U.S. patent application number 11/098889 was filed with the patent office on 2005-10-20 for methods of antagonizing morphogens.
This patent application is currently assigned to Curis, Inc.. Invention is credited to Heldin, Carl-Henrik, Miyazono, Kohei, Sampath, Kuber T., ten Dijke, Peter.
Application Number | 20050233379 11/098889 |
Document ID | / |
Family ID | 22889467 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050233379 |
Kind Code |
A1 |
ten Dijke, Peter ; et
al. |
October 20, 2005 |
Methods of antagonizing morphogens
Abstract
Disclosed are (1) nucleic acid sequences, amino acid sequences,
homologies, structural features and various other data
characterizing a morphogen cell surface receptors particularly
OP-1-binding cell surface receptors; (2) methods for producing
receptor proteins, including fragments thereof, using recombinant
DNA technology; (3) methods for identifying novel morphogen
receptors and their encoding DNAs; (4) methods and compositions for
identifying compounds capable of modulating endogenous morphogen
receptor levels; and (5) methods and compositions for identifying
morphogen receptor binding analogs useful in the design of
morphogen agonists and antagonists for therapeutic, diagnostic and
experimental uses.
Inventors: |
ten Dijke, Peter; (Uppsala,
SE) ; Heldin, Carl-Henrik; (Uppsala, SE) ;
Miyazono, Kohei; (Saitama, JP) ; Sampath, Kuber
T.; (Holliston, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Curis, Inc.
Cambridge
MA
Ludwig Institute for Cancer Research
New York
NY
|
Family ID: |
22889467 |
Appl. No.: |
11/098889 |
Filed: |
April 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11098889 |
Apr 4, 2005 |
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09982543 |
Oct 18, 2001 |
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09982543 |
Oct 18, 2001 |
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08448371 |
Jun 2, 1995 |
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6632618 |
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08448371 |
Jun 2, 1995 |
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PCT/US95/05467 |
Apr 28, 1995 |
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PCT/US95/05467 |
Apr 28, 1995 |
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08236428 |
Apr 29, 1994 |
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Current U.S.
Class: |
435/7.1 ;
530/324 |
Current CPC
Class: |
A61K 38/00 20130101;
G01N 33/6845 20130101; G01N 2500/04 20130101; C07K 14/51 20130101;
G01N 33/74 20130101; C07K 14/71 20130101; G01N 33/567 20130101;
G01N 33/6887 20130101; G01N 33/566 20130101; G01N 33/68 20130101;
G01N 2500/00 20130101 |
Class at
Publication: |
435/007.1 ;
530/324 |
International
Class: |
G01N 033/53; C07K
014/675 |
Claims
1-25. (canceled)
26. A method for antagonizing BMP-4 binding to a cell surface
receptor, the method comprising the step of: providing a cell
expressing a said cell-surface receptor with a protein having
binding specificity for a ligand binding domain of the cell-surface
receptor, said ligand binding domain defined by: (i) residues
24-152 of SEQ ID NO: 6 (ALK-3); (ii) residues 23-122 of SEQ ID NO:
8 (ALK-6); or (iii) a nucleotide sequence of a first nucleic acid
capable of hybridizing under stringent conditions with a second
nucleic acid comprising a sequence defined by nucleotides 256-552
of SEQ ID NO:7 (ALK-6), the stringent conditions being
hybridization in 40% formamide, 5.times.SSPE, 5.times. Denhardt's
Solution, 0.1% SDS at 37.degree. C. overnight, then washing in
0.1.times.SSPE, 0.1% SDS at 50.degree. C.; said protein sharing at
least 60% amino acid sequence identity or at least 70% homology
with residues 335-431 of the sequence defined by SEQ ID NO: 10
(OP-1), such that said protein, when provided to said cell, is
competent to interact specifically with said receptor, thereby
substantially inhibiting BMP-4 binding to said receptor.
27. (canceled)
28. The method of claim 26, wherein said protein is substantially
incapable of inducing a morphogen-mediated response.
29. The method of claim 28, wherein said morphogen mediated
response is an OP-1 mediated response.
30. The method of claim 26, wherein the cell further expresses
Daf-4.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 08/448,371 filed on Jun. 2, 1995.
FIELD OF THE INVENTION
[0002] This invention relates generally to the field of tissue
morphogenesis and more particularly to morphogenic protein-specific
cell surface receptors.
BACKGROUND OF THE INVENTION
[0003] Cell differentiation is the central characteristic of tissue
morphogenesis which initiates during embryogenesis, and continues
to various degrees throughout the life of an organism in adult
tissue repair and regeneration mechanisms. The degree of
morphogenesis in adult tissue varies among different tissues and is
related, among other things, to the degree of cell turnover in a
given tissue.
[0004] The cellular and molecular events which govern the stimulus
for differentiation of cells is an area of intensive research. In
the medical and veterinary fields, it is anticipated that the
discovery of the factor or factors which control cell
differentiation and tissue morphogenesis will advance significantly
medicine's ability to repair and regenerate diseased or damaged
mammalian tissues and organs. Particularly useful areas for human
and veterinary therapeutics include reconstructive surgery and in
the treatment of tissue degenerative diseases including arthritis,
emphysema, osteoporosis, cardiomyopathy, cirrhosis, degenerative
nerve diseases, inflammatory diseases, and cancer, and in the
regeneration of tissues, organs and limbs. (In this and related
applications, the terms "morphogenetic" and "morphogenic" are used
interchangeably.)
[0005] A number of different factors have been isolated in recent
years which appear to play a role in cell differentiation.
Recently, a distinct subfamily of the "superfamily" of structurally
related proteins referred to in the art as the "transforming growth
factor-b (TGF-.beta.) superfamily of proteins" have been identified
as true tissue morphogens.
[0006] The members of this distinct "subfamily" of true tissue
morphogenic proteins share substantial amino acid sequence homology
within their morphogenetically active C-terminal domains (at least
50% identity in the C-terminal 102 amino acid sequence), including
a conserved six or seven cysteine skeleton, and share the in vivo
activity of inducing tissue-specific morphogenesis in a variety of
organs and tissues. The proteins apparently contact and interact
with progenitor cells e.g., by binding suitable cell surface
molecules, predisposing or otherwise stimulating the cells to
proliferate and differentiate in a morphogenetically permissive
environment. These morphogenic proteins are capable of inducing the
developmental cascade of cellular and molecular events that
culminate in the formation of new organ-specific tissue, including
any vascularization, connective tissue formation, and nerve
innervation as required by the naturally occurring tissue. The
proteins have been shown to induce morphogenesis of both bone
cartilage and bone, as well as periodontal tissues, dentin, liver,
and neural tissue, including retinal tissue.
[0007] The true tissue morphogenic proteins identified to date
include proteins originally identified as bone inductive proteins.
These include OP-1, (osteogenic protein-1, also referred to in
related applications as "OP1"), its Drosophila homolog, 60A, with
which it shares 69% identity in the C-terminal "seven cysteine"
domain, and the related proteins-OP-2 (also referred to in related
applications as "OP2") and OP-3, both of which share approximately
70-75% identity with OP-1 in the C-terminal seven cysteine domain,
as well as BMP5, BMP6 and its murine homolog, Vgr-1, all of which
share greater than 85% identity with OP-1 in the C-terminal seven
cysteine domain, and the BMP6 Xenopus homolog, Vgl, which shares
approximately 57% identity with OP-1 in the C-terminal seven
cysteine domain. Other bone inductive proteins include the CBMP2
proteins (also referred to in the art as BMP2 and BMP4) and their
Drosophila homolog, DPP. Another tissue morphogenic protein is
GDF-1 (from mouse). See, for example, PCT documents US92/01968 and
US92/07358, the disclosures of which are incorporated herein by
reference.
[0008] As stated above, these true tissue morphogenic proteins are
recognized in the art as a distinct subfamily of proteins different
from other members of the TGF-.beta. superfamily in that they share
a high degree of sequence identity in the C-terminal domain and in
that the true tissue morphogenic proteins are able to induce, on
their own, the full cascade of events that result in formation of
functional tissue rather than merely inducing formation of fibrotic
(scar) tissue. Specifically, members of the family of morphogenic
proteins are capable of all of the following in a morphogenetically
permissive environment: stimulating cell proliferation and cell
differentiation, and supporting the growth and maintenance of
differentiated cells. The morphogenic proteins apparently may act
as endocrine, paracrine or autocrine factors.
[0009] The morphogenic proteins are capable of significant species
"crosstalk." That is, xenogenic (foreign species) homologs of these
proteins can substitute for one another in functional activity. For
example, DPP and 60A, two Drosophila proteins, can substitute for
their mammalian homologs, BMP2/4 and OP-1, respectively, and induce
endochondral bone formation at a non-bony site in a standard rat
bone formation assay. Similarly, BMP2 has been shown to rescue a
dpp.sup.- mutation in Drosophila. In their native form, however,
the proteins appear to be tissue-specific, each protein typically
being expressed in or provided to one or only a few tissues or,
alternatively, expressed only at particular times during
development. For example, GDF-1 appears to be expressed primarily
in neural tissue, while OP-2 appears to be expressed at relatively
high levels in early (e.g., 8-day) mouse embryos. The endogenous
morphogens may be synthesized by the cells on which they act, by
neighboring cells, or by cells of a distant tissue, the secreted
protein being transported to the cells to be acted on.
[0010] A particularly potent tissue morphogenic protein is OP-1.
This protein, and its xenogenic homologs, are expressed in a number
of tissues, primarily in tissues of urogenital origin, as well as
in bone, mammary and salivary gland tissue, reproductive tissues,
and gastrointestinal tract tissue. It is also expressed in
different tissues during embryogenesis, its presence coincident
with the onset of morphogenesis of that tissue.
[0011] The morphogenic protein signal transduction across a cell
membrane appears to occur as a result of specific binding
interaction with one or more cell surface receptors. Recent studies
on cell surface receptor binding of various members of the
TGF-.beta. protein superfamily suggests that the ligands can
mediate their activity by interaction with two different receptors,
referred to as Type I and Type II receptors to form a
hetero-complex. A cell surface bound beta-glycan also may enhance
the binding interaction. The Type I and Type II receptors are both
serine/threonine kinases, and share similar structures: an
intracellular domain that consists essentially of the kinase, a
short, extended hydrophobic sequence sufficient to span the
membrane one time, and an extracellular domain characterized by a
high concentration of conserved cysteines.
[0012] A number of Type II receptor sequences recently have been
identified. These include "TGF-.beta.R II" a TGF-.beta. Type II
receptor (Lin et al. (1992) Cell 68:775-785); and numerous
activin-binding receptors. See, for example, Mathews et al. (1991)
Cell 65:973-982 and international patent application WO 92/20793,
published Nov. 26, 1992, disclosing the "ActR II" sequence;
Attisano et al., (1992) Cell 68:97-108, disclosing the "ActR-IIB"
sequence; and Legerski et al. (1992) Biochem Biophys. Res. Commun
183:672-679. A different Type II receptor shown to have affinity
for activin is Atr-II (Childs et al. (1993) PNAS 90:9475-9479.) Two
Type II receptors have been identified in C. elegans, the daf-1
gene, (Georgi et al. (1990) Cell 61:635-645), having no known
ligand to date, and daf-4, which has been shown to bind BMP4, but
not activin or TGF-.beta. (Estevez, et al. (1993) Nature
365:644-649.)
[0013] Ten Dijke et al. disclose the cloning of six different Type
I cell surface receptors from murine and human cDNA libraries.
((1993) Oncogene 8:2879-2887, and Science (1994) 264:101-104. These
receptors, referenced as ALK-1 to ALK-6 ("activin receptor-like
kinases"), share significant sequence identities (60-79%) and
several have been identified as TGF-.beta. binding (ALK-5) or
activin binding (ALK-2, ALK-4) receptors. Xie et al. also report a
Drosophila Type I receptor encoded by the sax gene (Science (1994)
263:1756-1759). The authors suggest that the protein binds DPP.
[0014] To date, the Type I receptors with which the morphogenic
proteins described herein interact on the cell surface have not yet
been identified, and no Type II receptor has been described as
having binding affinity for OP-1 and its related sequences.
Identification of these cell surface molecules, with which the
morphogens interact and through which they may mediate their
biological effect, is anticipated to enhance elucidation of the
molecular mechanism of tissue morphogenesis and to enable
development of morphogen receptor binding "analogs", e.g.,
compounds (which may or may not be amino acid-based macromolecules)
capable of mimicing the binding affinity of a morphogen for its
receptor sufficiently to act either as a receptor binding agonist
or antagonist. These "analogs" have particular utility in
therapeutic, diagnostic and experimental research applications.
[0015] It is an object of this invention to provide nucleic acid
molecules and amino acid sequences encoding morphogenic protein
binding cell surface receptors, particularly OP-1-specific binding
receptor sequences. Another object is to provide methods for
identifying genes in a variety of species and/or tissues, and in a
variety of nucleic acid libraries encoding morphogenic protein
binding receptors, particularly receptors that bind OP-1. Still
another object is to provide means for designing biosynthetic
receptor-binding ligand analogs, particularly OP-1 analogs, and/or
for identifying natural-occurring ligand analogs, including
agonists and antagonists, using the receptor molecules described
herein, and analogs thereof. Another object is to provide
antagonists, including soluble receptor constructs comprising the
extracellular ligand-binding domain, which can modulate the
availability of OP1 for receptor binding in vivo. Another object is
to provide means and compositions for competing with
activin-receptor and BMP2/4-receptor interactions. Yet another
object is to provide means and compositions for ligand affinity
purification and for diagnostic detection and quantification of
ligands in a body fluid using OP1-specific cell surface receptors
and ligand-binding fragments thereof. Still another object is to
provides means and compositions for modulating the endogenous
expression or concentration of these receptor molecules. Yet
another object is to provide ligand-receptor complexes and analog
sequences thereof, as well as antibodies capable of identifying and
distinguishing the complex from its component proteins. Still
another object is to provide means and compositions for modulating
a morphogenesis in a mammal. These and other objects and features
of the invention will be apparent from the description, drawings
and claims which follow.
SUMMARY OF THE INVENTION
[0016] Type I and Type II cell surface receptor molecules capable
of specific binding affinity with true tissue morphogenic proteins,
particularly OP-1-related proteins, now have been identified.
Accordingly, the invention provides ligand-receptor complexes
comprising at least the ligand binding domain of these receptors
and OP-1 or an OP-1 receptor-binding analog as the ligand; means
for identifying and/or designing useful OP-1 receptor-binding
analogs and OP-1-binding-receptor analogs; and means for modulating
the tissue morphogenesis capability of a cell.
[0017] The morphogen cell surface receptors useful in this
invention are referred to in the art as Type I or Type II
serine/threonine kinase receptors. They share a conserved
structure, including an extracellular, ligand-binding domain
generally composed of about 100-130 amino acids (Type I receptors;
up to about 196 amino acids for Type II receptors), a transmembrane
domain sufficient to span a cellular membrane one time, and an
intracellular (cytoplasmic) domain having serine/threonine kinase
activity. The intact receptor is a single polypeptide chain of
about 500-550 amino acids and having an apparent molecular weight
of about 50-55 kDa.
[0018] Of particular utility in the methods and compositions of the
invention are the Type I cell surface receptors referenced herein
and in the literature as, ALK-2, ALK-3 and ALK-6, whose nucleic
acids and encoded amino acid sequences are represented by the
sequences in Seq. ID Nos. 3, 5 and 7 respectively, and which, as
demonstrated herein below, have specific binding affinity for OP1
and OP1-related analogs. Accordingly, in one embodiment, the
receptor sequences contemplated herein include OP-1 binding analogs
of the ALK-2. ALK-3 and ALK-6 proteins described herein.
[0019] As used herein, ligand-receptor binding specificity is
understood to mean a specific, saturable noncovalent interaction
between the ligand and the receptor, and which is subject to
competitive inhibition by a suitable competitor molecule. Preferred
binding affinities (defined as the amount of ligand required to
fill one-half (50%) of available receptor binding sites) are
described herein by dissociation constant (Kd). In one embodiment,
preferred binding affinities of the ligand-receptor complexes
described herein have a Kd of less than 10.sup.-7M, preferably less
than 5.times.10.sup.-7M, more preferably less than 10.sup.-8M. In
another preferred embodiment, the receptor molecules have little or
no substantial binding affinity for TGF-.beta..
[0020] As used herein, an "OP1-specific receptor analog" is
understood to mean a sequence variant of the ALK-2, ALK-3 or ALK-6
sequences which shares at least 40%, preferably at least 45%, and
most preferably at least 50%, amino acid identity in the
extracellular ligand binding domain with the sequence defined by
residues 23-122 of Seq. ID No. 7 (ALK-6), and which has
substantially the same binding affinity for OP1 as ALK-2, ALK-3 or
ALK-6. ALK-6 and ALK-3 share 46% amino acid sequence identity in
their ligand binding domains. Accordingly, in one preferred
embodiment, the OP1-specific receptor analogs share at least 46%
amino acid sequence identity with the extracellular, ligand binding
domains of ALK-6 or ALK-3.
[0021] As will be appreciated by those having ordinary skill in the
art, OP1-specific receptor analogs also can have binding affinity
for other, related morphogenic proteins. As used herein, an
OP1-specific receptor analog is understood to have substantially
the same binding affinity for OP-1 as ALK-2, ALK-3 or ALK-6 if it
can be competed successfully for OP-1 binding in a standard
competition assay with a known OP-1 binding receptor, e.g., with
ALK-2, ALK-3 or ALK-6. In one preferred embodiment, OP1-specific
receptor analogs have a binding affinity for OP-1 defined by a
dissociation constant of less than about 10.sup.-7 M, preferably
less than about 5.times.10.sup.-7M or 10.sup.-8 M. It is
anticipated however, that analogs having lower binding affinities,
e.g., on the order of 10.sup.-6M also will be useful. For example,
such analogs may be provided to an animal to modulate availability
of serum-soluble OP1 for receptor binding in vivo. Similarly, where
tight binding interaction is desired, for example as part of a
cancer therapy wherein the analog acts as a ligand-receptor
antagonist, preferred binding affinities may be on the order of
5.times.10.sup.8M.
[0022] In another embodiment, the OP-1 binding receptor analogs
contemplated by the invention include proteins encoded by nucleic
acids which hybridize with the DNA sequence encoding the
extracellular, ligand binding domain of ALK-2, ALK-3 or ALK-6 under
stringent hybridization conditions, and which have substantially
the same OP-1 binding affinity as ALK-2, ALK-3 or ALK-6. As used
herein, stringent hybridization conditions are as defined in the
art, (see, for example, Molecular Cloning: A Laboratory Manual,
Maniatis et al., eds. 2d. ed., Cold Spring Harbor Press, Cold
Spring Harbor, 1989.) An exemplary set of conditions is defined as:
hybridization in 40% formamide, 5.times.SSPE, 5.times. Denhardt's
Solution, and 0.1% SDS at 37.degree. C. overnight, and washing in
0.1.times.SSPE, 0.1% SDS at 50.degree. C.
[0023] In still another embodiment, the OP-1 binding receptor
analogs contemplated by the invention include part or all of a
serine/threonine kinase receptor encoded by a nucleic acid that can
be amplified with one or more primers derived from ALK-1 (Seq. ID
No. 1), ALK-2, ALK-3 or ALK-6 sequence in a standard PCR
(polymerase chain reaction) amplification scheme. In particular, a
primer or, most preferably, a pair of primers represented by any of
the sequences of SEQ ID Nos. 12-15 are envisioned to be
particularly useful. Use of primer pairs (e.g., SEQ. ID No. 12/15;
13/15; 14/15) are described in WO94/11502 (PCT/GB93/02367).
[0024] Useful OP1-specific receptor analogs include xenogenic
(foreign species) homologs of the murine and human ALK sequences
described herein, including those obtained from other mammalian
species, as well as other, eukaryotic, non-mammalian xenogenic
homologs. Also contemplated are biosynthetic constructs and
naturally-occurring sequence variants of ALK-2. ALK-3 and ALK-6,
provided these molecules, in all cases, share the appropriate
identity in the ligand binding domain, and bind OP-1 specifically
as defined herein. In one embodiment, sequence variants include
receptor analogs which have substantially the same binding affinity
for OP1 as ALK-2, ALK-3 or ALK-6 and which are recognized by an
antibody having binding specificity for ALK-2. ALK-3 or ALK-6.
[0025] In another embodiment the receptors and OP-1 binding
receptor analogs contemplated herein provide the means by which a
morphogen, e.g., OP-1, can mediate a cellular response. In one
embodiment these receptors include ALK-2, ALK-3, or ALK-6, or
sequence variants or OP-1 binding analogs thereof. In another
embodiment, ALK-1, including sequence variants thereof is
contemplated to participate in an OP-1 mediated cellular
response.
[0026] OP1-specific receptor analogs may be used as OP1
antagonists. For example, a soluble form of a receptor, e.g.,
consisting essentially of only the extracellular ligand-binding
domain, may be provided systemically to a mammal to bind to soluble
ligand, effectively competing with ligand binding to a cell surface
receptor, thereby modulating (reducing) the availability of free
ligand in vivo for cell surface binding.
[0027] The true tissue morphogenic proteins contemplated as useful
receptor ligands in the invention include OP-1 and OP-1
receptor-binding analogs. As used herein, an "OP-1 analog" or "OP-1
receptor-binding analog" is understood to include all molecules
able to functionally substitute for OP-1 in Type I receptor
binding, e.g., are able to successfully compete with OP-1 for
receptor binding in a standard competition assay. In one
embodiment, useful OP-1 receptor-binding analogs include molecules
whose binding affinity is defined by a dissociation constant of
less than about 5.times.10.sup.-6M, preferably less than about
10.sup.-7M or 5.times.10.sup.-7M. As for the OP-specific receptor
analogs above, both stronger and weaker binding affinities are
contemplated to be useful in particular applications. In one
preferred embodiment, these receptor-binding. OP-1 analogs also
bind OP-1 specific Type II serine/kinase receptors.
