Note: Descriptions are shown in the official language in which they were submitted.
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HUMAN DICKKOPF-RELATED PROTEIN AND NUCLEIC ACID
MOLECULES AND USES THEREFOR
Background of the Invention
Secreted proteins play an integral role in the formation, differentiation, and
maintenance of cells in multicellular organisms. For instance, secretory
proteins are
known in the art to be involved in signaling between cells which are not in
direct
contact. Such secreted signaling molecules are particularly important in the
development of vertebrate tissue during embryogenesis as well as in the
maintenance of
the differentiated state of adult tissues. For example, inductive interactions
that occur
between neighboring cell layers and tissues in the developing embryo are
largely
dependent on the existence and regulation of secreted signaling molecules. In
inductive
interactions, biochemical signals secreted by one cell population influence
the
developmental fate of a second cell population, typically by altering the fate
of the
second cell population. For example, the Wnt proteins are now recognized as
one of the
major families of developmentally important signaling molecules in organisms
ranging
from Drosophila to mice.
The Wnt gene family encode a large class of secreted proteins related to the
Intl /Wntl proto-oncogene and Drosophila wingless ("Wg"), a Drosophila Wntl
homologue, (Cadigan et al. (1997) Genes & Development 11:3286-3305). Wnts are
expressed in a variety of tissues and organs and are required for many
developmental
processes, including segmentation in Drosophila, endoderm development in
Caenorhabditis elegans, establishment of limb polarity, neural crest
differentiation,
kidney morphogenesis, sex determination, and brain development in mammals
(reviewed in Parr and McMahon (1994) Curr. Opinion Genetics & Devel. 4:523-
528;
Cadigan and Nusse, supra).
Recent studies in diverse organisms have led to identification of several
components of the Wnt signal transduction pathway in responding cells (Cadigan
and
Nusse, supra). Wnt signals are transduced by the Frizzled ("Fz") family of
seven
transmembrane domain receptors (Bhanot et al. (1996) Nature 382:225-230). The
resulting signal leads to the activation of the cytoplasmic protein
Dishevelled (Dsh) and
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stabilization of Armadillo/13-catenin (Perrimon (1994) Cell 76:781-784).
Negative
regulators of the Wnt pathway include glycogen synthase kinase 3 (GSK3)/shaggy
(Perrimon, supra), the tumor suppressor gene product adenomatous polyposis
coli
(APC) (Gumbiner (1997) Curr. Biol. 7:R443-436) and a novel protein, called
Axin
(Zeng et al. (1997) Cell 90:181-192). In the absence of a Wnt ligand, these
proteins
promote phosphorylation and then degradation off3-catenin, whereas Wnt
signaling
inactivates GSK3, thus preventing (3-catenin degradation. As a result, (3-
catenin is
translocated to the nucleus, where it forms a complex with TCF transcription
factors and
activates target gene expression (Cadigan and Nusse, supra). Deregulation of
this
pathway can lead to carcinogenesis (reviewed by Gumbiner, supra), emphasizing
the
long-recognized connection between Wnts, normal development and cancer. This
connection has been further established recently with the identification the c-
Myc proto-
oncogene as a target of Wnt signaling (He et al. (1998) Science 281:1509-
3512).
While the outcome of Wnt signaling may be influenced by multiple intracellular
regulatory mechanisms, recent studies have identified several classes of
secreted factors
which can modulate Wnt action outside of the cell. These include Cerberus, a
secreted
Wnt inhibitor implicated in head development (Bouwmeester et al. (1996) Nature
382:595-601), and a family of proteins related to the extracellular domain of
Frizzled.
These Frizzled-related proteins ("FRPs") (Rattner et al. (1997) Proc. Natl.
Acad. Sci.
USA 94:2859-2863), also known as secreted apoptosis-related proteins
("SARPs"), are
encoded by several independently discovered genes including FrzA/FRP1,
SDF5/FRP2,
FrzB/FRP3, FRP4 and Sizzled (Melkonyan etal. (1997) Proc. Natl. Acad. Sci. USA
94:13636-13641; Finch et al. (1997) Proc. Natl. Acad Sci. USA 94:6770-6775;
Wang et
al. (1997) Cell 88:747-766; Leyns etal. (1997) Cell 88:747-756; Mayr et al.
(1997)
Mech. Dev. 63:109-325; and Salic etal. (1997) Development 124:4739-4748).
These
proteins inhibit the ability of Xvvnt8 to induce a secondary axis in frog
embryos (for
review see Zorn (1997) Curr. Biol. 7:R501-504), and are thought to compete for
binding
of Wnt ligands to the Frizzled receptors. Data on binding of certain FRPs to
Xvvnt8
(Wang et al., (1997) Biochem. Biophys. Res. Comm. 236:502-504; and Leyns et
al.,
supra) and Wg corroborate this notion (Rattner et al., supra).
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It is now recognized that many of these families of signaling molecules have a
dual role to play in both the development of an organism as well as in
promoting or
maintaining the differentiated state of tissues in the adult animal.
Furthermore, major
families of signaling molecules have been implicated in controlling
proliferation of cells
in mature adult tissue, for example, during normal cell turnover in the adult
organism as
well as in tissue regeneration activated as a result of damage to the adult
tissue. Given
the important role of these signalling molecules such as the Wnts and FRPs in
both
developing and adult tissues, there exists a need for identifying novel
modulators of such
molecules for use in regulating a variety of cellular processes.
Summary of the Invention
The present invention is based, at least in part, on the discovery of nucleic
acid
molecules which encode a novel family of secreted human proteins, referred to
herein as
the human Dickkopf proteins or "hDkks" (formerly referred to as the "Cysteine-
Rich
Secreted Proteins", "CRSPs", "CRISPYs", or "CRSP proteins). The Dkk molecules
of
the present invention are useful as modulating agents in regulating a variety
of cellular
processes. Accordingly, in one aspect, this invention provides isolated
nucleic acid
molecules encoding Dkk proteins or biologically active portions thereof, as
well as
nucleic acid fragments suitable as primers or hybridization probes for the
detection of
Dick-encoding nucleic acids. In another aspect, this invention provides
isolated nucleic
acid molecules encoding Dkk-related proteins (e.g., Soggy proteins) or
biologically
active portions thereof, as well as nucleic acid fragments suitable as primers
or
hybridization probes for the detection of Dkk- or Soggy-encoding nucleic
acids.
In one embodiment, a Dkk nucleic acid molecule is 60% homologous to the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, the nucleotide sequence
of
the DNA insert of the plasmid deposited with ATCC as Accession Number 98452 or
complement thereof. In yet another embodiment, a Dkk nucleic acid molecule is
80%
homologous to the nucleotide sequence shown in SEQ ID NO:4, SEQ ID NO:6, or a
complement thereof. In yet another embodiment, a Dick nucleic acid molecule is
60%
homologous to the nucleotide sequence shown in SEQ ID NO:7, SEQ ID NO:9, or
the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
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Accession Number 98633, or a complement thereof. In yet another embodiment, a
Dkk
nucleic acid molecule is 85% homologous to the nucleotide sequence shown in
SEQ ID
NO:7, SEQ ID NO:9, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or a complement thereof. In yet
another embodiment, a Dkk nucleic acid molecule is 70% homologous to the
nucleotide
sequence shown in SEQ ID NO:20, SEQ ID NO:22 or a complement thereof. In yet
another embodiment, a nucleic acid molecule of the present invention (e.g., a
Dkk-related
nucleic acid molecule) is 90% homologous to the nucleotide sequence shown in
SEQ ID
NO:13, SEQ ID NO:15, or a complement thereof.
In a preferred embodiment, an isolated Dkk nucleic acid molecule has the
nucleotide sequence shown SEQ ID NO:3, or a complement thereof. In another
embodiment, a Dkk nucleic acid molecule further comprises nucleotides 1-37 of
SEQ ID
NO: 1. In yet another preferred embodiment, a Dkk nucleic acid molecule
further
comprises nucleotides 1088-2479 of SEQ ID NO: 1. In another preferred
embodiment,
an isolated Dkk nucleic acid molecule has the nucleotide sequence shown in SEQ
ID
NO:l.
In another preferred embodiment, an isolated Dick nucleic acid molecule has
the
nucleotide sequence shown SEQ ID NO:6, or a complement thereof. In another
embodiment, a Dick nucleic acid molecule further comprises nucleotides 1-124
of SEQ
ID NO:4. In yet another preferred embodiment, a Dick nucleic acid molecule
further
comprises nucleotides 797-848 of SEQ ID NO:4. In another preferred embodiment,
an
isolated Dick nucleic acid molecule has the nucleotide sequence shown in SEQ
ID NO:4.
In another preferred embodiment, an isolated Dick nucleic acid molecule has
the
nucleotide sequence shown SEQ ID NO:9, or a complement thereof. In another
embodiment, a Dkk nucleic acid molecule further comprises nucleotides 1-108 of
SEQ
ID NO:7. In yet another preferred embodiment, a Dick nucleic acid molecule
further
comprises nucleotides 907-1536 of SEQ ID NO:7. In another preferred
embodiment, an
isolated Dick nucleic acid molecule has the nucleotide sequence shown in SEQ
ID NO:7.
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In another preferred embodiment, an isolated Dkk nucleic acid molecule has the
nucleotide sequence shown SEQ ID NO:22, or a complement thereof. In another
embodiment, a Dkk nucleic acid molecule further comprises nucleotides 1-723 of
SEQ
ID NO:20. In yet another preferred embodiment, a Dkk nucleic acid molecule
further
comprises nucleotides 1501-3687 of SEQ ID NO:20. In yet another preferred
embodiment, an isolated Dkk nucleic acid molecule has the nucleotide sequence
shown
in SEQ ID NO:20.
In another preferred embodiment, an isolated nucleic acid molecule of the
present invention (e.g., a Dkk-related nucleic acid molecule) has the
nucleotide sequence
shown SEQ ID NO:15, or a complement thereof. In another embodiment, a nucleic
acid
molecule further comprises nucleotides 1-74 of SEQ ID NO:13. In yet another
preferred
embodiment, a nucleic acid molecule further comprises nucleotides 801-928 of
SEQ ID
NO:13. In yet another preferred embodiment, an isolated nucleic acid molecule
has the
nucleotide sequence shown in SEQ ID NO:13.
In another embodiment, a Dkk nucleic acid molecule includes a nucleotide
sequence encoding a protein having an amino acid sequence sufficiently
homologous to
the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID
NO:21. In another preferred embodiment, a Dkk nucleic acid molecule includes a
nucleotide sequence encoding a protein having an amino acid sequence at least
60%
homologous to the amino acid sequence of SEQ ID NO:2. In yet another preferred
embodiment, a Dkk nucleic acid molecule includes a nucleotide sequence
encoding a
protein having an amino acid sequence at least 60% homologous to the amino
acid
sequence of SEQ ID NO:5. In yet another preferred embodiment, a Dkk nucleic
acid
molecule includes a nucleotide sequence encoding a protein having an amino
acid
sequence at least 60% homologous to the amino acid sequence of SEQ ID NO:8. In
yet
another preferred embodiment, a Dkk nucleic acid molecule includes a
nucleotide
sequence encoding a protein having an amino acid sequence at least 75%
homologous to
the amino acid sequence of SEQ ID NO:8. In yet another preferred embodiment, a
Dkk
nucleic acid molecule includes a nucleotide sequence encoding a protein having
an
amino acid sequence at least 65% homologous to the amino acid sequence of SEQ
ID
NO:21. In another embodiment, a nucleic acid molecule of the present invention
(e.g., a
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Dkk-related nucleic acid molecule) includes a nucleotide sequence encoding a
protein
having an amino acid sequence sufficiently homologous to the amino acid
sequence of
SEQ ID NO:14 (e.g., encodes a protein having an amino acid sequence which is
60%
homologous to the amino acid sequence of SEQ ID NO:14).
In another embodiment, an isolated nucleic acid molecule of the present
invention encodes a Dick protein which includes a signal sequence and at least
one
cysteine-rich region, and is secreted. In another embodiment, an isolated
nucleic acid
molecule of the present invention encodes a Da protein which includes a signal
sequence and a cysteine-rich region, wherein the cysteine-rich region
comprises at least
one cysteine-rich domain, and is secreted. In yet another embodiment, a Dkk
nucleic
acid molecule encodes a Dkk protein and is a naturally occurring nucleotide
sequence.
In another embodiment, an isolated nucleic acid molecule of the present
invention encodes a Dkk-related protein (e.g., a Soggy protein) which includes
a signal
sequence, lacks cysteine-rich domains, and is secreted. In another embodiment,
an
isolated nucleic acid molecule of the present invention encodes a Dkk-related
protein
(e.g., a Soggy protein) which includes a signal sequence and a Soggy domain,
and is
secreted. In yet another embodiment, a nucleic acid molecule of the present
invention
encodes a Dkk-related protein and is a naturally occurring nucleotide
sequence.
Another embodiment of the invention features nucleic acid molecules which
specifically detect Dldc nucleic acid molecules relative to nucleic acid
molecules
encoding non-Dkk proteins (or specifically detect Dkk-related nucleic acid
molecules).
For example, in one embodiment, a nucleic acid molecule hybridizes under
stringent
conditions to a nucleic acid molecule consisting of nucleotides 470-2479 of
nucleotide
sequence shown in SEQ ID NO:1, to nucleotides 1-475 of nucleotide sequence
shown in
SEQ ID NO:4, or to nucleotides 1-600 of nucleotide sequence shown in SEQ ID
NO:7,
or hybridizes under stringent conditions to the nucleotide sequence of the DNA
insert of
the plasmid deposited with ATCC as Accession Number 98452, or to the
nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as Accession
Number
98633-
In another embodiment, the nucleic acid molecule
is at least 500 nucleotides in length and hybridizes under stringent
conditions to a
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nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1,
SEQ
ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20 or a complement thereof.
Another embodiment of the invention provides an isolated nucleic acid molecule
which is antisense to the coding strand of a Dkk nucleic acid or Dkk-related
nucleic
acid. Another embodiment of the invention provides an isolated nucleic acid
molecules
in a form suitable for expression of mRNA. In another embodiment, the isolated
nucleic
acid molecules are in a form suitable for expression of protein. In yet
another
embodiment, the isolated nucleic acid molecules are free from vector
seqeunces.
Another aspect of the invention provides a vector comprising a Dkk nucleic
acid
molecule or Dkk-related nucleic acid molecule. In certain embodiments, the
vector is a
recombinant expression vector. In another embodiment, the invention provides a
host
cell containing a vector of the invention. The invention also provides a
method for
producing a Dkk protein or Dkk-related protein by culturing in a suitable
medium, a host
cell of the invention containing a recombinant expression vector such that a
Dkk protein
or Dkk-related protein is produced.
Another aspect of this invention features isolated or recombinant Dkk proteins
and polypeptides or Dkk-related proteins and polypeptides. In one embodiment,
an
isolated Dkk protein has a signal sequence and a cysteine-rich region which
comprises
two cysteine-rich domains, and is secreted. In another embodiment, an isolated
Dkk
protein has an amino acid sequence sufficiently homologous to the amino acid
sequence
of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:21. In a preferred
embodiment, a Dkk protein has an amino acid sequence at least about 60%
homologous
to the amino acid sequence of SEQ ID NO:2. In another preferred embodiment, a
Dkk
protein has an amino acid sequence at least about 60% homologous to the amino
acid
sequence of SEQ ID NO:5. In another preferred embodiment, a Dkk protein has an
amino acid sequence at least about 60% homologous to the amino acid sequence
of SEQ
ID NO:8. In another preferred embodiment, a Dkk protein has an amino acid
sequence
at least about 75% homologous to the amino acid sequence of SEQ ID NO:8. In
another
preferred embodiment, a Dkk protein has an amino acid sequence at least about
65%
homologous to the amino acid sequence of SEQ ID NO:21. In another embodiment,
a
Dick protein has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID
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NO:8, or SEQ ID NO:21. In another preferred embodiment, a protein of the
present
invention has an amino acid sequence at least about 60% homologous to the
amino acid
sequence of SEQ ID NO:14. In another embodiment, a protein has the amino acid
sequence of SEQ ID NO:14.
Another embodiment of the invention features an isolated Dkk protein which is
encoded by a nucleic acid molecule having a nucleotide sequence at least about
60%
homologous to a nucleotide sequence of SEQ ID NO:1, or a complement thereof.
Another embodiment of the invention features an isolated Dkk protein which is
encoded
by a nucleic acid molecule having a nucleotide sequence at least about 80%
homologous
to a nucleotide sequence of SEQ ID NO:4, or a complement thereof. Another
embodiment of the invention features an isolated Dkk protein which is encoded
by a
nucleic acid molecule having a nucleotide sequence at least about 60%
homologous to a
nucleotide sequence of SEQ ID NO:7, or a complement thereof. Another
embodiment
of the invention features an isolated Dkk protein which is encoded by a
nucleic acid
molecule having a nucleotide sequence at least about 85% homologous to a
nucleotide
sequence of SEQ ID NO:7, or a complement thereof Another embodiment of the
invention features an isolated Dkk protein which is encoded by a nucleic acid
molecule
having a nucleotide sequence at least about 70% homologous to a nucleotide
sequence
of SEQ ID NO:20, or a complement thereof Another embodiment of the invention
features an isolated protein which is encoded by a nucleic acid molecule
having a
nucleotide sequence at least about 90% homologous to a nucleotide sequence of
SEQ ID
NO:13, or a complement thereof This invention further features an isolated
protein
which is encoded by a nucleic acid molecule having a nucleotide sequence which
hybridizes under stringent hybridization conditions to a nucleic acid molecule
comprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7,
SEQ ID NO:13, SEQ ID NO:20, or a complement thereof
The proteins of the present invention, or biologically active portions
thereof, can
be operatively linked to a non-Dkk polypeptide or non-Dkk-related polypeptide
to form
fusion proteins. The invention further features antibodies that specifically
bind Dkk or
Dick-related proteins, such as monoclonal or polyclonal antibodies. In
addition, the
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proteins or biologically active portions thereof can be incorporated into
pharmaceutical
compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting Dkk
expression (or the expression of a Dkk-related molecule) in a biological
sample by
contacting the biological sample with an agent capable of detecting a nucleic
acid
molecule, protein or polypeptide of the present invention such that the
presence of a Dkk
(of Dkk-related) nucleic acid molecule, protein or polypeptide is detected in
the
biological sample.
In another aspect, the present invention provides a method for detecting the
presence of a Dkk activity (or Dkk-related activity) in a biological sample by
contacting
the biological sample with an agent capable of detecting an indicator of Dkk
activity (or
Dkk-related activity) such that the presence of the activity is detected in
the biological
sample.
In another aspect, the invention provides a method for modulating Dkk activity
(or Dkk-related activity) comprising contacting the cell with an agent that
modulates the
activity such that the activity in the cell is modulated. In one embodiment,
the agent
inhibits Dkk activity (or Dkk-related activity). In another embodiment, the
agent
stimulates Dkk activity (or Dick-related activity). In one embodiment, the
agent is an
antibody that specifically binds to a Dkk (or Dkk-related) protein. In another
embodiment, the agent modulates expression of a protein (e.g., a Dkk or a Dkk-
related
protein) by modulating transcription of a gene or translation of a mRNA of the
present
invention. In yet another embodiment, the agent is a nucleic acid molecule
having a
nucleotide sequence that is antisense to the coding strand of a mRNA or gene
of the
present invention.
In one embodiment, the methods of the present invention are used to treat a
subject having a disorder characterized by aberrant expression or activity of
a protein or
nucleic acid of the invention by administering to the subject an agent which
is a
modulator of Dkk or a Dkk-related molecule. In one embodiment, the modulator
is a
Dkk or Dkk-related protein. In another embodiment the modulator is a Dick or
Dkk-
related nucleic acid molecule. In yet another embodiment, the modulator is an
antibody
peptide, peptidomimetic, or other small molecule. In a preferred embodiment,
the
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disorder characterized by aberrant protein or nucleic acid expression is a
developmental,
differentiative, or proliferative disorder.
The present invention also provides a diagnostic assay for identifying the
presence or absence of a genetic alteration characterized by at least one of
(i) aberrant
modification or mutation of a gene encoding a Dkk or Dkk-related protein; (ii)
mis-
regulation of said gene; and (iii) aberrant post-translational modification of
a Dkk or
Dkk-related protein, wherein a wild-type form of said gene encodes an protein
with a
Dkk or Dkk-related activity.
In another aspect the invention provides a method for identifying a compound
that binds to or modulates the activity of a Dkk or Dkk-related protein, by
providing a
indicator composition comprising a Dkk or Dkk-related protein having a
biological
activity, contacting the indicator composition with a test compound, and
determining the
effect of the test compound on the activity in the indicator composition to
identify a
compound that modulates the activity of a Dkk or Dkk-related protein.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
Brief Description of the Drawings
Figure 1A-B depicts the cDNA sequence and predicted amino acid sequence of
human Dkk-3. The nucleotide sequence corresponds to nucleic acids 1 to 2479 of
SEQ
ID NO:l. The amino acid sequence corresponds to amino acids 1 to 350 of SEQ ID
NO:2.
Figure 2 depicts the cDNA sequence and predicted amino acid sequence of
human Dkk-4. The nucleotide sequence corresponds to nucleic acids 1 to 848 of
SEQ
ID NO:4. The amino acid sequence corresponds to amino acids 1 to 224 of SEQ ID
NO:5.
Figure 3 depicts the cDNA sequence and predicted amino acid sequence of
human Dick-1. The nucleotide sequence corresponds to nucleic acids 1 to 1536
of SEQ
ID NO:7. The amino acid sequence corresponds to amino acids 1 to 266 of SEQ ID
NO:8.
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Figure 4A-B depicts the cDNA sequence and predicted amino acid sequence of
full-length human Dkk-2. The nucleotide sequence corresponds to nucleic acids
1 to
3687 of SEQ ID NO:20. The amino acid sequence corresponds to amino acids 1 to
259
of SEQ ID NO:21.
Figure 5A-B depicts the cDNA sequence and predicted amino acid sequence of
murine Dkk-3. The nucleotide sequence corresponds to nucleic acids 1 to 2380
of SEQ
ID NO:16. The amino acid sequence corresponds to amino acids 1 to 349 of SEQ
ID
NO:17.
Figure 6 depicts a multiple sequence alignment of the amino acid sequences of
hDkk-1 (corresponding the SEQ ID NO:8), mDlck-1 (having Accession No.
AF030433),
Xenopus Dkk-1 (xDklc-1") (having Accession No. AF030434), hDkk-2
(corresponding
to SEQ ID NO:21), hDkk-3 (corresponding to SEQ ID NO:2), mDlck-3
(corresponding
to SEQ ID NO:17), chicken Dklc-3 ("cD1c1c-3") (having Accession No. D26311),
and
hDick-4 (corresponding to SEQ ID NO:5). The alignment was performed using the
ClustalW algorithm as implemented in the GCG program PILEUP. The alignment
provides information regarding the relationship between the Dkk proteins of
the instant
invention. Predicted signal peptides are underlined, N-glycosylation sites are
indicated
by a thick bar, CRD-1 by an open box, CRD-2 by a shaded box. The proteolytic
cleavage site within hDldc4 is indicated by an arrow.
Figure 7 depicts the cDNA sequence and predicted amino acid sequence of
human Soggy. The nucleotide sequence corresponds to nucleic acids 1 to 928 of
SEQ
ID NO:13. The amino acid sequence corresponds to amino acids Ito 242 of SEQ ID
NO:14.
Figure 8 depicts the cDNA sequence and predicted amino acid sequence of
murine Soggy-1. The nucleotide sequence corresponds to nucleic acids 1 to 835
of SEQ
ID NO:26. The amino acid sequence corresponds to amino acids 1 to 230 of SEQ
ID
NO:27.
Figure 9 is a schematic diagram illustrating the Dklc and Dkk-related proteins
of
the instant invention. The figure depicts the structural domains of the human
Dicks and
Soggy. Signal peptides are indicated by darkened boxes. The cysteine-rich
domains of
* Trademark
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a DU cysteine-rich region are depicted as CRD-1 and CRD-2. Branches indicate
sites
of N-glycosylation.
Figure 10 depicts a multiple sequence alignment of hSoggy-1 (corresponding to
SEQ ID NO:14), murine Soggy-1 (corresponding to SEQ ID NO:27), hDkk-3
(corresponding to SEQ ID NO:2), and mDkk-3 (corresponding to SEQ ID NO:17).
The
alignment was generated as described in the legend to Figure 6. The alignment
provides
details regarding the relationship between the Dkk-3 and Soggy-1 proteins of
the instant
invention. Predicted signal peptides are underlined, N-glycosylation sites are
indicated
by a thick bar. CRD-1 and CRD-2 within Dkk-3 are indicated for reference by
open and
shaded boxes.
Figure 11 depicts a multiple sequence alignment of the carboxy-terminal
cysteine-rich domains of hDkk-1, hDkk-2, hDkk-3, hDkk-4 with human colipase
(having accession No. J02883). The carboxy-terminal cysteine-rich domains of
the Dkk
proteins are indicated by an open box. The alignment was generated using
PILEUP (gap
penalties of 12 for opening and 12 for extending). A minor adjustment was
necessary
since PILEUP inserts a single gap in hDkk-1 and hDkk-2 between Gly56 and
Ser57,
even with a gap opening penalty of 15. The conserved residues are indicated.
The
disulfide-bonding pattern typical for the colipase family and predicted for
the Dkk
family is indicated below the alignment.
Figure 12 is a schematic diagram depicting the relationship between the hDkk-3
nucleotide sequence (corresponding to SEQ ID NO:1) and those of RIG and RIG-
like 7-
1 (Accession Nos. U32331 and AF034208, respectively). Thick bars indicate
regions of
sequence identity between hDkk-3 and RIG or RIG-like 7-1 mRNAs. As between RIG
and hDkk-3, there exists a short region of identity within the 3' untranslated
regions of
the mRNAs when the mRNAs are aligned in reverse orientation. As between hDkk-3
and RIG-like 7-1, there exists a longer region of identity, however, RIG-like
7-1 lacks a
signal sequence and, accordingly, is not predicted to be secreted.
Detailed Description of the Invention
The present invention is based on the discovery of novel molecules, referred
to
herein as DU protein and nucleic acid molecules, which comprise a family of
molecules
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having certain conserved structural and functional features. The term "family"
when
referring to the protein and nucleic acid molecules of the invention is
intended to mean
two or more proteins or nucleic acid molecules having a common structural
domain and
having sufficient amino acid or nucleotide sequence homology as defined
herein. Such
family members can be naturally-occurring and can be from either the same or
different
species. For example, a family can contain a first protein of human origin, as
well as
other, distinct proteins of human origin or alternatively, can contain
homologues of non-
human origin. Members of a family may also have common functional
characteristics.
In one embodiment, a Dkk family member is identified based on the presence of
at least one "cysteine-rich domain" in the protein molecule or corresponding
amino acid
sequence. As defined herein, a "cysteine-rich domain" refers to a portion of a
Dkk
protein (e.g., hDkk-3) which is rich in cysteine residues. In a preferred
embodiment, a
"cysteine-rich domain" is a protein domain having an amino acid sequence of
about 45-
85 amino acids of which preferably 10 amino acids are cysteine residues
located at the
same relative amino acid position as the cysteine residues in human Dkk-3
having SEQ
ID NO:2 (e.g., amino acid residues 147-195 of SEQ ID NO:2). In another
embodiment,
a "cysteine-rich domain" has 30-100 amino acids, preferably about 35-95 amino
acids,
more preferably about 40-90 amino acids, more preferably about 50-80 amino
acids,
even more preferably about 55-75, 60-70, or 65 amino acids, of which at least
about 3-
20, preferably about 5-15, or more preferably about 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16,
17, 18, 19, or 20 amino acids are cysteine residues.
A preferred Dkk protein of the present invention has a first cysteine-rich
domain
("CRD-1") referred to herein as an "amino-terminal cysteine-rich domain" or "N-
terminal cysteine-rich domain" and a second cysteine-rich domain ("CRD-2"),
referred
to herein as a "carboxy-terminal cysteine-rich domain" or "C-terminal cysteine-
rich
domain". As defined herein, an "amino-terminal cysteine-rich domain" is a
protein
domain having an amino acid sequence of about 45-55 amino acids of which
preferably
10 amino acids are cysteine residues located at the same relative position as
the cysteine
residues in an amino-terminal cysteine-rich domain of human Dkk-3 having SEQ
ID
NO:2 (e.g., amino acid residues 147-195 of SEQ ID NO:2). In another
embodiment, an
"amino-terminal cysteine-rich domain" has 30-70, preferably 35-65, more
preferably
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,
about 40-60, and even more preferably about 46, 47, 48, 49, 50, 51, 52, 53, or
54 amino
acids, of which at least about 3-20, preferably about 5-15, or more preferably
about 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids are cysteine
residues. In a
preferred embodiment, an amino-terminal cysteine-rich domain has the following
consensus sequence: C - X(2) - D - X(2) - C - X(5) - C - X(8 -13) - C - X(2) -
C - X(6) -
C - X(5) - C - C - X(4) - C - X(4) - C (SEQ ID NO:23). The consensus sequences
described herein are described according to standard Prosit:Signature
designation (e.g.,
all amino acids are indicated according to their universal single letter
designation; X
designates any amino acid; X(n) designates any n amino acids, e.g., X (2)
designates any
2 amino acids; and [LIVM] indicates any one of the amino acids appearing
within the
brackets, e.g., any one of L, I, V, or M, in the alternative, any one of Leu,
Ile, Val, or
Met.)
As defined herein, a "carboxy-terminal cysteine-rich domain" is a protein
domain
having an amino acid sequence of about 80-85 amino acids of which preferably
10
amino acids are cysteine residues located at the same relative position as the
cysteine
residues in a carboxy-terminal cysteine-rich domain of human Dklc-3 having SEQ
ID
NO:2 (e.g., amino acid residues 201-284 of SEQ ID NO:2). In another
embodiment, a
"carboxy-terminal cysteine-rich domain" has 65-100, preferably 70-95, more
preferably
about 75-90, and even more preferably about 81, 82, 83, or 84 amino acids, of
which at
least about 3-20, preferably about 5-15, or more preferably about 6, 7, 8, 9,
10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20 amino acids are cysteine residues. In a
preferred
embodiment, a carboxy-terminal cysteine-rich domain has the following
consensus
sequence: C - X(4) - D - C - X(2) -G-X-C-C- X(8-10) - C - X - P - X(4) - G -
X(2) -
C - X(16-24) - C - X - C - X(2) - P - X(4) -0 - X(2) - C - X(16-24) - C - X -
C - X(2) -
G-L-X-C- X(10-17) - C (SEQ ID NO:24).
A preferred protein of the present invention is a hDkk-3 protein (human Dkk-3)
containing an amino-terminal cysteine-rich domain including about amino acids
147-
195 of SEQ ID NO:2, having 10 cysteine residues, and a carboxy-terminal
cysteine-rich
domain including about amino acids 201-284 of SEQ ID NO:2, having 10 cysteine
residues (the positions of the cysteine residues are depicted in Figure 6). In
another
embodiment, a hDidc-4 (human Dick-4) protein contains an amino-terminal
cysteine-rich
* Trademark
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domain including about amino acids 41-90 of SEQ ID NO:5, having 10 cysteine
residues, and a carboxy-terminal cysteine-rich domain including about amino
acids 138-
218 of SEQ ID NO:5, having 10 cysteine residues (the positions of the cysteine
residues
are depicted in Figure 6). In another embodiment, a hDlck-1 protein (human Dkk-
1)
contains an amino-terminal cysteine-rich domain including about amino acids 85-
138 of
SEQ ID NO:8, having 10 cysteine residues, and a carboxy-terminal cysteine-rich
domain
including about amino acids 182-263 of SEQ ID NO:8, having 10 cysteine
residues (the
positions of the cysteine residues are depicted in Figure 6). In another
embodiment, a
hDkk-2 protein (human Dkk-2) contains an amino-terminal cysteine-rich domain
including about amino acids 78-127 of SEQ ID NO:21, having 10 cysteine
residues, and
a carboxy-terminal cysteine-rich domain including about amino acids 176-256 of
SEQ
ID NO :21, having 10 cysteine residues (the positions of the cysteine residues
are
depicted in Figure 6).
Alignment of the human Dkk proteins with human colipase (having Accession
No. J02883) indicates that the carboxy-terminal cysteine-rich domains of the
human
Dkk proteins have a pattern of cysteines typical of colipase (Figure 11 and
Avarind and
Koonin, supra). Within colipase, these cysteine residues are involved in
disulfide
bonding which gives rise to a structure termed the "colipase fold". The
"colipase fold"
is typical of a range of small proteins which are involved in protein-protein
interactions
including, but not limited to the colipases, snake and scorpion toxins and
protease
inhibitors (Hubbard et al. (1997) Nucleic Acids Res. 25:236-239. These
proteins have a
series of short 13 strands with large connecting loops, which are held
together by
disulfide bonds. The disulfide-bonding pattern typical for colipase and
predicted for the
Dkk family is indicated below the alignment of Figure 11. Conserved
hydrophobic
residues between the Dkks and human colipase suggest that the Dkks, like the
colipases,
interact with lipids (e.g., Leu51 of human colipase, SEQ ID NO:25 which
corresponds
to Leu271 of hDkk-3 (SEQ ID NO:2); Leu200 of hDkk-4 (SEQ ID NO:5); Leu243 of
hDkk-1 (SEQ ID NO:8); and Leu237 of hDkk-2 (SEQ ID NO:21). The carboxy-
terminal cysteine-rich domain of the Dkk family, may function in the membrane
association of Dkk, which in turn may be required for the inhibition of Wnt
secretion or
Wnt:7 transmembrane receptor interaction. In addition, inhibition of Wnt
function by
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the Dkk family may be closely associated with the cell membrane and the
carboxy-
terminal cysteine-rich domain of the Dkk family may mediate this association.
Furthermore, the amino-terminal cysteine-rich domain of the Dkk family may
directly
interact with Wnt or its receptor. Accordingly, a preferred Dkk protein of the
present
invention comprises a carboxy-terminal cysteine-rich domain. In one
embodiment, a
Dkk protein comprising a carboxy-terminal cysteine-rich domain lacks the amino-
terminal cysteine-rich domain.
In a preferred embodiment, the cysteine residues of a cysteine-rich domain are
located at the same relative amino acid position as the cysteine residues in
human Dkk-3
having SEQ ID NO:2. In another preferred embodiment, the cysteine residues of
a
cysteine-rich domain are located at the same relative position as the cysteine
residues in
a cysteine-rich domain of human Dkk-3 having SEQ ID NO:2. For example, as
shown
in Figure 6, human Dkk-4 has at least about 10 cysteine residues located at
the same
relative amino acid position as the cysteine residues in human Dkk-3 having
SEQ ID
NO:2 (e.g., cys151 in Dkk-4, SEQ ID NO:5, is located at the same relative
amino acid
position as cys214 in Dkk-3, SEQ ID NO:2; cys156 in Dkk-4, SEQ ID NO:5, is
located
at the same relative amino acid position as cys219 in Dkk-3, SEQ ID NO:2; and
cys157
in Dkk-4, SEQ ID NO:5, is located at the same relative amino acid position as
cys220 in
Dkk-3, SEQ ID NO:2). Similarly, as shown in Figure 6, Dkk-1 has at least about
10
cysteine residues located at the same relative amino acid position as the
cysteine
residues in human Dkk-3 having SEQ ID NO:2. As also shown in Figure 6, Dkk-2
has
at least about 10 cysteine residues located at the same relative amino acid
position as the
cysteine residues in human Dkk-3 having SEQ ID NO:2. Table I sets forth at
least 20
cysteine residues in each of hDkk-4, hDkk-1, and hDkk-2 which are located in
the same
relative position as 20 cysteine residues in hDkk-3.
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Table I:
aa position aa position aa position aa position
cysteine in hDkk-3 in hDkk-4 in hDkk-1 in
hDkk-2
1 147 41 - 85 78
_
2 153 47 91 84 -
3 159 - 53 97 90
4 168 63 111 100
171 66 114 103
6 178 73 121 110
7 184 79 127 116
8 - 185 80 128 117
9 190 85 133 122
195 ' 90 138 127
11 208 - 145 ' 189 183
12 214 151 195 189
13 219 156 200 194
14 220 157 201 195
231 166 210 204
16 241 176 220 214
17 265 194 237 231
18 267 196 239 233
19 273 - 202 245 239
284 218 263 256
The first 10 rows of Table I contain 10 cysteine residues that are included
within
5 the first, or amino-terminal, cysteine-rich domain of each of hDkks-3, -
4, -1, and -2.
The last 10 rows of Table I contain 10 cysteine residues that are included
within the
second, or carboxy-terminal, cysteine-rich domain of each of hDlcks-3, -4, -1,
and -2.
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Preferred Dkk proteins have more than one cysteine-rich domain, more
preferably have at least two cysteine-rich domains and, thus, have a cysteine-
rich region.
As used herein, the term "cysteine-rich region" refers to a protein domain
which includes
at least two cysteine-rich domains and has an amino acid sequence of about 120-
200
amino acid residues of which at least about 20 of the amino acids are cysteine
residues.
In another embodiment, a "cysteine-rich region" has preferably about 140-180
amino
acid residues, and even more preferably at least about 135-175 amino acids of
which at
least about 10-30, preferably about 15-20, and more preferably about 16, 17,
18, or 19 of
the amino acids are cysteine residues. In a preferred embodiment, a cysteine-
rich region
is located in the C-terminal region of a Dkk protein. For example, in one
embodiment, a
hDkk-3 protein contains a cysteine rich region containing about amino acids
147-284 of
SEQ ID NO:2, having 20 cysteine residues at the positions indicated in Figure
6. In
another embodiment, a hDkk-4 protein contains a cysteine rich region
containing about
amino acids 41-218 of SEQ ID NO :5, having 20 cysteine residues at the
positions
indicated in Figure 6. In another embodiment, a hDkk-1 protein contains a
cysteine rich
region containing about amino acids 85-263 of SEQ ID NO:8, having 20 cysteine
residues at the positions indicated in Figure 6. In another embodiment, a hDkk-
2 protein
contains a cysteine rich region containing about amino acids 78-256 of SEQ ID
NO:21,
having 20 cysteine residues at the positions indicated in Figure 6.
In another embodiment, in addition to cysteine-rich domains, the cysteine-rich
region contains a spacer region which separates the first and second cysteine-
rich
domains. As used herein, the "spacer region" refers to amino acid residues
which are
located between the first and second cysteine-rich domains of a cysteine-rich
region and
includes amino acid residues located C-terminal to the first cysteine-rich
domain and N-
terminal to the second cysteine-rich domain. As defined herein, a "spacer
region" refers
to a protein domain of about 5-70 amino acids, preferably about 10-65 amino
acids,
more preferably about 15-60 amino acids, even more preferably about 20-55
amino
acids, and even more preferably about 25-50, 30-45 or 35-40 amino acids. For
example,
hDkk-3 protein contains a spacer region of about amino acids 196-200 of SEQ ID
NO:2;
hDkk-4 protein contains a spacer region of about amino acids 91-137 of SEQ ID
NO :5;
hDkk-1 protein contains a spacer region of about amino acids 139-181 of SEQ ID
NO:8;
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and hDkk-2 protein contains a spacer region of about amino acids 128-175 of
SEQ ID
NO:21. The spacer regions of hDkk-1, hDkk-2 and hDkk-4 are remarkably
conserved in
length (e.g., the spacer region of hDldc-1 consists of 43 amino acid residues,
the spacer
region of hDkk-2 consists of 48 amino acid residues and the spacer region of
hDkk-4
consists of 47 amino acid residues, suggesting that the close proximity of CRD-
1 and
CRD-2 is important in Dkk function. Accordingly, in one embodiment, the spacer
region functions to spacially restrict the separation of CRD-1 from CRD-2.
In another embodiment of the invention, the Dkk protein has at least one
cysteine-rich domain, preferably a cysteine-rich region, and a signal
sequence. As used
herein, a "signal sequence" refers to a peptide containing about 18-24 amino
acids which
occurs at the N-terminus of secretory and integral membrane proteins and which
contains at least about 40-70% hydrophobic amino acid residues (e.g., alanine,
valine,
leucine, isoleucine, phenylalanine, tyrosine, tryptophan, or proline). In
another
embodiment, a signal sequence contains at least about 8-34, 9-33, 10-32, 11-
31, 12-30,
13-29, 14-28 amino acid residues, preferably about 15-27 amino acid residues,
more
preferably about 16-26 amino acid residues, more preferably about 17-25 amino
acid
residues, and more preferably about 18-24, 19-23, 20-22, or 21 amino acid
residues, and
has at least about about 50-65%, and more preferably about 55-60% hydrophobic
amino
acid residues (e.g., alanine, valine, leucine, isoleucine, phenylalanine,
tyrosine,
tryptophan, or proline). Such a "signal sequence", also referred to in the art
as a "signal
peptide", serves to direct a protein containing such a sequence to a lipid
bilayer. For
example, in one embodiment, a hDkk-3 protein contains a signal sequence of
about
amino acids 1-23 of SEQ ID NO:2. In another embodiment, a hDkk-4 protein
contains a
signal sequence of about amino acids 1-19 of SEQ ID NO :5. In another
embodiment, a
hDkk-1 protein contains a signal sequence of about amino acids 1-20 of SEQ ID
NO:8.
In another embodiment, a hDkk-2 protein contains a signal sequence of about
amino
acids 1-33 of SEQ ID NO :21. A preferred Dkk protein of the present invention
is a
human protein (e.g., encoded by a nucleotide sequence correpsonding to a
naturally-
occurring human gene).
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Accordingly, one embodiment of the invention features a Dkk protein having at
least one cysteine-rich domain, preferably at least one cysteine-rich region.
Another
embodiment features a Dkk protein having at least one cysteine-rich region,
wherein the
cysteine-rich region includes at least one cysteine-rich domain. Another
embodiment
features a Dkk protein having at least one cysteine-rich region, wherein the
cysteine-rich
region includes at least two cysteine-rich domains. Another embodiment
features a
protein or domain within a protein having 20, 30, 40, 50, 60, 70, 80, 90, 95,
or 99%
homology to a cysteine-rich domain of a Dkk protein of the invention (e.g.,
hDkk-3,
hDkk-4, hDkk-1, or hDkk-2).
Yet another embodiment of the invention features a Dkk protein having at least
one cysteine-rich domain, preferably at least one cysteine-rich region and a
signal
peptide. Another embodiment features a Dkk protein having at least one
cysteine-rich
domain, preferably at least one cysteine-rich region and a signal peptide,
wherein the
cysteine-rich region includes at least two cysteine-rich domains. Another
embodiment
features a Dkk protein having at least one cysteine-rich domain, preferably at
least one
cysteine-rich region and a signal peptide, wherein the cysteine-rich region
includes at
least two cysteine-rich domains and a spacer.
Yet another aspect of the invention features Dkk proteins having domains
and/or
regions which are conserved among a subset of Dkk proteins but are not
necessarily
conserved among all Dkk family members. In one embodiment, a Dkk protein
(e.g.,
Dkk-3) has an "extended N-terminal region" which is extended in length as
compared to,
for example, the "N-terminal regions" of other Dkk family members (e.g., Dkk-
4, Dkk-
1, and Dkk-2). As defined herein, an "N-terminal region" of a Dkk proteins
consists of
amino acid residues found between the signal peptide and CRD-1 of a Dkk
protein.
Preferably, the first amino acid residue of an N-terminal region of Dkk is the
first
residue of a mature Dick protein and the last residue of an N-terminal region
of Dkk is
the residue preceeding the first cysteine residue of CRD-1. In a preferred
embodiment,
an N-terminal region is about 1-20 amino acid residues in length, preferably
about 21-
30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 101-110, 111-120, 121-
130, 131-
140, 141-150, 151-160 or more amino acid residues in length In contrast, an
"extended
N-terminal region" is at least about 71-80, 81-90, 91-100, 101-110, 111-120,
121-130,
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131-140, 141-150, 151-160 or more amino acid residues in length. For example,
in one
embodiment, a hDkk-4 protein includes an "N-terminal region" of about amino
acids 20-
40 of SEQ ID NO :5 (21 amino acid residues in length). In another embodiment,
a
hDkk-1 protein includes an N-terminal region of about amino acids 21-84 of SEQ
ID
NO:8 (64 amino acid residues in length). In another embodiment, a hDkk-2
protein
includes an "N-terminal region" of about amino acids 34-77 of SEQ ID NO:21 (44
amino acid residues in length). In another embodiment, a hDkk-3 protein has an
"extended N-terminal region" of about amino acids 23-146 of SEQ ID NO:2 (124
amino
acid residues in length).
In another embodiment, a Dick protein (e.g., Dkk-3) has an "acidic C-terminal
region" which incudes amino acid residues found C-terminal to CRD-2 of a Dkk
protein.
Preferably, the first amino acid residue of an acidic C-terminal region is the
residue
following the last cysteine of CRD-2 and the last residue of an acidic C-
terminal region
is the last residue of a Dkk protein. In a preferred embodiment, an acidic C-
terminal
region is about 65-66 amino acid residues in length and has about 27-25%
acidic amino
acid residues (e.g., glutamic acid or aspartic acid). In another preferred
embodiment, an
acidic C-terminal region is about 55-80 amino acid residues in length,
preferably about
60-75 amino acid residues in length, and more preferably about 64-70 amino
acid
residues in length and has about 21-35% acidic amino acid residues, preferably
about
23-33% acidic amino acid residues, and more preferably about 25-31% acidic
amino
acid residues. Preferably, an acidic C-terminal region is involved in protein-
protein
interactions. For example, in one embodiment, a hDkk-3 protein has an acidic C-
terminal region from about amino acids 285-350 of SEQ ID NO:2.
Preferred Dkk molecules of the present invention have an amino acid sequence
sufficiently homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID
NO:5,
SEQ ID NO:8, or SEQ ID NO:21. As used herein, the term "sufficiently
homologous"
refers to a first amino acid or nucleotide sequence which contains a
sufficient or
minimum number of identical or equivalent (e.g., an amino acid residue which
has a
similar side chain) amino acid residues or nucleotides to a second amino acid
or
nucleotide sequence such that the first and second amino acid or nucleotide
sequences
share common structural domains and/or a common functional activity. For
example,
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amino acid or nucleotide sequences which share common structural domains have
at
least about 40% homology, preferably 50% homology, more preferably 60%-70%
homology across the amino acid sequences of the domains and contain at least
one,
preferably two, more preferably three, and even more preferably four, five or
six
structural domains, are defined herein as sufficiently homologous.
Furthermore, amino
acid or nucleotide sequences which share at least 40%, preferably 50%, more
preferably
60, 70, or 80% homology and share a common functional activity are defined
herein as
sufficiently homologous.
