Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
USE OF A NOVEL DISINTEGRIN METALLOPROTEASE, MUTANTS,
FRAGMENTS AND THE LIKE
Field of the invention
The invention relates to a novel protein, its fragments and mutants and to its
use in detecting and testing drugs for ailments, including osteoarthritis and
others
characterized by up regulation of metalloproteases.
Back rg ound
A number of enzymes effect the breakdown of structural proteins and are
structurally related metalloproteases. These include human skin fibroblast
collagenase, human skin fibroblast gelatinase, human sputum collagenase and
gelatinase, and human stromelysin. See e.g., S.E. Whitham et al., Comparison
of
human stromeiysin and collagenase by cloning and sequence analysis" Biochem J.
240:913 ( 1986). See also G.I. Goldberg et al., "Human Fibroblast Collagenase"
J.
Biol. Chem. 261:660 (1986). Metal dependence (e.g., zinc) is a common feature
of
these structurally related enzymes known as "metalloproteases."
Controlled production and activity of these enzymes plays an important role in
the normal development of tissue architecture. In excess, however, these
enzymes can
cause pathologic destruction of connective tissues. See generally, J. Saus et
al., "The
Complete Primary Structure of Human Matrix Metalloprotease-3" J. Biol. Chem.
263:6742 (1988). Many of these are zinc-containing metalloprotease enzymes, as
are
the angiotensin-converting enzymes and the enkephalinases. Collagenase,
stromelysin and related enzymes are important in mediating the symptomatology
of a
number of diseases, including rheumatoid arthritis (Mullins, D. E., et al.,
Biochim
Biophys Acta (1983) 695:117-214); osteoarthritis (Henderson, B., et al., Drugs
of the
Future (1990) 15:495-508); the metastasis of tumor cells (ibid, Broadhurst, M.
J., et
al., European Patent Application 276.436 (published 1987), Reich, R., et al.,
48
Cancer Res 3307-3312 (1988); and various ulcerated conditions. Ulcerative
conditions can result in the cornea as the result of alkali burns or as a
result of
infection by Pseudomonas aeruginosa. :~canthamoeba, Hetpes simplex and
vaccinia
viruses.
CA 02281085 1999-08-10
CVO 98!37092 PCT/US98/03490
2
In fact, measurement of metalloproteases in cancer tissue suggests increased
levels of metalloproteases correlate with metastatic potential. See e.g., M.
J. Duffy et
al., "Assay of matrix metalloproteases types 8 and 9 by ELISA in human breast
cancer" Br. J. Cancer 71:1025 (1995).
Other conditions characterized by undesired metalloprotease activity include
periodontal disease, epidermolysis bullosa and scleritis. In view of the
involvement
of metalloproteases in a number of disease conditions, attempts have been made
to
prepare inhibitors to these enzymes. A number of such inhibitors are disclosed
in the
literature. The invention seeks to provide novel inhibitors, preferably
specific to this
protease, that have enhanced activity in treating diseases mediated or
modulated by
this protease.
Inhibitors of metalloproteases are useful in treating diseases caused, at
least in
part, by breakdown of structural proteins. A variety of inhibitors have been
prepared,
but there is a continuing need for metalloprotease inhibitor screens to design
drugs for
treating such diseases.
Given the involvement of matrix metalloproteases in a number of disease
conditions, attempts have been made to identify inhibitors of these enzymes.
For
Example TapI-2 and 1,10-phenanthroline are known metalloprotease inhibitors.
See,
e.g., J. Arribas et aL, "Diverse Cell Surface Protein Ectodomains Are Shed by
a
System Sensitive to Metalloprotease Inhibitors", J. Biol. Chem. 271:11376
(1996).
Metalloproteases are a broad class of proteins which have widely varied
functions. Disintegrins are zinc metalloproteases, abundant in snake venom.
Mammalian disintegrins are a family of proteins with about 18 known subgroups.
They act as cell adhesion disrupters and are also known to be active in
reproduction
(for example, in fertilization of the egg by the sperm, including fusion
thereof, and in
sperm maturation).
These proteases and many others are uncovered in molecular biology and
biochemistry. As a result, GenBank, a repository for gene sequences, provides
several
sequences of metalloproteases, including some said to encode fragments of
disintegrins. For example, GenBank accession # 248444 dated February 2S, 1994
discloses 2407 nucleotides of a rat gene said to be a rat disintegrin
metalloprotease
gene; GenBank accession # 248579 dated March 2, 1995 discloses i 824
nucleotides
of a partial sequence of a gene said to be a human disintegrin metalloprotease
gene;
GenBank accession # 221961 dated October 2S, 1994, discloses 2397 nucleotides
of a
partial sequence of a gene said to be a bovine zinc metalloprotease gene.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
3
Because there is such a wide variety of metalloproteases, there is a
continuing
need for i) methods that will specifically detect a particular
metalloprotease, as well as
ii) methods for identifying candidate inhibitors.
It would be advantageous to implicate metalloproteases in specific disease
states, and to use these metalloproteases as tools to detect and ultimately
cure, control
or design cures for such diseases.
OBJECTS OF THE INVENTION
It is an object of the present invention to provide a method for identifying
compounds capable of binding to the disintegrin protein.
It is also an object of the present invention to provide a host cell
comprising a
recombinant expression vector to the disintegrin protein and a recombinant
expression
vector encoding to the disintegrin protein.
It is also an object of the present invention to provide a method for
screening
for metalloprotease mediated diseases such as cancer, arthropothies (including
ankylosing spondolytis, rheumatiod arthritis, gouty arthritis (gout),
inflammatory
arthritis, Lyme disease and osteoarthrtis).
It is also an object of the present invention to provide an antibody to the
protein useful in the screen, in the isolation of the protein or as a
targeting moiety for
the protein.
SUMMARY OF THE INVENTION
This invention provides a method for identifying compounds capable of
binding to the disintegrin protein, and determining the amount and affinity of
a
compound capable of binding to the disintegrin protein in a sample.
This invention also provides a host cell comprising a recombinant expression
vector to the disintegrin protein and a recombinant expression vector encoding
to the
disintegrin protein and the human disintegrin metalloprotease protein,
fragment or
mutant thereof, useful for these purposes.
This invention also provides an in vivo or in vitro method for screening for
osteoarthritis and other metalloprotease based diseases, such as cancer,
capable of
manufacture and use in a kit form.
DETAILED DESCRIPTION
The term "gene" refers to a DN~~ ,equence that comprises control and coding
sequences necessary for the production of a mature protein or precursor
thereof. The
protein can be encoded by a full leyth cmiing sequence or by any portion of
the
coding sequence so long as the desired enm matic activity is retained.
The term "oligonucleotide" as u,r~3 hcrrin is defined as a molecule comprised
of two or more deoxyribonueleotides ur nhcmucleotides, usually more than three
(3).
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
4
and typically more than ten (10) and up to one hundred (100) or more (although
preferably between twenty and thirty). The exact size will depend on many
factors,
which in turn depends on the ultimate function or use of the oligonucleotide.
The
oligonucleotide may be generated in any manner, including chemical synthesis,
DNA
replication, restriction endonuclease digestion reverse transcription, or a
combination
thereof.
Because mononucleotides are reacted to make oligonucleotides in a manner
such that the 5' phosphate of one mononucleotide pentose ring is attached to
the 3'
oxygen of its neighbor in one direction via a phosphodiester linkage, an end
of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate is not
linked to the 3'
oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen
is not
linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used
herein, a nucleic acid sequence, even if internal to a larger oligonucleotide,
also may
be said to have 5' and 3' ends.
When two different, non-overlapping oligonucleotides anneal to different
regions of the same linear complementary nucleic acid sequence, and the 3' end
of one
oligonucleotide points towards the 5' end of the other, the former may be
called the
"upstream" oligonucleotide and the latter the "downstream" oligonucleotide.
The term "primer" refers to an oligonucleotide which is capable of acting as a
point of initiation of synthesis when placed under conditions in which primer
extension is initiated. An oligonucleotide "primer" may occur naturally, as in
a
purified restriction digest or may be produced synthetically.
A primer is selected to be "substantially" complementary to a strand of
specific
sequence of the template. A primer must be sufficiently complementary to
hybridize
with a template strand for primer elongation to occur. A primer sequence need
not
reflect the exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer, with the
remainder of
the primer sequence being substantially complementary to the strand. Non-
complementary bases or longer sequences can be interspersed into the primer,
provided that the primer sequence has sufficient complementarity with the
sequence
of the template to hybridize and thereby form a template primer complex for
synthesis
of the extension product of the primer.
"Hybridization" methods involve the annealing of a complementary sequence
to the target nucleic acid (the sequence to he detected). The ability of two
polymers of
nucleic acid containing complementary ,cquences to find each other and anneal
through base pairing interaction is a well-recognized phenomenon. The initial
observations of the "hybridization" procese by '~tarmur and Lane, Proc. Natl.
Acad.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
Sci. USA 46:453 ( 1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (
1960}
have been followed by the refinement of this process into an essential tool of
modern
biology. Nonetheless, a number of problems have prevented the wide scale use
of
hybridization as a tool in human diagnostics. Among the more formidable
problems
5 are: 1 ) the inefficiency of hybridization; 2) low concentration of specific
target
sequences in a mixture of genomic DNA; and 3) the hybridization of only
partially
complementary probes and targets.
With regard to efficiency, it is experimentally observed that only a fraction
of
the possible number of probe-target complexes are formed in a hybridization
reaction.
This is particularly true with short oligonucleotide probes (less than 100
bases in
length). There are three fundamental causes: a) hybridization cannot occur
because
of secondary and tertiary structure interactions; b) strands of DNA containing
the
target sequence have rehybridized (reannealed) to their complementary strand;
and c)
some target molecules are prevented from hybridization when they are used in
hybridization formats that immobilize the target nucleic acids to a solid
surface.
Even where the sequence of a probe is completely complementary to the
sequence of the target, i.e., the target's primary structure, the target
sequence must be
made accessible to the probe via rearrangements of higher-order structure.
These
higher-order structural rearrangements may concern either the secondary
structure or
tertiary structure of the molecule. Secondary structure is determined by
intramolecular bonding. In the case of DNA or RNA targets this consists of
hybridization within a single, continuous strand of bases (as opposed to
hybridization
between two different strands). Depending on the extent and position of
intramolecular bonding, the probe can be displaced from the target sequence
preventing hybridization.
Solution hybridization of oligonucleotide probes to denatured double-stranded
DNA is further complicated by the fact that the longer complementary target
strands
can renature or reatmeal. Again, hybridized probe is displaced by this
process. This
results in a low yield of hybridization ( low "coverage") relative to the
starting
concentrations of probe and target.
With regard to low target sequence concentration, the DNA fragment
containing the target sequence is usually in relatively low abundance in
genomic
DNA. This presents great technical ditiirulties: most conventional methods
that use
oligonucleotide probes lack the sensitiv nv nrcessary to detect hybridization
at such
low levels.
One attempt at a solution to the tarLrt sequence concentration problem is the
amplification of the detection sig:.° . Most often this entails placing
one or more
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
6
labels on an oligonucleotide probe. In the case of non-radioactive labels,
even the
highest affinity reagents have been found to be unsuitable for the detection
of single
copy genes in genomic DNA with oligonucleotide probes. See Wallace et ai.,
Biochimie 67:755 (1985). In the case of radioactive oligonucleotide probes,
only
extremely high specific activities are found to show satisfactory results. See
Studencki and Wallace, IINA 3:1 (1984) and Studencki et al., Human Genetics
37:42
( 1985).
K. B. Mullis et al., U.S. Patent Nos. 4,683,195 and 4,683,202, hereby
incorporated by reference, describe a method for increasing the concentration
of a
segment of a target sequence in a mixture of any DNA without cloning or
purification.
This process for amplifying the target sequence (which can be used in
conjunction
with the present invention to make target molecules) consists of introducing a
large
excess of two oligonucleotide primers to the DNA mixture containing the
desired
target sequence, followed by a precise sequence of thermal cycling in the
presence of
a DNA polymerase. The two primers are complementary to their respective
strands of
the double stranded target sequence. To effect amplification, the mixture is
denatured
and the primers then allowed to annealed to their complementary sequences
within the
target molecule. Following annealing, the primers are extended with a
polymerase so
as to form a new pair of complementary strands. The steps of denaturation,
primer
annealing, and primer extension can be repeated many times (i.e.,
denaturation,
annealing and extension constitute one "cycle;" there can be numerous
"cycles") to
obtain a high concentration of an amplified segment of the desired target
sequence.
The length of the amplified segment of the desired target sequence is
determined by
the relative positions of the primers with respect to each other, and
therefore, this
length is a controllable parameter. By virtue of the repeating aspect of the
process, the
method is referred to by the inventors as the "Polymerise Chain Reaction"
(hereinafter PCR). Because the desired amplified segments of the target
sequence
become the predominant sequences (in terms of concentration) in the mixture,
they are
said to be "PCR amplified."