[0028] The OP-1 analogs contemplated herein, all of which mimic the
binding activity of OP-1 or an OP-1-related protein sufficiently to
act as a substitute for OP-1 in receptor binding, can act as OP-1
agonists, capable of mimicking OP-1 both in receptor binding and in
inducing a transmembrane effect e.g., inducing threonine or
serine-specific phosphorylation following binding. Alternatively,
the OP-1 analog can act as an OP-1 antagonist, capable of mimicking
OP-1 in receptor binding only, but unable to induce a transmembrane
effect, thereby blocking the natural ligand from interacting with
its receptor, for example. Useful applications for antagonists
include their use as therapeutics to modulate uncontrolled
differentiated tissue growth, such as occurs in malignant
transformations such as in osteosarcomas or Paget's disease.
[0029] OP-1 analogs contemplated by the invention can be amino
acid-based, e.g. sequence variants of OP-1, or antibody-derived
sequences capable of is functionally mimicking OP-1 binding to an
OP-1-specific receptor. Examples of such antibodies may include
anti-idiotypic antibodies. In a specific embodiment, the
anti-idiotypic antibody mimics OP1 both in receptor binding and in
ability to induce a transmembrane effect. Alternatively, the OP-1
analogs can be composed in part or in whole of other chemical
structures, e.g., the analogs can be comprised in part or in whole
of nonproteinaceous molecules. In addition, the OP-1 analogs
contemplated can be naturally sourced or synthetically
produced.
[0030] As used herein, OP-1 related sequences include sequences
sharing at least 60%, preferably greater than 65% or even 70%
identity with the C-terminal 102 amino acid sequence of OP-1 as
defined in Seq ID NO.7, and which are able to substitute for OP-1
in ligand binding to ALK-2, ALK-3 or ALK-6, (e.g. able to compete
successfully with OP-1 for binding to one or more of these
receptors.) OP-1 related sequences contemplated by the invention
include xenogenic homologs (e.g., the Drosophila homolog 60A), and
the related sequences referenced herein and in the literature as
OP-2, OP-3, BMP5, BMP6 (and its xenogenic homolog Vgr-1.) OP-1
related sequences also include sequence variants encoded by a
nucleic acid which hybridizes with a DNA sequence comprising the
C-terminal 102 amino acids of Seq. ID No. 9 under stringent
hybridization conditions and which can substitute for OP1 in an
OP1-receptor binding assay. In another embodiment, an OP1 sequence
variant includes a protein which can substitute for OP1 in a
ligand-receptor binding assay and which is recognized by an
antibody having binding specificity for OP1.
[0031] As used herein, "amino acid sequence homology" is understood
to mean amino acid sequence similarity, and homologous sequences
sharing identical or similar amino acids, where similar amino acids
are conserved amino acids as defined by Dayoff et al., Atlas of
Protein Sequence and Structure; vol. 5, Suppl. 3, pp. 345-362 (M.
O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington D.C. 1978.)
Thus, a candidate sequence sharing 60% amino acid homology with a
reference sequence requires that, following alignment of the
candidate sequence with the reference sequence, 60% of the amino
acids in the candidate sequence are identical to the corresponding
amino acid in the reference sequence, or constitute a conserved
amino acid change thereto. "Amino acid sequence identity" is
understood to require identical amino acids between two aligned
sequences. Thus, a candidate sequence sharing 60% amino acid
identity with a reference sequence requires that, following
alignment of the candidate sequence with the reference sequence,
60% of the amino acids in the candidate sequence are identical to
the corresponding amino acid in the reference sequence.
[0032] As used herein, all receptor homologies and identities
calculated use ALK-6 as the reference sequence, with the
extracellular domain reference sequence constituting residues
23-122 of Seq. ID No.7; and the intracellular serine/threonine
kinase domain reference sequence constituting residues 206-495 of
Seq. ID No.7. Similarly, all OP-1 related protein homologies and
identities use OP-1 as the reference sequence, with the C-terminal
102 amino acids described in Seq. ID No. constituting the seven
cysteine domain.
[0033] Also as used herein, sequences are aligned for homology and
identity calculations as follows: Sequences are aligned by eye to
maximize sequence identity. Where receptor amino acid extracellular
domain sequences are compared, the alignment first maximizes
alignment of the cysteines present in the two sequences, then
modifies the alignment as necessary to maximize amino acid identity
and similarity between the two sequences. Where amino acid
intracellular domain sequences are compared, sequences are aligned
to maximize alignment of conserved amino acids in the kinase
domain, where conserved amino acids are those identified by boxes
in FIG. 3. The alignment then is modified as necessary to maximize
amino acid identity and similarity. In all cases, internal gaps and
amino acid insertions in the candidate sequence as aligned are
ignored when making the homology/identity calculation. Exemplary
alignments are illustrated in FIGS. 2 and 3 where the amino acid
sequences for the extracellular and intracellular domains,
respectively are presented in single letter format. In the figures
"gaps" created by sequence alignment are indicated by dashes.
[0034] In one aspect, the invention contemplates isolated
ligand-receptor complexes comprising OP-1 or an OP-1 analog as the
ligand in specific binding interaction with an OP-1 binding Type I
receptor or receptor analog, as defined herein. In another aspect,
the invention contemplates the ligand-receptor complex comprises
part or all of an OP-1 binding Type II receptor. Type II receptors
contemplated to be useful include Type II receptors defined in the
literature (referenced hereinabove) as having binding specificity
for activin or a bone morphogenic protein such as BMP-4. Such Type
II receptors include daf4, ActRII and AtrII. In still another
aspect, the ligand-receptor complex comprises both a Type I and a
Type II receptor and OP1, or an OP1 analog as the ligand. In all
complexes, the bound receptor can comprise just the extracellular,
ligand binding domain, or can also include part or all of the
transmembrane sequence, and/or the intracellular kinase domain.
Similarly, the OP-1 ligand may comprise just the receptor binding
sequence, longer sequences, including the mature dimeric species or
any soluble form of the protein or protein analog.
[0035] The OP-1 and OP-1 analogs described herein can interact
specifically with Type I and Type II receptors also known to
interact with other morphogenic proteins (e.g., BMP2/BMP4) and
activin. Thus invention also contemplates the use of OP-1 and OP-1
receptor-binding analogs as competitors of specific BMP-receptor
and activin-receptor interactions. As will be appreciated by those
having ordinary skill in the art, these binding competitors may act
as either agonists or antagonists (e.g., to inhibit an activin or
BMP-mediated cellular response).
[0036] In another aspect, the invention contemplates binding
partners having specific binding affinity for an epitope on the
ligand-receptor complex. In a preferred embodiment, the binding
partner can discriminate between the complex and the uncomplexed
ligand or receptor. In another embodiment, the binding partner has
little or no substantial binding affinity for the uncomplexed
ligand or receptor. In another preferred embodiment, the binding
partner is a binding protein, more preferably an antibody. These
antibodies may be monoclonal or polyclonal, may be intact molecules
or fragments thereof (e.g., Fab, Fab', (Fab)'.sub.2), or may be
biosynthetic derivatives, including, but not limited to, for
example, monoclonal fragments, such as single chain F.sub.v
fragments, referred to in the literature as sF.sub.vS, BABs and
SCAs, and chimeric monoclonals, in which portions of the
monoclonals are humanized (excluding those portions involved in
antigen recognition (e.g., complementarity determining regions,
"CDRs".) See, for example, U.S. Pat. Nos. 5,091,513 and 5,132,405,
the disclosures of which are incorporated herein by reference.
Biosynthetic chimeras, fragments and other antibody derivatives may
be synthesized using standard recombinant DNA methodology and/or
automated chemical nucleic acid synthesis methodology well
described in the art and as described below.
[0037] In still another aspect, the invention provides molecules
useful in the design and/or identification of receptor-binding
morphogenic protein analogs as described below, as well as kits and
methods, e,g., screening assays, for identifying these analogs. The
molecules useful in these assays can include part or all of the
receptor sequence of SEQ ID NO. 3, 5 or 7, including amino acid
sequence variants and OP-1 binding analogs and amino acid sequence
variants thereof.
[0038] As described above, sequence variants are contemplated to
have substantially the same binding affinity for OP-1 as the
receptors represented by the sequences in SEQ. ID Nos. 3-7. OP-1
binding receptor analogs include other, known or novel Type I or
Type II serine/threonine kinase receptors having binding affinity
and specificity for OP-1 as defined herein and which (1) share at
least 40% amino acid identity with residues 23-122 of Seq. ID No.
7, (2) are encoded by a nucleic acid that hybridizes under
stringent conditions with a nucleic acid comprising the sequence
defined by nucleotides 256-552 of Seq ID No. 7; or (3) are encoded
by a nucleic acid obtainable by amplification with one or more
primer sequences defined by Seq. ID Nos. 12-15. Currently preferred
for the assays of the invention are receptor sequences comprising
at least the sequence which defines the extracellular, ligand
binding domains of these proteins. The kits and assays may include
just Type I receptors or both Type I and Type II receptors.
Similarly, the kits and screening assays can be used in the design
and/or identification of OP1-specific receptor analogs. The OP-1
receptor-binding analogs and OP-1-binding receptor analogs thus
identified then can be produced in reasonable quantities using
standard recombinant expression or chemical synthesis technology
well known and characterized in the art. Alternatively, promising
candidates can be modified using standard biological or chemical
methodologies to, for example, enhance the binding affinity of the
candidate analog as described in Example 10, below, and the
preferred candidate derivative then produced in quantity.
[0039] In still another aspect, the receptor and/or OP1-specific
receptor analogs can be used in standard methodologies for affinity
purifying and/or quantifying OP1 and OP1 analogs. For example, the
receptor's ligand binding domain first may be immobilized on a
surface of a well or a chromatographic column; ligand in a sample
fluid then may be provided to the receptor under conditions to
allow specific binding; non-specific binding molecules then
removed, e.g., by washing, and the bound ligand then selectively
isolated and/or quantitated. Similarly, OP1 and OP1 analogs can be
used for affinity purifying and/or quantifying OP1-specific
receptors and receptor analogs. In one embodiment, the method is
useful in kits and assays for diagnostic purposes which detect the
presence and/or concentration of OP1 protein or related morphogen
in a body fluid sample including, without limitation, serum,
peritoneal fluid, spinal fluid, and breast exudate. The kits and
assays also can be used for detecting and/or quantitating
OP-1-specific receptors in a sample.
[0040] In still another aspect the invention comprises OP1-specific
receptors and OP-1-binding receptor analogs useful in screening
assays to identify organs, tissues and cell lines which express
OP1-specific receptors. These cells then can be used in screening
assays to identify ligands that modulate endogenous morphogen
receptor expression levels, including the density of receptors
expressed on a cell surface. Useful assay methodologies may be
modeled on those described in PCT US92/07359, and as described
below.
[0041] The invention thus relates to compositions and methods for
the use of morphogen-specific receptor sequences in diagnostic,
therapeutic and experimental procedures. Active receptors useful in
the compositions and methods of this invention can include
truncated or full length forms, as well as forms having varying
glycosylation patterns. Active receptors useful in the invention
also include chimeric constructs as described below. Active
OP1-specific receptors/analogs can be expressed from intact or
truncated genomic or cDNA, or from synthetic DNAs in procaryotic or
eucaryotic host cells, and purified, cleaved, refolded and oxidized
as necessary to form active molecules. Useful host cells include
prokaryotes, including E. coli and B. subtilis, and eukaryotic
cells, including mammalian cells, such as fibroblast 3T3 cells,
CHO, COS, melanoma or BSC cells, Hela and other human cells, the
insect/baculovirus system, as well as yeast and other microbial
host cell systems.
[0042] Thus, in view of this disclosure, skilled genetic engineers
now can, for example, identify and produce OP1-specific cell
surface receptors or analogs thereof; create and perform assays for
screening candidate OP1 receptor-binding analogs and evaluate
promising candidates and their progency in therapeutic regimes and
preclinical studies; modulate the availability of endogenous
morphogen for cell surface interactions; modulate endogenous
morphogen-specific cell surface receptor levels; elucidate the
signal transduction pathway induced by morphogen-cell surface
receptor binding; and modulate tissue morphogenesis in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] FIG. 1 is a schematic representation of the encoded ALK-2,
ALK-3, ALK-6 amino acid sequences, showing the signal sequence 10,
the transmembrane domain 12, the extracellular ligand binding
domain 14, and the intracellular serine/threonine kinase domain
16;
[0044] FIG. 2 is a homology alignment of the extracellular domains
of ALK-2, ALK-3, and ALK-6, aligned to maximize amino acid
identity, wherein conserved amino acids are identified by boxes and
conserved cysteines are indicated by asterisks; and
[0045] FIG. 3 is a homology alignment of the intracellular domain
of ALK-2, ALK-3 and ALK-6, aligned to maximize amino acid identity,
wherein conserved amino acids are boxed and the serine/threonine
kinase domain is indicated by arrows.
DETAILED DESCRIPTION
[0046] Disclosed herein are Type I and Type II receptors having
binding specificity for true tissue morphogenic proteins,
particularly OP1 and OP1-related proteins. It further has been
determined that OP1 binds to a broader range of receptors than
other known tissue morphogens or TGF-.beta. family members. The
Type I receptors disclosed herein, can be used together with OP1
and OP1 analogs for therapeutic, diagnostic and experimental uses
as described herein below. Moreover, soluble forms of the
OP1-binding receptor proteins, e.g., forms consisting essentially
of the extracellular domain or a fragment thereof sufficient to
bind OP1 with specificity, can be used as a soluble therapeutic
morphogen antagonist, as described below.
[0047] Following this disclosure, related OP1-specific receptors
are available, as are high and medium flux screening assays for
identifying OP1 analogs and OP1-specific receptor analogs. These
analogs can be naturally occurring molecules, or they can be
designed and biosynthetically created using a rational drug design
and an established structure/function analysis. The analogs can be
amino acid-based or can be composed in part or whole of
non-proteinaceous synthetic organic molecules. Useful analogs also
can include antibodies, preferably monoclonal antibodies (including
fragments thereof, e.g., Fab, Fab', and (Fab)'.sub.2), or synthetic
derivatives thereof, such as monoclonal single chain F.sub.v
fragments known in the art as sF.sub.vs, BABs, and SCAs (see
below), and bispecific antibodies or derivatives thereof. When
these antibodies mimic the binding activity of OP-1 to a cell
surface receptor without inducing the biological response OP-1 does
upon binding, the antibody can compete for OP-1 binding and act as
an antagonist. These antibodies or derivatives thereof also can
mimic OP-1 both in receptor binding and signal transduction, in
which case the antibody acts as an OP-1 agonist. The antibodies and
derivatives also can be used for inducing the morphogenic cellular
response by crosslinking receptors to morphogenic proteins,
particularly OP1 and OP1-related proteins to form either homo- or
hetero-complexes of the Type I and Type II receptors.
[0048] The OP1-binding receptor sequences described herein (ALK2,
ALK3, ALK6) also can be used to create chimeric sequences, wherein,
for example, part or all of either the extracellular domain or the
intracellular domain is a non-ALK sequence or is created from two
or more ALK sequences. These chimeric receptors can be synthesized
using standard recombinant DNA methodology and/or automated
chemical nucleic acid synthesis methodology well described in the
art and as disclosed below. Chimerics can be used, for example, in
OP1 analog assays, wherein the OP1-binding extracellular domain is
coupled to a non-ALK intracellular domain that is well
characterized and/or readily detectable as a second messenger
response system, as described below. Chimerics also can be used,
for example, in high flux OP1 analogs screens and as part of
purification protocols, wherein a soluble ligand binding domain of
an OP1-specific receptor is immobilized onto a support e.g., by
covalent or non-covalent interactions, with a chromatographic
matrix or the well surface of a 96-well plate. When immobilized
onto a chromatographic matrix surface, the receptor fragment can be
used in a protocol to isolate OP1 or OP1 analogs. When immobilized
on a well surface the receptor fragment is particularly useful in a
screening assay to identify receptor-binding OP1 analogs in a
standard competition assay.
[0049] The true tissue morphogenic proteins contemplated to be
useful in the methods and compositions of the invention include
forms having varying glycosylation patterns and varying N-termini.
The proteins can be naturally occurring or biosynthetically
derived, and can be produced by expression of recombinant DNA in
prokaryotic or eukaryotic host cells. The proteins are active as a
single species (e.g., as homodimers), or combined as a mixed
species. Useful sequences and eucaryotic and procaryotic expression
systems are well described in the art. See, for example, U.S. Pat.
Nos. 5,061,911 and 5,266,683 for useful expression systems.
[0050] Particularly contemplated herein are OP1 and OP1-related
sequences. Useful OP1 sequences are recited in U.S. Pat. Nos.
5,011,691; 5,018,753 and 5,266,683; in Ozkaynak et al. (1990) EMBO
J 9:2085-2093; and Sampath et al. (1993) PNAS 90: 6004-6008. OP-1
related sequences include xenogenic homologs, e.g.; 60A, from
Drosophila, Wharton et al. (1991) PNAS 88:9214-9218; and proteins
sharing greater than 60% identity with OP1 in the C-terminal seven
cysteine domain, preferably at least 65% identity. Examples of OP-1
related sequences include BMP5, BMP6 (and its species homolog
Vgr-1, Lyons et al. (1989) PNAS 86:4554-4558), Celeste, et al.
(1990) PNAS 87:9843-9847 and PCT international application
WO93/00432; OP-2 (Ozkaynak et al. (1992) J. Biol. Chem.
267:13198-13205) and OP-3 (PCT international application
WO94/06447). As will be appreciated by those having ordinary skill
in the art, chimeric constructs readily can be created using
standard molecular biology and mutagenesis techniques combining
various portions of different morphogenic protein sequences to
create a novel sequence, and these forms of the protein also are
contemplated herein.
[0051] A particularly preferred embodiment of the proteins
contemplated by the invention includes proteins whose amino acid
sequence in the cysteine-rich C-terminal domain has greater than
60% identity, and preferably greater than 65% identity with the
amino acid sequence of OPS(OP-1 sequence defining the C-terminal
conserved six cysteines, e.g., residues 335-431 of Seq. ID No.
9).
[0052] In another preferred aspect, the invention contemplates
osteogenic proteins comprising species of polypeptide chains having
the generic amino acid sequence herein referred to as "OPX" which
accommodates the homologies between the various identified species
of the osteogenic OP1 and OP2 proteins, and which is described by
the amino acid sequence presented below and in Sequence ID No.
11.
1 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe 1 5 10 Xaa Asp Leu Gly
Trp Xaa Asp Trp Xaa Ile 15 20 Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr
Cys 25 30 Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser 35 40 Xaa Met Asn
Ala Thr Asn His Ala Ile Xaa 45 50 Gln Xaa Leu Val His Xaa Xaa Xaa
Pro Xaa 55 60 Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr 65 70 Xaa Leu
Xaa Ala Xaa Ser Val Leu Tyr Xaa 75 80 Asp Xaa Ser Xaa Asn Val Ile
Leu Xaa Lys 85 90 Xaa Arg Asn Met Val Val Xaa Ala Cys Gly 95 100
Cys His,
[0053] and wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys
or Arg); Xaa at res. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu),
Xaa at res. 19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at
res. 26=(Ala or Ser); Xaa at res. 35=(Ala or Ser); Xaa at res.
39=(Asn or Asp); Xaa at res. 41=(Tyr or Cys); Xaa at res. 50=(Val
or Leu); Xaa at res. 52=(Ser or Thr); Xaa at res. 56=(Phe or Leu);
Xaa at res. 57=(Ile or Met); Xaa at res. 58=(Asn or Lys); Xaa at
res. 60=(Glu, Asp or Asn); Xaa at res. 61=(Thr, Ala or Val); Xaa at
res. 65=(Pro or Ala); Xaa at res. 71=(Gln or Lys); Xaa at res.
73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaa at res. 80=(Phe
or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res. 84=(Ser or Asn);
Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr or His); and Xaa
at res. 97=(Arg or Lys).
[0054] In still another preferred aspect, the invention
contemplates osteogenic proteins encoded by nucleic acids which
hybridize to DNA or RNA sequences encoding the C-terminal seven
cysteine domain of OP1 or OP2 under stringent hybridization
conditions.
[0055] A brief description of the various terms of OP-1 useful in
the invention is described below:
[0056] OP1 --Refers generically to the family of osteogenically
active proteins produced by expression of part or all of the hOP1
gene. Also referred to in related applications as "OPI" and
"OP-1".
[0057] OP1-PP--Amino acid sequence of human OP1 protein (prepro
form), Seq. ID No. 9, residues 1-431. Also referred to in related
applications as "OP1-PP" and "OPP".
[0058] OP1-18Ser--Amino acid sequence of mature human OP1 protein,
Seq. ID No. 9, residues 293-431. N-terminal amino acid is serine.
Originally identified as migrating at 18 kDa on SDS-PAGE in COS
cells. Depending on protein glycosylation pattern in different host
cells, also migrates at 23 kDa, 19 kDa and 17 kDa on SDS-PAGE. Also
referred to in related applications as "OP1-18."
[0059] OP1-16Ser; OP1-16Ala; OP1-16 Met; OP1-16 leu;
OP1-16Val--N-terminally truncated mature human OP1 protein species
defined, respectively, by residues 300-431; 316-431; 315-431;
313-431 and 318-431.
[0060] OPS--Amino acid sequence defining the C-terminal six
cysteine domain, residues 335-431 of Seq. ID No. 9.
[0061] OP7--Amino acid sequence defining the C-terminal seven
cysteine domain, residues 330-431 of Seq. ID No. 9.
[0062] Soluble form OP1--mature dimeric OP1 species having one or,
preferably two copies of pro domain, e.g. at least residues 158-292
of Seq. ID No. 9, preferably residues 48-292 or 30-292,
non-covalently complexed with the dimer.
[0063] The cloning procedure for obtaining OP1-binding ALK nucleic
acid sequences, means for expressing receptor sequences, as well as
other material aspects concerning the nature and utility of these
sequences, including how to make and how to use the subject matter
claimed, will be further understood from the following, which
constitutes the best mode currently contemplated for practicing the
invention.
EXAMPLE 1
Identification of ALK-1, ALK-2, ALK-3 and ALK-6
[0064] The cloning and characterization of ALK-I, -2, -3, and -6
receptors are described in detail in ten Dijke et al. (1993)
Oncogene 8:2879-2887; and (1994) Science 264:101-104. The general
structures of these proteins is described in FIG. 1, and the
sequence alignments between the ALK genes are shown in FIGS. 2 and
3. These molecules have similar domain structures: an N-terminal
predicted hydrophobic signal sequence (von Heijne (1986) Nucl.
Acids Res. 14: 4683-4690) is followed by a relatively small
extracellular cysteine-rich ligand binding domain, a single
hydrophobic transmembrane region (Kyte & Doolittle (1982) J.
