U.S. patent application number 10/107859 was filed with the patent office on 2002-08-22 for human heparanase polypeptide and cdna.
Invention is credited to Funakubo, Minako, Nakajima, Motowo.
Application Number | 20020115183 10/107859 |
Document ID | / |
Family ID | 10826705 |
Filed Date | 2002-08-22 |
United States Patent
Application |
20020115183 |
Kind Code |
A1 |
Nakajima, Motowo ; et
al. |
August 22, 2002 |
Human heparanase polypeptide and cDNA
Abstract
This invention relates to 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.
Inventors: |
Nakajima, Motowo;
(Ashiya-shi, JP) ; Funakubo, Minako;
(Minami-Ashigara-shi, JP) |
Correspondence
Address: |
THOMAS HOXIE
NOVARTIS CORPORATION
PATENT AND TRADEMARK DEPT
564 MORRIS AVENUE
SUMMIT
NJ
079011027
|
Family ID: |
10826705 |
Appl. No.: |
10/107859 |
Filed: |
March 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10107859 |
Mar 27, 2002 |
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09601777 |
Aug 7, 2000 |
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09601777 |
Aug 7, 2000 |
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PCT/EP99/00777 |
Feb 5, 1999 |
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Current U.S.
Class: |
435/200 ;
435/320.1; 435/325; 435/69.1; 435/84; 536/23.2 |
Current CPC
Class: |
A61K 48/00 20130101;
A61P 25/28 20180101; A61K 38/00 20130101; A61P 35/00 20180101; C12N
9/2402 20130101; C12Y 302/01166 20130101 |
Class at
Publication: |
435/200 ;
435/69.1; 435/320.1; 435/325; 435/84; 536/23.2 |
International
Class: |
C12P 019/26; C12N
009/24; C12P 021/02; C12N 005/06; C07H 021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 1998 |
GB |
9802725.3 |
Claims
1. A polypeptide showing the biological activity of human
heparanase obtainable from SV-40 transformed human fibroblast cell
line ATCC CCL 75.1, or a functional derivative, a functional
fragment or a functional analogue thereof.
2. A polypeptide according to claim 1 having the amino acid
sequence of amino acids 158 to 543 set forth in SEQ ID NO 2.
3. A polypeptide having the amino acid sequence of amino acids 1 to
543 set forth in SEQ ID NO 2:
4. A polynucleotide comprising a nucleotide sequence encoding a
polypeptide as defined in any of claims 1, 2 or 3.
5. A polynucleotide according to claim 4 comprising the
corresponding coding region contained in SEQ ID NO 1.
6. A hybrid vector comprising a polynucleotide as defined in any of
claims 4 or 5, said polynucleotide being operably linked to
suitable control sequences.
7. A hybrid vector according to claim 6, said hybrid vector being
an expression vector.
8. A host cell transformed with a hybrid vector as defined in claim
6 or claim 7.
9. A process for the preparation of a polypeptide as defined in any
of claims 1, 2 or 3, said process comprising chemical synthesis,
recombinant DNA technology or a combination of these methods.
10. A process according to claim 9, said process comprising (i)
cultivation of a host cell transformed with a hybrid vector as
defined in claim 7 under conditions suitable for performing
expression of the polypeptide, and (ii) isolation of the
thus-expressed polypeptide.
11. A process for the preparation of a polynucleotide as defined in
any of claims 4 or 5, said process comprising chemical synthesis,
recombinant DNA technology, polymerase chain reaction or a
combination of these methods.
12. An antibody which specifically recognizes and binds to a
polypeptide as defined in any of claims 1, 2 or 3.
13. A polypeptide according to any of claims 1, 2 or 3 for use in
medicine.
14. Use of a polypeptide according to any of claims 1, 2 or 3 in
the preparation of a pharmaceutical composition for the treatment
of a disease resulting from shortage or lack of said
polypeptide.
15. A pharmaceutical composition suitable for administration to a
warm-blooded animal inclusive man suffering from a disease
resulting from shortage or lack of a polypeptide as defined in any
of claims 1, 2 or 3, said composition comprising a polypeptide as
defined in any of claims 1, 2 or 3 together with at least one
pharmaceutically acceptable carrier and/or excipient.
16. A pharmaceutical composition suitable for administration to a
warm-blooded animal inclusive man suffering from a disease
resulting from excessive acitvity or overexpression of a
polypeptide as defined in any of claims 1, 2 or 3, said composition
comprising an antibody as defined in claim 12 together with at
least one pharmaceutically acceptable carrier and/or excipient.
17. A method of treatment of a disease resulting from shortage or
lack of a polypeptide as defined in any of claims 1, 2 or 3, said
method comprising administration of a suitable amount of a
polypeptide as defined in claim 1, 2 or 3.
18. A method of treatment of a disease resulting from excessive
acitvity or overexpression of a polypeptide as defined in any of
claims 1, 2 or 3, said method comprising administration of a
suitable amount of an antibody as defined in claim 12.
19. A method for identifying a substance capable of modulating the
biological activity or expression of a polypeptide as defined in
any of claims 1, 2 or 3 in a cell, said method comprising
contacting a polypeptide as defined in any of claims 1, 2 or 3, or
a functional derivative, a functional fragment or a functional
analogue thereof, or a cell capable of expressing a polypeptide as
defined in any of claims 1, 2 or 3, or a with at least one compound
or agent whose ability to modulate the biological activity or
expression of said polypeptide, functional derivative, functional
fragment or functional analogue is sought to be investigated, and
determining the change of the biological activity or the expression
of said polypeptide, derivative or fragment caused by the
substance.
20. An assay system for testing a substance for its capability of
binding to or having functional effects on a polypeptide as defined
in any of claims 1, 2 or 3 of, said assay system comprising a
polypeptide as defined in any of claims 1, 2 or 3, or a functional
derivative, a functional fragment or a functional analogue thereof,
or a cell expressing such a polypeptide, functional derivative,
functional fragment or functional analogue.
21. A substance obtainable by a method as defined in claim 19, said
substance being an agonist or an antagonist of a polypeptide as
defined in any of claims 1, 2 or 3.
22. An oligonucleotide or a derivative thereof, or a salt thereof
where salt-forming groups are present, which is specifically
hybridizable with the nucleotide sequence set forth in SEQ ID NO 1,
said oligonucleotide or derivative thereof comprising nucleoside
units or analogues of nucleoside units sufficient in number and
identity to allow such hybridization.
23. Use of a polynucleotide as defined in claim 4 in gene
therapy.
24. A method of diagnosis of conditions resulting from shortage or
lack of a polypeptide as defined in any of claims 1, 2 or 3, or
resulting from excessive acitvity or overexpression of a
polypeptide as defined in any of claims 1, 2 or 3, said method
comprising contacting cells or tissues or body fluids from an
animal inclusive man suspected of having such a condition with an
antibody as defined in claim 12 or a substance as defined in claim
21.
Description
FIELD OF THE INVENTION
[0001] 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
[0002] 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).
[0003] 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
.beta.-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 (gren 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.
[0004] 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.
[0005] 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 hisfidine rich glycoprotein (cHRG), taking advantage
that heparanase treated heparan sulfate fragment has low affinity
to cHRG.
[0006] Various methods for purifying heparanase have been disclosed
in WO 91 02977A 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.