With PCR, it is possible to amplify a single copy of a specific target
sequence
in genomic DNA to a level detectable by several different methodologies (e.g.,
hybridization with a labeled probe; incorporation of biotinylated primers
followed by
avidin-enzyme conjugate detection; incorporation of 32P labeled
deoxynucleotide
triphosphates, e.g., dCTP or dATP. into the amplified segment). In addition to
genomic DNA, any oligonucleotide acyurnce can be amplified with the
appropriate
set of primer molecules. In particular. the amplified segments created by the
PCR
process itself are, themselves, efficient templates for subsequent PCR
amplifications.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
7
The PCR amplification process is known to reach a plateau concentration of
specific target sequences of approximately 10-g M. A typical reaction volume
is 100
~1, which corresponds to a yield of 6 x 1011 double stranded product
molecules.
With regard to complementarily, it is important for some diagnostic
applications to determine whether the hybridization represents complete or
partial
complementarily. For example, where it is desired to detect simply the
presence or
absence of pathogen DNA or RNA (such as from a virus, bacterium, fungi,
mycoplasma, protozoan) it is only important that the hybridization method
ensures
hybridization when the relevant sequence is present; conditions can be
selected where
both partially complementary probes and completely complementary probes will
hybridize. Other diagnostic applications, however, may require that the
hybridization
method distinguish between partial and complete complementarily. It may be of
interest to detect genetic polymorphisms. For example, human hemoglobin is
composed, in part, of four polypeptide chains. Two of these chains are
identical
chains of 141 amino acids (alpha chains) and two of these chains are identical
chains
of 146 amino acids (beta chains). The gene encoding the beta chain is known to
exhibit polymorphism. The normal allele encodes a beta chain having glutamic
acid
at the sixth position. The mutant allele encodes a beta chain having valine at
the sixth
position. This difference in amino acids has a profound (most profound when
the
individual is homozygous for the mutant allele) physiological impact known
clinically
as sickle cell anemia. It is well known that the genetic basis of the amino
acid change
involves a single base difference between the normal allele DNA sequence and
the
mutant allele DNA sequence.
Unless combined with other techniques (such as restriction enzyme analysis),
methods that allow for the same level of hybridization in the case of both
partial as
well as complete complementarily are typically unsuited for such applications;
the
probe will hybridize to both the normal and variant target sequence.
Hybridization,
regardless of the method used, requires some degree of complementarily between
the
sequence being assayed (the target sequence) and the fragment of DNA used to
perform the test (the probe). (Of course, one can obtain binding without any
complementarily but this binding is nonspecific and to be avoided.)
The complement of a nucleic acid sequence as used herein refers to an
oligonucleotide which, when aligned with the nucleic acid sequence such that
the ~'
end of one sequence is paired with the 3' end of the other, is in
"antiparallel
association." Certain bases not commonly found in natural nucleic acids may be
included in the nucleic acids of the present invention and include, for
example.
inosine and 7-deazaguanine complementarily need not be perfect; stable
duplexes may
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
8
contain mismatched base ~ pairs or unmatched bases. Those skilled in the art
of
nucleic acid technology can determine duplex stability empirically considering
a
number of variables including, for example, the length of the oligonucleotide,
base
composition and sequence of the oligonucleotide, ionic strength and incidence
of
mismatched base pairs.
Stability of a nucleic acid duplex is measured by the melting temperature, or
"Tm." The Tm of a particular nucleic acid duplex under specific conditions is
the
temperature at which on average half of the base pairs have disassociated. The
equation for calculating the Tm of nucleic acids is well known in the art. As
indicated
by strand references, an estimate of the Tm value may be calculated by the
equation:
Tm - 81.5°C + 16.6 log M + .41 (%GC) - 0.61 (% form) - 500/L
where M is the molarity of monovalent cations, %GC is the percentage of
guanosine
and cytosine nucleotides in the DNA, %form is the percentage of formamide in
the
hybridization solution, and L = length of the hybrid in base pairs [See, e.g.,
Guide to
Molecular Cloning Techniques, Ed. S.L. Berger and A.R. Kimmel, in Methods in
Enzymology Vol. 152, 401 (1987)]. Other references include more sophisticated
computations which take structural as well as sequence characteristics into
account for
the calculation of Tm.
The term "probe" as used herein refers to a labeled oligonucleotide which
forms
a duplex structure with a sequence in another nucleic acid, due to
complementarity of
at least one sequence in the probe with a sequence in the other nucleic acid.
The term "label" as used herein refers to any atom or molecule which can be
used to provide a detectable (preferably quantifiable) signal, and which can
be
attached to a nucleic acid or protein. Labels may provide signals detectable
fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or
absorption,
magnetism, enzymatic activity, and the like. Such labels can be added to the
oligonucleotides of the present invention.
The terms "nucleic acid substrate" and nucleic acid template" are used herein
interchangeably and refer to a nucleic acid molecule which may comprise single-
or
double-stranded DNA or RNA.
The term "substantially single-stranded" when used in reference to a nucleic
acid substrate means that the substrate molecule exists primarily as a single
strand of
nucleic acid in contrast to a double-stranded substrate which exists as two
strands of
nucleic acid which are held together by inter-strand base pairing
interactions.
The term "sequence variation" a~ u;e~i herein refers to differences in nucleic
acid sequence between two nucleic am templates. For example, a wild-type
structural gene and a mutant form of this wild-type structural gene may vary
in
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
9
sequence by the presence of single base substitutions and/or deletions or
insertions of
one or more nucleotides. These two forms of the structural gene are said to
vary in
sequence from one another. A second mutant form of the structural gene may
exist.
This second mutant form is said to vary in sequence form both the wild-type
gene and
the first mutant form of the gene. It should be noted that, while the
invention does not
require that a comparison be made between one or more forms of a gene to
detect
sequence variations, such comparisons are possible with the oligo/solid
support matrix
of the present invention using particular hybridization conditions as
described in U.S.
Pat. Appl. Ser. No. 08/231,440, hereby incorporated by reference.
"Oligonucleotide primers matching or complementary to a gene sequence"
refers to oligonucleotide primers capable of facilitating the template-
dependent
synthesis of single or double-stranded nucleic acids. Oligonucleotide primers
matching or complementary to a gene sequence may be used in PCRs, reverse
transcriptase-PCR (RT-PCRs) and the like.
A "consensus gene sequence" refers to a gene sequence which is derived by
comparison of two or more gene sequences and which describes the nucleotides
most
often present in a given segment of the genes; the consensus sequence is the
canonical
sequence.
As used herein, the terms "protein" and "protease" refer to metalloprotease.
The term "metalloprotease" refers to a native metal dependent protease, a
fragment
thereof, a mutant or homologue which still retains its function. The invention
contemplates metalloproteases (or "disintegrins") from differing species, and
those
prepared by recombinant methods, in vitro methods, or standard peptide
synthesis.
Preferably the protein is a human disintegrin or mutant thereof. For the
purposes of
defining the mutants of the protein the preferred "native" protein is
partially described
in Gen Bank accession #Z48579, incorporated herein by reference and referred
to in
the sequence below. Homologue disintegrins include whole proteins with at
least
90% homology as understood by the art, or fragments thereof. It is recognized
that
some interspecies variation may occur including insertions or deletions which
may or
may not alter function. For example, a rat protein which is 95% homologous to
the
protein based on the peptide sequence, and a bovine protein (based on DNA
sequence)
being 97-98% homologous based on the first 300 base pairs are both considered
homologues. For reference GenBank accession #Z48444 dated February 25, 1994
discloses 2407 bases of a rat gene sail to be a rat disintegrin
metalloprotease gene:
GenBank accession #Z21961 dated ()ctui~r 2~. 1994, discloses 2397 bases of a
partial sequence of a gene said to be a h~» ine zinc metalloprotease gene.
Preferablv_
this metalloprotease is a human disinteLnn as Described below.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
The term "antibody" refers to an antibody to a disintegrin, or fragment
thereof.
These many be monoclonal or polyclonal, and can be from any of several
sources.
The invention also contemplates fragments of these antibodies made by any
method in
the protein or peptide art.
5 The term "disease screen" refers to a screen for a disease or disease state.
A
disease state is the physiological or cellular or biochemical manifestation of
the
disease. Preferably this screen is used on body tissues or fluids of an animal
or cell
culture, using standard techniques, such as ELISA. It also contemplates
"mapping" of
disease in a whole body, such as by labeled antibody as described above given
10 systemically: regardless of the detection method, preferable such detection
methods
include fluorescence, X-ray (including CAT scan), NMR (Including MRI), and the
like.
The term "compound screen" is related to the methods and screens related to
finding compounds, determining their affinity for the protease, or designing
or
selecting compounds based on the screen. In another embodiment, it
contemplates the
use of the three dimensional structure for drug design, preferable "rational
drug
design", as understood by the art. It may be preferred that the protease is in
"essentially pure form", which refers to a protein reasonably free of other
impurities,
so as to make it useful for experiments or characterization. Use of this
screening
method assists the skilled artisan in finding novel structures, whether made
by the
chemist or by nature, which bind to and preferably inhibit the protease. These
"inhibitors" may be useful in regulating or modulating the activity of the
protease, and
may be used to thus modulate the biological cascade that they function in.
This
approach affords new pharmaceutically useful compounds.
The term "disintegrin" refers to a disintegrin, a fragment thereof, a mutant
thereof or a homologue which still retains its function. This term
contemplates
aggrecanase, and other proteases which are involved in or modulate tissue
remodeling.
This contemplates disintegrins from dit~fering species, and those prepared by
recombinant methods, in vitro methods, or standard peptide synthesis.
Preferably the
protein is a human disintegrin or mutant thereof. For the purposes of defining
the
mutants, with reference to a protein is partially described in GenBank
accession #
248579, incorporated herein by reference and referred to in the sequence
below. SEQ
ID NO:I describes a fragment of that U~:~ sequence and its transcript and SEQ
ID
N0:2 describes the protein coded by the tune. I lomologue disintegrins include
whole
proteins with at least 90% homology as understood by the art, or fragments
thereof
For example, a rat protein which is 95° o humolugous to that of SEQ ID
N0:2 based
on the amino acid sequence derive ° :rom the DNA or cDNA sequence
containing
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
11
SEQ ID NO:1, and a bovine protein {similarly derived) being 97-98% homologous,
are both considered homologues. Thus homologous cDNAs cloned from other
organisms give rise to homologous proteins.
Likewise proteins may be considered homologues based on the amino acid
sequence alone. Practical limitations of amino acid sequencing would allow one
to
determine that a protein is homologous to another using, for example,
comparison of
the f rst SO amino acids of the protein. Hence 90% homology in would allow for
5
differing amino acids in the chain of the first 50 amino acids of the
homologous
protein.
The skilled artisan will appreciate that the degeneracy of the genetic code
provides for differing DNA sequences to provide the equivalent transcript, and
thus
the same protein. In certain cases preparing the DNA sequence, which encodes
for the
same protein, but differs from the native DNA include;
--- ease of sequencing or synthesis;
--- increased expression of the protein; and
--- preference of certain heterologous hosts for certain codons over others.
These practical considerations are widely known and provide embodiments
that may be advantageous to the user of the invention. Thus it is clearly
contemplated that the native DNA is not the only embodiment envisioned in this
invention.
In addition it is apparent to the skilled artisan that fragments of the
protein
may be used in screening, drug design and the like, and that the entire
protein may not
be required for the purposes of using the invention. Thus it is clearly
contemplated
that the skilled artisan will understand that the disclosure of the protein
and its uses
contemplates the useful peptide fragments.
The practical considerations of protein expression, purification yield,
stability,
solubility, and the like, are considered by the skilled artisan when choosing
whether to
use a fragment, and the fragment to be used. As a result, using routine
practices in the
art, the artisan can, given this disclosure practice the invention using
fragments of the
protein as well.
Thus, the present invention specifically contemplates the use of less than the
entire nucleic acid sequence for the gene and less than the entire amino acid
sequence
of the protein. Fragments of the protein may be used in screening, drug design
and
the like, and that the entire protein may not he required for the purposes of
using the
invention. The protein itself can be used to determine the binding activity of
small
moleeuies to the protein. Drug screening using enzymatic targets is used in
the art
and can be employed using automated. high throughput technologies.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
12
The protein or protease itself can be used to determine the binding activity
of
small molecules to the protein. Drug screening using enzymatic targets is used
in the
art and can be employed using automated, high throughput technologies.
The inhibition of disintegrin activity may be a predictor of efficacy in the
treatment of osteoarthritis, and other diseases involving degeneration of
articular
cartilage and other tissues having matrix degradation, such as tissue
remodeling and
the like.
Gene therany
Without being bound by theory it is thought that the metalloprotease is up
regulated during osteoarthritis in tissues. We have surprisingly found that a
human
disintegrin is up-regulated in human chondrocytes during osteoarthritic
conditions.
Inhibition of signal transduction mechanism is efficacious in disrupting the
cascade of
events in osteoarthritis and other diseases involving cartilage degeneration.