Mol. Biol. 157, 105-132) and a C-terminal intracellular portion,
which consists almost entirely of a kinase domain (FIG. 3).
[0065] The extracellular domains of these receptors, defined
essentially by residues 22-118 (SEQ. ID No. 1) for ALK-1; residues
16-123 (SEQ ID No. 3) for ALK-2; residues 24-152 (SEQ. ID No. 5)
for ALK-3; and residues 23-122 (SEQ ID No. 7) for ALK-6, have
cysteine-rich regions, but sequence similarity varies among the
proteins. For example, ALK-3 and ALK-6 share a high degree of
sequence similarity in their extracellular domains (46% identity)
whereas ALK-2 shows less similarity with ALK 3 or ALK6 (see FIG.
2.)
[0066] The positions of many of the cysteine residues in these
receptors can be aligned, indicating that the extracellular domains
likely adopt a similar structural configuration.
[0067] The intracellular domains of these receptors are
characterized by a serine/threonine kinase, defined essentially by
residues 204-494 (SEQ. ID. No. 1) for ALK-1; residues 210-510 (SEQ
ID No. 3) for ALK-2; residues 236-527 (SEQ ID No. 5) for ALK-3; and
residues 206-497 (SEQ ID No. 7) for ALK-6. The catalytic domains of
kinases can be divided into 12 subdomains with stretches of
conserved amino-acid residues. The key motifs are found in
serine/threonine kinase receptors indicating that they are
functional kinases. The consensus sequence for the binding of ATP
(Gly-X-Gly-X-X-Gly in subdomain I followed by a Lys residue further
downstream in subdomain II) is found in all the ALKS. Moreover,
ALK-1, ALK-2, ALK-3 and ALK-6 have the sequence motifs or similar
motifs HRDLKSKN (Subdomain VIB) and GTKRYMAPE (Subdomain VIII),
that are found in most of the serine/threonine kinase receptors and
can be used to distinguish them from tyrosine kinase receptors. Two
short inserts in the kinase domain (between subdomain VIA and VIB
and between X and XI are unique to members of this serine/threonine
kinase receptor family. In the intracellular domain, these regions,
together with the juxtamembrane part and C-terminal tail, are the
most divergent between family members.
[0068] Type II serine/threonine kinase receptors known in the art
are described and referenced herein above.
EXAMPLE 2
Receptor Expression
[0069] A. General Considerations
[0070] Receptor DNA, or a synthetic form thereof, can be inserted,
using conventional techniques well described in the art (see, for
example, Maniatis (1989) Molecular Cloning A Laboratory Manual),
into any of a variety of expression vectors and transfected into an
appropriate host cell to produce recombinant protein polypeptide
chains, including both full length and truncated forms thereof.
Shortened sequences, for example, can be used for the production of
soluble receptor fragments.
[0071] Useful host cells include E. coli, Saccharomyces cerevisiae,
Pichia pastoris, the insect/baculovirus cell system, myeloma cells,
and various other mammalian cells. The full length forms of the
proteins of this invention preferably are expressed in mammalian
cells, as disclosed herein. Soluble forms may be expressed from
both mammalian or bacterial cell systems. The vector additionally
may include various sequences to promote correct expression of the
recombinant protein, including transcription promoter and
termination sequences, enhancer sequences, preferred ribosome
binding site sequences, preferred mRNA leader sequences, preferred
protein processing sequences, preferred signal sequences for
protein secretion, and the like. The DNA sequence encoding the gene
of interest also may be manipulated to remove potentially
inhibiting sequences or to minimize unwanted secondary structure
formation. The recombinant morphogen receptor also may be expressed
as a fusion protein. After translation, the protein may be purified
from the cells themselves or recovered from the culture medium. The
DNA also may include sequences which aid in expression and/or
purification of the recombinant protein. One useful sequence for
example, is a hexa-His (His.sub.6) sequence, which adds a histidine
tail to allow affinity purification of the protein on an IMAC Cu2+
column (see below.)
[0072] For example, the DNA encoding the extracellular domain may
be inserted into a suitable expression vector for transformation
into a prokaryote host such as E. coli or B. subtilis, to produce a
soluble, morphogen binding fragment. The DNA may expressed directly
or may be expressed as part of a fusion protein having a readily
cleavable fusion junction. An exemplary protocol for prokaryote
expression using MR-1 DNA is provided below. Recombinant protein is
expressed in inclusion bodies and may be purified therefrom using
the technology disclosed in U.S. Pat. No. 5,013,653, for
example.
[0073] The DNA also may be expressed in a suitable mammalian host.
Useful hosts include fibroblast 3T3 cells, (e.g., NIH 3T3, from CRL
1658) COS (simian kidney ATCC, CRL-1650) or CHO (Chinese hamster
ovary) cells (e.g., CHO-DXB11, from Lawrence Chasin, Proc. Nat'l.
Acad. Sci. (1980) 77(7):4216-4222), mink-lung epithelial cells
(MV1Lu), human foreskin fibroblast cells, human glioblastoma cells,
and teratocarcinoma cells. Other useful eukaryotic cell systems
include yeast cells, the insect/baculovirus system or myeloma
cells.
[0074] To express an OP1-specific cell surface receptor, the DNA is
subcloned into an insertion site of a suitable, commercially
available vector along with suitable promoter/enhancer sequences
and 3' termination sequences. Useful promoter/enhancer sequence
combinations include the CMV promoter (human cytomegalovirus (MIE)
promoter) present, for example, on pCDM8, as well as the mammary
tumor virus promoter (MMTV) boosted by the Rous sarcoma virus LTR
enhancer sequence (e.g., from Clontech, Inc., Palo Alto). A useful
induceable promoter includes, for example, A Zn.sup.2+ induceable
promoter, such as the Zn.sup.2+ metallothionein promoter (Wrana et
al. (1992) Cell 71:1003-1014.) Other induceable promoters are well
known in the art and can be used with similar success. Expression
also may be further enhanced using transactivating enhancer
sequences. The plasmid also preferably contains an amplifiable
marker, such as DHFR under suitable promoter control, e.g., SV40
early promoter (ATCC #37148). Transfection, cell culturing, gene
amplification and protein expression conditions are standard
conditions, well known in the art, such as are described, for
example in Ausubel et al., ed., Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1989). Briefly, transfected
cells are cultured in medium containing 5-10% dialyzed fetal calf
serum (FCS), and stably transfected high expression cell lines
obtained by amplification and subcloning and evaluated by standard
Western and Northern blot analysis. Southern blots also can be used
to assess the state of integrated receptor sequences and the extent
of their copy number amplification.
[0075] The expressed protein then is purified using standard
procedures. A currently preferred methodology uses an affinity
column, such as a ligand affinity column or an antibody affinity
column, the bound material then washed, and receptor molecules
selectively eluted in a gradient of increasing ionic strength,
changes in pH or addition of mild denaturants. Alternatively, where
a useful anchor sequence has been added to the DNA, such as a
(His).sub.6 sequence, the column may be a standard affinity column
such as Cu.sup.2+ IMAC column. Here, for example, the cell culture
media containing the recombinant protein is passed over a Cu.sup.2+
IMAC column (for example, prepared with 25 mM imidazole). The bound
protein then is washed with a compatible solution and eluted with
EDTA. The anchor sequence can be removed by a standard chemical or
enzymatic procedure.
[0076] Mammalian cell expression is preferred where morphogen
receptor expression on a cell surface is desired. For example, cell
surface expression may be desired to test morphogen or morphogen
analog binding specificity for a cell surface receptor under in
vivo conditions. Cell surface expression also may be most
efficacious for medium flux cellular screen assays as described
below.
[0077] B.1 Exemplary Mammalian Cell Culture
[0078] The receptors tested in Examples 8 and 9 described below
were expressed in (1) COS-1 cells; (2) mink lung epithelial cells
(Mv1Lu); (3) AG1518 human foreskin fibroblasts; (4) MG-63 human
osteosarcoma cells; (5) PC12 rat pheochromocytoma cells (all
obtained from American Type Culture Collection, Rockville, Md.);
(6) U-1240 MG human glioblastoina cells (Bengt Westermark, et al.
(1988) Cancer Research 48:3910-3918); (7) Tera-2 teratocarcinoma
cells (clone 13, Thompson et al. (1984) J Cell Sci 72:37-64); (8)
MC3T3-E1 cells (Sudo et al. (1983) J. Cell Biol. 96:191-198, and
(9) ROS 17/2.8 rat osteosarcoma cells (Majeska et al. (1985)
Endocrinology 116:170-179. The ROS cells were cultured in Ham's F12
medium containing 14 mM HEPES buffer, 2.5 mM L-glutamine, 1.1 mM
CaCl.sub.2, 5% fetal bovine serum and antibiotics; MC3T3-E1 cells
were cultured in a-MEM with 10% fetal bovine serum and antibiotics,
and Tera-2 cells were cultured in 5% CO.sub.2 atmosphere at
37.degree. C. in a-MEM containing 10% fetal bovine serum, 100
units/ml of penicillin and 50 mg/ml of streptomycin, using
tissue-culture dishes pretreated with 0.1% swine skin gelatin
(Sigma) in phosphate-buffered saline. Unless otherwise specified,
cells were cultured in DMEM containing 10% fetal bovine serum and
antibiotics.
[0079] B.2 Transfection of cDNA
[0080] The receptors tested in Example 8.1 (ALK 1-6, daf-4) were
transfected as follows. Transient expression plasmids of ALK-1 to
-6 and daf-4 were generated by subcloning into an expression vector
(pSV7d, Truett et al. (1985) DNA 4:333-349) or into the pcDNA I
expression vector (Invitrogen, San Diego). For transient
transfection, COS-1 cells were transfected with 10 mg each of
plasmids by a calcium phosphate precipitation method using a
mammalian transfection kit (Stratagene, La Jolla), following the
manufacturer's protocol. One day after transfection, the cells were
used for the affinity labeling and cross-linking experiments.
EXAMPLE 3
Antibody Production
[0081] A. General Considerations
[0082] Antibodies capable of specifically binding the receptor
molecules, ligand molecules, or the ligand-receptor complex itself,
useful as analogs and useful in immunoassays and in the
immunopurification of morphogen receptors described may be obtained
as described below.
[0083] Where antibodies specific to the OP1-specific receptors are
desired, but which do not interfere with ligand binding, the
antigenic sequence preferably comprises the juxtamembrane sequence.
Where antibodies capable of competing for ligand binding are
desired, the ligand binding domain may be used as the antigen
source. Where antibodies to the complex are desired, the complex
itself preferably is used as the antigenic sequence and candidate
antibodies then tested for cross reactivity with uncomplexed ligand
and receptors versus the ligand-receptor complex. Finally,
bispecific antibodies may used to complex ligand to a cell surface
receptor (Type I or Type II) and/or to target an agent or ligand to
cells or tissue expressing a Type I or Type II morphogen-specific
receptor. Preferred bispecific antibody derived molecules are
single chain binding sites described in U.S. Pat. Nos. 5,091,513
and 5,132,405, the disclosures of which are incorporated
hereinabove by reference.
[0084] Antibodies useful as OP1 receptor-binding analogs may be
obtained using the receptor ligand binding domain as the immunogen
source and testing receptor-binding analogs for their ability to
compete with OP1 in a competition binding assay. Similarly, where
antibodies useful as OP1-specific receptor analogs are desired, OP1
is the immunogen source and the antibody candidate tested in a
competition assay with receptor protein.
[0085] Polyclonal antibodies specific for a morphogen receptor of
interest may be prepared generally as described below. Each rabbit
is given a primary immunization (e.g., 500 mg) of antigen in 0.1%
SDS mixed with 500 ml Complete Freund's Adjuvant. The antigen is
injected intradermally at multiple sites on the back and flanks of
the animal. The rabbit is boosted after a month with 500 mg of
antigen in the same manner using incomplete Freund's Adjuvant. Test
bleeds are taken from the ear vein seven days later. Two additional
boosts and test bleeds are performed at monthly intervals until
antibody against the antigenic sequence is detected in the serum
using a standard Western blot. Then, the rabbit is boosted monthly
with 100 mg/ml of antigen and bled (15 ml per bleed) at days seven
and ten after boosting.
[0086] Similarly, monoclonal antibodies specific for a given
morphogen receptor molecule of interest may be prepared as
described below: A mouse is given two injections of the antigenic
sequence. The protein preferably is recombinantly produced. Where
it is desired that the antibody recognize an epitope on the
morphogen binding surface of a receptor an antigenic fragment
derived from the extracellular domain preferably is provided. The
first injection contains 100 mg of antigen in complete Freund's
adjuvant and is given subcutaneously. The second injection contains
50 mg of antigen in incomplete adjuvant and is given
intraperitoneally. The mouse then receives a total of 230 mg of
antigen in four intraperitoneal injections at various times over an
eight month period. One week prior to fusion, the mouse is boosted
intraperitoneally with antigen (e.g., 100 mg) and may be
additionally boosted with an antigen-specific peptide conjugated to
bovine serum albumin with a suitable crosslinking agent. This boost
can be repeated five days (IP), four days (IP), three days (IP) and
one day (IV) prior to fusion. The mouse spleen cells then are fused
to commercially available myeloma cells at a ratio of 1:1 using PEG
1500 (Boehringer Mannheim, Germany), and the fused cells plated and
screened for ALK-specific antibodies, e.g., using ALK-2, ALK-3 or
ALK-6 as antigen. The cell fusion and monoclonal screening steps
readily are performed according to standard procedures well
described in standard texts widely available in the art. (See, for
example, Guide to Protein Purification Murray P. Deutscher, ed.,
Academic Press, San Diego, 1990.
[0087] B. Exemplary ALK-Scientific Antisera
[0088] Antibodies used in the assay of Example 8 were obtained as
follows. Rabbit antisera against ALK-1 to -6 were made against
synthetic peptides corresponding to the divergent, intracellular
juxtamembrane parts. (ALK-1: residues 119-141; ALK-2: residues
151-172; ALK-3 residues 181-202; ALK-6: residues 151-168.) Peptides
were synthesized with an Applied Biosystems 430 A Peptide
Synthesizer using t-butoxycarbonyl chemistry, and purified by
reverse phase HPLC. The synthetic peptides were coupled to keyhole
limpet hemocyanin (Calbiochem-Behring) using glutaraldehyde, as
decribed by Gullick et al. (1985) EMBO J 4: 2869-2877. The coupled
peptides then were mixed with Freund's adjuvant and used to
immunize rabbits using standard methodologies.
EXAMPLE 4
OP1-Receptor Binding Assays
[0089] Ligand binding specificity is determined by evaluating the
ability of a receptor molecule to bind a specific ligand, and the
ability of that ligand to compete against itself and other
molecules which bind the receptor. Useful ligands will have a
binding affinity for a soluble morphogen receptor extracellular
domain such that dissociation constant (Kd) is less than about
10.sup.-6M, preferably less than 5.times.10.sup.-7M. Where stronger
binding interaction is desired, preferred affinities are defined by
a Kd of 10.sup.-8-10.sup.-9M. OP1-related proteins are expected to
be able to bind with specificity to multiple different receptor
molecules, although likely with differing affinities.
[0090] Ligand binding specificity can be assayed as follows,
essentially following standard protocols well described in the art
and disclosed, for example, in Legerski et al. (1992) Biochem.
Biophys. Res. Comm 183:672-679 and Frakar et al., (1978) Biochem.
Biophys. Res. Comm. 80:849-857. In the ligand binding assays, a
ligand having a known, quantifiable affinity for a morphogen
receptor molecule of interest is labelled, typically by
radioiodination (.sup.125I), e.g., by chromogenic or fluorogenic
labeling, or by metabolic labelling, e.g., .sup.35S, and aliquots
of cells expressing the receptor on their surface are incubated
with the labelled ligand, in the presence of various concentrations
of unlabelled potential competitor ligand. In the assays described
in Examples 8 and 9, below, this competitor typically is the
candidate morphogen analog or an aliquot from a broth or extract
anticipated to contain a candidate morphogen analog.
[0091] Alternatively, a crosslinking agent may be used to
covalently link the ligand to the bound receptor, and the
crosslinked complex then immunoprecipitated with an antibody
specific to the ligand, receptor, or complex. (See, Example 8.)
[0092] A standard, exemplary protocol for determining binding
affinity is provided below. Briefly, cells expressing a receptor on
their cell surface are plated into 35 mM dishes and incubated for
48 hours in DMEM (Dulbecco's modified Eagle medium) plus 10% fetal
calf serum. Purified morphogen, here, e.g., OP-1, or an OP1-analog
is iodinated with Na.sup.125I by chloramine T oxidation, preferably
having a specific activity of about 50-100 mCi/mg, essentially
following the protocol of Frolik et al. (1984) J. Biol. Chem.
595:10995-11000. Labelled morphogen then is purified using standard
procedures, e.g., chromatographically. Plated cells then are washed
twice with physiologically buffered saline in the presence of 0.1%
BSA, and incubated at 22.degree. C. in the presence of BSA, buffer
and labelled morphogen (1 ng) and various concentrations (e.g.,
0-10 mg/ml) of unlabelled competitor, e.g., unlabelled morphogen or
candidate ligand analogs. Following binding, cells are washed three
times with cold buffer, solubilized in 0.5 ml of 0.5 N NaOH,
removed from the dish, and radioactivity determined by gamma or
scintillation counter. Data then are expressed as percent
inhibition, where 100% inhibition of specific binding is the
difference between binding in the absence of competitor and binding
in the presence of a 100-fold molar excess of unlabelled morphogen.
Binding parameters preferably are determined using a computer
program such as LIGAND (Munsun et al. (1980) Anal. Biochem.
107:220-259.)
[0093] Where the receptor cell surface binding domain is to be
provided as a soluble protein, the assay can be performed in
solution, most readily as an immunoprecipitation assay. In
currently preferred assays the morphogen molecule is labelled and
incubated with unlabelled receptor and candidate morphogen analogs.
Receptor-specific antibody then is provided to the solution to
precipitate the receptor-morphogen complex and the amount of
labelled morphogen in the precipitated complex determined using
standard detection means.
[0094] Where the receptor or ligand is to be used in an affinity
isolation protocol, the molecule preferably is immobilized on a
surface, preferably a matrix surface over which sample fluid will
flow, allowing the ligand of interest to bind, at letting
nonbinding components pass through as effluent. The complex then
can be removed intact or the ligand selectively removed with a
desired eluant.
[0095] 4.1 Screening Assay Considerations
[0096] In the analog screening assays described in Example 9 below,
the preferred protocol for assaying ligand-receptor binding is a
standard competition or radioimmunoassay (RIA). Here the OP1 is
labelled and the relative binding affinity of a candidate OP1
analog ligand in a sample is measured by quantitating the ability
of the candidate (unlabelled ligand analog) to inhibit binding of
the labelled ligand (competitor morphogen) by the receptor. In
performing the assay, fixed concentrations of receptor and labelled
morphogen are incubated in the absence and presence of unknown
samples containing candidate ligands. Sensitivity can be increased
by preincubating the receptor with candidate ligand before adding
the labelled morphogen. After the labelled competitor has been
added, sufficient time is allowed for adequate competitor binding,
and then free and bound labelled morphogen are separated, and one
or the other is measured. Useful morphogen labels include
radioactive labels, chromogenic or fluorogenic labels, and
conjugated enzymes having high turnover numbers, such as
horseradish peroxidase, alkaline phosphatase, or b-galactosidase,
used in combination with chemiluminescent or fluorogenic
substrates. In the same manner, OP1-specific receptor analogs can
be assayed for their affinity for OP1 in competition assays with
labelled OP1 specific receptors.
[0097] Assays for evaluating a candidate OP1 receptor-binding
analog's ability to mimic OP-1 in signal-transduction across a
membrane are exemplified in detail in Example 9.2, below. Briefly,
the assay involves use of a cell (1) known to express an
OP-1-specific receptor; or (2) which can be modified to express an
OP-1-specific receptor, and/or (3) which can induce an
OP-1-mediated cellular response. In the assay, the ability of a
candidate analog to induce an OP-1-specific cellular response is
monitored. Numerous OP-1 responsive cells and OP-1-mediated
inducible cellular and biochemical markers are known and described
in the art. Alternatively, and as exemplified below, an OP-1
inducible reporter gene system can be constructed and used to
advantage in the assay.
[0098] 4.2 Diagnostic Assay Considerations
[0099] The ability to detect morphogens in solution provides a
valuable tool for diagnostic assays, allowing one to monitor the
level of morphogen free in the body, e.g. in serum and other body
fluids. For example, OP-1 has been detected in a number of
different body fluids, including serum and spinal fluid, including
cerebro-spinal fluid, saliva, milk and other breast exudates. (See,
for example, PCT US92/07432, PCT US93/07231, WO94/06449).
[0100] As one example, OP-1 is an intimate participant in normal
bone growth and resorption. Thus, soluble OP-1 is expected to be
detected at higher concentrations in individuals experiencing high
bone formation, such as children, and at substantially lower levels
in individuals with abnormally low rates of bone formation, such as
patients with osteoporosis, aplastic bone disease, or osteopenia.
Monitoring the level of OP-1 in serum thus provides a means for
evaluating the status of bone tissue and bone homeostasis in an
individual, as well as a means for monitoring the efficacy of a
treatment to regenerate damaged or lost bone tissue. Alternatively,
the level of OP-1 in bone tissue can be assessed in a bone tissue
biopsy.
[0101] Similarly, OP-1 and other morphogens have been identified in
brain tissue. In particular, OP-1 is expressed and/or localized in
developing and adult rat brain and spinal cord tissue, in the
hippocampus, substantia nigra and the adendema glial cells, as well
as associated with astrocytes and the extracellular matrix
surrounding neuronal cell bodies. (See, PCTUS93/07331). Thus,
monitoring the level of OP-1 in spinal fluid or associated with a
nerve tissue biopsy can provide means for evaluating the status of
nerve tissue in an individual, as well as means for monitoring the
efficacy of a nerve regeration or repair therapy.
[0102] For serum assays, the serum preferably first is partially
purified to remove some of the excess, contaminating serum
proteins, such as serum albumin. Preferably the serum is extracted
by precipitation in ammonium sulfate (e.g., 45%) such that the
complex is precipitated. Further purification can be achieved using
purification strategies that take advantage of the differential
solubility of soluble morphogen complex or mature morphogens
relative to that of the other proteins present in serum. Further
purification also can be achieved by chromatographic techniques
well known in the art. The sample fluid then can be assayed for OP1
using the OP1-specific receptor(s) and binding assays as described
herein.
