U.S. patent application number 10/431438 was filed with the patent office on 2003-09-25 for avian and reptile derived polynucleotide encoding a polypeptide having heparanase activity.
This patent application is currently assigned to Insight Strategy & Marketing Ltd.. Invention is credited to Goldshmidt, Orit, Michal, Israel, Pecker, Iris, Vlodavsky, Israel, Zcharia, Eyal.
Application Number | 20030180788 10/431438 |
Document ID | / |
Family ID | 27099452 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180788 |
Kind Code |
A1 |
Goldshmidt, Orit ; et
al. |
September 25, 2003 |
Avian and reptile derived polynucleotide encoding a polypeptide
having heparanase activity
Abstract
Avian and reptile derived heparanase.
Inventors: |
Goldshmidt, Orit;
(Jerusalem, IL) ; Pecker, Iris; (Rishon LeZion,
IL) ; Vlodavsky, Israel; (Mevaseret Zion, IL)
; Michal, Israel; (Ashkelon, IL) ; Zcharia,
Eyal; (Jerusalem, IL) |
Correspondence
Address: |
G.E. Ehrlich (1995) LTD.
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Assignee: |
Insight Strategy & Marketing
Ltd.
Hadasit Medical Research Services and Development Ltd.
|
Family ID: |
27099452 |
Appl. No.: |
10/431438 |
Filed: |
May 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10431438 |
May 8, 2003 |
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09930218 |
Aug 16, 2001 |
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09930218 |
Aug 16, 2001 |
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09666390 |
Sep 20, 2000 |
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Current U.S.
Class: |
435/6.16 ;
435/200; 435/325; 435/349; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12Y 302/01166 20130101;
C07K 2319/00 20130101; C12N 9/2402 20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/200; 435/325; 435/349; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/24; C12N 005/06; C12P 021/02 |
Claims
What is claimed is:
1. A recombinant protein comprising a polypeptide being at least
75% similar to SEQ ID NO:4, as determined using the BESTFIT
software of the Wisconsin sequence analysis package, utilizing the
Smith and Waterman algorithm, where gap weight equals 8 and length
weight equals 2, average match equals 2.912 and average mismatch
equals -2.003, said polypeptide having heparanase catalytic
activity or said polypeptide is cleavable by a protease so as to
have said heparanase catalytic activity.
2. A recombinant protein comprising a polypeptide encoded by a
nucleic acid including a polynucleotide sequence being at lest 65%
identical to SEQ ID NO:10 or a portion thereof as determined using
the BESTFIT software of the Wisconsin sequence analysis package,
utilizing the Smith and Waterman algorithm, where gap weight equals
50, length weight equals 3, average match equals 10 and average
mismatch equals -9, said polypeptide having heparanase catalytic
activity or said polypeptide is cleavable by a protease so as to
have said heparanase catalytic activity.
3. A recombinant protein comprising a polypeptide being encoded by
a nucleic acid as set forth in SEQ ID NO:10.
4. A recombinant protein comprising a polypeptide encoded by a
polynucleotide sequence being hybridizable with SEQ ID NO:10 under
hybridization conditions of hybridization solution containing 10%
dextrane sulfate, 1 M NaCl, 1% SDS and 5.times.10.sup.6 cpm
.sup.32p labeled probe, at 65.degree. C., with a final wash
solution of 1.times. SSC and 0.1% SDS and final wash at 65.degree.
C., said polypeptide having heparanase catalytic activity or said
polypeptide is cleavable by a protease so as to have said
heparanase catalytic activity.
5. A pharmaceutical composition comprising, as an active
ingredient, the recombinant protein of claim 1 and a
pharmaceutically acceptable carrier.
6. A pharmaceutical composition comprising, as an active
ingredient, the recombinant protein of claim 2 and a
pharmaceutically acceptable carrier.
7. A pharmaceutical composition comprising, as an active
ingredient, the recombinant protein of claim 3 and a
pharmaceutically acceptable carrier.
8. A pharmaceutical composition comprising, as an active
ingredient, the recombinant protein of claim 4 and a
pharmaceutically acceptable carrier.
Description
[0001] This is a divisional of U.S. patent application Ser. No.
09/930,218, filed Aug. 16, 2001, which is a continuation-in-part of
U.S. patent application Ser. No. 09/666,390, filed Sep. 20,
2000.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to an avian and reptile
derived polynucleotide encoding a polypeptide having heparanase
catalytic activity. The present invention further relates to the
use of the signal peptide of avian and/or reptile heparanase for
expression of membrane associated and/or secreted proteins in
heterologous expression systems.
Glycosaminoglycans (GAGs)
[0003] GAGs are polymers of repeated disaccharide units consisting
of uronic acid and a hexosamine. Biosynthesis of GAGs except
hyaluronic acid is initiated from a core protein. Proteoglycans may
contain several GAG side chains from similar or different families.
GAGs are synthesized as homopolymers which may subsequently be
modified by N-deacetylation and N-sulfation, followed by
C5-epimerization of glucuronic acid to iduronic acid and
O-sulfation. The chemical composition of GAGs from various tissues
varies highly.
[0004] The natural metabolism of GAGs in animals is carried out by
hydrolysis. Generally, the GAGs are degraded in a two step
procedure. First the proteoglycans are internalized in endosomes,
where initial depolymerization of the GAG chain takes place. This
step is mainly hydrolytic and yields oligosaccharides. Further
degradation is carried out following fusion with lysosome, where
desulfation and exolytic depolymerization to monosaccharides take
place (42).
[0005] The only GAG degrading endolytic enzymes characterized so
far in animals are the hyaluronidases. The hyaluronidases are a
family of 1-4 endoglucosaminidases that depolymerize hyaluronic
acid and chondroitin sulfate. The cDNAs encoding sperm associated
PH-20 (Hyal3), and the lysosomal hyaluronidases Hyal 1 and Hyal 2
were cloned and published (27). These enzymes share an overall
homology of 40% and have different tissue specificities, cellular
localizations and pH optima for activity.
[0006] Exolytic hydrolases are better characterized, among which
are .beta.-glucoronidase, .alpha.-L-iduronidase, and
.beta.-N-acetylglucosami- nidase. In addition to hydrolysis of the
glycosidic bond of the polysaccharide chain, GAG degradation
involves desulfation, which is catalyzed by several lysosomal
sulfatases such as N-acetylgalactosamine-4- -sulfatase,
iduronate-2-sulfatase and heparin sulfamidase. Deficiency in any of
lysosomal GAG degrading enzymes results in a lysosomal storage
disease, mucopolysaccharidosis.
Glycosyl Hydrolases
[0007] Glycosyl hydrolases are a widespread group of enzymes that
hydrolyze the o-glycosidic bond between two or more carbohydrates
or between a carbohydrate and a noncarbohydrate moiety. The
enzymatic hydrolysis of glycosidic bond occurs by using major one
or two mechanisms leading to overall retention or inversion of the
anomeric configuration. In both mechanisms, catalysis involves two
residues: a proton donor and a nucleophile. Glycosyl hydrolyses
have been classified into 58 families based on amino acid
similarities. The glycosyl hydrolyses from families 1, 2, 5, 10,
17, 30, 35, 39 and 42 act on a large variety of substrates,
however, they all hydrolyze the glycosidic bond in a general acid
catalysis mechanism, with retention of the anomeric configuration.
The mechanism involves two glutamic acid residues, which are the
proton donor and the nucleophile, with an aspargine which always
precedes the proton donor. Analyses of a set of known 3D structures
from this group of enzymes revealed that their catalytic domains,
despite the low level of sequence identity, adopt a similar
(.alpha./.beta.) 8 fold with the proton donor and the nucleophile
located at the C-terminal ends of strands .beta.4 and .beta.7,
respectively. Mutations in the functional conserved amino acids of
lysosomal glycosyl hydrolases were identified in lysosomal storage
diseases.
[0008] Lysosomal glycosyl hydrolases including
.beta.-glucuronidase, .beta.-manosidase, .beta.-glucocerebrosidase,
.beta.-galactosidase and .alpha.-L-iduronidase, are all
exo-glycosyl hydrolases, belong to the GH-A clan and share a
similar catalytic site. However, many endo-glucanases from various
organisms, such as bacterial and fungal xylenases and cellulases
share this catalytic domain (1).
Heparan Sulfate Proteoglyeans (HSPGs)
[0009] HSPGs are ubiquitous macromolecules associated with the cell
surface and extracellular matrix (ECM) of a wide range of cells of
vertebrate and invertebrate tissues (3-7). The basic HSPG structure
consists of a protein core to which several linear heparan sulfate
chains are covalently attached. The polysaccharide chains are
typically composed of repeating hexuronic and D-glucosamine
disaccharide units that are substituted to a varying extent with N-
and O-linked sulfate moieties and N-linked acetyl groups (3-7).
Studies on the involvement of ECM molecules in cell attachment,
growth and differentiation revealed a central role of HSPGs in
embryonic morphogenesis, angiogenesis, metastasis, neurite
outgrowth and tissue repair (3-7). The heparan sulfate (HS) chains,
which are unique in their ability to bind a multitude of proteins,
ensure that a wide variety of effector molecules cling to the cell
surface (6-8). HSPGs are also prominent components of blood vessels
(5). In large vessels they are concentrated mostly in the intima
and inner media, whereas in capillaries they are found mainly in
the subendothelial basement membrane where they support
proliferating and migrating endothelial cells and stabilize the
structure of the capillary wall. The ability of HSPGs to interact
with ECM macromolecules such as collagen, laminin and fibronectin,
and with different attachment sites on plasma membranes suggests a
key role for this proteoglycan in the self-assembly and
insolubility of ECM components, as well as in cell adhesion and
locomotion. Cleavage of HS may therefore result in disassembly of
the subendothelial ECM and hence may play a decisive role in
extravasation of normal and malignant blood-borne cells (9-11). HS
catabolism is observed in inflammation, wound repair, diabetes, and
cancer metastasis, suggesting that enzymes, which degrade HS, play
important roles in pathologic processes.
Heparanase
[0010] Heparanase is a glycosylated enzyme that is involved in the
catabolism of certain glycosaminoglycans. It is an
endoglucuronidase that cleaves heparan sulfate at specific
intrachain sites (12-15). Interaction of T and B lymphocytes,
platelets, granulocytes, macrophages and mast cells with the
subendothelial extracellular matrix (ECM) is associated with
degradation of heparan sulfate by heparanase activity (16).
Placenta heparanase acts as an adhesion molecule or as a
degradative enzyme depending on the pH of the microenvironment
(17).
[0011] Heparanase is released from intracellular compartments
(e.g., lysosomes, specific granules) in response to various
activation signals (e.g., thrombin, calcium ionophores, immune
complexes, antigens and mitogens), suggesting its regulated
involvement in inflammation and cellular immunity responses
(16).
[0012] It was also demonstrated that heparanase can be readily
released from human neutrophils by 60 minutes incubation at
4.degree. C. in the absence of added stimuli (18).
[0013] Gelatinase, another ECM degrading enzyme, which is found in
tertiary granules of human neutrophils with heparanase, is secreted
from the neutrophils in response to phorbol 12-myristate 13-acetate
(PMA) treatment (19-20).
[0014] In contrast, various tumor cells appear to express and
secrete heparanase in a constitutive manner in correlation with
their metastatic potential (21).
[0015] Degradation of heparan sulfate by heparanase results in the
release of heparin-binding growth factors, enzymes and plasma
proteins that are sequestered by heparan sulfate in basement
membranes, extracellular matrices and cell surfaces (22-23).
[0016] Heparanase activity has been described in a number of cell
types including cultured skin fibroblasts, human neutrophils,
activated rat T-lymphocytes, normal and neoplastic murine
B-lymphocytes, human monocytes and human umbilical vein endothelial
cells, SK hepatoma cells, human placenta and human platelets.
[0017] A procedure for purification of natural heparanase was
reported for SK hepatoma cells and human placenta (U.S. Pat. No.
5,362,641) and for human platelets derived enzymes (62).
Cloning and Expression of the Human Heparanase Gene
[0018] The human hpa cDNA, which encodes human heparanase, was
cloned from human placenta. It contained an open reading frame,
which encodes a polypeptide of 543 amino acids with a calculated
molecular weight of 61,192 daltons (2). The cloning procedures are
described in length in U.S. patent application Ser. Nos.
08/922,170, 09/109,386, and 09/258,892, the latter is a
continuation-in-part of PCT/US98/17954, filed Aug. 31, 1998, all of
which are incorporated herein by reference. An identical cDNA
encoding human heparanase was isolated later on from hepatoma cell
line SK-hepl (54). From platelets (55, 57, PCT/US99/01489,
PCT/AU98/00898) and from SV40 transformed fibroblasts (56,
PCT/EP99/00777).
[0019] The genomic locus, which encodes heparanase, spans about 40
kb. It is composed of 12 exons separated by 11 introns and is
localized on human chromosome 4.
[0020] The ability of the hpa gene product to catalyze degradation
of heparan sulfate (HS) in vitro was examined by expressing the
entire open reading frame of hpa in High five and Sf21 insect
cells, and the mammalian human 293 embryonic kidney cell line
expression systems. Extracts of infected or transfected cells were
assayed for heparanase catalytic activity. For this purpose, cell
lysates were incubated with sulfate labeled, ECM-derived HSPG (peak
I), followed by gel filtration analysis (SEPHAROSE 6B) of the
reaction mixture. While the substrate alone consisted of high
molecular weight material, incubation of the HSPG substrate with
lysates of cells infected or transfected with hpa containing
vectors resulted in a complete conversion of the high molecular
weight substrate into low molecular weight labeled heparan sulfate
degradation fragments (see, for example, U.S. patent application
Ser. No. 09/071,618, which is incorporated herein by reference.
