U.S. patent application number 11/051267 was filed with the patent office on 2005-11-17 for transgenic non-human animals expressing a truncated activin type ii receptor.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Lee, Se-Jin, McPherron, Alexandra C..
Application Number | 20050257278 11/051267 |
Document ID | / |
Family ID | 25285564 |
Filed Date | 2005-11-17 |
United States Patent
Application |
20050257278 |
Kind Code |
A1 |
Lee, Se-Jin ; et
al. |
November 17, 2005 |
Transgenic non-human animals expressing a truncated activin type II
receptor
Abstract
The present invention provides a substantially purified growth
differentiation factor (GDF) receptor, including a GDF-8
(myostatin) receptor, as well as functional peptide portions
thereof. In addition, the invention provides a virtual
representation of a GDF receptor or a functional peptide portion
thereof. The present invention also provides a method of modulating
an effect of myostatin on a cell by contacting the cell with an
agent that affects myostatin signal transduction in the cell. In
addition, the invention provides a method of ameliorating the
severity of a pathologic condition, which is characterized, at
least in part, by an abnormal amount, development or metabolic
activity of muscle or adipose tissue in a subject, by modulating
myostatin signal transduction in a muscle cell or an adipose tissue
cell in the subject. The invention also provides a method of
modulating the growth of muscle tissue or adipose tissue in a
eukaryotic organism by administering an agent that affects
myostatin signal transduction to the organism.
Inventors: |
Lee, Se-Jin; (Baltimore,
MD) ; McPherron, Alexandra C.; (Baltimore,
MD) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
DLA PIPER RUDNICK GRAY CARY US LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
|
Family ID: |
25285564 |
Appl. No.: |
11/051267 |
Filed: |
February 3, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11051267 |
Feb 3, 2005 |
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09841730 |
Apr 24, 2001 |
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6891082 |
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09841730 |
Apr 24, 2001 |
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09626896 |
Jul 27, 2000 |
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6656475 |
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09626896 |
Jul 27, 2000 |
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09485046 |
May 5, 2000 |
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6696260 |
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09485046 |
May 5, 2000 |
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PCT/US98/15598 |
Jul 28, 1998 |
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60054461 |
Aug 1, 1997 |
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Current U.S.
Class: |
800/14 |
Current CPC
Class: |
A01K 67/0276 20130101;
A01K 2217/20 20130101; A61P 43/00 20180101; C07K 14/4703 20130101;
G01N 2500/00 20130101; A01K 2217/00 20130101; A01K 2267/0362
20130101; G01N 2333/495 20130101; A61P 3/10 20180101; C07K 14/71
20130101; A61P 1/14 20180101; A61P 21/04 20180101; C07K 14/475
20130101; C12N 2830/008 20130101; C12N 2830/85 20130101; C12N
2830/002 20130101; A01K 2227/30 20130101; A61P 21/06 20180101; A01K
2217/05 20130101; A61P 25/28 20180101; A61P 35/00 20180101; A01K
2267/0306 20130101; A61P 21/00 20180101; G01N 33/74 20130101; A01K
2227/105 20130101; A61K 38/00 20130101; A61K 38/1709 20130101; A01K
67/0275 20130101; A01K 2217/075 20130101; A01K 2267/03 20130101;
A61P 3/04 20180101; A61P 3/06 20180101; C12N 2800/30 20130101; A01K
67/0271 20130101; A01K 67/0278 20130101; A61P 9/12 20180101; C12N
15/8509 20130101; A01K 2207/15 20130101; A01K 2267/02 20130101 |
Class at
Publication: |
800/014 |
International
Class: |
A01K 067/027 |
Goverment Interests
[0002] This invention was made in part with government support
under Grant No. RO1HD35887 awarded by the National Institutes of
Health. The government has certain rights in this invention.
Claims
1-5. (canceled)
6. A non-human transgenic animal whose genome contains a nucleic
acid sequence comprising a myostatin prodomain and a
muscle-specific promoter operably linked and integrated into the
genome of the animal, wherein the nucleic acid sequence is
expressed so as to result in elevated levels of myostatin prodomain
and increased muscle mass in the animal as compared to a
corresponding corresponding nontransgenic animal.
7. The transgenic animal of claim 6, wherein the myostatin
prodomain comprises about amino acid residues 1 to 262 of a
promyostatin polypeptide selected from the group consisting of SEQ
ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, or a functional
peptide portion of said myostatin prodomain.
8. The transgenic animal of claim 6, wherein the myostatin
prodomain comprises an amino acid sequence selected from the group
consisting of: amino acid residues about 20 to 263 as set forth in
SEQ ID NO: 4; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 2; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 10; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 12; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 8; amino acid residues about 20 to 263 as set forth in
SEQ ID NO: 6; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 18; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 14; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 16; amino acid residues about 20 to 262 as set forth in
SEQ ID NO: 20; and a functional peptide portion thereof.
9. The transgenic animal of claim 6, wherein the myostatin
prodomain further comprises a myostatin signal peptide.
10. The transgenic animal of claim 6, wherein the muscle-specific
promoter is a myosin light chain promoter/enhancer.
11. A non-human transgenic animal whose genome contains a nucleic
acid sequence comprising a follistatin gene and a muscle-specific
promoter operably linked and integrated into the genome of the
animal, wherein the nucleic acid sequence is expressed so as to
result in elevated levels of follistatin and increased muscle mass
in the animal as compared to a corresponding corresponding
nontransgenic animal.
12-14. (canceled)
15. An expression cassette comprising a DNA segment encoding a
myostatin prodomain gene operably linked to a muscle-specific
control sequence.
16. The expression cassette of claim 15 wherein the muscle-specific
promoter is a myosin light chain promoter/enhancer.
17-19. (canceled)
20. A cell or cell line isolated from the animal of claim 1,
wherein said cell expresses truncated Activin Type II receptor,
myostatin prodomain, or follistatin, respectively.
21-39. (canceled)
40. A method of producing a chimeric non-human animal, the method
comprising: obtaining an ovum from animal ovaries; maturing the
ovum in vitro; fertilizing the mature ovum in vitro to form a
zygote; introducing into the zygote in vitro a nucleic acid
construct comprising in operable association a DNA sequence
encoding a truncated Activin Type II receptor, a myostatin
propeptide, or follistatin, and a regulatory sequence that promotes
expression of the DNA sequence encoding the polypeptide; maturing
the zygote to a preimplantation stage embryo in vitro; and
transplanting the embryo into a recipient female animal, wherein
the female animal gestates the embryo to produce a chimeric
animal.
41-42. (canceled)
43. A cell or cell line isolated from the animal of claim 6,
wherein said cell expresses truncated Activin Type II receptor,
myostatin prodomain, or follistatin, respectively.
44. A cell or cell line isolated from the animal of claim 11,
wherein said cell expresses truncated Activin Type II receptor,
myostatin prodomain, or follistatin, respectively.
Description
RELATED APPLICATION DATA
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/626,896, filed Jul. 27, 2000; which is a continuation-in-part of
U.S. Ser. No. 09/485,046, filed Jan. 31, 2000; which is based on
International Application PCT/US98/15598, filed Jul. 28, 1998;
which claims the benefit of U.S. Provisional Application Ser. No.
60/054,461, filed Aug. 1, 1997 (now abandoned), the entire contents
of each of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates generally to growth differentiation
factor (GDF) receptors, and more specifically to GDF-8 (myostatin)
receptors, to compositions that affect myostatin signal
transduction in a cell, and to methods of using such compositions
to modulate myostatin signal transduction in a cell.
BACKGROUND
[0004] The amount of time, effort and money spent in the United
States each year by individuals intent on losing weight is
staggering. For many of these individuals, the goal is not merely
to look better, but more importantly to avoid the inevitable
medical problems associated with being overweight.
[0005] Greater than half of the adult population in the United
States is considered to be overweight. Furthermore, twenty to
thirty percent of adult men and thirty to forty percent of adult
women in the United States are considered obese, with the highest
rates occurring among the poor and minorities. Obesity, which is
defined a being at least about twenty percent above the mean level
of adiposity, has dramatically increased in prevalence over the
past few decades and is becoming a major problem among the
pediatric population. Twenty percent of all children are now
considered overweight, a number that represents a doubling over the
past five years.
[0006] Obesity and the medical problems directly attributable to it
are a major cause of morbidity and mortality throughout the world.
Obesity is a major risk factor for the development of various
pathologic conditions, including atherosclerosis, hypertension,
heart attack, type II diabetes, gallbladder disease, and certain
cancers, and contributes to premature death. Heart disease is the
leading cause of mortality in the United States, and type II
diabetes afflicts over 16 million people in the United States and
is one of the leading causes of death by disease.
[0007] More than eighty percent of type II diabetes occurs in obese
persons. Although type II diabetes affects all races, it is
particularly prevalent among Native Americans, African Americans
and Hispanics. Significantly, type II diabetes, which used to occur
almost exclusively in adults over age forty, now occurs in
children, with reported cases having almost tripled over the last
five years. Type II diabetes, also called non-insulin dependent
diabetes, is characterized by reduced secretion of insulin in
response to glucose and by resistance of the body to the action of
insulin, even though insulin levels in the circulation generally
are normal or elevated. Type II diabetes affects the function of a
variety of different tissues and organs and can lead to vascular
disease, renal failure, retinopathy and neuropathy.
[0008] In contrast to the medical problems associated with obesity,
the severe weight loss that commonly occurs in patients with
certain chronic diseases also presents a challenge to medical
intervention. The molecular basis for this weight loss, referred to
as cachexia, is not well understood. It is clear, however, that
cachexia complicates management of such diseases and is associated
with a poor prognosis for the patients. The effects of cachexia are
evident in the wasting syndrome that occurs in cancer and AIDS
patients.
[0009] Although great efforts have been made in attempting to
elucidate the biological processes involved in regulating body
weight, the results have provided more fanfare than actual value.
For example, the discovery of leptin has been hailed as a
breakthrough in understanding the molecular basis for fat
accumulation in humans, and, with it, the promise of a cure for
obesity. Studies in animals indicated that leptin is involved in
transmitting internal signals regulating appetite, and suggested
leptin could be useful for treating humans suffering from obesity.
Progress in using leptin for treating obesity has been slow,
however, and, thus far, leptin has not met initial
expectations.
[0010] Treatment of the morbidly obese currently is limited to
surgery to remove portions of the intestine, thereby reducing the
amount of food (and calories) absorbed. For the moderately obese,
the only "treatment" is eating a healthy diet and exercising
regularly, a method that has proved modestly successful at best.
Thus, a need exists to identify the biological factors involved in
regulating body weight, including muscle development and fat
accumulation, such that methods for treating disorders such as
obesity and cachexia can be developed. The present invention
satisfies this need and provides additional advantages.
SUMMARY OF THE INVENTION
[0011] The present invention relates to a substantially purified
GDF receptor. A GDF receptor of the invention can be, for example,
a myostatin receptor, a GDF-11 receptor, or other GDF receptor. A
myostatin receptor, for example, interacts specifically at least
with myostatin, and also can interact specifically with one or a
few additional mature GDF peptides as well. Polynucleotides
encoding a GDF receptor, antibodies that specifically interact with
a GDF receptor, and the like also are provided.
[0012] The present invention also relates to a method of modulating
an effect of a GDF by affecting signal transduction effected by the
GDF. By way of example, a method of modulating an effect of
myostatin on a cell by contacting the cell with an agent that
affects myostatin signal transduction in the cell is provided. In
one embodiment, the agent alters a specific interaction of
myostatin with a myostatin receptor expressed by the cell, thereby
modulating myostatin signal transduction in the cell. The myostatin
receptor can be an activin receptor, or can be any other receptor
that can be contacted by a mature myostatin or functional peptide
portion thereof such that myostatin signal transduction is
activated. In another embodiment, the agent binds to a myostatin
receptor, thereby enhancing myostatin binding to the receptor or
competing with myostatin for the receptor. As such, the agent can
increase myostatin signal transduction, or can reduce or inhibit
myostatin signal transduction. In still another embodiment, the
agent acts intracellularly to alter myostatin signal transduction
in the cell.
[0013] An agent useful for modulating GDF signal transduction in a
cell can be a peptide, a peptidomimetic, a polynucleotide, a small
organic molecule, or any other agent, and can act as an agonist of
GDF signal transduction or as an antagonist of GDF signal
transduction. In one embodiment, the peptide agent alters a
specific interaction of myostatin with a myostatin receptor. Such a
peptide agent can be, for example, a peptide that binds or
otherwise sequesters myostatin, thereby affecting the ability of
myostatin to interact specifically with its receptor. Such agents
are exemplified by a mutant myostatin receptor, for example, a
soluble extracellular domain of a myostatin receptor, which can
specifically interact with myostatin; by a myostatin prodomain,
which can specifically interact with myostatin; and by a mutant
myostatin polypeptide that is resistant to proteolytic cleavage
into a prodomain and mature myostatin and can interact specifically
with myostatin, and are useful as myostatin signal transduction
antagonists, which reduce or inhibit myostatin signal transduction
in a cell.
[0014] In another embodiment, the peptide agent can specifically
interact with a myostatin receptor expressed by a cell, thereby
competing with myostatin for the receptor. Such a peptide agent is
exemplified by an anti-myostatin receptor antibody or by an
anti-idiotypic antibody of an anti-myostatin antibody. Such a
peptide agent provides the additional advantage that it can be
selected not only for its ability to interact specifically with a
myostatin receptor, thereby competing with myostatin for the
receptor, but can be further selected to have an ability to not
activate or not activate myostatin signal transduction. Thus, a
peptide agent that specifically interacts with a myostatin receptor
expressed by a cell, and activates myostatin dependent signal
transduction can be used as a myostatin agonist to increase
myostatin signal transduction in the cell, whereas a peptide agent
that specifically interacts with a myostatin receptor expressed by
a cell, but does not activate myostatin signal transduction can be
used as a myostatin antagonist to reduce or inhibit myostatin
signal transduction in the cell.
[0015] An agent useful in a method of the invention also can be a
polynucleotide. Generally, but not necessarily, the polynucleotide
is introduced into the cell, where it effects its function either
directly, or following transcription or translation or both. For
example, the polynucleotide agent can encode a peptide, which is
expressed in the cell and modulates myostatin activity. Such an
expressed peptide can be, for example, a mutant myostatin receptor
such as a soluble myostatin receptor extracellular domain; a
myostatin receptor extracellular domain operatively associated with
a membrane anchoring domain; or a mutant myostatin receptor lacking
protein kinase activity.
[0016] A peptide expressed from a polynucleotide agent also can be
a peptide that affects the level or activity of an intracellular
polypeptide component of a GDF signal transduction pathway. The
intracellular polypeptide can be, for example, an Smad polypeptide
such as a dominant negative Smad, which, as disclosed herein, can
affect myostatin signal transduction in a cell. Thus, a
polynucleotide agent can encode a dominant negative Smad 2, Smad 3
or Smad 4 polypeptide, which, upon expression in the cell, reduces
or inhibits myostatin signal transduction in the cell; or can
encode a Smad 6 or Smad 7 polypeptide, which, upon expression,
decreases myostatin signal transduction in the cell. A
polynucleotide agent also can encode an intracellular c-ski
polypeptide, the expression of which can reduce or inhibit
myostatin signal transduction.
[0017] A polynucleotide agent useful in a method of the invention
also can be, or can encode, an antisense molecule, a ribozyme or a
triplexing agent. For example, the polynucleotide can be (or can
encode) an antisense nucleotide sequence such as an antisense c-ski
nucleotide sequence, which can increase myostatin signal
transduction in a cell; or an antisense Smad nucleotide sequence,
which can increase myostatin signal transduction or can reduce or
inhibit myostatin signal transduction, depending on the particular
Smad antisense nucleotide sequence.
[0018] The present invention also relates to a method of
ameliorating the severity of a pathologic condition, which is
characterized, at least in part, by an abnormal amount, development
or metabolic activity of muscle or adipose tissue in a subject.
Such a method encompasses modulating GDF signal transduction in a
cell associated with the pathologic condition, for example,
modulating myostatin signal transduction in a muscle cell or an
adipose tissue cell in the subject. Various pathologic conditions
are amenable to amelioration using a method of the invention,
including, for example, wasting disorders such as cachexia,
anorexia, muscular dystrophies, neuromuscular diseases; and
metabolic disorders such as obesity and type II diabetes.
[0019] The present invention further relates to a method of
modulating the growth of muscle tissue or adipose tissue in a
eukaryotic organism by administering to the organism an agent that
affects signal transduction mediated by a GDF receptor. In one
embodiment, a method of modulating the growth of muscle tissue or
adipose tissue is performed by administering an agent that affects
myostatin signal transduction. In another embodiment, the agent
affects GDF-11 signal transduction, or myostatin and GDF-11 signal
transduction. The agent can be, for example, an agent alters the
specific interaction of myostatin with a myostatin receptor, an
agent that reduces or inhibits the specific interaction of
myostatin with a myostatin receptor, or any other agent as
disclosed herein. The eukaryotic organism can be a vertebrate, for
example, mammalian, avian or piscine organism, or can be an
invertebrate, for example, a mollusk such as a shrimp, a scallop, a
squid, an octopus, a snail, or a slug.
[0020] The present invention also relates to a method of
identifying an agent that specifically interacts with a growth
differentiation factor (GDF) receptor. Such a screening assay of
the invention can be performed, for example, by contacting a GDF
receptor with a test agent, and determining that the test agent
specifically interacts with the GDF receptor, thereby identifying
an agent that specifically interacts with a GDF receptor. The GDF
receptor can be any GDF receptor, particularly a myostatin
receptor, and the agent can be a GDF receptor agonist, which
increases GDF signal transduction, or a GDF receptor antagonist,
which reduces or inhibits GDF signal transduction. Such a method of
the invention is useful for screening a library of test agents,
particularly a combinatorial library of test agents.
[0021] The present invention also provides a virtual representation
of a GDF receptor or a functional peptide portion of a GDF
receptor, for example, a virtual representation of GDF 8 receptor
or GDF-11 receptor. In one embodiment, the virtual representation
includes an agent that interacts specifically with the GDF
receptor. As such, the invention further provides a method of
identifying an agent that interacts specifically with a growth
differentiation factor (GDF) receptor or a functional peptide
portion of a GDF receptor by using a computer system. For example,
the method can be performed by testing a virtual test agent for the
ability to interact specifically with a virtual GDF receptor or
functional peptide portion thereof; and detecting a specific
interaction of the virtual test agent with the virtual GDF receptor
or functional peptide portion thereof, thereby identifying an agent
that interacts specifically with a GDF receptor or functional
peptide portion thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows the amino acid sequences of murine promyostatin
(SEQ ID NO: 4); rat promyostatin (SEQ ID NO: 6); human promyostatin
(SEQ ID NO: 2); baboon promyostatin (SEQ ID NO: 10); bovine
promyostatin (SEQ ID NO: 12); porcine promyostatin (SEQ ID NO: 14);
ovine promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQ ID
NO: 8), turkey promyostatin (SEQ ID NO: 18); and zebrafish
promyostatin (SEQ ID NO: 20). Amino acids are numbered relative to
the human promyostatin (SEQ ID NO: 2). Dashed lines indicate gaps
introduced to maximize homology. Identical residues among sequences
are shaded.