[0103] For a tissue biopsy, cells can be collected and stained with
a labelled OP-1-specific antibody or receptor molecule.
Alternatively, the OP-1 protein selectively can be extracted and
quantitated as described above.
[0104] Morphogens useful in the binding/screening assays
contemplated herein include the soluble forms of the protein, e.g.,
the mature dimeric species complexed with one or two copies of the
pro domain, the mature dimeric species alone, and truncated forms
comprising essentially just the C-terminal active domain.
EXAMPLE 5
Transmembrane Signal Induction Assays/OP1 Mimetics
[0105] The kinase activity of the intracellular domains of the
OP1-specific receptors can be tested in an autophophorylation assay
as described by Mathews et al. (PCT/US92/03825, published Nov. 26,
1992). Briefly, the DNA fragment encoding at least the
intracellular kinase domain of an OP1-specific receptor is
subcloned into pGEX-2T (Smith et al. (1988) Gene 67:31-40) to
create a fusion protein between the putative kinase domain and
glutathione S-transferase (GST). The plasmid is introduced into E.
coli and the expressed fusion protein purified using glutathione
affinity chromatography. About 100-200 ng of fusion protein or
purified GST then are incubated with 25 mCi (gp.sup.32P) ATP in 50
mM tris, 10 mM MgCl.sub.2 buffer for 30 minutes at 37.degree. C.
Products then are analyzed by gel electrophoresis and
autoradiography. The fusion protein, but not GST alone, becomes
phophorylated, indicating that the kinase domain is functional.
Phosphoamino acid analysis then can be performed to determine the
predominant amino acid being phosphorylated. Similar assays can be
performed using similar fusion constructs expressed in mammalian
cells.
[0106] Various signaling transduction assays are provided in
Example 9, below. An assay also can be developed for testing kinase
activity transduction upon ligand binding using a ligand-induced
kinase activity assay known in the art. Here, the ability of OP-1
analog to induce phosphorylation upon binding to the receptor is
tested.
[0107] See, for example, various assays for measuring
ligand-induced kinase activity described by Accili et al. (1991) J.
Biol. Chem. 266:434-439 and Nakamura et al. (1992) J. Biol. Chem.
267: 18924-18928. For example, ligand-induced kinase activity
(e.g., receptor autophosphorylation) can be measured in vitro by
incubating purified receptor in the presence and absence of ligand
(here, OP1 or OP1 analog, e.g., 10.sup.-7M) under conditions
sufficient to allow binding of the ligand to the receptor, followed
by exposure to .sup.32P-ATP (e.g., 100 mCi in the presence of 10 mM
Tris-HCl (pH 7.6), 10 mM MgCl.sub.2, 10 mM MnCl.sub.2, 1 mM
dithiothreitol, 0.15M NaCl.sub.2, 0.1% Triton X-100 and 3%
glycerol) and the amount of phosphorylation measured, e.g., by SDS
polyacrylamide gel electrophoresis and autoradiography following
immunoprecipitation with antiphosphoserine, antiphosphothreonine or
antiphosphotyrosine antibody (e.g., commercially available or made
using standard antibody methodologies.) While a low level of
autophosphorylation may be detected in the absence of ligand,
incubation with ligand is anticipated to significantly increase
(e.g., 5-20 fold increase) the amount of phosphorylation
detected.
[0108] In another assay for detecting ligand-induced receptor
autophosphorylation, involving intact cells, receptor DNA is
transfected into a suitable host cell, e.g., a fibroblast, which
then is grown under standard conditions to create a confluent
monolayer of cells expressing the receptor on their cell surface.
On the day of the experiment, cells are incubated with or without
ligand (e.g., OP1 or OP1 analog, e.g., 10.sup.-7M) at 37.degree.
C., and then quickly washed with a "stopping solution" containing
ATP (e.g., 0.1H NaF, 4 mM EDTA, 10 mM ATP, 10 mM sodium
orthovanadate, 4 mM sodium pyrophosphate). The cells then are
frozen in a dry ice/ethanol bath, solubilized and the receptors
immunoprecipitated, e.g., with an antireceptor antibody, as
described herein. The immune complexes then are segregated, washed,
separated by gel electrophoresis using standard procedures and
transferred to a membrane for Western blot analysis using standard
procedures. Phosphorylation of the receptor then can be visualized
by immunodetection with a suitable antibody (e.g.,
antiphosphoserine, antiphosphothreonine or antiphosphotyrosine), as
described above. The bound antibody (e.g., bound antiphosphoserine,
antiphosphothreonine or antiphosphotyrosine) then can be detected
with .sup.125I labelled protein A, followed by autoradiography. The
amount of phosphorylated receptor detected is anticipated to be
significantly greater (5-20 fold increase) in receptors incubated
with ligand than receptors exposed to ATP in the absence of
ligand.
[0109] Ligand-induced receptor phosphorylation of exogenous
substrates similarly can be assayed essentially using the
methodology described herein. Here, a suitable substrate (e.g., a
synthetic polypeptide containing serine, threonine or tyrosine
amino acids) is provided to the receptor following ligand exposure
and prior to incubation with ATP. The substrate subsequently can be
segregated by immunoprecipitation with an antibody specific for the
substrate, and phosphorylation detected as described above. As for
autophosphorylation, the amount of phosphorylated substrate
detected following ligand incubation is anticipated to be greater
than that detected for substrates exposed to receptors in the
absence of ligand.
[0110] Alternatively, a reporter gene construct can be created to
assay transmembrane signal induction. Here, the expression control
elements for an OP-1 inducible protein marker is fused to the open
reading frame sequence for any reporter gene, and induction of the
reporter gene expression then assayed. Useful reporter genes
include the luciferase gene or GAL4 as well as other, easily
characterizable markers.
EXAMPLE 6
Chimeric Receptor Molecules
[0111] Chimeric receptor molecules, e.g., comprising an ALK or ALK
analog extracellular and transmembrane region and, for example,
part or all of an intracellular domain from another, different
receptor or an intracellular domain from a different cell surface
molecule, may be constructed using standard recombinant DNA
technology and/or an automated DNA synthesizer to construct the
desired sequence. As will be appreciated by persons skilled in the
art, useful junctions include sequences within the transmembrane
region and/or sequences at the junction of either the intracellular
or the extracellular domains. Also envisioned are chimers where the
extracellular domain or the intracellular domains themselves are
chimeric sequences.
[0112] Chimeric sequences are envisioned to be particularly useful
in screening assays to determine candidate binding ligands (e.g.,
OP1 analogs, see below), where the non-receptor intracellular
domain provides a suitable second messenger response system that is
easy to detect. Potentially useful other second messenger response
systems include those which, when activated, induce
phosphoinositide hydrolysis, adenylate cyclase, guanylate cyclase
or ion channels.
[0113] Chimeric receptor molecules have particular utility in gene
therapy protocols. For example, a population of cells expressing a
chimeric morphogen receptor molecule on their surface and competent
for expressing a desired phenotype can be implanted in a mammal at
a particular tissue locus. By careful choice of the ligand binding
domain used on these receptors a physician can administer to the
individual a morphogen agonist capable of: (1) binding to the
chimeric receptor alone and (2) stimulating the proliferation
and/or differentiation of the implanted cells without affecting
endogenous cell populations.
EXAMPLE 7
Considerations for Identifying Other OP1-Specific Receptors in
Nucleic Acid Libraries
[0114] Identification of ALK Type I receptors that can bind OP-1
allows one to identify other morphogen receptor sequences in
different species as well as in different tissues. The OP1-binding
ALK sequences themselves can be used as a probe or the sequence may
be modified to account for other potential codon usage (e.g., human
codon bias.) Currently preferred probe sequences are those which
encode the receptor's extracellular domain.
[0115] Probes based on the nucleic acid sequence of Seq. ID Nos.1,
3, 5 or 7 can be synthesized on commercially available DNA
synthesizers. e.g. Applied Biosystems model 381A, using standard
techniques, e.g. Gait, Oligonucleotide Synthesis: A Practical
Approach, (IRL Press, Washington D.C., 1984). It is preferable that
the probes are at least 8-50 bases long, more preferably 18-30
bases long. Probes can be labeled in a variety of ways standard in
the art, e.g. using radioactive, enzymatic or colormetric labels as
described, for example, by Berent et al, (May/June 1985)
Biotechniques: 208-220; and Jablonski et al, (1986) Nucleic Acids
Research 14: 6115-6128.
[0116] Preferably, low stringency conditions are employed when
screening a library for morphogen receptor sequences using a probe
derived from OP1-binding receptor. Preferred ALK-specific probes
are those corresponding to bases encoding the extracellular domain
("ECD"), or encoding a unique (nonhomologous) sequence within the
cytoplasmic domain. Useful probes may be designed from bases
encoding the juxtamembrane region, for example. The probe may be
further modified to use a preferred species codon bias.
Alternatively, probes derived from the serine/threonine kinase
domain can be used to identify new members of the receptor kinase
family which can be screened for OP1 binding using the methods
described in Example 8.
[0117] For example, for a probe of about 20-40 bases a typical
prehybridization, hybridization, and wash protocol is as
follows:
[0118] (1) prehybridization: incubate nitrocellulose filters
containing the denatured target DNA for 3-4 hours at 55.degree. C.
in 5.times. Denhardt's solution, 6.times.SSC (20.times.SSC consists
of 175 g NaCl, 88.2 g sodium citrate in 800 ml H.sub.2O adjusted to
pH. 7.0 with 10N NaOH), 0.1% SDS, and 100 mg/ml denatured salmon
sperm DNA,
[0119] (2) hybridization: incubate filters in prehybridization
solution plus probe at 42.degree. C. for 14-48 hours, (3) wash;
three 15 minutes washes in 6.times.SSC and 0.1% SDS at room
temperature, followed by a final 1-1.5 minute wash in 6.times.SSC
and 0.1% SDS at 55.degree. C. Other equivalent procedures, e.g.
employing organic solvents such as formamide, are well known in the
art.
[0120] Alternatively, morphogen receptor-specific DNA can be
amplified using a standard PCR (polymerase chain reaction)
methodology such as the one disclosed herein, to amplify
approximately 500 base pair fragments. As for the hybridization
screening probes described above, the primer sequences preferably
are derived from conserved sequences in the serine/threonine kinase
domain. The primers disclosed herein, in Seq. ID Nos. 12-15 are
envisioned to be particularly useful, particularly in
combination.
[0121] Examples of useful PCR amplifications, including the use of
the primers recited herein, are disclosed in ten Dijke, et al.
(1993) Oncogene 8:2879-2887 and (1994) Science 264:101-104, and
which also describe the isolation protocols for ALK-1, ALK-2, ALK-3
and ALK-6.
[0122] 7.1 Tissue Distribution of Morphogen Receptors
[0123] Determining the tissue distribution of OP1-specific
receptors can be used to identify tissue and cell sources which
express these receptors, to identify new, related OP1-specific
receptor molecules, as well as to identify target tissues for
OP1-receptor interactions under naturally occurring conditions. The
OP-1 specific receptor molecules (or their mRNA transcripts)
readily are identified in different tissues using standard
methodologies and minor modifications thereof in tissues where
expression may be low. For example, protein distribution can be
determined using standard Western blot analysis or
immunohistological detection techniques, and antibodies specific to
the morphogen receptor molecules of interest. Similarly, the
distribution of morphogen receptor transcripts can be determined
using standard Northern hybridization protocols and
transcript-specific probes or by in situ hybridization.
[0124] Any probe capable of hybridizing specifically to a
transcript, and distinguishing the transcript of interest from
other related transcripts can be used. Because the morphogen
receptors described herein likely share high sequence homology in
their intracellular domains, the tissue distribution of a specific
morphogen receptor transcript may best be determined using a probe
specific for the extracellular domain of the molecule. For example,
a particularly useful ALK-specific probe sequence is one derived
from a unique portion of the 5' coding sequence, the sequence
corresponding to the juxtamembrane region, or the 5' or 3'
noncoding sequences. The chosen fragment then is labelled using
standard means well known and described in the art and herein.
[0125] Using these receptor-specific probes, which can be
synthetically engineered or obtained from cloned sequences,
receptor transcripts can be identified and localized in various
tissues of various organisms, using standard methodologies well
known to those having ordinary skill in the art. A detailed
description of a suitable hybridization protocol is described in
Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn. 179:116-123,
and Ozkaynak, et al. (1992) J. Biol. Chem. 267:25220-25227.
Briefly, total RNA is prepared from various tissues (e.g., murine
embryo and developing and adult liver, kidney, testis, heart,
brain, thymus, stomach) by a standard methodologies such as by the
method of Chomczynski et al. ((1987) Anal. Biochem 162:156-159) and
described below. Poly (A)+ RNA is prepared by using oligo
(dT)-cellulose chromatography (e.g., Type 7, from Pharmacia LKB
Biotechnology, Inc.). Poly (A)+ RNA (generally 15 mg) from each
tissue is fractionated on a 1% agarose/formaldehyde gel and
transferred onto a Nytran membrane (Schleicher & Schuell).
Following the transfer, the membrane is baked at 80.degree. C. and
the RNA is cross-linked under UV light (generally 30 seconds at 1
mW/cm.sup.2). Prior to hybridization, the appropriate probe is
denatured by heating. The hybridization is carried out in a lucite
cylinder rotating in a roller bottle apparatus at approximately 1
rev/min for approximately 15 hours at 37.degree. C. using a
hybridization mix of 40% formamide, 5.times. Denhardts,
5.times.SSPE, and 0.1% SDS. Following hybridization, the
non-specific counts are washed off the filters in 0.1.times.SSPE,
0.1% SDS at 50.degree. C.
EXAMPLE 8
Demonstration that ALK-2, ALK-3 and ALK-6 are OP1-Binding
Receptors
[0126] The tissue morphogenic proteins OP1 and BMP4 were tested for
specific binding interaction with the ALK receptors in
receptor-transfected cells (where the receptor is over-expressed),
and in nontransfected cells. It previously was known that ALK-5
interacted specifically with TGF.beta.1 and ALK-2 and ALK-4
interacted specifically with activin. In the experiment, complexes
were crosslinked and immuno-precipitated with an ALK-specific
antibody as described below. To date, no binding with ALK-1 under
the conditions of this protocol have been detected.
[0127] Binding and affinity cross-linking using disuccinimidyl
suberate (Pierce Chemical Co.) were performed using standard
methods (e.g., Franzen et al. (1993) Cell 75:681-692 and Ichijo et
al. (1990) Exp. Cell Res. 187:10995-11000.) A typical protocol is
described below. Modifications from this protocol for individual
experiments were standard changes anticipated to produce the same
result as for the recited procedure. Briefly, cells in multi-well
plates were washed with binding buffer (e.g., phosphate buffered
saline containing 0.9 mM CaCl.sub.2, 0.49 mM MgCl.sub.2 and 1 mg/ml
bovine serum albumin (BSA)), incubated on ice in the same buffer
with labelled ligand, in the presence and absence of excess
unlabelled ligand for sufficient time for the reaction to
equilibrate (e.g. 3 hours.) Cells were washed and the crosslinking
was done in the binding buffer without BSA together with 0.28 mM
disuccinimidyl suberate for 15 min on ice. Cells were harvested by
addition of 1 ml of detachment buffer (10 mM Tris-HCl, pH 7.4, 1 mM
EDTA, 10% glycerol, 0.3 mM PMSF.) Cells then were pelleted by
centrifugation, then resuspended in 50 ml of solubilization buffer
(125 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% Triton X-100,
0.3 mM PMSF, 1% Trasylol) and incubated for 40 minutes on ice.
Cells were centrifuged again and supernatants subjected to analysis
by standard SDS-gel electrophoresis using 4%-15% polyacrylamide
gels, followed by autoradiography.
[0128] Cell lysates obtained following affinity cross-linking via
the general protocol described above were immunoprecipitated using
antisera against ALKs (e.g. raised against the ALK juxtamembrane
region), or directly analyzed by SDS-gel electrophoresis using
gradient gels consisting of 5-12% or 5-10% polyacrylamide. The gels
were fixed and dried, and then subjected to autoradiography or
analysis using phosphoImager (Molecular Dynamics).
[0129] 8.1 Binding of Op-1 and/or BMP-4 to ALKs in Transfected
Cells.
[0130] COS-1 cells transfected with ALK cDNA were tested for the
binding of .sup.125I-OP-1 and .sup.125I-BMP-4, in the presence or
absence of co-transfected Type II receptor DNA: daf-4 cDNA or
ActRII (Estevez et al. (1993) Nature 365:644-649 and Attisano et
al. (1992) Cell 68:97-108, disclosing the DNA sequence for these
Type II receptors and the disclosure of which is incorporated
herein by reference.) Since the cross-linked complexes were
sometimes difficult to visualize because of high background,
samples were immunoprecipitated by antisera against each ALK. The
results are presented in Table I below. In the Table, "N/T" means
"not tested". Binding was specific as determined by standard
competition assays. The values represented by "+/-", "+", "++",
"+++", and "-" are all qualitative descriptors of the relative
amount of radioactivity measured when the crosslinked molecules
were gel electrophoresed and subjected to autoradiography. More
radioactivity measured indicates a stronger binding interaction
detected. In the Table the strength of binding interaction is as
follows: +++>++>+>+/->-.
2 TABLE I .sup.125I OP1 .sup.125I BMP4 +daf4 -daf4 ActRII +daf4
-daf4 +ActRII ALK1 - - - ALK1 - - N/T ALK2 ++ +/- ++ ALK2 - - N/T
ALK3 ++ - +/- ALK3 +++ ++ N/T ALK4 - - - ALK4 - - N/T ALK5 - - -
ALK5 - - N/T ALK6 +++ ++ + ALK6 +++ +++ N/T
[0131] In the absence of daf-4, OP-1 bound to ALK-6, whereas BMP-4
bound to ALK-3 and ALK-6. Weaker binding of OP-1 to ALK-2 was also
observed. Other ALKs did not bind OP-1 or BMP-4 in the absence of
Daf-4. When ALK cDNAs were co-transfected with the daf-4 cDNA,
increased binding of OP-1 to ALK-2 and ALK-6 was seen. In addition,
ALK-3 also was found to bind OP-1 in the co-transfected cells.
Similarly, increased binding of BMP-4 to ALK-3 and ALK-6 could be
observed. Co-transfection of two different types of ALKs did not
further increase the binding of OP-1 or BMP-4. In cells
co-transfected with the DNA for ActRII and ALK-2, ALK-3 or ALK-6,
OP1-receptor binding was enhanced.
[0132] The sizes of the cross-linked complexes were slightly higher
for ALK-3 than for ALK-2 and ALK-6, consistent with its slightly
larger size. Complexes of about 95 kDa as well as multiple
components of 140-250 kDa were also co-immunoprecipitated with
certain of the ALKS.
[0133] In standard competition assays performed with the Type I
receptors in the presence and absence of the Type II receptors, the
binding of OP-1 and BMP-4 could be competed with excess amounts of
unlabeled OP-1, verifying the binding specificity of these
interactions where they occurred.
[0134] These results demonstrate that ALK-2, ALK-3 and ALK-6 can
serve as Type I receptors for OP-1. Notably, ligand binding
apparently can be enhanced in the presence of Type II receptors.
Moreover, OP-1 is able to interact with both a "bone morphogen"
Type II receptor (daf 4) and an "activin" Type II receptor
(ActRII), whereas, for example, activin only interacts with the
ActRII Type II receptor. The data indicate that OP1 has a broader
spectrum of receptor (Type I and Type II) binding affinities than
do other tissue morphogenic proteins, or other members of the
TGF-.beta. family. It is anticipated that OP1 will have specific
binding interactions with other activin-binding or "bone
morphogen-binding" Type II receptors.
[0135] The ability of OP-1 to bind to Type I, Type II receptors
having binding specificity for activin or BMP4 but not TGF-B,
indicates that OP-1 and OP-1 analogs will be useful as competitors
of activin or BMP4 binding to cell surface receptors. In
particular, OP-1 and OP-1 analogs will be useful for competing with
activin-ALK-2 binding and/or activin-ALK-2/ActRII (or other Type
II) receptor binding; and for competing with BMP4 (or BMP2)-ALK-6
binding, and/or BMP2/4-ALK-6/daf 4 (or other Type II) binding. The
OP-1 competitors may act as antagonists (e.g., binding competitors
unable to induce the signal transduction cascade upon binding) or
as agonists (e.g., able both to bind and induce the signal
transduction cascade).
[0136] 8.2 Identification of OP1-Specific Receptors in
Nontransfected Cells
[0137] ALK-5 has been shown to bind TGF-.beta.1, and ALKs 2, 4 bind
activin A with high affinities in nontransfected cells (ten Dijke,
Oncogene, (1993; Science, (1994) referenced herein above.) In the
present experiment, the binding affinity of OP1 and/or BMP4 to
receptors in nontransfected cells was demonstrated as follows. The
results corroborate the transfected cell data, verifying that OP1
interacts specifically with ALK-2, ALK-3 and ALK-6, but not ALK-4
or ALK-5.
[0138] MC3T3-E1 osteoblasts are well characterized cells known to
respond to OP-1 and BMP-4 in the induction of alkaline phosphatase
activity (Paralkar (1991) PNAS 8: 3397-3401.) These cells were
affinity labeled using .sup.125I-OP-1 as described herein above,
and cross-linked complexes of about 75 kDa were seen, which were
immunoprecipitated only with the ALK-2 antiserum. Tera-2
teratocarcinoma cells and Mv1Lu cells responded to OP-1 as measured
by production of plasminogen activator inhibitor-1 (PAI-1). Similar
to MC3T3-E1 cells, cross-linked complexes using .sup.125I-OP-1 in
Tera-2 teratocarcinoma were immunoprecipitated only by the ALK-2
antiserum.