As used interchangeably herein, a "Dkk activity", "biological activity of Dkk"
or
"functional activity of Dkk", refers to an activity exerted by a Dkk protein,
polypeptide
or nucleic acid molecule (e.g., an activity on a Dkk responsive cell) as
determined in
vivo, or in vitro, according to standard techniques. In one embodiment, a Dkk
activity is
a direct activity, such as an association with a Dkk-target molecule. As used
herein, a
"target molecule" is a molecule with which a Dkk protein binds or interacts in
nature,
such that Dkk-mediated function is acheived. A Dkk target molecule can be a
non-Dkk
molecule or a Dkk protein or polypeptide of the present invention. In an
exemplary
embodiment, a Dkk target molecule is a membrane-bound protein (e.g., a cell-
surface
receptor or "Dkk receptor") or a modified form of such a protein which has
been altered
such that the protein is soluble (e.g., recombinantly produced such that the
protein does
not express a membrane-binding domain). In another embodiment, a Dick target
is a
second soluble protein molecule (e.g., a "Dkk binding partner" or "Dkk
substrate"). In
such an exemplary embodiment, a Dkk binding partner can be a second soluble
non-Dkk
protein or a second Dkk protein molecule of the present invention.
Alternatively, a Dkk
activity is an indirect activity, such as a cellular signaling activity
mediated by
interaction of the Dkk protein with a second protein (e.g., a Dkk receptor).
As used
herein, the term "Dkk receptor" refers to a protein or protein complex, to
which a Dkk
protein, e.g., human Dkk, can bind. A receptor can be a cell surface receptor,
e.g., a
peptide, growth factor, or nuclear hormone receptor. Dkk receptors can be
isolated by
methods known in the art and further described herein. Interaction of a Dkk
protein with
a Dkk receptor can result in transduction of a signal from the cell surface to
the nucleus.
The signal transduced can be, an increase in intracellular calcium, an
increase in
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phosphatidylinositol or other molecule, and can result in, e.g., in
phosphorylation of
specific proteins, a modulation of gene transcription and any of the other
biological
activities set forth herein.
In a preferred embodiment, a Dkk activity is at least one or more of the
following
activities: (i) interaction of a Dkk protein with and/or binding to a second
molecule,
(e.g., a protein, such as a Dkk receptor, a soluble form of a Dkk receptor, a
receptor for a
member of the wnt family of signaling proteins, or a non-Dkk signaling
molecule, for
example, a lipid included in a cell membrane); (ii) interaction of a Dkk
protein with an
intracellular protein via a membrane-bound Dkk receptor; (iii) complex
formation
between a soluble Dkk protein and a second soluble Dkk binding partner (e.g.,
a non-
Dkk protein molecule or a second Dkk protein molecule); (iv) interaction with
other
extracellular proteins (e.g., regulation of wnt-dependent cellular adhesion to
extracellular
matrix components); (v) binding to and eliminating an undesirable molecule
(e.g., a
detoxifying activity or defense function); and/or (vi) an enzymatic activity.
In yet
another preferred embodiment, a Dkk activity is at least one or more of the
following
activities: (1) modulation of cellular signal transduction, either in vitro or
in vivo (e.g.,
modulation, e.g., antagonism, of the activity of members of the wnt family of
secreted
proteins or supression of wnt-dependent signal transduction, for example
suppression of
Wnt 2b, Wnt3 and/or Wnt8-dependent signal transduction by hDkk-1 and/or hDkk-
4);
(2) regulation of communication between cells (e.g., regulation of wnt-
dependent cell-
cell interactions); (3) regulation of expression of genes whose expression is
modulated
by binding of Dkk (e.g., hDkk-3) to a receptor; (4) regulation of gene
transcription in a
cell involved in development or differentiation, either in vitro or in vivo
(e.g., induction
of cellular differentiation); (5) regulation of gene transcription in a cell
involved in
development or differentiation, wherein at least one gene encodes a
differentiation-
specific protein; (6) regulation of gene transcription in a cell involved in
development or
differentaition, wherein at least one gene encodes a second secreted protein;
(7)
regulation of gene transcription in a cell involved in development or
differentiation,
wherein at least one gene encodes a signal transduction molecule; (8)
regulation of
cellular proliferation, either in vitro or in vivo (e.g., induction of
cellular proliferation or
inhibition of proliferation as in the case of supression of tumorigenesis
(e.g., suppression
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of glial cell tumor growth, for example, glioblastoma growth)); (9) formation
and
maintenance of ordered spatial arrangements of differentiated tissues in
vertebrates, both
adult and embryonic (e.g., induction of head formation during vertebrate
development or
maintenance of hematopoietic progenitor cells); (10) modulation of cell death,
such as
stimulation of cell survival; (11) regulating cell migration; and/or (12)
immune
modulation.
As referred to herein, "differentiation-specific proteins" include proteins
involved in the transition of a cell from the undifferentiated to the
differentiated
phenotype. For example, such proteins can be differentiation specific
structural proteins
or differentiation-specific transcription factors. Such differentiation-
specific proteins are
generally expressed at higher levels in cells which are making the transition
from the
undifferentiated to the differentiated phenotype (e.g., during embryonic
development or
during regeneration of mature tissue in the adult animal), or are expresed at
higher levels
in fully-differentiated or terminally-differentiated cells as compared to
their
undifferentiated counterparts. Also, as referred to herein, "differentiation-
specific
genes" include nucleic acid molecules which encode differentiation-specific
proteins.
Accordingly, another embodiment of the invention features isolated Dkk
proteins
and polypeptides having a Dkk activity. Preferred Dkk proteins have at least
one
cysteine-rich region and a Dkk activity. In another preferred embodiment, the
Dkk
protein has at least one cysteine-rich region, wherein the cysteine-rich
region comprises
at least one cysteine-rich domain, and a Mk activity. In another preferred
embodiment,
the Dkk protein has at least one cysteine-rich region, wherein the cysteine-
rich region
comprises at least two cysteine-rich domains, and a Dick activity. In yet
another
preferred embodiment, a Dkk protein further comprises a signal sequence. In
still
another preferred embodiment, a Dkk protein has a cysteine-rich region, a Dkk
activity,
and an amino acid sequence sufficiently homologous to an amino acid sequence
of SEQ
ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:21.
A preferred Dkk fragment comprises a carboxy-terminal cysteine-rich domain.
In one embodiment, a Dkk fragment comprises a carboxy-terminal cysteine-rich
domain
and retains a biological activity of a Dkk protein. In yet another embodiment,
a Dkk
fragment lacks an amino-terminal cysteine-rich domain.
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The human Dkk-3 cDNA, which is approximately 2479 nucleotides in length,
encodes a protein which is approximately 350 amino acid residues in length.
The
human Dkk-3 protein contains an N-terminal signal sequence and a cysteine-rich
region
comprising two cysteine-rich domains. A Dkk cysteine-rich region can be found
at
least, for example, from about amino acids 147-284 of SEQ ID NO:2. The hDkk-3
cysteine-rich region comprises an amino-terminal cysteine-rich domain from
about
amino acids 147-195 of SEQ ID NO:2 and a carboxy-terminal cysteine-rich domain
from about amino acids 201-284 of SEQ ID NO:2. The human Dkk-3 protein is a
secreted protein which further contains a signal sequence at about amino acids
1-21, 1-
22, 1-23, or 1-24 of SEQ ID NO:2. Accordingly, a mature human Dkk-3 protein
begins
at about amino acid residue 22, 23, 24, or 25 of SEQ ID NO:2 and is about 329,
328,
327, or 326 amino acids in length. The prediction of such a signal peptide can
be made,
for example, utilizing the computer algorithm SIGNALP (Nielsen, et al., (1997)
Protein
Engineering 10:1-6).
The human Dkk-4 cDNA, which is approximately 848 nucleotides in length,
encodes a protein which is approximately 224 amino acid residues in length.
The
human Dkk-4 protein contains an N-terminal signal sequence and a cysteine-rich
region
comprising two cysteine-rich domains. A Dkk cysteine-rich region can be found
at
least, for example, from about amino acids 41-218 of SEQ ID NO:5. The hDkk-4
cysteine-rich region comprises an amino-terminal cysteine-rich domain from
about
amino acids 41-90 of SEQ ID NO:5 and a carboxy-terminal cysteine-rich domain
from
about amino acids 138-218 of SEQ ID NO:5. The human Dkk-4 protein is a
secreted
protein which further contains a signal sequence at about amino acids 1-17, 1-
18, 1-19,
or 1-20 of SEQ ID NO:5. Accordingly, a mature human Dkk-4 protein begins at
about
amino acid residue 18, 19, 20, or 21 of SEQ ID NO:5 and is about 207, 206,
205, or 204
amino acids in length. A preferred fragment of hDkk-4 comprises amino acid
residues
134-224 of SEQ ID NO:5. In another embodiment, a preferred fragment of hDkk-4
consists of amino acid residues 134-224 of SEQ ID NO:5.
The human Dkk-1 cDNA, which is approximately 1536 nucleotides in length,
encodes a protein which is approximately 266 amino acid residues in length.
The
human Dkk-1 protein contains an N-terminal signal sequence and a cysteine-rich
region
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comprising two cysteine-rich domains. A Dkk cysteine-rich region can be found
at
least, for example, from about amino acids 85-263 of SEQ ID NO:8. The hDldc-1
cysteine-rich region comprises an amino-terminal cysteine-rich domain from
about
amino acids 85-138 of SEQ ID NO:8 and a carboxy-terminal cysteine-rich domain
from
about amino acids 182-263 of SEQ ID NO:8. The human Dkk-1 protein is a
secreted
protein which further contains a signal sequence at about amino acids 1-18, 1-
19, 1-20,
or 1-21 of SEQ ID NO:8. Accordingly, a mature human Dkk-1 protein begins at
about
amino acid residue 19, 20, 21, or 22 of SEQ ID NO:8 and is about 248, 247,
246, or 245
amino acids in length.
The human Dkk-2 cDNA, which is approximately 3687 nucleotides in length,
encodes a protein which is approximately 259 amino acid residues in length.
The
human Dkk-2 protein contains a cysteine-rich region comprising two cysteine-
rich
domains. A Dkk cysteine-rich region can be found at least, for example, from
about
amino acids 78-256 of SEQ ID NO:21. The hDkk-2 cysteine-rich region comprises
an
amino-terminal cysteine-rich domain from about amino acids 78-127 of SEQ ID
NO:21
and a carboxy-terminal cysteine-rich domain from about amino acids 176-256 of
SEQ
ID NO:21. The human Dkk-2 protein is a secreted protein which further contains
a
signal sequence at about amino acids 1-31, 1-32, 1-33, or 1-34 of SEQ ID
NO:21.
Accordingly, a mature human Dkk-2 protein begins at about amino acid residue
32, 33,
34, or 35 of SEQ ID NO:21 and is about 228, 227, 226, or 225 amino acids in
length.
Dkk proteins of the present invention can be used to identify additional Dkk-
related proteins or family members. For example, a protein having homology to
hDkk-3
was identified using the nucleotide sequence encoding the N-terminal unique
region of
hDkk-3 to search a nucleotide sequence database. A human cDNA clone (Accession
No.: AA397836) was identified from the dBEST database as having homology to
hDkk-
3 and was fully sequenced. The encoded protein is referred to herein as human
"Soggy-
1" or "Dkk-like-N". The nucleotide and predicted amino acid sequence of human
Soggy-1 are depicted in Figure 7. The nucleotide sequence of human Soggy-1
(SEQ ID
NO:13) encodes a protein having 242 amino acids (SEQ ID NO:14). The nucleotide
sequence of human Soggy-1 includes a 5' untranslated region containing
nucleotides 1-
74 of SEQ ID NO:13, a coding region containing nucleotides 75-800 of SEQ ID
NO:13
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(corresponding to nucleotides 1-726 of SEQ ID NO:15), and a 3' untranslated
region
containing nucleotides 801-928 of SEQ ID NO:13. The Soggy-I protein (amino
acid
residues 32-132) has 25% identity to an N-terminal domain of human Dkk-3
(consisting
of amino acid residues 22-140) as determined by ALIGN, Myers and Miller,
(1989)
CA BIOS, utilizing a PAM120 weight residue table, a gap length penalty of 12,
and a gap
penalty of 4.
Two murine cDNA clones were further identified from the database and fully
sequenced. Combining the sequence information from these two clones resulted
in a
full-length sequence for murine Soggy-1. The nucleotide sequence and predicted
amino
acid sequence of murine Soggy-1 are depicted in Figure 8. The nucleotide
sequence of
murine Soggy-1 (SEQ ID NO:26) encodes a protein having 230 amino acids (SEQ ID
NO:27). The nucleotide sequence of murine Soggy-1 includes a 5' untranslated
region
containing nucleotides 1-56 of SEQ ID NO:26, a coding region containing
nucleotides
57-746 of SEQ ID NO:26 (corresponding to SEQ ID NO:26), and a 3' untranslated
region containing nucleotides 747-835 of SEQ ID NO:26. Human and murine Soggy-
1
proteins display 59% overall identity. An alignment of human and murine Soggy
proteins to human and murine Dkk-3 proteins is depicted in Figure 10.
In one embodiment, a Soggy protein is identified based on the presence of at
least one soggy domain or "SGY" domain in the protein or corresponding nucleic
acid
molecule. As defined herein, a "SGY domain" includes a protein domain of a
Soggy
protein (e.g., hSoggy-1) having an amino acid sequence of about 45-56 amino
acids and
having at least about 25-40% identity with amino acid residues 90-140 of hDkk-
3
(leu90-glu140 of SEQ ID NO:2). In another embodiment, a "SGY domain" has 46-
55,
preferably 47-54, more preferably about 48-53, and even more preferably about
49-52 or
50-51 amino acids, and has at least about 27-38%, preferably about 28-37%,
more
preferably about 29-36%, even more preferably about 30-35%, and even more
preferably
about 31-34%, or 32-33% identity with amino acid residues 90-140 of hDlck-3
(Leu90-
Glu140 of SEQ ID NO:2). In yet another embodiment, a "SGY domain" has the
following consensus sequence: L - P - X(3) - H - X - E - X(7) -G-N - X- T-
X(3) - H -
X(4)-K-X-T-X-N-X(2)-G-X(4)-S-E-X-V-X(2)-S-X(4)-E(SEQID
NO:29). For example, human Soggy-1 has a SGY domain from about amino acid
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residues 81-131 (Leu 81-G1u131 of SEQ ID NO:14) having 33% identity with amino
acid residues 90-140 of hDkk-3 (leu90-glu140 of SEQ ID NO:2). Likewise, murine
Soggy-1 has a SGY domain from about amino acid residues 71-120 (Leu 71-G1u120
of
SEQ ID NO:27) having 33% identity with amino acid residues 90-140 of hDkk-3
(leu90-glu140 of SEQ ID NO:2). The SGY domains of human and murine Soggy-1 are
depicted by shaded boxes in Figure 10.
In another embodiment of the invention, a Soggy protein has at least one SGY
domain and a signal sequence. For example, in one embodiment, a hSoggy-1
protein
contains a signal sequence of about amino acids 1-29,1-30, 1-31, or 1-32 of
SEQ ID
NO:14. Accordingly, a mature hSoggy-1 protein begins at about amino acid
residue 30,
31, 32, or 33 of SEQ ID NO:14 and is about 213, 212, 211, or 210 amino acids
in length.
In another embodiment, a mSoggy-1 protein contains a signal sequence of about
amino
acids 1-19, 1-20, 1-21, or 1-22 of SEQ ID NO:27. Accordingly, a mature mSoggy-
1
protein begins at about amino acid residue 211, 210, 209, or 208 of SEQ ID
NO:28 and
is about 213, 212, 211, or 210 amino acids in length.
Various aspects of the invention are described in further detail in the
following
subsections:
I. Isolated Nucleic Acid Molecules
One aspect of the invention pertains to isolated nucleic acid molecules that
encode Dkk proteins or biologically active portions thereof, as well as
nucleic acid
fragments sufficient for use as hybridization probes to identify Dkk-encoding
nucleic
acids (e.g., Dkk mRNA) and fragments for use as PCR primers for the
amplification or
mutation of Dkk nucleic acid molecules. As used herein, the term "nucleic acid
molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and
RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid molecule can be single-stranded or double-
stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from other
nucleic
acid molecules which are present in the natural source of the nucleic acid.
Preferably, an
"isolated" nucleic acid is free of sequences which naturally flank the nucleic
acid (i.e.,
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sequences located at the 5' and 3' ends of the nucleic acid) in the genomic
DNA of the
organism from which the nucleic acid is derived. For example, in various
embodiments,
the isolated Dkk nucleic acid molecule can contain less than about 5 kb, 4kb,
3kb, 2kb, 1
kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic
acid
molecule in genomic DNA of the cell from which the nucleic acid is derived. An
isolated chromosome is not an isolated nucleic acid molecule as defined
herein.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium when produced
by
recombinant techniques, or substantially free of chemical precursors or other
chemicals
when chemically synthesized.
A nucleic acid molecule of the present invention, e.g., a nucleic acid
molecule
having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ
ID
NO:13, or SEQ ID NO:20, the nucleotide sequence of the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98452, or the nucleotide sequence of
the DNA
insert of the plasmid deposited with ATCC as Accession Number 98633,
or a portion thereof, can be isolated using standard molecular biology
techniques
and the sequence information provided herein. Using all or portion of the
nucleic acid
sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID
NO:20, or the nucleotide sequence of the DNA insert of the plasmid deposited
with
ATCC as Accession Number 98452, or the nucleotide sequence of the DNA insert
of the
plasmid deposited with ATCC as Accession Number 98633, as
a hybridization probe, Dkk nucleic acid molecules can be isolated using
standard
hybridization and cloning techniques (e.g., as described in Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989).
Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID
NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20, or the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
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Accession Number 98452 or,, the nucleotide sequence of the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98633, can be
isolated by the polymerase chain reaction (PCR) using synthetic
oligonucleotide primers
designed based upon the sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ
ID NO:13, or SEQ ID NO:20, or the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number 98452, or the nucleotide
sequence of
the DNA insert of the plasmid deposited with ATCC as Accession Number 98633.
A nucleic acid of the invention can be amplified using cDNA, mRNA or
alternatively, genomic DNA, as a template and appropriate oligonucleotide
primers
according to standard PCR amplification techniques. The nucleic acid so
amplified can
be cloned into an appropriate vector and characterized by DNA sequence
analysis.
Furthermore, oligonucleotides corresponding to Did( nucleotide sequences can
be
prepared by standard synthetic techniques, e.g., using an automated DNA
synthesizer.
In a preferred embodiment, an isolated nucleic acid molecule of the invention
comprises the nucleotide sequence shown in SEQ ID NO: 1. The sequence of SEQ
ID
NO:1 corresponds to the human Dkk-3 cDNA. This cDNA comprises sequences
encoding the human Dkk-3 protein e., "the coding region", from nucleotides 38-
1087),
as well as 5' untranslated sequences (nucleotides 1 to 37) and 3' untranslated
sequences
(nucleotides 1088 to 2479). Alternatively, the nucleic acid molecule can
comprise only
the coding region of SEQ ID NO:1 (e.g., nucleotides 38 to 1087, corresponding
to SEQ
ID NO:3). A plasmid containing the full-length nucleotide sequence encoding
hDkk-3
was deposited with the American Type Culture Collection (ATCC), presently in
Manassas Virginia, on June 11, 1997 and assigned Accession Number 98452.
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID NO:4. The sequence
of
SEQ ID NO:4 corresponds to the human Dkk-4 cDNA. This cDNA comprises
sequences encoding the human Dkk-4 protein (i. e. , "the coding region", from
nucleotides 125-796), as well as 5' untranslated sequences (nucleotides 1 to
124) and 3'
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untranslated sequences (nucleotides 797 to 848). Alternatively, the nucleic
acid
molecule can comprise only the coding region of SEQ ID NO:4 (e.g., nucleotides
125 to
796, corresponding to SEQ ID NO:6).
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID NO:7. The sequence
of
SEQ ID NO:7 corresponds to the human DIck-1 cDNA. This cDNA comprises
sequences encoding the human Dkk-1 protein (L e. , "the coding region", from
nucleotides 109-906), as well as 5' untranslated sequences (nucleotides 1 to
108) and 3'
untranslated sequences (nucleotides 907-1536). Alternatively, the nucleic acid
molecule
can comprise only the coding region of SEQ ID NO:7 (e.g., nucleotides 109-906,
corresponding to SEQ ID NO:9). A plasmid containing the full-length nucleotide
sequence encoding hDkk-1 was deposited with the American Type Culture
Collection
(ATCC), presently in Manassas Virginia, on January 16, 1998 and assigned
Accession
Number 98633.
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID NO:20. The
sequence of
SEQ ID NO:20 corresponds to the human Dkk-2 cDNA. This cDNA comprises
sequences encoding the human Dkk-2 protein (L e. , "the coding region", from
nucleotides 724-1500), 5' untranslated sequences (nucleotides 1-723), as well
as 3'
untranslated sequences (nucleotides 1501-3687). Alternatively, the nucleic
acid
molecule can comprise only the coding region of SEQ ID NO:20 (e.g.,
nucleotides 724
to1500, corresponding to SEQ ID NO:22). A plasmid, clone fthu133, containing
the
full-length nucleotide sequence encoding hDlck-2 was deposited with the
American
Type Culture Collection (ATCC), presently in Manassas Virginia, on March 2,
1999.
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises the nucleotide sequence shown in SEQ ID NO:13. The
sequence of
SEQ ID NO:13 corresponds to the human Soggy cDNA. This cDNA comprises
sequences encoding the human Soggy protein (i.e., "the coding region", from
nucleotides 75 to 800), as well as 5' untranslated sequences (nucleotides 1 to
74) and 3'
untranslated sequences (nucleotides 801 to 928). Alternatively, the nucleic
acid
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molecule can comprise only the coding region of SEQ ID NO:13 (e.g.,
nucleotides 75 to
800, corresponding to SEQ ID NO:15).
In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleic acid molecule which is a complement of the
nucleotide
sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or
SEQ ID NO:20, the nucleotide sequence of the DNA insert of the plasmid
deposited
with ATCC as Accession Number 98452, or the nucleotide sequence of the DNA
insert of
the plasmid deposited with ATCC as Accession Number 98633,
or a portion of any of these nucleotide sequences. A nucleic acid molecule
which is complementary to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20, or the nucleotide sequence
of
the DNA insert of the plasmid deposited with ATCC as Accession Number 98452,
or the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, is one which is sufficiently
complementary to the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4,
SEQ
ID NO:7, SEQ ID NO:13, or SEQ ID NO:20, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number 98452, or the
nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as Accession
Number
98633,
such that it can hybridize to the nucleotide
sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or
SEQ ID NO:20, or the nucleotide sequence of the DNA insert of the plasmid
deposited
with ATCC as Accession Number 98452, or the nucleotide sequence of the DNA
insert of
the plasmid deposited with ATCC as Accession Number 98633,
thereby forming a stable duplex.
In still another preferred embodiment, an isolated nucleic acid molecule of
the
present invention comprises a nucleotide sequence which is at least about 30-
35%,
preferably about 40-45%, more preferably about 50-55%, even more preferably
about
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60-65%, and even more preferably at least about 70-75%, 80-85%, 90-95% or more
homologous to the nucleotide sequences shown in SEQ ID NO:1, SEQ ID NO:3, SEQ
ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:20, or SEQ ID NO:22, the nucleotide sequence of the DNA insert of
the
plasmid deposited with ATCC as Accession Number 98452, or the nucleotide
sequence of
the DNA insert of the plasmid deposited with ATCC as Accession Number 98633,
or a portion of any of these nucleotide sequences.
In one aspect, the present invention features isolated nucleic acid molecules
which are linear (e.g., linear fragments of double-stranded DNA, linear
strands of single-
stranded DNA, single-stranded RNA molecules, and oligonucleotides). Another
aspect
of the present invention features circular nucleic acid molecules (e.g.,
double-standed
DNA molecules, for example, plasmid molecules including the nucleotide
sequences
shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:20, or SEQ ID NO:22, the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98452, or the nucleotide sequence of the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98633,
or a portion of any of these nucleotide sequences).
In one embodiment, the isolated nucleic acid molecules of the present
invention
are DNA molecules which are in a form suitable for expression (e.g., suitable
for
expression of corresponding messenger RNA or mRNA). In another embodiment, the
isolated nucleic acid molecules are DNA molecules which are in a form suitable
for
expression of corresponding protein (e.g., in a form, for example, in a
vector, which is
capable of expressing protein, e.g., in the appropriate orientation for
expression from
regulatory elements and/or in-frame with appropriate regulatory elements). In
another
embodiment, the isolated nucleic acids are in a form suitable for
determination of
nucleic acid sequence (e.g., in a form suitable for sequencing, for example,
is a
sequencing vector including a M13, T7, T3 and SP6 promoter. Examples of
sequencing
vectors include, but are not limited to pBluescripik(StratageneTm), pT7T3D
(PharmciaTM)
* Trademark
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and pCR2.1 (InVitrogen). In yet another embodiment, the isolated nucelic acid
molecules are free from vector sequences. In a preferred embodiment, an
isolated
nucleic acid molecule is free from sequencing vector sequences.
Moreover, the nucleic acid molecule of the invention can comprise only a
portion
of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID
NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number 98452, or the nucleotide sequence of
the DNA
insert of the plasmid deposited with ATCC as Accession Number 98633,
for example a fragment which can be used as a probe or
primer or a fragment encoding a biologically active portion of a Dkk protein
or Dkk-
related protein. The nucleotide sequence determined from the cloning of the
human Dkk
genes allows for the generation of probes and primers designed for use in
identifying
and/or cloning Dkk homologues in other cell types, e.g., from other tissues,
as well as
Dkk homologues from other mammals and Dkk-related proteins. The probe/primer
typically comprises substantially purified oligonucleotide. The
oligonucleotide typically
comprises a region of nucleotide sequence that hybridizes under stringent
conditions to
at least about 12, preferably about 25, more preferably about 40, 50 or 75
consecutive
nucleotides of a sense sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ
ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98452, or the nucleotide sequence of
the DNA
insert of the plasmid deposited with ATCC as Accession Number 98633,
of an anti-sense sequence of SEQ ID NO:1, SEQ ID NO:4,
SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number 98452, or the
nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as Accession
Number
98633,
or of a naturally occurring mutant of SEQ ID
NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as Accession
Number
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98452, Or the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC
as Accession Number 98633.
In an exemplary
embodiment, a nucleic acid molecule of the present invention comprises a
nucleotide
sequence which hybridizes under stringent hybridization conditions to a
nucleic acid
molecule consisting of nucleotides 470-2479 of SEQ ID NO:1 or to a nucleic
acid
molecule consisting of nucleotides 1-475 of SEQ ID NO :4.
Probes based on human nucleotide sequences (e.g., the human DIdc nucleotide
sequence) can be used to detect transcripts or genomic sequences encoding the
same or
homologous proteins. For instance, primers based on the nucleic acid
represented in
SEQ ID NOs:1 or 3 can be used in PCR reactions to clone Dkk homologs (e.g.,
hDkk-3
homologues). In a preferred embodiment of the invention, Dick homologs are
cloned by
PCR amplification (e.g., RT-PCR) using primers hybridizing to a portion of the
nucleotide sequence encoding the Dkk cysteine rich domain. Likewise, probes
based on
the subject Dick sequences can be used to detect transcripts or genomic
sequences
encoding the same or homologous proteins. In preferred embodiments, the probe
further
comprises a label group attached thereto, e.g., the label group can be a
radioisotope, a
fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be
used as
a part of a diagnostic test kit for identifying cells or tissue which
misexpress a Dkk
protein, such as by measuring a level of a 131(1c-encoding nucleic acid in a
sample of cells
from a subject e.g., detecting Dkk mRNA levels or determining whether a
genomic Dkk
gene has been mutated or deleted.
A nucleic acid fragment encoding a "biologically active portion of a Dick or
Dkk-
related protein" can be prepared by isolating a portion of SEQ ID NO:1, SEQ ID
NO:4,
SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number 98452, or the
nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as Accession
Number
98633, which encodes a polypeptide having a biological activity (the
biological activities
of the Dkk and Dick-related proteins have previously
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been described), expressing the encoded portion of the protein (e.g., by
recombinant
expression in vitro) and assessing the activity of the encoded portion of the
protein.
The invention further encompasses nucleic acid molecules that differ from the
nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID
NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number 98452, or the nucleotide sequence of
the DNA
insert of the plasmid deposited with ATCC as Accession Number 98633,
due to degeneracy of the genetic code and thus encode the
same proteins as those encoded by the nucleotide sequence shown in SEQ ID
NO:1,
SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence
of the DNA insert of the plasmid deposited with ATCC as Accession Number
98452,
or the nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as
Accession Number 98633.
In another embodiment, an isolated
nucleic acid molecule of the invention has a nucleotide sequence encoding a
protein
having an amino acid sequence shown in SEQ ID NO:2, SEQ ID NO: 5, SEQ ID NO:8,
SEQ ID NO:14, or SEQ ID NO:21.
In addition to the human nucleotide sequences shown in SEQ ID NO:1, SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number 98452, or
the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633,
it will be appreciated by those
skilled in the art that DNA sequence polymorphisms that lead to changes in the
amino
acid sequences of the Dkk or Dkk-related proteins may exist within a
population (e.g.,
the human population). Such genetic polymorphism in the Dkk or Dkk-related
genes
may exist among individuals within a population due to natural allelic
variation. As
used herein, the terms "gene" and "recombinant gene" refer to nucleic acid
molecules
comprising an open reading frame encoding a protein, preferably a mammalian
Dkk or
Dkk-related protein. Such natural allelic variations can typically result in 1-
5% variance
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in the nucleotide sequence of a Dkk or Dkk-related gene. Any and all such
nucleotide
variations and resulting amino acid polymorphisms in genes that are the result
of natural
allelic variation and that do not alter the functional activity of a Dkk or
Dkk-related
protein are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding Dkk or Dkk-related proteins from
other species, and thus which have a nucleotide sequence which differs from
the human
sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID
NO:20, the nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC
as Accession Number 98452, or the nucleotide sequence of the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98633, are
intended to be within the scope of the invention. For example, a murine Dkk-3
cDNA
has been identified based of the nucleotide sequence of human Dkk-3. The
nucleotide
sequence of murine Dkk-3 (SEQ ID NO:16) encodes a hDkk-3 protein having 349
amino acids. The nucleotide and amino acid sequences of murine Dkk-3 are
depicted in
Figure 5. The coding region of murine Dkk-3 is represented by SEQ ID NO:18. A
plasmid containing the full-length nucleotide sequence encoding mDkk-3 was
deposited
with the American Type Culture Collection (ATCC), presently in Manassas,
Virginia,
on January 16, 1998 and assigned Accession Number 98634. Likewise, a murine
Dkk-
related protein (Soggy-1) has been identified based of the nucleotide sequence
of human
Dkk-3. The nucleotide sequence of murine Soggy-1 (SEQ ID NO:26) encodes a
protein
having 230 amino acids (SEQ ID NO:27). The nucleotide and amino acid sequences
of
murine Soggy-1 are depicted in Figure 8. The coding region of murine Soggy-1
is
represented by SEQ ID NO:28.
Nucleic acid molecules corresponding to natural allelic variants and
homologues
of the Dkk or Dick-related cDNAs of the invention can be isolated based on
their
homology to the human nucleic acids disclosed herein using the human cDNA, or
a
portion thereof, as a hybridization probe according to standard hybridization
techniques
under stringent hybridization conditions. Examples of tissues and/or libraries
suitable
for isolation of the subject nucleic acids include brain, spinal chord and
heart tissue.
cDNA encoding a Dkk protein (e.g., a hDlck-3 protein) can be obtained by
isolating total
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,
mRNA from a cell, e.g., a vertebrate cell, a mammalian cell, or a human cell,
including
embryonic cells. Double stranded cDNAs can then be prepared from the total
mRNA,
and subsequently inserted into a suitable plasmid or bacteriophage vector
using any one
of a number of known techniques. The gene encoding a hDkk-3 protein can also
be
cloned using established polymerase chain reaction techniques in accordance
with the
nucleotide sequence information provided by the invention. The nucleic acid of
the
invention can be DNA or RNA or analogs thereof.
Accordingly, in another embodiment, an isolated nucleic acid molecule of the
invention is at least 15 nucleotides in length and hybridizes under stringent
conditions to
the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,
SEQ ID
NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number 98452, or
the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633.
In another embodiment, the nucleic
acid is at least 30, 50, 100, 250, 300, 400 or 500 nucleotides in length. As
used herein,
the term "hybridizes under stringent conditions" is intended to describe
conditions for
hybridization and washing under which nucleotide sequences at least 60%
homologous
to each other typically remain hybridized to each other. Preferably, the
conditions are
such that sequences at least about 70%, more preferably at least about 80%,
even more
preferably at least about 85% or 90% homologous to each other typically remain
hybridized to each other. Such stringent conditions are known to those skilled
in the art
and can be found in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y.
(1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization
conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at
about 45 C,
followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 C. Preferably,
an
isolated nucleic acid molecule of the invention that hybridizes under
stringent conditions
to the sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleic
acid
molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers
to an
RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g.,
encodes
a natural protein).
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In addition to naturally-occurring allelic variants of the Mk or Dkk-related
sequences that may exist in the population, the skilled artisan will further
appreciate that
changes can be introduced by mutation into the nucleotide sequences of SEQ ID
NO:1,
SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence
of the DNA insert of the plasmid deposited with ATCC as Accession Number
98452,
or the nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as
Accession Number 98633,
thereby leading to changes in the
amino acid sequence of the encoded Dick proteins, without altering the
functional ability
of the Dkk proteins. For example, nucleotide substitutions leading to amino
acid
substitutions (particularly conservative amino acid substitutions) at "non-
essential"
amino acid residues can be made in the sequence of SEQ ID NO:1, SEQ ID NO:4,
SEQ
ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert
of
the plasmid deposited with ATCC as Accession Number 98452, or the nucleotide
sequence
of the DNA insert of the plasmid deposited with ATCC as Accession Number
98633.
A "non-essential" amino acid residue is a residue that can be
altered from the wild-type sequence of Dldc (or wild-type Dick-related
sequence) (e.g.,
the sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ
ID NO:21) without altering the biological activity, whereas an "essential"
amino acid
residue is required for biological activity. For example, amino acid residues
that are
conserved among the Dkk or Dkk-related proteins of the present invention
(e.g., cysteine
residues within cysteine-rich domains), are predicted to be particularly
unamenable to
alteration. Furthermore, amino acid residues that are conserved between Dick
protein
and other proteins having cysteine-rich domains are not likely to be amenable
to
alteration.
Accordingly, another aspect of the invention pertains to nucleic acid
molecules
encoding Dick or Dkk-related proteins that contain changes in amino acid
residues that
are not essential for activity. Such proteins differ in amino acid sequence
from SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21 yet retain
biological activity. In one embodiment, the isolated nucleic acid molecule
comprises a
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nucleotide sequence encoding a protein, wherein the protein comprises an amino
acid
sequence at least about 60% homologous to the amino acid sequence of SEQ ID
NO:2,
SEQ ID NO:5, SEQ ID NO:8õ SEQ ID NO:14, or SEQ ID NO:21. Preferably, the
protein encoded by the nucleic acid molecule is at least about 65-70%
homologous to
SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, more
preferably at least about 75-80% homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ
ID
NO:8, SEQ ID NO:14, or SEQ ID N0:21, even more preferably at least about 85-
90%
homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID N0:14, or SEQ
ID N0:21, and most preferably at least about 95% homologous to SEQ ID NO:2,
SEQ
ID NO:5, SEQ ID NO:8, SEQ ID N0:14, or SEQ ID NO:21.
An isolated nucleic acid molecule encoding a DI& protein homologous to the
protein of SEQ ID NO:2, SEQ ID N0:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID
NO:21 can be created by introducing one or more nucleotide substitutions,
additions or
deletions into the nucleotide sequence of SEQ ID NO: I, SEQ ID NO:4, SEQ ID
NO:7,
SEQ ID N0:13, SEQ ID N0:20, the nucleotide sequence of the DNA insert of the
plasmid deposited with ATCC as Accession Number 98452, or the nucleotide
sequence of
the DNA insert of the plasmid deposited with ATCC as Accession Number 98633,
such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be introduced
into
SEQ ID NO:1, SEQ ID N0:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:20, the
nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98452, or the nucleotide sequence of the DNA insert of the
plasmid
deposited with ATCC as Accession Number 98633, by
standard techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
Preferably, conservative amino acid substitutions are made at one or more
predicted
non-essential amino acid residues. A "conservative amino acid substitution" is
one in
which the amino acid residue is replaced with an amino acid residue having a
similar
side chain. Families of amino acid residues having similar side chains have
been
defined in the art. These families include amino acids with basic side chains
(e.g.,
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lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,
glutamic acid),
uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine,
phenylalanine,
tryptophan, histidine). Thus, for example, a predicted nonessential amino acid
residue in
a Dkk protein (e.g., one not located in a cysteine-rich domain) is preferably
replaced
with another amino acid residue from the same side chain family.
Alternatively, in
another embodiment, mutations can be introduced randomly along all or part of
a Dick or
Dkk-related coding sequence, such as by saturation mutagenesis, and the
resultant
mutants can be screened for biological activity to identify mutants that
retain activity.
Following mutagenesis of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13,
SEQ ID NO:20, the nucleotide sequence of the DNA insert of the plasmid
deposited
with ATCC as Accession Number 98452, or the nucleotide sequence of the DNA
insert of
the plasmid deposited with ATCC as Accession Number 98633,
the encoded protein can be expressed recombinantly and the activity of the
protein can be determined.
In a preferred embodiment, a mutant Dick or Dick-related protein can be
assayed
for intracellular calcium, an increase in phosphatidylinositol or other
molecule, and can
result, e.g., in phosphorylation of specific proteins, a modulation of gene
transcription
and any of the other biological activities set forth herein.
In a preferred embodiment, a mutant Dkk or Dick-related protein can also be
assayed for the ability to (1) modulate cellular signal transduction, either
in vitro or in
vivo; (2) regulate communication between cells; (3) regulate expression of
genes whose
expression is modulated by binding of Dick (e.g., hDlck-3) to a receptor; (4)
regulate
gene transcription in a cell involved in development or differentiation,
either in vitro or
in vivo; (5) regulate cellular proliferation, either in vitro or in vivo; (6)
form and/or
maintain ordered spatial arrangements of differentiated tissues in
vertebrates; (7)
modulate cell death (e.g. cell survival); (8) regulate cell migration; and/or
(9) modulate
immune system function.
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In addition to the nucleic acid molecules encoding Dkk or Dkk-related proteins
described above, another aspect of the invention pertains to isolated nucleic
acid
molecules which are antisense thereto. An "antisense" nucleic acid comprises a
nucleotide sequence which is complementary to a "sense" nucleic acid encoding
a
protein, e.g., complementary to the coding strand of a double-stranded cDNA
molecule
or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid
can
hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be
complementary to an entire Dkk coding strand, or to only a portion thereof. In
one
embodiment, an antisense nucleic acid molecule is antisense to a "coding
region" of the
coding strand of a nucleotide sequence encoding Dkk. The term "coding region"
refers
to the region of the nucleotide sequence comprising codons which are
translated into
amino acid residues (e.g., the coding region of human Dkk-3 corresponds to SEQ
ID
NO:3, the coding region of human Dkk-4 corresponds to SEQ ID NO:6, the coding
region of human Dkk-1 corresponds to SEQ ID NO:9, the coding region of human
Dkk-
2 corresponds to SEQ ID NO:22, and the coding region of human Soggy
corresponds to
SEQ ID NO:15). In another embodiment, the antisense nucleic acid molecule is
antisense to a "noncoding region" of the coding strand of a nucleotide
sequence
encoding a Dkk or Dkk-related protein. The term "noncoding region" refers to
5' and 3'
sequences which flank the coding region that are not translated into amino
acids (i.e.,
also referred to as 5' and 3' untranslated regions).
Given the coding strand sequences disclosed herein (e.g., SEQ ID NO:3, SEQ ID
NO:6, SEQ ID NO:9, SEQ ID NO:15, or SEQ ID NO:22), antisense nucleic acids of
the
invention can be designed according to the rules of Watson and Crick base
pairing. The
antisense nucleic acid molecule can be complementary to the entire coding
region of a
Dkk or Dkk-related mRNA, but more preferably is an oligonucleotide which is
antisense
to only a portion of the coding or noncoding region of the mRNA. For example,
the
antisense oligonucleotide can be complementary to the region surrounding the
translation start site of Dkk mRNA. An antisense oligonucleotide can be, for
example,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90
nucleotides in
length. In a preferred embodiment, an oligonucleotide is about 30-90,
preferably about
40-80, more preferably about 50-70 nucleotides in length and is antisense to a
portion of
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SEQ ID NO:1 from about nucleotides 1-150. In another embodiment, an
oligonucleotide is antisense to a portion of SEQ ID NO:4 from about
nucleotides 25-
225. In another embodiment, an oligonucleotide is antisense to a portion of
SEQ ID
NO:7 from about nucleotides 1-200. In another embodiment, an oligonucleotide
is
antisense to a portion of SEQ ID NO:20 from about nucleotides 625-825. In yet
another
embodiment, an oligonucleotide is antisense to a portion of SEQ ID NO:13 from
about
nucleotides 1-175.
An antisense nucleic acid of the invention can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known in the art.
For
example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be
chemically
synthesized using naturally occurring nucleotides or variously modified
nucleotides
designed to increase the biological stability of the molecules or to increase
the physical
stability of the duplex formed between the antisense and sense nucleic acids,
e.g.,
phosphorothioate derivatives, acridine substituted nucleotides, can be used.
Alternatively, the antisense nucleic acid molecule can by synthesized to
increase
transport across cellular membranes, e.g., methylphosphonate derivatives. The
antisense
molecules can include a 3'-terminal cap (e.g., a 3'-aminopropyl modification),
a biotin
moiety, or even a 3'-3' terminal linkage.
Examples of modified nucleotides which can be used to generate the antisense
nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-
iodouracil,
hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-
carboxymethylaminomethy1-2-thiouridine, 5-carboxymethylaminomethyluracil,
dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-
methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-
methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-
methylguanine, 5-
methylaminomethyluracil, 5-methoxyaminomethy1-2-thiouracil, beta-D-
mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-
N6-
isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,
queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
methyluracil, uracil-5-
oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-
thiouracil, 3-(3-
amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
Alternatively, the
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antisense nucleic acid can be produced biologically using an expression vector
into
which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA
transcribed from the inserted nucleic acid will be of an antisense orientation
to a target
nucleic acid of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically
administered
to a subject or generated in situ such that they hybridize with or bind to
cellular mRNA
and/or genomic DNA encoding a Dkk or Dkk-related protein to thereby inhibit
expression of the protein, e.g., by inhibiting transcription and/or
translation. The
hybridization can be by conventional nucleotide complementarity to form a
stable
duplex, or, for example, in the case of an antisense nucleic acid molecule
which binds to
DNA duplexes, through specific interactions in the major groove of the double
helix.
An example of a route of administration of antisense nucleic acid molecules of
the
invention include direct injection at a tissue site. Alternatively, antisense
nucleic acid
molecules can be modified to target selected cells and then administered
systemically.
For example, for systemic administration, antisense molecules can be modified
such that
they specifically bind to receptors or antigens expressed on a selected cell
surface, e.g.,
by linking the antisense nucleic acid molecules to peptides or antibodies
which bind to
cell surface receptors or antigens. The antisense nucleic acid molecules can
also be
delivered to cells using the vectors described herein. To achieve sufficient
intracellular
concentrations of the antisense molecules, vector constructs in which the
antisense
nucleic acid molecule is placed under the control of a strong poi II or poi
III promoter
are preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention
is an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule
forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual 13-units, the strands run parallel to each other (Gaultier et al.,
(1987) Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-
o-
methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15:6131-6148) or
a
chimeric RNA-DNA analogue (Inoue et al., (1987) FEBS Lett. 215:327-330).
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In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity
which are
capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which
they
have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to
catalytically cleave Dkk or Dkk-related mRNA transcripts to thereby inhibit
translation
of Dkk or Dkk-related mRNA. A ribozyme having specificity for a Dkk- or Dkk-
related-encoding nucleic acid can be designed based upon the nucleotide
sequence of a
Dkk or Dkk-related cDNA disclosed herein (i.e., SEQ ID NO:1, SEQ ID NO:4, SEQ
ID
NO:7, SEQ ID NO:13, SEQ ID NO:20, the nucleotide sequence of the DNA insert of
the
plasmid deposited with ATCC as Accession Number 98452, the nucleotide sequence
of
the DNA insert of the plasmid deposited with ATCC as Accession Number 98633).
For
example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in
which the
nucleotide sequence of the active site is complementary to the nucleotide
sequence to be
cleaved in a Dkk-encoding mRNA. See, e.g., Cech etal., U.S. Patent No.
4,987,071;
and Cech etal., U.S. Patent No. 5,116,742. Alternatively, Dkk (or Dkk-related)
mRNA
can be used to select a catalytic RNA having a specific ribonuclease activity
from a pool
of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science
261:1411-
1418.
Alternatively, gene expression can be inhibited by targeting nucleotide
sequences
complementary to the regulatory region of the Dkk or Dkk-related gene (e.g.,
the
promoter and/or enhancers) to form triple helical structures that prevent
transcription of
the gene in target cells. See generally, Helene, C. (1991) Anticancer Drug
Des.
6(6):569-84; Helene, C. etal., (1992) Ann. N.Y. Acad. Sci. 660:27-36; and
Maher, L.J.
(1992) Bioassays 14(12):807-15.
In yet another embodiment, the nucleic acid molecules of the present invention
can be modified at the base moiety, sugar moiety or phosphate backbone to
improve,
e.g., the stability, hybridization, or solubility of the molecule. For
example, the
deoxyribose phosphate backbone of the nucleic acid molecules can be modified
to
generate peptide nucleic acids (see Hyrup B. etal. (1996) Bioorganic &
Medicinal
Chemistry 4 (1): 5-23). As used herein, the terms "peptide nucleic acids" or
"PNAs"
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refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose
phosphate
backbone is replaced by a pseudopeptide backbone and only the four natural
nucleobases
are retained. The neutral backbone of PNAs has been shown to allow for
specific
hybridization to DNA and RNA under conditions of low ionic strength. The
synthesis
of PNA oligomers can be performed using standard solid phase peptide synthesis
protocols as described in Hyrup B. etal. (1996) supra; Perry-O'Keefe etal.
PNAS 93:
14670-675.
PNAs of Dkk or Dkk-related nucleic acid molecules can be used therapeutic and
diagnostic applications. For example, PNAs can be used as antisense or
antigene agents
for sequence-specific modulation of gene expression by, for example, inducing
transcription or translation arrest or inhibiting replication. PNAs of Dkk or
Dkk-related
nucleic acid molecules can also be used in the analysis of single base pair
mutations in a
gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction
enzymes' when
used in combination with other enzymes, (e.g., Si nucleases (Hyrup B. (1996)
supra));
or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al.
(1996)
supra; Perry-O'Keefe supra).
In another embodiment, PNAs of Dkk can be modified, (e.g., to enhance their
stability or cellular uptake), by attaching lipophilic or other helper groups
to PNA, by
the formation of PNA-DNA chimeras, or by the use of liposomes or other
techniques of
drug delivery known in the art. For example, PNA-DNA chimeras of Dkk nucleic
acid
molecules can be generated which may combine the advantageous properties of
PNA
and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA
polymerases), to interact with the DNA portion while the PNA portion would
provide
high binding affinity and specificity. PNA-DNA chimeras can be linked using
linkers of
appropriate lengths selected in terms of base stacking, number of bonds
between the
nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA
chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J.
et al.