The
skilled artisan will recognize that if up-regulation is a cause of the onset
of arthritis,
then interfering with the activity of this gene may be useful in treating
osteoarthritis.
This is done by any of several methods, including gene (i.e., antisense)
therapy.
Purification of the protease
Media, cell extracts or inclusion bodies from mammalian, yeast, insect or
eukaryotic cells containing recombinant disintegrin or fragments of the full
length
protein are used for purification of disintegrin or fragments of disintegrin.
Solutions
consisting of denatured disintegrin may be refolded prior to purification
across
successive chromatographic resins or following the final stage of separation.
Media,
cell extracts, or solubilized disintegrin are prepared in the presence of one
or a
combination of detergents, denaturants or organic solvents, such as
octylglucoside,
urea or dimethylsulfoxide, as required. Ion exchange and hydrophobic
interaction
chromatography are used individually or in combination for the separation of
recombinant disintegrin from contaminating cell material. Such material is
applied to
the column and disintegrin is eluted by adjustment of pH, changes in ionic
strength,
addition of denaturant and/or use of organic solvent. Typically, solutions
containing
disintegrin are then passed over an antibody affinity column or ligand
affinity column
for site specific purification of disintegrin. The immunoaffinity column
contains an
antibody specific for disintegrin immobilized on a solid support such as
Sepharose 4B
(Pharmacia) or other similar materials. Preferably, the column is washed to
remove
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
13
unbound proteins and the disintegrin is eluted via low pH glycine buffer or
high ionic
strength. The ligand affinity column may have specificity for the active site
of
disintegrin or to a portion of the molecule adjacent or removed from the
active site.
The column is washed and disintegrin is eluted by addition of a competing
molecule
to the elution buffer. Preferably, a protease inhibitor cocktail containing
one or more
protease inhibitors, such as benzamidine, leupeptin, phosphoramidon,
phenylmethylsulfonyi fluoride, and 1,10-phenanthroline is present throughout
the
purification procedure. Various detergents such as octylthioglucoside and
Triton X-
100 or chemical agents such as glycerol may be added to increase disintegrin
solubility and stability. Final purification of the protein is achieved by gel
filtration
across a chromatographic support, if required.
Inhibitors of the protease
The protease of the invention can be used to find inhibitors of the protease.
Hence it is useful as a screening tool or for rational drug design. Without
being bound
by theory, the protease may modulate cellular remodeling and in fact may
enhance
extracellular matrix remodeling and thus enhance tissue breakdown. Hence
inhibition
of disintegrin provides a therapeutic route for treatment of diseases
characterized by
these processes.
In screening, a drug compound can be used to determine both the quality and
quantity of inhibition. As a result such screening provides information for
selection
of actives, preferably small molecule actives, which are useful in treating
these
diseases.
In therapy, inhibition of disintegrin metalloprotease activity via binding of
small molecular weight, synthetic metalloprotease inhibitors, such as those
used to
inhibit the matrix metalloproteases would be used to inhibit extracellular
matrix
remodeling.
Antibodies to the Qrotein
Metalloproteases can be targeted by conjugating a metalloprotease inhibitor to
a to an antibody or fragment thereof. ('t~njugation methods are known in the
art.
These antibodies are then useful both in th~raps and in monitoring the dosage
of the
inhibitors.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
14
The antibody of the invention can also be conjugated to solid supports. These
conjugates can be used as affinity reagents for the purification of a desired
metalloprotease, preferably a disintegrin.
In another aspect, the antibody of the invention is directly conjugated to a
label. As the antibody binds to the metalloprotease, the label can be used to
detect the
presence of relatively high levels of metalloprotease in vivo or in vitro cell
culture.
For example, targeting ligand which specifically reacts with a marker for the
intended target tissue can be used. Methods for coupling the invention
compound to
the targeting ligand are well known and are similar to those described below
for
coupling to carrier. The conjugates are formulated and administered as
described
above.
Preparation and Use of Antibodies:
Antibodies may be made by several methods, for example, the protein may be
injected into suitable (e.g., mammalian) subjects including mice, rabbits, and
the like.
Preferred protocols involve repeated injection of the immunogen in the
presence of
adjuvants according to a schedule which boosts production of antibodies in the
serum.
The titers of the immune serum can readily be measured using immunoassay
procedures, now standard in the art.
The antisera obtained can be used directly or monoclonal antibodies may be
obtained by harvesting the peripheral blood lymphocytes or the spleen of the
immunized animal and immortalizing the antibody-producing cells, followed by
identifying the suitable antibody producers using standard immunoassay
techniques.
Polyclonal or monoclonal preparations are useful in monitoring therapy or
prophylaxis regimens involving the compounds of the invention. Suitable
samples
such as those derived from blood, serum, urine, or saliva can be tested for
the
presence of the protein at various times during the treatment protocol using
standard
immunoassay techniques which employ the antibody preparations of the
invention.
These antibodies can also be coupled to labels such as scintigraphic labels,
e.g..
Tc-99 or I-131, using standard coupling methods. The labeled compounds are
administered to subjects to determine the locations of excess amounts of one
or more
metalloproteases in vivo. Hence a labele~f antibody to the protein would
operate as a
screening tool for such enhanced expression. indicating the disease.
The ability of the antibodies to hind mctalloprotease selectively is thus
taken
advantage of to map the distribution ol~ these enzymes in situ. The techniques
can also
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
be employed in histological procedures and the labeled antibodies can be used
in
competitive immunoassays.
Antibodies are advantageously coupled to other compounds or materials using
known methods. For example, materials having a carboxyl functionality, the
carboxyl
5 residue can be reduced to an aldehyde and coupled to carrier through
reaction with
side chain amino groups; optionally followed by reduction of imino linkage
formed.
The carboxyl residue can also be reacted with side chain amino groups using
condensing ' agents such as dicyclohexyl carbodiimide or other carbodiimide
dehydrating agents. Linker compounds can also be used to effect the coupling;
both
10 homobifunctional and heterobifunctional linkers are available from Pierce
Chemical
Company, Rockford, I11.
These antibodies, when conjugated to a suitable chromatography material are
useful in isolating the protein. Separation methods using affinity
chromatography are
well known in the art, and are within the purview of the skilled artisan.
15 Disease marker
As noted above, the present invention contemplates detecting expression of
metalloprotease genes in samples, including samples of diseased tissue. It is
not
intended that the present invention be limited by the nature of the source of
nucleic
acid (whether DNA or RNA); a variety of sources is contemplated, including but
not
limited to mammalian (e.g., cancer tissue, lymphocytes, etc.), sources.
Without being bound by theory, expression of genes, and preferably this gene
may have a restricted tissue distribution and its expression is up regulated
by potential
osteoarthritis mediators. Enhanced expression of this gene (and hence its
protein) for
example, in articular chondrocytes provides a marker to monitor the
development,
including the earliest, asymptomatic stages, and the progression of
osteoarthritis.
Hence an antibody raised to the protein would operate a screening tool for
such
enhanced expression, indicating the disease.
In addition, when used in a disease screen, antibodies can be conjugated to
chromophore or fluorophore containing materials, or can be conjugated to
enzymes
which produce chromophores or fluorophores in certain conditions. These
conjugating materials and methods are well known in the art. When used in this
manner detection of the protein by immunoassay is straightforward to the
skilled
artisan. Body fluids, (serum, urine, synch gal tluid) for example can be
screened in this
manner for calibration, and detection of Jwtnhution of metalloproteases, or
increased
levels of these proteases.
When used in this way the iwrntiun is a useful diagnostic and/or clinical
marker for metalloprotease mediate ° i,eaars. such as osteoarthritis or
other articular
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
16
cartilage degenerative diseases or other diseases characterized by degradation
or
remodeling of extracellular matrix. When disease is detected, it may be
treated before
the onset of symptom or debilitation.
Furthermore, such antibodies can be used to target diseased tissue, for
detection or treatment as described above.
Nucleic Acid Derived Tools
The nucleic acid content of cells consists of deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). The DNA contains the genetic blueprint of the cell.
RNA is
involved as an intermediary in the production of proteins based on the DNA
sequence.
RNA exists in three forms within cells, structural RNA (i.e., ribosomal RNA
"rRNA"), transfer RNA ("tRNA"), which is involved in translation, and
messenger
RNA ("mRNA"). Since the mRNA is the intermediate molecule between the genetic
information encoded in the DNA, and the corresponding proteins, the cell's
mRNA
component at any given time is representative of the physiological state of
the cell. In
order to study and utilize the molecular biology of the cell, it is therefore
important to
be able to purify W RNA, including purifying mRNA from the total nucleic acid
of a
sample.
The preparation of RNA is complicated by the presence of ribonucleases that
degrade RNA (e.g., T. Maniatis et al., Molecular Cloning, pp. 188-190, Cold
Spring
Harbor Laboratory [1982]). Furthermore, the preparation of amplifiable RNA is
made
difficult by the presence of ribonucleoproteins in association with RNA. (See,
R. J.
Slater, In: Techniques in Molecular Biology, J. M. Walter and W. Gaastra,
eds.,
Macmillan, NY, pp. 113-120 [1983]).
Typically, the steps involved in purification of nucleic acid from cells
include
1) cell lysis; 2) inactivation of cellular nucleases; and 3) separation of the
desired
nucleic acid form the cellular debris and other nucleic acid. Cell lysis may
be
achieved through various methods, including enzymatic, detergent or chaotropic
agent
treatment. Inactivation of cellular nucleases may be achieved by the use of
proteases
and/or the use of strong salts. Finally, separation of the desired nucleic
acid is
typically achieved by extraction of the nucleic acid with phenol or phenol-
chloroform;
this method partitions the sample into an aqueous phase (which contains the
nucleic
acids) and an organic phase (which contains other cellular components,
including
proteins). Commonly used protocols require the use of salts in conjunction
with
phenol (P. Chomczynski and N. Sacchi. ;anal. Biochem. 162:156 [1987]), or
employ a
centrifugation step to remove the protein i R. J. Slater, supra).
Once the nucleic acid fraction has hcen isolated from the cell, the structure
of
the mRNA molecule may be used to asaiat in the purification of mRNA from DNA
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
17
and other RNA molecules. Because the mRNA of higher organisms is usually
polyadenylated on its 3' end ("poly-A tail" or "poly-A track"), one means of
isolating
RNA from cells has been based on binding the poly-A tail with its
complementary
sequence (i.e., oligo-dT), that has been linked to a support such as
cellulose.
Commonly, the hybridized mRNA/oligo-dT is separated from the other components
present in the sample through centrifugation or, in the case of magnetic
formats,
exposure to a magnetic field. Once the hybridized mRNA/oligo-dT is separated
from
the other sample components, the mRNA is usually removed from the oligo-dT.
However, for some applications, the mRNA may remain bound to the oligo-dT that
is
70 linked to a solid support.
A wide variety of solid supports with linked oligo-dT have been developed
and are commercially available. Cellulose remains the most common support for
most oligo-dT systems, although formats with oligo-dT covalently linked to
latex
beads and paramagnetic particles have also been developed and are commercially
available. The paramagnetic particles may be used in a biotin-avidin system,
in which
biotinylated oligo-dT is annealed in solution to mRNA. The hybrids are then
captured
with streptavidin-coated paramagnetic particles, and separated using a
magnetic field.
In addition to these methods, variations exist, such as affinity purification
of
polyadenylated RNA from eukaryotic total RNA in a spun-column format. These
approaches allow for hybridization of poly-A mRNA, but vary in efficiency and
sensitivity.
In one embodiment, the mRNA is treated with reverse transcriptase to make
cDNA. The cDNA can be used in primer extension and PCR using the primers
described below. Thus, the present invention contemplates nucleic acid
molecules
detectable by primer extension suing the primers described below. Primer
extension
(and PCR for that matter) can be carried out under conditions (so-called "high
stringency conditions") such that only complementary nucleic acid will
hybridize (as
opposed to hybridization with partially complementary nucleic acid). These
conditions including annealing at or near the melting temperature of the
duplex.
Primers Directed To A Specific DisinteQrin Metalloprotease Gene
The invention provides a partial nucleic acid full length protein coding
region
sequence of a novel disintegrin metalloprotease gene useful for, among other
things,
the detection of disintegrin metalloprotease gene expression. In one
embodiment,
primers directed to a portion of this partial sequence are use to detect the
presence or
absence of the gene sequence. These primers can be also be used for the
identification
of a cDNA clone representing the entire gene. allowing for recombinant
expression in
CA 02281085 1999-08-10
'WO 98/37092 PCT/US98/03490
18
a host cell of the nucleic acid sequence encoding the disintegrin
metalloprotease or
fragments (or mutants) thereof.
Preferred primers are primer SEQ ID N0:9 (5'-AGCCTGTGTC-3') and SEQ
ID NO:10 (5'-AGCCTGTGTCTGAACCACT-3'). However, other primers can be
readily designed from the sequences set forth in SEQ ID NO:S and SEQ ID NO:1.
