KR20010023593A - Polynucleotide encoding a polypeptide having heparanase activity and expression of same in transduced cells - Google Patents

Abstract

The present application relates to a polynucleotide (hpa) encoding a polypeptide having heparanase activity, a vector comprising the same, a transduced cell expressing heparanase and a recombinant protein having heparanase activity.

Description

POLYNUCLEOTIDE ENCODING A POLYPEPTIDE HAVING HEPARANASE ACTIVITY AND EXPRESSION OF SAME IN TRANSDUCED CELLS} Polynucleotides Encoding Polypeptides Having Heparanase Activity and Their Expression in Transduced Cells

Heparan Sulfate Proteoglycans:

Heparan sulfate proteoglycans (HSPG) are commonly present macromolecules that are linked to the cell surface and extracellular matrix (ECM) of various cells of tissues of vertebrates and invertebrates (1-4). The basic structure of HSPG comprises a protein core to which several linear heparan sulfate chains are covalently attached. These polysaccharide chains typically consist of repeating hexaeuronic and D-glucoseamine disaccharide units, which are substituted to varying degrees by N- and O-linked sulfate residues and N-linked acetyl groups. Investigations of the involvement of ECM molecules in cell adhesion, growth and differentiation have shown that HSPG plays a central role in embryonic morphogenesis, angiogenesis, neural overgrowth and tissue repair (1-5). HSPG is the major component of blood vessels (3). They are mostly concentrated in the innermost and inner middle of many blood vessels, but in the capillary they are found primarily in the subendothelial basement membrane, where they help to proliferate and migrate endothelial cells and stabilize the structure of the capillary wall. The ability of HSPG to interact with ECM macromolecules such as collagen, laminin, and fibronectin and different attachment sites on the plasma membrane is such that proteoglycans play an important role in cell adhesion and movement as well as in self-assembly and insolubility of ECM components. Suggests. Thus, cleavage of heparan sulphate (HS) chains can lead to the degradation of endothelial ECM and thus play a critical role in the outflow of blood-mediated cells. HS degradation is observed in inflammation, wound repair, diabetes and cancer metastasis, suggesting that enzymes that degrade HS play an important role in the pathogenesis. Heparanase activity has been described for activated immune system cells and high metastatic cancer cells (6-8), but research has been limited because there is no biological tool to determine the possible causal role of heparanase in disease states.

Involvement of heparanase in tumor cell invasion and metastasis:

Circulating tumor cells detained in capillary layers of different organs invades endothelial cells for invasion into extravascular tissue (s) (where metastasis is achieved) and breaks down the underlying membrane of the underlying endothelial cells (9, 10). Metastatic tumor cells often attach to or near intercellular junctions between adjacent endothelial cells. This attachment of metastatic cells involves rupture of the junction, contraction of the endothelial cell boundaries and migration to exposed underlying BM through gaps in the endothelium (9). Once invasive cells are located between endothelial cells and BM, they must degrade the endothelial glycoproteins and proteoglycans of BM to migrate out of the vascular compartment. Several cellular enzymes (eg, collagenase IV, plasminogen activator, cathepsin B, elastase, etc.) are thought to be involved in the degradation of BM (10). Among these enzymes, enzymes that cleave HS at specific intrachain sites are endo-β-D-glucuronides (heparanases) (6, 8, 11). Expression of HS degraded heparanase was found to correlate with metastatic efficacy of mouse lymphoma (11), fibrosarcoma and melanoma (8) cells. Furthermore, increased concentrations of heparanase were detected in animals and melanoma patients (8) showing metastatic tumors and in tumor biopsies of cancer patients.

Control of cell proliferation and tumor progression by the local microenvironment, focused on the interaction of cells with extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells, was previously investigated by the inventors. The cultured ECM is very similar to the endothelial layer in vivo in its morphological appearance and molecular composition. It contains collagen (most types III and IV, lesser types I and V), proteoglycans (most heparan sulfate proteoglycans and dematane sulfate proteoglycans, smaller amounts of chondroitin sulfate proteoglycans), laminin, fibronectin, entaxin and elastin. (13, 14). The ability of cells to degrade HS in cultured ECM was studied by reacting the cells with metabolic sulfate labeled ECM, followed by gel invasion (Sepharose 6B) analysis of the degradation products released into the culture medium (11). Intact HSPG eluted after the pore volume of the column (Kav <0.2, Mr-0.5 × 10 ⁶ ), but labeled digested fragments of the HS side chain eluted closer to V _t of the column (0.5 <Kav <0.8). , Mr = 5-7 × 10 ³ ) (11).

The heparanese inhibitory effects of various non-aggregated species of heparin that may be effectively used to prevent the outflow of various blood-producing cells have also been investigated by the inventors. Inhibition of heparanase was achieved by heparin species containing at least 16 sugar units and having sulfate groups in both N and O positions. O-sulfate removal prevented the heparinase inhibitory effect of heparin, but O-sulfated and N-acetylated heparin had high inhibitory activity, provided that the N-substituted molecule had a molecular size of at least about 4,000 Daltons (7). Experimental treatment of animals with heparanase inhibitors (eg, non-anticoagulant species of heparin) has markedly demonstrated the development of lung metastasis induced by B16 melanoma, Lewis lung carcinoma and mammalian adenocarcinoma cells. (> 90%) decreased (7, 8, 16). Heparin fraction with high affinity for anti-thrombin III and heparin fraction with low affinity showed comparable high anti-metastatic activity. Plays an arbitrary role in metastatic properties (7).

Activity of Heparanase in Urine of Cancer Patients:

The heparanase activity of urine samples was screened to clarify the association between heparanase and the progression of tumors between human cancer and heparanase (16a). Although not all cancer patients, heparanase activity was detected in the urine of some patients. High levels of heparanase activity were detected in the urine of the invasive metastatic phase, but not in the urine of healthy donors.

Heparanase activity has been found in 20% of urine in general diabetics and in urine in patients with microalbuminin insulin dependent diabetes mellitus (IDDM), most likely due to diabetic kidney disease. It has been found to cause renal abnormalities in adults.

Possible relevance of heparanases in tumor angiogenesis:

Fibroblast growth factors are characterized in that they have a high affinity for heparin (17) and refers to a group of polypeptides structurally related to each other. They promote mitosis of vascular endothelial cells and are very strong potential angiogenic derivatives (17, 18). Basal fibroblast growth factor (bFGF) is extracted from in vitro generated endothelial ECM (19), or from the basal membrane of the cornea (20), and ECM has been proposed as a reservoir of bFGF. Immunohistochemical staining revealed the location of bFGF in the lowermost layer of various tissues and blood vessels (21). Although bFGF is present everywhere in normal tissues, endothelial cells proliferate in tissues rarely, suggesting that bFGF is somewhat isolated from the active site. Studies on the interaction between bFGF and ECM showed that bFGF binds to HSPG of ECM and is released into the active form by HS degrading enzymes (15, 20, 22). Heparanase activity is expressed by platelets, breast cells, neutrophils, and lymphoma cells, and is involved in the release of active bFGF from ECM and the lowermost layer (23), and heparanase activity acts on cell migration and infiltration In addition, it can induce an indirect neovascular response. From these results it can be inferred that ECM HSPG provides a natural reservoir of bFGF and heparin-binding growth promoters (24, 25). Substitution of bFGF from the reservoir between the lowermost membrane and the ECM reveals a novel mechanism for the induction of neovascularization in normal and pathological cases.

Recent studies have shown that heparin and HS are involved in bFGF binding to high affinity cell surface receptors and bFGF cell signaling (26,27). Moreover, the size of the HS needed to achieve the optimal effect is similar to the size of the HS fraction released by heparanase (28). Similar results (29) were obtained for vascular endothelial cell growth factor (VEGF), from which it can be inferred that the dual receptor mechanism including the cellular interaction of heparin-binding growth factor and HS works. Therefore, the suppression of endothelial cell growth factor of ECM prevents the vascular endothelial systemic action, and can be assumed to keep the endothelial cell conduction and vascular growth rate low. On the other hand, release of bFGF, which is stored in complex form with the HS fraction in ECM, induces localized endothelial metastasis and induces neovascular formation in processes such as wound healing, inflammation, and tumor development (24,25). ).

Expression of Heparanase by Immune System Cells:

Heparanase activity is associated with the ability to induce inflammation and autoimmune responses beyond the circulation of activated cells of the immune system. The interaction of platelets, granulocytes, T lymphocytes and B lymphocytes, macrophages and breast cells with endothelial ECM is associated with the degradation of HS by specific heparanase activity (6). Heparanases are released from intracellular compartments (eg lysosomes, specific granules, etc.) in response to various activation signals (eg thrombin, calcium ion transporters, immune complexes, antigens, mitotic promoters, etc.) and inflammation It is believed to be involved in the regulation of and cellular immunity.

Observations related to heparanases are reviewed in Ref. 6, some of which are listed below:

First, proteolytic activity (plasminogen activator) and heparanase synergistically participate in a series of ECM HSPG degradation by inflammatory leukocytes and malignant cells.

Second, a significant number of platelet heparanases exist in potential form in complex with chondroitin sulfate. Latent enzymes are activated by tumor cell-inducing factors and appear to promote cell invasion through the vascular endothelium during tumor metastasis.

Third, the release of platelet heparanases from α-granules is induced by strong stimulants (eg thrombin) but does not occur in response to platelet activity against ECM.

Fourth, neutrophil heparanase is readily released differentially in cell culture on ECM in response to critical activity.

Fifth, contacting neutrophils with ECM inhibits the release of harmful enzymes (protease, lysozyme) and oxygen radicals, but does not inhibit the release of enzymes that allow extravasation (heparanase, gelatinase). This protective role of subcutaneous endothelial ECM was observed when cells were stimulated with soluble factors but not phagocytic stimulants.

Sixth, intracellular heparanases are secreted within minutes after exposure of T cell lines to specific antigens.

Seventh, mitotic promoters (Con A, LPS) induce the synthesis and secretion of heparanase by normal T and B lymphocytes maintained in vitro. T lymphocyte heparanases are also induced by immunization with antigens in vivo.

Eighth, heparanase activity is only manifested by pre-B lymphomas and B-lymphomas, but not by plasma plasmoma and remaining normal B lymphocytes.

Ninth, heparanase activity was only expressed by activated macrophages in culture using ECM, with little or no enzyme released into the culture medium. Similar results were obtained with human myeloid leukemia cells induced to differentiate into mature macrophages.

Tenth, T-cell mediated delayed hypersensitivity and experimental autoimmunity are inhibited by small amounts of heparanases that interfere with the non-anticoagulant species of heparin (30).

Eleventh, heparanase activity exhibited by platelets, neutrophils and metastatic tumor cells releases active bFGF and the lowest layer membrane from ECM. Release of bFGF from the reservoir of ECM can elicit localized angiogenesis in processes such as wound healing, inflammation and tumor development.

Twelfth, the product of ECM generated by heparanases during degradation is tri-sulfate disaccharide that can inhibit T-cell mediated inflammation in vivo (31). This inhibition was associated with the inhibitory effect of disaccharides on the production of biologically active TNFα by activated T-cells in vitro (31).

Other Effective Prescription Applications:

Apart from their relevance in tumor cell metastasis, inflammation and autoimmunity, mammalian heparanases can be applied to regulate: bioavailability of heparin-binding growth factor 15; Cellular responses to heparin-binding growth factors (eg bFGF, VEGF) and cytokines (IL-8) (31a, 29); Cellular interaction with plasma lipoprotein 32; Cell susceptibility to certain viruses and some bacterial and protozoan infections (33, 31a, 33b); And decomposition of the amyloid plate 34. Thus, heparanase has proven to be useful for wound healing, angiogenesis, stenosis, atherosclerosis, inflammation, neurodegenerative diseases and viral infections. Mammalian heparanases can be used to neutralize plasma heparin as an effective substitute for protamine. Anti-heparanase antibodies can be applied to microsensitization, autoimmune lesions and immunodetection and diagnosis of kidney disease, plasma samples and body fluids in biopsy fragments. It is expected to be commonly used in basic research.

Identification of the hpa gene encoding for heparanase enzymes allows the production of recombinant enzymes in atypical expression systems. The availability of recombinant proteins is to enable them to resolve protein structural action relationships and to provide a means to develop new inhibitors.

Virus Infection:

The presence of heparin sulfate on the cell surface was a principle requirement for Herpes Simplex (33) and Dengue (33a) viruses to bind and infect cells. Thus, viral infection can be eliminated by removing the cell surface heparan sulfate by heparanase. In fact, treating cells with bacterial heparinase (decomposing heparan sulphate) or heparanese (heparan degrading) reduces the binding of two related animal herpes viruses to cells and causes the cells to be at least partially virally infected. It becomes resistant to (33). Some indicate that cell surface heparan sulphate is also associated with HIV infection 33b.

Neurodegenerative Diseases:

Heparan sulfate proteoglycans were identified in the prion protein amyloid version of Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape (34). Heparanase can break down these amyloid plates, which also contribute to the development of Alzheimer's disease.

Stenosis and Atherosclerosis:

Proliferation of arterial smooth muscle cells (SMCs) in response to epithelial damage and the accumulation of cholesterol-rich lipoproteins is fundamental in the pathogenesis of atherosclerosis and stenosis (35). Apart from its relevance in SMC proliferation (ie, low affinity receptors for heparin-binding growth factors), HS is also associated with lipoprotein binding, retention and uptake 36. HSPG and lipoprotein lipases participate in novel degenerative pathways that allow substantial cellular and interstitial accumulation of cholesterol rich lipoprotein 32. This pathway is expected to be highly atheromatous by promoting the accumulation of apoB and apoE packed lipoproteins (ie, LDL, VLDL, kilomicron), independent of feed inhibition by cell sterol content. Therefore, the removal of SMC HS by heparanases is expected to stop the development of stenosis and atherosclerosis by inhibiting SMC proliferation and lipid accumulation.

Thus, it is very advantageous to have a polynucleotide encoding a polypeptide having heparanase activity, a vector containing the same, a transformed cell expressing heparanase and a recombinant protein having heparanase activity.

Summary of the Invention

According to the present invention a polynucleotide encoding a polypeptide having heparanase activity, a vector containing the same, a transformed cell expressing heparanase and a recombinant protein having heparanase activity, which is then referred to as a hpa, hpa cDNA or hpa gene Is provided.

Cloning of the human hpa gene encoding heparanase and expression of recombinant heparanase by transfected host cells are recorded.

Purified preparations of heparanase isolated from human liver cancer cells were introduced for trypsin digestive digestion and microsequencing. The sequence of SEQ ID NO: 8 was used to screen the EST database for homologues of the corresponding reverse transcribed DNA sequence. Two closely related EST sequences were identified and subsequently found identical. All clones contained a 1020 bp insert, which included a 973 bp open reading frame followed by 27 bp of the 3 'non-transcribed region and the Poly A tail. Transcription starting sites were not identified.

