Human heparanase polypeptide and cDNA

Details Human heparanase polypeptide and cDNA Human heparanase polypeptide and cDNA

2000-08-07

2002-10-08

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S069100, C435S183000, C435S200000, C435S252300, C435S320100, C435S325000, C424S094610, C536S023200

Reexamination Certificate

active

06461848

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, a polypeptide of the present invention is “heparanase” obtainable from the human SV40-transformed fibroblast cell line ATCC CCL 75.1. The heparanase is an endoglucuronidase capable of specifically degrading heparan sulfate into 6 to 20 kDa fragments. The invention also relates to vectors and host cells, comprising a polynucleotide of the invention. Furthermore, the present invention relates to antibodies directed to polypeptides according to the present invention, to pharmaceutical compositions comprising such antibodies or polypeptides, and to assay systems suitable for identifiying agonists or antagonists of such polypeptides.
BACKGROUND OF THE INVENTION
Heparanase was first identified in murine metastatic melanoma cells by Nakajima et al. (Nakajima et al., Science 220: 611-613, 1983). They concluded the heparan sulfate degrading enzyme responsible is an endoglucuronidase, cleaving linkage between GIcA and GIcNAc, and named it heparanase (Nakajima et al., J. Biol. Chem. 259: 2283-2290, 1984). The heparanase is a hydrolase distinguished from Flavobacterium heparitinase and heparinase (Ototani, N. et al., Carbohydrate Res. 88: 291-303, 1981) which are eliminases capable of specifically degrading heparan sulfate and heparin, respectively into di- and tetra-saccharides (Nakajima et al., J. Biol. Chem. 259: 2283-2290, 1984).
Heparanase-like activity has been found in several normal and tumor cells and tissues as reviewed by Nakajima et al. (J. Cell. Biochem. 36: 157-167, 1988). According to the reports from various laboratories the existance of at least three different types of heparin and/or heparan sulfate-degrading endoglucuronidase has been predicted. The melanoma heparanase degrades heparan sulfate but is not active on heparin. The human platelet heparanase depolymerizes both heparin and heparan sulfate and cleaves &bgr;-glucuronidic linkages in the antithrombin-binding domain of heparin (Thunberg et al., J. Biol. Chem. 257:10278-10282, 1982). Another endoglucuronidase from mastocytoma cells catalyzes the depolymerization of macromolecular heparin proteoglycans into fragments similar in size to commercial heparin (Ögren and Lindahl, J. Biol. Chem. 250:2690-2697,1975). The mastocytoma enzyme has little or no activity against heparan sulfate and does not cleave the antithrombin-binding regions of heparin.
The enzymatic characteristics of heparanase have been studied in several laboratories. Nakajima et al. found that heparanase does not degrade highly sulfated heparin of porcine mucosa and bovine lung, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, and hyaluronic acid (Nakajima et al., J. Biol. Chem. 259: 2283-2290,1984). They also reported that heparanase is inhibited by heparin described above but not by an exoglucuronidase inhibitor, 1,4-saccharolactone (Nakajima et al., J. Biol. Chem. 259: 2283-2290,1984). Highly sulfated heparan sulfate produced by vascular endothelial cells is relatively resistant to heparanase as compared with bovine lung and kidney heparan sulfate and is cleaved by heparanase into large molecular size fragments (Nakajima et al., J. Biol. Chem. 259: 2283-2290, 1984). Thus they suggested the domain structures of heparan sulfate recognized by heparanase. Bai, X. et al (J. Biol. Chem. 272: 23172-23179, 1997) have recently shown with a use of a mutant cell line of CHO cell that 2-O-sulfate uronic acid is important for heparanase recognition of heparan sulfate and its enzymatic activity. Bame, K. J. et al (J. Biol. Chem. 272: 2245-2251, 1997) have predicted the existance of two types of heparanase, one cleaving near the reducing end and the other cleaving near the non-reducing end of highly sulfated region of heparan sulfate. Schmidtchen, A. et al. (Eur. J. Biochem. 223: 211-221, 1994) have also proposed the model of heparanase cleavage site from experiments that heparanase treatment generated low-sulfated, GIcNAc-containg heparan sulfate fragments of approximately 7 kDa in molecular size.
