Processing

Please wait...

Settings

Settings

Goto Application

1. WO2002024863 - AVIAN AND REPTILE DERIVED POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

[ EN ]

AVIAN AND REPTILE DERIVED POLYNUCLEOTIDE ENCODING A
POLYPEPTIDE HAVING HEPARANASE ACTIVITY

FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to an avian and reptile derived polynucleotide encoding a polypeptide having heparanase catalytic activity. The present invention further relates to the use of the signal peptide of avian and/or reptile heparanase for expression of membrane associated and/or secreted proteins in heterologous expression systems.
Gly cos aminogly cans (GAGs)
GAGs are polymers of repeated disaccharide units consisting of uronic acid and a hexosamine. Biosynthesis of GAGs except hyaluronic acid is initiated from a core protein. Proteoglycans may contain several GAG side chains from similar or different families. GAGs are synthesized as homopolymers which may subsequently be modified by N-deacetylation and N-sulfation, followed by C5-epimerization of glucuronic acid to iduronic acid and O-sulfation. The chemical composition of GAGs from various tissues varies highly.
The natural metabolism of GAGs in animals is carried out by hydrolysis. Generally, the GAGs are degraded in a two step procedure. First the proteoglycans are internalized in endosomes, where initial depolymerization of the GAG chain takes place. This step is mainly hydrolytic and yields oligosaccharides. Further degradation is carried out following fusion with lysosome, where desulfation and exolytic depolymerization to monosaccharides take place (42).
The only GAG degrading endolytic enzymes characterized so far in animals are the hyaluronidases. The hyaluronidases are a family of 1-4 endoglucosaminidases that depolymerize hyaluronic acid and chondroitin sulfate. The cDNAs encoding sperm associated PH-20 (Hyal3), and the lysosomal hyaluronidases Hyal 1 and Hyal 2 were cloned and published (27).

These enzymes share an overall homology of 40 % and have different tissue specificities, cellular localizations and pH optima for activity.
Exolytic hydrolases are better characterized, among which are β-glucoronidase, α-L-iduronidase, and β-N-acetylglucosaminidase. In addition to hydrolysis of the glycosidic bond of the polysaccharide chain, GAG degradation involves desulfation, which is catalyzed by several lysosomal sulfatases such as N-acetylgalactosamine-4-sulfatase, iduronate-2-sulfatase and heparin sulfamidase. Deficiency in any of lysosomal GAG degrading enzymes results in a lysosomal storage disease, mucopolysaccharidosis.
Gly cosy I hydrolases
Glycosyl hydrolases are a widespread group of enzymes that hydrolyze the o-glycosidic bond between two or more carbohydrates or between a carbohydrate and a noncarbohydrate moiety. The enzymatic hydrolysis of glycosidic bond occurs by using major one or two mechanisms leading to overall retention or inversion of the anomeric configuration. In both mechanisms, catalysis involves two residues: a proton donor and a nucleophile. Glycosyl hydrolyses have been classified into 58 families based on amino acid similarities. The glycosyl hydrolyses from families 1, 2, 5, 10, 17, 30, 35, 39 and 42 act on a large variety of substrates, however, they all hydrolyze the glycosidic bond in a general acid catalysis mechanism, with retention of the anomeric configuration. The mechanism involves two glutamic acid residues, which are the proton donor and the nucleophile, with an aspargine which always precedes the proton donor. Analyses of a set of known 3D structures from this group of enzymes revealed that their catalytic domains, despite the low level of sequence identity, adopt a similar (α/β) 8 fold with the proton donor and the nucleophile located at the C-terminal ends of strands β4 and β7, respectively. Mutations in the functional conserved amino acids of lysosomal glycosyl hydrolases were identified in lysosomal storage diseases.

Lysosomal glycosyl hydrolases including β-glucuronidase, β-manosidase, β-glucocerebrosidase, β-galactosidase and α-L-iduronidase, are all exo-glycosyl hydrolases, belong to the GH-A clan and share a similar catalytic site. However, many endo-glucanases from various organisms, such as bacterial and fungal xylenases and cellulases share this catalytic domain (1).
Heparan sulfate proteoglycans (HSPGs)
HSPGs are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (3-7). The basic HSPG structure consists of a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and 0-linked sulfate moieties and N-linked acetyl groups (3-7). Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPGs in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair (3-7). The heparan sulfate (HS) chains, which are unique in their ability to bind a multitude of proteins, ensure that a wide variety of effector molecules cling to the cell surface (6-8). HSPGs are also prominent components of blood vessels (5). In large 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 HSPGs 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 HS may therefore result in disassembly of the subendothelial ECM and hence may play a decisive role in extravasation of normal and malignant blood-borne cells (9-11). 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
Heparanase is a glycosylated enzyme that is involved in the catabolism of certain glycosaminoglycans. It is an endoglucuronidase that cleaves heparan sulfate at specific intrachain sites (12-15). Interaction of T and B lymphocytes, platelets, granulocytes, macrophages and mast cells with the subendothelial extracellular matrix (ECM) is associated with degradation of heparan sulfate by. heparanase activity (16). Placenta heparanase acts as an adhesion molecule or as a degradative enzyme depending on the pH of the microenviromnent (17).
Heparanase is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophores, immune complexes, antigens and mitogens), suggesting its regulated involvement in inflammation and cellular immunity responses (16).
It was also demonstrated that heparanase can be readily released from human neutrophils by 60 minutes incubation at 4 °C in the absence of added stimuli (18).
Gelatinase, another ECM degrading enzyme, which is found in tertiary granules of human neutrophils with heparanase, is secreted from the neutrophils in response to phorbol 12-myristate 13-acetate (PMA) treatment (19-20).
In contrast, various tumor cells appear to express and secrete heparanase in a constitutive manner in correlation with their metastatic potential (21).
Degradation of heparan sulfate by heparanase results in the release of heparin-binding growth factors, enzymes and plasma proteins that are sequestered by heparan sulfate in basement membranes, extracellular matrices and cell surfaces (22-23).
Heparanase activity has been described in a number of cell types including cultured skin fibroblasts, human neutrophils, activated rat T-lymphocytes, normal and neoplastic murine B-lymphocytes, human monocytes and human umbilical vein endothelial cells, SK hepatoma cells, human placenta and human platelets.
A procedure for purification of natural heparanase was reported for SK hepatoma cells and human placenta (U.S. Pat. No. 5,362,641) and for human platelets derived enzymes (62).
Cloning and expression of the human heparanase gene
The human hpa cDNA, which encodes human heparanase, was cloned from human placenta. It contained an open reading frame, which encodes a polypeptide of 543 amino acids with a calculated molecular weight of 61,192 daltons (2). The cloning procedures are described in length in U.S. Pat. Application Nos. 08/922,170, 09/109,386, and 09/258,892, the latter is a continuation-in-part of PCT/US98/17954, filed August 31, 1998, all of which are incorporated herein by reference. An identical cDNA encoding human heparanase was isolated later on from hepatoma cell line SK-hepl (54). From platelets (55 ,57, PCT/US99/01489, PCT/AU98/00898) and from SV40 transformed fibroblasts (56, PCT/EP99/00777).
The genomic locus, which encodes heparanase, spans about 40 kb. It is composed of 12 exons separated by 11 introns and is localized on human chromosome 4.
The ability of the hpa gene product to catalyze degradation of heparan sulfate (HS) in vitro was examined by expressing the entire open reading frame of hpa in High five and Sf21 insect cells, and the mammalian human 293 embryonic kidney cell line expression systems. Extracts of infected or transfected cells were assayed for heparanase catalytic activity. For this purpose, cell lysates were incubated with sulfate labeled, ECM-derived HSPG (peak I), followed by gel filtration analysis (Sepharose 6B) of the reaction mixture. While the substrate alone consisted of high molecular weight material, incubation of the HSPG substrate with lysates of cells infected or transfected with hpa containing vectors resulted in a complete conversion of the high molecular weight substrate into low molecular weight labeled heparan sulfate degradation fragments (see, for example, U.S. Pat. Application No. 09/071,618, which is incorporated herein by reference.
In other experiments, it was demonstrated that the heparanase enzyme expressed by cells infected with a pFhpa virus is capable of degrading HS complexed to other macromolecular constituents (e.g., fibronectin, laminin, collagen) present in a naturally produced intact ECM (see U.S. Pat. Application

No. 09/109,386, which is incorporated herein by reference), in a manner similar to that reported for highly metastatic tumor cells or activated cells of the immune system (7, 8).
In human primary fibroblasts transfected with the heparanase cDNA the enzyme was localized to the lysosomes.
Preferential expression of the hpa gene in human breast and hepatocellular
carcinomas
Semi-quantitative RT-PCR was employed to evaluate the expression of the hpa gene by human breast carcinoma cell lines exhibiting different degrees of metastasis. A marked increase in hpa gene expression is observed which correlates to metastatic capacity of non-metastatic MCF-7 breast carcinoma, moderately metastatic MDA 231 and highly metastatic MDA 435 breast carcinoma cell lines. Significantly, the differential pattern of the hpa gene expression correlated with the pattern of heparanase activity.
Expression of the hpa gene in human breast carcinoma was demonstrated by in situ hybridization to archival paraffin embedded human breast tissue.