Cloning of the lost 5 'end of hpa was performed by PCR amplification of DNA from the placental Marathon RACE cDNA complex and the linker of the complex using primers selected corresponding to the EST clone sequence. A 900 bp PCR fraction was obtained that partially overlaps with the identified 3 'encoding EST clone. The linked cDNA fraction (hpa), 1721 bp long (SEQ ID NO: 9) contained an open reading frame encoding a polypeptide of 543 amino acids (SEQ ID NO: 10) with a calculated molecular weight of 61,192 Daltons.

Cloning the extended 5 'sequence was possible from the SK-hep1 cell line by PCR amplification using marathon RACE. The 5 'extended sequence of SK-hep1 hpa cDNA was assembled with the sequence of hpa cDNA isolated from the human placenta (SEQ ID NO: 9). The assembled sequence contained an open reading frame, SEQ ID NO: 14 and SEQ ID NO: 16, which contained a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 Daltons as shown in SEQ ID NO: 15 and SEQ ID NO: 17. It was.

The ability of the hpa gene product to catalyze the degradation of heparan sulphate in an in vitro test was tested by expressing the full open reading frame of hpa in insect cells using a baculovirus expression system. Extracts of cells infected with virus containing the hpa gene and conditioned media demonstrated a large amount of heparan sulfate degradation activity towards soluble ECM-induced HSPG and no manipulation ECM. Degradation activity was inhibited by heparin, another substrate of heparanase. Cells infected with similar constructs that did not contain any hpa genes did not have this activity at all, nor did they have infected cells. The ability of heparanases expressed from the extended 5 'clone towards heparin has been demonstrated in mammalian expression systems.

Expression patterns of hpa RNA in various tissues and cell lines were investigated using RT-PCR. It has been discovered that it is expressed only in tissues and cells already known to have heparanase activity.

A panel of monochromosomal human / CHO and human / mouse somatic hybrids was used to localize the human heparanase gene to human chromosome 4. The newly isolated heparanase sequence was used to identify the chromosomal region containing the human heparanase gene in the chromosome spread.

According to a further feature in a preferred embodiment of the invention described below, there is provided a polynucleotide fraction comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

According to still further features in the described preferred embodiments, the polynucleotide fraction comprises nucleotides 63 to 1691 of SEQ ID NO: 9 or nucleotides 139 to 1869 of SEQ ID NO: 14, encoding the entire human heparanase enzyme.

According to still further features in the described preferred embodiments, a polynucleotide fraction is provided comprising a polynucleotide sequence capable of hybridizing with hpa cDNA, in particular nucleotides 1 to 721 of SEQ ID NO: 9.

According to still further features in the described preferred embodiments the polynucleotide sequence encoding a polypeptide having heparanase activity is at least 60% homologous, preferably at least 70% homologous, more preferably at least 80%, most Preferably at least 90% is shared with SEQ ID NO: 9 or SEQ ID NO: 14.

According to still further features in the preferred embodiments described, the polynucleotide fraction according to the invention comprises a part (fraction) of SEQ ID NO: 9 or SEQ ID NO: 14. For example, such a fraction may comprise a fraction of SEQ ID NO: 9 encoding a polypeptide having nucleotides 63 to 721 of SEQ ID NO: 9 and / or heparanase catalytic activity.

According to still further features in the preferred embodiments described, the polypeptide encoded by the polynucleotide fraction comprises the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15 or a functional part thereof.

According to still further features in the described preferred embodiments, the polynucleotide sequence encodes a polypeptide having heparanase activity, which is at least 60% homologous, preferably at least 70% homologous, more preferably at least 80% Most preferably at least 90% with SEQ ID NO: 10 or SEQ ID NO: 15.

According to still further features in the described preferred embodiments, the polynucleotide fraction encodes a polypeptide having heparanase activity and consequently an allelic species of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 15 and And / or inducible variants. Any such variant may be considered homologous.

According to still further features in the described preferred embodiments a single stranded polynucleotide fraction comprising a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity as described above This is provided.

According to still further features in the described preferred embodiments, a vector is provided comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

The vector can be of any suitable type and includes, but is not limited to, phage, virus, plasmid, phagemid, cosmid, backmid or even artificial chromosomes. The polynucleotide sequence encoding the polypeptide having heparanase catalytic activity may comprise any of the polynucleotide fractions described above.

According to still further features in the described preferred embodiments, a host cell is provided comprising an exogenous polynucleotide fraction comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

The exogenous polynucleotide fraction can be any of the fractions described above. Host cells can be of any type, for example prokaryotic cells, eukaryotic cells, cell lines or some cells of a multinuclear organism (eg, cells of transgenic organisms).

According to still further features in the described preferred embodiments, a recombinant protein is provided comprising a polypeptide having heparanase catalytic activity.

According to still further features in the described preferred embodiments, there is provided a pharmaceutical composition comprising as an active ingredient a recombinant protein having heparanase catalytic activity.

According to still further features in the described preferred embodiments, there is provided a medical device comprising a medical device containing as an active ingredient a recombinant protein having heparanase catalytic activity.

According to still further features in the described preferred embodiments, there is provided a heparanase overexpression system comprising cell overexpression heparanase catalytic activity.

According to still further features in the preferred embodiments described, (a) hybridizing a chromosomal spread with a tagged polynucleotide probe encoding heparanases; (b) washing the chromosome spread to remove non-hybrid formed probe excess; And (c) examining a signal associated with the hybridized tailed polynucleotide probe, a method for identifying a chromosomal region comprising a human heparanase gene in a chromosome spread is provided, wherein the detected signal is Chromosomal region containing human heparanase gene).

The present invention can be used to develop new medicines for inhibiting tumor cell metastasis, inflammation and autoimmunity. Identifying the hpa gene encoding for the heparanase enzyme allows the production of recombinant enzymes in atypical expression systems.

The present invention relates to polynucleotides encoding polypeptides having heparanase activity (hereinafter referred to herein as hpa), vectors comprising the same and transduced cells expressing heparanases. The invention further relates to recombinant proteins having heparanase activity.

The invention will now be described with reference to the following attached drawings for purposes of illustration only:

1 shows the nucleotide sequence and the deduced amino acid sequence of hpa cDNA. A single nucleotide difference and the resulting amino acid substituents (Tyr to Phe) at positions 799 (A to T) between the Expressed Sequence Tag (EST) and the PCR amplified cDNA (reverse transcribed RNA) were substituted above and below Each is indicated in units. Cysteine residues and poly adenylated consensus sequences are underlined. The asterisk is the stop codon TGA.

Figure 2 shows degradation of soluble sulfate labeled HSPG substrates by the rise of High Five cells infected with pFhpa2 virus. Resates of high five cells infected with pFhpa2 virus (●) or high five cells infected with control pF2 virus (□) were incubated with sulfate-labeled ECM-induced soluble HSPG (peak I) (18 h, 37 ° C.). . Culture medium was then introduced to gel filtration on Sepharose 6B.

Low molecular weight HS digestion fractions (Peak II) were prepared by culturing only with pFhpa2 infected cells, but no HSPG substrate () was degraded by the lysate of pF2 infected cells.

3A and 3B show the degradation of soluble sulfate labeled HSPG substrate by the culture medium of pFhpa2 and pFhpa4 infected cells. Culture medium of high five cells infected with pFhpa2 (3a) or pFhpa4 (3b) virus, or high five cells infected with control virus (□) was treated with sulfate-labeled ECM-induced soluble HSPG (Peke I, ◇). Incubated together (18 hours, 37 ° C.). The incubation medium was then gel filtered over Sepharose 6B. Low molecular weight HS digestion fractions (Peak II) were prepared with incubation with only hpa gene containing virus. The HSPG substrate was not degraded at all by the medium of cells infected with the control virus.

4 shows size fractionation of heparanase activity expressed by pFhpa2 infected cells. Medium of pFhpa2 infected high five cells was applied on 50 kDa cleaved membranes. Heparanase activity (conversion of peak I substrate (◇) to peak II HS digestion fraction) was found in the high molecular weight (> 50 kDa) (●), low molecular weight (<50 kDa) (○) compartments.

5 a and b depict the effect of heparin on heparanase activity expressed by high five cells infected with pFhpa2 and pFhpa4. Soluble HSPG derived from sulfate-labeled ECMs by dividing the culture medium of high five cells infected with pFhpa2 (5a) and pFhpa4 (5b) with or without 10 μg // ml heparin (Δ) and without (▲) Incubated with peaks I, ◇) (18 hours, 37 ° C). In the presence of heparin, a potential inhibitor of heparanase activity, no production of low molecular weight HS degradation fractions was observed (6, 7).

Figures 6a and b show the degradation of non-engineered ECM sulfate-labeled by virus-infected high-five and Sf21 cells. High five cells (6a) and Sf21 (6b) cells were placed in sulfate-labeled ECM plates and infected with pFhpa4 (() or control pF1 (□) virus (48 hours, 28 ° C). Control uninfected Sf21 cells (R) were also placed in labeled ECM plates. The pH of the culture medium was adjusted to 6.0-6.2 and then incubated at 37 ° C. for 24 hours. Sulfate medium material released into the culture medium was analyzed by gel filtration on Sepharose 6B. HS degradation fraction was generated only in cells infected with virus containing hpa.

Figures 7a and b show the degradation of sulphate labeled no manipulation ECM by virus infected cells. High five cells (7a) and Sf21 cells (7b) were placed in sulfite-labeled ECM plates and infected with pFhpa4 (●) or control pF1 (□) virus (48 hours, 28 ° C). Control uninfected Sf21 cells (R) were also placed in labeled ECM plates. The pH of the culture medium was 6.0 to 6.2, and then incubated at 28 ° C. for 48 hours. Sulfate labeled medium material released into the culture medium was analyzed by gel filtration on Sepharose 6B. HS degradation fraction was generated only in cells infected with virus containing hpa.

8a and b show the degradation of sulfate-labeled, tamper-free ECM by the culture medium of pFhpa4 infected cells. Intact culture medium of high five cells (8a) and Sf21 cells (8b) infected with pFhpa4 (●) or control pF1 (□) virus was incubated with sulfate labeled ECM (48 h, 37 ° C., pH 6.0). ). ECM was also incubated with the culture medium of control uninfected Sf21 cells (R). Sulfate labeled material released into the reaction mixture was analyzed by gel filtration. The activity of heparanase was detected only in the culture medium of cells infected with pFhpa4.

9 shows the effect of heparin on heparanase activity in culture medium of pFhpa4 infected cells. Sulfate labeled ECM was incubated for 24 hours at pH 6.0 and 37 ° C. in culture medium of pFhpa4 infected high five (9a) and Sf21 (9b) in the absence or presence of 10 μg / ml heparin. Sulfate labeled material released into the culture medium was gel filtered on Sepharose 6B. In the presence of heparin, heparanase activity (preparation of peak II HS degraded segments) was completely inhibited.

10A-B relate to the purification of recombinant heparanese in heparin-sepharose. Culture medium of SF21 cells infected with pFhpa4 virus was subjected to heparin-sepharose chromatography. Elution of the fractions was carried out in a NaCl gradient (◇) of 0.35-2 M. Heparanase activity in the eluted fractions is illustrated in FIG. 10A (•). Fractions 15 to 28 were subjected to 15% SDS-polyacrylamide gel electrophoresis and then developed with silver nitrate. Correlation between major protein bands (molecular weight about 63,000) and heparanase activity in fractions 19-24.

11A-B illustrate purification of recombinant heparanese in a Superdex 75 gel filtration column. The active fractions eluted from heparin Sepharose (FIG. 10A) were collected, concentrated and applied to a Superdex 75 FPLC column. Fractions were collected and droplets of each fraction were tested for heparanase activity (c, FIG. 11A), analyzed by SDS-polyacrylamide gel electrophoresis, and then developed with silver nitrate (FIG. 11B). Correlation between the appearance of major protein bands (molecular weight of about 63,000) and heparanase activity was seen in fractions 4-7.

12A-E show that the hpa gene is expressed by RT-PCR with human RNA from tissues 12a, tissues other than human embryos 12b and total RNA from cell lines 12c to e of different origins. When using a control reaction without hpa specific primers (I), primers for the GAPDH housekeeping gene (II), and reverse transcriptase indicating the presence of other contamination in genomic DNA or RNA samples (III) RT-PCT product. M represents the DNA molecular weight marker VI (Beringer Menheim). In FIG. 12A, lane 1 is neutrophil cell (adult) and lane 2 is muscle, lane 3 is thymus, lane 4 is heart and lane 5 is adrenal. In FIG. 12B, lane 1 is kidney, lane 2 is 8 week placenta, lane 3 is 11 week placenta, lanes 4-7 are mole (mole in full foam). Lane 8 is cytotrophic cell (freshly separated), lane 9 is cytotrophic cell (1.5 hours in vitro), lane 10 is cytotrophic cell (6 hours in vitro), Lane 11 is cytotrophic cells (18 hours in vitro) and lane 12 is cytotrophic cells (48 hours in vitro). In FIG. 12C, lane 1 is a JAR bladder cell line, lane 2 is a cell line of NCITT testicular cancer, lane 3 is a SW-480 human liver cancer cell line, and lane 4 is HTR (cytotrophic cell transformed with SV40). Lane 5 is a carcinoma cell line of HPTLP-I cancer cells, and lane 6 is a cell line of EJ-28 bladder carcinoma. In FIG. 12D, lane 1 is SK-hep-1 human liver cancer cell line, lane 2 is DAMI human megakaryocyte cell line, lane 3 is DAMI cell line + PMA, lane 4 is CHRF cell line + PMA, 5 Burn lanes are CHRF cell lines. In FIG. 12E, lane 1 is aortic endothelial cells of ABAE bovine, lane 2 is a cell line of 1063 human ovary, lane 3 is a human breast cancer MDA435 cell line, and lane 4 is a human breast cancer MDA231 cell line.

FIG. 13 shows the nucleotide sequence of human hpa and 80% homology to the EST cDNA segment of the mouse (SEQ ID NO: 13) (starting at nucleotide 1066 of SEQ ID NO: 9) of the human hpa. It is a comparison. Aligned terminal codons are underlined.

14 shows the chromosomal location of the hpa gene. DNA derived from cell hybrids of celiac and PCR products of genomic DNA of hamster, mouse and human were isolated on 0.7% agarose gel and amplified with hpa specific primers. Lane 1 is lambda DNA digested with BstEII, lane 2 is a control without DNA, and lanes 3 to 29 are PCR amplification products. Lanes 3 to 5 are genomic DNA of human, mouse and hamster, respectively. Lanes 6-29 are human monochromosomal celiac cell hybrids showing chromosomes 1-22, X and Y, respectively. Lane 30 is lambda DNA digested with BstEII. Amplification products of about 2.8 Kb are observed in lanes 5 and 9, indicating DNA derived from cell hybrids carrying human genomic DNA and human chromosome 4, respectively. These results demonstrate that the hpa gene is located on human chromosome 4.

The present invention provides polynucleotides interchangeably referred to below as hpa, hpa cDNA or hpa genes encoding polypeptides having heparanase activity, vectors comprising the same, transduced cells expressing heparanase and It relates to a recombinant protein having heparanase activity.

