U.S. patent application number 09/972473 was filed with the patent office on 2004-11-25 for novel human dickkopf-related protein and nucleic acid molecules and uses therefor.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to McCarthy, Sean A..
Application Number | 20040234515 09/972473 |
Document ID | / |
Family ID | 33459227 |
Filed Date | 2004-11-25 |
United States Patent
Application |
20040234515 |
Kind Code |
A9 |
McCarthy, Sean A. |
November 25, 2004 |
Novel human dickkopf-related protein and nucleic acid molecules and
uses therefor
Abstract
Novel Dkk and Dkk-related polypeptides, proteins, and nucleic
acid molecules are disclosed. In addition to isolated, full-length
Dkk and Dkk-related proteins, the invention further provides
isolated fusion proteins, antigenic peptides and antibodies. The
invention also provides Dkk and Dkk-related nucleic acid molecules,
recombinant expression vectors containing a nucleic acid molecule
of the invention, host cells into which the expression vectors have
been introduced and non-human transgenic animals in which a Dkk and
Dkk-related gene has been introduced or disrupted. Diagnostic,
screening and therapeutic methods utilizing compositions of the
invention are also provided.
Inventors: |
McCarthy, Sean A.; (San
Diego, CA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 0068312 A1 |
April 10, 2003 |
|
|
Family ID: |
33459227 |
Appl. No.: |
09/972473 |
Filed: |
October 4, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09972473 |
Oct 4, 2001 |
|
|
|
09263022 |
Mar 5, 1999 |
|
|
|
09263022 |
Mar 5, 1999 |
|
|
|
PCT/US98/07894 |
Apr 16, 1998 |
|
|
|
PCT/US98/07894 |
Apr 16, 1998 |
|
|
|
09009802 |
Jan 20, 1998 |
|
|
|
PCT/US98/07894 |
Apr 16, 1998 |
|
|
|
08842898 |
Apr 17, 1997 |
|
|
|
08842898 |
Apr 17, 1997 |
|
|
|
08843704 |
Apr 16, 1997 |
|
|
|
60071589 |
Jan 15, 1998 |
|
|
|
Current U.S.
Class: |
424/94.5 ;
435/194; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
Y10S 530/867 20130101;
Y10S 530/866 20130101; C12Q 2600/136 20130101; C07K 2319/00
20130101; C12Q 2600/106 20130101; A61K 38/00 20130101; C07K 14/4718
20130101; C07K 14/47 20130101; C12Q 1/6886 20130101 |
Class at
Publication: |
424/094.5 ;
435/194; 435/069.1; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C07H 021/04; A61K
038/51; C12N 009/12; C12P 021/02; C12N 005/06 |
Claims
What is claimed:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence which is at least 60% homologous to a nucleotide sequence
of 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, SEQ ID
NO:22, the DNA insert of the plasmid deposited with ATCC as
Accession Number 98452, the DNA insert of the plasmid deposited
with ATCC as Accession Number 98633, or the nucleotide sequence of
the DNA insert of the plasmid deposited with ATCC as Accession
Number ______, or a complement thereof, b) a nucleic acid molecule
comprising a fragment of at least 1000 nucleotides of a nucleic
acid comprising the nucleotide sequence of 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, SEQ ID NO: 22, the DNA insert of
the plasmid deposited with ATCC as Accession Number 98452 , the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, or a complement
thereof; c) a nucleic acid molecule which encodes a polypeptide
comprising 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, the amino acid sequence encoded by
the DNA insert of the plasmid deposited with ATCC as Accession
Number 98452, the amino acid sequence encoded by the DNA insert of
the plasmid deposited with ATCC as Accession Number 98633, or the
amino acid sequence encoded by the DNA insert of the plasmid
deposited with the ATCC as Accession Number ______; d) a nucleic
acid molecule which encodes a fragment of a polypeptide comprising
the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8,
SEQ ID NO:14, SEQ ID NO:21, the polypeptide encoded by the DNA
insert of the plasmid deposited with ATCC as Accession Number
98452, the polypeptide encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, wherein the fragment comprises at least 15
contiguous amino acid residues of the amino acid sequence of SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:21, the
polypeptide encoded by the DNA insert of the plasmid deposited with
ATCC as Accession Number 98452, the polypeptide encoded by the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the polypeptide encoded by the DNA insert of the plasmid
deposited with the ATCC as Accession Number ______; and e) a
nucleic acid molecule which encodes a naturally occurring allelic
variant of a polypeptide comprising the amino acid sequence of SEQ
ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:21, the
polypeptide encoded by the DNA insert of the plasmid deposited with
ATCC as Accession Number 98452, the polypeptide encoded by the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, wherein the nucleic
acid molecule hybridizes to a nucleic acid molecule comprising 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, SEQ ID NO:22,the
DNA insert of the plasmid deposited with ATCC as Accession Number
98452, the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the DNA insert of the plasmid deposited
with Accession Number ______ under stringent conditions.
2. The isolated nucleic acid molecule of claim 1 which is selected
from the group consisting of: a) a nucleic acid molecule comprising
the nucleotide sequence of 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, SEQ ID NO:22, the DNA insert of the plasmid deposited
with ATCC as Accession Number 98452, the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, or a complement thereof, and b) a nucleic
acid molecule which encodes a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14,
SEQ ID NO:21, the polypeptide encoded by the DNA insert of the
plasmid deposited with ATCC as Accession Number 98452, the
polypeptide encoded by the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the polypeptide encoded by the
DNA insert of the plasmid deposited with the ATCC as Accession
Number ______.
3. The nucleic acid molecule of claim 1 further comprising vector
nucleic acid sequences.
4. The nucleic acid molecule of claim 1 further comprising nucleic
acid sequences encoding a heterologous polypeptide.
5. A host cell which contains the nucleic acid molecule of claim
1.
6. The host cell of claim 5 which is a mammalian host cell.
7. A non-human mammalian host cell containing the nucleic acid
molecule of claim 1.
8. An isolated polypeptide selected from the group consisting of:
a) a fragment of a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID
NO:21, the polypeptide encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number 98452, the polypeptide
encoded by the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
______, wherein the fragment comprises at least 15 contiguous amino
acids of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ
ID NO:21, the polypeptide encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number 98452, the polypeptide
encoded by the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the polypeptide encoded by the DNA
insert of the plasmid deposited with the ATCC as Accession Number
______; b) a naturally occurring allelic variant of a polypeptide
comprising the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ
ID NO:8, SEQ ID NO:14, SEQ ID NO:21, the polypeptide encoded by the
DNA insert of the plasmid deposited with ATCC as Accession Number
98452, the polypeptide encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, wherein the polypeptide is encoded by a
nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising 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, SEQ
ID NO:22, the DNA insert of the plasmid deposited with ATCC as
Accession Number 98452, the DNA insert of the plasmid deposited
with ATCC as Accession Number 98633, or the DNA insert of the
plasmid deposited with the ATCC as Accession Number ______ under
stringent conditions; c) a polypeptide which is encoded by a
nucleic acid molecule comprising a nucleotide sequence which is at
least 60% homologous to a nucleic acid comprising the nucleotide
sequence of 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, SEQ
ID NO:22, the DNA insert of the plasmid deposited with ATCC as
Accession Number 98452, the DNA insert of the plasmid deposited
with ATCC as Accession Number 98633, or the DNA insert of the
plasmid deposited with ATCC as Accession Number ______; and d) a
polypeptide comprising an amino acid sequence which is at least 60%
homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5,
SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:21, the polypeptide encoded by
the DNA insert of the plasmid deposited with ATCC as Accession
Number 98452, the polypeptide encoded by the DNA insert of the
plasmid deposited with ATCC as Accession Number 98633, or the
polypeptide encoded by the DNA insert of the plasmid deposited with
ATCC as Accession Number ______.
9. The isolated polypeptide of claim 8 comprising the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14,
SEQ ID NO:21, the polypeptide encoded by the DNA insert of the
plasmid deposited with ATCC as Accession Number 98452, the
polypeptide encoded by the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the polypeptide encoded by the
DNA insert of the plasmid deposited with ATCC as Accession Number
______.
10. The polypeptide of claim 8 further comprising heterologous
amino acid sequences.
11. An antibody which selectively binds to a polypeptide of claim
8.
12. A method for producing a polypeptide selected from the group
consisting of: a) a polypeptide comprising the amino acid sequence
of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID
NO:21, the polypeptide encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number 98452, the polypeptide
encoded by the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the polypeptide encoded by the DNA
insert of the plasmid deposited with ATCC as Accession Number
______; b) a fragment of a polypeptide comprising the amino acid
sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14,
SEQ ID NO:21, the polypeptide encoded by the DNA insert of the
plasmid deposited with ATCC as Accession Number 98452, the
polypeptide encoded by the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, wherein the fragment comprises at least 15 contiguous amino
acids of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ
ID NO:21, the polypeptide encoded by the DNA insert of the plasmid
deposited with ATCC as Accession Number 98452, the polypeptide
encoded by the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the polypeptide encoded by the DNA
insert of the plasmid deposited with ATCC as Accession Number
______; and c) a naturally occurring allelic variant of a
polypeptide comprising the amino acid sequence of SEQ ID NO:2, SEQ
ID NO:5, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:21, the polypeptide
encoded by the DNA insert of the plasmid deposited with ATCC as
Accession Number 98452, the polypeptide encoded by the DNA insert
of the plasmid deposited with ATCC as Accession Number 98633, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______, wherein the polypeptide is
encoded by a nucleic acid molecule which hybridizes to a nucleic
acid molecule comprising 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, SEQ ID NO:22, 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, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______ under stringent conditions; comprising culturing the host
cell of claim 5 under conditions in which the nucleic acid molecule
is expressed.
13. A method for detecting the presence of a polypeptide of claim 8
in a sample comprising: a) contacting the sample with a compound
which selectively binds to the polypeptide; and b) determining
whether the compound binds to the polypeptide in the sample to
thereby detect the presence of a polypeptide of claim 8 in the
sample.
14. The method of claim 13, wherein the compound which binds to the
polypeptide is an antibody.
15. A kit comprising a compound which selectively binds to a
polypeptide of claim 8 and instructions for use.
16. A method for detecting the presence of a nucleic acid molecule
in claim 1 in a sample comprising: a) contacting the sample with a
nucleic acid probe or primer which selectively hybridizes to the
nucleic acid molecule; and b) determining whether the nucleic acid
probe or primer binds to a nucleic acid molecule in the sample to
thereby detect the presence of a nucleic acid molecule of claim 1
in the sample.
17. The method of claim 16, wherein the sample comprises mRNA
molecules and is contacted with a nucleic acid probe.