(1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be
synthesized on a solid support using standard phosphoramidite coupling
chemistry and
modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-
thymidine
phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag,
M. et
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al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a
stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3'
DNA
segment (Finn P.J. et al. (1996) supra). Alternatively, chimeric moleclues can
be
synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al.
(1975)
Bioorganic Med. Chem. Lett. 5: 1119-11124).
In other embodiments, the oligonucleotide may include other appended groups
such as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating
transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc.
Natl. Acad.
Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA
84:648-652;
PCT Publication No. W088/09810, published December 15, 1988) or the blood-
brain
barrier (see, e.g., PCT Publication No. W089/10134, published April 25, 1988).
In
addition, oligonucleotides can be modified with hybridization-triggered
cleavage agents
(See, e.g., Krol etal. (1988) BioTechniques 6:958-976) or intercalating
agents. (See,
e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may
be
conjugated to another molecule, (e.g., a peptide, hybridization triggered
cross-linking
agent, transport agent, or hybridization-triggered cleavage agent).
II. Isolated Dkk Proteins and Anti-Dkk Antibodies
One aspect of the invention pertains to isolated Dkk proteins, Dkk-related
proteins and biologically active portions thereof, as well as polypeptide
fragments
suitable for use as immunogens to raise antibodies. In one embodiment, native
Dkk or
Dkk-related proteins can be isolated from cells or tissue sources by an
appropriate
purification scheme using standard protein purification techniques. In another
embodiment, proteins are produced by recombinant DNA techniques. Alternative
to
recombinant expression, a Dkk or Dkk-related protein or polypeptide can be
synthesized
chemically using standard peptide synthesis techniques.
An "isolated" or "purified" protein or biologically active portion thereof is
substantially free of cellular material or other contaminating proteins from
the cell or
tissue source from which the Dkk or Dkk-related protein is derived, or
substantially free
from chemical precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes preparations of
protein in
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which the protein is separated from cellular components of the cells from
which it is
isolated or recombinantly produced. In one embodiment, the language
"substantially
free of cellular material" includes preparations of Dkk or Dkk-related protein
having less
than about 30% (by dry weight) of non-Dkk protein or non-Dkk-related protein
(also
referred to herein as a "contaminating protein"), more preferably less than
about 20% of
non-Dkk protein or non-Dkk-related protein, still more preferably less than
about 10%
of non-Dkk protein or non-Dkk-related protein, and most preferably less than
about 5%
non-Dkk protein or non-Dkk-related protein. When the Dkk or Dkk-related
protein or
biologically active portion thereof is recombinantly produced, it is also
preferably
substantially free of culture medium, i.e., culture medium represents less
than about
20%, more preferably less than about 10%, and most preferably less than about
5% of
the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals"
includes preparations of Dkk or Dkk-related protein in which the protein is
separated
from chemical precursors or other chemicals which are involved in the
synthesis of the
protein. In one embodiment, the language "substantially free of chemical
precursors or
other chemicals" includes preparations of Dkk protein having less than about
30% (by
dry weight) of chemical precursors, non-Dkk chemicals, or non-Dick-related
chemicals,
more preferably less than about 20% chemical precursors, non-Dick chemicals,
or non-
Dick-related chemicals, still more preferably less than about 10% chemical
precursors,
non-Dkk chemicals, or non-Dkk-related chemicals, and most preferably less than
about
5% chemical precursors, non-Dkk chemicals, or non-Dkk-related chemicals.
Biologically active portions of a Dkk or Dkk-related protein include peptides
comprising amino acid sequences sufficiently homologous to or derived from the
amino
acid sequence of the Dick or Dkk-related protein, e.g., the amino acid
sequence shown in
SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, which
include less amino acids than the full length proteins, and exhibit at least
one activity of
a Dkk or Dkk-related protein. Typically, biologically active portions comprise
a domain
or motif with at least one activity of the Dkk or Dkk-related protein. A
biologically
active portion of a protein can be a polypeptide which is, for example, 10,
25, 50, 100 or
more amino acids in length.
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In one embodiment, a biologically active portion of a Dkk protein comprises at
least a cysteine-rich region. In another embodiment, a biologically active
portion of a
Dkk protein comprises at least a cysteine-rich region, wherein the cysteine-
rich region
includes at least one cysteine-rich domain. In yet another embodiment, a
biologically
active portion of a Dkk protein comprises at least a signal sequence.
In another embodiment, a biologically active portion of a Dkk-related protein
(e.g., a Soggy protein) comprises at least a Soggy domain. In yet another
embodiment, a
biologically active portion of a Dkk-related protein comprises at least a
signal sequence.
In an alternative embodiment, a biologically active portion of a Dkk or Dkk-
related protein comprises an amino acid sequence lacking a signal sequence.
It is to be understood that a preferred biologically active portion of a Dkk
or
Dkk-related protein of the present invention may contain at least one of the
above-
identified structural domains. A more preferred biologically active portion of
a Dkk or
Dkk-related protein may contain at least two of the above-identified
structural domains.
An even more preferred biologically active portion of a protein may contain at
least
three of the above-identified structural domains. A particularly preferred
biologically
active portion of a protein of the present invention may contain at least four
of the
above-identified structural domains.
Moreover, other biologically active portions, in which other regions of the
protein are deleted, can be prepared by recombinant techniques and evaluated
for one or
more of the functional activities of a native Dkk or Dkk-related protein.
In a preferred embodiment, the Dkk protein has an amino acid sequence shown
in SEQ ID NO:2 or an amino acid sequence at least about 55% homologous to SEQ
ID
NO:2. In another preferred embodiment, the Dkk protein has an amino acid
sequence
shown in SEQ ID NO:5 or an amino acid sequence at least about 35% homologous
to
SEQ ID NO:5. In another preferred embodiment, the Dkk protein has an amino
acid
sequence shown in SEQ ID NO:8 or an amino acid sequence at least about 85%
homologous to SEQ ID NO:8. In another preferred embodiment, the Dkk protein
has an
amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence at least
about
35% homologous to SEQ ID NO:21. In another preferred embodiment, the protein
has
an amino acid sequence shown in SEQ ID NO:14 or an amino acid sequence at
least
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about 60% homologous to SEQ ID NO:14. In still another preferred embodiment, a
protein of the present invention comprises an amino acid sequence which is at
least
about 30-35%, preferably about 40-45%, more preferably about 50-55%, even more
preferably about 60-65%, and even more preferably at least about 70-75%, 80-
85%, 90-
95% or more homologous to the amino acid sequences shown in SEQ ID NO:2, SEQ
ID
NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21.
In other embodiments, the protein is substantially homologous to SEQ ID NO:2,
SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, and, preferably,
retains the functional activity of the protein of SEQ ID NO:2, SEQ ID NO:5,
SEQ ID
NO:8, SEQ ID NO:14, or SEQ ID NO:21, yet differs in amino acid sequence due to
natural allelic variation or mutagenesis, as described in detail in subsection
I above.
Accordingly, in another embodiment, the protein is a protein which comprises
an amino
acid sequence at least about 60% homologous to the amino acid sequence of SEQ
ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21 and, preferably,
retains the functional activity of the proteins of SEQ ID NO:2, SEQ ID NO:5,
SEQ ID
NO:8, SEQ ID NO:14, or SEQ ID NO:11, respectively. Preferably, the protein is
at
least about 70% homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID
NO:14, or SEQ ID NO:21, more preferably at least about 80% homologous to SEQ
ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21, even more
preferably at least about 90% homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID
NO:8, SEQ ID NO:14, or SEQ ID NO:21, and most preferably at least about 95% or
more homologous to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or
SEQ ID NO:21.
To determine the percent homology of two amino acid sequences or of two
nucleic acids, the sequences are aligned for optimal comparison purposes
(e.g., gaps can
be introduced in the sequence of a first amino acid or nucleic acid sequence
for optimal
alignment with a second amino or nucleic acid sequence and non-homologous
sequences
can be disregarded for comparison purposes). In one embodiment, an alignment
is a
global alignment, e.g., an overall sequence alignment. In another embodiment,
an
alignment is a local alignment. In a preferred embodiment, the length of a
sequence
aligned for comparison purposes is at least 30%, preferably at least 40%, more
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preferably at least 50%, even more preferably at least 60%, and even more
preferably at
least 70%, 80%, or 90% of the length of the reference sequence to which it is
aligned
(e.g., when aligning a second sequence to the DU amino acid sequence of SEQ ID
NO:2, at least 105, preferably at least 145, more preferably at least 175,
even more
preferably at least 210, and even more preferably at least 245, 280 or 315
amino acid
residues are aligned). The amino acid residues or nucleotides at corresponding
amino
acid positions or nucleotide positions are then compared. When a position in
the first
sequence is occupied by the same amino acid residue or nucleotide as the
corresponding
position in the second sequence, then the molecules are identical at that
position (as used
herein amino acid or nucleic acid "identity" is equivalent to amino acid or
nucleic acid
"homology"). The percent identity between the two sequences is a function of
the
number of identical positions shared by the sequences, taking into account the
number of
gaps, and the length of each gap, which need to be introduced for optimal
alignment of
the two sequences.
The comparison of sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm. In a embodiment,
the
percent identity between two amino acid sequences is determined using the
Needleman
and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been
incorporated
into the GAP program in the GCG software package (Accelrys Software Inc., San
Diego, CA),
using either a Blossotrr62 matrix or a PAM250 matrix, and a gap weight of 16,
14, 12,
10, 8,6, 5, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another
embodiment, the
percent identity between two nucleotide sequences is determined using the GAP
program in the GCG software package (Accelrys Software Inc., San Deigo, CA),
using a
NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight
of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between
two amino
acid or nucleotide sequences is determined using the algorithm of E. Meyers
and W.
Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN
program
(version 2.0), using a PAM120 weight residue table, a gap length penalty of 12
and a
gap penalty of 4.
=
* Trademark
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-
The nucleic acid and protein sequences of the present invention can further be
used as a "query sequence" to perform a search against public databases to,
for example,
identify other family members or related sequences. Such searches can be
performed
using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.
(1990)J.
Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the
NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences
homologous to Dick nucleic acid molecules of the invention. BLAST protein
searches
can be performed with the XBLAST program, score = 50, wordlength =3 to obtain
amino acid sequences homologous to Dick protein molecules of the invention. To
obtain
gapped alignments for comparison purposes, Gapped BLAST can be utilized as
described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters of the
respective
programs (e.g., XBLAST and NBLAST) can be used.
The invention also provides Dick or Dick-related chimeric or fusion proteins.
As
used herein, a "chimeric protein" or "fusion protein" comprises a Didc or Dkk-
related
polypeptide operatively linked to a non-Dkk polypeptide or non-Dkk-related
polypeptide. A "Dkk polypeptide" or "Dkk-related polypeptide" refers to a
polypeptide
having an amino acid sequence corresponding to Didc or a Dkk-related protein,
whereas
a "non-Dkk polypeptide" or "non-Dick-related polypeptide" refers to a
polypeptide
having an amino acid sequence corresponding to a protein which is not
substantially
homologous to the Dkk or Dkk-related protein, e.g., a protein which is
different from the
Dkk or Dick-related protein and which is derived from the same or a different
organism.
Within a Dkk or Dkk-related fusion protein the Dkk or Dick-related polypeptide
can
correspond to all or a portion of a Didc or Dkk-related protein. In a
preferred
embodiment, a Dick or Dick-related fusion protein comprises at least one
biologically
active portion of a Ink protein. In another preferred embodiment, a Dldc or
Dkk-related
fusion protein comprises at least two biologically active portions of a Dkk or
Dkk-
related protein. In another preferred embodiment, a Didc or Dkk-related fusion
protein
comprises at least three biologically active portions of a Dkk or Dick-related
protein.
Within the fusion protein, the term "operatively linked" is intended to
indicate that the
Dick or Dick-related polypeptide and the non-Dkk or non-Dkk-related
polypeptide are
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fused in-frame to each other. The non-Dkk or non-Dkk-related polypeptide can
be fused
to the N-terminus or C-terminus of the Dkk or Dkk-related polypeptide.
For example, in one embodiment, the fusion protein is a GST-Dkk fusion protein
in which the Dkk sequences are fused to the C-terminus of the UST sequences.
Such
fusion proteins can facilitate the purification of recombinant Dkk.
In another embodiment, the fusion protein is a Dkk or Dkk-related protein
containing a heterologous signal sequence at its N-terminus. For example, the
native
Dkk signal sequence (i.e, about amino acids 1 to 23 of SEQ ID NO:2) can be
removed
and replaced with a signal sequence from another protein. In certain host
cells (e.g.,
mammalian host cells), expression and/or secretion of Dkk or Dkk-related
proteins can
be increased through use of a heterologous signal sequence.
In yet another embodiment, the fusion protein is a Dkk-immunoglobulin fusion
protein in which the Dkk sequences comprising primarily the Dkk cysteine-rich
regions
are fused to sequences derived from a member of the immunoglobulin protein
family.
Soluble derivatives have also been made of cell surface glycoproteins in the
immunoglobulin gene superfamily consisting of an extracellular domain of the
cell
surface glycoprotein fused to an immunoglobulin constant (Fc) region (see
e.g., Capon,
et al. (1989) Nature 337:525-531 and Capon U.S. Patents 5,116,964 and
5,428,130
[CD4-IgG1 constructs]; Linsley, P.S. et al. (1991)J Exp. Med. 173:721-730 [a
CD28-
IgGl construct and a B7-1-IgG1 construct]; and Linsley, P.S. et al. (1991)J.
Exp. Med
174:561-569 and U.S. Patent 5,434,131[a CTLA4-IgG1]). Such fusion proteins
have
proven useful for modulating receptor-ligand interactions. Soluble derivatives
of cell
surface proteins of the tumor necrosis factor receptor (TNFR) superfamily
proteins have
been made consisting of an extracellular domain of the cell surface receptor
fused to an
immunoglobulin constant (Fc) region (See for example Moreland etal. (1997) N.
Engl.
J. Med. 337(3):141-147; van der Poll etal. (1997) Blood 89(10):3727-3734; and
Ammann etal. (1997) J. Clin. Invest. 99(7):1699-1703.)
The Dkk-immunoglobulin fusion proteins of the invention can be incorporated
into pharmaceutical compositions and administered to a subject to inhibit an
interaction
between a Dkk ligand and a Dkk receptor on the surface of a cell, to thereby
suppress
Dkk-mediated signal transduction in vivo. The Dkk-immunoglobulin fusion
proteins
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can be used to affect the bioavailability of a Dick cognate receptor.
Inhibition of the Dkk
ligand/Dkk interaction may be useful therapeutically for both the treatment of
differentiative or proliferative disorders, as well as modulating (e.g.,
promoting or
inhibiting) developmental responses, cell adhesion, and/or cell fate.
Moreover, the Dkk-
immunoglobulin fusion proteins of the invention can be used as immunogens to
produce
anti-Dkk antibodies in a subject, to purify Dkk ligands and in screening
assays to
identify molecules which inhibit the interaction of Dkk with a Dkk ligand.
Preferably, a Dkk or Dkk-related chimeric or fusion protein of the invention
is
produced by standard recombinant DNA techniques. For example, DNA fragments
coding for the different polypeptide sequences are ligated together in-frame
in
accordance with conventional techniques, for example by employing blunt-ended
or
stagger-ended termini for ligation, restriction enzyme digestion to provide
for
appropriate termini, filling-in of cohesive ends as appropriate, alkaline
phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. In another
embodiment,
the fusion gene can be synthesized by conventional techniques including
automated
DNA synthesizers. Alternatively, PCR amplification of gene fragments can be
carried
out using anchor primers which give rise to complementary overhangs between
two
consecutive gene fragments which can subsequently be annealed and reamplified
to
generate a chimeric gene sequence (see, for example, Current Protocols in
Molecular
Biology, eds. Ausubel et al., John Wiley & Sons: 1992). Moreover, many
expression
vectors are commercially available that already encode a fusion moiety (e.g.,
a GST
polypeptide). A Dkk-encoding nucleic acid or nucleic acid encoding a Dkk-
related
protein can be cloned into such an expression vector such that the fusion
moiety is
linked in-frame to the protein.
The present invention also pertains to variants of the Dkk or Dkk-related
proteins which function as either agonists (mimetics) or as antagonists.
Variants of the
Dkk or Dkk-related proteins can be generated by mutagenesis, e.g., discrete
point
mutation or truncation of a Dkk or Dkk-related protein. An agonist of the Dkk
or Dkk-
related proteins can retain substantially the same, or a subset, of the
biological activities
of the naturally occurring form of a Dkk or Dkk-related protein. An antagonist
of a Dkk
or Dkk-related protein can inhibit one or more of the activities of the
naturally occurring
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form of the protein by, for example, competitively binding to a downstream or
upstream
member of a cellular signaling cascade which includes the Dkk or Dkk-related
protein.
Thus, specific biological effects can be elicited by treatment with a variant
of limited
function. In one embodiment, treatment of a subject with a variant having a
subset of
the biological activities of the naturally occurring form of the protein has
fewer side
effects in a subject relative to treatment with the naturally occurring form
of the Dkk or
Dkk-related protein.
In one embodiment, variants of a Dkk or Dkk-related protein which function as
either agonists (mimetics) or as antagonists can be identified by screening
combinatorial
libraries of mutants, e.g., truncation mutants, of a Dkk or Dkk-related
protein for protein
agonist or antagonist activity. In one embodiment, a variegated library of Dkk
or Dkk-
related variants is generated by combinatorial mutagenesis at the nucleic acid
level and
is encoded by a variegated gene library. A variegated library of Dkk or Dkk-
related
variants can be produced by, for example, enzymatically ligating a mixture of
synthetic
oligonucleotides into gene sequences such that a degenerate set of potential
Dkk or Dkk-
related sequences is expressible as individual polypeptides, or alternatively,
as a set of
larger fusion proteins (e.g., for phage display) containing the set of Dkk or
Dkk-related
sequences therein. There are a variety of methods which can be used to produce
libraries of potential Dkk or Dick-related variants from a degenerate
oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be performed in
an
automatic DNA synthesizer, and the synthetic gene then ligated into an
appropriate
expression vector. Use of a degenerate set of genes allows for the provision,
in one
mixture, of all of the sequences encoding the desired set of potential Dkk or
Dkk-related
sequences. Methods for synthesizing degenerate oligonucleotides are known in
the art
(see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura etal., (1984) Annu.
Rev.
Biochem. 53:323; Itakura etal., (1984) Science 198:1056; Ike etal., (1983)
Nucleic Acid
Res. 11:477.
In addition, libraries of fragments of a Dkk or Dkk-related protein coding
sequence can be used to generate a variegated population of Dick or Dick-
related
fragments for screening and subsequent selection of variants of a Dkk or Dkk-
related
protein. In one embodiment, a library of coding sequence fragments can be
generated by
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treating a double stranded PCR fragment of a Dkk coding sequence with a
nuclease
under conditions wherein nicking occurs only about once per molecule,
denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA which can
include sense/antisense pairs from different nicked products, removing single
stranded
portions from reformed duplexes by treatment with Si nuclease, and ligating
the
resulting fragment library into an expression vector. By this method, an
expression
library can be derived which encodes N-terminal, C-terminal and internal
fragments of
various sizes of the Dkk protein.
Several techniques are known in the art for screening gene products of
combinatorial libraries made by point mutations or truncation, and for
screening cDNA
libraries for gene products having a selected property. Such techniques are
adaptable for
rapid screening of the gene libraries generated by the combinatorial
mutagenesis of Dkk
or Dkk-related proteins. The most widely used techniques, which are amenable
to high
through-put analysis, for screening large gene libraries typically include
cloning the
gene library into replicable expression vectors, transforming appropriate
cells with the
resulting library of vectors, and expressing the combinatorial genes under
conditions in
which detection of a desired activity facilitates isolation of the vector
encoding the gene
whose product was detected. Recrusive ensemble mutagenesis (REM), a new
technique
which enhances the frequency of functional mutants in the libraries, can be
used in
combination with the screening assays to identify Dkk variants (Arkin and
Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al., (1993) Protein Engineering 6(3):327-
331).
In one embodiment, cell based assays can be exploited to analyze a variegated
Dkk or Dkk-related library. For example, a library of expression vectors can
be
transfected into a cell line which ordinarily responds to a particular ligand
in a Dkk-
dependent manner. The transfected cells are then contacted with the ligand and
the
effect of expression of the mutant on signaling by the ligand can be detected,
e.g., by
measuring any of a number of immune cell responses. Plasmid DNA can then be
recovered from the cells which score for inhibition, or alternatively,
potentiation of
ligand induction, and the individual clones further characterized.
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An isolated Dkk protein, Dkk-related protein, or a portion or fragment
thereof,
can be used as an immunogen to generate antibodies that bind Dkk or Dkk-
related
proteins using standard techniques for polyclonal and monoclonal antibody
preparation.
A full-length Dldc or Dkk-related protein can be used or, alternatively, the
invention
provides antigenic peptide fragments for use as immunogens. The antigenic
peptide of
Dkk comprises at least 8 amino acid residues of the amino acid sequence shown
in SEQ
ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID NO:21 and
encompasses an epitope of Dkk or Dkk-related protein such that an antibody
raised
against the peptide forms a specific immune complex with the protein.
Preferably, the
antigenic peptide comprises at least 10 amino acid residues, more preferably
at least 15
amino acid residues, even more preferably at least 20 amino acid residues, and
most
preferably at least 30 amino acid residues.
Preferred epitopes encompassed by the antigenic peptide are regions of Dkk or
Dkk-related proteins that are located on the surface of the protein, e.g.,
hydrophilic
regions.
A Dkk or Dkk-related immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal)
with the
immunogen. An appropriate immunogenic preparation can contain, for example,
recombinantly expressed Dkk or Dkk-related protein or a chemically synthesized
Dkk or
Dkk-related polypeptide. The preparation can further include an adjuvant, such
as
Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic Dkk preparation, for
example,
induces a polyclonal anti-Dkk antibody response.
Accordingly, another aspect of the invention pertains to anti-Dkk antibodies
as
well as antobodies to Dkk-related proteins. The term "antibody" as used herein
refers to
immunoglobulin molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site which
specifically binds
(immunoreacts with) an antigen, such as Dkk or Dkk-related antigens. Examples
of
immunologically active portions of immunoglobulin molecules include F(ab) and
F(abt)2 fragments which can be generated by treating the antibody with an
enzyme such
as pepsin. The invention provides polyclonal and monoclonal antibodies that
bind Dkk
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or Dkk-related polypeptides. The term "monoclonal antibody" or "monoclonal
antibody
composition", as used herein, refers to a population of antibody molecules
that contain
only one species of an antigen binding site capable of immunoreacting with a
particular
epitope of Dkk or a or Dick-related protein. A monoclonal antibody composition
thus
typically displays a single binding affinity for a particular Dkk or Dkk-
related protein
with which it immunoreacts.
Polyclonal antibodies can be prepared as described above by immunizing a
suitable subject with a Dkk or Dkk-related immunogen. The antibody titer in
the
immunized subject can be monitored over time by standard techniques, such as
with an
enzyme linked immunosorbent assay (ELISA) using immobilized Dkk or Dkk-related
protein. If desired, the antibody molecules directed against Dkk or Dkk-
related protein
can be isolated from the mammal (e.g., from the blood) and further purified by
well
known techniques, such as protein A chromatography to obtain the IgG fraction.
At an
appropriate time after immunization, e.g., when the antibody titers are
highest, antibody-
producing cells can be obtained from the subject and used to prepare
monoclonal
antibodies by standard techniques, such as the hybridoma technique originally
described
by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al.,
(1981)J.
Immunol. 127:539-46; Brown etal., (1980)J Biol. Chem .255:4980-83; Yeh etal.,
(1976) PNAS 76:2927-31; and Yeh et al., (1982) Int. J Cancer 29:269-75), the
more
recent human B cell hybridoma technique (Kozbor etal., (1983) Immunol Today
4:72),
the EBV-hybridoma technique (Cole etal., (1985), Monoclonal Antibodies and
Cancer
Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology
for
producing monoclonal antibody hybridomas is well known (see generally R. H.
Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses,
Plenum
Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol.
Med.,
54:387-402; M. L. Gefter etal., (1977) Somatic Cell Genet. 3:231-36). Briefly,
an
immortal cell line (typically a myeloma) is fused to lymphocytes (typically
splenocytes)
from a mammal immunized with a Dkk or Dkk-related immunogen as described
above,
and the culture supernatants of the resulting hybridoma cells are screened to
identify a
hybridoma producing a monoclonal antibody that binds Dkk or Dkk-related
protein.
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Any of the many well known protocols used for fusing lymphocytes and
immortalized cell lines can be applied for the purpose of generating a
monoclonal
antibody (see, e.g., G. Galfre et al., (1977) Nature 266:55052; Gefter et al.,
Somatic Cell
Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth,
Monoclonal
Antibodies, cited supra). Moreover, the ordinarily skilled worker will
appreciate that
there are many variations of such methods which also would be useful.
Typically, the
immortal cell line (e.g., a myeloma cell line) is derived from the same
mammalian
species as the lymphocytes. For example, murine hybridomas can be made by
fusing
lymphocytes from a mouse immunized with an immunogenic preparation of the
present
invention with an immortalized mouse cell line. Preferred immortal cell lines
are mouse
myeloma cell lines that are sensitive to culture medium containing
hypoxanthine,
aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell
lines
can be used as a fusion partner according to standard techniques, e.g., the P3-
NS1/1-
Ag4-1, P3-x63-Ag8.653 or Sp2/0-Ag14 myeloma lines. These myeloma lines are
available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to
mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting
from
the fusion are then selected using HAT medium, which kills unfused and
unproductively
fused myeloma cells (unfused splenocytes die after several days because they
are not
transformed). Hybridoma cells producing a monoclonal antibody of the invention
are
detected by screening the hybridoma culture supernatants for antibodies that
bind Dkk or
Dkk-related protein, e.g., using a standard ELISA assay.
Alternative to preparing monoclonal antibody-secreting hybridomas, a
monoclonal antibody can be identified and isolated by screening a recombinant
combinatorial immunoglobulin library (e.g., an antibody phage display library)
with
Dkk or Dkk-related protein to thereby isolate immunoglobulin library members
that bind
Dkk or Dkk-related protein. Kits for generating and screening phage display
libraries
are commercially available (e.g., the Pharmacia Recombinant Phage Antibody
System,
Catalog No. 27-9400-01; and the Stratagene Surj'Z4PTM Phage Display Kit,
Catalog No.
240612). Additionally, examples of methods and reagents particularly amenable
for use
in generating and screening antibody display library can be found in, for
example,
Ladner et al., U.S. Patent No. 5,223,409; Kang et al., PCT International
Publication No.
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WO 92/18619; Dower etal., PCT International Publication No. WO 91/17271;
Winter
et al., PCT International Publication WO 92/20791; Markland et al., PCT
International
Publication No. WO 92/15679; Breitling et al., PCT International Publication
WO
93/01288; McCafferty et al., PCT International Publication No. WO 92/01047;
Garrard
et al., PCT International Publication No. WO 92/09690; Ladner et al., PCT
International
Publication No. WO 90/02809; Fuchs etal., (1991) Bio/Technology 9:1370-1372;
Hay
etal., (1992) Hum. Antibod Hybridomas 3:81-85; Huse etal., (1989) Science
246:1275-
1281; Griffiths etal., (1993) EMBO J12:725-734; Hawkins etal., (1992) 1 Mol.
BioL
226:889-896; Clarkson etal., (1991) Nature 352:624-628; Gram etal., (1992)
PNAS
89:3576-3580; Garrad et al., (1991) Bio/Technology 9:1373-1377; Hoogenboom
etal.,
(1991) Nuc. Acid Res. 19:4133-4137; Barbas etal., (1991) PNAS 88:7978-7982;
and
McCafferty et al., Nature (1990) 348:552-554.
Additionally, recombinant antibodies, such as chimeric and humanized
monoclonal antibodies, comprising both human and non-human portions, which can
be
made using standard recombinant DNA techniques, are within the scope of the
invention. Such chimeric and humanized monoclonal antibodies can be produced
by
recombinant DNA techniques known in the art, for example using methods
described in
Robinson et al., International Application No. PCT/US86/02269; Akira, et al.,
European
Patent Application 184,187; Taniguchi, M., European Patent Application
171,496;
Morrison et al., European Patent Application 173,494; Neuberger et al., PCT
International Publication No. WO 86/01533; Cabilly etal., U.S. Patent No.
4,816,567;
Cabilly etal., European Patent Application 125,023; Better etal., (1988)
Science
240:1041-1043; Liu etal., (1987) PNAS 84:3439-3443; Liu etal., (1987)1 Immunot
139:3521-3526; Sun etal., (1987) PNAS 84:214-218; Nishimura et al., (1987)
Canc.
Res. 47:999-1005; Wood etal., (1985) Nature 314:446-449; and Shaw etal.,
(1988) 1
Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-
1207; Oi et
al., (1986) BioTechniques 4:214; Winter U.S. Patent 5,225,539; Jones etal.,
(1986)
Nature 321:552-525; Verhoeyan etal., (1988) Science 239:1534; and Beidler
etal.,
(1988) 1 Immunol. 141:4053-4060.
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An antibody (e.g., monoclonal antibody) can be used to isolate Dkk or Dkk-
related protein by standard techniques, such as affinity chromatography or
immunoprecipitation. Anantibody can facilitate the purification of natural Dkk
or Dkk-
related protein from cells and of recombinantly produced Dkk or Dkk-related
protein
expressed in host cells. Moreover, an antibody can be used to detect Dkk or
Dkk-related
protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate
the abundance
and pattern of expression of the Dkk or Dkk-related protein. Antibodies can be
used
diagnostically to monitor protein levels in tissue as part of a clinical
testing procedure,
e.g., to, for example, determine the efficacy of a given treatment regimen.
Detection can
be facilitated by coupling (i.e., physically linking) the antibody to a
detectable
substance. Examples of detectable substances include various enzymes,
prosthetic
groups, fluorescent materials, luminescent materials, bioluminescent
materials, and
radioactive materials. Examples of suitable enzymes include horseradish
peroxidase,
alkaline phosphatase, 13-galactosidase, or acetylcholinesterase; examples of
suitable
prosthetic group complexes include streptavidin/biotin and avidin/biotin;
examples of
suitable fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride
or
phycoerythrin; an example of a luminescent material includes luminol; examples
of
bioluminescent materials include luciferase, luciferin, and aequorin, and
examples of
suitable radioactive material include 1251, 1311, 35S or 3H.
III. Recombinant Expression Vectors and Host Cells
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid encoding Dkk or a nucleic acid encoding a
Dkk-
related protein (or a portion thereof). As used herein, the term "vector"
refers to a
nucleic acid molecule capable of transporting another nucleic acid to which it
has been
linked. One type of vector is a "plasmid", which refers to a circular double
stranded
DNA loop into which additional DNA segments can be ligated. Another type of
vector
is a viral vector, wherein additional DNA segments can be ligated into the
viral genome.
Certain vectors are capable of autonomous replication in a host cell into
which they are
introduced (e.g., bacterial vectors having a bacterial origin of replication
and episomal
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mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are
integrated into the genome of a host cell upon introduction into the host
cell, and thereby
are replicated along with the host genome. Moreover, certain vectors are
capable of
directing the expression of genes to which they are operatively linked. Such
vectors are
referred to herein as "expression vectors". In general, expression vectors of
utility in
recombinant DNA techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" can be used interchangeably as the
plasmid is the
most commonly used form of vector. However, the invention is intended to
include
such other forms of expression vectors, such as viral vectors (e.g.,
replication defective
retroviruses, adenoviruses and adeno-associated viruses), which serve
equivalent
functions.
The recombinant expression vectors of the invention comprise a nucleic acid of
the invention in a form suitable for expression of the nucleic acid in a host
cell, which
means that the recombinant expression vectors include one or more regulatory
sequences, selected on the basis of the host cells to be used for expression,
which is
operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant
expression vector, "operably linked" is intended to mean that the nucleotide
sequence of
interest is linked to the regulatory sequence(s) in a manner which allows for
expression
of the nucleotide sequence (e.g., in an in vitro transcription/translation
system or in a
host cell when the vector is introduced into the host cell). The term
"regulatory
sequence" is intended to includes promoters, enhancers and other expression
control
elements (e.g., polyadenylation signals). Such regulatory sequences are
described, for
example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, CA (1990). Regulatory sequences include those which
direct constitutive expression of a nucleotide sequence in many types of host
cell and
those which direct expression of the nucleotide sequence only in certain host
cells (e.g.,
tissue-specific regulatory sequences). It will be appreciated by those skilled
in the art
that the design of the expression vector can depend on such factors as the
choice of the
host cell to be transformed, the level of expression of protein desired, etc.
The
expression vectors of the invention can be introduced into host cells to
thereby produce
proteins or peptides, including fusion proteins or peptides, encoded by
nucleic acids as
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described herein (e.g., Dkk proteins, Dkk-related proteins, mutant forms of
Dkk or Dkk-
related proteins, fusion proteins, etc.).
The recombinant expression vectors of the invention can be designed for
expression of Dkk or Dkk-related proteins in prokaryotic or eukaryotic cells.
For
example, Dkk can be expressed in bacterial cells such as E. coil, insect cells
(using
baculovirus expression vectors) yeast cells or mammalian cells. Suitable host
cells are
discussed further in Goeddel, Gene Expression Technology: Methods in
Enzymology
185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant
expression
vector can be transcribed and translated in vitro, for example using T7
promoter
regulatory sequences and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coil
with
vectors containing constitutive or inducible promotors directing the
expression of either
fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a
protein
encoded therein, usually to the amino terminus of the recombinant protein.
Such fusion
vectors typically serve three purposes: I) to increase expression of
recombinant protein;
2) to increase the solubility of the recombinant protein; and 3) to aid in the
purification
of the recombinant protein by acting as a ligand in affinity purification.
Often, in fusion
expression vectors, a proteolytic cleavage site is introduced at the junction
of the fusion
moiety and the recombinant protein to enable separation of the recombinant
protein from
the fusion moiety subsequent to purification of the fusion protein. Such
enzymes, and
their cognate recognition sequences, include Factor Xa, thrombin and
enterokinase.
Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith,
D.B.
and Johnson, K.S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly,
MA)
and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase
(GST),
maltose E binding protein, or protein A, respectively, to the target
recombinant protein.
Purified fusion proteins can be utilized in activity assays, in ligand binding
(e.g.,
direct assays or competitive assays described in detail below), to generate
antibodies
specific for Dkk or Dkk-related proteins, as examples. In a preferred
embodiment, a
Dkk or Dkk-related fusion expressed in a retroviral expression vector of the
present
invention can be utilized to infect bone marrow cells which are subsequently
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transplanted into irradiated recipients. The pathology of the subject
recipient is then
examined after sufficient time has passed (e.g six (6) weeks).
Examples of suitable inducible non-fusion E. coli expression vectors include
pTrc (Amann et al., (1988) Gene 69:301-315) and pET lid (Studier etal., Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
California (1990) 60-89). Target gene expression from the pTrc vector relies
on host
RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target
gene
expression from the pET lid vector relies on transcription from a T7 gn 10-lac
fusion
promoter mediated by a coexpressed viral RNA polymerase (T7 gni). This viral
polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a
resident X.
prophage harboring a T7 gni gene under the transcriptional control of the
lacUV 5
promoter.
One strategy to maximize recombinant protein expression in E. coli is to
express
the protein in a host bacteria with an impaired capacity to proteolytically
cleave the
recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, California (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an
expression vector so that the individual codons for each amino acid are those
preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res.
20:2111-2118).
Such alteration of nucleic acid sequences of the invention can be carried out
by standard
DNA synthesis techniques.
In another embodiment, the Dkk or Dkk-related expression vector is a yeast
expression vector. Examples of vectors for expression in yeast S. cerivisae
include
pYepSecl (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and
Herskowitz,
(1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego,
CA).
Alternatively, Dkk or Dkk-related protein can be expressed in insect cells
using
baculovirus expression vectors. Baculovirus vectors available for expression
of proteins
in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith
etal., (1983) Mol.
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology
170:31-39).
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In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC
(Kaufman etal., (1987) EMBO J. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral regulatory
elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression systems for
both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989.
In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a
particular cell type
(e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-
specific regulatory elements are known in the art. Non-limiting examples of
suitable
tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.,
(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji etal., (1983)
Cell
33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) PNAS 86:5473-5477),
pancreas-specific promoters (Edlund etal., (1985) Science 230:912-916), and
mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316
and
European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed, for example the murine hox promoters (Kessel and Gruss
(1990)
Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman
(1989)
Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector comprising a
DNA molecule of the invention cloned into the expression vector in an
antisense
orientation. That is, the DNA molecule is operatively linked to a regulatory
sequence in
a manner which allows for expression (by transcription of the DNA molecule) of
an
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RNA molecule which is antisense to Dkk mRNA or a Dkk-related mRNA. Regulatory
sequences operatively linked to a nucleic acid cloned in the antisense
orientation can be
chosen which direct the continuous expression of the antisense RNA molecule in
a
variety of cell types, for instance viral promoters and/or enhancers, or
regulatory
sequences can be chosen which direct constitutive, tissue specific or cell
type specific
expression of antisense RNA. The antisense expression vector can be in the
form of a
recombinant plasmid, phagemid or attenuated virus in which antisense nucleic
acids are
produced under the control of a high efficiency regulatory region, the
activity of which
can be determined by the cell type into which the vector is introduced. For a
discussion
of the regulation of gene expression using antisense genes see Weintraub, H.
etal.,
Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in
Genetics,
Vol. 1(1) 1986.
Another aspect of the invention pertains to host cells into which a
recombinant
expression vector of the invention has been introduced. The terms "host cell"
and
"recombinant host cell" are used interchangeably herein. It is understood that
such
terms refer not only to the particular subject cell but to the progeny or
potential progeny
of such a cell. Because certain modifications may occur in succeeding
generations due
to either mutation or environmental influences, such progeny may not, in fact,
be
identical to the parent cell, but are still included within the scope of the
term as used
herein.
A host cell can be any prokaryotic or eukaryotic cell. For example, Dick
protein
can be expressed in bacterial cells such as E. coli, insect cells, yeast or
mammalian cells
(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host
cells are
known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell,
including
calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, or electroporation. Suitable methods for
transforming or
transfecting host cells can be found in Sambrook, et al., (Molecular Cloning:
A
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Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred
selectable markers include those which confer resistance to drugs, such as
G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding Dkk or can be
introduced
on a separate vector. Cells stably transfected with the introduced nucleic
acid can be
identified by drug selection (e.g., cells that have incorporated the
selectable marker gene
will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell in
culture, can be used to produce (i.e., express) a Dkk or Dkk-related protein.
Accordingly, the invention further provides methods for producing Dkk or Dkk-
related
proteins using the host cells of the invention. In one embodiment, the method
comprises
culturing the host cell of invention (into which a recombinant expression
vector
encoding Dkk or a Dkk-related protein has been introduced) in a suitable
medium such
that protein is produced. In another embodiment, the method further comprises
isolating
Dkk or a Dkk-related protein from the medium or the host cell.
The host cells of the invention can also be used to produce nonhuman
transgenic
animals. For example, in one embodiment, a host cell of the invention is a
fertilized
oocyte or an embryonic stem cell into which Dkk-coding sequences (or Dkk-
related
coding sequences) have been introduced. Such host cells can then be used to
create non-
human transgenic animals in which exogenous Dkk sequences (or Dkk-related
sequences) have been introduced into their genome or homologous recombinant
animals
in which endogenous Dkk sequences (or Dkk-related sequences) have been
altered.
Such animals are useful for studying the function and/or activity of Dick or
Dkk-related
proteins and for identifying and/or evaluating modulators of Dkk or Dick-
related protein
activity. As used herein, a "transgenic animal" is a non-human animal,
preferably a
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mammal, more preferably a rodent such as a rat or mouse, in which one or more
of the
cells of the animal includes a transgene. Other examples of transgenic animals
include
non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A
transgene
is exogenous DNA which is integrated into the genome of a cell from which a
transgenic
animal develops and which remains in the genome of the mature animal, thereby
directing the expression of an encoded gene product in one or more cell types
or tissues
of the transgenic animal. As used herein, a "homologous recombinant animal" is
a non-
human animal, preferably a mammal, more preferably a mouse, in which an
endogenous
Dkk or Dkk-related gene has been altered by homologous recombination between
the
endogenous gene and an exogenous DNA molecule introduced into a cell of the
animal,
e.g., an embryonic cell of the animal, prior to development of the animal.
A transgenic animal of the invention can be created, for example, by
introducing
Dkk-encoding nucleic acid into the male pronuclei of a fertilized oocyte,
e.g., by
microinjection, retroviral infection, and allowing the oocyte to develop in a
pseudopregnant female foster animal. The human Dkk cDNA sequence of SEQ ID
NO:1, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:20 can be introduced as a
transgene
into the genome of a non-human animal. Alternatively, a nonhuman homologue of
a
human Dkk gene, such as a mouse Dkk gene, can be isolated based on
hybridization to
the human Dkk cDNA (described further in subsection I above) and used as a
transgene.
Intronic sequences and polyadenylation signals can also be included in the
transgene to
increase the efficiency of expression of the transgene. A tissue-specific
regulatory
sequence(s) can be operably linked to the Dkk transgene to direct expression
of Dkk
protein to particular cells. Methods for generating transgenic animals via
embryo
manipulation and microinjection, particularly animals such as mice, have
become
conventional in the art and are described, for example, in U.S. Patent Nos.
4,736,866 and
4,870,009, both by Leder etal., U.S. Patent No. 4,873,191 by Wagner etal., and
in
Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory
Press,
Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of
other
transgenic animals. A transgenic founder animal can be identified based upon
the
presence of the Dkk transgene in its genome and/or expression of Dkk mRNA in
tissues
or cells of the animals. A transgenic founder animal can then be used to breed
additional
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animals carrying the transgene. Moreover, transgenic animals carrying a
transgene
encoding Dick can further be bred to other transgenic animals carrying other
transgenes.
To create a homologous recombinant animal, a vector is prepared which contains
at least a portion of a Dkk gene into which a deletion, addition or
substitution has been
introduced to thereby alter, e.g., functionally disrupt, the Dick gene. The
Dkk gene can
be a human gene (e.g., the cDNA of SEQ ID NO:3, SEQ ID NO: 6, SEQ ID NO:9 or
SEQ ID NO:22), but more preferably, is a non-human homologue of a human Dick
gene.
For example, a mouse Dkk gene of SEQ ID NO:16 can be used to construct a
homologous recombination vector suitable for altering an endogenous Dkk gene
in the
mouse genome. In a preferred embodiment, the vector is designed such that,
upon
homologous recombination, the endogenous Dkk gene is functionally disrupted
(i.e., no
longer encodes a functional protein; also referred to as a "knock out"
vector).
Alternatively, the vector can be designed such that, upon homologous
recombination,
the endogenous Dkk gene is mutated or otherwise altered but still encodes
functional
protein (e.g., the upstream regulatory region can be altered to thereby alter
the
expression of the endogenous Dkk protein). In the homologous recombination
vector,
the altered portion of the Dkk gene is flanked at its 5' and 3' ends by
additional nucleic
acid of the Dkk gene to allow for homologous recombination to occur between
the
exogenous Dkk gene carried by the vector and an endogenous Dkk gene in an
embryonic
stem cell. The additional flanking Dkk nucleic acid is of sufficient length
for successful
homologous recombination with the endogenous gene. Typically, several
kilobases of
flanking DNA (both at the 5' and 3' ends) are included in the vector (see
e.g., Thomas,
K.R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous
recombination vectors). The vector is introduced into an embryonic stem cell
line (e.g.,
by electroporation) and cells in which the introduced Dkk gene has
homologously
recombined with the endogenous Dldc gene are selected (see e.g., Li, E. et
al., (1992)
Cell 69:915). The selected cells are then injected into a blastocyst of an
animal (e.g., a
mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas
and
Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. (IRL, Oxford,
1987)
pp. 113-152). A chimeric embryo can then be implanted into a suitable
pseudopregnant
female foster animal and the embryo brought to term. Progeny harboring the
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homologously recombined DNA in their germ cells can be used to breed animals
in
which all cells of the animal contain the homologously recombined DNA by
germline
transmission of the transgene. Methods for constructing homologous
recombination
vectors and homologous recombinant animals are described further in Bradley,
A.
(1991) Current Opinion in Biotechnology 2:823-829 and in PCT International
Publication Nos.: WO 90/11354 by Le Mouellec etal.; WO 91/01140 by Smithies et
al.; WO 92/0968 by Zijlstra etal.; and WO 93/04169 by Berns etal. It is also
within the
scope of the present invention to practice the above-described transgenic
methodology
utilizing nucleic acid molecules which encode Dkk-related proteins.
In another embodiment, transgenic non-humans animals can be produced which
contain selected systems which allow for regulated expression of the
transgene. One
example of such a system is the cre/loxP recombinase system of bacteriophage
Pl. For
a description of the cre/loxP recombinase system, see, e.g., Lakso et al.,
(1992) PNAS
89:6232-6236. Another example of a recombinase system is the FLP recombinase
system of Saccharomyces cerevisiae (O'Gorman etal., (1991) Science 251:1351-
1355.
If a cre/loxP recombinase system is used to regulate expression of the
transgene, animals
containing transgenes encoding both the Cre recombinase and a selected protein
are
required. Such animals can be provided through the construction of "double"
transgenic
animals, e.g., by mating two transgenic animals, one containing a transgene
encoding a
selected protein and the other containing a transgene encoding a recombinase.
Clones of the non-human transgenic animals described herein can also be
produced according to the methods described in Wilmut, I. etal., (1997) Nature
385:810-813. In brief, a cell, e.g., a somatic cell, from the transgenic
animal can be
isolated and induced to exit the growth cycle and enter Go phase. The
quiescent cell can
then be fused, e.g., through the use of electrical pulses, to an enucleated
oocyte from an
animal of the same species from which the quiescent cell is isolated. The
recontructed
oocyte is then cultured such that it develops to morula or blastocyte and then
transferred
to pseudopregnant female foster animal. The offspring borne of this female
foster
animal will be a clone of the animal from which the cell, e.g., the somatic
cell, is
isolated.
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IV. Pharmaceutical Compositions
The Dkk and Dkk-related nucleic acid molecules, Dkk and Dkk-related proteins,
and anti-Dkk or anti-Dkk-related protein antibodies (also referred to herein
as "active
compounds") of the invention can be incorporated into pharmaceutical
compositions
suitable for administration. Such compositions typically comprise the nucleic
acid
molecule, protein, or antibody and a pharmaceutically acceptable carrier. As
used herein
the language "pharmaceutically acceptable carrier" is intended to include any
and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents, and the like, compatible with pharmaceutical
administration.
The use of such media and agents for pharmaceutically active substances is
well known
in the art. Except insofar as any conventional media or agent is incompatible
with the
active compound, use thereof in the compositions is contemplated.
Supplementary
active compounds can also be incorporated into the compositions.