[0021] In other experiments, it was demonstrated that the
heparanase enzyme expressed by cells infected with a pFhpa virus is
capable of degrading HS complexed to other macromotecular
constituents (e.g., fibronectin, laminin, collagen) present in a
naturally produced intact ECM (see U.S. patent application Ser. No.
09/109,386, which is incorporated herein by reference), in a manner
similar to that reported for highly metastatic tumor cells or
activated cells of the immune system (7, 8).
[0022] In human primary fibroblasts transfected with the heparanase
cDNA the enzyme was localized to the lysosomes.
Preferential Expression of the hpa Gene in Human Breast and
Hepatocellular Carcinomas
[0023] Semi-quantitative RT-PCR was employed to evaluate the
expression of the hpa gene by human breast carcinoma cell lines
exhibiting different degrees of metastasis. A marked increase in
hpa gene expression is observed which correlates to metastatic
capacity of non-metastatic MCF-7 breast carcinoma, moderately
metastatic MDA 231 and highly metastatic MDA 435 breast carcinoma
cell lines. Significantly, the differential pattern of the hpa gene
expression correlated with the pattern of heparanase activity.
[0024] Expression of the hpa gene in human breast carcinoma was
demonstrated by in situ hybridization to archival paraffin embedded
human breast tissue. Hybridization of the heparanase antisense
riboprobe to invasive duct carcinoma tissue sections resulted in a
massive positive staining localized specifically to the carcinoma
cells. The hpa gene was also expressed in areas adjacent to the
carcinoma showing fibrocystic changes. Normal breast tissue derived
from reduction mammoplasty failed to express the hpa transcript.
High expression of the hpa gene was also observed in tissue
sections derived from human hepatocellular carcinoma specimens but
not in normal adult liver tissue. Furthermore, tissue specimens
derived from adenocarcinoma of the ovary, squamous cell carcinoma
of the cervix and colon adenocarcinoma exhibited strong staining
with the hpa RNA probe, as compared to a very low staining of the
hpa mRNA in the respective non-malignant control tissues (2).
[0025] A preferential expression of heparanase in human tumors
versus the corresponding normal tissues was also noted by
immunohistochemical staining of paraffin embedded sections with
monoclonal anti-heparanase antibodies. Positive cytoplasmic
staining was found in neoplastic cells of the colon carcinoma and
in dysplastic epithelial cells of a tubulovillous adenoma found in
the same specimen while there was little or no staining of the
normal looking colon epithelium located away from the carcinoma. Of
particular significance was an intense immunostaining of colon
adenocarcinoma cells that had metastasized into lymph nodes, lung
and liver, as compared to the surrounding normal tissues (58).
Latent and Active Forms of the Heparanase Protein
[0026] The apparent molecular size of the recombinant enzyme
produced in the baculovirus expression system was about 65 kDa.
This heparanase polypeptide contains 6 potential N-glycosylation
sites. Following deglycosylation by treatment with peptide
N-glycosidase, the protein appeared as a 57 kDa band. This
molecular weight corresponds to the deduced molecular mass (61,192
daltons) of the 543 amino acid polypeptide encoded by the full
length hpa cDNA after cleavage of the predicted 3 kDa signal
peptide. No further reduction in the apparent size of the
N-deglycosylated protein was observed following concurrent
O-glycosidase and neuraminidase treatment. Deglycosylation had no
detectable effect on enzymatic activity.
[0027] Unlike the baculovirus enzyme, expression of the full length
heparanase polypeptide in mammalian cells (e.g., 293 kidney cells,
CHO) yielded a major protein of about 50 kDa and a minor about 65
kDa protein in cell lysates. Comparison of the enzymatic activity
of the two forms, using a semi-quantitative gel filtration assay,
revealed that the 50 kDa enzyme is at least 100-fold more active
than the 65 kDa form, which activity may be attributed to minute
contamination by the 50 kDa protein in the analyzed samples. A
similar difference was observed when the specific activity of the
recombinant 65 kDa baculovirus enzyme was compared to that of the
50 kDa heparanase preparations purified from human platelets,
SK-hep-1 cells, or placenta. These results suggest that the 50 kDa
protein is a mature processed form of a latent heparanase
precursor. Amino terminal sequencing of the platelet heparanase
indicated that cleavage occurs between amino acids Gln.sup.157 and
Lys.sup.158. As indicated by the hydropathic plot of heparanase,
this site is located within a hydrophillic peak, which is likely to
be exposed and hence accessible to proteases.
Involvement of Heparanase in Tumor Cell Invasion and Metastasis
[0028] Circulating tumor cells arrested in the capillary beds often
attach at or near the intercellular junctions between adjacent
endothelial cells. Such attachment of the metastatic cells is
followed by rupture of the junctions, retraction of the endothelial
cell borders and migration through the breach in the endothelium
toward the exposed underlying base membrane (BM) (24). Once located
between endothelial cells and the BM, the invading cells must
degrade the subendothelial glycoproteins and proteoglycans of the
BM in order to migrate out of the vascular compartment. Several
cellular enzymes (e.g., collagenase IV, plasminogen activator,
cathepsin B, elastase, etc.) are thought to be involved in
degradation of BM (25). Among these enzymes is heparanase that
cleaves HS at specific intrachain sites (16, 11). Expression of a
HS degrading heparanase was found to correlate with the metastatic
potential of mouse lymphoma (26), fibrosarcoma and melanoma (21)
cells. Moreover, elevated levels of heparanase were detected in
sera from metastatic tumor bearing animals and melanoma patients
(21) and in tumor biopsies of cancer patients (12).
[0029] The inhibitory effect of various non-anticoagulant species
of heparin on heparanase was examined in view of their potential
use in preventing extravasation of blood-borne cells. Treatment of
experimental animals with heparanase inhibitors markedly reduced
(>90%) the incidence of lung metastases induced by B16 melanoma,
Lewis lung carcinoma and mammary adenocarcinoma cells (12, 13, 28).
Heparin fractions with high and low affinity to anti-thrombin III
exhibited a comparable high anti-metastatic activity, indicating
that the heparanase inhibiting activity of heparin, rather than its
anticoagulant activity, plays a role in the anti-metastatic
properties of the polysaccharide (12).
[0030] The direct role of heparanase in cancer metastasis was
demonstrated by two experimental systems. The murine T-lymphoma
cell line Eb has no detectable heparanase activity. Whether the
introduction of the hpa gene into Eb cells would confer a
metastatic behavior on these cells was investigated. To this
purpose, Eb cells were transfected with a full length human hpa
cDNA. Stable transfected cells showed high expression of the
heparanase mRNA and enzyme activity. These hpa and mock transfected
Eb cells were injected subcutaneously into DBA/2 mice and mice were
tested for survival time and liver metastases. All mice (n=20)
injected with mock transfected cells survived during the first 4
weeks of the experiment, while 50% mortality was observed in mice
inoculated with Eb cells transfected with the hpa cDNA. The liver
of mice inoculated with hpa transfected cells was infiltrated with
numerous Eb lymphoma cells, as was evident both by macroscopic
evaluation of the liver surface and microscopic examination of
tissue sections. In contrast, metastatic lesions could not be
detected by gross examination of the liver of mice inoculated with
mock transfected control Eb cells. Few or no lymphoma cells were
found to infiltrate the liver tissue. In a different model of tumor
metastasis, transient transfection of the heparanase gene into low
metastatic B16-Fl mouse melanoma cells followed by i.v.
inoculation, resulted in a 4- to 5-fold increase in lung
metastases.
[0031] Finally, heparanase externally adhered to B16-Fl melanoma
cells increased the level of lung metastases in C57BL mice as
compared to control mice (see U.S. patent application Ser. No.
09/260,037 which is incorporated herein by reference).
Possible Involvement of Heparanase in Tumor Angiogenesis
[0032] Fibroblast growth factors are a family of structurally
related polypeptides characterized by high affinity to heparin
(29). They are highly mitogenic for vascular endothelial cells and
are among the most potent inducers of neovascularization (29-30).
Basic fibroblast growth factor (bFGF) has been extracted from a
subendothelial ECM produced in vitro (31) and from basement
membranes of the cornea (32), suggesting that ECM may serve as a
reservoir for bFGF. Immunohistochemical staining revealed the
localization of bFGF in basement membranes of diverse tissues and
blood vessels (23). Despite the ubiquitous presence of bFGF in
normal tissues, endothelial cell proliferation in these tissues is
usually very low, suggesting that bFGF is somehow sequestered from
its site of action. Studies on the interaction of bFGF with ECM
revealed that bFGF binds to HSPG in the ECM and can be released in
an active form by HS degrading enzymes (33, 32, 34). It was
demonstrated that heparanase activity expressed by platelets, mast
cells, neutrophils, and lymphoma cells is involved in release of
active bFGF from ECM and basement membranes (35), suggesting that
heparanase activity may not only function in cell migration and
invasion, but may also elicit an indirect neovascular response.
These results suggest that the ECM HSPG provides a natural storage
depot for bFGF and possibly other heparin-binding growth promoting
factors (36, 37). Displacement of bFGF from its storage within
basement membranes and ECM may therefore provide a novel mechanism
for induction of neovascularization in normal and pathological
situations.
[0033] Recent studies indicate that heparin and HS are involved in
binding of bFGF to high affinity cell surface receptors and in bFGF
cell signaling (38, 39). Moreover, the size of HS required for
optimal effect was similar to that of HS fragments released by
heparanase (40). Similar results were obtained with vascular
endothelial cells growth factor (VEGF) (41), suggesting the
operation of a dual receptor mechanism involving HS in cell
interaction with heparin-binding growth factors. It is therefore
proposed that restriction of endothelial cell growth factors in ECM
prevents their systemic action on the vascular endothelium, thus
maintaining a very low rate of endothelial cells turnover and
vessel growth. On the other hand, release of bFGF from storage in
ECM as a complex with HS fragment, may elicit localized endothelial
cell proliferation and neovascularization in processes such as
wound healing, inflammation and tumor development (36,37).
The Involvement of Heparanase in Other Physiological Processes and
its Potential Therapeutic Applications
[0034] Apart from its involvement in tumor cell metastasis,
inflammation and autoimmunity, mammalian heparanase may be applied
to modulate bioavailability of heparin-binding growth factors;
cellular responses to heparin-binding growth factors (e.g., bFGF,
VEGF) and cytokines (IL-8) (44, 41); cell interaction with plasma
lipoproteins (49); cellular susceptibility to certain viral and
some bacterial and protozoa infections (45-47); and disintegration
of amyloid plaques (48).
[0035] Viral Infection: The presence of heparan sulfate on cell
surfaces have been shown to be the principal requirement for the
binding of Herpes Simplex (45) and Dengue (46) viruses to cells and
for subsequent infection of the cells. Removal of the cell surface
heparan sulfate by heparanase may therefore abolish virus
infection. In fact, treatment of cells with bacterial heparitinase
(degrading heparan sulfate) or heparinase (degrading heparan)
reduced the binding of two related animal herpes viruses to cells
and rendered the cells at least partially resistant to virus
infection (45). There are some indications that the cell surface
heparan sulfate is also involved in HIV infection (47).
[0036] Neurodegenerative Diseases: Heparan sulfate proteoglycans
were identified in the prion protein amyloid plaques of
Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape
(48). Heparanase may disintegrate these amyloid plaques, which are
also thought to play a role in the pathogenesis of Alzheimer's
disease.
[0037] Restenosis and Atherosclerosis: Proliferation of arterial
smooth muscle cells (SMCs) in response to endothelial injury and
accumulation of cholesterol rich lipoproteins are basic events in
the pathogenesis of atherosclerosis and restenosis (50). Apart from
its involvement in SMC proliferation as a low affinity receptor for
heparin-binding growth factors, HS is also involved in lipoprotein
binding, retention and uptake (51). It was demonstrated that HSPG
and lipoprotein lipase participate in a novel catabolic pathway
that may allow substantial cellular and interstitial accumulation
of cholesterol rich lipoproteins (49). The latter pathway is
expected to be highly atherogenic by promoting accumulation of apoB
and apoE rich lipoproteins (e.g., LDL, VLDL, chylomicrons),
independent of feed back inhibition by the cellular cholesterol
content. Removal of SMC HS by heparanase is therefore expected to
inhibit both SMC proliferation and lipid accumulation and thus may
halt the progression of restenosis and atherosclerosis.
[0038] Pulmonary Diseases: The data obtained from the literature
suggests a possible role for GAGs degrading enzymes, such as, but
not limited to, heparanases, connective tissue activating peptide,
heparinases, hyluronidases, sulfatases and chondroitinases, in
reducing the viscosity of sinuses and airway secretions with
associated implications on curtailing the rate of infection and
inflammation. The sputum from CF patients contains at least 3%
GAGs, thus contributing to its volume and viscous properties. We
have shown that heparanase reduces the viscosity of sputum of
Cystic fibrosis (CF) patients (U.S. patent application Ser. No.
09/046,475). Recombinant heparanase has been shown to reduce
viscosity of sputum of CF patients (see, U.S. patent application
Ser. No. 09/046,475).
[0039] In summary, heparanase may thus prove useful for conditions
such as wound healing, angiogenesis, restenosis, atherosclerosis,
inflammation, neurodegenerative diseases and viral infections.
Mammalian heparanase can be used to neutralize plasma heparin, as a
potential replacement of protamine. Anti-heparanase antibodies may
be applied for immunodetection and diagnosis of micrometastases,
autoimmune lesions and renal failure in biopsy specimens, plasma
samples, and body fluids.