18. A kit comprising a compound which selectively hybridizes to a
nucleic acid molecule of claim 1 and instructions for use.
19. A method for identifying a compound which binds to a
polypeptide of claim 8 comprising: a) contacting the polypeptide,
or a cell expressing the polypeptide with a test compound; and b)
determining whether the polypeptide binds to the test compound.
20. The method of claim 19, wherein the binding of the test
compound to the polypeptide is detected by a method selected from
the group consisting of: a) detection of binding by direct
detection of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; and c) detection of
binding using an assay for Dkk activity.
21. A method of modulating the activity of a polypeptide of claim 8
comprising contacting the polypeptide or a cell expressing the
polypeptide with a compound which binds to the polypeptide in a
sufficient concentration to modulate the activity of the
polypeptide.
22. A method for identifying a compound which modulates the
activity of a polypeptide of claim 8 comprising: a) contacting a
polypeptide of claim 8 with a test compound; and b) determining the
effect of the test compound on the activity of the polypeptide to
thereby identify a compound which modulates the activity of the
polypeptide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of prior-filed
application PCT Application No. PCT/US98/07894, filed Apr. 16,
1998, which in turn claims the benefit of prior-filed application
U.S. patent application Ser. No. 09/009,802, filed Jan. 20, 1998.
U.S. patent application Ser. No. 09/009,802 claims the benefit of
prior-filed U.S. patent application Ser. No. 60/071,589, filed Jan.
15, 1998 and of prior-filed U.S. patent application Ser. No.
08/842,898, filed Apr. 17, 1997. U.S. patent application Ser. No.
08/842,898 is a continuation-in-part of U.S. patent application
Ser. No. 08/843,704, filed Apr. 16, 1997. The contents of each of
the above-referenced patent applications are incorporated herein by
this reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] The Wnt gene family encode a large class of secreted
proteins related to the Int1/Wnt1 proto-oncogene and Drosophila
wingless ("Wg"), a Drosophila Wnt1 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).
[0004] 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 stabilization of Armadillo/.beta.-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 of .beta.-catenin, whereas Wnt signaling
inactivates GSK3, thus preventing .beta.-catenin degradation. As a
result, .beta.-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
protooncogene as a target of Wnt signaling (He et al. (1998)
Science 281:1509-3512).
[0005] 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 et al. (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 et al. (1997) Cell 88:747-756;
Mayr et al. (1997) Mech. Dev. 63:109-325; and Salic et al. (1997)
Development 124:4739-4748). These proteins inhibit the ability of
Xwnt8 to induce a secondary axis in frog embryos (for review see
Zom (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 Xwnt8 (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).
[0006] 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
[0007] 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 Dkk-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.
[0008] 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 Dkk
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 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 the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number ______, 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.
[0009] 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:1.
[0010] In another preferred embodiment, an isolated Dkk nucleic
acid molecule has the nucleotide sequence shown SEQ ID NO:6, or a
complement thereof. In another embodiment, a Dkk nucleic acid
molecule further comprises nucleotides 1-124 of SEQ ID NO:4. In yet
another preferred embodiment, a Dkk nucleic acid molecule further
comprises nucleotides 797-848 of SEQ ID NO:4. In another preferred
embodiment, an isolated Dkk nucleic acid molecule has the
nucleotide sequence shown in SEQ ID NO:4.
[0011] In another preferred embodiment, an isolated Dkk 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 Dkk nucleic acid molecule further
comprises nucleotides 907-1536 of SEQ ID NO:7. In another preferred
embodiment, an isolated Dkk nucleic acid molecule has the
nucleotide sequence shown in SEQ ID NO:7.
[0012] 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.
[0013] 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.
[0014] 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 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).
[0015] In another embodiment, an isolated nucleic acid molecule of
the present invention encodes a Dkk 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 Dkk 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.
[0016] 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.
[0017] Another embodiment of the invention features nucleic acid
molecules which specifically detect Dkk 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, to the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCCas Accession Number 98633,
or to the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______. In another
embodiment, the nucleic acid molecule is at least 500 nucleotides
in length and hybridizes under stringent conditions to a 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.
[0018] 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.
[0019] 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.
[0020] 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 Dkk protein has 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 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.
[0021] 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.
[0022] 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 Dkk-related proteins, such as monoclonal or polyclonal
antibodies. In addition, the proteins or biologically active
portions thereof can be incorporated into pharmaceutical
compositions, which optionally include pharmaceutically acceptable
carriers.
[0023] 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.
[0024] 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.
[0025] 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 Dkk-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.
[0026] 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 Dkk 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 disorder characterized by aberrant protein or nucleic acid
expression is a developmental, differentiative, or proliferative
disorder.
[0027] 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.
[0028] 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.
[0029] Other features and advantages of the invention will be
apparent from the following detailed description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 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:1. The amino acid sequence
corresponds to amino acids 1 to 350 of SEQ ID NO:2.
[0031] FIG. 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.
[0032] FIG. 3 depicts the cDNA sequence and predicted amino acid
sequence of human Dkk-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.
[0033] FIG. 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.
[0034] FIG. 5A-B depicts the cDNA sequence and predicted amino acid
sequence of murine Dkk-3. The nucleotide sequence corresponds to
nucleic acids 1 to 2381 of SEQ ID NO:16. The amino acid sequence
corresponds to amino acids 1 to 349 of SEQ ID NO:17.
[0035] FIG. 6 depicts a multiple sequence alignment of the amino
acid sequences of hDkk-1 (corresponding the SEQ ID NO:8), mDkk-1
(having Accession No. AF030434), Xenopus Dkk-1 ("xDkk-1") (having
Accession No. AF030433), hDkk-2 (corresponding to SEQ ID NO:21),
hDkk-3 (corresponding to SEQ ID NO:2), mDkk-3 (corresponding to SEQ
ID NO:17), chicken Dkk-3 ("cDkk-3") (having Accession No. D2631 1),
and hDkk-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 hDkk4 is indicated by an
arrow.
[0036] FIG. 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 1 to 242 of SEQ ID NO:14.
[0037] FIG. 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.
[0038] FIG. 9 is a schematic diagram illustrating the Dkk and
Dkk-related proteins of the instant invention. The figure depicts
the structural domains of the human Dkks and Soggy. Signal peptides
are indicated by darkened boxes. The cysteine-rich domains of a Dkk
cysteine-rich region are depicted as CRD-1 and CRD-2. Branches
indicate sites of N-glycosylation.
[0039] FIG. 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 FIG. 6. The alignment provides details
regarding the relationship between the Dkk-3 and Soggy-I 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.
[0040] FIG. 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.
[0041] FIG. 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
[0042] The present invention is based on the discovery of novel
molecules, referred to herein as Dkk protein and nucleic acid
molecules, which comprise a family of molecules 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.
[0043] 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.
[0044] 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
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 Prosite
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 Dkk-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)-G-X(2)-C-X(16-24)-C-X-C-X(2)-G-L-X-C-X-
(10-17)-C (SEQ ID NO:24).
[0045] 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 FIG. 6). In another embodiment, a hDkk-4 (human Dkk-4)
protein contains an amino-terminal cysteine-rich 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
FIG. 6). In another embodiment, a hDkk-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 FIG. 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 IO 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 FIG. 6).
[0046] 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 (FIG. 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 .beta.
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 FIG. 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 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.
[0047] 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 FIG. 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 FIG. 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 FIG.
6, Dkk-2 has at least about 10cysteine 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.
1TABLE 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 5 171 66 114 103 6 178 73 121
110 7 184 79 127 116 8 185 80 128 117 9 190 85 133 122 10 195 90
138 127 11 208 145 189 183 12 214 151 195 189 13 219 156 200 194 14
220 157 201 195 15 231 166 210 204 16 241 176 220 214 17 265 194
237 231 18 267 196 239 233 19 273 202 245 239 20 284 218 263
256
[0048] The first 10 rows of Table I contain 10 cysteine residues
that are included within 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 hDkks-3, -4, -1, and -2.
[0049] 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 FIG. 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 FIG. 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 FIG. 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 FIG. 6.
[0050] 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; 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 hDkk-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.
[0051] 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).
[0052] 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).
[0053] 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.
[0054] 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 Dkk 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, 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).
[0055] In another embodiment, a Dkk 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.
[0056] 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, 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.
[0057] 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 Dkk
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 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.
[0058] 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 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.
[0059] 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.
[0060] 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 Dkk 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 Dkk 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.
[0061] 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.
[0062] 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).
[0063] 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.
[0064] 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 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 hDkk-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.
[0065] 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.
[0066] 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 FIG. 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
(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-1 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)
CABIOS, utilizing a PAM120 weight residue table, a gap length
penalty of 12, and a gap penalty of 4.
[0067] 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 FIG. 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 FIG. 10.
[0068] 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 hDkk-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 (SEQ ID NO:29). For example, human
Soggy-I has a SGY domain from about amino acid residues 81-131 (Leu
81-Glu131 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-Glu120 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 FIG. 10.
[0069] 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.
[0070] Various aspects of the invention are described in further
detail in the following subsections:
[0071] I. Isolated Nucleic Acid Molecules
[0072] 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.
[0073] 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., 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, 4 kb, 3
kb, 2 kb, 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.
[0074] 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, 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, the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number 98633, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______, 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, N.Y., 1989).
[0075] 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 Accession Number 98452, the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, 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, the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______.
[0076] 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 Dkk nucleotide
sequences can be prepared by standard synthetic techniques, e.g.,
using an automated DNA synthesizer.
[0077] 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 (i.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 Va., on Jun. 11, 1997 and
assigned Accession Number 98452.
[0078] 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' 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).
[0079] 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 Dkk-1 cDNA. This cDNA comprises sequences encoding the human
Dkk-1 protein (i.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 Va., on Jan.
16, 1998 and assigned Accession Number 98633.
[0080] 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 (i.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 to 1500,
corresponding to SEQ ID NO:22). A plasmid, clone fthul33,
containing the full-length nucleotide sequence encoding hDkk-2 was
deposited with the American Type Culture Collection (ATCC),
presently in Manassas Va., on Mar. 2, 1999 and assigned Accession
Number ______.
[0081] 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 molecule can comprise only the
coding region of SEQ ID NO:13 (e.g., nucleotides 75 to 800,
corresponding to SEQ ID NO:15).
[0082] 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, 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, the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
_____, 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, the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
_____, thereby forming a stable duplex.
[0083] 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 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, or a portion of any
of these nucleotide sequences.
[0084] 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 5
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, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, or a portion of any
of these nucleotide sequences).
[0085] 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 20 vectors include, but are not limited to pBluescript
(Stratagene.TM.), pT7T3D (Pharmcia.TM.) 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.
[0086] 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, 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, the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
_____, 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 98452, the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number 98633, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______. 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.