A pharmaceutical composition of the invention is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,
inhalation),
transdermal (topical), transmucosal, and rectal administration. Solutions or
suspensions
used for parenteral, intradermal, or subcutaneous application can include the
following
components: a sterile diluent such as water for injection, saline solution,
fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants
such as
ascorbic acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents for the
adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or
bases,
such as hydrochloric acid or sodium hydroxide. The parenteral preparation can
be
enclosed in ampoules, disposable syringes or multiple dose vials made of glass
or
plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous
solutions (where water soluble) or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological saline,
bacteriostatic
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water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline
(PBS). In
all cases, the composition must be sterile and should be fluid to the extent
that easy
syringability exists. It must be stable under the conditions of manufacture
and storage
and must be preserved against the contaminating action of microorganisms such
as
bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
can be maintained, for example, by the use of a coating such as lecithin, by
the
maintenance of the required particle size in the case of dispersion and by the
use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol,
sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be
brought about by including in the composition an agent which delays
absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a Dkk protein, Dkk-related protein or antibody) in the
required amount
in an appropriate solvent with one or a combination of ingredients enumerated
above, as
required, followed by filtered sterilization. Generally, dispersions are
prepared by
incorporating the active compound into a sterile vehicle which contains a
basic
dispersion medium and the required other ingredients from those enumerated
above. In
the case of sterile powders for the preparation of sterile injectable
solutions, the
preferred methods of preparation are vacuum drying and freeze-drying which
yields a
powder of the active ingredient plus any additional desired ingredient from a
previously
sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They
can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral
therapeutic administration, the active compound can be incorporated with
excipients and
used in the form of tablets, troches, or capsules. Oral compositions can also
be prepared
using a fluid carrier for use as a mouthwash, wherein the compound in the
fluid carrier is
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applied orally and swished and expectorated or swallowed. Pharmaceutically
compatible binding agents, and/or adjuvant materials can be included as part
of the
composition. The tablets, pills, capsules, troches and the like can contain
any of the
following ingredients, or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as
starch or
lactose, a disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant
such as magnesium stearate or Sterot4; a glidant such as colloidal silicon
dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of
an
aerosol spray from pressured container or dispenser which contains a suitable
propellant,
e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be 'by transmucosal or transdermal means. For
= = . = .
. . - " = ,
transmucosal.or transdermal administratiOn, penetrants appropriate. to the
barrier to be
'pernyieated reused in the formulation. uclh penetrants are gerierally`known
in the art,
and include, for example, for transmucosal administration, detergents, bile
salts, and
- _
fusidic acid derivati* Transmucosal administration can be accomplished through
the
-
µuse of nasal sprays or Suppositories. For transdermal administration, the
active
.compounds are formulated into ointr4nts,'salves; gels, 'or creams as
$enerally known in
the At.
, . The compounds can also-be=prepared in the form of
supp9sitoriesSe.g., with
conventional suppository bases such as cocoa butter and other glycerides) or
retention
enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that will
protect the compound against rapid elimination from the body, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Methods for preparation of such formulations will be apparent to those skilled
in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to
infected
* Trademark
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cells with monoclonal antibodies to viral antigens) can also be used as
pharmaceutically
acceptable carriers. These can be prepared according to methods known to those
skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit form
as used herein refers to physically discrete units suited as unitary dosages
for the subject
to be treated; each unit containing a predetermined quantity of active
compound
calculated to produce the desired therapeutic effect in association with the
required
pharmaceutical carrier. The specification for the dosage unit forms of the
invention are
dictated by and directly dependent on the unique characteristics of the active
compound
and the particular therapeutic effect to be achieved, and the limitations
inherent in the art
of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental animals,
e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio
LD50/ED50. Compounds which exhibit large therapeutic indices are preferred.
While
compounds that exhibit toxic side effects may be used, care should be taken to
design a
delivery system that targets such compounds to the site of affected tissue in
order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and animal studies can be used
in
formulating a range of dosage for use in humans. The dosage of such compounds
lies
preferably within a range of circulating concentrations that include the ED50
with little
or no toxicity. The dosage may vary within this range depending upon the
dosage form
employed and the route of administration utilized. For any compound used in
the
method of the invention, the therapeutically effective dose can be estimated
initially
from cell culture assays. A dose may be formulated in animal models to achieve
a
circulating plasma concentration range that includes the IC50 (i.e., the
concentration of
the test compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more accurately
determine
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useful doses in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
The nucleic acid molecules of the invention can be inserted into vectors and
used
as gene therapy vectors. Gene therapy vectors can be delivered to a subject
by, for
example, intravenous injection, local administration (see U.S. Patent
5,328,470) or by
stereotactic injection (see e.g., Chen et al., (1994) PNAS 91:3054-3057). The
pharmaceutical preparation of the gene therapy vector can include the gene
therapy
vector in an acceptable diluent, or can comprise a slow release matrix in
which the gene
delivery vehicle is imbedded. Alternatively, where the complete gene delivery
vector
can be produced intact from recombinant cells, e.g., retroviral vectors, the
pharmaceutical preparation can include one or more cells which produce the
gene
delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
V. Uses and Methods of the Invention
The molecules of the present invention (e.g., nucleic acid molecules,
proteins,
protein homologues, and antibodies described herein) can be used in one or
more of the
following methods: a) screening assays; b) predictive medicine (e.g.,
diagnostic assays,
prognostic assays, monitoring clinical trials, and pharmacogenetics); and c)
methods of
treatment (e.g., therapeutic and prophylactic). As described herein, a Dkk
protein of the
invention has one or more of the following activities: intracellular calcium,
an increase
in phosphatidylinositol or other molecule, and can result, e.g., in
phosphorylation of
specific proteins, a modulation of gene transcription and any of the other
biological
activities set forth herein.
In a preferred embodiment, a Dkk activity is at least one or more of the
following
activities: (i) interaction of a Dkk protein with and/or binding to a second
molecule,
(e.g., a protein, such as a Dkk (e.g., hDkk-3) receptor, a soluble form of a
Dkk receptor,
a receptor for a member of the wnt family of signaling proteins, or a non-Dkk
signaling
molecule); (ii) interaction of a Dkk protein with an intracellular protein via
a membrane-
bound Dkk receptor; (iii) complex formation between a soluble Dkk protein and
a
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second soluble Dkk binding partner (e.g., a non-Did( protein molecule or a
second Dkk
protein molecule); (iv) interaction with other extracellular proteins (e.g.,
regulation of
wnt-dependent cellular adhesion to extracellular matrix components); (v)
binding to and
eliminating an undesirable molecule (e.g., a detoxifying activity or defense
function);
and/or (vi) an enzymatic activity, and can can thus be used in, for example,
(1)
modulation of cellular signal transduction, either in vitro or in vivo (e.g.,
antagonism of
the activity of members of the wnt family of secreted proteins or supression
of wnt-
dependent signal transduction); (2) regulation of communication between cells
(e.g.,
regulation of wnt-dependent cell-cell interactions); (3) regulation of
expression of genes
whose expression is modulated by binding of Dkk (e.g., hDkk-3) to a receptor;
(4)
regulation of gene transcription in a cell involved in development or
differentiation,
either in vitro or in vivo (e.g., induction of cellular differentiation); (5)
regulation of
gene transcription in a cell involved in development or differentiation,
wherein at least
one gene encodes a differentiation-specific protein; (6) regulation of gene
transcription
in a cell involved in development or differentaition, wherein at least one
gene encodes a
second secreted protein; (7) regulation of gene transcription in a cell
involved in
development or differentiation, wherein at least one gene encodes a signal
transduction
molecule; (8) regulation of cellular proliferation, either in vitro or in vivo
(e.g., induction
of cellular proliferation or inhibition of proliferation, for example,
inhibition of
tumorigenesis (e.g., inhibition of glioblastoma proliferation)); (9) formation
and
maintenance of ordered spatial arrangements of differentiated tissues in
vertebrates, both
adult and embryonic (e.g., induction of head formation during vertebrate
development or
maintenance of hematopoietic progenitor cells); (10) modulation of cell death,
such as
stimulation of cell survival; (11) regulating cell migration; and/or (12)
immune
modulation.
Accordingly one embodiment of the present invention involves a method of use
(e.g., a diagnostic assay, prognostic assay, or a prophylactic/therapeutic
method of
treatment) wherein a molecule of the present invention (e.g., a Dkk protein,
Dkk nucleic
acid, or a Dick modulator) is used, for example, to diagnose, prognose and/or
treat a
disease and/or condition in which any of the aforementioned activities (i.e.,
activities (i)
- (vi) and (1) - (12) in the above paragraph) is indicated. In another
embodiment, the
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present invention involves a method of use (e.g., a diagnostic assay,
prognostic assay, or
a prophylactic/therapeutic method of treatment) wherein a molecule of the
present
invention (e.g., a Dkk protein, Dkk nucleic acid, or a Dkk modulator) is used,
for
example, for the diagnosis, prognosis, and/or treatment of subjects,
preferably a human
subject, in which any of the aforementioned activities is pathologically
perturbed. In a
preferred embodiment, the methods of use (e.g., diagnostic assays, prognostic
assays, or
prophylactic/therapeutic methods of treatment) involve administering to a
subject,
preferably a human subject, a molecule of the present invention (e.g., a Dkk
protein,
Dkk nucleic acid, or a Dkk modulator) for the diagnosis, prognosis, and/or
therapeutic
treatment. In another embodiment, the methods of use (e.g., diagnostic assays,
prognostic assays, or prophylactic/therapeutic methods of treatment) involve
administering to a human subject a molecule of the present invention (e.g., a
Dkk
protein, Dkk nucleic acid, or a Dkk modulator).
Other embodiments of the invention pertain to the use of isolated nucleic acid
molecules of the invention can be used, for example, to express Dkk or Dkk-
related
protein (e.g., via a recombinant expression vector in a host cell in gene
therapy
applications), to detect Dkk or Dkk-related mRNA (e.g., in a biological
sample) or a
genetic alteration in a Dkk or Dick-related gene, and to modulate Dkk or Dkk-
related
activity, as described further below. In addition, the Dkk or Dkk-related
proteins can be
used to screen drugs or compounds which modulate the Dkk activity as well as
to treat
disorders characterized by insufficient or excessive production of Dkk or Dkk-
related
protein or production of Dkk or Dkk-related protein forms which have decreased
or
aberrant activity compared to Dkk or Dkk-related wild type protein (e.g.,
developmental
disorders or proliferative diseases such as cancer as well as diseases, ocular
disorders
(e.g., blindness) conditions or disorders characterized by abnormal cell
differentiation
and/or survival, an abnormal extracellular structure, or an abnormality in a
defense
mechanism). Moreover, the antibodies of the invention can be used to detect
and isolate
Dick or Dkk-related proteins, regulate the bioavailability of Dkk or Dick-
related proteins,
and modulate Dkk or Dkk-related activity. The term "an aberrant activity", as
applied to
an activity of a protein such as Dkk (e.g., hDkk-3), refers to an activity
which differs
from the activity of the wild-type or native protein or which differs from the
activity of
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the protein in a healthy subject. An activity of a protein can be aberrant
because it is
stronger than the activity of its native counterpart. Alternatively, an
activity can be
aberrant because it is weaker or absent related to the activity of its native
counterpart.
An aberrant activity can also be a change in an activity. For example an
aberrant protein
can interact with a different protein relative to its native counterpart. A
cell can have an
aberrant Dkk (e.g., hDkk-3) activity due to overexpression or underexpression
of the
gene encoding Dkk.
A. Screening Assays:
The invention provides a method (also referred to herein as a "screening
assay")
for identifying modulators, i.e., candidate or test compounds or agents (e.g.,
peptides,
peptidomimetics, small molecules or other drugs) which bind to Dkk or Dick-
related
proteins or have a stimulatory or inhibitory effect on, for example, Dkk or
Dkk-related
expression or activity. Modulators can include, for example, agonists and/or
antagonists. The term "agonist", as used herein, is meant to refer to an agent
that mimics
or upregulates (e.g. potentiates or supplements) a Dkk or Dkk-related (e.g.,
hDkk-3)
bioactivity. An agonist can be a compound which mimics a bioactivity of a Dkk
or
Dkk-related protein, such as transduction of a signal from a Dkk receptor, by,
e.g.,
interacting with a hDkk-3 receptor. An agonist can also be a compound that
upregulates
expression of a Dkk or Dkk-related gene. An agonist can also be a compound
which
modulates the expression or activity of a protein which is located downstream,
for
example, of a Dkk receptor, thereby mimicking or enhancing the effect of
binding of
Dkk to a Dkk receptor.
"Antagonist" as used herein is meant to refer to an agent that inhibits,
decreases
or suppresses a bioactivity (e.g., hDkk-3). An antagonist can be a compound
which
decreases signalling from a Dkk or Dkk-related protein, e.g., a compound that
is capable
of binding to hDkk-3 or to a hDkk-3 receptor. A preferred antagonist inhibits
the
interaction between a Dkk or Dick-related protein and another molecule, such
as a Dkk
receptor. Alternatively, an antagonist can be a compound that downregulates
expression
of a Dkk or Dkk-related gene. An antagonist can also be a compound which
modulates
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the expression or activity of a protein which is located downstream of a Dkk
receptor,
thereby antagonizing the effect of binding of Dkk to a Dkk receptor.
In one embodiment, the invention provides assays for screening candidate or
test
compounds which bind to or modulate the activity of a Dkk or Dkk-related
protein or
polypeptide or biologically active portion thereof In another embodiment, the
invention
provides assays for screening candidate or test compounds which bind to or
modulate
the activity of a Dkk receptor. The test compounds of the present invention
can be
obtained using any of the numerous approaches in combinatorial library methods
known
in the art, including: biological libraries; spatially addressable parallel
solid phase or
solution phase libraries; synthetic library methods requiring deconvolution;
the 'one-
bead one-compound' library method; and synthetic library methods using
affinity
chromatography selection. The biological library approach is limited to
peptide
libraries, while the other four approaches are applicable to peptide, non-
peptide
oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer
Drug
Des. 12:145).
Examples of methods for the synthesis of molecular libraries can be found in
the
art, for example in: DeWitt etal. (1993) Proc. Natl. Acad. Sci. U.S.A.
90:6909; Erb et
al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann etal. (1994). J.
Med.
Chem. 37:2678; Cho et al., (1993) Science 261:1303; Carrell etal. (1994)
Angew.
Chem. Int. Ed. Engl. 33:2059; Carell etal. (1994) Angew. Chem. Int. Ed. Engl.
33:2061;
and in Gallop et al. (1994) J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten (1992)
Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips
(Fodor
(1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner
USP
'409), plasmids (Cull etal. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on
phage
(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-
406);
(Cwirla etal. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) 1
MoL Biol.
222 :301-310); (Ladner supra.).
In one embodiment, an assay is a cell-based assay in which a cell which
expresses a Dkk receptor on the cell surface is contacted with a test compound
and the
ability of the test compound to bind to a Dkk receptor determined. The cell,
for
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example, can be of mammalian origin or a yeast cell. Determining the ability
of the test
compound to bind to a Dkk receptor can be accomplished, for example, by
coupling the
test compound with a radioisotope or enzymatic label such that binding of the
test
compound to the Dick receptor can be determined by detecting the labeled
compound in
a complex. For example, test compounds can be labeled with 1251, 35s, 14C, or
3H,
either directly or indirectly, and the radioisotope detected by direct
counting of
radioemmission or by scintillation counting. Alternatively, test compounds can
be
enzymatically labeled with, for example, horseradish peroxidase, alkaline
phosphatase,
or luciferase, and the enzymatic label detected by determination of conversion
of an
appropriate substrate to product.
It is also within the scope of this invention to determine the ability of a
test
compound to interact with a Dick receptor without the labeling of any of the
interactants.
For example, a microphysiometer can be used to detect the interaction of a
test
compound with a Dkk receptor without the labeling of either the test compound
or the
receptor. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used
herein, a
"microphysiometer" (e.g., CytosensorTM) is an analytical instrument that
measures the
rate at which a cell acidifies its environment using a light-addressable
potentiometric
sensor (LAPS). Changes in this acidification rate can be used as an indicator
of the
interaction between ligand and receptor.
In a preferred embodiment, the assay comprises contacting a cell which
expresses a Dkk receptor on the cell surface with a Dkk protein or
biologically-active
portion thereof, to form an assay mixture, contacting the assay mixture with a
test
compound, and determining the ability of the test compound to interact with a
Dick
receptor, wherein determining the ability of the test compound to interact
with a Dkk
receptor comprises determining the ability of the test compound to
preferentially bind to
the Dkk receptor as compared to the ability of Dkk, or a biologically active
portion
thereof, to bind to the receptor.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell expressing a Dkk target molecule with a test compound and determining the
ability
of the test compound to modulate (e.g. stimulate or inhibit) the activity of
the Dkk target
molecule. Determining the ability of the test compound to modulate the
activity of a
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Dkk target molecule can be accomplished, for example, by determining the
ability of the
Dkk protein to bind to or interact with the Dkk target molecule.
Determining the ability of the Dkk protein to bind to or interact with a Dkk
target
molecule can be accomplished by one of the methods described above for
determining
direct binding. In a preferred embodiment, determining the ability of the Dkk
protein to
bind to or interact with a Dkk target molecule can be accomplished by
determining the
activity of the target molecule. For example, the activity of the target
molecule can be
determined by detecting induction of a cellular second messenger of the target
(i.e.
intracellular Ca2 +, diacylglycerol, 1P3, etc.), detecting catalytic/enzymatic
activity of
the target an appropriate substrate, detecting the induction of a reporter
gene (comprising
a Dkk-responsive regulatory element operatively linked to a nucleic acid
encoding a
detectable marker, e.g., luciferase), or detecting a cellular response, for
example,
development, differentiation or rate of proliferation.
In yet another embodiment, an assay of the present invention is a cell-free
assay
in which a Dkk or Dkk-related protein or biologically active portion thereof
is contacted
with a test compound and the ability of the test compound to bind to the Dkk
or Dkk-
related protein or biologically active portion thereof is determined. Binding
of the test
compound to the Dkk or Dkk-related protein can be determined either directly
or
indirectly as described above. In a preferred embodiment, the assay includes
contacting
the Dkk or Dkk-related protein or biologically active portion thereof with a
known
compound which binds Dkk or the Dkk-related protein to form an assay mixture,
contacting the assay mixture with a test compound, and determining the ability
of the
test compound to interact with a Dkk or Dkk-related protein, wherein
determining the
ability of the test compound to interact with a Dkk or Dkk-related protein
comprises
determining the ability of the test compound to preferentially bind to Dkk or
a Dkk-
related protein or biologically active portion thereof as compared to the
known
compound.
In another embodiment, the assay is a cell-free assay in which a Dkk or Dkk-
related protein or biologically active portion thereof is contacted with a
test compound
and the ability of the test compound to modulate (e.g., stimulate or inhibit)
the activity
of the Dick or Dkk-related protein or biologically active portion thereof is
determined.
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Determining the ability of the test compound to modulate the activity of a Dkk
or Dkk-
related protein can be accomplished, for example, by determining the ability
of the Dkk
or Dkk-related protein to bind to a target molecule (e.g., a Dkk-target
molecule) by one
of the methods described above for determining direct binding. Determining the
ability
of the Dkk or Dkk-related protein to bind to a target molecule can also be
accomplished
using a technology such as real-time Biomolocular Interaction Analysis (BIA).
Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et
al.
(1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, "BIA" is a
technology for
studying biospecific interactions in real time, without labeling any of the
interactants
(e.g., BIAcoreTm). Changes in the optical phenomenon surface plasmon resonance
(SPR)
can be used as an indication of real-time reactions between biological
molecules.
In an alternative embodiment, determining the ability of the test compound to
modulate the activity of a Mk or Dick-related protein can be accomplished by
determining the ability of the Dkk or Dkk-related protein to further modulate
the activity
of a target molecule (e.g., a Dkk-target molecule). For example, the
catalytic/enzymatic
activity of the target molecule on an appropriate substrate can be determined
as
previously described.
In yet another embodiment, the cell-free assay involves contacting a Dkk or
Dkk-
related protein or biologically active portion thereof with a known compound
which
binds the Dick or Dkk-related protein to form an assay mixture, contacting the
assay
mixture with a test compound, and determining the ability of the test compound
to
interact with the Dkk or Dkk-related protein, wherein determining the ability
of the test
compound to interact with the Dkk or Dkk-related protein comprises determining
the
ability of the Dkk or Dkk-related protein to preferentially bind to or
modulate the
activity of a target molecule (e.g., a Dkk target molecule).
In many drug screening programs which test libraries of compounds and natural
extracts, high throughput assays are desirable in order to maximize the number
of
compounds surveyed in a given period of time. Assays which are performed in
cell-free
systems, such as may be derived with purified or semi-purified proteins, are
often
preferred as "primary" screens in that they can be generated to permit rapid
development
and relatively easy detection of an alteration in a molecular target which is
mediated by
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a test compound. Moreover, the effects of cellular toxicity and/or
bioavailability of the
test compound can be generally ignored in the in vitro system, the assay
instead being
focused primarily on the effect of the drug on the molecular target as may be
manifest in
an alteration of binding affinity with upstream or downstream elements.
Accordingly, in
an exemplary screening assay of the present invention, the compound of
interest is
contacted with a Dkk (e.g., hDkk-3) protein or a Dkk (e.g., hDlck-3) binding
partner,
e.g., a receptor. The receptor can be soluble or the receptor can be present
on a cell
surface. To the mixture of the compound and the Dkk protein or Dkk binding
partner is
then added a composition containing a Dkk binding partner or a Dkk protein,
respectively. Detection and quantification of complexes of Dkk proteins and
Dick
binding partners provide a means for determining a compound's efficacy at
inhibiting (or
potentiating) complex formation between Dkk and a binding partner. The
efficacy of
the compound can be assessed by generating dose response curves from data
obtained
using various concentrations of the test compound. Moreover, a control assay
can also
be performed to provide a baseline for comparison. In the control assay,
isolated and
purified Dkk polypeptide or binding partner is added to a composition
containing the
Dkk binding partner or Dkk polypeptide, and the formation of a complex is
quantitated
in the absence of the test compound.
The cell-free assays of the present invention are amenable to use of both
soluble
and/or membrane-bound forms of isolated proteins (e.g. Dkk proteins or
biologically
active portions thereof or Dkk target molecules). In the case of cell-free
assays in which
a membrane-bound form an isolated protein is used (e.g., a Dkk target molecule
or
receptor) it may be desirable to utilize a solubilizing agent such that the
membrane-
bound form of the isolated protein is maintained in solution. Examples of such
solubilizing agents include non-ionic detergents such as n-octylglucoside, n-
dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-
methylglucamide, Triton X-100, Triton X-114, Thesit ,
Isotridecypoly(ethylene
glycol ether), 34(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate
(CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate
(CHAPSO), or N-dodecy1=N,N-dimethy1-3-ammonio-1-propane sulfonate.
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In more than one embodiment of the above assay methods of the present
invention, it may be desirable to immobilize either Dkk, a Dkk-related protein
or a target
molecule to facilitate separation of complexed from uncomplexed forms of one
or both
of the proteins, as well as to accommodate automation of the assay. Binding of
a test
compound to a Dkk or Dkk-related protein, or interaction of a Did( or Dkk-
related
protein with a target molecule in the presence and absence of a candidate
compound, can
be accomplished in any vessel suitable for containing the reactants. Examples
of such
vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In
one
embodiment, a fusion protein can be provided which adds a domain that allows
one or
both of the proteins to be bound to a matrix. For example, glutathione-S-
transferase/
Dkk fusion proteins or glutathione-S-transferase/target fusion proteins can be
adsorbed
onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or
glutathione
derivatized microtitre plates, which are then combined with the test compound
or the test
compound and either the non-adsorbed target protein or Dkk protein, and the
mixture
incubated under conditions conducive to complex formation (e.g., at
physiological
conditions for salt and pH). Following incubation, the beads or microtitre
plate wells are
washed to remove any unbound components, the matrix immobilized in the case of
beads, complex determined either directly or indirectly, for example, as
described above.
Alternatively, the complexes can be dissociated from the matrix, and the level
of Dkk
binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening assays of the invention. For example, either a Dkk protein, Dick-
related
protein, or a Dkk target molecule can be immobilized utilizing conjugation of
biotin and
streptavidin. Biotinylated protein or target molecules can be prepared from
biotin-NHS
(N-hydroxy-succinimide) using techniques well known in the art (e.g.,
biotinylation kit,
Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-
coated 96
well plates (Pierce Chemical). Alternatively, antibodies reactive with Dkk,
Dkk-related
protein, or target molecules but which do not interfere with binding of the
protein to its
target molecule can be derivatized to the wells of the plate, and unbound
target, Dkk, or
Dkk-related protein trapped in the wells by antibody conjugation. Methods for
detecting
such complexes, in addition to those described above for the GST-immobilized
* Trademark
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complexes, include immunodetection of complexes using antibodies reactive with
the
Dkk or Dkk-related protein or target molecule, as well as enzyme-linked assays
which
rely on detecting an enzymatic activity associated with the Dkk or Dkk-related
protein or
target molecule.
In another embodiment, modulators of Dkk or Dkk-related expression are
identified in a method wherein a cell is contacted with a candidate compound
and the
expression of Dkk or Dkk-related mRNA or protein in the cell is determined.
The level
of expression of mRNA or protein in the presence of the candidate compound is
compared to the level of expression of mRNA or protein in the absence of the
candidate
compound. The candidate compound can then be identified as a modulator of Dkk
or
Dkk-related expression based on this comparison. For example, when expression
of
Dkk mRNA or protein is greater (statistically significantly greater) in the
presence of the
candidate compound than in its absence, the candidate compound is identified
as a
stimulator of Dkk mRNA or protein expression. Alternatively, when expression
of Dkk
mRNA or protein is less (statistically significantly less) in the presence of
the candidate
compound than in its absence, the candidate compound is identified as an
inhibitor of
Dkk mRNA or protein expression. The level of Dkk or Dick-related mRNA or
protein
expression in the cells can be determined by methods described herein for
detecting Dkk
mRNA or protein.
In yet another aspect of the invention, the Dkk or Dkk-related proteins can be
used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see,
e.g., U.S. Patent
No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.
Biol.
Chem. 268:12046-12054; Bartel et al., (1993) Biotechniques 14:920-924;
Iwabuchi et
al. (1993) Oncogene 8:1693-1696; and Brent W094/10300), to identify other
proteins,
which bind to or interact with Dkk or Dkk-related proteins ("binding proteins"
or "bp")
and modulate Dkk or Dkk-related activity. Suchbinding proteins are also likely
to be
involved in the propagation of signals by the Dkk or Dkk-related proteins as,
for
example, downstream elements of a Dkk-mediated signaling pathway.
Alternatively,
such binding proteins are likely to be cell-surface molecules associated with
non-Dkk
expressing cells, wherein such binding proteins are involved in signal
transduction.
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The two-hybrid system is based on the modular nature of most transcription
factors, which consist of separable DNA-binding and activation domains.
Briefly, the
assay utilizes two different DNA constructs. In one construct, the gene that
codes for a
Dkk protein is fused to a gene encoding the DNA binding domain of a known
transcription factor (e.g., GAL-4). In the other construct, a DNA sequence,
from a
library of DNA sequences, that encodes an unidentified protein ("prey" or
"sample") is
fused to a gene that codes for the activation domain of the known
transcription factor. If
the "bait" and the "prey" proteins are able to interact, in vivo, forming a
Dkk-dependent
complex, the DNA-binding and activation domains of the transcription factor
are
brought into close proximity. This proximity allows transcription of a
reporter gene
(e.g., LacZ) which is operably linked to a transcriptional regulatory site
responsive to the
transcription factor. Expression of the reporter gene can be detected and cell
colonies
containing the functional transcription factor can be isolated and used to
obtain the
cloned gene which encodes the protein which interacts with the Dkk or Dkk-
related
protein.
This invention further pertains to novel agents identified by the above-
described
screening assays and to processes for producing such agents by use of these
assays.
Accordingly, in one embodiment, the present invention includes a compound or
agent
obtainable by a method comprising the steps of any one of the aformentioned
screening
assays (e.g., cell-based assays or cell-free assays). For example, in one
embodiment, the
invention includes a compound or agent obtainable by a method comprising
contacting a
cell which expresses a target molecule with a test compound and the
determining the
ability of the test compound to bind to, or modulate the activity of, the
target molecule.
In another embodiment, the invention includes a compound or agent obtainable
by a
method comprising contacting a cell which expresses a target molecule with a
Dkk or
Dkk-related protein or biologically-active portion thereof, to form an assay
mixture,
contacting the assay mixture with a test compound, and determining the ability
of the
test compound to interact with, or modulate the activity of, the target
molecule. In
another embodiment, the invention includes a compound or agent obtainable by a
method comprising contacting a Dkk or Dkk-related protein or biologically
active
portion thereof with a test compound and determining the ability of the test
compound to
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bind to, or modulate (e.g., stimulate or inhibit) the activity of, the Dkk or
Dkk-related
protein or biologically active portion thereof. In yet another embodiment, the
present
invention includes a compound or agent obtainable by a method comprising
contacting a
Dkk or Dkk-related protein or biologically active portion thereof with a known
compound which binds the Dkk or Dkk-related protein to form an assay mixture,
contacting the assay mixture with a test compound, and determining the ability
of the
test compound to interact with, or modulate the activity of the Dkk or Dkk-
related
protein.
Accordingly, it is within the scope of this invention to further use an agent
identified as described herein in an appropriate animal model. For example, an
agent
identified as described herein (e.g., a Dkk modulating agent, an antisense
Dick nucleic
acid molecule, a Dkk-specific antibody, or a Dkk-binding partner) can be used
in an
animal model to determine the efficacy, toxicity, or side effects of treatment
with such
an agent. Alternatively, an agent identified as described herein can be used
in an animal
model to determine the mechanism of action of such an agent.
The present inventon also pertains to uses of novel agents identified by the
above-described screening assays for diagnoses, prognoses, and treatments as
described
herein. Accordingly, it is within the scope of the present invention to use
such agents in
the design, formulation, synthesis, manufacture, and/or production of a drug
or
pharmaceutical composition for use in diagnosis, prognosis, or treatment, as
described
herein. For example, in one embodiment, the present invention includes a
method of
synthesizing or producing a drug or pharmaceutical composition by reference to
the
structure and/or properties of a compound obtainable by one of the above-
described
screening assays. For example, a drug or pharmaceutical composition can be
synthesized based on the structure and/or properties of a compound obtained by
a
method in which a cell which expresses a target molecule (e.g., a Dkk target
molecule)
is contacted with a test compound and the ability of the test compound to bind
to, or
modulate the activity of, the target molecule is determined. In another
exemplary
embodiment, the present invention includes a method of synthesizing or
producing a
drug or pharmaceutical composition based on the structure and/or properties of
a
compound obtainable by a method in which a Dkk or Dick-related protein or
biologically
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active portion thereof is contacted with a test compound and the ability of
the test
compound to bind to, or modulate (e.g., stimulate or inhibit) the activity of,
the Dkk or
Dick-related protein or biologically active portion thereof is determined.
B. Detection Assays
Portions or fragments of the cDNA sequences identified herein (and the
corresponding complete gene sequences) can be used in numerous ways as
polynucleotide reagents. For example, these sequences can be used to: (i) map
their
respective genes on a chromosome; and, thus, locate gene regions associated
with
genetic disease; (ii) identify an individual from a minute biological sample
(tissue
typing); and (iii) aid in forensic identification of a biological sample.
These applications
are described in the subsections below.
1. Chromosome Mapping
Once the sequence (or a portion of the sequence) of a gene has been isolated,
this
sequence can be used to map the location of the gene on a chromosome. This
process is
called chromosome mapping. Accordingly, portions or fragments of the Dkk or
Dkk-
related nucleotide sequences, described herein, can be used to map the
location of the
Dkk or Dkk-related genes on a chromosome. The mapping of the Dkk or Dkk-
related
sequences to chromosomes is an important first step in correlating these
sequences with
genes associated with disease.
Briefly, Dkk or Dkk-related genes can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp in length) from the Dkk or Dkk-related
nucleotide
sequences. Computer analysis of the Dkk or Dkk-related sequences can be used
to
predict primers that do not span more than one exon in the genomic DNA, thus
complicating the amplification process. These primers can then be used for PCR
screening of somatic cell hybrids containing individual human chromosomes.
Only
those hybrids containing the human gene corresponding to the Dick or Dkk-
related
sequences will yield an amplified fragment.
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Somatic cell hybrids are prepared by fusing somatic cells from different
mammals (e.g., human and mouse cells). As hybrids of human and mouse cells
grow
and divide, they gradually lose human chromosomes in random order, but retain
the
mouse chromosomes. By using media in which mouse cells cannot grow, because
they
lack a particular enzyme, but human cells can, the one human chromosome that
contains
the gene encoding the needed enzyme, will be retained. By using various media,
panels
of hybrid cell lines can be established. Each cell line in a panel contains
either a single
human chromosome or a small number of human chromosomes, and a full set of
mouse
chromosomes, allowing easy mapping of individual genes to specific human
chromosomes. (D'Eustachio P. et al., (1983) Science 220:919-924). Somatic cell
hybrids containing only fragments of human chromosomes can also be produced by
using human chromosomes with translocations and deletions.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular sequence to a particular chromosome. Three or more sequences can be
assigned per day using a single thermal cycler. Using the Dkk or Dkk-related
nucleotide
sequences to design oligonucleotide primers, sublocalization can be achieved
with
panels of fragments from specific chromosomes. Other mapping strategies which
can
similarly be used to map a 9o, 1 p, or lv sequence to its chromosome include
in situ
hybridization (described in Fan, Y. et al., (1990) PNAS, 87:6223-27), pre-
screening with
labeled flow-sorted chromosomes, and pre-selection by hybridization to
chromosome
specific cDNA libraries.
Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase
chromosomal spread can further be used to provide a precise chromosomal
location in
one step. Chromosome spreads can be made using cells whose division has been
blocked in metaphase by a chemical such as colcemid that disrupts the mitotic
spindle.
The chromosomes can be treated briefly with trypsin, and then stained with
Giemsa. A
pattern of light and dark bands develops on each chromosome, so that the
chromosomes
can be identified individually. The FISH technique can be used with a DNA
sequence
as short as 500 or 600 bases. However, clones larger than 1,000 bases have a
higher
likelihood of binding to a unique chromosomal location with sufficient signal
intensity
for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases
will
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suffice to get good results at a reasonable amount of time. For a review of
this
technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York 1988).
Reagents for chromosome mapping can be used individually to mark a single
chromosome or a single site on that chromosome, or panels of reagents can be
used for
marking multiple sites and/or multiple chromosomes. Reagents corresponding to
noncoding regions of the genes actually are preferred for mapping purposes.
Coding
sequences are more likely to be conserved within gene families, thus
increasing the
chance of cross hybridizations during chromosomal mapping.
Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic map
data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance
in
Man, available on-line through Johns Hopkins University Welch Medical
Library). The
relationship between a gene and a disease, mapped to the same chromosomal
region, can
then be identified through linkage analysis (co-inheritance of physically
adjacent genes),
described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.
Moreover, differences in the DNA sequences between individuals affected and
unaffected with a disease associated with a Dkk or Dkk-related gene, can be
determined.
If a mutation is observed in some or all of the affected individuals but not
in any
unaffected individuals, then the mutation is likely to be the causative agent
of the
particular disease. Comparison of affected and unaffected individuals
generally involves
first looking for structural alterations in the chromosomes, such as deletions
or
translocations that are visible from chromosome spreads or detectable using
PCR based
on that DNA sequence. Ultimately, complete sequencing of genes from several
individuals can be performed to confirm the presence of a mutation and to
distinguish
mutations from polymorphisms.
2. Tissue Typing
The Dkk or Dkk-related sequences of the present invention can also be used to
identify individuals from minute biological samples. The United States
military, for
example, is considering the use of restriction fragment length polymorphism
(RFLP) for
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identification of its personnel. In this technique, an individual's genomic
DNA is
digested with one or more restriction enzymes, and probed on a Southern blot
to yield
unique bands for identification. This method does not suffer from the current
limitations
of "Dog Tags" which can be lost, switched, or stolen, making positive
identification
difficult. The sequences of the present invention are useful as additional DNA
markers
for RFLP (described in U.S. Patent 5,272,057).
Furthermore, the sequences of the present invention can be used to provide an
alternative technique which determines the actual base-by-base DNA sequence of
selected portions of an individual's genome. Thus, the Dkk or Dkk-related
nucleotide
sequences described herein can be used to prepare two PCR primers from the 5'
and 3'
ends of the sequences. These primers can then be used to amplify an
individual's DNA
and subsequently sequence it.
Panels of corresponding DNA sequences from individuals, prepared in this
manner, can provide unique individual identifications, as each individual will
have a
unique set of such DNA sequences due to allelic differences. The sequences of
the
present invention can be used to obtain such identification sequences from
individuals
and from tissue. The Dkk or Dkk-related nucleotide sequences of the invention
uniquely
represent portions of the human genome. Allelic variation occurs to some
degree in the
coding regions of these sequences, and to a greater degree in the noncoding
regions. It
is estimated that allelic variation between individual humans occurs with a
frequency of
about once per each 500 bases. Each of the sequences described herein can, to
some
degree, be used as a standard against which DNA from an individual can be
compared
for identification purposes. Because greater numbers of polymorphisms occur in
the
noncoding regions, fewer sequences are necessary to differentiate individuals.
The
noncoding sequences of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13,
or SEQ ID NO:20, can comfortably provide positive individual identification
with a
panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified
sequence
of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:3, SEQ
ID
NO:6, SEQ ID NO:9, SEQ ID NO:15 or SEQ ID NO:22 are used, a more appropriate
number of primers for positive individual identification would be 500-2,000.
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If a panel of reagents from Dkk or Dkk-related nucleotide sequences described
herein is used to generate a unique identification database for an individual,
those same
reagents can later be used to identify tissue from that individual. Using the
unique
identification database, positive identification of the individual, living or
dead, can be
made from extremely small tissue samples.
3. Use of Partial Dkk or Dkk-related Sequences in Forensic Biology
DNA-based identification techniques can also be used in forensic biology.
Forensic biology is a scientific field employing genetic typing of biological
evidence
found at a crime scene as a means for positively identifying, for example, a
perpetrator
of a crime. To make such an identification, PCR technology can be used to
amplify
DNA sequences taken from very small biological samples such as tissues, e.g.,
hair or
skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene.
The amplified
sequence can then be compared to a standard, thereby allowing identification
of the
origin of the biological sample.
The sequences of the present invention can be used to provide polynucleotide
reagents, e.g., PCR primers, targeted to specific loci in the human genome,
which can
enhance the reliability of DNA-based forensic identifications by, for example,
providing
another "identification marker" (i.e. another DNA sequence that is unique to a
particular
individual). As mentioned above, actual base sequence information can be used
for
identification as an accurate alternative to patterns formed by restriction
enzyme
generated fragments. Sequences targeted to noncoding regions of SEQ ID NOs:1,
SEQ
ID NO:4, SEQ ID NO:7, SEQ ID NO:13, or SEQ ID NO:20 are particularly
appropriate
for this use as greater numbers of polymorphisms occur in the noncoding
regions,
making it easier to differentiate individuals using this technique. Examples
of
polynucleotide reagents include the Dkk nucleotide sequences or portions
thereof, e.g.,
fragments derived from the noncoding regions of SEQ ID NO:1, SEQ ID NO:4, SEQ
ID
NO:7, SEQ ID NO:13, or SEQ ID NO:20, having a length of at least 20 bases,
preferably at least 30 bases.
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The Dkk or Dkk-related nucleotide sequences described herein can further be
used to provide polynucleotide reagents, e.g., labeled or labelable probes
which can be
used in, for example, an in situ hybridization technique, to identify a
specific tissue, e.g.,
brain tissue. This can be very useful in cases where a forensic pathologist is
presented
with a tissue of unknown origin. Panels of such Dkk or Dkk-related probes can
be used
to identify tissue by species and/or by organ type.
In a similar fashion, these reagents, e.g., Dkk or Dkk-related primers or
probes
can be used to screen tissue culture for contamination (i.e. screen for the
presence of a
mixture of different types of cells in a culture).
C. Predictive Medicine:
The present invention also pertains to the field of predictive medicine in
which
diagnostic assays, prognostic assays, and monitoring clinical trials are used
for
prognostic (predictive) purposes to thereby treat an individual
prophylactically.
Accordingly, one aspect of the present invention relates to diagnostic assays
for
determining Dkk or Dkk-related protein and/or nucleic acid expression as well
as Dkk or
Dkk-related activity, in the context of a biological sample (e.g., blood,
serum, cells,
tissue) to thereby determine whether an individual is afflicted with a disease
or disorder,
or is at risk of developing a disorder, associated with aberrant Dkk or Dkk-
related
expression or activity, such as aberrant cell proliferation, differentiation,
and/or survival
resulting for example in a neurodegenerative disease (e.g., Alzheimer's
disease,
Parkinson's disease, Huntington's chorea, amylotrophic lateral sclerosis and
the like, as
well as spinocerebellar degenerations) or cancer (for example, cancers of the
epithelia
(e.g., carcinomas of the pancreas, stomach, liver, secretory glands (e.g.,
adenocarcinoma) bladder, lung, breast, skin (e.g., malignant melanoma),
reproductive
tract including prostate gland, ovary, cervix and uterus); cancers of the
hematopoietic
and immune system (e.g., leukemias and lymphomas); cancers of the central
nervous,
brain system and eye (e.g., gliomas, glioblastoma, neuroblastoma and
retinoblastoma);
and cancers of connective tissues, bone, muscles and vasculature (e.g.,
sarcomas)). The
invention also provides for prognostic (or predictive) assays for determining
whether an
individual is at risk of developing a disorder associated with Dkk or Dkk-
related protein,
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_
nucleic acid expression or activity. For example, mutations in a Dick or Dkk-
related
gene can be assayed in a biological sample. Such assays can be used for
prognostic or
predictive purpose to thereby phophylactically treat an individual prior to
the onset of a
disorder characterized by or associated with Dkk or Dkk-related protein,
nucleic acid
expression or activity.
Another aspect of the invention pertains to monitoring the influence of agents
(e.g., drugs, compounds) on the expression or activity of Dick or Dkk-related
in clinical
trials.
These and other agents are described in further detail in the following
sections.
=
1. Diagnostic Assays
An exemplary method for detecting the presence or absence of Dkk or Dkk-
related protein or nucleic acid in a biological sample involves obtaining a
biological
sample from a test subject and contacting the biological sample with a
compound or an
agent capable of detecting Dkk or Dkk-related protein or nucleic acid (e.g.,
mRNA,
genomic DNA) that encodes Dick or Dick-related protein such that the presence
of Dldc
or Dkk-related protein or nucleic acid is detected in the biological sample. A
preferred
agent for detecting Dkk or Dick-related mRNA or genomic DNA is a labeled
nucleic
acid probe capable of hybridizing to Dkk or Dkk-related mRNA or genomic DNA.
The
nucleic acid probe can be, for example, a full-length Dick nucleic acid, such
as the
nucleic acid of SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:13, SEQ ID
NO:20, the DNA insert of the plasmid deposited with ATCC as Accession Number
98452, or the DNA insert of the plasmid deposited with ATCC as Accession
Number
98633,
or a portion thereof, such as an oligonucleotide of
at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to
specifically
hybridize under stringent conditions to Dick or Dkk-related mRNA or genomic
DNA.
Other suitable probes for use in the diagnostic assays of the invention are
described
herein.
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A preferred agent for detecting Dkk or Dkk-related protein is an antibody
capable of binding to the protein, preferably an antibody with a detectable
label.
Antibodies can be polyclonal, or more preferably, monoclonal. An intact
antibody, or a
fragment thereof (e.g., Fab or F(ab')2) can be used. The term "labeled", with
regard to
the probe or antibody, is intended to encompass direct labeling of the probe
or antibody
by coupling (i.e., physically linking) a detectable substance to the probe or
antibody, as
well as indirect labeling of the probe or antibody by reactivity with another
reagent that
is directly labeled. Examples of indirect labeling include detection of a
primary
antibody using a fluorescently labeled secondary antibody and end-labeling of
a DNA
probe with biotin such that it can be detected with fluorescently labeled
streptavidin.
The term "biological sample" is intended to include tissues, cells and
biological fluids
isolated from a subject, as well as tissues, cells and fluids present within a
subject. That
is, the detection method of the invention can be used to detect Dkk or Dkk-
related
mRNA, protein, or genomic DNA in a biological sample in vitro as well as in
vivo. For
example, in vitro techniques for detection of Dkk or Dkk-related mRNA include
Northern hybridizations and in situ hybridizations. In vitro techniques for
detection of
Dkk or Dkk-related protein include enzyme linked immunosorbent assays
(ELISAs),
Western blots, immunoprecipitations and immunofluorescence. In vitro
techniques for
detection of Dkk or Dkk-related genomic DNA include Southern hybridizations.
Furthermore, in vivo techniques for detection of Dkk or Dkk-related protein
include
introducing into a subject a labeled antibody. For example, the antibody can
be labeled
with a radioactive marker whose presence and location in a subject can be
detected by
standard imaging techniques.
In one embodiment, the biological sample contains protein molecules from the
test subject. Alternatively, the biological sample can contain mRNA molecules
from the
test subject or genomic DNA molecules from the test subject. A preferred
biological
sample is a serum sample isolated by conventional means from a subject.
In another embodiment, the methods further involve obtaining a control
biological sample from a control subject, contacting the control sample with a
compound or agent capable of detecting Dkk or Dkk-related protein, mRNA, or
genomic
DNA, such that the presence of Dkk or Dkk-related protein, mRNA or genomic DNA
is
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detected in the biological sample, and comparing the presence of Dkk or Dkk-
related
protein, mRNA or genomic DNA in the control sample with the presence of Dldc
or
Dkk-related protein, mRNA or genomic DNA in the test sample.
The invention also encompasses kits for detecting the presence of Dkk or a Dkk-
related protein in a biological sample. For example, the kit can comprise a
labeled
compound or agent capable of detecting Dkk or Dkk-related protein or mRNA in a
biological sample; means for determining the amount of Dkk or Dkk-related
protein or
mRNA in the sample; and means for comparing the amount of Dkk or Dkk-related
protein or mRNA in the sample with a standard. The compound or agent can be
packaged in a suitable container. The kit can further comprise instructions
for using the
kit to detect Dkk or Dkk-related protein or nucleic acid.
2. Prognostic Assays
The diagnostic methods described herein can furthermore be utilized to
identify
subjects having or at risk of developing a disease or disorder associated with
aberrant
Dkk expression or activity. For example, the assays described herein, such as
the
preceding diagnostic assays or the following assays, can be utilized to
identify a subject
having or at risk of developing a disorder associated with Dkk or Dkk-related
protein,
nucleic acid expression or activity such as a proliferative disorder, a
differentiative or
developmental disorder, a hematopoietic disorder as well as diseases,
conditions or
disorders characterized by abnormal cell survival, abnormal extracellular
structure, or an
abnormality in a defense mechanism. Alternatively, the prognostic assays can
be
utilized to identify a subject having or at risk for developing a
differentiative or
proliferative disease (e.g., cancer). Thus, the present invention provides a
method for
identifying a disease or disorder associated with aberrant Dkk or Dkk-related
expression
or activity in which a test sample is obtained from a subject and Dkk or Dkk-
related
protein or nucleic acid (e.g., mRNA, genomic DNA) is detected, wherein the
presence of
Dkk or Dkk-related protein or nucleic acid is diagnostic for a subject having
or at risk of
developing a disease or disorder associated with aberrant Dick or Dkk-related
expression
or activity. As used herein, a "test sample" refers to a biological sample
obtained from a
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subject of interest. For example, a test sample can be a biological fluid
(e.g., serum),
cell sample, or tissue.