Various methods for detecting heparanase activity are reported including (i) polyacrylamide gel electrophoresis (Nakajima, M. et al., Science 220: 611-613, 1983), (ii) gel filtration chromatography (Nakajima, M. et al., J. Biol. Chem. 259: 2283-2290, 1984), (iii) high speed gel permeation chromatography (Irimura, T. et al., Anal. Biochem, 130: 461-468, 1983) (iv) solid-phase substrates of heparanase (Nakajima, M. et al., Anal. Biochem. 157: 162-171, 1986; U.S. Pat. No. 4,859,581), (v) radio-labeled and florescein-labeled heparan sulfate for detection of heparanase activity (U.S. Pat. No. 4,859,581, WO 9504158A), (vi) use of chicken histidine rich glycoprotein (cHRG), taking advantage that heparanase treated heparan sulfate fragment has low affinity to cHRG.
Various methods for purifying heparanase have been disclosed in WO 9102977A and WO 9504158A: the former is a method for preparation of the native heparanase by using chromatographic procedure, and the later is a method for purifying heparanase having activity of endo-N-acetylglucosaminidase.
Biochemical, biological, and pathological studies of heparan sulfate proteoglycans have led to examine the role of heparanase in various diseases. Heparan sulfate is a major component of basement membranes which are continuous sheets of extracellular matrices separating parenchymal cells from underlying interstitial connective tissues. Basement membranes have characteristic permeability and play a role in maintaining normal tissue architecture. Heparan sulfate proteoglycans promote basal lamina matrix assembly by enhancing the interactions of collagenous and noncollagenous protein components while protecting them against proteolytic attack. Heparan sulfate is also a real barrier against cationic and large molecules in the basement membrane. Thus, the destruction of heparan sulfate proteoglycan barrier is an important step during the penetration of basement membranes by both normal and tumor cells (Nakajima, M. et al., J. Cell. Biochem.
36:157-167, 1988).
Most cancer mortality is the result of metastasis to regional and distant metastases. Metastasis formation occurs via a sequential and complex series of unique interactions between tumor cells and normal host cells and tissues. During the metastasis formation migrating tumor cells confront natural barriers such as connective tissues and basement membranes. The ability of malignant cells to penetrate these barriers depends on the presence of tumor and/or host enzymes capable of degrading stromal and basement membrane components. In fact several tumor cell-associated proteinases and glycosidases have been implicated in the tumor cell invasion and metastasis and their activities correlate with metastatic potential in several types of malignant cells. The enzymatic degradation of heparan sulfate proteoglycans in vascular subendothelial basement membranes followed by release of heparan sulfate fragments are achieved by metastatic tumor cells, angiogenic endothelial cells, and inflammatory cells. A good correlation between heparanase activity and metastatic potential has been found in several types of malignant tumors such as melanoma, T cell lymphoma, fibrosarcoma, and rhabdomyosarcoma (Nakajima et al., Science220: 611, 1983; Vlodavsky et al., Cancer Res. 43: 2704, 1983; Ricoveri and Cappelletti, Cancer Res. 46: 3855, 1986; Becker et al., J. Natl. Cancer Inst. 77: 417, 1986).
Similar observations have been reported from several other laboratories using different types of tumors as reviewed by Nakajima et al. (J. Cell. Biochem. 36: 157-167, 1988) and Vlodavsky et al. (Cancer Metastasis Rev., 9: 203-226, 1990), suggesting that heparanase plays a critical role in cell penetration through vascular basement membranes during the blood-borne metastasis, angiogenesis, and inflammatory cell migration. Therefore, hep

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