[0087] Probes based on human nucleotide sequences (e.g. the human
Dkk 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, Dkk 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 Dkk 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 Dkk-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.
[0088] A nucleic acid fragment encoding a "biologically active
portion of a Dkk 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, the
nucleotide sequence of the DNA insert of the plasmid deposited with
ATCC as Accession Number 98633, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, which encodes a polypeptide having a biological activity
(the biological activities of the Dkk and Dkk-related proteins have
previously 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.
[0089] 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, the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number 98633, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______, 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, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______. 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.
[0090] 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, 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 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.
[0091] 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, the nucleotide sequence of the DNA insert
of the plasmid deposited with ATCC as Accession Number 98633, or
the nucleotide sequence of the DNA insert of the plasmid deposited
with ATCC as Accession Number ______, 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 FIG. 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, Va., on Jan. 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 FIG. 8. The
coding region of murine Soggy-1 is represented by SEQ ID NO:28.
[0092] Nucleic acid molecules corresponding to natural allelic
variants and homologues of the Dkk or Dkk-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 hDkk-3 protein) can be
obtained by isolating total 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.
[0093] 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, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number _____. 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 6.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in 0.2 .times.
SSC, 0.1% SDS at 50-65.degree. 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).
[0094] In addition to naturally-occurring allelic variants of the
Dkk 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, the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
98633, or the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number ______, thereby leading to
changes in the amino acid sequence of the encoded Dkk 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, the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
______. A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of Dkk (or wild-type
Dkk-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 Dkk protein and other proteins having cysteine-rich domains
are not likely to be amenable to alteration.
[0095] Accordingly, another aspect of the invention pertains to
nucleic acid molecules encoding Dkk 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 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:8, 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 NO: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 NO:14,
or SEQ ID NO:21, and most preferably at least about 95% homologous
to SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:14, or SEQ ID
NO:21.
[0096] An isolated nucleic acid molecule encoding a Dkk protein
homologous to the protein of SEQ ID NO:2, SEQ ID NO: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: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, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, 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 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, or the nucleotide sequence of the
DNA insert of the plasmid deposited with ATCC as Accession Number
______, by standard techniques, such as site-directed mutagenesis
and PCR-mediated mutagenesis.
[0097] 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., 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 Dkk 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.
[0098] 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, the nucleotide sequence of the DNA insert of the plasmid
deposited with ATCC as Accession Number 98633, or the nucleotide
sequence of the DNA insert of the plasmid deposited with ATCC as
Accession Number ______, the encoded protein can be expressed
recombinantly and the activity of the protein can be
determined.
[0099] In a preferred embodiment, a mutant Dkk or Dkk-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.
[0100] In a preferred embodiment, a mutant Dkk or Dkk-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 Dkk (e.g.,
hDkk-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.
[0101] 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).
[0102] 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 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.
[0103] 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.
[0104] 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-carboxymethylaminomethyl-2-thiouridin- e,
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-methoxyaminomethyl-2-thiour- acil,
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 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).
[0105] 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 pol II or pol III promoter are
preferred.
[0106] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .alpha.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .beta.-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).
[0107] 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 et al., U.S. Pat. No. 4,987,071;
and Cech et al., U.S. Pat. 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.
[0108] 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. et al., (1992) Ann. N. Y Acad.
Sci. 660:27-36; and Maher, L. J. (1992) Bioassays
14(12):807-15.
[0109] 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. et al.
(1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used
herein, the terms "peptide nucleic acids" or "PNAs" 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. et al. (1996) supra;
Perry-O'Keefe et al. PNAS 93: 14670-675.
[0110] 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., S1 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).
[0111] 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-thy- midine
phosphoramidite, can be used as a between the PNA and the 5' end of
DNA (Mag, M. et 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).
[0112] 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 Dec.
15, 1988) or the blood-brain barrier (see, e.g., PCT Publication
No. W089/10134, published Apr. 25, 1988). In addition,
oligonucleotides can be modified with hybridization-triggered
cleavage agents (See, e.g., Krol et al. (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).
[0113] II. Isolated Dkk Proteins and Anti-Dkk Antibodies
[0114] 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.
[0115] 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 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.
[0116] 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-Dkk-related chemicals, more
preferably less than about 20% chemical precursors, non-Dkk
chemicals, or non-Dkk-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.
[0117] 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 Dkk 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.
[0118] 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.
[0119] 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.
[0120] In an alternative embodiment, a biologically active portion
of a Dkk or Dkk-related protein comprises an amino acid sequence
lacking a signal sequence.
[0121] 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.
[0122] 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.
[0123] 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 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.
[0124] 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.
[0125] 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 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 Dkk
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.
[0126] 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
(available at http://www.gcg.com), using either a Blossom 62 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
(available at http://www.gcg.com), 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.
[0127] 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 Dkk 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 Dkk 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. See
http://www.ncbi.nlm.nih.gov.
[0128] The invention also provides Dkk or Dkk-related chimeric or
fusion proteins. As used herein, a "chimeric protein" or "fusion
protein" comprises a Dkk 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 Dkk or a
Dkk-related protein, whereas a "non-Dkk polypeptide" or
"non-Dkk-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 Dkk-related protein and
which is derived from the same or a different organism. Within a
Dkk or Dkk-related fusion protein the Dkk or Dkk-related
polypeptide can correspond to all or a portion of a Dkk or
Dkk-related protein. In a preferred embodiment, a Dkk or
Dkk-related fusion protein comprises at least one biologically
active portion of a Dkk protein. In another preferred embodiment, a
Dkk or Dkk-related fusion protein comprises at least two
biologically active portions of a Dkk or Dkk-related protein. In
another preferred embodiment, a Dkk or Dkk-related fusion protein
comprises at least three biologically active portions of a Dkk or
Dkk-related protein. Within the fusion protein, the term
"operatively linked" is intended to indicate that the Dkk or
Dkk-related polypeptide and the non-Dkk or non-Dkk-related
polypeptide are 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.
[0129] 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 GST sequences. Such fusion proteins can
facilitate the purification of recombinant Dkk.
[0130] 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.
[0131] 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-IgG1 construct and a B7-1-IgG1
construct]; and Linsley, P. S. et al. (1991) J Exp. Med.
174:561-569 and U.S. Pat. No. 5,434,131[a CTLA4-IgGl]). 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 et al. (1997) N. Engl. J. Med. 337(3):141-147; van
der Poll et al. (1997) Blood 89(10):3727-3734; and Ammann et al.
(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 can be
used to affect the bioavailability of a Dkk 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.
[0132] 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.
[0133] 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 form of the protein by, for
example, competitively binding to a downstream or upstream member
of 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.
[0134] 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 Dkk-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
et al., (1984) Annu. Rev. Biochem. 53:323; Itakura et al, (1984)
Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477.
[0135] In addition, libraries of fragments of a Dkk or Dkk-related
protein coding sequence can be used to generate a variegated
population of Dkk or Dkk-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 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 S1 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.
[0136] 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 (1 992) PNAS 89:7811-7815; Delgrave et al., (1 993) Protein
Engineering 6(3):327-33 1).
[0137] 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.
[0138] 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 Dkk 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.
[0139] 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.
[0140] 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.
[0141] 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(ab').sub.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 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
Dkk-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.
[0142] 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 et al., (1980) J Biol.
Chem .255:4980-83; Yeh et al., (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 et al, (1983) Immunol Today 4:72), the
EBV-hybridoma technique (Cole et al, (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, N.Y. (1980); E. A. Lerner (1981)
Yale J Biol. Med., 54:387-402; M. L. Gefter et al., (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.
[0143] 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 1 5 antibody (see, e.g., G.
Galfre et al., (1 977) 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/O-Agl4 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.
[0144] 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 SurfZAP.TM. 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. Pat. No.
5,223,409; Kang et al., PCT International Publication No. WO
92/18619; Dower et al., 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
et al., (1991) Bio/Technology 9:1370-1372; Hay et al., (1992) Hum.
Antibod. Hybridomas 3:81-85; Huse et al., (1989) Science
246:1275-1281; Griffiths et al., (1993) EMBO J 12:725-734; Hawkins
et al., (1992) J 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 et al., (1991)
Nuc. Acid Res. 19:4133-4137; Barbas et al., (1991) PNAS
88:7978-7982; and McCafferty et al., Nature (1990) 348:552-554.
[0145] 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 et al, U.S. Pat.
No. 4,816,567; Cabilly et al., European Patent Application 125,023;
Better et al., (1988) Science 240:1041-1043; Liu et al., (1987)
PNAS 84:3439-3443; Liu et al., (1987) J. ImmunoL. 139:3521-3526;
Sun et al., (1987) PNAS 84:214-218; Nishimura et al., (1987) Canc.
Res. 47:999-1005; Wood et al., (1985) Nature 314:446-449; and Shaw
et al., (1988) J Natl. Cancer Inst. 80:1553-1559); Morrison, S. L.
(1985) Science 229:1202-1207; Oi et al., (1986) BioTechniques
4:214; Winter U.S. Pat. No. 5,225,539; Jones et al., (1986) Nature
321:552-525; Verhoeyan et al., (1988) Science 239:1534; and Beidler
et al., (1988) J. Immunol. 141:4053-4060.
[0146] 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, .beta.-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 .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0147] III. Recombinant Expression Vectors and Host Cells
[0148] 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 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.
[0149] 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, Calif. (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 described herein
(e.g., Dkk proteins, Dkk-related proteins, mutant forms of Dkk or
Dkk-related proteins, fusion proteins, etc.).
[0150] 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. coli, 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, Calif.
(1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro, for example using T7 promoter
regulatory sequences and T7 polymerase.
[0151] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters 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: 1) 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, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase
(GST), maltose E binding protein, or protein A, respectively, to
the target recombinant protein.
[0152] 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 transplanted into irradiated
recipients. The pathology of the subject recipient is then examined
after sufficient time has passed (e.g six (6) weeks).
[0153] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (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 11d vector relies on
transcription from a T7 gn I 0-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident
.lambda. prophage harboring a T7 gn1 gene under the transcriptional
control of the lacUV 5 promoter.
[0154] 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, Calif. (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.
[0155] 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 et al., (1987) Gene 54:113-123), pYES2
(Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen
Corp, San Diego, Calif.).
[0156] 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 et al.,
(1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and
Summers (1989) Virology 170:31-39).
[0157] 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 et al., (1
987) 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, N.Y., 1989.
[0158] 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 et al., (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 et al., (1985)
Science 230:912-916), and mammary gland-specific promoters (e.g.,
milk whey promoter; U.S. Pat. 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
oc-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.
3:537-546).
[0159] 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 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. et al., Antisense RNA as a molecular tool for genetic
analysis, Reviews--Trends in Genetics, Vol. l(1) 1986.