[0023] FIG. 2 shows the amino acid sequences of murine promyostatin
(SEQ ID NO: 4) and zebrafish promyostatin (SEQ ID NO: 20), and
portions of the amino acid sequences of salmon allele 1
promyostatin (SEQ ID NO: 27; "salmon 1") and salmon allele 2
promyostatin (SEQ ID NO: 29; "salmon 2"). Amino acid position
relative to human promyostatin is indicated to left of each row
(compare FIG. 1; first amino acid of salmon1 corresponds to human
promyostatin 218; first amino acid of salmon2 corresponds to human
promyostatin 239). Dashed lines indicate gaps introduced to
maximize homology. Relative amino acid positions, including gaps,
is indicated along top of each row. Identical residues among
sequences are shaded.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides a substantially purified
peptide portion of a promyostatin polypeptide. Promyostatin, which
previously has been referred to as growth differentiation factor-8
(GDF-8), comprises an amino terminal prodomain and a C-terminal
mature myostatin peptide (see U.S. Pat. No. 5,827,733). Myostatin
activity is effected by the mature myostatin peptide following its
cleavage from promyostatin. Thus, promyostatin is a precursor
polypeptide that is proteolytically cleaved to produce active
myostatin. As disclosed herein, the myostatin prodomain can inhibit
myostatin activity, GDF-11 activity, or both.
[0025] The present invention also provides a substantially purified
peptide portion of a pro-GDF-11 polypeptide. Pro-GDF-11, which
previously has been referred to generally as GDF-11, comprises an
amino terminal prodomain and a C-terminal mature GDF-11 peptide
(see International Publication No. WO 98/35019, which is
incorporated herein by reference). GDF-11 activity is effected by
the mature GDF-11 peptide following its cleavage from pro-GDF-11.
Thus, pro-GDF-11, like promyostatin, is a precursor polypeptide
that is proteolytically cleaved to produce active GDF-11. As
disclosed herein, the GDF-11 prodomain can inhibit GDF-11 activity,
myostatin activity, or both.
[0026] Promyostatin and pro-GDF-11 are members of the transforming
growth factor-.theta. (TGF-.theta.) superfamily, which consists of
multifunctional polypeptides that control proliferation,
differentiation, and other functions in various cell types. The
TGF-.quadrature. superfamily, which encompasses a group of
structurally-related proteins that affect a wide range of
differentiation processes during embryonic development, includes,
for example, Mullerian inhibiting substance (MIS), which is
required for normal male sex development (Behringer et al., Nature
345:167, 1990), Drosophila decapentaplegic (DPP) gene product,
which is required for dorsal-ventral axis formation and
morphogenesis of the imaginal disks (Padgett et al., Nature
325:81-84, 1987), the Xenopus Vg-1 gene product, which localizes to
the vegetal pole of eggs (Weeks et al., Cell 51:861-867, 1987), the
activins (Mason et al., Biochem. Biophys. Res. Comm. 135:957-964,
1986), which can induce the formation of mesoderm and anterior
structures in Xenopus embryos (Thomsen et al., Cell 63:485, 1990),
and the bone morphogenic proteins (BMPs, osteogenin, OP-1), which
can induce de novo cartilage and bone formation (Sampath et al., J.
Biol. Chem. 265:13198, 1990). The TGF-.theta. family members can
influence a variety of differentiation processes, including
adipogenesis, myogenesis, chondrogenesis, hematopoiesis, and
epithelial cell differentiation (Massague, Cell 49:437, 1987;
Massague, Ann. Rev. Biochem. 67:753-791, 1998; each of which is
incorporated herein by reference).
[0027] Many of the TGF-.theta. family members have regulatory
effects (positive or negative) on other peptide growth factors. In
particular, certain members of the TGF-.theta. superfamily have
expression patterns or possess activities that relate to the
function of the nervous system. For example, the inhibins and
activins are expressed in the brain (Meunier et al., Proc. Natl.
Acad. Sci., USA 85:247, 1988; Sawchenko et al., Nature 334:615,
1988), and activin can function as a nerve cell survival molecule
(Schubert et al., Nature 344:868, 1990). Another family member,
growth differentiation factor-1 (GDF-1), is nervous system-specific
in its expression pattern (Lee, Proc. Natl. Acad. Sci., USA
88:4250, 1991), and other family members such as Vgr-1 (Lyons et
al., Proc. Natl. Acad. Sci., USA 86:4554, 1989; Jones et al.,
Development 111:531, 1991), OP-1 (Ozkaynak et al., J. Biol. Chem.
267:25220, 1992), and BMP-4 (Jones et al., Development 111:531,
1991), are also expressed in the nervous system. Because skeletal
muscle produces a factor or factors that promote the survival of
motor neurons (Brown, Trends Neurosci. 7:10, 1984), the expression
of myostatin (GDF-8) and GDF-11 in muscle suggests that myostatin
and GDF-11 can be trophic factors for neurons. As such, methods for
modulating the activity of myostatin, GDF-11, or both can be useful
for treating neurodegenerative diseases such as amyotrophic lateral
sclerosis or muscular dystrophy, or for maintaining cells or
tissues in culture prior to transplantation.
[0028] The proteins of the TGF-.theta. family are synthesized as
large precursor proteins, which subsequently undergo proteolytic
cleavage at a cluster of basic residues approximately 110 to 140
amino acids from the C-terminus, resulting in the formation of a
prodomain peptide and a C-terminal mature peptide. The C-terminal
mature peptides of the members of this family of proteins are
structurally related, and the different family members can be
classified into distinct subgroups based on the extent of their
homology. Although the homologies within particular subgroups range
from 70% to 90% amino acid sequence identity, the homologies
between subgroups are significantly lower, generally ranging from
20% to 50%. In each case, the active species appears to be a
disulfide-linked dimer of C-terminal peptide fragments.
[0029] Promyostatin and pro-GDF-11 polypeptides have been
identified in mammalian, avian and piscine species, and myostatin
is active in various other species, including vertebrates and
invertebrates. During embryonic development and in adult animals,
myostatin, for example, is expressed specifically by cells in the
myogenic lineage (McPherron et al., Nature 387:83-90, 1997, which
is incorporated herein by reference). During early embryogenesis,
myostatin is expressed by cells in the myotome compartment of
developing somites. At later embryonic stages and in adult animals,
myostatin is expressed widely in skeletal muscle tissue, although
the levels of expression vary considerably from muscle to muscle.
Myostatin expression also is detected in adipose tissue, although
at lower levels than in muscle. Similarly, GDF-11 is expressed in
skeletal muscle and adipose tissue, as well as in adult thymus,
spleen and uterus, and also is expressed in brain at various stages
of development.
[0030] Promyostatin polypeptides from various species share
substantial sequence identity, and the amino acid sequences of
human, murine, rat and chicken mature myostatin C-terminal sequence
are 100% identical (see FIG. 1). Promyostatin polypeptides are
exemplified herein (see FIG. 1) by human promyostatin (SEQ ID NO:
2); murine promyostatin (SEQ ID NO: 4); rat promyostatin (SEQ ID
NO: 6); baboon promyostatin (SEQ ID NO: 10); bovine promyostatin
(SEQ ID NO: 12); porcine promyostatin (SEQ ID NO: 14); ovine
promyostatin (SEQ ID NO: 16); chicken promyostatin (SEQ ID NO: 8),
turkey promyostatin (SEQ ID NO: 18); and zebrafish promyostatin
(SEQ ID NO: 20). Promyostatin polypeptides also are exemplified
herein by a polypeptide comprising the portions of salmon allele 1
(SEQ ID NO: 27; "salmon 1") and of salmon allele 2 (SEQ ID NO: 29;
"salmon2"; see FIG. 2). Nucleic acid molecules encoding these
promyostatin polypeptides are disclosed herein as SEQ ID NOS: 1, 3,
5, 9, 11, 13, 15, 7, 17, 19, 26 and 28, respectively (see, also,
McPherron and Lee, Proc. Natl. Acad. Sci., USA 94:12457, 1997,
which is incorporated herein by reference). A pro-GDF-11
polypeptide is exemplified herein by human pro-GDF-11 (SEQ ID NO:
25), which is encoded by SEQ ID NO: 24.
[0031] In view of the extensive conservation among promyostatin
polypeptides, particularly among species as diverse as humans and
fish, it would be a routine matter to obtain polynucleotides
encoding myostatin from any species, including the remainders of
the salmon1 and salmon2 sequences, and to identify promyostatin or
myostatin expression in any species. In particular, the mature
myostatin sequence shares significant homology to other members of
the TGF-.theta. superfamily, and myostatin contains most of the
residues that are highly conserved among the other family members
and in other species. Furthermore, myostatin, like the
TGF-.quadrature.s and inhibin .quadrature.s, contains an extra pair
of cysteine residues in addition to the seven cysteine residues
present in virtually all other family members. Myostatin is most
homologous to Vgr-1 (45% sequence identity). Like other members of
the TGF-.theta. superfamily, myostatin is synthesized as a larger
precursor promyostatin polypeptide that is proteolytic cleaved into
an active myostatin peptide.
[0032] Polynucleotides encoding promyostatin polypeptides of
various organisms can be identified using well known procedures and
algorithms based on identity (or homology) to the disclosed
sequences. Homology or identity is often measured using sequence
analysis software such as the Sequence Analysis Software Package of
the Genetics Computer Group (University of Wisconsin Biotechnology
Center, 1710 University Avenue, Madison, Wis. 53705). Such software
matches similar sequences by assigning degrees of homology to
various deletions, substitutions and other modifications. The terms
"homology" and "identity," when used herein in the context of two
or more nucleic acids or polypeptide sequences, refer to two or
more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or of nucleotides that
are the same when compared and aligned for maximum correspondence
over a comparison window or designated region as measured using any
number of sequence comparison algorithms or by manual alignment and
visual inspection.
[0033] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0034] The term "comparison window" is used broadly herein to
include reference to a segment of any one of the number of
contiguous positions, for example, about 20 to 600 positions, for
example, amino acid or nucleotide position, usually about 50 to
about 200 positions, more usually about 100 to about 150 positions,
in which a sequence may be compared to a reference sequence of the
same number of contiguous positions after the two sequences are
optimally aligned. Methods of alignment of sequence for comparison
are well-known in the art. Optimal alignment of sequences for
comparison can be conducted, for example, by the local homology
algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1981), by
the homology alignment algorithm of Needleman and Wunsch (J. Mol.
Biol. 48:443, 1970), by the search for similarity method of Person
and Lipman (Proc. Natl. Acad. Sci., USA 85:2444, 1988), each of
which is incorporated herein by reference; by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.); or by manual
alignment and visual inspection. Other algorithms for determining
homology or identity include, for example, in addition to a BLAST
program (Basic Local Alignment Search Tool at the National Center
for Biological Information), ALIGN, AMAS (Analysis of Multiply
Aligned Sequences), AMPS (Protein Multiple Sequence Alignment),
ASSET (Aligned Segment Statistical Evaluation Tool), BANDS,
BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis Node),
BLIMPS (BLocks IMProved Searcher), FASTA, Intervals & Points,
BMB, CLUSTAL V, CLUSTAL W, CONSENSUS, LCONSENSUS, WCONSENSUS,
Smith-Waterman algorithm, DARWIN, Las Vegas algorithm, FNAT (Forced
Nucleotide Alignment Tool), Framealign, Framesearch, DYNAMIC,
FILTER, FSAP (Fristensky Sequence Analysis Package), GAP (Global
Alignment Program), GENAL, GIBBS, GenQuest, ISSC (Sensitive
Sequence Comparison), LALIGN (Local Sequence Alignment), LCP (Local
Content Program), MACAW (Multiple Alignment Construction &
Analysis Workbench), MAP (Multiple Alignment Program), MBLKP,
MBLKN, PIMA (Pattern-Induced Multi-sequence Alignment), SAGA
(Sequence Alignment by Genetic Algorithm) and WHAT-IF. Such
alignment programs can also be used to screen genome databases to
identify polynucleotide sequences having substantially identical
sequences.
[0035] A number of genome databases are available for comparison,
including, for example, a substantial portion of the human genome
is available as part of the Human Genome Sequencing Project (J.
Roach,
http://weber.u.Washington.edu/.about.roach/human_genome_progress
2.html). In addition, at least twenty-one genomes have been
sequenced in their entirety, including, for example, M. genitalium,
M. jannaschii, H. influenzae, E. coli, yeast (S. cerevisiae), and
D. melanogaster. Significant progress has also been made in
sequencing the genomes of model organism such as mouse, C. elegans,
and Arabadopsis sp. Several databases containing genomic
information annotated with some functional information are
maintained by different organizations, and are accessible via the
internet, for example, http://wwwtigr.org/tdb;
http://www.genetics.wisc.edu;
http://genome-www.stanford.edu/.about.ball;
http://hiv-web.lanl.gov; http://www.ncbi.nlm.nih.gov;
http://www.ebi.ac.uk; http://Pasteur.fr/other/biology; and
http://www.genome.wi.mit.edu.
[0036] One example of a useful algorithm is BLAST and BLAST 2.0
algorithms, which are described by Altschul et al. (Nucleic Acids
Res. 25:3389-3402, 1977; J. Mol. Biol. 215:403-410, 1990, each of
which is incorporated herein by reference). Software for performing
BLAST analyses is publicly available through the National Center
for Biotechnology Information (http://www.ncbi.nlm.nih.gov). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra, 1977, 1990). These initial
neighborhood word hits act as seeds for initiating searches to find
longer HSPs containing them. The word hits are extended in both
directions along each sequence for as far as the cumulative
alignment score can be increased. Cumulative scores are calculated
using, for nucleotide sequences, the parameters M (reward score for
a pair of matching residues; always >0). For amino acid
sequences, a scoring matrix is used to calculate the cumulative
score. Extension of the word hits in each direction are halted
when: the cumulative alignment score falls off by the quantity X
from its maximum achieved value; the cumulative score goes to zero
or below, due to the accumulation of one or more negative-scoring
residue alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T, and X determine the sensitivity
and speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff and Henikoff, Proc. Natl. Acad. Sci., USA 89:10915, 1989)
alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a
comparison of both strands.
[0037] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, for example, Karlin and
Altschul, Proc. Natl. Acad. Sci., USA 90:5873, 1993, which is
incorporated herein by reference). One measure of similarity
provided by BLAST algorithm is the smallest sum probability (P(N)),
which provides an indication of the probability by which a match
between two nucleotide or amino acid sequences would occur by
chance. For example, a nucleic acid is considered similar to a
references sequence if the smallest sum probability in a comparison
of the test nucleic acid to the reference nucleic acid is less than
about 0.2, more preferably less than about 0.01, and most
preferably less than about 0.001.
[0038] In one embodiment, protein and nucleic acid sequence
homologies are evaluated using the Basic Local Alignment Search
Tool ("BLAST"). In particular, five specific BLAST programs are
used to perform the following task:
[0039] (1) BLASTP and BLAST3 compare an amino acid query sequence
against a protein sequence database;
[0040] (2) BLASTN compares a nucleotide query sequence against a
nucleotide sequence database;
[0041] (3) BLASTX compares the six-frame conceptual translation
products of a query nucleotide sequence (both strands) against a
protein sequence database;
[0042] (4) TBLASTN compares a query protein sequence against a
nucleotide sequence database translated in all six reading frames
(both strands); and
[0043] (5) TBLASTX compares the six-frame translations of a
nucleotide query sequence against the six-frame translations of a
nucleotide sequence database.
[0044] The BLAST programs identify homologous sequences by
identifying similar segments, which are referred to herein as
"high-scoring segment pairs," between a query amino or nucleic acid
sequence and a test sequence which is preferably obtained from a
protein or nucleic acid sequence database. High-scoring segment
pairs are preferably identified (i.e., aligned) by means of a
scoring matrix, many of which are known in the art. Preferably, the
scoring matrix used is the BLOSUM62 matrix (Gonnet et al., Science
256:1443-1445, 1992; Henikoff and Henikoff, Proteins 17:49-61,
1993, each of which is incorporated herein by reference). Less
preferably, the PAM or PAM250 matrices may also be used (Schwartz
and Dayhoff, eds., "Matrices for Detecting Distance Relationships:
Atlas of Protein Sequence and Structure" (Washington, National
Biomedical Research Foundation 1978)). BLAST programs are
accessible through the U.S. National Library of Medicine, for
example, at www.ncbi.nlm.nih.gov.
[0045] The parameters used with the above algorithms may be adapted
depending on the sequence length and degree of homology studied. In
some embodiments, the parameters may be the default parameters used
by the algorithms in the absence of instructions from the user.
[0046] A polynucleotide encoding a promyostatin can be derived from
any organism, including, for example, mouse, rat, cow, pig, human,
chicken, turkey, zebrafish, salmon, finfish, other aquatic
organisms and other species. Examples of aquatic organisms include
those belonging to the class Piscina, such as trout, char, ayu,
carp, crucian carp, goldfish, roach, whitebait, eel, conger eel,
sardine, flying fish, sea bass, sea bream, parrot bass, snapper,
mackerel, horse mackerel, tuna, bonito, yellowtail, rockfish,
fluke, sole, flounder, blowfish, filefish; those belonging to the
class Cephalopoda, such as squid, cuttlefish, octopus; those
belonging to the class Pelecypoda, such as clams (e.g., hardshell,
Manila, Quahog, Surf, Soft-shell); cockles, mussels, periwinkles;
scallops (e.g., sea, bay, calloo); conch, snails, sea cucumbers;
ark shell; oysters (e.g., C. virginica, Gulf, New Zealand,
Pacific); those belonging to the class Gastropoda such as turban
shell, abalone (e.g. green, pink, red); and those belonging to the
class Crustacea such as lobster, including but not limited to
Spiny, Rock, and American; prawn; shrimp, including but not limited
to M. rosenbergii, P. styllrolls, P. indicus, P. jeponious, P.
monodon, P. vannemel, M. ensis, S. melantho, N. norvegious, cold
water shrimp; crab, including, but not limited to, Blue, rook,
stone, king, queen, snow, brown, dungeness, Jonah, Mangrove,
soft-shelled; squilla, krill, langostinos; crayfish/crawfish,
including, but not limited, to Blue, Marron, Red Claw, Red Swamp,
Soft-shelled, white; Annelida; Chordata, including, but not limited
to, reptiles such as alligators and turtles; Amphibia, including
frogs; and Echinodermata, including, but not limited to, sea
urchins.
[0047] The present invention provides substantially purified
peptide portions of a promyostatin polypeptide and substantially
purified peptide portions of a pro-GDF-11 polypeptide. As used
herein, reference to a "pro-GDF," for example, promyostatin or
pro-GDF-11, means the full length polypeptide, including the amino
terminal prodomain and the carboxy terminal biologically active GDF
peptide. In addition, the prodomain includes a signal peptide
(leader sequence), which comprises about the first 15 to 30 amino
acids at the amino terminus of the prodomain. The signal peptide
can be cleaved from the full length pro-GDF polypeptide, which can
be further cleaved at an Arg-Xaa-Xaa-Arg (SEQ ID NO: 21)
proteolytic cleavage site.