[0040] There is thus a widely recognized need for, and it would be
highly advantageous to have, additional molecules with glycosyl
hydrolase activity, because such molecules may exhibit greater
specific activity toward certain substrates or different substrate
specificity than the known heparanase.
SUMMARY OF THE INVENTION
[0041] According to one aspect of the present invention there is
provided An isolated nucleic acid comprising a genomic,
complementary or composite polynucleotide sequence which (a)
encodes a polypeptide which is at least 75% similar to SEQ ID NO:4
or a portion thereof as determined using the BESTFIT software of
the Wisconsin sequence analysis package, utilizing the Smith and
Waterman algorithm, where gap weight equals 8 and length weight
equals 2, average match equals 2.912 and average mismatch equals
-2.003; (b) is at lest 65% identical to SEQ ID NO:10 or a portion
thereof as determined using the BESTFIT software of the Wisconsin
sequence analysis package, utilizing the Smith and Waterman
algorithm, where gap weight equals 50, length weight equals 3,
average match equals 10 and average mismatch equals -9; (c) is as
set forth in SEQ ID NO: 10 or a portion thereof; and/or (d) is
hybridizable with SEQ ID NO:10 or a portion thereof under
hybridization conditions of hybridization solution containing 10%
dextrane sulfate, 1 M NaCl, 1% SDS and 5.times.10.sup.6 cpm
.sup.32p labeled probe, at 65.degree. C., with a final wash
solution of 1.times. SSC and 0.1% SDS and final wash at 65.degree.
C.
[0042] According to a preferred embodiment of the present invention
the polynucleotide encodes a polypeptide which has heparanase
catalytic activity or which is cleavable by a protease so as to
have the heparanase catalytic activity.
[0043] According to another aspect of the present invention there
is provided a nucleic acid construct comprising any of the
polynucleotides of the present invention in a sense or antisense
orientation with respect to expression regulatory sequences of the
construct.
[0044] According to yet another aspect of the present invention
there is provided a cell transformed or transfected with
polynucleotides or constructs of the present invention.
[0045] According to still another aspect of the present invention
there is provided an oligonucleotide of at least 17 bases
specifically hybridizable with the isolated nucleic acid described
herein and which is not hybridizable with any mammalian heparanase
cDNA.
[0046] According to an additional aspect of the present invention
there is provided a pair of oligonucleotides each of at least 17
bases specifically hybridizable with the isolated nucleic acid
described herein in an opposite orientation so as to direct
exponential amplification of a portion thereof in a nucleic acid
amplification reaction, and which are not hybridizable with any
mammalian heparanase cDNA.
[0047] According to yet an additional aspect of the present
invention there is provided a nucleic acid amplification product
obtained using the pair of primers described herein.
[0048] According to yet a further aspect of the present invention
there is provided a nucleic acid construct comprising a first
polynucleotide encoding a signal peptide of avian or reptile
heparanase and an in frame, second polynucleotide encoding a
membrane targeted or secreted polypeptide.
[0049] According to still a further aspect of the present invention
there is provided a nucleic acid construct comprising a first
polynucleotide encoding an avian or reptile heparanase signal
peptide, e.g., a peptide as set forth at positions 1 to 19 of SEQ
ID NO:4, and an in frame, second polynucleotide encoding a membrane
targeted or secreted polypeptide.
[0050] Preferably, the targeted or secreted polypeptide is human
heparanase.
[0051] According to still an additional aspect of the present
invention there is provided a method of expressing a protein of
interest in a cell, the method comprising transforming the cell
with a nucleic acid construct that comprises a first polynucleotide
encoding a signal peptide of avian or reptile heparanase and an in
frame, second polynucleotide encoding a membrane targeted or
secreted polypeptide; and culturing the cell under suitable growth
conditions.
[0052] As used herein the term "transforming" refers to any and all
methods of permanent or transient introduction of foreign nucleic
acids into cells, such as for example, plasmid transformation,
phage infection, gene knock-in and the like.
[0053] According to yet an additional aspect of the present
invention there is provided a recombinant protein comprising a
polypeptide (a) which is at least 75% similar to SEQ ID NO:4 or a
portion thereof as determined using the BESTFIT software of the
Wisconsin sequence analysis package, utilizing the Smith and
Waterman algorithm, where gap weight equals 8 and length weight
equals 2, average match equals 2.912 and average mismatch equals
-2.003; (b) encoded by a nucleic acid including a genomic,
complementary or composite polynucleotide sequence being at lest
65% identical to SEQ ID NO:10 or a portion thereof as determined
using the BESTFIT software of the Wisconsin sequence analysis
package, utilizing the Smith and Waterman algorithm, where gap
weight equals 50, length weight equals 3, average match equals 10
and average mismatch equals -9; (c) encoded by a nucleic acid as
set forth in SEQ ID NO:10 or a portion thereof; and/or encoded by a
nucleic acid including a genomic, complementary or composite
polynucleotide sequence being hybridizable with SEQ ID NO:10 or a
portion thereof under hybridization conditions of hybridization
solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and
5.times.10.sup.6 cpm .sup.32p labeled probe, at 65.degree. C., with
a final wash solution of 1.times. SSC and 0.1% SDS and final wash
at 65.degree. C.
[0054] According to further features in preferred embodiments of
the invention described below, the polypeptide has heparanase
catalytic activity or the polypeptide is cleavable by a protease so
as to have the heparanase catalytic activity.
[0055] According to a further aspect of the present invention there
is provided a pharmaceutical composition comprising, as an active
ingredient, the recombinant protein of described herein and a
pharmaceutically acceptable carrier.
[0056] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
novel polynucleotides which encode novel polypeptides having
heparanase catalytic activity and which can be used to intervene
with pathologies associated with impaired heparin-binding growth
factors, cellular responses to heparin-binding growth factors and
cytokines, cell interaction with plasma lipoproteins, cellular
susceptibility to viral, protozoa and bacterial infections or
disintegration of neurodegenerative plaques, all as is further
delineated in the background section above.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057] The invention is herein described, by way of example only,
with reference to the accompanying drawings. With specific
reference now to the drawings in detail, it is stressed that the
particulars shown are by way of example and for purposes of
illustrative discussion of the preferred embodiments of the present
invention only, and are presented in the cause of providing what is
believed to be the most useful and readily understood description
of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the
invention in more detail than is necessary for a fundamental
understanding of the invention, the description taken with the
drawings making apparent to those skilled in the art how the
several forms of the invention may be embodied in practice.
[0058] In the drawings:
[0059] FIG. 1a presents an alignment of the amino acid sequences
of, mouse (SEQ ID NO:1), rat (SEQ ID NO:2), human (SEQ ID NO:3) and
chicken (SEQ ID NO:4) heparanases. The amino acid sequences were
determined by sequence analysis of the isolated cDNAs. The amino
acids, which are identical in heparanases from the four organisms,
are marked with an asterisk, conserved differences are marked by
double or single dots. The putative two catalytic residues, the
proton donor and the nucleophile are bolded. The signal peptide of
human heparanase and the putative signal peptides of chicken, mouse
and rat heparanases are underlined. The cleavage site of the 50 kDa
mature protein and the borders of the associated 8 kDa peptide are
pointed by arrows. Multiple alignment was generated by
ClustalW.
[0060] FIG. 1b presents the chicken heparanase coding sequence (SEQ
ID NO:10) and its translation product (SEQ ID NO:4).
[0061] FIG. 2 shows western analysis of heparanase secreted by Eb
lymphoma cells transfected with chicken heparanase cDNA (Chk-hpa)
and human heparanase cDNA (Hum-hpa). Heparanase was partially
purified (SP-SEPHAROSE) from serum free medium conditioned by Eb
lymphoma cells transfected with Chk-hpa (lane 1), Hum-hpa (lane 2),
or plasmid alone (lane 3). Protein samples were subjected to 10%
SDS/PAGE and western blot analysis applying polyclonal rabbit
anti-heparanase antibodies and ECL visualization. Protein bands
correspond to the 58 kDa and 45 kDa forms of the chicken enzyme vs.
the 65 kDa and 50 kDa latent and active human heparanase forms.
[0062] FIGS. 3a-c demonstrate heparanase activity in cell lysates,
intact cells and medium conditioned by Eb cells transfected with
chicken vs. human heparanases. Eb mouse lymphoma cells transfected
with Chk-hpa (.box-solid.), Hum-hpa (.tangle-solidup.) and control
vector (+) were maintained (24 h, 2.times.106 cells/ml) in serum
free RPMI medium. Intact cells (3a), conditioned media (3b) and
lysates (3c) of 2.times.10.sup.6 cells were then tested for
heparanase activity. For this purpose, 1 ml conditioned medium and
2.times.10.sup.6 intact or lysed cells were incubated (24 h,
37.degree. C., pH 6.2) in serum free medium with sulfate labeled
ECM. Labeled degradation fragments released into the incubation
medium were analyzed by gel filtration on SEPHAROSE 6B. Nearly
intact heparan sulfate proteoglycans elute next to V.sub.0 (peak I,
fractions 1-10) whereas heparan sulfate degradation products elute
toward the V.sub.t of the column (peak II, fractions 15-35). A much
higher heparanase activity was expressed by intact lymphoma cells
(3a) and even more was secreted into the conditioned medium (3b) of
cells transfected with the Chk-hpa as compared to cells transfected
with the Hum-hpa. In contrast, there was no difference in
heparanase activity found in the corresponding cell lysates
(3c).
[0063] FIG. 4 presents a comparison of heparanase activities of
partially purified chicken and human heparanases. Chicken and human
heparanases were partially purified (SP SEPHAROSE) from serum free
medium conditioned by stable hpa transfected Eb lymphoma cells.
Equal amounts (60 ng/ml) of partially purified chicken
(.box-solid.) and human (.tangle-solidup.) heparanases were
incubated (24 h, 37.degree. C., pH 6.2) with sulfate labeled ECM.
Labeled degradation fragments released into the incubation medium
were analyzed by gel filtration on SEPHAROSE 6B. Both enzymes
exhibit a similar apparent specific activity, as indicated by an
almost identical elution pattern of HS degradation products.
[0064] FIGS. 5a-d demonstrates the cellular localization of chicken
(Chk-hpa) and chimeric (chimeric-hpa) heparanases vs. human (H-hpa)
heparanase. C6 rat glioma cells were transfected with chicken (a),
human (c), or chimeric (b) heparanase cDNAs. Pooled populations of
stable transfected cells were subjected to indirect
immunofluorescence staining with monoclonal anti-heparanase
antibodies (mAb 130) followed by Cy-3 conjugated goat anti-mouse
antibody, as described in the Examples section that follows. Mock
transfected C6 glioma cells (d) were used as control and showed no
staining. Chk-hpa (a) and chimeric-hpa (c) transfected cells
exhibited intense staining associated mostly with the cell membrane
(arrow), while cells transfected with H-hpa cDNA (b) displayed
primarily a peri-nuclear granular staining (arrow). Bar=10
.mu.M.
[0065] FIG. 6 presents the comparison between heparanases of human,
chicken, mouse and rat. Percents of identity between the coding
nucleotide sequences appear in the upper block. Percents of
identity and similarity between the amino acid sequences appear in
the lower right and lower left blocks, respectively. The nucleotide
sequence was determined for each one of the four species and the
amino acid sequence was deduced from the cDNA sequence.
[0066] FIG. 7 demonstrates the secretion of chicken and chimeric
heparanases. Eb mouse lymphoma cells were stable transfected with
Chk-hpa (.box-solid.), H-hpa (.diamond.), or chimeric-hpa
(.circle-solid.). Serum free medium conditioned by these cells was
incubated (24 h, 37.degree. C., pH 6.2) with sulfate labeled ECM
and tested for heparanase activity. Mock transfected Eb lymphoma
cells (.largecircle.) were used as control.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0067] The present invention is of an avian or reptile derived
polynucleotide encoding a polypeptide having heparanase catalytic
activity which can be used in a variety of medical applications.
Specifically, the present invention can be used to intervene with
pathologies associated with impaired heparin-binding growth
factors, cellular responses to heparin-binding growth factors and
cytokines, cell interaction with plasma lipoproteins, cellular
susceptibility to viral, protozoa and bacterial infections or
disintegration of neurodegenerative plaques, all as is further
delineated in the background section above. The present invention
is further of chimeric nucleic acids encoding, in frame, the signal
peptide sequence of avian or reptile heparanase and a protein of
interest, such as human heparanase.
[0068] The principles, operation and uses of the present invention
may be better understood with reference to the drawings and
accompanying descriptions.
[0069] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details set forth in the following
description or illustrated in the examples. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0070] In an attempt to isolate an avian heparanase encoding
polynucleotide, cDNA libraries and reverse transcribed mRNA from
avian tissue were screened via hybridization and polymerase chain
reaction techniques using mammalian derived probes and
oligonucleotides, yet with no successes even under the mildest
hybridization conditions. The human heparanase amino acid sequence
was thereafter used to screen EST databases for homology to a
chicken unidentified mRNA sequences. Following extensive screening,
a single related chicken EST was identified which shared 60.5%
homology with the 276 bp at the 3' end of the human heparanase
coding sequence. This sequence encoded a truncated open reading
frame of 91 amino acids, 74% homologous to human heparanase,
followed by 325 nucleotides 3' untranslated region (UTR). The
heparanase homologous sequence was derived from chicken activated T
cells cDNA, clone pat.pk0039.c8.f 5', mRNA sequence, accession No.
AI980994.