Hybridization of the heparanase antisense riboprobe to invasive duct carcinoma tissue sections resulted in a massive positive staining localized specifically to the carcinoma cells. The hpa gene was also expressed in areas adjacent to the carcinoma showing fibrocystic changes. Normal breast tissue derived from reduction mammoplasty failed to express the hpa transcript. High expression of the hpa gene was also observed in tissue sections derived from human hepatocellular carcinoma specimens but not in normal adult liver tissue. Furthermore, tissue specimens derived from adenocarcinoma of the ovary, squamous cell carcinoma of the cervix and colon adenocarcinoma exhibited strong staining with the hpa RNA probe, as compared to a very low staining of the hpa mRNA in the respective non-malignant control tissues (2).
A preferential expression of heparanase in human tumors versus the corresponding normal tissues was also noted by immunohistochemical staining of paraffin embedded sections with monoclonal anti-heparanase antibodies. Positive cytoplasmic staining was found in neoplastic cells of the colon carcinoma and in dysplastic epithelial cells of a tubulovillous adenoma found in the same specimen while there was little or no staining of the normal looking colon epithelium located away from the carcinoma. Of particular significance was an intense immunostaining of colon adenocarcinoma cells that had metastasized into lymph nodes, lung and liver, as compared to the surrounding normal tissues (58).
Latent and active forms of the heparanase protein
The apparent molecular size of the recombinant enzyme produced in the baculovirus expression system was about 65 kDa. This heparanase polypeptide contains 6 potential N-glycosylation sites. Following deglycosylation by treatment with peptide N-glycosidase, the protein appeared as a 57 kDa band. This molecular weight corresponds to the deduced molecular mass (61,192 daltons) of the 543 amino acid polypeptide encoded by the full length hpa cDNA after cleavage of the predicted 3 kDa signal peptide. No further reduction in the apparent size of the N-deglycosylated protein was observed following concurrent O-glycosidase and neuraminidase treatment. Deglycosylation had no detectable effect on enzymatic activity.
Unlike the baculovirus enzyme, expression of the full length heparanase polypeptide in mammalian cells (e.g., 293 kidney cells, CHO) yielded a major protein of about 50 kDa and a minor about 65 kDa protein in cell lysates. Comparison of the enzymatic activity of the two forms, using a semi-quantitative gel filtration assay, revealed that the 50 kDa enzyme is at least 100-fold more active than the 65 kDa form, which activity may be attributed to minute contamination by the 50 kDa protein in the analyzed samples. A similar difference was observed when the specific activity of the recombinant 65 kDa baculovirus enzyme was compared to that of the 50 kDa heparanase preparations purified from human platelets, SK-hep-1 cells, or placenta. These results suggest that the 50 kDa protein is a mature processed form of a latent heparanase precursor. Amino terminal sequencing of the platelet heparanase indicated that cleavage occurs between amino acids Gln1^ and Lysl58 As indicated by the hydropathic plot of heparanase, this site is located within a hydrophillic peak, which is likely to be exposed and hence accessible to proteases.
Involvement of Heparanase in Tumor Cell Invasion and Metastasis
Circulating tumor cells arrested in the capillary beds 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 base membrane (BM) (24). 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 (25). Among these enzymes is heparanase that cleaves HS at specific intrachain sites (16, 11). Expression of a HS degrading heparanase was found to correlate with the metastatic potential of mouse lymphoma (26), fibrosarcoma and melanoma (21) cells. Moreover, elevated levels of heparanase were detected in sera from metastatic tumor bearing animals and melanoma patients (21) and in tumor biopsies of cancer patients (12).
The inhibitory effect of various non-anticoagulant species of heparin on heparanase was examined in view of their potential use in preventing extravasation of blood-borne cells. Treatment of experimental animals with heparanase inhibitors markedly reduced (> 90 %) the incidence of lung metastases induced by B16 melanoma, Lewis lung carcinoma and mammary adenocarcinoma cells (12, 13, 28). 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 of the polysaccharide (12).
The direct role of heparanase in cancer metastasis was demonstrated by two experimental systems. The murine T-lymphoma cell line Eb has no detectable heparanase activity. Whether the introduction of the hpa gene into Eb cells would confer a metastatic behavior on these cells was investigated. To this purpose, Eb cells were transfected with a full length human hpa cDNA. Stable transfected cells showed high expression of the heparanase mRNA and enzyme activity. These hpa and mock transfected Eb cells were injected subcutaneously into DBA/2 mice and mice were tested for survival time and liver metastases. All mice (n=20) injected with mock transfected cells survived during the first 4 weeks of the experiment, while 50% mortality was observed in mice inoculated with Eb cells transfected with the hpa cDNA. The liver of mice inoculated with hpa transfected cells was infiltrated with numerous Eb lymphoma cells, as was evident both by macroscopic evaluation of the liver surface and microscopic examination of tissue sections. In contrast, metastatic lesions could not be detected by gross examination of the liver of mice inoculated with mock transfected control Eb cells. Few or no lymphoma cells were found to infiltrate the liver tissue. In a different model of tumor metastasis, transient transfection of the heparanase gene into low metastatic B16-F1 mouse melanoma cells followed by i.v. inoculation, resulted in a 4- to 5-fold increase in lung metastases.
Finally, heparanase externally adhered to B16-F1 melanoma cells increased the level of lung metastases in C57BL mice as compared to control mice (see U.S. Pat. Application No. 09/260,037 which is incorporated herein by reference).
Possible involvement of heparanase in tumor angiogenesis
Fibroblast growth factors are a family of structurally related polypeptides characterized by high affinity to heparin (29). They are highly mitogenic for vascular endothelial cells and are among the most potent inducers of neovascularization (29-30). Basic fibroblast growth factor (bFGF) has been extracted from a subendothelial ECM produced in vitro (31) and from basement membranes of the cornea (32), 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 (23). 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 (33, 32, 34). 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 (35), 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 (36, 37). 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 (38, 39). Moreover, the size of HS required for optimal effect was similar to that of HS fragments released by heparanase (40). Similar results were obtained with vascular endothelial cells growth factor (VEGF) (41), 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 (36,37).
The involvement of heparanase in other physiological processes and its
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; cellular responses to heparin-binding growth factors (e.g., bFGF, VEGF) and cytokines (IL-8) (44, 41); cell interaction with plasma lipoproteins (49); cellular susceptibility to certain viral and some bacterial and protozoa infections (45-47); and disintegration of amyloid plaques (48).
Viral Infection: The presence of heparan sulfate on cell surfaces have been shown to be the principal requirement for the binding of Herpes Simplex (45) and Dengue (46) 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 (45). There are some indications that the cell surface heparan sulfate is also involved in HIV infection (47).
Neurodegenerative diseases: Heparan sulfate proteoglycans were identified in the prion protein amyloid plaques of Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape (48). 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 (50). Apart from its involvement in SMC proliferation as a low affinity receptor for heparin-binding growth factors, HS is also involved in lipoprotein binding, retention and uptake (51). 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 (49). The latter pathway is expected to be highly atherogenic by promoting accumulation of apoB and apoE rich lipoproteins (e.g., LDL, VLDL, chylomicrons), independent of feed back inhibition by the cellular cholesterol 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.
Pulmonary diseases: The data obtained from the literature suggests a possible role for GAGs degrading enzymes, such as, but not limited to, heparanases, connective tissue activating peptide, heparinases, hyluronidases, sulfatases and chondroitinases, in reducing the viscosity of sinuses and airway secretions with associated implications on curtailing the rate of infection and inflammation. The sputum from CF patients contains at least 3 % GAGs, thus contributing to its volume and viscous properties. We have shown that heparanase reduces the viscosity of sputum of Cystic fibrosis (CF) patients (U.S. Pat. application No. 09/046,475). Recombinant heparanase has been shown to reduce viscosity of sputum of CF patients (see, U.S. Pat. application No. 09/046,475).
In summary, 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.
There is thus a widely recognized need for, and it would be highly advantageous to have, additional molecules with glycosyl hydrolase activity, because such molecules may exhibit greater specific activity toward certain substrates or different substrate specificity than the known heparanase.

SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided An isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence which (a) encodes a polypeptide which is at least 75 % similar to SEQ ID NO:4 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 8 and length weight equals 2, average match equals 2.912 and average mismatch equals -2.003; (b) is at lest 65 % identical to SEQ ID NO: 10 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9; (c) is as set forth in SEQ ID NO: 10 or a portion thereof; and/or (d) is hybridizable with SEQ ID NO: 10 or a portion thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10^ cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 65 °C.
According to a preferred embodiment of the present invention the polynucleotide encodes a polypeptide which has heparanase catalytic activity or which is cleavable by a protease so as to have the heparanase catalytic activity.

According to another aspect of the present mvention there is provided a nucleic acid construct comprising any of the polynucleotides of the present invention in a sense or antisense orientation with respect to expression regulatory sequences of the construct.
According to yet another aspect of the present invention there is provided a cell transformed or transfected with polynucleotides or constructs of the present invention.
According to still another aspect of the present invention there is provided an oligonucleotide of at least 17 bases specifically hybridizable with the isolated nucleic acid described herein and which is not hybridizable with any mammalian heparanase cDNA.
According to an additional aspect of the present invention there is provided a pair of oligonucleotides each of at least 17 bases specifically hybridizable with the isolated nucleic acid described herein in an opposite orientation so as to direct exponential amplification of a portion thereof in a nucleic acid amplification reaction, and which are not hybridizable with any mammalian heparanase cDNA..
According to yet an additional aspect of the present invention there is provided a nucleic acid amplification product obtained using the pair of primers described herein.
According to yet a further aspect of the present invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a signal peptide of avian or reptile heparanase and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide.
According to still a further aspect of the present invention there is provided a nucleic acid construct comprising a first polynucleotide encoding an avian or reptile heparanase signal peptide, e.g., a peptide as set forth at positions

1 to 19 of SEQ ID NO:4, and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide.
Preferably, the targeted or secreted polypeptide is human heparanase.

According to still an additional aspect of the present invention there is provided a method of expressing a protein of interest in a cell, the method comprising transforming the cell with a nucleic acid construct that comprises a first polynucleotide encoding a signal peptide of avian or reptile heparanase and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide; and culturing the cell under suitable growth conditions.
As used herein the term "transforming" refers to any and all methods of permanent or transient introduction of foreign nucleic acids into cells, such as for example, plasmid transfoπnation, phage infection, gene knock-in and the like.
According to yet an additional aspect of the present invention there is provided a recombinant protein comprising a polypeptide (a) which is at least 75 % similar to SEQ ID NO:4 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 8 and length weight equals 2, average match equals 2.912 and average mismatch equals -2.003; (b) encoded by a nucleic acid including a genomic, complementary or composite polynucleotide sequence being at lest 65 % identical to SEQ ID NO: 10 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9; (c) encoded by a nucleic acid as set forth in SEQ ID NO: 10 or a portion thereof; and/or encoded by a nucleic acid including a genomic, complementary or composite polynucleotide sequence being hybridizable with SEQ ID NO: 10 or a portion thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 3 p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 65 °C.
According to further features in preferred embodiments of the invention described below, the polypeptide has heparanase catalytic activity or the polypeptide is cleavable by a protease so as to have the heparanase catalytic activity.
According to a further aspect of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, the recombinant protein of described herein and a pharmaceutically acceptable carrier.
The present invention successfully addresses the shortcomings of the presently known configurations by providing novel polynucleotides which encode novel polypeptides having heparanase catalytic activity and which can be used to intervene with pathologies associated with impaired 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, all as is further delineated in the background section above.

BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.
In the drawings:
Figure la presents an alignment of the amino acid sequences of, mouse (SEQ ID NO: 1), rat (SEQ ID NO:2), human (SEQ ID NO:3) and chicken (SEQ ID NO:4) heparanases. The amino acid sequences were determined by sequence analysis of the isolated cDNAs. The amino acids, which are identical in heparanases from the four organisms, are marked with an asterisk, conserved differences are marked by double or single dots. The putative two catalytic residues, the proton donor and the nucleophile are bolded. The signal peptide of human heparanase and the putative signal peptides of chicken, mouse and rat heparanases are underlined. The cleavage site of the 50 kDa mature protein and the borders of the associated 8 kDa peptide are pointed by arrows. Multiple alignment was generated by ClustalW.
Figure lb presents the chicken heparanase coding sequence (SEQ ID

NO: 10) and its translation product (SEQ ID NO:4).
Figure 2 shows western analysis of heparanase secreted by Eb lymphoma cells transfected with chicken heparanase cDNA (Chk-hpa) and human heparanase cDNA (Eum-hpa). Heparanase was partially purified (SP-Sepharose) from serum free medium conditioned by Eb lymphoma cells transfected with Chk-hpa (lane 1), Jrlum-hpa (lane 2), or plasmid alone (lane 3). Protein samples were subjected to 10 % SDS/PAGE and western blot analysis applying polyclonal rabbit anti-heparanase antibodies and ECL visualization. Protein bands correspond to the 58 kDa and 45 kDa forms of the chicken enzyme vs. the 65 kDa and 50 kDa latent and active human heparanase forms.
Figures 3a-c demonstrate heparanase activity in cell lysates, intact cells and medium conditioned by Eb cells transfected with chicken vs. human heparanases. Eb mouse lymphoma cells transfected with Chk-hpa (■ ), ϊlum-hpa (A) and control vector (+) were maintained (24 h, 2 x 106 cells/ml) in serum free RPMI medium. Intact cells (3 a), conditioned media (3b) and lysates (3 c) of 2 x lθ6 cells were then tested for heparanase activity. For this purpose, 1 ml conditioned medium and 2x10^ intact or lysed cells were incubated (24 h, 37 °C, pH 6.2) in serum free medium with sulfate labeled ECM. Labeled degradation fragments released into the incubation medium were analyzed by gel filtration on Sepharose 6B. Nearly intact heparan sulfate proteoglycans elute next to VQ (peak I, fractions 1-10) whereas heparan sulfate degradation products elute toward the Vf of the column (peak II, fractions 15-35). A much higher heparanase activity was expressed by intact lymphoma cells (3 a) and even more was secreted into the conditioned medium (3b) of cells transfected with the Chk-hpa as compared to cells transfected with the Hum-hpa. In contrast, there was no difference in heparanase activity found in the corresponding cell lysates (3c).
Figure 4 presents a comparison of heparanase activities of partially purified chicken and human heparanases. Chicken and human heparanases were partially purified (SP Sepharose) from serum free medium conditioned by stable hpa transfected Eb lymphoma cells. Equal amounts (60 ng/ml) of partially purified chicken (■ ) and human (*) heparanases were incubated (24 h, 37 °C, pH 6.2) with sulfate labeled ECM. Labeled degradation fragments released into the incubation medium were analyzed by gel filtration on Sepharose 6B. Both enzymes exhibit a similar apparent specific activity, as indicated by an almost identical elution pattern of HS degradation products.

Figures 5a-d demonstrates the cellular localization of chicken (Chk-hpa) and chimeric (chimeric- zpα) heparanases vs. human (H-hpa) heparanase. C6 rat glioma cells were transfected with chicken (a), human (c), or chimeric (b) heparanase cDNAs. Pooled populations of stable transfected cells were subjected to indirect immunofluorescence staining with monoclonal anti-heparanase antibodies (mAb 130) followed by Cy-3 conjugated goat anti-mouse antibody, as described in the Examples section that follows. Mock transfected C6 glioma cells (d) were used as control and showed no staining. Chk-hpa (a) and chimeric-/z/r>α (c) transfected cells exhibited intense staining associated mostly with the cell membrane (arrow), while cells transfected with Η-hpa cDNA (b) displayed primarily a peri-nuclear granular staining (arrow). Bar = 10 μM.