[0048] Reference herein to amino acid residues is made with respect
to the full length pro-GDF polypeptides as shown in FIGS. 1 and 2
(see, also, Sequence Listing). It should also be recognized that
reference is made herein to particular peptides beginning or ending
at "about" a particular amino acid residue. The term "about" is
used in this context because it is recognized that a particular
protease can cleave a pro-GDF polypeptide at or immediately
adjacent to a proteolytic cleavage recognition site, or one or a
few amino acids from the recognition site. As such, reference, for
example, to a myostatin prodomain having a sequence of about amino
acid residues 1 to 263 of SEQ ID NO: 4 would include an amino
terminal peptide portion of promyostatin that includes the signal
peptide and has a carboxy terminus ending at amino acid residue 257
to amino acid residue 269, preferably at amino acid residue 260 to
amino acid residue 266.
[0049] Similarly, the signal peptide can be cleaved at any position
from about amino acid residue 15 to 30 of a pro-GDF polypeptide,
for example, at residue 15, 20, 25 or 30, without affecting the
function, for example, of a remaining prodomain. Thus, for
convenience, reference is made generally herein to a peptide
portion of a pro-GDF polypeptide, from which the signal peptide has
been cleaved, as beginning at about amino acid residue 20. However,
it will be recognized that cleavage of the signal peptide can be at
any amino acid position within about the first 15 to 30 amino
terminal amino acids of a pro-GDF polypeptide. As such, reference,
for example, to a myostatin prodomain having a sequence of about
amino acid residues 20 to 263 of SEQ ID NO: 4 would include a
peptide portion of promyostatin that lacks about the first 15 to 30
amino acids of promyostatin, comprising the signal peptide, and
that has a carboxy terminus ending at amino acid residue 257 to
amino acid residue 269, preferably at amino acid residue 260 to
amino acid residue 266.
[0050] In general, reference is made herein to a pro-GDF
polypeptide or a GDF prodomain as beginning at about amino acid 1.
In view of the above disclosure, however, it will be recognized
that such pro-GDF polypeptides or GDF prodomains that lack the
signal peptide also are encompassed within the present invention.
Further in this respect, it should be recognized that the presence
or absence of a signal peptide in a peptide of the invention can
influence, for example, the compartments of a cell through which a
peptide, for example, a myostatin prodomain will traverse and to
which the peptide ultimately will localize, including whether the
peptide will be secreted from the cell. Thus, the present invention
further provides a substantially purified signal peptide portion of
a pro-GDF polypeptide. As disclosed herein, such a signal peptide
can be used to target an agent, particularly a peptide agent, to
the same cellular compartments as the naturally occurring GDF from
which the signal peptide is derived.
[0051] The term "peptide" or "peptide portion" is used broadly
herein to mean two or more amino acids linked by a peptide bond.
The term "fragment" or "proteolytic fragment" also is used herein
to refer to a product that can be produced by a proteolytic
reaction on a polypeptide, i.e., a peptide produced upon cleavage
of a peptide bond in the polypeptide. Although the term
"proteolytic fragment" is used generally herein to refer to a
peptide that can be produced by a proteolytic reaction, it should
be recognized that the fragment need not necessarily be produced by
a proteolytic reaction, but also can be produced using methods of
chemical synthesis or methods of recombinant DNA technology, as
discussed in greater detail below, to produce a synthetic peptide
that is equivalent to a proteolytic fragment. In view of the
disclosed homology of promyostatin with other members of the
TGF-.theta. superfamily, it will be recognized that a peptide of
the invention is characterized, in part, in that it is not present
in previously disclosed members of this superfamily. Whether a
peptide portion of a promyostatin or pro-GDF-11 polypeptide is
present in a previously disclosed member of the TGF-.theta.
superfamily readily can be determined using the computer algorithms
described above.
[0052] Generally, a peptide of the invention contains at least
about six amino acids, usually contains about ten amino acids, and
can contain fifteen or more amino acids, particularly twenty or
more amino acids. It should be recognized that the term "peptide"
is not used herein to suggest a particular size or number of amino
acids comprising the molecule, and that a peptide of the invention
can contain up to several amino acid residues or more. For example,
a full length mature C-terminal myostatin peptide contains more
than 100 amino acids and a full length prodomain peptide can
contain more than 260 amino acids.
[0053] As used herein, the term "substantially purified" or
"substantially pure" or "isolated" means that the molecule being
referred to, for example, a peptide or a polynucleotide, is in a
form that is relatively free of proteins, nucleic acids, lipids,
carbohydrates or other materials with which it is naturally
associated. Generally, a substantially pure peptide,
polynucleotide, or other molecule constitutes at least twenty
percent of a sample, generally constitutes at least about fifty
percent of a sample, usually constitutes at least about eighty
percent of a sample, and particularly constitutes about ninety
percent or ninety-five percent or more of a sample. A determination
that a peptide or a polynucleotide of the invention is
substantially pure can be made using well known methods, for
example, by performing electrophoresis and identifying the
particular molecule as a relatively discrete band. A substantially
pure polynucleotide, for example, can be obtained by cloning the
polynucleotide, or by chemical or enzymatic synthesis. A
substantially pure peptide can be obtained, for example, by a
method of chemical synthesis, or using methods of protein
purification, followed by proteolysis and, if desired, further
purification by chromatographic or electrophoretic methods.
[0054] A peptide of the invention can be identified by comparison
to a promyostatin or pro-GDF-11 sequence and determining that the
amino acid sequence of the peptide is contained within the
promyostatin or pro-GDF-11 polypeptide sequence, respectively. It
should be recognized, however, that a peptide of the invention need
not be identical to a corresponding amino acid sequence of
promyostatin or pro-GDF-11. Thus, a peptide of the invention can
correspond to an amino acid sequence of a promyostatin polypeptide,
for example, but can vary from a naturally occurring sequence, for
example, by containing one or more D-amino acids in place of a
corresponding L-amino acid; or by containing one or more amino acid
analogs, for example, an amino acid that has been derivatized or
otherwise modified at its reactive side chain. Similarly, one or
more peptide bonds in the peptide can be modified. In addition, a
reactive group at the amino terminus or the carboxy terminus or
both can be modified. Such peptides can be modified, for example,
to have improved stability to a protease, an oxidizing agent or
other reactive material the peptide may encounter in a biological
environment, and, therefore, can be particularly useful in
performing a method of the invention. Of course, the peptides can
be modified to have decreased stability in a biological environment
such that the period of time the peptide is active in the
environment is reduced.
[0055] The sequence of a peptide of the invention also can be
modified in comparison to the corresponding sequence in a
promyostatin or pro-GDF-11 polypeptide by incorporating a
conservative amino acid substitution for one or a few amino acids
in the peptide. Conservative amino acid substitutions include the
replacement of one amino acid residue with another amino acid
residue having relatively the same chemical characteristics, for
example, the substitution of one hydrophobic residue such as
isoleucine, valine, leucine or methionine for another, or the
substitution of one polar residue for another, for example,
substitution of arginine for lysine; or of glutamic for aspartic
acid; or of glutamine for asparagine; or the like. Examples of
positions of a promyostatin polypeptide that can be modified are
evident from examination of FIG. 1, which shows various amino acid
differences in the myostatin prodomain and mature myostatin peptide
that do not substantially affect promyostatin or myostatin
activity.
[0056] The present invention also provides a substantially purified
proteolytic fragment of a growth differentiation factor (GDF)
polypeptide (a pro-GDF polypeptide) or a functional peptide portion
thereof. Proteolytic fragments of a pro-GDF polypeptide are
exemplified herein by proteolytic fragments of a promyostatin
polypeptide and proteolytic fragments of a pro-GDF-11 polypeptide.
As disclosed herein, a peptide portion of a pro-GDF polypeptide
that is equivalent to a proteolytic fragment of a pro-GDF can be
produced by a chemical method or a recombinant DNA method. In view
of the present disclosure, proteolytic fragments of other GDF
polypeptides readily can be made and used.
[0057] In general, peptides corresponding to proteolytic fragments
of a pro-GDF polypeptide are exemplified by a carboxy terminal
(C-terminal) mature GDF fragment, which can interact specifically
with a GDF receptor and affect GDF signal transduction, and an
amino terminal prodomain fragment, which can include a signal
peptide and, as disclosed herein, can interact specifically with a
pro-GDF polypeptide or mature GDF peptide and affect its ability to
effect GDF signal transduction. For example, proteolytic fragments
of a promyostatin polypeptide include a C-terminal mature myostatin
peptide, which can interact specifically with a myostatin receptor
and induce myostatin signal transduction; and an amino terminal
prodomain fragment, which can interact specifically with myostatin,
thereby reducing or inhibiting the ability of myostatin to induce
myostatin signal transduction.
[0058] A proteolytic fragment of a pro-GDF polypeptide, or a
functional peptide portion thereof, is characterized, in part, by
having or affecting an activity associated with the stimulation or
inhibition of GDF signal transduction. For example, a promyostatin
polypeptide or functional peptide portion thereof can have
myostatin receptor binding activity, myostatin signal transduction
stimulatory or inhibitory activity, myostatin binding activity,
promyostatin binding activity, or a combination thereof. Thus, the
term "functional peptide portion," when used herein in reference to
a pro-GDF polypeptide, means a peptide portion of the pro-GDF
polypeptide that can interact specifically with its receptor and
stimulate or inhibit GDF signal transduction; that can interact
specifically with a mature GDF or a pro-GDF; or that exhibits
cellular localization activity, i.e., the activity of a signal
peptide. It should be recognized that a functional peptide portion
of full length mature myostatin peptide, for example, need not have
the same activity of the mature myostatin, including the ability to
stimulate myostatin signal transduction, since functional peptide
portions of the mature peptide can have, for example, an ability to
specifically interact with a myostatin receptor without also having
the ability to activate the signal transduction pathway. Methods
for identifying such a functional peptide portion of a pro-GDF
polypeptide, which can be useful as a myostatin antagonist, are
disclosed herein or otherwise known in the art. Thus, in one
embodiment, a functional peptide portion of a promyostatin
polypeptide can interact specifically with a myostatin receptor,
and can act as an agonist to stimulate myostatin signal
transduction or as an antagonist to reduce or inhibit myostatin
signal transduction.
[0059] In another embodiment, a functional peptide portion of a
promyostatin polypeptide can interact specifically with a
promyostatin polypeptide or with a mature myostatin peptide,
thereby blocking myostatin signal transduction. Such a functional
peptide portion of promyostatin can act, for example, by preventing
cleavage of a promyostatin polypeptide to mature myostatin; by
forming a complex with a mature myostatin peptide; or by some other
mechanism. Where a peptide-myostatin complex is formed, the complex
can block myostatin signal transduction, for example, by reducing
or inhibiting the ability of the myostatin to interact specifically
with its receptor, or by binding to the receptor in the form that
lacks the ability to induce myostatin signal transduction.
[0060] Proteolytic fragments of a pro-GDF polypeptide can be
produced by cleavage of the polypeptide at a proteolytic cleavage
site having a consensus amino acid sequence Arg-Xaa-Xaa-Arg (SEQ ID
NO: 21). Such proteolytic recognition sites are exemplified by the
Arg-Ser-Arg-Arg (SEQ ID NO: 22) sequence shown as amino acid
residues 263 to 266 in SEQ ID NO: 1 (promyostatin) or amino acid
residues 295 to 298 of SEQ ID NO: 25 (human pro-GDF-11; see, also,
relative positions 267 to 270 of FIG. 2), and by the
Arg-Ile-Arg-Arg (SEQ ID NO: 23) sequence shown as amino acid
residues 263 to 266 in SEQ ID NO: 20.
[0061] In addition to the proteolytic cleavage site for the signal
peptide, promyostatin polypeptides, for example, contain two
additional potential proteolytic processing sites (Lys-Arg and
Arg-Arg). Cleavage of a promyostatin polypeptide at or near the
latter proteolytic processing site, which is contained within the
consensus Arg-Xaa-Xaa-Arg (SEQ ID NO: 21) proteolytic cleavage
recognition site (see, for example, amino acid residues 263 to 266
of SEQ ID NO: 2), generates a biologically active C-terminal mature
human myostatin fragment. The exemplified full length mature
myostatin peptides contain about 103 to about 109 amino acids and
have a predicted molecular weight of approximately 12,400 daltons
(Da). In addition, myostatin can form dimers, which have an
expected molecular weight of about 23 to 30 kiloDaltons (kDa). The
dimers can be myostatin homodimers or can be heterodimers, for
example, with GDF-11 or another GDF or TGF-.theta. family
member.
[0062] A proteolytic fragment of the invention is exemplified by a
GDF prodomain, for example, a myostatin prodomain, which includes
about amino acid residues 20 to 262 of a promyostatin polypeptide,
or a functional peptide portion thereof, or a GDF-11 prodomain,
which includes about amino acid residues 20 to 295 of a pro-GDF-11
polypeptide, or a functional peptide portion thereof, each of which
can further contain the signal peptide comprising about amino acids
1 to 20 of the respective pro-GDF polypeptide. Myostatin prodomains
are further exemplified by about amino acid residues 20 to 263 as
set forth in SEQ ID NO: 4 and SEQ ID NO: 6; as well as by about
amino acid residues about 20 to 262 as set forth in SEQ ID NO: 2,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 20, which can be produced by
proteolytic cleavage of a corresponding promyostatin polypeptide,
can be chemically synthesized, or can be expressed from a
recombinant polynucleotide encoding the proteolytic fragment. A
functional peptide portion of a myostatin prodomain is exemplified
by a peptide portion of a myostatin prodomain that can interact
specifically with myostatin or with promyostatin. A GDF-11
prodomain is exemplified by about amino acid residues 20 to 295 of
SEQ ID NO: 25, which can further include the signal peptide
comprising about amino acid residues 1 to 20 of SEQ ID NO: 25, and
a functional peptide portion of a GDF-11 prodomain is exemplified
by a peptide portion of a GDF-11 prodomain that can specifically
interact with mature GDF-11 or a pro-GDF-11 polypeptide.
Preferably, the functional peptide portion of a GDF prodomain
inhibits the ability of the corresponding GDF or a related GDF to
stimulate signal transduction, for example, by reducing or
inhibiting the ability of the GDF to interact specifically with its
receptor, or by binding to the receptor as an inactive complex. In
one embodiment, the present invention provides a functional
fragment of a pro-GDF polypeptide, particularly a functional
fragment of a GDF prodomain, operably linked to a GDF signal
peptide, preferably a myostatin signal peptide or a GDF-11 signal
peptide comprising about the first 15 to 30 amino terminal amino
acids of promyostatin or pro-GDF-11, respectively.
[0063] As disclosed herein, a myostatin prodomain or GDF-11
prodomain can interact with mature myostatin, GDF-11, or both,
thereby reducing or inhibiting the ability of the mature GDF to
interact specifically with its receptor (see Examples 7 and 8).
Thus, a functional peptide portion of a myostatin prodomain, for
example, can be obtained by examining peptide portions of a
myostatin prodomain using methods as provided herein, and
identifying functional peptide portions of the prodomain that can
interact specifically with myostatin or with promyostatin and can
reduce or inhibit the ability of myostatin to interact specifically
with a myostatin receptor or to stimulate myostatin signal
transduction.
[0064] A functional peptide portion of a myostatin prodomain that
can specifically interact with myostatin, or a functional peptide
portion of another GDF prodomain, also can be identified using any
of various assays known to be useful for identifying specific
protein-protein interactions. Such assays include, for example,
methods of gel electrophoresis, affinity chromatography, the two
hybrid system of Fields and Song (Nature 340:245-246, 1989; see,
also, U.S. Pat. No. 5,283,173; Fearon et al., Proc. Natl. Acad.
Sci., USA 89:7958-7962, 1992; Chien et al., Proc. Natl. Acad. Sci.
USA 88:9578-9582, 1991; Young, Biol. Reprod. 58:302-311 (1998),
each of which is incorporated herein by reference), the reverse two
hybrid assay (Leanna and Hannink, Nucl. Acids Res. 24:3341-3347,
1996, which is incorporated herein by reference), the repressed
transactivator system (U.S. Pat. No. 5,885,779, which is
incorporated herein by reference), the phage display system
(Lowman, Ann. Rev. Biophys. Biomol. Struct. 26:401-424, 1997, which
is incorporated herein by reference), GST/HIS pull down assays,
mutant operators (WO 98/01879, which is incorporated herein by
reference), the protein recruitment system (U.S. Pat. No.
5,776,689, which is incorporated herein by reference), and the like
(see, for example, Mathis, Clin. Chem. 41:139-147, 1995 Lam,
Anticancer Drug Res. 12:145-167, 1997; Phizicky et al., Microbiol.
Rev. 59:94-123, 1995; each of which is incorporated herein by
reference).
[0065] A functional peptide portion of a GDF prodomain also can be
identified using methods of molecular modeling. For example, an
amino acid sequence of a mature myostatin peptide can be entered
into a computer system having appropriate modeling software, and a
three dimensional representation of the myostatin ("virtual
myostatin") can be produced. A promyostatin amino acid sequence
also can be entered into the computer system, such that the
modeling software can simulate portions of the promyostatin
sequence, for example, portions of the prodomain, and can identify
those peptide portions of the prodomain that can interact
specifically with the virtual myostatin. A base line for a specific
interaction can be predefined by modeling the virtual myostatin and
a full length promyostatin prodomain, and identifying the amino
acid residues in the virtual myostatin that are "contacted" by the
prodomain, since such an interaction is known to inhibit the
activity of the myostatin.
[0066] It should be recognized that such methods, including two
hybrid assays and molecular modeling methods, also can be used to
identify other specifically interacting molecules encompassed
within the present invention. Thus, methods such as the two hybrid
assay can be used to identify a GDF receptor such as a myostatin
receptor using, for example, a myostatin peptide or a peptide
portion thereof that specifically interacts with an Act RIIA or Act
RIIB receptor as one binding component of the assay, and
identifying a GDF receptor, which specifically interacts with the
myostatin peptide. Similarly, methods of molecular modeling can be
used to identify an agent that interacts specifically with a mature
GDF peptide such as mature myostatin, or with a GDF receptor and,
therefore, can be useful as an agonist or an antagonist of signal
transduction mediated by the GDF or the GDF receptor. Such an agent
can be, for example, a functional peptide portion of a myostatin
prodomain or GDF-11 prodomain, or a chemical agent that mimics the
action of the GDF prodomain.
[0067] Modeling systems useful for the purposes disclosed herein
can be based on structural information obtained, for example, by
crystallographic analysis or nuclear magnetic resonance analysis,
or on primary sequence information (see, for example, Dunbrack et
al., "Meeting review: the Second meeting on the Critical Assessment
of Techniques for Protein Structure Prediction (CASP2) (Asilomar,
Calif., December 13-16, 1996). Fold Des. 2 (2): R27-42, (1997);
Fischer and Eisenberg, Protein Sci. 5:947-55, 1996; (see, also,
U.S. Pat. No. 5,436,850); Havel, Prog. Biophys. Mol. Biol.
56:43-78, 1991; Lichtarge et al., J. Mol. Biol. 274:325-37, 1997;
Matsumoto et al., J. Biol. Chem. 270:19524-31, 1995; Sali et al.,
J. Biol. Chem. 268:9023-34, 1993; Sali, Molec. Med. Today 1:270-7,
1995a; Sali, Curr. Opin. Biotechnol. 6:437-51, 1995b; Sali et al.,
Proteins 23: 318-26, 1995c; Sali, Nature Struct. Biol. 5:1029-1032,
1998; U.S. Pat. No. 5,933,819; U.S. Pat. No. 5,265,030, each of
which is incorporated herein by reference).