[0007] 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).
[0008] 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; Biacker et al., J. Natl. Cancer Inst. 77: 417,
1986).
[0009] 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,
heparanase inhibition leads to suppression of tumor cell
extravasation and angiogenesis resulting in the inhibition of tumor
metastasis. U.S. Pat. No. 5,262,403A describes glycosaminoglycan
derivatives and their use as inhibitors of tumor invasiveness of
metastatic profusion. U.S. Pat. No. 4,882,318 describes heparin
derivatives as inhibitors of tumor metastasis and angiogenesis.
[0010] Various molecules such as fibroblast growth factor (FGF),
antithbrombin III, platelet factor IV, vascular endothelial growth
factor (VEGIF), interferon-gamma (IFN-g), hepatocyto growth factor
(HGF), kinases, phosphatases lipoprotein lipase, IP-10, herpes
simplex virus type I are known to bind to heparan sulfate. Among
all, extensive studies on interaction of bFGF and heparan sulfate
have been reported from various laboratories(for review:
Schlessinger, J. et al. Cell. 83: 357-360, 1995). Modulations of
the interactions and bioavailability of these molecules by
heparanase have been reported by several groups. Whitelock. J. M.
et al. (J. Biol. Chem. 271: 10079-10086, 1996) have shown
heparanase as the most efficient agent among the enzymes they have
tested (plasimin, collagenase, thrombin, and stromelysin) to
release bound growth factors from perlecari, one of the heparan
sulfate proteoglycans. They have speculated the release of bFGF
from heparan sulfate oligosaccaride chains would lead to
regeneration of tissues at sites of injury in the wound healing
process.
[0011] Hoogewerf. A. J. et al. have identified heparanase from
human platelets and examined its enzymatic characteristics (J.
Biol. Chem. 270: 3268-3277, 1995). The N-terminal amino acid
sequence of heparanase from human platelets, which they identified,
revealed it as Connective Tissue Activating Peptide-III (CTAP-III),
one of a CXC Chemokine family, and its mode of action was an
endo-N-acetylgluco saminidase, degrading heparan sulfate into
dissachrides. In their discussion they referred to the dual
function -of CTAP-III as both a heparanase and a neutrophil
chemoattractant, suggesting its functions in various pathologies.
For example, heparanase activity of the chemokine could
down-regulate inflammation by degrading focal sites of chemokine
anchoring on the surface of the inflammed endothelium. In vascular
pathologies, degradation of vascular heparan sulfate by CTAP-III
would remove antithrombin III binding sites and promote
thrombogenesis.
[0012] Lider et al. have shown a disaccharide that inhibits tumor
necrosis factor alpha is formed from the extracellular matrix by
the enzyme heparanase (Proc. Natl. Acad. Sci. U.S.A. 92: 5037-5041,
1995). When T-cells are activated by the antigens presented on
antigen-presenting cells, they produce effector molecules, such as
an inflammatory cytokines, tumor necrosis factor-alpha (TNF-a) and
an enzyme, heparanase. As a consequence, heparanase disrupts
heparan sulfate molecules in extracellular matricies and/or cell
surfaces and produces dissaccharides of heparan sulfate, which in
turn down-regulates the inflammatory activity of activated T-cells.
The negative feedback control of T-cell mediated inflammation by
heparanase implies that administering such enzyme molecules
therapeutically might succeed in immune system modulation.
[0013] Gilat, D. et al. have shown that the heparanase serves as a
T-cell adhesion molecule at physiological pH (J. Exp. Med. 181:
1929-1934, 1995). At a physiological pH, the relatively quiescent
enzyme, heparanase appears to act as a lectin-like proadhesive
molecule that can organize the recruitment of resting T cells in
extravascular loci. Therefore, the heparanase-mediated ECM-anchored
CD4+ T cells could readily respond to costimulatory signals
elicited by specifically activated adjacent immune cells.
[0014] Beta-amyloid is a major component of the senile plaques
characteristic of Alzheimer's disease (Snow, A. D. et al., Neuron
12: 219.-234, 1996). Histochemical and immunocytochemical studies
have shown that heparan sulfate proteoglycans and
glycosaminoglycans colocalize with beta-arnyloid protein in senile
plaques of Alzheimer's disease. McCubbin et al (Biochem J. 256:
775-783, 1988) have shown that heparan sulfate removed all the
random coil structure of serum amyloid polypeptide and converted
into beta-sheet beta-turn conformation leading to aggregation of
amyloid-beta fibrils. Glypican, one of heparan sulfate
proteoglycans was shown to binds to the amyloid precursor protein
of Alzheimer's disease and inhibits amyloici precursor
protein-induced neurite outgrowth (Williamson et al., J. Biol.
Chem. 271: 312115-31221, 1996). These findings suggest that one
mechanism to prevent the complex formation of beta-amyloid with
heparan sulfate proteoglycan that lead to deposition of amyloid
would be to degrade the proteoglycan. Small molecules, which mimic
elements of the heparan sulfate structure, can interfere with both
in vivo induction and persistence of amyloid protein, and
furthermore can interfere with the induction of amiloid beta-sheet
formation and the amyloid-beta fibrinogenesis in vitro by heparan
sulfate (Kisilevsky et al., Nature Med. 1: 143-148, 1995). Thus,
heparanase capable of degrading heparan sulfate can be used as a
therapeutic tool to prevent beta-amyloid deposition in senile
plaques.
[0015] In view of the various features of enzymes showing
heparanase-like activities, which are well suited for use in the
pharmaceutical field, there is an ongoing need to provide further
polypeptides showing such activities. Preferably, such polypeptides
show an advantageous behaviour compared to that of known enzymes.
Moreover, there is a need for providing a polynucleotide encoding
such a polypeptide, in order to be able to prepare sufficient
amounts of such a polypeptide by means of recombinant expression
technologies.
SUMMARY OF INVENTION
[0016] The present invention relates to polypeptide showing the
biological activity of human heparanase obtainable from the SV-40
transformed human fibroblast cell line ATCC CCL 75.1, or a
functional derivative, a functional fragment or a functional
analogue thereof.
[0017] The present invention further relates to a polynucleotide
comprising a nucleotide sequence encoding a such a polypeptide.
[0018] In another aspect the invention relates to a process for
preparing such a polypeptide or such a polynucleotide.
[0019] Moreover, the present invention relates to a hybrid vector
comprising such a polynucleotide, and to a host cell transformed
with such a hybrid vector.
[0020] It is a further object of the present invention to provide
an antibody specifically recognizing and binding to such a
polypeptide and to a method of diagnosis utilizing such an
antibody.
[0021] Further objects of the present invention relate to
pharmaceutical compositions comprising such a polypeptide or
antibody, and to a method of treatment comprising administration of
such a polypeptide or antibody.
[0022] In a yet further aspect the present invention provides an
assay system and a method for identifying a substance capable of
modulating the biological activity or expression of such a
polypeptide, and substances obtainable by such a method.
[0023] Another aspect of the present application relates to an
oligonucleotide or a derivative thereof capable of specifically
hybridizing with a polynucleotide according to the present
invention.
[0024] In a further aspect, the present invention is directed to
the use of such a polynucleotide in gene therapy.