Figure 6 presents the comparison between heparanases of human, chicken, mouse and rat. Percents of identity between the coding nucleotide sequences appear in the upper block. Percents of identity and similarity between the amino acid sequences appear in the lower right and lower left blocks, respectively. The nucleotide sequence was determined for each one of the four species and the amino acid sequence was deduced from the cDNA sequence.
Figure 7 demonstrates the secretion of chicken and chimeric heparanases. Eb mouse lymphoma cells were stable transfected with Chk-hpa (■), H-hpa (? ), or chmiQήc-hpa (? ). Serum free medium conditioned by these cells was incubated (24 h, 37 °C, pH 6.2) with sulfate labeled ECM and tested for heparanase activity. Mock transfected Eb lymphoma cells (O) were used as control.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is of an avian or reptile derived polynucleotide encoding a polypeptide having heparanase catalytic activity which can be used in a variety of medical applications. Specifically, the present invention can be used to intervene with pathologies associated with impaired 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, all as is further delineated in the background section above. The present invention is further of chimeric nucleic acids encoding, in frame, the signal peptide sequence of avian or reptile heparanase and a protein of interest, such as human heparanase.
The principles, operation and uses of the present invention may be better understood with reference to the drawings and accompanying descriptions.
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 set forth in the following description or illustrated in the examples. 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.
In an attempt to isolate an avian heparanase encoding polynucleotide, cDNA libraries and reverse transcribed mRNA from avian tissue were screened via hybridization and polymerase chain reaction techniques using mammalian derived probes and oligonucleotides, yet with no successes even under the mildest hybridization conditions. The human heparanase amino acid sequence was thereafter used to screen EST databases for homology to a chicken unidentified mRNA sequences. Following extensive screening, a single related chicken EST was identified which shared 60.5 % homology with the 276 bp at the 3' end of the human heparanase coding sequence. This sequence encoded a truncated open reading frame of 91 amino acids, 74 % homologous to human heparanase, followed by 325 nucleotides 3' untranslated region (UTR). The heparanase homologous sequence was derived from chicken activated T cells cDNA, clone pat.pk0039.c8.f 5', mRNA sequence, accession No. AI980994.
In order to isolate a full length clone and to test whether it is indeed heparanase, chicken kidney mRNA was subjected to 5' RACE (rapid amplification of cDNA ends). The gene specific PCR primers were designed according to the EST described above. A DNA fragment of approximately 1,600 bp was obtained, partially overlapping with the identified 3' encoding EST clone. The entire cDNA cloned, in pGEM-T Easy Vector, was designated Chk-hpa. The complete cDNA (Chk-hpa) is 1,609 bp long (SEQ ID NO: 10), and it contains an open reading frame that encodes a polypeptide of 523 amino acids (SEQ ID NO:4) with a calculated molecular weight of 58,842 Daltons. Analysis of the amino acid sequence and of the hydropathic profile of the protein indicates a hydrophobic amino acid tail at the N-terminus. A signal peptide is predicted to span the N-terminal 19 amino acids.

The overall homology between the chicken and the human heparanase coding sequences is 62 %. The similarity between the chicken and the human heparanases is 69 % (of which 61 % amino acid sequence identity). The heparanase is synthesized as a latent, 65 kDa precursor and is then processed to an active mature 50 kDa form. Based on the homology to human heparanase the chicken heparanase is cleaved between Trpl36 nd Lys^. According to Fairbank et al. the precursor is cleaved at three sites to form a heterodimer of a 50 kDa polypeptide (the mature form) that is associated with a 8 kDa peptide. The putative chicken 8 kDa peptide spans amino acids Glu^O to Glu94. The mature heparanases of various organisms share high homology while the pro-peptides are relatively diverse.
Structure prediction of human heparanase suggests a (α/β)8 Tim barrel fold typical of family 10 glycosyl hydrolases. According to this prediction the active site involves two glutamic acid residues, which are the proton donor and the nucleophile, with an aspargine always preceding the proton donor. The proton donor in human heparanase is Glu^25 ancι the nucleophile is Glu343 The conservation of the amino acid sequence flanking these residues supports the identification of the active site. Based on the homology the proton donor of chicken heparanase is Glu204 an^ the nucleophile is Glu323.
Chicken heparanase is slightly more similar to human heparanase than to mouse and rat heparanases. As expected, the homology among mammals is far higher than that of mammals with chicken.
The ability of the Chk-Hpa product to catalyze degradation of heparan sulfate (HS) in vitro was determined by expressing the entire open reading frame of Chk-hpα in mammalian cells lacking heparanase activity. Expression of heparanase in the transfected cells was confirmed by RT-PCR. Chicken heparanase transcript was detected only in Chk-hpα transfected cells.
Heparanase activity was assayed in cells transfected with Chk-hpa as compared to mock transfected cells. High activity was observed in intact cells, conditioned media and cell lysates of Chk-hpa transfectants while no activity was observed in the mock transfected cells.
The heparan sulfate degradation activity of Eb cells transfected with Chk-hpa cDNA was compared with that of Eb cells transfected with human hpa (Hum-hpά). The activity of chicken heparanase was higher than that of human heparanase in intact cells and in conditioned media, however, in cell extracts the activity of the two enzymes was similar. This suggests that the chicken heparanase is unexpectedly preferentially secreted as is compared to the mammalian enzyme.
In order to compare the activity of the chicken and human heparanases, the enzymes were partially purified from conditioned media of Chk-hpa and Hum- zpα transfected cells. Western blot analysis of the partially purified chicken heparanase showed a major protein of 58 kDa, which corresponds to the heparanase precursor and a minor protein of 45 kDa, which corresponds to the mature form. The human heparanase fraction contained the equivalent human 65 kDa heparanase precursor and 50 kDa mature forms. The difference in molecular weight between chicken and human heparanases is mainly due to a different glycosylation pattern.
Activity of the two enzymes was compared using equal amounts of the partially purified enzymes in a semi-quantitative assay. The specific activity of chicken and human heparanases was found to be similar.
Cells transfected with Chk-hpa exhibited intense staining associated with the cell membrane while only a weak signal was observed in the cytoplasm. In contrast, in cells transfected with Kum-hpa heparanase is localized to peri-nuclear vesicles. A similar pattern was observed in transiently transfected human primary fibroblasts, where chicken heparanase was associated with cell membrane while human heparanase was localized to peri-nuclear vesicles, which were identified as lysosomes. The signal peptides of the chicken and human heparanases share no significant homology. It appears that the signal peptide of chicken heparanase unexpectedly targets the enzyme to the cell surface of mammalian cells while the signal peptide of human heparanase targets the enzyme to lysosomes. Indeed, replacing the signal peptide of human heparanase with that of chicken heparanase resulted in improved secretion and membrane localization of the human heparanase.
Thus, according to one aspect of the present invention there is provided

An isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence which (a) encodes a polypeptide which is at least 75 % similar to SEQ ID NO:4 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 8 and length weight equals 2, average match equals 2.912 and average mismatch equals -2.003; (b) is at lest 65 % identical to SEQ ID NO: 10 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9; (c) is as set forth in SEQ ID NO: 10 or a portion thereof; and or (d) is hybridizable with SEQ ID NO: 10 or a portion thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 10^ cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 65 °C. Under these hybridization conditions the chicken heparanase cDNA fails to hybridize with any mammalian heparanase.
The phrase "composite polynucleotide sequence" refers to a sequence which includes exonal sequences required to encode the polypeptide having heparanase activity, as well as any number of intronal sequences. The intronal sequences can be of any source and typically will include conserved splicing signal sequences. Such intronal sequences may further include cis acting expression regulatory elements.
Thus, this aspect of the present invention encompasses (i) polynucleotides as set forth in SEQ ID NO: 10; (ii) fragments thereof; (iii) sequences hybridizable therewith; (iv) sequences homologous thereto, such as reptile derived sequences; (v) sequences encoding similar polypeptides with different codon usage; (vi) altered sequences characterized by mutations, such as deletion, insertion or substitution of one or more nucleotides, either naturally occurring or man induced, either randomly or in a targeted fashion.
It will be appreciated in this respect that avian and reptiles are evolutionary closely related. As such, using the polynucleotides described herein, one of ordinary skills in the art, would be motivated and readily capable of screening a reptile cDNA library or use other methods routinely employed to isolate related genes from closely related species, to thereby clone full length cDNAs or genomic DNAs from any avian or reptile.
The heparanase sequence described herein can be used to study the catalytic mechanism of heparanase. Carefully selected site directed mutagenesis can be employed to provide modified heparanase proteins having modified characteristics in terms of, for example, substrate specificity, sensitivity to inhibitors, etc.
According to a preferred embodiment of the present invention the polynucleotide encodes a polypeptide which has heparanase catalytic activity or which is cleavable by a protease so as to have the heparanase catalytic activity. Removal of a 19 amino acid long signal peptide of chicken heparanase is demonstrated in the Examples section that follows.
The term "heparanase catalytic activity" or its equivalent term "heparanase activity" both refer to a mammalian endoglycosidase hydrolyzing activity which is specific for heparin 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).
According to another aspect of the present invention there is provided a nucleic acid construct comprising any of the polynucleotides of the present invention in a sense or antisense orientation with respect to expression regulatory sequences of the construct.