Before describing one or more aspects of the invention in detail, it will be understood that the invention is not limited to the details of the structure and arrangement of components set forth in the following description or the drawings. The invention may be carried out or carried out in various ways, or may be other aspects. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and not of limitation.

The present invention can be used to develop treatments for various diseases, to develop diagnostic methods for such diseases, and in particular to provide novel methods for basic research in the medical and biological fields.

In particular, the present invention can be used for the development of novel drugs for inhibiting cancer cell metastasis, inflammation and autoimmunity. Identification of the hpa gene encoding for the heparanase enzyme enables the production of recombinant enzymes in heterologous expression systems.

In addition, the present invention relates to bioavailability of heparin-binding growth factors, cellular responses to heparin-binding growth factors (eg bFGF, VEGF) and cytokines (IL-8), cellular responses to plasma lipoproteins, viruses, protozoa And cellular susceptibility to other bacterial infections, and the division of neurodegenerative plaques. Thus, recombinant hepparaanases may treat wound healing, angiogenesis, stenosis, atherosclerosis, inflammation, neurodegenerative diseases (eg Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease). -Jakob disease), scrape and Alzheimer's disease) and potential viruses for infection by certain viruses and some bacteria and protozoa. Recombinant heparanese can be used to neutralize plasma heparin as a potential substitute for protamine.

As used herein, “modulate” means substantially inhibiting, slowing or reversing the progress of a disease, substantially ameliorating clinical signs of a disease or condition, or the appearance of clinical signs of a disease or condition. To prevent this. Thus, the term "modulator" refers to a medicament capable of controlling a disease or condition. Modulation of viruses, protozoa and bacterial infections reduces the activity of any virus, bacteria or protozoa, and / or a virus, bacterium that reduces or prevents infection by a virus, bacterium or protozoa of a subject, such as a human or sub-animal Or any effect that substantially inhibits, prevents or reduces the stage of the life cycle of protozoa.

As used herein, the term "wound" refers to any injury to any part of the subject's body, including, for example, thermal burns, chemical burns, burns caused by radiation, excessive exposure to ultraviolet light. Acute diseases such as burns due to exposure (eg, sunburn); Damage to body tissues, such as the perineum, for example, due to childbirth, ongoing damage during medical practices such as perineal incisions, trauma-induced injuries including incisions (these injuries persist in motor vehicles and other mechanical accidents And damages caused by rifles, knives and other weapons), and chronic diseases such as post ulcer damage as well as compression ulcers, pressure sores, diseases related to diabetes and circulation disorders, and all forms of acne. However, this is not limitative.

Anti-heparanase antibodies that arise against recombinant enzymes are useful for immunodetection and microtransition in biopsy specimens, plasma samples and body fluids, diagnosis of autoimmune injury and renal failure. Such antibodies can also be used as neutralizing agents for heparanase activity.

Cloning the human hpa gene encoding herpanase by infected cells and expressing recombinant herrapase are reported herein. This is the first mammalian heparanase cloned.

Purification method of heparanase isolated from human liver cancer cells is trypsin digestion and microsequencer application.

The identified YGPDVGQPR (SEQ ID NO: 8) sequence was used to screen the EST base for homologues for the corresponding reverse translated DNA. Two closely related EST sequences were identified and then found identical.

Two clones included an open reading frame of 973 bp and a 1020 bp insert comprising a 27 bp and 3 'untranslated region of the poly A tail, but no translation start site was identified.

Cloning of the missing 5 'ends was performed by PCR amplifying DNA from the Placenta Marathon RACE cDNA combination using primers selected according to the linker of the EST clone sequence and combination.

A 900 bp PCR segment was obtained that partially overlaps with the identified 3 ′ coding EST clone. The linked cDNA segment (hpa) (1721 bp length, SEQ ID NO: 9) is a polypeptide of 543 amino acids (SEQ ID NO: 10) (calculated molecular weight: 61,192 Daltons), as shown in FIG. 1 and SEQ ID NO: 11. Included is an open reading frame for coding

A single nucleotide difference at positions 799 (A-T) between the EST clone and the PCR amplified cDNA was observed. This difference results in a single amino acid substitution (Tyr to Phe) (FIG. 1). In addition, published EST sequences containing unidentified nucleotides (following the DNA sequences of both EST crones) were separated into two nucleotides (G at position 1630 and C at position 1631, respectively).

The ability of the hpa gene product to catalyze the degradation of heparan sulphate in an in vitro test was assessed by expressing a complete open reading frame in infected cells using a baculovirus expression system.

Extracts and conditioned media of virus infected cells containing the hpa gene showed high levels of heparan sulfate degradation activity on both soluble ECM-induced HSPG and intact ECM inhibited by heparin, while the hpa gene Cells that did not contain but infected with similar constructs showed no activity and uninfected cells did not.

Expression patterns of hpa RNA in various tissues and cell lines were assessed using RT-PCR. It has been found to be expressed only in tissues and cells already known to have heparanase activity.

Cloning the extended 5 'sequence was possible from human SK-hep-1 cell line by PCR amplification using marathon RACE. The 5 'extended sequence of SK-hep1 hpa cDNA was assembled into hpa cDNA (SEQ ID NO: 9) isolated from human placenta. The assembled sequence is an open reading frame of SEQ ID NO: 14 and SEQ ID NO: 16, which encodes a polypeptide of 592 amino acids with a calculated molecular weight of 66,407 daltons, as shown by SEQ ID NO: 15 and SEQ ID NO: 17. It includes. This open reading frame appears to be responsible for the expression of catalytically active heparanases in mammalian cell expression systems. The expressed heparanase was detectable by anti-herapanase antibodies in Western blot assay.

A panel of human / CHO and human / mouse placental cell hybrids of monochromosomes was used to locate the human heparanase gene on human chromosome 4. Thus, the newly isolated heparanase sequence can be used to identify chromosomal regions containing human heparanase genes in distributed chromosomes.

Accordingly, according to the present invention, there is provided a polynucleotide segment (both single stranded or double stranded DNA or RNA) comprising a polynucleotide sequence encoding a polypeptide having heparase catalyzed activity.

Both "heparanase catalytic activity" or the equivalent term "heparanase activity" are bacterial enzymes (heparanase I, II) that degrade heparin or heparan sulfate by the method of β-removal reaction 37. In contrast to the activity of and III), it relates to the endoglycosidase hydrolytic activity of mammals specific for heparan or heparan sulfate proteoglycan substrates.

In a preferred embodiment of the invention, the polynucleotide segment is nucleotide 63-1691 of SEQ ID NO: 9 or nucleotides 139-1869 of SEQ ID NO: 14, which encodes a whole human heparanase enzyme.

However, since this is the first publication of an open reading frame (ORF) that codes for any mammalian heparanase, the scope of the present invention is not limited to human heparanase. Using hpa cDNA, some or synthetic oligonucleotides designed according to these sequences allow one skilled in the art to identify genomes and / or cDNA clones containing similar sequences from other mammals.

Accordingly, the present invention further relates to hpa cDNA, in particular base nucleotide conjugation under stringent or tolerant hybrid conditions; which can hybridize to nucleotides 1-721 of SEQ ID NO: 9; for example, Sambrool, J., Fritsch, EF, Maniatis, T. (1989) Molecular Cloning.A Laboratory Manual.cold Spring Harbor Laboratory Press, New York.) Relates to a polynucleotide segment comprising a polynucleotide sequence.

In fact, it encodes a polypeptide having heparanase activity and is at least 60% homologous to SEQ ID NO: 9 or SEQ ID NO: 14, preferably at least 70% homologous, more preferably at least 80% homologous, most Preferably any polynucleotide sequence that shares at least 90% homology is within the scope of the present invention.

The polynucleotide sequence according to the invention may comprise any part of SEQ ID NO: 9 or SEQ ID NO: 14. For example, it may comprise nucleotides 63-721 of the novel sequence SEQ ID NO: 9. However, it can include any sequence of SEQ ID NO: 9 or SEQ ID NO: 14 encoding a polypeptide having heparanase catalysis activity.

When the phrase “encoding a polypeptide having heparanase catalytic activity” is used herein and in the claims section below, it refers to the ability to direct the synthesis of the polypeptide and, if necessary, the following subsequent translation. Post-modification (eg, but not limited to proteolysis (removing signal peptide and pro- or preprotein sequences), methionine modification, glycosylation, alkylation (eg methylation), acetylation, etc.). For example, it is catalytically active in the degradation of ECM and HS related cell surfaces.

In a preferred embodiment of the invention, the polypeptide encoded by the polynucleotide segment comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 15 and a portion of their action, ie a portion retaining heparanase catalytic activity.

However, any polynucleotide segment encoding a polypeptide having heparanase activity is within the scope of the present invention. Thus, the polypeptide may be an allele, species and / or derived variant of the amino acid sequence according to SEQ ID NO: 10 or SEQ ID NO: 15 or a functional part thereof.

In fact, at least 60% homology, preferably at least 70% homology, more preferably at least 80% homology, most preferably at least 100% homologous to the polypeptide coding or having SEQ ID NO: 10 or SEQ ID NO: 15 having heparase activity Any polynucleotide having 90% homology is within the scope of the present invention.

The present invention also provides a single strand of polynucleotide segments comprising a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having a heparanase catalysis activity as described above. As used herein, the term "complementary" refers to base confrontation.

Segments of polynucleotides of a single strand can be DNA or RNA or can include nucleotide analogues (eg, thiolated nucleotides), which can be synthetic oligonucleotides or can be prepared by a transgenic host cell, It may be of any desired length to provide base pairings (eg, 8 or 10, preferably longer nucleotides), and may include inappropriate pairs that do not limit base pairings.

In addition, the present invention relates to providing a vector having a polynucleotide sequence encoding a polypeptide that exhibits heparanase catalysis activity.

The vector can be in any form. This may be a bacteriophage or a virus that infects eukaryotic cells that infects bacteria or a virus that infects eukaryotic cells. It may also be a plasmid, phagemid, cosmid or artificial chromosome. The polynucleotide sequence encoding the polypeptide having heparanase catalysis activity may comprise some of the polynucleotide segments described above.

Further, the present invention relates to providing a host cell comprising an exogenous polynucleotide segment encoding a polypeptide having heparanase catalysis activity.

The exogenous polynucleotide segment can be any of the segments described above. The host cell can be in any form. It can be a protozoan cell, eukaryotic cell, cell line or cell as part of an organism. Exogenous polynucleotide segments may be present permanently or temporarily in cells. Stated differently, transduced cells obtained by the following stable or transient transfection, transformation or transduction are within the scope of the present invention. As used herein, the term “exogenous” refers to the introduction of a polynucleotide segment externally into a cell. Among them, if it is present in one of any number of copies, it may be integrated into one or more chromosomes at any position or exist as an external chromosomal material.

Further, the present invention is directed to providing a heparanase overexpression system comprising cells overexpressing heparanase catalysis activity. Cells are transiently or stably transfected or transformed into any suitable vector comprising a polynucleotide sequence encoding a polypeptide having heparanase activity and a suitable promoter and enhancer sequence for direct overexpression of heparanase. May be a host cell. However, the overexpressed cells can also be the product of an injection (eg, the same recombination) of the promoter and / or enhancer sequence downstream to the endogenous heparanase gene of the expressing cell, which can be directly overexpressed from the endogenous gene. . The term “overexpression” as used in the specification herein and in the claims below, refers to a degree of expression that is greater than the basic degree of expression that typically characterizes cells designated under the same conditions.

Further, the present invention relates to providing a recombinant protein comprising a polypeptide having heparanase catalysis activity.

Recombinant proteins may be purified and / or mixed with additives by any conventional protein purification process that is closely related to homogeneity. Recombinant protein can be prepared using any of the cells described above. Recombinant protein may be in any form. It may be in crystallized form, dehydrated powder form or in solution form. Recombinant proteins are useful for protruding both anti-heparanase antibodies, antibodies of multiple or monoclonal, and may be useful as anti-heparanase inhibitors or as screening active ingredients in drug screening assays or systems. It may be useful to obtain.

Further, the present invention relates to providing a pharmaceutical composition comprising as an active ingredient a recombinant protein having heparanase catalysis activity as the active ingredient.

Formulations for topical administration include, but are not limited to, lotions, ointments, gels, creams, suppositories, eye drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powdered or oily substrates, pressure sensitive adhesives and the like may be necessary or desirable. Coated condoms, stents, active pads and other medical devices may be useful. Indeed, the scope of the present invention includes any medical device as a medical device comprising a recombinant protein having heparanase catalysis activity as an active ingredient.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules or tablets. Tackifiers, diluents, flavors, dispersion aids, emulsifiers or binders may be preferred.

Formulations for parenteral administration include, but are not limited to, buffers, diluents and other suitable additives.

Dosing may depend on the variety and sensitivity of the condition to be treated, and may generally be administered one or more times daily, and the duration of treatment lasts for days to months, or when treatment is effective or until the disease state is reduced. do. One skilled in the art can readily determine the optimal dosage, method of administration and number of repetitions.

In addition, the present invention provides a method for identifying a chromosomal region comprising a human heparanase gene in a distributed chromosome. This method is carried out according to the following steps: In the first step, the distributed chromosomes (both distributed mutually or transfected) are hybridized to the indicated polynucleotide probes encoding heparanases. The display is preferably a fluorescent display. In a second step according to the method, the distributed chromosomes are washed to remove excess non-hybridized probes. Finally, find a signal associated with the hybridized indicated polynucleotide probe, where the detected signal represents a chromosomal region comprising the human heparanase gene. Those skilled in the art will know how to use the sequences mentioned herein in appropriate labeling reactions and how to use labeled probes to detect chromosomal regions containing human heparanase genes using in situ hybrids.

With reference to the following examples together with the above description, it will be described without limiting the invention.

Details of the following protocols and experiments can be found in the following examples:

Purification and Characterization of Heparanases from Human Liver Cancer Cell Lines and Human Placenta: The human liver cancer cell line (SK-Hep-1) has been selected as a source for purification of human tumor induced heparanases. Purification is described in US Pat. No. 5,362,641 to Fuk, which is incorporated herein by reference. Briefly, 500 liters of 5 × 10 ¹¹ cells were grown in suspension and the heparanese enzyme was purified about 240,000-fold by applying the following steps: (i) in a NaCl gradient of 0.3-1.4 M at pH 6.0. Cation (CM-Sephadex) chromatography is performed; (ii) performing cation exchange (CM- Sephadex) chromatography at pH 7.4 using a 0.3 to 1.1 M NaCl gradient in the presence of 0.1% CHAPS; (iii) performing heparin-sepharose chromatography at pH 7.4 using a gradient of 0.35 to 1.1 M NaCl in the presence of 0.1% CHAPS; (iv) performing ConA-Sepharose chromatography using 0.25M α-methylmannoside at water pH 6.0 in the presence of a buffer comprising 0.1% CHAPS and 1M NaCl; (v) HPLC cation exchange (Mono-S) chromatography was performed at pH 7.4 using a NaCl gradient of 0.25-1 M in the presence of 0.1% CHAPS.