[0160] 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.
[0161] A host cell can be any prokaryotic or eukaryotic cell. For
example, Dkk 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.
[0162] 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
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),
and other laboratory manuals.
[0163] 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).
[0164] 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.
[0165] 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 Dkk
or Dkk-related proteins and for identifying and/or evaluating
modulators of Dkk or Dkk-related protein activity. As used herein,
a "transgenic animal" is a non-human animal, preferably a 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.
[0166] 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.
Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat.
No. 4,873,191 by Wagner et al, 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 animals carrying the transgene. Moreover, transgenic
animals carrying a transgene encoding Dkk can further be bred to
other transgenic animals carrying other transgenes.
[0167] 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 Dkk 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 Dkk 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
Dkk 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 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
et al.; WO 91/01140 by Smithies.et al.; WO 92/0968 by Zijlstra et
al.; and WO 93/04169 by Berns et al. 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.
[0168] 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 P1. For a description
of the cre/loxP recombinase system, see, e.g., Lakso et al., (1992)
PiVAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et
al., (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.
[0169] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut,
I. et al., (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 G.sub.o 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.
[0170] IV. Pharmaceutical Compositions
[0171] 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.
[0172] 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.
[0173] 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 water, Cremophor EL.TM. (BASF, Parsippany, N.J.) 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.
[0174] 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.
[0175] 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
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 Sterotes; 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.
[0176] 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.
[0177] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0178] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0179] 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 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. Pat. No.
4,522,811.
[0180] 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.
[0181] 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.
[0182] 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 useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0183] 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. Pat. No. 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.
[0184] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0185] V. Uses and Methods of the Invention
[0186] 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.
[0187] 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 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, 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.
[0188] 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
Dkk 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 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).
[0189] 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 Dkk-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 Dkk or Dkk-related proteins, regulate
the bioavailability of Dkk or Dkk-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
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.
[0190] A. Screening Assays:
[0191] 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 Dkk-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.
[0192] "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 Dkk-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 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.
[0193] 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).
[0194] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J Med. Chem.
37:2678; Cho et al., (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0195] 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 et al (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 et al. (1990) Proc. Natl. Acad. Sci.
87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner
supra.).
[0196] 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 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 Dkk
receptor can be determined by detecting the labeled compound in a
complex. For example, test compounds can be labeled with .sup.125I,
.sup.35S, .sup.14C, or .sup.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.
[0197] It is also within the scope of this invention to determine
the ability of a test compound to interact with a Dkk 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.
[0198] 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 Dkk
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.
[0199] 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 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.
[0200] 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 Ca.sup.2+, diacylglycerol, IP.sub.3, 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.
[0201] 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.
[0202] 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 Dkk or Dkk-related protein or biologically active portion
thereof is determined. 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., BIAcore.TM.). Changes in the optical
phenomenon surface plasmon resonance (SPR) can be used as an
indication of real-time reactions between biological molecules.
[0203] In an alternative embodiment, determining the ability of the
test compound to modulate the activity of a Dkk or Dkk-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.
[0204] 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 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 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).
[0205] 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 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.; hDkk-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 Dkk 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.
[0206] 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-methylglucamid- e,
Triton.RTM. X-100, Triton.RTM. X-114, Thesit.RTM.,
Isotridecypoly(ethylene glycol ether).sub.n,
3-[(3-cholamidopropyl)dimeth- ylamminio]-1-propane sulfonate
(CHAPS), 3-[(3-cholamidopropyl)dimethylammi-
nio]-2-hydroxy-1-propane sulfonate (CHAPSO), or
N-dodecyl=N,N-dimethyl-3-a- mmonio-1-propane sulfonate.
[0207] 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 Dkk 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.
[0208] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either a Dkk protein, Dkk-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
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.
[0209] 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 Dkk-related mRNA or protein
expression in the cells can be determined by methods described
herein for detecting Dkk mRNA or protein.
[0210] 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. Pat. 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
WO94/10300), 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.
[0211] 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.
[0212] 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 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.
[0213] 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 Dkk
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.
[0214] 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
Dkk-related protein or biologically 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 Qr Dkk-related protein or biologically active portion
thereof is determined.
[0215] B. Detection Assays
[0216] 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.
[0217] 1. Chromosome Mapping
[0218] 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.
[0219] 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 Dkk or Dkk-related sequences will
yield an amplified fragment.
[0220] 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.
[0221] 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, 1p, or 1v
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.
[0222] 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 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).
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 2. Tissue Typing
[0227] 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
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.
Pat.5,272,057).
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 3. Use of Partial Dkk or Dkk-related Sequences in Forensic
Biology
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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).
[0236] C. Predictive Medicine:
[0237] 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, nucleic acid
expression or activity. For example, mutations in a Dkk 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.
[0238] Another aspect of the invention pertains to monitoring the
influence of agents., (e.g., drugs, compounds) on the expression or
activity of Dkk or Dkk-related in clinical trials.
[0239] These and other agents are described in further detail in
the following sections.
[0240] 1. Diagnostic Assays
[0241] 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 Dkk or Dkk-related protein
such that the presence of Dkk or Dkk-related protein or nucleic
acid is detected in the biological sample. A preferred agent for
detecting Dkk or Dkk-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 Dkk 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, the DNA insert of the plasmid deposited with ATCC as
Accession Number 98633, or the nucleotide sequence of the DNA
insert of the plasmid deposited with ATCC as Accession Number
______, 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
Dkk or Dkk-related mRNA or genomic DNA. Other suitable probes for
use in the diagnostic assays of the invention are described
herein.
[0242] 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').sub.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.
[0243] 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.
[0244] 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 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 Dkk or Dkk-related protein, mRNA or genomic DNA in
the test sample.
[0245] 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.
[0246] 2. Prognostic Assays
[0247] 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 Dkk or Dkk-related expression or activity.
As used herein, a "test sample" refers to a biological sample
obtained from a subject of interest. For example, a test sample can
be a biological fluid (e.g., serum), cell sample, or tissue.
[0248] 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 Dkk-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.)
[0249] 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 Dkk or Dkk-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 Dkk-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 alteration in the level of a messenger RNA
transcript of a Dkk or Dkk-related gene, 6) aberrant modification
of a Dkk or Dkk-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 Dkk or Dkk-related-protein, 9)
allelic loss of a Dkk or Dkk-related gene, and 10) inappropriate
post-translational modification of a Dkk or Dkk-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.
[0250] 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. Pat. 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.
[0251] 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
schemes are especially useful for the detection of nucleic acid
molecules if such molecules are present in very low numbers.
[0252] 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. Pat. No.
5,498,531) can be used to score for the presence of specific
mutations by development or loss of a ribozyme cleavage site.
[0253] 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. et al. (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.
[0254] 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 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) Appl. Biochem. Biotechnol. 38:147-159).
[0255] 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 et al. (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 S1 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 et al. (1988) Proc.
Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol.
217:286-295. In a preferred embodiment, the control DNA or RNA can
be labeled for detection.
[0256] 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. coli 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,
and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, for example, U.S. Pat.
No. 5,459,039.
[0257] 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) MutatRes
285:125-144; and Hayashi (1992) GenetAnal Tech Appl 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).
[0258] 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).
[0259] 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 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.
[0260] 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) Mol. 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.
[0261] 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.
[0262] 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.
[0263] 3. Monitoring of Effects During Clinical Trials
[0264] 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 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 Dkk 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.
[0265] 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.
[0266] 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 Dkk 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, ie. 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.
[0267] C. Methods of Treatment:
[0268] 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 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 Dkk 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.
[0269] 1. Prophylactic Methods
[0270] 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.
[0271] 2. Therapeutic Methods
[0272] 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 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 Dkk-related protein or nucleic acid molecule is
expressed.
[0273] 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).
[0274] 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.
[0275] 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.
[0276] 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.
[0277] 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, hamartoma, and
mesenchymal tumors).
[0278] 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).
[0279] 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).
[0280] 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).
[0281] 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, 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).
[0282] 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).
[0283] 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.
[0284] 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).
[0285] 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.
[0286] 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.
[0287] 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).
[0288] In another embodiment, Dkk-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), 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).
[0289] 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-I polypeptides,
nucleic acids, or modulators thereof, can be used to treat
infertility due to, for example, spermatogenetic failure.
[0290] 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 downregulates) 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.
[0291] 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
Dkk-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
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.
[0292] Stimulation of Dkk or Dkk-related activity is desirable in
situations in which Dkk or Dkk-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.)
[0293] 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.
[0294] Hyperproliferative diseases can be treated with Dkk or
Dkk-related (e.g., hDkk-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.
[0295] 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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, 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).
[0301] 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.
[0302] 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.
[0303] Yet other Dkk or Dkk-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 Dkk.
[0304] 3. Pharmacogenomics
[0305] The Dkk or Dkk-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., Dkk or
Dkk-related gene expression) as identified by a screening assay
described 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 Dkk-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.
[0306] 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.
[0307] 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 "bi-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 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.
[0308] 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.
[0309] 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 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.
[0310] 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.
[0311] 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.
[0312] 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
[0313] 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.
Example 1
Isolation And Characterization of Human hDkk-3 CDNA
[0314] 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.
[0315] Isolation of a Human Dkk-3 cDNA
[0316] 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.
[0317] Briefly, a randomly primed cDNA library using mRNA prepared
from human fetal brain tissue (Clontech, Palo Alto Calif.) was made
by using the Stratagene-ZAP-cDNA Synthesis.TM. 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 Wizard.TM. DNA purification kit
(Promega). DNA was transfected into COS-7 cells with
lipofectamine.TM. (Gibco-BRL). After 48 hours incubation the COS
cell supernatants were assayed for alkaline phosphatase on a Wallac
Micro-Beta scintillation counter using the Phospha-Light.TM. 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.
[0318] 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 Ziplox.TM. human fetal brain cDNA library
using conventional hybridization techniques (Sambrook et al.,
supra). The nucleotide sequence encoding the full length human
Dkk-3 protein is shown in FIG. 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 shown in
FIG. 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.
[0319] Analysis of Human hDkk-3
[0320] 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 hDkk-3 has
been identified using a Signal Sequence Trap system, indicates that
hDkk-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).
[0321] Examination of the cDNA sequence depicted in FIG. 1 shows
that human Dkk-3 is particularly rich in cysteine residues. As
shown in FIG. 1, hDkk-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.
[0322] A BLAST search (Altschul et al., (1990) J. Mol. Biol.
215:403) of the nucleotide and the amino acid sequences of hDkk-3
has revealed that hDkk-3 is similar to a chicken cDNA encoding a
protein of unknown function having GenBank Accession No. D263 11.