DETAILED DESCRIPTION OF THE INVENTION
[0025] In accordance with one aspect of the present invention,
there is provided a polypeptide showing the biological activity of
human heparanase obtainable from SV-40 transformed human fibroblast
cell line ATCC CCL 75.1, or a functional derivative, a functional
fragment or a functional analogue thereof. In particular, such a
polypeptide is an endoglucuronidase capable of specifically
cleaving heparan sulfate into fragments having a size range of from
about 6 kDa to about 20 kDa. Said human cell line is the cell line
WI-38 VA13 subline 2RA, available from ATCC (American Type Culture
Collection) under accession number ATCC CCL 75.1. It is an SV40
virus-transformed derivative of the WI-38 fibroblast cell line
(ATCC CCL 75), which is a diploid cell line from normal embryonic
(3-month gestation) lung tissue of a Caucasian female.
[0026] In a preferred embodiment thereof a polypeptide according to
the present invention is the mature form of the polypeptide having
the amino acid sequence set forth in SEQ ID NO 2. In particular,
the mature polypeptide has the amino acid sequence of amino acids
158 to 543 set forth in SEQ ID NO 2.
[0027] The polypeptide having the amino acid sequence set forth in
SEQ ID NO 2 (i.e. amino acids 1 to 543) is the predicted precursor
form of the protein where approximately the first 24 amino acids
represent the leader sequence and the first 157 amino acids are the
prosequence. Such a precursor polypeptide forms another aspect of
the present invention.
[0028] The present invention further provides a process for the
preparation of a polypeptide of the invention, said process
comprising chemical synthesis, recombinant DNA technology or a
combination of these methods.
[0029] In accordance with another aspect of the present invention,
there is provided an isolated nucleic acid molecule
(polynucleotide) comprising a nucleotide sequence encoding a
polypeptide according to the present invention, i.e. a polypeptide
showing the biological activity of human heparanase obtainable from
SV-40 transformed human fibroblast cell line ATCC CCL 75.1, or
coding for the amino acid sequence of a functional derivative, a
functional fragment or a functional analogue thereof. Preferably
such a polynucleotide comprises a nucleotide sequence encoding such
a polypeptide having the amino acid sequence of amino acids 158 to
543 set forth in SEQ ID NO 2, said polypeptide being the mature
form of the heparanase of the present invention. In another
embodiment such polynucleotide comprises a nucleotide sequence
encoding a polypeptide having the amino acid sequence of amino
acids 1 to 543 set forth in SEQ ID NO 2, said polypeptide being the
precursor form of the heparanase of the present invention. Another
preferred embodiment of the present invention relates to a
polynucleotide having the nucleotide sequence set forth in SEQ ID
NO 1.
[0030] A polynucleotide encoding a polypeptide of the present
invention may be obtained from a cDNA library derived from human
carcinoma cells, placenta, peripheral blood leucocytes, or lung. In
particular, the polynucleotide described herein is isolated from a
cDNA library derived from human SV-40 transformed fibroblast cell
line ATCC CCL 75.1. The cDNA insert is 3726 base pairs (bp) in
length and contains an open reading frame encoding a protein 543
amino acids in length of which approximately the first 24 amino
acids represent the leader sequence and first 157 amino acids
represent the prosequence. Thus, the mature form of the polypeptide
of the present invention consists of 386 amino acids after the 157
amino acid prosequence (which includes the approximately 24 amino
acid leader sequence) is cleaved. The polypeptide may be found in
lysosomes of, or extracellularly near, carcinoma cells.
[0031] The polynucleotide of the present invention may be in the
form of RNA or in the form of DNA, which RNA includes mRNA and
pre-mRNA, and which DNA includes cDNA, genomic DNA and synthetic
DNA. The DNA may be double-stranded or single-stranded, and if
single stranded may be the coding strand or non-coding (anti-sense)
strand. The coding sequence which encodes the mature polypeptide or
the precursor form may be identical to the coding sequence
contained in SEQ ID NO 1, or may be different from that coding
sequence, as a result of the redundancy or degeneracy of the
genetic code, but encodes the same, mature polypeptide or the
precursor form thereof as the cDNA of SEQ ID NO 1.
[0032] In preferred embodiments, the polynucleotide according to
the present invention may include: only the coding sequence for
mature polypeptide; the coding sequence for the leader or secretory
sequence or a proprotein sequence; the coding suquence for the
mature polypeptide (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequence 5',
and/or 3' of the coding sequence for the mature polypeptide.
[0033] Thus, the term "polynucleotide comprising a nucleotide
sequence encoding a polypeptide" encompasses a polynucleotide which
includes only coding sequence for the polypeptide as well as a
polynucleotide which includes one or more additional coding and/or
non-coding sequences.
[0034] The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequence of SEQ ID NO 2. The variant of the polynucleotide may
be a naturally occurring allelic variant of the polynucleotide or a
non-naturally occurring variant of the polynucleotide. The present
invention also relates to polynucleotide probes constructed form
the polynucleotide sequence of SEQ ID NO 1 or a segment of the
sequence of SEQ ID No 1 amplified by the PCR method, which could be
utilized to screen an above mentioned cDNA library to deduce the
polypeptide of the present invention.
[0035] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide of the present invention as
well as variants of such polynucleotides which variants encode for
a fragment, derivative or analog of such a polypeptide. Such
nucleotide variants include deletion variants, substitution
variants and addition or insertion variants.
[0036] As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequence of SEQ ID NO 1. As known in the art, an allelic
variant is an alternate form of a polynucleotide sequence which may
have a substitution, deletion or addition of one or more
nucleotides, which does not substantially alter the function of the
encoded polypeptide.
[0037] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide sequence which aids in
expression and secretion of a polypeptide from a host cell, for
example, a controlling transport of a polypeptide from the cell.
The polypeptide having a leader sequence is a preprotein and may
have the leader sequence cleaved by the host cell to form a mature
form of the polypeptide. The polynucleotides may also encode for a
pro-protein which is the mature protein plus additional N-terminal
and C-terminal amino acid residues. A mature protein having a
prosequence is a pro-protein and may in some cases be an inactive
form of the protein. Once the prosequence is cleaved an active
mature protein remains.
[0038] Thus, for example, the polynucleotide of the present
invention may encode for a mature protein, or for a protein having
a prosequence or for a protein having both a presequence (leader
sequence) and a prosequence.
[0039] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker-sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hebxa-histidine tag
supplied by a pProEX-HTb (Gibco BRL) vector to provide for
purification of the mature polypeptide fused to the marker in the
case of a bacterial host, or, for example, the marker sequence may
be a hemagglutinin (HA) tag when a mammalian host, e.g. COS7 cells,
is used. The HA tag corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson, I., et al. Cell, 37:767
(1984)).
[0040] A polynucleotide according to the present invention can be
prepared by a process comprising chemical synthesis, recombinant
DNA technology, polymerase chain reaction or a combination of these
methods. Such a process forms a further aspect of the present
invention.
[0041] The terms "functional fragment," "functional derivative" and
"functional analogue" when referring to a polypeptide according to
the present invention, means a polypeptide which retains
essentially the same biological function or activity as such
polypeptide. Thus, an analogue may include a proprotein portion to
produce an active mature polypeptide.
[0042] The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a recombinant polypeptide.
