U.S. patent application number 10/068134 was filed with the patent office on 2002-10-24 for 22012, a novel human carboxypeptidase.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Kapeller-Libermann, Rosana, MacBeth, Kyle J., Williamson, Mark.
Application Number | 20020156264 10/068134 |
Document ID | / |
Family ID | 23355185 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020156264 |
Kind Code |
A1 |
Kapeller-Libermann, Rosana ;
et al. |
October 24, 2002 |
22012, a novel human carboxypeptidase
Abstract
The present invention relates to a newly identified human
carboxypeptidase. The invention also relates to polynucleotides
encoding the carboxypeptidase. The invention further relates to
methods using the carboxypeptidase polypeptides and polynucleotides
as a target for diagnosis and treatment in carboxypeptidase-related
disorders. The invention further relates to drug-screening methods
using the carboxypeptidase polypeptides and polynucleotides to
identify agonists and antagonists for diagnosis and treatment. The
invention further encompasses agonists and antagonists based on the
carboxypeptidase polypeptides and polynucleotides. The invention
further relates to procedures for producing the carboxypeptidase
polypeptides and polynucleotides.
Inventors: |
Kapeller-Libermann, Rosana;
(Chestnut Hill, MA) ; MacBeth, Kyle J.; (Boston,
MA) ; Williamson, Mark; (Saugus, MA) |
Correspondence
Address: |
ALSTON & BIRD LLP
BANK OF AMERICA PLAZA
101 SOUTH TRYON STREET, SUITE 4000
CHARLOTTE
NC
28280-4000
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
23355185 |
Appl. No.: |
10/068134 |
Filed: |
February 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10068134 |
Feb 6, 2002 |
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09345469 |
Jun 30, 1999 |
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6369210 |
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Current U.S.
Class: |
536/23.2 ;
435/226; 435/320.1; 435/325; 435/69.1 |
Current CPC
Class: |
A61P 19/04 20180101;
A61P 9/10 20180101; C07K 2319/00 20130101; C12N 9/48 20130101; A61P
9/00 20180101; A61P 1/00 20180101; A61P 9/02 20180101; A61P 25/28
20180101; A61P 7/04 20180101; A61P 9/12 20180101; A61P 25/14
20180101; A61P 35/00 20180101; A61P 33/02 20180101; A61P 39/02
20180101; A61P 7/02 20180101; A61P 1/16 20180101; A61P 31/04
20180101; A61P 25/00 20180101; A61P 29/00 20180101; A61P 37/08
20180101; A61P 11/00 20180101; A61P 43/00 20180101; A61P 35/04
20180101; A61P 1/12 20180101; A61P 1/04 20180101; A61P 3/02
20180101; A61K 38/00 20130101; A61P 33/00 20180101; A61P 15/06
20180101; A61P 31/18 20180101; A61P 31/22 20180101; A61P 25/16
20180101; A61P 3/00 20180101; A61P 3/10 20180101; A61P 37/02
20180101 |
Class at
Publication: |
536/23.2 ;
435/226; 435/320.1; 435/325; 435/69.1 |
International
Class: |
C12N 009/64; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
That which is claimed:
1. An isolated nucleic acid molecule selected from the group
consisting of: a) a nucleic acid molecule comprising a nucleotide
sequence having at least 85% sequence identity to the nucleotide
sequence of SEQ ID NO:2, or the nucleotide sequence of the cDNA
insert of the plasmid deposited with ATCC as Accession Number
PTA-1643, wherein said nucleotide sequence encodes a polypeptide
having carboxypeptidase activity; b) a nucleic acid molecule
comprising a fragment of at least 50 contiguous nucleotides of the
nucleotide sequence of SEQ ID NO:2, or the nucleotide sequence of
the cDNA insert of the plasmid deposited with ATCC as Accession
Number PTA-1643, c) a nucleic acid molecule which encodes a
fragment of a polypeptide comprising the amino acid sequence of SEQ
ID NO:1, or the amino acid sequence encoded by the cDNA insert of
the plasmid deposited with the ATCC as Accession Number PTA-1643,
wherein the fragment comprises at least 15 contiguous amino acids
of SEQ ID NO:2, or the amino acid sequence encoded by the cDNA
insert of the plasmid deposited with the ATCC as Accession Number
PTA-1643, d) a nucleic acid molecule which encodes a polypeptide
having carboxypeptidase activity comprising the amino acid sequence
of SEQ ID NO:1, or the amino acid sequence encoded by the cDNA
insert of the plasmid deposited with the ATCC as Accession Number
PTA-1643, wherein the nucleic acid molecule hybridizes to a nucleic
acid molecule comprising the complement of SEQ ID NO:2 under
stringent conditions; and e) a nucleic acid molecule comprising the
complement of a), b), c), or d).
2. The isolated nucleic acid molecule of claim 1, comprising a
nucleotide sequence having at least 95% sequence identity to the
nucleotide sequence of SEQ ID NO:2, or the nucleotide sequence of
the cDNA insert of the plasmid deposited with ATCC as Accession
Number PTA-1643, wherein said nucleotide sequence encodes a
polypeptide having carboxypeptidase activity, or a complement
thereof.
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
3.
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 polypeptide having carboxypeptidase activity which is encoded
by a nucleic acid molecule comprising a nucleotide sequence having
at least 85% identity to a nucleic acid comprising the nucleotide
sequence of SEQ ID NO:2, or the nucleotide sequence of the cDNA
insert of the plasmid deposited with ATCC as Accession Number
PTA-1643; b) a polypeptide having carboxypeptidase activity,
wherein the polypeptide is encoded by a nucleic acid molecule which
hybridizes to a nucleic acid molecule comprising the complement of
SEQ ID NO:2, or the nucleotide sequence of the cDNA insert of the
plasmid deposited with ATCC as Accession Number PTA-1643 under
stringent conditions; c) a fragment of a polypeptide comprising the
amino acid sequence of SEQ ID NO: 1, or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with the ATCC
as Accession Number PTA-1643, wherein the fragment comprises at
least 15 contiguous amino acids of SEQ ID NO: 1; and d) a
polypeptide having at least 85% sequence identity to the amino acid
sequence SEQ ID NO: 1, wherein the polypeptide has carboxypeptidase
activity.
9. The isolated polypeptide of claim 8 comprising the amino acid
sequence of SEQ ID NO:1.
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 a) a polypeptide comprising a fragment of the amino acid
sequence of SEQ ID NO: 1, or the amino acid sequence encoded by the
cDNA insert of the plasmid deposited with the ATCC as Accession
Number PTA-1643, wherein the fragment comprises at least 15
contiguous amino acids of SEQ ID NO:1, or the amino acid sequence
encoded by the cDNA insert of the plasmid deposited with the ATCC
as Accession Number PTA-1643; c) a polypeptide having
carboxypeptidase activity, wherein the polypeptide is encoded by a
nucleic acid molecule which hybridizes to a nucleic acid molecule
comprising the complement of SEQ ID NO:2, or the nucleotide
sequence of the cDNA insert of the plasmid deposited with ATCC as
Accession Number PTA-1643, d) a polypeptide having at least 85%
sequence identity to the amino acid sequence of SEQ ID NO:2,
wherein said polypeptide has carboxypeptidase activity; 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 a polypeptide of claim 8; and b)
determining whether the compound binds to the polypeptide 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
of claim 1 in a sample, comprising the steps of: 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.
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 the steps of: a) contacting a
polypeptide, or a cell expressing a polypeptide of claim 8 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
detecting of test compound/polypeptide binding; b) detection of
binding using a competition binding assay; c) detection of binding
using an assay for carboxypeptidase activity.
21. A method for modulating the activity of a polypeptide of claim
8 comprising contacting a polypeptide or a cell expressing a
polypeptide of claim 8 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 that modulates the activity of the
polypeptide.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/345,469, filed Jun. 30, 1999, which is hereby incorporated
in its entirety by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a newly identified human
carboxypeptidase. The invention also relates to polynucleotides
encoding the carboxypeptidase. The invention further relates to
methods using the carboxypeptidase polypeptides and polynucleotides
as a target for diagnosis and treatment in carboxypeptidase-related
disorders. The invention further relates to drug-screening methods
using the carboxypeptidase polypeptides and polynucleotides to
identify agonists and antagonists for diagnosis and treatment. The
invention further encompasses agonists and antagonists based on the
carboxypeptidase polypeptides and polynucleotides. The invention
further relates to procedures for producing the carboxypeptidase
polypeptides and polynucleotides.
BACKGROUND OF THE INVENTION
[0003] Proteolytic enzymes are involved in many cellular processes.
The carboxypeptidase family of enzymes catalyzes the cleavage of
C-terminal amino acids of peptides and proteins, altering their
biological activity. Lysosomal carboxypeptidase enzymes are highly
concentrated in lysosomes, but may also be active extracellularly
after their release from lysosomes in soluble form or bound to
transmembrane or other membrane-associated proteins.
Carboxypeptidases may cleave peptides in a sequence-specific
manner. For example, prolylcarboxypeptidases cleave only peptides
linked to proline residues (for example, des-Arg9-bradykinin,
angiotensin II). There is also evidence that these enzymes are
involved in terminating signal transduction by inactivating peptide
ligands after receptor endocytosis.
[0004] In contrast to endoproteases which cleave internal peptide
bonds of proteins and polypeptides, carboxypeptidases (CPs)
catalyze the cleavage of only the C-terminal peptide bond,
releasing one amino acid at a time. The two main groups of CPs
include serine CPs and metallo-CPs, the serine CPs containing a
signature trio of Ser, Asp, His in the active site. This trio is
also contained in prolylendopeptidase serine proteases. Serine CPs
include polycarboxypeptidase (PRCP) also referred to as
angiotensinase C; and deamidase, also referred to as cathepsin A
and lysosomal protective protein. See Skidgel et al. (1998)
Immunological Reviews 161:129-141.
[0005] Metallo-CPs contain a signature glutamic acid as the primary
catalytic residue and require zinc-binding for activity.
Metallo-CPs can be grouped by substrate specificity into CPA and
CPB types; the CPA type preferentially cleaving C-terminal
hydrophobic residues, and the CPB type cleaving only peptides with
C-terminal basic Arg or Lys residues. See R. A. Skidgel (1993) In:
Hooper NM, ed. Zinc Metalloproteases in Health and Disease, London:
Taylor & Francis, Ltd., p. 241-283.
[0006] CPM is a B type carboxypeptidase which is anchored on cell
membranes via gylcosylphosphatidylinositol (GPI) association with
its mildly hydrophobic stretch of 15 C-terminal amino acids. As in
many other proteins sharing this anchoring mechanism, CPM is
released from the membrane by bacterial
phosphatidylinositol-specific phospholipase C. Human CPM is a
glycoprotein of 426 amino acid residues with 43% identity to human
intracellular secretory granular CP (CPE), 41% with the active 50
kDa subunit of human plasma CPN, and 15% with bovine pancreatic CPA
or CPB. The active sites of these CPs contain conserved amino acid
residues corresponding to the zinc binding residues
His.sup.66Glu.sup.69 and His.sup.173, substrate binding residues
Arg.sup.137 and Tyr.sup.242, and the catalytic Glu.sup.264, as
designated for CPM. Sequence homologies around these conserved
residues is high, with an identity between CPs M, E and N of
approximately 70-90%. See Tan et al. (1989) J. Biol. Chem.
264:13165-13170; Deddish et al. (1990) J. Biol. Chem.
265:15083-15089; R. A. Skidgel (1993) In: Hooper NM, ed. Zinc
Metalloproteases in Health and Disease, London: Taylor &
Francis, Ltd., p. 241-283. CPM has been mapped to the chromosomal
location of chromosome 12q13-q15 which is associated with a variety
of solid tumors.
[0007] The optimal pH range of CPM is in the neutral range of
6.5-7.5. As no endogenous inhibitors are known for CPM, the enzyme
is considered to be constitutively active. Synthetic inhibitors
including Arg analogs DL-2
mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA) and
guanidinoethylmercaptosuccinic acid (GEMSA) inhibit CPM. See R. A.
Skidgel (1991) In: Conn PM, ed. Methods in Neurosciences: Peptide
Technology Vol. 6, Orlando: Academic Press, p. 373-385; Plummer et
al. (1981) Biochem. Biophys. Res. Comm. 98: 448-254.
[0008] As with other B type regulatory CPs, CPM cleaves only
C-terminal Arg or Lys residues; however, CPM has a preference for
the C-terminal Arg. The penultimate amino acid also affects the
rate of hydrolysis. Naturally occurring peptide substrates of CPM
include bradykinin, Arg.sup.6- and Lys.sup.6 enkephalins, dynorphin
A.sup.1-13 and epidermal growth factor (EGF). See Sidgel et al.
(1989) J. Biol. Chem. 264:2236-2241; McGwire et al. (1995) J. Biol.
Chem. 270:17154-17158.
[0009] CPM is primarily found on the plasma membrane, with highest
levels found in lung and placenta. It is also present in kidney,
blood vessels, intestine, brain and peripheral nerves. See R. A.
Skidgel (1988) Trends Pharm. Sci. 9:299-304; Skidgel et al. (1984)
Biochem. Pharmacol. 33: 3471-3478; Skidgel et al. (1991) FASEB J.
5: 1578; Nagae et al. (1992) J. Neurochem. 59:2201-2212; Nagae et
al. (1993) Am. J. Respir. Cell Mol. Biol. 9:221-229. Expression of
CPM is responsive to differentiation of monocytes and lymphocytes.
See de Saint-Vis et al. (1995) Blood 86:1098-1105; Rehli et al.
(1995) J. Biol. Chem. 270:15644-15649.
[0010] CPM participates in the control of peptide hormone activity
at the cell surface and degradation of extracellular proteins and
peptides. It catalyzes the second step in prohormone processing and
removes C-terminal Arg or Lys residues from peptides released from
prohormones. CPM functions as a soluble enzyme after its release
from the plasma membrane and may function in the plasma membrane
form to control peptide receptor activities. CPM can regulate
receptor specificity of kinins by cleaving the C-terminal
ARG.sup.9, for example, from bradykinin. The intact bradykinin
binds the B2 receptor. The cleaved bradykinin
(des-ARG.sup.9-bradykinin). Des-ARG.sup.9-bradykinin also binds the
B1 receptors: stimulates IL-1 and tumor necrosis factor release
from macrophages. Regulation of the B1 receptor is associated with
injury or inflammation. CPM may also be involved with other
inflammatory mediators, such as anaphylatoxin C5a which mediates
histamine release. In addition, CPM may metabolize growth factors
containing terminal Arg or Lys, such as EGF, EGF-like peptides,
nerve growth factor (NGF) amphiregulin, hepatocyte growth factor,
erythropoietin, and macrophage-stimulating protein. In the lung,
varying levels of CPM are associated with pneumocystic or bacterial
pneumonia or lung cancer, and in the placenta, CPM may protect the
fetus from maternally derived peptides. See R. A. Skidgel (1992) J.
Cardiovasc. Pharmacol. 20(Suppl. 9):S4-S9; Bhoola et al. (1992)
Pharmacol. Rev. 44:1-80; R. A. Skidgel (1993) In: Hooper NM, ed.
Zinc Metalloproteases in Health and Disease, London: Taylor &
Francis, Ltd., p. 241-283; Dragovic et al. (1995) Am. J. Respir.
Crit. Care Med. 152:760-764; Nagae et al. (1992) J. Neurochem.
59:2201-2212; MacFadden et al. (1988) FASEB J. 2:1179
(Abstract).
[0011] Another B-type regulatory CP metalloprotein is CPD, a
membrane-bound glycoprotein. Human CPD is a protein of 1,377 amino
acids with 75% identity with duck GP180 and 90% identity with rat
CPD. Human CPD contains two hydrophobic regions located at the C-
and N-termini. A 55-60 residue cytoplasmic domain is highly
conserved among duck, human and rat sequences and may be
significant in intracellular sorting, protein-protein interactions
or endocytosis. CPD contains three tandem CP homology domains
numbered sequentially from the N- to the C-terminus, and thereby
may contain more than one active site. See Tan et al. (1997)
Biochem. J. 327:81-87; Skidgel et al. (1993) In: Robertson JLS
Nicholls MG, eds. The Renin Angiotensin System, Vol 1, London:
Gower Medical Publishing, p. 10.1-10.10. CPD is located on human
chromosome 17, 17P, 11.1-17q, 11.2.
[0012] CPD is primarily found on intracellular membranes, mainly in
the Golgi, with some CPD found on the plasma membrane. The tissue
distribution of CPD is wide and includes most duck tissues and
mammalian tissues as well, including brain, pituitary, placenta,
pancreas, adrenal, kidney, lung, heart, spleen, intestine, ovary,
and testes. See McGwire et al. (1997) Life Sci. 60:715-724; Song et
al. (1995) J. Biol. Chem. 270:25007-25013; Xin et al. (1997) DNA
Cell Biol. 16:897-909; Tan et al. (1997) Biochem. J. 327:81-87;
Song et al. (1996) J. Biol. Chem. 271:28884-28889.
[0013] The function of CPD is speculated to include peptide and
protein processing in the constitutive secretory pathway after
endoprotease cleavage of precursor proteins. The enzyme has an
acidic pH optimum. Mammalian CPD may act as a hepatitis B virus
binding protein, similar to the duck CPD. See R. A. Skidgel (1998)
Immunological Reviews 161:129-141.
[0014] Serine CPs include PRCP and deamidase. PRCP cloned from a
human kidney library indicates a glycoprotein of 51 kDa.sup.3; and
containing 496 amino acids, including a 30 residue signal peptide
and a 15 residue propeptide. See Tan et al. (1993) J. Biol. Chem.
268:16631-16638. A serine repeat is found in the C-terminal half,
similar to the serine repeat of a yeast CP encoded by the KEX1
gene.
[0015] PRCP has an acidic pH optimum for synthetic peptide
substrates, but retains activity at neutral ranges with longer
naturally occurring peptides. PRCP cleaves peptides only if the
penultimate residue is proline. The enzyme does not cleave
Pro-Pro-COOH or (OH)-Pro-Pro-COOH bond. See Odya et al. (1978) J.
Biol. Chem. 253:5927-5931. Substrates of PRCP include
des-Arg.sup.9-bradykinin and angiotensin II.
[0016] PRCP may be involved in terminating signal transduction by
inactivating peptide ligands after receptor endocytosis. PRCP is
contained in lysosomes and released in response to stimulation. The
enzyme is widely distributed and found in human placenta, lung,
liver, and kidney.
[0017] Another serine CP, deamidase, is likely a 94 kDa homodimer
of 52 kDa subunits. Human platelet deamidase is activated by
cleavage of a 14 amino acid fragment from the C-terminus. The
enzyme binds and maintains activity and stability of
.beta.-galactocidase and neuraminidase in lysosomes, a defect of
which is associated with severe galactosialidosis. See Bonten et
al. (1995) J. Biol Chem. 270:26441-26445; Galjart et al. (1988)
Cell 54:755-764; D'Azzo et al. (1982) Proc. Natl. Acad. Sci.