[0068] The crystal structure coordinates of a promyostatin
polypeptide or a GDF receptor can be used to design compounds that
bind to the protein and alter its physical or physiological
properties in a variety of ways. The structure coordinates of the
protein can also be used to computationally screen small molecule
data bases for agents that bind to the polypeptide to develop
modulating or binding agents, which can act as agonists or
antagonists of GDF signal transduction. Such agents can be
identified by computer fitting kinetic data using standard
equations (see, for example, Segel, "Enzyme Kinetics" (J. Wiley
& Sons 1975), which is incorporated herein by reference).
[0069] Methods of using crystal structure data to design inhibitors
or binding agents are known in the art. For example, GDF receptor
coordinates can be superimposed onto other available coordinates of
similar receptors, including receptors having a bound inhibitor, to
provide an approximation of the way the inhibitor interacts with
the receptor. Computer programs employed in the practice of
rational drug design also can be used to identify compounds that
reproduce interaction characteristics similar to those found, for
example, between a mature myostatin and a co-crystallized myostatin
prodomain. Detailed knowledge of the nature of the specific
interactions allows for the modification of compounds to alter or
improve solubility, pharmacokinetics, and the like, without
affecting binding activity.
[0070] Computer programs for carrying out the activities necessary
to design agents using crystal structure information are well
known. Examples of such programs include, Catalyst
Databases.TM.--an information retrieval program accessing chemical
databases such as BioByte Master File, Derwent WDI and ACD;
Catalyst/HYPO.TM.--generates models of compounds and hypotheses to
explain variations of activity with the structure of drug
candidates; Ludi.TM.--fits molecules into the active site of a
protein by identifying and matching complementary polar and
hydrophobic groups; and Leapfrog.TM.--"grows" new ligands using a
genetic algorithm with parameters under the control of the
user.
[0071] Various general purpose machines can be used with such
programs, or it may be more convenient to construct more
specialized apparatus to perform the operations. Generally, the
embodiment is implemented in one or more computer programs
executing on programmable systems each comprising at least one
processor, at least one data storage system (including volatile and
non-volatile memory and/or storage elements), at least one input
device, and at least one output device. The program is executed on
the processor to perform the functions described herein.
[0072] Each such program can be implemented in any desired computer
language, including, for example, machine, assembly, high level
procedural, or object oriented programming languages, to
communicate with a computer system. In any case, the language may
be a compiled or interpreted language. The computer program will
typically be stored on a storage media or device, for example, a
ROM, CD-ROM, magnetic or optical media, or the like, that is
readable by a general or special purpose programmable computer, for
configuring and operating the computer when the storage media or
device is read by the computer to perform the procedures described
herein. The system may also be considered to be implemented as a
computer-readable storage medium, configured with a computer
program, where the storage medium so configured causes a computer
to operate in a specific and predefined manner to perform the
functions described herein.
[0073] Embodiments of the invention include systems, for example,
internet based systems, particularly computer systems which store
and manipulate coordinate information obtained by crystallographic
or NMR analysis, or amino acid or nucleotide sequence information,
as disclosed herein. As used herein, the term "computer system"
refers to the hardware components, software components, and data
storage components used to analyze coordinates or sequences as set
forth herein. The computer system typically includes a processor
for processing, accessing and manipulating the sequence data. The
processor can be any well known type of central processing unit,
for example, a Pentium II or Pentium III processor from Intel
Corporation, or a similar processor from Sun, Motorola, Compaq,
Advanced MicroDevices or International Business Machines.
[0074] Typically the computer system is a general purpose system
that comprises the processor and one or more internal data storage
components for storing data, and one or more data retrieving
devices for retrieving the data stored on the data storage
components. A skilled artisan can readily appreciate that any one
of the currently available computer systems are suitable.
[0075] In one embodiment, the computer system includes a processor
connected to a bus, which is connected to a main memory, preferably
implemented as RAM, and one or more internal data storage devices
such as a hard drive or other computer readable media having data
recorded thereon. In some embodiments, the computer system further
includes one or more data retrieving devices for reading the data
stored on the internal data storage devices.
[0076] The data retrieving device may represent, for example, a
floppy disk drive, a compact disk drive, a magnetic tape drive, or
a modem capable of connection to a remote data storage system
(e.g., via the internet). In some embodiments, the internal data
storage device is a removable computer readable medium such as a
floppy disk, a compact disk, a magnetic tape, etc. containing
control logic and/or data recorded thereon. The computer system may
advantageously include or be programmed by appropriate software for
reading the control logic and/or the data from the data storage
component once inserted in the data retrieving device.
[0077] The computer system generally include a display, which is
used to display output to a computer user. It should also be noted
that the computer system can be linked to other computer systems in
a network or wide area network to provide centralized access to the
computer system.
[0078] Where it is desired to identify a chemical entity that
interacts specifically with myostatin or with a GDF receptor, any
of several methods to screen chemical entities or fragments for
their ability to interact specifically with the molecule can be
used. This process may begin by visual inspection, for example, of
myostatin and a myostatin prodomain on the computer screen.
Selected peptide portions of the prodomain, or chemical entities
that can act as mimics, then can be positioned in a variety of
orientations, or docked, within an individual binding site of the
myostatin. Docking can be accomplished using software such as
Quanta and Sybyl, followed by energy minimization and molecular
dynamics with standard molecular mechanics forcefields, such as
CHARMM and AMBER.
[0079] Specialized computer programs can be particularly useful for
selecting peptide portions of a prodomain, or chemical entities
useful, for example, as a GDF receptor agonist or antagonist. Such
programs include, for example, GRID (Goodford, J. Med. Chem.,
28:849-857, 1985; available from Oxford University, Oxford, UK);
MCSS (Miranker and Karplus, Proteins: Structure. Function and
Genetics 11:29-34, 1991, available from Molecular Simulations,
Burlington Mass.); AUTODOCK (Goodsell and Olsen, Proteins:
Structure. Function, and Genetics 8:195-202, 1990, available from
Scripps Research Institute, La Jolla Calif.); DOCK (Kuntz, et al.,
J. Mol. Biol. 161:269-288, 1982, available from University of
California, San Francisco Calif.), each of which is incorporated
herein by reference.
[0080] Suitable peptides or agents that have been selected can be
assembled into a single compound or binding agent. Assembly can be
performed by visual inspection of the relationship of the fragments
to each other on the three-dimensional image displayed on a
computer screen, followed by manual model building using software
such as Quanta or Sybyl. Useful programs to aid one of skill in the
art in connecting the individual chemical entities or fragments
include, for example, CAVEAT (Bartlett et al, Special Pub., Royal
Chem. Soc. 78:182-196, 1989, available from the University of
California, Berkeley Calif.); 3D Database systems such as MACCS-3D
(MDL Information Systems, San Leandro Calif.; for review, see
Martin, J. Med. Chem. 35:2145-2154, 1992); HOOK (available from
Molecular Simulations, Burlington, Mass.), each of which is
incorporated herein by reference.
[0081] In addition to the method of building or identifying such
specifically interacting agents in a step-wise fashion, one
fragment or chemical entity at a time as described above, the
agents can be designed as a whole or de novo using either an empty
active site or, optionally, including some portions of a known
agent that specifically interacts, for example, a full length
myostatin prodomain, which interacts specifically with myostatin.
Such methods include, for example, LUDI (Bohm, J. Comp. Aid. Molec.
Design 6:61-78, 1992, available from Biosym Technologies, San Diego
Calif.); LEGEND (Nishibata and Itai, Tetrahedron 47:8985, 1991,
available from Molecular Simulations, Burlington Mass.); LeapFrog
(available from Tripos Associates, St. Louis Mo.), and those
described by Cohen et al. (J. Med. Chem. 33:883-894, 1990) and by
Navia and Murcko, Curr. Opin. Struct. Biol. 2:202-210, 1992, each
of which is incorporated herein by reference).
[0082] Specific computer software is available in the art to
evaluate compound deformation energy and electrostatic interaction.
Examples of programs designed for such uses include Gaussian 92,
revision C (Frisch, Gaussian, Inc., Pittsburgh Pa., 1992); AMBER,
version 4.0 (Kollman, University of California at San Francisco,
1994); QUANTA/CHARMM (Molecular Simulations, Inc., Burlington
Mass., 1994); and Insight II/Discover (Biosysm Technologies Inc.,
San Diego Calif., 1994). These programs may be implemented using,
for example, a Silicon Graphics workstation, IRIS 4D/35 or IBM
RISC/6000 workstation model 550. Other hardware systems and
software packages will be known to those skilled in the art of
which the speed and capacity are continually modified.
[0083] A molecular modeling process for identifying an agent that
interacts specifically with a molecule of interest, for example,
with a mature GDF peptides such as mature myostatin, or with a GDF
receptor can be performed as disclosed herein. In a first step, a
virtual representation of a target molecule, for example,
myostatin, is performed. Thus, in one embodiment, the present
invention provides a virtual representation of a target molecule,
wherein the target molecule is selected from a pro-GDF polypeptide,
for example, promyostatin; a peptide portion of a pro-GDF
polypeptide; a GDF receptor; and a relevant domain of a GDF
receptor, for example, a GDF binding domain. The virtual
representation of the target molecule can be displayed or can be
maintained in a computer system memory. The process begins at a
start state, comprising the virtual target molecule, then moves to
a state wherein a database containing one or more virtual test
molecules stored to a memory in the computer system. As discussed
above, the memory can be any type of memory, including RAM or an
internal storage device.
[0084] The process then moves to a state wherein the ability of a
virtual first test molecule to specifically interact with the
virtual target molecule is determined, wherein the database
containing the virtual test molecule, which can be one of a
population of test molecules, is opened for analysis of the an
interaction of the virtual target molecule and virtual test
molecule, and the analysis is made. A determination of a specific
interaction can be made based on calculations performed by software
maintained in the computer system, or by comparison to a
predetermined specific interaction, which can be stored in a memory
in the computer system and accessed as appropriate.
[0085] The process then moves to a state wherein, where a specific
interaction is detected, the virtual test molecule is displayed, or
is stored in a second database on the computer. If appropriate, the
process is repeated for the virtual target molecule and a second
virtual test molecule, a third virtual test molecule, and so on, as
desired.
[0086] If a determination is made that a virtual test molecule
specifically interacts with the virtual target molecule, the
identified virtual test molecule is moved from the database and can
be displayed to the user. This state notifies the user that the
molecule with the displayed name or structure interacts
specifically with the target molecule within the constraints that
were entered. Once the name of the identified test molecule is
displayed to the user, the process moves to a decision state,
wherein a determination is made whether more virtual test molecules
exist in the database or are to be examined. If no more molecules
exist in the database, then the process terminates at an end state.
However, if more test molecules exist in the database, then the
process moves to a state, wherein a pointer is moved to the next
test molecule in the database so that it can be examined for
specific binding activity. In this manner, the new molecule is
examined for the ability to interact specifically with the virtual
target molecule.
[0087] Such methods as described above can be used in various
aspects encompassed within the claimed invention. Thus, the methods
can be used to identify a peptide portion of a promyostatin
prodomain that can interact specifically with myostatin and reduce
or inhibit the ability of myostatin to interact with its receptor
or otherwise affect the ability of the myostatin to effect signal
transduction. Similarly, the methods can be used to identify small
organic molecules that mimic the action of a GDF prodomain, thereby
reducing or inhibiting myostatin or GDF-11 signal transduction. The
methods also can be used to identify agents that interact
specifically with a GDF receptor, for example, an Act RIIA, Act
RIIB or other GDF receptor, such agents being useful as GDF
receptor agonists or antagonists, which can modulate GDF signal
transduction in a cell. In addition, the methods provide a means to
identify previously unknown pro-GDF polypeptides or GDF receptors,
for example, by identifying conserved structural features of the
particular polypeptides.
[0088] Similar to other members of the TGF-.theta. superfamily,
active GDF peptides are expressed as precursor polypeptides, which
are cleaved to a mature, biologically active form. Accordingly, in
still another embodiment, the proteolytic fragment of a pro-GDF
polypeptide is a mature GDF peptide, or a functional peptide
portion of a mature GDF peptide, where, as discussed above, the
functional peptide portion can have the activity of a GDF agonist
or antagonist. The proteolytic fragment can be a mature C-terminal
myostatin peptide, which includes about amino acid residues 268 to
374 of a promyostatin polypeptide (see FIG. 1; see, also, FIG. 2),
or a mature C-terminal GDF-11 peptide, which includes about amino
acid residues 299 to 407 of a pro-GDF-11 polypeptide. Full length
mature myostatin peptides are exemplified by amino acid residues
about 268 to 375 as set forth in SEQ ID NO: 4 and SEQ ID NO: 6; by
amino acid residues about 267 to 374 as set forth in SEQ ID NO: 2,
SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID
NO: 14, SEQ ID NO: 16, and SEQ ID NO: 20, and by amino acid
residues about 49 to 157 of SEQ ID NO: 27 and amino acid residues
about 28 to 136 of SEQ ID NO: 29. A full length mature GDF-11
peptide is exemplified by amino acid residues about 299 to 407 of
SEQ ID NO: 25. Functional peptide portions of the mature GDF
peptides are exemplified by peptide portions of mature myostatin or
mature GDF-11 that have an agonist or antagonist activity with
respect to the activity of a mature GDF peptide. Preferably, the
mature GDF peptide activity is an ability to interact specifically
with its receptor.
[0089] As disclosed herein, a mature myostatin peptide (referred to
herein generally as "myostatin") can induce myostatin signal
transduction activity by interacting specifically with a myostatin
receptor expressed on the surface of a cell (see Example 7). Thus,
a functional peptide portion of myostatin can be obtained by
examining peptide portions of a mature myostatin peptide using a
method as described herein (Example 7) or otherwise known in the
art, and identifying functional peptide portions of myostatin that
specifically interact with a myostatin receptor, for example, an
activin type IIA receptor (Act RIIA) or Act RIIB receptor expressed
on a cell (Act RIIA, Cell 65:973-982, 1991; Act RIIB Cell
68:97-108, 1992, both herein incorporated by reference in their
entirety).
[0090] A myostatin prodomain can reduce or inhibit myostatin signal
transduction activity. In one embodiment, the myostatin prodomain
can interact specifically with myostatin, thereby reducing or
inhibiting the ability of the myostatin peptide to interact
specifically with its receptor. As disclosed herein, a precursor
promyostatin also lacks the ability to interact specifically with a
myostatin receptor, and, therefore, mutations in promyostatin that
reduce or inhibit the ability of promyostatin to be cleaved into
mature myostatin provide a means to reduce or inhibit myostatin
signal transduction. Accordingly, in another embodiment, the
present invention provides a mutant pro-GDF polypeptide, which
contains one or more amino acid mutations that disrupt proteolytic
cleavage of the mutant pro-GDF to an active mature GDF peptide.
[0091] A mutant pro-GDF polypeptide of the invention can have a
mutation that affects cleavage at a proteolytic cleavage site such
as the consensus proteolytic cleavage recognition site
Arg-Xaa-Xaa-Arg (SEQ ID NO: 21), which is present in pro-GDF
polypeptides. Thus, the mutation can be a mutation of an Arg
residue of SEQ ID NO: 21, such that a mutant promyostatin, for
example, cannot be cleaved into a myostatin prodomain and a mature
myostatin peptide. However, the mutation also can be at a site
other than the proteolytic cleavage site, and can alter the ability
of the protease to bind to the pro-GDF polypeptide so as to effect
proteolysis at the cleavage site. A mutant pro-GDF polypeptide of
the invention, for example, a mutant promyostatin or mutant
pro-GDF-11 can have a dominant negative activity with respect to
myostatin or GDF-11 and, therefore, can be useful for reducing or
inhibiting myostatin or GDF-11 signal transduction in a cell.
[0092] The present invention also provides a substantially purified
polynucleotide, which encodes a peptide portion of a promyostatin
polypeptide or a mutant promyostatin, or a peptide portion of a
pro-GDF-11 polypeptide or mutant pro-GDF-11, as described above. As
discussed in greater detail below, the invention also provides
polynucleotides useful as agents for modulating the affect of
myostatin on a cell, and further provides a polynucleotide encoding
a GDF receptor, or functional peptide portion thereof. Examples of
such polynucleotides are provided in the following disclosure. As
such, it should be recognized that the following disclosure is
relevant to the various embodiments of the invention as disclosed
herein.
[0093] The term "polynucleotide" is used broadly herein to mean a
sequence of two or more deoxyribonucleotides or ribonucleotides
that are linked together by a phosphodiester bond. As such, the
term "polynucleotide" includes RNA and DNA, which can be a gene or
a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid
sequence, or the like, and can be single stranded or double
stranded, as well as a DNA/RNA hybrid. Furthermore, the term
"polynucleotide" as used herein includes naturally occurring
nucleic acid molecules, which can be isolated from a cell, as well
as synthetic molecules, which can be prepared, for example, by
methods of chemical synthesis or by enzymatic methods such as by
the polymerase chain reaction (PCR). In various embodiments, a
polynucleotide of the invention can contain nucleoside or
nucleotide analogs, or a backbone bond other than a phosphodiester
bond (see above).
[0094] In general, the nucleotides comprising a polynucleotide are
naturally occurring deoxyribonucleotides, such as adenine,
cytosine, guanine or thymine linked to 2'-deoxyribose, or
ribonucleotides such as adenine, cytosine, guanine or uracil linked
to ribose. However, a polynucleotide also can contain nucleotide
analogs, including non-naturally occurring synthetic nucleotides or
modified naturally occurring nucleotides. Such nucleotide analogs
are well known in the art and commercially available, as are
polynucleotides containing such nucleotide analogs (Lin et al.,
Nucl. Acids Res. 22:5220-5234 (1994); Jellinek et al., Biochemistry
34:11363-11372 (1995); Pagratis et al., Nature Biotechnol. 15:68-73
(1997), each of which is incorporated herein by reference).
[0095] The covalent bond linking the nucleotides of a
polynucleotide generally is a phosphodiester bond. However, the
covalent bond also can be any of numerous other bonds, including a
thiodiester bond, a phosphorothioate bond, a peptide-like bond or
any other bond known to those in the art as useful for linking
nucleotides to produce synthetic polynucleotides (see, for example,
Tam et al., Nucl. Acids Res. 22:977-986 (1994); Ecker and Crooke,
BioTechnology 13:351360 (1995), each of which is incorporated
herein by reference). The incorporation of non-naturally occurring
nucleotide analogs or bonds linking the nucleotides or analogs can
be particularly useful where the polynucleotide is to be exposed to
an environment that can contain a nucleolytic activity, including,
for example, a tissue culture medium or upon administration to a
living subject, since the modified polynucleotides can be less
susceptible to degradation.
[0096] A polynucleotide comprising naturally occurring nucleotides
and phosphodiester bonds can be chemically synthesized or can be
produced using recombinant DNA methods, using an appropriate
polynucleotide as a template. In comparison, a polynucleotide
comprising nucleotide analogs or covalent bonds other than
phosphodiester bonds generally will be chemically synthesized,
although an enzyme such as T7 polymerase can incorporate certain
types of nucleotide analogs into a polynucleotide and, therefore,
can be used to produce such a polynucleotide recombinantly from an
appropriate template (Jellinek et al., supra, 1995).