Method of Comnarine Bioloeical Samples by Differential Disnlay
Successful amplification can be confirmed by characterization of the
products) from the reaction. It is not intended that the present invention be
limited
by the method by which extension products or PCR products are detected. In one
embodiment, the PCR products are analyzed by high resolution agarose gel
electrophoresis using 2% agarose gels (BRL) and the amplified DNA fragments
are
visualized by ethidium bromide staining and UV transillumination. The present
invention contemplates, in one embodiment, using electrophoresis to confirm
product
formation and compare the results between samples.
Hence, the present invention contemplates detection of sequences of the novel
disintegrin metalloprotease gene in mixtures of nucleic acid (e.g., cDNA or RT-
mRNA). By carrying out PCR on a mixture of nucleic acid and running the
products
on gels, nucleic acid comprising a sequence that is defined by the primers is
"isolated." The product can thereafter be "purified" by cutting the band from
the gel
(or by other suitable methods such as electroelution).
Synopsis of the Sequence Listing
For the aid of the reader, the inter-relation of the sequence listings are
described hereinbelow:
SEQ ID NO:1 is a fragmentary DNA sequence, and is part of SEQ ID N0:3.
The first base (Cytosine or C) of SEQ ID NO:I is base 940 of SEQ ID N0:3. The
DNA sequences are identical where they overlap.
SEQ ID N0:2 and SEQ ID N0:4, are the expressed amino acid sequences of
SEQ ID NO:1 and SEQ ID N0:3 respectively. The first amino acid of SEQ ID N0:2,
Gln, is the 309th amino acid in SEQ ID NO:.1. The two sequences are homologous
to
the carboxy terminus of the protein.
SEQ ID N0:7 is a sense strand of DNA provided by differential display
experiments. The first base of SEQ ID N():7 corresponds to base 1371 of SEQ ID
NO:1, and to base 2310 of SEQ ID NO:.~. l~hese sequences are homologous for
452
bases, to base 1822 of SEQ ID NO:1 and to hase 2761 of SEQ ID N0:3. The
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
19
difference in the last two bases of SEQ ID NO:1 and SEQ ID N0:3 may be due to
errors in sequencing or a common replicatory error found in PCR, or may be
part of a
cloning vector. SEQ ID N0:7 continues some 284 bases beyond the homology, and
thus well beyond the terminus of SEQ ID NO:1 and SEQ ID N0:3.
In addition, bases 477 to 716 of SEQ ID N0:7 are the SEQ ID NO 6. SEQ ID
NO 6 is the sense strand of SEQ ID NO:S, which is an antisense strand found
via
differential display cl9oning. Hence SEQ ID NO: 6 shows the DNA orientation as
it
would appear in the mRNA. These two sequences are found near the 3' end of
this
gene.
1 o Although bases 452 to the 3' end of SEQ ID N0:7 differ from SEQ ID NO: l
and SEQ ID N0:3, SEQ ID N0:7 is nonetheless valid. It is essential to note
that the
expressed peptide sequence is not affected by this difference. It is likely
these bases
do not appear in SEQ ID NO:1 and SEQ ID N0:3 because of the use of an
alternative
polyadenylation signal. .
SEQ ID NO 8 is a novel full length DNA sequence. SEQ ID N0:9 is the novel
expressed protein of SEQ ID N0:8. SEQ ID N0:9 differs from SEQ ID N0:4 in that
amino acids 162 (Ser)-213 (Tyr) of SEQ ID NO: 4 is replaced by a single
residue,
Asn, at position 162 of SEQ ID~N0:9. That change is reflected in the DNA by a
deletion bases 501-654 for a total of 153 bases, leaving the reading frame
intact but
changing one residue and deleting the 51 amino acids present in SEQ ID N0:4.
SEQ ID NO: i 0 and SEQ ID NO: I i are antisense primers useful in PCR, and
are the inverse of the 3' terminus of SEQ ID N0:7, other sequences for primers
are
discernible by the skilled artisan using sequences referred to herein.
EXAMPLES
The following non-limiting examples illustrate a preferred embodiment of
the present invention, and briefly describe the uses of the present invention.
These
examples'~a're provided for the guidance of the skilled artisan, and do not
limit the
invention in any way. Armed with this disclosure and these examples the
skilled
artisan is capable of making and using the claimed invention.
Standard starting materials arr u,c~l for these examples. Many of these
materials are known and commercially available. For example, E. coli CJ236 and
JM101 are known strains, pUB 1 ( 0 i; a known plasmid and Kunkel method
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
mutagenesis is also well known in the art. In addition certain cell lines and
cDNA
may be commercially available, for example U-937, available from Clontech
Inc.,
Palo Alto, California.
Variants may be made by expression systems and by various methods in
5 various hosts, these methods are within the scope of the practice of the
skilled artisan
in molecular biology, biochemistry or other arts related to biotechnology.
Example 1
RNA is isolated from unstimulated and interleukin-1 stimulated cultures of
normal human articular chondrocytes. The RNA is reverse transcribed into cDNA.
10 The cDNA is subjected to a modified differential display procedure using a
series of
random primers.
PCR samples generated from both stimulated and unstimulated chondrocytes
are electrophoresed in adjacent lanes on polyacrylamide gels. The
differentially
expressed band is excised from the gel, cloned, and sequenced. The
differential
15 expression of the gene is confirmed by RNAase protection and nuclear run on
experiments.
Example 2
A novel partial human cDNA coding the protein is cloned from primary
cultures of interleukin-1 stimulated human articular (femoral head)
chondrocytes,
20 using known methods. The same sequence is found, and the gene completed by
screening of human cDNA libraries to obtain full length clones.
Example 3
The cloned DNA of example 2 is placed in pUB 110 using known methods.
This plasmid is used to transform E. coli and provides a template for site-
directed mutagenesis to create new mutants. Kunkel method mutagenesis was
performed altering the Gln 1 to Ala.
Example 4
~i25I~ disintegrin antibody is prepared using IODOBEADS (Pierce, Rockford, IL;
immobilized chloramine-T on nonporous polystyrene beads). Lyophilized antibody
(2
pg) is taken up in 50 wl of 10 mM acetic acid and added to 450 ~tl of
phosphate
buffered saline (PBS) (Sigma, St. Louis, V10) on ice. To the tube is added 500
Curie of I25I (Amersham, Arlington Ilrights, IL) (2200Ci/mmol) in 5 pl, and
one
IODOBEAD. The reaction is incubated on ice for 10 min with occasional shaking.
The reaction is then terminated by removal of the reaction from the IODOBEAD.
To
remove unreacted I25I, the mixture is appl~r~i to a PD-10 gel filtration
column.
F~ample 5
CA 02281085 1999-08-10
w0 98/37092 PCT/US98/03490
21
A fluorogenic disintegrin metalloprotease substrate peptide (Bachem, Guelph
Mills, King of Prussia, Pa) is mixed with the disintegrin and change in the
fluorescence is evaluated at 2 min, as a control. Then the fluorogenic peptide
is
mixed with the disintegrin in the presence of the compound (metalloprotease
inhibitor) in evaluation in a separate run, with evaluation at various time
points over 2
to 12 hours. Data are evaluated using standard methodology to provide relative
binding of the evaluated compound.
Example 6
0.5 ml of synovial fluid from the left knee of a patient is withdrawn and
tested
for elevated levels disintegrin by ELISA. The results indicate higher than
normal
disintegrin level. The patient is prescribed a prophylactic dose of a
disintegrin
inhibitor administered orally over time or is administered an injection of
same in the
left knee before leaving the clinician's office.
Example 7
Inhibition of extracellular matrix remodeling is explored via inhibition of
disintegrin metalloprotease activity. Using a small molecular weight,
synthetic
metalloprotease inhibitor, such as those used to inhibit the matrix
metalloproteases,
tissue integrity and proteoglycan is monitored.
A sample of IL-I stimulated bovine nasal cartilage derived articular cartilage
is
grown in a 1 micromolar solution of a small molecular weight disintegrin
inhibitor.
The experiment is controlled and compared to an identical culture grown with
no
inhibitor.
The assay of the culture after 7 days shows that the inhibited culture has
less
tissue breakdown and less proteoglycan present in the serum of the culture.
The result
is consistent with the inhibited aggrecanase activity. Inhibition of
aggrecanase would
inhibit tissue breakdown and reduce the release of proteoglycan.
Example 8
Inhibition of proteolytic processing resulting in the release from the
membrane
bound form of the disintegrin metalloprotease domain inhibits "second
messenger"
signaling of the membrane bound disintegrin molecule. Such second messenger
signaling would result in cellular phenotypic changes, changes in gene
expression.
changes in mitotic activity, and the like.
Cells known to contain disintegrin are treated with a serine protease.
Proteins
released from the cell are measured by standard methods. Specifically the
metalloprotease activity is monitored via literature methods. The amount of
metalloprotease released is correlated to thc; amount of serine protease used
to treat the
cells.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
22
Increases, versus control, in src tyrosine kinase activity are measured by
Western blot analysis of intracellular proteins using monoclonal antibodies
specific
for phosphotyrosine following cleavage and release of the disintegrin
metalloprotease.
Controls are cells that have not been treated with serine protease.
src tyrosine kinase activity in the cell (or is it cell culture) is measured
by
literature methods. Release of the metalloprotease domain of the disintegrin
is also
monitored via literature methods. There is a direct correlation between
release of the
metalloprotease domain and increases in intracellular src tyrosine kinase
activity.
This result is consistent with stimulation of disintegrin-mediated cell
signaling by
stimulation of the src tyrosine kinase cascade.
Example 9
Integrin binding is measured with a peptide containing the sequence RGD.
Inhibition of intercellular adhesion molecules, or extracellular matrix
components
results in the inhibition of phenotypic changes, including changes in cell
shape,
associated with such interactions. Integrin binding is measured via
competitive assay,
using cellular changes in shape visible via microscopy. The peptide inhibits
such
cellular changes.
This result is consistent with competition with or blocking of the interaction
of
disintegrin. The RGD peptide inhibits cellular changes in chondrocytes. The
osteoarthritis phenotype, characterized by increased matrix synthesis and
accelerated
matrix metalloprotease activity does not occur. Other readily assayable
cellular
changes can be used to monitor this result, including gene expression, changes
in
mitotic activity, and the like.
Exaarple 10
A small molecular weight metalloprotease inhibitor is used to treat a tissue
culture according to the method of Example 7. The release of TNF-a from the
cell
membrane is measured by literature methods: The inhibitor of Example 7 also
decreases the amount of TNF-a secreted from the cell membrane.
Hence it is contemplated that inhibition of disintegrin metalloprotease
activity
will result in the inhibition of a disintegrin associated inflammation cascade
and
secretase activity. It is contemplated that monitoring the release of
cytokines or IL-1
from the cell membrane, and the like, will produce the same result.
Example 11
Differential Display Screening for Disease
RNA is isolated from unstimulated and interleukin-1 stimulated cultures of
normal human articular chondrocytes. The RNA is reverse transcribed into cDNA.
The cDNA is subjected to amplification (PCR) using the above-named primers.
PCR
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
23
samples generated from both stimulated and unstimulated chondrocytes are
electrophoresed in adjacent lanes on polyacrylamide gels. A differentially
expressed
band {i.e., a band found only in the stimulated cells and not expressed at
significant or
detectable levels in the unstimulated cells) is excised from the gel, cloned,
and
partially sequenced. The partial sequence is shown in SEQ ID NO:~. the
sequence is
found to exhibit approximately 60% homology to a rat metalloprotease (see
above).
The sequence is found to exhibit approximately 85% homology to a human
metalloprotease (see Gen Bank Accession #Z48597, see Figure 2).
Example 12
Screening for Metastatic Potential of Tumors
Cancer tissue is tested for metalloprotease gene expression. The above-named
primers are used in PCR on extracted nucleic acid from the sample. High levels
of
transcripts suggest metastatic potential.
Example 13
~ 5 Drue Screen for Expression Inhibitors
Candidate inhibitors of metalloprotease gene expression are screened in vitro.
Interleukin-1 stimulated cultures of normal human articular chondrocytes are
exposed
in vitro to candidate inhibitors. The RNA is isolated and reverse transcribed
into
cDNA. the cDNA is subjected to amplification (PCR) using the above-named
primers. PCR samples generated from both chondrocytes exposed to inhibitors
and
uninhibited chondrocytes are electrophoreses in adjacent lanes on
polyacrylamide
gels. Reduced levels of PCR product identifies an inhibitor.
Example 14
Drug Screen For Metalloprotease Inhibitors
Candidate inhibitors of the metalloprotease itself are screened in vitro. The
culture supernatant of Interleukin-1 stimulated cultures of normal human
articular
chondrocytes are assayed on suitable metalloprotease substrates (e.g., matrix
proteins)
in the presence and absence of candidate inhibitors. Known inhibitors are used
as
controls (e.g., 1,10-phenanthroline available commercially from Sigma Co., St.
Louis). Reduced levels of substrate (e.g., lluorogenic disintegrin
metallaprotease
substrate) degradation identifies an inhibitor.