79:4535-4539. The gene for the human deamidase is mapped to
chromosome 20 at q13.1.
[0018] Deamidase cleaves various peptides containing C-terminal or
penultimate hydrophobic residues including substance P, angiotensin
I, bradykinin, endothelin, and fMet-Leu-Phe. Like PRCP, deamidase
is also found in lysosomes, and distributed in human placenta,
lung, liver, and kidney. Like PRCP, deamidase is implicated in
blocking part of the signal transduction pathway stimulated by
peptides. Bradykinin, containing a C-terminal Arg.sup.9 and a
penultimate hydrophobic amino acid Phe.sup.8, is cleaved by
deamidase. Similarly, angiotensin, containing a C-terminal His and
a penultimate Phe, is cleaved by deamidase. Accordingly, deamidase
is implicated in termination of bradykinin activity on the B2
receptor to generate a B1 receptor agonist. Deamidase may also have
a role in chemotaxis and in metabolism of the anti-cancer growth
factor antagonist. See Skidgel et al. (1998) Immunological Reviews
161:129-141; Jackman et al (1990) J. Biol. Chem. 265:11265-11272;
Jackman et al. (1995) Am. J. Respir. Cell Mol. Biol. 13:196-204;
Hinek et al. (1996) Biol. Chem. 377:471-480; Jones et al. (1995)
Peptides 16:777-783; Cummings et al. (1995) Biochem Pharmacol.
49:1709-1712.
[0019] Given the wide distribution and various physiological and
pathological roles of carboxypeptidases, methods and compositions
directed at regulating levels of these enzymes are useful for
regulating peptide hormone activity, modulating metabolism of
substance P, angiotensin I, angiotensin II, bradykinin, and
endothelin, and regulation of signal transduction by inactivation
of peptide ligands subsequent to receptor endocytosis.
[0020] Accordingly, carboxypeptidases are a major target for drug
action and development. Therefore, it is valuable to the field of
pharmaceutical development to identify and characterize previously
unknown carboxypeptidases. The present invention advances the state
of the art by providing a previously unidentified human
carboxypeptidase.
SUMMARY OF THE INVENTION
[0021] It is an object of the invention to identify novel
carboxypeptidases.
[0022] It is a further object of the invention to provide novel
carboxypeptidase polypeptides that are useful as reagents or
targets in carboxypeptidase assays applicable to treatment and
diagnosis of carboxypeptidase-related disorders.
[0023] It is a further object of the invention to provide
polynucleotides corresponding to the novel carboxypeptidase
polypeptides that are useful as targets and reagents in
carboxypeptidase assays applicable to treatment and diagnosis of
carboxypeptidase-related disorders and useful for producing novel
carboxypeptidase polypeptides by recombinant methods.
[0024] A specific object of the invention is to identify compounds
that act as agonists and antagonists and modulate the expression of
the novel carboxypeptidase.
[0025] A further specific object of the invention is to provide
compounds that modulate expression of the carboxypeptidase for
treatment and diagnosis of carboxypeptidase-related disorders.
[0026] The invention is thus based on the identification of a novel
human carboxypeptidase. The amino acid sequence is shown in SEQ ID
NO: 1. The nucleotide sequence is shown as SEQ ID NO:2.
[0027] The invention provides isolated carboxypeptidase
polypeptides, including a polypeptide having the amino acid
sequence shown in SEQ ID NO:1 or the amino acid sequence encoded by
the cDNA deposited as ATCC No. PTA-1643 on Apr. 5, 2000 ("the
deposited cDNA").
[0028] The invention also provides isolated carboxypeptidase
nucleic acid molecules having the sequence shown in SEQ ID NO:2 or
in the deposited cDNA.
[0029] The invention also provides variant polypeptides having an
amino acid sequence that is substantially homologous to the amino
acid sequence shown in SEQ ID NO:1 or encoded by the deposited
cDNA.
[0030] The invention also provides variant nucleic acid sequences
that are substantially homologous to the nucleotide sequence shown
in SEQ ID NO:2 or in the deposited cDNA.
[0031] The invention also provides fragments of the polypeptide
shown in SEQ ID NO:1 and nucleotide sequence shown in SEQ ID NO:2,
as well as substantially homologous fragments of the polypeptide or
nucleic acid.
[0032] The invention further provides nucleic acid constructs
comprising the nucleic acid molecules described herein. In a
preferred embodiment, the nucleic acid molecules of the invention
are operatively linked to a regulatory sequence.
[0033] The invention also provides vectors and host cells for
expressing the carboxypeptidase nucleic acid molecules and
polypeptides, and particularly recombinant vectors and host
cells.
[0034] The invention also provides methods of making the vectors
and host cells and methods for using them to produce the
carboxypeptidase nucleic acid molecules and polypeptides.
[0035] The invention also provides antibodies or antigen-binding
fragments thereof that selectively bind the carboxypeptidase
polypeptides and fragments.
[0036] The invention also provides methods of screening for
compounds that modulate expression or activity of the
carboxypeptidase polypeptides or nucleic acid (RNA or DNA).
[0037] The invention also provides a process for modulating
carboxypeptidase polypeptide or nucleic acid expression or
activity, especially using the screened compounds. Modulation may
be used to treat conditions related to aberrant activity or
expression of the carboxypeptidase polypeptides or nucleic
acids.
[0038] The invention also provides assays for determining the
activity of or the presence or absence of the carboxypeptidase
polypeptides or nucleic acid molecules in a biological sample,
including for disease diagnosis.
[0039] The invention also provides assays for determining the
presence of a mutation in the polypeptides or nucleic acid
molecules, including for disease diagnosis.
[0040] In still a further embodiment, the invention provides a
computer readable means containing the nucleotide and/or amino acid
sequences of the nucleic acids and polypeptides of the invention,
respectively.
DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 shows the carboxypeptidase nucleotide sequence (SEQ
ID NO:2) and the deduced amino acid sequence (SEQ ID NO:1).
[0042] FIG. 2 shows a comparison of the carboxypeptidase against
the Prosite database of protein patterns, specifically showing a
high score against the prolyloligopeptidase family (SEQ ID NO:3)
and an alpha/beta hydrolase (SEQ ID NO:4).
[0043] FIG. 3 shows an analysis of the carboxypeptidase amino acid
sequence: .alpha..beta.turn and coil regions; hydrophilicity;
amphipathic regions; flexible regions; antigenic index; and surface
probability plot.
[0044] FIG. 4 shows a hydrophobicity plot of the carboxypeptidase
(SEQ ID NO:1).
[0045] FIG. 5 shows an analysis of the carboxypeptidase open
reading frame for amino acids corresponding to specific functional
sites. Glycosylation sites, protein kinase C phosphorylation sites,
casein kinase II phosphorylation sites, N-myristoylation sites, and
leucine zipper pattern of SEQ ID NO:1 are shown.
[0046] FIG. 6 shows RNA expression of the carboxypeptidase in
normal and tumor tissues.
[0047] FIG. 7 shows RNA expression of the carboxypeptidase in
normal human tissues.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Polypeptides
[0049] The invention is based on the discovery of a novel human
carboxypeptidase. Specifically, an expressed sequence tag (EST) was
selected based on homology to carboxypeptidase sequences. This EST
was used to design primers based on sequences that it contains and
used to identify a cDNA found in osteoblast, brain, small
intestine, heart, and prostate cDNA libraries. Positive clones were
sequenced and the overlapping fragments were assembled. Analysis of
the assembled sequence revealed that the cloned cDNA molecule
encodes a carboxypeptidase.
[0050] The invention thus relates to a novel carboxypeptidase
having the deduced amino acid sequence shown in FIG. 1 (SEQ ID NO:
1) or having the amino acid sequence encoded by the deposited cDNA,
ATCC No. PTA-1643.
[0051] The deposit will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms. The deposit is provided as a convenience to those
of skill in the art and is not an admission that a deposit is
required under 35 U.S.C. .sctn. 112. The deposited sequence, as
well as the polypeptide encoded by the sequence, is incorporated
herein by reference and controls in the event of any conflict, such
as a sequencing error, with description in this application.
[0052] The carboxypeptidase of the invention has homology to the
family of prolylendopeptidases. The catalytic triad signature, SER
ASP HIS, is found in the protein of the invention as well as in
prolylcarboxypeptidase (angiotensinase C NOBF [P42785], PCP). PCP
is classified as belonging to the prolylendopeptidase and serine
carboxypeptidase family. The amino acids around the SER in PCP is
consistent with a prolylendopeptidase. The ones found in the
polypeptide of the invention are more similar to the ones found in
the serine carboxypeptidase family.
[0053] "Carboxypeptidase polypeptide" or "carboxypeptidase protein"
refers to the polypeptides in SEQ ID NO:1 or encoded by the
deposited cDNA. The term "carboxypeptidase protein" or
"carboxypeptidase polypeptide", however, further includes the
numerous variants described herein, as well as fragments derived
from the full-length carboxypeptidase and variants.
[0054] Tissues and/or cells in which the carboxypeptidase is found
include, but are not limited to, those found in the figures herein.
In particular, the carboxypeptidase is expressed in prostate,
breast, skeletal muscle, brain, testis, thyroid, fetal kidney,
fetal liver, and fetal heart tissues. Disease expression is
associated with colon carcinoma, breast carcinoma, and lung
squamous cell carcinoma. Expression has been observed in HCT116, a
variant cell line isolated from a colon carcinoma, HT29, an
adenocarcinoma line, KM12, a weakly metastatic colorectal carcinoma
cell line, and HTC8, a colon carcinoma cell line. Expression has
also been observed in the breast carcinoma cell line MDA-231,
MCF-7, HMEC, ZR-75, and MDA-435. Up-regulation has been observed in
HCT116, DLD-1 (adenocarcinoma of the sigmoid colon), HT29, SW480
(adenocarcinoma from metastatic lymph node), SW620 (adenocarcinoma
from metastatic lymph node), and KMI2. Thus, this carboxypeptidase
is overexpressed in a number of breast, lung and colon tumors,
including colon carcinoma, lung adenocarcinoma, small cell lung
carcinoma, and colon metastatic tissue. Elevated levels in clinical
cancerous tumors and not in noncancerous normal tissues indicate a
cellular proteolytic imbalance in these tissues. Accordingly,
expression of the carboxypeptidase is relevant to carcinogenesis,
including invasion and metastasis.
[0055] The present invention thus provides an isolated or purified
carboxypeptidase polypeptide and variants and fragments
thereof.
[0056] Based on a BLAST search, highest homology was shown to a
human prolylcarboxypeptidase.
[0057] As used herein, a polypeptide is said to be "isolated" or
"purified" when it is substantially free of cellular material when
it is isolated from recombinant and non-recombinant cells, or free
of chemical precursors or other chemicals when it is chemically
synthesized. A polypeptide, however, can be joined to another
polypeptide with which it is not normally associated in a cell and
still be considered "isolated" or "purified."
[0058] The carboxypeptidase polypeptides can be purified to
homogeneity. It is understood, however, that preparations in which
the polypeptide is not purified to homogeneity are useful and
considered to contain an isolated form of the polypeptide. The
critical feature is that the preparation allows for the desired
function of the polypeptide, even in the presence of considerable
amounts of other components. Thus, the invention encompasses
various degrees of purity.
[0059] In one embodiment, the language "substantially free of
cellular material" includes preparations of the carboxypeptidase
having less than about 30% (by dry weight) other proteins (i.e.,
contaminating protein), less than about 20% other proteins, less
than about 10% other proteins, or less than about 5% other
proteins. When the polypeptide is recombinantly produced, it can
also be substantially free of culture medium, i.e., culture medium
represents less than about 20%, less than about 10%, or less than
about 5% of the volume of the protein preparation.
[0060] A carboxypeptidase polypeptide is also considered to be
isolated when it is part of a membrane preparation or is purified
and then reconstituted with membrane vesicles or liposomes.
[0061] The language "substantially free of chemical precursors or
other chemicals" includes preparations of the carboxypeptidase
polypeptide in which it is separated from chemical precursors or
other chemicals that are involved in its synthesis. In one
embodiment, the language "substantially free of chemical precursors
or other chemicals" includes preparations of the polypeptide having
less than about 30% (by dry weight) chemical precursors or other
chemicals, less than about 20% chemical precursors or other
chemicals, less than about 10% chemical precursors or other
chemicals, or less than about 5% chemical precursors or other
chemicals.
[0062] In one embodiment, the carboxypeptidase polypeptide
comprises the amino acid sequence shown in SEQ ID NO:1. However,
the invention also encompasses sequence variants. Variants include
a substantially homologous protein encoded by the same genetic
locus in an organism, i.e., an allelic variant. The
carboxypeptidase has been mapped to human chromosome 9 at 9q33-34.
Nearby known genes include DYT1, CAIN, VAV2, NOTCH1, ABL1, SDHD,
TSC1, SURF1, RPL7A, RXRA, COL5A1, PAEP, ABC2, N14, LCN1, CACNA1B,
FCN1. Nearby mutations/loci include Human-DYS, DYSAUTONOMIA,
FAMILIAL; MUSCULAR DYSTROPHY, LIMB-GIRDLE, TYPE 2H; AFD1,
ACROFACIAL DYSOSTOSIS 1, BDB1, NAGER TYPE; BRACHYDACTYLY, TYPE B1;
ALS4, AMYOTROPHIC LATERAL SCLEROSIS 4, JUVENILE.
[0063] A possible locus in the mouse is on chromosome 2 and
possibly chromosome 4. In the mouse, the Scc2 locus controls
susceptibility to 1,2 dimethylhydrazine-induced colon tumors. The
following loci in the mouse are relevant: Mouse Chr2- Scc2, colon
tumor susceptibility 2; stu, stumbler; Sd, Danforth's short tail;
stb, stubby; us, urogenital syndrome; ebo, ebouriffe; sar,
sarcosinemia autosomal recessive; Lsr1, listeria resistance; Anth2,
resistance to Bacillus anthracis 2; mdm, muscular dystrophy with
myositis. See Moen, C. J. et al. Proc. Natl. Acad. Sci. U.S.A. 93
(1996).
[0064] Variants also encompass proteins derived from other genetic
loci in an organism, but having substantial homology to the
carboxypeptidase of SEQ ID NO:1. Variants also include proteins
substantially homologous to the carboxypeptidase but derived from
another organism, i.e., an ortholog. Variants also include proteins
that are substantially homologous to the carboxypeptidase that are
produced by chemical synthesis. Variants also include proteins that
are substantially homologous to the carboxypeptidase that are
produced by recombinant methods. It is understood, however, that
variants exclude any amino acid sequences disclosed prior to the
invention.
[0065] As used herein, two proteins (or a region of the proteins)
are substantially homologous when the amino acid sequences are at
least about 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, typically at
least about 80-85%, and most typically at least about 90-95% or
more homologous. A substantially homologous amino acid sequence,
according to the present invention, will be encoded by a nucleic
acid sequence hybridizing to the nucleic acid sequence, or portion
thereof, of the sequence shown in SEQ ID NO:2 under stringent
conditions as more fully described below.
[0066] To determine the percent identity of two amino acid
sequences or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). In a
preferred embodiment, the length of a reference 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 (e.g., when aligning a second sequence to
the amino acid sequences herein having 502 amino acid residues, at
least 165, preferably at least 200, more preferably at least 250,
even more preferably at least 300, and even more preferably at
least 350, 400, 450, and 500 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.
[0067] The invention also encompasses polypeptides having a lower
degree of identity but having sufficient similarity so as to
perform one or more of the same functions performed by the
carboxypeptidase. Similarity is determined by conserved amino acid
substitution. Such substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Conservative substitutions are likely to be
phenotypically silent. Typically seen as conservative substitutions
are the replacements, one for another, among the aliphatic amino
acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residues
Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution between the amide residues Asn and Gln, exchange of
the basic residues Lys and Arg and replacements among the aromatic
residues Phe, Tyr. Guidance concerning which amino acid changes are
likely to be phenotypically silent are found in Bowie et al.,
Science 247:1306-1310 (1990).
1TABLE 1 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Giutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alamne Serine Threonine
Methionine Glycine
[0068] The comparison of sequences and determination of percent
identity and similarity between two sequences can be accomplished
using a mathematical algorithm. (Computational Molecular Biology,
Lesk, A. M., ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., eds., M Stockton Press, New York, 1991).
[0069] A preferred, non-limiting example of such a mathematical
algorithm is described in Karlin et al. (1993) Proc. Natl. Acad.
Sci. USA 90:5873-5877. Such an algorithm is incorporated into the
NBLAST and XBLAST programs (version 2.0) as described in Altschul
et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST
and Gapped BLAST programs, the default parameters of the respective
programs (e.g., NBLAST) can be used. See www.ncbi.nlm.nih.gov. In
one embodiment, parameters for sequence comparison can be set at
score=100, wordlength=12, or can be varied (e.g., W=5 or W=20).
[0070] In a preferred embodiment, the percent identity between two
amino acid sequences is determined using the Needleman et al.
(1970) (J. Mol. Biol. 48:444-453) algorithm which has been
incorporated into the GAP program in the GCG software package
(available at www.gcg.com), using either a BLOSUM 62 matrix or a
PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a
length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred
embodiment, the percent identity between two nucleotide sequences
is determined using the GAP program in the GCG software package
(Devereux et al. (1984) Nucleic Acids Res. 12(1):387) (available at
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.
[0071] Another preferred, non-limiting example of a mathematical
algorithm utilized for the comparison of sequences is the algorithm
of Myers and Miller, CABIOS (1989). Such an algorithm is
incorporated into the ALIGN program (version 2.0) which is part of
the CGC sequence alignment software package. When utilizing the
ALIGN program for comparing amino acid sequences, a PAM120 weight
residue table, a gap length penalty of 12, and a gap penalty of 4
can be used. Additional algorithms for sequence analysis are known
in the art and include ADVANCE and ADAM as described in Torellis et
al. (1994) Comput. Appl. Biosci. 10:3-5; and FASTA described in
Pearson et al. (1988) PNAS 85:2444-8.
[0072] A variant polypeptide can differ in amino acid sequence by
one or more substitutions, deletions, insertions, inversions,
fusions, and truncations or a combination of any of these.
[0073] Variant polypeptides can be fully functional or can lack
function in one or more activities. Thus, in the present case,
variations can affect the function, for example, of one or more of
the regions relating to peptide binding, specificity, or
hydrolysis, regulatory/allosteric regions, regions involved in
membrane association, regions involved in modification or
activation of the carboxypeptidase, such as glycosylation,
phosphorylation, and myristoylation, and any metal binding
regions.
[0074] Fully functional variants typically contain only
conservative variation or variation in non-critical residues or in
non-critical regions. Functional variants can also contain
substitution of similar amino acids, which results in no change or
an insignificant change in function. Alternatively, such
substitutions may positively or negatively affect function to some
degree.
[0075] Non-functional variants typically contain one or more
non-conservative amino acid substitutions, deletions, insertions,
inversions, or truncation or a substitution, insertion, inversion,
or deletion in a critical residue or critical region.