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 et al., (1996) Int. J.
Dev. Biol. 40:531). hDkk-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
hDkk-3 which is located between amino acids 147 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.
[0323] 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. U3233 1 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 FIG. 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 .about.100% identical
to residues 2479 to 2153 of SEQ ID NO:1). RIG-like 7-1 is
homologous to hDkk-3 accross a longer region (e.g., 97% identical
from about nucleotides 316 to 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 hDkk-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 (11p15.1) implicated in the
development of human malignant astrocytoma (Ligon et al., supra)
further indicates a role for these genes in tumorigenesis.
[0324] Human hDkk-3 protein has also some amino acid sequence
similarity to metallothionein, particularly in the cyteine-rich
domain.
[0325] Tissue Distribution of hDkk-3 mRNA
[0326] For Northern blots, all hybridizations were to Clontech
Multiple Tissue Northern Blots and were performed in ExpressHyb
solution (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 0.2.times. SSC/0.2%
SDS and exposed to autoradiographic film. Hybridizations of a
control .beta.-actin cDNA probe consistently demonstrated even
loading of the Northern blots. The results of hybridization of the
probe to various mRNA samples are described below.
[0327] Hybridization of a Clontech Fetal Multiple Tissue Northern
(NFTN) blot (Clontech, LaJolla, Calif.) containing RNA from fetal
brain, lung, liver, and kidney indicated the presence of high
levels of hDkk-3 mRNA (.about.2.5 kb) 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.
[0328] Hybridization of a Clontech human Multiple Tissue Northern
(MTN) blot (Clontech, LaJolla, Calif.) 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. J. Dev. Biol. 40:531).
[0329] Further hybridization of a Clontech human Multiple Tissue
Northern (MTN) blot (Clontech, LaJolla, Calif.) 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.
[0330] 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
[0331] 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.
[0332] Identification of a Murine Dkk-3 cDNA
[0333] 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
FmmbO59s 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.
[0334] Tissue Distribution of mDkk-3 mRNA
[0335] 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
TissueTek.TM. O.C.T Compound (Sakura Finetek U.S.A., Inc.,
Torrance, Calif.), frozen on dry ice, and stored at -80.degree. C.
Cryostat serial sections (8 .mu.m) were thaw mounted on Superfrost
PlUS.TM. slides (VWR Scientific, West Chester, Pa.) and air dried
on a slide warmer at 40.degree. 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-HCl (TEA, pH 8.0), incubated in 0.25% acetic
anhydride-TEA for 10 minutes and rinsed in DEPC-2.times. 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.
[0336] Antisense and sense RNA transcripts were prepared by in
vitro transcription (Riboprobe Gemini System.TM., Promega) of PCR
amplified cDNA templates. Template amplification primers were as
follows;
[0337] mDkk-3 forward 5'-CAGTGAGTGCTGTGGAGACC-3' (SEQ ID NO:30),
and 1 5 reverse 5'-TCTTCAGTCAGGCTCCTCTC-3' (SEQ ID NO:31).
[0338] Probes were labeled with .sup.35S-UTP (NEN) and purified on
G-25 spin columns (Pharmacia). The hybridization cocktail
contained: 50% formamide, 10% dextran sulfate, 0.1% sodium dodecyl
sulfate (SDS), 0.1% sodium thiosulfate, IX Denhardt's solution, 0.6
M NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM dithiothreitol
(DTT), 0.1 mg/ml sheared salmon sperm, 50 .mu.g/ml yeast tRNA, 0.5
mg/ml yeast total RNA, and .sup.35S-UTP labeled probe at a
concentration of 5.times.10.sup.7 c.p.m./100 .mu.l of final
hybridization solution; 100 .mu.l 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.degree. C. for
18 h. After hybridization, slides were washed with 2.times. SSC.
Sections were then sequentially incubated at 37.degree. C. in TNE
(a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1
mM EDTA), for 10 minutes, in TNE with 10 ug/ml RNase A for 30
minutes, and finally in TNE for 10 minutes. Slides were then rinsed
with 2.times. SSC at room temperature, washed in 2.times. SSC at
50.degree. C. for 1 h, 0.2.times. SSC at 55.degree. C. for 1 h, and
0.2.times. SSC at 60.degree. 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.
Example 3
Secretion and Post-Translational Modification of Dkk-3
[0339] 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.
[0340] hDkk-3 Expression Constructs
[0341] Expression constructs for two forms of hDkk-3 were prepared
using the mammalian expression vector pMET-stop. Form-1 comprised a
cDNA incorporating the complete 350aa hDkk-3 protein coding
sequence (hDkk-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 SalI restriction sites.
[0342] Trial Transfection--Small Scale Expression
[0343] Expression constructs for hDkk-3flag.long and
hDkk-3flag.short were transfected into 293T cells using 10 .mu.l of
lipofectamine (GIBCO/BRL) and 2 .mu.g of DNA per well of a 6-well
plate of cells which were 70-80% confluent. After 5 hours at
37.degree. C., cells were fed with 1 ml of 20%FCS/DMEM. After
incubation overnight at 37.degree. C., cells were conditioned in 1
ml OptiMEM for 48 hours at 37.degree. 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 hDkk-3 tested are greater than predicted from the amino
acid sequence, suggesting that the hDkk-3 proteins secreted by 293T
cells may be glycosylated. This is consistent with the presence of
four potential sites for N-linked 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).
[0344] Deglycosylation of hDkk-3
[0345] 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, 1 mL samples of 293T cell
supernatants collected 72 hours after transfection with the
appropriate constructs were incubated with 50 .mu.L anti-flag M2
agarose beads (Sigma) for 16 hrs at 4.degree. 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 .mu.L of 2OmM sodium
phosphate, pH 7.5, 50 mM 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 10 .mu.L aliquots
which were diluted with 10 .mu.L incubation buffer, 5.mu.L 5% NP-40
and then with either 5.mu.L N-Glycanase (Oxford Glycosystems) in
enzyme buffer (20 mM Tris-HCl, lmM EDTA, 50 mM NaCl, 0.02% sodium
azide pH 7.5) or with enzyme buffer alone as control. After 18
hours at 37.degree. 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 (Biomax
MR2 film, Kodak).
[0346] 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 hDkk-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 N-Glycanase treatment. This form was only
observed after overnight incubation of the samples and may be a
non-specific degradation product.
[0347] Large Scale hDkk-3 Protein Production
[0348] For scale-up of hDkk-3flag.long protein expression,
30.times.150 mM plates of 293T cells at 70-80% confluence were
transfected with 27 .mu.g DNA, 100 .mu.l lipofectamine in 18 ml
OptiMEM for 5 hours at 37.degree. C. 18 ml of 10%FCS/DMEM was added
to each plate and incubated overnight at 37.degree. 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.degree. C. for 72 hours. Conditioned medium was
harvested, spun at 4000 rpm for 30 min. at 4.degree. C., and
filtered through a 0.45 micron filter unit. 1100 ml was passed over
a 1.6.times.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
[0349] 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.
[0350] Isolation and Analysis of a Human Dkk-4 cDNA
[0351] 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 FIG. 2.
[0352] 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.
[0353] Tissue Distribution of hDkk-4
[0354] 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 hDkk-4 specific PCR
primers. Using such primers, products were identified in libraries
prepared from cerebellum, activated human T-lymphocytes, lung and
esophagus.
[0355] Secretion and Post-Translational Modification of human
Dkk-4
[0356] 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.
[0357] To determine the possible cause of heterogeneity in the size
of secreted hDkk-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.times.30 mm, PE Biosystems);
the column was then washed with PBS, pH 7.4 and flag-tagged protein
eluted with 200 mM glycine, pH 3.0. Eluted fractions with 280nm
absorbance greater than background were analyzed by SDS-PAGE and
western blot. Purified Dkk-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 (Spherogel micro PTH
3-micron column) and determined by separation and peak height as
compared to standards.
[0358] 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:38) 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 11 4/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.
[0359] 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) hDkk-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.
Example 5
Isolation and Characterization of hDkk-1
[0360] 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.
[0361] Identification of a Human Dkk-1 cDNA
[0362] 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 FIG. 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.
[0363] Tissue Distribution of hDkk-1
[0364] 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 .about.1.8 kb hDkkl mRNA was detected at high levels in
human placenta, but not in other tissues tested.
[0365] Secretion and Post-Translational Modification of hDkk-1
[0366] Flag epitope-tagged human Dkk-1 protein was transiently
overexpressed in 293T cells and analyzed as described previously.
hDkk-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 kD. 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.
Example 6
Isolation and Characterization of hDkk-2
[0367] In this example, the isolation and characterization of the
gene encoding human Dkk-2 (also referred to as "hDkk-2", "Cysteine
Rich Secreted Protein-4", "CRSP-4" or "CRISPY-4") is described.
[0368] Isolation of a Human Dkk-2 cDNA
[0369] Using the hDkk-3 sequence to query the dbEST database, a
clone having similarity to a portion of hDkk-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:1 1. 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 fthul33 was deposited
with the ATCC as Accession No. ______. hDkk-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.
[0370] Tissue Distribution of hDkk-2
[0371] 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
hDkk-2. Of the tissues tested, hDkk-2 mRNA expression was highest
in heart, brain, placenta, lung, and skeletal muscle. hDkk-2
transcripts of approximately 4.0 and 4.5 kb were observed.
[0372] Secretion and Post-translational Modification of hDkk-2
[0373] 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
[0374] 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.
[0375] Identification of a Human and Murine Soggy-1 cDNAs
[0376] 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 FIG. 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 FIG. 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, 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 (FIG. 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").
[0377] Tissue Distribution of Soggy-1
[0378] 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.
[0379] In situ analysis was performed as described in Example 1.
For detection of murine Soggy-1, the following primers were
used:
[0380] mSoggyforward 5'-ACCTGCAA TGTGTCGACTGAG-3' (SEQ ID NO:32),
and reverse 5'-CACTTACAGCTGTTGGGATG-3' (SEQ ID NO:33).
[0381] 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
E13.5-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.
[0382] Secretion and Post-Translational Modification of Soggy
Proteins
[0383] 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
[0384] 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-1 correspond to a murine
and Xenopus proteins set forth in Glinks et al., supra, and having
Accession Nos: AF030433 and AF030434, respectively. Likewise cDkk-3
has Accession No. D263 11
2TABLE II 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: CLF- hDkk-3 hDkk-4 hDkk-1 hDkk-2 mDkk-1 xDkk-1 EST 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
[0385]
3TABLE III 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: CLF- hDkk-3 hDkk-4 hDkk-1
hDkk-2 mDkk-1 xDkk-1 EST 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
[0386]
4TABLE IV 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
[0387] 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 FIG. 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
FIG. 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 hDkk-3 and hSoggy-1, and sites of N-glycosylation are
indicated.