[0071] In order to isolate a full length clone and to test whether
it is indeed heparanase, chicken kidney mRNA was subjected to 5'
RACE SYSTEM (rapid amplification of cDNA ends). The gene specific
PCR primers were designed according to the EST described above. A
DNA fragment of approximately 1,600 bp was obtained, partially
overlapping with the identified 3' encoding EST clone. The entire
cDNA cloned, in pGEM-T EASY VECTOR, was designated Chk-hpa. The
complete cDNA (Chk-hpa) is 1,609 bp long (SEQ ID NO:10), and it
contains an open reading frame that encodes a polypeptide of 523
amino acids (SEQ ID NO:4) with a calculated molecular weight of
58,842 Daltons. Analysis of the amino acid sequence and of the
hydropathic profile of the protein indicates a hydrophobic amino
acid tail at the N-terminus. A signal peptide is predicted to span
the N-terminal 19 amino acids.
[0072] The overall homology between the chicken and the human
heparanase coding sequences is 62%. The similarity between the
chicken and the human heparanases is 69% (of which 61% amino acid
sequence identity). The heparanase is synthesized as a latent, 65
kDa precursor and is then processed to an active mature 50 kDa
form. Based on the homology to human heparanase the chicken
heparanase is cleaved between Trp.sup.136 and Lys.sup.137.
According to Fairbank et al. the precursor is cleaved at three
sites to form a heterodimer of a 50 kDa polypeptide (the mature
form) that is associated with a 8 kDa peptide. The putative chicken
8 kDa peptide spans amino acids Glu.sup.10 to Glu.sup.94. The
mature heparanases of various organisms share high homology while
the pro-peptides are relatively diverse.
[0073] Structure prediction of human heparanase suggests a
(.alpha./.beta.)8 Tim barrel fold typical of family 10 glycosyl
hydrolases. According to this prediction the active site involves
two glutamic acid residues, which are the proton donor and the
nucleophile, with an aspargine always preceding the proton donor.
The proton donor in human heparanase is Glu.sup.225 and the
nucleophile is Glu.sup.343. The conservation of the amino acid
sequence flanking these residues supports the identification of the
active site. Based on the homology the proton donor of chicken
heparanase is Glu.sup.204 and the nucleophile is Glu.sup.323.
[0074] Chicken heparanase is slightly more similar to human
heparanase than to mouse and rat heparanases. As expected, the
homology among mammals is far higher than that of mammals with
chicken.
[0075] The ability of the Chk-Hpa product to catalyze degradation
of heparan sulfate (HS) in vitro was determined by expressing the
entire open reading frame of Chk-hpa in mammalian cells lacking
heparanase activity. Expression of heparanase in the transfected
cells was confirmed by RT-PCR. Chicken heparanase transcript was
detected only in Chk-hpa transfected cells.
[0076] Heparanase activity was assayed in cells transfected with
Chk-hpa as compared to mock transfected cells. High activity was
observed in intact cells, conditioned media and cell lysates of
Chk-hpa transfectants while no activity was observed in the mock
transfected cells.
[0077] The heparan sulfate degradation activity of Eb cells
transfected with Chk-hpa cDNA was compared with that of Eb cells
transfected with human hpa (Hum-hpa). The activity of chicken
heparanase was higher than that of human heparanase in intact cells
and in conditioned media, however, in cell extracts the activity of
the two enzymes was similar. This suggests that the chicken
heparanase is unexpectedly preferentially secreted as is compared
to the mammalian enzyme.
[0078] In order to compare the activity of the chicken and human
heparanases, the enzymes were partially purified from conditioned
media of Chk-hpa and Hum-hpa transfected cells. Western blot
analysis of the partially purified chicken heparanase showed a
major protein of 58 kDa, which corresponds to the heparanase
precursor and a minor protein of 45 kDa, which corresponds to the
mature form. The human heparanase fraction contained the equivalent
human 65 kDa heparanase precursor and 50 kDa mature forms. The
difference in molecular weight between chicken and human
heparanases is mainly due to a different glycosylation pattern.
[0079] Activity of the two enzymes was compared using equal amounts
of the partially purified enzymes in a semi-quantitative assay. The
specific activity of chicken and human heparanases was found to be
similar.
[0080] Cells transfected with Chk-hpa exhibited intense staining
associated with the cell membrane while only a weak signal was
observed in the cytoplasm. In contrast, in cells transfected with
Hum-hpa heparanase is localized to peri-nuclear vesicles. A similar
pattern was observed in transiently transfected human primary
fibroblasts, where chicken heparanase was associated with cell
membrane while human heparanase was localized to peri-nuclear
vesicles, which were identified as lysosomes. The signal peptides
of the chicken and human heparanases share no significant homology.
It appears that the signal peptide of chicken heparanase
unexpectedly targets the enzyme to the cell surface of mammalian
cells while the signal peptide of human heparanase targets the
enzyme to lysosomes. Indeed, replacing the signal peptide of human
heparanase with that of chicken heparanase resulted in improved
secretion and membrane localization of the human heparanase.
[0081] Thus, according to one aspect of the present invention there
is provided An isolated nucleic acid comprising a genomic,
complementary or composite polynucleotide sequence which (a)
encodes a polypeptide which is at least 75% similar to SEQ ID NO:4
or a portion thereof as determined using the BESTFIT software of
the Wisconsin sequence analysis package, utilizing the Smith and
Waterman algorithm, where gap weight equals 8 and length weight
equals 2, average match equals 2.912 and average mismatch equals
-2.003; (b) is at lest 65% identical to SEQ ID NO:10 or a portion
thereof as determined using the BESTFIT software of the Wisconsin
sequence analysis package, utilizing the Smith and Waterman
algorithm, where gap weight equals 50, length weight equals 3,
average match equals 10 and average mismatch equals -9; (c) is as
set forth in SEQ ID NO:10 or a portion thereof; and/or (d) is
hybridizable with SEQ ID NO:10 or a portion thereof under
hybridization conditions of hybridization solution containing 10%
dextrane sulfate, 1 M NaCl, 1% SDS and 5.times.10.sup.6 cpm
.sup.32p labeled probe, at 65.degree. C., with a final wash
solution of 1.times. SSC and 0.1% SDS and final wash at 65.degree.
C. Under these hybridization conditions the chicken heparanase cDNA
fails to hybridize with any mammalian heparanase.
[0082] The phrase "composite polynucleotide sequence" refers to a
sequence which includes exonal sequences required to encode the
polypeptide having heparanase activity, as well as any number of
intronal sequences. The intronal sequences can be of any source and
typically will include conserved splicing signal sequences. Such
intronal sequences may further include cis acting expression
regulatory elements.
[0083] Thus, this aspect of the present invention encompasses (i)
polynucleotides as set forth in SEQ ID NO:10; (ii) fragments
thereof; (iii) sequences hybridizable therewith; (iv) sequences
homologous thereto, such as reptile derived sequences; (v)
sequences encoding similar polypeptides with different codon usage;
(vi) altered sequences characterized by mutations, such as
deletion, insertion or substitution of one or more nucleotides,
either naturally occurring or man induced, either randomly or in a
targeted fashion.
[0084] It will be appreciated in this respect that avian and
reptiles are evolutionary closely related. As such, using the
polynucleotides described herein, one of ordinary skills in the
art, would be motivated and readily capable of screening a reptile
cDNA library or use other methods routinely employed to isolate
related genes from closely related species, to thereby clone full
length cDNAs or genomic DNAs from any avian or reptile.
[0085] The heparanase sequence described herein can be used to
study the catalytic mechanism of heparanase. Carefully selected
site directed mutagenesis can be employed to provide modified
heparanase proteins having modified characteristics in terms of,
for example, substrate specificity, sensitivity to inhibitors,
etc.
[0086] According to a preferred embodiment of the present invention
the polynucleotide encodes a polypeptide which has heparanase
catalytic activity or which is cleavable by a protease so as to
have the heparanase catalytic activity. Removal of a 19 amino acid
long signal peptide of chicken heparanase is demonstrated in the
Examples section that follows.
[0087] The term "heparanase catalytic activity" or its equivalent
term "heparanase activity" both refer to a mammalian
endoglycosidase hydrolyzing activity which is specific for heparin
or heparan sulfate proteoglycan substrates, as opposed to the
activity of bacterial enzymes (heparinase I, II and III) which
degrade heparin or heparan sulfate by means of .beta.-elimination
(37).
[0088] According to another aspect of the present invention there
is provided a nucleic acid construct comprising any of the
polynucleotides of the present invention in a sense or antisense
orientation with respect to expression regulatory sequences of the
construct.
[0089] According to a preferred embodiment the nucleic acid
construct according to this aspect of the present invention
includes a promoter for regulating the expression of the isolated
nucleic acid in a sense or antisense orientation. Such promoters
are known to be cis-acting sequence elements required for
transcription as they serve to bind DNA dependent RNA polymerase
which transcribes sequences present downstream thereof. Such down
stream sequences can be in either one of two possible orientations
to result in the transcription of sense RNA which is translatable
by the ribozyme machinery or antisense RNA which typically does not
contain translatable sequences, yet can duplex or triplex with
endogenous sequences, either mRNA or chromosomal DNA and hamper
gene expression, all as further detailed hereinunder.
[0090] While the isolated nucleic acid described herein is an
essential element of the invention, it is modular and can be used
in different contexts. The promoter of choice that is used in
conjunction with this invention is of secondary importance, and
will comprise any suitable promoter. It will be appreciated by one
skilled in the art, however, that it is necessary to make sure that
the transcription start site(s) will be located upstream of an open
reading frame. In a preferred embodiment of the present invention,
the promoter that is selected comprises an element that is active
in the particular host cells of interest. These elements may be
selected from transcriptional regulators that activate the
transcription of genes essential for the survival of these cells in
conditions of stress or starvation, including the heat shock
proteins.
[0091] A construct according to the present invention preferably
further includes an appropriate selectable marker. In a more
preferred embodiment according to the present invention the
construct further includes an origin of replication. In another
most preferred embodiment according to the present invention the
construct is a shuttle vector, which can propagate both in E. coli
(wherein the construct comprises an appropriate selectable marker
and origin of replication) and be compatible for propagation in
cells, or integration in the genome, of an organism of choice. The
construct according to this aspect of the present invention can be,
for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a
virus or an artificial chromosome.
[0092] The polynucleotide encoding heparanase can be permanently or
transiently present in the cell. In other words, genetically
modified cells obtained following stable or transient transfection,
transformation or transduction are all within the scope of the
present invention. The polynucleotide can be present in the cell in
low copy (say 1-5 copies) or high copy number (say 5-50 copies or
more). It may be integrated in one or more chromosomes at any
location or be present as an extrachromosomal material.
[0093] Alternatively, the nucleic acid construct according to this
aspect of the present invention further includes a positive and a
negative selection markers and may therefore be employed for
selecting for homologous recombination events, including, but not
limited to, homologous recombination employed in knock-in and
knock-out procedures. One ordinarily skilled in the art can readily
design a knock-out or knock-in constructs including both positive
and negative selection genes for efficiently selecting transfected
embryonic stem cells that underwent a homologous recombination
event with the construct. Such cells can be introduced into
developing embryos to generate chimeras, the offspring thereof can
be tested for carrying the knock-out or knock-in constructs.
Additional detail can be found in Fukushige, S. and Ikeda, J.E.:
Trapping of mammalian promoters by Cre-lox site-specific
recombination. DNA Res 3 (1996) 73-80; Bedell, M. A., Jenkins, N.
A. and Copeland, N. G.: Mouse models of human disease. Part I:
Techniques and resources for genetic analysis in mice. Genes and
Development 11 (1997) 1-11; Bermingham, J. J., Scherer, S. S.,
O'Connell, S., Arroyo, E., Kalla, K. A., Powell, F. L. and
Rosenfeld, M. G.: Tst-1/Oct-6/SCIP regulates a unique step in
peripheral myelination and is required for normal respiration.
Genes Dev 10 (1996) 1751-62, which are incorporated herein by
reference.
[0094] According to yet another aspect of the present invention
there is provided a cell transformed or transfected with
polynucleotides or constructs of the present invention. The cell
according to this aspect of the present invention can be a
eukaryote cell of a multicellular organism, such as, but not
limited to, a mammalian, avian, reptile or insect cell, a eukaryote
cell of a unicellular organism, such as yeast or a prokaryote cell,
such as a bacteria cell, e.g., an E. coli cell. Methods of
transforming and transfecting each of these cells are well known in
the art. Such procedures are detailed in many experimental
procedure text books such as "Molecular Cloning: A laboratory
Manual" Sambrook et al., (1989); "Current Protocols in Molecular
Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al.,
"Current Protocols in Molecular Biology", John Wiley and Sons,
Baltimore, Md. (1989); Perbal, "A Practical Guide to Molecular
Cloning", John Wiley & Sons, New York (1988); Watson et al.,
"Recombinant DNA", Scientific American Books, New York; Birren et
al. (eds).
[0095] The present invention is further directed at providing a
heparanase over-expression system which includes a cell
overexpressing heparanase catalytic activity. The cell may be a
genetically modified host cell transiently or stably transfected or
transformed with any suitable vector which includes a
polynucleotide sequence encoding a polypeptide having heparanase
activity and a suitable promoter and enhancer sequences to direct
over-expression of heparanase. However, the overexpressing cell may
also be a product of an insertion (e.g., via homologous
recombination) of a promoter and/or enhancer sequence downstream to
the endogenous heparanase gene of the expressing cell, which will
direct over-expression from the endogenous gene.
[0096] The term "over-expression" as used herein in the
specification and claims below refers to a level of expression
which is higher than a basal level of expression typically
characterizing a given cell under otherwise identical
conditions.