Furthermore, the prognostic assays described herein can be used to determine
whether a subject can be administered an agent (e.g., an agonist, antagonist,
peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug
candidate)
to treat a disease or disorder associated with aberrant Dkk or Dkk-related
expression or
activity. For example, such methods can be used to determine whether a subject
can be
effectively treated with an agent for a disorder, such as a proliferative
disorder, a
differentiative or developmental disorder, a hematopoietic disorder, as well
disorders
characterized by abnormal cell survival, an abnormal extracellular structure,
or an
abnormality in a defense mechanism. Alternatively, such methods can be used to
determine whether a subject can be effectively treated with an agent for a
differentiative
or proliferative disease (e.g., cancer). Thus, the present invention provides
methods for
determining whether a subject can be effectively treated with an agent for a
disorder
associated with aberrant Dkk or Dick-related expression or activity in which a
test
sample is obtained and Dkk or Dkk-related protein or nucleic acid expression
or activity
is detected (e.g., wherein the abundance of Dkk or Dkk-related protein or
nucleic acid
expression or activity is diagnostic for a subject that can be administered
the agent to
treat a disorder associated with aberrant Dkk or Dkk-related expression or
activity.)
The methods of the invention can also be used to detect genetic alterations in
a
Dkk or Dkk-related gene, thereby determining if a subject with the altered
gene is at risk
for a disorder characterized by aberrant development, aberrant cellular
differentiation,
aberrant cellular proliferation or an aberrant hematopoietic response. In
preferred
embodiments, the methods include detecting, in a sample of cells from the
subject, the
presence or absence of a genetic alteration characterized by at least one of
an alteration
affecting the integrity of a gene encoding a Dick or Dick-related-protein, or
the mis-
expression of the Dkk or Dkk-related gene. For example, such genetic
alterations can be
detected by ascertaining the existence of at least one of 1) a deletion of one
or more
nucleotides from a Dkk or Dick-related gene; 2) an addition of one or more
nucleotides
to a Dkk or Dkk-related gene; 3) a substitution of one or more nucleotides of
a Dkk or
Dkk-related gene, 4) a chromosomal rearrangement of a Dkk or Dkk-related gene;
5) an
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alteration in the level of a messenger RNA transcript of a Dkk or Dkk-related
gene, 6)
aberrant modification of a Dkk or Dick-related gene, such as of the
methylation pattern
of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a
messenger
RNA transcript of a Dkk or Dkk-related gene, 8) a non-wild type level of a DU
or Dkk-
related-protein, 9) allelic loss of a Dkk or Dkk-related gene, and 10)
inappropriate post-
translational modification of a Dick or DU-related-protein. As described
herein, there
are a large number of assay techniques known in the art which can be used for
detecting
alterations in a Dkk or Dkk-related gene. A preferred biological sample is a
tissue or
serum sample isolated by conventional means from a subject.
In certain embodiments, detection of the alteration involves the use of a
probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos.
4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively,
in a
ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science
241:1077-1080;
and Nakazawa et al. (1994) PNAS 91:360-364), the latter of which can be
particularly
useful for detecting point mutations in the Dkk or Dkk-related-gene (see
Abravaya et al.
(1995) Nucleic Acids Res .23:675-682). This method can include the steps of
collecting
a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA
or both)
from the cells of the sample, contacting the nucleic acid sample with one or
more
primers which specifically hybridize to a Dkk or Dkk-related gene under
conditions such
that hybridization and amplification of the Dkk or Dkk-related-gene (if
present) occurs,
and detecting the presence or absence of an amplification product, or
detecting the size
of the amplification product and comparing the length to a control sample. It
is
anticipated that PCR and/or LCR may be desirable to use as a preliminary
amplification
step in conjunction with any of the techniques used for detecting mutations
described
herein.
Alternative amplification methods include: self sustained sequence replication
(Guatelli, J.C. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878),
transcriptional
amplification system (Kwoh, D.Y. et al., 1989, Proc. Natl. Acad. Sci. USA
86:1173-
*
1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1197),
or any
other nucleic acid amplification method, followed by the detection of the
amplified
molecules using techniques well known to those of skill in the art. These
detection
* Trademark
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schemes are especially useful for the detection of nucleic acid molecules if
such
molecules are present in very low numbers.
In an alternative embodiment, mutations in a Dkk or Dkk-related gene from a
sample cell can be identified by alterations in restriction enzyme cleavage
patterns. For
example, sample and control DNA is isolated, amplified (optionally), digested
with one
or more restriction endonucleases, and fragment length sizes are determined by
gel
electrophoresis and compared. Differences in fragment length sizes between
sample and
control DNA indicates mutations in the sample DNA. Moreover, the use of
sequence
specific ribozymes (see, for example, U.S. Patent No. 5,498,531) can be used
to score
for the presence of specific mutations by development or loss of a ribozyme
cleavage
site.
In other embodiments, genetic mutations in a Dkk or Dkk-related gene can be
identified by hybridizing a sample and control nucleic acids, e.g., DNA or
RNA, to high
density arrays containing hundreds or thousands of oligonucleotides probes
(Cronin,
M.T. et al. (1996) Human Mutation 7: 244-255; Kozal, M.J. etal. (1996) Nature
Medicine 2: 753-759). For example, genetic mutations in Dkk can be identified
in two
dimensional arrays containing light-generated DNA probes as described in
Cronin, M.T.
et al. supra. Briefly, a first hybridization array of probes can be used to
scan through
long stretches of DNA in a sample and control to identify base changes between
the
sequences by making linear arrays of sequential ovelapping probes. This step
allows the
identification of point mutations. This step is followed by a second
hybridization array
that allows the characterization of specific mutations by using smaller,
specialized probe
arrays complementary to all variants or mutations detected. Each mutation
array is
composed of parallel probe sets, one complementary to the wild-type gene and
the other
complementary to the mutant gene.
In yet another embodiment, any of a variety of sequencing reactions known in
the art can be used to directly sequence the Dkk or Dkk-related gene and
detect
mutations by comparing the sequence of the sample Dkk or Dkk-related sequence
with
the corresponding wild-type (control) sequence. Examples of sequencing
reactions
include those based on techniques developed by Maxim and Gilbert ((1977) PNAS
74:560) or Sanger ((1977) PNAS 74:5463). It is also contemplated that any of a
variety
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of automated sequencing procedures can be utilized when performing the
diagnostic
assays ((1995) Biotechniques 19:448), including sequencing by mass
spectrometry (see,
e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv.
Chromatogr. 36:127-162; and Griffin et al. (1993) App!. Biochem. Biotechnol.
38:147-
159).
Other methods for detecting mutations in the Dkk or Dkk-related gene include
methods in which protection from cleavage agents is used to detect mismatched
bases in
RNA/RNA or RNA/DNA heteroduplexes (Myers etal. (1985) Science 230:1242). In
general, the art technique of "mismatch cleavage" starts by providing
heteroduplexes of
formed by hybridizing (labeled) RNA or DNA containing the wild-type Dkk or Dkk-
related sequence with potentially mutant RNA or DNA obtained from a tissue
sample.
The double-stranded duplexes are treated with an agent which cleaves single-
stranded
regions of the duplex such as which will exist due to basepair mismatches
between the
control and sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with Si nuclease to enzymatically digesting
the
mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes
can be treated with hydroxylamine or osmium tetroxide and with piperidine in
order to
digest mismatched regions. After digestion of the mismatched regions, the
resulting
material is then separated by size on denaturing polyacrylamide gels to
determine the
site of mutation. See, for example, Cotton etal. (1988) Proc. Natl Acad Sci
USA
85:4397; Saleeba etal. (1992) Methods Enzymol. 217:286-295. In a preferred
embodiment, the control DNA or RNA can be labeled for detection.
In still another embodiment, the mismatch cleavage reaction employs one or
more proteins that recognize mismatched base pairs in double-stranded DNA (so
called
"DNA mismatch repair" enzymes) in defined systems for detecting and mapping
point
mutations in Dkk cDNAs obtained from samples of cells. For example, the mutY
enzyme of E. coil cleaves A at G/A mismatches and the thymidine DNA
glycosylase
from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis
15:1657-1662). According to an exemplary embodiment, a probe based on a Dkk
sequence, e.g., a wild-type Dkk sequence, is hybridized to a cDNA or other DNA
product from a test cell(s). The duplex is treated with a DNA mismatch repair
enzyme,
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and the cleavage products, if any, can be detected from electrophoresis
protocols or the
like. See, for example, U.S. Patent No. 5,459,039.
In other embodiments, alterations in electrophoretic mobility will be used to
identify mutations in Dkk or Dkk-related genes. For example, single strand
conformation polymorphism (SSCP) may be used to detect differences in
electrophoretic
mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc
Natl.
Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and
Hayashi
(1992) Genet Anal Tech App! 9:73-79). Single-stranded DNA fragments of sample
and
control Dkk or Dkk-related nucleic acids will be denatured and allowed to
renature. The
secondary structure of single-stranded nucleic acids varies according to
sequence, the
resulting alteration in electrophoretic mobility enables the detection of even
a single
base change. The DNA fragments may be labeled or detected with labeled probes.
The
sensitivity of the assay may be enhanced by using RNA (rather than DNA), in
which the
secondary structure is more sensitive to a change in sequence. In a preferred
embodiment, the subject method utilizes heteroduplex analysis to separate
double
stranded heteroduplex molecules on the basis of changes in electrophoretic
mobility
(Keen et al. (1991) Trends Genet 7:5).
In yet another embodiment the movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed using
denaturing
gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When
DGGE is used as the method of analysis, DNA will be modified to insure that it
does not
completely denature, for example by adding a GC clamp of approximately 40 bp
of
high-melting GC-rich DNA by PCR. In a further embodiment, a temperature
gradient is
used in place of a denaturing gradient to identify differences in the mobility
of control
and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
Examples of other techniques for detecting point mutations include, but are
not
limited to, selective oligonucleotide hybridization, selective amplification,
or selective
primer extension. For example, oligonucleotide primers may be prepared in
which the
known mutation is placed centrally and then hybridized to target DNA under
conditions
which permit hybridization only if a perfect match is found (Saiki et al.
(1986) Nature
324:163); Saiki et al. (1989) Proc. Natl Acad Sci USA 86:6230). Such allele
specific
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oligonucleotides are hybridized to PCR amplified target DNA or a number of
different
mutations when the oligonucleotides are attached to the hybridizing membrane
and
hybridized with labeled target DNA.
Alternatively, allele specific amplification technology which depends on
selective PCR amplification may be used in conjunction with the instant
invention.
Oligonucleotides used as primers for specific amplification may carry the
mutation of
interest in the center of the molecule (so that amplification depends on
differential
hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the
extreme 3
' end of one primer where, under appropriate conditions, mismatch can prevent,
or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it
may be
desirable to introduce a novel restriction site in the region of the mutation
to create
cleavage-based detection (Gasparini et al. (1992) Mot. Cell Probes 6:1). It is
anticipated
that in certain embodiments amplification may also be performed using Taq
ligase for
amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases,
ligation
will occur only if there is a perfect match at the 3' end of the 5' sequence
making it
possible to detect the presence of a known mutation at a specific site by
looking for the
presence or absence of amplification.
The methods described herein may be performed, for example, by utilizing pre-
packaged diagnostic kits comprising at least one probe nucleic acid or
antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings
to diagnose
patients exhibiting symptoms or family history of a disease or illness
involving a Dkk
gene.
Furthermore, any cell type or tissue in which Dkk or a Dkk-related sequence is
expressed may be utilized in the prognostic assays described herein.
3. Monitoring of Effects During Clinical Trials
Monitoring the influence of agents (e.g., drugs, compounds) on the expression
or
activity of Dkk or Dkk-related molecule (e.g., modulation of cellular signal
transduction,
regulation of gene transcription in a cell involved in development or
differentiation,
regulation of cellular proliferation) can be applied not only in basic drug
screening, but
also in clinical trials. For example, the effectiveness of an agent determined
by a
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screening assay as described herein to increase Dkk or Dkk-related gene
expression,
protein levels, or upregulate Dkk or Dkk-related activity, can be monitored in
clinical
trials of subjects exhibiting decreased Dkk or Dkk-related gene expression,
protein
levels, or downregulated Dkk or Dkk-related activity. Alternatively, the
effectiveness of
an agent determined by a screening assay to decrease Dkk or Dkk-related gene
expression, protein levels, or downregulate Dkk or Dkk-related activity, can
be
monitored in clinical trials of subjects exhibiting increased Dkk or Dkk-
related gene
expression, protein levels, or upregulated Dkk or Dkk-related activity. In
such clinical
trials, the expression or activity of Dick or Dkk-related and, preferably,
other genes that
have been implicated in, for example, a proliferative disorder can be used as
a "read out"
or markers of the phenotype of a particular cell.
For example, and not by way of limitation, genes, including Dkk and Dkk-
related genes, that are modulated in cells by treatment with an agent (e.g.,
compound,
drug or small molecule) which modulates Dkk or Dkk-related activity (e.g.,
identified in
a screening assay as described herein) can be identified. Thus, to study the
effect of
agents on proliferative disorders, developmental or differentiative disorder,
hematopoietic disorder as well disorders characterized by abnormal cell
differentiation
and/or survival, an abnormal extracellular structure, or an abnormality in a
defense
mechanism, for example, in a clinical trial, cells can be isolated and RNA
prepared and
analyzed for the levels of expression of Dkk, Dkk-related, and other genes
implicated in
the proliferative disorder, developmental or differentiative disorder,
hematopoietic
disorder as well as disorders characterized by abnormal cell differentiation
and/or
survival, an abnormal extracellular structure, or an abnormality in a defense
mechanism,
respectively. The levels of gene expression (i.e., a gene expression pattern)
can be
quantified by Northern blot analysis or RT-PCR, as described herein, or
alternatively by
measuring the amount of protein produced, by one of the methods as described
herein,
or by measuring the levels of activity of Dkk, Dkk-related, or other genes. In
this way,
the gene expression pattern can serve as a marker, indicative of the
physiological
response of the cells to the agent. Accordingly, this response state may be
determined
before, and at various points during treatment of the individual with the
agent.
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In a preferred embodiment, the present invention provides a method for
monitoring the effectiveness of treatment of a subject with an agent (e.g., an
agonist,
antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or
other drug
candidate identified by the screening assays described herein) comprising the
steps of (i)
obtaining a pre-administration sample from a subject prior to administration
of the
agent; (ii) detecting the level of expression of a Dkk or Dkk-related protein,
mRNA, or
genomic DNA in the preadministration sample; (iii) obtaining one or more post-
administration samples from the subject; (iv) detecting the level of
expression or activity
of the Dkk or Dkk-related protein, mRNA, or genomic DNA in the post-
administration
samples; (v) comparing the level of expression or activity of the Dkk or Dkk-
related
protein, mRNA, or genomic DNA in the pre-administration sample with the Didc
or
Dkk-related protein, mRNA, or genomic DNA in the post administration sample or
samples; and (vi) altering the administration of the agent to the subject
accordingly. For
example, increased administration of the agent may be desirable to increase
the
expression or activity of Dkk or Dkk-related nucleic acid or protein to higher
levels than
detected, i.e., to increase the effectiveness of the agent. Alternatively,
decreased
administration of the agent may be desirable to decrease expression or
activity of Dkk or
Dkk-related nucleic acid or protein to lower levels than detected, i.e. to
decrease the
effectiveness of the agent. According to such an embodiment, Dkk or Dkk-
related
expression or activity may be used as an indicator of the effectiveness of an
agent, even
in the absence of an observable phenotypic response.
C. Methods of Treatment:
The present invention provides for both prophylactic and therapeutic methods
of
treating a subject at risk of (or susceptible to) a disorder or having a
disorder associated
with aberrant Dkk or Dkk-related expression or activity. With regards to both
prophylactic and therapeutic methods of treatment, such treatments may be
specifically
tailored or modified, based on knowledge obtained from the field of
pharmacogenomics.
"Pharmacogenomics", as used herein, refers to the application of genomics
technologies
such as gene sequencing, statistical genetics, and gene expression analysis to
drugs in
clinical development and on the market. More specifically, the term refers the
study of
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how a patient's genes determine his or her response to a drug (e.g., a
patient's "drug
response phenotype", or "drug response genotype") Thus, another aspect of the
invention provides methods for tailoring an individual's prophylactic or
therapeutic
treatment with either the Dkk or Dkk-related molecules of the present
invention or Da
or Dkk-related modulators according to that individual's drug response
genotype.
Pharmacogenomics allows a clinician or physician to target prophylactic or
therapeutic
treatments to patients who will most benefit from the treatment and to avoid
treatment of
patients who will experience toxic drug-related side effects.
1. Prophylactic Methods
In one aspect, the invention provides a method for preventing in a subject, a
disease or condition associated with an aberrant Dkk or Dkk-related expression
or
activity, by administering to the subject an agent which modulates Dkk or Dkk-
related
expression or at least one Dkk or Dkk-related activity. Subjects at risk for a
disease
which is caused or contributed to by aberrant Dkk or Dkk-related expression or
activity
can be identified by, for example, any or a combination of diagnostic or
prognostic
assays as described herein. Administration of a prophylactic agent can occur
prior to the
manifestation of symptoms characteristic of the Dkk or Dkk-related aberrancy,
such that
a disease or disorder is prevented or, alternatively, delayed in its
progression.
Depending on the type of Dkk or Dkk-related aberrancy, for example, an agonist
or
antagonist agent can be used for treating the subject. The appropriate agent
can be
determined based on screening assays described herein. The prophylactic
methods of
the present invention are further discussed in the following subsections.
2. Therapeutic Methods
Another aspect of the invention pertains to methods of modulating Dkk or Dkk-
related expression or activity for therapeutic purposes. The modulatory method
of the
invention involves contacting a cell with an agent that modulates one or more
of the
activities of Dkk or Dkk-related protein activity associated with the cell. An
agent that
modulates Dkk or Dkk-related protein activity can be an agent as described
herein, such
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as a nucleic acid or a protein, a naturally-occurring target molecule of a Dkk
or Dkk-
related protein, a peptide, a Dkk or Dkk-related peptidomimetic, or other
small
molecule. In one embodiment, the agent stimulates one or more Dkk or Dkk-
related
protein activity. Examples of such stimulatory agents include active Dkk or
Dkk-related
protein and a nucleic acid molecule encoding Dkk or Dkk-related that has been
introduced into the cell. In another embodiment, the agent inhibits one or
more Dkk or
Dkk-related protein activity. Examples of such inhibitory agents include
antisense Dkk
or Dkk-related nucleic acid molecules and antibodies. These modulatory methods
can
be performed in vitro (e.g., by culturing the cell with the agent) or,
alternatively, in vivo
(e.g., by administering the agent to a subject). As such, the present
invention provides
methods of treating an individual afflicted with a disease or disorder
characterized by
aberrant expression or activity of a Dkk or Dkk-related protein or nucleic
acid molecule.
The present invention also provides methods of modulating the function,
morphology,
proliferation, and/or differentiation of cells in the tissues in which a Dkk
or Dkk-related
protein or nucleic acid molecule is expressed. Alternatively, Dkk or Dkk-
related
polypeptides, nucleic acids, and modulators thereof, can be used to treat
disorders
associated with abnormal or aberrant metabolism or function of cells in the
tissues in
which the Dkk or Dick-related protein or nucleic acid molecule is expressed.
For example, tissues in which Dkk-3 is expressed include embryonic eye, bone,
and cartilage, fetal brain, lung, and kidney, and adult heart (in particular,
atrioventricular
valves and atrial myocytes), eye (in particular, the integrating bipolar and
ganglion cells
of the retina, the ciliary body, and lens epithelium), brain (in particular,
neurons of the
cortex and hippocampus), placenta, lung, and skeletal muscle. Accordingly, Dkk-
3
polypeptides, nucleic acids, or modulators thereof, can be used to treat
cardiovascular
disorders, such as ischemic heart disease (e.g., angina pectoris, myocardial
infarction,
and chronic ischemic heart disease), hypertensive heart disease, pulmonary
heart
disease, valvular heart disease (e.g., rheumatic fever and rheumatic heart
disease,
endocarditis, mitral valve prolapse, and aortic valve stenosis), congenital
heart disease
(e.g., valvular and vascular obstructive lesions, atrial or ventricular septal
defect, and
patent ductus arteriosus), or myocardial disease (e.g., myocarditis,
congestive
cardiomyopathy, and hypertrophic cariomyopathy).
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In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulators
thereof, can be used to treat optic disorders such as diseases associated with
amaurosis
(e.g., a. fugax and a. albuminuric) diseases associated with amblyopia,
glaucoma, optic
neuropathy (e.g., ischemic neuropathy, optic neuritis, and infiltrative
neuropathy),
opthalmia (e.g., o. catarrhal, trachoma, o. neuroparalytic, and conjunctiva),
visual
disorders resulting from systemic disease or disorders of other tissues (e.g.,
diabetes
mellitus, hyperthyroidism, and vitamin A or riboflavin deficiency), or tumors,
neoplasms, and metastases.
In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulators
thereof, can be used to treat disorders of the brain, such as cerebral edema,
senile
dementia of the Alzeimer type, epilepsy, amnesia, hydrocephalus, brain
herniations,
iatrogenic disease (due to, e.g., infection, toxins, or drugs), inflammations
(e.g.. bacterial
and viral meningitis, encephalitis, and cerebral toxoplasmosis),
cerebrovascular diseases
(e.g., hypoxia, ischemia, and infarction, intracranial hemorrhage and vascular
malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial
tumors,
neuronal tumors, tumors of pineal cells, meningeal tumors, primary and
secondary
lymphomas, intracranial tumors, and medulloblastoma), and to treat injury or
trauma to
the brain.
In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulators
thereof, can be used to treat placental disorders, such as toxemia of
pregnancy (e.g.,
preeclampsia and eclampsia), placentitis, or spontaneous abortion.
In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulators
thereof, can be used to treat pulmonary disorders, such as atelectasis,
pulmonary
congestion or edema, chronic obstructive airway disease (e.g., emphysema,
chronic
bronchitis, bronchial asthma, and bronchiectasis), diffuse interstitial
diseases (e.g.,
sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's
syndrome,
idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis,
desquamative
interstitial pneumonitis, chronic interstitial pneumonia, fibrosing
alveolitis, hamman-
rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis,
Wegener's
granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), or tumors
(e.g.,
bronchogenic carcinoma, bronchioloalveolar carcinoma, bronchial carcinoid,
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hamartoma, and mesenchymal tumors).
In another embodiment, Dkk-3 polypeptides, nucleic acids, or modulators
thereof, can be used to treat disorders of skeletal muscle, such as muscular
atrophy (due
to, e.g., denervation, malnutrition, loss of blood supply, or neuromuscular
disease, e.g.,
amyotonia congenita, amyotrophic lateral sclerosis of Charcot, and progressive
muscular
atrophy of Aran-Duchenne), myositis (due to, e.g., bacterial, viral, fungal or
parasitic
infection), muscular dystrophies (e.g., Duchenne type, Becker type,
facioscapulohumeral, limb-girdle, myotonic dystrophy, and ocular myopathy),
myasthenia gravis, or tumors and tumor-like lesions of muscles (e.g.,
traumatic myositis
ossificans, desmoids, musculoaponeurotic fibromatosis, Dupuytren's
contracture,
nodular (pseudosarcomatous) fasciitis, rhadomyoma, rhabdomyosarcoma, and
granular
cell myoblastomas).
Tissues in which Dkk-4 is expressed include cerebellum, activated human T-
lymphocytes, lung, and esophagus. Accordingly, in one embodiment, Dkk-4
polypeptides, nucleic acids, or modulators thereof, can be used to treat
disorders of the
cerebellum, such as disturbances of synergy (e.g., asynergia or limb ataxia,
dysmetria,
decomposition of movement, hypermetria, hypometria, dysdiadochokinesia,
hypotonia,
tremor, dysarthria, nystagmus), disturbances of equilibrium (due to, e.g., a
lesion
involving the vestibulocerebellum), disturbances of gait stance, or tone (due
to, e.g., a
lesion or degeneration of the spinocerebellum), or tumors (e.g., astrocytoma
and
medulloblastoma).
In another embodiment, Dkk-4 polypeptides, nucleic acids, or modulators
thereof, can be used to treat lymphocytic disorders, such as lymphopenia,
lymphocytosis, acute and chronic lymphadenitis, malignant lymphomas (e.g., Non-
Hodgkin's lymphomas, Hodgkin's lymphomas, leukemias, multiple myeloma,
histiocytoses, and angioimmunoblastic lymphadenopathy).
In another embodiment, Dkk-4 polypeptides, nucleic acids, or modulators
thereof, can be used to treat pulmonary disorders, such as atelectasis,
pulmonary
congestion or edema, chronic obstructive airway disease (e.g., emphysema,
chronic
bronchitis, bronchial asthma, and bronchiectasis), diffuse interstitial
diseases (e.g.,
sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's
syndrome,
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idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis,
desquamative
interstitial pneumonitis, chronic interstitial pneumonia, fibrosing
alveolitis, hamman-
rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis,
Wegener's
granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), or tumors
(e.g.,
bronchogenic carcinoma, bronchiolovlveolar carcinoma, bronchial carcinoid,
hamartoma, and mesenchymal tumors).
In another embodiment, Dkk-4 polypeptides, nucleic acids, or modulators
thereof, can be used to treat esophageal disorders, such as neuromuscular
disturbances
(e.g., achalasia, annular narrowings, Schatzki's rings, hiatal hernia, Mallory-
Weiss
syndrome), esophagitis (due to e.g., bacteremia, viremia, fungal infections,
uremia,
graft-versus-host disease, chemotherapy, radiation, and prolonged gastric
intubation),
diverticula (e.g., Zenker's diverticulum), systemic sclerosis, varices (due
to, e.g., portal
hypertension, systemic amyloidosis and sarcoidosis), or tumors or neoplasms
(e.g.,
leimyoma, fibromas, lipomas, hemangiomas, lymphangiomas, squamous papillomas,
adenocarcinomas and undifferentiated carcinomas, and sarcomas).
Dkk-1 is highly expressed, for example, in placenta. Accordingly, Dkk-1
polypeptides, nucleic acids, or modulators thereof, can be used to treat
placental
disorders, such as toxemia of pregnancy (e.g., preeclampsia and eclampsia),
placentitis,
or spontaneous abortion.
Tissues in which Dkk-2 is expressed include, for example, heart, brain,
placenta,
lung, and skeletal muscle. Accordingly, Dkk-2 polypeptides, nucleic acids, or
modulators thereof, can be used to treat cardiovascular disorders, such as
ischemic heart
disease (e.g., angina pectoris, myocardial infarction, and chronic ischemic
heart disease),
hypertensive heart disease, pulmonary heart disease, valvular heart disease
(e.g.,
rheumatic fever and rheumatic heart disease, endocarditis, mitral valve
prolapse, and
aortic valve stenosis), congenital heart disease (e.g., valvular and vascular
obstructive
lesions, atrial or ventricular septal defect, and patent ductus arteriosus),
or myocardial
disease (e.g., myocarditis, congestive cardiomyopathy, and hypertrophic
cariomyopathy).
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In another embodiment, Dkk-2 polypeptides, nucleic acids, or modulators
thereof, can be used to treat disorders of the brain, such as cerebral edema,
senile
dementia of the Alzeimer type, epilepsy, amnesia, hydrocephalus, brain
herniations,
iatrogenic disease (due to, e.g., infection, toxins, or drugs), inflammations
(e.g., bacterial
and viral meningitis, encephalitis, and cerebral toxoplasmosis),
cerebrovascular diseases
(e.g., hypoxia, ischemia, and infarction, intracranial hemorrhage and vascular
malformations, and hypertensive encephalopathy), and tumors (e.g., neuroglial
tumors,
neuronal tumors, tumors of pineal cells, meningeal tumors, primary and
secondary
lymphomas, intracranial tumors, and medulloblastoma), and to treat injury or
trauma to
the brain.
In another embodiment, Dkk-2 polypeptides, nucleic acids, or modulators
thereof, can be used to treat placental disorders, such as toxemia of
pregnancy (e.g.,
preeclampsia and eclampsia), placentitis, or spontaneous abortion.
In another embodiment, Dkk-2 polypeptides, nucleic acids, or modulators
thereof, can be used to treat pulmonary disorders, such as atelectasis,
pulmonary
congestion or edema, chronic obstructive airway disease (e.g., emphysema,
chronic
bronchitis, bronchial asthma, and bronchiectasis), diffuse interstitial
diseases (e.g.,
sarcoidosis, pneumoconiosis, hypersensitivity pneumonitis, Goodpasture's
syndrome,
idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis,
desquamative
interstitial pneumonitis, chronic interstitial pneumonia, fibrosing
alveolitis, hamman-
rich syndrome, pulmonary eosinophilia, diffuse interstitial fibrosis,
Wegener's
granulomatosis, lymphomatoid granulomatosis, and lipid pneumonia), or tumors
(e.g.,
bronchogenic carcinoma, bronchiolovlveolar carcinoma, bronchial carcinoid,
hamartoma, and mesenchymal tumors).
In another embodiment, Dklc-2 polypeptides, nucleic acids, or modulators
thereof, can be used to treat disorders of skeletal muscle, such as muscular
atrophy (due
to, e.g., denervation, malnutrition, loss of blood supply, or neuromuscular
disease, e.g.,
amyotonia congenita, amyotrophic lateral sclerosis of Charcot, and progressive
muscular
atrophy of Aran-Duchenne), myositis (due to, e.g., bacterial, viral, fungal or
parasitic
infection), muscular dystrophies (e.g., Duchenne type, Becker type,
facioscapulohumeral, limb-girdle, myotonic dystrophy, and ocular myopathy),
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myasthenia gravis, or tumors and tumor-like lesions of muscles (e.g.,
traumatic myositis
ossificans, desmoids, musculoaponeurotic fibromatosis, Dupuytren's
contracture,
nodular (pseudosarcomatous) fasciitis, rhadomyoma, rhabdomyosarcoma, and
granular
cell myoblastomas).
Soggy-1 is expressed in, for example, testis (e.g., spermatogenic epithelium
of
the seminiferous tubules, spermatogonia) and in embryonic developing dorsal
root
ganglia, cartilage primordium of the nasal septum, and the eye. Accordingly,
Soggy-1
polypeptides, nucleic acids, or modulators thereof, can be used to treat
testicular
disorders, such as unilateral testicular enlargment (e.g., nontuberculous,
granulomatous
orchitis), inflammatory diseases resulting in testicular dysfunction (e.g.,
gonorrhea and
mumps), and tumors (e.g., germ cell tumors, interstitial cell tumors,
androblastoma,
testicular lymphoma and adenomatoid tumors). In another embodiment, Soggy-1
polypeptides, nucleic acids, or modulators thereof, can be used to treat
infertility due to,
for example, spermatogenetic failure.
In one aspect, the above-described methods involve administering an agent
(e.g.,
an agent identified by a screening assay described herein), or combination of
agents that
modulates (e.g., upregulates or dovvnregulates) Dkk or Dkk-related expression
or
activity. In another embodiment, the method involves administering a Dkk or
Dkk-
related protein or nucleic acid molecule as therapy to compensate for reduced
or aberrant
Dkk or Dkk-related expression or activity.
A preferred embodiment of the present invention involves a method for
treatment
of a disease or disorder associated with a Dkk or Dkk-related protein which
includes the
step of administering a therapeutically effective amount of an antibody to a
Dkk or Dick-
related protein to a subject. As defined herein, a therapeutically effective
amount of
antibody (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body
weight,
preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20
mg/kg
body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to
8 mg/kg,
4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate
that
certain factors may influence the dosage required to effectively treat a
subject, including
but not limited to the severity of the disease or disorder, previous
treatments, the general
health and/or age of the subject, and other diseases present. Moreover,
treatment of a
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subject with a therapeutically effective amount of an antibody can include a
single
treatment or, preferably, can include a series of treatments. In a preferred
example, a
subject is treated with antibody in the range of between about 0.1 to 20 mg/kg
body
weight, one time per week for between about 1 to 10 weeks, preferably between
2 to 8
weeks, more preferably between about 3 to 7 weeks, and even more preferably
for about
4, 5, or 6 weeks. It will also be appreciated that the effective dosage of
antibody used
for treatment may increase or decrease over the course of a particular
treatment.
Changes in dosage may result from the results of diagnostic assays as
described herein.
Stimulation of Dkk or Dkk-related activity is desirable in situations in which
Dkk or Dick-related activity is abnormally downregulated and/or in which
increased Dkk
or Dkk-related activity is likely to have a beneficial effect. Likewise,
inhibition of Dkk
or Dkk-related activity is desirable in situations in which Dkk or Dkk-related
activity is
abnormally upregulated and/or in which decreased Dkk or Dkk-related activity
is likely
to have a beneficial effect. One example of such a situation is where a
subject has a
disorder characterized by aberrant development or cellular differentiation.
Another
example of such a situation is where the subject has a proliferative disease
(e.g., cancer)
or a neurogenerative disorder. Yet another example of such a situation is
where it is
desireable to acheive tissue regeneration in a subject (e.g., where a subject
has
undergone brain or spinal cord injury and it is desirable to regenerate
neuronal tissue in a
regulated manner.)
Accordingly, in one embodiment, the disease is a disease characterized by an
abnormal cell proliferation, differentiation, and/or survival. For example,
the disease
can be a hyper-or hypoproliferative disease. The invention also provides
methods for
treating diseases characterized by an abnormal cell proliferation,
differentiation, and/or
survival in a subject, which are not characterized by an abnormal Dkk or Dkk-
related
activity (e.g., hDkk-3 activity). In fact, since Dkk is likely to be capable
of modulating
the proliferative state of a cell (i.e., state of proliferation,
differentiation, and or survival
of a cell), Dkk can regulate disease wherein the abnormal proliferative state
of a cell
results from a defect other than an abnormal Dkk activity.
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Hyperproliferative diseases can be treated with Dkk or Dkk-related (e.g.,
hDlck-
3) therapeutics include neoplastic and hyperplastic diseases, such as various
forms of
cancers and leukemias, and fibroproliferative disorders. Other
hyperproliferative
diseases that can be treated or prevented with the subject Dkk or Dkk-related
therapeutics (e.g. hDkk-3 therapeutics) include malignant conditions,
premalignant
conditions, and benign conditions. The condition to be treated or prevented
can be a
solid tumor, such as a tumor arising in an epithelial tissue. Accordingly,
treatment of
such a cancer could comprise administration to the subject of a Dkk or Dkk-
related
therapeutic decreasing the interaction of Dkk with a Dkk receptor. Other
cancers that
can be treated or prevented with a Dkk or Dkk-related protein include cancers
of the
epithelia (e.g., carcinomas of the pancreas, kidney, stomach, colon, esophagus
liver,
secretory glands (e.g., adenocarcinoma) bladder, lung, breast, skin (e.g.,
malignant
melanoma, seminoma squamous adenocarcinoma), reproductive tract including
prostate
gland, testis, ovary, cervix and uterus); cancers of the hematopoietic and
immune system
(e.g., leukemias and lymphomas); cancers of the central nervous, brain system
and eye
(e.g., malignant astrocytoma, gliomas, neuroblastoma and retinoblastoma); and
cancers
of connective tissues, bone, heart, muscles and vasculature (e.g., sarcomas,
for example,
osteosarcoma). Additional solid tumors within the scope of the invention
include those
that can be found in a medical textbook.
The condition to be treated or prevented can also be a soluble tumor, such as
leukemia, either chronic or acute, including chronic or acute myelogenous
leukemia,
chronic or acute lymphocytic leukemia, promyelocytic leukemia, monocytic
leukemia,
myelomonocytic leukemia, and erythroleukemia. Yet other proliferative
disorders that
can be treated with a Dkk or Dkk-related therapeutic of the invention include
heavy
chain disease, multiple myeloma, lymphoma, e.g., Hodgkin's lymphoma and non-
Hodgkin's lymphoma, and Waldenstroem's macroglobulemia.
Diseases or conditions characterized by a solid or soluble tumor can be
treated by
administrating a Dkk or Dkk-related therapeutic either locally or
systemically, such that
aberrant cell proliferation is inhibited or decreased. Methods for
administering the
compounds of the invention are further described below.
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The invention also provides methods for preventing the formation and/or
development of tumors. For example, the development of a tumor can be preceded
by
the presence of a specific lesion, such as a pre-neoplastic lesion, e.g.,
hyperplasia,
metaplasia, and dysplasia, which can be detected, e.g., by cytologic methods.
Such
lesions can be found, e.g., in epithelial tissue. Thus, the invention provides
a method for
inhibiting progression of such a lesion into a neoplastic lesion, comprising
administering
to the subject having a preneoplastic lesion an amount of a Dkk or Dkk-related
therapeutic sufficient to inhibit progression of the preneoplastic lesion into
a neoplastic
lesion.
The invention also provides for methods for treating or preventing diseases or
conditions in which proliferation of cells is desired. For example, Dkk or Dkk-
related
therapeutics can be used to stimulate tissue repair or wound healing, such as
after
surgery or to stimulate tissue healing from burns. Other diseases in which
proliferation
of cells is desired are hypoproliferative diseases, i.e., diseases
characterized by an
abnormally low proliferation of certain cells.
In yet another embodiment, the invention provides a method for treating or
preventing diseases or conditions characterized by aberrant cell
differentiation.
Accordingly, the invention provides methods for stimulating cellular
differentiation in
conditions characterized by an inhibition of normal cell differentiation which
may or
may not be accompanied by excessive proliferation. Alternatively, Dkk or Dkk-
related
therapeutics can be used to inhibit differentiation of specific cells.
In a preferred method, the aberrantly proliferating and/or differentiating
cell is a
cell present in the nervous system. A role for Dkk in the nervous system is
suggested at
least in part from the fact that human Dkk-3 is expressed in human fetal
brain.
Accordingly, the invention provides methods for treating diseases or
conditions
associated with a central or peripheral nervous system. For example, the
invention
provides methods for treating lesions of the nervous system associated with an
aberrant
proliferation, differentiation or survival of any of the following cells:
cells of the central
nervous system including neurons and glial cells (e.g., astrocytes and
oligodendrocytes)
and supporting cells of peripheral neurons (e.g., Schwann cells and satellite
cells).
Disorders of the nervous system include, but are not limited to: spinal cord
injuries,
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brain injuries, brain tumors (e.g., astrocytic tumors, for example,
astrocytomas and
glioblastomas), lesions associated with surgery, ischemic lesions, malignant
lesions,
infectious lesions, degenerative lesions (e.g., Parkinson's disease,
Alzheimer's disease,
Huntington's chorea, amyotrophic lateral sclerosis), demyelinlating diseases
(e.g.,
multiple sclerosis, human immunodeficiency associated myelopathy, transverse
myelopathy, progressive multifocal leukoencephalopathy, pontine myelinolysis),
motor
neuron injuries, progressive spinal muscular atrophy, progressive bulbar
palsy, primary
lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar
paralysis of
childhood (i.e., Fazio-Londe syndrome), poliomyelitis, and hereditary
motorsensory
neuropathy (i.e., Charcot-Marie-Tooth disease).
In another embodiment, the invention provides a method for enhancing the
survival and/or stimulating proliferation and/or differentiation of cells and
tissues in
vitro. In a preferred embodiment, Dkk or Dkk-related therapeutics are used to
promote
tissue regeneration and/or repair (e.g., to treat nerve injury). For example,
tissues from a
subject can be obtained and grown in vitro in the presence of a Dkk or Dkk-
related
therapeutic, such that the tissue cells are stimulated to proliferate and/or
differentiate.
The tissue can then be readministered to the subject.
Among the approaches which may be used to ameliorate disease symptoms
involving an aberrant Dkk or Dkk-related activity and/or an abnormal cell
proliferation,
differentiation, and/or survival, are, for example, antisense, ribozyme, and
triple helix
molecules described above. Examples of suitable compounds include the
antagonists,
agonists or homologues described in detail above.
Yet other Dkk or Dick-related therapeutics consist of a first peptide
comprising a
Dkk or Dkk-related peptide capable of binding to a Dkk receptor, and a second
peptide
which is cytotoxic. Such therapeutics can be used to specifically target and
lyse cells
expressing or overexpressing a receptor for Dick.
3. Pharmacogenomics
The Dick or Dick-related molecules of the present invention, as well as
agents, or
modulators which have a stimulatory or inhibitory effect on Dkk or Dkk-related
activity
(e.g., Dick or Dkk-related gene expression) as identified by a screening assay
described
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herein can be administered to individuals to treat (prophylactically or
therapeutically)
disorders (e.g., proliferative or developmental disorders) associated with
aberrant Dkk or
Dkk-related activity. In conjunction with such treatment, pharmacogenomics
(i.e., the
study of the relationship between an individual's genotype and that
individual's response
to a foreign compound or drug) may be considered. Differences in metabolism of
therapeutics can lead to severe toxicity or therapeutic failure by altering
the relation
between dose and blood concentration of the pharmacologically active drug.
Thus, a
physician or clinician may consider applying knowledge obtained in relevant
pharmacogenomics studies in determining whether to administer a Dkk or Dick-
related
molecule or Dkk or Dkk-related modulator as well as tailoring the dosage
and/or
therapeutic regimen of treatment with a Dkk or Dkk-related molecule or Dkk or
Dkk-
related modulator.
Pharmacogenomics deals with clinically significant hereditary variations in
the
response to drugs due to altered drug disposition and abnormal action in
affected
persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-11)
:983-
985 and Linder, M.W., Clin Chem, 1997, 43(2):254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic conditions
transmitted as a
single factor altering the way drugs act on the body (altered drug action) or
genetic
conditions transmitted as single factors altering the way the body acts on
drugs (altered
drug metabolism). These pharmacogenetic conditions can occur either as rare
genetic
defects or as naturally-occurring polymorphisms. For example, glucose-6-
phosphate
dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the
main clinical complication is haemolysis after ingestion of oxidant drugs
(anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
One pharmacogenomics approach to identifying genes that predict drug
response, known as "a genome-wide association", relies primarily on a high-
resolution
map of the human genome consisting of already known gene-related markers
(e.g., a
allelic" gene marker map which consists of 60,000-100,000 polymorphic or
variable
sites on the human genome, each of which has two variants.) Such a high-
resolution
genetic map can be compared to a map of the genome of each of a statistically
significant number of patients taking part in a Phase II/III drug trial to
identify markers
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associated with a particular observed drug response or side effect.
Alternatively, such a
high resolution map can be generated from a combination of some ten-million
known
single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a
"SNP" is a common alteration that occurs in a single nucleotide base in a
stretch of
DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may
be involved in a disease process, however, the vast majority may not be
disease-
associated. Given a genetic map based on the occurrence of such SNPs,
individuals can
be grouped into genetic categories depending on a particular pattern of SNPs
in their
individual genome. In such a manner, treatment regimens can be tailored to
groups of
genetically similar individuals, taking into account traits that may be common
among
such genetically similar individuals.
Alternatively, a method termed the "candidate gene approach", can be utilized
to
identify genes that predict drug response. According to this method, if a gene
that
encodes a drugs target is known (e.g., a Dkk protein or Dkk receptor of the
present
invention), all common variants of that gene can be fairly easily identified
in the
population and it can be determined if having one version of the gene versus
another is
associated with a particular drug response.
As an illustrative embodiment, the activity of drug metabolizing enzymes is a
major determinant of both the intensity and duration of drug action. The
discovery of
genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase
2 (NAT
2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation
as to why some patients do not obtain the expected drug effects or show
exaggerated
drug response and serious toxicity after taking the standard and safe dose of
a drug.
These polymorphisms are expressed in two phenotypes in the population, the
extensive
metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different
among
different populations. For example, the gene coding for CYP2D6 is highly
polymorphic
and several mutations have been identified in PM, which all lead to the
absence of
functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently
experience exaggerated drug response and side effects when they receive
standard doses.
If a metabolite is the active therapeutic moiety, PM show no therapeutic
response, as
demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed
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metabolite morphine. The other extreme are the so called ultra-rapid
metabolizers who
do not respond to standard doses. Recently, the molecular basis of ultra-rapid
metabolism has been identified to be due to CYP2D6 gene amplification.
Alternatively, a method termed the "gene expression profiling", can be
utilized to
identify genes that predict drug response. For example, the gene expression of
an
animal dosed with a drug (e.g., a Dkk molecule or Dkk modulator of the present
invention) can give an indication whether gene pathways related to toxicity
have been
turned on.
Information generated from more than one of the above pharmacogenomics
approaches can be used to determine appropriate dosage and treatment regimens
for
prophylactic or therapeutic treatment an individual. This knowledge, when
applied to
dosing or drug selection, can avoid adverse reactions or therapeutic failure
and thus
enhance therapeutic or prophylactic efficiency when treating a subject with a
Dkk
molecule or Dkk or Dkk-related modulator, such as a modulator identified by
one of the
exemplary screening assays described herein.
This invention is further illustrated by the following examples which should
not
be construed as limiting. The contents of all references, patents and
published patent
applications cited throughout this application are hereby incorporated by
reference.
EXAMPLES
The invention is based, at least in part, on the discovery of a family of
genes
encoding human cysteine-rich secreted proteins which are related to Xenopus
Dickkopf
(Dkk) proteins. This family includes hDkk-1, hDkk-2, hDkk-3, and hDkk-4. hDkks
1-4
contain two highly conserved cysteine-rich domains (CRDs), the most C-terminal
of
which demonstrates similarity to the colipase protein family. The invention is
based
also in part on the discovery of a family of Dkk-related proteins, referred to
as Soggy
proteins, as well as the genes encoding Soggy proteins. Soggy-1 is a novel
secreted
protein which is related to the N-terminal region of Dkk-3 but lacks CRDs. The
following examples illustrate the structure and function of each of these
novel human
secreted proteins.
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-
Example 1: Isolation And Characterization of Human hDkk-3 cDNA
In this example, the isolation and characterization of the gene encoding human
Dkk-3 (also referred to as "hDkk-3", "Cysteine Rich Secreted Protein-1", "CRSP-
1"
"CRISPY-1" or "TANGO 59") is described.
Isolation of a Human Dkk-3 cDNA
The invention is based at least in part on the discovery of a human gene
encoding
a secreted protein, referred to herein as human Dickkopf-3 (hDkk-3). A partial
cDNA
was isolated using a Signal Sequence Trap method. This methodology takes
advantage
of the fact that molecules such as Dkk have an amino terminal signal sequence
which
directs certain secreted and membrane-bound proteins through the cellular
secretory
apparatus.
Briefly, a randomly primed cDNA library using mRNA prepared from human
fetal brain tissue (Clontech, Palo Alto CA) was made by using the Stratagene-
ZAP-
cDNA Synthesis."'" kit, (catalog #20041). The cDNA was ligated into the
mammalian
expression vector pTrap adjacent to a cDNA encoding placental alkaline
phosphatase
lacking a secretory signal. The plasmids were transformed into E. coli and DNA
was
prepared using the WizardTM DNA purification kit (Promega). DNA was
transfected into
COS-7 cells with lipofectamineTM (Gibco-BRL). After 48 hours incubation the
COS cell
supernatants were assayed for alkaline phosphatase on a Wallac Micro-Beta
scintillation
counter using the Pbospha-LightTM kit (Tropix Inc. Catalog #BP300). The
individual
plasmid DNAs scoring positive in the COS cell Alkaline Phosphatase secretion
assay
were further analyzed by DNA sequencing using standard procedures.