[0076] As indicated, variants can be naturally-occurring or can be
made by recombinant means or chemical synthesis to provide useful
and novel characteristics for the carboxypeptidase polypeptide.
This includes preventing immunogenicity from pharmaceutical
formulations by preventing protein aggregation.
[0077] Useful variations further include alteration of catalytic
activity. For example, one embodiment involves a variation at the
binding site that results in binding but not hydrolysis, or slower
hydrolysis, of substrate. A further useful variation at the same
site can result in altered affinity for substrate. Useful
variations also include changes that provide for affinity for
another substrate. Another useful variation includes one that
prevents modification of the carboxypeptidase. Another useful
variation includes variation in the region that provides for
altered membrane association. Another useful variation provides a
fusion protein in which one or more regions are operationally fused
to one or more regions from another carboxypeptidase.
[0078] Amino acids that are essential for function can be
identified by methods known in the art, such as site-directed
mutagenesis or alanine-scanning mutagenesis (Cunningham et al.
(1985) Science 244:1081-1085). The latter procedure introduces
single alanine mutations at every residue in the molecule. The
resulting mutant molecules are then tested for biological activity,
such as peptide hydrolysis in vitro or peptide-dependent in vitro
activity, such as proliferative activity. Sites that are critical
for binding can also be determined by structural analysis such as
crystallization, nuclear magnetic resonance or photoaffinity
labeling (Smith et al. (1992) J. Mol. Biol. 224:899-904; de Vos et
al. (1992) Science 255:306-312).
[0079] Substantial homology can be to the entire nucleic acid or
amino acid sequence or to fragments of these sequences.
[0080] The invention thus also includes polypeptide fragments of
the carboxypeptidase. Fragments can be derived from the amino acid
sequence shown in SEQ ID NO:1. However, the invention also
encompasses fragments of the variants of the carboxypeptidase as
described herein.
[0081] The fragments to which the invention pertains, however, are
not to be construed as encompassing fragments that may be disclosed
prior to the present invention.
[0082] Accordingly, a fragment can comprise at least about 11, 12,
13, 14, 15, 20, 25, 30, 35, 40, 45, 50 or more contiguous amino
acids. Fragments can retain one or more of the biological
activities of the protein, for example the ability to bind to or
hydrolyze peptides, as well as fragments that can be used as an
immunogen to generate carboxypeptidase antibodies.
[0083] Biologically active fragments (peptides which are, for
example, 5, 7, 10, 12, 15, 20, 30, 35, 36, 37, 38, 39, 40, 50, 100
or more amino acids in length) can comprise a region or motif,
e.g., regions relating to peptide binding, specificity, or
hydrolysis, regulatory/allosteric regions, regions involved in
membrane association, regions involved in modification or
activation of the carboxypeptidase, such as glycosylation,
phosphorylation, and myristoylation, and any metal binding
regions.
[0084] Such regions or motifs can be identified by means of routine
computerized homology searching procedures.
[0085] Fragments, for example, can extend in one or both directions
from the functional site to encompass 5, 10, 15, 20, 30, 40, 50, or
up to 100 amino acids. Further, fragments can include sub-fragments
of the specific domains mentioned above, which sub-fragments retain
the function of the region or domain from which they are
derived.
[0086] These regions can be identified by well-known methods
involving computerized homology analysis.
[0087] The invention also provides fragments with immunogenic
properties. These contain an epitope-bearing portion of the
carboxypeptidase and variants. These epitope-bearing peptides are
useful to raise antibodies that bind specifically to a
carboxypeptidase polypeptide or region or fragment. These peptides
can contain at least 10, 12, at least 14, or between at least about
15 to about 30 amino acids.
[0088] Non-limiting examples of antigenic polypeptides that can be
used to generate antibodies include but are not limited to peptides
derived from an extracellular region. Regions having a high
antigenicity index are shown in FIG. 3. However,
intracellularly-made antibodies ("intrabodies") are also
encompassed, which would recognize intracellular peptide
regions.
[0089] The epitope-bearing carboxypeptidase polypeptides may be
produced by any conventional means (Houghten, R. A. (1985) Proc.
Natl. Acad. Sci. USA 82:5131-5135). Simultaneous multiple peptide
synthesis is described in U.S. Pat. No. 4,631,211.
[0090] Fragments can be discrete (not fused to other amino acids or
polypeptides) or can be within a larger polypeptide. Further,
several fragments can be comprised within a single larger
polypeptide. In one embodiment a fragment designed for expression
in a host can have heterologous pre- and pro-polypeptide regions
fused to the amino terminus of the carboxypeptidase fragment and an
additional region fused to the carboxyl terminus of the
fragment.
[0091] The invention thus provides chimeric or fusion proteins.
These comprise a carboxypeptidase peptide sequence operatively
linked to a heterologous peptide having an amino acid sequence not
substantially homologous to the carboxypeptidase. "Operatively
linked" indicates that the carboxypeptidase peptide and the
heterologous peptide are fused in-frame. The heterologous peptide
can be fused to the N-terminus or C-terminus of the
carboxypeptidase or can be internally located.
[0092] In one embodiment the fusion protein does not affect
carboxypeptidase function per se. For example, the fusion protein
can be a GST-fusion protein in which the carboxypeptidase sequences
are fused to the C-terminus of the GST sequences. Other types of
fusion proteins include, but are not limited to, enzymatic fusion
proteins, for example beta-galactosidase fusions, yeast two-hybrid
GAL4 fusions, poly-His fusions and Ig fusions. Such fusion
proteins, particularly poly-His fusions, can facilitate the
purification of recombinant carboxypeptidase. In certain host cells
(e.g., mammalian host cells), expression and/or secretion of a
protein can be increased by using a heterologous signal sequence.
Therefore, in another embodiment, the fusion protein contains a
heterologous signal sequence at its N-terminus.
[0093] EP-A-O 464 533 discloses fusion proteins comprising various
portions of immunoglobulin constant regions. The Fc is useful in
therapy and diagnosis and thus results, for example, in improved
pharmacokinetic properties (EP-A 0232 262). In drug discovery, for
example, human proteins have been fused with Fc portions for the
purpose of high-throughput screening assays to identify antagonists
(Bennett et al. (1995) J. Mol. Recog. 8:52-58 (1995) and Johanson
et al. J. Biol. Chem. 270:9459-9471). Thus, this invention also
encompasses soluble fusion proteins containing a carboxypeptidase
polypeptide and various portions of the constant regions of heavy
or light chains of immunoglobulins of various subclass (IgG, IgM,
IgA, IgE). Preferred as immunoglobulin is the constant part of the
heavy chain of human IgG, particularly IgG1, where fusion takes
place at the hinge region. For some uses it is desirable to remove
the Fc after the fusion protein has been used for its intended
purpose, for example when the fusion protein is to be used as
antigen for immunizations. In a particular embodiment, the Fc part
can be removed in a simple way by a cleavage sequence, which is
also incorporated and can be cleaved with factor Xa.
[0094] A chimeric or fusion protein can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for
the different protein sequences are ligated together in-frame in
accordance with conventional techniques. 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 re-amplified to
generate a chimeric gene sequence (see Ausubel et al. (1992)
Current Protocols in Molecular Biology). Moreover, many expression
vectors are commercially available that already encode a fusion
moiety (e.g., a GST protein). A carboxypeptidase-encoding nucleic
acid can be cloned into such an expression vector such that the
fusion moiety is linked in-frame to the carboxypeptidase.
[0095] Another form of fusion protein is one that directly affects
carboxypeptidase functions. Accordingly, a carboxypeptidase
polypeptide is encompassed by the present invention in which one or
more of the carboxypeptidase regions (or parts thereof) has been
replaced by homologous regions (or parts thereof) from another
carboxypeptidase. Accordingly, various permutations are possible.
For example, the active site region, or subregion thereof, can be
replaced with the active site region or subregion from another
carboxypeptidase. As a further example, the membrane-associated
region, or parts thereof, can be replaced. Thus, chimeric
carboxypeptidases can be formed in which one or more of the native
regions has been replaced by another.
[0096] It is understood, however, that sites could be derived from
carboxypeptidases that occur in the mammalian genome but which have
not yet been discovered or characterized. Such sites include, but
are not limited to, regions relating to peptide binding,
specificity, or hydrolysis, regulatory/allosteric regions, regions
involved in membrane association, regions involved in modification
or activation of the carboxypeptidase, such as glycosylation,
phosphorylation, and myristoylation, and any metal binding regions,
and any other functional site disclosed herein.
[0097] The isolated carboxypeptidase protein can be purified from
cells that naturally express it, such as from those disclosed
herein, especially purified from cells that have been altered to
express it (recombinant), or synthesized using known protein
synthesis methods. Preferred tissues in which the carboxypeptidase
is expressed include, but are not limited to, prostate, breast,
skeletal muscle, brain, testis, and thyroid. Cells also include
colon, breast, and lung carcinoma.
[0098] In one embodiment, the protein is produced by recombinant
DNA techniques. For example, a nucleic acid molecule encoding the
carboxypeptidase polypeptide is cloned into an expression vector,
the expression vector introduced into a host cell and the protein
expressed in the host cell. The protein can then be isolated from
the cells by an appropriate purification scheme using standard
protein purification techniques. Polypeptides often contain amino
acids other than the 20 amino acids commonly referred to as the 20
naturally-occurring amino acids. Further, many amino acids,
including the terminal amino acids, may be modified by natural
processes, such as processing and other post-translational
modifications, or by chemical modification techniques well known in
the art. Common modifications that occur naturally in polypeptides
are described in basic texts, detailed monographs, and the research
literature, and they are well known to those of skill in the
art.
[0099] Accordingly, the polypeptides also encompass derivatives or
analogs in which a substituted amino acid residue is not one
encoded by the genetic code, in which a substituent group is
included, in which the mature polypeptide is fused with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol), or in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence for purification of
the mature polypeptide or a pro-protein sequence.
[0100] Known modifications include, but are not limited to,
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphatidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
crosslinks, formation of cystine, formation of pyroglutamate,
formylation, gamma carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, proteolytic processing, phosphorylation, prenylation,
racemization, selenoylation, sulfation, transfer-RNA mediated
addition of amino acids to proteins such as arginylation, and
ubiquitination.
[0101] Such modifications are well-known to those of skill in the
art and have been described in great detail in the scientific
literature. Several particularly common modifications,
glycosylation, lipid attachment, sulfation, gamma-carboxylation of
glutamic acid residues, hydroxylation and ADP-ribosylation, for
instance, are described in most basic texts, such as
Proteins--Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993). Many
detailed reviews are available on this subject, such as by Wold,
F., Posttranslational Covalent Modification of Proteins, B. C.
Johnson, Ed., Academic Press, New York 1-12 (1983); Seifter et al.
(1990) Meth. Enzymol. 182: 626-646) and Rattan et al. (1992) Ann.
N. Y. Acad. Sci. 663:48-62).
[0102] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translation events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides may be synthesized by
non-translational natural processes and by synthetic methods.
[0103] Modifications can occur anywhere in a polypeptide, including
the peptide backbone, the amino acid side-chains and the amino or
carboxyl termini. Blockage of the amino or carboxyl group in a
polypeptide, or both, by a covalent modification, is common in
naturally-occurring and synthetic polypeptides. For instance, the
aminoterminal residue of polypeptides made in E. coli, prior to
proteolytic processing, ahnost invariably will be
N-formylmethionine.
[0104] The modifications can be a function of how the protein is
made. For recombinant polypeptides, for example, the modifications
will be determined by the host cell posttranslational modification
capacity and the modification signals in the polypeptide amino acid
sequence. Accordingly, when glycosylation is desired, a polypeptide
should be expressed in a glycosylating host, generally a eukaryotic
cell. Insect cells often carry out the same posttranslational
glycosylations as mammalian cells and, for this reason, insect cell
expression systems have been developed to efficiently express
mammalian proteins having native patterns of glycosylation. Similar
considerations apply to other modifications.
[0105] The same type of modification may be present in the same or
varying degree at several sites in a given polypeptide. Also, a
given polypeptide may contain more than one type of
modification.
[0106] Polypeptide Uses
[0107] The protein sequences of the present invention can 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 the 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 the proteins 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 www.ncbi.nlm.nih.gov.
[0108] The carboxypeptidase polypeptides are useful for producing
antibodies specific for the carboxypeptidase, regions, or
fragments. Regions having a high antigenicity index score are shown
in FIG. 3.
[0109] The carboxypeptidase polypeptides are useful for biological
assays related to carboxypeptidases. Such assays involve any of the
known carboxypeptidase functions or activities or properties useful
for diagnosis and treatment of carboxypeptidase-related
conditions.
[0110] The carboxypeptidase polypeptides are also useful in drug
screening assays, in cell-based or cell-free systems. Cell-based
systems can be native, i.e., cells that normally express the
carboxypeptidase, as a biopsy or expanded in cell culture. In one
embodiment, however, cell-based assays involve recombinant host
cells expressing the carboxypeptidase.
[0111] Determining the ability of the test compound to interact
with the carboxypeptidase can also comprise determining the ability
of the test compound to preferentially bind to the polypeptide as
compared to the ability of a known binding molecule to bind to the
polypeptide.
[0112] The polypeptides can be used to identify compounds that
modulate carboxypeptidase activity. Such compounds, for example,
can increase or decrease affinity or rate of binding to substrate,
compete with substrate for binding to the carboxypeptidase, or
displace substrate bound to the carboxypeptidase. Both
carboxypeptidase and appropriate variants and fragments can be used
in high-throughput screens to assay candidate compounds for the
ability to bind to the carboxypeptidase. These compounds can be
further screened against a functional carboxypeptidase to determine
the effect of the compound on the carboxypeptidase activity.
Compounds can be identified that activate (agonist) or inactivate
(antagonist) the carboxypeptidase to a desired degree. 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.
[0113] The carboxypeptidase polypeptides can be used to screen a
compound for the ability to stimulate or inhibit interaction
between the carboxypeptidase protein and a target molecule that
normally interacts with the carboxypeptidase protein. The assay
includes the steps of combining the carboxypeptidase protein with a
candidate compound under conditions that allow the carboxypeptidase
protein or fragment to interact with the target molecule, and to
detect the formation of a complex between the carboxypeptidase
protein and the target or to detect the biochemical consequence of
the interaction with the carboxypeptidase and the target, such as
any of the associated effects of peptide hydrolysis.
[0114] Determining the ability of the carboxypeptidase to bind to a
target molecule can also be accomplished using a technology such as
real-time Bimolecular Interaction Analysis (BIA). Sjolander et al.
(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.
[0115] 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 polypeptide libraries, while the
other four approaches are applicable to polypeptide, non-peptide
oligomer or small molecule libraries of compounds (Lam, K. S.
(1997) Anticancer Drug Des. 12:145).
[0116] 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. USA 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; Carell 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. 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 U.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat.
No. '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. 97:6378-6382); (Felici (1991) J. Mol.
Biol. 222:301-310); (Ladner supra).
[0117] Candidate compounds include, for example, 1) peptides such
as soluble peptides, including Ig-tailed fusion peptides and
members of random peptide libraries (see, e.g., Lam et al. (1991)
Nature 354:82-84; Houghten et al. (1991) Nature 354:84-86) and
combinatorial chemistry-derived molecular libraries made of D-
and/or L- configuration amino acids; 2) phosphopeptides (e.g.,
members of random and partially degenerate, directed phosphopeptide
libraries, see, e.g., Songyang et al. (1993) Cell 72:767-778); 3)
antibodies (e.g., polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric, and single chain antibodies as well as
Fab, F(ab').sub.2, Fab expression library fragments, and
epitope-binding fragments of antibodies); and 4) small organic and
inorganic molecules (e.g., molecules obtained from combinatorial
and natural product libraries).
[0118] One candidate compound is a soluble full-length
carboxypeptidase or fragment that competes for substrate binding.
Other candidate compounds include mutant carboxypeptidases or
appropriate fragments containing mutations that affect
carboxypeptidase function and thus compete for substrate.
Accordingly, a fragment that competes for substrate, for example
with a higher affinity, or a fragment that binds the substrate but
does not degrade it, is encompassed by the invention.
[0119] The invention provides other end points to identify
compounds that modulate (stimulate or inhibit) carboxypeptidase
activity. The assays typically involve an assay of cellular events
that indicate carboxypeptidase activity. Thus, the expression of
genes that are up- or down-regulated in response to
carboxypeptidase activity can be assayed. In one embodiment, the
regulatory region of such genes can be operably linked to a marker
that is easily detectable, such as luciferase. Alternatively,
modification of the carboxypeptidase could also be measured.
[0120] Any of the biological or biochemical functions mediated by
the carboxypeptidase can be used as an endpoint assay. These
include all of the biochemical or biochemical/biological events
described herein, in the references cited herein, incorporated by
reference for these endpoint assay targets, and other functions
known to those of ordinary skill in the art.
[0121] In the case of the carboxypeptidase, assays for specific end
points can include assays for peptide hydrolysis (e.g., amino acid
production or change in substrate peptide size).
[0122] Binding and/or activating compounds can also be screened by
using chimeric carboxypeptidase proteins in which one or more
regions/domains, segments, sites, and the like, as disclosed
herein, or parts thereof, can be replaced by their heterologous
counterparts derived from other carboxypeptidases. For example, a
catalytic region can be used that interacts with a different
specificity and/or affinity than the native carboxypeptidase.
Accordingly, a different set of cellular components is available as
an end-point assay for activation. Alternatively, a
membrane-associated portion or subregions can be replaced with the
membrane portion or subregions specific to a host cell that is
different from the host cell from which the native carboxypeptidase
is derived. This allows for assays to be performed in other than
the specific host cell from which the carboxypeptidase is derived.
Alternatively, a heterologous substrate recognition sequence can
replace the native sequence. This can result in having an effect on
a different cellular pathway. Accordingly, a different set of
cellular components is available as an endpoint assay for
activation. Activation can also be detected by a reporter gene
containing an easily detectable coding region operably linked to a
transcriptional regulatory sequence that is part of the native
pathway.
[0123] The carboxypeptidase polypeptides are also useful in
competition binding assays in methods designed to discover
compounds that interact with the carboxypeptidase. Thus, a compound
is exposed to a carboxypeptidase polypeptide under conditions that
allow the compound to bind or to otherwise interact with the
polypeptide. Soluble carboxypeptidase polypeptide is also added to
the mixture. If the test compound interacts with the soluble
carboxypeptidase polypeptide, it decreases the amount of complex
formed or activity from the carboxypeptidase target. This type of
assay is particularly useful in cases in which compounds are sought
that interact with specific regions of the carboxypeptidase. Thus,
the soluble polypeptide that competes with the target
carboxypeptidase region is designed to contain peptide sequences
corresponding to the region of interest.