[0097] Where a polynucleotide encodes a peptide, for example, a
peptide portion of promyostatin or a peptide agent, the coding
sequence generally is contained in a vector and is operatively
linked to appropriate regulatory elements, including, if desired, a
tissue specific promoter or enhancer. The encoded peptide can be
further operatively linked, for example, to peptide tag such as a
His-6 tag or the like, which can facilitate identification of
expression of the agent in the target cell. A polyhistidine tag
peptide such as His-6 can be detected using a divalent cation such
as nickel ion, cobalt ion, or the like. Additional peptide tags
include, for example, a FLAG epitope, which can be detected using
an anti-FLAG antibody (see, for example, Hopp et al., BioTechnology
6:1204 (1988); U.S. Pat. No. 5,011,912, each of which is
incorporated herein by reference); a c-myc epitope, which can be
detected using an antibody specific for the epitope; biotin, which
can be detected using streptavidin or avidin; and glutathione
S-transferase, which can be detected using glutathione. Such tags
can provide the additional advantage that they can facilitate
isolation of the operatively linked peptide or peptide agent, for
example, where it is desired to obtain a substantially purified
peptide corresponding to a proteolytic fragment of a myostatin
polypeptide.
[0098] As used herein, the term "operatively linked" or
"operatively associated" means that two or more molecules are
positioned with respect to each other such that they act as a
single unit and effect a function attributable to one or both
molecules or a combination thereof. For example, a polynucleotide
sequence encoding a peptide of the invention can be operatively
linked to a regulatory element, in which case the regulatory
element confers its regulatory effect on the polynucleotide
similarly to the way in which the regulatory element would effect a
polynucleotide sequence with which it normally is associated with
in a cell. A first polynucleotide coding sequence also can be
operatively linked to a second (or more) coding sequence such that
a chimeric polypeptide can be expressed from the operatively linked
coding sequences. The chimeric polypeptide can be a fusion
polypeptide, in which the two (or more) encoded peptides are
translated into a single polypeptide, i.e., are covalently bound
through a peptide bond; or can be translated as two discrete
peptides that, upon translation, can operatively associate with
each other to form a stable complex.
[0099] A chimeric polypeptide generally demonstrates some or all of
the characteristics of each of its peptide components. As such, a
chimeric polypeptide can be particularly useful in performing
methods of the invention, as disclosed herein. For example, in one
embodiment, a method of the invention can modulate myostatin signal
transduction in a cell. Thus, where one peptide component of a
chimeric polypeptide encodes a cell compartment localization domain
and a second peptide component encodes a dominant negative Smad
polypeptide, the functional chimeric polypeptide can be
translocated to the cell compartment designated by the cell
compartment localization domain and can have the dominant negative
activity of the Smad polypeptide, thereby modulating myostatin
signal transduction in the cell.
[0100] Cell compartmentalization domains are well known and
include, for example, a plasma membrane localization domain, a
nuclear localization signal, a mitochondrial membrane localization
signal, an endoplasmic reticulum localization signal, or the like
(see, for example, Hancock et al., EMBO J. 10:4033-4039, 1991; Buss
et al., Mol. Cell. Biol. 8:3960-3963, 1988; U.S. Pat. No. 5,776,689
each of which is incorporated herein by reference). Such a domain
can be useful to target an agent to a particular compartment in the
cell, or to target the agent for secretion from a cell. For
example, the kinase domain of a myostatin receptor such as Act RIIB
generally is associated with the inner surface of the plasma
membrane. Thus, a chimeric polypeptide comprising a dominant
negative myostatin receptor kinase domain, for example, a dominant
negative Act RIIB receptor, which lacks kinase activity, can
further comprise a plasma membrane localization domain, thereby
localizing the dominant negative Act RIIB kinase domain to the
inner cell membrane.
[0101] As disclosed herein, a pro-GDF signal peptide has cellular
localization activity. As used herein, the term "cellular
localization activity" refers to the ability of a signal peptide to
direct translocation of a peptide operably linked thereto to one or
more specific intracellular compartments or to direct secretion of
the molecule from the cell. As such, a pro-GDF signal peptide can
be particularly useful for directing translocation of a peptide or
other agent operably linked to the signal peptide to the same
intracellular compartments as a naturally expressed GDF having
substantially the same signal peptide. Furthermore, the signal
peptide, for example, a promyostatin signal peptide comprising
about the first 15 to 30 amino acids of promyostatin, can direct
secretion of an operably linked agent from the cell through the
same pathway as the naturally occurring pro-GDF having comprising
the signal peptide. Thus, particularly useful agents for performing
a method of the invention include a GDF prodomain or functional
peptide portion thereof that is operably linked to a GDF signal
peptide, preferably a promyostatin or pro-GDF-11 signal
peptide.
[0102] A polynucleotide of the invention, including a
polynucleotide agent useful in performing a method of the
invention, can be contacted directly with a target cell. For
example, oligonucleotides useful as antisense molecules, ribozymes,
or triplexing agents can be directly contacted with a target cell,
whereupon the enter the cell and effect their function. A
polynucleotide agent also can interact specifically with a
polypeptide, for example, a myostatin receptor (or myostatin),
thereby altering the ability of myostatin to interact specifically
with the receptor. Such polynucleotides, as well as methods of
making and identifying such polynucleotides, are disclosed herein
or otherwise well known in the art (see, for example,
O.quadrature.Connell et al., Proc. Natl. Acad. Sci., USA
93:5883-5887, 1996; Tuerk and Gold, Science 249:505-510, 1990; Gold
et al., Ann. Rev. Biochem. 64:763-797, 1995; each of which is
incorporated herein by reference).
[0103] A polynucleotide of the invention, which can encode a
peptide portion of a pro-GDF polypeptide such as promyostatin, or
can encode a mutant promyostatin polypeptide, or can encode a GDF
receptor or functional peptide portion thereof, or can be a
polynucleotide agent useful in performing a method of the
invention, can be contained in a vector, which can facilitate
manipulation of the polynucleotide, including introduction of the
polynucleotide into a target cell. The vector can be a cloning
vector, which is useful for maintaining the polynucleotide, or can
be an expression vector, which contains, in addition to the
polynucleotide, regulatory elements useful for expressing the
polynucleotide and, where the polynucleotide encodes a peptide, for
expressing the encoded peptide in a particular cell. An expression
vector can contain the expression elements necessary to achieve,
for example, sustained transcription of the encoding
polynucleotide, or the regulatory elements can be operatively
linked to the polynucleotide prior to its being cloned into the
vector.
[0104] An expression vector (or the polynucleotide) generally
contains or encodes a promoter sequence, which can provide
constitutive or, if desired, inducible or tissue specific or
developmental stage specific expression of the encoding
polynucleotide, a poly-A recognition sequence, and a ribosome
recognition site or internal ribosome entry site, or other
regulatory elements such as an enhancer, which can be tissue
specific. The vector also can contain elements required for
replication in a prokaryotic or eukaryotic host system or both, as
desired. Such vectors, which include plasmid vectors and viral
vectors such as bacteriophage, baculovirus, retrovirus, lentivirus,
adenovirus, vaccinia virus, semliki forest virus and
adeno-associated virus vectors, are well known and can be purchased
from a commercial source (Promega, Madison Wis.; Stratagene, La
Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or can be constructed by
one skilled in the art (see, for example, Meth. Enzymol., Vol. 185,
Goeddel, ed. (Academic Press, Inc., 1990); Jolly, Canc. Gene Ther.
1:51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25:37-42, 1993;
Kirshenbaum et al., J. Clin. Invest. 92:381-387, 1993; each of
which is incorporated herein by reference).
[0105] A tetracycline (tet) inducible promoter can be particularly
useful for driving expression of a polynucleotide of the invention,
for example, a polynucleotide encoding a dominant negative form of
myostatin, in which the proteolytic processing site has been
mutated, or encoding a myostatin prodomain, which can form a
complex with a mature myostatin peptide, or encoding a dominant
negative form of a GDF receptor. Upon administration of
tetracycline, or a tetracycline analog, to a subject containing a
polynucleotide operatively linked to a tet inducible promoter,
expression of the encoded peptide is induced, whereby the peptide
can effect its activity, for example, whereby a peptide agent can
reduce or inhibit myostatin signal transduction. Such a method can
be used, for example, to induce muscle hypertrophy in an adult
organism.
[0106] The polynucleotide also can be operatively linked to tissue
specific regulatory element, for example, a muscle cell specific
regulatory element, such that expression of an encoded peptide is
restricted to the muscle cells in an individual, or to muscle cells
in a mixed population of cells in culture, for example, an organ
culture. Muscle cell specific regulatory elements including, for
example, the muscle creatine kinase promoter (Sternberg et al.,
Mol. Cell. Biol. 8:2896-2909, 1988, which is incorporated herein by
reference) and the myosin light chain enhancer/promoter (Donoghue
et al., Proc. Natl. Acad. Sci., USA 88:5847-5851, 1991, which is
incorporated herein by reference) are well known in the art.
[0107] Viral expression vectors can be particularly useful for
introducing a polynucleotide into a cell, particularly a cell in a
subject. Viral vectors provide the advantage that they can infect
host cells with relatively high efficiency and can infect specific
cell types. For example, a polynucleotide encoding a myostatin
prodomain or functional peptide portion thereof can be cloned into
a baculovirus vector, which then can be used to infect an insect
host cell, thereby providing a means to produce large amounts of
the encoded prodomain. The viral vector also can be derived from a
virus that infects cells of an organism of interest, for example,
vertebrate host cells such as mammalian, avian or piscine host
cells. Viral vectors can be particularly useful for introducing a
polynucleotide useful in performing a method of the invention into
a target cell. Viral vectors have been developed for use in
particular host systems, particularly mammalian systems and
include, for example, retroviral vectors, other lentivirus vectors
such as those based on the human immunodeficiency virus (HIV),
adenovirus vectors, adeno-associated virus vectors, herpesvirus
vectors, vaccinia virus vectors, and the like (see Miller and
Rosman, BioTechniques 7:980-990, 1992; Anderson et al., Nature
392:25-30 Suppl., 1998; Verma and Somia, Nature 389:239-242, 1997;
Wilson, New Engl. J. Med. 334:1185-1187 (1996), each of which is
incorporated herein by reference).
[0108] When retroviruses, for example, are used for gene transfer,
replication competent retroviruses theoretically can develop due to
recombination of retroviral vector and viral gene sequences in the
packaging cell line utilized to produce the retroviral vector.
Packaging cell lines in which the production of replication
competent virus by recombination has been reduced or eliminated can
be used to minimize the likelihood that a replication competent
retrovirus will be produced. All retroviral vector supernatants
used to infect cells are screened for replication competent virus
by standard assays such as PCR and reverse transcriptase assays.
Retroviral vectors allow for integration of a heterologous gene
into a host cell genome, which allows for the gene to be passed to
daughter cells following cell division.
[0109] A polynucleotide, which can be contained in a vector, can be
introduced into a cell by any of a variety of methods known in the
art (Sambrook et al., Molecular Cloning: A laboratory manual (Cold
Spring Harbor Laboratory Press 1989); Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1987, and supplements through 1995), each of which is incorporated
herein by reference). Such methods include, for example,
transfection, lipofection, microinjection, electroporation and,
with viral vectors, infection; and can include the use of
liposomes, microemulsions or the like, which can facilitate
introduction of the polynucleotide into the cell and can protect
the polynucleotide from degradation prior to its introduction into
the cell. The selection of a particular method will depend, for
example, on the cell into which the polynucleotide is to be
introduced, as well as whether the cell is isolated in culture, or
is in a tissue or organ in culture or in situ.
[0110] Introduction of a polynucleotide into a cell by infection
with a viral vector is particularly advantageous in that it can
efficiently introduce the nucleic acid molecule into a cell ex vivo
or in vivo (see, for example, U.S. Pat. No. 5,399,346, which is
incorporated herein by reference). Moreover, viruses are very
specialized and can be selected as vectors based on an ability to
infect and propagate in one or a few specific cell types. Thus,
their natural specificity can be used to target the nucleic acid
molecule contained in the vector to specific cell types. As such, a
vector based on an HIV can be used to infect T cells, a vector
based on an adenovirus can be used, for example, to infect
respiratory epithelial cells, a vector based on a herpesvirus can
be used to infect neuronal cells, and the like. Other vectors, such
as adeno-associated viruses can have greater host cell range and,
therefore, can be used to infect various cell types, although viral
or non-viral vectors also can be modified with specific receptors
or ligands to alter target specificity through receptor mediated
events.
[0111] The present invention also provides antibodies that
specifically bind a peptide portion of a promyostatin polypeptide
or a mutant promyostatin polypeptide. Particularly useful
antibodies of the invention include antibodies that specifically
bind a myostatin prodomain, or a functional peptide portion
thereof, and antibodies that bind a promyostatin polypeptide and
reduce or inhibit proteolytic cleavage of the promyostatin to a
mature myostatin peptide. In addition, an antibody of the invention
can be an antibody that specifically binds a GDF receptor, or
functional peptide portion thereof, as described below. Methods of
preparing and isolating an antibody of the invention are described
in greater detail below, the disclosure of which is incorporated
herein by reference.
[0112] Myostatin is essential for proper regulation of skeletal
muscle mass. As compared to wild type mice, myostatin knock-out
mice, which lack myostatin, have two to three times the amount of
muscle due to a combination of hyperplasia and hypertrophy. As
disclosed herein, myostatin knock-out mice also have a dramatic
reduction in fat accumulation due, at least in part, to an
increased anabolic state of skeletal muscle tissue throughout the
body. Conversely, overexpression of myostatin in nude mice induced
a wasting syndrome that resembles the cachectic state observed in
human patients suffering from chronic diseases such as cancer or
AIDS. As further disclosed herein, myostatin activity can be
mediated through a signal transduction having the characteristics
of the Smad signal transduction pathway. Accordingly, the invention
provides methods of modulating an effect of myostatin on a cell by
contacting the cell with an agent that affects myostatin signal
transduction in the cell.
[0113] As used herein, the term "modulate," when used in reference
to an effect of myostatin on a cell, means that myostatin signal
transduction in the cell either is increased or is reduced or
inhibited. The terms "increase" and "reduce or inhibit" are used in
reference to a baseline level of myostatin signal transduction
activity, which can be the level of activity of the signal
transduction pathway in the absence of myostatin, or the level of
activity in a normal cell in the presence of myostatin. For
example, the myostatin signal transduction pathway exhibits a
particular activity in a muscle cell contacted with myostatin, and,
upon further contacting the muscle cell with a myostatin prodomain,
myostatin signal transduction activity can be reduced or inhibited.
As such, a myostatin prodomain is an agent useful for reducing or
inhibiting myostatin signal transduction. Similarly, a prodomain of
another GDF family member such as a GDF-11 prodomain, or of another
TGF-.theta. family member such as an activin prodomain, MIS
prodomain, or the like, can be useful for reducing myostatin signal
transduction. The terms "reduce or inhibit" are used together
herein because it is recognized that, in some cases, the level of
myostatin signal transduction can be reduced below a level that can
be detected by a particular assay. As such, it may not be
determinable using such an assay as to whether a low level of
myostatin signal transduction remains, or whether the signal
transduction is completely inhibited.
[0114] As used herein, the term "myostatin signal transduction"
refers to the series of events, generally a series of
protein-protein interactions, that occurs in a cell due to the
specific interaction of myostatin with a myostatin receptor
expressed on the surface of the cell. As such, myostatin signal
transduction can be detected, for example, by detecting a specific
interaction of myostatin with its receptor on a cell, by detecting
phosphorylation of one or more polypeptides involved in a myostatin
signal transduction pathway in the cell, by detecting expression of
one or more genes that are specifically induced due to myostatin
signal transduction, or by detecting a phenotypic change that
occurs in response to myostatin signal transduction (see Examples).
As disclosed herein, an agent useful in a method of the invention
can act as an agonist to stimulate myostatin signal transduction or
as an antagonist to reduce or inhibit myostatin signal
transduction.
[0115] The methods of the present invention are exemplified
generally herein with respect to myostatin. It should be
recognized, however, that the methods of the invention can more
broadly encompass modulating an effect of other GDF peptides, for
example, GDF-11, on a cell by contacting the cell with an agent
that affects signal transduction due to the GDF in the cell.
Methods of practicing the full scope of the invention will be
readily known in view of the present disclosure, which includes,
for example, methods for identifying GDF receptors, methods for
identifying agents that modulate signal transduction due to a
specific interaction of the GDF with its receptor, and the
like.
[0116] A myostatin signal transduction pathway is exemplified
herein by the Smad pathway, which is initiated upon myostatin
specifically interacting with the extracellular domain of an
activin type II receptor and propagated through interactions of
intracellular polypeptides, including Smad proteins, in the cell.
In general, myostatin signal transduction is associated with
phosphorylation or dephosphorylation of specific intracellular
polypeptides such as Smad polypeptides. Thus, myostatin signal
transduction in a cell can be detected by detecting an increased
level of phosphorylation of one or more Smad polypeptides in the
presence of myostatin as compared to the level of phosphorylation
of the polypeptides in the absence of myostatin. A method of the
invention provides a means to increase or decrease myostatin signal
transduction and, therefore, the level of phosphorylation of an
Smad polypeptide involved in a myostatin signal transduction
pathway will be increased above a normal level or decreased below a
level expected in the presence of myostatin, respectively.
[0117] A method of the invention can be performed, for example, by
contacting under suitable conditions a target cell and an agent
that affects myostatin signal transduction in the cell. Suitable
conditions can be provided by placing the cell, which can be an
isolated cell or can be a component of a tissue or organ, in an
appropriate culture medium, or by contacting the cell in situ in an
organism. For example, a medium containing the cell can be
contacted with an agent the affects the ability of myostatin to
specifically interact with a myostatin receptor expressed on the
cell, or with an agent that affects a myostatin signal transduction
pathway in the cell. In general, the cell is a component of a
tissue or organ in a subject, in which case contacting the cell can
comprise administering the agent to the subject. However, the cell
also can be manipulated in culture, then can be maintained in
culture, administered to a subject, or used to produce a transgenic
nonhuman animal.
[0118] An agent useful in a method of the invention can be any type
of molecule, for example, a polynucleotide, a peptide, a
peptidomimetic, peptoids such as vinylogous peptoids, a small
organic molecule, or the like, and can act in any of various ways
to affect myostatin signal transduction. The agent can act
extracellularly by binding myostatin or a myostatin receptor such
as an activin receptor, thereby altering the ability of myostatin
to specifically interact with its receptor, or can act
intracellularly to alter myostatin signal transduction in the cell.
In addition, the agent can be an agonist, which mimics or enhances
the effect of myostatin on a cell, for example, the ability of
myostatin to specifically interact with its receptor, thereby
increasing myostatin signal transduction in the cell; or can be an
antagonist, which can reduces or inhibits the effect of myostatin
on a cell, thereby reducing or inhibiting myostatin signal
transduction in the cell.