Using a partial cDNA isolated by the above-described method (clone Amhb3c2),
a full length cDNA encoding human Dkk-3 was isolated from a lambda ZiploxTM
human
fetal brain cDNA library using conventional hybridization techniques (Sambrook
et at,
supra). The nucleotide sequence encoding the full length human Dkk-3 protein
is
shown in Figure 1 and is set forth as SEQ ID NO: 1. The full length protein
encoded by
this nucleic acid is comprised of about 350 amino acids and has the amino acid
sequence
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shown in Figure 1 and set forth as SEQ ID NO:2. The coding portion (open
reading
frame) of SEQ ID NO:1 is set forth as SEQ ID NO:3. DNA for the clone Fmhb059
was
deposited with the ATCC as Accession No. 98452.
Analysis of Human hDkk-3
Determination of the hydrophobicity profile of human Dkk-3 having the amino
acid sequence set forth in SEQ ID NO:2 indicated the presence of a hydrophobic
region
from about amino acid 1 to about amino acid 22 of SEQ ID NO:2. Further
analysis of
the amino acid sequence SEQ ID NO:2 using a signal peptide prediction program
predicted the presence of a signal peptide from about amino acid 1 to about
amino acid
22 of SEQ ID NO:2. Accordingly, the mature hDkk-3 protein includes about 328
amino
acids spanning from about amino acid 23 to about amino acid 350 of SEQ ID
NO:2.
The presence of the signal sequence, in addition to the fact that hDlck-3 has
been
identified using a Signal Sequence Trap system, indicates that hDklc-3 is a
secreted
protein. Furthermore, the prediction of such a signal peptide and signal
peptide cleavage
site can be made, for example, utilizing the computer algorithm SIGNALP
(Nielsen, et
al., (1997) Protein Engineering 10:1-6).
Examination of the cDNA sequence depicted in Figure 1 shows that human Dkk-
3 is particularly rich in cysteine residues. As shown in Figure 1, hDklc-3
contains 20
cysteine residues located between amino acid 147 and amino acid 284 of SEQ ID
NO: 2.
This region has been termed the cysteine-rich region. These cysteine residues
can form
10 disulfide bridges.
A BLAST search (Altschul et al., (1990)J. Mol. Biol. 215:403) of the
nucleotide
and the amino acid sequences of hDldc-3 has revealed that hDlck-3 is similar
to a chicken
cDNA encoding a protein of unknown function having GenBank Accession No.
D26311. This cDNA was isolated from a chicken lens cDNA library and was shown
to
be expressed in lens fibers and lens epithelium, but not in neural retina nor
in liver cells.
(Sawada etal., (1996) Int. J. Dev. Biol. 40:531). hDldc-3 and the chicken
protein have
56% amino acid sequence identity and 72% amino acid sequence similarity. The
amino
acid sequence similarity between the chicken protein and human Dkk-3 is
particularly
high in the cysteine-rich domain of hDlck-3 which is located between amino
acids 147
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and 284 of SEQ ID NO: 2. In particular, the 20 cysteine residues of hDkk-3
located in
this region are present in the chicken protein.
Two genes recently identified in a screen for suppressors of glioblastoma
formation (Ligon et al. (1997) Oncogene 14:1075-1081) also show homology to
hDkk-
3. These genes, RIG ("Regulated In Glioblastoma") and RIG-like 7-1 (GenBank
Accession Nos. U32331 and AF034208, respectively) were identified in a
differential
screen for mRNAs regulated by the introduction of a normal copy of chromosome
10
into a glioblastoma cell line harboring a deletion in chromosome 10 that
promotes
tumorigenesis. A schematic diagram summarizing the relationship between the
sequences of the hDkk-3 and the RIG genes is presented as Figure 12. The
indicated
region of identity between hDkk-3 and RIG comprises a short portion of the 3'
UTR of
the human Dkk-3 mRNA (e.g., RIG mRNA is ¨100% identical to residues 2479 to
2153
of SEQ ID NO: I). RIG-like 7-1 is homologous to hDklc-3 accross a longer
region (e.g.,
97% identical from about nucleotides 31610 2438 of SEQ ID NO:1) although the
encoded RIG-like 7-1 protein lacks the Dkk N-terminal signal sequence and is
not
therefore predicted to be a secreted protein. These data associate hDkk-3 with
human
glioblastoma and suggest that hDkk-3 may be important in the suppression of
the
tumorigenic phenotype. A role in glioblastoma is also consistent with the high
level of
hDklc-3 mRNA expression observed in human brain tissue. In addition, the co-
localization of the hDkk-3, RIG and RIG-like genes to a region of chromosome
11
(1 1p15.1) implicated in the development of human malignant astrocytoma (Ligon
et al.,
supra) further indicates a role for these genes in tumorigenesis.
Human hDkk-3 protein has also some amino acid sequence similarity to
metallothionein, particularly in the cyteine-rich domain.
Tissue Distribution of hDkk-3 mRNA
For Northern blots, all hybridizations were to Clontech Multiple Tissue
Northern
Blots and were performed in ExpressHyb*sollition (Clontech) for 1-20 hours.
All probes
were prepared by random primed radiolabelling (Prime-It, Stratagene). Blots
were
washed sequentially to a final stringency of 0.2x SSC/0.2% SDS and exposed to
autoradiographic film. Hybridizations of a control p-actin cDNA probe
consistently
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demonstrated even loading of the Northern blots. The results of hybridization
of the
probe to various mRNA samples are described below.
Hybridization of a Clontech Fetal Multiple Tissue Northern (MTN) blot
(Clontech, LaJolla, CA) containing RNA from fetal brain, lung, liver, and
kidney
indicated the presence of high levels of hDkk-3 mRNA (-2.5kb) in fetal brain,
lung, and
slightly lower levels of hDkk-3 mRNA in fetal kidney. However, no significant
level of
hDkk-3 mRNA was found in fetal liver.
Hybridization of a Clontech human Multiple Tissue Northern (MTN) blot
(Clontech, LaJolla, CA) containing RNA from adult heart, brain, placenta,
lung, liver,
skeletal muscle, kidney, and pancreas with a human Dkk-3 probe indicated the
presence
of high levels of hDkk-3 mRNA in heart, slightly lower levels in brain, and
much lower
levels in placenta and lung. Some hDkk-3 mRNA was also found in adult skeletal
muscle. However, no significant levels of hDkk-3 mRNA was observed in adult
liver,
kidney, or pancreas. Interestingly, the chicken gene which is homologous to
hDkk-3
was not expressed at detectable levels in liver either (Sawada et al., (1996)
Int. I Dev.
Biol. 40:531).
Further hybridization of a Clontech human Multiple Tissue Northern (MTN) blot
(Clontech, LaJolla, CA) including RNA from bone marrow, adrenal gland,
trachea,
lymph node, spinal cord, thyroid, and stomach revealed high levels of
expression of
hDkk-3 in mRNA isolated from adult spinal cord, and lower level expression in
adrenal
gland, trachea, thyroid, and stomach.
Thus, hDkk-3 is expressed in a tissue specific manner, with the strongest
expression observed in brain, heart, and spinal cord.
Example 2: Isolation And Characterization of mDkk-3 cDNA
In this example, the isolation and characterization of the gene encoding
murine
Dkk-3 (also referred to as "mDkk-3", "murine Cysteine Rich Secreted Protein-
1",
"murine CRSP-1" or "murine CRISPY-1") is described.
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Identification of a Murine Dkk-3 cDNA
A full length mDkk-3 cDNA was identified by comparison of the hDkk-3
sequence to a proprietary EST Database using the BLAST-X algorithm. A single
clone
identified in a adult mouse brain cDNA library was obtained and sequenced
fully. DNA
for the clone Fmmb059s was deposited with the ATCC as Accession No. 98634.
mDkk-
3 is predicted to have a signal peptide from residues 1 to 23 of SEQ ID NO:17,
cleavage
of which results in a mature protein having 326 amino acids in length
corresponding to
amino acids 24 to 349 of SEQ ID NO:17.
Tissue Distribution of mDkk-3 mRNA
To determine the expression pattern of mDkk-3, in situ hybridization was
performed as follows. Normal mouse embryos and adult mouse tissues were
collected
from C57BL/6 mice, embedded in TissueTekTm 0.C.T Compound (Sakura Finetek
U.S.A., Inc., Torrance, CA), frozen on dry ice, and stored at -80 C. Cryostat
serial
sections (8).1m) were thaw mounted on Superfrost PlusTM slides (VWR
Scientific, West
Chester, PA.) and air dried on a slide warmer at 40 C for 20 minutes. Sections
were
then fixed with 4% formaldehyde in DEPC treated 0.1 M phosphate-buffered
saline
(PBS, pH 7.5) at room temperature for 10 minutes and rinsed twice in DEPC-PBS.
Sections were rinsed in 0.1 M triethanolamine-HC1 (TEA, pH 8.0) , incubated in
0.25%
acetic anhydride-TEA for 10 minutes and rinsed in DEPC-2X SSC (standard sodium
citrate). Sections were dehydrated through a series of graded ethanols,
incubated in
100% chloroform for 5 minutes, rinsed in 100% and 95% ethanol for 1 minutes
and air
dried.
Antisense and sense RNA transcripts were prepared by in vitro transcription
(Riboprobe Gemini SystemTM, Promega) of PCR amplified cDNA templates. Template
amplification primers were as follows;
mDkk-3 forward 5'-CAGTGAGTGCTGTGGAGACC-3' (SEQ ID NO:30), and
reverse 5'-TCTTCAGTCAGGCTCCTCTC-3' (SEQ ID NO:31).
Probes were labeled with 35S-UTP (NEN) and purified on G-25 spin columns
(Pharmacia). The hybridization cocktail contained: 50% formamide, 10% dextran
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sulfate, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium thiosulfate, lx
Denhardt's
solution, 0.6 M NaC1, 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM dithiothreitol
(DTT), 0.1 mg/ml sheared salmon sperm, 50 pg/m1 yeast tRNA, 0.5 mg/ml yeast
total
RNA, and 35S-UTP labeled probe at a concentration of 5 X 107 c.p.m./100 1 of
final
hybridization solution; 100 I of hybridization solution was put on each
section. The
sections were then covered with a glass coverslip and incubated in a humidifed
chamber
at 55 C for 18 h. After hybridization, slides were washed with 2 X SSC.
Sections were
then sequentially incubated at 37 C in TNE (a solution containing 10 mM Tris-
HC1 (pH
7.6), 500 mM NaC1, and 1 mM EDTA), for 10 minutes, in TNE with 1 Oug/m1RNase A
for 30 minutes, and finally in TNE for 10 minutes. Slides were then rinsed
with 2 X
SSC at room temperature, washed in 2 X SSC at 50 C for 1 h, 0.2 X SSC at 55 C
for lh,
and 0.2 X SSC at 60 C for 1 h. Sections were dehydrated with a series of
graded
concentrations of ethanol 0.3 M ammonium acetate, air dried and exposed to
Kodak
Biomax MRTM scientific imaging film for 6 days at room temperature.
mDkk-3 expression in the brain was found to be highly localized to the cortex
and hippocampus but was not observed in the dentate gyrus. Higher power
magnification confirmed the mDkk-3 mRNA was localized to neurons within these
structures. In the adult eye, mDkk-3 mRNA was found to be highly expressed in
the
retina, ciliary body, and lens epithelium. Expression in the retina was
localized to the
integrating bipolar and ganglion cells. In adult heart, mDkk-3 was detected in
the
atrioventricular valves and also in myocytes of the atria. Expression was
highly
restricted to the atria and noticeably absent from ventricular tissue. High
level
expression of mDkk3 mRNA was also observed in developing eye, bone and
cartilage in
day 14 embryos. These findings corroborate and extend the northern analysis of
hDkk-3
mRNA expression in human tissues and also suggest that Dkk-3 may play a role
in bone
and ocular physiology in addition to functions in neural and cardiac tissues.
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Example 3: Secretion and Post-Translational Modification of Dkk-3
This example describes the secretion and post-translational modification
(e.g.,
glycosylation and processing) of hDkk-3 as well as methods for small and large
scale
purification of hDkk-3.
hDkk-3 Expression Constructs
Expression constructs for two forms of hDklc-3 were prepared using the
mammalian expression vector pMET-stop. Form-1 comprised a cDNA incorporating
the complete 350aa hDkk-3 protein coding sequence (hDklc-3flag.long) and form-
2
comprised the entire hDkk-3 protein coding sequence except for the final 18
amino acids
(hDkk-3flag.short). A C-terminal sequence encoding the FLAG epitope (DYKDDDDK)
(SEQ ID NO:19) was added to both hDkk-3 forms for ease of detection and
purification.
hDkk-3flag cDNAs were generated by PCR from a full length hDkk-3 cDNA template
and ligated into pMET-stop using EcoR1 and Sall restriction sites.
Trial Transfection - Small Scale Expression
Expression constructs for hDkk-3flaglong and hDkk-3flag.short were
transfected into 293T cells using 10 of lipofectamine (GIBCO/BRL) and 2 lig of
DNA per well of a 6-well plate of cells which were 70-80% confluent. After 5
hours at
37 C, cells were fed with lml of 20%FCS/DMEM. After incubation overnight at 37
C,
cells were conditioned in lml OptiMEM for 48 hours at 37 C. Samples of
supernatant
and cell pellets were solubilized in boiling SDS-PAGE gel buffer, run out on a
4-20%
SDS-PAGE gel, transferred to a nylon membrane and probed with the anti-FLAG
monoclonal antibody M2. Samples from both supernatant and pellet samples
showed
significant immunoreactivity within a molecular weight range of 40-65 kDa on
autoradiographic film using a HRP conjugated secondary antibody and ECL
detection
reagents. Thus, both forms of hDkk-3 tested are secreted from 293T cells
thereby
confirming experimentally that hDkk-3 is a secreted protein. It should be
noted that the
molecular weights of both forms of hDlck-3 tested are greater than predicted
from the
amino acid sequence, suggesting that the hDldc-3 proteins secreted by 293T
cells may be
glycosylated. This is consistent with the presence of four potential sites for
N-linked
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glycosylation in the hDkk-3 protein (e.g., at about amino acids 96-99, 106-
109, 121-124,
and 204-207 of SEQ ID NO:2).
Deglycosylation of hDklc-3
Given the heterogenous nature of secreted human Dkk-3, the effect of N-
Glycanase treatment on the mobility of secreted flag-tagged hDkk-3 was
studied.
Briefly, lmL samples of 293T cell supernatants collected 72 hours after
transfection
with the appropriate constructs were incubated with 501.1.L anti-flag M2
agarose beads
(Sigma) for 16hrs at 4 C. Beads were washed with PBS (pH7.4) containing,
sequentially, 0.1%, 0.05% and 0.01% Triton X-100. The beads were resuspended
in 20[1
L of 20mM sodium phosphate, pH 7.5, 50mM EDTA, 0.02%sodium azide, (incubation
buffer) together with 0.5% SDS, 5% 2-mercaptoethanol and boiled for 2 minutes.
The
supernatant was split into equal 101.IL aliquots which were diluted with 104
incubation
buffer, 54 5% NP-40 and then with either 54 N-Glycanase (Oxford Glycosystems)
in
enzyme buffer (20mM Tris-HC1, 1mM EDTA, 50mM NaCl, 0.02% sodium azide pH
7.5) or with enzyme buffer alone as control. After 18 hours at 37 C, samples
were
boiled in equal volumes of SDS-PAGE buffer and analyzed by SDS-PAGE and
Western
blotting. For western analysis, samples were electroblotted onto PVDF (Novex)
after
SDS-PAGE on 4-20% gradient gels, probed with M2 anti-flag antibody (1:500,
Sigma)
followed by HRP conjugated sheep anti-mouse IgG (1:5000, Amersham), developed
with chemiluminescent reagents (Renaissance, Dupont) and exposed to
autoradiography
film (BiomaZMR2 film, Kodak).
Utilizing the above-described methodology, it was determined that hDkk-3
protein displayed a significant increase in mobility following N-Glycanase
treatment.
The major 45-65 kD form of soluble hDkk-3 was observed as two species of 45-55
and
40 kD following deglycosylation. This finding is consistent with the presence
of
multiple potential sites of N-linked glycosylation in the hDkk-3 protein. The
reason for
the heterogeneity of deglycosylated hDldc-3 reflects either proteolytic
processing or
incomplete removal of carbohydrate from one or more attachment sites. A 30 kD
hDkk3
species was also observed in these experiments, the mobility of which was
unaltered by
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N-Glycanase treatment. This form was only observed after overnight incubation
of the
samples and may be a non-specific degradation product.
Large Scale hDkk-3 Protein Production
For scale-up of hDkk-3flag.long protein expression, 30 x 150mM plates of 293T
cells at 70-80% confluence were transfected with 27 lig DNA, 100 pi
lipofectamine in
18m1 OptiMEM for 5 hours at 37 C. 18 ml of 10%FCS/DMEM was added to each plate
and incubated overnight at 37 C. 24 hours after the start of transfection,
transfection
supernatant was aspirated and 35 mls OptiMEM was added to each plate and the
plates
incubated at 37 C for 72 hours. Conditioned medium was harvested, spun at 4000
rpm
for 30 min. at 4 C, and filtered through a 0.45 micron filter unit. 1100 ml
was passed
over a 1.6 x 10 cm anti-FLAG M2 affinity column pre-equilibrated in PBS pH7.4
buffer
at a flow rate of 2.0 ml per minute. After washing with 200 ml of PBS pH 7.4
buffer,
bound material was eluted by a step of 200 mM Glycine pH 3.0 buffer and 0.5 ml
fractions collected. Upon elution, a significant protein peak was detected by
absorbance
at 280nm. Samples corresponding to conditioned medium, flow through and eluted
fractions were analyzed by Coomassie blue and silver stained SDS-PAGE and by
western blot analysis as described above. Significant immunoreactivity within
a
molecular weight range of 40-65 kDa was detected in conditioned medium and
eluted
fractions but not in the flow through sample, indicating that the secreted
hDkk-
3flag.long protein bound to the affinity column specifically and was eluted
efficiently by
the described conditions. Coomassie blue staining of SDS-PAGE gels suggested
that
the predominant immunoreactive protein constituted >90% of the protein present
in the
bound and eluted protein peak. Peak fractions of eluted protein were pooled
and
dialysed against Phosphate Buffered Saline resulting in a 4 ml volume of
recombinant
hDkk-3flag.long protein at a concentration of approximately lmg/ml.
Example 4: Isolation and Characterization of hDkk-4
In this example, the isolation and characterization of the gene encoding human
Dkk-4 (also referred to as "hDkk-4", "Cysteine Rich Secreted Protein-2", "CRSP-
2" or
"CRISPY 2") is described.
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Isolation and Analysis of a Human Dkk-4 cDNA
To identify novel proteins related to hDkk-3, the human Dkk-3 amino acid
sequence was used to search the dbEST database using TBLASTN (WashUversion,
2.0,
BLOSUM62 search matrix). A dbEST clone with accession number AA565546 was
identified as having homology to a portion of the hDkk-3 cDNA. This clone was
obtained from the IMAGE consortium and sequenced fully to define the entire
hDkk-4
sequence depicted in Figure 2.
Determination of the hydrophobicity profile of human Dkk-4 having the amino
acid sequence set forth in SEQ ID NO:5 indicated the presence of a hydrophobic
region
from about amino acid 1 to about amino acid 19 of SEQ ID NO:5. Further
analysis of
the amino acid sequence SEQ ID NO:5 using a signal peptide prediction program
predicted the presence of a signal peptide from about amino acid 1 to about
amino acid
19 of SEQ ID NO:5. Accordingly, the mature hDkk-4 protein includes about 205
amino
acids spanning from about amino acid 20 to about amino acid 224 of SEQ ID
NO:5.
Tissue Distribution of hDkk-4
hDkk-4 mRNA was undetectable by Northern analysis in all adult and fetal
human tissues examined. Accordingly, a survey was performed of a cDNA library
panel
by PCR with hDlck-4 specific PCR primers. Using such primers, products were
identified in libraries prepared from cerebellum, activated human T-
lymphocytes, lung
and esophagus.
Secretion and Post-Translational Modification of human Dkk-4
Flag epitope-tagged human Dkk-4 protein was transiently overexpressed in 293T
cells and analyzed as described previously for hDkk-3. Soluble hDkk-4 was
consistently
detected as three major immunoreactive species of approximately 40 kD [form
(i)], 30-
32 kD [form (ii)] and 15-17 kD [form (iii)]. Neither form (i), (ii) or (iii)
was
significantly affected by N-glycanase treatment, consistent with the absence
of N-
glycosylation sites from the protein.
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To determine the possible cause of heterogeneity in the size of secreted hDldc-
4,
Edman N-terminal sequencing of anti-flag affinity purified material
corresponding to
bands (i), (ii) and (iii) was performed. Briefly, flag-tagged Dkk-4 protein
was isolated
by passing the conditioned media over an M2-biotin (Sigma)/streptavidin Poros
column
(2.1 X 30 mm, PE Biosystems); the column was then washed with PBS, pH 7.4 and
flag-tagged protein eluted with 200 mM glycine, pH 3Ø Eluted fractions with
280nm
absorbance greater than background were analyzed by SDS-PAGE and western blot.
Purified DIdc-4 protein bound to PVDF membrane after SDS-PAGE and
electroblotting
was sequenced for N-terminal amino acid analysis on a PE Applied Biosystems
Model
494 Procise instrument using Edman-based chemistry protein sequencing. The
amino
acid residues were analyzed by HPLC (Spherogermicro PTH 3-micron column) and
determined by separation and peak height as compared to standards.
The N-terminal sequence of band (i) was found to be XVLDFNNIRS (SEQ ID
NO:34) which corresponds exactly to the predicted signal peptide cleavage site
(between
Ala-18 and Leu-19). Because the same band is identified by anti-flag
antibodies, which
recognize the C-terminal epitope tag, band (i) was thus identified as the full
length,
mature hDkk-4 protein. The band (iii) N-terminal sequence was found to be
SQGRKGQEGS (SEQ ID NO:35) which corresponds to CRD-2 cleaved at the dibasic
site Lys132/Lys133 (e.g., Lys113/Lys114 of the mature protein following
cleavage of
the a 19 amino acid signal sequence or Lys 114/Lys 115 following cleavage of a
18
amino acid signal sequence). These data obtained for bands (i) and (iii)
indicate clearly
that hDkk4 is proteolytically processed by 293T cells, resulting in the
release of CRD-2
(a 91 amino acid biologically-active fragment) from the full length protein.
Moreover, the three major species migrated similarly on SDS-PAGE conducted
under either reducing or non-reducing conditions. Thus, each of the major C-
terminal
(anti-flag immunoreactive) hDklc-4 species exist as independent proteolytic
fragments
that are not covalently linked via disulfide bonds to other subunits or
complex
components when secreted from 293T cells.
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Example 5: Isolation and Characterization of hDkk-1
In this example, the isolation and characterization of the gene encoding human
Dkk-1 (also referred to as "hDkk-1", "Cysteine Rich Secreted Protein-3", "CRSP-
3" or
"CRISPY-3") is described.
Identification of a Human Dkk-1 cDNA
Searching a proprietary database of EST information using the sequence of
hDkk-3, an hDkk-1 partial sequence was found corresponding to a clone from a
human
fetal kidney cDNA library having the identification code jthKb075a10. This
clone was
sequenced further and to define the entire hDkk-1 sequence depicted in Figure
3. DNA
for the clone jthKb075a10 was deposited with the ATCC as Accession No. 98633.
hDkk-1 has a predicted signal peptide from about amino acid residue 1 to 20 of
SEQ ID
NO:8, cleavage of which results in a mature protein having 246 amino acid
resudues in
length and corresponding to amino acid residues 21 to 266 of SEQ ID NO:8.
Tissue Distribution of hDkk-1
Northern blot analysis of various tissues including heart, brain, placenta,
lung,
liver, skeletal muscle, kidney, and pancreas was performed as previously
described
using a probe specific for hDkk-1. A ¨1.8 kb hDkkl mRNA was detected at high
levels
in human placenta, but not in other tissues tested.
Secretion and Post-Translational Modification of hDkk-1
Flag epitope-tagged human Dkk-1 protein was transiently overexpressed in 293T
cells and analyzed as described previously. hDla-1 was efficiently secreted
from
mammalian cells and was readily detected in conditioned medium of transfected
cells.
Mature secreted hDkk-1 migrated with a molecular weight of approximately 42-50
IcD.
Treatment with N-Glycanase had no significant effect on the mobility of
soluble hDkk-
1. Although hDkk-1 contains one potential site of N-linked glycosylation at
its extreme
C-terminus (e.g., at amino acids 256-259 of SEQ ID N08), this site is not
conserved in
Xenopus Dkk-1 (Glinka et al., supra) and appears not to be a major site of
carbohydrate
addition in 293T cells.
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Example 6: Isolation and Characterization of hDkk-2
In this example, the isolation and characterization of the gene encoding human
Dkk-2 (also referred to as "hDkk-2", "Cysteine Rich Secreted Protein4", "CRSP-
4" or
"CRISPY-4") is described.
Isolation of a Human Dkk-2 cDNA
Using the hDkk-3 sequence to query the dbEST database, a clone having
similarity to a portion of hDldc-3 was identified having Accession No. W55979.
This
clone was subsequently obtained from the IMAGE consortium and sequenced to
define
a partial hDkk-2 sequence set forth as SEQ ID NO:10. This cDNA comprises a
coding
region from nucleotides 1-537, as well as 3' untranslated sequences
(nucleotides 538 to
702). The coding region alone is set forth as SEQ ID NO:12. The predicted
amino acid
sequence corresponds to amino acids 1 to 179 of SEQ ID NO:11. A cDNA encoding
full length hDkk-2 was isolated from a human fetal lung lambda Ziplox
libraries by
conventional plaque hybridization (Sambrook et al., 1989) and fully sequenced.
The
full-length nucleotide sequence is set forth as SEQ ID NO:20 and the predicted
amino
acid sequence is set forth as SEQ ID NO:21. The coding region alone is set
forth as
SEQ ID NO:22. The predicted amino acid sequence corresponds to amino acids 1
to
259 of SEQ ID NO:21. DNA for the clone fthul 33 was deposited with the ATCC .
hDklc-2 has a predicted signal peptide from about amino acid
residue 1 to 33 of SEQ ID NO:21, cleavage of which results in a mature protein
having
226 amino acid resudues in length and corresponding to amino acid residues 34
to 259
of SEQ ID NO:21.
Tissue Distribution of hDkk-2
Northern blot analysis of various tissues (e.g., heart, brain, skeletal
muscle,
colon, thymus, spleen, kidney, liver, small intestine, placenta, lung, and
peripheral blood
leukocytes) was performed as previously described using a probe specific for
hDlck-2.
Of the tissues tested, hDkk-2 mRNA expression was highest in heart, brain,
placenta,
lung, and skeletal muscle. hDklc-2 transcripts of approximately 4.0 and 4.5 kb
were
observed.
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Secretion and Post-translational Modification of hDkk-2
Flag epitope-tagged human Dkk-2 protein was transiently overexpressed in 293T
cells and analyzed as described previously. Soluble hDkk-2 was detected as a
major
species of 15-17 kD, closely similar in size to form (iii) of hDkk-4.
Additional minor
forms of hDkk-2 were also observed in certain experiments in the range of 20-
21kD.
Deglycosylation of hDkk-2 was not studied since the protein sequence lacks
potential N-
glycosylation sites. By comparison with the data presented in Example 4
regarding the
dibasic proteolytic cleavage site in the hDkk-4 protein sequences, it is
predicted that the
major 15-17 kD form of hDkk-2 detected in these experiments corresponds to CRD-
2, as
was the case for hDkk-4.
Example 7: Isolation of Soggy proteins
In this example, the isolation and characterization of the gene encoding human
and murine Soggy-1 (also referred to as "Cysteine Rich Secreted Protein-N" or
"CRISP-
N") is described.
Identification of a Human and Murine Soggy-1 cDNAs
Human Soggy-1 was identified as a novel protein with similarity to the N-
terminal domain of hDkk3. A human partial sequence was identified in the dbEST
database for a clone having the accession number AA397836. This clone was
obtained
from the IMAGE collection and sequenced fully to define the entire human Soggy-
1
sequence depicted in Figure 7. Two murine partial sequences were likewise
identified in
the dBEST database. The clones were obtained from the IMAGE consortium and
sequenced. The entire murine Soggy-1 sequence is depicted in Figure 8. Human
and
murine Soggy cDNAs encode proteins of 242aa and 230aa, respectively, and are
predicted to be secreted owing to the presence of N-terminal signal peptides.
hSoggy-1
has a predicted signal peptide from about amino acid residue 1 to 30 of SEQ ID
NO:14,
cleavage of which results in a mature protein having 194 amino acid resudues
in length
and corresponding to amino acid residues 31 to 224 of SEQ ID NO:14. mSoggy-1
has a
predicted signal peptide from about amino acid residue 1 to 20 of SEQ ID NO
:27,
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cleavage of which results in a mature protein having 210 amino acid resudues
in length
and corresponding to amino acid residues 21 to 230 of SEQ ID NO:27. Human and
murine Soggy proteins display 59% overall identity although significant amino
acid
identities are seen beyond this domain that extend into the CRDs of Dkk-3
(Figure 10).
However, cysteine residues are not conserved within these domains and the
residues
shared by Soggy and Dkk-3 are poorly conserved in other Dkks indicating that
the
sequence relationship between these proteins is unique. Homology is most
obvious
within a 51 amino acid region in which 33% identity is observed between
hSoggy,
mSoggy, hDkk-3 and mDkk-3. This 51 amino acid domain is referred to herein as
an
SGY domain. Human and mouse Soggy-1 proteins each possess 2 sites of potential
N-
linked glycosylation which are within the SGY domain and are also conserved
with
Dkk3. (e.g., NNTL, corresponding to amino acid residues 97-100 of SEQ ID NO:14
and
NKTG corresponding to amino acid residues 112-115 of SEQ ID NO:14). In
contrast to
other Dkks, the C-terminal domain of Soggy-1 shows no similarity to other
protein
sequences in the public databases nor does it contain any cysteine residues.
Soggy was
so named in view of its lack of CRDs compared to hDkk-3, which had been
previously
designated Cysteine Rich Secreted Protein-1 ("CRISPY-1").
Tissue Distribution of Soggy-1
To investigate Soggy-1 mRNA expression, a mouse cDNA probe was used on
murine Nothern blots. A lkb mSoggy-1 mRNA was expressed at very high levels in
testis and, interestingly, demonstrated transient expression during mouse
embryogenesis.
Soggy-1 mRNA, which was undetectable at day 7 of gestation, was transiently
expressed at day 11 and day 15, after which the expression level declined to
undetectable levels. Thus, mSoggy-1 displays a developmentally regulated
pattern of
expression.
In situ analysis was performed as described in Example 1. For detection of
murine Soggy-1 ,the following primers were used:
mSoggy forward 5'-ACCTGCAATGTGTCGACTGAG-3' (SEQ ID NO:32), and
reverse 5'-CACTTACAGCTGTTGGGATG -3' (SEQ ID NO:33).
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Consistent with the Northern analysis, very high level expression of Soggy-1
mRNA was observed by in situ analysis in adult testis. Upon high
magnification,
Soggy-1 mRNA was found to be expressed at high levels in the spermatogenic
epithelium of the seminiferous tubules and in the spermatogonia at various
stages of
development. A series of saggital sections of mouse embryos from El 3.5 - E
17.5 and
post-natal day 1.5 pups were also analyzed. In E15.5 embryos, Soggy-1 mRNA
transcripts were localized to the developing dorsal root ganglia (DRGs) and
also found
in the cartilage primordium of the nasal septum. Soggy-1 expression was also
seen in
the eye from E13.5 to E16.5, as observed for mDkk-3. Expression of Soggy-1
mRNA
at various stages of development is consistent with the northern analysis
described above
and suggests that Soggy-1 may play a role in multiple stages of development.
Secretion and Post-Translational Modification of Soggy Proteins
Flag epitope-tagged human Soggy-1 protein was transiently overexpressed in
293T cells and analysed as previously described. hSoggy was efficiently
secreted from
transfected 293T cells and migrated with a molecular weight of approximately
40-50
kD. Given the heterogenous nature of secreted human Soggy-1, the effect of N-
Glycanase treatment on the mobility of secreted flag-tagged hSoggy-1 was
studied.
hSoggy displayed a 5-10 kD decrease in apparent molecular weight after N-
Glycanase
treatment, consistent with the presence of 2 potential sites of N-
glycosylation in the
protein.
Example 8: Structure of the Dkk Family proteins and Dkk-Related Proteins
The amino acid and nucleotide homology between Dkk family members and
Dkk-related proteins is set forth in the following tables. Where indicated,
mDkk-1 and
xDkk-lcorrespond to a murine and Xenopus proteins set forth in Glinks etal.,
supra,
and having Accession Nos: AF030433 and AF030434, respectively. Likewise cDkk-3
has Accession No. D26311
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Table II sets forth overall sequence identities as determined using the ALIGN
program,
(version 2.0) using a PAM120 weight residue table, a gap length penalty of 12,
and a
gap penalty of 4:
hDkk-3 hDkk-4 hDkk-1 hDkk-2 mDkk-1 xDkk-1 CLFEST
hDkk-3 100 16.0 18.6 15.1 18.5 16.5 53.0
hDkk-4 100 33.7 35.2 32.6 33.7 16.2
hDkk-1 100 33.1 80.2 53.5 17.4
hDkk-2 100 30.5 33.7 12.5
Table III sets forth nucleic acid identities as determined using the using the
Wilbur
Lipman DNA alignment program, Ktuple: 3; Gap Penalty: 3; Window: 20:
hDkk-3 hDkk-4 hDkk-1 hDkk-2 mDkk-1 xDkk-1 CLFEST
hDkk-3 100 30.0 37.2 34.7 31.5 45.4 58.8
hDkk-4 100 43.0 35.9 38.8 38.4 36.7
hDkk-1 100 59.3 66.4 53.7 32.1
hDkk-2 100 38.8 38.4 36.7
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Table IV sets forth local amino acid identities as determined using the Smith-
Waterman
algorithm as implemented in the program Bestfit of the GCG package, with gap
penalties of 8 for opening and 1 for extending:
hDkk-1 =
mDkk-1 82
xDkk-1 64 63
hDkk-2 50 48 47
hDkk-3 39 37 37 37.
mDkk-3 36 33 38 40 83
cDkk-3 34 31 35 36 61 60
hDkk-4 45 43 47 46 40 39 34
hDkk-1 mDkk-1 xDkk-1 hDkk-2 hDkk-3 mDkk-3 cDkk-3 hDkk-4
A multiple alignment of the amino acid sequences of hDkk-1, hDkk-2, hDkk-3,
hDkk-4, mDkk-1, mDkk-3, xDkk-1, and cDkk-3 is shown in Figure 6. Predicted
signal
peptides are underlined, N-glycosylation sites are indicated by a thick bar,
CRD-1 by an
open box and CRD-2 by a shaded box. The proteolytic cleavage site within hDkk4
is
indicated by a double asterisk. The domain structure of the full length human
Dkk
proteins of the present invention as well as human Soggy are schematically
illustrated in
Figure 9. Signal peptides (darkened boxes), Cysteine Rich Domain 1 ("CRD-1")
(also
referred to as the "amino-terminal cysteine-rich domain"), Cysteine Rich
Domain 2
("CRD-2") (also referred to as the "carboxy-terminal cysteine-rich domain"),
the soggy
domain (SGY) within hDlck-3 and hSoggy-1, and sites of N-glycosylation are
indicated.
As demonstrated at least in Figures 6 and 9, human Mks 1 through 4 each
possess an N-terminal signal peptide and contain two conserved cysteine-rich
domains
(CRDs) separated by a linker or spacer region. Each CRD possesses 10 conserved
cysteine residues. The second CRD has elsewhere been described as a colipase-
like
domain because the positions of the ten conserved cysteines in this domain
have been
shown to be closely similar to those in proteins of the colipase family
(Aravind and
Koonin, supra). Conservation of CRD-1 and CRD-2 suggests important functions
for
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these domains. In contrast to the CRDs, the linker or spacer region that joins
CRD-1
and CRD-2 is highly variable between hDkks, being notably larger in hDkk-1, -2
and -4
(50-55aa) as compared to Dkk-3 (12aa). Four potential sites of N-linked
glycosylation =
are present in hDldc3 and are conserved in chicken and mouse Dkk-3. These
sites are
not conserved in other Dick famiy members. hDkIcl possesses one potential N-
glycosylation site located close to the C-terminus of the protein which is
conserved in
murine Dkk-1 but not in Xenopus Dkk-1 (Fig. 6). In addition, each hDlck
possesses
several potential dibasic proteolytic cleavage sites, suggesting the proteins
may be
subject to post-translational processing. hDk1c3 is the most divergent of the
four human
Dicks and possesses an extended N-terminal unique region which precedes CR0-1
and
an extended C-terminal unique region which is highly acidic.
Example 9: Effects of hDkks and Soggy on Wnt-induced axis duplication in
Xenopus embryos
This Example describes the functional activities of the hDkk and Soggy
proteins
of the present invention.
Xenopus embryo culture and RNA microinjections
Eggs were obtained from Xenopus females injected with 700 units of human
chorionic gonadotropin, fertilized in vitro and cultured in 0.1 x MMR (Newport
and
Kirschner (1982) Cell 30:675-686). Embryonic stages were determned according
to
Nieuwkoop and Faber (1967) Normal table of Xenopus laevis (Daudin) Amsterdam:
North Holland Publ. All cDNAs were subcloned into pCS2 vector (Rupp et al.
(1984)
Genes & Development 8:1311-1323), and capped mRNAs were synthesized in vitro
as
described (Krieg and Melton (1984) Nucleic Acids Res. 12:7057-7070, using the
Message Machine kit (Ambion). The following plasmids were used as templates
for
mRNA synthesis: hDklc-1-pCS2, hDkk-2-pCS2, hDkk-3-pCS2, hDkk-4-pCS2, hSoggy-
pCS2, Xwnt8 (Christian et al., (1991) Development 111:1045-1055), Xwnt2B
(Landesman and Sokol (1997) Mech. Dev. 61:1199-209), Xwnt3a (Wolda et al.
(1993)
Dev. Biol. 155:46-5), Xfz8-pXT7 (Itoh etal. (1998) Mech. Devel. 74:145-157),
Xdsh-
pXT7 (Sokol, etal. (1995) Mech. Devel. 74:145-157). Protein expression from
all
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pCS2-Dkk constructs was confirmed by in vitro transcription and translation
(TNT,
Promega). For secondary axis induction, a single ventral blastomere of 4- or 8-
cell
embryos was injected with 10n1 of a solution containing 2-4pg of Xwnt8 mRNA,
2.5-
5pg of Xwnt3a mRNA or 10pg of Xwnt2B mRNA as described (Itoh et al. (1995)
supra.). The effect of DIck RNAs was tested by coinjecting Wnt mRNAs with
2.5ng of
hDkk mRNAs. For studies of Frizzled and Dhshevelled, 5ng Fz8 and lng Xdsh
mRNAs
were injected as indicated. After injections, embryos were cultured in 3%
Ficoll 400
(Pharmacia), 0.5xMMR. Secondary axes were scored at stage 35 as complete, when
they
contained anterior neuroectodermal derivatives including pronounced cement
gland and
eyes, and as partial, when the secondary neural tube with melanocytes, but
without head
structures, was apparent.
Inhibition of Secondary Axis Induction by hDkk-1 and hDkk-4 in Xenopus Embryos
hDkk-1 or hDkk-2 mRNAs were coinjected with Xwnt8 mRNA into single
ventral blastomeres of 4- or 8-cell embryos. Injected embryos were cultured
for 2 days
and secondary axes were scored based on external morphology. Xwnt8 injected
embryos displayed complete axis duplication, which was inhibited by co-
injection with
mRNAs encoding hDkk-1 and hDkk-4. To determine whether hDkks interacted with
specific Wnt ligands, several different Wnts were assayed in combination with
hDkk-1
or hDkk-4 for secondary axis formation. hDkk-1 and hDkk-4 inhibited axis
duplication
in response to Xwnt3a and Xwnt2b in addition to Xwnt8. hDkk-1 consistently
demonstrated stronger inhibition of Wnt signaling than hDkk-4. Thus, hDkk-1
and
hDkk -4 do not show any clear selectivity for the Wnt ligands used in this
study. This
compares to the FRPs, which also show little specificity with respect to their
ability to
inhibit Wnts (Leyns etal. (1997) supra; Wang et al. (1997) supra; Salic et al.
(1997)
supra; Mayr et al. (1997) supra; Finch etal. (1997) supra).
To investigate the mechanism by which hDkk-1 and hDkk-4 inhibit Wnt
signaling, Dkk mRNAs were coinjected with Xdsh, a downstream component of the
Wnt
signaling pathway (Itoh et al. (1998) supra). hDkks-1 and -4 did not block
secondary
axis formation by Xdsh, indicating that Dkks function upstream of, or parallel
with,
Xdsh signaling. Similar findings have been reported previously for xDkk-1
(Glinka et
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al. (1998) supra). It was also determined whether hDkks could antagonize
signaling by
Xenopus Frizzled-8 (Xfz8), which can also induce a secondary axis through Wnt
signaling (Itoh etal. (1998) supra). Neither hDkk-1 or hDkk-4 inhibited the
axis-
inducing activity of Xfz8 mRNA. This data, taken together with the fact that
hDkk-1
and hDkk-4 are secreted, indicate that Dkks antagonize Wnt signaling at a
point
upstream of Wnt receptors.
Assay for Inhibition of Secondary Axis Induction by hDkk-2, hDkk-3 and hSoggy-
1 in
Xenopus Embryos
hDkk-2, hDkk-3 or Soggy mRNAs were coinjected with Xwnt8 mRNA into
single ventral blastomeres of 4- or 8-cell embryos and secondary axes were
scored after
two days as described for hDkk-1 and hDkk-4. Injection of mRNAs encoding hDkk-
2,
hDkk-3 or hSoggy-1 had no effect on Xwnt8-induced axis duplication. The
ability of
hDkk-2, hDkk-3 and hSoggy-1 to interact with specific Wnt ligands was also
determined
as described previously. hDkk-2, hDkk-3 and hSoggy-1 were inactive against
each of
the three Wnts tested. The lack of activity of hDkk-2, hDkk-3 and hSoggy-1
suggests
that these proteins antagonize other members of the Wnt superfamily not tested
here, or
that they perform functions distinct from Wnt inhibition.
Example 10: Preparation of Antibodies Specific for hDkk and hSoggy Proteins
This example describes the making of polyclonal antibodies specific for hDkk-
1, hDkk-4, hDkk-1, hDlck-2, and hSoggy-1.
Peptides were synthesized using Fmoc solid phase methodology utilizing MAP
resin technology which increases the antigenic response (Tarn (1988) Proc.
Natl.
Acad. Sci. USA 85:5409-5413. For each protein, the peptides used for
immunization
are listed below:
hDkk-3
peptide #44 FREVEELMEDTQHKL
peptide #46 GSFMEEVRQELEDLE
hDkk-4
peptide #91 HAEGTTGHPVQENQP
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hDkk-1
peptide #93 GNKYQTIDNYQPYPC
hDkk-2
peptide #56 GHYSNHDLGWQNLGR
hSoggy-1
peptide #58 LQAIRDGLRKGTHKD
Peptides were designed to meet at least the following criteria: (1) not
included
within the cysteine-rich domain; (2) not including an N-glycosylation site;
and (3)
hydrophilic (e.g., solvent exposed).
Antibodies were generated in New Zealand white rabbits over a 10-week
period. The immunogen includes KLH-peptide emulsified by mixing with an equal
volume of Freund's Adjuvant, and injected into three subcutaneous dorsal
sites, for a
total of 0.1mg peptide per immunization. Animals were bled from the articular
artery.
The blood was allowed to clot and the serum collected by centrifugation. The
serum is
stored at -20 C.
For purification, peptide antigens were immobilized on an activated support.
Antisera was passed through the sera column and then washed. Specific
antibodies
were eluted via a pH gradient, collected and stored in a borate buffer (0.125M
total
borate) at ¨0.25mg/m1. The anti-peptide titers were determined using ELISA
methodology with free peptide bound in solid phase (1n/we11). Detection was
obtained using biotinylated anti-rabbit IgG, HRP-SA conjugate, and ABTS.
All antibodies performed well in ELISA assays. Anti-peptide #44, #46, and
#58 are particularly useful for detection of hDkk-3 and hSoggy-1,
respectively, as
determined by western blotting of supernatants from hDkk-3- and hSoggy-l-
transfected 293T cells.
The Dkk family comprises a novel family of secreted proteins which to date
includes hDkk-1, hDkk-2, hDkk-3, hDkk-4, xDkk-1, mDkk-1 and cDkk-1.
Structurally, Dkks 1-4 are related by several conserved features. Firstly, all
four
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proteins are secreted proteins. Secondly, Dkks 1-4 each possess two distinct
cysteine
rich domains. Each domain contains 10 conserved cysteine residues, and these
domains are highly conserved between family members. The C-terminal cysteine
rich
domain, referred to as CRD-2, bears significant similarity to proteins of the
colipase
family and sequence conservation among the Dkks is greatest within CRD-2
(Aravind
and Koonin, supra). This may reflect a need for Dkks to interact with lipids
in order
to regulate Wnt function, since Wnt proteins remain tightly associated with
the cell
surface.
Despite the similarities between Dkks 1-4, notable differences between these
family members appear with regard to their mRNA expression patterns. In adult
human tissues hDkk-1 and hDkk-4 showed highly restricted mRNA expression
patterns while hDkk-2 and hDkk-3 are more widely expressed. Murine Dkk-3 mRNA
was found to be restricted to the myocytes of the atria in the heart, neurons
of the
cortex and hippocampus in the brain and also to the retinal neurons and lens
epithelium in the eye. Such specific expression patterns reflect localized
action of the
Dkks as regulators of Wnt activity and/or that of other signaling molecules.
Different
Wnt family members have been shown to have divergent patterns of mRNA
expression in adult and embryonic mammalian tissues. For example, murine Wnts
4,
7a and 7b are expressed in brain and lung, whereas Wnt6 is highly expressed in
testis
(Gavin etal., 1990). Wnts 5b and 13 are more broadly expressed (Gavin etal.
(1990)
supra; Katoh et al. (1996) supra). Although Wnts have been studied mostly in
the
context of their roles in embryonic development and tumorigensis, the
expression of
many family members in normal adult human and mouse tissues, together with
their
regulators such as the Dkks, suggests that these signaling proteins play
important roles
in normal tissue homeostasis.