[0139] On the other hand, cross-linked complexes using
.sup.125I-OP-1 to Mv1Lu cells were immunoprecipitated by ALK-2 as
well as ALK-3 and ALK-6 antisera. Mv1Lu cells are known to express
ALK-4 and ALK-5 (Ebner (1993) Science 260:1344-1348), but
cross-linked complexes with .sup.125I-OP-1 were not precipitated by
antisera against these receptors. Similarly, cross-linked complexes
in U-1240 MG glioblastoma cells were immunoprecipitated by ALK-2
and ALK-6 antisera, and weakly by ALK-3 antiserum. In contrast,
cross-linking of .sup.125I-OP-1 to AG1518 human foreskin
fibroblasts yielded weak immunoprecipitated components only by
ALK-3 antiserum. Type II receptor-like components of about 95 kDa
as well as high molecular weight complexes of 140-250 kDa
co-immunoprecipitated with certain ALKs in the Tera-2 cells, Mv1Lu
cells and U-1240 MG cells.
[0140] Receptors for BMP-4 have also been investigated using
nontransfected cells. Cross-linked complexes using .sup.125I-BMP-4
to MC3T3-E1 cells and AG1518 human foreskin fibroblasts were
immunoprecipitated only by ALK-3. On the other hand, cross-linking
of .sup.125I-BMP-4 to Tera-2 cells did not yield any
immunoprecipitated components by antisera against ALKs.
[0141] .sup.125I-OP-1 and/or .sup.125I-BMP-4 also were demonstrated
by affinity cross-linking to interact specifically with receptors
in other BMP-responsive cells, e.g. MG-63 osteosarcoma cells and
PC12 pheochromocytoma cells. A summary of the binding of ALKs to
OP-1 or BMP-4 in different cell types is shown in Table II, below.
In the Table, "N/T" means not tested, and receptors presented in
brackets indicate comparatively lower quantities of radioactive
complexes detected.
3TABLE II Cell lines Binding of OP-1 Binding of BMP-4 Mouse
osteoblasts ALK-2 ALK-3 (MC3T3-E1 Mink lung epithelial ALK-2, -3,
-6 N/T cells (Mv1Lu) Human glioblastoma ALK2, [-3], -6 N/T Human
teratocarcinoma ALK2 -- (Tera-2) Human foreskin [ALK3] ALK3
fibroblasts (AG1518) Rat osteosarcoma ALK2, [-3] N/T.
(ROS17/2.8)
EXAMPLE 9
OP1, OP1-Specific Receptor Analog Screening Assays
[0142] The present invention is useful to determine whether a
ligand, such as a known or putative drug, is capable of binding to
and/or activating an OP1-specific cell surface receptor as
described herein. Ligands capable of specific binding interaction
with a given OP1-specific receptor (e.g., ALK-2, ALK-3, ALK-6) are
referred to herein as OP1 analogs and can be used for therapeutic
and diagnostic applications. Some analogs will have the ability to
stimulate morphogenetic activity in the cell, mimicking both the
receptor binding and signal transducing activity of OP1. These are
referred to OP1 agonists or mimetics. Others will have strong
binding affinity but will not stimulate morphogenesis, these are
OP1 antagonists. The analogs can be amino acid-based, or they can
be composed of non-proteinaceous chemical structures.
[0143] The methods and kits described below similarly can be used
to identify OP1-specific receptor analogs, capable of mimicking the
binding affinity of ALK-2, ALK-3 or ALK-6 for OP1. The analogs can
be provided to a mammal to interact with serum-soluble OP1,
effectively sequestering the protein and modulating its
availability for cell surface interaction.
[0144] Transfection of an isolated clone encoding a morphogen
receptor into the cell systems described above provides an assay
system for the ability of ligands to bind to and/or to activate the
receptor encoded by the isolated DNA molecule. Transfection
systems, such as those described above, are useful as living cell
cultures for competitive binding assays between known or candidate
drugs and ligands which bind to the receptor and compete with the
binding of known morphogens, which are labeled by radioactive,
enzymatic, spectroscopic or other reagents. Membrane preparations
containing the receptor and isolated from transfected cells are
also useful in these competitive binding assays. Alternatively, and
currently preferred, purified receptor molecules or their ligand
binding extracellular domains can be plated onto a microtiter well
surface, in a modification of a sandwich assay, e.g., as a
competition assay, such as an RIA, described above. Finally, as
described above, solution assays, and using only the receptor
extracellular domain, also may be used to advantage in these
assays. Functional assays of second messenger systems or their
sequelae in transfection systems act as assays for binding affinity
and efficacy in the activation of receptor function or efficacy in
the antagonism of receptor function. Such a transfection system
constitutes a "drug discovery system", useful for the
identification of natural or synthetic compounds with potential for
drug development that can be further modified or used directly as
therapeutic compounds to activate or inhibit the natural functions
of the receptor encoded by the isolated DNA molecule.
[0145] Once such candidate drugs (e.g., OP-1 or receptor-binding
analogs thereof) are identified, they can be produced in
reasonable, useful quantities using standard methodologies known in
the art. Amino acid-based molecules can be encoded by synthetic
nucleic acid molecules, and expressed in a recombinant expression
system as described herein above or in the art. Alternatively, such
molecules can be chemically synthesized, e.g., by means of an
automated Peptide synthesizer, for example. Non-amino acid-based
molecules can be produced by standard organic chemical synthesis
procedures. Where the candidate molecule is of undetermined
structure, or composition, its composition readily can be
determined by, for example, mass spectroscopy. Two approaches to
identifying analogs typically are practiced in the art: high flux
screens and rational design of ligand mimetics. High flux screens
typically screen naturally sourced materials or chemical banks for
their ability to bind a protein of interest, here, e.g., the
receptor. Typically, compounds are obtained from a range of
sources, e.g., chemical banks, microbial broths, plant and animal
extracts, and the like. In a high flux screen typically, purified
receptor, preferably the soluble, ligand binding extracellular
domain, is plated onto a microtiter well surface and a standard
volume of a sample solution to be tested then is added. Also added
is a standard volume having a known quantity of a purified ligand
known to bind the receptor with specificity. Preferably the ligand
is labelled with a substance that is readily detectable by
automated means (e.g., radiolabel, chromophoric, fluorometric,
enzymatic or spectroscopic label.) The wells then are washed and
the amount of label remaining after washing or the amount of label
remaining associated with the receptor then is detected. Positive
scores are identified by the ability of the test substance to
prevent interaction of the labelled ligand with the receptor. The
screening assays can be performed without undue experimentation,
using standard molecular and cell biology tools in common use in
the art. For example, screening assays can be performed in standard
96-well plates. Fifteen such plates reasonably can be set up at a
time to perform multiple screening assays in parallel. Thus, with
only 10-11 reiterations of the screening assay 15,625 (5.sup.6)
compounds can be screened for their binding affinity. Even allowing
for a maximum incubation time of 2 hours, all 15,625 compounds
reasonably can be assayed in a matter of days.
[0146] High flux screens exploit both the high degree of
specificity of the labelled ligand for its receptor, as well as
high throughput capacity of computer driven robotics and computer
handling of data. Candidate analogs identified in this manner, then
can be analyzed structurally and this information used to design
and to synthesize analogs having enhanced potency, increased
duration of action, increased selectivity and reduced side effects.
Candidates also can be used in a rational design program as
described below. Finally, candidate analogs also can be tested to
determine morphogenetic effect, if any, as described below.
[0147] The second approach to the identification of analogs uses a
rational design approach to create molecules capable of mimicking
the binding effect of OP1 with an OP1-specific receptor. Here the
relevant structure for receptor binding is analyzed to identify
critical sequences and structures necessary for binding activity
and this information can be used to design and synthesize minimal
size morphogen analogs. As for candidate compounds in the high flux
assay, design candidates can be tested for receptor binding
activity as described above. As described above, a candidate
sequence can be further modified by, for example standard
biological or chemical mutagenesis techniques to create a candidate
derivative having, for example, enhanced binding affinity or
another preferred characteristic.
[0148] Antibodies capable of interacting specifically with the
receptor and competing with OP1 binding also can be used as an
analog. Antibodies can be generated as described above.
[0149] OP1 analogs may be evaluated for their ability to mimic OP1
or to inhibit OP1 binding (e.g., agonists or antagonists) by
monitoring the effect of the analogs on cells bearing an
OP1-specific receptor (e.g., ALK-2, ALK-3 or ALK-6.) OP1 agonists
are anticipated to have utility in any application where tissue
morphogenesis is desired, such as in the regeneration of damaged
tissue resulting from mechanical or chemical trauma, degenerative
diseases, tissue destruction resulting from chronic inflammation,
cirrhosis, inflammatory diseases, cancer and the like, and in the
regeneration of tissues, organs and limbs. OP1 antagonists are
envisioned to have utility in applications where tissue
morphogenesis is to be limited as, for example, in the treatment of
malignant transformations including, but not limited to,
osteosarcomas and Paget's disease.
[0150] Several exemplary systems for assaying the ability of a
candidate analog transduce an OP-1-specific signal across the
cellular membrane are described below.
[0151] 9.1 Induction of Osteoblast Differentiation Markers
[0152] For example, OP1 is known to preferentially induce
differentiation of progenitor cells, including embryonic
mesenchymal cells and primary osteoblasts (see, for example, PCT
US92/07432) As one example, OP1 analogs can be tested for their
ability to induce differentiation of primary osteoblasts, by
measuring the ability of these analogs to induce production of
alkaline phosphatase, PTH-mediated cAMP and osteocalcin, all of
which are induced when primary osteoblasts are exposed to OP-1, 60A
or DPP.
[0153] Briefly, the assays may be performed as follows. In this and
all examples involving osteoblast cultures, rat osteoblast-enriched
primary cultures preferably are used. Although these cultures are
heterogeneous in that the individual cells are at different stages
of differentiation, these cultures are believed to more accurately
reflect the metabolism and function of osteoblasts in vivo than
osteoblast cultures obtained from established cell lines. Unless
otherwise indicated, all chemicals referenced are standard,
commercially available reagents, readily available from a number of
sources, including Sigma Chemical, Co., St. Louis; Calbiochem,
Corp., San Diego and Aldrich Chemical Co., Milwaukee.
[0154] Rat osteoblast-enriched primary cultures are prepared by
sequential collagenase digestion of newborn suture-free rat
calvaria (e.g., from 1-2 day-old animals, Long-Evans strain,
Charles River Laboratories, Wilmington, Mass.), following standard
procedures, such as are described, for example, in Wong et al.,
(1975) PNAS 72:3167-3171. Rat osteoblast single cell suspensions
then are plated onto a multi-well plate (e.g., a 24 well plate) at
a concentration of 50,000 osteoblasts per well in alpha MEM
(modified Eagle's medium, Gibco, Inc., Long Island) containing 10%
FBS (fetal bovine serum), L-glutamine and penicillin/streptomycin.
The cells are incubated for 24 hours at 37.degree. C., at which
time the growth medium is replaced with alpha MEM containing 1% FBS
and the cells incubated for an additional 24 hours so that the
cells are in serum-deprived growth medium at the time of the
experiment.
[0155] Alkaline Phosphatase Induction of Osteoblasts
[0156] The cultured cells in serum-free medium are incubated with
OP1, OP1 analog or a negative control, using a range of
concentrations. For example, 0.1, 1.0, 10.0, 40.0 or 80.0 ng
OP-1/ml medium typically are used. 72 hours after the incubation
period the cell layer is extracted with 0.5 ml of 1% Triton X-100.
The resultant cell extract then, is centrifuged, and 100 ml of the
extract is added to 90 ml of paranitrosophenylphospate
(PNPP)/glycerine mixture and incubated for 30 minutes in a
37.degree. C. water bath and the reaction stopped with 100 ml NaOH.
The samples then are run through a plate reader (e.g., Dynatech
MR700 plate reader, and absorbance measured at 400 nm, using
p-nitrophenol as a standard) to determine the presence and amount
of alkaline phosphate activity. Protein concentrations are
determined by the Biorad method. Alkaline phosphatase activity is
calculated in units/mg protein, where 1 unit=1 nmol p-nitrophenol
liberated/30 minutes at 37.degree. C. OP-1 induces a five-fold
increase in the specific activity of alkaline phosphate by this
method. Agonists are expected to have similar induction effects.
Antagonists should inhibit or otherwise interfere with OP1 binding,
and diminished alkaline phophatase induction should result when the
assay is performed with an antagonist in the presence of a limiting
amount of OP1.
[0157] Induction of PTH-Mediated cAMP.
[0158] The effect of a morphogen analog on parathyroid
hormone-mediated cAMP production in rat osteoblasts in vitro may be
demonstrated as follows.
[0159] Rat osteoblasts are prepared and cultured in a multiwell
plate as described above. The cultured cells then are divided into
three groups: (1) wells which receive, for example, 1.0, 10.0 and
40.0 ng OP-1/ml medium); (2) wells which receive the candidate
analog at various concentration ranges; and (3) a control group
which receives no additional factors. The plate is then incubated
for another 72 hours. At the end of the 72 hours the cells are
treated with medium containing 0.5% bovine serum albumin (BSA) and
1 mM 3-isobutyl-1-methylxanthine for 20 minutes followed by the
addition into half of the wells of human recombinant parathyroid
hormone (hPTH, Sigma, St. Louis) at a concentration of 200 ng/ml
for 10 minutes. The cell layer then is extracted from each well
with 0.5 ml of 1% Triton X-100. The CAMP levels then are determined
using a radioimmunoassay kit (e.g., Amersham, Arlington Heights,
Ill.). OP-1 doubles cAMP production in the presence of PTH.
Agonists are expected to have similar induction effects.
Antagonists are expected to inhibit or otherwise interfere with OP1
binding, and diminished cAMP production should result when the
assay is performed with an antagonist in the presence of limiting
the amount of OP1.
[0160] Induction of Osteocalcin Production
[0161] Osteocalcin is a bone-specific protein synthesized by
osteoblasts which plays an integral role in the rate of bone
mineralization in vivo. Circulating levels of osteocalcin in: serum
are used as a marker for osteoblast activity and bone formation in
vivo. Induction of osteocalcin synthesis in osteoblast-enriched
cultures can be used to demonstrate morphogenic efficacy in
vitro.
[0162] Rat osteoblasts are prepared and cultured in a multi-well
plate as above. In this experiment the medium is supplemented with
10% FBS, and on day 2, cells are fed with fresh medium supplemented
with fresh 10 mM b-glycerophosphate (Sigma, Inc.). Beginning on day
5 and twice weekly thereafter, cells are fed with a complete
mineralization medium containing all of the above components plus
fresh L(+)-ascorbate, at a final concentration of 50 mg/ml medium.
OP1 or OP1 analog then is added to the wells directly, e.g. in 50%
acetonitrile (or 50% ethanol) containing 0.1% trifluoroacetic acid
(TFA), at no more than 5 ml OP1/ml medium. Control wells receive
solvent vehicle only. The cells then are re-fed and the conditioned
medium sample diluted 1:1 in standard radioimmunoassay buffer
containing standard protease inhibitors and stored at -20.degree.
C. until assayed for osteocalcin. Osteocalcin synthesis is measured
by standard radioimmunoassay using a commercially available
osteocalcin-specific antibody and can be confirmed by Northern blot
analysis to calculate the amount of osteocalcin mRNA produced in
the presence and absence of, OP-1 or OP1 analog. OP-1 induces a
dose-dependent increase in osteocalcin production (5-fold increase
using 25 ng of OP-1 protein/ml), and a 20-fold increase in
osteocalcin mRNA. Agonists are expected to have similar induction
effects; antagonists are expected to inhibit or otherwise interfere
with OP1 binding, thereby substantially interfering with
osteocalcin induction in the presence of a limiting amount of
OP1.
[0163] Mineralization is determined on long term cultures (13 day)
using a modified von Kossa staining technique on fixed cell layers:
cells are fixed in fresh 4% paraformaldehyde at 23.degree. C. for
10 min, following rinsing cold 0.9% NaCl. Fixed cells then are
stained for endogenous alkaline phosphatase at pH 9.5 for 10 min,
using a commercially available kit (Sigma, Inc.) Purple stained
cells then are dehydrated with methanol and air dried. After 30 min
incubation in 3% AgNO.sub.3 in the dark, H.sub.2O-rinsed samples
are exposed for 30 sec to 254 nm UV light to develop the black
silver-stained phosphate nodules. Individual mineralized foci (at
least 20 mm in size) are counted under a dissecting microscope and
expressed as nodules/culture. OP-1 induces a 20-fold increase in
initial mineralization rate. Agonists are expected to have similar
induction effects; antagonists are expected to inhibit or otherwise
interfere with OP1 binding, thereby inhibiting mineralization
induction in the presence of a limiting amount of OP1.
[0164] 9.2 Induction of a Constructed Reporter Gene
[0165] Alternatively, a reporter gene construct can be used to
determine the ability of candidate molecule to induce signal
transduction across a membrane following receptor binding. For
example, PAI-1 protein, (Plasminogen Activator Inhibitor-1)
expression can be induced by OP-1 in Mv1Lu-cells (see above). Also,
as demontrated above, these cells express ALK-2, -3 and -6 surface
receptors. In addition, preliminary studies indicate that ALK-1,
when overexpressed in a chemically mutagenized derivative of these
cells, also apparently mediates PAI-1 induction in the presence of
OP1.
[0166] Accordingly. PAI-1 promoter elements can be fused to a
reporter gene and induction of the reporter gene monitored
following incubation of the transfected cell with a candidate
analog. As one example, the luciferase reporter gene can be used,
in, for example, the construct p3TP-Lux described by Wrana et al.
(1992) Cell 71: 1003-1014 and Attisano et al. (1993) Cell 74:
671-680. This reporter gene construct includes a region of the
human PAI-1 gene promoter in combination with three sets of
tetradecanoyl phorbol-acetate responsive elements upstream of the
lucifrase open reading frame.
[0167] In a typical assay, transfected cells starved in DMEM
containing 0.1% fetal bovine serum and antibiotics (e.g., 100
units/ml penicillin and 50 .mu.g/ml streptomycin) for 6 hrs., and
then exposed to ligand for 24 hr. Luciferase activity in the cell
lysate then is measured using a luminometer in the luciferase assay
system, according to the manufacturer's protocol (Promega) In Mv1Lu
mutant cells, "R mutant" cells co-transfected with ALK-2 and Act
RII, OP1 mediated induction of luciferase activity.
[0168] 9.3 Inhibition of Epithelial Cell Proliferation
[0169] OP1 is known to inhibit epithelial cells. Thus, the ability
of a candidate analog to inhibit cell proliferation, as measured by
3H-thymidine uptake by an epithelial cell can be used in an assay
to evaluate signal transduction activity of the candidate. Analogs
competent to inhibit epithelial cell growth are contemplated to
have particular utility in therapeutic applications where
limitation of a proliferating cell population is desired. Such
applications include chemotherapies and radiation therapies where
limiting the growth of a normally proliferating population of cells
can protect these cells from the cytotoxic effects of these cancer
therapies. (see e.g. WO94/06420). In addition, psoriasis and other
tissue disorders resulting from uncontrolled cell proliferation,
including benign and malignant neoplasties, can be modulated by use
of an OP1 analog.
[0170] As an example, mink lung epithelial cell growth is inhibited
by OP-1. (see, PCT US93/08885; WO94/06420.) As described above,
derivatives of these cells [e.g., "R-4 mutants", clone 4-2, Laiho
et al. (1990) J. Biol. Chem. 265: 18518-18524] can be transfected
with DNA encoding OP1-specific receptors and induced to express
these receptors. The transfected cells, then can be assayed for a
candidate analog's ability to block cell growth. As one example,
when R-4 cells are transfected with ALK-3 under a
Zn.sup.2+-inducible promoter, and induced to express the receptor
following induction with ZnCl.sub.2, cell growth can be inhibited
in the presence of OP1 in a dose dependent manner. Preliminary
experiments with ALK-1 indicates that this receptor also can
mediate this OP-1-specific effect.
[0171] In a typical assay, cells are seeded in 24-well cell culture
plates at a density of 10.sup.4 cells per well in DMEM with 10%
FBS, and incubated overnight. The medium is replaced with DMEM
containing 0.2% FBS and 100 uM ZnLC.sub.2, and the cells are
incubated for 5 h, after which the medium is replaced with fresh
DMEM containing 0.2% FBS, 100 uM ZnCL.sub.2 and various
concentrations of OP-1 or an analog candidate. After 16 h of
incubation, 0.25 ci of [.sup.3H]thymidine (Amersham) are added and
the cells incubated for an additional 2 h. Thereafter, the cells
are fixed in 10% trichloroacetic acid for more than 15 min on ice,
and solubilized with 1 M NaOH. The cell extracts are neutralized
with 1 M HCl and .sup.3H radioactivity determined in a liquid
scintillation counter.
EXAMPLE 10
Screening Assay for Compounds which Alter Endogenous OP1 Receptor
Expression Levels
[0172] Candidate compound(s) which can be administered to affect
the level of a given endogenous OP1 receptor can be found using the
following screening assay, in which the level of OP1 receptor
production by a cell type which produces measurable levels of the
receptor is determined by incubating the cell in culture with and
without the candidate compound, in order to assess the effects of
the compound on the cell. This also can be accomplished by
detection of the OP1 receptor either at the protein level by
Western blot or immunolocalization, or at the RNA level by Northern
blot or in situ hydridization. The protocol is based on a procedure
for identifying compounds which alter endogenous levels of OP1
expression, a detailed description also may be found in PCT US
92/07359, incorporated herein by reference.
[0173] Cell cultures of, for example, bone, brain, intestine, lung,
heart, eye, breast, gonads, kidney, adrenals, urinary bladder,
brain, or other organs, may be prepared as described widely in the
literature. For example, kidneys may be explanted from neonatal or
new born or young or adult rodents (mouse or rat) and used in organ
culture as whole or sliced (1-4 mm) tissues. Primary tissue
cultures and established cell lines, also derived from kidney,
adrenals, urinary, bladder, brain, mammary, or other tissues may be
established in multiwell plates (6 well or 24 well) according to
conventional cell culture techniques, and are cultured in the
absence or presence of serum for a period of time (1-7 days). Cells
can be cultured, for example, in Dulbecco's Modified Eagle medium
(Gibco, Long Island, N.Y.) containing serum (e.g., fetal calf serum
at 1%-10%, Gibco) or in serum-deprived medium, as desired, or in
defined medium (e.g., containing insulin, transferrin, glucose,
albumin, or other growth factors).