[0097] According to still another aspect of the present invention
there is provided an oligonucleotide of at least 17, at least 18,
at least 19, at least 20, at least 22, at least 25, at least 30 or
at least 40 bases specifically hybridizable with the isolated
nucleic acid described herein and which is not hybridizable with
any mammalian heparanase cDNA.
[0098] Hybridization of shorter nucleic acids (below 200 bp in
length, e.g. 17-40 bp in length) is effected by stringent, moderate
or mild hybridization, wherein stringent hybridization is effected
by a hybridization solution of 6.times. SSC and 1% SDS or 3 M
TMACI, 0.01 M sodium phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5%
SDS, 100 .mu.g/ml denatured salmon sperm DNA and 0.1% nonfat dried
milk, hybridization temperature of 1-1.5.degree. C. below the
T.sub.m, final wash solution of 3 M TMACI, 0.01 M sodium phosphate
(pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5.degree. C. below
the T.sub.m; moderate hybridization is effected by a hybridization
solution of 6.times. SSC and 0.1% SDS or 3 M TMACI, 0.01 M sodium
phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 .mu.g/ml
denatured salmon sperm DNA and 0.1% nonfat dried milk,
hybridization temperature of 2-2.5.degree. C. below the T.sub.m,
final wash solution of 3 M TMACI, 0.01 M sodium phosphate (pH 6.8),
1 mM EDTA (pH 7.6), 0.5% SDS at 1-1.5.degree. C. below the T.sub.m,
final wash solution of 6.times. SSC, and final wash at 22.degree.
C.; whereas mild hybridization is effected by a hybridization
solution of 6.times. SSC and 1% SDS or 3 M TMACI, 0.01 M sodium
phosphate (pH 6.8), 1 mM EDTA (pH 7.6), 0.5% SDS, 100 .mu.g/ml
denatured salmon sperm DNA and 0.1% nonfat dried milk,
hybridization temperature of 37.degree. C., final wash solution of
6.times. SSC and final wash at 22.degree. C.
[0099] According to an additional aspect of the present invention
there is provided a pair of oligonucleotides each of at least 17,
at least 18, at least 19, at least 20, at least 22, at least 25, at
least 30 or at least 40 bases specifically hybridizable with the
isolated nucleic acid described herein in an opposite orientation
so as to direct exponential amplification of a portion thereof in a
nucleic acid amplification reaction, such as a polymerase chain
reaction, and which are not hybridizable with any mammalian
heparanase cDNA.
[0100] The polymerase chain reaction and other nucleic acid
amplification reactions are well known in the art and require no
further description herein. The pair of oligonucleotides according
to this aspect of the present invention are preferably selected to
have compatible melting temperatures (T.sub.m), e.g., melting
temperatures which differ by less than that 7.degree. C.,
preferably less than 5.degree. C., more preferably less than
4.degree. C., most preferably less than 3.degree. C., ideally
between 3.degree. C. and zero .degree. C.
[0101] Consequently, according to yet an additional aspect of the
present invention there is provided a nucleic acid amplification
product obtained using the pair of primers described herein. Such a
nucleic acid amplification product can be isolated by gel
electrophoresis or any other size based separation technique.
Alternatively, such a nucleic acid amplification product can be
isolated by affinity separation, either stranded affinity or
sequence affinity. In addition, once isolated, such a product can
be further genetically manipulated by restriction, ligation and the
like.
[0102] According to yet a further aspect of the present invention
there is provided a nucleic acid construct comprising a first
polynucleotide encoding a signal peptide of chicken and/or avian
heparanase, such as the peptide set forth at positions 1 to 19 of
SEQ ID NO:4, and an in frame, second polynucleotide encoding a
membrane targeted or secreted polypeptide. The chimeric polypeptide
resulting from the expression of the open reading frame of this
construct will be preferentially directed to the cell membrane or
secreted outside the cell, depending on the nature of the
polypeptide. Any polypeptide can be fused to the signal peptide of
the invention, including, but not limited to, any enzyme, e.g.,
human heparanase, hormone, receptor, immunoglobulin, structural
protein and the like. Cells transformed with a chimeric construct
as herein described are grown under suitable culturing conditions
and the protein of interest (the polypeptide) is extracted
therefrom or from the growth medium to which it is secreted.
[0103] According to still an additional aspect of the present
invention there is provided a recombinant protein comprising a
polypeptide (a) which is at least 75% similar to SEQ ID NO:4 or a
portion thereof as determined using the BESTFIT software of the
Wisconsin sequence analysis package, utilizing the Smith and
Waterman algorithm, where gap weight equals 8 and length weight
equals 2, average match equals 2.912 and average mismatch equals
-2.003; (b) encoded by a nucleic acid including a genomic,
complementary or composite polynucleotide sequence being at lest
65% identical to SEQ ID NO:10 or a portion thereof as determined
using the BESTFIT software of the Wisconsin sequence analysis
package, utilizing the Smith and Waterman algorithm, where gap
weight equals 50, length weight equals 3, average match equals 10
and average mismatch equals -9; (c) encoded by a nucleic acid as
set forth in SEQ ID NO:10 or a portion thereof; and/or encoded by a
nucleic acid including a genomic, complementary or composite
polynucleotide sequence being hybridizable with SEQ ID NO:10 or a
portion thereof under hybridization conditions of hybridization
solution containing 10% dextrane sulfate, 1 M NaCl, 1% SDS and
5.times.10.sup.6 cpm .sup.32p labeled probe, at 65.degree. C., with
a final wash solution of 1.times. SSC and 0.1% SDS and final wash
at 65.degree. C.
[0104] Thus, this aspect of the present invention encompasses (i) a
polypeptide as set forth in SEQ ID NO:4; (ii) fragments thereof;
(iii) polypeptides similar (identical+homologous acids) thereto;
and (iv) altered polypeptides characterized by mutations, such as
deletion, insertion or substitution of one or more amino acids,
either naturally occurring or man induced, either randomly or in a
targeted fashion.
[0105] According to further features in preferred embodiments of
the invention described below, the polypeptide has heparanase
catalytic activity or the polypeptide is cleavable by a protease so
as to have the heparanase catalytic activity.
[0106] The recombinant protein of the present invention may be
purified by any conventional protein purification procedure close
to homogeneity and/or be mixed with additives. The recombinant
protein may be manufactured using any of the genetically modified
cells described above, which include any of the expression nucleic
acid constructs described herein. The recombinant protein may be in
any form. It may be in a crystallized form, a dehydrated powder
form or in solution. The recombinant protein may be useful in
obtaining pure heparanase, which in turn may be useful in eliciting
anti-heparanase antibodies, either poly or monoclonal antibodies,
and as a screening active ingredient in an anti-heparanase
inhibitors or drugs screening assay or system.
[0107] According to a further aspect of the present invention there
is provided a pharmaceutical composition comprising, as an active
ingredient, the recombinant protein of described herein and a
pharmaceutically acceptable carrier.
[0108] The heparanase according to the present invention can be
administered to an organism per se, or in a pharmaceutical
composition where it is mixed with suitable carriers or
excipients.
[0109] As used herein a "pharmaceutical composition" refers to
active or activatable heparanase, with other chemical components
such as physiologically suitable carriers and excipients. The
purpose of a pharmaceutical composition is to facilitate
administration of an active ingredient to an organism.
[0110] Herein the term "active ingredient" refers to active or
activatable heparanase accountable for a biological effect.
[0111] Hereinafter, the terms "physiologically acceptable carrier"
and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the active
ingredient.
[0112] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of the active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0113] Techniques for formulation and administration of active
ingredients may be found in "Remington's Pharmaceutical Sciences,"
Mack Publishing Co., Easton, Pa., latest edition, which is
incorporated herein by reference.
[0114] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, intestinal or parenteral delivery,
including intramuscular, subcutaneous and intramedullary injections
as well as intrathecal, direct intraventricular, intravenous,
inrtaperitoneal, intranasal, or intraocular injections.
[0115] Alternately, one may administer the active ingredient in a
local rather than systemic manner, for example, via injection of
the active ingredient directly into a solid tumor often in a depot
or slow release formulation, such as described below.
[0116] Furthermore, one may administer the active ingredient in a
targeted drug delivery system, for example, in a liposome coated
with a tumor specific antibody. The liposomes will be targeted to
and taken up selectively by the tumor.
[0117] Pharmaceutical compositions of the present invention may be
manufactured by processes well known in the art, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes.
[0118] Pharmaceutical compositions for use in accordance with the
present invention thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries, which facilitate processing of the
active ingredient into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0119] For injection, the active ingredient of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0120] For administration by inhalation, the active ingredient for
use according to the present invention are conveniently delivered
in the form of an aerosol spray presentation from a pressurized
pack or a nebulizer with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichloro-tetrafluoroethane or carbon dioxide. In the case of a
pressurized aerosol, the dosage unit may be determined by providing
a valve to deliver a metered amount. Capsules and cartridges of,
e.g., gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the active ingredient and a
suitable powder base such as lactose or starch.
[0121] The active ingredient described herein may be formulated for
parenteral administration, e.g., by bolus injection or continues
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multidose containers with
optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0122] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredient may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acids esters such as
ethyl oleate, triglycerides or liposomes. Aqueous injection
suspensions may contain substances, which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol or
dextran. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the active
ingredient to allow for the preparation of highly concentrated
solutions.
[0123] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water, before use.
[0124] The active ingredient of the present invention may also be
formulated for local administration, such as a depot preparation.
Such long acting formulations may be administered by implantation
(for example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the preparation may be formulated
with suitable polymeric or hydrophobic materials (for example, as
an emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives such as sparingly soluble salts.
Formulations for topical administration may include, but are not
limited to, lotions, suspensions, ointments gels, creams, drops,
liquids, sprays emulsions and powders.
[0125] The pharmaceutical compositions herein described may also
comprise suitable solid of gel phase carriers or excipients.
Examples of such carriers or excipients include, but are not
limited to, calcium carbonate, calcium phosphate, various sugars,
starches, cellulose derivatives, gelatin and polymers such as
polyethylene glycols.
[0126] Pharmaceutical compositions suitable for use in context of
the present invention include compositions wherein the active
ingredients are contained in an amount effective to achieve the
intended purpose. More specifically, a therapeutically effective
amount means an amount the active ingredient effective to prevent,
alleviate or ameliorate symptoms of disease or prolong the survival
of the subject being treated.
[0127] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0128] The therapeutically effective amount or dose can be
estimated initially from cell culture assays. For example, a dose
can be formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50 as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans.
[0129] Toxicity and therapeutic efficacy of the active ingredient
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., by
determining the IC.sub.50 and the LD.sub.50 (lethal dose causing
death in 50% of the tested animals) for the active ingredient. The
data obtained from these cell culture assays and animal studies can
be used in formulating a range of dosage for use in human. The
dosage may vary depending upon the dosage form employed and the
route of administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See e.g., Fingl, et al., 1975,
in "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1).
[0130] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0131] Compositions of the present invention may, if desired, be
presented in a pack or dispenser device, such as an FDA approved
kit, which may contain one or more unit dosage forms containing the
active ingredient. The pack may, for example, comprise metal or
plastic foil, such as a blister pack. The pack or dispenser device
may be accompanied by instructions for administration. The pack or
dispenser may also be accompanied by a notice associated with the
container in a form prescribed by a governmental agency regulating
the manufacture, use or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the
compositions or human or veterinary administration. Such notice,
for example, may be of labeling approved by the U.S. Food and Drug
Administration for prescription drugs or of an approved product
insert. Compositions comprising a preparation of the invention
formulated in a compatible pharmaceutical carrier may also be
prepared, placed in an appropriate container, and labeled for
treatment of an indicated condition.
[0132] The present invention can be used to develop treatments for
various diseases, to develop diagnostic assays for these diseases
and to provide new tools for basic and directed research especially
in the fields of medicine and biology.
[0133] Specifically, the present invention can be used to develop
new drugs to inhibit tumor cell metastasis, inflammation and
autoimmunity. The identification of the hpa gene encoding for the
heparanase enzyme from avian enables the production of a
recombinant enzyme in heterologous expression systems.
[0134] Furthermore, the present invention can be used to modulate
bioavailability of heparin-binding growth factors, cellular
responses to heparin-binding growth factors (e.g., bFGF, VEGF) and
cytokines (e.g., IL-8), cell interaction with plasma lipoproteins,
cellular susceptibility to viral, protozoa and some bacterial
infections, and disintegration of neurodegenerative plaques.
Recombinant heparanase offers a potential treatment for wound
healing, angiogenesis, restenosis, atherosclerosis, inflammation,
neurodegenerative diseases (such as, for example,
Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease, Scrape
and Alzheimer's disease) and certain viral and some bacterial and
protozoa infections. Recombinant heparanase can be used to
neutralize plasma heparin, as a potential replacement of
protamine.
[0135] As used herein, the term "modulate" includes substantially
inhibiting, slowing or reversing the progression of a disease,
substantially ameliorating clinical symptoms of a disease or
condition, or substantially preventing the appearance of clinical
symptoms of a disease or condition. A "modulator" therefore
includes an agent which may modulate a disease or condition.
Modulation of viral, protozoa and bacterial infections includes any
effect which substantially interrupts, prevents or reduces any
viral, bacterial or protozoa activity and/or stage of the virus,
bacterium or protozoon life cycle, or which reduces or prevents
infection by the virus, bacterium or protozoon in a subject, such
as a human or lower animal.