Marked differences in the post-translational processing of different human Dkk
proteins was also observed. hDkk-3 is secreted from 293T cells as a
heterogeneously
glycosylated protein, whereas Dkk-1, 2 and 4 proteins show no evidence of
glycosylation. This is consistent with sequence analysis that identifies 4
potential sites
of N-glycosylation in the hDkk-3 protein but no sites in either hDkk-2 or
hDlck-4. A
single putative site in hDkk-1 does not appear to be utilized in 293T cells
and may
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well not be a significant site for N-linked carbohydrate addition in view of
its C-
terminal location and lack of conservation with xDkk-1. C-terminal proteolysis
of
hDkk4 in 293T cells was also characterized. Dkk proteins contain multiple
potential
sites of proteolytic processing. Secreted hDkk-4 was consistently detected as
three
major C-terminal fragments. N-terminal sequencing identified two of these as
mature,
full length hDkk4 and CRD-2, which was derived from the full length protein by
a
specific proteolytic event at lysines 132 and 133. Thus, the hDkk-4 CRD-2 is
released
from the full length protein upon expression in 293T cells. Similar processing
of
hDkk4 in COS cells has been observed.
Within the Dkk family, Dkks 1, 2 and 4 display closest similarity whereas
Dkk-3 is more distantly related. For example, Dkk-3 contains a linker region
connecting CRD-1 and CRD-2 which is shorter than in other Dkks. Dkk-3 also
possesses extended N-and C-terminal regions compared to other Dkks. Within the
Dkk-3 N-terminal unique region, a distinct domain has been identified that is
also
found in Soggy (the SGY domain). The SGY domains of human and mouse Soggy-1
and Dkk-3 proteins contain two conserved sites of N-linked glycosylation.
Protein
expression studies confirm that, like hDkk3, hSoggy is secreted as a
glycoprotein.
Murine Soggy-1 is highly expressed in adult testis and also displays a highly
restricted
mRNA expression in El 5 -El 6 mouse embryos, being localized mainly to the
cartilage
primordia within the developing vertebrae/developing dorsal root ganglia.
Soggy
mRNA was also detected at high levels in the developing eye, similar to Dkk-3.
This
developmentally regulated pattern of Soggy expression overlaps with that of
Dkk-3
suggesting that Soggy may play a role in the regulation of Dkk-3 function.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
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SEQUENCE LISTING
<110> MILLENNIUM PHARMACEUTICALS, INC.
<120> HUMAN DICKKOPF-RELATED PROTEIN AND NUCLEIC ACID
MOLECULES AND USES THEREFOR
<130> 08-892661CA
<140>
<141> 2000-03-03
<150> 09/263,022
<151> 1999-03-05
<160> 38
<170> PatentIn Ver. 2.0
<210> 1
<211> 2479
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (38)..(1087)
<220>
<223> 'n' at position 1146 may be any nucleotide
<400> 1
ggcacgaggg ggcggcggct gcgggcgcag agcggag atg cag cgg ctt ggg gcc 55
Met Gin Arg Leu Gly Ala
1 5
acc ctg ctg tgc ctg ctg ctg gcg gcg gcg gtc ccc acg gcc ccc gcg 103
Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala Val Pro Thr Ala Pro Ala
15 20
ccc gct ccg acg gcg acc tcg gct cca gtc aag ccc ggc ccg gct ctc 151
Pro Ala Pro Thr Ala Thr Ser Ala Pro Val Lys Pro Gly Pro Ala Leu
25 30 35
agc tac ccg cag gag gag gcc acc ctc aat gag atg ttc cgc gag gtt 199
Ser Tyr Pro Gin Glu Glu Ala Thr Leu Asn Glu Met Phe Arg Glu Val
40 45 50
gag gaa ctg atg gag gac acg cag cac aaa ttg cgc agc gcg gtg gaa 247
Glu Glu Leu Met Glu Asp Thr Gin His Lys Leu Arg Ser Ala Val Glu
55 60 65 70
gag atg gag gca gaa gaa gct gct gct aaa gca tca tca gaa gtg aac 295
Glu Met Glu Ala Glu Glu Ala Ala Ala Lys Ala Ser Ser Glu Val Asn
75 80 85
ctg gca aac tta cct ccc agc tat cac aat gag acc aac aca gac acg 343
Leu Ala Asn Leu Pro Pro Ser Tyr His Asn Glu Thr Asn Thr Asp Thr
90 95 100
aac gtt gga aat aat acc atc cat gtg cac cga gaa att cac aag ata 391
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Asn Val Gly Asn Asn Thr Ile His Val His Arg Glu Ile His Lys Ile
105 110 115
acc aac aac cag act gga caa atg gtc ttt tca gag aca gtt atc aca 439
Thr Asn Asn Gin Thr Gly Gin Met Val Phe Ser Glu Thr Val Ile Thr
120 125 130
tct gtg gga gac gaa gaa ggc aga agg agc cac gag tgc atc atc gac 487
Ser Val Gly Asp Glu Glu Gly Arg Arg Ser His Glu Cys Ile Ile Asp
135 140 145 150
gag gac tgt ggg ccc agc atg tac tgc cag ttt gcc agc ttc cag tac 535
Glu Asp Cys Gly Pro Ser Met Tyr Cys Gin Phe Ala Ser Phe Gin Tyr
155 160 165
acc tgc cag cca tgc cgg ggc cag agg atg ctc tgc acc cgg gac agt 583
Thr Cys Gin Pro Cys Arg Gly Gin Arg Met Leu Cys Thr Arg Asp Ser
170 175 180
gag tgc tgt gga gac cag ctg tgt gtc tgg ggt cac tgc acc aaa atg 631
Glu Cys Cys Gly Asp Gin Leu Cys Val Trp Gly His Cys Thr Lys Met
185 190 195
gcc acc agg ggc agc sat ggg acc atc tgt gac aac cag agg gac tgc 679
Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys Asp Asn Gin Arg Asp Cys
200 205 210
cag cog ggg ctg tgc tgt gcc ttc cag aga ggc ctg ctg ttc cct gtg 727
Gin Pro Gly Leu Cys Cys Ala Phe Gin Arg Gly Leu Leu Phe Pro Val
215 220 225 230
tgc aca ccc ctg ccc gtg gag ggc gag ctt tgc cat gac ccc gcc agc 775
Cys Thr Pro Leu Pro Val Glu Gly Glu Leu Cys His Asp Pro Ala Ser
235 240 245
cgg ctt ctg gac ctc atc acc tgg gag cta gag cct gat gga gcc ttg 823
Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu Glu Pro Asp Gly Ala Leu
250 255 260
gac cga tgc cct tgt gcc agt ggc ctc ctc tgc cag ccc cac agc cac 871
Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu Cys Gin Pro His Ser His
265 270 275
agc ctg gtg tat gtg tgc sag cog acc ttc gtg ggg agc cgt gac caa 919
Ser Leu Val Tyr Val Cys Lys Pro Thr Phe Val Gly Ser Arg Asp Gin
280 285 290
gat ggg gag atc ctg ctg ccc aga gag gtc ccc gat gag tat gas gtt 967
Asp Gly Glu Ile Leu Leu Pro Arg Glu Val Pro Asp Glu Tyr Glu Val
295 300 305 310
ggc agc ttc atg gag gag gtg cgc cag gag ctg gag gac ctg gag agg 1015
Gly Ser Phe Met Glu Glu Val Arg Gin Glu Leu Glu Asp Leu Glu Arg
315 320 325
agc ctg act gaa gag atg gcg ctg agg gag cot gcg got gcc gcc got 1063
Ser Leu Thr Glu Glu Met Ala Leu Arg Glu Pro Ala Ala Ala Ala Ala
330 335 340
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 3 -
gca ctg ctg gga agg gaa gag att tagatctgga ccaggctgtg ggtagatgtg 1117
Ala Leu Leu Gly Arg Glu Glu Ile
345 350
caatagaaat agctaattta tttccccang tgtgtgcttt aagcgtgggc tgaccaggct 1177
tcttcctaca tcttcttccc agtaagtttc ccctctggct tgacagcatg aggtgttgtg 1237
catttgttca gctcccccag gctgttctcc aggcttcaca gtctggtgct tgggagagtc 1297
aggcagggtt aaactgcagg agcagtttgc cacccctgtc cagattattg gctgctttgc 1357
ctctaccagt tggcagacag ccgtttgttc tacatggctt tgataattgt ttgaggggag 1417
gagatggaaa caatgtggag tctccctctg attggttttg gggaaatgtg gagaagagtg 1477
ccctgctttg caaacatcaa cctggcaaaa atgcaacaaa tgaattttcc acgcagttct 1537
ttccatgggc ataggtaagc tgtgccttca gctgttgcag atgaaatgtt ctgttcaccc 1597
tgcattacat gtgtttattc atccagcagt gttgctcagc tcctacctct gtgccagggc 1657
agcattttca tatccaagat caattccctc tctcagcaca gcctggggag ggggtcattg 1717
ttctcctcgt ccatcaggga tttcagaggc tcagagactg caagctgctt gcccaagtca 1777
cacagctagt gaagaccaga gcagtttcat ctggttgtga ctctaagctc agtgctctct 1837
ccactacccc acaccagcct tggtgccacc aaaagtgctc cccaaaagga aggagaatgg 1897
gatttttctt ttgaggcatg cacatctgga attaaggtca aactaattct cacatcoctc 1957
taaaagtaaa ctactgttag gaacagcagt gttctcacag tgtggggcag ccgtocttct 2017
aatgaagaca atgatattga cactgtccct ctttggcagt tgcattagta actttgaaag 2077
gtatatgact gagcgtagca tacaggttaa cctgcagaaa cagtacttag gtaattgtag 2137
ggcgaggatt ataaatgaaa tttgcaaaat cacttagcag caactgaaga caattatcaa 2197
ccacgtggag aaaatcaaac cgagcagggc tgtgtgaaac atggttgtaa tatgcgactg 2257
cgaacactga actctacgcc actccacaaa tgatgttttc aggtgtcatg gactgttgcc 2317
accatgtatt catccagagt tcttaaagtt taaagttgca catgattgta taagcatgct 2377
ttctttgagt tttaaattat gtataaacat aagttgcatt tagaaatcaa gcataaatca 2437
cttcaactgc taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 2479
<210> 2
<211> 350
<212> PRT
<213> Homo sapiens
<400> 2
Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala
1 5 10 15
CA 02366062 2001-09-13
W000/52047
PCT/US00/05452
-4-
Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val
20 25 30
Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn
35 40 45
Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys
50 55 60
Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys
65 70 75 80
Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn
85 90 95
Glu Thr Asn Thr Asp Thr Asn Val Gly Asn Asn Thr Ile His Val His
100 105 110
Arg Glu Ile His Lys Ile Thr Asn Asn Gln Thr Gly Gln Met Val Phe
115 120 125
Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser
130 135 140
His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln
145 150 155 160
Phe Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met
165 170 175
Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp
180 185 190
Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys
195 200 205
Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg
210 215 220
Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
225 230 235 240
Cys His Asp Pro Ala Ser Arg Leu Leu Asp Leu Ile Thr Trp Glu Leu
245 250 255
Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu
260 265 270
Cys Gln Pro His Ser His Ser Leu Val Tyr Val Cys Lys Pro Thr Phe
275 280 285
Val Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val
290 295 300
Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu
305 310 315 320
Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala Leu Arg Glu
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-5-
325 330 335
Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Arg Glu Glu Ile
340 345 350
<210> 3
<211> 1050
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(1050)
<400> 3
atg cag cgg ctt ggg gcc acc ctg ctg tgc ctg ctg ctg gcg gcg gcg 48
Met Gin Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala
1 5 10 15
gtc ccc acg gcc ccc gcg ccc gct cog acg gcg acc tcg gct cca gtc 96
Val Pro Thr Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val
20 25 30
aag ccc ggc cog gct ctc ago tac ccg cag gag gag gcc acc ctc aat 144
Lys Pro Gly Pro Ala Leu Ser Tyr Pro Gin Glu Glu Ala Thr Leu Asn
35 40 45
gag atg ttc cgc gag gtt gag gaa ctg atg gag gac acg cag cac aaa 192
Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gin His Lys
50 55 60
ttg cgc ago gcg gtg gaa gag atg gag gca gaa gaa gct gct gct aaa 240
Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys
65 70 75 80
gca tca tca gaa gtg aac ctg gca aac tta cct ccc ago tat cac aat 288
Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro Ser Tyr His Asn
85 - 90 95
gag acc aac aca gac acg aac gtt gga aat aat acc atc cat gtg cac 336
Glu Thr Asn Thr Asp Thr Asn Val Gly Asn Asn Thr Ile His Val His
100 105 110
cga gaa att cac aag ata acc aac aac cag act gga caa atg gtc ttt 384
Arg Glu Ile His Lys Ile Thr Asn Asn Gin Thr Gly Gin Met Val Phe
115 120 125
tca gag aca gtt atc aca tot gtg gga gac gaa gaa ggc aga agg ago 432
Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Arg Arg Ser
130 135 140
cac gag tgc atc atc gac gag gac tgt ggg ccc ago atg tac tgc cag 480
His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gin
145 150 155 160
ttt gcc ago ttc cag tac acc tgc cag cca tgc cgg ggc cag agg atg 528
Phe Ala Ser Phe Gin Tyr Thr Cys Gin Pro Cys Arg Gly Gin Arg Met
165 170 175
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-6-
ctc tgc acc cgg gac agt gag tgc tgt gga gac cag ctg tgt gtc tgg 576
Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gin Leu Cys Val Trp
180 185 190
ggt cac tgc acc aaa atg gcc acc agg ggc ago aat ggg acc atc tgt 624
Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys
195 200 205
gac aac cag agg gac tgc cag ccg ggg ctg tgc tgt gcc ttc cag aga 672
Asp Asn Gin Arg Asp Cys Gin Pro Gly Leu Cys Cys Ala Phe Gin Arg
210 215 220
ggc ctg ctg ttc cct gtg tgc aca ccc ctg ccc gtg gag ggc gag ctt 720
Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
225 230 235 240
tgc cat gac ccc gcc ago cgg ctt ctg gac ctc atc acc tgg gag cta 768
Cys His Asp Pro Ala Ser Arg Leta Leu Asp Leu Ile Thr Trp Glu Leu
245 250 255
gag cot gat gga gcc ttg gac cga tgc cot tgt gcc agt ggc ctc ctc 816
Glu Pro Asp Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu
260 265 270
tgc cag ccc cac ago cac ago ctg gtg tat gtg tgc aag cog acc ttc 864
Cys Gin Pro His Ser His Ser Leu Val Tyr Val Cys Lys Pro Thr Phe
275 280 285
gtg ggg ago cgt gac caa gat ggg gag atc ctg ctg ccc aga gag gtc 912
Val Gly Ser Arg Asp Gin Asp Gly Glu Ile Leu Leu Pro Arg Glu Val
290 295 300
ccc gat gag tat gaa gtt ggc ago ttc atg gag gag gtg cgc cag gag 960
Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gin Glu
305 310 315 320
ctg gag gac ctg gag agg ago ctg act gaa gag atg gcg ctg agg gag 1008
Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala Leu Arg Glu
325 330 335
cot gcg got gcc gcc got gca ctg ctg gga agg gaa gag att 1050
Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Arg Glu Glu Ile
340 345 350
<210> 4
<211> 848
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (125)..(796)
<400> 4
gaattcggca cgagagacga cgtgctgagc tgccagctta gtggaagctc tgctctgggt 60
ggagagcagc ctcgctttgg tgacgcacag tgctgggacc ctccaggagc cccgggattg 120
CA 02366062 2001-09-13
VA) 00/52047
PCT/US00/05452
-7-
aagg atg gtg gcg gcc gtc ctg ctg ggg ctg agc tgg ctc tgc tct ccc 169
Met Val Ala Ala Val Leu Leu Gly Leu Ser Trp Leu Cys Ser Pro
1 5 10 15
ctg gga gct ctg gtc ctg gac ttc aac aac atc agg ago tct gct gac 217
Leu Gly Ala Leu Val Leu Asp Phe Asn Asn Ile Arg Ser Ser Ala Asp
20 25 30
ctg cat ggg gcc cgg aag ggc tca cag tgc ctg tct gac acg gac tgc 265
Leu His Gly Ala Arg Lys Gly Ser Gin Cys Leu Ser Asp Thr Asp Cys
35 40 45
aat acc aga aag ttc tgc ctc cag ccc cgc gat gag aag cog ttc tgt 313
Asn Thr Arg Lys Phe Cys Leu Gin Pro Arg Asp Glu Lys Pro Phe Cys
50 55 60
gct aca tgt cgt ggg ttg cgg agg agg tgc cag cga gat gcc atg tgc 361
Ala Thr Cys Arg Gly Leu Arg Arg Arg Cys Gin Arg Asp Ala Met Cys
65 70 75
tgc cot ggg aca ctc tgt gtg aac gat gtt tgt act acg atg gaa gat 409
Cys Pro Gly Thr Leu Cys Val Asn Asp Val Cys Thr Thr Met Glu Asp
80 85 90 95
gca acc cca ata tta gaa agg cag ctt gat gag caa gat ggc aca cat 457
Ala Thr Pro Ile Leu Glu Arg Gin Leu Asp Glu Gin Asp Gly Thr His
100 105 110
gca gaa gga aca act ggg cac cca gtc cag gaa aac caa ccc aaa agg 505
Ala Glu Gly Thr Thr Gly His Pro Val Gin Glu Asn Gin Pro Lys Arg
115 120 125
aag cca agt att aag aaa tca caa ggc agg aag gga caa gag gga gaa 553
Lys Pro Ser Ile Lys Lys Ser Gin Gly Arg Lys Gly Gin Glu Gly Glu
130 135 140
agt tgt ctg aga act ttt gac tgt ggc cct gga ctt tgc tgt gct cgt 601
Ser Cys Leu Arg Thr Phe Asp Cys Gly Pro Gly Leu Cys Cys Ala Arg
145 150 155
cat ttt tgg acg aaa att tgt aag cca gtc ctt ttg gag gga cag gtc 649
His Phe Trp Thr Lys Ile Cys Lys Pro Val Leu Leu Glu Gly Gin Val
160 165 170 175
tgc tcc aga aga ggg cat aaa gac act gct caa gct cca gaa atc ttc 697
Cys Ser Arg Arg Gly His Lys Asp Thr Ala Gin Ala Pro Glu Ile Phe
180 185 190
cag cgt tgc gac tgt ggc cot gga cta ctg tgt cga ago caa ttg acc 745
Gin Arg Cys Asp Cys Gly Pro Gly Leu Leu Cys Arg Ser Gin Leu Thr
195 200 205
ago aat cgg cag cat gct cga tta aga gta tgc caa aaa ata gaa aag 793
Ser Asn Arg Gin His Ala Arg Leu Arg Val Cys Gin Lys Ile Glu Lys
210 215 220
cta taaatatttc aaaataaaga agaatccaca ttgcaaaaaa aaaaaaaaaa aa 848
Leu
CA 02366062 2001-09-13
VA) 00/52047
PCT/US00/05452
-8-
<210> 5
<211> 224
<212> PRT
<213> Homo sapiens
<400> 5
Met Val Ala Ala Val Leu Leu Gly Leu Ser Trp Leu Cys Ser Pro Leu
1 5 10 15
Gly Ala Leu Val Leu Asp Phe Asn Asn Ile Arg Ser Ser Ala Asp Leu
20 25 30
His Gly Ala Arg Lys Gly Ser Gln Cys Leu Ser Asp Thr Asp Cys Asn
35 40 45
Thr Arg Lys Phe Cys Leu Gln Pro Arg Asp Glu Lys Pro Phe Cys Ala
50 55 60
Thr Cys Arg Gly Leu Arg Arg Arg Cys Gln Arg Asp Ala Met Cys Cys
65 70 75 80
Pro Gly Thr Leu Cys Val Asn Asp Val Cys Thr Thr Met Glu Asp Ala
85 90 95
Thr Pro Ile Leu Glu Arg Gln Leu Asp Glu Gln Asp Gly Thr His Ala
100 105 110
Glu Gly Thr Thr Gly His Pro Val Gin Glu Asn Gln Pro Lys Arg Lys
115 120 125
Pro Ser Ile Lys Lys Ser Gln Gly Arg Lys Gly Gln Glu Gly Glu Ser
130 135 140
Cys Leu Arg Thr Phe Asp Cys Gly Pro Gly Leu Cys Cys Ala Arg His
145 150 155 160
Phe Trp Thr Lys Ile Cys Lys Pro Val Leu Leu Glu Gly Gln Val Cys
165 170 175
Ser Arg Arg Gly His Lys Asp Thr Ala Gln Ala Pro Glu Ile Phe Gln
180 185 190
Arg Cys Asp Cys Gly Pro Gly Leu Leu Cys Arg Ser Gln Leu Thr Ser
195 200 205
Asn Arg Gln His Ala Arg Leu Arg Val Cys Gln Lys Ile Glu Lys Leu
210 215 220
<210> 6
<211> 672
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(672)
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 9 -
<4 0 0> 6
atg gtg gcg gcc gtc ctg ctg ggg ctg agc tgg ctc tgc tct ccc ctg 48
Met Val Ala Ala Val Leu Leu Gly Leu Ser Trp Leu Cys Ser Pro Leu
1 5 10 15
gga gct ctg gtc ctg gac ttc aac aac atc agg agc tct gct gac ctg 96
Gly Ala Leu Val Leu Asp Phe Asn Asn Ile Arg Ser Ser Ala Asp Leu
20 25 30
cat ggg gcc cgg aag ggc tca cag tgc ctg tct gac acg gac tgc aat 144
His Gly Ala Arg Lys Gly Ser Gin Cys Leu Ser Asp Thr Asp Cys Asn
35 40 45
acc aga aag ttc tgc ctc cag ccc cgc gat gag aag ccg ttc tgt gct 192
Thr Arg Lys Phe Cys Leu Gln Pro Arg Asp Glu Lys Pro Phe Cys Ala
50 55 60
aca tgt cgt ggg ttg cgg agg agg tgc cag cga gat gcc atg tgc tgc 240
Thr Cys Arg Gly Leu Arg Arg Arg Cys Gin Arg Asp Ala Met Cys Cys
65 70 75 80
cot ggg aca ctc tgt gtg aac gat gtt tgt act acg atg gaa gat gca 288
Pro Gly Thr Leu Cys Val Asn Asp Val Cys Thr Thr Met Glu Asp Ala
85 90 95
acc cca ata tta gaa agg cag ctt gat gag caa gat ggc aca cat gca 336
Thr Pro Ile Leu Glu Arg Gin Leu Asp Glu Gin Asp Gly Thr His Ala
100 105 110
gaa gga aca act ggg cac cca gtc cag gaa aac caa ccc aaa agg aag 384
Glu Gly Thr Thr Gly His Pro Val Gin Glu Asn Gin Pro Lys Arg Lys
115 120 125
cca agt att aag aaa tca caa ggc agg aag gga caa gag gga gaa agt 432
Pro Ser Ile Lys Lys Ser Gin Gly Arg Lys Gly Gin Glu Gly Glu Ser
130 135 140
tgt ctg aga act ttt gac tgt ggc cot gga ctt tgc tgt gct cgt cat 480
Cys Leu Arg Thr Phe Asp Cys Gly Pro Gly Leu Cys Cys Ala Arg His
145 150 155 160
ttt tgg acg aaa att tgt aag cca gtc ctt ttg gag gga cag gtc tgc 528
Phe Trp Thr Lys Ile Cys Lys Pro Val Leu Leu Glu Gly Gin Val Cys
165 170 175
too aga aga ggg cat aaa gac act gct caa gct cca gaa atc ttc cag 576
Ser Arg Arg Gly His Lys Asp Thr Ala Gin Ala Pro Glu Ile Phe Gin
180 185 190
cgt tgc gac tgt ggc cct gga cta ctg tgt cga agc caa ttg acc agc 624
Arg Cys Asp Cys Gly Pro Gly Leu Leu Cys Arg Ser Gin Leu Thr Ser
195 200 205
aat cgg cag cat gct cga tta aga gta tgc caa aaa ata gaa aag cta 672
Asn Arg Gin His Ala Arg Leu Arg Val Cys Gin Lys Ile Glu Lys Leu
210 215 220
CA 02366062 2001-09-13
va) 011152047
PCMS00/05452
-10-
<210> 7
<211> 1536
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (109)..(906)
<400> 7
gtcgacccac gcgtccgcgg acgcgtgggc ggcacggttt cgtggggacc caggcttgca 60
aagtgacggt cattttctct ttctttctcc ctcttgagtc cttctgag atg atg gct 117
Met Met Ala
1
ctg ggc gca gcg gga got acc cgg gtc ttt gtc gcg atg gta gcg gcg 165
Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val Ala Met Val Ala Ala
10 15
got ctc ggc ggc cac cot ctg ctg gga gtg ago gcc acc ttg aac tog 213
Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr Leu Asn Ser
20 25 30 35
gtt ctc aat too aac got atc aag aac ctg ccc cca ccg ctg ggc ggc 261
Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro. Pro Pro Leu Gly Gly
40 45 50
got gcg ggg cac cca ggc tot gca gtc ago goo gcg ccg gga atc ctg 309
Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro Gly Ile Leu
55 60 65
tac ccg ggc ggg aat aag tac cag acc att gac aac tac cag ccg tac 357
Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr Gln Pro Tyr
70 75 80
ccg tgc gca gag gac gag gag tgc ggc act gat gag tac tgc got agt 405
Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr Cys Ala Ser
85 90 95
coo acc cgc gga ggg gac gca ggc gtg caa atc tgt ctc goo tgc agg 453
Pro Thr Arg Gly Gly Asp Ala Gly Val Gln Ile Cys Leu Ala Cys Arg
100 105 110 115
aag cgc cga aaa cgc tgc atg cgt cac got atg tgc tgc ccc ggg aat 501
Lys Arg Arg Lys Arg Cys Met Arg His Ala Met Cys Cys Pro Gly Asn
120 125 130
tac tgc aaa aat gga ata tgt gtg tot tot gat caa aat cat ttc cga 549
Tyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp Gln Asn His Phe Arg
135 140 145
gga gas att gag gaa acc atc act gas ago ttt ggt aat gat cat ago 597
Gly Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn Asp His Ser
150 155 160
acc ttg gat ggg tat too aga aga acc acc ttg tot tca aaa atg tat 645
Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser Lys Met Tyr
165 170 175
CA 02366062 2001-09-13
WO 00/52047 PCT/US00/05452
-11-
cac acc aaa gga caa gaa ggt tct gtt tgt ctc cgg tca tca gac tgt 693
His Thr Lys Gly Gin Glu Gly Ser Val Cys Leu Arg Ser Ser Asp Cys
180 185 190 195
gcc tca gga ttg tgt tgt gct aga cac ttc tgg tcc aag atc tgt aaa 741
Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys Ile Cys Lys
200 205 210
cct gtc ctg aaa gaa ggt caa gtg tgt acc aag cat agg aga aaa ggc 789
Pro Val Leu Lys Glu Gly Gin Val Cys Thr Lys His Arg Arg Lys Gly
215 220 225
tct cat gga cta gaa ata ttc cag cgt tgt tac tgt gga gaa ggt ctg 837
Ser His Gly Leu Glu Ile Phe Gin Arg Cys Tyr Cys Gly Glu Gly Leu
230 235 240
tct tgc cgg ata cag aaa gat cac cat caa gcc agt aat tct tct agg 885
Ser Cys Arg Ile Gin Lys Asp His His Gin Ala Ser Asn Ser Ser Arg
245 250 255
ctt cac act tgt cag aga cac taaaccagct atccaaatgc agtgaactcc 936
Leu His Thr Cys Gin Arg His
260 265
ttttatataa tagatgctat gaaaaccttt tatgaccttc atcaactcaa tcctaaggat 996
atacaagttc tgtggtttca gttaagcatt ccaataacac cttccaaaaa cctggagtgt 1056
aagagctttg tttctttatg gaactcccct gtgattgcag taaattactg tattgtaaat 1116
tctcagtgtg gcacttacct gtaaatgcaa tgaaactttt aattattttt ctaaaggtgc 1176
tgcactgcct atttttcctc ttgttatgta aatttttgta cacattgatt gttatcttga 1236
ctgacaaata ttctatattg aactgaagta aatcatttca gcttatagtt cttaaaagca 1296
taacccttta ccccatttaa ttctagagtc tagaacgcaa ggatctcttg gaatgacaaa 1356
tgataggtac ctaaaatgta acatgaaaat actagcttat tttctgaaat gtactatctt 1416
aatgcttaaa ttatatttcc ctttaggctg tgatagtttt tgaaataaaa tttaacattt 1476
aatatcatga aatgttataa gtagacataa aaaaaaaaaa aaaaaaaaaa gggcggccgc 1536
<210> 8
<211> 266
<212> PRT
<213> Homo sapiens
<400> 8
Met Met Ala Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val Ala Met
1 5 10 15
Val Ala Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr
20 25 30
Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-12-
35 40 45
Leu Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro
50 55 60
Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gin Thr Ile Asp Asn Tyr
65 70 75 80
Gin Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr
85 90 95
Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly Val Gln Ile Cys Leu
100 105 110
Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Ala Met Cys Cys
115 120 125
Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp Gin Asn
130 135 140
His Phe Arg Gly Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn
145 150 155 160
Asp His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser
165 170 175
Lys Met Tyr His Thr Lys Gly Gin Glu Gly Ser Val Cys Leu Arg Ser
180 185 190
Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys
195 200 205
Ile Cys Lys Pro Val Leu Lys Glu Gly Gin Val Cys Thr Lys His Arg
210 215 220
Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gin Arg Cys Tyr Cys Gly
225 230 235 240
Glu Gly Leu Ser Cys Arg Ile Gin Lys Asp His His Gin Ala Ser Asn
245 250 255
Ser Ser Arg Leu His Thr Cys Gin Arg His
260 265
<210> 9
<211> 798
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(798)
<400> 9
atg atg gct ctg ggc gca gcg gga gct acc cgg gtc ttt gtc gcg atg 48
Met Met Ala Leu Gly Ala Ala Gly Ala Thr Arg Val Phe Val Ala Met
1 5 10 15
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-13-
gta gcg gcg gct ctc ggc ggc cac cot ctg ctg gga gtg ago gcc acc 96
Val Ala Ala Ala Leu Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr
20 25 30
ttg aac tog gtt ctc aat too aac got atc aag aac ctg coo cca cog 144
Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro
35 40 45
ctg ggc ggc got gcg ggg cac cca ggc tot gca gtc ago gcc gcg cog 192
Leu Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro
50 55 60
gga atc ctg tac cog ggc ggg aat aag tac cag acc att gac aac tac 240
Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gin Thr Ile Asp Asn Tyr
65 70 75 80
cag cog tac cog tgc gca gag gac gag gag tgc ggc act gat gag tac 288
Gln Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu Tyr
85 90 95
tgc got agt ccc acc cgc gga ggg gac gca ggc gtg caa atc tgt ctc 336
Cys Ala Ser Pro Thr Arg Gly Gly Asp Ala Gly Val Gin Ile Cys Leu
100 105 110
gcc tgc agg aag cgc cga aaa cgc tgc atg cgt cac got atg tgc tgc 384
Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Arg His Ala Met Cys Cys
115 120 125
coo ggg aat tac tgc aaa aat gga ata tgt gtg tot tot gat caa aat 432
Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp Gin Asn
130 135 140
cat ttc cga gga gaa att gag gaa acc atc act gaa ago ttt ggt aat 480
His Phe Arg Gly Glu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn
145 150 155 160
gat cat ago acc ttg gat ggg tat too aga aga acc acc ttg tot tca 528
Asp His Ser Thr Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser
165 170 175
aaa atg tat cac acc aaa gga caa gaa ggt tot gtt tgt ctc cgg tca 576
Lys Met Tyr His Thr Lys Gly Gin Glu Gly Ser Val Cys Leu Arg Ser
180 185 190
tca gac tgt gcc tca gga ttg tgt tgt got aga cac ttc tgg too aag 624
Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys
195 200 205
atc tgt aaa cot gtc ctg aaa gaa ggt caa gtg tgt acc aag cat agg 672
Ile Cys Lys Pro Val Leu Lys Glu Gly Gin Val Cys Thr Lys His Arg
210 215 220
aga aaa ggc tot cat gga cta gaa ata ttc cag cgt tgt tac tgt gga 720
Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gin Arg Cys Tyr Cys Gly
225 230 235 240
gaa ggt ctg tot tgc cgg ata cag aaa gat cac cat caa gcc agt aat 768
Glu Gly Leu Ser Cys Arg Ile Gin Lys Asp His His Gin Ala Ser Asn
245 250 255
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-14-
tct tct agg ctt cac act tgt cag aga cac 798
Ser Ser Arg Leu His Thr Cys Gin Arg His
260 265
<210> 10
<211> 702
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(537)
<400> 10
gaa ttc ggc acg agg gtt ggg agg tat tgc cac agt ccc cac caa gga 48
Glu Phe Gly Thr Arg Val Gly Arg Tyr Cys His Ser Pro His Gin Gly
1 5 10 15
tca tog gcc tgc atg gtg tgt cgg aga aaa aag aag cgc tgc cac cga 96
Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys His Arg
20 25 30
gat ggc atg tgc tgc ccc agt acc cgc tgc aat aat ggc atc tgt atc 144
Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly Ile Cys Ile
35 40 45
cca gtt act gaa ago atc tta acc cot cac atc cog got ctg gat ggt 192
Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro Ala Leu Asp Gly
50 55 60
act cgg cac aga gat cga sac cac ggt cat tac tca aac cat gac ttg 240
Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His Asp Leu
65 70 75 80
gga tgg cag aat cta gga aga cca cac act aag atg tca cat ata aaa 288
Gly Trp Gin Asn Leu Gly Arg Pro His Thr Lys Met Ser His Ile Lys
85 90 95
ggg cat gaa gga gac ccc tgc cta cga tca tca gac tgc att gaa ggg 336
Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp Cys Ile Glu Gly
100 105 110
ttt tgc tgt got cgt cat ttc tgg acc aaa atc tgc aaa cca gtg ctc 384
Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys Lys Pro Val Leu
115 120 125
cat cag ggg gaa gtc tgt acc aaa caa cgc aag aag ggt tot cat ggg 432
His Gin Gly Glu Val Cys Thr Lys Gin Arg Lys Lys Gly Ser His Gly
130 135 140
ctg gaa att ttc cag cgt tgc gac tgt gcg aag ggc ctg tot tgc aaa 480
Leu Glu Ile Phe Gin Arg Cys Asp Cys Ala Lys Gly Leu Ser Cys Lys
145 150 155 160
gta tgg aaa gat gcc acc tac tcc tcc aaa gcc aga ctc cat gtg tgt 528
Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg Leu His Val Cys
165 170 175
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 15 -
cag aaa aft tgatcaccat tgaggaacat catcaattgc agactgtgaa 577
Gin Lys Ile
gttgtgtatt taatgcatta tagcatggtg gaaaataagg ttcagatgca gaagaatggc 637
taaaataaga aacgtgataa gaatatagat gatcacaaaa aaaaaaaaaa aaaagatgcg 697
gccgc 702
<210> 11
<211> 179
<212> PRT
<213> Homo sapiens
<400> 11
Glu Phe Gly Thr Arg Val Gly Arg Tyr Cys His Ser Pro His Gin Gly
1 5 10 15.
Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys His Arg
20 25 30
Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly Ile Cys Ile
35 40 45
Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro Ala Leu Asp Gly
50 55 60
Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His Asp Leu
65 70 75 80
Gly Trp Gin Asn Leu Gly Arg Pro His Thr Lys Met Ser His Ile Lys
85 90 95
Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp Cys Ile Glu Gly
100 105 110
Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys Lys Pro Val Leu
115 120 125
His Gin Gly Glu Val Cys Thr Lys Gin Arg Lys Lys Gly Ser His Gly
130 135 140
Leu Glu Ile Phe Gin Arg Cys Asp Cys Ala Lys Gly Leu Ser Cys Lys
145 150 155 160
Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg Leu His Val Cys
165 170 175
Gin Lys Ile
<210> 12
<211> 537
<212> DNA
<213> Homo sapiens
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-16-
<220>
<221> CDS
<222> (1)..(537)
<400> 12
gaa ttc ggc acg agg gtt ggg agg tat tgc cac agt ccc cac caa gga 48
Glu Phe Gly Thr Arg Val Gly Arg Tyr Cys His Ser Pro His Gin Gly
1 5 10 15
tca tog gcc tgc atg gtg tgt cgg aga aaa aag aag cgc tgc cac cga 96
Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys His Arg
20 25 30
gat ggc atg tgc tgc ccc agt acc cgc tgc aat aat ggc atc tgt atc 144
Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly Ile Cys Ile
35 40 45
cca gtt act gaa ago atc tta acc cot cac atc cog got ctg gat ggt 192
Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro Ala Leu Asp Gly
50 55 60
act cgg cac aga gat cga aac cac ggt cat tac tca aac cat gac ttg 240
Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His Asp Leu
65 70 75 80
gga tgg cag aat cta gga aga cca cac act aag atg tca cat ata aaa 288
Gly Trp Gin Asn Leu Gly Arg Pro His Thr Lys Met Ser His Ile Lys
85 90 95
ggg cat gaa gga gac ccc tgc cta cga tca tca gac tgc att gaa ggg 336
Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp Cys Ile Glu Gly
100 105 110
ttt tgc tgt got cgt cat ttc tgg acc aaa atc tgc aaa cca gtg ctc 384
Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys Lys Pro Val Leu
115 120 125
cat cag ggg gaa gtc tgt acc aaa caa cgc aag aag ggt tot cat ggg 432
His Gin Gly Glu Val Cys Thr Lys Gin Arg Lys Lys Gly Ser His Gly
130 135 140
ctg gaa att ttc cag cgt tgc gac tgt gcg aag ggc ctg tot tgc aaa 480
Leu Glu Ile Phe Gin Arg Cys Asp Cys Ala Lys Gly Leu Ser Cys Lys
145 150 155 160
gta tgg aaa gat gcc acc tac too too aaa gcc aga ctc cat gtg tgt 528
Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg Leu His Val Cys
165 170 175
cag aaa att 537
Gin Lys Ile
<210> 13
<211> 928
<212> DNA
<213> Homo sapiens
<220>
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-17-
<221> CDS
<222> (75)..(800)
<400> 13
ctcgaggcca aaattcggca cgaggccggg ctgtggtcta gcataaaggc ggagcccaga 60
agaaggggcg gggt atg gga gaa gcc tcc cca cot gcc ccc gca agg cgg 110
Met Gly Glu Ala Ser Pro Pro Ala Pro Ala Arg Arg
1 5 10
cat ctg ctg gtc ctg ctg ctg ctc ctc tct acc ctg gtg atc ccc too 158
His Leu Leu Val Leu Leu Leu Leu Leu Ser Thr Leu Val Ile Pro Ser
15 20 25
got gca got cot atc cat gat got gac gcc caa gag ago too ttg ggt 206
Ala Ala Ala Pro Ile His Asp Ala Asp Ala Gin Glu Ser Ser Leu Gly
30 35 40
ctc aca ggc ctc cag ago cta ctc caa ggc ttc ago cga ctt ttc ctg 254
Leu Thr Gly Leu Gin Ser Leu Leu Gin Gly Phe Ser Arg Leu Phe Leu
45 50 55 60
aaa ggt aac ctg ctt cgg ggc ata gac ago tta ttc tot gcc ccc atg 302
Lys Gly Asn Leu Leu Arg Gly Ile Asp Ser Leu Phe Ser Ala Pro Met
65 70 75
gac ttc cgg ggc ctc cot ggg aac tac cac aaa gag gag aac cag gag 350
Asp Phe Arg Gly Leu Pro Gly Asn Tyr His Lys Glu Glu Asn Gin Glu
80 85 90
cac cag ctg ggg aac aac acc ctc too ago cac ctc cag atc gac aag 398
His Gin Leu Gly Asn Asn Thr Leu Ser Ser His Leu Gin Ile Asp Lys
95 100 105
atg acc gac aac aag aca gga gag gtg ctg atc too gag aat gtg gtg 446
Met Thr Asp Asn Lys Thr Gly Glu Val Leu Ile Ser Glu Asn Val Val
110 115 120
gca too att caa cca gcg gag ggg ago ttc gag ggt gat ttg aag gta 494
Ala Ser Ile Gin Pro Ala Glu Gly Ser Phe Glu Gly Asp Leu Lys Val
125 130 135 140
ccc agg atg gag gag aag gag gcc ctg gta coo atc cag aag gcc acg 542
Pro Arg Met Glu Glu Lys Glu Ala Leu Val Pro Ile Gin Lys Ala Thr
145 150 155
gac ago ttc cac aca gaa ctc cat coo cgg gtg gcc ttc tgg atc att 590
Asp Ser Phe His Thr Glu Leu His Pro Arg Val Ala Phe Trp Ile Ile
160 165 170
aag ctg cca cgg cgg agg too cac cag gat gcc ctg gag ggc ggc cac 638
Lys Leu Pro Arg Arg Arg Ser His Gin Asp Ala Leu Glu Gly Gly His
175 180 185
tgg ctc ago gag aag cga cac cgc ctg cag gcc atc cgg gat gga ctc 686
Trp Leu Ser Glu Lys Arg His Arg Leu Gin Ala Ile Arg Asp Gly Leu
190 195 200
cgc aag ggg acc cac aag gac gtc cta gaa gag ggg acc gag ago too 734
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-18-
Arg Lys Gly Thr His Lys Asp Val Leu Glu Glu Gly Thr Glu Ser Ser
205 210 215 220
tcc cac tcc agg ctg tcc ccc cga aag acc cac tta ctg tac atc ctc 782
Ser His Ser Arg Leu Ser Pro Arg Lys Thr His Leu Leu Tyr Ile Leu
225 230 235
agg ccc tct cgg cag ctg taggggtggg gaccggggag cacctgcctg 830
Arg Pro Ser Arg Gin Leu
240
tagcccccat cagaccctgc cccaagcacc atatggaaat aaagttcttt cttacatcta 890
aaaaaaaaaa aaaaaaaaaa aaaaaaattg gcggccgc 928
<210> 14
<211> 242
<212> PRT
<213> Homo sapiens
<400> 14
Met Gly Glu Ala Ser Pro Pro Ala Pro Ala Arg Arg His Leu Leu Val
1 5 10 15
Leu Leu Leu Leu Leu Ser Thr Leu Val Ile Pro Ser Ala Ala Ala Pro
20 25 30
Ile His Asp Ala Asp Ala Gin Glu Ser Ser Leu Gly Leu Thr Gly Leu
35 40 45
Gin Ser Leu Leu Gin Gly Phe Ser Arg Leu Phe Leu Lys Gly Asn Leu
50 55 60
Leu Arg Gly Ile Asp Ser Leu Phe Ser Ala Pro Met Asp Phe Arg Gly
65 70 75 80
Leu Pro Gly Asn Tyr His Lys Glu Glu Asn Gin Glu His Gin Leu Gly
85 90 95
Asn Asn Thr Leu Ser Ser His Leu Gin Ile Asp Lys Met Thr Asp Asn
100 105 110
Lys Thr Gly Glu Val Leu Ile Ser Glu Asn Val Val Ala Ser Ile Gin
115 120 125
Pro Ala Glu Gly Ser Phe Glu Gly Asp Leu Lys Val Pro Arg Met Glu
130 135 140
Glu Lys Glu Ala Leu Val Pro Ile Gin Lys Ala Thr Asp Ser Phe His
145 150 155 160
Thr Glu Leu His Pro Arg Val Ala Phe Trp Ile Ile Lys Leu Pro Arg
165 170 175
Arg Arg Ser His Gin Asp Ala Leu Glu Gly Gly His Trp Leu Ser Glu
180 185 190
Lys Arg His Arg Leu Gin Ala Ile Arg Asp Gly Leu Arg Lys Gly Thr
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-19-
195 200 205
His Lys Asp Val Leu Glu Glu Gly Thr Glu Ser Ser Ser His Ser Arg
210 215 220
Leu Ser Pro Arg Lys Thr His Leu Leu Tyr Ile Leu Arg Pro Ser Arg
225 230 235 240
Gin Leu
<210> 15
<211> 726
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(726)
<400> 15
atg gga gaa gcc tcc cca cct gcc ccc gca agg cgg cat ctg ctg gtc 48
Met Gly Glu Ala Ser Pro Pro Ala Pro Ala Arg Arg His Leu Leu Val
1 5 10 15
ctg ctg ctg ctc ctc tct acc ctg gtg atc ccc tcc gct gca gct cct 96
Leu Leu Leu Leu Leu Ser Thr Leu Val Ile Pro Ser Ala Ala Ala Pro
20 25 30
atc cat gat gct gac gcc caa gag agc tcc ttg ggt ctc aca ggc ctc 144
Ile His Asp Ala Asp Ala Gin Glu Ser Ser Leu Gly Leu Thr Gly Leu
35 40 45
cag agc cta ctc caa ggc ttc agc cga ctt ttc ctg aaa ggt aac ctg 192
Gin Ser Leu Leu Gin Gly Phe Ser Arg Leu Phe Leu Lys Gly Asn Leu
50 55 60
ctt cgg ggc ata gac agc tta ttc tct gcc ccc atg gac ttc cgg ggc 240
Leu Arg Gly Ile Asp Ser Leu Phe Ser Ala Pro Met Asp Phe Arg Gly
65 70 75 80
ctc Oct ggg aac tac cac aaa gag gag aac cag gag cac cag ctg ggg 288
Leu Pro Gly Asn Tyr His Lys Glu Glu Asn Gin Glu His Gin Leu Gly
85 90 95
aac aac acc ctc tcc agc cac ctc cag atc gac aag atg acc gac aac 336
Asn Asn Thr Leu Ser Ser His Leu Gin Ile Asp Lys Met Thr Asp Asn
100 105 110
aag aca gga gag gtg ctg atc tcc gag aat gtg gtg gca tcc att caa 384
Lys Thr Gly Glu Val Leu Ile Ser Glu Asn Val Val Ala Ser Ile Gin
115 120 125
cca gcg gag ggg agc ttc gag ggt gat ttg aag gta ccc agg atg gag 432
Pro Ala Glu Gly Ser Phe Glu Gly Asp Leu Lys Val Pro Arg Met Glu
130 135 140
gag aag gag gcc ctg gta ccc atc cag aag gcc acg gac agc ttc cac 480
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-20-
Glu Lys Glu Ala Leu Val Pro Ile Gin Lys Ala Thr Asp Ser Phe His
145 150 155 160
aca gaa ctc cat ccc cgg gtg gcc ttc tgg atc att aag ctg cca cgg 528
Thr Glu Leu His Pro Arg Val Ala Phe Trp Ile Ile Lys Leu Pro Arg
165 170 175
cgg agg tcc cac cag gat gcc ctg gag ggc ggc cac tgg ctc agc gag 576
Arg Arg Ser His Gin Asp Ala Leu Glu Gly Gly His Trp Leu Ser Glu
180 185 190
aag cga cac cgc ctg cag gcc atc cgg gat gga ctc cgc aag ggg acc 624
Lys Arg His Arg Leu Gin Ala Ile Arg Asp Gly Leu Arg Lys Gly Thr
195 200 205
cac aag gac gtc cta gaa gag ggg acc gag agc tcc tcc cac tcc agg 672
His Lys Asp Val Leu Glu Glu Gly Thr Glu Ser Ser Ser His Ser Arg
210 215 220
ctg tcc ccc cga aag acc cac tta ctg tac atc ctc agg ccc tct cgg 720
Leu Ser Pro Arg Lys Thr His Leu Leu Tyr Ile Leu Arg Pro Ser Arg
225 230 235 240
cag ctg 726
Gin Leu
<210> 16
<211> 2380
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (109)..(1155)
<400> 26
gtcgacccac gcgtccgctg tggcagccca gctaccggtc gtgaccagat ccagcttgca 60
gctcagcttt gttcattcga attgggcggc ggccagcgcg gaacaaac atg cag cgg 117
Met Gin Arg
1
ctc ggg ggt att ttg ctg tgt aca ctg ctg gcg gcg gcg gtc ccc act 165
Leu Gly Gly Ile Leu Leu Cys Thr Leu Leu Ala Ala Ala Val Pro Thr
10 15
gct cct gct cct tcc ccg acg gtc act tgg act ccg gcg gag ccg ggc 213
Ala Pro Ala Pro Ser Pro Thr Val Thr Trp Thr Pro Ala Glu Pro Gly
20 25 30 35
cca gct ctc aac tac cct cag gag gaa gct acg ctc aat gag atg ttt 261
Pro Ala Leu Asn Tyr Pro Gin Glu Glu Ala Thr Leu Asn Glu Met Phe
40 45 50
cga gag gtg gag gag ctg atg gaa gac act cag cac aaa ctg cgc agt 309
Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gin His Lys Leu Arg Ser
55 60 65
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-21-
gcc gtg gag gag atg gag gcg gaa gaa gca gct gct aaa acg tcc tct 357
Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys Thr Ser Ser
70 75 80
gag gtg aac ctg gca agc tta cct ccc aac tat cac aat gag acc agc 405
Glu Val Asn Leu Ala Ser Leu Pro Pro Asn Tyr His Asn Glu Thr Ser
85 90 95
acg gag acc agg gtg gga aat aac aca gtc cat gtg cac cag gaa gtt 453
Thr Glu Thr Arg Val Gly Asn Asn Thr Val His Val His Gln Glu Val
100 105 110 115
cac aag ata acc aac aac cag agt gga cag gtg gtc ttt tct gag aca 501
His Lys Ile Thr Asn Asn Gln Ser Gly Gln Val Val Phe Ser Glu Thr
120 125 130
gtc att aca tct gta ggg gat gaa gaa ggc aag agg agc cat gaa tgt 549
Val Ile Thr Ser Val Gly Asp Glu Glu Gly Lys Arg Ser His Glu Cys
135 140 145
atc att gat gaa gac tgt ggg ccc acc agg tac tgc cag ttc tcc agc 597
Ile Ile Asp Glu Asp Cys Gly Pro Thr Arg Tyr Cys Gln Phe Ser Ser
150 155 160
ttc aag tac acc tgc cag cca tgc cgg gac cag cag atg cta tgc acc 645
Phe Lys Tyr Thr Cys Gln Pro Cys Arg Asp Gln Gln Met Leu Cys Thr
165 170 175
cga gac agt gag tgc tgt gga gac cag ctg tgt gcc tgg ggt cac tgc 693
Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Ala Trp Gly His Cys
180 185 190 195
acc caa aag gcc acc aaa ggt ggc aat ggg acc atc tgt gac aac cag 741
Thr Gln Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile Cys Asp Asn Gln
200 205 210
agg gat tgc cag cct ggc ctg tgt tgt gcc ttc caa aga ggc ctg ctg 789
Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg Gly Leu Leu
215 220 225
ttc ccc gtg tgc aca ccc ctg ccc gtg gag gga gag ctc tgc cat gac 837
Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu Cys His Asp
230 235 240
ccc acc agc cag ctg ctg gat ctc atc acc tgg gaa ctg gag cct gaa 885
Pro Thr Ser Gln Leu Leu Asp Leu Ile Thr Trp Glu Leu Glu Pro Glu
245 250 255
gga gct ttg gac cga tgc ccc tgc gcc agt ggc ctc cta tgc cag cca 933
Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu Cys Gln Pro
260 265 270 275
cac agc cac agt ctg gtg tac atg tgc aag cca gcc ttc gtg ggc agc 981
His Ser His Ser Leu Val Tyr Met Cys Lys Pro Ala Phe Val Gly Ser
280 285 290
cat gac cac agt gag gag agc cag ctg ccc agg gag gcc ccg gat gag 1029
His Asp His Ser Glu Glu Ser Gln Leu Pro Arg Glu Ala Pro Asp Glu
295 300 305
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-22-
tac gaa gat gtt ggc ttc ata ggg gaa gtg cgc cag gag ctg gaa gac 1077
Tyr Glu Asp Val Gly Phe Ile Gly Glu Val Arg Gin Glu Leu Glu Asp
310 315 320
ctg gag cgg agc cta gcc cag gag atg gca ttt gag ggg cct gcc cct 1125
Leu Glu Arg Ser Leu Ala Gin Glu Net Ala Phe Glu Gly Pro Ala Pro
325 330 335
gtg gag tca cta ggc gga gag gag gag att taggcccaga cccagctgag 1175
Val Glu Ser Leu Gly Gly Glu Glu Glu Ile
340 345
tcactggtag atgtgcaata gaaatggcta atttattttc ccaggagtgt ccccaagtgt 1235
ggaatggccg cagctccttc ccagtagctt ttcctctggc ttgacaaggt acagtgcagt 1295
acatttcttc cagccgccct gcttctctga cttgggaaag acaggcatgg cgggtaaggg 1355
cagcggtgag tcgtccctcg ctgttgctag aaacgctgtc ttgttcttca tggatggaag 1415
atttgtttga agggagagga tgggaagggg tgaagtctgc tcatgatgga tttgggggat 1475
acagggagga ggatgcctgc cttgcagacg tggacttggc aaaatgtaac ctttgctttt 1535
gtcttgcgcc gctcccatgg gctgaggcag tggctacaca agagctatgc tgctctgtgg 1595
cctcccacat attcatccct gtgtttcagc tcctacctca ctgtcagcac agcccttcat 1655
agccacgccc cctcttgctc accacagcct aggaggggac cagaggggac ttctctcaga 1715
gccccatgct ctctctctca accccatacc agcctctgtg ccagcgacag tccttccaaa 1775
tggagggagt gaaatccttt ggtttaatta ttttctcctt caaggcacgc ctgccactaa 1835
ggtcaggctg acttgcatgt ccctctaacg ttcgtagcag tgtggtggac actgtcttcc 1895
accgactgct tcaatacctc tgaaagccag tgctcggagt gcagttcgtg taaattaatt 1955
tgcaggaagt atacttggct aattgtaggg ctaggattgt gaatgaaatt tgcaaagtcg 2015
cttagcaaca atggaaagcc tttctcagtc acaccgagaa gtcacaacca agccaggttg 2075
tgtagagtac agctgtgaca tacagacaga agaaggctgg gctggatgtc aggcctcaga 2135
tgacggtttc aggtgccagg aactattacc attctgtatc tatccagagt tattaaaatt 2195
gaaagttgca cacatttgta taagcatgcc tttctcctga gttttaaatt atatgtatac 2255
acaaacatgt ggccctcaaa gatcatgcac aaaccactac tctttgctaa ttcttggact 2315
tttctctttg attttcaata aatacaaatc cccttcatgc aaaaaaaaaa aaaaagggcg 2375
gccgc 2380
<210> 17
<211> 349
<212> PRT
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 23 -
<213> Homo sapiens
<400> 17
Met Gln Arg Leu Gly Gly Ile Leu Leu Cys Thr Leu Leu Ala Ala Ala
1 5 10 15
Val Pro Thr Ala Pro Ala Pro Ser Pro Thr Val Thr Trp Thr Pro Ala
20 25 30
Glu Pro Gly Pro Ala Leu Asn Tyr Pro Gln Glu Glu Ala Thr Leu Asn
35 40 45
Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys
50 55 60
Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys
65 70 75 80
Thr Ser Ser Glu Val Asn Leu Ala Ser Leu Pro Pro Asn Tyr His Asn
85 90 95
Glu Thr Ser Thr Glu Thr Arg Val Gly Asn Asn Thr Val His Val His
100 105 110
Gln Glu Val His Lys Ile Thr Asn Asn Gln Ser Gly Gln Val Val Phe
115 120 125
Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Lys Arg Ser
130 135 140
His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Thr Arg Tyr Cys Gln
145 150 155 160
Phe Ser Ser Phe Lys Tyr Thr Cys Gln Pro Cys Arg Asp Gln Gln Met
165 170 175
Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Ala Trp
180 185 190
Gly His Cys Thr Gln Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile Cys
195 200 205
Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln Arg
210 215 220
Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
225 230 235 240
Cys His Asp Pro Thr Ser Gln Leu Leu Asp Leu Ile Thr Trp Glu Leu
245 250 255
Glu Pro Glu Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu
260 265 270
Cys Gln Pro His Ser His Ser Leu Val Tyr Met Cys Lys Pro Ala Phe
275 280 285
Val Gly Ser His Asp His Ser Glu Glu Ser Gln Leu Pro Arg Glu Ala
290 295 300
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-24-
Pro Asp Glu Tyr Glu Asp Val Gly Phe Ile Gly Glu Val Arg Gin Glu
305 310 315 320
Leu Glu Asp Leu Glu Arg Ser Leu Ala Gin Glu Met Ala Phe Glu Gly
325 330 335
Pro Ala Pro Val Glu Ser Leu Gly Gly Glu Glu Glu Ile
340 345
<210> 18
<211> 1047
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(1047)
<400> 18
atg cag cgg ctc ggg ggt att ttg ctg tgt aca ctg ctg gcg gcg gcg 48
Met Gin Arg Leu Gly Gly Ile Leu Leu Cys Thr Leu Leu Ala Ala Ala
1 5 10 15
gtc ccc act gct cct gct cct tcc ccg acg gtc act tgg act ccg gcg 96
Val Pro Thr Ala Pro Ala Pro Ser Pro Thr Val Thr Trp Thr Pro Ala
20 25 30
gag ccg ggc cca gct ctc aac tac cct cag gag gaa gct acg ctc aat 144
Glu Pro Gly Pro Ala Leu Asn Tyr Pro Gin Glu Glu Ala Thr Leu Asn
35 40 45
gag atg ttt cga gag gtg gag gag ctg atg gaa gac act cag cac aaa 192
Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys
50 55 60
ctg cgc agt gcc gtg gag gag atg gag gcg gaa gaa gca gct gct aaa 240
Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala Ala Lys
65 70 75 80
acg tcc tct gag gtg aac ctg gca agc tta cct ccc aac tat cac aat 288
Thr Ser Ser Glu Val Asn Leu Ala Ser Leu Pro Pro Asn Tyr His Asn
85 90 95
gag acc agc acg gag acc agg gtg gga aat aac aca gtc cat gtg cac 336
Glu Thr Ser Thr Glu Thr Arg Val Gly Asn Asn Thr Val His Val His
100 105 110
cag gaa gtt cac aag ata acc aac aac cag agt gga cag gtg gtc ttt 384
Gin Glu Val His Lys Ile Thr Asn Asn Gin Ser Gly Gin Val Val Phe
115 120 125
tct gag aca gtc att aca tct gta ggg gat gaa gaa ggc aag agg agc 432
Ser Glu Thr Val Ile Thr Ser Val Gly Asp Glu Glu Gly Lys Arg Ser
130 135 140
cat gaa tgt atc att gat gaa gac tgt ggg ccc acc agg tac tgc cag 480
His Glu Cys Ile Ile Asp Glu Asp Cys Gly Pro Thr Arg Tyr Cys Gin
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-25 -
145 150 155 160
ttc tcc agc ttc aag tac acc tgc cag cca tgc cgg gac cag cag atg 528
Phe Ser Ser Phe Lys Tyr Thr Cys Gin Pro Cys Arg Asp Gin Gin Met
165 170 175
cta tgc acc cga gac agt gag tgc tgt gga gac cag ctg tgt gcc tgg 576
Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gin Leu Cys Ala Trp
180 185 190
ggt cac tgc acc caa aag gcc acc aaa ggt ggc aat ggg acc atc tgt 624
Gly His Cys Thr Gin Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile Cys
195 200 205
gac aac cag agg gat tgc cag cot ggc ctg tgt tgt gcc ttc caa aga 672
Asp Asn Gin Arg Asp Cys Gin Pro Gly Leu Cys Cys Ala Phe Gin Arg
210 215 220
ggc ctg ctg ttc ccc gtg tgc aca ccc ctg ccc gtg gag gga gag ctc 720
Gly Leu Leu Phe Pro Val Cys Thr Pro Leu Pro Val Glu Gly Glu Leu
225 230 235 240
tgc cat gac ccc acc ago cag ctg ctg gat ctc atc acc tgg gaa ctg 768
Cys His Asp Pro Thr Ser Gin Leu Leu Asp Leu Ile Thr Trp Glu Leu
245 250 255
gag cct gaa gga got ttg gac cga tgc ccc tgc gcc agt ggc ctc cta 816
Glu Pro Glu Gly Ala Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu
260 265 270
tgc cag cca cac ago cac agt ctg gtg tac atg tgc aag cca gcc ttc 864
Cys Gin Pro His Ser His Ser Leu Val Tyr Met Cys Lys Pro Ala Phe
275 280 285
gtg ggc ago cat gac cac agt gag gag ago cag ctg ccc agg gag gcc 912
Val Gly Ser His Asp His Ser Glu Glu Ser Gin Leu Pro Arg Glu Ala
290 295 300
ccg gat gag tac gaa gat gtt ggc ttc ata ggg gaa gtg cgc cag gag 960
Pro Asp Glu Tyr Glu Asp Val Gly Phe Ile Gly Glu Val Arg Gin Glu
305 310 315 320
ctg gaa gac ctg gag cgg ago cta gcc cag gag atg gca ttt gag ggg 1008
Leu Glu Asp Leu Glu Arg Ser Leu Ala Gin Glu Met Ala Phe Glu Gly
325 330 335
=
cot gcc cot gtg gag tca cta ggc gga gag gag gag att 1047
Pro Ala Pro Val Glu Ser Leu Gly Gly Glu Glu Glu Ile
340 345
<210> 19
<211> 8
<212> PRT
<213> synthtic construct
<400> 19
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
CA 02366062 2001-09-13
WO 00/52047 PCT/US00/05452
-26-
<210> 20
<211> 3696
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (712)..(1500)
<400> 20
gtcgacccac gcgtccggcg ggagcccgcg gcgagcgtag cgcaagtccg ctccctaggc 60
atcgctgcgc tggcagcgat tcgctgtctc ttgtgagtca ggggacaacg cttcggggca 120
actgtgagtg cgcgtgtggg ggacctcgat tctcttcaga tctcgaggat tcggtccggg 180
gacgtctcct gatcccctac taaagcgcct gctaactttg aaaaggagca ctgtgtcctg 240
caaagtttga cacataaagg ataggaaaag agaggagaga aaagcaactg ag-ttgaagga 300
gaaggagctg atgcgggcct cctgatcaat taagaggaga gttaaaccgc cgagatcccg 360
gcgggaccaa ggaggtgcgg ggcaagaagg aacggaagcg gtgcgatcca cagggctggg 420
ttttcttgca ccttgggtca cgcctccttg gcgagaaagc gcctcgcatt tgattgcttc 480
cagttattgc agaacttcct gtcctggtgg agaagcgggt ctcgcttggg ttccgctaat 540
ttctgtcctg aggcgtgaga ctgagttcat agggtcctgg gtccccgaac caggaagggt 600
tgagggaaca caatctgcaa gcccccgcga cccaagtgag gggccccgtg ttggggtcct 660
ccctccottt gcattcccac ccctccgggc tttgcgtctt cctggggacc c cot cgc 717
Pro Arg
1
cgg gag atg gcc gcg ttg atg cgg ago aag gat tog too tgc tgc ctg 765
Arg Glu Met Ala Ala Leu Met Arg Ser Lys Asp Ser Ser Cys Cys Leu
10 15
ctc cta ctg gcc gcg gtg ctg atg gtg gag agc tca cag atc ggc agt 813
Leu Leu Leu Ala Ala Val Leu Met Val Glu Ser Ser Gln Ile Gly Ser
20 25 30
tog cgg gcc aaa ctc aac too atc aag too tct ctg ggc ggg gag acg 861
Ser Arg Ala Lys Leu Asn Ser Ile Lys Ser Ser Leu Gly Gly Glu Thr
35 40 45 50
cct ggt cag goo goo aat cga tot gcg ggc atg tac caa gga ctg gca 909
Pro Gly Gln Ala Ala Asn Arg Ser Ala Gly Met Tyr Gln Gly Leu Ala
55 60 65
ttc ggc ggc agt aag aag ggc aaa aac ctg ggg cag goo tac cct tgt 957
Phe Gly Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Ala Tyr Pro Cys
70 75 80
ago agt gat aag gag tgt gaa gtt ggg agg tat tgc cac agt coo cac 1005
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-27-
Ser Ser Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser Pro His
85 90 95
caa gga tca tog gcc tgc atg gtg tgt cgg aga aaa aag aag cgc tgc 1053
Gin Gly Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys
100 105 110
cac cga gat ggc atg tgc tgc ccc agt acc cgc tgc aat aat ggc atc 1101
His Arg Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly Ile
115 120 125 130
tgt atc cca gtt act gaa ago atc tta acc cot cac atc cog gct ctg 1149
Cys Ile Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro Ala Leu
135 140 145
gat ggt act cgg cac aga gat cga aac cac ggt cat tac tca aac cat 1197
Asp Gly Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His
150 155 160
gac ttg gga tgg cag aat cta gga aga cca cac act aag atg tca cat 1245
Asp Leu Gly Trp Gin Asn Leu Gly Arg Pro His Thr Lys Met Ser His
165 170 175
ata aaa ggg cat gaa gga gac ccc tgc cta cga tca tca gac tgc att 1293
Ile Lys Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp Cys Ile
180 185 190
gaa ggg ttt tgc tgt gct cgt cat ttc tgg acc aaa atc tgc aaa cca 1341
Glu Gly Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys Lys Pro
195 200 205 210
gtg ctc cat cag ggg gaa gtc tgt acc aaa caa cgc aag aag ggt tot 1389
Val Leu His Gin Gly Glu Val Cys Thr Lys Gin Arg Lys Lys Gly Ser
215 220 225
cat ggg ctg gaa att ttc cag cgt tgc gac tgt gcg aag ggc ctg tot 1437
His Gly Leu Glu Ile Phe Gin Arg Cys Asp Cys Ala Lys Gly Leu Ser
230 235 240
tgc aaa gta tgg aaa gat gcc acc tac too too aaa gcc aga ctc cat 1485
Cys Lys Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg Leu His
245 250 255
gtg tgt cag aaa att tgatcaccat tgaggaacat catcaattgc agactgtgaa 1540
Val Cys Gin Lys Ile
260
gttgtgtatt taatgcatta tagcatggtg gaaaataagg ttcagatgca gaagaatggc 1600
taaaataaga aacgtgataa gaatatagat gatcacaaaa agggagaaag aaaacatgaa 1660
ctgaatagat tagaatgggt gacaaatgca gtgcagccag tgtttccatt atgcaacttg 1720
tctatgtaaa taatgtacac atttgtggaa aatgctatta ttaagagaac aagcacacag 1780
tggaaattac tgatgagtag catgtgactt tccaagagtt taggttgtgc tggaggagag 1840
gtttccttca gattgctgat tgcttataca aataacctac atgccagatt tctattcaac 1900
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-28-
gttagagttt aacaaaatac tcctagaata acttgttata caataggttc taaaaataaa 1960
attgctaaac aagaaatgaa aacatggagc attgttaatt tacaacagaa aattaccttt 2020
tgatttgtaa cactacttct gctgttcaat caagagtctt ggtagataag aaaaaaatca 2080
gtcaatattt ccaaataatt gcaaaataat ggccagttgt ttaggaaggc ctttaggaag 2140
acaaataaat aacaaacaaa cagccacaaa tacttttttt tcaaaatttt agttttacct 2200
gtaattaata agaactgata caagacaaaa acagttcctt cagattctac ggaatgacag 2260
tatatctctc tttatcctat gtgattcctg ctctgaatgc attatatttt ccaaagtata 2320
cccataaatt gtgactagta aaatacttac acagagcaga attttcacag atggcaaaaa 2380
aatttaaaga tgtccaatat atgtgggaaa agagctaaca gagagatcat tatttcttaa 2440
agattggcca taacctgtat tttgatagaa ttagattggt aaatacatgt attcatacat 2500
actctgtggt aatagagact tgagctggat ctgtactgca ctggagtaag caagaaaatt 2560
gggaaaactt tttcgtttgt tcaggttttg gcaacacata gatcatatgt ctgaggcaca 2620
agttggctgt tcatctttga aaccagggga tgcacagtct aaatgaatat ctgcatggga 2680
tttgtatcat aatatttact atgcagatga attcagtgtg aggtcctgtg tccgtactat 2740
cctcaaatta tttattttat agtgctgaga tcctcaaata atctcaattt caggaggttt 2800
cacaaaatgg actcctgaag tagacagagt agtgaggttt cattgccctc tataagcttc 2860
tgactagcca atggcatcat ccaattttct tcccaaacct ctgcagcatc tgctttattg 2920
ccaaagggct agtttcggtt ttctgcagcc attgcggtta aaaaatataa gtaggataac 2980
ttgtaaaacc tgcatattgc taatctatag acaccacagt ttctaaattc tttgaaacca 3040
ctttactact ttttttaaac ttaactcagt tctaaatact ttgtctggag cacaaaacaa 3100
taaaaggtta tcttatagtt gtgactttaa acttttgtag accacaattc actttttagt 3160
tttcttttac ttaaatccca tctgcagtct caaatttaag ttctcccagt agagattgag 3220
tttgagcctg tatatctatt aaaaatttca acttcccaca tatatttact aagatgatta 3280
agacttacat tttctgcaca ggtctgcaaa aacaaaaatt ataaactagt ccatccaaga 3340
accaaagttt gtataaacag gttgctataa gcttggtgaa atgaaaatgg aacatttcaa 3400
tcaaacattt cctatataac aattattata tttacaattt ggtttctgca atatttttct 3460
tatgtccacc cttttaaaaa ttattatttg aagtaattta tttacaggaa atgttaatga 3520
gatgtatttt cttatagaga tatttcttac agaaagcttt gtagcagaat atatttgcag 3580
ctattgactt tgtaatttag gaaaaatgta taataagata aaatctatta aatttttctc 3640
ctctaaaaac tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagggc ggccgc 3696
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-29-
<210> 21
<211> 263
<212> PRT
<213> Homo sapiens
<400> 21
Pro Arg Arg Glu Met Ala Ala Leu Met Arg Ser Lys Asp Ser Ser Cys
1 5 10 15
Cys Leu Leu Leu Leu Ala Ala Val Leu Met Val Glu Ser Ser Gln Ile
20 25 30
Gly Ser Ser Arg Ala Lys Leu Asn Ser Ile Lys Ser Ser Leu Gly Gly
35 40 45
Glu Thr Pro Gly Gln Ala Ala Asn Arg Ser Ala Gly Met Tyr Gln Gly
50 55 60
Leu Ala Phe Gly Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Ala Tyr
65 70 75 80
Pro Cys Ser Ser Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser
85 90 95
Pro His Gln Gly Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys
100 105 110
Arg Cys His Arg Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn
115 120 125
Gly Ile Cys Ile Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro
130 135 140
Ala Leu Asp Gly Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser
145 150 155 160
Asn His Asp Leu Gly Trp Gln Asn Leu Gly Arg Pro His Thr Lys Met
165 170 175
Ser His Ile Lys Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp
180 185 190
Cys Ile Glu Gly Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys
195 200 205
Lys Pro Val Leu His Gln Gly Glu Val Cys Thr Lys Gln Arg Lys Lys
210 215 220
Gly Ser His Gly Leu Glu Ile Phe Gln Arg Cys Asp Cys Ala Lys Gly
225 230 235 240
Leu Ser Cys Lys Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg
245 250 255
Leu His Val Cys Gln Lys Ile
260
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 30 -
<210> 22
<211> 789
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(789)
<400> 22
cct cgc cgg gag atg gcc gcg ttg atg cgg agc aag gat tcg tcc tgc 48
Pro Arg Arg Glu Met Ala Ala Leu Met Arg Ser Lys Asp Ser Ser Cys
1 5 10 15
tgc ctg ctc cta ctg gcc gcg gtg ctg atg gtg gag agc tca cag atc 96
Cys Leu Leu Leu Leu Ala Ala Val Leu Met Val Glu Ser Ser Gin Ile
20 25 30
ggc agt tcg cgg gcc aaa ctc aac tcc atc aag tcc tct ctg ggc ggg 144
Gly Ser Ser Arg Ala Lys Leu Asn Ser Ile Lys Ser Ser Leu Gly Gly
35 40 45
gag acg cot ggt cag gcc gcc aat cga tct gcg ggc atg tac caa gga 192
Glu Thr Pro Gly Gin Ala Ala Asn Arg Ser Ala Gly Met Tyr Gin Gly
50 55 60
ctg gca ttc ggc ggc agt aag aag ggc aaa aac ctg ggg cag gcc tac 240
Leu Ala Phe Gly Gly Ser Lys Lys Gly Lys Asn Leu Gly Gin Ala Tyr
65 70 75 80
cot tgt agc agt gat aag gag tgt gaa gtt ggg agg tat tgc cac agt 288
Pro Cys Ser Ser Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser
85 90 95
ccc cac caa gga tca tcg gcc tgc atg gtg tgt cgg aga aaa aag aag 336
Pro His Gin Gly Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys
100 105 110
cgc tgc cac cga gat ggc atg tgc tgc ccc agt acc cgc tgc aat aat 384
Arg Cys His Arg Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn
115 120 125
ggc atc tgt atc cca gtt act gaa agc atc tta acc cot cac atc ccg 432
Gly Ile Cys Ile Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro
130 135 140
got ctg gat ggt act cgg cac aga gat cga aac cac ggt cat tac tca 480
Ala Leu Asp Gly Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser
145 150 155 160
aac cat gac ttg gga tgg cag aat cta gga aga cca cac act aag atg 528
Asn His Asp Leu Gly Trp Gin Asn Leu Gly Arg Pro His Thr Lys Met
165 170 175
tca cat ata aaa ggg cat gaa gga gac ccc tgc cta cga tca tca gac 576
Ser His Ile Lys Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp
180 185 190
CA 02366062 2001-09-13
MA) 00/52047 PCT/US00/05452
-31 -
tgc att gaa ggg ttt tgc tgt gct cgt cat ttc tgg acc aaa atc tgc 624
Cys Ile Glu Gly Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys
195 200 205
aaa cca gtg ctc cat cag ggg gaa gtc tgt acc aaa caa cgc aag aag 672
Lys Pro Val Leu His Gin Gly Glu Val Cys Thr Lys Gin Arg Lys Lys
210 215 220
ggt tct cat ggg ctg gaa att ttc cag cgt tgc gac tgt gcg aag ggc 720
Gly Ser His Gly Leu Glu Ile Phe Gin Arg Cys Asp Cys Ala Lys Gly
225 230 235 240
ctg tct tgc aaa gta tgg aaa gat gcc acc tac tcc tcc aaa gcc aga 768
Leu Ser Cys Lys Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg
245 250 255
ctc cat gtg tgt cag aaa att 789
Leu His Val Cys Gin Lys Ile
260
<210> 23
<211> 54
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: consensus
sequence
<220>
<223> Xaa's at positions
2,3,5,6,8-12,14-26,28,29,31-36,38-42,45-48, and
50-53 may be any amino acid
<220>
<223> Xaa's at postions 22-26 may be absent
<400> 23
Cys Xaa Xaa Asp Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Cys Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa Xaa
35 40 45
Cys Xaa Xaa Xaa Xaa Cys
<210> 24
<211> 123
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: consensus
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 32 -
sequence
<220>
<223> Xaa's at positions
2,5,8,9,11,14-23,25,27-30,32,33,35-53,60,62,63,65,
68,70,71,73-96,98,100,101,104, and 106-122 may be
any amino acid
<220>
<223> Xaa's at positions 22,23,51-58,89-96 and 116-122
may be absent
<400> 24
Cys Xaa Xaa Xaa Xaa Asp Cys Xaa Xaa Gly Xaa Cys Cys Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Gly Xaa
20 25 30
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys Xaa Xaa Pro
50 55 60
Xaa Xaa Xaa Xaa Gly Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Cys Xaa Cys Xaa Xaa Gly Leu Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa
100 105 110
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
115 120
<210> 25
<211> 87
<212> PRT
<213> Homo sapiens
<400> 25
Ile Asn Leu Glu Asn Gly Glu Leu Cys Met Asn Ser Ala Gin Cys Lys
1 5 10 15
Ser Asn Cys Cys Gin His Ser Ser Ala Leu Gly Leu Ala Arg Cys Thr
20 25 30
Ser Met Ala Ser Glu Asn Ser Glu Cys Ser Val Lys Thr Leu Tyr Gly
35 40 45
Ile Tyr Tyr Lys Cys Pro Cys Glu Arg Gly Leu Thr Cys Glu Gly Asp
50 55 60
Lys Thr Ile Val Gly Ser Ile Thr Asn Thr Asn Phe Gly Ile Cys His
65 70 75 80
CA 02366062 2001-09-13
VA) 00/52047 PCT/US00/05452
-33-
Asp Ala Gly Arg Ser Lys Gin
<210> 26
<211> 835
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (57)..(746)
<400> 25
gaattcggca cgaggcagaa ggcgcgaatg aaggcaaagc ctcccaccca cctgca atg 59
Met
1
tgt cga ctg agg gtc ttg ctg ctg ctg ctc ccc ttg gcc ttc gtg tcc 107
Cys Arg Leu Arg Val Leu Leu Leu Leu Leu Pro Leu Ala Phe Val Ser
5 10 15
tcc tot gct ctc ccc atc cat gat gtc gac tct cag cag aac acc tcc 155
Ser Ser Ala Leu Pro Ile His Asp Val Asp Ser Gin Gin Asn Thr Ser
20 25 30
ggg ttc ctg ggc ctt cag agg ctt ctc caa ago ttt agt cga ctg ttc 203
Gly Phe Leu Gly Leu Gin Arg Leu Leu Gin Ser Phe Ser Arg Leu Phe
35 40 45
cta aaa aat gac ctg cta cga gac ctg gac aac ttc ttc tcc tcc ccc 251
Leu Lys Asn Asp Leu Leu Arg Asp Leu Asp Asn Phe Phe Ser Ser Pro
50 55 60 65
atg gac ttc cga gac ctt cot agg aac ttc cat cag gaa gag aac cag 299
Met Asp Phe Arg Asp Leu Pro Arg Asn Phe His Gin Glu Glu Asn Gin
70 75 80
gag cac aga atg ggc aac cat acc ctc tcc ago cac cta cag ata gac 347
Glu His Arg Met Gly Asn His Thr Leu Ser Ser His Leu Gin Ile Asp
85 90 95
aag gtg act gac aac cag aca ggg gag gtg cac atc tog gag aaa gtc 395
Lys Val Thr Asp Asn Gin Thr Gly Glu Val His Ile Ser Glu Lys Val
100 105 110
gag gcc tcc att gag cca gaa cgg aac cog gaa ggg gac tgg aag gtt 443
Glu Ala Ser Ile Glu Pro Glu Arg Asn Pro Glu Gly Asp Trp Lys Val
115 120 125
ccc aaa gta gaa gca aaa gag ccc cog gtg cct gtg cag aag gtc acc 491
Pro Lys Val Glu Ala Lys Glu Pro Pro Val Pro Val Gin Lys Val Thr
130 135 140 145
gac ago ttg cac cca gag ccc cgg cag gtg got ttc tgg atc atg aag 539
Asp Ser Leu His Pro Glu Pro Arg Gin Val Ala Phe Trp Ile Met Lys
150 155 160
atg cca agg cgg agg acc cag ccc gat gtc cag gat gga ggc cgc tgg 587
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
- 34 -
Met Pro Arg Arg Arg Thr Gin Pro Asp Val Gin Asp Gly Gly Arg Trp
165 170 175
ctc ata gaa aag cga cat cgc atg cag gcc atc cgg gat ggg ctc cgt 635
Leu Ile Glu Lys Arg His Arg Met Gin Ala Ile Arg Asp Gly Leu Arg
180 185 190
gga ggc gcc cgt gag gac agc ctg gag gat ggg gtc cat atc ccc caa 683
Gly Gly Ala Arg Glu Asp Ser Leu Glu Asp Gly Val His Ile Pro Gin
195 200 205
cac gcc aag ctg cct gtc aga aag aca cac ttt ctc tac atc ctc agg 731
His Ala Lys Leu Pro Val Arg Lys Thr His Phe Leu Tyr Ile Leu Arg
210 215 220 225
cca tcc caa cag ctg taagtgggga ccagatgtcc cacaccctac cccaacacca 786
Pro Ser Gin Gin Leu
230
tatggaaata aaggttttct tacatctaaa aaaaaaaaaa aaaaaaaaa 835
<210> 27
<211> 230
<212> PRT
<213> Mus musculus
<400> 27
Met Cys Arg Leu Arg Val Leu Leu Leu Leu Leu Pro Leu Ala Phe Val
1 5 10 15
Ser Ser Ser Ala Leu Pro Ile His Asp Val Asp Ser Gin Gin Asn Thr
20 25 30
Ser Gly Phe Leu Gly Leu Gin Arg Leu Leu Gin Ser Phe Ser Arg Leu
35 40 45
Phe Leu Lys Asn Asp Leu Leu Arg Asp Lou Asp Asn Phe Phe Ser Ser
50 55 60
Pro Met Asp Phe Arg Asp Leu Pro Arg Asn Phe His Gin Glu Glu Asn
65 70 75 80
Gin Glu His Arg Met Gly Asn His Thr Leu Ser Ser His Leu Gin Ile
85 90 95
Asp Lys Val Thr Asp Asn Gin Thr Gly Glu Val His Ile Ser Glu Lys
100 105 110
Val Glu Ala Ser Ile Glu Pro Glu Arg Asn Pro Glu Gly Asp Trp Lys
115 120 125
Val Pro Lys Val Glu Ala Lys Glu Pro Pro Val Pro Val Gin Lys Val
130 135 140
Thr Asp Ser Leu His Pro Glu Pro Arg Gin Val Ala Phe Trp Ile Met
145 150 155 160
Lys Met Pro Arg Arg Arg Thr Gin Pro Asp Val Gin Asp Gly Gly Arg
CA 02366062 2001-09-13
VA) 00/52047
PCT/US00/05452
- 35 -
165 170 175
Trp Leu Ile Glu Lys Arg His Arg Met Gin Ala Ile Arg Asp Gly Leu
180 185 190
Arg Gly Gly Ala Arg Glu Asp Ser Leu Glu Asp Gly Val His Ile Pro
195 200 205
Gln His Ala Lys Leu Pro Val Arg Lys Thr His Phe Leu Tyr Ile Leu
210 215 220
Arg Pro Ser Gin Gin Leu
225 230
<210> 28
<211> 690
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (1)..(690)
<400> 28
atg tgt cga ctg agg gtc ttg ctg ctg ctg ctc ccc ttg gcc ttc gtg 48
Met Cys Arg Leu Arg Val Leu Leu Leu Leu Leu Pro Leu Ala Phe Val
1 5 10 15
tcc tcc tot gct ctc ccc atc cat gat gtc gac tot cag cag aac acc 96
Ser Ser Ser Ala Leu Pro Ile His Asp Val Asp Ser Gin Gin Asn Thr
20 25 30
tcc ggg ttc ctg ggc ctt cag agg ctt ctc caa ago ttt agt cga ctg 144
Ser Gly Phe Leu Gly Leu Gin Arg Leu Leu Gin Ser Phe Ser Arg Leu
35 40 45
ttc cta aaa aat gac ctg cta cga gac ctg gac aac ttc ttc tcc tcc 192
Phe Leu Lys Asn Asp Leu Leu Arg Asp Leu Asp Asn Phe Phe Ser Ser
50 55 60
ccc atg gac ttc cga gac ctt cct agg aac ttc cat cag gaa gag aac 240
Pro Met Asp Phe Arg Asp Leu Pro Arg Asn Phe His Gin Glu Glu Asn
65 70 75 80
cag gag cac aga atg ggc aac cat acc ctc tcc ago cac cta cag ata 288
Gin Glu His Arg Met Gly Asn His Thr Leu Ser Ser His Leu Gin Ile
85 90 95
gac aag gtg act gac aac cag aca ggg gag gtg cac atc tog gag aaa 336
Asp Lys Val Thr Asp Asn Gin Thr Gly Glu Val His Ile Ser Glu Lys
100 105 110
gtc gag gcc tcc att gag cca gaa cgg aac cog gaa ggg gac tgg aag 384
Val Glu Ala Ser Ile Glu Pro Glu Arg Asn Pro Glu Gly Asp Trp Lys
115 120 125
gtt ccc aaa gta gaa gca aaa gag ccc cog gtg cot gtg cag aag gtc 432
Val Pro Lys Val Glu Ala Lys Glu Pro Pro Val Pro Val Gin Lys Val
CA 02366062 2001-09-13
VW) 00/52047 PCMS00/05452
-36 -
130 135 140
acc gac agc ttg cac cca gag ccc cgg cag gtg gct ttc tgg atc atg 480
Thr Asp Ser Leu His Pro Glu Pro Arg Gin Val Ala Phe Trp Ile Met
145 150 155 160
aag atg cca agg cgg agg acc cag ccc gat gtc cag gat gga ggc cgc 528
Lys Met Pro Arg Arg Arg Thr Gin Pro Asp Val Gin Asp Gly Gly Arg
165 170 175
tgg ctc ata gaa aag cga cat cgc atg cag gcc atc cgg gat ggg ctc 576
Trp Leu Ile Glu Lys Arg His Arg Met Gin Ala Ile Arg Asp Gly Leu
180 185 190
cgt gga ggc gcc cgt gag gac agc ctg gag gat ggg gtc cat atc ccc 624
Arg Gly Gly Ala Arg Glu Asp Ser Leu Glu Asp Gly Val His Ile Pro
195 200 205
caa cac gcc aag ctg cct gtc aga aag aca cac ttt ctc tac atc ctc 672
Gin His Ala Lys Leu Pro Val Arg Lys Thr His Phe Leu Tyr Ile Leu
210 215 220
agg cca tcc caa cag ctg 690
Arg Pro Ser Gin Gin Leu
225 230
<210> 29
<211> 51
<212> PRT
<213> Artificial Sequence
<220>
<223> Xaa at positions
3-5,7,9-15,18,20-22,24-27,29,31,33,34,36-39,42,44,
45, and 47-50 may be any amino acid
<220>
<223> Description of Artificial Sequence: consensus
sequence
<400> 29
Leu Pro Xaa Xaa Xaa His Xaa Glu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly
1 5 10 15
Asn Xaa Thr Xaa Xaa Xaa His Xaa Xaa Xaa Xaa Lys Xaa Thr Xaa Asn
20 25 30
Xaa Xaa Gly Xaa Xaa Xaa Xaa Ser Glu Xaa Val Xaa Xaa Ser Xaa Xaa
35 40 45
Xaa Xaa Glu
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-37-
<220>
<223> Description of Artificial Sequence: primer
<400> 30
cagtgagtgc tgtggagacc 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 31
tcttcagtca ggctcctctc 20
<210> 32
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 32
acctgcaatg tgtcgactga g 21
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 33
cacttacagc tgttgggatg 20
<210> 34
<211> 10
<212> PRT
<213> Homo sapiens
<220>
<223> Xaa at position I may be any amino acid
<400> 34
Xaa Val Leu Asp She Asn Asn Ile Arg Ser
1 5 10
<210> 35
<211> 10
CA 02366062 2001-09-13
. V/0 00/52047
PCT/US00/05452
- 38 -
<212> PRT
<213> Homo sapiens
<400> 35
Ser Gin Gly Arg Lys Gly Gin Glu Gly Ser
1 5 10
<210> 36
<211> 272
<212> PRT
<213> Mus musculus
<400> 36
Met Met Val Val Cys Ala Pro Ala Ala Val Arg Phe Leu Ala Val Phe
1 5 10 15
Thr Met Met Ala Leu Cys Ser Leu Pro Leu Leu Gly Ala Ser Ala Thr
20 25 30
Leu Asn Ser Val Leu Ile Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro
35 40 45
Pro Leu Gly Gly Ala Gly Gly Gin Pro Gly Ser Ala Val Ser Val Ala
50 55 60
Pro Gly Val Leu Tyr Glu Gly Gly Asn Lys Tyr Gln Thr Leu Asp Asn
65 70 75 80
Tyr Gin Pro Tyr Pro Cys Ala Glu Asp Glu Glu Cys Gly Ser Asp Glu
85 90 95
Tyr Cys Ser Ser Pro Ser Arg Gly Ala Ala Gly Val Gly Gly Val Gin
100 105 110
Ile Cys Leu Ala Cys Arg Lys Arg Arg Lys Arg Cys Met Thr His Ala
115 120 125
Met Cys Cys Pro Gly Asn Tyr Cys Lys Asn Gly Ile Cys Met Pro Ser
130 135 140
Asp His Ser His Phe Pro Arg Gly Glu Ile Glu Glu Ser Ile Ile Glu
145 150 155 160
Asn Leu Gly Asn Asp His Asn Ala Ala Ala Gly Asp Gly Tyr Pro Arg
165 170 175
Arg Thr Thr Leu Thr Ser Lys Ile Tyr His Thr Lys Gly Gin Glu Gly
180 185 190
Ser Val Cys Leu Arg Ser Ser Asp Cys Ala Ala Gly Leu Cys Cys Ala
195 200 205
Arg His Phe Trp Ser Lys Ile Cys Lys Pro Val Leu Lys Glu Gly Gin
210 215 220
Val Cys Thr Lys His Lys Arg Lys Gly Ser His Gly Leu Glu Ile Phe
225 230 235 240
CA 02366062 2001-09-13
WO 00/52047
PCT/US00/05452
-39-
Gln Arg Cys Tyr Cys Gly Glu Gly Leu Ala Cys Arg Ile Gin Lys Asp
245 250 255
His His Gin Ala Ser Asn Ser Ser Arg Leu His Thr Cys Gin Arg His
260 265 270
<210> 37
<211> 259
<212> PRT
<213> Xenopus laevis
<400> 37
Met Gly Ser Asn Met Phe Pro Val Pro Leu Ile Val Phe Trp Gly Phe
1 5 10 15
Ile Leu Asp Gly Ala Leu Gly Phe Val Met Met Thr Asn Ser Asn Ser
20 25 30
Ile Lys Asn Val Pro Ala Ala Pro Ala Gly Gin Pro Ile Gly Tyr Tyr
35 40 45
Pro Val Ser Val Ser Pro Asp Ser Leu Tyr Asp Ile Ala Asn Lys Tyr
50 55 60
Gin Pro Leu Asp Ala Tyr Pro Leu Tyr Ser Cys Thr Glu Asp Asp Asp
65 70 75 80
Cys Ala Leu Asp Glu Phe Cys His Ser Ser Arg Asn Gly Asn Ser Leu
85 90 95
Val Cys Leu Ala Cys Arg Lys Arg Arg Lys Arg Cys Leu Arg Asp Ala
100 105 110
Met Cys Cys Thr Gly Asn Tyr Cys Ser Asn Gly Ile Cys Val Pro Val
115 120 125
Glu Gln Asp Gin Glu Arg Phe Gin His Gin Gly Tyr Leu Glu Glu Thr
130 135 140
Ile Leu Glu Asn Tyr Asn Asn Ala Asp His Ala Thr Met Asp Thr His
145 150 155 160
Ser Lys Leu Thr Thr Ser Pro Ser Gly Met Gin Pro Phe Lys Gly Arg
165 170 175
Asp Gly Asp Val Cys Leu Arg Ser Thr Asp Cys Ala Pro Gly Leu Cys
180 185 190
Cys Ala Arg His Phe Trp Ser Lys Ile Cys Lys Pro Val Leu Asp Glu
195 200 205
Gly Gin Val Cys Thr Lys His Arg Arg Lys Gly Ser His Gly Leu Glu
210 215 220
Ile Phe Gin Arg Cys His Cys Gly Ala Gly Leu Ser Cys Arg Leu Gin
225 230 235 240
Lys Gly Glu Phe Thr Thr Val Pro Lys Thr Ser Arg Leu His Thr Cys
CA 02366062 2001-09-13
VA) 00/52047 PCT/US00/05452 -
- 40 -
245 250 255
Gin Arg His
<210> 38
<211> 350
<212> PRT
<213> Gallus gallus
<400> 38
Met Arg Arg Gly Glu Gly Pro Ala Pro Arg Arg Arg Trp Leu Leu Leu
1 5 10 15
Leu Ala Val Leu Ala Ala Leu Cys Cys Ala Ala Ala Gly Ser Gly Gly
20 25 30
Arg Arg Arg Ala Ala Ser Leu Gly Glu Met Leu Arg Glu Val Glu Ala
35 40 45
Leu Met Glu Asp Thr Gin His Lys Leu Arg Asn Ala Val Gin Glu Met
50 55 60
Glu Ala Glu Glu Glu Gly Ala Lys Lys Leu Ser Glu Val Asn Phe Glu
65 70 75 80
Asn Leu Pro Pro Thr Tyr His Asn Glu Ser Asn Thr Glu Thr Arg Ile
85 90 95
Gly Asn Lys Thr Val Gin Thr His Gin Glu Ile Asp Lys Val Thr Asp
100 105 110
Asn Arg Thr Gly Ser Thr Ile Phe Ser Glu Thr Ile Ile Thr Ser Ile
115 120 125
Lys Gly Gly Glu Asn Lys Arg Asn His Glu Cys Ile Ile Asp Glu Asp
130 135 140
Cys Glu Thr Gly Lys Tyr Cys Gin Phe Ser Thr Phe Glu Tyr Lys Cys
145 150 155 160
Gin Pro Cys Lys Thr Gin His Thr His Cys Ser Arg Asp Val Glu Cys
165 170 175
Cys Gly Asp Gin Leu Cys Val Trp Gly Glu Cys Arg Lys Ala Thr Ser
180 185 190
Arg Gly Glu Asn Gly Thr Ile Cys Glu Asn Gin His Asp Cys Asn Pro
195 200 205
Gly Thr Cys Cys Ala Phe Gin Lys Glu Leu Leu Phe Pro Val Cys Thr
210 215 220
Pro Leu Pro Glu Glu Gly Glu Pro Cys His Asp Pro Ser Asn Arg Leu
225 230 235 240
Leu Asn Leu lie Thr Trp Glu Leu Glu Pro Asp Gly Val Leu Glu Arg
245 250 255
CA 02366062 2001-09-13
W000/52047
PCT/US00/05452 -
-41-
Cys Pro Cys Ala Ser Gly Leu Ile Cys Gin Pro Gin Ser Ser His Ser
260 265 270
Thr Thr Ser Val Cys Glu Leu Ser Ser Asn Glu Thr Arg Lys Asn Glu
275 280 285
Lys Glu Asp Pro Leu Asn Met Asp Glu Met Pro Phe Ile Ser Leu Ile
290 295 300
Pro Arg Asp Ile Leu Ser Asp Tyr Glu Glu Ser Ser Val Ile Gin Glu
305 310 315 320
Val Arg Lys Glu Leu Glu Ser Leu Glu Asp Gin Ala Gly Val Lys Ser
325 330 335
Glu His Asp Pro Ala His Asp Leu Phe Leu Gly Asp Glu Ile
340 345 350