[0043] The functional fragment, derivative or analog of a
polypeptide of the present invention may be (i) one in which one or
more of the amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code, or (ii) one in which one or more
of the amino acid residues includes a substituent group, or (iii)
one in which the mature polypeptide is fused with another compound,
such as a compound to increase the half-life of the polypeptide
(for example, polyethylene glycol), or (iv) one in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0044] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity. The term "isolated" means
that the material is removed from its original environment (e.g.,
the natural environment if it is naturally occurring). For example,
a naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide (e.g. a
DNA or a RNA molecule) or polypeptide, separated from some or all
of the coexisting materials in the natural system, is isolated. A
polynucleotide of the present invention could be a part of a vector
and/or such a polynucleotide or polypeptide could be a part- of a
composition, and still be isolated in that such a vector or
composition is not a part of its natural environment.
[0045] The present invention also relates to a hybrid vector
comprising a polynucleotides of the present invention, said hybrid
vector being operably linked to suitable control sequences.
Preferably, such a hybrid vector is an expression vector.
Furthermore, the present invention relates to a host cell
transformed with a hybrid vector of the present invention, i.e.
which host cell is genetically engineered with such Et hybrid
vector. In another aspect, the present invention relates to a
recombinant process for the preparation of a polypeptide of the
present invention, said process comprising (i) cultivation of a
host cell transformed with a hybrid vector of the present invention
under conditions suitable for performing expression of the
polypeptide, and (ii) isolation of the thus-expressed
polypeptide.
[0046] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vectors or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc.
[0047] The engineered host cells can be cultured in conventional
nutrient media modified as appropriate for activating promotes,
selecting transformants or amplifying the Heparanase gene. The
culture conditions, such as temperature, pH and the like, are those
used with the host cell selected for expression, and will be
apparent to the ordinarily skilled art.
[0048] The polynucleotide of the present invention may be employed
for producing a polypeptide by recombinant techniques. Thus, for
example, the polynucleotide sequence may be included in any one of
a variety of expression vehicles, in particular vectors or plasmids
for expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences, e.g., derivatives of
SV40; bacterial plasmids; phage DNA; yeast plasmid; vectors derived
from combinations of plasmids and phage DNA, viral DNA such as
vaccinia, adenovirus, fowl pox virus, baculovirus, and
pseudorabies. However, any other plasmid or vector may be used as
long as it is replicable and viable in the host.
[0049] As hereinabove indicated, the appropriate DNA sequence may
be inserted into the vector by a variety of procedures. In general,
the DNA sequence is inserted into appropriate restriction
endonuclease sites by procedures known in the art.
[0050] The DNA sequence in the expression vector is operatively
linked to (an) appropriate expression control sequence(s)
(promoter) to direct mRNA synthesis. As representative examples of
such promoters, there may be mentioned: LTR or SV40 promoter, the
E.coli. lac or trp, the expression of genes in procaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription teminator. The vector may also include appropriate
sequences for amplifying expression.
[0051] In addition, the expression vectors preferably contain a
gene to provide a phenotypic trait for selection of transformed
host cells such as dihydrofolate reductase or neomycin resistance
for eukaryotic cell culture, or such as tetracycline or ampicillin
resistance in E.coli.
[0052] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein. As representative
examples of an appropriate host, there may be mentioned: bacterial
cells, such as E.coli, Salmonella typhimurium; Streptomyces; fungal
cells, such a s yeast; insect cells such as Drosophila and Sf9;
animal cells such as CHO and COS; plant cells, etc. The selection
of an appropriate host is deemed to be within the scope of those
skilled in the art from the teachings herein.
[0053] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverese orientation. The
construct further comprises regulatory sequences, including, for
example, a promoter, operably linked to the sequence. Large numbers
of suitable vectors and promoters are known to those of skill in
the art, and are commercially available. The following vectors are
provided by way of example. Bacterial:pQE70, pQE60, pQE-9
(Oiagen)pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a,
pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3,
pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat; pOG44,
pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
However, any other plasmid or vector may be used as long as it is
replicable and viable in the host.
[0054] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particularly named bacterial promoters include lac, lacZ, T3, T7,
gpt, lambda PR, PI and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs
from retrovirus, and mouse metallothionein-l. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art. In a further embodiment, the present
invention relates to host cells containing the above-described
construct. The host cell can be a higher eukaryotic cell such as
mammalian cell, or a lower eukaryotic cell, such as a yeast cell,
e.g. S. cerevisiae, or the host cell can be a prokaryotic cell,
such as a bacterial cell, e.g. E. coli. Introduction of the
construct into the host cell can be effected by clacium phospahe
transfection, DEAE-Dextran mediated trasfection, or electorporation
(Davis, L., Dibner, M., Battey I., Basic Methods in Molecular
Biology, 1986)).
[0055] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0056] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA costructs of
the present invention. Appropriate cloning an expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook. Et al., Molecular Cloning: A Laboratory Manual, Second
Edition, (Cold Spring Harbor, N.Y., 1989), the disclosure of which
is hereby incorporated by reference.
[0057] Transcription of a DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 19 to 300 bp, that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin (bp 100 to
270), a cytomegalovirus early promoter enhancer, a polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers.
[0058] Generally, recombinant expression vectors; will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S cerevisiae TRP1 gene, and a promoter derived
form a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes. such as 3-phosphoglycerate
kinase (PGK), alpha factor, acid phosphatase or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of a translated protein into the periplasmic
space or extracellular medium. Optionally, the heterologous
sequence can encode a fusion protein including an N-terminal
identification peptide imparing desired characteristics, e.g.,
stabilization or simplified purificatio of expressed recominant
product.
[0059] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vetor and to,
if desirable,, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E.coli, Bacillus
subilis, Salmonellatyphimurium and various species within the
genera Pseudomonas, Streptomyce, and Staphylococcus, although
others may also be employed as a matter of choice.
[0060] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBK322 (ATCC37017). Such commercial
vectors include, for, example, PKK223-3 (Pharmacia) and GEM1
(Promega Biotec, Madison, Wis., USA). These pBR3222 ,"backbone"
sections are combined with an appropriate promoter and structural
sequence to be expressed. Various mammalian cell culture systems
can also be employed to express recombint protein. Examples of
mammalian expression systems include the COS-7 lines of monkey
kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and
other cell lines capable of expressing a compatible vector, for
example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian
expression vectors will comprise an origin of replication, a
suitable promoter and enhancer, and also any necessary ribosome
binding sites, polyadenylation site, splice donor and acceptor
sites, transcriptional termination sequences, and 5' flaking
nontranscribed sequences. DNA sequences derived from the SV40 viral
genome for example, SV40 origin, early promoter, enhancer, splice,
and polyadenylation sites may be used to provide the required
nontranscribed genetic elements.
[0061] A thus-expressed polypeptide of the present invention is
recovered and purified from recombinant cell cultures by methods
used heretofore, including detergent homoginates, Heparin-Sepharose
chromatography, cation exchange chromatography, Con A-Sepharose
chromatography, gel-filtration chromatography, and hydrophobic
interaction chromatography.