[0136] As used herein, the term "wound" includes any injury to any
portion of the body of a subject including, but not limited to,
acute conditions such as thermal burns, chemical burns, radiation
burns, burns caused by excess exposure to ultraviolet radiation
such as sunburn, damage to bodily tissues such as the perineum as a
result of labor and childbirth, including injuries sustained during
medical procedures such as episiotomies, trauma-induced injuries
including cuts, those injuries sustained in automobile and other
mechanical accidents, and those caused by bullets, knives and other
weapons, and post-surgical injuries, as well as chronic conditions
such as pressure sores, bedsores, conditions related to diabetes
and poor circulation, and all types of acne, etc.
[0137] The present invention successfully addresses the
shortcomings of the presently known configurations by providing
novel polynucleotides which encode novel polypeptides having
heparanase catalytic activity and which can be used to intervene
with pathological procedures associated with impaired
heparin-binding growth factors, cellular responses to
heparin-binding growth factors and cytokines, cell interaction with
plasma lipoproteins, cellular susceptibility to viral, protozoa and
bacterial infections or disintegration of neurodegenerative
plaques, all as is further delineated in the background section
above.
[0138] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0139] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
[0140] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorpotaed by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
MATERIALS AND EXPERIMENTAL PROCEDURES
[0141] Cells: The methylcholanthrene-induced non-metastatic Eb
(L5178Y) T-lymphoma cells (clone 737) were provided by V.
Schirrmacher (DKFZ, Heidelberg, Germany). The cells were routinely
transplanted as ascites tumors in syngeneic female DBA2/J mice.
Alternatively, they were grown in RPMI 1640 (Life Technologies
Inc., Rockville, Md., USA) supplemented with .beta.-mercaptoethanol
(5.times.10.sup.-5 M) and 10% FCS. C6 rat Glioma cells were
obtained from Dr. E. Keshet (Hadassah Medical School Jerusalem
Israel). Cells were cultured in DMEM (4.5 g glucose/liter)
containing 10% fetal calf serum. Cells were dissociated with a
solution of 0.05% trypsin, 0.02% EDTA, 0.01 M sodium phosphate, pH
7.4 and were subcultured at a `split` ratio of 1:10.
[0142] Heparanase Activity: Cell lysates, intact cells, conditioned
media and serum free conditioned media were incubated 24 hours at
37.degree. C., pH 6.2-6.6, with .sup.35S-labeled ECM in the
presence of 20 mM phosphate buffer (pH 6.2). The incubation medium
was centrifuged and the supernatant was analyzed by gel filtration
on a SEPHAROSE CL-6B column (0.9.times.30 cm). Fractions (0.2 ml)
were eluted with PBS and their radioactivity was measured. Nearly
intact HSPGs was eluted next to just after the V.sub.0
(K.sub.av<0.2, peak I, fractions 1-10) whereas degradation
fragments of HS side chains were eluted from SEPHAROSE 6B at
0.5<K.sub.av<0.8 (peak II, fractions 15-35).
[0143] Cloning of Chk-hpa cDNA: The amino acid sequence of human
heparanase was used to screen EST databases for homology to a
chicken unidentified mRNA sequences. Following extensive searches,
a single chicken derived EST suspected as heparanase related was
identified, which shared only 60.5% sequence homology with a 276 bp
nucleotide stretch at the 3' end of the human heparanase coding
sequence. The full-length chicken heparanase cDNA was isolated from
chicken kidney mRNA. To this end, mRNA was isolated from fresh
chicken kidney using POLYATTRACT mRNA Isolation System III
(Promega, USA). The method for amplification of 5' ends was
developed according to the principle of the 5' RACE SYSTEM (rapid
amplification of cDNA ends) System of GibcoBRL. Chicken kidney mRNA
was reverse transcribed (RT) using SUPERSCRIPT II (Gibco BRL) and
oligo dT(.sub.15) (SEQ ID NO:5) as a primer. Following RT the cDNA
was extended by 3' C-tailing using terminal deoxynucleotidyl
transferase (TdT) (Promega). PCR amplification used EXPAND HIGH
FIDELITY enzyme (Boehringer). The primers used for amplification
were:
[0144] First step: 5' primer, complementary to the C tail: AP1
5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3' SEQ ID NO:6 and the 3'
gene specific primer ChkL1: 5'-GACTCCTCAAGCATTCCCTCAG-3' (SEQ ID
NO:7). Second cycle: 5' nested primer nested AP2:
5'-GGCCACGCGTCGACTAGTACG-3' (SEQ ID NO:8) and a nested, gene
specific 3' primer ChkL2 5'-AGCCCTGTTACTCTGCGTGC- TC-3' (SEQ ID
NO:9). The gene specific primers ChkL1 and ChkL2 were selected
according to the sequence of the EST.
[0145] PCR program of both first and second cycles was as follows:
94.degree. C. 3 minutes, followed by 30 cycles of: 94.degree. C. 30
seconds, 64.degree. C. 1 minute and 72.degree. C. 3 minutes, and
finally 72.degree. C., 7 minutes.
[0146] The resulting 1.8-kb PCR product was cloned into the pGEM-T
EASY VECTOR (Promega, USA).
[0147] DNA Sequencing: Sequence determination used vector-specific
and gene-specific primers, with an automated DNA sequencer (ABI
PRISM.TM. model 310 Genetic Analyzer). Each nucleotide was read
from at least two independent primers, and from several clones.
[0148] Computer Analysis of Sequence: Database searches for
sequence similarities were performed using the NCBI Blast network
service. Sequence analysis and alignment of DNA and protein
sequences were done using the DNA sequence analysis software
package developed by the Genetic Computer Group (GCG) at the
University of Wisconsin. Multiple alignment was generated by
ClustalW.
[0149] Immunostaining: Cells were seeded on round cover slips in 4
well plates for 24 hours. Cell were then washed with PBS, fixed
with 100% chilled (-20.degree. C.) MetOH for 3 minutes. Following
fixation cells were washed with PBS 5 times and intrinsic
fluorescence was blocked with 50 mM NH.sub.4Cl for 5 minutes. Cells
were washed with PBS 3 times, incubated with 5% goat serum for 30
minutes and washed with PBS twice. Slides were then incubated with
anti heparanase monoclonal antibody HP-130, 8 .mu.g/ml for 2 hours
at room temperature, washed with PBS 5 times and then incubated
with the second antibody Cy-3 conjugated goat anti mouse IgG
(Jackson) for 1 hour at room temperature. Slides were washed with
PBS 8 times, and mounting solution (90% glycerol in PBS ) was
added. The monoclonal antibody HP-130 was generated against human
heparanase as described in U.S. patent application No.
08/922,170.
[0150] Generation of a Chimeric Chicken-Human Heparanase Gene: The
N-terminal coding portion of the human hpa (H-hpa ) cDNA encoding
the signal peptide was replaced by a corresponding sequence of the
chicken hpa cDNA. To this end, the Chk-hpa signal peptide coding
sequence was amplified using specific primers (KPN/SPU:
5'-CGGGGTACCCGATGCTGGTGCT-3' (SEQ ID NO:11); SPL:
5'-AGGTCCACGACGTCCTGTGCCGTC CGCCTCG-3', (SEQ ID NO:12)). A H-hpa
cDNA region encoding a segment extending from the first amino acid
downstream the H-hpa signal peptide to the BamHI restriction site
was amplified, using H-hpa specific primers (HU:
5'-CGAGGCGGACGGCACAGGACGTCGTGGACCT-3' (SEQ ID NO:13; H/BHIL:
5'-CCACATCAGGAGGGATGGATCC-3' (SEQ ID NO:14). The PCR products were
combined by means of primer extension and PCR amplification. The
resulting fragment was then cloned in frame into a pcCDNA3 plasmid
(Invitrogen, NV Leek, Netherlands) containing the H-hpa cDNA
downstream the BamHI site, generating a chimeric construct in which
the Chk-hpa signal peptide precedes the H-hpa. The chimeric gene
was validated by sequencing. SEQ ID NO:15 deleniates the chimeric
cDNA, whereas SEQ ID NO:16 deleniates the amino acid sequence of
the chimeric heparanase protein.
EXPERIMENTAL RESULTS
[0151] Failures in Cloning the Chicken Heparanase cDNA: Cloning of
chicken heparanase was attempted based on minute homology to human
heparanase. cDNA library of chicken kidney was screened using low
stringency hybridization conditions and human hpa cDNA as a probe.
No specific hybridization signal was observed and no hpa homologous
clones could be isolated following the screening. A different
approach utilized PCR primers of human hpa for amplification of the
heparanase cDNA from chicken kidney. Human hpa primers failed to
amplify chicken heparanase using annealing temperature as low as
37.degree. C.
[0152] Cloning the Chicken Heparanase cDNA: The human heparanase
amino acid sequence was used to screen EST databases for homology
to a chicken unidentified mRNA sequences. Following extensive
screening, a single related chicken EST was identified which shared
60.5% homology with the 276 bp at the 3' end of the human
heparanase coding sequence . This sequence encoded a truncated open
reading frame of 91 amino acids, 74% homologous to human
heparanase, followed by 325 nucleotides 3' untranslated region
(UTR). The heparanase homologous sequence was derived from chicken
activated T cells cDNA, clone pat.pk0039.c8.f 5', mRNA sequence,
accession # AI980994.
[0153] In order to isolate a full length clone and to test whether
it is indeed heparanase, chicken kidney mRNA was subjected to 5'
RACE SYSTEM (rapid amplification of cDNA ends). The gene specific
PCR primers were designed according to the EST described above. A
DNA fragment of approximately 1,600 bp was obtained, partially
overlapping with the identified 3' encoding EST clone. The entire
cDNA cloned, in pGEM-T EASY VECTOR, was designated Chk-hpa. The
complete cDNA (Chk-hpa) is 1,609 bp long (SEQ ID NO:10, FIG. 1b),
and it contains an open reading frame that encodes a polypeptide of
523 amino acids (SEQ ID NO:4, FIG. 1a-b) with a calculated
molecular weight of 58,842 Daltons. Analysis of the amino acid
sequence and of the hydropathic profile of the protein indicates a
hydrophobic amino acid tail (FIG. 1a underlined) at the N-terminus.
A signal peptide is predicted to span the N-terminal 19 amino
acids.
[0154] The overall homology between the chicken and the human hpa
coding sequences is 62%. The similarity between the chicken and the
human heparanases is 69% (of which 61% amino acid sequence
identity). The heparanase is synthesized as a latent, 65 kDa
precursor and is then processed to an active mature 50 kDa form.
Based on the homology to human heparanase the chicken heparanase is
cleaved between Trp.sup.136 and Lys.sup.137. According to Fairbank
et al. (57) the precursor is cleaved at three sites to form a
heterodimer of a 50 kDa polypeptide (the mature form) that is
associated with a 8 kDa peptide. The putative chicken 8 kDa peptide
spans amino acids Glu.sup.10 to Glu.sup.94 (FIG. 1a). The mature
heparanases of various organisms share high homology while the
pro-peptides are relatively diverse (FIG. 1a).
[0155] Structure prediction of human heparanase suggests a
(.alpha./.beta.)8 Tim barrel fold typical of family 10 glycosyl
hydrolases. According to this prediction the active site involves
two glutamic acid residues, which are the proton donor and the
nucleophile, with an aspargine always preceding the proton donor.
The proton donor in human heparanase is Glu.sup.225 and the
nucleophile is Glu.sup.343. The conservation of the amino acid
sequence flanking these residues supports the identification of the
active site. Based on the homology the proton donor of chicken
heparanase is Glu.sup.204 and the nucleophile is Glu.sup.323.
[0156] The comparison between the nucleotide as well as the amino
acid sequences of the all the heparanases published so far is
presented in FIG. 6. Chicken heparanase is slightly more similar to
human heparanase than to mouse and rat heparanases. As expected,
the homology among mammals is far higher than that of mammals with
chicken.
[0157] It is therefore yet to be determined whether the
polynucleotide isolated from chicken indeed encodes a protein
having heparanase catalytic activity. This is shown below.
[0158] Functional Expression of Recombinant Chicken Heparanase in
Mammalian Cells: The ability of the Chk-Hpa product to catalyze
degradation of heparan sulfate (HS) in vitro was determined by
expressing the entire open reading frame of Chk-hpa in mammalian
cells lacking heparanase activity. Mouse Eb-lymphoma and rat
C6-glioma cells were transfected with the pcDNA3 plasmid vector
containing the chicken heparanase cDNA (Chk-hpa) or with a control
empty plasmid (mock transfected). Stable transfectants were then
selected for further analysis. Expression of heparanase in the
transfected cells was confirmed by RT-PCR. Chicken heparanase
transcript was detected only in Chk-hpa transfected cells.
[0159] Heparanase activity was assayed in cells transfected with
Chk-hpa as compared to mock transfected cells. As shown in FIGS.
3a-c high activity was observed in intact cells, conditioned media
and cell lysates of Chk-hpa transfectants while no activity was
observed in the mock transfected cells.
[0160] The heparan sulfate degradation activity of Eb cells
transfected with Chk-hpa cDNA was compared with that of Eb cells
transfected with human hpa (Hum-hpa). As shown in FIGS. 3a-c the
activity of chicken heparanase was higher than that of human
heparanase in intact cells and in conditioned media, however, in
cell extracts the activity of the two enzymes was similar. This
suggests that the chicken heparanase is unexpectedly preferentially
secreted.
[0161] In order to compare the activity of the chicken and human
heparanases, the enzymes were partially purified from conditioned
media of Chk-hpa and Hum-hpa transfected cells. Western blot
analysis of the partially purified chicken heparanase showed a
major protein of 58 kDa, which corresponds to the heparanase
precursor and a minor protein of 45 kDa, which corresponds to the
mature form. The human heparanase fraction contained the equivalent
human 65 kDa heparanase precursor and 50 kDa mature forms (FIG. 2).