[0124] Another type of competition-binding assay can be used to
discover compounds that interact with specific functional sites. As
an example, peptide substrate and a candidate compound can be added
to a sample of the carboxypeptidase. Compounds that interact with
the carboxypeptidase at the same site as the peptide will reduce
the amount of complex formed between the carboxypeptidase and the
peptide. Accordingly, it is possible to discover a compound that
specifically prevents interaction between the carboxypeptidase and
the peptide. Another example involves adding a candidate compound
to a sample of carboxypeptidase and peptide substrate. A compound
that competes with the peptide substrate will reduce the amount of
hydrolysis or binding of the substrate to the carboxypeptidase.
Accordingly, compounds can be discovered that directly interact
with the carboxypeptidase and compete with the substrate. Such
assays can involve any other component that interacts with the
carboxypeptidase.
[0125] To perform cell free drug screening assays, it is desirable
to immobilize either the carboxypeptidase, or fragment, or its
target molecule to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay.
[0126] Techniques for immobilizing proteins on matrices can be used
in the drug screening assays. In one embodiment, a fusion protein
can be provided which adds a domain that allows the protein to be
bound to a matrix. For example,
glutathione-S-transferase/carboxypeptidase 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 cell lysates (e.g., .sup.35S-labeled) and
the candidate compound, and the mixture incubated under conditions
conducive to complex formation (e.g., at physiological conditions
for salt and pH). Following incubation, the beads are washed to
remove any unbound label, and the matrix immobilized and radiolabel
determined directly, or in the supernatant after the complexes is
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of
carboxypeptidase-binding protein found in the bead fraction
quantitated from the gel using standard electrophoretic techniques.
For example, either the polypeptide or its target molecule can be
immobilized utilizing conjugation of biotin and streptavidin using
techniques well known in the art. Alternatively, antibodies
reactive with the protein but which do not interfere with binding
of the protein to its target molecule can be derivatized to the
wells of the plate, and the protein trapped in the wells by
antibody conjugation. Preparations of a carboxypeptidase-binding
target component, such as peptide substrate, and a candidate
compound are incubated in the carboxypeptidase-presenting wells and
the amount of complex trapped in the well can be quantitated.
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
carboxypeptidase target molecule, or which are reactive with
carboxypeptidase and compete with the target molecule; as well as
enzyme-linked assays which rely on detecting an enzymatic activity
associated with the target molecule.
[0127] Modulators of carboxypeptidase activity identified according
to these drug screening assays can be used to treat a subject with
a disorder related to the carboxypeptidase, by treating cells that
express the carboxypeptidase, such as those disclosed here.
Preferred tissues in which the carboxypeptidase is expressed
include, but are not limited to, prostate, breast, skeletal muscle,
brain, testes, thyroid, and fetal liver, kidney, and heart.
Further, preferred tissues include, but are not limited to, colon
and breast carcinoma, and lung carcinoma, especially squamous cell
carcinoma. In addition, this carboxypeptidase is overexpressed in
breast, lung and colon tumors as disclosed herein. Accordingly,
expression of the carboxypeptidase is especially relevant to cancer
treatment, including invasion and metastasis. These methods of
treatment include the steps of administering the modulators of
carboxypeptidase activity in a pharmaceutical composition as
described herein, to a subject in need of such treatment.
[0128] Further disorders in which the carboxypeptidase expression
is relevant include, but are not limited to, any disorders
involving the cells in which the carboxypeptidase is expressed as
disclosed herein.
[0129] Disorders involving the lung include, but are not limited
to, congenital anomalies; atelectasis; diseases of vascular origin,
such as pulmonary congestion and edema, including hemodynamic
pulmonary edema and edema caused by microvascular injury, adult
respiratory distress syndrome (diffuse alveolar damage), pulmonary
embolism, hemorrhage, and infarction, and pulmonary hypertension
and vascular sclerosis; chronic obstructive pulmonary disease, such
as emphysema, chronic bronchitis, bronchial asthma, and
bronchiectasis; diffuse interstitial (infiltrative, restrictive)
diseases, such as pneumoconioses, sarcoidosis, idiopathic pulmonary
fibrosis, desquamative interstitial pneumonitis, hypersensitivity
pneumonitis, pulmonary eosinophilia (pulmonary infiltration with
eosinophilia), Bronchiolitis obliterans-organizing pneumonia,
diffuse pulmonary hemorrhage syndromes, including Goodpasture
syndrome, idiopathic pulmonary hemosiderosis and other hemorrhagic
syndromes, pulmonary involvement in collagen vascular disorders,
and pulmonary alveolar proteinosis; complications of therapies,
such as drug-induced lung disease, radiation-induced lung disease,
and lung transplantation; tumors, such as bronchogenic carcinoma,
including paraneoplastic syndromes, bronchioloalveolar carcinoma,
neuroendocrine tumors, such as bronchial carcinoid, miscellaneous
tumors, and metastatic tumors; pathologies of the pleura, including
inflammatory pleural effusions, noninflammatory pleural effusions,
pneumothorax, and pleural tumors, including solitary fibrous tumors
(pleural fibroma) and malignant mesothelioma.
[0130] Disorders involving the colon include, but are not limited
to, congenital anomalies, such as atresia and stenosis, Meckel
diverticulum, congenital aganglionic megacolon-Hirschsprung
disease; enterocolitis, such as diarrhea and dysentery, infectious
enterocolitis, including viral gastroenteritis, bacterial
enterocolitis, necrotizing enterocolitis, antibiotic-associated
colitis (pseudomembranous colitis), and collagenous and lymphocytic
colitis, miscellaneous intestinal inflammatory disorders, including
parasites and protozoa, acquired immunodeficiency syndrome,
transplantation, drug-induced intestinal injury, radiation
enterocolitis, neutropenic colitis (typhlitis), and diversion
colitis; idiopathic inflammatory bowel disease, such as Crohn
disease and ulcerative colitis; tumors of the colon, such as
non-neoplastic polyps, adenomas, familial syndromes, colorectal
carcinogenesis, colorectal carcinoma, and carcinoid tumors.
[0131] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, a.sub.1-antitrypsin deficiency,
and neonatal hepatitis; intrahepatic biliary tract disease, such as
secondary biliary cirrhosis, primary biliary cirrhosis, primary
sclerosing cholangitis, and anomalies of the biliary tree;
circulatory disorders, such as impaired blood flow into the liver,
including hepatic artery compromise and portal vein obstruction and
thrombosis, impaired blood flow through the liver, including
passive congestion and centrilobular necrosis and peliosis hepatis,
hepatic vein outflow obstruction, including hepatic vein thrombosis
(Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease
associated with pregnancy, such as preeclampsia and eclampsia,
acute fatty liver of pregnancy, and intrehepatic cholestasis of
pregnancy; hepatic complications of organ or bone marrow
transplantation, such as drug toxicity after bone marrow
transplantation, graft-versus-host disease and liver rejection, and
nonimmunologic damage to liver allografts; tumors and tumorous
conditions, such as nodular hyperplasias, adenomas, and malignant
tumors, including primary carcinoma of the liver and metastatic
tumors.
[0132] Disorders involving the brain include, but are limited to,
disorders involving neurons, and disorders involving glia, such as
astrocytes, oligodendrocytes, ependyrnal cells, and microglia;
cerebral edema, raised intracranial pressure and herniation, and
hydrocephalus; malformations and developmental diseases, such as
neural tube defects, forebrain anomalies, posterior fossa
anomalies, and syringomyelia and hydromyelia; perinatal brain
injury; cerebrovascular diseases, such as those related to hypoxia,
ischemia, and infarction, including hypotension, hypoperfusion, and
low-flow states--global cerebral ischemia and focal cerebral
ischemia--infarction from obstruction of local blood supply,
intracranial hemorrhage, including intracerebral (intraparenchymal)
hemorrhage, subarachnoid hemorrhage and ruptured berry aneurysms,
and vascular malformations, hypertensive cerebrovascular disease,
including lacunar infarcts, slit hemorrhages, and hypertensive
encephalopathy; infections, such as acute meningitis, including
acute pyogenic (bacterial) meningitis and acute aseptic (viral)
meningitis, acute focal suppurative infections, including brain
abscess, subdural empyema, and extradural abscess, chronic
bacterial meningoencephalitis, including tuberculosis and
mycobacterioses, neurosyphilis, and neuroborreliosis (Lyme
disease), viral meningoencephalitis, including arthropod-bome
(Arbo) viral encephalitis, Herpes simplex virus Type 1, Herpes
simplex virus Type 2, Varicalla-zoster virus (Herpes zoster),
cytomegalovirus, poliomyelitis, rabies, and human immunodeficiency
virus 1, including HIV-1 meningoencephalitis (subacute
encephalitis), vacuolar myelopathy, AIDS-associated myopathy,
peripheral neuropathy, and AIDS in children, progressive multifocal
leukoencephalopathy, subacute sclerosing panencephalitis, fungal
meningoencephalitis, other infectious diseases of the nervous
system; transmissible spongiform encephalopathies (prion diseases);
demyelinating diseases, including multiple sclerosis, multiple
sclerosis variants, acute disseminated encephalomyelitis and acute
necrotizing hemorrhagic encephalomyelitis, and other diseases with
demyelination; degenerative diseases, such as degenerative diseases
affecting the cerebral cortex, including Alzheimer disease and Pick
disease, degenerative diseases of basal ganglia and brain stem,
including Parkinsonism, idiopathic Parkinson disease (paralysis
agitans), progressive supranuclear palsy, corticobasal degenration,
multiple system atrophy, including striatonigral degenration,
Shy-Drager syndrome, and olivopontocerebellar atrophy, and
Huntington disease; spinocerebellar degenerations, including
spinocerebellar ataxias, including Friedreich ataxia, and
ataxia-telanglectasia, degenerative diseases affecting motor
neurons, including amyotrophic lateral sclerosis (motor neuron
disease), bulbospinal atrophy (Kennedy syndrome), and spinal
muscular atrophy; inborn errors of metabolism, such as
leukodystrophies, including Krabbe disease, metachromatic
leukodystrophy, adrenoleukodystrophy, Pelizaeus-Merzbacher disease,
and Canavan disease, mitochondrial encephalomyopathies, including
Leigh disease and other mitochondrial encephalomyopathies; toxic
and acquired metabolic diseases, including vitamin deficiencies
such as thiamine (vitamin B.sub.1) deficiency and vitamin B.sub.12
deficiency, neurologic sequelae of metabolic disturbances,
including hypoglycemia, hyperglycemia, and hepatic encephatopathy,
toxic disorders, including carbon monoxide, methanol, ethanol, and
radiation, including combined methotrexate and radiation-induced
injury; tumors, such as gliomas, including astrocytoma, including
fibrillary (diffuse) astrocytoma and glioblastoma multiforme,
pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and brain
stem glioma, oligodendroglioma, and ependymoma and related
paraventricular mass lesions, neuronal tumors, poorly
differentiated neoplasms, including medulloblastoma, other
parenchymal tumors, including primary brain lymphoma, germ cell
tumors, and pineal parenchymal tumors, meningiomas, metastatic
tumors, paraneoplastic syndromes, peripheral nerve sheath tumors,
including schwannoma, neurofibroma, and malignant peripheral nerve
sheath tumor (malignant schwannoma), and neurocutaneous syndromes
(phakomatoses), including neurofibromotosis, including Type 1
neurofibromatosis (NF1) and TYPE 2 neurofibromatosis (NF2),
tuberous sclerosis, and Von Hippel-Lindau disease.
[0133] Disorders involving the heart, include but are not limited
to, heart failure, including but not limited to, cardiac
hypertrophy, left-sided heart failure, and right-sided heart
failure; ischemic heart disease, including but not limited to
angina pectoris, myocardial infarction, chronic ischemic heart
disease, and sudden cardiac death; hypertensive heart disease,
including but not limited to, systemic (left-sided) hypertensive
heart disease and pulmonary (right-sided) hypertensive heart
disease; valvular heart disease, including but not limited to,
valvular degeneration caused by calcification, such as calcific
aortic stenosis, calcification of a congenitally bicuspid aortic
valve, and mitral annular calcification, and myxomatous
degeneration of the mitral valve (mitral valve prolapse), rheumatic
fever and rheumatic heart disease, infective endocarditis, and
noninfected vegetations, such as nonbacterial thrombotic
endocarditis and endocarditis of systemic lupus erythematosus
(Libman-Sacks disease), carcinoid heart disease, and complications
of artificial valves; myocardial disease, including but not limited
to dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive
cardiomyopathy, and myocarditis; pericardial disease, including but
not limited to, pericardial effusion and hemopericardium and
pericarditis, including acute pericarditis and healed pericarditis,
and rheumatoid heart disease; neoplastic heart disease, including
but not limited to, primary cardiac tumors, such as myxoma, lipoma,
papillary fibroelastoma, rhabdomyoma, and sarcoma, and cardiac
effects of noncardiac neoplasms; congenital heart disease,
including but not limited to, left-to-right shunts--late cyanosis,
such as atrial septal defect, ventricular septal defect, patent
ductus arteriosus, and atrioventricular septal defect,
right-to-left shunts--early cyanosis, such as tetralogy of fallot,
transposition of great arteries, truncus arteriosus, tricuspid
atresia, and total anomalous pulmonary venous connection,
obstructive congenital anomalies, such as coarctation of aorta,
pulmonary stenosis and atresia, and aortic stenosis and atresia,
and disorders involving cardiac transplantation.
[0134] The carboxypeptidase polypeptides are thus useful for
treating a carboxypeptidase-associated disorder characterized by
aberrant expression or activity of a carboxypeptidase. In one
embodiment, the method involves 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) expression or activity of the protein. In another
embodiment, the method involves administering the carboxypeptidase
as therapy to compensate for reduced or aberrant expression or
activity of the protein.
[0135] Methods for treatment include but are not limited to the use
of soluble carboxypeptidase or fragments of the carboxypeptidase
protein that compete for substrate. These carboxypeptidases or
fragments can have a higher affinity for the target so as to
provide effective competition.
[0136] Stimulation of activity is desirable in situations in which
the protein is abnormally downregulated and/or in which increased
activity is likely to have a beneficial effect. Likewise,
inhibition of activity is desirable in situations in which the
protein is abnormally upregulated and/or in which decreased
activity is likely to have a beneficial effect. In one example of
such a situation, a subject has a disorder characterized by
aberrant development or cellular differentiation. In another
example, the subject has a proliferative disease (e.g., cancer) or
a disorder characterized by an aberrant hematopoietic response. In
another example, it is desirable to achieve 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).
[0137] In yet another aspect of the invention, the proteins of the
invention 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 WO
94/10300), to identify other proteins (captured proteins) which
bind to or interact with the proteins of the invention and modulate
their activity.
[0138] The carboxypeptidase polypeptides also are useful to provide
a target for diagnosing a disease or predisposition to disease
related to the carboxypeptidase, including, but not limited to,
those diseases discussed herein, and particularly breast, colon and
lung carcinoma. Targets are useful for diagnosing a disease or
predisposition to disease mediated by the carboxypeptidase,
especially in the tissues shown in those found herein, especially
in prostate, breast, skeletal muscle, brain, testis, thyroid, and
carcinomas, such as in colon, breast, and lung. Accordingly,
methods are provided for detecting the presence, or levels of, the
carboxypeptidase in a cell, tissue, or organism. The method
involves contacting a biological sample with a compound capable of
interacting with the carboxypeptidase such that the interaction can
be detected.
[0139] One agent for detecting carboxypeptidase is an antibody
capable of selectively binding to carboxypeptidase. A biological
sample includes tissues, cells and biological fluids isolated from
a subject, as well as tissues, cells and fluids present within a
subject.
[0140] The carboxypeptidase also provides a target for diagnosing
active disease, or predisposition to disease, in a patient having a
variant carboxypeptidase. Thus, carboxypeptidase can be isolated
from a biological sample and assayed for the presence of a genetic
mutation that results in an aberrant protein. This includes amino
acid substitution, deletion, insertion, rearrangement, (as the
result of aberrant splicing events), and inappropriate
post-translational modification. Analytic methods include altered
electrophoretic mobility, altered tryptic peptide digest, altered
carboxypeptidase activity in cell-based or cell-free assay,
alteration in substrate binding or degradation, antibody-binding
pattern, altered isoelectric point, direct amino acid sequencing,
and any other of the known assay techniques useful for detecting
mutations in a protein in general or in a carboxypeptidase
specifically.
[0141] In vitro techniques for detection of carboxypeptidase
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. Alternatively, the
protein can be detected in vivo in a subject by introducing into
the subject a labeled anti-carboxypeptidase 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. Particularly useful are methods, which detect
the allelic variant of the carboxypeptidase expressed in a subject,
and methods, which detect fragments of the carboxypeptidase in a
sample.
[0142] The carboxypeptidase polypeptides are also useful in
pharmacogenomic analysis. Pharmacogenomics deal 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. (1996) Clin. Exp. Pharmacol. Physiol.
23(10-11):983-985, and Linder, M. W. (1997) Clin. Chem.
43(2):254-266. The clinical outcomes of these variations result in
severe toxicity of therapeutic drugs in certain individuals or
therapeutic failure of drugs in certain individuals as a result of
individual variation in metabolism. Thus, the genotype of the
individual can determine the way a therapeutic compound acts on the
body or the way the body metabolizes the compound. Further, the
activity of drug metabolizing enzymes affects both the intensity
and duration of drug action. Thus, the pharmacogenomics of the
individual permit the selection of effective compounds and
effective dosages of such compounds for prophylactic or therapeutic
treatment based on the individual's genotype. The discovery of
genetic polymorphisms in some drug metabolizing enzymes has
explained why some patients do not obtain the expected drug
effects, show an exaggerated drug effect, or experience serious
toxicity from standard drug dosages. Polymorphisms can be expressed
in the phenotype of the extensive metabolizer and the phenotype of
the poor metabolizer. Accordingly, genetic polymorphism may lead to
allelic protein variants of the carboxypeptidase in which one or
more of the carboxypeptidase functions in one population is
different from those in another population. The polypeptides thus
allow a target to ascertain a genetic predisposition that can
affect treatment modality. Thus, in a peptide-based treatment,
polymorphism may give rise to catalytic regions that are more or
less active. Accordingly, dosage would necessarily be modified to
maximize the therapeutic effect within a given population
containing the polymorphism. As an alternative to genotyping,
specific polymorphic polypeptides could be identified.
[0143] The carboxypeptidase polypeptides are also useful for
monitoring therapeutic effects during clinical trials and other
treatment. Thus, the therapeutic effectiveness of an agent that is
designed to increase or decrease gene expression, protein levels or
carboxypeptidase activity can be monitored over the course of
treatment using the carboxypeptidase polypeptides as an end-point
target. The monitoring can be, for example, as follows: (i)
obtaining a pre-administration sample from a subject prior to
administration of the agent; (ii) detecting the level of expression
or activity of the protein in the pre-administration sample; (iii)
obtaining one or more post-administration samples from the subject;
(iv) detecting the level of expression or activity of the protein
in the post-administration samples; (v) comparing the level of
expression or activity of the protein in the pre-administration
sample with the protein in the post-administration sample or
samples; and (vi) increasing or decreasing the administration of
the agent to the subject accordingly.