[0062] A polypeptide of the present invention may be purified
products naturally expressed from a high expressing cell line, or a
product of chemical synthetic procedures, or produced by
recombinant techniques from a prokaryotic or eukaryotic host (for
example, by bacterial, yeast, higher plant, insect and mammalian
cells in culture). Depending upon the host employed in a
recombinant production procedure, a polypeptide of the present
invention may be glycosylated with mammalian or other eukaryotic
carbohydrates or may be non-glycosylated. A polypeptide of the
invention may also include an initial methionine amino acid
residue.
[0063] The polynucleotides of the present invention are also
valuable for chromosome identification. The polynucleotide is
specifically targeted to and can hybridize with a particular
location on an individual human chromosome. Moreover, there is a
current need for identifying particular sites on the chromosome.
Few chromosome marking reagents based on actual sequence data
(repeat polymorphism's) are presently available for marking
chromosomal location. The mapping of DNAs to chromosomes according
to the present invention is an important first step in correlating
those sequences with genes associated with diseases.
[0064] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 b bp) from the cDNA Computer analysis
of the cDNA is used to rapidly select primers that do not span more
than one exon in the genomic DNA, thus complicating the
amplification process. These primers are then used for PCR
screening of somatic cell hybrids containing individual human
chromosomes. Only those hybrids containing the human gene
corresponding to the primer will yield an amplified fragment.
[0065] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0066] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA as short as 500 or 600 bases; however, clones larger than
2,000 bp have a higher likelihood of binding to a unique
chromosomal location with sufficient signal intensity for simple
detection. FISH requires use-of the clone from which the EST was
derived, and the longer the better. For example, 2,000 bp is good,
4,000 is better, and more than 4,000 is probably not necessary to
get good results a reasonable percentage of the time. For a review
of this technique, see Verrna et at., Human Chromosomes* a Manual
of Basic Techniques. Pergamon Press, New York (1988).
[0067] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. (Such data are found, for
example, in V. McKusck, Mendelian Inheritance in Man (available on
line through Johns Hopkins University welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physicall adjacent genes).
[0068] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0069] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb)
[0070] Comparison of affected and unaffected individuals generally
involves first looking for structural alterations in the
chromosomes, such as deletions or translocations that are visible
form chromosome spreads or detectable using PCR based on that cDNA
sequence. Ultimately, complete sequencing of gened from several
individuals is required to confirm the presence of a mutation and
to distinguish mutations from polymorphisms.
[0071] In another aspect the present invention relates to an
oligonucleotide or a derivative thereof, or a salt thereof where
salt-forming groups are present, which is specifically hybridizable
with the nucleotide sequence set forth in SEQ ID NO 1, said
oligonucleotide or derivative thereof comprising nucleoside units
or analogues of nucleoside units sufficient in number and identity
to allow such hybridization.
[0072] Such an oligonucleotide may have a length of, e.g., from
about 5 to about 100 or to even several hundred nucleoside units or
analogs thereof, depending on the intended use.
[0073] An oligonucleotide of the invention may be used as a cloning
or sequencing primer or probe. Another use relates to stimulating
and inhibiting a polypeptide of the present invention in vivo by
the use of sense or anti-sense technology. These technology can be
used to control gene expression through triple-helix formation on
double-stranded DNA or antisense-mechanisms on RNA, both of which
methods are based on binding of such an oligonucleotide to DNA or
RNA. For example, the 5' coding portion of the polynucleotide
sequence, which encodes for the mature polypeptide of the present
invention, is used to design an antisense RNA oligonucleotide of
from about 10 to about 40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the
gene involved in transcription (for triple-helix technology see Lee
et al, Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science,
241:456 (1988); and Dervan et al, Science, 251:1360 (1991)),
thereby preventing transcription and the production of Heparanase.
The antisense RNA oligonucleotide hybridizes to the mRNA in vivo
and blocks translation of an mRNA molecule into a polypeptide of
the present invention (for antisense--technology see Okano, J.
Neuroch., 56:569 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.
(1988)).
[0074] Alternatively, the oligonucleotides described above can be
delivered to cells by procedures in the art such that the
anti-sense RNA or DNA may be expressed in vivo to inhibit
production of a polypeptide of the present invention in the manner
described above.
[0075] Antisense constructs to a polypeptide of the invention,
therefore inhibit the action of such a polypeptide and may be used
for treating certain disorders, for example, cancer and cancer
metastasis, since elevated levels of such a polypeptide is found in
highly metastatic cell lines.
[0076] The polypeptides, their functional fragments., derivatives
or analogs thereof, or a cell expressing them can be used as an
immunogen to produce antibodies thereto.
[0077] Therfore, the present invention relates to an antibody which
specifically recognizes and binds to a polypeptide of the
invention.
[0078] Such an antibody can be, for example, a polyclonal or a
monoclonal antibody. The present invention also includes chimeric,
single chain and humanized antibodies, as well as Fab fragments, or
the product of and Fab expressing library. Various procedures known
in the art may be used for the production of such antibodies and
fragments.
[0079] Antibodies generated against a polypeptide of the present
invention can be obtained by direct injection of the polypeptide
into an animal or by administering the polypeptide to an animal,
preferably human. The antibody so obtained will then bind the
polypeptide itself. In this manner, even a sequence encoding only a
fragment of the polypeptide can be used to generate antibodies
binding the whole native polypeptide. Such an antibody can then be
used to isolate the polypeptide from tissue expressing that
polypeptide. For preparation of monoclonal antibodies, any
technique which provides antibodies produced by continuous cell
line cultures can be used. Examples include the hybridoma technique
(Kohler and Milstein, 1975, Nature, 256:49597), the trioma
technique, the human B-cell hybridoma technique (Kozbor et al.,
1985, Imunology Today 4:72), and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole, et al., 1985, in
Monoclonal Antibodies and Cancer Theraph, Alan R. Liss, Inc.,
pp.77-96).
[0080] Techniques described for the production of single chain
antibodies (e.g. U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention.
[0081] An antibodie specific to a polypeptide of the present
invention may further be used to inhibit the biological action of
the polypeptide by binding to the polypeptide. In this manner, the
antibodies may be used in therapy, for example, to treat cancer
since mRNA of such a polypeptide and the polypeptide itself is
increased in SV-40 transformed fibroblasts.
[0082] Further, such antibodies can detect the presence or absence
of a polypeptide of the present invention and the level of
concentration of such polypeptide and, therefore, are useful as
diagnostic markers for the diagnosis; of disorders such as cancer,
cancer metastasis, and angiogenesis.
[0083] Hence, the present invention relates to a method of
diagnosis of conditions resulting from shortage or lack of a
polypeptide of the present invention, in particular of a
polypeptide showing the biological activity of human heparanase
obtainable from the SV-40 transformed human fibroblast cell line
ATCC CCL 75.1, or a functional derivative, a functional fragment or
a functional analogue thereof, inclusive the respective preferred
embodiments thereof, or resulting from excessive acitvity or
overexpression of such a polypeptide, said method comprising
contacting cells or tissues or body fluids from an animal inclusive
man suspected of having such a condition with an antibody of the
present invention.