The active fractions were collected, precipitated with TCA and the precipitate was subjected to SDS polyacrylamide gel electrophoresis and / or trypsin digestion and reversed phase HPLC. Trypsin peptides of the purified proteins were separated by reverse phase HPLC (C8 column) and homogeneous peaks were amino acid sequenced.

Purified enzyme was applied to reverse phase HPLC and N-terminal amino acid sequencing using an amino acid sequencer (Applied Biosystems).

Cells: Cultures of bovine corneal endothelial cells (BCEC) were obtained from bovine eye as previously described (19, 38). Parent cultures were maintained in DMEM (1 g glucose / liter) filled with 10% neonatal serum and 5% FCS. bFGF (1 ng / ml) was added every other day during the phase of active cell growth (13, 14).

Preparation of dishes coated with ECM: BCEC (2-5th pass) was plated in 4-well plates at an initial density of 2 x 10 ⁵ cells / ml, and sulfate added with 5% dextran T-40 for 12 days Incubated in Fischer medium without. After seeding, Na ₂ ³⁵ SO ₄ (25 μCi / ml) was added on days 1 and 5, and the cultures were incubated with labels without changing medium. Endothelial ECM was exposed by dissolving the cell layer with PBS containing 0.5% Triton X-100 and 20 mM NH ₄ OH (5 min, room temperature) by dissolving the four washes with PBS. The ECM was left intact without cell debris and firmly attached to the entire area of the tissue culture dish (19, 22).

To prepare soluble sulfate labeled proteoglycans (peak I material), ECM was digested with trypsin (25 μg / ml, 6 hours, 37 ° C.), the digest was concentrated by reverse dialysis and the concentrated material was sepharose ( Sepharose) was applied onto a 6B gel filtration column. The resulting high molecular weight material (Kav &lt; 0.2, peak I) was collected. More than 80% of the labeled material was shown to consist of heparan sulfate proteoglycans (11, 39).

HEPA Raney Izu activity: Cells (1 × 10 ^6/35 mm dish), cell lysates or conditioned medium for the cultured on top of the ³⁵ S- labeled ECM in the presence of 20 mM phosphate buffer (pH 6.2) (18 sigan , 27 ° C.). Cell lysates and conditioned media were also incubated with sulfate labeled peak I material (10-20 μl). Culture medium was harvested, centrifuged (18,000 × g, 4 ° C., 3 min) and sulfate labeled material was analyzed by gel filtration on Sepharose CL-6B column (0.9 × 30 cm). Fractions (0.2 ml) were eluted with PBS at a flow rate of 5 ml / hour and counted for radioactivity using a Bio-fluor scintillation fluid. Exclusion volume (V ₀ ) is indicated by blue dextran and total inclusion volume (V _t ) is indicated by phenol red. The latter appears to move with the free sulfate (7, 11, 23). Digestion fragments of the HS side chain were eluted from Sepharose 6B at 0.5 &lt; Kav &lt; 0.8 (peak II) (7, 11, 23). Nearly native HSPG released by trypsin (and only low during incubation with PBS) eluted close to V ₀ (Kav <0.2, peak I). The recovery of the labeled material applied on the column ranges from 85 to 95% in other experiments (11). Each experiment was performed three or more times and the change in elution position (Kav value) did not exceed +/- 15%.

Cloning of hpa cDNA: cDNA clones 257548 and 260138 were identified in I.M.A.G.E. From the Association (Huntsville Memorial Parkway SW 2130, Alabama). cDNA was first cloned at the EcoRI and NotI cloning sites in the plasmid vector pT3TD-Pac. These clones are reported to be somewhat different, but DNA sequencing demonstrated that these clones were identical to each other. Marathon RACE (fast amplification of cDNA termini) The human placental (poly-A) cDNA complex is provided by Professor Yossi Shiloh of Tel Aviv University. This complex is free of vectors because the duplex, partially single stranded adapter comprises reverse transcription cDNA fragments attached on both sides. The structure of the specific composites used is described in reference 39a.

Amplification of the hp3 PCR fragment was performed according to the protocol provided by Clontech laboratories. The template for use in the amplification was a sample obtained from the complex. Primers used for amplification are as follows:

First step: 5'-primer: API: 5'-CCATCCTAATACGACTCACTATAGGGC-3 ', SEQ ID NO: 1; 3'-primer: HPL229: 5'-GTAGTGATGCCATGTAACTGAATC-3 ', SEQ ID NO: 2.

Second step: nested 5′-primer: AP2: 5′-ACTCACTATAGGGCTCGAGCG GC-3 ′, SEQ ID NO: 3; Nested 3'-primer: HPL171: 5'-GCATCTTAGCCGTCTTTCTTCG-3 ', SEQ ID NO: 4. HPL229 and HPL171 were selected according to the sequence of the EST clone. These include nucleotides 993-956 and 876-897 of SEQ ID NO: 9, respectively.

The PCR program was performed 30 times at 94 ℃-4 minutes, 94 ℃-40 seconds, 62 ℃-1 minutes, 72 ℃-2.5 minutes. Amplification was performed with Expand High Fidelity (Boeringer Mannheim). The resulting about 900 bp hp3 PCR product was digested with BfrI and PvuII. Clone 257548 (phpa1) was digested with EcoRI, then terminally charged and then further digested with BfrI. The pvuII-BfrI fragment of the hp3 PCR product is then cloned into the blunt end-BfrI end of clone phpa1, which has the full cDNA cloned in the pT3T7-pac vector, called phpa2.

DNA Sequencing: Sequencing was performed with vector specific and gene specific primers using an automated DNA sequencer (Applied Biosystems, Model 373A). Each nucleotide was read from two or more independent primers.

Computer Analysis of Sequences: Database studies for sequence similarity were performed using Blast network service. Sequence and alignment of DNA and protein sequences was performed using a DNA sequence analysis software package developed by the Genetic Computer Group (GCG) of the University of Wisconsin.

RT-PCR: RNA was prepared using TRI-Reagent (Molecular research center Inc.) according to manufacturer's instructions. 1.25 μg was taken for reverse transcription reaction using MuMLV reverse transscriptase (Gibco BRL) and Oligo (dT) ₁₅ primer, SEQ ID NO: 5 (Promega). Amplification of the resulting first strand cDNA was performed with Taq polymerase (promega). The following primers were used:

HPU-355: nucleotides 372-394 in 5'-TTCGATCCCAAGAAGGAATCAAC-3 ', SEQ ID NO: 6, SEQ ID NO: 9 or SEQ ID NO: 11.

HPL-229: nucleotides 993-956 in 5'-GTAGTGATGCCATGTAACTGAATC-3 ', SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11.

PCR program: 94 ° C.-4 min, then 94 ° C.-40 sec, 62 ° C.-1 min, 72 ° C.-1 min 30 times.

Expression of Recombinant Heparanase in Insect Cells: Cells, High Five and Sf2 insect cell lines were maintained as single layer culture in SF900II-SFM medium (GibcoBRL).

Recombinant Baculovirus: A recombinant virus containing the hpa gene was prepared using the Bac versus Bac system (GibcoBRL). The delivery vector pFastBac was digested with SalI and NotI and linked with a 1.7 kb fragment of phpa2 digested with XhoI and NotI. The resulting plasmid was called pFasthpa2. The same plasmid, referred to as pFasthpa4, was prepared as a duplicate, both independently used for further experiments. Recombinant bacmids were generated with pFasthpa2, pFasthpa4 and pFastBac according to the manufacturer's instructions. The latter was used as negative control. Recombinant bacmid DNA was transfected into Sf21 insect cells. Five days after transfection, recombinant viruses were obtained and infected with high five insect cells of 3 × 10 ⁶ cells in a T-25 flask. Cells were obtained 2-3 days after infection. 4 × 10 ⁶ cells were centrifuged and resuspended in reaction buffer containing 20 mM phosphate citrate buffer, 50 mM NaCl. Cells were frozen and thawed three times and lysates were stored at -80 ° C. The conditioned medium was stored at 4 ° C.

Partial Purification of Recombinant Heparanese: Partial purification of recombinant heparanese was performed by heparin-Sepharose column chromatography followed by Superdex 75 column gel filtration. Culture medium (150 ml) of Sf21 cells infected with pFhpa4 virus was subjected to heparin-sepharose chromatography. Elution of 1 ml fractions was performed with a 0.35 to 2 M NaCl gradient in the presence of 0.1% CHAPS and 1 mM DTT in 10 mM sodium acetate buffer (pH 5.0). 25 μl samples of each fraction were tested for heparanase activity. Heparanase activity was eluted in the range of 0.65 to 1.1 M NaCl (fractions 18 to 26, FIG. 10A). 5 μl of each fraction was stained with 15% SDS-polyacrylamide gel electrophoresis followed by silver nitrate. The active fraction eluted from heparin-sepharose (FIG. 10A) was collected and concentrated on YM3 cleaved membrane (× 6). 0.5 ml of concentrated material was applied onto a 30 ml Superdex 75 FPLC column equilibrated with 10 mM sodium acetate buffer (pH 5.0) containing 0.8 M NaCl, 1 mM DTT and 0.1% CHAPS. Fractions (0.56 ml) were collected at a flow rate of 0.75 ml / min. Aliquots of each fraction were tested for heparanase activity, SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining (FIG. 11B).

Example 1

cloning of hpa genes

Purified fractions of heparanese (SK-hep-1) isolated from human liver cancer cells were trypsin digested and microsequenced. An EST (expressed sequence tag) database was screened for homology to the retranslated DNA sequence corresponding to the obtained peptide. Two EST sequences (Accession Numbers N41349 and N45367) contained a DNA sequence encoding peptide YGPDVGQPR (SEQ ID NO: 8). These two sequences were derived from clones 257548 and 260138 (I.M.A.G.E.association) prepared from 8-9 week placental cDNA libraries (Soares). The two clones found to be identical contained an open read flame (ORF) of 973 bp, followed by a 1020 bp insert comprising a poly ′ tail and a 3 ′ unread region of 27 bp. Detoxification start site (AUG) was not identified at the 5 'end of these clones.

Cloning of the missing 5 'end was performed by PCR amplification of DNA from placental marathon RACE cDNA complex. A 900 bp fragment (referred to as hp3) was obtained which partially overlaps with the identified 3 'encoding EST clone.

The linked cDNA fragment (1721 bp in length) (SEQ ID NO: 9) encodes a 543 amino acid polypeptide (SEQ ID NO: 10) with a calculated molecular weight of 61,192 Dalton as shown in FIG. 1 and SEQ ID NO: 11. It contained an open read flame. The 3 'end of the partial cDNA inserts contained in clones 257548 and 260138 started at SEQ ID NO: 9 and nucleotide G ⁷²¹ in FIG.

As also shown in FIG. 1, there was a single sequence mismatch between the EST clone and the PCR amplified sequence, which resulted in an amino acid substitution from Tyr ²⁴⁶ in EST to Phe ²⁴⁶ in amplified cDNA. The nucleotide sequence of the PCR amplified cDNA fragment was demonstrated from two independent amplification products. The new gene was referred to as hpa.

As indicated above, the 3 'end of the partial cDNA insert contained in EST clones 257548 and 260138 started at nucleotide 721 of hpa (SEQ ID NO: 9). The ability of hpa cDNA to form a stable secondary structure, eg, a stem and loop structure containing nucleotide extension in the vicinity of position 721, was investigated using computer modeling. Stable stem and loop structures have been found to contain nucleotides 698-724 (SEQ ID NO: 9) and are prone to formation. In addition, a high GC content of up to 70% is characteristic of the 5 'terminal region of the hpa gene as compared to only about 40% in the 3' region. This finding may explain the early termination, thus lacking the 5 'end in the EST clone.

To investigate the ability of the hpa gene product to promote the degradation of heparan sulphate in an in vitro assay, the entire open reading frame was expressed in insect cells using the Baculovirus expression system. Extracts of cells infected with viruses containing the hpa gene demonstrate high levels of heparan sulfate degradation activity, but cells infected with similar constructs that do not contain the hpa gene show no activity and do not have uninfected cells. These results are also illustrated in the following examples.

Example 2

Degradation of Soluble ECM-induced HSPG

Monolayer cultures of high five cells were infected with recombinant baculoviruses containing the pFasthpa plasmid, or control virus containing the plasmid without insertion. Cells were obtained, frozen and thawed three times and lysed in heparanase reaction buffer. The lysates were then incubated with sulfate labeled, ECM induced HSPG (Peak I) (18 hours, 37 ° C.) and the reaction mixture was gel filtered analyzed (Sepharose 6B).

As shown in FIG. 2, the substrate contained mostly high molecular weight (Mr) material, eluted close to V ₀ alone (peak I, fractions 5-20, Kav <0.35). Similar elution patterns were obtained when the HSPG substrate was incubated with lysates of cells infected with the control virus. In contrast, incubating HSPG substrates with lysates of cells infected with virus containing hpa completely converted the high Mr substrates into low Mr labeled degradation fragments (peak II, fractions 22-35, 0.5 <Kav <0.75). .

The fragment eluted with peak II is (i) 5-6 times smaller than the native heparan sulfate side chain released from ECM by treatment with alkaline borohydride or papain (Kav about 0.33) and (ii) papain or chondroitinase. It has been shown to be a degradation product of heparan sulphate because it is resistant to further degradation to ABC and is easy to deamine with nitric acid (6, 11).

Similar results (not shown) were obtained in Sf21 cells. Again, heparanase activity was detected in cells infected with virus containing hpa (pFhpa), but not in those containing no control virus (pF). This result was obtained from two independently generated recombinant viruses. Lysates of control uninfected high five cells did not degrade the HSPG substrate.

In subsequent experiments, labeled HSPG substrates were incubated in media regulated by infected high five or Sf21 cells.

As shown in FIGS. 3A-B, heparanase activity found by converting a high Mr peak I substrate to a low Mr peak II representing HS degraded fragments was found in the culture medium of cells infected with pFhpa2 or pFhpa4 virus, but control It was not found in either pF1 or pF2 virus. No heparase activity was detected in the culture medium of control uninfected high five or Sf21 cells.

The medium of cells infected with the pFhpa4 virus was passed through a 50 kDa cleaved membrane to obtain a recombinant heparanese enzyme of the original molecular weight. As described in FIG. 4, all enzymatic activity was retained in the upper compartment and no activity in the flow passage (<50 kDa) material. This result is consistent with the expected molecular weight of the hpa gene product.

In order to further characterize the hpa product as an inhibitory effect of heparin, a potent inhibitor 40 of heparanase mediated HS degradation was investigated.

As described in FIGS. 5A-B, conversion of the peak I substrate to the peak II HS digested fragments was completely removed in the presence of heparin.

Overall, these results show that heparanase enzymes are expressed in active form by insect cells infected with baculoviruses containing the newly identified human hpa gene.

Example 3

Disassembly of HSPG from Intact ECM

Next, the original infected insect cells were examined for their ability to degrade HS in native, naturally produced ECM. For this purpose, high five or Sf21 cells were seeded on metabolic sulfate labeled ECM and then infected with pFhpa4 or pF2 virus (48 h, 28 ° C.). The pH of the medium was then adjusted to 6.2-6.4 and the cells were further incubated with labeled ECM for 48 hours at 28 ° C. or for 24 hours at 37 ° C. Sulfate labeled material released into the culture medium was analyzed by gel filtration on Sepharose 6B.