Example t5
A 1400 BP clone is isolated via itan~iar~i screening techniques from U-937, a
monocyte-like cell cDNA line library. I~h~ initial sequence is a truncated
clone,
missing a portion of the 5' end. The ~' en~i i, Lenerated using 5' R.A.C.E.
(Rapid
Amplification of 5 c-DNA Ends, see t~~~r raample, Chapter 4 (pages 28-38), and
CA 02281085 1999-08-10
WO 98/3?092 PCT/US98/03490
24
references therein of PCR Protocols A Guide to Methods and Annlications,
Innis, et
al, eds. 1990 Academic Press), a known technique, generating a 1600 by clone
containing the remaining 5' sequence. These two sequences together provide SEQ
ID
N0:8, from which the peptide sequence is derived.
Example 16
Primers SEQ ID N0:9 (5'-AGCCTGTGTC-3') and SEQ ID NO:10 (5'-
AGCCTGTGTCTGAACCACT-3') are used in differential display of mRNA (ddrd-
PCR). 2-5 ng of sscDNA is used in the PCR. The reaction is precooled 0.2 p.l
thin-
walled tubes on ice. Each tube containing, SOmM TrisHCl (pH 8.5), SOmM KC1,
1.5
mM MgCl2 1 mM of each dNTP, 2-5 ng of sscDNA, 1 Opmoles of each primer above,
OS. ftl of a-P33 dCTP (10 ~Ci/~l, Amersham} and water to 20 ~1. The mixture is
subjected to 35 cycles of denaturation (94°C for 30 sec.), annealing
(36°C for 30 sec.)
and extension (72°C for 1 min.) using a Perkin-Elmer System 2400
Thermal Cycler
(Perking-Elmer, Norwalk, CT).
By this method, IL-1 treated chondrocytes expressed the mRNA associated
with this gene, while the untreated (no IL-1) control chondrocytes expressed
no
detectable mRNA.
Example 17
Assay system amenable to hi hg througl~ut screenine
The protease activity of disintegrin is measured in a kinetic enzyme
inhibition
assay using a fluorescent substrate. Using cloned disintegrin enzyme, and a
small
MW fluorescently labeled protein as the substrate. Enzyme activity is
quantified by
measurement of fluorescence after cleavage of the substrate molecule at room
temperature. This assay simple and very easy to automate.
Using standard techniques, this assay is adapted to 96 or 384 well plates.
All references described herein are hereby incorporated by reference.
While particular embodiments of the subject invention have been described, it
will be obvious to those skilled in the art that various changes. and
modifications of
the subject invention can be made without departing from the spirit and scope
of the
invention. It is intended to cover, in the ap~ndrd claims, all such
modifications that
are within the scope of this invention.
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
SEQUENCE LISTING
(1) GENERAL INFORMATION:
5
(i) APPLICANT: TINDAL, MICHAEL H
HAQQI, TARIQ M
(ii) TITLE OF INVENTION: USE OF A NOVEL DISINTEGRIN
METALLOPROTEASE, ITS MUTANTS, FRAGMENTS AND THE LIKE
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: THE PROCTER & GAMBLE COMPANY
(B) STREET: 8700 MASON-MONTGOMERY ROAD
(C) CI2'Y: MASON
(D) STATE: OH
(Ey COUNTRY: USA
(F) ZIP: 45040-9462
(vy COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
25 (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
3O (B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION
(A) NAME: HAKE, RICHARD A
(B) REGISTRATION NUMBER: ?',~.13
(C) REFERENCE/DOCKET NUMBER ~;g06,
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
26
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 513/622-0087
(B) TELEFAX: 513/622-0270
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1824 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(H) LOCATION: 2..1477
2~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
C CAG ACC ACA GAC TTC TCC GGA ATC CGT AAC ATC AGT TTC ATG GTG 46
Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val
1 5 10 35
AAA CGC ATA AGA ATC AAT ACA ACT GCT GAT GAG AAG GAC CCT ACA AAT 94
Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn
3~ 20 25 30
CCT TTC CGT TTC CCA AAT ATT AGT GTG GAG AAG TTT CTG GAA TTG AAT 142
Pro Phe Arg Phe Pro Asn Ile Ser Val ~:u ~ys Phe Leu Glu Leu Asn
40 45
TCT GAG CAG AAT CAT GAT GAC TAC TGT ".'.'~ ~CC TAT GTC TTC ACA GAC 190
Ser Glu Gln Asn His Asp Asp Tyr Cys ~PU Ala Tyr Val Phe Thr Asp
CA 02281085 1999-08-10
'WO 98/37092 PCT/US98/03490
27
50 55 60
CGA GAT TTT GAT GAT GGC GTA CTT GGT CTG GCT TGG GTT GGA GCA CCT 238
Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro
65 70 75
TCA GGA TCT GGA GGA ATA 286
AGC TGT GAA AAA AGT
AAA CTC TAT TCA
GAT
Ser Gly Ser Gly Gly Ile Lys Ser Lys Leu Tyr Ser
Ser Cys Glu Asp
80 85 9p g5
GGT AAG AAG TCC TTA AAC ATT ATT ACT GTT CAG AAC 334
AAG ACT GGA TAT
Gly Lys Lys Ser Leu Asn Ile Ile Thr Val Gln Asn
Lys Thr Gly Tyr
100 105 110
GGG TCT GTA CCT CCC AAA CAC ATT ACT TTT GCT CAC 382
CAT GTC TCT GAA
Gly Ser Val Pro Pro Lys His Ile Thr Phe Ala His
His Val Ser Glu
115 120 125
GTT GGA AAC TTT GGA TCC GAT TCT GGA ACA GAG TGC 430
CAT CCA CAT ACA
Val Gly Asn Phe Gly Ser Asp Ser Gly Thr Glu Cys
His Pro His Thr
130 135 140
CCA GGA TCT AAG AAT TTG AAA GAA AAT GGC AAT TAC 478
GAA GGT CAA ATC
Pro Gly Ser Lys Asn Leu Lys Glu Asn Gly Asn Tyr
Glu Gly Gln Ile
145 150 155
ATG TAT AGA GCA ACA TCT AAA CTT AAC AAC AAT AAA 526
GCA GGG GAC TTC
Met Tyr Arg Ala Thr Ser Lys Leu Asn Asn Asn Lys
Ala Gly Asp Phe
160 165 170 175
TCA CTC AGT ATT AGA AAT CAA GTT CTT GAG AAG AAG 574
TGT ATA AGC AGA
Ser Leu Ser Ile Arg Asn Gln Val Leu Glu Lys Lys
Cys Ile Ser Arg
180 185 190
AAC AAC TTT GTT GAA TCT C~T A':T TGT GGA AAT 622
TGT GGC CAA GGA ATG
Asn Asn Phe Val Glu Ser Pro Ile Cys Gly Asn Gly
Cys Gly Gln Met
195 200 205
CA 02281085 1999-08-10
'WO 98/37092 PCT/US98/03490
28
GTA GAA CAA GGT GAA GAA TGT GAT TGT GGC TAT AGT GAC CAG TGT AAA 670
Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys
210 215 220
GAT GAA TGC TGC TTC GAT GCA AAT CAA CCA GAG GGA AGA AAA TGC AAA 718
Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys
225 230 235
1O CTG AAA CCT GGG AAA CAG TGC AGT CCA AGT CAA GGT CCT TGT TGT ACA 766
Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr
240 245 250 255
GCA CAG TGT GCA TTC AAG TCA AAG TCT GAG AAG TGT CGG GAT GAT TCA 814
Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser
260 265 270
GAC TGT GCA AGG GAA GGA ATA TGT AAT GGC TTC ACA GCT CTC TGC CCA 862
Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro
2~ 275 280 285
GCA TCT GAC CCT AAA CCA AAC TTC ACA GAC TGT AAT AGG CAT ACA CAA 910
Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln
290 295 300
GTG TGC ATT AAT GGG CAA TGT GCA GGT TCT ATC TGT GAG AAA TAT GGC 958
Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly
305 310 315
TTA GAG GAG TGT ACG TGT GCC AGT TCT CA: .~GC AAA GAT GAT AAA GAA 1006
Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu
320 325 330 335
TTA TGC CAT GTA TGC TGT ATG AAG AAA :.'.~ -)AC ACA TCA ACT TGT GCC 1054
Leu Cys His Val Cys Cys Met Lys Lys !"~- :,sp Pro Ser Thr Cys Ala
340 'i: 350
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
29
AGT ACA GGG TCT GTG CAG TGG AGT AGG CAC TTC AGT GGT CGA ACC ATC 1102
Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile
355 360 365
ACC CTG CAA CCT GGA TCC CCT TGC AAC GAT TTT AGA GGT TAC TGT GAT 1150
Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp
370 375 380
GTT TTC ATG CGG TGC AGA TTA GTA GAT GCT GAT GGT CCT CTA GCT AGG 1198
VaI Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg
385 390 395
CTT AAA AAA GCA ATT TTT AGT CCA GAG CTC TAT GAA AAC ATT GCT GAA 1246
Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu
400 405 410 415
TGG ATT GTG GCT CAT TGG TGG GCA GTA TTA CTT ATG GGA ATT GCT CTG 1294
Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly Ile Ala Leu
420 425 430
ATC ATG CTA ATG GCT GGA TTT ATT AAG ATA TGC AGT GTT CAT ACT CCA 1342
Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro
435 440 445
AGT AGT AAT CCA AAG TTG CCT CCT CCT AAA CCA CTT CCA GGC ACT TTA 1390
Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu
450 455 460
AAG AGG AGG AGA CCT CCA CAG CCC ATT CAG CAA CCC CAG CGT CAG CGG 1438
Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg
465 470 475
CCC CGA GAG AGT TAT CAA ATG GGA CAC A:'~ AGA CGC TAA CTGCAGCTTT 1487
Pro Arg Glu Ser Tyr Gln Met Gly His uA~_ Arg Arg
480 485 a;0
TGCCTTGGTT CTTCCTAGTG CCTACAATGG GA~w,".":'.'A CTCCAAAGAG AAACCTATTA 1547
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
AGTCATCATC TCCAAACTAA ACCCTCACAA GTAACAGTTG AAGAAAAAAT GGCAAGAGAT 1607
CATATCCTCA GACCAGGTGG AATTACTTAA ATTTTAAAGC CTGAAAATTC CAATTTGGGG 1667
5
GTGGGAGGTG GAAAAGGAAC CCAATTTTCT TATGAACAGA TATTTTTAAC TTAATGGCAC 1727
AAAGTCTTAG AATATTATTA TGTGCCCCGT GTTCCCTGTT CTTCGTTGCT GCATTTTCTT 1787
'IO CACTTGCAGG CAAACTTGGC TCTCAATAAA CTTTTCG 1824
(2) INFORMATION FOR SEQ ID N0:2:
15 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 492 amino acids
(H) TYPE: amino acid
(D) TOPOLOGY: linear
20 (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser Phe Met Val Lys
25 1 5 10 15
Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp Pro Thr Asn Pro
20 25 30
3~ Phe Arg Phe Pro Asn Ile Ser Val Glu Las Phe Leu Glu Leu Asn Ser
40 45
Glu Gln Asn His Asp Asp Tyr Cys Leu A:i '.'~;: Val Phe Thr Asp Arg
50 55 50
Asp Phe Asp Asp Gly Val Leu Gly Leu :,. n :':p ~Jal Gly Ala Pro Ser
65 70 ~5 80
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
31
Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp Gly
85 90 95
Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr Gly
100 105 110
Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu Val
115 120 125
Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr Pro
130 135 140
Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile Met
145 150 155 160
Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe Ser
165 170 175
Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg Asn
180 185 190
Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly Asn Gly Met Val
195 200 205
Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp Gln Cys Lys Asp
210 215 220
Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys Leu
225 230 235 240
Lys Pro Gly Lys,Gln Cys Ser Pro Ser ~:~ ~~ly Pro Cys Cys Thr Ala
245 _ ~., 255
Gln Cys Ala Phe Lys Ser Lys Ser Glu W:s =ys Arg Asp Asp Ser Asp
260 265 270
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
32
Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro Ala
275 280 285
Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln Val
290 295 300
Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly Leu
305 310 315 320
Glu Giu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu Leu
325 330 335
Cys His Val Cys Cars Met Lys Lys Met Asp Pro Ser Thr Cys Ala Ser
340 345 350
Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile Thr
355 360 365
Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp Val
370 375 380
Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg Leu
3B5 390 395 400
Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn Ile Ala Glu Trp
405 410 415
Ile Val Ala His Trp Txp Ala Val Leu Leu Met Gly Ile Ala Leu Ile
420 425 430
Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro Ser
435 440 445
Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu Lys
450 455 460
Arg Arg Arg Pro Pro Gln Pro ile Gln Gln Pro Gln Arg Gln Arg Pro
CA 02281085 1999-08-10
WO 98/37092 PCTNS98/03490
33
465 470 475 480
Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg
485 490
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2763 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 17..2419
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GGCGGCGGCA CGGAAG ATG GTG TTG CTG AGA GTG TTA ATT CTG CTC CTC 99
Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu
495 500
TCC TGG GCG GCG GGG ATG GGA GGT CAG TAT GGG AAT CCT TTA AAT AAA 97
Ser Trp Ala Ala Gly Met Gly Gly Gln Tyr Giy Asn Pro Leu Asn Lys
3~ 505 510 515
TAT ATC AGA CAT TAT GAA GGA TTA TCT TA: AAT GTG GAT TCA TTA CAC 145
Tyr Ile Arg His Tyr Glu Gly Leu Ser '.~~r :,sn 'Jal Asp Ser Leu His
520 525 ~.30 535
CAA AAA CAC CAG CGT GCC AAA AGA GCA ~.~ '.':'A CAT GAA GAC CAA TTT 193
Gln Lys His Gln Arg Ala Lys Arg Ala ~:~: ,er His Glu Asp Gln Phe
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
34
540 545 550
TTA CGT CTA GAT CAT GCC CAT CAT TTC AAC CTA CGA 241
TTC GGA AGA ATG
Leu Arg Leu Asp His Ala His His Phe Asn Leu Arg
Phe Gly Arg Met
555 560 565
AAG AGG GAC ACT CTT TTC AGT TTT AAA GTA GAA ACA 289
TCC GAT GAA TCA
Lys Arg Asp Thr Leu Phe Ser Phe Lys Val Glu Thr
Ser Asp Glu Ser
570 575 580
AAT AAA GTA CTT TAT GAT ACC ATT TAC ACT GGA CAT 337
GAT TCT CAT ATT
Asn Lys Val Leu Tyr Asp Thr Ile Tyr Thr Gly His
Asp Ser His Ile
585 590 595
TAT GGT GAA GAA AGT TTT AGC TCT GTT ATT GAT GGA 385
GGA CAT GGG AGA
Tyr Gly Glu Glu Ser Phe Ser Ser Val Ile Asp Gly
Gly His Gly Arg
600 605 610 615
TTT GAA GGA TTC CAG ACT CGT ACA TTT TAT GTT GAG 433
ATC GGT GGC CCA
Phe Glu Gly Phe Gln Thr Arg Thr Phe Tyr Val Glu
Ile Gly Gly Pro
620 625 630
GCA GAG AGA TAT AAA GAC CGA CCA TTT CAC TCT GTC 481
ATT ACT CTG ATT
Ala Glu Arg Tyr Lys Asp Arg Pro Phe His Ser Val
Ile Thr Leu Ile
635 640 645
TAT CAT GAA GAT ATT AGT GAA AAA CTG AGG CTT AGA 529
GAT AGG CTT AAA
Tyr His Glu Asp Ile Ser Glu Lys Leu Arg Leu Arg
Asp Arg Leu Lys
650 655 660
CTT ATG TCA CTT TTG TGG ACC TGT TTA CCC TGT GCT 577
GAG TCC T.~T CTT
Leu Met Ser Leu Leu Trp Thr Cys Leu Pro Cys Ala
Glu Ser Cys Leu
665 670 675.