According to a preferred embodiment the nucleic acid construct according to this aspect of the present invention includes a promoter for regulating the expression of the isolated nucleic acid in a sense or antisense orientation. Such promoters are known to be cz-y-acting sequence elements required for transcription as they serve to bind DNA dependent RNA polymerase which transcribes sequences present downstream thereof. Such down stream sequences can be in either one of two possible orientations to result in the transcription of sense RNA which is translatable by the ribozyme machinery or antisense RNA which typically does not contain translatable sequences, yet can duplex or triplex with endogenous sequences, either mRNA or chromosomal DNA and hamper gene expression, all as further detailed hereinunder.
While the isolated nucleic acid described herein is an essential element of the invention, it is modular and can be used in different contexts. The promoter of choice that is used in conjunction with this invention is of secondary importance, and will comprise any suitable promoter. It will be appreciated by one skilled in the art, however, that it is necessary to make sure that the transcription start site(s) will be located upstream of an open reading frame. In a preferred embodiment of the present invention, the promoter that is selected comprises an element that is active in the particular host cells of interest. These elements may be selected from transcriptional regulators that activate the transcription of genes essential for the survival of these cells in conditions of stress or starvation, including the heat shock proteins.
A construct according to the present invention preferably further includes an appropriate selectable marker. In a more preferred embodiment according to the present invention the construct further includes an origin of replication. In another most preferred embodiment according to the present invention the construct is a shuttle vector, which can propagate both in E. coli (wherein the construct comprises an appropriate selectable marker and origin of replication) and be compatible for propagation in cells, or integration in the genome, of an organism of choice. The construct according to this aspect of the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
The polynucleotide encoding heparanase can be permanently or transiently present in the cell. In other words, genetically modified cells obtained following stable or transient transfection, transformation or transduction are all within the scope of the present invention. The polynucleotide can be present in the cell in low copy (say 1-5 copies) or high copy number (say 5-50 copies or more). It may be integrated in one or more chromosomes at any location or be present as an extrachromosomal material.
Alternatively, the nucleic acid construct according to this aspect of the present invention further includes a positive and a negative selection markers and may therefore be employed for selecting for homologous recombination events, including, but not limited to, homologous recombination employed in knock- in and knock-out procedures. One ordinarily skilled in the art can readily design a knock-out or knock-in constructs including both positive and negative selection genes for efficiently selecting transfected embryonic stem cells that underwent a homologous recombination event with the construct. Such cells can be introduced into developing embryos to generate chimeras, the offspring thereof can be tested for carrying the knock-out or knock-in constructs. Additional detail can be found in Fukushige, S. and Ikeda, J.E.: Trapping of mammalian promoters by Cre-lox site-specific recombination. DNA Res 3 (1996) 73-80; Bedell, M.A., Jenkins, N.A. and Copeland, N.G.: Mouse models of human disease. Part I: Techniques and resources for genetic analysis in mice. Genes and Development 11 (1997) 1-11; Beπningham, J.J., Scherer, S.S., O'Connell, S., Arroyo, E., Kalla, K.A., Powell, F.L. and Rosenfeld, M.G.: Tst-l/Oct-6/SCIP regulates a unique step in peripheral myelination and is required for normal respiration. Genes Dev 10 (1996) 1751-62, which are incorporated herein by reference.

According to yet another aspect of the present invention there is provided a cell transformed or transfected with polynucleotides or constructs of the present invention. The cell according to this aspect of the present invention can be a eukaryote cell of a multicellular organism, such as, but not limited to, a mammalian, avian, reptile or insect cell, a eukaryote cell of a unicellular organism, such as yeast or a prokaryote cell, such as a bacteria cell, e.g., an E. coli cell. Methods of transforming and transfecting each of these cells are well known in the art. Such procedures are detailed in many experimental procedure text books such as "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds).
The present invention is further directed at providing a heparanase over-expression system which includes a cell overexpressing heparanase catalytic activity. The cell may be a genetically modified 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 over-expression 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 over-expression from the endogenous gene.
The term "over-expression" 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.

According to still another aspect of the present invention there is provided an oligonucleotide of at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40 bases specifically hybridizable with the isolated nucleic acid described herein and which is not hybridizable with any mammalian heparanase cDNA.
Hybridization of shorter nucleic acids (below 200 bp in length, e.g. 17-40 bp in length) is effected by stringent, moderate or mild hybridization, wherein stringent hybridization is effected by a hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 1 - 1.5 °C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm; moderate hybridization is effected by a hybridization solution of 6 x SSC and 0.1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 2 - 2.5 °C below the Tm, final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS at 1 - 1.5 °C below the Tm, final wash solution of 6 x SSC, and final wash at 22 °C; whereas mild hybridization is effected by a hybridization solution of 6 x SSC and 1 % SDS or 3 M TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5 % SDS, 100 μg/ml denatured salmon sperm DNA and 0.1 % nonfat dried milk, hybridization temperature of 37 °C, final wash solution of 6 x SSC and final wash at 22 °C.
According to an additional aspect of the present invention there is provided a pair of oligonucleotides each of at least 17, at least 18, at least 19, at least 20, at least 22, at least 25, at least 30 or at least 40 bases specifically hybridizable with the isolated nucleic acid described herein in an opposite orientation so as to direct exponential amplification of a portion thereof in a nucleic acid amplification reaction, such as a polymerase chain reaction, and which are not hybridizable with any mammalian heparanase cDNA.
The polymerase chain reaction and other nucleic acid amplification reactions are well known in the art and require no further description herein. The pair of oligonucleotides according to this aspect of the present invention are preferably selected to have compatible melting temperatures (Tm), e.g., melting temperatures which differ by less than that 7 °C, preferably less than 5 °C, more preferably less than 4 °C, most preferably less than 3 °C, ideally between 3 ° C and zero °C.
Consequently, according to yet an additional aspect of the present invention there is provided a nucleic acid amplification product obtained using the pair of primers described herein. Such a nucleic acid amplification product can be isolated by gel electrophoresis or any other size based separation technique. Alternatively, such a nucleic acid amplification product can be isolated by affinity separation, either stranded affinity or sequence affinity. In addition, once isolated, such a product can be further genetically manipulated by restriction, ligation and the like.
According to yet a further aspect of the present invention there is provided a nucleic acid construct comprising a first polynucleotide encoding a signal peptide of chicken and/or avian heparanase, such as the peptide set forth at positions 1 to 19 of SEQ ID NO:4, and an in frame, second polynucleotide encoding a membrane targeted or secreted polypeptide. The chimeric polypeptide resulting from the expression of the open reading frame of this construct will be preferentially directed to the cell membrane or secreted outside the cell, depending on the nature of the polypeptide. Any polypeptide can be fused to the signal peptide of the invention, including, but not limited to, any enzyme, e.g., human heparanase, hormone, receptor, immunoglobulm, structural protein and the like. Cells transformed with a chimeric construct as herein described are grown under suitable culturing conditions and the protein of interest (the polypeptide) is extracted therefrom or from the growth medium to which it is secreted.
According to still an additional aspect of the present invention there is provided a recombinant protein comprising a polypeptide (a) which is at least 75 % similar to SEQ ID NO:4 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 8 and length weight equals 2, average match equals 2.912 and average mismatch equals -2.003; (b) encoded by a nucleic acid including a genomic, complementary or composite polynucleotide sequence being at lest 65 % identical to SEQ ID NO: 10 or a portion thereof as determined using the BestFit software of the Wisconsin sequence analysis package, utilizing the Smith and Waterman algorithm, where gap weight equals 50, length weight equals 3, average match equals 10 and average mismatch equals -9; (c) encoded by a nucleic acid as set forth in SEQ ID NO: 10 or a portion thereof; and/or encoded by a nucleic acid including a genomic, complementary or composite polynucleotide sequence being hybridizable with SEQ ID NO: 10 or a portion thereof under hybridization conditions of hybridization solution containing 10 % dextrane sulfate, 1 M NaCl, 1 % SDS and 5 x 106 cpm 32p labeled probe, at 65 °C, with a final wash solution of 1 x SSC and 0.1 % SDS and final wash at 65 °C.
Thus, this aspect of the present invention encompasses (i) a polypeptide as set forth in SEQ ID NO:4; (ii) fragments thereof; (iii) polypeptides similar (identical + homologous acids) thereto; and (iv) altered polypeptides characterized by mutations, such as deletion, insertion or substitution of one or more amino acids, either naturally occurring or man induced, either randomly or in a targeted fashion.
According to further features in preferred embodiments of the invention described below, the polypeptide has heparanase catalytic activity or the polypeptide is cleavable by a protease so as to have the heparanase catalytic activity.