[0084] In a further aspect, the present invention relates to a
method for identifying a substance capable of modulating the
biological activity or expression of a polypeptide of the present
invention, said method comprising contacting such a polypeptide or
a functional derivative, a functional fragment or a functional
analogue thereof, or a cell capable of expressing such a
polypeptide, functional derivative, functional fragment or
functional analogue, with at least one compound or agent whose
ability to modulate the biological activity or expression of said
polypeptide is sought to be investigated, and determining the
change of the biological activity or the expression of said
polypeptide, derivative, fragment or analogue caused by the
substance.
[0085] Another embodiment of the present invention relates to an
assay system for testing a substance for its capability of binding
to or having functional effects on a polypeptide of the present
invention, said assay system comprising a polypeptide as of the
present invention, or a functional derivative, a functional
fragment or a functional analogue thereof, or a cell expressing
such a polypeptide, functional derivative, functional fragment or
functional analogue.
[0086] In this context, the present invention is also directed to
substance obtainable by an identification-method as described
above, said substance being an agonist or an antagonist of a
polypeptide of the present invention.
[0087] Thus, the present invention is also directed to antagonists
and inhibitors of a polypeptide of the present invention. The
antagonists and inhibitors are those substances which inhibit or
eliminate the function of such a polypeptide. The present invention
further relates to agonists and stimulators of a polypeptide of the
present invention. The agonists and stimulators are those
substances which enhance the function or activity or the expression
of such a polypeptide.
[0088] For example, an antagonist may bind to a polypeptide of the
present invention and inhibit or eliminates its function. The
antagonist, for example, could be an antibody against the
polypeptide which eliminated the activity of Heparanase by binding
to Heparanase, or in some cases the antagonist may be an
oligonucleotide. An example of an inhibitor is a small molecule
inhibitor which inactivates the polypeptide by binding to and
occupying the catalytic site, thereby making the catalytic site
inaccessible to a substrate, such that the biological activity of
Heparanase is prevented. Examples of small molecule inhibitors
include but are not limited to small carbohydrate or
carbohydrate-like molecules.
[0089] In these ways, the antagonists and inhibitors may be used to
treat cancer, angiogenesis by preventing heparanase from
functioning tic break down extracellular matrix and release heparan
sulfate from extracellular matrix and cell surface.
[0090] The antagonists and inhibitors may be employed in a
composition with a pharmaceutically acceptable carrier, including
but not limited to saline, buffered saline, dextrose, wate,
glycerol, ethanol and combinations thereof. Administration of
inhibitors of the polypeptides of the present invention are
preferably systemic. Suramin, a polysulfonated naphthylurea, was
shown to strongly inhibit murin melanoma heparanase and its
invation (Nakajima, M. et al. J. Biol. Chem. 266, p9661-9666,
1991). 2,3-O-desulfated heparin was shown to inhibit heparanase
activity, tumor growth of a subcutaneous human pancreatic
(Ca-Pan-2) adenocarcinoma in nude mice and prolonged the survival
times of C57BU6N mice in a B16-F10 melanoma experimental lung
metastasis assay (Lapierre, F. et al. Glycobiology vol. 6, 335-366,
1996).
[0091] In particular, the present invention also relates to an
assay for identifying the above-mentioned substances, e.g. small
molecule inhibitors, which are specific to the polypeptides and
prevent them from functioning or prevent their expression. Either
natural carbohydrate substrates or synthetic carbohydrates would be
used to assess endo-glycosidase activity of the polypeptide, and
the ability of inhibitors to prevent this activity could be the
basis for a screen to identify compounds that have therapeutic
activity in disorders of excessive activity or overexpression of a
polypeptide according to the present invention.
[0092] In particular, the present invention also relates to an
assay for identifying the above-mentioned substances, e.g. small
molecule stimulators, which are specific to the polypeptides and
enhance its function or expression. Either natural carbohydrate
substrates or synthetic carbohydrates would be used to assess
endo-glycosidase activity of the d the ability of stimulators to
enhance this activity could be the basis for a screen to identify
compounds that have therapeutic activity in disorders which are
resultant of shortage or lack of a polypeptide according to the
present invention.
[0093] A further aspect relates to a polypeptide according to the
present invention for use in medicine. In particular, the invention
relates to the use of a polypeptide according to the present
invention in the preparation of a pharmaceutical composition for
the treatment of a disease resulting from shortage or lack of said
polypeptide. Instead of a polypeptide of the present invention, an
agonist of the polypeptide or an expression inducer/enhancer of
such a polypeptide may be used for the medicinal purposes. Such
diseases are, for example, trauma, autoimmune diseases, skin
diseases, cardiovascular diseases and nervous system diseases
including Alzheimer's disease.
[0094] Another aspect relates to an antibody according to the
present invention for use medicine. In particular, the invention
relates to the use of an antibody according to the present
invention in the preparation of a pharmaceutical composition for
the treatment of a disease resulting from excessive acitvity or
overexpression of a polypeptide of a polypeptide according to the
present invention. Instead of a antibody of the present invention,
an antagonist of the polypeptide or an expression inhibitor of such
a polypeptide may be used for the medicinal purposes. Such diseases
are. for example, cancer, cancer metastasis, angiogenesis and
inflammation including arthritis.
[0095] The invention moreover is directed to a pharmaceutical
composition suitable for administration to a warm-blooded animal
inclusive man suffering from a disease resulting from shortage or
lack of a polypeptide of the present invention, said composition
comprising such a polypeptide together with at least one
pharmaceutically acceptable carrier and/or excipient. In this
context, the invention is directed to a method of treatment of a
disease resulting from shortage or lack of a polypeptide of the
invention said method comprising administration of a suitable
amount of such a polypeptide. As mentioned above, instead of such a
polypeptide said composition or method of treatment may comprise or
utilize an agonist of the polypeptide or an expression
inducer/enhancer of such a polypeptide.
[0096] Furthermore, the invention is directed to a pharmaceutical
composition suitable for administration to a warm-blooded animal
inclusive man suffering from a disease resulting from excessive
acitvity or overexpression of a polypeptide of the present
invention, said composition comprising an antibody of the present
invention together with at least one pharmaceutically acceptable
carrier and/or excipient. In this context, the invention relates to
a method of treatment of a disease resulting from excessive
acitvity or overexpression of a polypeptide of the present
invention, said method comprising administration of a suitable
amount of an antibody of the present invention. As mentioned above,
instead of such an antibody said composition or method of treatment
may comprise or utilize an antagonist of the polypeptide or an
expression inhibitor of such a polypeptide.
[0097] Further ingredients, i.e. a carrier or excipient, of a
pharmaceutical composition of the present invention may be those
known in the art, in particular those as described herein
above.
[0098] In another aspect the present invention relates to a
polynucleotide of the invention for use in gene therapy.
[0099] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0100] In order to facilitate understanding of the following
examples, certain frequently occurring methods and/or terms will be
described.
[0101] "Plasmids" are designated by a lower case preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0102] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirement were used as would be known to the ordinarily
skilled artisan. Fro analytical purpose, typically 1 microg of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 microl of buffer solution. For the pupose of isolating DNA
fragments for plasmid construction, typically 5 to 50 microg of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0103] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acis Res., 8*4057 (1980).
[0104] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary strande
polydeoxynucleotide strands which may be chemically
synthesized.
[0105] Such synthetic oligonucleotides have no 5' phosphate and
thus will not ligate to another oligonucleotide without adding a
phosphate with an ATP in the presence of a kinas. A synthetic
oligonucleotide will ligate to a fragment that has not been
dephosphorylated.