[0119] As used herein, the term "specific interaction" or
"specifically binds" or the like means that two molecules form a
complex that is relatively stable under physiologic conditions. The
term is used herein in reference to various interactions,
including, for example, the interaction of myostatin and a
myostatin receptor, the interaction of the intracellular components
of a myostatin signal transduction pathway, the interaction of an
antibody and its antigen, and the interaction of a myostatin
prodomain with myostatin. A specific interaction can be
characterized by a dissociation constant of at least about
1.times.10-6 M, generally at least about 1.times.10-7 M, usually at
least about 1.times.10-8 M, and particularly at least about
1.times.10-9 M or 1.times.10-10 M or greater. A specific
interaction generally is stable under physiological conditions,
including, for example, conditions that occur in a living
individual such as a human or other vertebrate or invertebrate, as
well as conditions that occur in a cell culture such as used for
maintaining mammalian cells or cells from another vertebrate
organism or an invertebrate organism. In addition, a specific
interaction such as the extracellular interaction of a myostatin
prodomain and myostatin generally is stable under conditions such
as those used for aquaculture of a commercially valuable marine
organism. Methods for determining whether two molecules interact
specifically are well known and include, for example, equilibrium
dialysis, surface plasmon resonance, and the like.
[0120] An agent that alters a specific interaction of myostatin
with its receptor can act, for example, by binding to myostatin
such that it cannot interact specifically with its cellular
receptor, by competing with myostatin for binding to its receptor,
or by otherwise by-passing the requirement that myostatin
specifically interact with its receptor in order to induce
myostatin signal transduction. A truncated myostatin receptor such
as a soluble extracellular domain of a myostatin receptor is an
example of an agent that can bind myostatin, thereby sequestering
myostatin and reducing or inhibiting its ability to interact
specifically with a cell surface myostatin receptor. A myostatin
prodomain or functional peptide portion thereof is another example
of an agent that can bind myostatin, thereby reducing or inhibiting
the ability of the myostatin to interact specifically with a cell
surface myostatin receptor. Such myostatin antagonists are useful
in practicing a method of the invention, particularly for reducing
or inhibiting myostatin signal transduction in a cell.
[0121] Follistatin is another example of an agent that can bind to
myostatin, thereby reducing or inhibiting the ability of myostatin
to interact specifically with its receptor. Follistatin can bind to
and inhibit the activity of various TGF-.beta. family members,
including myostatin (GDF-8; U.S. Pat. No. 6,004,937) and GDF-11
(Gamer et al., Devel. Biol. 208:222-232, 1999) and, therefore, can
be used for performing a method as disclosed. Although the use of
follistatin for modulating the effects of myostatin previously has
been described (U.S. Pat. No. 6,004,937), it was not known, prior
to the present disclosure, that follistatin reduces or inhibits the
ability of myostatin to interact specifically with a myostatin
receptor such as Act RIIB.
[0122] An agent useful in a method of the invention also can
interact with a cellular myostatin receptor, thereby competing with
myostatin for the receptor. Such an agent can be, for example, an
antibody that specifically binds a cell surface myostatin receptor,
including all or a portion of the myostatin binding domain, thereby
preventing myostatin from interacting specifically with the
receptor. Such an anti-myostatin receptor antibody can be selected
for its ability to specifically bind the receptor without
activating myostatin signal transduction and, therefore, can be
useful as a myostatin antagonist for reducing or inhibiting
myostatin signal transduction; or can be selected for its ability
to specifically bind the receptor and activate myostatin signal
transduction, thus acting as a myostatin agonist. The antibody can
be raised using a myostatin receptor, or the extracellular domain
of the receptor, as an immunogen, or can be an anti-idiotype
antibody, which is raised against an anti-myostatin antibody and
mimics myostatin. Anti-GDF receptor antibodies are discussed in
greater detail below.
[0123] An agent useful in a method of the invention also can be an
agent that reduces or inhibits proteolytic cleavage of a pro-GDF
polypeptide to an active mature GDF peptide, thereby reducing or
inhibiting GDF signal transduction. Such an agent can be a protease
inhibitor, particularly one that inhibits the activity of a
protease that recognizes and cleaves an Arg-Xaa-Xaa-Arg (SEQ ID NO:
21) proteolytic recognition site. Where the pro-GDF is
promyostatin, an anti-myostatin antibody that reduces or inhibits
the specific binding of a protease to the Arg-Xaa-Xaa-Arg (SEQ ID
NO: 21) proteolytic cleavage site in myostatin also can be used
reduce or inhibit proteolysis of promyostatin, thereby reducing the
amount of mature myostatin produced. Such an antibody can bind the
proteolytic cleavage site, or can bind some other site on the
pro-GDF polypeptide such that binding of and cleavage by the
protease is reduced or inhibited.
[0124] In addition, an agent useful in a method of the invention
can be a mutant myostatin receptor, which, for example, lacks
myostatin signal transduction activity in response to myostatin
binding, or has constitutive myostatin signal transduction
activity. For example, a mutant myostatin receptor can have a point
mutation, a deletion, or the like in its kinase domain such that
the receptor lacks kinase activity. Such a dominant negative mutant
myostatin receptor lacks the ability to transmit myostatin signal
transduction despite the fact that it can specifically bind
myostatin.
[0125] An agent useful in a method of the invention also can
modulate the level or activity of an intracellular polypeptide
involved in a myostatin signal transduction pathway. As disclosed
herein, regulation of muscle growth by myostatin can involve
components of a signal transduction pathway that is activated by
activin type II receptors (see Examples 7 and 9; see, also, Example
14). Myostatin specifically interacts with activin type IIB
receptors (Act RIIB) expressed in COS cells in culture (Example 7).
The low affinity of binding indicates that myostatin binding to Act
RIIB in vivo may involve other factors, similar to TGF-.beta.,
which has significantly higher affinity for the type II receptor
when the type I receptor also is present (Attisano et al., Cell
75:671-680, 1993), or to other systems that require other molecules
for presenting the ligand to the signaling receptor (Massague,
supra, 1998; Wang et al., Cell 67:795-805, 1991).
[0126] The specific interaction of myostatin with Act RIIB
indicates that myostatin signal transduction can involve components
of the Smad signal transduction pathway. Thus, the Smad signal
transduction pathway provides a target for modulating the effect of
myostatin on a cell, and agents that affect the Smad pathway can be
useful for modulating myostatin signal transduction in a cell.
[0127] Agents useful for modulating the level or activity of
intracellular polypeptide components of a GDF signal transduction
include agonists, which can increase signal transduction activity,
and antagonists, which can reduce or inhibit signal transduction
activity. With respect to myostatin, for example, agents that can
increase myostatin signal transduction activity are exemplified by
phosphatase inhibitors, which can reduce or inhibit
dephosphorylation of Smad polypeptides, thereby prolonging the
signal transducing activity of the Smad. Dominant negative Smad 6
or Smad 7 polypeptides, which can negate the inhibitory effect of
Smad 6 and Smad 7 on myostatin signal transduction, are additional
examples of agents that can increase myostatin signal transduction
activity by increasing Smad signal transduction.
[0128] Antagonist agents that can reduce or inhibit myostatin
signal transduction activity are exemplified by dominant negative
Smad polypeptides such as dominant negative Smad 2, Smad 3 or Smad
4, in which the C-terminal phosphorylation sites have been mutated.
The inhibitory Smad polypeptides such as Smad 6 and Smad 7, which
inhibit the activation of Smad 2 and Smad 3; and a c-ski
polypeptide, which binds Smad polypeptides and inhibits signal
transduction, are additional examples of antagonists useful for
reducing or inhibiting myostatin signal transduction by decreasing
Smad signal transduction.
[0129] Where the agent that acts intracellularly is a peptide, it
can be contacted with the cell directly, or a polynucleotide
encoding the peptide (or polypeptide) can be introduced into the
cell and the peptide can be expressed in the cell. It is recognized
that some of the peptides useful in a method of the invention are
relatively large and, therefore, may not readily traverse a cell
membrane. However, various methods are known for introducing a
peptide into a cell. The selection of a method for introducing such
a peptide into a cell will depend, in part, on the characteristics
of the target cell, into which the polypeptide is to be provided.
For example, where the target cells, or a few cell types including
the target cells, express a receptor, which, upon binding a
particular ligand, is internalized into the cell, the peptide agent
can be operatively associated with the ligand. Upon binding to the
receptor, the peptide is translocated into the cell by
receptor-mediated endocytosis. The peptide agent also can be
encapsulated in a liposome or formulated in a lipid complex, which
can facilitate entry of the peptide into the cell, and can be
further modified to express a receptor (or ligand), as above. The
peptide agent also can be introduced into a cell by engineering the
peptide to contain a protein transduction domain such as the human
immunodeficiency virus TAT protein transduction domain, which
facilitates translocation of the peptide into the cell (see
Schwarze et al., Science 285:1569-1572 (1999), which is
incorporated herein by reference; see, also, Derossi et al., J.
Biol. Chem. 271:18188 (1996)).
[0130] The target cell also can be contacted with a polynucleotide
encoding the peptide agent, which can be expressed in the cell. The
expressed peptide agent can be a mutant GDF receptor or peptide
portion thereof. Example of a mutant GDF receptor include a
kinase-deficient form of a myostatin receptor such as a dominant
negative Act RIIA or Act RIIB, which can, but need not, have the
ability to specifically bind a ligand (e.g., myostatin); and a
truncated myostatin or other GDF receptor, such as a soluble form
of a myostatin receptor, which binds myostatin, thereby
sequestering it from interacting specifically with a cellular
myostatin receptor; a dominant-negative form of a Smad polypeptide
such as a dominant negative Smad 3, in which the C-terminal
phosphorylation sites have been mutated (Liu et al., Proc. Natl.
Acad. Sci., USA 94:10669-10674, 1997); a Smad 7 polypeptide, which
inhibits the activation of Smad 2 and Smad 3 (Heldin et al., Nature
390:465-471, 1997); or a c-ski polypeptide, which can bind a Smad
polypeptide and inhibit signal transduction by the Smad (Suprave et
al., Genes Devel. 4:1462-1472, 1990).
[0131] Expression of a c-ski peptide agent in a cell can be
particularly useful for modulating myostatin signal transduction.
Mice lacking c-ski show a severe reduction in skeletal muscle mass
(Berk et al., Genes Devel. 11:2029-2039, 1997), whereas transgenic
mice that overexpress c-ski in muscle show dramatic muscle
hypertrophy (Suprave et al., supra, 1990). c-ski interacts with and
blocks the activity of certain Smad proteins, including Smad 2,
Smad 3 and Smad 4, which mediate signaling of TGF-.beta. and
activin type II receptors (Luo et al., Genes Devel. 13:2196-1106,
1999; Stroschein et al., Science 286:771-774, 1999; Sun et al.,
Mol. Cell 4:499-509, 1999a; Sun et al., Proc. Natl. Acad. Sci., USA
96:112442-12447, 1999b; Akiyoshi et al., J. Biol. Chem. 274:35269,
1999). Thus, in view of the present disclosure that myostatin
activity can be mediated through Act RIIB binding, it will be
recognized that the activity of myostatin, or of any GDF that
utilizes an Smad pathway, can be modulated by increasing or
decreasing c-ski expression in a target cell.
[0132] An agent useful in a method of the invention can be a
polynucleotide, which can be contacted with or introduced into a
cell as described above. Generally, but not necessarily, the
polynucleotide is introduced into the cell, where it effects its
function either directly, or following transcription or translation
or both. For example, as discussed above, the polynucleotide can
encode a peptide agent, which is expressed in the cell and
modulates myostatin activity. Such an expressed peptide can be, for
example, a mutant promyostatin polypeptide, which cannot be cleaved
into active myostatin; or can be a mutant myostatin receptor, for
example, a truncated myostatin receptor extracellular domain; a
myostatin receptor extracellular domain operatively associated with
a membrane anchoring domain; or a mutant myostatin receptor lacking
protein kinase activity. Methods for introducing a polynucleotide
into a cell are exemplified below or otherwise known in the
art.
[0133] A polynucleotide agent useful in a method of the invention
also can be, or can encode, an antisense molecule, a ribozyme or a
triplexing agent. For example, the polynucleotide can be (or can
encode) an antisense nucleotide sequence such as an antisense c-ski
nucleotide sequence, which can act as an agonist to increase
myostatin signal transduction in a cell; or an antisense Smad
nucleotide sequence, which can act either as an agonist to increase
myostatin signal transduction or as antagonist to reduce or inhibit
myostatin signal transduction, depending on the particular Smad
antisense nucleotide sequence. Such polynucleotides can be
contacted directly with a target cell and, upon uptake by the cell,
can effect their antisense, ribozyme or triplexing activity; or can
be encoded by a polynucleotide that is introduced into a cell,
whereupon the polynucleotide is expressed to produce, for example,
an antisense RNA molecule or ribozyme, which effects its
activity.
[0134] An antisense polynucleotide, ribozyme or triplexing agent is
complementary to a target sequence, which can be a DNA or RNA
sequence, for example, messenger RNA, and can be a coding sequence,
a nucleotide sequence comprising an intron-exon junction, a
regulatory sequence such as a Shine-Delgarno sequence, or the like.
The degree of complementarity is such that the polynucleotide, for
example, an antisense polynucleotide, can interact specifically
with the target sequence in a cell. Depending on the total length
of the antisense or other polynucleotide, one or a few mismatches
with respect to the target sequence can be tolerated without losing
the specificity of the polynucleotide for its target sequence.
Thus, few if any mismatches would be tolerated in an antisense
molecule consisting, for example, of 20 nucleotides, whereas
several mismatches will not affect the hybridization efficiency of
an antisense molecule that is complementary, for example, to the
full length of a target mRNA encoding a cellular polypeptide. The
number of mismatches that can be tolerated can be estimated, for
example, using well known formulas for determining hybridization
kinetics (see Sambrook et al., supra, 1989) or can be determined
empirically using methods as disclosed herein or otherwise known in
the art, particularly by determining that the presence of the
antisense polynucleotide, ribozyme, or triplexing agent in a cell
decreases the level of the target sequence or the expression of a
polypeptide encoded by the target sequence in the cell.
[0135] A polynucleotide useful as an antisense molecule, a ribozyme
or a triplexing agent can inhibit translation or cleave the nucleic
acid molecule, thereby modulating myostatin signal transduction in
a cell. An antisense molecule, for example, can bind to an mRNA to
form a double stranded molecule that cannot be translated in a
cell. Antisense oligonucleotides of at least about 15 to 25
nucleotides are preferred since they are easily synthesized and can
hybridize specifically with a target sequence, although longer
antisense molecules can be expressed from a polynucleotide
introduced into the target cell. Specific nucleotide sequences
useful as antisense molecules can be identified using well known
methods, for example, gene walking methods (see, for example,
Seimiya et al., J. Biol. Chem. 272:4631-4636 (1997), which is
incorporated herein by reference). Where the antisense molecule is
contacted directly with a target cell, it can be operatively
associated with a chemically reactive group such as iron-linked
EDTA, which cleaves a target RNA at the site of hybridization. A
triplexing agent, in comparison, can stall transcription (Maher et
al., Antisense Res. Devel. 1:227 (1991); Helene, Anticancer Drug
Design 6:569 (1991)). Thus, a triplexing agent can be designed to
recognize, for example, a sequence of a Smad gene regulatory
element, thereby reducing or inhibiting the expression of a Smad
polypeptide in the cell, thereby modulating myostatin signal
transduction in a target cell.
[0136] The present invention also provides a method of identifying
an agent that can alter the effect of a GDF such myostatin on a
cell, particularly agents that can alter the ability of the GDF to
interact specifically with its cellular receptor. Such agents can
act by increasing or decreasing the ability of the GDF to interact
specifically with its receptor and, therefore, are useful for
increasing or decreasing GDF signal transduction, respectively. A
screening method of the invention is exemplified herein using a
myostatin receptor, for example, an activin type II receptor such
as Act RIIA or Act RIIB.
[0137] A screening method of the invention can be performed, for
example, by contacting under suitable conditions myostatin, or a
functional peptide portion thereof, a myostatin receptor such as
Act RIIA or Act RIIB, and an agent to be tested. The myostatin, the
receptor and the agent can be contacted in any order as desired. As
such, the screening method can be used to identify agents that can
competitively or non-competitively inhibit myostatin binding to the
receptor, agents that can mediate or enhance myostatin binding to
the receptor, agents that can induce dissociation of specifically
bound myostatin from the receptor, and agents that otherwise affect
the ability of myostatin to induce signal transduction, such agents
having agonist or antagonist activity. Appropriate control
reactions are performed to confirm that the action of the agent is
specific with respect to the myostatin or other GDF receptor.
[0138] Suitable conditions for performing a screening method of the
invention can be any conditions that allow myostatin to
specifically interact with its receptor, including methods as
disclosed herein (see Examples 7 and 9) or otherwise known in the
art. Thus, suitable conditions for performing the screening assay
can be, for example, in vitro conditions using a substantially
purified myostatin receptor; cell culture conditions, utilizing a
cell that normally expresses a myostatin receptor, for example, an
adipocyte or a muscle cell, or a cell that has been genetically
modified to express a functional myostatin receptor on its surface;
or in situ conditions as occur in an organism.
[0139] A screening method of the invention also can be performed
using the methods of molecular modeling as described above. The
utilization of a molecular modeling method provides a convenient,
cost effective means to identify those agents, among a large
population such as a combinatorial library of potential agents,
that are most likely to interact specifically with a GDF receptor,
thereby reducing the number of potential agents that need to be
screened using a biological assay. Upon identifying agents that
interact specifically with a GDF receptor such as Act RIIB using a
molecular modeling method, the selected agents can be examined for
the ability to modulate an effect of a GDF such as myostatin on a
cell using the methods disclosed herein.
[0140] The ability of a test agent to modulate an effect of
myostatin can be detected using methods as disclosed herein (see
Examples 7 and 9) or otherwise known in the art. The term "test
agent" or "test molecule" is used broadly herein to mean any agent
that is being examined for agonist or antagonist activity in a
method of the invention. Although the method generally is used as a
screening assay to identify previously unknown molecules that can
act as agonist or antagonist agents as described herein, the
methods also can be used to confirm that a agent known to have a
particular activity in fact has the activity, for example, in
standardizing the activity of the agent.
[0141] A method of the invention can be performed, for example, by
contacting myostatin with a cell that has been genetically modified
to express an Act RIIB receptor, and determining the effect of an
agent, for example, a dominant negative Act RIIB, by examining
phosphorylation of a Smad polypeptide involved in the myostatin
signal transduction pathway. If desired, the cell can be further
genetically modified to contain a reporter nucleotide sequence, the
expression of which is dependent on the myostatin signal
transduction pathway, for example, on activation of the Smad
pathway, and the effect of the test agent can be determined by
comparing expression of the reporter nucleotide sequence in the
presence and absence of the agent, the myostatin, or both.
Expression of the reporter nucleotide sequence can be detected, for
example, by detecting an RNA transcript of the reporter nucleotide
sequence, or by detecting a polypeptide encoded by the reporter
nucleotide sequence. A polypeptide reporter can be, for example, a
.theta.-lactamase, chloramphenicol acetyltransferase, adenosine
deaminase, aminoglycoside phosphotransferase, dihydrofolate
reductase, hygromycin-B phosphotransferase, thymidine kinase,
.theta.-galactosidase, luciferase or xanthine guanine
phosphoribosyltransferase polypeptide or the like, and can be
detected, for example, by detecting radioactivity, luminescence,
chemiluminescence, fluorescence, enzymatic activity, or specific
binding due to the reporter polypeptide.