As shown in FIGS. 6A-B and 7A-B, incubating ECM with cells infected with control pF2 virus was a label consisting mostly of high (> 90%) high Mr fragments (peak I) eluting at or near V ₀ . The released material was released constantly. It has previously been shown that the proteolytic activity present in the ECM itself and / or the ECM exerted by the cells is responsible for the release of high Mr material (6). This nearly intact HSPG provides a soluble substrate for subsequent degradation by heparanases, which is also manifested by the relatively large amount of peak I material that accumulates when the heparanase enzyme is inhibited by heparin (6 , 7, 12, FIG. 9). On the other hand, culturing the labeled ECM with cells infected with the pFhpa4 virus resulted in the formation of low Mr sulfate labeled fragments (Peak II, 0.5 〈Kav ≦ 0.75) regardless of whether the infected cells were cultured with ECM at 28 ° C. or 37 ° C. To release 60-70% of the ECM-related radioactivity. Uninfected Sf21 or high five cells in the control intact state did not degrade the ECM HS side chain.

In subsequent experiments, as described in FIGS. 8A-B, high five and Sf21 cells were infected with pFhpa4 or control pF1 virus (96 h, 28 ° C.), and the culture medium was incubated with sulfate labeled ECM. Low Mr HS digested fragments were released from ECM only when incubated in media controlled by pFhpa4 infected cells. As shown in FIG. 9, the generation of these fragments was removed in the presence of heparin. No activity of heparanase was detected in the culture medium of control uninfected cells. These results indicate that other macromolecular components of the native ECM, which are naturally produced in a manner similar to that reported by the herapanase enzyme expressed by cells infected with pFhpa4 virus, for highly metastable tumor cells or activated cells of the immune system. (Ie, fibronectin, laminin, collagen), which shows the ability to degrade HS (6, 7).

Example 4

Purification of Recombinant Heparanase

Recombinant heparanese was partially purified from the media of pFhpa4 infected Sf21 cells by heparin-sepharose chromatography (FIG. 10A) followed by gel filtration of the active fractions collected on FPLC Superdex 75 column (FIG. 11A). Proteins up to 63 kDa were observed and their amount detected by silver stained SDS-polyacrylamide gel electrophoresis correlated with heparanase activity in the relevant column fractions (FIGS. 10B and 11B, respectively). This protein was not detected in the culture medium of cells infected with control pF1 and similar fractionation on Sephalin-Sepharose was performed (not shown).

Example 5

Expression of hpa genes in multiple cell types, organs and tissues

Referring now to FIGS. 12A-E, RT-PCR was applied to assess expression of hpa genes by various cell types and tissues. For this purpose, total RNA was reverse transcribed and amplified. Expected 585 bp in length of cDNA was clearly evident in freshly isolated and short-lived (1.5-48 hours) cultured human placental cytotrophic cells (FIG. 12A) as well as in human kidney, placenta (weeks 8 and 11) and tissue tissue. As described, they are all known to express high heparanase activity (41). hpa transcription was also expressed by normal human neutrophils (FIG. 12B). In contrast, expression of hpa mRNA could not be detected in embryonic human muscle tissue, thymus, heart and adrenal gland (FIG. 12B). Multiple (but not all) hpa genes Human bladder cancer cell line (FIG. 12C), SK liver cancer (SK-hep-1), ovarian cancer (OV 1063), breast cancer (435, 231), melanoma and megakaryocytes (DAMI , CHRF) human cell line (Fig. 12D-E).

The above described expression pattern of hpa transcription was determined to have a very good correlation with the level of heparanase activity measured in various tissues and cell types.

Example 6

hpa homology genes

The EST database was screened for sequences homologous to the hpa gene. Three mouse ESTs were identified (accession number Aa177901 from mouse spleen, Aa067997 from mouse skin, Aa47943 from mouse fetus), partially open reading flame (lacking the 5 'end) and 195 bp of 3' undecrypted Assembled with 824 bp cDNA fragment containing the region (SEQ ID NO: 13). As shown in Figure 13, the coding region is 80% similar to the 3 'end of the hpa cDNA sequence. These ESTs will be cDNA fragments of the mouse hpa homologs that encode for mouse herrapase.

The study of concordant protein domains has shown that the amino terminal phase between herapanase and several precursor proteins such as procollagen alpha 1 precursor, tyrosine-protein kinase-RYK, fibulin-1, insulin-like growth factor binding protein and others Revealed the same sex. The amino termini are very hydrophobic and contain potential intermembrane domains. Homology to known signal peptide sequences indicated that it could act as a signal peptide for protein positioning.

Example 7

Isolation of Extended 5 ′ Terminal of hpa cDNA from Human SK-hep 1 Cell Line

The 5 'end of the hpa cDNA was isolated from the human SK-hep 1 cell line by PCR amplification using a Marathon RACE (propagation amplification of cDNA ends) kit (Clontech). All RNA was prepared from SK-hep 1 cells using TRI-reagent (Molecular research center Inc.) according to the manufacturer's instructions. Poly A + RNA was isolated using mRNA Separator Kit (Clontech).

Marathon RACE SK-hep 1 cDNA complex was prepared according to manufacturer's recommendations. First, hpa specific antisense primer hpl-629: 5'-CCCCAGGAGCAGCAGCATCAG-3 ', sequence corresponding to adapter specific primer AP1: 5'-CCATCCTAATACGACTCACTATAGGGC-3', SEQ ID NO: 1, and nucleotides 119-99 of SEQ ID NO: 9 Amplify circularly using No. 19. The resulting PCR product was then followed by hpa specific antisense nested primers corresponding to nucleotides 83-63 of adapter specific nested primers AP2: 5'-ACTCACTATAGGGCTCGAGCGGC-3 ', SEQ ID NO: 3, and SEQ ID NO: 9. hpl-666: 5'AGGCTTCGAGCGCAGCAGCAT-3 ', SEQ ID NO: 20, to amplify circularly. The PCR program is as follows: 30 min of cycles of 68 ° C. for 4 seconds at 90 ° C. for 30 seconds were performed after 1 minute of starting at a high temperature of 94 ° C. The resulting 300 bp DNA fragment was extracted from agarose gel and cloned into vector pGEM-T Easy (Promega). The resulting recombinant plasmid was designated pHPSK1.

By measuring the nucleotide sequence of the pHPSK1 insert, it was found that it contained 62 nucleotides (SEQ ID NO: 9) at the 5 'end of the placental hpa cDNA and an additional 178 nucleotides upstream (where the sequence numbers of the first 178 nucleotides were 13 and 15).

A single nucleotide mismatch between the SK-hep1 cDNA and the placental cDNA was found to appear. The "T" derivative at position 9 (SEQ ID NO: 9) of placental cDNA was replaced with the "C" derivative at corresponding position 187 (SEQ ID NO: 14) of SK-hep1 cDNA.

This discrepancy is easily caused by mutations at the 5 'end of the placental cDNA clone, as confirmed by sequencing of some additional cDNA clones isolated from the placenta, which result in SK containing C at position 9 of SEQ ID NO: 9 -hep1 cDNA.

The 5 'extended sequence of SK-hep1 hpa cDNA was assembled with the sequence of hpa cDNA (SEQ ID NO: 9) isolated from the human placenta. The associated sequence contained an open reading frame encoding a polypeptide of 592 amino acids calculated at a molecular weight of 66,407 daltons as shown in SEQ ID NOs: 14 and 15. The open read frame is placed on the side of the 5 'untranslated region (UTR) of 93 bp.

Example 8

Isolation of Upstream Genomic Regions of the hpa Gene

The upstream region of the hpa gene was isolated using the Genome Walker kit (Clontech) according to the producer's recommendations. The kit includes five human genomic DNA samples digested with different restriction endonuclease producing blunt ends, respectively.

Blunt-terminated DNA fragments were bound to some single helix adapter. Genomic DNA samples were PCR amplified using adapter specific primers and gene specific primers. Amplification was performed using Expand High Fidelity (Boehringer Mannheim).

First, hpa specific antisense primers hpl-666: 5'-AGGCTTCGAGCGCAGCAGCAT-3 'corresponding to ap1 primer: 5'-GTAATACGACTCACTATAGGGC-3', SEQ ID NO: 21, and nucleotides 83-63 of SEQ ID NO: 9 Amplified circularly using 20. The PCR program was as follows: 36 cycles of 94 ° C. for 40 seconds and 67 ° C. for 4 minutes were performed after a high temperature of 94 ° C. for 3 minutes.

The PCR product of the first amplification was diluted 1:50. 1 μl of the diluted sample was nested adapter specific primers ap2: 5′-ACTATAGGGCACGCGTGGT-3 ′, SEQ ID NO: 22, and hpa specific antisense primers hpl-690: corresponding to nucleotides 62-42 of SEQ ID NO: 9. 5'-CTTGGGCTCACCTGGCTGCTC-3 ', SEQ ID NO: 23, was used as a template for the second amplification process. The resulting amplification product was analyzed using agarose gel electrophoresis. Five different PCR products were obtained from five amplification reactions. Approximately 750 bp of DNA fragment obtained from the SspI digested DNA sample was gel extracted. Purified fragments were incorporated into plasmid vector pGEM-T Easy (Promega). The resulting presynthesis plasmid was designated pGHP6905 and the nucleotide sequence of the hpa insert was measured.

Some sequences of 594 nucleotides are shown in SEQ ID NO: 18. The final nucleotide in SEQ ID NO: 14 corresponds to nucleotide 93 in SEQ ID: 13. The DNA sequence in SEQ ID NO: 18 contains 501 nucleotides of the genomic upstream region that is predicted to contain the 5 'region of the hpa cDNA, and the promoter region of the hpa gene.

Example 9

Expression of 592 amino acid HPA polypeptides in human 293 cell line

A 592 amino acid open reading frame (SEQ ID NOs: 14 and 15) was prepared by conjugating 110bp corresponding to the 5 'end of SK-hep 1 hpa cDNA with placental cDNA. More specifically, the marathon RACE-PCR amplification product of placental hpa DNA was digested with SacI and about 1 kb fragment was incorporated into the SacI-digested pGHP 6905 plasmid. The resulting plasmid was digested using EarI and AatII. The viscous end of EarI was blunted and an EarI / blunt-AatII fragment of about 280 bp was isolated. The fragments were ligated with pFasthpa digested with the end blunted EcoRI and further digested with AatII using Klenow fragments. The resulting plasmid contained an 1827 bp open reading frame, 31 bp 3 'UTR and 21 bpdml 5' UTP insert. The plasmid was designated pFastLhpa.

The stray expression vectors were prepared to induce the expression of 592 amino acid heparanase polypeptides in human cells. hpa cDNA was digested with prom pFastLhap using BssHII and NotI. The resulting 1850 bp BssHII-NotI fragment was ligated into the mammalian fragment vector pSI (promega) digested with MluI and NotI. The resulting recombinant plasmid pSIhpaMet2 was transferred to human 293 fetal kidney cell line.

Transient expression of 592 amino acid heparanases was examined by western blot analysis, and enzyme activity was tested using a gel transfer assay. All of these procedures are verbosely described in US patent application Ser. No. 09 / 071,739, filed May 1, 1998, which is fully disclosed and incorporated herein by reference. Cells were cultured for 3 days and then metastasized. The cultured cells are resuspended in digestion buffer containing 150 mM NaCl, 50 mM Tris (pH 7.5), 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail (Boehringer Manaim). I was. 40 μg of protein extract was used to separate on SDS-PAGE. Proteins were transferred onto PVDF Hybond-P membrane (Amersham). Membranes were incubated with affinity purified-polyclonal anti-heparanase antibodies as described in US Patent Application Serial No. 09 / 071,739. Not only was a major band of about 50 kDa found in the metastasized cells, but a minor band of about 65 kDa was found in the metastasized cells. Similar patterns have been found in extracts of cells that have been metastasized using pShpa as discussed in US Patent Application Serial No. 09 / 071,739. These two bands represent two forms of recombinant heparanase protein produced by metastasized cells. A 65 kDa protein is probably representative of the heparanase precursor, while a 50 kDa protein is thought to represent a progressive or mature form.

The catalytic activity of the recombinant protein expressed in pShpaMet2 transferred cells was tested by gel transfer assay. Cell extracts of metastasized cells and cell extracts of pseudo-metastatic cells were heparin at 37 ° C. in the presence of 20 mM phosphate citrate buffer (pH 5.4), 1 mM CaCl ₂ , 1 mM DTT and 50 mM NaCl (each Incubated overnight using 6 μg per reaction). The reaction mixture was then separated on 10% polyacrylamide gel. The catalytic activity of recombinant heparanese was clearly demonstrated to move rapidly compared to heparin molecules cultured with the metastasized cell extract when compared to the control. Faster migration indicates the loss of high molecular weight heparin molecules and the production of low molecular weight degradable products.

Example 10

Chromosome localization of the hpa gene

Chromosomal mapping of the hpa gene was performed using a panel of monochromosomal human / CHO and human / mouse somatic cell hybrids, obtained from the UK HGMP Research Center (Cambridge, UK).

Forty ng of each somatic cell hydride DNA sample was subjected to PCR amplification using the following hpa primers: hpu565 5'-AGCTCTGTAGATGTGCTATACAC-3 ', SEQ ID NO: 24, corresponding to nucleotides 564-586 of SEQ ID NO: 9 And antisense primer hpl 171 corresponding to nucleotides 897-876 of SEQ ID NO: 9 '5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO: 25.

The PCR program is as follows: run at a high temperature of 94 ° C. for 3 minutes starting, followed by seven cycles of 94 ° C. for 45 seconds, 66 ° C. for 1 minute, 68 ° C. for 5 minutes, followed by 45 seconds The cycle of 94 ° C., 62 ° C. for 1 minute, 68 ° C. for 5 minutes was repeated 30 times and finally extended at 72 ° C. for 10 minutes.

The reaction was performed using Expand long PCR (Möllinger Mannheim). The resulting amplification product was analyzed using agarose gel interlock. As demonstrated in FIG. 14, a single band of about 2.8 Kb was obtained from chromosome 4 as well as control human genomic DNA. Amplification products of 2.8 kb are predicted based on amplification of genomic hpa clones (data not shown). No amplification products were obtained in control DNA samples of hamsters and mice or in somatic hybrids of other human chromosomes.

Although the present invention has been described in connection with specific embodiments, numerous other methods, modified methods, and modified methods will be apparent to those skilled in the art. Accordingly, it is intended to embrace such other methods, variations and variations as fall within the spirit and broad scope of the appended claims.

List of references cited herein

1.Wight, TN, Kinsella, MG and Qwarnstromn, EE, "The role of proteoglycans in cell adhesion. , migration and proliferation. Curr. Opin. Cell Biol., 4, 793-801 (1992).

