell is an insect cell. 41. The host cell of claim 27, wherein said cell is an insect cell. 42. A method of identifying a chromosome region harboring a human heparanase gene in a chromosome spread comprising the steps of: (a) hybridizing the chromosome spread with a tagged polynucleotyde probe encoding heparanase; (b) washing the chromosome spread, thereby removing excess of non- hybridized probe; and (c) searching for signals associated with said hybridized tagged polynucleotyde probe, wherein detected signals being indicative of a chromosome region harboring a human heparanase gene.

Inventors: FEINSTEIN ELENA, PECKER IRIS, VLODAVSKY ISRAEL, FEINSTEIN, ELENA, PECKER, IRIS, VLODAVSKY, ISRAEL
Applicants: FEINSTEIN ELENA, FRIEDMAN MARK M, HADASIT MED RES SERVICE, INSIGHT STRATEGY & MARKETING, PECKER IRIS, VLODAVSKY ISRAEL, FEINSTEIN, ELENA, FRIEDMAN, MARK, M., HADASIT MEDICAL RESEARCH SERVICES & DEVELOPMENT LTD., INSIGHT STRATEGY & MARKETING LTD., PECKER, IRIS, VLODAVSKY, ISRAEL
Application year: 1998
Main classification: C12N15
IPC codes: A61K38, A61K39, A61L27, A61P9, A61P17, A61P29, A61P35, A61P37, A61P43, C12N1, C12N5, C12N9, C12N15, C12Q1, G01N33