[0142] A screening method of the invention provides the advantage
that it can be adapted to high throughput analysis and, therefore,
can be used to screen combinatorial libraries of test agents in
order to identify those agents that can modulate an effect of
myostatin on a cell, including those agents that can alter a
specific interaction of myostatin and a myostatin receptor. Methods
for preparing a combinatorial library of molecules that can be
tested for a desired activity are well known in the art and
include, for example, methods of making a phage display library of
peptides, which can be constrained peptides (see, for example, U.S.
Pat. No. 5,622,699; U.S. Pat. No. 5,206,347; Scott and Smith,
Science 249:386-390, 1992; Markland et al., Gene 109:13-19, 1991;
each of which is incorporated herein by reference); a peptide
library (U.S. Pat. No. 5,264,563, which is incorporated herein by
reference); a peptidomimetic library (Blondelle et al., Trends
Anal. Chem. 14:83-92, 1995; a nucleic acid library
(O.quadrature.Connell et al., supra, 1996; Tuerk and Gold, supra,
1990; Gold et al., supra, 1995; each of which is incorporated
herein by reference); an oligosaccharide library (York et al.,
Carb. Res., 285:99-128, 1996; Liang et al., Science, 274:1520-1522,
1996; Ding et al., Adv. Expt. Med. Biol., 376:261-269, 1995; each
of which is incorporated herein by reference); a lipoprotein
library (de Kruif et al., FEBS Lett., 399:232-236, 1996, which is
incorporated herein by reference); a glycoprotein or glycolipid
library (Karaoglu et al., J. Cell Biol., 130:567-577, 1995, which
is incorporated herein by reference); or a chemical library
containing, for example, drugs or other pharmaceutical agents
(Gordon et al., J. Med. Chem., 37:1385-1401, 1994; Ecker and
Crooke, Bio/Technology, 13:351-360, 1995; each of which is
incorporated herein by reference). Polynucleotides can be
particularly useful as agents that can modulate a specific
interaction of myostatin and its receptor because nucleic acid
molecules having binding specificity for cellular targets,
including cellular polypeptides, exist naturally, and because
synthetic molecules having such specificity can be readily prepared
and identified (see, for example, U.S. Pat. No. 5,750,342, which is
incorporated herein by reference).
[0143] In view of the present disclosure, it will be recognized
that various animal model systems can be used as research tools to
identify agents useful for practicing a method of the invention.
For example, transgenic mice or other experimental animals can be
prepared using the various myostatin inhibitor constructs disclosed
herein, and the transgenic non-human organism can be examined
directly to determine the effect produced by expressing various
levels of a particular agent in the organism. In addition, the
transgenic organism, for example, a transgenic mouse, can be
crossbred with other mice, for example, with ob/ob, db/db, or
agouti lethal yellow mutant mice, to determine optimal levels of
expression of a myostatin inhibitor useful for treating or
preventing a disorder such as obesity, type II diabetes, or the
like. As such, the present invention provides transgenic non-human
organisms, particularly transgenic organisms containing a
polynucleotide encoding a myostatin prodomain, which can include
the myostatin signal peptide, a pro-peptide (see Examples) or a
polynucleotide encoding a mutant promyostatin polypeptide. Further,
the invention provides transgenic non-human organisms that express
high levels of follistatin or that express a dominant negative Act
IIB receptor (see Examples). Such organisms exhibit dramatic
increases in muscle mass, similar to that seen in myostatin
knock-out mice (see for example, U.S. Pat. No. 5,994,618, herein
incorporated by reference). As discussed herein, such animal models
are important to identify agents for enhancing muscle growth for
therapeutic purposes and agricultural applications. Methods of
producing transgenic non-human animals are known in the art (see
for Example U.S. Pat. Nos. 6,140,552; 5,998,698; 6,218,596, all of
which are herein incorporated by reference).
[0144] A myostatin polynucleotide of the invention is derived from
any organism, including mouse, rat, cow, pig, human, chicken,
ovine, turkey, finfish and other aquatic organisms and other
species. Such polynucleotides can be used to make transgenic
animals as described herein. Examples of aquatic animals for which
transgenics can be made (and from which myostatin polynucleotide
can be derived) include those belonging to the class Piscine such
as salmon, trout, char, ayu, carp, crucian carp, goldfish, roach,
whitebait, eel, conger eel, sardine, zebrafish, flying fish, sea
bass, sea bream, parrot bass, snapper, mackerel, horse mackerel,
tuna, bonito, yellowtail, rockfish, fluke, sole, flounder,
blowfish, filefish; those belonging to the class Cephalopoda such
as squid, cuttlefish, octopus; those belonging to the class
Pelecypoda such as clam (e.g., hardshell, Manila, Quahog, Surf,
Soft-shell); cockles, mussels, periwinkles; scallops (e.g., sea,
bay, calloo); conch, snails, sea cucumbers; ark shell; oyster
(e.g., C. virginica, Gulf, New Zealand, Pacific); those belonging
to the class Gastropoda such as turban shell, abalone (e.g. green,
pink, red); and those belonging to the class Crustacea such as
lobster, including but not limited to Spiny, Rock, and American;
prawn; shrimp, including but not limited to M. rosenbergii, P.
styllrolls, P. indicus, P. jeponious, P. monodon, P. vannemel, M.
ensis, S. melantho, N. norvegious, cold water shrimp; crab,
including but not limited to Blue, rook, stone, king, queen, snow,
brown, dungeness, Jonah, Mangrove, soft-shelled; squilla, krill,
langostinos; crayfish/crawfish, including but not limited to Blue,
Marron, Red Claw, Red Swamp, Soft-shelled, white; Annelida;
Chordates, including but not limited to reptiles, such as
alligators and turtles; and Amphibians, including frogs; and
Echinoderms, including but not limited to sea urchins.
[0145] Various methods are known for producing a transgenic animal.
In one method, an embryo at the pronuclear stage (a "one cell
embryo") is harvested from a female and the transgene is
microinjected into the embryo, in which case the transgene will be
chromosomally integrated into the germ cells and somatic cells of
the resulting mature animal. In another method, embryonic stem
cells are isolated and the transgene is incorporated into the stem
cells by electroporation, plasmid transfection or microinjection;
the stem cells are then reintroduced into the embryo, where they
colonize and contribute to the germ line. Methods for
microinjection of polynucleotides into mammalian species are
described, for example, in U.S. Pat. No. 4,873,191, which is
incorporated herein by reference. In yet another method, embryonic
cells are infected with a retrovirus containing the transgene,
whereby the germ cells of the embryo have the transgene
chromosomally integrated therein.
[0146] When the animals to be made transgenic are avian,
microinjection into the pronucleus of the fertilized egg is
problematic because avian fertilized ova generally go through cell
division for the first twenty hours in the oviduct and, therefore,
the pronucleus is inaccessible. Thus, the retrovirus infection
method is preferred for making transgenic avian species (see U.S.
Pat. No. 5,162,215, which is incorporated herein by reference). If
microinjection is to be used with avian species, however, the
embryo can be obtained from a sacrificed hen approximately 2.5
hours after the laying of the previous laid egg, the transgene is
microinjected into the cytoplasm of the germinal disc and the
embryo is cultured in a host shell until maturity (Love et al.,
Biotechnology 12, 1994). When the animals to be made transgenic are
bovine or porcine, microinjection can be hampered by the opacity of
the ova, thereby making the nuclei difficult to identify by
traditional differential interference-contrast microscopy. To
overcome this problem, the ova first can be centrifuged to
segregate the pronuclei for better visualization.
[0147] Non-human transgenic animals of the invention can be bovine,
porcine, ovine, piscine, avian or other animals. The transgene can
be introduced into embryonal target cells at various developmental
stages, and different methods are selected depending on the stage
of development of the embryonal target cell. The zygote is the best
target for microinjection. The use of zygotes as a target for gene
transfer has a major advantage in that the injected DNA can
incorporate into the host gene before the first cleavage (Brinster
et al., Proc. Natl. Acad. Sci., USA 82:4438-4442, 1985). As a
consequence, all cells of the transgenic non-human animal carry the
incorporated transgene, thus contributing to efficient transmission
of the transgene to offspring of the founder, since 50% of the germ
cells will harbor the transgene.
[0148] A transgenic animal can be produced by crossbreeding two
chimeric animals, each of which includes exogenous genetic material
within cells used in reproduction. Twenty-five percent of the
resulting offspring will be transgenic animals that are homozygous
for the exogenous genetic material, 50% of the resulting animals
will be heterozygous, and the remaining 25% will lack the exogenous
genetic material and have a wild type phenotype.
[0149] In the microinjection method, the transgene is digested and
purified free from any vector DNA, for example, by gel
electrophoresis. The transgene can include an operatively
associated promoter, which interacts with cellular proteins
involved in transcription, and provides for constitutive
expression, tissue specific expression, developmental stage
specific expression, or the like. Such promoters include those from
cytomegalovirus (CMV), Moloney leukemia virus (MLV), and herpes
virus, as well as those from the genes encoding metallothionein,
skeletal actin, Phosphenolpyruvate carboxylase (PEPCK),
phosphoglycerate (PGK), dihydrofolate reductase (DHFR), and
thymidine kinase (TK). Promoters from viral long terminal repeats
(LTRs) such as Rous sarcoma virus LTR also can be employed. When
the animals to be made transgenic are avian, preferred promoters
include those for the chicken .quadrature.-globin gene, chicken
lysozyme gene, and avian leukosis virus. Constructs useful in
plasmid transfection of embryonic stem cells will employ additional
regulatory elements, including, for example, enhancer elements to
stimulate transcription, splice acceptors, termination and
polyadenylation signals, ribosome binding sites to permit
translation, and the like.
[0150] In the retroviral infection method, the developing non-human
embryo can be cultured in vitro to the blastocyst stage. During
this time, the blastomeres can be targets for retroviral infection
(Jaenich, Proc. Natl. Acad. Sci, USA 73:1260-1264, 1976). Efficient
infection of the blastomeres is obtained by enzymatic treatment to
remove the zona pellucida (Hogan et al., Manipulating the Mouse
Embryo (Cold Spring Harbor Laboratory Press, 1986). The viral
vector system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al., Proc. Natl. Acad. Sci., USA 82:6927-6931, 1985; Van der Putten
et al., Proc. Natl. Acad. Sci, USA 82:6148-6152, 1985).
Transfection is easily and efficiently obtained by culturing the
blastomeres on a monolayer of virus producing cells (Van der Putten
et al., supra, 1985; Stewart et al., EMBO J. 6:383-388, 1987).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al., Nature 298:623-628, 1982). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic nonhuman animal.
Further, the founder can contain various retroviral insertions of
the transgene at different positions in the genome, which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line, albeit with low
efficiency, by intrauterine retroviral infection of the
mid-gestation embryo (Jahner et al., supra, 1982).
[0151] Embryonal stem cell (ES) also can be targeted for
introduction of the transgene. ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. Nature 292:154-156, 1981; Bradley et al., Nature
309:255-258, 1984; Gossler et al., Proc. Natl. Acad. Sci., USA
83:9065-9069, 1986; Robertson et al., Nature 322:445-448, 1986).
Transgenes can be efficiently introduced into the ES cells by DNA
transfection or by retrovirus mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts
from a nonhuman animal. The ES cells thereafter colonize the embryo
and contribute to the germ line of the resulting chimeric animal
(see Jaenisch, Science 240:1468-1474, 1988).
[0152] As disclosed herein, myostatin can exert its activity, at
least in part, through the Smad signal transduction pathway, and
myostatin expression can be associated with various pathological
conditions. As such, the present invention provides new targets for
the treatment of various pathological conditions associated with
myostatin, including metabolic conditions such as obesity and type
II diabetes. Accordingly, the present invention provides methods
for ameliorating the severity of a pathological condition in a
subject, wherein the pathologic condition is characterized at least
in part by an abnormal amount, development or metabolic activity of
muscle or adipose tissue, by modulating myostatin signal
transduction in a muscle cell or adipose tissue cell in the
subject.
[0153] Myostatin functions as a negative regulator of muscle growth
(McPherron et al., supra, 1997). Myostatin knock-out mice weighed
approximately 25% to 30% more than wild type littermates, and this
increase in body weight in the mice examined resulted entirely from
a dramatic increase in skeletal muscle tissue weight. In mice
lacking myostatin, the skeletal muscles weighed about 2 to 3 times
as much as the corresponding muscles of wild type littermates. This
increased muscle weight in the homozygous knock-out mice resulted
from a combination of hyperplasia and hypertrophy.
[0154] As disclosed herein, heterozygous myostatin knock-out mice
also have increased skeletal muscle mass, although to a lesser
extent than that observed in homozygous mutant mice, thus
demonstrating that myostatin acts in a dose-dependent manner in
vivo (see Example 1). Furthermore, overexpression of myostatin in
animals had the opposite effect with respect to muscle growth. For
example, nude mice carrying myostatin-expressing tumors developed a
wasting syndrome characterized by a dramatic loss of muscle and fat
weight (see Example 8). This syndrome in the nude mice resembled
the cachectic state that occurs in patients with chronic diseases
such as cancer or AIDS.
[0155] The serum levels of myostatin immunoreactive material have
been correlated with the status of patients with respect to muscle
wasting (Gonzalez-Kadavid et al., Proc. Natl. Acad. Med., USA
95:14938-14943, 1998, which is incorporated herein by reference).
Thus, patients with AIDS, who also showed signs of cachexia as
measured by loss of total body weight, had slightly increased serum
levels of myostatin immunoreactive material compared to either
normal males without AIDS or to AIDS patients that did not have
weight loss. However, the interpretation of these results was
complicated because the myostatin immunoreactive material detected
in the serum samples did not have the mobility on SDS gels that was
expected for authentic processed myostatin.
[0156] As disclosed herein, myostatin not only affects muscle mass,
but also affects the overall metabolism of an organism. For
example, myostatin is expressed in adipose tissue, and myostatin
deficient mice have a dramatic reduction in fat accumulation as the
animals age (see Examples II and III). Although no mechanism for
myostatin action is proposed herein, the effect of myostatin can be
direct effect of myostatin on adipose tissue, or can be an indirect
effect caused by the lack of myostatin activity on skeletal muscle
tissue. Regardless of the mechanism, the overall anabolic effect on
muscle tissue that results in response to decreased myostatin
activity can alter the overall metabolism of the organism and
affect the storage of energy in the form of fat, as demonstrated by
the introduction of a myostatin mutation into an obese mouse strain
(agouti lethal yellow (Ay) mice), which suppressed fat accumulation
by five-fold (see Example 5). Abnormal glucose metabolism also was
partially suppressed in agouti mice containing the myostatin
mutation. These results demonstrate that methods that inhibition of
myostatin can be used to treat or prevent metabolic diseases such
as obesity and type II diabetes.
[0157] The methods of the invention are useful, for example, for
ameliorating the severity of various pathologic conditions,
including, for example, the cachexia associated with chronic
diseases such as cancer (see Norton et al., Crit. Rev. Oncol.
Hematol. 7:289-327, 1987), as well as conditions such as type II
diabetes, obesity, and other metabolic disorders. As used herein,
the term "pathologic condition" refers to a disorder that is
characterized, at least in part, by an abnormal amount, development
or metabolic activity of muscle or adipose tissue. Such pathologic
conditions, which include, for example, obesity; conditions
associated with obesity, for example, atherosclerosis,
hypertension, and myocardial infarction; muscle wasting disorders
such as muscular dystrophy, neuromuscular diseases, cachexia, and
anorexia; and metabolic disorders such as type II diabetes, which
generally, but not necessarily, is associated with obesity, are
particularly amenable to treatment using a method of the
invention.
[0158] As used herein, the term "abnormal," when used in reference
to the amount, development or metabolic activity of muscle or
adipose tissue, is used in a relative sense in comparison to an
amount, development or metabolic activity that a skilled clinician
or other relevant artisan would recognize as being normal or ideal.
Such normal or ideal values are known to the clinician and are
based on average values generally observed or desired in a healthy
individual in a corresponding population. For example, the
clinician would know that obesity is associated with a body weight
that is about twenty percent above an "ideal" weight range for a
person of a particular height and body type. However, the clinician
would recognize that a body builder is not necessarily obese simply
by virtue of having a body weight that is twenty percent or more
above the weight expected for a person of the same height and body
type in an otherwise corresponding population. Similarly, the
artisan would know that a patient presenting with what appears to
abnormally decreased muscle activity could be identified as having
abnormal muscle development, for example, by subjecting the patient
to various strength tests and comparing the results with those
expected for an average healthy individual in a corresponding
population.
[0159] A method of the invention can ameliorate the severity of a
pathologic condition that is characterized, at least in part, by an
abnormal amount, development or metabolic activity in muscle or
adipose tissue, by modulating myostatin signal transduction in a
muscle or adipose tissue cell associated with the etiology of the
condition. As used herein, the term "ameliorate," when used in
reference to the severity of a pathologic condition, means that
signs or symptoms associated with the condition are lessened. The
signs or symptoms to be monitored will be characteristic of a
particular pathologic condition and will be well known to skilled
clinician, as will the methods for monitoring the signs and
conditions. For example, where the pathologic condition is type II
diabetes, the skilled clinician can monitor the glucose levels,
glucose clearance rates, and the like in the subject. Where the
pathologic condition is obesity or cachexia, the clinician can
simply monitor the subject's body weight.
[0160] The agent to be administered to the subject is administered
under conditions that facilitate contact of the agent with the
target cell and, if appropriate, entry into the cell. Entry of a
polynucleotide agent into a cell, for example, can be facilitated
by incorporating the polynucleotide into a viral vector that can
infect the cells. If a viral vector specific for the cell type is
not available, the vector can be modified to express a receptor (or
ligand) specific for a ligand (or receptor) expressed on the target
cell, or can be encapsulated within a liposome, which also can be
modified to include such a ligand (or receptor). A peptide agent
can be introduced into a cell by various methods, including, for
example, by engineering the peptide to contain a protein
transduction domain such as the human immunodeficiency virus TAT
protein transduction domain, which can facilitate translocation of
the peptide into the cell (see Schwarze et al., supra, 1999;
Derossi et al., supra, 1996).
[0161] The presence of the agent in the target cell can be
identified directly, for example, by operatively linking a
detectable label to the agent, by using an antibody specific for
the agent, particularly a peptide agent, or by detecting a
downstream effect due to the agent, for example, decreased
phosphorylation of an Smad polypeptide in the cell. An agent can be
labeled so as to be detectable using methods well known in the art
(Hermanson, "Bioconjugate Techniques" (Academic Press 1996), which
is incorporated herein by reference; see, also, Harlow and Lane,
supra, 1988). For example, a peptide or polynucleotide agent can be
labeled with various detectable moieties including a radiolabel, an
enzyme such as alkaline phosphatase, biotin, a fluorochrome, and
the like. Where the agent is contained in a kit, the reagents for
labeling the agent also can be included in the kit, or the reagents
can be purchased separately from a commercial source.
[0162] An agent useful in a method of the invention can be
administered to the site of the pathologic condition, or can be
administered by any method that provides the target cells with the
polynucleotide or peptide. As used herein, the term "target cells"
means muscle cells or adipocytes that are to be contacted with the
agent. For administration to a living subject, the agent generally
is formulated in a pharmaceutical composition suitable for
administration to the subject. Thus, the invention provides
pharmaceutical compositions containing an agent, which is useful
for modulating myostatin signal transduction in a cell, in a
pharmaceutically acceptable carrier. As such, the agents are useful
as medicaments for treating a subject suffering from a pathological
condition as defined herein.