[0174] Cell samples for testing the level of OP1 receptor
production are collected periodically and evaluated for receptor
production by immunoblot analysis (Sambrook et al., eds., 1989,
Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y.), or, alternatively, a portion of the cell culture itself can
be collected periodically and used to prepare polyA+ RNA for mRNA
analysis by Northern blot analysis. To monitor de novo receptor
synthesis, some cultures are labeled according to conventional
procedures with an .sup.35S-methionine/.sup.35S-cysteine mixture
for 6-24 hours and then evaluated to quantitate receptor synthesis
by conventional immunoassay methods. Alternatively, anti-receptor
antibodies may be labelled and incubated with the cells or cell
lysates, and the bound complexes detected and quantitated by
conventional means, such as those described hereinabove. Northern
blots may be performed using a portion of the OP1 receptor coding
sequence to create hybridization probes, and following the RNA
hybridization protocol described herein.
EXAMPLE 11
General Formulation/Administration Considerations
[0175] The analogs and constructs described herein can be provided
to an individual as part of a therapy to enhance, inhibit, or
othewise modulate the in vivo binding interaction between OP1 and
one or more OP1-specific cell surface receptors. The molecules then
comprise part of a pharmaceutical composition as described herein
below and can be administered by any suitable means, preferably
directly or systemically, e.g., parenterally or orally. Where the
therapeutic molecule is to be provided directly (e.g., locally, as
by injection, to a desired tissue site), or parenterally, such as
by intravenous, subcutaneous, intramuscular, intraorbital,
ophthalmic, intraventricular, intracranial, intracapsular,
intraspinal, intracisternal, intraperitoneal, buccal, rectal,
vaginal, intranasal or by aerosol administration, the therapeutic
preferably comprises part of an aqueous solution. The solution
preferably is physiologically acceptable so that in addition to
delivery of the desired morphogen to the patient, the solution does
not otherwise adversely affect the patient's electrolyte and volume
balance. The aqueous medium for the therapeutic molecule thus may
comprise normal physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4 or
other pharmaceutically acceptable salts thereof.
[0176] Useful solutions for oral or parenteral administration can
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, (Gennaro, A., ed.), Mack Pub., 1990. Formulations may
include, for example, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like. Formulations for direct administration, in particular,
can include glycerol and other compositions of high viscosity.
Biocompatible, preferably bioresorbable polymers, including, for
example, hyaluronic acid, collagen, tricalcium phosphate,
polybutyrate, polylactide, polyglycolide and lactide/glycolide
copolymers, may be useful excipients to control the release of the
morphogen in vivo.
[0177] Other potentially useful parenteral delivery systems for
these therapeutic molecules include ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems,
and liposomes. Formulations for inhalation administration may
contain as excipients, for example, lactose, or can be aqueous
solutions containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or oily solutions for administration
in the form of nasal drops, or as a gel to be applied
intranasally.
[0178] Alternatively, the morphogens described herein may be
administered orally.
[0179] The therapeutic molecules also can be associated with means
for targeting the therapeutic to a desired tissue. For example,
tetracycline and diphosphonates (bisphosphonates) are known to bind
to bone mineral, particularly at zones of bone remodeling, when
they are provided systemically in a mammal. Accordingly, these
molecules may be included as useful agents for targeting
therapeutics to bone tissue. Alternatively, an antibody or other
binding protein that interacts specifically with a surface molecule
on the desired target tissue cells also can be used. Such targeting
molecules further can be covalently associated to the therapeutic
molecule e.g., by chemical crosslinking, or by using standard
genetic engineering means to create, for example, an acid labile
bond such as an Asp-Pro linkage. Useful targeting molecules can be
designed, for example, using the single chain binding site
technology disclosed, for example, in U.S. Pat. No. 5,091,513.
[0180] Finally, therapeutic molecules can be administered alone or
in combination with other molecules known to have a beneficial
effect on tissue morphogenesis, including molecules capable of
tissue repair and regeneration and/or inhibiting inflammation.
Examples of useful cofactors for stimulating bone tissue growth in
osteoporotic individuals, for example, include but are not limited
to, vitamin D.sub.3, calcitonin, prostaglandins, parathyroid
hormone, dexamethasone, estrogen and IGF-I or IGF-II. Useful
cofactors for nerve tissue repair and regeneration can include
nerve growth factors. Other useful cofactors include
symptom-alleviating cofactors, including antiseptics, antibiotics,
antiviral and antifungal agents and analgesics and anesthetics.
[0181] Therapeutic molecules further can be formulated into
pharmaceutical compositions by admixture with pharmaceutically
acceptable nontoxic excipients and carriers. As noted above, such
compositions can be prepared for parenteral administration,
particularly in the form of liquid solutions or suspensions; for
oral administration, particularly in the form of tablets or
capsules; or intranasally, particularly in the form of powders,
nasal drops or aerosols. Where adhesion to a tissue surface is
desired the composition may include the morphogen dispersed in a
fibrinogen-thrombin composition or other bioadhesive such as is
disclosed, for example in PCT US91/09275, the disclosure of which
is incorporated herein by reference. The composition then can be
painted, sprayed or otherwise applied to the desired tissue
surface.
[0182] The compositions can be formulated for parenteral or oral
administration to humans or other mammals in therapeutically
effective amounts, e.g. amounts which provide appropriate
concentrations of the analog to target tissue for a time sufficient
to induce the desired effect.
[0183] Where the analog is to be used as part of a transplant
procedure, it can be provided to the living tissue or organ to be
transplanted prior to removal of tissue or organ from the donor.
The analog may be provided to the donor host directly, as by
injection of a formulation comprising the analog into the tissue,
or indirectly, e.g., by oral or parenteral administration, using
any of the means described above.
[0184] Alternatively or, in addition, once removed from the donor,
the organ or living tissue can be placed in a preservation solution
containing the therapeutic molecule. In addition, the recipient
also preferably is provided with the analog just prior to, or
concommitant with, transplantation. In all cases, the analog can be
administered directly to the tissue at risk, as by injection to the
tissue, or it may be provided systemically, either by oral or
parenteral administration, using any of the methods and
formulations described herein and/or known in the art.
[0185] Where the therapeutic molecule comprises part of a tissue or
organ preservation solution, any commercially available
preservation solution can be used to advantage. For example, useful
solutions known in the art include Collins solution, Wisconsin
solution, Belzer solution, Eurocollins solution and lactated
Ringer's solution. Generally, an organ preservation solution
usually possesses one or more of the following properties: (a) an
osmotic pressure substantially equal to that of the inside of a
mammalian cell, (solutions typically are hyperosmolar and have K+
and/or Mg++ ions present in an amount sufficient to produce an
osmotic pressure slightly higher than the inside of a mammalian
cell); (b) the solution typically is capable of maintaining
substantially normal ATP levels in the cells; and (c) the solution
usually allows optimum maintenance of glucose metabolism in the
cells. Organ preservation solutions also may contain
anticoagulants, energy sources such as glucose, fructose and other
sugars, metabolites, heavy metal chelators, glycerol and other
materials of high viscosity to enhance survival at low
temperatures, free oxygen radical inhibiting and/or scavenging
agents and a pH indicator. A detailed description of preservation
solutions and useful components can be found, for example, in U.S.
Pat. No. 5,002,965, the disclosure of which is incorporated herein
by reference.
[0186] As will be appreciated by those skilled in the art, the
concentration of the compounds described in a therapeutic
composition will vary depending upon a number of factors, including
the dosage of the drug to be administered, the chemical
characteristics (e.g., hydrophobicity) of the compounds employed,
and the route of administration. The preferred dosage of drug to be
administered also is likely to depend on such variables as the type
and extent of tissue loss or defect, the overall health status of
the particular patient, the relative biological efficacy of the
compound selected, the formulation of the compound, the presence
and types of excipients in the formulation, and the route of
administration. In general terms, the therapeutic molecules of this
invention may be provided to and individual where typical dose
ranges are from about 10 ng/kg to about 1 g/kg of body weight per
day; a preferred dose range being from about 0.1 mg/kg to 100 mg/kg
of body weight. No obvious morphogen-induced pathological lesions
are induced when mature morphogen (e.g., OP-1, 20 mg) is
administered daily to normal growing rats for 21 consecutive days.
Moreover, 10 mg systemic injections of morphogen (e.g., OP-1)
injected daily for 10 days into normal newborn mice does not
produce any gross abnormalities.
Other Embodiments
[0187] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
Sequence CWU 1
1
15 1 1509 DNA Homo sapiens CDS (1)..(1509) 1 atg acc ttg ggc tcc
ccc agg aaa ggc ctt ctg atg ctg ctg atg gcc 48 Met Thr Leu Gly Ser
Pro Arg Lys Gly Leu Leu Met Leu Leu Met Ala 1 5 10 15 ttg gtg acc
cag gga gac cct gtg aag ccg tct cgg ggc ccg ctg gtg 96 Leu Val Thr
Gln Gly Asp Pro Val Lys Pro Ser Arg Gly Pro Leu Val 20 25 30 acc
tgc acg tgt gag agc cca cat tgc aag ggg cct acc tgc cgg ggg 144 Thr
Cys Thr Cys Glu Ser Pro His Cys Lys Gly Pro Thr Cys Arg Gly 35 40
45 gcc tgg tgc aca gta gtg ctg gtg cgg gag gag ggg agg cac ccc cag
192 Ala Trp Cys Thr Val Val Leu Val Arg Glu Glu Gly Arg His Pro Gln
50 55 60 gaa cat cgg ggc tgc ggg aac ttg cac agg gag ctc tgc agg
ggg cgc 240 Glu His Arg Gly Cys Gly Asn Leu His Arg Glu Leu Cys Arg
Gly Arg 65 70 75 80 ccc acc gag ttc gtc aac cac tac tgc tgc gac agc
cac ctc tgc aac 288 Pro Thr Glu Phe Val Asn His Tyr Cys Cys Asp Ser
His Leu Cys Asn 85 90 95 cac aac gtg tcc ctg gtg ctg gag gcc acc
caa cct cct tcg gag cag 336 His Asn Val Ser Leu Val Leu Glu Ala Thr
Gln Pro Pro Ser Glu Gln 100 105 110 ccg gga aca gat ggc cag ctg gcc
ctg atc ctg ggc ccc gtg ctg gcc 384 Pro Gly Thr Asp Gly Gln Leu Ala
Leu Ile Leu Gly Pro Val Leu Ala 115 120 125 ttg ctg gcc ctg gtg gcc
ctg ggt gtc ctg ggc ctg tgg cat gtc cga 432 Leu Leu Ala Leu Val Ala
Leu Gly Val Leu Gly Leu Trp His Val Arg 130 135 140 cgg agg cag gag
aag cag cgt ggc ctg cac agc gag ctg gga gag tcc 480 Arg Arg Gln Glu
Lys Gln Arg Gly Leu His Ser Glu Leu Gly Glu Ser 145 150 155 160 agt
ctc atc ctg aaa gca tct gag cag ggc gac acg atg ttg ggg gac 528 Ser
Leu Ile Leu Lys Ala Ser Glu Gln Gly Asp Thr Met Leu Gly Asp 165 170
175 ctc ctg gac agt gac tgc acc aca ggg agt ggc tca ggg ctc ccc ttc
576 Leu Leu Asp Ser Asp Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe
180 185 190 ctg gtg cag agg aca gtg gca cgg cag gtt gcc ttg gtg gag
tgt gtg 624 Leu Val Gln Arg Thr Val Ala Arg Gln Val Ala Leu Val Glu
Cys Val 195 200 205 gga aaa ggc cgc tat ggc gaa gtg tgg cgg ggc ttg
tgg cac ggt gag 672 Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu
Trp His Gly Glu 210 215 220 agt gtg gcc gtc aag atc ttc tcc tcg agg
gat gaa cag tcc tgg ttc 720 Ser Val Ala Val Lys Ile Phe Ser Ser Arg
Asp Glu Gln Ser Trp Phe 225 230 235 240 cgg gag act gag atc tat aac
aca gta ttg ctc aga cac gac aac atc 768 Arg Glu Thr Glu Ile Tyr Asn
Thr Val Leu Leu Arg His Asp Asn Ile 245 250 255 cta ggc ttc atc gcc
tca gac atg acc tcc cgc aac tcg agc acg cag 816 Leu Gly Phe Ile Ala
Ser Asp Met Thr Ser Arg Asn Ser Ser Thr Gln 260 265 270 ctg tgg ctc
atc acg cac tac cac gag cac ggc tcc ctc tac gac ttt 864 Leu Trp Leu
Ile Thr His Tyr His Glu His Gly Ser Leu Tyr Asp Phe 275 280 285 ctg
cag aga cag acg ctg gag ccc cat ctg gct ctg agg cta gct gtg 912 Leu
Gln Arg Gln Thr Leu Glu Pro His Leu Ala Leu Arg Leu Ala Val 290 295
300 tcc gcg gca tgc ggc ctg gcg cac ctg cac gtg gag atc ttc ggt aca
960 Ser Ala Ala Cys Gly Leu Ala His Leu His Val Glu Ile Phe Gly Thr
305 310 315 320 cag ggc aaa cca gcc att gcc cac cgc gac ttc aag agc
cgc aat gtg 1008 Gln Gly Lys Pro Ala Ile Ala His Arg Asp Phe Lys
Ser Arg Asn Val 325 330 335 ctg gtc aag agc aac ctg cag tgt tgc atc
gcc gac ctg ggc ctg gct 1056 Leu Val Lys Ser Asn Leu Gln Cys Cys
Ile Ala Asp Leu Gly Leu Ala 340 345 350 gtg atg cac tca cag ggc agc
gat tac ctg gac atc ggc aac aac ccg 1104 Val Met His Ser Gln Gly
Ser Asp Tyr Leu Asp Ile Gly Asn Asn Pro 355 360 365 aga gtg ggc acc
aag cgg tac atg gca ccc gag gtg ctg gac gag cag 1152 Arg Val Gly
Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Gln 370 375 380 atc
cgc acg gac tgc ttt gag tcc tac aag tgg act gac atc tgg gcc 1200
Ile Arg Thr Asp Cys Phe Glu Ser Tyr Lys Trp Thr Asp Ile Trp Ala 385
390 395 400 ttt ggc ctg gtg ctg tgg gag att gcc cgc cgg acc atc gtg
aat ggc 1248 Phe Gly Leu Val Leu Trp Glu Ile Ala Arg Arg Thr Ile
Val Asn Gly 405 410 415 atc gtg gag gac tat aga cca ccc ttc tat gat
gtg gtg ccc aat gac 1296 Ile Val Glu Asp Tyr Arg Pro Pro Phe Tyr
Asp Val Val Pro Asn Asp 420 425 430 ccc agc ttt gag gac atg aag aag
gtg gtg tgt gtg gat cag cag acc 1344 Pro Ser Phe Glu Asp Met Lys
Lys Val Val Cys Val Asp Gln Gln Thr 435 440 445 ccc acc atc cct aac
cgg ctg gct gca gac ccg gtc ctc tca ggc cta 1392 Pro Thr Ile Pro
Asn Arg Leu Ala Ala Asp Pro Val Leu Ser Gly Leu 450 455 460 gct cag
atg atg cgg gag tgc tgg tac cca aac ccc tct gcc cga ctc 1440 Ala
Gln Met Met Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala Arg Leu 465 470
475 480 acc gcg ctg cgg atc aag aag aca cta caa aaa att agc aac agt
cca 1488 Thr Ala Leu Arg Ile Lys Lys Thr Leu Gln Lys Ile Ser Asn
Ser Pro 485 490 495 gag aag cct aaa gtg att caa 1509 Glu Lys Pro
Lys Val Ile Gln 500 2 503 PRT Homo sapiens 2 Met Thr Leu Gly Ser
Pro Arg Lys Gly Leu Leu Met Leu Leu Met Ala 1 5 10 15 Leu Val Thr
Gln Gly Asp Pro Val Lys Pro Ser Arg Gly Pro Leu Val 20 25 30 Thr
Cys Thr Cys Glu Ser Pro His Cys Lys Gly Pro Thr Cys Arg Gly 35 40
45 Ala Trp Cys Thr Val Val Leu Val Arg Glu Glu Gly Arg His Pro Gln
50 55 60 Glu His Arg Gly Cys Gly Asn Leu His Arg Glu Leu Cys Arg
Gly Arg 65 70 75 80 Pro Thr Glu Phe Val Asn His Tyr Cys Cys Asp Ser
His Leu Cys Asn 85 90 95 His Asn Val Ser Leu Val Leu Glu Ala Thr
Gln Pro Pro Ser Glu Gln 100 105 110 Pro Gly Thr Asp Gly Gln Leu Ala
Leu Ile Leu Gly Pro Val Leu Ala 115 120 125 Leu Leu Ala Leu Val Ala
Leu Gly Val Leu Gly Leu Trp His Val Arg 130 135 140 Arg Arg Gln Glu
Lys Gln Arg Gly Leu His Ser Glu Leu Gly Glu Ser 145 150 155 160 Ser
Leu Ile Leu Lys Ala Ser Glu Gln Gly Asp Thr Met Leu Gly Asp 165 170
175 Leu Leu Asp Ser Asp Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe
180 185 190 Leu Val Gln Arg Thr Val Ala Arg Gln Val Ala Leu Val Glu
Cys Val 195 200 205 Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu
Trp His Gly Glu 210 215 220 Ser Val Ala Val Lys Ile Phe Ser Ser Arg
Asp Glu Gln Ser Trp Phe 225 230 235 240 Arg Glu Thr Glu Ile Tyr Asn
Thr Val Leu Leu Arg His Asp Asn Ile 245 250 255 Leu Gly Phe Ile Ala
Ser Asp Met Thr Ser Arg Asn Ser Ser Thr Gln 260 265 270 Leu Trp Leu
Ile Thr His Tyr His Glu His Gly Ser Leu Tyr Asp Phe 275 280 285 Leu
Gln Arg Gln Thr Leu Glu Pro His Leu Ala Leu Arg Leu Ala Val 290 295
300 Ser Ala Ala Cys Gly Leu Ala His Leu His Val Glu Ile Phe Gly Thr
305 310 315 320 Gln Gly Lys Pro Ala Ile Ala His Arg Asp Phe Lys Ser
Arg Asn Val 325 330 335 Leu Val Lys Ser Asn Leu Gln Cys Cys Ile Ala
Asp Leu Gly Leu Ala 340 345 350 Val Met His Ser Gln Gly Ser Asp Tyr
Leu Asp Ile Gly Asn Asn Pro 355 360 365 Arg Val Gly Thr Lys Arg Tyr
Met Ala Pro Glu Val Leu Asp Glu Gln 370 375 380 Ile Arg Thr Asp Cys