[0144] Antibodies
[0145] The invention also provides antibodies that selectively bind
to the carboxypeptidase and its variants and fragments. An antibody
is considered to selectively bind, even if it also binds to other
proteins that are not substantially homologous with the
carboxypeptidase. These other proteins share homology with a
fragment or domain of the carboxypeptidase. This conservation in
specific regions gives rise to antibodies that bind to both
proteins by virtue of the homologous sequence. In this case, it
would be understood that antibody binding to the carboxypeptidase
is still selective.
[0146] To generate antibodies, an isolated carboxypeptidase
polypeptide is used as an immunogen to generate antibodies using
standard techniques for polyclonal and monoclonal antibody
preparation. Either the full-length protein or antigenic peptide
fragment can be used. Regions having a high antigenicity index are
shown in FIG. 3.
[0147] Antibodies are preferably prepared from these regions or
from discrete fragments in these regions. However, antibodies can
be prepared from any region of the peptide as described herein. A
preferred fragment produces an antibody that diminishes or
completely prevents substrate hydrolysis or binding. Antibodies can
be developed against the entire carboxypeptidase or regions/domains
of the carboxypeptidase as described herein. Antibodies can also be
developed against specific functional sites as disclosed
herein.
[0148] The antigenic peptide can comprise a contiguous sequence of
at least 12, 14, 15, or 30 amino acid residues. In one embodiment,
fragments correspond to regions that are located on the surface of
the protein, e.g., hydrophilic regions. These fragments are not to
be construed, however, as encompassing any fragments, which may be
disclosed prior to the invention.
[0149] Antibodies can be polyclonal or monoclonal. An intact
antibody, or a fragment thereof (e.g. Fab or F(ab').sub.2) can be
used.
[0150] 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.
[0151] An appropriate immunogenic preparation can be derived from
native, recombinantly expressed, or chemically synthesized
peptides.
[0152] Antibody Uses
[0153] The antibodies can be used to isolate a carboxypeptidase by
standard techniques, such as affinity chromatography or
immunoprecipitation. The antibodies can facilitate the purification
of the natural carboxypeptidase from cells and recombinantly
produced carboxypeptidase expressed in host cells.
[0154] The antibodies are useful to detect the presence of
carboxypeptidase in cells or tissues to determine the pattern of
expression of the carboxypeptidase among various tissues in an
organism and over the course of normal development.
[0155] The antibodies can be used to detect carboxypeptidase in
situ, in vitro, or in a cell lysate or supernatant in order to
evaluate the abundance and pattern of expression.
[0156] The antibodies can be used to assess abnormal tissue
distribution or abnormal expression during development.
[0157] Antibody detection of circulating fragments of the full
length carboxypeptidase can be used to identify carboxypeptidase
turnover.
[0158] Further, the antibodies can be used to assess
carboxypeptidase expression in disease states such as in active
stages of the disease or in an individual with a predisposition
toward disease related to carboxypeptidase function. When a
disorder is caused by an inappropriate tissue distribution,
developmental expression, or level of expression of the
carboxypeptidase protein, the antibody can be prepared against the
normal carboxypeptidase protein. If a disorder is characterized by
a specific mutation in the carboxypeptidase, antibodies specific
for this mutant protein can be used to assay for the presence of
the specific mutant carboxypeptidase. However, intracellularly-made
antibodies ("intrabodies") are also encompassed, which would
recognize intracellular carboxypeptidase peptide regions.
[0159] The antibodies can also be used to assess normal and
aberrant subcellular localization of cells in the various tissues
in an organism. Antibodies can be developed against the whole
carboxypeptidase or portions of the carboxypeptidase, for example,
the catalytic region.
[0160] The diagnostic uses can be applied, not only in genetic
testing, but also in monitoring a treatment modality. Accordingly,
where treatment is ultimately aimed at correcting carboxypeptidase
expression level or the presence of aberrant carboxypeptidases and
aberrant tissue distribution or developmental expression,
antibodies directed against the carboxypeptidase or relevant
fragments can be used to monitor therapeutic efficacy.
[0161] Antibodies accordingly 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.
[0162] Additionally, antibodies are useful in pharmacogenomic
analysis. Thus, antibodies prepared against polymorphic
carboxypeptidase can be used to identify individuals that require
modified treatment modalities.
[0163] The antibodies are also useful as diagnostic tools as an
immunological marker for aberrant carboxypeptidase analyzed by
electrophoretic mobility, isoelectric point, tryptic peptide
digest, and other physical assays known to those in the art.
[0164] The antibodies are also useful for tissue typing. Thus,
where a specific carboxypeptidase has been correlated with
expression in a specific tissue, antibodies that are specific for
this carboxypeptidase can be used to identify a tissue type.
[0165] The antibodies are also useful in forensic identification.
Accordingly, where an individual has been correlated with a
specific genetic polymorphism resulting in a specific polymorphic
protein, an antibody specific for the polymorphic protein can be
used as an aid in identification.
[0166] The antibodies are also useful for inhibiting
carboxypeptidase function, for example, blocking the catalytic
site.
[0167] These uses can also be applied in a therapeutic context in
which treatment involves inhibiting carboxypeptidase function. An
antibody can be used, for example, to block peptide substrate
binding. Antibodies can be prepared against specific fragments
containing sites required for function or against intact
carboxypeptidase associated with a cell.
[0168] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. For an overview of this
technology for producing human antibodies, see Lonberg et al.
(1995) Int. Rev. Immunol. 13:65-93. For a detailed discussion of
this technology for producing human antibodies and human monoclonal
antibodies and protocols for producing such antibodies, e.g., U.S.
Pat. No. 5,625,126; 5,633,425; 5,569,825; 5,661,016; and
5,545,806.
[0169] The invention also encompasses kits for using antibodies to
detect the presence of a carboxypeptidase protein in a biological
sample. The kit can comprise antibodies such as a labeled or
labelable antibody and a compound or agent for detecting
carboxypeptidase in a biological sample; means for determining the
amount of carboxypeptidase in the sample; and means for comparing
the amount of carboxypeptidase 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
carboxypeptidase.
[0170] Polynucleotides
[0171] The nucleotide sequences in SEQ ID NO:2 were obtained by
sequencing the deposited human cDNA. Accordingly, the sequence of
the deposited clone is controlling as to any discrepancies between
the two and any reference to the sequence of SEQ ID NO:2 includes
reference to the sequence of the deposited cDNA.
[0172] The specifically disclosed cDNA comprises the coding region
and 5' and 3' untranslated sequences in SEQ ID NO:2.
[0173] The nucleic acid is expressed in those tissues as disclosed
herein. In particular, the carboxypeptidase is expressed in
prostate, breast, skeletal muscle, brain, testis, thyroid, and
carcinomas, such as in colon, breast, and lung. In addition, this
carboxypeptidase is overexpressed in a number of breast, lung and
colon tumor lines as disclosed herein.
[0174] The invention provides isolated polynucleotides encoding the
novel carboxypeptidases. The term "carboxypeptidase polynucleotide"
or "carboxypeptidase nucleic acid" refers to the sequence shown in
SEQ ID NO:2 or in the deposited cDNA. The term "carboxypeptidase
polynucleotide" or "carboxypeptidase nucleic acid" further includes
variants and fragments of the carboxypeptidase polynucleotides.
[0175] An "isolated" carboxypeptidase nucleic acid is one that is
separated from other nucleic acid present in the natural source of
the carboxypeptidase nucleic acid. Preferably, an "isolated"
nucleic acid is free of sequences which naturally flank the
carboxypeptidase 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. However, there can be some
flanking nucleotide sequences, for example up to about 5KB. The
important point is that the carboxypeptidase nucleic acid is
isolated from flanking sequences such that it can be subjected to
the specific manipulations described herein, such as recombinant
expression, preparation of probes and primers, and other uses
specific to the carboxypeptidase nucleic acid sequences.
[0176] Moreover, an "isolated" nucleic acid molecule, such as a
cDNA or RNA molecule, can be substantially free of other cellular
material, or culture medium when produced by recombinant
techniques, or chemical precursors or other chemicals when
chemically synthesized. However, the nucleic acid molecule can be
fused to other coding or regulatory sequences and still be
considered isolated.
[0177] In some instances, the isolated material will form part of a
composition (for example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0178] For example, recombinant DNA molecules contained in a vector
are considered isolated. Further examples of isolated DNA molecules
include recombinant DNA molecules maintained in heterologous host
cells or purified (partially or substantially) DNA molecules in
solution. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the isolated DNA molecules of the present invention.
Isolated nucleic acid molecules according to the present invention
further include such molecules produced synthetically.
[0179] In some instances, the isolated material will form part of a
composition (or example, a crude extract containing other
substances), buffer system or reagent mix. In other circumstances,
the material may be purified to essential homogeneity, for example
as determined by PAGE or column chromatography such as HPLC.
Preferably, an isolated nucleic acid comprises at least about 50,
80 or 90% (on a molar basis) of all macromolecular species
present.
[0180] The carboxypeptidase polynucleotides can encode the mature
protein plus additional amino or carboxyterminal amino acids, or
amino acids interior to the mature polypeptide (when the mature
form has more than one polypeptide chain, for instance). Such
sequences may play a role in processing of a protein from precursor
to a mature form, facilitate protein trafficking, prolong or
shorten protein half-life or facilitate manipulation of a protein
for assay or production, among other things. As generally is the
case in situ, the additional amino acids may be processed away from
the mature protein by cellular enzymes.
[0181] The carboxypeptidase polynucleotides include, but are not
limited to, the sequence encoding the mature polypeptide alone, the
sequence encoding the mature polypeptide and additional coding
sequences, such as a leader or secretory sequence (e.g., a pre-pro
or pro-protein sequence), the sequence encoding the mature
polypeptide, with or without the additional coding sequences, plus
additional non-coding sequences, for example introns and non-coding
5' and 3' sequences such as transcribed but non-translated
sequences that play a role in transcription, mRNA processing
(including splicing and polyadenylation signals), ribosome binding
and stability of mRNA. In addition, the polynucleotide may be fused
to a marker sequence encoding, for example, a peptide that
facilitates purification.
[0182] Carboxypeptidase polynucleotides can be in the form of RNA,
such as mRNA, or in the form DNA, including cDNA and genomic DNA
obtained by cloning or produced by chemical synthetic techniques or
by a combination thereof. The nucleic acid, especially DNA, can be
double-stranded or single-stranded. Single-stranded nucleic acid
can be the coding strand (sense strand) or the non-coding strand
(anti-sense strand).
[0183] Carboxypeptidase nucleic acid can comprise the nucleotide
sequences shown in SEQ ID NO:2, corresponding to human osteoblast
or brain cDNA.
[0184] In one embodiment, the carboxypeptidase nucleic acid
comprises only the coding region.
[0185] The invention further provides variant carboxypeptidase
polynucleotides, and fragments thereof, that differ from the
nucleotide sequence shown in SEQ ID NO:2 due to degeneracy of the
genetic code and thus encode the same protein as that encoded by
the nucleotide sequence shown in SEQ ID NO:2.
[0186] The invention also provides carboxypeptidase nucleic acid
molecules encoding the variant polypeptides described herein. Such
polynucleotides may be naturally occurring, such as allelic
variants (same locus), homologs (different locus), and orthologs
(different organism), or may be constructed by recombinant DNA
methods or by chemical synthesis. Such non-naturally occurring
variants may be made by mutagenesis techniques, including those
applied to polynucleotides, cells, or organisms. Accordingly, as
discussed above, the variants can contain nucleotide substitutions,
deletions, inversions and insertions.
[0187] Typically, variants have a substantial identity with a
nucleic acid molecules of SEQ ID NO:2 and the complements thereof
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions.
[0188] Orthologs, homologs, and allelic variants can be identified
using methods well known in the art. These variants comprise a
nucleotide sequence encoding a carboxypeptidase that is typically
at least about 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, more
typically at least about 80-85%, and most typically at least about
90-95% or more homologous to the nucleotide sequence shown in SEQ
ID NO:2 or a fragment of this sequence. Such nucleic acid molecules
can readily be identified as being able to hybridize under
stringent conditions, to the nucleotide sequence shown in SEQ ID
NO:2 or a fragment of the sequence. It is understood that stringent
hybridization does not indicate substantial homology where it is
due to general homology, such as poly A sequences, or sequences
common to all or most proteins, all carboxypeptidases, all prolyl
carboxypeptidases or all seryl carboxypeptidases. Moreover, it is
understood that variants do not include any of the nucleic acid
sequences that may have been disclosed prior to the invention.
[0189] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences encoding a polypeptide
at least about 50-55% homologous to each other typically remain
hybridized to each other. The conditions can be such that sequences
at least about 60-65%, at least about 70%, at least about 75%, at
least about 80%, at least about 90%, at least about 95% or more
identical to each other remain hybridized to one another. 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, incorporated by reference.
One 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. In another non-limiting example,
nucleic acid molecules are allowed to hybridize in 6.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more low stringency washes in 0.2.times. SSC/0.1% SDS at
room temperature, or by one or more moderate stringency washes in
0.2.times. SSC/0. 1% SDS at 42.degree. C., or washed in 0.2.times.
SSC/0.1% SDS at 65.degree. C. for high stringency. In one
embodiment, an isolated nucleic acid molecule 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).
[0190] As understood by those of ordinary skill, the exact
conditions can be determined empirically and depend on ionic
strength, temperature and the concentration of destabilizing agents
such as formamide or denaturing agents such as SDS. Other factors
considered in determining the desired hybridization conditions
include the length of the nucleic acid sequences, base composition,
percent mismatch between the hybridizing sequences and the
frequency of occurrence of subsets of the sequences within other
non-identical sequences. Thus, equivalent conditions can be
determined by varying one or more of these parameters while
maintaining a similar degree of identity or similarity between the
two nucleic acid molecules.
[0191] The present invention also provides isolated nucleic acids
that contain a single or double stranded fragment or portion that
hybridizes under stringent conditions to the nucleotide sequence of
SEQ ID NO:2 or the complement of SEQ ID NO:2. In one embodiment,
the nucleic acid consists of a portion of the nucleotide sequence
of SEQ ID NO:2 and the complement of SEQ ID NO:2. The nucleic acid
fragments of the invention are at least about 15, preferably at
least about 18, 20, 23 or 25 nucleotides, and can be 30, 40, 50,
100, 200, 500 or more nucleotides in length. Longer fragments, for
example, 30 or more nucleotides in length, which encode antigenic
proteins or polypeptides described herein are useful.
[0192] As discussed, the invention provides polynucleotides that
comprise a fragment of the full-length carboxypeptidase
polynucleotides. The fragment can be single or double-stranded and
can comprise DNA or RNA. The fragment can be derived from either
the coding or the non-coding sequence.
[0193] In another embodiment an isolated carboxypeptidase nucleic
acid encodes the entire coding region. In another embodiment the
isolated carboxypeptidase nucleic acid encodes a sequence
corresponding to the mature protein that may be from about amino
acid 6 to the last amino acid. Other fragments include nucleotide
sequences encoding the amino acid fragments described herein.
[0194] Thus, carboxypeptidase nucleic acid fragments further
include sequences corresponding to the regions/domains described
herein, subregions also described, and specific functional sites.
Carboxypeptidase nucleic acid fragments also include combinations
of the regions/domains, segments, and other functional sites
described above. A person of ordinary skill in the art would be
aware of the many permutations that are possible.
[0195] Where the location of the domains or sites have been
predicted by computer analysis, one of ordinary sill would
appreciate that the amino acid residues constituting these domains
can vary depending on the criteria used to define the domains.
[0196] However, it is understood that a carboxypeptidase fragment
includes any nucleic acid sequence that does not include the entire
gene.
[0197] The invention also provides carboxypeptidase nucleic acid
fragments that encode epitope bearing regions of the
carboxypeptidase proteins described herein.
[0198] Fragments from about 1-660 can comprise about 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, or more
nucleotides.
[0199] Nucleic acid fragments, according to the present invention,
are not to be construed as encompassing those fragments that may
have been disclosed prior to the invention.
[0200] Polynucleotide Uses
[0201] The nucleotide sequences of the present invention can be
used as a "query sequence" to perform a search against public
databases, for example, to 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 protein searches can be performed with the
XBLAST program, score=50, wordlength=3 to obtain amino acid
sequences homologous to the proteins 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.
[0202] The nucleic acid fragments of the invention provide probes
or primers in assays such as those described below. "Probes" are
oligonucleotides that hybridize in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
polypeptide nucleic acids, as described in Nielsen et al. (1991)
Science 254:1497-1500. Typically, a probe comprises a region of
nucleotide sequence that hybridizes under highly stringent
conditions to at least about 15, typically about 20-25, and more
typically about 40, 50 or 75 consecutive nucleotides of the nucleic
acid sequence shown in SEQ ID NO:2 and the complements thereof.
More typically, the probe further comprises a label, e.g.,
radioisotope, fluorescent compound, enzyme, or enzyme
co-factor.
[0203] As used herein, the term "primer" refers to a
single-stranded oligonucleotide which acts as a point of initiation
of template-directed DNA synthesis using well-known methods (e.g.,
PCR, LCR) including, but not limited to those described herein. The
appropriate length of the primer depends on the particular use, but
typically ranges from about 15 to 30 nucleotides. The term "primer
site" refers to the area of the target DNA to which a primer
hybridizes. The term "primer pair" refers to a set of primers
including a 5' (upstream) primer that hybridizes with the 5' end of
the nucleic acid sequence to be amplified and a 3' (downstream)
primer that hybridizes with the complement of the sequence to be
amplified.
[0204] The carboxypeptidase polynucleotides are thus useful for
probes, primers, and in biological assays.
[0205] Where the polynucleotides are used to assess
carboxypeptidase properties or functions, such as in the assays
described herein, all or less than all of the entire cDNA can be
useful. Assays specifically directed to carboxypeptidase functions,
such as assessing agonist or antagonist activity, encompass the use
of known fragments. Further, diagnostic methods for assessing
carboxypeptidase function can also be practiced with any fragment,
including those fragments that may have been known prior to the
invention. Similarly, in methods involving treatment of
carboxypeptidase dysfunction, all fragments are encompassed
including those, which may have been known in the art.
[0206] The carboxypeptidase polynucleotides are useful as a
hybridization probe for cDNA and genomic DNA to isolate a
full-length cDNA and genomic clones encoding the polypeptides
described in SEQ ID NO:1 and to isolate cDNA and genomic clones
that correspond to variants producing the same polypeptides shown
in SEQ ID NO:1 or the other variants described herein. Variants can
be isolated from the same tissue and organism from which the
polypeptides shown in SEQ ID NO:1 were isolated, different tissues
from the same organism, or from different organisms. This method is
useful for isolating genes and cDNA that are
developmentally-controlled and therefore may be expressed in the
same tissue or different tissues at different points in the
development of an organism.
[0207] The probe can correspond to any sequence along the entire
length of the gene encoding the carboxypeptidase. Accordingly, it
could be derived from 5' noncoding regions, the coding region, and
3' noncoding regions.