The recombinant protein of the present invention 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 of the genetically modified cells described above, which include any of the expression nucleic acid constructs described herein. 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.
According to a further aspect of the present invention there is provided a pharmaceutical composition comprising, as an active ingredient, the recombinant protein of described herein and a pharmaceutically acceptable carrier.
The heparanase according to the present invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
As used herein a "pharmaceutical composition" refers to active or activatable heparanase, with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to an organism.
Herein the term "active ingredient" refers to active or activatable heparanase accountable for a biological effect.
Hereinafter, the terms "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the active ingredient.
Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of the active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
Techniques for formulation and administration of active ingredients may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.
Suitable routes of administration may, for example, include oral, rectal, transmucosal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intravenfricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
Alternately, one may administer the active ingredient in a local rather than systemic manner, for example, via injection of the active ingredient directly into a solid tumor often in a depot or slow release formulation, such as described below.
Furthermore, one may administer the active ingredient in a targeted drug delivery system, for example, in a liposome coated with a tumor specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.
Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredient into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredient of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
For administration by inhalation, the active ingredient for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the active ingredient and a suitable powder base such as lactose or starch.
The active ingredient described herein may be formulated for parenteral administration, e.g., by bolus injection or continuos infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredient may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredient to allow for the preparation of highly concentrated solutions.
Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The active ingredient of the present invention may also be formulated for local administration, such as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the preparation may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives such as sparingly soluble salts. Formulations for topical administration may include, but are not limited to, lotions, suspensions, ointments gels, creams, drops, liquids, sprays emulsions and powders.
The pharmaceutical compositions herein described may also comprise suitable solid of gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin and polymers such as polyethylene glycols.
Pharmaceutical compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount the active ingredient effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
The therapeutically effective amount or dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
. Toxicity and therapeutic efficacy of the active ingredient described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 and the LD50 (lethal dose causing death in 50 % of the tested animals) for the active ingredient. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.i).

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

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 and directed 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 from avian 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 (e.g., IL-8), cell interaction with plasma lipoproteins, cellular susceptibility to viral, protozoa and some bacterial infections, and disintegration of neurodegenerative plaques. Recombinant heparanase offers 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.
The present invention successfully addresses the shortcomings of the presently known configurations by providing novel polynucleotides which encode novel polypeptides having heparanase catalytic activity and which can be used to intervene with pathological procedures associated with impaired 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, all as is further delineated in the background section above.
Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES
Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course

Manual" CSHL Press (1996); all of which are incorpotaed by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND EXPERIMENTAL PROCEDURES
Cells: The methylcholanthrene-induced non-metastatic Eb (L5178Y) T-lymphoma cells (clone 737) were provided by V. Schirrmacher (DKFZ, Heidelberg, Germany). The cells were routinely transplanted as ascites tumors in syngeneic female DBA2/J mice. Alternatively, they were grown in RPMI 1640 (Life Technologies) supplemented with β-mercaptoethanol (5 x 10"5 M) and 10 % FCS. C6 rat Glioma cells were obtained from Dr. E. Keshet (Hadassah Medical School Jerusalem Israel). Cells were cultured in DMEM (4.5 g glucose/liter) containing 10 % fetal calf serum. Cells were dissociated with a solution of 0.05 % trypsin, 0.02 % EDTA, 0.01 M sodium phosphate, pH 7.4 and were subcultured at a 'split' ratio of 1 : 10.
Heparanase activity: Cell lysates, intact cells, conditioned media and serum free conditioned media were incubated 24 hours at 37 °C, pH 6.2-6.6, with S-labeled ECM in the presence of 20 mM phosphate buffer (pH 6.2). The incubation medium was centrifuged and the supernatant was analyzed by gel filtration on a Sepharose CL-6B column (0.9 x 30 cm). Fractions (0.2 ml) were eluted with PBS and their radioactivity was measured. Nearly intact HSPGs was eluted next to just after the V0 (Kav < 0.2, peak I, fractions 1-10) whereas degradation fragments of HS side chains were eluted from Sepharose 6B at 0.5 < Kav < 0.8 (peak II, fractions 15-35).
Cloning of Chk-hpa cDNA: The amino acid sequence of human heparanase was used to screen EST databases for homology to a chicken unidentified mRNA sequences. Following extensive searches, a single chicken derived EST suspected as heparanase related was identified, which shared only 60.5 % sequence homology with a 276 bp nucleotide stretch at the 3' end of the human heparanase coding sequence. The full-length chicken heparanase cDNA was isolated from chicken kidney mRNA. To this end, mRNA was isolated from fresh chicken kidney using PlyATtract mRNA Isolation System III (Promega, USA). The method for amplification of 5' ends was developed according to the principle of the 5' RACE (rapid amplification of cDNA ends) System of GibcoBRL. Chicken kidney mRNA was reverse transcribed (RT) using Superscript II (Gibco BRL) and oligo dT(15) (SEQ ID NO:5) as a primer. Following RT the cDNA was extended by 3' C-tailing using terminal deoxynucleotidyl transferase (TdT) (Promega). PCR amplification used Expand High Fidelity enzyme (Boehringer). The primers used for amplification were: First step: 5' primer, complementary to the C tail: API 5'-GGCCACGCGTCGACTAGTACGGGI IGGGIIGGGI IG-3' SEQ ID NO: 6 and the 3' gene specific primer ChkLl: 5'-GACTCCTCAAGCATTCCCTCAG-3' (SEQ ID NO:7). Second cycle: 5' nested primer nested AP2: 5'-GGCCACGCGTCGACTAGTACG-3' (SEQ ID NO:8) and a nested, gene specific 3' primer ChkL2 5'-AGCCCTGTTACTCTGCGTGCTC-3' (SEQ ID NO:9). The gene specific primers ChkLl and ChkL2 were selected according to the sequence of the EST.
PCR program of both first and second cycles was as follows: 94 °C 3 minutes, followed by 30 cycles of: 94 °C 30 seconds, 64 °C 1 minute and 72 °C 3 minutes, and finally 72 °C, 7 minutes.
The resulting 1.8-kb PCR product was cloned into the pGEM-T Easy Vector (Promega, USA).
DNA Sequencing: Sequence determination used vector-specific and gene-specific primers, with an automated DNA sequencer (ABI Prism™ model 310 Genetic Analyzer). Each nucleotide was read from at least two independent primers, and from several clones.

Computer analysis of sequence: Database searches for sequence similarities were performed using the NCBI 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. Multiple alignment was generated by ClustalW.
Immunostaining: Cells were seeded on round cover slips in 4 well plates for 24 hours. Cell were then washed with PBS, fixed with 100 % chilled (-20 °C) MetOH for 3 minutes. Following fixation cells were washed with PBS 5 times and intrinsic fluorescence was blocked with 50 mM NH4CI for 5 minutes. Cells were washed with PBS 3 times, incubated with 5 % goat serum for 30 minutes and washed with PBS twice. Slides were then incubated with anti heparanase monoclonal antibody HP- 130, 8 μg/ml for 2 hours at room temperature, washed with PBS 5 times and then incubated with the second antibody Cy-3 conjugated goat anti mouse IgG (Jackson) for 1 hour at room temperature. Slides were washed with PBS 8 times, and mounting solution (90 % glycerol in PBS ) was added. The monoclonal antibody HP- 130 was generated against human heparanase as described in U.S. Pat. Application No. 08/922,170.
Generation of a chimeric chicken-human heparanase gene: The