[0106] "Ligation" refers to the process of forming phosphodiester
bonds between two double strande nucleic acids. Unless otherwise
provided, ligation may be accomplished using known buffers and
conditions with 10 units of T4 DNA ligase per 0.5 microg of
approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation is performed as described
in the methods of Graham, F. and Van Der Eb, A., Virology,
52:456-457 (1973).
EXAMPLES
Example 1
Expression and Purification of a Polypeptide of the Present
Invention
[0107] The DNA sequence encoding the polypeptide of the present
invention as outlined in SEQ ID NO 1 is initially amplified using
PCR oligonucleotide primers corresponding to the 5' and 3' end of
the DNA sequence to synthesize insertion fragments. The 5'
oligonucleotide primer has the sequence of
5'-GGAMTTCAGCAGCCAGGTGAGCCCAAG-3' containing an EcoRI restriction
enzyme site followed by 20 nucleotides of the polypeptide coding
sequence starting from the methionine start codon. The 3' primer
sequence was 5'-ACTCGAGGATGCAAGCAGCMCTTTGGC,-3' containing a
complementary sequence to Xhol site and the last 21 nucleotides of
the polypeptide coding sequence. The restriction enzyme sites
correspond to the restriction enzyme sites on the pFastBacl (Gibco
BRL, Rockville, Md., U.S.A.). The plasmid vector encodes antibiotic
resistance (Ampr), a bacterial origin of replication (ori), and
bacterial transposon Tn7. The pFastBaci vector is digested with
EcoRI and Xhol and the insertion fragments are ligated into the
vector maintaing the reading frame. The ligation mixture is then
used to transform the E. coli strain DH10B. The transformants are
selected by their ability to grow on LB plates containing Amp.
Clones containing the desired construct is then transposed to
Bacmid DNA by transfoming DH10Bac with the recombinant plasmid
vector containg desired heparanase gene. Transposed Bacmid DNA
containing heparanase gene is selected by Blue/White selection by
Bluo-gal on LB plate containg Kan, Gen, Tet, Bluo-gal, and IPTG.
Clones Bacmid DNA containing desired gene is isolated, then,
transfected to insect cell line Sf9 cells with CellFECTIN reagent
(also available from GIBCO BRL). Harvesting of recombinant
baculovirus is performed 3 days after the transfection. Expression
of the polypeptide is carried out by infecting insect Tn cells with
recombinant baculoviruses containing the heparanase-coding gene.
Culture supernatants of infected Tn cells are collected, and
heparanase is purified by affinity chromatography. Briefly, the
supernatant is loaded onto a heparin-Sepharose column and then
heparanase is eluted from the column by gradient solution of 0.15
to 1.0 M of NaCl.
Example 2
Assay for Identifying Agonist and Antagonist of a Polypeptide of
the Present Invention
[0108] For heparanase substrates, heparan sulfate labeled with
fluorescein isothiocyante (FITC) is used. Briefly, 5 mg of heparan
sulfate from bovine kidney (available from Seikagaku Kougyou Ltd.
Tokyo, Japan) is mixed with 5 mg of FITC in 0.1 M sodium carbonate
(pH 9.5), and incubated with gentle mixing at 4.degree. C., for 12
hours. The FITC-labeled heparan sulfate is fractionated by gel
filtration using a Sephacyl S-300HR column (Pharmacia Biotech,
Inc.) equilibrated with 25 mM Tris-HCl, 150 mM NaCl. One to five
micrograms of FITC-labeled heparan sulfate is mixed with the
purified polypeptide, which is prepared as described in the example
1, in the presence (Dr absence of agonist/antagonist and then
incubated at 37.degree. C. for 1-2 hours. The enzyme reaction is
terminated by heating at 95.degree. C. for 5 minutes. The
inactivated mixture is analyzed by high-speed gel permeation
chromatography using a TSKgelG3000PWXL column (available from TOSOH
Ltd. Tokyo, Japan). The inhibitory activity of antagonist and the
stimulatory activity of agonist are assessed by the amount of
non-degraded heparan sulfate detected by a fluorescence monitor.
Sequence CWU 1
1
2 1 3726 DNA Human 1 cagcgctgct ccccgggcgc tcctccccgg gcgctcctcc
ccaggcctcc cgggcgcttg 60 gatcccggcc atctccgcac ccttcaagtg
ggtgtgggtg atttcctggc ggggggagca 120 gccaggtgag cccaagatgc
tgctgcgctc gaagcctgcg ctgccgccgc cgctgatgct 180 gctgctcctg
gggccgctgg gtcccctctc ccctggcgcc ctgccccgac ctgcgcaagc 240
acaggacgtc gtggacctgg acttcttcac ccaggagccg ctgcacctgg tgagcccctc
300 gttcctgtcc gtcaccattg acgccaacct ggccacggac ccgcggttcc
tcatcctcct 360 gggttctcca aagcttcgta ccttggccag aggcttgtct
cctgcgtacc tgaggtttgg 420 tggcaccaag acagacttcc taattttcga
tcccaagaag gaatcaacct ttgaagagag 480 aagttactgg caatctcaag
tcaaccagga tatttgcaaa tatggatcca tccctcctga 540 tgtggaggag
aagttacggt tggaatggcc ctaccaggag caattgctac tccgagaaca 600
ctaccagaaa aagttcaaga acagcaccta ctcaagaagc tctgtagatg tgctatacac
660 ttttgcaaac tgctcaggac tggacttgat ctttggccta aatgcgttat
taagaacagc 720 agatttgcag tggaacagtt ctaatgctca gttgctcctg
gactactgct cttccaaggg 780 gtataacatt tcttgggaac taggcaatga
acctaacagt ttccttaaga aggctgatat 840 tttcatcaat gggtcgcagt
taggagaaga ttttattcaa ttgcataaac ttctaagaaa 900 gtccaccttc
aaaaatgcaa aactctatgg tcctgatgtt ggtcagcctc gaagaaagac 960
ggctaagatg ctgaagagct tcctgaaggc tggtggagaa gtgattgatt cagttacatg
1020 gcatcactac tatttgaatg gacggactgc taccagggaa gattttctaa
accctgatgt 1080 attggacatt tttatttcat ctgtgcaaaa agttttccag
gtggttgaga gcaccaggcc 1140 tggcaagaag gtctggttag gagaaacaag
ctctgcatat ggaggcggag cgcccttgct 1200 atccgacacc tttgcagctg
gctttatgtg gctggataaa ttgggcctgt cagcccgaat 1260 gggaatagaa
gtggtgatga ggcaagtatt ctttggagca ggaaactacc atttagtgga 1320
tgaaaacttc gatcctttac ctgattattg gctatctctt ctgttcaaga aattggtggg
1380 caccaaggtg ttaatggcaa gcgtgcaagg ttcaaagaga aggaagcttc