CTG CTT CAC TCA AAG AAA GCT '._'." CAC TGC CTT 625
TGG GTA H:.:' TAC TTC
Leu Leu His Ser Lys Lys Ala ..__- His Cys Leu
Trp Val :,.,.. Tyr Phe
680 685 ~i0 695
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
AAG GAT TTC TGG GGC TTT TCT GAA ATC TAC TAT CCC CAT AAA TAC GGT 673
Lys Asp Phe Trp Gly Phe Ser Glu Ile Tyr Tyr Pro His Lys Tyr Gly
700 705 710
5
CCT CAG GGC GGC TGT GCA GAT CAT TCA GTA TTT GAA AGA ATG AGG AAA 721
Pro Gln Gly Gly Cys Ala Asp His Ser Val Phe Glu Arg Met Arg Lys
715 720 725
1O TAC CAG ATG ACT GGT GTA GAG GAA GTA ACA CAG ATA CCT CAA GAA GAA 769
Tyr Gln Met Thr Gly Val Glu Glu Val Thr Gln Ile Pro Gln Glu Glu
730 735 740
CAT GCT GCT AAT GGT CCA GAA CTT CTG AGG AAA AGA CGT ACA ACT TCA g17
15 His Ala Ala Asn Gly Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser
745 750 755
GCT GAA AAA AAT ACT TGT CAG CTT TAT ATT CAG ACT GAT CAT TTG TTC 865
Ala Glu Lys Asn Thr Cys Gln Leu Tyr Ile Gln Thr Asp His Leu Phe
2O 760 765 770 775
TTT AAA TAT TAC GGA ACA CGA GAA GCT GTG ATT GCC CAG ATA TCC AGT 913
Phe Lys Tyr Tyr Gly Thr Arg Glu Ala Val :le Ala Gln Ile Ser Ser
780 785 790
CAT GTT AAA GCG ATT GAT ACA ATT TAC CAG ACC ACA GAC TTC TCC GGA 961
His Val Lys Ala Ile Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly
795 800 805
3O ATC CGT AAC ATC AGT TTC ATG GTG AAA ;:~' ~,:'A AGA ATC AAT ACA ACT 1009
Ile Arg Asn Ile Ser Phe Met Val Lys Ara :.e Arg Ile Asn Thr Thr
810 815 g20
GCT GAT GAG AAG GAC CCT ACA AAT CC'.' " ' ,. '""'C CCA AAT ATT AGT 1057
Ala Asp Glu Lys Asp Pro Thr Asn Pry a ":o she Pro Asn Ile Ser
825 830 935
CA 02281085 1999-08-10
WO 98/37092 PCT/tJS98/03490
36
GTG GAG AAG TTT CTG GAA TTG AAT CAG AAT CAT GAT GAC 1105
TCT GAG TAC
Val Glu Lys Phe Leu Glu Leu Asn Gln Asn His Asp Asp
Ser Glu Tyr
840 845 850 855
TGT TTG GCC TAT GTC TTC ACA GAC TTT GAT GAT GGC GTA 1153
CGA GAT CTT
Cys Leu Ala Tyr Val Phe Thr Asp Phe Asp Asp Gly Val
Arg Asp Leu
860 865 g70
GGT CTG GCT TGG GTT GGA GCA CCT AGC TCT GGA GGA ATA 1201
TCA GGA TGT
Gly Leu Ala Trp Val Gly Ala Pro Ser Ser Gly Gly Ile
Ser Gly Cys
875 880 885
GAA AAA AGT AAA CTC TAT TCA GAT AAG AAG TCC TTA AAC 1249
GGT AAG ACT
Glu Lys Ser Lys Leu Tyr Ser Asp Lys Lys Ser Leu Asn
Gly Lys Thr
15890 895 900
GGA ATT ATT ACT GTT CAG AAC TAT CAT GTA CCT CCC AAA 1297
GGG TCT GTC
Gly Ile Ile Thr Val Gln Asn Tyr His Val Pro Pro Lys
Gly Ser Val
905 910 915
2~
TCT CAC ATT ACT TTT GCT CAC GAA CAT AAC TTT GGA TCC 1345
GTT GGA CCA
Ser His Ile Thr Phe Ala His Glu His Asn Phe Gly Ser
Val Gly Pro
920 925 930 935
2~JCAT GAT TCT GGA ACA GAG TGC ACA GAA TCT AAG AAT TTG 1393
CCA GGA GGT
His Asp Ser Gly Thr Glu Cys Thr Glu Ser Lys Asn Leu
Pro Gly Gly
940 945 950
CAA AAA GAA AAT GGC AAT TAC ATC ATG TAT GCA AGA GCA ACA TCT GGG 1441
3~ Gln Lys Glu Asn Gly Asn Tyr Ile Met '.'~fr Ala Arg Ala Thr Ser Gly
955 960 965
GAC AAA CTT AAC AAC AAT AAA TTC TCA ".". -,T AGT ATT AGA AAT ATA 1489
Asp Lys Leu Asn Asn Asn Lys Phe Se: ._ . ~~,~s Ser Ile Arg Asn Ile
35 970 975 980
AGC CAA GTT CTT GAG AAG AAG AGA AAC A~,_ :',, :'TT GTT GAA TCT GGC 1537
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
37
Ser Gln Val Leu Glu Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly
985 990 995
CAA CCT ATT TGT GGA AAT GGA ATG GTA GAA CAA GGT GAA GAA TGT GAT 1585
Gln Pro Ile Cys Gly Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp
1000 1005 1010 1015
TGT GGC TAT AGT GAC CAG TGT AAA GAT GAA TGC TGC TTC GAT GCA AAT 1633
Cys Gly Tyr Ser Asp Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn
1020 1025 1030
CAA CCA GAG GGA AGA AAA TGC AAA CTG AAA CCT GGG AAA CAG TGC AGT 1681
Gln Pro Glu Gly Arg Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser
1035 1040 1045
CCA AGT CAA GGT CCT TGT TGT ACA GCA CAG TGT GCA TTC AAG TCA AAG 1729
Pro Ser Gln Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys
1050 1055 1060
ZO TCT GAG AAG TGT CGG GAT GAT TCA GAC TGT GCA AGG GAA GGA ATA TGT 1777
Ser Glu Lys Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys
1065 1070 1075
AAT GGC TTC ACA GCT CTC TGC CCA GCA TCT GAC CCT AAA CCA AAC TTC 1825
Asn Gly Phe Thr Ala Leu Cars Pro Ala Ser Asp Pro Lys Pro Asn Phe
1080 1085 1090 1095
ACA GAC TGT AAT AGG CAT ACA CAA GTG TGC ATT AAT GGG CAA TGT GCA 1873
Thr Asp Cys Asn Arg His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala
3~ 1100 1105 1110
GGT TCT ATC TGT GAG AAA TAT GGC TTA GAG GAG TGT ACG TGT GCC AGT 1921
Gly Ser Ile Cys Glu Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser
1115 1120 1125
TCT GAT GGC AAA GAT GAT AAA GAA TTA ~;~C CAT GTA TGC TGT ATG AAG 1969
Ser Asp Gly Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
38
1130 1135 1140
AAA ATG GAC CCA TCA ACT TGT GCC AGT ACA GGG TCT GTG CAG TGG AGT 2017
Lys Met Asp Pro Ser Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser
1145 1150 1155
AGG CAC TTC AGT GGT CGA ACC CTGCAA CCTTGC 2065
ATC ACC CCT
GGA
TCC
Arg His Phe Ser Gly Arg Thr LeuGln Gly ProCys
Ile Thr Pro Ser
1160 1165 1170 1175
AAC GAT TTT AGA GGT TAC TGT TTCATG TGC TTAGTA 2113
GAT GTT CGG AGA
Asn Asp Phe Arg Gly Tyr Cys PheMet Cys LeuVal
Asp Va1 Arg Arg
1180 1185 1190
GAT GCT GAT GGT CCT CTA GCT AAAAAA ATT AGTCCA 2161
AGG CTT GCA TTT
Asp Ala Asp Gly Pro Leu Ala LysLys Ile SerPro
Arg Leu Ala Phe
1195 1200 1205
GAG CTC TAT GAA AAC ATT GCT ATTGTG CAT TGGGCA 2209
GAA TGG GCT TGG
Glu Leu Tyr Glu Asn Ile Ala IleVal His TrpAla
Glu Trp Ala Trp
1210 1215 1220
GTA TTA CTT ATG GGA ATT GCT ATGCTA GCT TTTATT 2257
CTG ATC ATG GGA
Val Leu Leu Met Gly Ile Ala MetLeu Ala PheIle
Leu Ile Met Gly
1225 1230 1235
AAG ATA TGC AGT GTT CAT ACT AGTAAT AAG CCTCCT 2305
CCA AGT CCA TTG
Lys Ile Cys Ser Val His Thr SerAsn Lys ProPro
Pro Ser Pro Leu
1240 1245 1250 1255
CCT AAA CCA CTT CCA GGC ACT A~~:.AGG CCT CAGCCC 2353
TTA AAG AGA CCA
Pro Lys Pro Leu Pro Gly Thr A:-~Arg Pro GlnPro
Leu Lys Arg Pro
1260 :_> 1270
ATT CAG CAA CCC CAG CGT CAG CGG CCC "::. ':,~ :,:,T TAT CAA ATG GGA 2401
Ile Gln Gln Pro Gln Arg Gln Arg Pro A:o ~:u 3er Tyr Gln Met Gly
1275 128~. 1285
CA 02281085 1999-08-10
R'O 98/3'1092 PCT/US98/03490
39
CAC ATG AGA CGC T AACTGCAGCT TTTGCCTTGG TTCTTCCTAG TGCCTACAAT 2454
His Met Arg Arg
1290
GGGAAAACTT CACTCCAAAG AGAAACCTAT TAAGTCATCA TCTCCAAACT AAACCCTCAC 2514
AAGTAACAGT TGAAGAAAAA ATGGCAAGAG ATCATATCCT CAGACCAGGT GGAATTACTT 2574
1O AAATTTTAAA GCCTGAAAATTCCAATTTGGGGGTGGGAGGTGGAAAAGGAACCCAATTTT2634
CTTATGAACA GATATTTTTAACTTAATGGCACAAAGTCTTAGAATATTATTATGTGCCCC2694
GTGTTCCCTG TTCTTCGTTG CTGCATTTTC TTCACTTGCA GGCAAACTTG GCTCTCAATA 2754
AACTTTTCG 2763
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 799 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly
1 5 ~~ 15
Met Gly Gly Gln Tyr Gly Asn Pro Leu :.s~~. :.r s Tyr Ile Arg His Tyr
20 ~S 30
Glu Gly Leu Ser Tyr Asn Val Asp Se: :. _ ..:s ~:n Lys His Gln Arg
35 40 45
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe
55 60
His Ala His Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser
65 70 75 80
Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp
85 90 95
Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly
100 105 110
Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile
115 120 125
Gln Thr Arg Gly Gay Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile
130 135 140
Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp
145 150 155 160
Ile Ser Glu Arg Leu Lys Leu Arg Leu Arg Lys Leu Met Ser Leu Glu
165 170 175
Leu Trp Thr Ser Cys Cys Leu Pro Cys Ala Leu Leu Leu His Ser Trp
180 185 190
Lys Lys Ala Val Asn Ser His Cys Leu Tyr Phe Lys Asp Phe Trp Gly
195 200 205
Phe Ser Glu Ile Tyr Tyr Pro His Lys '.', ~:y Pro Gln Gly Gly Cys
210 215 220
Ala Asp His Ser Val Phe Glu Arg Men ;,: ~ :., s '.'yr Gln Met Thr Gly
225 230 _?~ 240
CA 02281085 1999-08-10
WO 98/37092 PCT/US98103490
41
Val Glu Glu Va1 Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn G1y
245 250 255
Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser Ala Glu Lys Asn Thr
260 265 270
Cys Gln Leu Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly
275 280 285
Thr Arg Glu Ala Val Ile Ala Gln Ile Ser Ser His Val Lys Ala Ile
290 295 300
Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn Ile Ser
305 310 315 320
Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu Lys Asp
325 330 335
Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu
340 345 350
Glu Leu Asn Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val
355 360 365