[0208] The nucleic acid probe can be, for example, the full-length
cDNA of SEQ ID NO:2, or a fragment thereof, such as an
oligonucleotide of at least 12, 15, 30, 50, 100, 250 or 500
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to mRNA or DNA.
[0209] Fragments of the polynucleotides described herein are also
useful to synthesize larger fragments or full-length
polynucleotides described herein. For example, a fragment can be
hybridized to any portion of an MRNA and a larger or full-length
cDNA can be produced.
[0210] The fragments are also useful to synthesize antisense
molecules of desired length and sequence.
[0211] Antisense nucleic acids of the invention can be designed
using the nucleotide sequences of SEQ ID NO:2, and 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 and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluraci- l, 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-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopenten- yladenine,
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).
[0212] Additionally, the nucleic acid molecules of the 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 acids can be modified to generate peptide
nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal
Chemistry 4:5). 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 et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl
Acad. Sci. USA 93:14670. PNAs can be further modified, e.g., to
enhance their stability, specificity 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. The synthesis 6f
PNA-DNA chimeras can be performed as described in Hyrup (1996),
supra, Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63, Mag et
al. (1989) Nucleic Acids Res. 17:5973, and Peterser et al. (1975)
Bioorganic Med. Chem. Lett. 5:1119.
[0213] The nucleic acid molecules and fragments of the invention
can also include other appended groups such as peptides (e.g., for
targeting host cell carboxypeptidases in vivo), or agents
facilitating transport across the cell membrane (see, e.g.,
Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;
Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT
Publication No. WO 88/0918) or the blood brain barrier (see, e.g.,
PCT Publication No. WO 89/10134). In addition, oligonucleotides can
be modified with hybridization-triggered cleavage agents (see,
e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating
agents (see, e.g., Zon (1988) Pharm Res. 5:539-549).
[0214] The carboxypeptidase polynucleotides are also useful as
primers for PCR to amplify any given region of a carboxypeptidase
polynucleotide.
[0215] The carboxypeptidase polynucleotides are also useful for
constructing recombinant vectors. Such vectors include expression
vectors that express a portion of, or all of, the carboxypeptidase
polypeptides. Vectors also include insertion vectors, used to
integrate into another polynucleotide sequence, such as into the
cellular genome, to alter in situ expression of carboxypeptidase
genes and gene products. For example, an endogenous
carboxypeptidase coding sequence can be replaced via homologous
recombination with all or part of the coding region containing one
or more specifically introduced mutations.
[0216] The carboxypeptidase polynucleotides are also useful for
expressing antigenic portions of the carboxypeptidase proteins.
[0217] The carboxypeptidase polynucleotides are also useful as
probes for determining the chromosomal positions of the
carboxypeptidase polynucleotides by means of in situ hybridization
methods, such as FISH. (For a review of this technique, see Verma
et al. (1988) Human Chromosomes: A Manual of Basic Techniques
(Pergamon Press, New York), and PCR mapping of somatic cell
hybrids. The mapping of the sequences to chromosomes is an
important first step in correlating these sequences with genes
associated with disease.
[0218] 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.
[0219] 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 et al. ((1987) Nature 325:783-787).
[0220] Moreover, differences in the DNA sequences between
individuals affected with or free of a disease associated with a
specified 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.
[0221] The carboxypeptidase polynucleotide probes are also useful
to determine patterns of the presence of the gene encoding the
carboxypeptidases and their variants with respect to tissue
distribution, for example, whether gene duplication has occurred
and whether the duplication occurs in all or only a subset of
tissues. The genes can be naturally occurring or can have been
introduced into a cell, tissue, or organism exogenously.
[0222] The carboxypeptidase polynucleotides are also useful for
designing ribozymes corresponding to all, or a part, of the MRNA
produced from genes encoding the polynucleotides described
herein.
[0223] The carboxypeptidase polynucleotides are also useful for
constructing host cells expressing a part, or all, of the
carboxypeptidase polynucleotides and polypeptides.
[0224] The carboxypeptidase polynucleotides are also useful for
constructing transgenic animals expressing all, or a part, of the
carboxypeptidase polynucleotides and polypeptides.
[0225] The carboxypeptidase polynucleotides are also useful for
making vectors that express part, or all, of the carboxypeptidase
polypeptides.
[0226] The carboxypeptidase polynucleotides are also useful as
hybridization probes for determining the level of carboxypeptidase
nucleic acid expression. Accordingly, the probes can be used to
detect the presence of, or to determine levels of, carboxypeptidase
nucleic acid in cells, tissues, and in organisms. The nucleic acid
whose level is determined can be DNA or RNA. Accordingly, probes
corresponding to the polypeptides described herein can be used to
assess gene copy number in a given cell, tissue, or organism. This
is particularly relevant in cases in which there has been an
amplification of the carboxypeptidase genes.
[0227] Alternatively, the probe can be used in an in situ
hybridization context to assess the position of extra copies of the
carboxypeptidase genes, as on extrachromosomal elements or as
integrated into chromosomes in which the carboxypeptidase gene is
not normally found, for example as a homogeneously staining
region.
[0228] These uses are relevant for diagnosis of disorders involving
an increase or decrease in carboxypeptidase expression relative to
normal, such as a proliferative disorder, a differentiative or
developmental disorder, or a hematopoietic disorder.
[0229] Disorders in which the carboxypeptidase expression is
relevant include, but are not limited to, breast, colon and lung
carcinoma, and to disorders involving the tissues in which the gene
is expressed, especially as disclosed herein.
[0230] Thus, the present invention provides a method for
identifying a disease or disorder associated with aberrant
expression or activity of carboxypeptidase nucleic acid, in which a
test sample is obtained from a subject and nucleic acid (e.g.,
mRNA, genomic DNA) is detected, wherein the presence of the nucleic
acid is diagnostic for a subject having or at risk of developing a
disease or disorder associated with aberrant expression or activity
of the nucleic acid.
[0231] One aspect of the invention relates to diagnostic assays for
determining nucleic acid expression as well as activity in the
context of a biological sample (e.g., blood, serum, cells, tissue)
to determine whether an individual has a disease or disorder, or is
at risk of developing a disease or disorder, associated with
aberrant nucleic acid expression or activity. Such assays can be
used for prognostic or predictive purpose to thereby
prophylactically treat an individual prior to the onset of a
disorder characterized by or associated with expression or activity
of the nucleic acid molecules.
[0232] In vitro techniques for detection of mRNA include Northern
hybridizations and in situ hybridizations. In vitro techniques for
detecting DNA includes Southern hybridizations and in situ
hybridization.
[0233] Probes can be used as a part of a diagnostic test kit for
identifying cells or tissues that express the carboxypeptidase,
such as by measuring the level of a carboxypeptidase-coding nucleic
acid in a sample of cells from a subject e.g., mRNA or genomic DNA,
or determining if the carboxypeptidase gene has been mutated.
[0234] Nucleic acid expression assays are useful for drug screening
to identify compounds that modulate carboxypeptidase nucleic acid
expression (e.g., antisense, polypeptides, peptidomimetics, small
molecules or other drugs). A cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of the mRNA in the presence of the candidate compound is
compared to the level of expression of the MRNA in the absence of
the candidate compound. The candidate compound can then be
idenfified as a modulator of nucleic acid expression based on this
comparison and be used, for example to treat a disorder
characterized by aberrant nucleic acid expression. The modulator
can bind to the nucleic acid or indirectly modulate expression,
such as by interacting with other cellular components that affect
nucleic acid expression 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 gent to a
subject) in patients or in transgenic animals.
[0235] The invention thus provides a method for identifying a
compound that can be used to treat a disorder associated with
nucleic acid expression of the carboxypeptidase gene. The method
typically includes assaying the ability of the compound to modulate
the expression of the carboxypeptidase nucleic acid and thus
identifying a compound that can be used to treat a disorder
characterized by undesired carboxypeptidase nucleic acid
expression.
[0236] The assays can be performed in cell-based and cell-free
systems. Cell-based assays include cells naturally expressing the
carboxypeptidase nucleic acid or recombinant cells genetically
engineered to express specific nucleic acid sequences.
[0237] Alternatively, candidate compounds can be assayed in vivo in
patients or in transgenic animals.
[0238] The assay for carboxypeptidase nucleic acid expression can
involve direct assay of nucleic acid levels, such as mRNA levels,
or on collateral compounds involved in the pathway. Further, the
expression of genes that are up- or down-regulated in response to
the carboxypeptidase pathway can also be assayed. In this
embodiment the regulatory regions of these genes can be operably
linked to a reporter gene such as luciferase.
[0239] Thus, modulators of carboxypeptidase gene expression can be
identified in a method wherein a cell is contacted with a candidate
compound and the expression of mRNA determined. The level of
expression of carboxypeptidase mRNA in the presence of the
candidate compound is compared to the level of expression of
carboxypeptidase MRNA in the absence of the candidate compound. The
candidate compound can then be identified as a modulator of nucleic
acid expression based on this comparison and be used, for example
to treat a disorder characterized by aberrant nucleic acid
expression. When expression of mRNA is statistically significantly
greater in the presence of the candidate compound than in its
absence, the candidate compound is identified as a stimulator of
nucleic acid expression. When nucleic acid expression is
statistically significantly less in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of nucleic acid expression.
[0240] Accordingly, the invention provides methods of treatment,
with the nucleic acid as a target, using a compound identified
through drug screening as a gene modulator to modulate
carboxypeptidase nucleic acid expression. Modulation includes both
up-regulation (i.e. activation or agonization) or down-regulation
(suppression or antagonization) or effects on nucleic acid activity
(e.g. when nucleic acid is mutated or improperly modified).
Treatment is of disorders characterized by aberrant expression or
activity of the nucleic acid.
[0241] Disorders in which the carboxypeptidase expression is
relevant include, but are not limited to, breast, colon and lung
carcinoma, and to disorders involving the tissues in which the gene
is expressed, especially as disclosed herein.
[0242] Alternatively, a modulator for carboxypeptidase nucleic acid
expression can be a small molecule or drug identified using the
screening assays described herein as long as the drug or small
molecule inhibits the carboxypeptidase nucleic acid expression.
[0243] The carboxypeptidase polynucleotides are also useful for
monitoring the effectiveness of modulating compounds on the
expression or activity of the carboxypeptidase gene in clinical
trials or in a treatment regimen. Thus, the gene expression pattern
can serve as a barometer for the continuing effectiveness of
treatment with the compound, particularly with compounds to which a
patient can develop resistance. The gene expression pattern can
also serve as a marker indicative of a physiological response of
the affected cells to the compound. Accordingly, such monitoring
would allow either increased administration of the compound or the
administration of alternative compounds to which the patient has
not become resistant. Similarly, if the level of nucleic acid
expression falls below a desirable level, administration of the
compound could be commensurately decreased.
[0244] Monitoring can be, for example, as follows: (i) obtaining a
pre-administration sample from a subject prior to administration of
the agent; (ii) detecting the level of expression of a specified
mRNA or genomic DNA of the invention in the pre-administration
sample; (iii) obtaining one or more post-administration samples
from the subject; (iv) detecting the level of expression or
activity of the mRNA or genomic DNA in the post-administration
samples; (v) comparing the level of expression or activity of the
mRNA or genomic DNA in the pre-administration sample with the MRNA
or genomic DNA in the post-administration sample or samples; and
(vi) increasing or decreasing the administration of the agent to
the subject accordingly.
[0245] The carboxypeptidase polynucleotides are also useful in
diagnostic assays for qualitative changes in carboxypeptidase
nucleic acid, and particularly in qualitative changes that lead to
pathology. The polynucleotides can be used to detect mutations in
carboxypeptidase genes and gene expression products such as mRNA.
The polynucleotides can be used as hybridization probes to detect
naturally-occurring genetic mutations in the carboxypeptidase gene
and thereby to determine whether a subject with the mutation is at
risk for a disorder caused by the mutation. Mutations include
deletion, addition, or substitution of one or more nucleotides in
the gene, chromosomal rearrangement, such as inversion or
transposition, modification of genomic DNA, such as aberrant
methylation patterns or changes in gene copy number, such as
amplification. Detection of a mutated form of the carboxypeptidase
gene associated with a dysfunction provides a diagnostic tool for
an active disease or susceptibility to disease when the disease
results from overexpression, underexpression, or altered expression
of a carboxypeptidase.
[0246] Mutations in the carboxypeptidase gene can be detected at
the nucleic acid level by a variety of techniques. Genomic DNA can
be analyzed directly or can be amplified by using PCR prior to
analysis. RNA or cDNA can be used in the same way.
[0247] In certain embodiments, detection of the mutation 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
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
gene under conditions such that hybridization and amplification of
the 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.
Deletions and insertions can be detected by a change in size of the
amplified product compared to the normal genotype. Point mutations
can be identified by hybridizing amplified DNA to normal RNA or
antisense DNA sequences.
[0248] 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.
[0249] Alternative amplification methods include: self sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al. (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.
[0250] Alternatively, mutations in a carboxypeptidase gene can be
directly identified, for example, by alterations in restriction
enzyme digestion patterns determined by gel electrophoresis.
[0251] Further, sequence-specific ribozymes (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.
[0252] Perfectly matched sequences can be distinguished from
mismatched sequences by nuclease cleavage digestion assays or by
differences in melting temperature.
[0253] Sequence changes at specific locations can also be assessed
by nuclease protection assays such as RNase and SI protection or
the chemical cleavage method.
[0254] Furthermore, sequence differences between a mutant
carboxypeptidase gene and a wild-type gene can be determined by
direct DNA sequencing. 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 gene include
methods in which protection from cleavage agents is used to detect
mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al.
(1985) Science 230:1242); Cotton et al. (1988) PNAS 85:4397;
Saleeba et al. (1992) Meth. Enzymol. 21 7:286-295), electrophoretic
mobility of mutant and wild type nucleic acid is compared (Orita et
al. (1989) PNAS 86:2766; Cotton et al. (1993) Mutat. Res.
285:125-144; and Hayashi et al. (1992) Genet. Anal. Tech. Appl.
9:73-79), and movement of mutant or wild-type fragments in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (Myers et al. (1985)
Nature 313:495). 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 one 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).
Examples of other techniques for detecting point mutations include,
selective oligonucleotide hybridization, selective amplification,
and selective primer extension.
[0256] In other embodiments, genetic mutations 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
oligonucleotide probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations can be identified in two dimensional
arrays containing light-generated DNA probes as described in Cronin
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 overlapping 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.
[0257] The carboxypeptidase polynucleotides are also useful for
testing an individual for a genotype that while not necessarily
causing the disease, nevertheless affects the treatment modality.
Thus, the polynucleotides can be used to study the relationship
between an individual's genotype and the individual's response to a
compound used for treatment (pharmacogenomic relationship). In the
present case, for example, a mutation in the carboxypeptidase gene
that results in altered affinity for substrate could result in an
excessive or decreased drug effect with standard concentrations of
substrate. Accordingly, the carboxypeptidase polynucleotides
described herein can be used to assess the mutation content of the
gene in an individual in order to select an appropriate compound or
dosage regimen for treatment.
[0258] Thus polynucleotides displaying genetic variations that
affect treatment provide a diagnostic target that can be used to
tailor treatment in an individual. Accordingly, the production of
recombinant cells and animals containing these polymorphisms allow
effective clinical design of treatment compounds and dosage
regimens.
[0259] The methods can involve obtaining a control biological
sample from a control subject, contacting the control sample with a
compound or agent capable of detecting mRNA, or genomic DNA, such
that the presence of mRNA or genomic DNA is detected in the
biological sample, and comparing the presence of mRNA or genomic
DNA in the control sample with the presence of mRNA or genomic DNA
in the test sample.
[0260] The carboxypeptidase polynucleotides are also useful for
chromosome identification when the sequence is identified with an
individual chromosome and to a particular location on the
chromosome. First, the DNA sequence is matched to the chromosome by
in situ or other chromosome-specific hybridization. Sequences can
also be correlated to specific chromosomes by preparing PCR primers
that can be used for PCR screening of somatic cell hybrids
containing individual chromosomes from the desired species. Only
hybrids containing the chromosome containing the gene homologous to
the primer will yield an amplified fragment. Sublocalization can be
achieved using chromosomal fragments. Other strategies include
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to chromosome-specific libraries. Further mapping
strategies include fluorescence in situ hybridization, which allows
hybridization with probes shorter than those traditionally used.
Reagents for chromosome mapping can be used individually to mark a
single chromosome or a single site on the 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.
[0261] The carboxypeptidase polynucleotides can also be used to
identify individuals from small biological samples. This can be
done for example using restriction fragment-length polymorphism
(RFLP) to identify an individual. Thus, the polynucleotides
described herein are useful as DNA markers for RFLP (See U.S. Pat.
No. 5,272,057).
[0262] Furthermore, the carboxypeptidase sequence can be used to
provide an alternative technique, which determines the actual DNA
sequence of selected fragments in the genome of an individual.
Thus, the carboxypeptidase 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 DNA from an
individual for subsequent sequencing.
[0263] 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. It is estimated that allelic variation in humans
occurs with a frequency of about once per each 500 bases. Allelic
variation occurs to some degree in the coding regions of these
sequences, and to a greater degree in the noncoding regions. The
carboxypeptidase sequences can be used to obtain such
identification sequences from individuals and from tissue. The
sequences represent unique fragments of the human genome. 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.
[0264] If a panel of reagents from the sequences 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.
[0265] The carboxypeptidase polynucleotides can also be used in
forensic identification procedures. PCR technology can be used to
amplify DNA sequences taken from very small biological samples,
such as a single hair follicle, body fluids (e.g. blood, saliva, or
semen). The amplified sequence can then be compared to a standard
allowing identification of the origin of the sample.
[0266] The carboxypeptidase polynucleotides can thus 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 described
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 the
noncoding region are particularly useful since greater polymorphism
occurs in the noncoding regions, making it easier to differentiate
individuals using this technique.
[0267] The carboxypeptidase polynucleotides 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. This is useful in cases
in which a forensic pathologist is presented with a tissue of
unknown origin. Panels of carboxypeptidase probes can be used to
identify tissue by species and/or by organ type.
[0268] In a similar fashion, these primers and 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).
[0269] Alternatively, the carboxypeptidase polynucleotides can be
used directly to block transcription or translation of
carboxypeptidase gene sequences by means of antisense or ribozyme
constructs. Thus, in a disorder characterized by abnormally high or
undesirable carboxypeptidase gene expression, nucleic acids can be
directly used for treatment.
[0270] The carboxypeptidase polynucleotides are thus useful as
antisense constructs to control carboxypeptidase gene expression in
cells, tissues, and organisms. A DNA antisense polynucleotide is
designed to be complementary to a region of the gene involved in
transcription, preventing transcription and hence production of
carboxypeptidase protein. An antisense RNA or DNA polynucleotide
would hybridize to the mRNA and thus block translation of MRNA into
carboxypeptidase protein.