N-terminal coding portion of the human hpa (H-hpa ) cDNA encoding the signal peptide was replaced by a corresponding sequence of the chicken hpa cDNA. To this end, the Chk-hpa signal peptide coding sequence was amplified using specific primers (KPN/SPU: 5'-CGGGGTACCCGATGCTGGTGCT-3' (SEQ ID NO:l l); SPL: 5'-AGGTCCACGACGTCCTGTGCCGTC CGCCTCG-3', (SEQ ID NO:12)). A R-hpa cDNA region encoding a segment extending from the first amino acid downstream the H-hpa signal peptide to the BamHI restriction site was amplified, using H-hpa specific primers (HU: 5'-CGAGGCGGACGGCACAGGACGTCGTGGACCT-3' (SEQ ID NO:13; H/BHIL: 5'-CCACATCAGGAGGGATGGATCC-3' (SEQ ID NO:14). The PCR products were combined by means of primer extension and PCR amplification. The resulting fragment was then cloned in frame into a pcCDNA3 plasmid (Invitrogen, NV Leek, Netherlands) containing the H-hpa cDNA downstream the BamRΪ site, generating a chimeric construct in which the Chk-hpa signal peptide precedes the H-hpa. The chimeric gene was validated by sequencing. SEQ ID NO: 15 deleniates the chimeric cDNA, whereas SEQ ID NO: 16 deleniates the amino acid sequence of the chimeric heparanase protein.
EXPERIMENTAL RESULTS
Failures in cloning the chicken heparanase cDNA: Cloning of chicken heparanase was attempted based on minute homology to human heparanase. cDNA library of chicken kidney was screened using low stringency hybridization conditions and human hpa cDNA as a probe. No specific hybridization signal was observed and no hpa homologous clones could be isolated following the screening. A different approach utilized PCR primers of human hpa for amplification of the heparanase cDNA from chicken kidney. Human hpa primers failed to amplify chicken heparanase using annealing temperature as low as 37 °C.
Cloning the chicken heparanase cDNA: The human heparanase amino acid sequence was used to screen EST databases for homology to a chicken unidentified mRNA sequences. Following extensive screening, a single related chicken EST was identified which shared 60.5 % homology with the 276 bp at the 3' end of the human heparanase coding sequence . This sequence encoded a truncated open reading frame of 91 amino acids, 74 % homologous to human heparanase, followed by 325 nucleotides 3' untranslated region (UTR). The heparanase homologous sequence was derived from chicken activated T cells cDNA, clone pat.pk0039.c8.f 5', mRNA sequence, accession # AI980994.
In order to isolate a full length clone and to test whether it is indeed heparanase, chicken kidney mRNA was subjected to 5' RACE (rapid amplification of cDNA ends). The gene specific PCR primers were designed according to the EST described above. A DNA fragment of approximately 1,600 bp was obtained, partially overlapping with the identified 3' encoding EST clone. The entire cDNA cloned, in pGEM-T Easy Vector, was designated Chk-hpa. The complete cDNA (Chk-hpa) is 1,609 bp long (SEQ ID NO:10, Figure lb), and it contains an open reading frame that encodes a polypeptide of 523 amino acids (SEQ ID NO:4, Figure la-b) with a calculated molecular weight of 58,842 Daltons. Analysis of the amino acid sequence and of the hydropathic profile of the protein indicates a hydrophobic amino acid tail (Figure la underlined) at the N-terminus. A signal peptide is predicted to span the N-terminal 19 amino acids.
The overall homology between the chicken and the human hpa coding sequences is 62 %. The similarity between the chicken and the human heparanases is 69 % (of which 61 % amino acid sequence identity). The heparanase is synthesized as a latent, 65 kDa precursor and is then processed to an active mature 50 kDa form. Based on the homology to human heparanase the chicken heparanase is cleaved between Trp*36 and Lysl37. According to Fairbank et al. (57) the precursor is cleaved at three sites to form a heterodimer of a 50 kDa polypeptide (the mature form) that is associated with a 8 kDa peptide. The putative chicken 8 kDa peptide spans amino acids GlulO to Glu^4 (Figure la). The mature heparanases of various organisms share high homology while the pro-peptides are relatively diverse (Figure la).
Structure prediction of human heparanase suggests a (α/β)8 Tim barrel fold typical of family 10 glycosyl hydrolases. According to this prediction the active site involves two glutamic acid residues, which are the proton donor and the nucleophile, with an aspargine always preceding the proton donor. The proton donor in human heparanase is Glu225 d the nucleophile is Glu343. The conservation of the amino acid sequence flanking these residues supports the identification of the active site. Based on the homology the proton donor of chicken heparanase is Glu 0 ^^ me nucleophile is Glu32 .
The comparison between the nucleotide as well as the amino acid sequences of the all the heparanases published so far is presented in Figure 6.

Chicken heparanase is slightly more similar to human heparanase than to mouse and rat heparanases. As expected, the homology among mammals is far higher than that of mammals with chicken.
It is therefore yet to be determined whether the polynucleotide isolated from chicken indeed encodes a protein having heparanase catalytic activity. This is shown below.
Functional expression of recombinant chicken heparanase in mammalian cells: The ability of the Chk-Hpa product to catalyze degradation of heparan sulfate (HS) in vitro was deteπnined by expressing the entire open reading frame of C\ k-hpa in mammalian cells lacking heparanase activity. Mouse Eb-lymphoma and rat C6-glioma cells were transfected with the pcDNA3 plasmid vector containing the chicken heparanase cDNA (Chk-hpa) or with a control empty plasmid (mock transfected). Stable transfectants were then selected for further analysis. Expression of heparanase in the transfected cells was confirmed by RT-PCR. Chicken heparanase transcript was detected only in Chk-hpa transfected cells.
Heparanase activity was assayed in cells transfected with Chk-hpa as compared to mock transfected cells. As shown in Figures 3a-c high activity was observed in intact cells, conditioned media and cell lysates of Chk-hpa transfectants while no activity was observed in the mock transfected cells.
The heparan sulfate degradation activity of Eb cells transfected with Chk-hpa cDNA was compared with that of Eb cells transfected with human hpa (Hum-Zzpα). As shown in Figures 3a-c the activity of chicken heparanase was higher than that of human heparanase in intact cells and in conditioned media, however, in cell extracts the activity of the two enzymes was similar. This suggests that the chicken heparanase is unexpectedly preferentially secreted.
In order to compare the activity of the chicken and human heparanases, the enzymes were partially purified from conditioned media of Chk-hpa and H m-hpa transfected cells. Western blot analysis of the partially purified chicken heparanase showed a major protein of 58 kDa, which corresponds to the heparanase precursor and a minor protein of 45 kDa, which corresponds to the mature form. The human heparanase fraction contained the equivalent human 65 kDa heparanase precursor and 50 kDa mature forms (Figure 2). The difference in molecular weight between chicken and human heparanases is mainly due to a different glycosylation pattern.
Activity of the two enzymes was compared using equal amounts of the partially purified enzymes in a semi-quantitative assay. As shown in Figure 4 the specific activity of chicken and human heparanases is similar.
Localization of chicken heparanase in transfected cells: C-6 glioma cells stably transfected with the chicken or human heparanase cDNAs were grown in four well chamber slides and were subjected to indirect immunofluorescence staining with the anti-human heparanase mAb 130 (15). These antibodies cross-react with the chicken enzyme. Confocal fluorescence microscopy revealed that C-6 glioma cells transfected with the Chk-hpa cDNA exhibited an intense granular staining of the heparanase protein mostly associated with the cell surface. Preferential localization of the chicken heparanase was noted in areas of cell to cell contacts (Figure 5a, arrow). Unlike this pattern of immunostaining, C-6 glioma cells overexpressing the human heparanase displayed primarily a peri-nuclear granular staining pattern with almost no detectable surface localization of the enzyme (Figure 5c). A similar pattern was observed in transiently transfected human primary fibroblasts, where chicken heparanase was associated with cell membrane while human heparanase was localized to peri-nuclear vesicles, which were identified as lysosomes. The chicken and human heparanase cDNAs were also expressed in homologous cells (i.e., QT6 quail fibrosarcoma and Huh7 human hepatocarcinoma cells, respectively), resulting in an immunostaining pattern similar to that observed with the transfected C-6 rat glioma cells.
The signal peptides of the chicken and human heparanases share no significant homology. It appears that the signal peptide of chicken heparanase targets the enzyme to the cell surface of mammalian cells while the signal peptide of human heparanase targets the enzyme to lysosomes.
One may take advantage of the unexpected membrane targeting feature of chicken heparanase signal peptide for targeting other proteins to cell membrane.
Chimeric chicken-human heparanase gene: The results described above indicate that the chicken heparanase is more readily secreted into the incubation medium and/or retained on the cell surface, as compared with the human enzyme, most likely due to the marked difference between the respective signal peptide sequences. In order to further study this unexpected observation, a chimeric construct was generated, composed of the chicken signal peptide fused to the human cDNA downstream nucleotide 105. Briefly, chicken specific primers were used to amplify the chicken signal sequence which was then fused by means of primer extension to the human hpa sequence, replacing its signal peptide, as described in Experimental Procedures above. The chimeric construct was subcloned into pcDNA3 plasmid which was then used to stable transfect Eb mouse lymphoma and C-6 rat glioma cells. Serum free medium conditioned for 24 hours by Eb cells transfected with the chimeric construct (chimeric-/z/-->α, SEQ ID NOs:15 and 16) was tested for heparanase activity. As shown in Figure 7, cells transfected with the chimeric enzyme were comparable to cells transfected with Chk-hpa in their ability to secrete the heparanase enzyme into the culture medium. In contrast, little or no heparanase activity was detected in medium conditioned by H-hpa transfected cells (Figure 7), indicating that secretion of the enzyme is in fact driven by the chicken signal peptide sequence. Similar results were obtained with C-6 glioma cells.
Cellular localization of chimeric heparanase enzymes: The cell surface targeting of the chicken heparanase signal peptide was also demonstrated by the cellular localization of chimeric heparanase. Immunostaining of C-6 glioma cells transfected with the chimeric heparanase revealed preferential surface localization pattern (Figure 5b), similar to that of cells expressing the chicken heparanase (5a). Mock transfected glioma cells showed no staining (Figure 5d). The results of the swapping experiment emphasize that the pronounced difference in cellular localization of the chicken and human heparanases is due primarily to the marked difference in sequence, length and hydrophobic properties of the respective signal peptides. The preferential cell surface association of the chicken and chimeric heparanases is in accordance with the higher HS degrading activity expressed by intact cells overexpressing the chicken or chimeric enzymes vs. the human heparanase.

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 of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.