[0388] As demonstrated at least in FIGS. 6 and 9, human Dkks 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 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 hDkk3 and
are conserved in chicken and mouse Dkk-3. These sites are not
conserved in other Dkk famiy members. hDkk1 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 hDkk possesses several potential dibasic
proteolytic cleavage sites, suggesting the proteins may be subject
to post-translational processing. hDkk3 is the most divergent of
the four human Dkks and possesses an extended N-terminal unique
region which precedes CRD-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
[0389] This Example describes the functional activities of the hDkk
and Soggy proteins of the present invention.
[0390] Xenopus Embryo Culture and RNA Microinjections
[0391] Eggs were obtained from Xenopus females injected with 700
units of human chorionic gonadotropin, fertilized in vitro and
cultured in 0.1.times. 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: hDkk-1-pCS2, hDkk-2-pCS2, hDkk-3-pCS2, hDkk-4-pCS2,
hSoggy-pCS2, Xwnt8 (Christian etal, (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 et al. (1998) Mech. Devel. 74:145-157), Xdsh-pXT7
(Sokol, et al. (1995) Mech. Devel. 74:145-157). Protein expression
from all 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 10 nl of a solution containing 2-4pg of Xwnt8 mRNA,
2.5-5 pg of Xwnt3a mRNA or 10 pg of Xwnt2B mRNA as described (Itoh
et al. (1995) supra.). The effect of Dkk RNAs was tested by
coinjecting Wnt mRNAs with 2.5 ng of hDkk mRNAs. For studies of
Frizzled and Dhshevelled, 5 ng Fz8 and 1 ng Xdsh mRNAs were
injected as indicated. After injections, embryos were cultured in
3% Ficoll 400 (Pharmacia), 0.5.times. MMR. 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.
[0392] Inhibition of Secondary Axis Induction by hDkk-1 and hDkk-4
in Xenopus Embryos
[0393] 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 et al. (1997) supra; Wang et al. (1997) supra;
Salic et al. (1997) supra; Mayr et al. (1997) supra; Finch et al.
(1997) supra).
[0394] 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 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 et al. (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.
[0395] Assay for Inhibition of Secondary Axis Induction by hDkk-2,
hDkk-3 and hSoggy-1 in Xenopus Embryos
[0396] 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
[0397] This example describes the making of polyclonal antibodies
specific for hDkk-1, hDkk-4, hDkk-1, hDkk-2, and hSoggy-1.
[0398] 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:
5 hDkk-3 peptide #44 FREVEELMEDTQHKL peptide #46 GSFMEEVRQELEDLE
hDkk-4 peptide #91 HAEGTTGHPVQENQP hDkk-1 peptide #93
GNKYQTIDNYQPYPC hDkk-2 peptide #56 GHYSNHDLGWQNLGR hSoggy-1 peptide
#58 LQAIRDGLRKGTHKD
[0399] 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).
[0400] Antibodies were generated in New Zealand white rabbits over
a 1 0-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 0.1 mg 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.degree. C.
[0401] 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
.about.0.25mg/ml. The anti-peptide titers were determined using
ELISA methodology with free peptide bound in solid phase (1
.mu.g/well). Detection was obtained using biotinylated anti-rabbit
IgG, HRP-SA conjugate, and ABTS.
[0402] 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-1-transfected 293T cells.
[0403] 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 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.
[0404] 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 et al., 1990). Wnts 5b and 13 are more broadly
expressed (Gavin et al. (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.
[0405] 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 hDkk-4. A single putative site in hDkk-1 does not appear
to be utilized in 293T cells and may 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.
[0406] 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 E15-E16 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.
[0407] Equivalents
[0408] 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.
Sequence CWU 1
1
38 1 2479 DNA Homo sapiens CDS (38)..(1087) 'n' at position 1146
may be any nucleotide 1 ggcacgaggg ggcggcggct gcgggcgcag agcggag
atg cag cgg ctt ggg gcc 55 Met Gln 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 10 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 Gln 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 Gln 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 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 Gln Thr Gly
Gln 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 Gln Phe Ala Ser Phe Gln Tyr 155 160
165 acc tgc cag cca tgc cgg ggc cag agg atg ctc tgc acc cgg gac agt
583 Thr Cys Gln Pro Cys Arg Gly Gln 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 Gln Leu Cys Val Trp Gly His Cys Thr
Lys Met 185 190 195 gcc acc agg ggc agc aat ggg acc atc tgt gac aac
cag agg gac tgc 679 Ala Thr Arg Gly Ser Asn Gly Thr Ile Cys Asp Asn
Gln Arg Asp Cys 200 205 210 cag ccg ggg ctg tgc tgt gcc ttc cag aga
ggc ctg ctg ttc cct gtg 727 Gln Pro Gly Leu Cys Cys Ala Phe Gln 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 Gln Pro His Ser His 265 270 275 agc
ctg gtg tat gtg tgc aag ccg acc ttc gtg ggg agc cgt gac caa 919 Ser
Leu Val Tyr Val Cys Lys Pro Thr Phe Val Gly Ser Arg Asp Gln 280 285
290 gat ggg gag atc ctg ctg ccc aga gag gtc ccc gat gag tat gaa 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 Gln Glu Leu Glu
Asp Leu Glu Arg 315 320 325 agc ctg act gaa gag atg gcg ctg agg gag
cct gcg gct gcc gcc gct 1063 Ser Leu Thr Glu Glu Met Ala Leu Arg
Glu Pro Ala Ala Ala Ala Ala 330 335 340 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 cacatccctc 1957 taaaagtaaa ctactgttag
gaacagcagt gttctcacag tgtggggcag ccgtccttct 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 2 350 PRT Homo
sapiens 2 Met Gln Arg Leu Gly Ala Thr Leu Leu Cys Leu Leu Leu Ala
Ala Ala 1 5 10 15 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 325 330 335 Pro Ala Ala
Ala Ala Ala Ala Leu Leu Gly Arg Glu Glu Ile 340 345 350 3 1050 DNA
Homo sapiens CDS (1)..(1050) 3 atg cag cgg ctt ggg gcc acc ctg ctg
tgc ctg ctg ctg gcg gcg gcg 48 Met Gln 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 ccg 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 ccg gct
ctc agc tac ccg cag gag gag gcc acc ctc aat 144 Lys Pro Gly Pro Ala
Leu Ser Tyr Pro Gln 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 Gln His Lys 50 55 60 ttg
cgc agc 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 agc 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 Gln Thr
Gly Gln Met Val Phe 115 120 125 tca gag aca gtt atc aca tct gtg gga
gac gaa gaa ggc aga agg agc 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 agc atg tac tgc cag 480 His Glu Cys Ile Ile Asp Glu
Asp Cys Gly Pro Ser Met Tyr Cys Gln 145 150 155 160 ttt gcc agc ttc
cag tac acc tgc cag cca tgc cgg ggc cag agg atg 528 Phe Ala Ser Phe
Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met 165 170 175 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 Gln Leu Cys Val Trp 180 185 190
ggt cac tgc acc aaa atg gcc acc agg ggc agc 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 Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln
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 agc cgg ctt ctg gac
ctc atc acc tgg gag cta 768 Cys His Asp Pro Ala Ser Arg Leu Leu Asp
Leu Ile Thr Trp Glu Leu 245 250 255 gag cct gat gga gcc ttg gac cga
tgc cct 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 agc cac
agc ctg gtg tat gtg tgc aag ccg acc ttc 864 Cys Gln Pro His Ser His
Ser Leu Val Tyr Val Cys Lys Pro Thr Phe 275 280 285 gtg ggg agc cgt
gac caa gat ggg gag atc ctg ctg ccc aga gag gtc 912 Val Gly Ser Arg
Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val 290 295 300 ccc gat
gag tat gaa gtt ggc agc ttc atg gag gag gtg cgc cag gag 960 Pro Asp
Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu 305 310 315
320 ctg gag gac ctg gag agg agc 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 cct gcg gct gcc gcc gct 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 4 848 DNA Homo sapiens CDS (125)..(796) 4 gaattcggca
cgagagacga cgtgctgagc tgccagctta gtggaagctc tgctctgggt 60
ggagagcagc ctcgctttgg tgacgcacag tgctgggacc ctccaggagc cccgggattg
120 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 agc 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 Gln Cys Leu Ser
Asp Thr Asp Cys 35 40 45 aat acc aga aag ttc tgc ctc cag ccc cgc
gat gag aag ccg ttc tgt 313 Asn Thr Arg Lys Phe Cys Leu Gln 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 Gln Arg Asp Ala Met Cys 65 70 75 tgc cct 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 Gln Leu Asp Glu Gln 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 Gln Glu Asn Gln 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 Gln Gly Arg Lys Gly Gln 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 Gln 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
Gln Ala Pro Glu Ile Phe 180 185 190 cag cgt tgc gac tgt ggc cct gga
cta ctg tgt cga agc caa ttg acc 745 Gln Arg Cys Asp Cys Gly Pro Gly
Leu Leu Cys Arg Ser Gln Leu Thr 195 200 205 agc aat cgg cag cat gct
cga tta aga gta tgc caa aaa ata gaa aag 793 Ser Asn Arg Gln His Ala
Arg Leu Arg Val Cys Gln Lys Ile Glu Lys 210 215 220 cta taaatatttc
aaaataaaga agaatccaca ttgcaaaaaa aaaaaaaaaa aa 848 Leu 5 224 PRT
Homo sapiens 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 Gln 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 6
672 DNA Homo sapiens CDS (1)..(672) 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 Gln 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 Gln
Arg Asp Ala Met Cys Cys 65 70 75 80 cct 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
Gln Leu Asp Glu Gln 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 Gln Glu Asn Gln 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 Gln Gly Arg Lys Gly Gln Glu Gly Glu Ser 130 135 140
tgt ctg aga act ttt gac tgt ggc cct 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 Gln
Val Cys 165 170 175 tcc 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 Gln Ala Pro
Glu Ile Phe Gln 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 Gln 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 Gln His Ala Arg Leu Arg
Val Cys Gln Lys Ile Glu Lys Leu 210 215 220 7 1536 DNA Homo sapiens