[0271] Examples of antisense molecules useful to inhibit nucleic
acid expression include antisense molecules complementary to a
fragment of the 5' untranslated region of SEQ ID NO:2 which also
includes the start codon and antisense molecules which are
complementary to a fragment of the 3' untranslated region of SEQ ID
NO:2. Alternatively, a class of antisense molecules can be used to
inactivate mRNA in order to decrease expression of carboxypeptidase
nucleic acid. Accordingly, these molecules can treat a disorder
characterized by abnormal or undesired carboxypeptidase nucleic
acid expression. This technique involves cleavage by means of
ribozymes containing nucleotide sequences complementary to one or
more regions in the MRNA that attenuate the ability of the mRNA to
be translated. Possible regions include coding regions and
particularly coding regions corresponding to the catalytic and
other functional activities of the carboxypeptidase protein.
[0272] The carboxypeptidase polynucleotides also provide vectors
for gene therapy in patients containing cells that are aberrant in
carboxypeptidase gene expression. Thus, recombinant cells, which
include the patient's cells that have been engineered ex vivo and
returned to the patient, are introduced into an individual where
the cells produce the desired carboxypeptidase protein to treat the
individual.
[0273] The invention also encompasses kits for detecting the
presence of a carboxypeptidase nucleic acid in a biological sample.
For example, the kit can comprise reagents such as a labeled or
labelable nucleic acid or agent capable of detecting
carboxypeptidase nucleic acid in a biological sample; means for
determining the amount of carboxypeptidase nucleic acid in the
sample; and means for comparing the amount of carboxypeptidase
nucleic acid 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 carboxypeptidase
mRNA or DNA.
[0274] Computer Readable Means
[0275] The nucleotide or amino acid sequences of the invention are
also provided in a variety of mediums to facilitate use thereof. As
used herein, "provided" refers to a manufacture, other than an
isolated nucleic acid or amino acid molecule, which contains a
nucleotide or amino acid sequence of the present invention. Such a
manufacture provides the nucleotide or amino acid sequences, or a
subset thereof (e.g., a subset of open reading frames (ORFs)) in a
form which allows a skilled artisan to examine the manufacture
using means not directly applicable to examining the nucleotide or
amino acid sequences, or a subset thereof, as they exists in nature
or in purified form.
[0276] In one application of this embodiment, a nucleotide or amino
acid sequence of the present invention can be recorded on computer
readable media. As used herein, "computer readable media" refers to
any medium that can be read and accessed directly by a computer.
Such media include, but are not limited to: magnetic storage media,
such as floppy discs, hard disc storage medium, and magnetic tape;
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. The skilled artisan will readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide or amino acid sequence
of the present invention.
[0277] As used herein, "recorded" refers to a process for storing
information on computer readable medium. The skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate manufactures
comprising the nucleotide or amino acid sequence information of the
present invention.
[0278] A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide or amino acid sequence of the present
invention. The choice of the data storage structure will generally
be based on the means chosen to access the stored information. In
addition, a variety of data processor programs and formats can be
used to store the nucleotide sequence information of the present
invention on computer readable medium. The sequence information can
be represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. The
skilled artisan can readily adapt any number of dataprocessor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0279] By providing the nucleotide or amino acid sequences of the
invention in computer readable form, the skilled artisan can
routinely access the sequence information for a variety of
purposes. For example, one skilled in the art can use the
nucleotide or amino acid sequences of the invention in computer
readable form to compare a target sequence or target structural
motif with the sequence information stored within the data storage
means. Search means are used to identify fragments or regions of
the sequences of the invention which match a particular target
sequence or target motif.
[0280] As used herein, a "target sequence" can be any DNA or amino
acid sequence of six or more nucleotides or two or more amino
acids. A skilled artisan can readily recognize that the longer a
target sequence is, the less likely a target sequence will be
present as a random occurrence in the database. The most preferred
sequence length of a target sequence is from about 10 to 100 amino
acids or from about 30 to 300 nucleotide residues. However, it is
well recognized that commercially important fragments, such as
sequence fragments involved in gene expression and protein
processing, may be of shorter length.
[0281] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to, enzyme
active sites and signal sequences. Nucleic acid target motifs
include, but are not limited to, promoter sequences, hairpin
structures and inducible expression elements (protein binding
sequences).
[0282] Computer software is publicly available which allows a
skilled artisan to access sequence information provided in a
computer readable medium for analysis and comparison to other
sequences. A variety of known algorithms are disclosed publicly and
a variety of commercially available software for conducting search
means are and can be used in the computer-based systems of the
present invention. Examples of such software includes, but is not
limited to, MacPattern (EMBL), BLASTN and BLASTX (NCBIA).
[0283] For example, software which implements the BLAST (Altschul
et al. (1990) J. Mol. Biol. 215:403-410) and BLAZE (Brutlag et al.
(1993) Comp. Chem. 17:203-207) search algorithms on a Sybase system
can be used to identify open reading frames (ORFs) of the sequences
of the invention which contain homology to ORFs or proteins from
other libraries. Such ORFs are protein encoding fragments and are
useful in producing commercially important proteins such as enzymes
used in various reactions and in the production of commercially
useful metabolites. Vectors/Host Cells The invention also provides
vectors containing the carboxypeptidase polynucleotides. The term
"vector" refers to a vehicle, preferably a nucleic acid molecule
that can transport the carboxypeptidase polynucleotides. When the
vector is a nucleic acid molecule, the carboxypeptidase
polynucleotides are covalently linked to the vector nucleic acid.
With this aspect of the invention, the vector includes a plasmid,
single or double stranded phage, a single or double stranded RNA or
DNA viral vector, or artificial chromosome, such as a BAC, PAC,
YAC, OR MAC.
[0284] A vector can be maintained in the host cell as an
extrachromosomal element where it replicates and produces
additional copies of the carboxypeptidase polynucleotides.
Alternatively, the vector may integrate into the host cell genome
and produce additional copies of the carboxypeptidase
polynucleotides when the host cell replicates.
[0285] The invention provides vectors for the maintenance (cloning
vectors) or vectors for expression (expression vectors) of the
carboxypeptidase polynucleotides. The vectors can function in
procaryotic or eukaryotic cells or in both (shuttle vectors).
[0286] Expression vectors contain cis-acting regulatory regions
that are operably linked in the vector to the carboxypeptidase
polynucleotides such that transcription of the polynucleotides is
allowed in a host cell. The polynucleotides can be introduced into
the host cell with a separate polynucleotide capable of affecting
transcription. Thus, the second polynucleotide may provide a
trans-acting factor interacting with the cis-regulatory control
region to allow transcription of the carboxypeptidase
polynucleotides from the vector. Alternatively, a trans-acting
factor may be supplied by the host cell. Finally, a trans-acting
factor can be produced from the vector itself.
[0287] It is understood, however, that in some embodiments,
transcription and/or translation of the carboxypeptidase
polynucleotides can occur in a cell-free system.
[0288] The regulatory sequence to which the polynucleotides
described herein can be operably linked include promoters for
directing mRNA transcription. These include, but are not limited
to, the left promoter from bacteriophage .lambda., the lac, TRP,
and TAC promoters from E. coli, the early and late promoters from
SV40, the CMV immediate early promoter, the adenovirus early and
late promoters, and retrovirus long-terminal repeats.
[0289] In addition to control regions that promote transcription,
expression vectors may also include regions that modulate
transcription, such as repressor binding sites and enhancers.
Examples include the SV40 enhancer, the cytomegalovirus immediate
early enhancer, polyoma enhancer, adenovirus enhancers, and
retrovirus LTR enhancers.
[0290] In addition to containing sites for transcription initiation
and control, expression vectors can also contain sequences
necessary for transcription termination and, in the transcribed
region a ribosome binding site for translation. Other regulatory
control elements for expression include initiation and termination
codons as well as polyadenylation signals. The person of ordinary
skill in the art would be aware of the numerous regulatory
sequences that are useful in expression vectors. Such regulatory
sequences are described, for example, in Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.).
[0291] A variety of expression vectors can be used to express a
carboxypeptidase polynucleotide. Such vectors include chromosomal,
episomal, and virus-derived vectors, for example vectors derived
from bacterial plasmids, from bacteriophage, from yeast episomes,
from yeast chromosomal elements, including yeast artificial
chromosomes, from viruses such as baculoviruses, papovaviruses such
as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies
viruses, and retroviruses. Vectors may also be derived from
combinations of these sources such as those derived from plasmid
and bacteriophage genetic elements, e.g. cosmids and phagemids.
Appropriate cloning and expression vectors for prokaryotic and
eukaryotic hosts are described in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd. ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
[0292] The regulatory sequence may provide constitutive expression
in one or more host cells (i.e. tissue specific) or may provide for
inducible expression in one or more cell types such as by
temperature, nutrient additive, or exogenous factor such as a
hormone or other ligand. A variety of vectors providing for
constitutive and inducible expression in prokaryotic and eukaryotic
hosts are well known to those of ordinary skill in the art.
[0293] The carboxypeptidase polynucleotides can be inserted into
the vector nucleic acid by well-known methodology. Generally, the
DNA sequence that will ultimately be expressed is joined to an
expression vector by cleaving the DNA sequence and the expression
vector with one or more restriction enzymes and then ligating the
fragments together. Procedures for restriction enzyme digestion and
ligation are well known to those of ordinary skill in the art.
[0294] The vector containing the appropriate polynucleotide can be
introduced into an appropriate host cell for propagation or
expression using well-known techniques. Bacterial cells include,
but are not limited to, E. coli, Streptomyces, and Salmonella
typhimurium. Eukaryotic cells include, but are not limited to,
yeast, insect cells such as Drosophila, animal cells such as COS
and CHO cells, and plant cells.
[0295] As described herein, it may be desirable to express the
polypeptide as a fusion protein. Accordingly, the invention
provides fusion vectors that allow for the production of the
carboxypeptidase polypeptides. Fusion vectors can increase the
expression of a recombinant protein, increase the solubility of the
recombinant protein, and aid in the purification of the protein by
acting for example as a ligand for affinity purification. A
proteolytic cleavage site may be introduced at the junction of the
fusion moiety so that the desired polypeptide can ultimately be
separated from the fusion moiety. Proteolytic enzymes include, but
are not limited to, factor Xa, thrombin, and enterokinase. Typical
fusion expression vectors include pGEX (Smith et al. (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. 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. (1990) Gene
Expression Technology: Methods in Enzymology 185:60-89).
[0296] Recombinant protein expression can be maximized in a host
bacteria by providing a genetic background wherein the host cell
has an impaired capacity to proteolytically cleave the recombinant
protein. (Gottesman, S. (1990) Gene Expression Technology: Methods
in Enzymology 185, Academic Press, San Diego, Calif. 119-128).
Alternatively, the sequence of the polynucleotide of interest can
be altered to provide preferential codon usage for a specific host
cell, for example E. coli. (Wada et al. (1992) Nucleic Acids Res.
20:2111-2118).
[0297] The carboxypeptidase polynucleotides can also be expressed
by expression vectors that are operative in yeast. Examples of
vectors for expression in yeast e.g., S. cerevisiae include
pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan et
al. (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987) Gene
54:113-123), and pYES2 (Invitrogen Corporation, San Diego,
Calif.).
[0298] The carboxypeptidase polynucleotides can also be expressed
in insect cells using, for example, 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 et al. (1989) Virology 170:31-39).
[0299] In certain embodiments of the invention, the polynucleotides
described herein are expressed in mammalian cells using mammalian
expression vectors. Examples of mammalian expression vectors
include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman
et al. (1987) EMBO J. 6:187-195).
[0300] The expression vectors listed herein are provided by way of
example only of the well-known vectors available to those of
ordinary skill in the art that would be useful to express the
carboxypeptidase polynucleotides. The person of ordinary skill in
the art would be aware of other vectors suitable for maintenance
propagation or expression of the polynucleotides described herein.
These are found for example in Sambrook et al. (1989) Molecular
Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.
[0301] The invention also encompasses vectors in which the nucleic
acid sequences described herein are cloned into the vector in
reverse orientation, but operably linked to a regulatory sequence
that permits transcription of antisense RNA. Thus, an antisense
transcript can be produced to all, or to a portion, of the
polynucleotide sequences described herein, including both coding
and non-coding regions. Expression of this antisense RNA is subject
to each of the parameters described above in relation to expression
of the sense RNA (regulatory sequences, constitutive or inducible
expression, tissue-specific expression).
[0302] The invention also relates to recombinant host cells
containing the vectors described herein. Host cells therefore
include prokaryotic cells, lower eukaryotic cells such as yeast,
other eukaryotic cells such as insect cells, and higher eukaryotic
cells such as mammalian cells.
[0303] The recombinant host cells are prepared by introducing the
vector constructs described herein into the cells by techniques
readily available to the person of ordinary skill in the art. These
include, but are not limited to, calcium phosphate transfection,
DEAE-dextran-mediated transfection, cationic lipid-mediated
transfection, electroporation, transduction, infection,
lipofection, and other techniques such as those found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.).
[0304] Host cells can contain more than one vector. Thus, different
nucleotide sequences can be introduced on different vectors of the
same cell. Similarly, the carboxypeptidase polynucleotides can be
introduced either alone or with other polynucleotides that are not
related to the carboxypeptidase polynucleotides such as those
providing trans-acting factors for expression vectors. When more
than one vector is introduced into a cell, the vectors can be
introduced independently, co-introduced or joined to the
carboxypeptidase polynucleotide vector.
[0305] In the case of bacteriophage and viral vectors, these can be
introduced into cells as packaged or encapsulated virus by standard
procedures for infection and transduction. Viral vectors can be
replication-competent or replication-defective. In the case in
which viral replication is defective, replication will occur in
host cells providing functions that complement the defects.
[0306] Vectors generally include selectable markers that enable the
selection of the subpopulation of cells that contain the
recombinant vector constructs. The marker can be contained in the
same vector that contains the polynucleotides described herein or
may be on a separate vector. Markers include tetracycline or
ampicillin-resistance genes for prokaryotic host cells and
dihydrofolate reductase or neomycin resistance for eukaryotic host
cells. However, any marker that provides selection for a phenotypic
trait will be effective.
[0307] While the mature proteins can be produced in bacteria,
yeast, mammalian cells, and other cells under the control of the
appropriate regulatory sequences, cell-free transcription and
translation systems can also be used to produce these proteins
using RNA derived from the DNA constructs described herein.
[0308] Where secretion of the polypeptide is desired, appropriate
secretion signals are incorporated into the vector. The signal
sequence can be endogenous to the carboxypeptidase polypeptides or
heterologous to these polypeptides. Where the polypeptide is not
secreted into the medium, the protein can be isolated from the host
cell by standard disruption procedures, including freeze thaw,
sonication, mechanical disruption, use of lysing agents and the
like. The polypeptide can then be recovered and purified by
well-known purification methods including ammonium sulfate
precipitation, acid extraction, anion or cationic exchange
chromatography, phosphocellulose chromatography,
hydrophobic-interaction chromatography, affinity chromatography,
hydroxylapatite chromatography, lectin chromatography, or high
performance liquid chromatography.
[0309] It is also understood that depending upon the host cell in
recombinant production of the polypeptides described herein, the
polypeptides can have various glycosylation patterns, depending
upon the cell, or maybe non-glycosylated as when produced in
bacteria. In addition, the polypeptides may include an initial
modified methionine in some cases as a result of a host-mediated
process.
[0310] Uses of Vectors and Host Cells
[0311] It is understood that "host cells" and "recombinant host
cells" refer not only to the particular subject cell but also 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.
[0312] The host cells expressing the polypeptides described herein,
and particularly recombinant host cells, have a variety of uses.
First, the cells are useful for producing carboxypeptidase proteins
or polypeptides that can be further purified to produce desired
amounts of carboxypeptidase protein or fragments. Thus, host cells
containing expression vectors are useful for polypeptide
production.
[0313] Host cells are also useful for conducting cell-based assays
involving the carboxypeptidase or carboxypeptidase fragments. Thus,
a recombinant host cell expressing a native carboxypeptidase is
useful to assay for compounds that stimulate or inhibit
carboxypeptidase function.
[0314] Host cells are also useful for identifying carboxypeptidase
mutants in which these functions are affected. If the mutants
naturally occur and give rise to a pathology, host cells containing
the mutations are useful to assay compounds that have a desired
effect on the mutant carboxypeptidase (for example, stimulating or
inhibiting function) which may not be indicated by their effect on
the native carboxypeptidase.
[0315] Recombinant host cells are also useful for expressing the
chimeric polypeptides described herein to assess compounds that
activate or suppress activation by means of a heterologous domain,
segment, site, and the like, as disclosed herein.
[0316] Further, mutant carboxypeptidases can be designed in which
one or more of the various functions is engineered to be increased
or decreased (e.g., peptide substrate binding) and used to augment
or replace carboxypeptidase proteins in an individual. Thus, host
cells can provide a therapeutic benefit by replacing an aberrant
carboxypeptidase or providing an aberrant carboxypeptidase that
provides a therapeutic result. In one embodiment, the cells provide
carboxypeptidases that are abnormally active.
[0317] In another embodiment, the cells provide carboxypeptidases
that are abnormally inactive. These carboxypeptidases can compete
with endogenous carboxypeptidases in the individual.
[0318] In another embodiment, cells expressing carboxypeptidases
that cannot be activated, are introduced into an individual in
order to compete with endogenous carboxypeptidases for substrate.
For example, in the case in which excessive substrate is part of a
treatment modality, it may be necessary to inactivate this molecule
at a specific point in treatment. Providing cells that compete for
the molecule, but which cannot be affected by carboxypeptidase
activation would be beneficial.
[0319] Homologously recombinant host cells can also be produced
that allow the in situ alteration of endogenous carboxypeptidase
polynucleotide sequences in a host cell genome. This technology is
more fully described in WO 93/09222, WO 91/12650 and U.S. Pat. No.
5,641,670. Briefly, specific polynucleotide sequences corresponding
to the carboxypeptidase polynucleotides or sequences proximal or
distal to a carboxypeptidase gene are allowed to integrate into a
host cell genome by homologous recombination where expression of
the gene can be affected. In one embodiment, regulatory sequences
are introduced that either increase or decrease expression of an
endogenous sequence. Accordingly, a carboxypeptidase protein can be
produced in a cell not normally producing it, or increased
expression of carboxypeptidase protein can result in a cell
normally producing the protein at a specific level. Alternatively,
the entire gene can be deleted. Still further, specific mutations
can be introduced into any desired region of the gene to produce
mutant carboxypeptidase proteins. Such mutations could be
introduced, for example, into the specific regions disclosed
herein.
[0320] In one embodiment, the host cell can be a fertilized oocyte
or embryonic stem cell that can be used to produce a transgenic
animal containing the altered carboxypeptidase gene. Alternatively,
the host cell can be a stem cell or other early tissue precursor
that gives rise to a specific subset of cells and can be used to
produce transgenic tissues in an animal. See also Thomas et al.,
Cell 51:503 (1987) 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 gene
has homologously recombined with the endogenous carboxypeptidase
gene is 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; WO 91/01140; and
WO 93/04169.