Phe Thr Asp Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val
370 375 380
Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu
385 390 395 400
Tyr Ser Asp Gly Lys Lys Lys Ser Leu As..~,':hr Gly Ile Ile Thr Val
405 .i:~ 415
Gln Asn Tyr Gly Ser His Val Pro Pr. :., ':ai Ser His Ile Thr Phe
420 4430
Ala His Glu Val Gly His Asn Phe G1,~ - . .:o ais Asp Ser Gly Thr
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
42
435 440 445
Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly
450 455 460
Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn
465 470 475 480
Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser G1n Val Leu Glu
485 490 495
Lys Lys Arg Asn Asn Cys Phe Val Glu Ser Gly Gln Pro Ile Cys Gly
500 505 510
Asn Gly Met Val Glu Gln Gly Glu Glu Cys Asp Cys Gly Tyr Ser Asp
515 520 525
Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg
530 535 540
Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro
545 550 555 560
Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg
565 570 575
Asp Asp Ser Asp Gars Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala
580 5B5 590
Leu Cys Pro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg
595 600 605
His Thr Gln Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu
610 615 620
Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp
625 630 535 640
CA 02281085 1999-08-10
'GVO 98/37092 PCT/US98/03490
43
Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser
645 650 655
Thr Cys Ala Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly
660 665 670
Arg Thr Ile Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly
675 680 685
Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Va1 Asp Ala Asp Gly Pro
690 695 700
Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr Glu Asn
705 7IO 71s 7zo
Ile Ala Glu Trp Ile Val Ala His Trp Trp Ala Val Leu Leu Met Gly
725 730 735
Ile Ala Leu Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val
740 745 750
His Thr Pro Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro
755 760 765
Gly Thr Leu Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln
770 775 780
Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Giy His Met Arg Arg
785 790 '95
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 base pair
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
44
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: DNA (genomic)
(iv) ANTI-SENSE: YES
1O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
AATACCACCA TTCTCTGTTA TCCTGAGTAT GTCAATTAAA CAGTAATTTT TAATTAAGAG 60
CGGAAAAATT TTATAATACA AAGAAACATC CATATTGCAA TTTCTGTTTA CAATTGCACA 120
CAGAAGTACA GTGTACGTAA GAAATACATG TCTGCATATA ACAAGGTATG TACATTGGCA 180
AGTGATGTCT CCAATGTTGA GGTGGTCGAG CCTCCTAGCC TTGATTGGCA GTTGAAAAA 239
ZO (2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ :J .. _.
TTTTTCAACT GCCAATCAAG GCTAGGAGGC :'_.., ';,'_'. =AACATTGGA GACATCACTT 60
GCCAATGTAC ATACCTTGTT ATATGCAGAC A:'-~'.v,.""-"' "ACGTACACT GTACTTCTGT 120
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
GTGCAATTGT AAACAGAAAT TGCAATATGG ATGTTTCTTT GTATTATAAA ATTTTTCCGC 180
TCTTAATTAA AAATTACTGT TTAATTGACA TACTCAGGAT AACAGAGAAT GGTGGTATT 239
5
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 736 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:7:
AACCACTTCC AGGCACTTTAAAGAGGAGGAGACCTCCACAGCCCATTCAG CAACCCCAGC60
GTCAGCGGCC CCGAGAGAGTTATCAAATGGGACACATGAGACGCTAACTG CAGCTTTTGC120
CTTGGTTCTT CCTAGTGCCTACAATGGGAAAACTTCACTCCAAAGAGAAA CCTATTAAGT180
CATCATCTCC AAACTAAACCCTCACAAGTAACAGTTGAAGAAAAAATGGC AAGAGATCAT240
ATCCTCAGAC CAGGTGGAATTACTTAAATTTTAAAGCCTGAAAATTCCAA TTTGGGGGTG300
GGAGGTGGAA AAGGAACCCAATTTTCTTATGAA.'A:;A'.'ATTTTTAACTTA ATGGCACAAA360
GTCTTAGAAT ATTATTATGTGCCCCGTGTTC"'. ~GTTGCTGCA TTTTCTTCAC420
.'."_..
TTGCAGGCAA ACTTGGCTCTCAATAAACTT:' . :'TGAAATAAA TATATTTTTT4
., , 8
, ~;,a,A 0
TCAACTGCCA ATCAAGGCTAGGAGGCTCG:._.,. ATTGGAGACA ATCACTTGCC540
~ "."'F,A~
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
46
AATGTACATA CCTTGTTATA TGCAGACATG TATTTCTTAC GTACACTGTA CTTCTGTGTG 600
CAATTGTAAA CAGAAATTGC AATATGGATG TTTCTTTGTA TTATAAAATT TTTCCGCTCT 660
TAATTAAAAA TTACTGTTTA ATTGACATAC TCAGGATAAC AGAGAATGGT GGTATTCAGT 720
GGTTCAGACA CAGGCT 736
'IO (2) INFORMATION FOR SEQ ID NO: B:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2625 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 17..2263
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
GGCGGCGGCA CGGAAG ATG GTG TTG CTG AGA GTG TTA ATT CTG CTC CTC 49
Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu
goo gos glo
TCC TGG GCG GCG GGG ATG GGA GGT CAG ".'A".' ~GG AAT CCT TTA AAT AAA 97
Ser Trp Ala Ala Gly Met Gly Gly G1.~. ~, ~:y Asn Pro Leu Asn Lys
815 ~_ g25
TAT ATC AGA CAT TAT GAA GGA TTA TC': '. , ' :,~, ; GTG GAT TCA TTA CAC 14 5
Tyr Ile Arg His Tyr Glu Gly Leu Ser '.'. Asn Val Asp Ser Leu His
CA 02281085 1999-08-10
VVO 98/37092 PCT/US98/03490
47
83D 835 840
CAA AAA CAC CAG CGT GCC GCA TCA CAT GAA GAC CAA 193
AAA AGA GTC TTT
Gln Lys His Gln Arg Ala Ala Ser His Glu Asp Gln
Lys Arg Val Phe
845 850 855
TTA CGT CTA GAT TTC CAT GGA CAT TTC AAC CTA CGA 241
GCC CAT AGA ATG
Leu Arg Leu Asp Phe His Gly His Phe Asn Leu Arg
Ala His Arg Met
860 865 870
AAG AGG GAC ACT TCC CTT GAT TTT AAA GTA GAA ACA 289
TTC AGT GAA TCA
Lys Arg Asp Thr Ser Leu Asp Phe Lys Val Glu Thr
Phe Ser Glu Ser
875 8B0 885 890
AAT AAA GTA CTT GAT TAT TCT ATT TAC ACT GGA CAT 337
GAT ACC CAT ATT
Asn Lys Val Leu Asp Tyr Ser Ile Tyr Thr Gly His
Asp Thr His Ile
895 900 905
TAT GGT GAA GAA GGA AGT CAT TCT GTT ATT GAT GGA 385
TTT AGC GGG AGA
Tyr Gly Glu Glu Gly Ser His Ser Val Ile Asp Gly
Phe Ser Gly Arg
910 915 920
TTT GAA GGA TTC ATC CAG GGT ACA TTT TAT GTT GAG 433
ACT CGT GGC CCA
Phe Glu Gly Phe Ile Gln Gly Thr Phe Tyr Val Glu
Thr Arg Gly Pro
925 930 935
GCA GAG AGA TAT ATT AAA ACT CCA TTT CAC TCT GTC 481
GAC CGA CTG ATT
Ala Glu Arg Tyr Ile Lys Thr Pro Phe His Ser Val
Asp Arg Leu Ile
940 945 950
TAT CAT GAA GAT GAT ATT AAC TAT CCC CAT AAA TAC GGT CCT CAG GGC 529
Tyr His Glu Asp Asp Ile Asn Tyr Pro His Lys Tyr Gly Pro Gln Gly
955 96D 965 970
GGC TGT GCA GAT CAT TCA GTA TTT GAA AGA ATG AGG AAA TAC CAG ATG 577
Gly Cys Ala Asp His Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met
975 y9D 985
CA 02281085 1999-08-10
WO 98/37092 PCT/US98103490
48
ACT GGT GTA GAG GAA GTA ACA CAG ATA CCT CAA GAA GAA CAT GCT GCT 625
Thr Gly Val Glu Glu Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala
990 995 1000
AAT GGT CCA GAA CTT CTG AGG AAA AGA CGT ACA ACT TCA GCT GAA AAA 673
Asn Gly Pro Glu Leu Leu Arg Lys Arg Arg Thr Thr Ser Ala Glu Lys
1005 1010 1015
AAT ACT TGT CAG CTT TAT ATT CAG ACT GAT CAT TTG TTC TTT AAA TAT 721
Asn Thr Cys Gln Leu Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr
1020 1025 1030
TAC GGA ACA CGA GAA GCT GTG ATT GCC CAG ATA TCC AGT CAT GTT AAA 769
~5 Tyr Gly Thr Arg Glu Ala Val Ile Ala Gln Ile Ser Ser His Val Lys
1035 1040 1045 1050
GCG ATT GAT ACA ATT TAC CAG ACC ACA GAC TTC TCC GGA ATC CGT AAC 817
Ala Ile Asp Thr Ile Tyr Gln Thr Thr Asp Phe Ser Gly Ile Arg Asn
20 1055 1060 1065
ATC AGT TTC ATG GTG AAA CGC ATA AGA ATC AAT ACA ACT GCT GAT GAG 865
Ile Ser Phe Met Val Lys Arg Ile Arg Ile Asn Thr Thr Ala Asp Glu
1070 1075 1080
AAG GAC CCT ACA AAT CCT TTC CGT TTC CCA AAT ATT AGT GTG GAG AAG 913
Lys Asp Pro Thr Asn Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys
1085 1090 1095
TTT CTG GAA TTG AAT TCT GAG CAG AAT CAT ~AT GAC TAC TGT TTG GCC 961
Phe Leu Glu Leu Asn Ser Glu Gln Asn 4:s Asp Asp Tyr Cys Leu Ala
1100 1105 1110
TAT GTC TTC ACA GAC CGA GAT TTT GA'." .,. ~_ ~'."A CTT GGT CTG GCT 1009
Tyr Val Phe Thr Asp Arg Asp Phe Asp :.:::: . ':31 Leu Gly Leu Ala
1115 1120 ::~5 1130
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
49
TGG GTT GGA GCA CCT TCA GGA AGC TCT GGA GGA ATA TGT GAA AAA AGT 1057
Trp Val Gly Ala Pro Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser
1135 1140 1145
AAA CTC TAT TCA GAT GGT AAG AAG AAG TCC TTA AAC ACT GGA ATT ATT 1105
Lys Leu Tyr Ser Asp Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile
1150 1155 1160
ACT GTT CAG AAC TAT GGG TCT CAT GTA CCT CCC AAA GTC TCT CAC ATT 1153
Thr Val Gln Asn Tyr Gly Ser His Val Pro Pro Lys Val Ser His Ile
1165 1170 1175
ACT TTT GCT CAC GAA GTT GGA CAT AAC TTT GGA TCC CCA CAT GAT TCT 1201
Thr Phe Ala His Glu Val Gly His Asn Phe Gly Ser Pro His Asp Ser
lleo llas 1190