CDS (109)..(906) 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 gct 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 5 10 15 gct ctc ggc
ggc cac cct ctg ctg gga gtg agc gcc acc ttg aac tcg 213 Ala Leu Gly
Gly His Pro Leu Leu Gly Val Ser Ala Thr Leu Asn Ser 20 25 30 35 gtt
ctc aat tcc aac gct 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 gct gcg ggg cac cca ggc tct gca gtc agc gcc 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 gct agt 405 Pro Cys Ala Glu Asp Glu Glu Cys Gly Thr Asp Glu
Tyr Cys Ala Ser 85 90 95 ccc acc cgc gga ggg gac gca ggc gtg caa
atc tgt ctc gcc 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 gct 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 tct tct 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 gaa att
gag gaa acc atc act gaa agc ttt ggt aat gat cat agc 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 tcc aga aga acc acc ttg tct tca aaa atg tat 645 Thr
Leu Asp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser Lys Met Tyr 165 170
175 cac acc aaa gga caa gaa ggt tct gtt tgt ctc cgg tca tca gac tgt
693 His Thr Lys Gly Gln 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 Gln 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 Gln 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 Gln Lys Asp
His His Gln Ala Ser Asn Ser Ser Arg 245 250 255 ctt cac act tgt cag
aga cac taaaccagct atccaaatgc agtgaactcc 936 Leu His Thr Cys Gln
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 8 266
PRT Homo sapiens 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 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 Gln Thr Ile Asp Asn Tyr 65 70 75 80 Gln 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 Gln 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 Gln 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 Gln Val Cys Thr Lys His Arg 210 215 220
Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gln Arg Cys Tyr Cys Gly 225
230 235 240 Glu Gly Leu Ser Cys Arg Ile Gln Lys Asp His His Gln Ala
Ser Asn 245 250 255 Ser Ser Arg Leu His Thr Cys Gln Arg His 260 265
9 798 DNA Homo sapiens CDS (1)..(798) 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 gta gcg gcg gct ctc
ggc ggc cac cct ctg ctg gga gtg agc gcc acc 96 Val Ala Ala Ala Leu
Gly Gly His Pro Leu Leu Gly Val Ser Ala Thr 20 25 30 ttg aac tcg
gtt ctc aat tcc aac gct atc aag aac ctg ccc cca ccg 144 Leu Asn Ser
Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro Pro Pro 35 40 45 ctg
ggc ggc gct gcg ggg cac cca ggc tct gca gtc agc gcc gcg ccg 192 Leu
Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala Pro 50 55
60 gga atc ctg tac ccg ggc ggg aat aag tac cag acc att gac aac tac
240 Gly Ile Leu Tyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr
65 70 75 80 cag ccg tac ccg 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 gct 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
Gln Ile Cys Leu 100 105 110 gcc tgc agg aag cgc cga aaa cgc tgc atg
cgt cac gct atg tgc tgc 384 Ala Cys Arg Lys Arg Arg Lys Arg Cys Met
Arg His Ala Met Cys Cys 115 120 125 ccc ggg aat tac tgc aaa aat gga
ata tgt gtg tct tct gat caa aat 432 Pro Gly Asn Tyr Cys Lys Asn Gly
Ile Cys Val Ser Ser Asp Gln Asn 130 135 140 cat ttc cga gga gaa att
gag gaa acc atc act gaa agc 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 agc
acc ttg gat ggg tat tcc aga aga acc acc ttg tct 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 tct gtt tgt ctc cgg tca 576 Lys
Met Tyr His Thr Lys Gly Gln Glu Gly Ser Val Cys Leu Arg Ser 180 185
190 tca gac tgt gcc tca gga ttg tgt tgt gct aga cac ttc tgg tcc aag
624 Ser Asp Cys Ala Ser Gly Leu Cys Cys Ala Arg His Phe Trp Ser Lys
195 200 205 atc tgt aaa cct gtc ctg aaa gaa ggt caa gtg tgt acc aag
cat agg 672 Ile Cys Lys Pro Val Leu Lys Glu Gly Gln Val Cys Thr Lys
His Arg 210 215 220 aga aaa ggc tct cat gga cta gaa ata ttc cag cgt
tgt tac tgt gga 720 Arg Lys Gly Ser His Gly Leu Glu Ile Phe Gln Arg
Cys Tyr Cys Gly 225 230 235 240 gaa ggt ctg tct tgc cgg ata cag aaa
gat cac cat caa gcc agt aat 768 Glu Gly Leu Ser Cys Arg Ile Gln Lys
Asp His His Gln Ala Ser Asn 245 250 255 tct tct agg ctt cac act tgt
cag aga cac 798 Ser Ser Arg Leu His Thr Cys Gln Arg His 260 265 10
702 DNA Homo sapiens CDS (1)..(537) 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 Gln Gly 1 5 10 15 tca tcg 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 agc atc tta acc cct cac atc ccg gct 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 Gln 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 gct 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 tct cat ggg 432 His Gln Gly Glu Val Cys Thr Lys
Gln Arg Lys Lys Gly Ser His Gly 130 135 140 ctg gaa att ttc cag cgt
tgc gac tgt gcg aag ggc ctg tct tgc aaa 480 Leu Glu Ile Phe Gln 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 cag
aaa att tgatcaccat tgaggaacat catcaattgc agactgtgaa 577 Gln Lys Ile
gttgtgtatt taatgcatta tagcatggtg gaaaataagg ttcagatgca gaagaatggc
637 taaaataaga aacgtgataa gaatatagat gatcacaaaa aaaaaaaaaa
aaaagatgcg 697 gccgc 702 11 179 PRT Homo sapiens 11 Glu Phe Gly Thr
Arg Val Gly Arg Tyr Cys His Ser Pro His Gln 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 Gln 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 Gln Gly Glu Val
Cys Thr Lys Gln Arg Lys Lys Gly Ser His Gly 130 135 140 Leu Glu Ile
Phe Gln 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 Gln Lys Ile 12 537 DNA Homo sapiens CDS (1)..(537) 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 Gln Gly 1 5 10
15 tca tcg 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 agc atc tta acc cct cac atc ccg
gct 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 Gln 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 gct
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 tct cat ggg 432 His Gln
Gly Glu Val Cys Thr Lys Gln Arg Lys Lys Gly Ser His Gly 130 135 140
ctg gaa att ttc cag cgt tgc gac tgt gcg aag ggc ctg tct tgc aaa 480
Leu Glu Ile Phe Gln 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 cag aaa att 537 Gln Lys Ile 13 928 DNA Homo
sapiens CDS (75)..(800) 13 ctcgaggcca aaattcggca cgaggccggg
ctgtggtcta gcataaaggc ggagcccaga 60 agaaggggcg gggt atg gga gaa gcc
tcc cca cct 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 tcc 158 His Leu Leu Val Leu Leu Leu Leu Leu Ser Thr
Leu Val Ile Pro Ser 15 20 25 gct gca gct cct
atc cat gat gct gac gcc caa gag agc tcc ttg ggt 206 Ala Ala Ala Pro
Ile His Asp Ala Asp Ala Gln Glu Ser Ser Leu Gly 30 35 40 ctc aca
ggc ctc cag agc cta ctc caa ggc ttc agc cga ctt ttc ctg 254 Leu Thr
Gly Leu Gln Ser Leu Leu Gln Gly Phe Ser Arg Leu Phe Leu 45 50 55 60
aaa ggt aac ctg ctt cgg ggc ata gac agc tta ttc tct 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 cct 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 Gln
Glu 80 85 90 cac cag ctg ggg aac aac acc ctc tcc agc cac ctc cag
atc gac aag 398 His Gln Leu Gly Asn Asn Thr Leu Ser Ser His Leu Gln
Ile Asp Lys 95 100 105 atg acc gac aac aag aca gga gag gtg ctg atc
tcc 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 tcc att caa cca gcg gag ggg agc
ttc gag ggt gat ttg aag gta 494 Ala Ser Ile Gln 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 ccc atc cag aag gcc acg 542 Pro Arg Met Glu Glu Lys
Glu Ala Leu Val Pro Ile Gln Lys Ala Thr 145 150 155 gac agc ttc cac
aca gaa ctc cat ccc 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 tcc cac cag gat gcc ctg gag ggc ggc cac 638 Lys Leu
Pro Arg Arg Arg Ser His Gln Asp Ala Leu Glu Gly Gly His 175 180 185
tgg ctc agc gag aag cga cac cgc ctg cag gcc atc cgg gat gga ctc 686
Trp Leu Ser Glu Lys Arg His Arg Leu Gln Ala Ile Arg Asp Gly Leu 190
195 200 cgc aag ggg acc cac aag gac gtc cta gaa gag ggg acc gag agc
tcc 734 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 Gln Leu 240 tagcccccat
cagaccctgc cccaagcacc atatggaaat aaagttcttt cttacatcta 890
aaaaaaaaaa aaaaaaaaaa aaaaaaattg gcggccgc 928 14 242 PRT Homo
sapiens 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 Gln Glu Ser Ser
Leu Gly Leu Thr Gly Leu 35 40 45 Gln Ser Leu Leu Gln 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 Gln Glu His Gln Leu Gly 85 90 95 Asn Asn
Thr Leu Ser Ser His Leu Gln 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 Gln 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 Gln 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 Gln Asp Ala Leu
Glu Gly Gly His Trp Leu Ser Glu 180 185 190 Lys Arg His Arg Leu Gln
Ala Ile Arg Asp Gly Leu Arg Lys Gly Thr 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 Gln Leu 15 726 DNA Homo sapiens CDS (1)..(726) 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 Gln 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 Gln Ser Leu Leu Gln 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 cct 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
Gln Glu His Gln 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
Gln 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 Gln 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 Glu Lys
Glu Ala Leu Val Pro Ile Gln 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 Gln 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 Gln 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 Gln Leu
16 2380 DNA Homo sapiens CDS (109)..(1155) 16 gtcgacccac gcgtccgctg
tggcagccca gctaccggtc gtgaccagat ccagcttgca 60 gctcagcttt
gttcattcga attgggcggc ggccagcgcg gaacaaac atg cag cgg 117 Met Gln
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 5 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 Gln 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
Gln His Lys Leu Arg Ser 55 60 65 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 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 Gln 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 Gln Glu Met 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 17 349 PRT Homo sapiens 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 Pro Asp
Glu Tyr Glu Asp Val Gly Phe Ile Gly Glu Val Arg Gln Glu 305 310 315
320 Leu Glu Asp Leu Glu Arg Ser Leu Ala Gln Glu Met Ala Phe Glu Gly
325 330 335 Pro Ala Pro Val Glu Ser Leu Gly Gly Glu Glu Glu Ile 340
345 18 1047 DNA Homo sapiens CDS (1)..(1047) 18 atg cag cgg ctc ggg
ggt att ttg ctg tgt aca ctg ctg gcg gcg gcg 48 Met Gln 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 Gln 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 Gln Glu Val His Lys Ile Thr Asn