Phe Glu Ser Tyr Lys Trp Thr Asp Ile Trp Ala 385 390 395 400 Phe Gly
Leu Val Leu Trp Glu Ile Ala Arg Arg Thr Ile Val Asn Gly 405 410 415
Ile Val Glu Asp Tyr Arg Pro Pro Phe Tyr Asp Val Val Pro Asn Asp 420
425 430 Pro Ser Phe Glu Asp Met Lys Lys Val Val Cys Val Asp Gln Gln
Thr 435 440 445 Pro Thr Ile Pro Asn Arg Leu Ala Ala Asp Pro Val Leu
Ser Gly Leu 450 455 460 Ala Gln Met Met Arg Glu Cys Trp Tyr Pro Asn
Pro Ser Ala Arg Leu 465 470 475 480 Thr Ala Leu Arg Ile Lys Lys Thr
Leu Gln Lys Ile Ser Asn Ser Pro 485 490 495 Glu Lys Pro Lys Val Ile
Gln 500 3 2724 DNA Homo sapiens CDS (104)..(1633) 3 ctccgagtac
cccagtgacc agagtgagag aagctctgaa cgagggcacg cggcttgaag 60
gactgtgggc agatgtgacc aagagcctgc attaagttgt aca atg gta gat gga 115
Met Val Asp Gly 1 gtg atg att ctt cct gtg ctt atc atg att gct ctc
ccc tcc cct agt 163 Val Met Ile Leu Pro Val Leu Ile Met Ile Ala Leu
Pro Ser Pro Ser 5 10 15 20 atg gaa gat gag aag ccc aag gtc aac ccc
aaa ctc tac atg tgt gtg 211 Met Glu Asp Glu Lys Pro Lys Val Asn Pro
Lys Leu Tyr Met Cys Val 25 30 35 tgt gaa ggt ctc tcc tgc ggt aat
gag gac cac tgt gaa ggc cag cag 259 Cys Glu Gly Leu Ser Cys Gly Asn
Glu Asp His Cys Glu Gly Gln Gln 40 45 50 tgc ttt tcc tca ctg agc
atc aac gat ggc ttc cac gtc tac cag aaa 307 Cys Phe Ser Ser Leu Ser
Ile Asn Asp Gly Phe His Val Tyr Gln Lys 55 60 65 ggc tgc ttc cag
gtt tat gag cag gga aag atg acc tgt aag acc ccg 355 Gly Cys Phe Gln
Val Tyr Glu Gln Gly Lys Met Thr Cys Lys Thr Pro 70 75 80 ccg tcc
cct ggc caa gct gtg gag tgc tgc caa ggg gac tgg tgt aac 403 Pro Ser
Pro Gly Gln Ala Val Glu Cys Cys Gln Gly Asp Trp Cys Asn 85 90 95
100 agg aac atc acg gcc cag ctg ccc act aaa gga aaa tcc ttc cct gga
451 Arg Asn Ile Thr Ala Gln Leu Pro Thr Lys Gly Lys Ser Phe Pro Gly
105 110 115 aca cag aat ttc cac ttg gag gtt ggc ctc att att ctc tct
gta gtg 499 Thr Gln Asn Phe His Leu Glu Val Gly Leu Ile Ile Leu Ser
Val Val 120 125 130 ttc gca gta tgt ctt tta gcc tgc ctg ctg gga gtt
gct ctc cga aaa 547 Phe Ala Val Cys Leu Leu Ala Cys Leu Leu Gly Val
Ala Leu Arg Lys 135 140 145 ttt aaa agg cgc aac caa gaa cgc ctc aat
ccc cga gac gtg gag tat 595 Phe Lys Arg Arg Asn Gln Glu Arg Leu Asn
Pro Arg Asp Val Glu Tyr 150 155 160 ggc act atc gaa ggg ctc atc acc
acc aat gtt gga gac agc act tta 643 Gly Thr Ile Glu Gly Leu Ile Thr
Thr Asn Val Gly Asp Ser Thr Leu 165 170 175 180 gca gat tta ttg gat
cat tcg tgt aca tca gga agt ggc tct ggt ctt 691 Ala Asp Leu Leu Asp
His Ser Cys Thr Ser Gly Ser Gly Ser Gly Leu 185 190 195 cct ttt ctg
gta caa aga aca gtg gct cgc cag att aca ctg ttg gag 739 Pro Phe Leu
Val Gln Arg Thr Val Ala Arg Gln Ile Thr Leu Leu Glu 200 205 210 tgt
gtc ggg aaa ggc agg tat ggt gag gtg tgg agg ggc agc tgg caa 787 Cys
Val Gly Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Ser Trp Gln 215 220
225 ggg gaa aat gtt gcc gtg aag atc ttc tcc tcc cgt gat gag aag tca
835 Gly Glu Asn Val Ala Val Lys Ile Phe Ser Ser Arg Asp Glu Lys Ser
230 235 240 tgg ttc agg gaa acg gaa ttg tac aac act gtg atg ctg agg
cat gaa 883 Trp Phe Arg Glu Thr Glu Leu Tyr Asn Thr Val Met Leu Arg
His Glu 245 250 255 260 aat atc tta ggt ttc att gct tca gac atg aca
tca aga cac tcc agt 931 Asn Ile Leu Gly Phe Ile Ala Ser Asp Met Thr
Ser Arg His Ser Ser 265 270 275 acc cag ctg tgg tta att aca cat tat
cat gaa atg gga tcg ttg tac 979 Thr Gln Leu Trp Leu Ile Thr His Tyr
His Glu Met Gly Ser Leu Tyr 280 285 290 gac tat ctt cag ctt act act
ctg gat aca gtt agc tgc ctt cga ata 1027 Asp Tyr Leu Gln Leu Thr
Thr Leu Asp Thr Val Ser Cys Leu Arg Ile 295 300 305 gtg ctg tcc ata
gct agt ggt ctt gca cat ttg cac ata gag ata ttt 1075 Val Leu Ser
Ile Ala Ser Gly Leu Ala His Leu His Ile Glu Ile Phe 310 315 320 ggg
acc caa ggg aaa cca gcc att gcc cat cga gat tta aag agc aaa 1123
Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325
330 335 340 aat att ctg gtt aag aag aat gga cag tgt tgc ata gca gat
ttg ggc 1171 Asn Ile Leu Val Lys Lys Asn Gly Gln Cys Cys Ile Ala
Asp Leu Gly 345 350 355 ctg gca gtc atg cat tcc cag agc acc aat cag
ctt gat gtg ggg aac 1219 Leu Ala Val Met His Ser Gln Ser Thr Asn
Gln Leu Asp Val Gly Asn 360 365 370 aat ccc cgt gtg ggc acc aag cgc
tac atg gcc ccc gaa gtt cta gat 1267 Asn Pro Arg Val Gly Thr Lys
Arg Tyr Met Ala Pro Glu Val Leu Asp 375 380 385 gaa acc atc cag gtg
gat tgt ttc gat tct tat aaa agg gtc gat att 1315 Glu Thr Ile Gln
Val Asp Cys Phe Asp Ser Tyr Lys Arg Val Asp Ile 390 395 400 tgg gcc
ttt gga ctt gtt ttg tgg gaa gtg gcc agg cgg atg gtg agc 1363 Trp
Ala Phe Gly Leu Val Leu Trp Glu Val Ala Arg Arg Met Val Ser 405 410
415 420 aat ggt ata gtg gag gat tac aag cca ccg ttc tac gat gtg gtt
ccc 1411 Asn Gly Ile Val Glu Asp Tyr Lys Pro Pro Phe Tyr Asp Val
Val Pro 425 430 435 aat gac cca agt ttt gaa gat atg agg aag gta gtc
tgt gtg gat caa 1459 Asn Asp Pro Ser Phe Glu Asp Met Arg Lys Val
Val Cys Val Asp Gln 440 445 450 caa agg cca aac ata ccc aac aga tgg
ttc tca gac ccg aca tta acc 1507 Gln Arg Pro Asn Ile Pro Asn Arg
Trp Phe Ser Asp Pro Thr Leu Thr 455 460 465 tct ctg gcc aag cta atg
aaa gaa tgc tgg tat caa aat cca tcc gca 1555 Ser Leu Ala Lys Leu
Met Lys Glu Cys Trp Tyr Gln Asn Pro Ser Ala 470 475 480 aga ctc aca
gca ctg cgt atc aaa aag act ttg acc aaa att gat aat 1603 Arg Leu
Thr Ala Leu Arg Ile Lys Lys Thr Leu Thr Lys Ile Asp Asn 485 490 495
500 tcc ctc gac aaa ttg aaa act gac tgt tga cattttcata gtgtcaagaa
1653 Ser Leu Asp Lys Leu Lys Thr Asp Cys 505 ggaagatttg acgttgttgt
cattgtccag ctgggaccta atgctggcct gactggttgt 1713 cagaatggaa
tccatctgtc tccctcccca aatggctgct ttgacaaggc agacgtcgta 1773
cccagccatg tgttggggag acatcaaaac caccctaacc tcgctcgatg actgtgaact
1833 gggcatttca cgaactgttc acactgcaga gactaatgtt ggacagacac
tgttgcaaag 1893 gtagggactg gaggaacaca gagaaatcct aaaagagatc
tgggcattaa gtcagtggct 1953 ttgcatagct ttcacaagtc tcctagacac
tccccacggg aaactcaagg aggtggtgaa 2013 tttttaatca gcaatattgc
ctgtgcttct cttctttatt gcactaggaa ttctttgcat 2073 tccttacttg
cactgttact cttaatttta aagacccaac ttgccaaaat gttggctgcg 2133
tactccactg gtctgtcttt ggataatagg aattcaattt ggcaaaacaa aatgtaatgt
2193 cagactttgc tgcattttac acatgtgctg atgtttacaa tgatgccgaa
cattaggaat 2253 tgtttataca caactttgca aattatttat tacttgtgca
cttagtagtt tttacaaaac 2313 tgctttgtgc atatgttaaa gcttattttt
atgtggtctt atgattttat tacagaaatg 2373 tttttaacac tatactctaa
aatggacatt ttcttttatt atcagttaaa atcacatttt 2433 aagtgcttca
catttgtatg tgtgtagact gtaacttttt ttcagttcat atgcagaacg 2493
tatttagcca ttacccacgt gacaccaccg aatatattat cgatttagaa gcaaagattt
2553 cagtagaatt ttagtcctga acgctacggg gaaaatgcat tttcttcaga
attatccatt 2613 acgtgcattt aaactctgcc agaaaaaaat aactattttg
ttttaatcta ctttttgtat 2673 ttagtagtta tttgtataaa ttaaataaac
tgttttcaag tcaaaaaaaa a 2724 4 509 PRT Homo sapiens 4 Met Val Asp
Gly Val Met Ile Leu Pro Val Leu Ile Met Ile Ala Leu 1 5 10 15 Pro
Ser Pro Ser Met Glu Asp Glu Lys Pro Lys Val Asn Pro Lys Leu 20 25
30 Tyr Met Cys Val Cys Glu Gly Leu Ser Cys Gly Asn Glu Asp His Cys
35 40 45 Glu Gly Gln Gln Cys Phe Ser Ser Leu Ser Ile Asn Asp Gly
Phe His 50 55 60 Val Tyr Gln Lys Gly Cys Phe Gln Val Tyr Glu Gln
Gly Lys Met Thr 65 70 75 80 Cys Lys Thr Pro Pro Ser Pro Gly Gln
Ala
Val Glu Cys Cys Gln Gly 85 90 95 Asp Trp Cys Asn Arg Asn Ile Thr
Ala Gln Leu Pro Thr Lys Gly Lys 100 105 110 Ser Phe Pro Gly Thr Gln
Asn Phe His Leu Glu Val Gly Leu Ile Ile 115 120 125 Leu Ser Val Val
Phe Ala Val Cys Leu Leu Ala Cys Leu Leu Gly Val 130 135 140 Ala Leu
Arg Lys Phe Lys Arg Arg Asn Gln Glu Arg Leu Asn Pro Arg 145 150 155
160 Asp Val Glu Tyr Gly Thr Ile Glu Gly Leu Ile Thr Thr Asn Val Gly
165 170 175 Asp Ser Thr Leu Ala Asp Leu Leu Asp His Ser Cys Thr Ser
Gly Ser 180 185 190 Gly Ser Gly Leu Pro Phe Leu Val Gln Arg Thr Val
Ala Arg Gln Ile 195 200 205 Thr Leu Leu Glu Cys Val Gly Lys Gly Arg
Tyr Gly Glu Val Trp Arg 210 215 220 Gly Ser Trp Gln Gly Glu Asn Val
Ala Val Lys Ile Phe Ser Ser Arg 225 230 235 240 Asp Glu Lys Ser Trp
Phe Arg Glu Thr Glu Leu Tyr Asn Thr Val Met 245 250 255 Leu Arg His
Glu Asn Ile Leu Gly Phe Ile Ala Ser Asp Met Thr Ser 260 265 270 Arg
His Ser Ser Thr Gln Leu Trp Leu Ile Thr His Tyr His Glu Met 275 280
285 Gly Ser Leu Tyr Asp Tyr Leu Gln Leu Thr Thr Leu Asp Thr Val Ser
290 295 300 Cys Leu Arg Ile Val Leu Ser Ile Ala Ser Gly Leu Ala His
Leu His 305 310 315 320 Ile Glu Ile Phe Gly Thr Gln Gly Lys Pro Ala
Ile Ala His Arg Asp 325 330 335 Leu Lys Ser Lys Asn Ile Leu Val Lys
Lys Asn Gly Gln Cys Cys Ile 340 345 350 Ala Asp Leu Gly Leu Ala Val
Met His Ser Gln Ser Thr Asn Gln Leu 355 360 365 Asp Val Gly Asn Asn
Pro Arg Val Gly Thr Lys Arg Tyr Met Ala Pro 370 375 380 Glu Val Leu
Asp Glu Thr Ile Gln Val Asp Cys Phe Asp Ser Tyr Lys 385 390 395 400
Arg Val Asp Ile Trp Ala Phe Gly Leu Val Leu Trp Glu Val Ala Arg 405
410 415 Arg Met Val Ser Asn Gly Ile Val Glu Asp Tyr Lys Pro Pro Phe
Tyr 420 425 430 Asp Val Val Pro Asn Asp Pro Ser Phe Glu Asp Met Arg
Lys Val Val 435 440 445 Cys Val Asp Gln Gln Arg Pro Asn Ile Pro Asn
Arg Trp Phe Ser Asp 450 455 460 Pro Thr Leu Thr Ser Leu Ala Lys Leu
Met Lys Glu Cys Trp Tyr Gln 465 470 475 480 Asn Pro Ser Ala Arg Leu
Thr Ala Leu Arg Ile Lys Lys Thr Leu Thr 485 490 495 Lys Ile Asp Asn
Ser Leu Asp Lys Leu Lys Thr Asp Cys 500 505 5 2932 DNA Homo sapiens
CDS (310)..(1908) 5 gctccgcgcc gagggctgga ggatgcgttc cctggggtcc
ggacttatga aaatatgcat 60 cagtttaata ctgtcttgga attcatgaga
tggaagcata ggtcaaagct gtttggagaa 120 aatcagaagt acagttttat
ctagccacat cttggaggag tcgtaagaaa gcagtgggag 180 ttgaagtcat
tgtcaagtgc ttgcgatctt ttacaagaaa atctcactga atgatagtca 240
tttaaattgg tgaagtagca agaccaatta ttaaaggtga cagtacacag gaaacattac
300 aattgaaca atg act cag cta tac att tac atc aga tta ttg gga gcc
tat 351 Met Thr Gln Leu Tyr Ile Tyr Ile Arg Leu Leu Gly Ala Tyr 1 5
10 ttg ttc atc att tct cgt gtt caa gga cag aat ctg gat agt atg ctt
399 Leu Phe Ile Ile Ser Arg Val Gln Gly Gln Asn Leu Asp Ser Met Leu
15 20 25 30 cat ggc act ggg atg aaa tca gac tcc gac cag aaa aag tca
gaa aat 447 His Gly Thr Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser
Glu Asn 35 40 45 gga gta acc tta gca cca gag gat acc ttg cct ttt
tta aag tgc tat 495 Gly Val Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe
Leu Lys Cys Tyr 50 55 60 tgc tca ggg cac tgt cca gat gat gct att
aat aac aca tgc ata act 543 Cys Ser Gly His Cys Pro Asp Asp Ala Ile
Asn Asn Thr Cys Ile Thr 65 70 75 aat gga cat tgc ttt gcc atc ata
gaa gaa gat gac cag gga gaa acc 591 Asn Gly His Cys Phe Ala Ile Ile
Glu Glu Asp Asp Gln Gly Glu Thr 80 85 90 aca tta gct tca ggg tgt
atg aaa tat gaa gga tct gat ttt cag tgc 639 Thr Leu Ala Ser Gly Cys
Met Lys Tyr Glu Gly Ser Asp Phe Gln Cys 95 100 105 110 aaa gat tct
cca aaa gcc cag cta cgc cgg aca ata gaa tgt tgt cgg 687 Lys Asp Ser
Pro Lys Ala Gln Leu Arg Arg Thr Ile Glu Cys Cys Arg 115 120 125 acc
aat tta tgt aac cag tat ttg caa ccc aca ctg ccc cct gtt gtc 735 Thr
Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr Leu Pro Pro Val Val 130 135
140 ata ggt ccg ttt ttt gat ggc agc att cga tgg ctg gtt ttg ctc att
783 Ile Gly Pro Phe Phe Asp Gly Ser Ile Arg Trp Leu Val Leu Leu Ile
145 150 155 tct atg gct gtc tgc ata att gct atg atc atc ttc tcc agc
tgc ttt 831 Ser Met Ala Val Cys Ile Ile Ala Met Ile Ile Phe Ser Ser
Cys Phe 160 165 170 tgt tac aaa cat tat tgc aag agc atc tca agc aga
cgt cgt tac aat 879 Cys Tyr Lys His Tyr Cys Lys Ser Ile Ser Ser Arg
Arg Arg Tyr Asn 175 180 185 190 cgt gat ttg gaa cag gat gaa gca ttt
att cca gtt gga gaa tca cta 927 Arg Asp Leu Glu Gln Asp Glu Ala Phe
Ile Pro Val Gly Glu Ser Leu 195 200 205 aaa gac ctt att gac cag tca
caa agt tct ggt agt ggg tct gga cta 975 Lys Asp Leu Ile Asp Gln Ser
Gln Ser Ser Gly Ser Gly Ser Gly Leu 210 215 220 cct tta ttg gtt cag
cga act att gcc aaa cag att cag atg gtc cgg 1023 Pro Leu Leu Val
Gln Arg Thr Ile Ala Lys Gln Ile Gln Met Val Arg 225 230 235 caa gtt
ggt aaa ggc cga tat gga gaa gta tgg atg ggc aaa tgg cgt 1071 Gln
Val Gly Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 240 245
250 ggc gaa aaa gtg gcg gtg aaa gta ttc ttt acc act gaa gaa gcc agc
1119 Gly Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala
Ser 255 260 265 270 tgg ttt cga gaa aca gaa atc tac caa act gtg cta
atg cgc cat gaa 1167 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val
Leu Met Arg His Glu 275 280 285 aac ata ctt ggt ttc ata gcg gca gac
att aaa ggt aca ggt tcc tgg 1215 Asn Ile Leu Gly Phe Ile Ala Ala
Asp Ile Lys Gly Thr Gly Ser Trp 290 295 300 act cag ctc tat ttg att
act gat tac cat gaa aat gga tct ctc tat 1263 Thr Gln Leu Tyr Leu
Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr 305 310 315 gac ttc ctg
aaa tgt gct aca ctg gac acc aga gcc ctg ctt aaa ttg 1311 Asp Phe
Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys Leu 320 325 330
gct tat tca gct gcc tgt ggt ctg tgc cac ctg cac aca gaa att tat
1359 Ala Tyr Ser Ala Ala Cys Gly Leu Cys His Leu His Thr Glu Ile
Tyr 335 340 345 350 ggc acc caa gga aag ccc gca att gct cat cga gac
cta aag agc aaa 1407 Gly Thr Gln Gly Lys Pro Ala Ile Ala His Arg
Asp Leu Lys Ser Lys 355 360 365 aac atc ctc atc aag aaa aat ggg agt
tgc tgc att gct gac ctg ggc 1455 Asn Ile Leu Ile Lys Lys Asn Gly
Ser Cys Cys Ile Ala Asp Leu Gly 370 375 380 ctt gct gtt aaa ttc aac
agt gac aca aat gaa gtt gat gtg ccc ttg 1503 Leu Ala Val Lys Phe
Asn Ser Asp Thr Asn Glu Val Asp Val Pro Leu 385 390 395 aat acc agg
gtg ggc acc aaa cgc tac atg gct ccc gaa gtg ctg gac 1551 Asn Thr
Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp 400 405 410
gaa agc ctg aac aaa aac cac ttc cag ccc tac atc atg gct gac atc
1599 Glu Ser Leu Asn Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp
Ile 415 420 425 430 tac agc ttc ggc cta atc att tgg gag atg gct cgt
cgt tgt atc aca 1647 Tyr Ser Phe Gly Leu Ile Ile Trp Glu Met Ala
Arg Arg Cys Ile Thr 435 440 445 gga ggg atc gtg gaa gaa tac caa ttg
cca tat tac aac atg gta ccg 1695 Gly Gly Ile Val Glu Glu Tyr Gln
Leu Pro Tyr Tyr Asn Met Val Pro 450 455 460 agt gat ccg tca tac gaa
gat atg cgt gag gtt gtg tgt gtc aaa cgt 1743 Ser Asp Pro Ser Tyr
Glu Asp Met Arg Glu Val Val Cys Val Lys Arg 465 470 475 ttg cgg cca
att gtg tct aat cgg tgg aac agt gat gaa tgt cta cga 1791 Leu Arg
Pro Ile Val Ser Asn Arg Trp Asn Ser Asp Glu Cys Leu Arg 480 485 490
gca gtt ttg aag cta atg tca gaa tgc tgg gcc cac aat cca gcc tcc
1839 Ala Val Leu Lys Leu Met Ser Glu Cys Trp Ala His Asn Pro Ala
Ser 495 500 505 510 aga ctc aca gca ttg aga att aag aag acg ctt gcc
aag atg gtt gaa 1887 Arg Leu Thr Ala Leu Arg Ile Lys Lys Thr Leu
Ala Lys Met Val Glu 515 520 525 tcc caa gat gta aaa atc tga
tggttaaacc atcggaggag aaactctaga 1938 Ser Gln Asp Val Lys Ile 530
ctgcaagaac tgtttttacc catggcatgg gtggaattag agtggaataa ggatgttaac
1998 ttggttctca gactctttct tcactacgtg ttcacaggct gctaatatta
aacctttcag 2058 tactcttatt aggatacaag ctgggaactt ctaaacactt
cattctttat atatggacag 2118 ctttatttta aatgtggttt ttgatgcctt
tttttaagtg ggtttttatg aactgcatca 2178 agacttcaat cctgattagt
gtctccagtc aagctctggg tactgaattg cctgttcata 2238 aaacggtgct
ttctgtgaaa gccttaagaa gataaatgag cgcagcagag atggagaaat 2298
agactttgcc ttttacctga gacattcagt tcgtttgtat tctacctttg taaaacagcc
2358 tatagatgat gatgtgtttg ggatactgct tattttatga tagtttgtcc
tgtgtcctta 2418 gtgatgtgtg tgtgtctcca tgcacatgca cgccgggatt
cctctgctgc catttgaatt 2478 agaagaaaat aatttatatg catgcacagg
aagatattgg tggccggtgg ttttgtgctt 2538 taaaaatgca atatctgacc
aagattcgcc aatctcatac aagccattta ctttgcaagt 2598 gagatagctt
ccccaccagc tttatttttt aacatgaaag ctgatgccaa ggccaaaaga 2658
agtttaaagc atctgtaaat ttggactgtt ttccttcaac caccattttt tttgtggtta
2718 ttatttttgt cacggaaagc atcctctcca aagttggagc ttctattgcc
atgaaccatg 2778 cttacaaaga aagcacttct tattgaagtg aattcctgca
tttgatagca atgtaagtgc 2838 ctataaccat gttctatatt ctttattctc
agtaactttt aaaagggaag ttatttatat 2898 tttgtgtata atgtgcttta
tttgcaaatc accc 2932 6 532 PRT Homo sapiens 6 Met Thr Gln Leu Tyr
Ile Tyr Ile Arg Leu Leu Gly Ala Tyr Leu Phe 1 5 10 15 Ile Ile Ser
Arg Val Gln Gly Gln Asn Leu Asp Ser Met Leu His Gly 20 25 30 Thr
Gly Met Lys Ser Asp Ser Asp Gln Lys Lys Ser Glu Asn Gly Val 35 40
45 Thr Leu Ala Pro Glu Asp Thr Leu Pro Phe Leu Lys Cys Tyr Cys Ser
50 55 60 Gly His Cys Pro Asp Asp Ala Ile Asn Asn Thr Cys Ile Thr
Asn Gly 65 70 75 80 His Cys Phe Ala Ile Ile Glu Glu Asp Asp Gln Gly
Glu Thr Thr Leu 85 90 95 Ala Ser Gly Cys Met Lys Tyr Glu Gly Ser
Asp Phe Gln Cys Lys Asp 100 105 110 Ser Pro Lys Ala Gln Leu Arg Arg
Thr Ile Glu Cys Cys Arg Thr Asn 115 120 125 Leu Cys Asn Gln Tyr Leu
Gln Pro Thr Leu Pro Pro Val Val Ile Gly 130 135 140 Pro Phe Phe Asp
Gly Ser Ile Arg Trp Leu Val Leu Leu Ile Ser Met 145 150 155 160 Ala
Val Cys Ile Ile Ala Met Ile Ile Phe Ser Ser Cys Phe Cys Tyr 165 170
175 Lys His Tyr Cys Lys Ser Ile Ser Ser Arg Arg Arg Tyr Asn Arg Asp
180 185 190 Leu Glu Gln Asp Glu Ala Phe Ile Pro Val Gly Glu Ser Leu
Lys Asp 195 200 205 Leu Ile Asp Gln Ser Gln Ser Ser Gly Ser Gly Ser
Gly Leu Pro Leu 210 215 220 Leu Val Gln Arg Thr Ile Ala Lys Gln Ile