GGA ACA GAG TGC ACA CCA GGA GAA TCT AAG AAT TTG GGT CAA AAA GAA 1249
Gly Thr Glu Cys Thr Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu
1195 1200 1205 1210
AAT GGC AAT TAC ATC ATG TAT GCA AGA GCA ACA TCT GGG GAC AAA CTT 1297
Asn Gly Asn Tyr Ile Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu
1215 1220 1225
AAC AAC AAT AAA TTC TCA CTC TGT AGT ATT AGA AAT ATA AGC CAA GTT 1345
Asn Asn Asn Lys Phe Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val
1230 1235 1240
CTT GAG AAG AAG AGA AAC AAC TGT TTT GTT GAA TCT GGC CAA CCT ATT 1393
Leu Glu Lys Lys Arg Asn Asn Cys Phe :'3i Clu Ser Gly Gln Pro Ile
1245 1250 1255
TGT GGA AAT GGA ATG GTA GAA CAA GGT .=_~ ~AA TGT GAT TGT GGC TAT 1441
Cys Gly Asn Gly Met Val Glu Gln G:: .. ... Cys Asp Cys Gly Tyr
1260 1265 :270
AGT GAC CAG TGT AAA GAT GAA TGC T~:~ --~ ~. ACA AAT CAA CCA GAG 1489
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
Ser Asp Gln Cys Lys Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu
1275 1280 1285 1290
GGA AGA AAA TGC AAA CTG AAA CCT GGG AAA CAG TGC AGT CCA AGT CAA 1537
5 Gly Arg Lys Cys Lys Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln
1295 1300 1305
GGT CCT TGT TGT ACA GCA CAG TGT GCA TTC AAG TCA AAG TCT GAG AAG 1585
Gly Pro Cys Cys Thr Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys
1310 1315 1320
TGT CGG GAT GAT TCA GAC TGT GCA AGG GAA GGA ATA TGT AAT GGC TTC 1633
Cys Arg Asp Asp Ser Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe
1325 1330 1335
ACA GCT CTC TGC CCA GCA TCT GAC CCT AAA CCA AAC TTC ACA GAC TGT 1681
Thr Ala Leu Cys fro Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys
1340 1345 1350
ZO AAT CAT ACA CAA GTG TGC GGG CAA TGT TCT ATC 1729
AGG ATT AAT GCA GGT
Asn Arg His Thr Gln Val Cys Gly Gln Cys Ser Ile
Ile Asn Ala Gly
1355 1360 1365 1370
TGT GAG AAA TAT GGC TTA GAG GAG TGT ACG TGT GCC AGT TCT GAT GGC 1777
Cys Glu Lys Tyr Gly Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly
1375 1380 1385
AAA GAT GAT AAA GAA TTA TGC CAT GTA TGC TGT ATG AAG AAA ATG GAC 1825
Lys Asp Asp Lys Glu Leu Cys His Val Cys Cys Met Lys Lys Met Asp
1390 1395 1400
CCA TCA ACT TGT GCC AGT ACA GGG TC'." ~.~ ':,~ EGG AGT AGG CAC TTC 1873
Pro Ser Thr Cys Ala Ser Thr Gly Ser : ~. ~... :'rp Ser Arg His Phe
1405 1410 1415
AGT GGT CGA ACC ATC ACC CTG CAA C~_".' :~:, ." ~ ~~T TGC AAC GAT TTT 1921
Ser Gly Arg Thr Ile Thr Leu Gln " ~ , ~.~r 2ro Cys Asn Asp Phe
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
51
1420 1425 1430
AGA GGT TAC TGT GAT GTT TTC ATG CGG TGC AGA TTA GTA GAT GCT GAT 1969
Arg Giy Tyr Cys Asp Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp
1435 1440 1445 1450
GGT CCT CTA GCT AGG CTT AAA AAA GCA ATT TTT AGT CCA GAG CTC TAT 2017
Gly Pro Leu Ala Arg Leu Lys Lys Ala Ile Phe Ser Pro Glu Leu Tyr
1455 1460 1465
GAA AAC ATT GCT GAA TGG ATT CAT TGG GCA GTA TTA 2065
GTG GCT TGG CTT
Glu Asn Ile Ala Glu Trp Ile HisTrp Trp Ala Val Leu
Val Ala Leu
1470 1475 1480
ATG GGA ATT GCT CTG ATC ATG GCTGGA TTT ATT AAG ATA 2113
CTA ATG TGC
Met Gly Ile Ala Leu Ile Met AlaGly Phe Ile Lys Ile
Leu Met Cys
1485 1490 1495
AGT GTT CAT ACT CCA AGT AGT AAGTTG CCT CCT CCT AAA 2161
AAT CCA CCA
Ser Val His Thr Pro Ser Ser LysLeu Pro Pro Pro Lys
Asn Pro Pro
1500 1505 1510
CTT CCA GGC ACT TTA AAG AGG CCTCCA CAG CCC ATT CAG 2209
AGG AGA CAA
Leu Pro Gly Thr Leu Lys Arg ProPro Gln Pro Ile Gln
Arg Arg Gln
Z~J1515 1520 15251530
CCC CAG CGT CAG CGG CCC CGA GAG AGT TAT CAA ATG GGA CAC ATG AGA 2257
Pro Gln Arg Gln Arg Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg
1535 1540 1545
CGC TAA CTGCAGCTTT TGCCTTGGTT CTTCC':AG'."G CCTACAATGG GAAAACTTCA 2313
Arg
CTCCAAAGAG AAACCTATTA AGTCATCATC '."'_"'~,:,:.~".'AA ACCCTCACAA GTAACAGTTG
2373
AAGAAAAAAT GGCAAGAGAT CATATCCTCA GAC':..;.:,'::,~:, AATTACTTAA ATTTTAAAGC 2433
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
52
CTGAAAATTC CAATTTGGGG GTGGGAGGTG GAAAAGGAAC CCAATTTTCT TATGAACAGA 2493
TATTTTTAAC TTAATGGCAC AAAGTCTTAG AATATTATTA TGTGCCCCGT GTTCCCTGTT 2553
CTTCGTTGCT GCATTTTCTT CACTTGCAGG CAAACTTGGC TCTCAATAAA CTTTTACCAC 2613
P~AAAAAAAAA AA
2625
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 749 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
2O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Met Val Leu Leu Arg Val Leu Ile Leu Leu Leu Ser Trp Ala Ala Gly
1 5 10 15
Met Gly Gly Gln Tyr Gly Asn Pro Leu Asn Lys Tyr Ile Arg His Tyr
20 25 30
Glu Gly Leu Ser Tyr Asn Val Asp Ser Leu His Gln Lys His Gln Arg
40 45
Ala Lys Arg Ala Val Ser His Glu Asp Gln Phe Leu Arg Leu Asp Phe
50 55 60
His Ala His Gly Arg His Phe Asn Leu Arg Met Lys Arg Asp Thr Ser
65 70 75 80
Leu Phe Ser Asp Glu Phe Lys Val Glu Thr Ser Asn Lys Val Leu Asp
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
53
85 90 95
Tyr Asp Thr Ser His Ile Tyr Thr Gly His Ile Tyr Gly Glu Glu Gly
100 105 110
Ser Phe Ser His Gly Ser Val Ile Asp Gly Arg Phe Glu Gly Phe Ile
115 120 125
Gln Thr Arg Gly Gly Thr Phe Tyr Val Glu Pro Ala Glu Arg Tyr Ile
130 135 140
Lys Asp Arg Thr Leu Pro Phe His Ser Val Ile Tyr His Glu Asp Asp
145 150 155 160
Ile Asn Tyr Pro His Lys Tyr Gly Pro Gln Gly Gly Cys Ala Asp His
165 170 175
Ser Val Phe Glu Arg Met Arg Lys Tyr Gln Met Thr Gly Val Glu Glu
180 185 190
2~
Val Thr Gln Ile Pro Gln Glu Glu His Ala Ala Asn Gly Pro Glu Leu
195 200 205
Leu Arg Lys Arg Arg Thr Thr Ser Ala G1u Lys Asn Thr Cys G1n Leu
zlo 21s 22a
Tyr Ile Gln Thr Asp His Leu Phe Phe Lys Tyr Tyr Gly Thr Arg Glu
225 230 235 240
3~ Ala Val Ile Ala Gln Ile Ser Ser His ':a: ~ys Ala Ile Asp Thr Ile
245 :50 255
Tyr Gln Thr Thr Asp Phe Ser Gly Ile :,:~ Asn Ile Ser Phe Met Val
260 265 270
Lys Arg Ile Arg Ile Asn Thr Thr Ala ~:~c ~:u :.ys Asp Pro Thr Asn
275 280 285
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
54
Pro Phe Arg Phe Pro Asn Ile Ser Val Glu Lys Phe Leu Glu Leu Asn
290 295 300
Ser Glu Gln Asn His Asp Asp Tyr Cys Leu Ala Tyr Val Phe Thr Asp
305 310 315 320
Arg Asp Phe Asp Asp Gly Val Leu Gly Leu Ala Trp Val Gly Ala Pro
325 330 335
Ser Gly Ser Ser Gly Gly Ile Cys Glu Lys Ser Lys Leu Tyr Ser Asp
340 345 350
Gly Lys Lys Lys Ser Leu Asn Thr Gly Ile Ile Thr Val Gln Asn Tyr
355 360 365
Gly Ser His Val Pro Pro Lys Val Ser His Ile Thr Phe Ala His Glu
370 375 380
2~ Val Gly His Asn Phe Gly Ser Pro His Asp Ser Gly Thr Glu Cys Thr
385 390 395 400
Pro Gly Glu Ser Lys Asn Leu Gly Gln Lys Glu Asn Gly Asn Tyr Ile
405 410 415
Met Tyr Ala Arg Ala Thr Ser Gly Asp Lys Leu Asn Asn Asn Lys Phe
420 425 430
Ser Leu Cys Ser Ile Arg Asn Ile Ser Gln Val Leu Glu Lys Lys Arg
435 440 445
Asn Asn Cys Phe Val Glu Ser Gly Gln r.-~ 'le Cys Gly Asn Gly Met
450 455 460
Val Glu Gln Gly Glu Glu Cys Asp Cys ~ . - Ser Asp Gln Cys Lys
465 470 ~ ,5 480
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
Asp Glu Cys Cys Phe Asp Ala Asn Gln Pro Glu Gly Arg Lys Cys Lys
485 490 495
Leu Lys Pro Gly Lys Gln Cys Ser Pro Ser Gln Gly Pro Cys Cys Thr
5 500 505 510
Ala Gln Cys Ala Phe Lys Ser Lys Ser Glu Lys Cys Arg Asp Asp Ser
515 520 525
10 Asp Cys Ala Arg Glu Gly Ile Cys Asn Gly Phe Thr Ala Leu Cys Pro
530 535 540
Ala Ser Asp Pro Lys Pro Asn Phe Thr Asp Cys Asn Arg His Thr Gln
545 550 555 560
Val Cys Ile Asn Gly Gln Cys Ala Gly Ser Ile Cys Glu Lys Tyr Gly
565 570 575
Leu Glu Glu Cys Thr Cys Ala Ser Ser Asp Gly Lys Asp Asp Lys Glu
580 585 590
Leu Cys His Val Cys Cys Met Lys Lys Met Asp Pro Ser Thr Cys Ala
595 600 605
Ser Thr Gly Ser Val Gln Trp Ser Arg His Phe Ser Gly Arg Thr Ile
610 615 620
Thr Leu Gln Pro Gly Ser Pro Cys Asn Asp Phe Arg Gly Tyr Cys Asp
625 630 635 640
Val Phe Met Arg Cys Arg Leu Val Asp Ala Asp Gly Pro Leu Ala Arg
645 « J 655
Leu Lys Lys Ala Ile Phe Ser Pro Glu ~~. '.'~,- Flu Asn Ile Ala Glu
s6o 66s s7o
Trp Ile Val Ala His Trp Trp Ala ~.. _ ~.~~ :..eu Met Gly Ile Ala Leu
CA 02281085 1999-08-10
WO 98/37092 PCT/US98/03490
56
675 680 685
Ile Met Leu Met Ala Gly Phe Ile Lys Ile Cys Ser Val His Thr Pro
690 695 700
Ser Ser Asn Pro Lys Leu Pro Pro Pro Lys Pro Leu Pro Gly Thr Leu
705 710 715 720
Lys Arg Arg Arg Pro Pro Gln Pro Ile Gln Gln Pro Gln Arg Gln Arg
725 730 735
Pro Arg Glu Ser Tyr Gln Met Gly His Met Arg Arg
740 745
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AGCCTGTGTC 10
(2) INFORMATION FOR SEQ ID NO:11:
3O (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genom:~
CA 02281085 1999-08-10
WO 98/37092 PCT/~JS98/03490
57
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AGCCTGTGTC TGAACCACT I9