REFERENCES CITED
(Additional references are cited in the text)
1. Durand, P., Lehn, P., Callebaut, I., Fabrega, S., Henrissat, B. and Mornon, J.P. (1997). Active-site motifs of lysosomal acid hydrolases: invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis. Glycobiology, 7(2), 277-284.
2. Vlodavsky, I., Friedmann, Y., Elkin, M., Aingorn, H., Atzmon, R., Ishai-Michaeli, R., Bitan, M., Pappo, O., Peretz, T., Michal, I., Spector, L and Pecker I. (1999). Mammalian heparanase: gene cloning, expression and function in tumor progression and metastasis. Nature Medicine (In press)
3. Wight, T.N., Kinsella, M.G., and Qwamstromn, E.E. (1992). The role of proteoglycans in cell adhesion, migration and proliferation. Curr. Opin. Cell Biol., 4, 793-801.
4. Jackson, R.L., Busch, S.J., and Cardin, A.L. (1991). Glycosaminoglycans: Molecular properties, protein interactions and role in physiological processes. Physiol. Rev., 71, 481-539.
5. Wight, T.N. (1989). Cell biology of arterial proteoglycans. Arteriosclerosis, 9, 1-20.
6. Kjellen, L., and Lindahl, U. (1991). Proteoglycans: structures and interactions. Annu. Rev. Biochem., 60, 443-475.
7. Ruoslahti, E., and Yamaguchi, Y. (1991). Proteoglycans as modulators of growth factor activities. Cell, 64, 867-869.
8. Vlodavsky, I., Bar-Shavit, R, Korner, G., and Fuks, Z. (1993). Extracellular matrix-bound growth factors, enzymes and plasma proteins. In Basement membranes: Cellular and molecular aspects (eds. D.H. Rohrbach and R. Timpl), pp 327-343. Academic press Inc., Orlando, Fl.

9. Vlodavsky, I., Bar-Shavit, R., Korner, G., and Fuks, Z. (1993). Extracellular matrix-bound growth factors, enzymes and plasma proteins.

In Basement membranes: Cellular and molecular aspects (eds. D.H.

Rohrbach and R. Timpl), pp 327-343. Academic press Inc., Orlando, Fl.

10. Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O., Naparstek, Y., Cohen, I.R., and Fuks, Z. (1992). Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion & Metastasis, 12, 112-127.
11. Nakajima, M., Irimura, T., and Nicolson, G.L. (1988). Heparanase and tumor metastasis. J. Cell. Biochem., 36, 157-167.
12. Vlodavsky, I. et al. Invasion Metastasis 1995, 14:290-302.
13. Nakagima, M. et al J. Cell. Biochem. 1988, 36:157-167.
14. Vlodavsky, I. et al. Cancer res. 1983, 43:2704-2711.
15. Vlodavsky, I. et al. J. Med 1988, 24:464-470.
16. Vlodavsky, I. et al. Invasion and Metastasis 12:112-127.
17. Gilat, D. et al. J. Exp. Med. 1995, 181 :1929-1934.
18. Matzner et al. 1985, J. Clin. Invest. 10:1306-1313.
19. Mollinedo, F. et al. Biochem. J. 1997, 327:917-923.
20. Murphy, G. et al. Biochem. J. 1990, 192:517-525.
21. Nakajima, M. et al. J. Cell. Biochem. 1988, 36(2):157-167.
22. Ishai-Michaeli R. wt al. Cell Reg. 1990, 1:833-842.
23. Cardon-Cardo C. et al. Lab. Inrest. 1990, 63:832-840.
24. Nicolson, G.L. (1988). Organ specificity of tumor metastasis: Role of preferential adhesion, invasion and growth of malignant cells at specific secondary sites. Cancer Met. Rev., 7, 143-188.
25. Liotta, L.A., Rao, C.N., and Barsky, S.H. (1983). Tumor invasion and the extracellular matrix. Lab. Invest., 49, 639-649.
26. Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and Schirrmacher, V. (1983). Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell metastasis. Cancer Res., 43, 2704-2711.

27. Sun L, Feusi E, Sibalic A, Beck-Schimmer B, Wuthrich RP (1998). xpression profile of hyaluronidase mRNA transcripts in the kidney and in renal cells. Kidney Blood Press Res;21(6):413-8
28. Parish, CR, Coombe, D.R., Jakobsen, K.B., and Underwood, P.A. (1987). Evidence that sulphated polysaccharides inhibit tumor metastasis by blocking tumor cell-derived heparanase. Int. J. Cancer, 40, 511-517.
29. Burgess, W.H., and Maciag, T. (1989). The heparin-binding (fibroblast) growth factor family of proteins. Annu. Rev. Biochem., 58, 575-606.

30. Folkman, J., and Klagsbrun, M. (1987). Angiogenic factors. Science, 235, 442-447.
31. Vlodavsky, I., Folkman, J., Sullivan, R., Fridman, R., Ishai-Michaelli, R., Sasse, J., and Klagsbrun, M. (1987). Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. .Proc. Natl. Acad. Sci. USA, 84, 2292-2296.
32. Folkman, J., Klagsbrun, M., Sasse, J., Wadzinski, M., Ingber, D., and Vlodavsky, I. (1980). A heparin-binding angiogenic protein - basic fibroblast growth factor - is stored within basement membrane. Am. J. Pathol., 130, 393-400.
33. 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.
34. Ishai-Michaeli, R, Svahn, C.-M., Chajek-Shaul, T., Korner, G., Ekre, H.-P., and Vlodavsky, I. (1992). Importance of size and sulfation of heparin in release of basic fibroblast factor from the vascular endo helium and extracellular matrix. Biochemistry, 31, 2080-2088.
35. Ishai-Michaeli, R, Eldor, A., and Vlodavsky, I. (1990). Heparanase activity expressed by platelets, neutrophils and lymphoma cells releases active fibroblast growth factor from extracellular matrix. Cell Reg., 1, 833-842.

36. Vlodavsky, I., Bar-Shavit, R., Ishai-Michaeli, R., Bashkin, P., and Fuks, Z. (1991). Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism? Trends Biochem. Sci., 16, 268-271.
37. Vlodavsky, I., Bar-Shavit, R, Korner, G., and Fuks, Z. (1993). Extracellular matrix-bound growth factors, enzymes and plasma proteins. In Basement membranes: Cellular and molecular aspects (eds. D.H. Rohrbach and R. Timpl), pp327-343. Academic press Inc., Orlando, Fl.

38. Yayon, A., Klagsbrun, M., Esko, J.D., Leder, P., and Ornitz, D.M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell, 64, 841-848.

39. Spivak-Kroizman, T., Lemmon, M.A., Dikic, I., Ladbury, J.E., Pinchasi, D., Huang, J., Jaye, M., Crumley, G., Schlessinger, J., and Lax, I. (1994). Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerizatio, activation, and cell proliferation. Cell, 79, 1015-1024.
40. Ornitz, D.M., Herr, A.B., Nilsson, M., West, a., J., Svahn, C.-M., and Waksman, G. (1995). FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides. Science, 268, 432-436.

41. Gitay-Goren, H., Soker, S., Vlodavsky, I., and Neufeld, G. (1992). Cell surface associated heparin-like molecules are required for the binding of vascular endothelial growth factor (VEGF) to its cell surface receptors. J. Biol. Chem., 267, 6093-6098.
42. Ernst S, Langer R, Cooney CL, Sasisekharan R. (1995). Enzymatic degradation of glycosaminoglycans. Crit Rev Biochem Mol Biol ;30(5):387-444
43. Current protocols in molecular biology (1994-1999). Ausubel, F.M., Brent, R, Kingston, R.E., Moore D.D., Seidman, J.G., Smith, J.A., Struhl, K. Eds. John Wiley & Sons, Inc.

44. 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.
45. Shieh, M-T., Wundunn, D., Montgomery, RL, 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.
46. 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.
47. Putnak, J.R., Kanesa-Thasan, N., and Innis, B.L. (1997). A putative cellular receptor for dengue viruses. Nature Medicine 3, 828-829.
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.
49. 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.

50. Ross, R. (1993). The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature (Lond.)., 362:801-809.
51. 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.

52. Current protocols in protein science (1996-1999). Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W. and Wingfield, P.T. Eds. John Wiley & Sons Inc.

53. Freeman C and Parish CR (1998) Human platelet heparanase: purification, characterization and catalytic activity. : Biochem J Mar 15;330 ( Pt 3):1341-50.
54. Kussie et al. (1999) Biochem Biophys Res Commun. 261(1): 183-7.
55. Hullet et al. (1999) Nature Medicine 5(7):803-809.
56. Toyoshima and Nakajima (1999). J. Biol. Chem. 274(34):24153-24160.

57. Fairbanks et al. (1999) J. Biol. Chem. 274(42):29587-29590.
58. Friedmann et al. (2000) Am J Pathol (in press).