2. Jackson, RL, Busch, SJ, and Cardin, AL. "Glycosaminoglycans: Molecular properties (Glycosaminoglycans: Molecular properties) , protein interactions and role in physiological processes. Physiol. Rev., 71, 481-539 (1991).

3. Wight, “Cell biology of arterial proteoglycans. Arteriosclerosis, 9, 1-20 (1989)”.

4. Kjellen, L. and Lindhl, U., "Proteoglycans: structures and interactions. Annu. Rev. Biochem., 60, 443-475 ( 1991).

5. Ruoslahti, E. and Yamaguchi, Y., “Proteoglycans as modulators of growth factor activities. Cell, 64, 867-869. (1991) ".

6. Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A., Matsner, Y., Ishai-Michaeli , R.), Levi, E., Bashkin, P., Leader (Lider, O.), Naparstek, Y., Cohen, InR., And Fuchs ( Fuks, Z., "Expression of heparanase by platelets and circulating cells of the immune system: Possible involvement in diapedesis and extravasation. Invasion & Metastasis, 12, 112-127 (1993).

7. Blodowski, Mohsen, M., leader, Ishai-Mikaeli, Elk (Ekre, J.-P.), Svahn, CM, Vigoda, M. and Peretz ( Peretz, T., "Inhibition of tumor metastasis by heparanase inhibitiong species of heparin. Invasion & Metastasis, 14, 290-302 (1995)".

8. Nakajima (M.), Irimura, T. and Nicolson, GL, "Heparanase and tumor metastasis, J. Cell, Biochem., 36, 157-167 (1988) ".

9. Nicholson et al., "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 (1988).

10. Liotta, LA, Rao, CN and Barsky, SH, "Tumor invasion and the extracellular matrix. Lab. Invest., 49, 639. -649 (1983) ".

11. Sulfated in Endothelial Extracellular Cytoplasm, Blodowski, Fuchs, Bar-Ner, M., Ariv, Y. and Schirmacher, V. Lymphoma cell mediated degradation of sulfated proteoglycans in the subendothelial extracellular matrix: Relationship to tumor cell matstasis. Cancer Res., 43, 2704-2711 ( 1983) ".

12. Hepa in Tumor Metastasis and Angiogenesis by Blodowski, Ishai-Mikaelli, Var-Ner, Fridman, R., Horowitz, AT, Fuchs and Biran, S. Involvement of heparanase in tumor metstasis and angiogenesis. Is. J. Med., 24, 464-470 (1988).

13. Blodsky, Liu (GM) and Gospodarowicz, D., et al., "Phase, Growth Behavior and Migration Activity of Human Tumor Cells Retained on Extracellular Cytoplasm for Plastics. Morphological appearance, growth behavior and migratory activity of human tumor cells maintained on extracellular matrix vs. plastic). Cell, 19, 607-616 (1980) ".

14. Gospodarowitz, Delgado, D. and Blodowski, "Permissive effect of the extracellular matrix on cell proliferation in-vitro" Proc. Natl. Acad. Sci. USA., 77, 4094-4098 (1980).

15. Bashkin, Doctrow, S., Klagsbrum, M., Svahn, CM, Folkman, J., and Blodsky et al. Basic fibroblast growth factor binds to subendothelial extracellular matrix and is released by heparitinase and heparin-like molecules.Biochemistry, 28, 1737- 1743 (1989) ".

16. Sulfated polysaccharides block tumor cell-induced heparanases by Parish, CR, Coombe, DR, Jakobsen, KB and Underwood, PA. Evidence that sulphated polysaccharides inhibit tumor metastasis by blocking tumor cell-derived heparanase. Int. J. Cancer, 40, 511-517 (1987) ".

16a. Blodowski, Hua-quan Miao., Benezra, M., Leader, Bar-Shavit, R., Schmidt, A., and Peretz (Pervolvez, T.), "Involvement of the extracellular matrix, heparan sulfate proteoglycans and heparan sulfate degrading in angiogenesis and metastasis, extracellular cytoplasm, heparan sulfate polythioglycan and heparan sulfate degrading enzyme enzymes in angiogenesis and metastasis. In: Tumor Angiogenesis. Eds. CE Lewis, R. Bicknell & N. Ferrara. Oxford University Press, Oxford UK, pp. 125-140 (1997).

17. The heprin-binding (fibroblast) growth factor family of proteins of Burges, WH and Maciag, T., Annu. Rev. Biochem., 58, 575-606 (1989).

18. Angiogenic factors, Polksman and Klagsbrun, M., Science, 235, 442-447 (1987).

19. Blodowski, Polkman, Sullivan, R., Friedman, Ishai-Mikaelli, Sasse, J., and Kragsbrunn, "Endothelial Cell-derived Basal Fibroblast Growth Factors". : Endothelial cell-derived basic fibroblast growth factor: Synthesis and deposition into subendothelial extracellular matrix. Proc. Natl. Acad. Sci. USA, 84, 2292-2296 (1987) ".

20. Polkman, Kragsbrunn, Sase, Wadzinski, M., Ingber, D. and Blodowski, "Heparin-binding angiogenic proteins-basal fibroblast growth factor- A heparin-binding angiogenic protein-basic fibroblast growth factor-is stored within basedment membrane. Am. J. Pathol., 130, 393-400 (1980) ".

21. Cardon-Cardo (C.), Blodowski, Hymovtz-Fredmann Haimovitz-Fredman, Hicklin, D. and Fuchs, "Basic Fibroblast Growth in Normal Human Tissues" Expression of basic fibroblast growth factor in normal human tissues. Lab. Invest., 63, 832-840 (1990) ".

22. Ishai-Mikaelli, Svahn, C.-M., Chaekek-Shaul, T., Korner, G., Ekre, H.-p. And the importance of importation of size and sulfation of heparin in release of basic fibroblast factor from the vascular in the release of basic fibroblast factor from vascular endothelial and extracellular cytoplasm. endothelium and extracellular matrix). Biochemixtry, 31, 2080-2088 (1992).

23. Ishai-Mikaelli, Eldo and Blodowski, "Heparanase activity expressed by platelets, which are expressed by platelets, neutrophils and lymphocytes, releasing active fibroblast growth factor from extracellular cytoplasm. , neutrophils and lymphoma cells releases active fibroblast growth factor from extracellular matrix). Cell Reg., 1, 833-842 (1990) ".

24. Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism by Bladowski, Bar-Schavit, Ishai-Mikaelli, Washkin and Fuchs. Trends Biochem. Sci., 16, 268-271 (1991).

25. Extracellular matrix-bound growth factors, enzymes and plasma proteins by Bladowski, Bar-Schavit, Corner and Fuchs. In Basement membranes: Cellular and molecular aspects (eds. DH Rohrbach and R. Timpl), pp 327-343. Academic press Inc., Orlando, Fl (1993).

26. Yaon, A., Kragsbrunn, Esko, JD, Leather, P. and Oritz, DM, "Heparin-like molecules present on the cell surface are based Cell suface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell, 64, 841-848 (1991). ) ".

27.Spivak-Kroizman (T.), Lemon (Lemmon, MA), Dikic (I.), Ladbury, JE, Pinchasi (Pinchasi, D.), Hung ( Hung, J., Jaye, M., Crumley, G., Schlessinger, J., and Lax, I., “Heparin-derived oligomers of FGF molecules. Hearin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell, 79, 1015-1024 (1994). "

28. Ornitz, DM, Herr, AB, Nielsson, M., West, a., J., Svahn, C.-M., and Waxman ( Waksman, G., "FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides." Science, 268 , 432-436 (1995).

29. The cell surface associated heparin-like molecules of Kitay-Goren, H., Soker, S., Blodowski, and Neufeld, G. describe the growth of vascular endothelium. 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 (1992).

30. Leader, Baharav, E., Mekori, Y., Miller, T., Naparstek, Y., Blodowski and Cohen, IR Suppression of experimental autoimmune diseases and prololongation of allograft survival by treatment of animals with heparinoid by treating animals with heparinoid inhibitors of T lymphocytes heparanases. inhibiors of T lymphocyte heparanase. J. Clin. Invest., 83, 752-756 (1989).

31.Leaders, Cahalon, L., Gilat, D., Hershkovitz, R., Siegel, D., Margalit, R., Shoseyov, O. and Cohn, IR, et al., "Disaccharides that inhibit tumor necrosis factor α are formed from extracellular cytoplasm by enzyme heparanase (A disaccharide that inhibits tumor necrosis). factor α is formed from the extracellular matrix by the enzyme heparanase). Proc. Natl. Acad. Sci. USA., 92, 5037-5041 (1995).

31a. Rapraeger, A., Krufka, A. and Olwin, BR, "Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. for bFGF-mediated fibroblast growth and myoblast differentiation. Science, 252, 1705-1708 (1991).

32. Eisenberg, S., Sehayek, E., Olivecrona, T., and Blodowski, report “Lipo on Heparan Sulfate on Cell Surface and Extracellular Cytoplasm. Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix. J. Clin. Invest., 90, 2013-2021 (1992).

33. Cell surface receptors for the Herpes simplex virus by Shehie, MT., Wundunn, D., Montgomery, RI, Esco and Spear, PGJ. Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol., 116, 1273-1281 (1992).

33a. By Chen, Y., Maguire, T., Hileman, RE, Fromm, JR, Esco, Linhardt, RJ and Marks, RM. Dengue virus infectivity depends on envelope protein binding to target cell heapran sulfate. Nature Medicine 3, 866-871 (1997).

33b. Putak, JR, Kanesa-Thasan, N, n, and Innes, BL, "A putative cellular receptor for dengue viruses. Nature Medicine 3, 828-829 (1997).

34. Narindrasorasak (S.), Lowery, D., Gonzalez-DeWhitt, P., Poorman, RA, Greenberg, B. ), High affinity interactions between the Alzheimer's beta-amyloid precursor protein and the Alzheimer's β-amyloid precursor and teparan sulfate prothioglycans basement membrane form of the paran sulfate proteoglycan. J. Bil. Chem., 266, 12878-83 (1991).

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

36. Zhong-Sheng, J., Walter, J., Brecht, R., Miranda, D., Mahmood Hussain, M., Inner Role of Heparan Sulfate Prothioglycans in Binding and Adsorption of Apolipoprotein E-Rich Residual Lipoprotein by Cultured Cells, Innerarity (TL) and Mahley, WR of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells). J. Biol. Chem., 268, 10160-10167 (1993).

37. Enzymatic Degradation of Glycosaminoglycans by Ernst, S., Langer, R., Cooney, Ch. L. and Sasisekharan, R. (Enzymatic degradation of glycosaminoglycans). Critical Reviews in Biochemistry and Molecular Biology, 30 (5), 387-444 (1995).

38. Stimulation of corneal endothelial by fibroblasts and endothelial growth factors by Gospodarowitz, Mescher, AL. And Birddwell, CR. cell proliferation in vitro by fibroblast and epidermal growth factors. Exp Eye Res 25, 75-89 (1977).

39. Activation of platelet heparinase by tumor cell-derived factors by Haimovitz-Friedman, Falcone (DJ), Eldo, Schirmacher, V., Blodowski and Fuchs. of platelet heparitinase by tumor cell-derived factors. Blood, 78, 789-796 (1991).

39a. Savitsky (K.), Platzer (M.), Uziel (T.), Gilad (S.), Sartiel (A.), Rosenthal (Rosental, A.), Elroy-Stein, O., Siloh, Y., and Rotman, G., et al. "Ataxia-telangi etasia: structural diversity of untranslated sequences Ataxia-telangiectasia: structural diversity of untranslated sequences suggests complex post-translational regulation of ATM gene expression. Nucleic Acids Res. 25 (9), 1678-1684 (1997) " .

40. Var-Ner, Eldo, Wasserman, L., Matzner, Y. and Blodowski, "Extracellular intercellular interstitial heparan sulfate by modified and non-anticoagulant heparin species. Inhibition of heparanase mediated degradation of extracellular matrix heparan sulfate by modified and non-anticoagulant heparin species. Blood, 70, 551-557 (1987) ".

41.Goshen, R., Hochberg, A., Corner, Levi, E., Ishii-Mikaeli, Elkin, M., de Grot, N.) and Bladovski's "Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts." Mol. Human Reprod. 2 , 679-684 (1996) ".

<110> Insight strategy and marketing LTD; Hadasit medical research services and development LTD

〈120〉 Polynucleotide encoding a polypeptide having heparanase activity

and expression of same in transduced cells

〈130〉 1

〈150〉 US 08 / 922,170

<151> 1997-09-02

〈150〉 US 09 / 109,386

<151> 1998-07-02

〈160〉 23

〈170〉 KOPATIN 1.5

〈210〉 1

<211> 27

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 5'-primer

<400> 1

ccatcctaat acgactcact atagggc 27

〈210〉 2

<211> 24

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 3'-primer

<400> 2

gtagtgatgc catgtaactg aatc 24

〈210〉 3

<211> 23

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 5'-primer

<400> 3

actcactata gggctcgagc ggc 23

〈210〉 4

<211> 22

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 3'-primer

<400> 4

gcatcttagc cgtctttctt cg 22

<210> 5

<211> 15

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> oligo (dT) 15 primer

<400> 5

tttttttttt ttttt 15

〈210〉 6

<211> 23

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> primer

<400> 6

ttcgatccca agaaggaatc aac 23

〈210〉 7

<211> 24

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> primer

<400> 7

gtagtgatgc catgtaactg aatc 24

<210> 8

<211> 9

<212> PRT

〈213〉 Artificial Sequence

〈220〉

223 DNA fragment of heparanase from human hepatoma cell

<400> 8

Tyr Gly Pro Asp Val Gly Gln Pro Arg

1 5

〈210〉 9

<211> 1721

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 hpa cDNA from human placenta