Gln Met Val Arg Gln Val 225 230 235 240 Gly Lys Gly Arg Tyr Gly Glu
Val Trp Met Gly Lys Trp Arg Gly Glu 245 250 255 Lys Val Ala Val Lys
Val Phe Phe Thr Thr Glu Glu Ala Ser Trp Phe 260 265 270 Arg Glu Thr
Glu Ile Tyr Gln Thr Val Leu Met Arg His Glu Asn Ile 275 280 285 Leu
Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly Ser Trp Thr Gln 290 295
300 Leu Tyr Leu Ile Thr Asp Tyr His Glu Asn Gly Ser Leu Tyr Asp Phe
305 310 315 320 Leu Lys Cys Ala Thr Leu Asp Thr Arg Ala Leu Leu Lys
Leu Ala Tyr 325 330 335 Ser Ala Ala Cys Gly Leu Cys His Leu His Thr
Glu Ile Tyr Gly Thr 340 345 350 Gln Gly Lys Pro Ala Ile Ala His Arg
Asp Leu Lys Ser Lys Asn Ile 355 360 365 Leu Ile Lys Lys Asn Gly Ser
Cys Cys Ile Ala Asp Leu Gly Leu Ala 370 375 380 Val Lys Phe Asn Ser
Asp Thr Asn Glu Val Asp Val Pro Leu Asn Thr 385 390 395 400 Arg Val
Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu Asp Glu Ser 405 410 415
Leu Asn Lys Asn His Phe Gln Pro Tyr Ile Met Ala Asp Ile Tyr Ser 420
425 430 Phe Gly Leu Ile Ile Trp Glu Met Ala Arg Arg Cys Ile Thr Gly
Gly 435 440 445 Ile Val Glu Glu Tyr Gln Leu Pro Tyr Tyr Asn Met Val
Pro Ser Asp 450 455 460 Pro Ser Tyr Glu Asp Met Arg Glu Val Val Cys
Val Lys Arg Leu Arg 465 470 475 480 Pro Ile Val Ser Asn Arg Trp Asn
Ser Asp Glu Cys Leu Arg Ala Val 485 490 495 Leu Lys Leu Met Ser Glu
Cys Trp Ala His Asn Pro Ala Ser Arg Leu 500 505 510 Thr Ala Leu Arg
Ile Lys Lys Thr Leu Ala Lys Met Val Glu Ser Gln 515 520 525 Asp Val
Lys Ile 530 7 1952 DNA Homo sapiens CDS (187)..(1695) 7 aagcggcggc
agaagttgcc ggcgtggtgc tcgtagtgag ggcgcggagg acccgggacc 60
tgggaagcgg cggcgggtta acttcggctg aatcacaacc atttggcgct gagctatgac
120 aagagagcaa acaaaaagtt aaaggagcaa cccggccata agtgaagaga
gaagtttatt 180 gataac atg ctc tta cga agc tct gga aaa tta aat gtg
ggc acc aag 228 Met Leu Leu Arg Ser Ser Gly Lys Leu Asn Val Gly Thr
Lys 1 5 10 aag gag gat gga gag agt aca gcc ccc acc cct cgg ccc aag
atc cta 276 Lys Glu Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro Lys
Ile Leu 15 20 25 30 cgt tgt aaa tgc cac cac cac tgt ccg gaa gac tca
gtc aac aat atc 324 Arg Cys Lys Cys His His His Cys Pro Glu Asp Ser
Val Asn Asn Ile 35 40 45 tgc agc aca gat ggg tac tgc ttc acg atg
ata gaa gaa gat gac tct 372 Cys Ser Thr Asp Gly Tyr Cys Phe Thr Met
Ile Glu Glu Asp Asp Ser 50 55 60 gga atg cct gtt gtc acc tct gga
tgt cta gga cta gaa ggg tca gat 420 Gly Met Pro Val Val Thr Ser Gly
Cys Leu Gly Leu Glu Gly Ser Asp 65 70 75 ttt caa tgt cgt gac act
ccc att cct cat caa aga aga tca att gaa 468 Phe Gln Cys Arg Asp Thr
Pro Ile Pro His Gln Arg Arg Ser Ile Glu 80 85 90 tgc tgc aca gaa
agg aat gag tgt aat aaa gac ctc cac ccc act ctg 516 Cys Cys Thr Glu
Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu 95 100 105 110 cct
cct ctc aag gac aga gat ttt gtt gat ggg ccc ata cac cac aag 564 Pro
Pro Leu Lys Asp Arg Asp Phe Val Asp Gly Pro Ile His His Lys 115 120
125 gcc ttg ctt atc tct gtg act gtc tgt agt tta ctc ttg gtc ctc att
612 Ala Leu Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile
130 135 140 att tta ttc tgt tac ttc agg tat aaa aga caa gaa gcc cga
cct cgg 660 Ile Leu Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Ala Arg
Pro Arg 145 150 155 tac agc att ggg ctg gag cag gac gag aca tac att
cct cct gga gag 708 Tyr Ser Ile Gly Leu Glu Gln Asp Glu Thr Tyr Ile
Pro Pro Gly Glu 160 165 170 tcc ctg aga gac ttg atc gag cag tct cag
agc tcg gga agt gga tca 756 Ser Leu Arg Asp Leu Ile Glu Gln Ser Gln
Ser Ser Gly Ser Gly Ser 175 180 185 190 ggc ctc cct ctg ctg gtc caa
agg aca ata gct aag caa att cag atg 804 Gly Leu Pro Leu Leu Val Gln
Arg Thr Ile Ala Lys Gln Ile Gln Met 195 200 205 gtg aag cag att gga
aaa ggc cgc tat ggc gag gtg tgg atg gga aag 852 Val Lys Gln Ile Gly
Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys 210 215 220 tgg cgt gga
gaa aag gtg gct gtg aaa gtg ttc ttc acc acg gag gaa 900 Trp Arg
Gly
Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu 225 230 235 gcc
agc tgg ttc cga gag act gag ata tat cag acg gtc ctg atg cgg 948 Ala
Ser Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg 240 245
250 cat gag aat att ctg ggg ttc att gct gca gat atc aaa ggg act ggg
996 His Glu Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr Gly
255 260 265 270 tcc tgg act cag ttg tac ctc atc aca gac tat cat gaa
aac ggc tcc 1044 Ser Trp Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His
Glu Asn Gly Ser 275 280 285 ctt tat gac tat ctg aaa tcc acc acc tta
gac gca aag tcc atg ctg 1092 Leu Tyr Asp Tyr Leu Lys Ser Thr Thr
Leu Asp Ala Lys Ser Met Leu 290 295 300 aag cta gcc tac tcc tct gtc
agc ggc cta tgc cat tta cac acg gaa 1140 Lys Leu Ala Tyr Ser Ser
Val Ser Gly Leu Cys His Leu His Thr Glu 305 310 315 atc ttt agc act
caa ggc aag cca gca atc gcc cat cga gac ttg aaa 1188 Ile Phe Ser
Thr Gln Gly Lys Pro Ala Ile Ala His Arg Asp Leu Lys 320 325 330 agt
aaa aac atc ctg gtg aag aaa aat gga act tgc tgc ata gca gac 1236
Ser Lys Asn Ile Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp 335
340 345 350 ctg ggc ttg gct gtc aag ttc att agt gac aca aat gag gtt
gac atc 1284 Leu Gly Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu
Val Asp Ile 355 360 365 cca ccc aac acc cgg gtt ggc acc aag cgc tat
atg cct cca gaa gtg 1332 Pro Pro Asn Thr Arg Val Gly Thr Lys Arg
Tyr Met Pro Pro Glu Val 370 375 380 ctg gac gag agc ttg aat aga aac
cat ttc cag tcc tac att atg gct 1380 Leu Asp Glu Ser Leu Asn Arg
Asn His Phe Gln Ser Tyr Ile Met Ala 385 390 395 gac atg tac agc ttt
gga ctc atc ctc tgg gag att gca agg aga tgt 1428 Asp Met Tyr Ser
Phe Gly Leu Ile Leu Trp Glu Ile Ala Arg Arg Cys 400 405 410 gtt tct
gga ggt ata gtg gaa gaa tac cag ctt ccc tat cac gac ctg 1476 Val
Ser Gly Gly Ile Val Glu Glu Tyr Gln Leu Pro Tyr His Asp Leu 415 420
425 430 gtg ccc agt gac cct tct tat gag gac atg aga gaa att gtg tgc
atg 1524 Val Pro Ser Asp Pro Ser Tyr Glu Asp Met Arg Glu Ile Val
Cys Met 435 440 445 aag aag tta cgg cct tca ttc ccc aat cga tgg agc
agt gat gag tgt 1572 Lys Lys Leu Arg Pro Ser Phe Pro Asn Arg Trp
Ser Ser Asp Glu Cys 450 455 460 ctc agg cag atg ggg aag ctt atg aca
gag tgc tgg gcg cag aat cct 1620 Leu Arg Gln Met Gly Lys Leu Met
Thr Glu Cys Trp Ala Gln Asn Pro 465 470 475 gcc tcc agg ctg acg gcc
ctg aga gtt aag aaa acc ctt gcc aaa atg 1668 Ala Ser Arg Leu Thr
Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met 480 485 490 tca gag tcc
cag gac att aaa ctc tga cgtcagatac ttgtggacag 1715 Ser Glu Ser Gln
Asp Ile Lys Leu 495 500 agcaagaatt tcacagaagc atcgttagcc caagccttga
acgttagcct actgcccagt 1775 gagttcagac tttcctggaa gagagcacgg
tgggcagaca cagaggaacc cagaaacacg 1835 gattcatcat ggctttctga
ggaggagaaa ctgtttgggt aacttgttca agatatgatg 1895 catgttgctt
tctaagaaag ccctgtattt tgaattacca tttttttata aaaaaaa 1952 8 502 PRT
Homo sapiens 8 Met Leu Leu Arg Ser Ser Gly Lys Leu Asn Val Gly Thr
Lys Lys Glu 1 5 10 15 Asp Gly Glu Ser Thr Ala Pro Thr Pro Arg Pro
Lys Ile Leu Arg Cys 20 25 30 Lys Cys His His His Cys Pro Glu Asp
Ser Val Asn Asn Ile Cys Ser 35 40 45 Thr Asp Gly Tyr Cys Phe Thr
Met Ile Glu Glu Asp Asp Ser Gly Met 50 55 60 Pro Val Val Thr Ser
Gly Cys Leu Gly Leu Glu Gly Ser Asp Phe Gln 65 70 75 80 Cys Arg Asp
Thr Pro Ile Pro His Gln Arg Arg Ser Ile Glu Cys Cys 85 90 95 Thr
Glu Arg Asn Glu Cys Asn Lys Asp Leu His Pro Thr Leu Pro Pro 100 105
110 Leu Lys Asp Arg Asp Phe Val Asp Gly Pro Ile His His Lys Ala Leu
115 120 125 Leu Ile Ser Val Thr Val Cys Ser Leu Leu Leu Val Leu Ile
Ile Leu 130 135 140 Phe Cys Tyr Phe Arg Tyr Lys Arg Gln Glu Ala Arg
Pro Arg Tyr Ser 145 150 155 160 Ile Gly Leu Glu Gln Asp Glu Thr Tyr
Ile Pro Pro Gly Glu Ser Leu 165 170 175 Arg Asp Leu Ile Glu Gln Ser
Gln Ser Ser Gly Ser Gly Ser Gly Leu 180 185 190 Pro Leu Leu Val Gln
Arg Thr Ile Ala Lys Gln Ile Gln Met Val Lys 195 200 205 Gln Ile Gly
Lys Gly Arg Tyr Gly Glu Val Trp Met Gly Lys Trp Arg 210 215 220 Gly
Glu Lys Val Ala Val Lys Val Phe Phe Thr Thr Glu Glu Ala Ser 225 230
235 240 Trp Phe Arg Glu Thr Glu Ile Tyr Gln Thr Val Leu Met Arg His
Glu 245 250 255 Asn Ile Leu Gly Phe Ile Ala Ala Asp Ile Lys Gly Thr
Gly Ser Trp 260 265 270 Thr Gln Leu Tyr Leu Ile Thr Asp Tyr His Glu
Asn Gly Ser Leu Tyr 275 280 285 Asp Tyr Leu Lys Ser Thr Thr Leu Asp
Ala Lys Ser Met Leu Lys Leu 290 295 300 Ala Tyr Ser Ser Val Ser Gly
Leu Cys His Leu His Thr Glu Ile Phe 305 310 315 320 Ser Thr Gln Gly
Lys Pro Ala Ile Ala His Arg Asp Leu Lys Ser Lys 325 330 335 Asn Ile
Leu Val Lys Lys Asn Gly Thr Cys Cys Ile Ala Asp Leu Gly 340 345 350
Leu Ala Val Lys Phe Ile Ser Asp Thr Asn Glu Val Asp Ile Pro Pro 355
360 365 Asn Thr Arg Val Gly Thr Lys Arg Tyr Met Pro Pro Glu Val Leu
Asp 370 375 380 Glu Ser Leu Asn Arg Asn His Phe Gln Ser Tyr Ile Met
Ala Asp Met 385 390 395 400 Tyr Ser Phe Gly Leu Ile Leu Trp Glu Ile
Ala Arg Arg Cys Val Ser 405 410 415 Gly Gly Ile Val Glu Glu Tyr Gln
Leu Pro Tyr His Asp Leu Val Pro 420 425 430 Ser Asp Pro Ser Tyr Glu
Asp Met Arg Glu Ile Val Cys Met Lys Lys 435 440 445 Leu Arg Pro Ser
Phe Pro Asn Arg Trp Ser Ser Asp Glu Cys Leu Arg 450 455 460 Gln Met
Gly Lys Leu Met Thr Glu Cys Trp Ala Gln Asn Pro Ala Ser 465 470 475
480 Arg Leu Thr Ala Leu Arg Val Lys Lys Thr Leu Ala Lys Met Ser Glu
485 490 495 Ser Gln Asp Ile Lys Leu 500 9 1822 DNA Homo sapiens CDS
(49)..(1341) 9 ggtgcgggcc cggagcccgg agcccgggta gcgcgtagag ccggcgcg
atg cac gtg 57 Met His Val 1 cgc tca ctg cga gct gcg gcg ccg cac
agc ttc gtg gcg ctc tgg gca 105 Arg Ser Leu Arg Ala Ala Ala Pro His
Ser Phe Val Ala Leu Trp Ala 5 10 15 ccc ctg ttc ctg ctg cgc tcc gcc
ctg gcc gac ttc agc ctg gac aac 153 Pro Leu Phe Leu Leu Arg Ser Ala
Leu Ala Asp Phe Ser Leu Asp Asn 20 25 30 35 gag gtg cac tcg agc ttc
atc cac cgg cgc ctc cgc agc cag gag cgg 201 Glu Val His Ser Ser Phe
Ile His Arg Arg Leu Arg Ser Gln Glu Arg 40 45 50 cgg gag atg cag
cgc gag atc ctc tcc att ttg ggc ttg ccc cac cgc 249 Arg Glu Met Gln
Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg 55 60 65 ccg cgc
ccg cac ctc cag ggc aag cac aac tcg gca ccc atg ttc atg 297 Pro Arg
Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met 70 75 80
ctg gac ctg tac aac gcc atg gcg gtg gag gag ggc ggc ggg ccc ggc 345
Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 85
90 95 ggc cag ggc ttc tcc tac ccc tac aag gcc gtc ttc agt acc cag
ggc 393 Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln
Gly 100 105 110 115 ccc cct ctg gcc agc ctg caa gat agc cat ttc ctc
acc gac gcc gac 441 Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu
Thr Asp Ala Asp 120 125 130 atg gtc atg agc ttc gtc aac ctc gtg gaa
cat gac aag gaa ttc ttc 489 Met Val Met Ser Phe Val Asn Leu Val Glu
His Asp Lys Glu Phe Phe 135 140 145 cac cca cgc tac cac cat cga gag
ttc cgg ttt gat ctt tcc aag atc 537 His Pro Arg Tyr His His Arg Glu
Phe Arg Phe Asp Leu Ser Lys Ile 150 155 160 cca gaa ggg gaa gct gtc
acg gca gcc gaa ttc cgg atc tac aag gac 585 Pro Glu Gly Glu Ala Val
Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp 165 170 175 tac atc cgg gaa
cgc ttc gac aat gag acg ttc cgg atc agc gtt tat 633 Tyr Ile Arg Glu
Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr 180 185 190 195 cag
gtg ctc cag gag cac ttg ggc agg gaa tcg gat ctc ttc ctg ctc 681 Gln
Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 200 205
210 gac agc cgt acc ctc tgg gcc tcg gag gag ggc tgg ctg gtg ttt gac
729 Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp
215 220 225 atc aca gcc acc agc aac cac tgg gtg gtc aat ccg cgg cac
aac ctg 777 Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His
Asn Leu 230 235 240 ggc ctg cag ctc tcg gtg gag acg ctg gat ggg cag
agc atc aac ccc 825 Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln
Ser Ile Asn Pro 245 250 255 aag ttg gcg ggc ctg att ggg cgg cac ggg
ccc cag aac aag cag ccc 873 Lys Leu Ala Gly Leu Ile Gly Arg His Gly
Pro Gln Asn Lys Gln Pro 260 265 270 275 ttc atg gtg gct ttc ttc aag
gcc acg gag gtc cac ttc cgc agc atc 921 Phe Met Val Ala Phe Phe Lys
Ala Thr Glu Val His Phe Arg Ser Ile 280 285 290 cgg tcc acg ggg agc
aaa cag cgc agc cag aac cgc tcc aag acg ccc 969 Arg Ser Thr Gly Ser
Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro 295 300 305 aag aac cag
gaa gcc ctg cgg atg gcc aac gtg gca gag aac agc agc 1017 Lys Asn
Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 310 315 320
agc gac cag agg cag gcc tgt aag aag cac gag ctg tat gtc agc ttc
1065 Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser
Phe 325 330 335 cga gac ctg ggc tgg cag gac tgg atc atc gcg cct gaa
ggc tac gcc 1113 Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro
Glu Gly Tyr Ala 340 345 350 355 gcc tac tac tgt gag ggg gag tgt gcc
ttc cct ctg aac tcc tac atg 1161 Ala Tyr Tyr Cys Glu Gly Glu Cys
Ala Phe Pro Leu Asn Ser Tyr Met 360 365 370 aac gcc acc aac cac gcc
atc gtg cag acg ctg gtc cac ttc atc aac 1209 Asn Ala Thr Asn His
Ala Ile Val Gln Thr Leu Val His Phe Ile Asn 375 380 385 ccg gaa acg
gtg ccc aag ccc tgc tgt gcg ccc acg cag ctc aat gcc 1257 Pro Glu
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala 390 395 400
atc tcc gtc ctc tac ttc gat gac agc tcc aac gtc atc ctg aag aaa
1305 Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys
Lys 405 410 415 tac aga aac atg gtg gtc cgg gcc tgt ggc tgc cac
tagctcctcc 1351 Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420
425 430 gagaattcag accctttggg gccaagtttt tctggatcct ccattgctcg
ccttggccag 1411 gaaccagcag accaactgcc ttttgtgaga ccttcccctc
cctatcccca actttaaagg 1471 tgtgagagta ttaggaaaca tgagcagcat
atggcttttg atcagttttt cagtggcagc 1531 atccaatgaa caagatccta
caagctgtgc aggcaaaacc tagcaggaaa aaaaaacaac 1591 gcataaagaa
aaatggccgg gccaggtcat tggctgggaa gtctcagcca tgcacggact 1651
cgtttccaga ggtaattatg agcgcctacc agccaggcca cccagccgtg ggaggaaggg
1711 ggcgtggcaa ggggtgggca cattggtgtc tgtgcgaaag gaaaattgac
ccggaagttc 1771 ctgtaataaa tgtcacaata aaacgaatga atgaaaaaaa
aaaaaaaaaa a 1822 10 431 PRT Homo sapiens 10 Met His Val Arg Ser
Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 15 Leu Trp Ala
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 30 Leu
Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 35 40
45 Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu
50 55 60 Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser
Ala Pro 65 70 75 80 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val
Glu Glu Gly Gly 85 90 95 Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro
Tyr Lys Ala Val Phe Ser 100 105 110 Thr Gln Gly Pro Pro Leu Ala Ser
Leu Gln Asp Ser His Phe Leu Thr 115 120 125 Asp Ala Asp Met Val Met
Ser Phe Val Asn Leu Val Glu His Asp Lys 130 135 140 Glu Phe Phe His
Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 145 150 155 160 Ser
Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile 165 170
175 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile
180 185 190 Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser
Asp Leu 195 200 205 Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu
Glu Gly Trp Leu 210 215 220 Val Phe Asp Ile Thr Ala Thr Ser Asn His
Trp Val Val Asn Pro Arg 225 230 235 240 His Asn Leu Gly Leu Gln Leu
Ser Val Glu Thr Leu Asp Gly Gln Ser 245 250 255 Ile Asn Pro Lys Leu
Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn 260 265 270 Lys Gln Pro
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 275 280 285 Arg
Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser 290 295
300 Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu
305 310 315 320 Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His
Glu Leu Tyr 325 330 335 Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp
Ile Ile Ala Pro Glu 340 345 350 Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly
Glu Cys Ala Phe Pro Leu Asn 355 360 365 Ser Tyr Met Asn Ala Thr Asn
His Ala Ile Val Gln Thr Leu Val His 370 375 380 Phe Ile Asn Pro Glu
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385 390 395 400 Leu Asn
Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415
Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425
430 11 102 PRT Artificial Sequence Description of Artificial
Sequence Generic sequence OPX 11 Cys Xaa Xaa His Glu Leu Tyr Val
Xaa Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 15 Asp Trp Xaa Ile Ala Pro
Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 20 25 30 Glu Cys Xaa Phe
Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 35 40 45 Ile Xaa
Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 50 55 60
Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 65
70 75 80 Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met
Val Val 85 90 95 Xaa Ala Cys Gly Cys His 100 12 28 DNA Artificial
Sequence Primer 12 gcggatcctg ttgtgaaggn aatatgtg 28 13 24 DNA
Artificial Sequence Primer 13 gcgatccgtc gcagtcaaaa tttt 24 14 26
DNA Artificial Sequence Primer 14 gcggatccgc gatatattaa aagcaa 26
15 20 DNA Artificial Sequence Primer 15 cggaattctg gtgccatata
20
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