[0321] The genetically engineered host cells can be used to produce
non-human transgenic animals. A transgenic animal is preferably a
mammal, for example a rodent, such as a rat or mouse, in which one
or more of the cells of the animal include a transgene. 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 in one or more cell types or tissues of the
transgenic animal. These animals are useful for studying the
function of a carboxypeptidase protein and identifying and
evaluating modulators of carboxypeptidase protein activity.
[0322] Other examples of transgenic animals include non-human
primates, sheep, dogs, cows, goats, chickens, and amphibians.
[0323] In one embodiment, a host cell is a fertilized oocyte or an
embryonic stem cell into which carboxypeptidase polynucleotide
sequences have been introduced.
[0324] A transgenic animal can be produced by introducing 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. Any of the
carboxypeptidase nucleotide sequences can be introduced as a
transgene into the genome of a non-human animal, such as a
mouse.
[0325] Any of the regulatory or other sequences useful in
expression vectors can form part of the transgenic sequence. This
includes intronic sequences and polyadenylation signals, if not
already included. A tissue-specific regulatory sequence(s) can be
operably linked to the transgene to direct expression of the
carboxypeptidase protein to particular cells.
[0326] 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 transgene
in its genome and/or expression of transgenic 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 can further be bred to
other transgenic animals carrying other transgenes. A transgenic
animal also includes animals in which the entire animal or tissues
in the animal have been produced using the homologously recombinant
host cells described herein.
[0327] In another embodiment, transgenic non-human 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)
PNAS 89:6232-6236. Another example of a recombinase system is the
FLP recombinase system of S. 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 is
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.
[0328] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT International Publication
Nos. WO 97/07668 and WO 97/07669. 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 reconstructed oocyte is then
cultured such that it develops to morula or blastocyst and then
transferred to a pseudopregnant female foster animal. The offspring
born of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0329] Transgenic animals containing recombinant cells that express
the polypeptides described herein are useful to conduct the assays
described herein in an in vivo context. Accordingly, the various
physiological factors that are present in vivo and that could
affect, for example, substrate binding and hydrolysis or
carboxypeptidase activation, may not be evident from in vitro
cell-free or cell-based assays. Accordingly, it is useful to
provide non-human transgenic animals to assay in vivo
carboxypeptidase function, including substrate interaction, the
effect of specific mutant carboxypeptidases on carboxypeptidase
function and substrate interaction, and the effect of chimeric
carboxypeptidases. It is also possible to assess the effect of null
mutations, that is mutations that substantially or completely
eliminate one or more carboxypeptidase functions.
[0330] Pharmaceutical Compositions
[0331] The carboxypeptidase nucleic acid molecules, protein,
modulators of the protein, and antibodies (also referred to herein
as "active compounds") can be incorporated into pharmaceutical
compositions suitable for administration to a subject, e.g., a
human. Such compositions typically comprise the nucleic acid
molecule, protein, modulator, or antibody and a pharmaceutically
acceptable carrier.
[0332] The term "administer" is used in its broadest sense and
includes any method of introducing the compositions of the present
invention into a subject. This includes producing polypeptides or
polynucleotides in vivo as by transcription or translation in vivo
of polynucleotides that have been exogenously introduced into a
subject. Thus, polypeptides or nucleic acids produced in the
subject from the exogenous compositions are encompassed in the term
"administer."
[0333] 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, such media can be used in the compositions of the
invention. Supplementary active compounds can also be incorporated
into the compositions. A pharmaceutical composition of the
invention is formulated to be compatible with its intended route of
administration. Examples of routes of administration include
parenteral, e.g., intravenous, intradermal, subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration. Solutions or suspensions used for parenteral,
intradermal, or subcutaneous application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. pH can be adjusted with acids or
bases, such as hydrochloric acid or sodium hydroxide. The
parenteral preparation can be enclosed in ampules, disposable
syringes or multiple dose vials made of glass or plastic.
[0334] 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 ELTM (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 polyethylene 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 mannitol, 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.
[0335] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a carboxypeptidase protein
or anti- carboxypeptidase 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.
[0336] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For oral administration, the agent can be
contained in enteric forms to survive the stomach or further coated
or mixed to be released in a particular region of the GI tract by
known methods. 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.
[0337] 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.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] 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.
[0342] 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 (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.
[0343] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0344] This invention may be embodied in many different forms and
should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will filly convey the invention to those skilled in the
art. Many modifications and other embodiments of the invention will
come to mind in one skilled in the art to which this invention
pertains having the benefit of the teachings presented in the
foregoing description. Although specific terms are employed, they
are used as in the art unless otherwise indicated.
Sequence CWU 1
1
4 1 492 PRT Homo sapiens 1 Met Gly Ser Ala Pro Trp Ala Pro Val Leu
Leu Leu Ala Leu Gly Leu 1 5 10 15 Arg Gly Leu Gln Ala Gly Ala Arg
Arg Ala Pro Asp Pro Gly Phe Gln 20 25 30 Glu Arg Phe Phe Gln Gln
Arg Leu Asp His Phe Asn Phe Glu Arg Phe 35 40 45 Gly Asn Lys Thr
Phe Pro Gln Arg Phe Leu Val Ser Asp Arg Phe Trp 50 55 60 Val Arg
Gly Glu Gly Pro Ile Phe Phe Tyr Thr Gly Asn Glu Gly Asp 65 70 75 80
Val Trp Ala Phe Ala Asn Asn Ser Gly Phe Val Ala Glu Leu Ala Ala 85
90 95 Glu Arg Gly Ala Leu Leu Val Phe Ala Glu His Arg Tyr Tyr Gly
Lys 100 105 110 Ser Leu Pro Phe Gly Ala Gln Ser Thr Gln Arg Gly His
Thr Glu Leu 115 120 125 Leu Thr Val Glu Gln Ala Leu Ala Asp Phe Ala
Glu Leu Leu Arg Ala 130 135 140 Leu Arg Arg Asp Leu Gly Ala Gln Asp
Ala Pro Ala Ile Ala Phe Gly 145 150 155 160 Gly Ser Tyr Gly Gly Met
Leu Ser Ala Tyr Leu Arg Met Lys Tyr Pro 165 170 175 His Leu Val Ala
Gly Ala Leu Ala Ala Ser Ala Pro Val Leu Ala Val 180 185 190 Ala Gly
Leu Gly Asp Ser Asn Gln Phe Phe Arg Asp Val Thr Ala Asp 195 200 205
Phe Glu Gly Gln Ser Pro Lys Cys Thr Gln Gly Val Arg Glu Ala Phe 210
215 220 Arg Gln Ile Lys Asp Leu Phe Leu Gln Gly Ala Tyr Asp Thr Val
Arg 225 230 235 240 Trp Glu Phe Gly Thr Cys Gln Pro Leu Ser Asp Glu
Lys Asp Leu Thr 245 250 255 Gln Leu Phe Met Phe Ala Arg Asn Ala Phe
Thr Val Leu Ala Met Met 260 265 270 Asp Tyr Pro Tyr Pro Thr Asp Phe
Leu Gly Pro Leu Pro Ala Asn Pro 275 280 285 Val Lys Val Gly Cys Asp
Arg Leu Leu Ser Glu Ala Gln Arg Ile Thr 290 295 300 Gly Leu Arg Ala
Leu Ala Gly Leu Val Tyr Asn Ala Ser Gly Ser Glu 305 310 315 320 His
Cys Tyr Asp Ile Tyr Arg Leu Tyr His Ser Cys Ala Asp Pro Thr 325 330
335 Gly Cys Gly Thr Gly Pro Asp Ala Arg Ala Trp Asp Tyr Gln Ala Cys
340 345 350 Thr Glu Ile Asn Leu Thr Phe Ala Ser Asn Asn Val Thr Asp
Met Phe 355 360 365 Pro Asp Leu Pro Phe Thr Asp Glu Leu Arg Gln Arg
Tyr Cys Leu Asp 370 375 380 Thr Trp Gly Val Trp Pro Arg Pro Asp Trp
Leu Leu Thr Ser Phe Trp 385 390 395 400 Gly Gly Asp Leu Arg Ala Ala
Ser Asn Ile Ile Phe Ser Asn Gly Asn 405 410 415 Leu Asp Pro Trp Ala
Gly Gly Gly Ile Arg Arg Asn Leu Ser Ala Ser 420 425 430 Val Ile Ala
Val Thr Ile Gln Gly Gly Ala His His Leu Asp Leu Arg 435 440 445 Ala
Ser His Pro Glu Asp Pro Ala Ser Val Val Glu Ala Arg Lys Leu 450 455
460 Glu Ala Thr Ile Ile Gly Glu Trp Val Lys Ala Ala Arg Arg Glu Gln
465 470 475 480 Gln Pro Ala Leu Arg Gly Gly Pro Arg Leu Ser Leu 485
490 2 1653 DNA Homo sapiens CDS (19)...(1494) 2 cgtccggcgg aaggcgac
atg ggc tcc gct ccc tgg gcc ccg gtc ctg ctg 51 Met Gly Ser Ala Pro
Trp Ala Pro Val Leu Leu 1 5 10 ctg gcg ctc ggg ctg cgc ggc ctc cag
gcg ggg gcc cgc agg gcc ccg 99 Leu Ala Leu Gly Leu Arg Gly Leu Gln
Ala Gly Ala Arg Arg Ala Pro 15 20 25 gac ccc ggc ttc cag gag cgc
ttc ttc cag cag cgt ctg gac cac ttc 147 Asp Pro Gly Phe Gln Glu Arg
Phe Phe Gln Gln Arg Leu Asp His Phe 30 35 40 aac ttc gag cgc ttc
ggc aac aag acc ttc cct cag cgc ttc ctg gtg 195 Asn Phe Glu Arg Phe
Gly Asn Lys Thr Phe Pro Gln Arg Phe Leu Val 45 50 55 tcg gac agg
ttc tgg gtc cgg ggc gag ggg ccc atc ttc ttc tac act 243 Ser Asp Arg
Phe Trp Val Arg Gly Glu Gly Pro Ile Phe Phe Tyr Thr 60 65 70 75 ggg
aac gag ggc gac gtg tgg gcc ttc gcc aac aac tcg ggc ttc gtc 291 Gly
Asn Glu Gly Asp Val Trp Ala Phe Ala Asn Asn Ser Gly Phe Val 80 85
90 gcg gag ctg gcg gcc gag cgg ggg gct cta ctg gtc ttc gcg gag cac
339 Ala Glu Leu Ala Ala Glu Arg Gly Ala Leu Leu Val Phe Ala Glu His
95 100 105 cgc tac tac ggg aag tcg ctg ccg ttc ggt gcg cag tcc acg
cag cgc 387 Arg Tyr Tyr Gly Lys Ser Leu Pro Phe Gly Ala Gln Ser Thr
Gln Arg 110 115 120 ggg cac acg gag ctg ctg acg gtg gag cag gcc ctg
gcc gac ttc gca 435 Gly His Thr Glu Leu Leu Thr Val Glu Gln Ala Leu
Ala Asp Phe Ala 125 130 135 gag ctg ctc cgc gcg cta cga cgc gac ctc
ggg gcc cag gat gcc ccc 483 Glu Leu Leu Arg Ala Leu Arg Arg Asp Leu
Gly Ala Gln Asp Ala Pro 140 145 150 155 gcc atc gcc ttc ggt gga agt
tat ggg ggg atg ctc agt gcc tac ctg 531 Ala Ile Ala Phe Gly Gly Ser
Tyr Gly Gly Met Leu Ser Ala Tyr Leu 160 165 170 agg atg aag tat ccc
cac ctg gtg gcg ggg gcg ctg gcg gcc agc gcg 579 Arg Met Lys Tyr Pro
His Leu Val Ala Gly Ala Leu Ala Ala Ser Ala 175 180 185 ccc gtt cta
gct gtg gca ggc ctc ggc gac tcc aac cag ttc ttc cgg 627 Pro Val Leu
Ala Val Ala Gly Leu Gly Asp Ser Asn Gln Phe Phe Arg 190 195 200 gac
gtc acg gcg gac ttt gag ggc cag agt ccc aaa tgc acc cag ggt 675 Asp
Val Thr Ala Asp Phe Glu Gly Gln Ser Pro Lys Cys Thr Gln Gly 205 210
215 gtg cgg gaa gcg ttc cga cag atc aag gac ttg ttc cta cag gga gcc
723 Val Arg Glu Ala Phe Arg Gln Ile Lys Asp Leu Phe Leu Gln Gly Ala
220 225 230 235 tac gac acg gtc cgc tgg gag ttc ggc acc tgc cag ccg
ctg tca gac 771 Tyr Asp Thr Val Arg Trp Glu Phe Gly Thr Cys Gln Pro
Leu Ser Asp 240 245 250 gag aag gac ctg acc cag ctc ttc atg ttc gcc
cgg aat gcc ttc acc 819 Glu Lys Asp Leu Thr Gln Leu Phe Met Phe Ala
Arg Asn Ala Phe Thr 255 260 265 gtg ctg gcc atg atg gac tac ccc tac
ccc act gac ttc ctg ggt ccc 867 Val Leu Ala Met Met Asp Tyr Pro Tyr
Pro Thr Asp Phe Leu Gly Pro 270 275 280 ctc cct gcc aac ccc gtc aag
gtg ggc tgt gat cgg ctg ctg agt gag 915 Leu Pro Ala Asn Pro Val Lys
Val Gly Cys Asp Arg Leu Leu Ser Glu 285 290 295 gcc cag agg atc acg
ggg ctg cga gca ctg gca ggg ctg gtc tac aac 963 Ala Gln Arg Ile Thr
Gly Leu Arg Ala Leu Ala Gly Leu Val Tyr Asn 300 305 310 315 gcc tcg
ggc tcc gag cac tgc tac gac atc tac cgg ctc tac cac agc 1011 Ala
Ser Gly Ser Glu His Cys Tyr Asp Ile Tyr Arg Leu Tyr His Ser 320 325
330 tgt gct gac ccc act ggc tgc ggc acc ggc ccc gac gcc agg gcc tgg
1059 Cys Ala Asp Pro Thr Gly Cys Gly Thr Gly Pro Asp Ala Arg Ala
Trp 335 340 345 gac tac cag gcc tgc acc gag atc aac ctg acc ttc gcc
agc aac aat 1107 Asp Tyr Gln Ala Cys Thr Glu Ile Asn Leu Thr Phe
Ala Ser Asn Asn 350 355 360 gtg acc gat atg ttc ccc gac ctg ccc ttc
act gac gag ctc cgc cag 1155 Val Thr Asp Met Phe Pro Asp Leu Pro
Phe Thr Asp Glu Leu Arg Gln 365 370 375 cgg tac tgc ctg gac acc tgg
ggc gtg tgg ccc cgg ccc gac tgg ctg 1203 Arg Tyr Cys Leu Asp Thr
Trp Gly Val Trp Pro Arg Pro Asp Trp Leu 380 385 390 395 ctg acc agc
ttc tgg ggg ggt gat ctc aga gcc gcc agc aac atc atc 1251 Leu Thr
Ser Phe Trp Gly Gly Asp Leu Arg Ala Ala Ser Asn Ile Ile 400 405 410
ttc tcc aac ggg aac ctg gac ccc tgg gca ggg ggc ggg att cgg agg
1299 Phe Ser Asn Gly Asn Leu Asp Pro Trp Ala Gly Gly Gly Ile Arg
Arg 415 420 425 aac ctg agt gcc tca gtc atc gcc gtc acc atc cag ggg
gga gcg cac 1347 Asn Leu Ser Ala Ser Val Ile Ala Val Thr Ile Gln
Gly Gly Ala His 430 435 440 cac ctc gac ctc aga gcc tcc cac cca gaa
gat cct gct tcc gtg gtt 1395 His Leu Asp Leu Arg Ala Ser His Pro
Glu Asp Pro Ala Ser Val Val 445 450 455 gag gcg cgg aag ctg gag gcc
acc atc atc ggc gag tgg gta aag gca 1443 Glu Ala Arg Lys Leu Glu
Ala Thr Ile Ile Gly Glu Trp Val Lys Ala 460 465 470 475 gcc agg cgt
gag cag cag cca gct ctg cgt ggg ggg ccc aga ctc agc 1491 Ala Arg
Arg Glu Gln Gln Pro Ala Leu Arg Gly Gly Pro Arg Leu Ser 480 485 490
ctc tgagcacagg actggagggg tctcaaggct cctcatggag tgggggcttc 1544 Leu
actcaagcag ctggcggcag agggaagggg ctgaataaac gcctggaggc ctggcaaaaa
1604 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1653 3
10 PRT Artificial Sequence consensus sequence for the
prolyloligopeptidase family from the Prosite database of protein
patterns 3 Ile Phe Gly Gly Ser Asn Gly Gly Leu Leu 1 5 10 4 251 PRT
Artificial Sequence consensus sequence for the alpha/beta hydrolase
family from the Prosite database of protein patterns 4 Phe Arg Val
Ile Ala Leu Asp Leu Arg Gly Phe Gly Glu Ser Ser Arg 1 5 10 15 Pro
Ser Asp Leu Ala Asp Tyr Arg Phe Asp Asp Leu Ala Glu Asp Leu 20 25
30 Glu Ala Leu Leu Asp Ala Leu Gly Leu Asp Lys Pro Val Ile Leu Val
35 40 45 Gly His Ser Met Gly Gly Ala Leu Ala Ala Ala Tyr Ala Ala
Lys Tyr 50 55 60 Pro Glu Glu Arg Val Lys Ala Leu Val Leu Val Ser
Thr Pro Ala Pro 65 70 75 80 Ala Gly Leu Ser Ser Arg Leu Phe Pro Arg
Leu Gly Asn Leu Glu Gly 85 90 95 Leu Leu Leu Ala Asn Phe Phe Asn
Arg Leu Ser Arg Ser Val Glu Ala 100 105 110 Leu Leu Gly Arg Ala Leu
Lys Gln Phe Phe Leu Leu Gly Arg Pro Phe 115 120 125 Val Ser Asp Phe
Leu Lys Gln Ala Glu Asp Trp Leu Ser Ser Leu Ala 130 135 140 Arg Pro
Gly Glu Thr Asp Gly Gly Asp Gly Leu Leu Gly Tyr Ala Val 145 150 155
160 Ala Leu Gly Lys Leu Leu Gln Trp Asp Arg Ser Ala Leu Lys Asp Ile
165 170 175 Lys Val Pro Thr Leu Val Ile Trp Gly Asp Asp Asp Pro Leu
Val Pro 180 185 190 Leu Lys Ala Ser Glu Lys Leu Ser Ala Leu Phe Pro
Asn Ala Glu Val 195 200 205 Val Val Ile Asp Asp Ala Gly His Leu Ala
Leu Leu Glu Lys Pro Glu 210 215 220 Glu Val Ala Glu Leu Ile Lys Phe
Leu Ala Leu Ser Thr Asn Asx Ile 225 230 235 240 Arg Asp Ala Leu Ser
Thr Asn Asx Ile Arg Asp 245 250
* * * * *
References