<400> 9

ctagagcttt cgactctccg ctgcgcggca gctggcgggg ggagcagcca ggtgagccca 60

agatgctgct gcgctcgaag cctgcgctgc cgccgccgct gatgctgctg ctcctggggc 120

cgctgggtcc cctctcccct ggcgccctgc cccgacctgc gcaagcacag gacgtcgtgg 180

acctggactt cttcacccag gagccgctgc acctggtgag cccctcgttc ctgtccgtca 240

ccattgacgc caacctggcc acggacccgc ggttcctcat cctcctgggt tctccaaagc 300

ttcgtacctt ggccagaggc ttgtctcctg cgtacctgag gtttggtggc accaagacag 360

acttcctaat tttcgatccc aagaaggaat caacctttga agagagaagt tactggcaat 420

ctcaagtcaa ccaggatatt tgcaaatatg gatccatccc tcctgatgtg gaggagaagt 480

tacggttgga atggccctac caggagcaat tgctactccg agaacactac cagaaaaagt 540

tcaagaacag cacctactca agaagctctg tagatgtgct atacactttt gcaaactgct 600

caggactgga cttgatcttt ggcctaaatg cgttattaag aacagcagat ttgcagtgga 660

acagttctaa tgctcagttg ctcctggact actgctcttc caaggggtat aacatttctt 720

gggaactagg caatgaacct aacagtttcc ttaagaaggc tgatattttc atcaatgggt 780

cgcagttagg agaagattat attcaattgc ataaacttct aagaaagtcc accttcaaaa 840

atgcaaaact ctatggtcct gatgttggtc agcctcgaag aaagacggct aagatgctga 900

agagcttcct gaaggctggt ggagaagtga ttgattcagt tacatggcat cactactatt 960

tgaatggacg gactgctacc agggaagatt ttctaaaccc tgatgtattg gacattttta 1020

tttcatctgt gcaaaaagtt ttccaggtgg ttgagagcac caggcctggc aagaaggtct 1080

ggttaggaga aacaagctct gcatatggag gcggagcgcc cttgctatcc gacacctttg 1140

cagctggctt tatgtggctg gataaattgg gcctgtcagc ccgaatggga atagaagtgg 1200

tgatgaggca agtattcttt ggagcaggaa actaccattt agtggatgaa aacttcgatc 1260

ctttacctga ttattggcta tctcttctgt tcaagaaatt ggtgggcacc aaggtgttaa 1320

tggcaagcgt gcaaggttca aagagaagga agcttcgagt ataccttcat tgcacaaaca 1380

ctgacaatcc aaggtataaa gaaggagatt taactctgta tgccataaac ctccataacg 1440

tcaccaagta cttgcggtta ccctatcctt tttctaacaa gcaagtggat aaataccttc 1500

taagaccttt gggacctcat ggattacttt ccaaatctgt ccaactcaat ggtctaactc 1560

taaagatggt ggatgatcaa accttgccac ctttaatgga aaaacctctc cggccaggaa 1620

gttcactggg cttgccagct ttctcatata gtttttttgt gataagaaat gccaaagttg 1680

ctgcttgcat ctgaaaataa aatatactag tcctgacact g 1721

<210> 10

<211> 543

<212> PRT

〈213〉 Artificial Sequence

〈220〉

223 translated protein of SEQ ID NO: 9

<400> 10

Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu

1 5 10 15

Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro

20 25 30

Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro

35 40 45

Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn

50 55 60

Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu

65 70 75 80

Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly

85 90 95

Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe

100 105 110

Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys

115 120 125

Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp

130 135 140

Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe

145 150 155 160

Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe

165 170 175

Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu

180 185 190

Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu

195 200 205

Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn

210 215 220

Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser

225 230 235 240

Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser

245 250 255

Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg

260 265 270

Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu

275 280 285

Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr

290 295 300

Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile

305 310 315 320

Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly

325 330 335

Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala

340 345 350

Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys

355 360 365

Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val

370 375 380

Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro

385 390 395 400

Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr

405 410 415

Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg

420 425 430

Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly

435 440 445

Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu

450 455 460

Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu

465 470 475 480

Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn

485 490 495

Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met

500 505 510

Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser

515 520 525

Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile

530 535 540

<210> 11

<211> 1721

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 open reading frame

〈220〉

<221> CDS

222 (63) .. (1691)

<400> 11

ctagagcttt cgactctccg ctgcgcggca gctggcgggg ggagcagcca ggtgagccca 60

ag atg ctg ctg cgc tcg aag cct gcg ctg ccg ccg ccg ctg atg 104

Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met

1 5 10

ctg ctg ctc ctg ggg ccg ctg ggt ccc ctc tcc cct ggc gcc ctg ccc 152

Leu Leu Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro

15 20 25 30

cga cct gcg caa gca cag gac gtc gtg gac ctg gac ttc ttc acc cag 200

Arg Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln

35 40 45

gag ccg ctg cac ctg gtg agc ccc tcg ttc ctg tcc gtc acc att gac 248

Glu Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp

50 55 60

gcc aac ctg gcc acg gac ccg cgg ttc ctc atc ctc ctg ggt tct cca 296

Ala Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro

65 70 75

aag ctt cgt acc ttg gcc aga ggc ttg tct cct gcg tac ctg agg ttt 344

Lys Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe

80 85 90

ggt ggc acc aag aca gac ttc cta att ttc gat ccc aag aag gaa tca 392

Gly Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser

95 100 105 110

acc ttt gaa gag aga agt tac tgg caa tct caa gtc aac cag gat att 440

Thr Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile

115 120 125

tgc aaa tat gga tcc atc cct cct gat gtg gag gag aag tta cgg ttg 488

Cys Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu

130 135 140

gaa tgg ccc tac cag gag caa ttg cta ctc cga gaa cac tac cag aaa 536

Glu Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys

145 150 155

aag ttc aag aac agc acc tac tca aga agc tct gta gat gtg cta tac 584

Lys Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr

160 165 170

act ttt gca aac tgc tca gga ctg gac ttg atc ttt ggc cta aat gcg 632

Thr Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala

175 180 185 190

tta tta aga aca gca gat ttg cag tgg aac agt tct aat gct cag ttg 680

Leu Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu

195 200 205

ctc ctg gac tac tgc tct tcc aag ggg tat aac att tct tgg gaa cta 728

Leu Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu

210 215 220

ggc aat gaa cct aac agt ttc ctt aag aag gct gat att ttc atc aat 776

Gly Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn

225 230 235

ggg tcg cag tta gga gaa gat tat att caa ttg cat aaa ctt cta aga 824

Gly Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg

240 245 250

aag tcc acc ttc aaa aat gca aaa ctc tat ggt cct gat gtt ggt cag 872

Lys Ser Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln

255 260 265 270

cct cga aga aag acg gct aag atg ctg aag agc ttc ctg aag gct ggt 920

Pro Arg Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly

275 280 285

gga gaa gtg att gat tca gtt aca tgg cat cac tac tat ttg aat gga 968

Gly Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly

290 295 300

cgg act gct acc agg gaa gat ttt cta aac cct gat gta ttg gac att 1016

Arg Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile

305 310 315

ttt att tca tct gtg caa aaa gtt ttc cag gtg gtt gag agc acc agg 1064

Phe Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg

320 325 330

cct ggc aag aag gtc tgg tta gga gaa aca agc tct gca tat gga ggc 1112

Pro Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly

335 340 345 350

gga gcg ccc ttg cta tcc gac acc ttt gca gct ggc ttt atg tgg ctg 1160

Gly Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu

355 360 365

gat aaa ttg ggc ctg tca gcc cga atg gga ata gaa gtg gtg atg agg 1208

Asp Lys Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg

370 375 380

caa gta ttc ttt gga gca gga aac tac cat tta gtg gat gaa aac ttc 1256

Gln Val Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe

385 390 395

gat cct tta cct gat tat tgg cta tct ctt ctg ttc aag aaa ttg gtg 1304

Asp Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val

400 405 410

ggc acc aag gtg tta atg gca agc gtg caa ggt tca aag aga agg aag 1352

Gly Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys

415 420 425 430

ctt cga gta tac ctt cat tgc aca aac act gac aat cca agg tat aaa 1400

Leu Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys

435 440 445

gaa gga gat tta act ctg tat gcc ata aac ctc cat aac gtc acc aag 1448

Glu Gly Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys

450 455 460

tac ttg cgg tta ccc tat cct ttt tct aac aag caa gtg gat aaa tac 1496

Tyr Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr

465 470 475

ctt cta aga cct ttg gga cct cat gga tta ctt tcc aaa tct gtc caa 1544

Leu Leu Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln

480 485 490

ctc aat ggt cta act cta aag atg gtg gat gat caa acc ttg cca cct 1592

Leu Asn Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro

495 500 505 510

tta atg gaa aaa cct ctc cgg cca gga agt tca ctg ggc ttg cca gct 1640

Leu Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala

515 520 525

ttc tca tat agt ttt ttt gtg ata aga aat gcc aaa gtt gct gct tgc 1688

Phe Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys

530 535 540

atc tgaaaataa aatatactag tcctgacact g 1721

Ile

<210> 12

<211> 543

<212> PRT

〈213〉 Artificial Sequence

<400> 12

Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu

1 5 10 15

Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro

20 25 30

Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro

35 40 45

Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn

50 55 60

Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu

65 70 75 80

Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly

85 90 95

Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe

100 105 110

Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys

115 120 125

Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp

130 135 140

Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe

145 150 155 160

Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe

165 170 175

Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu

180 185 190

Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu

195 200 205

Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn

210 215 220

Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser

225 230 235 240

Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser

245 250 255

Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg

260 265 270

Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu

275 280 285

Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr

290 295 300

Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile

305 310 315 320

Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly

325 330 335

Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala

340 345 350

Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys

355 360 365

Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val

370 375 380

Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro

385 390 395 400

Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr

405 410 415

Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg

420 425 430

Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly

435 440 445

Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu

450 455 460

Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu

465 470 475 480

Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn

485 490 495

Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met

500 505 510

Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser

515 520 525

Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile

530 535 540

〈210〉 13

<211> 824

<212> DNA

〈213〉 Artificial Sequence

〈220〉

223 translated protein of SEQ ID NO: 9

〈400〉 13

ctggcaagaa ggtctggttg ggagagacga gctcagctta cggtggcggt gcacccttgc 60

tgtccaacac ctttgcagct ggctttatgt ggctggataa attgggcctg tcagcccaga 120

tgggcataga agtcgtgatg aggcaggtgt tcttcggagc aggcaactac cacttagtgg 180

atgaaaactt tgagccttta cctgattact ggctctctct tctgttcaag aaactggtag 240

gtcccagggt gttactgtca agagtgaaag gcccagacag gagcaaactc cgagtgtatc 300

tccactgcac taacgtctat cacccacgat atcaggaagg agatctaact ctgtatgtcc 360

tgaacctcca taatgtcacc aagcacttga aggtaccgcc tccgttgttc aggaaaccag 420

tggatacgta ccttctgaag ccttcggggc cggatggatt actttccaaa tctgtccaac 480

tgaacggtca aattctgaag atggtggatg agcagaccct gccagctttg acagaaaaac 540

ctctccccgc aggaagtgca ctaagcctgc ctgccttttc ctatggtttt tttgtcataa 600

gaaatgccaa aatcgctgct tgtatatgaa aataaaaggc atacggtacc cctgagacaa 660

aagccgaggg gggtgttatt cataaaacaa aaccctagtt taggaggcca cctccttgcc 720

gagttccaga gcttcgggag ggtggggtac acttcagtat tacattcagt gtggtgttct 780

ctctaagaag aatactgcag gtggtgacag ttaatagcac tgtg 824

〈210〉 14

<211> 1899

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> mouse EST cDNA fragment

<400> 14

gggaaagcga gcaaggaagt aggagagagc cgggcaggcg gggcggggtt ggattgggag 60

cagtgggagg gatgcagaag aggagtggga gggatggagg gcgcagtggg aggggtgagg 120

aggcgtaacg gggcggagga aaggagaaaa gggcgctggg gctcggcggg aggaagtgct 180

agagctctcg actctccgct gcgcggcagc tggcgggggg agcagccagg tgagcccaag 240

atgctgctgc gctcgaagcc tgcgctgccg ccgccgctga tgctgctgct cctggggccg 300

ctgggtcccc tctcccctgg cgccctgccc cgacctgcgc aagcacagga cgtcgtggac 360

ctggacttct tcacccagga gccgctgcac ctggtgagcc cctcgttcct gtccgtcacc 420

attgacgcca acctggccac ggacccgcgg ttcctcatcc tcctgggttc tccaaagctt 480

cgtaccttgg ccagaggctt gtctcctgcg tacctgaggt ttggtggcac caagacagac 540

ttcctaattt tcgatcccaa gaaggaatca acctttgaag agagaagtta ctggcaatct 600

caagtcaacc aggatatttg caaatatgga tccatccctc ctgatgtgga ggagaagtta 660

cggttggaat ggccctacca ggagcaattg ctactccgag aacactacca gaaaaagttc 720

aagaacagca cctactcaag aagctctgta gatgtgctat acacttttgc aaactgctca 780

ggactggact tgatctttgg cctaaatgcg ttattaagaa cagcagattt gcagtggaac 840

agttctaatg ctcagttgct cctggactac tgctcttcca aggggtataa catttcttgg 900

gaactaggca atgaacctaa cagtttcctt aagaaggctg atattttcat caatgggtcg 960

cagttaggag aagattatat tcaattgcat aaacttctaa gaaagtccac cttcaaaaat 1020

gcaaaactct atggtcctga tgttggtcag cctcgaagaa agacggctaa gatgctgaag 1080

agcttcctga aggctggtgg agaagtgatt gattcagtta catggcatca ctactatttg 1140

aatggacgga ctgctaccag ggaagatttt ctaaaccctg atgtattgga catttttatt 1200

tcatctgtgc aaaaagtttt ccaggtggtt gagagcacca ggcctggcaa gaaggtctgg 1260

ttaggagaaa caagctctgc atatggaggc ggagcgccct tgctatccga cacctttgca 1320

gctggcttta tgtggctgga taaattgggc ctgtcagccc gaatgggaat agaagtggtg 1380

atgaggcaag tattctttgg agcaggaaac taccatttag tggatgaaaa cttcgatcct 1440

ttacctgatt attggctatc tcttctgttc aagaaattgg tgggcaccaa ggtgttaatg 1500

gcaagcgtgc aaggttcaaa gagaaggaag cttcgagtat accttcattg cacaaacact 1560

gacaatccaa ggtataaaga aggagattta actctgtatg ccataaacct ccataacgtc 1620

accaagtact tgcggttacc ctatcctttt tctaacaagc aagtggataa ataccttcta 1680

agacctttgg gacctcatgg attactttcc aaatctgtcc aactcaatgg tctaactcta 1740

aagatggtgg atgatcaaac cttgccacct ttaatggaaa aacctctccg gccaggaagt 1800

tcactgggct tgccagcttt ctcatatagt ttttttgtga taagaaatgc caaagttgct 1860

gcttgcatct gaaaataaaa tatactagtc ctgacactg 1899

<210> 15

<211> 592

<212> PRT

〈213〉 Artificial Sequence

〈220〉

〈223〉 open reading frame

<400> 15

Met Glu Gly Ala Val Gly Gly Val Arg Arg Arg Asn Gly Ala Glu Glu

1 5 10 15

Arg Arg Lys Gly Arg Trp Gly Ser Ala Gly Gly Ser Ala Arg Ala Leu

20 25 30

Asp Ser Pro Leu Arg Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro

35 40 45

Lys Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu

50 55 60

Leu Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg

65 70 75 80

Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu

85 90 95

Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala

100 105 110

Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys

115 120 125

Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly

130 135 140

Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr

145 150 155 160

Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys

165 170 175

Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu

180 185 190

Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys

195 200 205

Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr

210 215 220

Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gla Leu Asn Ala Leu

225 230 235 240

Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu

245 250 255

Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly

260 265 270

Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly

275 280 285

Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys

290 295 300

Ser Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro

305 310 315 320

Arg Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly

325 330 335

Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg

340 345 350

Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe

355 360 365

Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro

370 375 380

Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly

385 390 395 400

Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp

405 410 415

Lys Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln

420 425 430

Val Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp

435 440 445

Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly

450 455 460

Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu

465 470 475 480

Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu

485 490 495

Gly Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr

500 505 510

Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu

515 520 525

Leu Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu

530 535 540

Asn Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu

545 550 555 560

Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe

565 570 575

Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile

580 585 590

<210> 16

<211> 1899

<212> DNA

〈213〉 Artificial Sequence

〈220〉

〈223〉 open reading frame

〈220〉

<221> CDS

222 (94) .. (1869)