gagtatacct 1440 tcattgcaca aacactgaca atccaaggta taaagaagga
gatttaactc tgtatgccat 1500 aaacctccat aatgtcacca agtacttgcg
gttaccctat cctttttcta acaagcaagt 1560 ggataaatac cttctaagac
ctttgggacc tcatggatta ctttccaaat ctgtccaact 1620 caatggtcta
actctaaaga tggtggatga tcaaaccttg ccacctttaa tggaaaaacc 1680
tctccggcca ggaagttcac tgggcttgcc agctttctca tatagttttt ttgtgataag
1740 aaatgccaaa gttgctgctt gcatctgaaa ataaaatata ctagtcctga
cactgaattt 1800 ttcaagtata ctaagagtaa agcaactcaa gttataggaa
aggaagcaga taccttgcaa 1860 agcaactagt gggtgcttga gagacactgg
gacactgtca gtgctagatt tagcacagta 1920 ttttgatctc gctaggtaga
acactgctaa taataatagc taataatacc ttgttccaaa 1980 tactgcttag
cattttgcat gttttacttt tatctaaagt tttgttttgt tttattattt 2040
atttatttat ttattttgtg acggagagag attccatctc aaaaaaacaa gttattaaaa
2100 atgtatatga atgctcctaa tatggtcagg aagcaaggaa gcgaaggata
tattatgagt 2160 tttaagaagg tgcttagctg tatatttatc tttcaaaatg
tattagaaga ttttagaatt 2220 ctttccttca tgtgccatct ctacaggcac
ccatcagaaa aagcatactg ccgttaccgt 2280 gaaactggtt gtaaaagaga
aactatctat ttgcacctta aaagacagct agattttgct 2340 gattttcttc
tttcggtttt ctttgtcagc aataatatgt gagaggacag attgttagat 2400
atgatagtat aaaaaatggt taatgacaat tcagaggcga ggagattctg taaacttaaa
2460 attactataa atgaaattga tttgtcaaga ggataaattt tagaaaacac
ccaatacctt 2520 ataactgtct gttaatgctt gctttttctc tacctttctt
ccttgtttca gttgggaagc 2580 ttttggctgc aagtaacaga aactcctaat
tcaaatggct taagcaataa ggaaatgtat 2640 attcccacat aactagacgt
tcaaacaggc caggctccag cacttcagta cgtcaccagg 2700 ggatctgggt
tcttcccagc tctctgctct gccatcttta gcgctggctt cattctcaga 2760
ctctggtagc atgatggctg tagctgtttc atgggcccct tcaaacctca tagcaaccag
2820 aggaagaaaa tgagccattt tttgagtctc cttcatagac ttgaataact
ctttttcaga 2880 gcttctcaca gcaaacctct cctcatgtct cctcatgtct
tattgttcag aaatgggtaa 2940 tgtggccatt tcaccagtca ctgccaacaa
caacgaggtt cctataattg tctctgagta 3000 accctttgga atggagaggg
tgttggtcag tctacaaact gaacactgca gttctgcgct 3060 ttttaccagt
gaaaaaatgt aattattttc ccctcttaag gattaatatt cttcaaatgt 3120
atgcctgtta tggatatagt atctttaaaa ttttttattt taatagcttt aggggtacac
3180 actttttgct tacaggggtg aattgtgtag tggtgaagac tcggctttta
atgtacttgt 3240 cacctgagtg atgtacattg tacccaatag gtaatttttc
atccattacc ctccttccgc 3300 cctcttccct tctgagtctc caacatccct
tataccactg tgtatgttct tgtgtaccta 3360 cagctaagct tccacttata
agtgagaaca tgcagtattt ggttttccat tcctgagtta 3420 cttcccttag
gataacagcc cccagttccg tccaagttgc tgcaaaatac attattcttc 3480
tttatggctg agtaatagtc catggtacat atataccaca ttttctttat ccacttatca
3540 gttgatggac acttaggtta attccattca atttcattca atttaagtat
atttgtaagg 3600 agctaaagct gaaaattaaa ttttagatct ttcaatactc
ttaaatttta tatgtaagtg 3660 gtttttatat tttcacattt gaaataaagt
aatttttata accttgaaaa aaaaaaaaaa 3720 aaaaaa 3726 2 588 PRT Human 2
Ser Ala Ala Pro Arg Ala Leu Leu Pro Gly Arg Ser Ser Pro Gly Leu 1 5
10 15 Pro Gly Ala Trp Ile Pro Ala Ile Ser Ala Pro Phe Lys Trp Val
Trp 20 25 30 Val Ile Ser Trp Arg Gly Glu Gln Pro Gly Glu Pro Lys
Met Leu Leu 35 40 45 Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met
Leu Leu Leu Leu Gly 50 55 60 Pro Leu Gly Pro Leu Ser Pro Gly Ala
Leu Pro Arg Pro Ala Gln Ala 65 70 75 80 Gln Asp Val Val Asp Leu Asp
Phe Phe Thr Gln Glu Pro Leu His Leu 85 90 95 Val Ser Pro Ser Phe
Leu Ser Val Thr Ile Asp Ala Asn Leu Ala Thr 100 105 110 Asp Pro Arg
Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu Arg Thr Leu 115 120 125 Ala
Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr 130 135
140 Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe Glu Glu Arg
145 150 155 160 Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys
Tyr Gly Ser 165 170 175 Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu
Glu Trp Pro Tyr Gln 180 185 190 Glu Gln Leu Leu Leu Arg Glu His Tyr
Gln Lys Lys Phe Lys Asn Ser 195 200 205 Thr Tyr Ser Arg Ser Ser Val
Asp Val Leu Tyr Thr Phe Ala Asn Cys 210 215 220 Ser Gly Leu Asp Leu
Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Ala 225 230 235 240 Asp Leu
Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu Asp Tyr Cys 245 250 255
Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn 260
265 270 Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser Gln Leu
Gly 275 280 285 Glu Asp Phe Ile Gln Leu His Lys Leu Leu Arg Lys Ser
Thr Phe Lys 290 295 300 Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln
Pro Arg Arg Lys Thr 305 310 315 320 Ala Lys Met Leu Lys Ser Phe Leu
Lys Ala Gly Gly Glu Val Ile Asp 325 330 335 Ser Val Thr Trp His His
Tyr Tyr Leu Asn Gly Arg Thr Ala Thr Arg 340 345 350 Glu Asp Phe Leu
Asn Pro Asp Val Leu Asp Ile Phe Ile Ser Ser Val 355 360 365 Gln Lys
Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly Lys Lys Val 370 375 380
Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu Leu 385
390 395 400 Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys Leu
Gly Leu 405 410 415 Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln
Val Phe Phe Gly 420 425 430 Ala Gly Asn Tyr His Leu Val Asp Glu Asn
Phe Asp Pro Leu Pro Asp 435 440 445 Tyr Trp Leu Ser Leu Leu Phe Lys
Lys Leu Val Gly Thr Lys Val Leu 450 455 460 Met Ala Ser Val Gln Gly
Ser Lys Arg Arg Lys Leu Arg Val Tyr Leu 465 470 475 480 His Cys Thr
Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly Asp Leu Thr 485 490 495 Leu
Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu Arg Leu Pro 500 505
510 Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu Arg Pro Leu
515 520 525 Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn Gly
Leu Thr 530 535 540 Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu
Met Glu Lys Pro 545 550 555 560 Leu Arg Pro Gly Ser Ser Leu Gly Leu
Pro Ala Phe Ser Tyr Ser Phe 565 570 575 Phe Val Ile Arg Asn Ala Lys
Val Ala Ala Cys Ile 580 585
* * * * *