Asn Gln Ser Gly Gln 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 Gln 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 Gln Pro Cys Arg Asp Gln Gln 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 Gln 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 Gln Lys Ala Thr Lys Gly Gly Asn Gly Thr Ile Cys 195 200 205 gac
aac cag agg gat tgc cag cct ggc ctg tgt tgt gcc ttc caa aga 672 Asp
Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys Ala Phe Gln 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 agc cag ctg ctg gat ctc atc acc
tgg gaa ctg 768 Cys His Asp Pro Thr Ser Gln Leu Leu Asp Leu Ile Thr
Trp Glu Leu 245 250 255 gag cct gaa gga gct 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 agc cac agt ctg gtg
tac atg tgc aag cca gcc ttc 864 Cys Gln Pro His Ser His Ser Leu Val
Tyr Met Cys Lys Pro Ala Phe 275 280 285 gtg ggc agc cat gac cac agt
gag gag agc cag ctg ccc agg gag gcc 912 Val Gly Ser His Asp His Ser
Glu Glu Ser Gln 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 Gln Glu 305 310 315 320 ctg gaa
gac ctg gag cgg agc cta gcc cag gag atg gca ttt gag ggg 1008 Leu
Glu Asp Leu Glu Arg Ser Leu Ala Gln Glu Met Ala Phe Glu Gly 325 330
335 cct gcc cct 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 19 8 PRT
synthtic construct 19 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 20 3696
DNA Homo sapiens CDS (712)..(1500) 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 agttgaagga 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 ccctcccttt gcattcccac ccctccgggc
tttgcgtctt cctggggacc c cct cgc 717 Pro Arg 1 cgg gag atg gcc gcg
ttg atg cgg agc aag gat tcg tcc tgc tgc ctg 765 Arg Glu Met Ala Ala
Leu Met Arg Ser Lys Asp Ser Ser Cys Cys Leu 5 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 tcg cgg
gcc aaa ctc aac tcc atc aag tcc 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 gcc gcc aat cga tct 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 gcc tac cct
tgt 957 Phe Gly Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Ala Tyr Pro
Cys 70 75 80 agc agt gat aag gag tgt gaa gtt ggg agg tat tgc cac
agt ccc cac 1005 Ser Ser Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys
His Ser Pro His 85 90 95 caa gga tca tcg gcc tgc atg gtg tgt cgg
aga aaa aag aag cgc tgc 1053 Gln 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 agc atc tta acc cct cac atc ccg 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 Gln 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 tct 1389 Val Leu
His Gln Gly Glu Val Cys Thr Lys Gln Arg Lys Lys Gly Ser 215 220 225
cat ggg ctg gaa att ttc cag cgt tgc gac tgt gcg aag ggc ctg tct
1437 His Gly Leu Glu Ile Phe Gln Arg Cys Asp Cys Ala Lys Gly Leu
Ser 230 235 240 tgc aaa gta tgg aaa gat gcc acc tac tcc tcc 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 Gln 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
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 21 263 PRT
Homo sapiens 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 22 789 DNA Homo
sapiens CDS (1)..(789) 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 Gln 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 cct ggt
cag gcc gcc aat cga tct gcg ggc atg tac caa gga 192 Glu Thr Pro Gly
Gln Ala Ala Asn Arg Ser Ala Gly Met Tyr Gln 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 Gln Ala Tyr 65 70 75 80
cct 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 Gln 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 cct cac atc ccg 432 Gly Ile Cys Ile Pro Val Thr Glu Ser Ile Leu
Thr Pro His Ile Pro 130 135 140 gct 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
Gln 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 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 Gln Gly Glu Val Cys Thr Lys Gln 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 Gln 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 Gln Lys Ile 260 23 54 PRT Artificial Sequence Description
of Artificial Sequence consensus sequence 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 50 24 123 PRT Artificial Sequence
Description of Artificial Sequence consensus sequence 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 25 87 PRT Homo sapiens 25 Ile Asn
Leu Glu Asn Gly Glu Leu Cys Met Asn Ser Ala Gln Cys Lys 1 5 10 15
Ser Asn Cys Cys Gln 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 Asp Ala Gly Arg Ser Lys Gln 85 26
835 DNA Mus musculus CDS (57)..(746) 26 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 tct
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 Gln Gln Asn Thr Ser 20 25 30
ggg ttc ctg ggc ctt cag agg ctt ctc caa agc ttt agt cga ctg ttc 203
Gly Phe Leu Gly Leu Gln Arg Leu Leu Gln 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 cct agg aac ttc cat cag gaa
gag aac cag 299 Met Asp Phe Arg Asp Leu Pro Arg Asn Phe His Gln Glu
Glu Asn Gln 70 75 80 gag cac aga atg ggc aac cat acc ctc tcc agc
cac cta cag ata gac 347 Glu His Arg Met Gly Asn His Thr Leu Ser Ser
His Leu Gln Ile Asp 85 90 95 aag gtg act gac aac cag aca ggg gag
gtg cac atc tcg gag aaa gtc 395 Lys Val Thr Asp Asn Gln Thr Gly Glu
Val His Ile Ser Glu Lys Val 100 105 110 gag gcc tcc att gag cca gaa
cgg aac ccg 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 ccg gtg cct gtg cag aag gtc
acc 491 Pro Lys Val Glu Ala Lys Glu Pro Pro Val Pro Val Gln Lys Val
Thr 130 135 140 145 gac agc ttg cac cca gag ccc cgg cag gtg gct ttc
tgg atc atg aag 539 Asp Ser Leu His Pro Glu Pro Arg Gln 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 Met Pro Arg Arg Arg Thr Gln Pro Asp Val
Gln 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
Gln 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 Gln 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 Gln Gln Leu 230 tatggaaata aaggttttct tacatctaaa aaaaaaaaaa
aaaaaaaaa 835 27 230 PRT Mus musculus 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 Gln Gln Asn Thr 20 25 30 Ser Gly
Phe Leu Gly Leu Gln Arg Leu Leu Gln Ser Phe Ser Arg Leu 35 40 45
Phe Leu Lys Asn Asp Leu Leu Arg Asp Leu Asp Asn Phe Phe Ser Ser 50
55 60 Pro Met Asp Phe Arg Asp Leu Pro Arg Asn Phe His Gln Glu Glu
Asn 65 70 75 80 Gln Glu His Arg Met Gly Asn His Thr Leu Ser Ser His
Leu Gln Ile 85 90 95 Asp Lys Val Thr Asp Asn Gln 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 Gln Lys Val 130 135 140 Thr Asp Ser Leu His
Pro Glu Pro Arg Gln Val Ala Phe Trp Ile Met 145 150 155 160 Lys Met
Pro Arg Arg Arg Thr Gln Pro Asp Val Gln Asp Gly Gly Arg 165 170 175
Trp Leu Ile Glu Lys Arg His Arg Met Gln 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 Gln Gln Leu 225 230 28 690 DNA
Mus musculus CDS (1)..(690) 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 tct gct ctc ccc atc
cat gat gtc gac tct cag cag aac acc 96 Ser Ser Ser Ala Leu Pro Ile
His Asp Val Asp Ser Gln Gln Asn Thr 20 25 30 tcc ggg ttc ctg ggc
ctt cag agg ctt ctc caa agc ttt agt cga ctg 144 Ser Gly Phe Leu Gly
Leu Gln Arg Leu Leu Gln 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 Gln Glu Glu Asn 65 70
75 80 cag gag cac aga atg ggc aac cat acc ctc tcc agc cac cta cag
ata 288 Gln Glu His Arg Met Gly Asn His Thr Leu Ser Ser His Leu Gln
Ile 85 90 95 gac aag gtg act gac aac cag aca ggg gag gtg cac atc
tcg gag aaa 336 Asp Lys Val Thr Asp Asn Gln Thr Gly Glu Val His Ile
Ser Glu Lys 100 105 110 gtc gag gcc tcc att gag cca gaa cgg aac ccg
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
ccg gtg cct gtg cag aag gtc 432 Val Pro Lys Val Glu Ala Lys Glu Pro
Pro Val Pro Val Gln Lys Val 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 Gln 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 Gln Pro Asp Val Gln 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 Gln 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 Gln 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 Gln Gln Leu
225 230 29 51 PRT Artificial Sequence 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 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 50 30
20 DNA Artificial Sequence Description of Artificial Sequence
primer 30 cagtgagtgc tgtggagacc 20 31 20 DNA Artificial Sequence
Description of Artificial Sequence primer 31 tcttcagtca ggctcctctc
20 32 21 DNA Artificial Sequence Description of Artificial Sequence
primer 32 acctgcaatg tgtcgactga g 21 33 20 DNA Artificial Sequence
Description of Artificial Sequence primer 33 cacttacagc tgttgggatg
20 34 10 PRT Homo sapiens Xaa at position 1 may be any amino acid
34 Xaa Val Leu Asp Phe Asn Asn Ile Arg Ser 1 5 10 35 10 PRT Homo
sapiens 35 Ser Gln Gly Arg Lys Gly Gln Glu Gly Ser 1 5 10 36 272
PRT Mus musculus 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 Gln 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 Gln
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 Gln 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 Gln 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 Gln 210 215 220
Val Cys Thr Lys His Lys Arg Lys Gly Ser His Gly Leu Glu Ile Phe 225
230 235 240 Gln Arg Cys Tyr Cys Gly Glu Gly Leu Ala Cys Arg Ile Gln
Lys Asp 245 250 255 His His Gln Ala Ser Asn Ser Ser Arg Leu His Thr
Cys Gln Arg His 260 265 270 37 259 PRT Xenopus laevis 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 Gln 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 Gln 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
Gln Glu Arg Phe Gln His Gln 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 Gln 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 Gln Val Cys Thr Lys His Arg Arg
Lys Gly Ser His Gly Leu Glu 210 215 220 Ile Phe Gln Arg Cys His Cys
Gly Ala Gly Leu Ser Cys Arg Leu Gln 225 230 235 240 Lys Gly Glu Phe
Thr Thr Val Pro Lys Thr Ser Arg Leu His Thr Cys 245 250 255 Gln Arg
His 38 350 PRT Gallus gallus 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 Gln His Lys Leu Arg Asn Ala Val Gln 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 Gln Thr His Gln 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
Gln Phe Ser Thr Phe Glu Tyr Lys Cys 145 150 155 160 Gln Pro Cys Lys
Thr Gln His Thr His Cys Ser Arg Asp Val Glu Cys 165 170 175 Cys Gly
Asp Gln 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 Gln His Asp Cys Asn Pro 195
200 205 Gly Thr Cys Cys Ala Phe Gln 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 Ile Thr Trp Glu Leu Glu Pro
Asp Gly Val Leu Glu Arg 245 250 255 Cys Pro Cys Ala Ser Gly Leu Ile
Cys Gln Pro Gln 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 Gln Glu 305 310 315
320 Val Arg Lys Glu Leu Glu Ser Leu Glu Asp Gln 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
* * * * *
References