<400> 16

gggaaagcga gcaaggaagt aggagagagc cgggcaggcg gggcggggtt ggattgggag 60

cagtgggagg gatgcagaag aggagtggga ggg atg gag ggc gca gtg 108

Met Glu Gly Ala Val

1 5

gga ggg gtg agg agg cgt aac ggg gcg gag gaa agg aga aaa ggg cgc 156

Gly Gly Val Arg Arg Arg Asn Gly Ala Glu Glu Arg Arg Lys Gly Arg

10 15 20

tgg ggc tcg gcg gga gga agt gct aga gct ctc gac tct ccg ctg cgc 204

Trp Gly Ser Ala Gly Gly Ser Ala Arg Ala Leu Asp Ser Pro Leu Arg

25 30 35

ggc agc tgg cgg ggg gag cag cca ggt gag ccc aag atg ctg ctg cgc 252

Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro Lys Met Leu Leu Arg

40 45 50

tcg aag cct gcg ctg ccg ccg ccg ctg atg ctg ctg ctc ctg ggg ccg 300

Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu Leu Leu Gly Pro

55 60 65

ctg ggt ccc ctc tcc cct ggc gcc ctg ccc cga cct gcg caa gca cag 348

Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro Ala Gln Ala Gln

70 75 80 85

gac gtc gtg gac ctg gac ttc ttc acc cag gag ccg ctg cac ctg gtg 396

Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro Leu His Leu Val

90 95 100

agc ccc tcg ttc ctg tcc gtc acc att gac gcc aac ctg gcc acg gac 444

Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn Leu Ala Thr Asp

105 110 115

ccg cgg ttc ctc atc ctc ctg ggt tct cca aag ctt cgt acc ttg gcc 492

Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu Arg Thr Leu Ala

120 125 130

aga ggc ttg tct cct gcg tac ctg agg ttt ggt ggc acc aag aca gac 540

Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr Asp

135 140 145

ttc cta att ttc gat ccc aag aag gaa tca acc ttt gaa gag aga agt 588

Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe Glu Glu Arg Ser

150 155 160 165

tac tgg caa tct caa gtc aac cag gat att tgc aaa tat gga tcc atc 636

Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys Tyr Gly Ser Ile

170 175 180

cct cct gat gtg gag gag aag tta cgg ttg gaa tgg ccc tac cag gag 684

Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp Pro Tyr Gln Glu

185 190 195

caa ttg cta ctc cga gaa cac tac cag aaa aag ttc aag aac agc acc 732

Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe Lys Asn Ser Thr

200 205 210

tac tca aga agc tct gta gat gtg cta tac act ttt gca aac tgc tca 780

Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe Ala Asn Cys Ser

215 220 225

gga ctg gac ttg atc ttt ggc cta aat gcg tta tta aga aca gca gat 828

Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Ala Asp

230 235 240 245

ttg cag tgg aac agt tct aat gct cag ttg ctc ctg gac tac tgc tct 876

Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu Asp Tyr Cys Ser

250 255 260

tcc aag ggg tat aac att tct tgg gaa cta ggc aat gaa cct aac agt 924

Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser

265 270 275

ttc ctt aag aag gct gat att ttc atc aat ggg tcg cag tta gga gaa 972

Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser Gln Leu Gly Glu

280 285 290

gat tat att caa ttg cat aaa ctt cta aga aag tcc acc ttc aaa aat 1020

Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser Thr Phe Lys Asn

295 300 305

gca aaa ctc tat ggt cct gat gtt ggt cag cct cga aga aag acg gct 1068

Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg Arg Lys Thr Ala

310 315 320 325

aag atg ctg aag agc ttc ctg aag gct ggt gga gaa gtg att gat tca 1116

Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu Val Ile Asp Ser

330 335 340

gtt aca tgg cat cac tac tat ttg aat gga cgg act gct acc agg gaa 1164

Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr Ala Thr Arg Glu

345 350 355

gat ttt cta aac cct gat gta ttg gac att ttt att tca tct gtg caa 1212

Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile Ser Ser Val Gln

360 365 370

aaa gtt ttc cag gtg gtt gag agc acc agg cct ggc aag aag gtc tgg 1260

Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly Lys Lys Val Trp

375 380 385

tta gga gaa aca agc tct gca tat gga ggc gga gcg ccc ttg cta tcc 1308

Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu Leu Ser

390 395 400 405

gac acc ttt gca gct ggc ttt atg tgg ctg gat aaa ttg ggc ctg tca 1356

Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys Leu Gly Leu Ser

410 415 420

gcc cga atg gga ata gaa gtg gtg atg agg caa gta ttc ttt gga gca 1404

Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val Phe Phe Gly Ala

425 430 435

gga aac tac cat tta gtg gat gaa aac ttc gat cct tta cct gat tat 1452

Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro Leu Pro Asp Tyr

440 445 450

tgg cta tct ctt ctg ttc aag aaa ttg gtg ggc acc aag gtg tta atg 1500

Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr Lys Val Leu Met

455 460 465

gca agc gtg caa ggt tca aag aga agg aag ctt cga gta tac ctt cat 1548

Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg Val Tyr Leu His

470 475 480 485

tgc aca aac act gac aat cca agg tat aaa gaa gga gat tta act ctg 1596

Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly Asp Leu Thr Leu

490 495 500

tat gcc ata aac ctc cat aac gtc acc aag tac ttg cgg tta ccc tat 1644

Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu Arg Leu Pro Tyr

505 510 515

cct ttt tct aac aag caa gtg gat aaa tac ctt cta aga cct ttg gga 1692

Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu Arg Pro Leu Gly

520 525 530

cct cat gga tta ctt tcc aaa tct gtc caa ctc aat ggt cta act cta 1740

Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn Gly Leu Thr Leu

535 540 545

aag atg gtg gat gat caa acc ttg cca cct tta atg gaa aaa cct ctc 1788

Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met Glu Lys Pro Leu

550 555 560 565

cgg cca gga agt tca ctg ggc ttg cca gct ttc tca tat agt ttt ttt 1836

Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser Tyr Ser Phe Phe

570 575 580

gtg ata aga aat gcc aaa gtt gct gct tgc atc t gaaaataaaa 1880

Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile

585 590

tatactagtc ctgacactg 1899

〈210〉 17

<211> 592

<212> PRT

〈213〉 Artificial Sequence

〈400〉 17

Met Glu Gly Ala Val Gly Gly Val Arg Arg Arg Asn Gly Ala Glu Glu

1 5 10 15

Arg Arg Lys Gly Arg Trp Gly Ser Ala Gly Gly Ser Ala Arg Ala Leu

20 25 30

Asp Ser Pro Leu Arg Gly Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro

35 40 45

Lys Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu

50 55 60

Leu Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg

65 70 75 80

Pro Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu

85 90 95

Pro Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala

100 105 110

Asn Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys

115 120 125

Leu Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly

130 135 140

Gly Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr

145 150 155 160

Phe Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys

165 170 175

Lys Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu

180 185 190

Trp Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys

195 200 205

Phe Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr

210 215 220

Phe Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gla Leu Asn Ala Leu

225 230 235 240

Leu Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu

245 250 255

Leu Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly

260 265 270

Asn Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly

275 280 285

Ser Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys

290 295 300

Ser Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro

305 310 315 320

Arg Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly

325 330 335

Glu Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg

340 345 350

Thr Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe

355 360 365

Ile Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro

370 375 380

Gly Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly

385 390 395 400

Ala Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp

405 410 415

Lys Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln

420 425 430

Val Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp

435 440 445

Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly

450 455 460

Thr Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu

465 470 475 480

Arg Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu

485 490 495

Gly Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr

500 505 510

Leu Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu

515 520 525

Leu Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu

530 535 540

Asn Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu

545 550 555 560

Met Glu Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe

565 570 575

Ser Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile

580 585 590

〈210〉 18

<211> 594

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> DNA fragment

〈400〉 18

attactatag ggcacgcgtg gtcgacggcc cgggctggta ttgtcttaat gagaagttga 60

taaagaattt tgggtggttg atctctttcc agctgcagtt tagcgtatgc tgaggccaga 120

ttttttcagg caaaagtaaa atacctgaga aactgcctgg ccagaggaca atcagatttt 180

ggctggctca agtgacaagc aagtgtttat aagctagatg ggagaggaag ggatgaatac 240

tccattggag gctttactcg agggtcagag ggatacccgg cgccatcaga atgggatctg 300

ggagtcggaa acgctgggtt cccacgagag cgcgcagaac acgtgcgtca ggaagcctgg 360

tccgggatgc ccagcgctgc tccccgggcg ctcctccccg ggcgctcctc cccaggcctc 420

ccgggcgctt ggatcccggc catctccgca cccttcaagt gggtgtgggt gatttcgtaa 480

gtgaacgtga ccgccaccgg ggggaaagcg agcaaggaag taggagagag ccgggcaggc 540

ggggcggggt tggattggga gcagtgggag ggatgcagaa gaggagtggg aggg 594

〈210〉 19

<211> 21

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> hpa specific antisense primer hpl-629

〈400〉 19

ccccaggagc agcagcatca g 21

<210> 20

<211> 21

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> hpa specific antisence nested primer hpl-666

<400> 20

aggcttcgag cgcagcagca t 21

〈210〉 21

<211> 22

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> ap1 primer

<400> 21

gtaatacgac tcactatagg gc 22

〈210〉 22

<211> 19

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> nested adapter specific primer ap2

<400> 22

actatagggc acgcgtggt 19

<210> 23

<211> 21

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> hpa specific antisense primer hpl-690

<400> 23

cttgggctca cctggctgct c 21

<210> 24

<211> 23

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> hpa primer hpu565

<400> 24

agctctgtag atgtgctata cac 23

<210> 25

<211> 22

<212> DNA

〈213〉 Artificial Sequence

〈220〉

<223> antisense primer hpl171

<400> 25

gcatcttagc cgtctttctt cg 22

Claims (42)

A polynucleotide fragment comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The method of claim 1, A polynucleotide fragment wherein the polynucleotide sequence comprises nucleotides 63-1691 of SEQ ID NO: 9, or nucleotides 139-1869 of SEQ ID NO: 14. The method of claim 1, A polynucleotide fragment wherein the polynucleotide sequence comprises nucleotides 63-721 of SEQ ID NO: 9. The method of claim 1, A polynucleotide fragment, wherein the polynucleotide is set forth in SEQ ID NO: 9 or SEQ ID NO: 14. The method of claim 1, A polynucleotide fragment in which the polynucleotide sequence comprises a segment of SEQ ID NO: 9 or SEQ ID NO: 14, wherein the segment encodes a polypeptide having heparanase catalytic activity. The method of claim 1, A polynucleotide fragment, wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 15. The method of claim 1, A polynucleotide fragment wherein the polypeptide comprises a segment of SEQ ID NO: 10 or SEQ ID NO: 15, wherein the segment contains heparanase catalytic activity. The method of claim 1, A polynucleotide fragment wherein the polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. A single stranded polynucleotide fragment comprising a polynucleotide sequence complementary to at least a portion of a polynucleotide strand encoding a polypeptide having heparanase catalytic activity. The method of claim 9, A polynucleotide fragment wherein the polynucleotide sequence comprises at least a portion of SEQ ID NO: 9 or SEQ ID NO: 14. A vector comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The method of claim 11, A vector wherein the polynucleotide sequence comprises nucleotides 63-1691 of SEQ ID NO: 9, or nucleotides 139-1869 of SEQ ID NO: 14. The method of claim 11, A vector wherein the polynucleotide sequence comprises nucleotides 63-721 of SEQ ID NO: 9. The method of claim 11, A vector wherein the polynucleotide sequence is set forth in SEQ ID NO: 9 or SEQ ID NO: 14. The method of claim 11, A vector wherein the polynucleotide sequence comprises a segment of SEQ ID NO: 9 or SEQ ID NO: 14, wherein said segment encodes a polypeptide having heparanase catalytic activity. The method of claim 11, A vector wherein the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 15. The method of claim 11, A polypeptide comprising a segment of SEQ ID NO: 10 or SEQ ID NO: 15, wherein the segment contains heparanase catalytic activity. The method of claim 11, A vector wherein the polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. A host cell comprising an exogenous polynucleotide fragment comprising a polynucleotide sequence encoding a polypeptide having heparanase catalytic activity. The method of claim 19, A host cell wherein the polynucleotide sequence comprises nucleotides 63-1691 of SEQ ID NO: 9, or nucleotides 139-1869 of SEQ ID NO: 14. The method of claim 19, A host cell wherein the polynucleotide sequence comprises nucleotides 63-721 of SEQ ID NO: 9. The method of claim 19, A host cell, wherein the polynucleotide sequence is set forth in SEQ ID NO: 9 or SEQ ID NO: 14. The method of claim 19, A host cell in which the polynucleotide sequence comprises a segment of SEQ ID NO: 9 or SEQ ID NO: 14, wherein the segment encodes a polypeptide having heparanase catalytic activity. The method of claim 19, A host cell in which the polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 15. The method of claim 19, A host cell wherein the polypeptide comprises a segment of SEQ ID NO: 10 or SEQ ID NO: 15, wherein the segment contains heparanase catalytic activity. The method of claim 19, A host cell wherein the polynucleotide sequence is selected from the group consisting of double stranded DNA, single stranded DNA and RNA. Host cells expressing recombinant heparanase. A recombinant protein comprising a polypeptide having heparanase catalytic activity. The method of claim 28, A recombinant protein wherein the polypeptide comprises a segment of SEQ ID NO: 10 or SEQ ID NO: 15. A polynucleotide fragment comprising a polynucleotide sequence capable of hybridizing with nucleotides 1-721 of SEQ ID NO: 9. The polynucleotide sequence as set forth in SEQ ID NO: 9 or SEQ ID NO: 14. A polynucleotide sequence homologous to SEQ ID NO: 9 or SEQ ID NO: 14. Amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO: 15. An amino acid sequence homologous to SEQ ID NO: 10 or SEQ ID NO: 15. A pharmaceutical composition comprising a recombinant protein having heparanase catalytic activity as an active ingredient. Heparanase overexpression system comprising cells overexpressing heparanase catalytic activity. Cell response to heparin-binding growth factor, heparin-binding growth factor and cytokines, interaction of cells with plasma lipoproteins, viruses, protozoa and bacterial infections, comprising as a active ingredient a recombinant protein having heparanase catalytic activity Modulators for the disruption of cell sensitive or neurodegenerative plaques. A pharmaceutical device comprising a medicament device containing as an active ingredient a recombinant protein having heparanase catalytic activity. The method of claim 11, A vector that is a baculovirus vector. The method of claim 19, Host cells that are insect cells. The method of claim 27, Host cells that are insect cells. (a) hybridizing a chromosome spread with a tagged polynucleotide probe encoding heparanases; (b) washing the chromosome spread to remove excess probes that are not hybridized; And (c) examining a signal associated with the hybridized tagged polynucleotide probe, wherein the detected signal represents a chromosomal region containing the human heparanase gene. A method for identifying chromosomal regions containing human heparanase genes in chromosomal spreads.