The difference in molecular weight between chicken and human
heparanases is mainly due to a different glycosylation pattern.
[0162] Activity of the two enzymes was compared using equal amounts
of the partially purified enzymes in a semi-quantitative assay. As
shown in FIG. 4 the specific activity of chicken and human
heparanases is similar.
[0163] Localization of Chicken Heparanase in Transfected Cells: C-6
glioma cells stably transfected with the chicken or human
heparanase cDNAs were grown in four well chamber slides and were
subjected to indirect immunofluorescence staining with the
anti-human heparanase mAb 130 (15). These antibodies cross-react
with the chicken enzyme. Confocal fluorescence microscopy revealed
that C-6 glioma cells transfected with the Chk-hpa cDNA exhibited
an intense granular staining of the heparanase protein mostly
associated with the cell surface. Preferential localization of the
chicken heparanase was noted in areas of cell to cell contacts
(FIG. 5a, arrow). Unlike this pattern of immunostaining, C-6 glioma
cells overexpressing the human heparanase displayed primarily a
peri-nuclear granular staining pattern with almost no detectable
surface localization of the enzyme (FIG. 5c). A similar pattern was
observed in transiently transfected human primary fibroblasts,
where chicken heparanase was associated with cell membrane while
human heparanase was localized to peri-nuclear vesicles, which were
identified as lysosomes. The chicken and human heparanase cDNAs
were also expressed in homologous cells (i.e., QT6 quail
fibrosarcoma and Huh7 human hepatocarcinoma cells, respectively),
resulting in an immunostaining pattern similar to that observed
with the transfected C-6 rat glioma cells.
[0164] The signal peptides of the chicken and human heparanases
share no significant homology. It appears that the signal peptide
of chicken heparanase targets the enzyme to the cell surface of
mammalian cells while the signal peptide of human heparanase
targets the enzyme to lysosomes.
[0165] One may take advantage of the unexpected membrane targeting
feature of chicken heparanase signal peptide for targeting other
proteins to cell membrane.
[0166] Chimeric Chicken-Human Heparanase Gene: The results
described above indicate that the chicken heparanase is more
readily secreted into the incubation medium and/or retained on the
cell surface, as compared with the human enzyme, most likely due to
the marked difference between the respective signal peptide
sequences. In order to further study this unexpected observation, a
chimeric construct was generated, composed of the chicken signal
peptide fused to the human cDNA downstream nucleotide 105. Briefly,
chicken specific primers were used to amplify the chicken signal
sequence which was then fused by means of primer extension to the
human hpa sequence, replacing its signal peptide, as described in
Experimental Procedures above. The chimeric construct was subcloned
into pcDNA3 plasmid which was then used to stable transfect Eb
mouse lymphoma and C-6 rat glioma cells. Serum free medium
conditioned for 24 hours by Eb cells transfected with the chimeric
construct (chimeric-hpa, SEQ ID NOs:15 and 16) was tested for
heparanase activity. As shown in FIG. 7, cells transfected with the
chimeric enzyme were comparable to cells transfected with Chk-hpa
in their ability to secrete the heparanase enzyme into the culture
medium. In contrast, little or no heparanase activity was detected
in medium conditioned by H-hpa transfected cells (FIG. 7),
indicating that secretion of the enzyme is in fact driven by the
chicken signal peptide sequence. Similar results were obtained with
C-6 glioma cells.
[0167] Cellular Localization of Chimeric Heparanase Enzymes: The
cell surface targeting of the chicken heparanase signal peptide was
also demonstrated by the cellular localization of chimeric
heparanase. Immunostaining of C-6 glioma cells transfected with the
chimeric heparanase revealed preferential surface localization
pattern (FIG. 5b), similar to that of cells expressing the chicken
heparanase (5a). Mock transfected glioma cells showed no staining
(FIG. 5d). The results of the swapping experiment emphasize that
the pronounced difference in cellular localization of the chicken
and human heparanases is due primarily to the marked difference in
sequence, length and hydrophobic properties of the respective
signal peptides. The preferential cell surface association of the
chicken and chimeric heparanases is in accordance with the higher
HS degrading activity expressed by intact cells overexpressing the
chicken or chimeric enzymes vs. the human heparanase.
[0168] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
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Sequence CWU 1
1
16 1 535 PRT Mus musculus 1 Met Leu Arg Leu Leu Leu Leu Trp Leu Trp
Gly Pro Leu Gly Ala Leu 1 5 10 15 Ala Gln Gly Ala Pro Ala Gly Thr
Ala Pro Thr Asp Asp Val Val Asp 20 25 30 Leu Glu Phe Tyr Thr Lys
Arg Pro Leu Arg Ser Val Ser Pro Ser Phe 35 40 45 Leu Ser Ile Thr
Ile Asp Ala Ser Leu Ala Thr Asp Pro Arg Phe Leu 50 55 60 Thr Phe
Leu Gly Ser Pro Arg Leu Arg Ala Leu Ala Arg Gly Leu Ser 65 70 75 80
Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr Asp Phe Leu Ile Phe 85
90 95 Asp Pro Asp Lys Glu Pro Thr Ser Glu Glu Arg Ser Tyr Trp Lys
Ser 100 105 110 Gln Val Asn His Asp Ile Cys Arg Ser Glu Pro Val Ser
Ala Ala Val 115 120 125 Leu Arg Lys Leu Gln Val Glu Trp Pro Phe Gln
Glu Leu Leu Leu Leu 130 135 140 Arg Glu Gln Tyr Gln Lys Glu Phe Lys
Asn Ser Thr Tyr Ser Arg Ser 145 150 155 160 Ser Val Asp Met Leu Tyr
Ser Phe Ala Lys Cys Ser Gly Leu Asp Leu 165 170 175 Ile Phe Gly Leu
Asn Ala Leu Leu Arg Thr Pro Asp Leu Arg Trp Asn 180 185 190 Ser Ser
Asn Ala Gln Leu Leu Leu Asp Tyr Cys Ser Ser Lys Gly Tyr 195 200 205
Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe Trp Lys Lys 210
215 220 Ala His Ile Leu Ile Asp Gly Leu Gln Leu Gly Glu Asp Phe Val
Glu 225 230 235 240 Leu His Lys Leu Leu Gln Arg Ser Ala Phe Gln Asn
Ala Lys Leu Tyr 245 250 255 Gly Pro Asp Ile Gly Gln Pro Arg Gly Lys
Thr Val Lys Leu Leu Arg 260 265 270 Ser Phe Leu Lys Ala Gly Gly Glu
Val Ile Asp Ser Leu Thr Trp His 275 280 285 His Tyr Tyr Leu Asn Gly
Arg Ile Ala Thr Lys Glu Asp Phe Leu Ser 290 295 300 Ser Asp Ala Leu
Asp Thr Phe Ile Leu Ser Val Gln Lys Ile Leu Lys 305 310 315 320 Val
Thr Lys Glu Ile Thr Pro Gly Lys Lys Val Trp Leu Gly Glu Thr 325 330
335 Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu Leu Ser Asn Thr Phe Ala
340 345 350 Ala Gly Phe Met Trp Leu Asp Lys Leu Gly Leu Ser Ala Gln
Met Gly 355 360 365 Ile Glu Val Val Met Arg Gln Val Phe Phe Gly Ala
Gly Asn Tyr His 370 375 380 Leu Val Asp Glu Asn Phe Glu Pro Leu Pro
Asp Tyr Trp Leu Ser Leu 385 390 395 400 Leu Phe Lys Lys Leu Val Gly
Pro Arg Val Leu Leu Ser Arg Val Lys 405 410 415 Gly Pro Asp Arg Ser
Lys Leu Arg Val Tyr Leu His Cys Thr Asn Val 420 425 430 Tyr His Pro
Arg Tyr Gln Glu Gly Asp Leu Thr Leu Tyr Val Leu Asn 435 440 445 Leu
His Asn Val Thr Lys His Leu Lys Val Pro Pro Pro Leu Phe Arg 450 455
460 Lys Pro Val Asp Thr Tyr Leu Leu Lys Pro Ser Gly Pro Asp Gly Leu
465 470 475 480 Leu Ser Lys Ser Val Gln Leu Asn Gly Gln Ile Leu Lys
Met Val Asp 485 490 495 Glu Gln Thr Leu Pro Ala Leu Thr Glu Lys Pro
Leu Pro Ala Gly Ser 500 505 510 Ala Leu Ser Leu Pro Ala Phe Ser Tyr
Gly Phe Phe Val Ile Arg Asn 515 520 525 Ala Lys Ile Ala Ala Cys Ile
530 535 2 536 PRT Rattus rattus 2 Met Leu Arg Pro Leu Leu Leu Leu
Trp Leu Trp Gly Arg Leu Arg Ala 1 5 10 15 Leu Thr Gln Gly Thr Pro
Ala Gly Thr Ala Pro Thr Lys Asp Val Val 20 25 30 Asp Leu Glu Phe
Tyr Thr Lys Arg Leu Phe Gln Ser Val Ser Pro Ser 35 40 45 Phe Leu
Ser Ile Thr Ile Asp Ala Ser Leu Ala Thr Asp Pro Arg Phe 50 55 60
Leu Thr Phe Leu Gly Ser Pro Arg Leu Arg Ala Leu Ala Arg Gly Leu 65
70 75 80 Ser Pro Ala Tyr Leu Arg Phe Gly Gly Thr Lys Thr Asp Phe
Leu Ile 85 90 95 Phe Asp Pro Asn Lys Glu Pro Thr Ser Glu Glu Arg
Ser Tyr Trp Gln 100 105 110 Ser Gln Asp Asn Asn Asp Ile Cys Gly Ser
Glu Arg Val Ser Ala Asp 115 120 125 Val Leu Arg Lys Leu Gln Met Glu
Trp Pro Phe Gln Glu Leu Leu Leu 130 135 140 Leu Arg Glu Gln Tyr Gln
Arg Glu Phe Lys Asn Ser Thr Tyr Ser Arg 145 150 155 160 Ser Ser Val
Asp Met Leu Tyr Ser Phe Ala Lys Cys Ser Arg Leu Asp 165 170 175 Leu
Ile Phe Gly Leu Asn Ala Leu Leu Arg Thr Pro Asp Leu Arg Trp 180 185
190 Asn Ser Ser Asn Ala Gln Leu Leu Leu Asn Tyr Cys Ser Ser Lys Gly
195 200 205 Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe
Trp Lys 210 215 220 Lys Ala Gln Ile Ser Ile Asp Gly Leu Gln Leu Gly
Glu Asp Phe Val 225 230 235 240 Glu Leu His Lys Leu Leu Gln Lys Ser
Ala Phe Gln Asn Ala Lys Leu 245 250 255 Tyr Gly Pro Asp Ile Gly Gln
Pro Arg Gly Lys Thr Val Lys Leu Leu 260 265 270 Arg Ser Phe Leu Lys
Ala Gly Gly Glu Val Ile Asp Ser Leu Thr Trp 275 280 285 His His Tyr
Tyr Leu Asn Gly Arg Val Ala Thr Lys Glu Asp Phe Leu 290 295 300 Ser
Ser Asp Val Leu Asp Thr Phe Ile Leu Ser Val Gln Lys Ile Leu 305 310
315 320 Lys Val Thr Lys Glu Met Thr Pro Gly Lys Lys Val Trp Leu Gly
Glu 325 330 335 Thr Ser Ser Ala Tyr Gly Gly Gly Ala Pro Leu Leu Ser
Asn Thr Phe 340 345 350 Ala Ala Gly Phe Met Trp Leu Asp Lys Leu Gly
Leu Ser Ala Gln Leu 355 360 365 Gly Ile Glu Val Val Met Arg Gln Val
Phe Phe Gly Ala Gly Asn Tyr 370 375 380 His Leu Val Asp Glu Asn Phe
Glu Pro Leu Pro Asp Tyr Trp Leu Ser 385 390 395 400 Leu Leu Phe Lys
Lys Leu Val Gly Pro Lys Val Leu Met Ser Arg Val 405 410 415 Lys Gly
Pro Asp Arg Ser Lys Leu Arg Val Tyr Leu His Cys Thr Asn 420 425 430
Val Tyr His Pro Arg Tyr Arg Glu Gly Asp Leu Thr Leu Tyr Val Leu 435
440 445 Asn Leu His Asn Val Thr Lys His Leu Lys Leu Pro Pro Pro Met
Phe 450 455 460 Ser Arg Pro Val Asp Lys Tyr Leu Leu Lys Pro Phe Gly
Ser Asp Gly 465 470 475 480 Leu Leu Ser Lys Ser Val Gln Leu Asn Gly
Gln Thr Leu Lys Met Val 485 490 495 Asp Glu Gln Thr Leu Pro Ala Leu
Thr Glu Lys Pro Leu Pro Ala Gly 500 505 510 Ser Ser Leu Ser Val Pro
Ala Phe Ser Tyr Gly Phe Phe Val Ile Arg 515 520 525 Asn Ala Lys Ile
Ala Ala Cys Ile 530 535 3 543 PRT Homo sapiens 3 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
4 523 PRT Gallus gallus 4 Met Leu Val Leu Leu Leu Leu Val Leu Leu
Leu Ala Val Pro Pro Arg 1 5 10 15 Arg Thr Ala Glu Leu Gln Leu Gly
Leu Arg Glu Pro Ile Gly Ala Val 20 25 30 Ser Pro Ala Phe Leu Ser
Leu Thr Leu Asp Ala Ser Leu Ala Arg Asp 35 40 45 Pro Arg Phe Val
Ala Leu Leu Arg His Pro Lys Leu His Thr Leu Ala 50 55 60 Ser Gly
Leu Ser Pro Gly Phe Leu Arg Phe Gly Gly Thr Ser Thr Asp 65 70 75 80
Phe Leu Ile Phe Asn Pro Asn Lys Asp Ser Thr Trp Glu Glu Lys Val 85
90 95 Leu Ser Glu Phe Gln Ala Lys Asp Val Cys Glu Ala Trp Pro Ser
Phe 100 105 110 Ala Val Val Pro Lys Leu Leu Leu Thr Gln Trp Pro Leu
Gln Glu Lys 115 120 125 Leu Leu Leu Ala Glu His Ser Trp Lys Lys His
Lys Asn Thr Thr Ile 130 135 140 Thr Arg Ser Thr Leu Asp Ile Leu His
Thr Phe Ala Ser Ser Ser Gly 145 150 155 160 Phe Arg Leu Val Phe Gly
Leu Asn Ala Leu Leu Arg Arg Ala Gly Leu 165 170 175 Gln Trp Asp Ser
Ser Asn Ala Lys Gln Leu Leu Gly Tyr Cys Ala Gln 180 185 190 Arg Ser
Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn Ser Phe 195 200 205
Arg Lys Lys Ser Gly Ile Cys Ile Asp Gly Phe Gln Leu Gly Arg Asp 210
215 220 Phe Val His Leu Arg Gln Leu Leu Ser Gln His Pro Leu Tyr Arg
His 225 230 235 240 Ala Glu Leu Tyr Gly Leu Asp Val Gly Gln Pro Arg
Lys His Thr Gln 245 250 255 His Leu Leu Arg Ser Phe Met Lys Ser Gly
Gly Lys Ala Ile Asp Ser 260 265 270 Val Thr Trp His His Tyr Tyr Val
Asn Gly Arg Ser Ala Thr Arg Glu 275 280 285 Asp Phe Leu Ser Pro Glu
Val Leu Asp Ser Phe Ala Thr Ala Ile His 290 295 300 Asp Val Leu Gly
Ile Val Glu Ala Thr Val Pro Gly Lys Lys Val Trp 305 310 315 320 Leu
Gly Glu Thr Gly Ser Ala Tyr Gly Gly Gly Ala Pro Gln Leu Ser 325 330
335 Asn Thr Tyr Val Ala Gly Phe Met Trp Leu Asp Lys Leu Gly Leu Ala
340 345 350 Ala Arg Arg Gly Ile Asp Val Val Met Arg Gln Val Ser Phe
Gly Ala 355 360 365 Gly Ser Tyr His Leu Val Asp Ala Gly Phe Lys Pro
Leu Pro Asp Tyr 370 375 380 Trp Leu Ser Leu Leu Tyr Lys Arg Leu Val
Gly Thr Arg Val Leu Gln 385 390 395 400 Ala Ser Val Glu Gln Ala Asp
Ala Arg Arg Pro Arg Val Tyr Leu His 405 410 415 Cys Thr Asn Pro Arg
His Pro Lys Tyr Arg Glu Gly Asp Val Thr Leu 420 425 430 Phe Ala Leu
Asn Leu Ser Asn Val Thr Gln Ser Leu Gln Leu Pro Lys 435 440 445 Gln
Leu Trp Ser Lys Ser Val Asp Gln Tyr Leu Leu Leu Pro His Gly 450 455
460 Lys Asp Ser Ile Leu Ser Arg Glu Val Gln Leu Asn Gly Arg Leu Leu
465 470 475 480 Gln Met Val Asp Asp Glu Thr Leu Pro Ala Leu His Glu
Met Ala Leu 485 490 495 Ala Pro Gly Ser Thr Leu Gly Leu Pro Ala Phe
Ser Tyr Gly Phe Tyr 500 505 510 Val Ile Arg Asn Ala Lys Ala Ile Ala
Cys Ile 515 520 5 15 DNA Artificial sequence synthetic
polynucleotide 5 tttttttttt ttttt 15 6 36 DNA Artificial sequence
synthetic polynucleotide 6 ggccacgcgt cgactagtac gggnngggnn gggnng
36 7 22 DNA Artificial sequence synthetic polynucleotide 7
gactcctcaa gcattccctc ag 22 8 21 DNA Artificial sequence synthetic
polynucleotide 8 ggccacgcgt cgactagtac g 21 9 22 DNA Artificial
sequence synthetic polynucleotide 9 agccctgtta ctctgcgtgc tc 22 10
1605 DNA Gallus gallus 10 aaggtgagaa ggaggaggaa ggatgctggt
gctgctgctg ctcgtgctgc tgctcgctgt 60 gccgccgagg cggacggcag
agctgcagct ggggctgcgg gaacccatcg gggcggtaag 120 cccagccttc
ctctctctta cactggacgc cagcttggcc cgtgacccgc gctttgttgc 180
cctgctcaga caccccaagc tgcacactct ggccagtggg ctctccccag gcttcctcag
240 gtttggtggc accagtacag atttcctgat cttcaatccc aacaaagatt
caacttggga 300 agagaaagtc ttgtcggaat ttcaggccaa ggatgtgtgt
gaagcgtggc ccagctttgc 360 tgtggttcca aagctgctgc tcacccagtg
gcccctccag gagaaactgc tcctcgctga 420 acattcctgg aaaaagcaca
aaaacaccac cattacaagg agcacgctgg acatcctcca 480 cacgttcgcc
agcagctcag gcttccgcct ggtgtttggg ctgaacgcac tgctgcgcag 540
ggctggcctg cagtgggaca gctccaacgc caagcagctg ctgggctact gtgcacagcg
600 cagctacaac atctcctggg agctgggtaa tgagcccaac agcttcagga
agaagtcggg 660 catctgcatc gatggcttcc agttgggacg tgatttcgtc
cacctgcggc agctcctgag 720 ccagcacccc ctgtaccgac acgctgagct
gtacggcctc gacgtggggc agccccgcaa 780 gcacacccag cacctgctca
gaagcttcat gaaatctgga gggaaggcga ttgactcggt 840 cacctggcac
cactactatg tgaatggccg aagtgcaacg agggaggatt tcctgagccc 900
tgaagtgctg gactcctttg ccactgccat acacgatgtc ctggggatcg tggaagcaac
960 ggtgcccggc aagaaggtat ggctgggtga gaccggctcg gcctacggcg
ggggggcccc 1020 ccagctctcc aacacctatg tggccggctt catgtggctg
gacaagctgg ggttggcggc 1080 tcggcgtggc attgatgtgg tgatgaggca
ggtctccttt ggtgctggca gctatcacct 1140 ggtggatgcc ggcttcaagc
ccttgccgga ctactggctg tcactgctat acaagaggct 1200 ggtgggcacc
cgggtactac aggccagcgt ggagcaagcg gatgcgcggc gcccgcgggt 1260
ctacctgcac
tgcaccaacc cccggcaccc caaataccgg gaaggggatg tgacactgtt 1320
tgccttgaac ctctccaacg tgacccagag cttgcagctg cctaagcagt tgtggagtaa
1380 gagtgtggat cagtacctgc tgctgcccca cggcaaggac agcatcctgt
ccagagaggt 1440 gcagctgaat ggccgcctac tgcagatggt ggacgatgag
acactccccg cgctgcacga 1500 gatggccctt gcccctggca gcacgctcgg
cctgccagcc ttctcttacg gtttctacgt 1560 gatcaggaac gctaaggcta
ttgcttgcat ttgagcacgc agagt 1605 11 22 DNA Artificial sequence
synthetic polynucleotide 11 cggggtaccc gatgctggtg ct 22 12 31 DNA
Artificial sequence synthetic polynucleotide 12 aggtccacga
cgtcctgtgc cgtccgcctc g 31 13 31 DNA Artificial sequence synthetic
polynucleotide 13 cgaggcggac ggcacaggac gtcgtggacc t 31 14 22 DNA
Artificial sequence synthetic polynucleotide 14 ccacatcagg
agggatggat cc 22 15 1584 DNA Artificial sequence Chicken signal
peptide/Human heparanase chimera coding sequence 15 atgctggtgc
tgctgctgct cgtgctgctg ctcgctgtgc cgccgaggcg gacggcacag 60
gacgtcgtgg acctggactt cttcacccag gagccgctgc acctggtgag cccctcgttc
120 ctgtccgtca ccattgacgc caacctggcc acggacccgc ggttcctcat
cctcctgggt 180 tctccaaagc ttcgtacctt ggccagaggc ttgtctcctg
cgtacctgag gtttggtggc 240 accaagacag acttcctaat tttcgatccc
aagaaggaat caacctttga agagagaagt 300 tactggcaat ctcaagtcaa
ccaggatatt tgcaaatatg gatccatccc tcctgatgtg 360 gaggagaagt
tacggttgga atggccctac caggagcaat tgctactccg agaacactac 420
cagaaaaagt tcaagaacag cacctactca agaagctctg tagatgtgct atacactttt
480 gcaaactgct caggactgga cttgatcttt ggcctaaatg cgttattaag
aacagcagat 540 ttgcagtgga acagttctaa tgctcagttg ctcctggact
actgctcttc caaggggtat 600 aacatttctt gggaactagg caatgaacct
aacagtttcc ttaagaaggc tgatattttc 660 atcaatgggt cgcagttagg
agaagatttt attcaattgc ataaacttct aagaaagtcc 720 accttcaaaa
atgcaaaact ctatggtcct gatgttggtc agcctcgaag aaagacggct 780
aagatgctga agagcttcct gaaggctggt ggagaagtga ttgattcagt tacatggcat
840 cactactatt tgaatggacg gactgctacc agggaagatt ttctaaaccc
tgatgtattg 900 gacattttta tttcatctgt gcaaaaagtt ttccaggtgg
ttgagagcac caggcctggc 960 aagaaggtct ggttaggaga aacaagctct
gcatatggag gcggagcgcc cttgctatcc 1020 gacacctttg cagctggctt
tatgtggctg gataaattgg gcctgtcagc ccgaatggga 1080 atagaagtgg
tgatgaggca agtattcttt ggagcaggaa actaccattt agtggatgaa 1140
aacttcgatc ctttacctga ttattggcta tctcttctgt tcaagaaatt ggtgggcacc
1200 aaggtgttaa tggcaagcgt gcaaggttca aagagaagga agcttcgagt
ataccttcat 1260 tgcacaaaca ctgacaatcc aaggtataaa gaaggagatt
taactctgta tgccataaac 1320 ctccataacg tcaccaagta cttgcggtta
ccctatcctt tttctaacaa gcaagtggat 1380 aaataccttc taagaccttt
gggacctcat ggattacttt ccaaatctgt ccaactcaat 1440 ggtctaactc
taaagatggt ggatgatcaa accttgccac ctttaatgga aaaacctctc 1500
cggccaggaa gttcactggg cttgccagct ttctcatata gtttttttgt gataagaaat
1560 gccaaagttg ctgcttgcat ctga 1584 16 527 PRT Artificial sequence
Chicken signal peptide/Human heparanase chimera protein sequence 16
Met Leu Val Leu Leu Leu Leu Val Leu Leu Leu Ala Val Pro Pro Arg 1 5
10 15 Arg Thr Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu
Pro 20 25 30 Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile
Asp Ala Asn 35 40 45 Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu
Gly Ser Pro Lys Leu 50 55 60 Arg Thr Leu Ala Arg Gly Leu Ser Pro
Ala Tyr Leu Arg Phe Gly Gly 65 70 75 80 Thr Lys Thr Asp Phe Leu Ile
Phe Asp Pro Lys Lys Glu Ser Thr Phe 85 90 95 Glu Glu Arg Ser Tyr
Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys 100 105 110 Tyr Gly Ser
Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp 115 120 125 Pro
Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe 130 135
140 Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe
145 150 155 160 Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn
Ala Leu Leu 165 170 175 Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn
Ala Gln Leu Leu Leu 180 185 190 Asp Tyr Cys Ser Ser Lys Gly Tyr Asn
Ile Ser Trp Glu Leu Gly Asn 195 200 205 Glu Pro Asn Ser Phe Leu Lys
Lys Ala Asp Ile Phe Ile Asn Gly Ser 210 215 220 Gln Leu Gly Glu Asp
Phe Ile Gln Leu His Lys Leu Leu Arg Lys Ser 225 230 235 240 Thr Phe
Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg 245 250 255
Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu 260
265 270 Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg
Thr 275 280 285 Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp
Ile Phe Ile 290 295 300 Ser Ser Val Gln Lys Val Phe Gln Val Val Glu
Ser Thr Arg Pro Gly 305 310 315 320 Lys Lys Val Trp Leu Gly Glu Thr
Ser Ser Ala Tyr Gly Gly Gly Ala 325 330 335 Pro Leu Leu Ser Asp Thr
Phe Ala Ala Gly Phe Met Trp Leu Asp Lys 340 345 350 Leu Gly Leu Ser
Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val 355 360 365 Phe Phe
Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro 370 375 380
Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr 385
390 395 400 Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys
Leu Arg 405 410 415 Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg
Tyr Lys Glu Gly 420 425 430 Asp Leu Thr Leu Tyr Ala Ile Asn Leu His
Asn Val Thr Lys Tyr Leu 435 440 445 Arg Leu Pro Tyr Pro Phe Ser Asn
Lys Gln Val Asp Lys Tyr Leu Leu 450 455 460 Arg Pro Leu Gly Pro His
Gly Leu Leu Ser Lys Ser Val Gln Leu Asn 465 470 475 480 Gly Leu Thr
Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met 485 490 495 Glu
Lys Pro Leu Arg Pro Gly Ser Ser Leu Gly Leu Pro Ala Phe Ser 500 505
510 Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile 515
520 525
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