[0163] Pharmaceutically acceptable carriers are well known in the
art and include, for example, aqueous solutions such as water or
physiologically buffered saline or other solvents or vehicles such
as glycols, glycerol, oils such as olive oil or injectable organic
esters. A pharmaceutically acceptable carrier can contain
physiologically acceptable compounds that act, for example, to
stabilize or to increase the absorption of the conjugate. Such
physiologically acceptable compounds include, for example,
carbohydrates, such as glucose, sucrose or dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins or other stabilizers or excipients. One
skilled in the art would know that the choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable
compound, depends, for example, on the physico-chemical
characteristics of the therapeutic agent and on the route of
administration of the composition, which can be, for example,
orally or parenterally such as intravenously, and by injection,
intubation, or other such method known in the art. The
pharmaceutical composition also can contain a second reagent such
as a diagnostic reagent, nutritional substance, toxin, or
therapeutic agent, for example, a cancer chemotherapeutic
agent.
[0164] The agent can be incorporated within an encapsulating
material such as into an oil-in-water emulsion, a microemulsion,
micelle, mixed micelle, liposome, microsphere or other polymer
matrix (see, for example, Gregoriadis, Liposome Technology, Vol. 1
(CRC Press, Boca Raton, Fla. 1984); Fraley, et al., Trends Biochem.
Sci., 6:77 (1981), each of which is incorporated herein by
reference). Liposomes, for example, which consist of phospholipids
or other lipids, are nontoxic, physiologically acceptable and
metabolizable carriers that are relatively simple to make and
administer. "Stealth" liposomes (see, for example, U.S. Pat. Nos.
5,882,679; 5,395,619; and 5,225,212, each of which is incorporated
herein by reference) are an example of such encapsulating materials
particularly useful for preparing a pharmaceutical composition
useful for practicing a method of the invention, and other "masked"
liposomes similarly can be used, such liposomes extending the time
that the therapeutic agent remain in the circulation. Cationic
liposomes, for example, also can be modified with specific
receptors or ligands (Morishita et al., J. Clin. Invest.,
91:2580-2585 (1993), which is incorporated herein by reference). In
addition, a polynucleotide agent can be introduced into a cell
using, for example, adenovirus-polylysine DNA complexes (see, for
example, Michael et al., J. Biol. Chem. 268:6866-6869 (1993), which
is incorporated herein by reference).
[0165] The route of administration of a pharmaceutical composition
containing an agent that alters myostatin signal transduction will
depend, in part, on the chemical structure of the molecule.
Polypeptides and polynucleotides, for example, are not particularly
useful when administered orally because they can be degraded in the
digestive tract. However, methods for chemically modifying
polypeptides, for example, to render them less susceptible to
degradation by endogenous proteases or more absorbable through the
alimentary tract are well known (see, for example, Blondelle et
al., supra, 1995; Ecker and Crook, supra, 1995). In addition, a
peptide agent can be prepared using D-amino acids, or can contain
one or more domains based on peptidomimetics, which are organic
molecules that mimic the structure of peptide domain; or based on a
peptoid such as a vinylogous peptoid.
[0166] A pharmaceutical composition as disclosed herein can be
administered to an individual by various routes including, for
example, orally or parenterally, such as intravenously,
intramuscularly, subcutaneously, intraorbitally, intracapsularly,
intraperitoneally, intrarectally, intracistemally or by passive or
facilitated absorption through the skin using, for example, a skin
patch or transdermal iontophoresis, respectively. Furthermore, the
pharmaceutical composition can be administered by injection,
intubation, orally or topically, the latter of which can be
passive, for example, by direct application of an ointment, or
active, for example, using a nasal spray or inhalant, in which case
one component of the composition is an appropriate propellant. A
pharmaceutical composition also can be administered to the site of
a pathologic condition, for example, intravenously or
intra-arterially into a blood vessel supplying a tumor.
[0167] The total amount of an agent to be administered in
practicing a method of the invention can be administered to a
subject as a single dose, either as a bolus or by infusion over a
relatively short period of time, or can be administered using a
fractionated treatment protocol, in which multiple doses are
administered over a prolonged period of time. One skilled in the
art would know that the amount of the pharmaceutical composition to
treat a pathologic condition in a subject depends on many factors
including the age and general health of the subject as well as the
route of administration and the number of treatments to be
administered. In view of these factors, the skilled artisan would
adjust the particular dose as necessary. In general, the
formulation of the pharmaceutical composition and the routes and
frequency of administration are determined, initially, using Phase
I and Phase II clinical trials.
[0168] The pharmaceutical composition can be formulated for oral
formulation, such as a tablet, or a solution or suspension form; or
can comprise an admixture with an organic or inorganic carrier or
excipient suitable for enteral or parenteral applications, and can
be compounded, for example, with the usual non-toxic,
pharmaceutically acceptable carriers for tablets, pellets,
capsules, suppositories, solutions, emulsions, suspensions, or
other form suitable for use. The carriers, in addition to those
disclosed above, can include glucose, lactose, mannose, gum acacia,
gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn
starch, keratin, colloidal silica, potato starch, urea, medium
chain length triglycerides, dextrans, and other carriers suitable
for use in manufacturing preparations, in solid, semisolid, or
liquid form. In addition auxiliary, stabilizing, thickening or
coloring agents and perfumes can be used, for example a stabilizing
dry agent such as triulose (see, for example, U.S. Pat. No.
5,314,695).
[0169] The present invention also provides a method of modulating
the growth of muscle tissue or adipose tissue in a subject. As
disclosed herein, GDF receptors such as Act RIIA and Act RIIB are
involved in mediating the effects of a GDF such as myostatin, which
is involved in muscle tissue and adipose tissue formation. Thus, in
one embodiment, a method of modulating the growth of muscle tissue
or adipose tissue includes affecting signal transduction from a GDF
receptor, such as an activin receptor, e.g., Act RIIA or Act RIIB.
Such a method can be performed by contacting cells of the tissue,
or expressing in the cells, a mutant GDF receptor, which has
dominant negative activity, constitutive activity, or the like.
[0170] In another embodiment, a method of modulating the growth of
muscle tissue or adipose tissue in an organism is performed by
administering to the organism an agent that affects myostatin
signal transduction. Preferably, the agent is or encodes a
myostatin prodomain or a mutant promyostatin polypeptide, either of
which can include a myostatin signal peptide. As used herein, the
term "growth" is used in a relative sense in referring to the mass
of muscle tissue or mass of adipose tissue in an organism that has
been subjected to a method of the invention as compared to a
corresponding organism that has not been subjected to a method of
the invention. Thus, where a method of the invention is performed
such that myostatin signal transduction has been reduced or
inhibited, it will be recognized that the growth of muscle tissue
in the organism would result in an increased muscle mass in the
organism as compared to the muscle mass of a corresponding organism
(or population of organisms) in which myostatin signal transduction
had not been so effected.
[0171] A method of the invention can be useful for increasing the
muscle mass or reducing the fat content of an organism or both. For
example, where such a method is performed on an organism that is
useful as a food source, the protein content of the food can be
increased, the cholesterol level can be decreased, and the quality
of the foodstuff can be improved. A method of the invention also
can be useful for decreasing the growth of muscle tissue in an
organism, for example, an organism that is detrimental to an
environment, such that the organism is less able to compete in the
environment. Thus, a method of the invention can be performed on
any eukaryotic organism that expresses myostatin, including a
vertebrate organism, for example, mammalian, avian or piscine
organism, or can be an invertebrate organism, for example, a
mollusk, echinoderm, gastropod or cephalopod.
[0172] The agent can be any agent that alters myostatin signal
transduction, as disclosed herein, and can be administered to the
organism in any convenient manner. For example, where the organism
to be treated are fish, shrimp, scallops, or the like, which are
grown in aquaculture, the agent can be added to the water in which
the organisms are maintained or can be included in their food,
particularly where the agent is a soluble peptide or a small
organic molecule.
[0173] Where the agent used in a method of the invention is a
polynucleotide that encodes a peptide agent, an antisense agent, or
the like, germ cells of a non-human organism containing the
polynucleotide can be selected and transgenic organisms expressing
the agent can be produced. Preferably, the polynucleotide is under
the control of an inducible regulatory element, such that the agent
encoded by the polynucleotide can be expressed at a time and for a
duration as desired. Accordingly, the present invention provides
transgenic nonhuman organisms, as well as food products produced by
these organisms. Such food products have increased nutritional
value because of the increase in muscle tissue. The transgenic
non-human animals can be any species as disclosed herein, including
vertebrate organisms such as cattle, pigs, sheep, chicken, turkey
and fish, and invertebrate species such as shrimp, lobster, crabs,
squid, oysters and abalone.
[0174] The regulation of TGF-.theta. family members and their
specific interactions with cell surface receptors are beginning to
be elucidated. Thus, coexpression of the prodomain of a TGF-.theta.
family member with a mature region of another member of the
TGF-.theta. family is associated with intracellular dimerization
and secretion of biologically active homodimers occur (Gray et al.,
Science 247:1328, 1990). For example, use of the BMP-2 prodomain
with the BMP-4 mature region led to dramatically improved
expression of mature BMP-4 (Hammonds et al., (Mol. Endocrinol.
5:149, 1991). For most of the family members that have been
studied, the homodimeric species are biologically active, whereas
for other family members such as the inhibins (Ling et al., Nature
321:779, 1986) and the TGF-.theta.s (Cheifetz et al., Cell 48:409,
1987), heterodimers also have been detected and appear to have
different biological properties than the respective homodimers.
[0175] Receptor-ligand interaction studies have revealed a great
deal of information as to how cells respond to external stimuli,
and have led to the development of therapeutically important
compounds such as erythropoietin, the colony stimulating factors,
and PDGF. Thus, continual efforts have been made at identifying the
receptors that mediate the action of the TGF-.theta. family
members. As disclosed herein, myostatin specifically interacts with
an activin type II receptor. The identification of this interaction
provides targets for identifying antagonists and agonists useful
for agricultural and human therapeutic purposes, for example, for
treating in various pathological conditions such as obesity, type
II diabetes, and cachexia. The identification of this specific
interaction also provides a means to identify other myostatin
receptors, as well as the specific receptors of other growth
differentiation factors. Accordingly, the present invention
provides GDF receptors, which specifically interact with a GDF or
combination of GDFs, for example, with myostatin, GDF-11, or
both.
[0176] A GDF receptor of the invention is exemplified herein by a
myostatin receptor, particularly an activin type II receptor, which
specifically interacts with myostatin and with GDF-11. However,
myostatin receptors that specifically interact with myostatin, but
not GDF-11, also are encompassed within the present invention, as
are GDF-11 receptors that specifically interact with GDF-11 but not
myostatin, and the like. For convenience of discussion, the
receptors of the invention are referred to herein generally as a
"GDF receptor" and are exemplified by a myostatin receptor, which
is a receptor that specifically interacts at least with myostatin.
As such, while reference is made generally to a specific
interaction of myostatin with a myostatin receptor, it will be
recognized that the present disclosure more broadly encompasses any
GDF receptor, including a GDF-11 receptor, which specifically
interacts at least with GDF-11.
[0177] A recombinant cell line that expresses a GDF receptor
polypeptide also is provided, as are antibodies that specifically
bind the receptor, substantially purified polynucleotides that
encode the receptor, and substantially purified GDF receptor
polypeptides. Peptide portions of a GDF receptor also are provided,
including, for example, soluble extracellular domains of a GDF
receptor such as a myostatin receptor, which, as disclosed herein,
can alter the specific interaction of myostatin with a cellular
myostatin receptor; a constitutively active intracellular kinase
domain of a GDF receptor, which can induce, stimulate or otherwise
maintain GDF signal transduction in a cell; or other truncated
portion of a GDF receptor having an ability to modulate myostatin
or other GDF signal transduction.
[0178] The invention also provides methods for identifying a GDF
receptor polypeptide, including methods of screening genomic or
cDNA libraries, which can be expression libraries, using nucleotide
probes or antibody probes; methods of screening cells that are
responsive to and, therefore, express the receptor, using, for
example, a GDF such as myostatin or a functional peptide portion
thereof; two hybrid assays, as described above, using, for example,
the GDF peptide as a component of one hybrid and peptides expressed
from a cDNA library, which is prepared from cells expressing a
receptor for the GDF, as components of the second hybrids, and the
like.
[0179] As described above, agents that specifically interact with a
GDF receptor, for example, a myostatin receptor such as Act RIIB
can be identified by using the receptor to screen for such agents.
Conversely, an agent that has been identified as having the ability
to specifically interact with a myostatin receptor such as the Act
RIIB receptor, can be used to screen for additional myostatin
receptors or other GDF receptors. Such a method can include
incubating components such as the agent (or myostatin or other GDF)
and a cell expressing a GDF receptor, which can be a truncated
membrane bound receptor or a soluble receptor, under conditions
sufficient to allow the agent (or GDF) to interact specifically
with the receptor; measuring the agent (or GDF) bound to the
receptor; and isolating the receptor. A method of molecular
modeling as described above also can be useful as a screening
method for identifying a GDF receptor, or a functional peptide
portion thereof.
[0180] Non-human transgenic animals that have a phenotype
characterized by expression of a GDF receptor also are provided,
the phenotype being conferred by a transgene contained in the
somatic and germ cells of the animal. The transgene comprises a
polynucleotide encoding the GDF receptor, for example, myostatin
receptor, polypeptide. Methods of producing such transgenic animals
are disclosed herein or otherwise known in the art.
[0181] The present invention provides a substantially purified
polynucleotide encoding all or a peptide portion of a GDF receptor.
Although a GDF receptor is exemplified herein by an activin type II
receptor, polynucleotides encoding activin type II receptors
previously have been described (U.S. Pat. No. 5,885,794). Thus, it
should be recognized that such activin type II receptors are not
encompassed within the present invention (Massague, supra, 1998;
Heldin et al., supra, 1997). Similarly, activin type I receptors,
including Act RIB; TGF-.alpha. receptors, including TGF-.theta. RI
and TGF-.theta. RII; and BMP receptors, including BMP RIA, BMP RIB
and BMP RII, have been described and are well known in the art
(Massague, supra, 1998; Heldin et al., supra, 1997) and, therefore,
are not encompassed within the GDF receptors of the invention.
[0182] A polynucleotide of the invention can encode a polypeptide
having a myostatin receptor activity, for example, myostatin
binding activity, or can encode a mutant myostatin receptor, for
example, a mutant myostatin receptor having a mutation in a kinase
domain, such that the mutant acts as a dominant negative myostatin
receptor (see above). Thus, a polynucleotide of the invention can
be a naturally occurring, synthetic, or intentionally manipulated
polynucleotide. For example, portions of the mRNA sequence can be
altered due to alternate RNA splicing patterns or the use of
alternate promoters for RNA transcription. As another example, the
polynucleotide can be subjected to site directed mutagenesis. The
polynucleotide also can be antisense nucleotide sequence. GDF
receptor polynucleotides of the invention include sequences that
are degenerate as a result of the genetic code. There are 20
natural amino acids, most of which are specified by more than one
codon. Therefore, all degenerate nucleotide sequences are included
within the invention, provided the amino acid sequence of the GDF
receptor polypeptide encoded by the polynucleotide is functionally
unchanged. Also included are nucleotide sequences that encode
myostatin receptor polypeptides.
[0183] Oligonucleotide portions of a polynucleotide encoding a GDF
receptor of the invention also are encompassed within the present
invention. Such oligonucleotides generally are at least about 15
bases in length, which is sufficient to permit the oligonucleotide
to selectively hybridize to a polynucleotide encoding the receptor,
and can be at least about 18 nucleotides or 21 nucleotides or more
in length. As used herein, the term "selective hybridization" or
"selectively hybridize" refers to hybridization under moderately
stringent or highly stringent physiological conditions, which can
distinguish related nucleotide sequences from unrelated nucleotide
sequences.
[0184] In nucleic acid hybridization reactions, the conditions used
to achieve a particular level of stringency will vary, depending on
the nature of the nucleic acids being hybridized. For example, the
length, degree of complementarity, nucleotide sequence composition
(for example, relative GC:AT content), and nucleic acid type, i.e.,
whether the oligonucleotide or the target nucleic acid sequence is
DNA or RNA, can be considered in selecting hybridization
conditions. An additional consideration is whether one of the
nucleic acids is immobilized, for example, on a filter. Methods for
selecting appropriate stringency conditions can be determined
empirically or estimated using various formulas, and are well known
in the art (see, for example, Sambrook et al., supra, 1989).
[0185] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions, for
example, high stringency conditions, or each of the conditions can
be used, for example, for 10 to 15 minutes each, in the order
listed above, repeating any or all of the steps listed.
[0186] A GDF receptor-encoding polynucleotide of the invention can
be obtained by any of several methods. For example, the
polynucleotide can be isolated using hybridization or computer
based techniques, as are well known in the art. These methods
include, but are not limited to, 1) hybridization of genomic or
cDNA libraries with probes to detect homologous nucleotide
sequences; 2) antibody screening of expression libraries to detect
cloned DNA fragments with shared structural features; 3) polymerase
chain reaction (PCR) on genomic DNA or cDNA using primers capable
of annealing to the DNA sequence of interest; 4) computer searches
of sequence databases for similar sequences (see above); 5)
differential screening of a subtracted DNA library; and 6) two
hybrid assays using, for example, a mature GDF peptide in one of
the hybrids.
[0187] In view of the present disclosure that an activin receptor
specifically interacts with myostatin, oligonucleotide probes can
be designed based on the sequence encoding an activin receptor, for
example, a sequence encoding the extracellular domain, which binds
myostatin, and used to screen a library prepared from cells such as
muscle cells or adipocytes, which are responsive to myostatin, thus
facilitating identification of a polynucleotide encoding a
myostatin receptor. Selected clones can be further screened, for
example, by subcloning the inserts into an expression vector and,
following expression of the cloned sequences, screening the
expressed polypeptides using myostatin.
[0188] A polynucleotide of the invention, for example, a
polynucleotide encoding a myostatin receptor, can be derived from a
vertebrate species, including a mammalian, avian, or piscine
species, or from an invertebrate species. Screening procedures that
rely on nucleic acid hybridization allow the isolation any gene
sequence from any organism, provided the appropriate probe is
available. Oligonucleotide probes that correspond to a part of the
sequence encoding the protein in question can be synthesized
chemically. This requires that short, oligopeptide stretches of
amino acid sequence are known. A polynucleotide sequence encoding
the receptor can be deduced from the genetic code, taking into
account the degeneracy of the genetic code. Thus, mixed addition
reactions can be performed when the sequence is degenerate. This
includes a heterogeneous mixture of denatured double stranded DNA.
For such screening, hybridization is preferably performed on either
single stranded DNA or denatured double stranded DNA. Hybridization
is particularly useful in the detection of cDNA clones derived from
sources where an extremely low amount of mRNA sequences relating to
the polypeptide of interest are present. Thus, by using stringent
hybridization conditions directed to avoid nonspecific binding,
autoradiographic visualization can be used to identify a specific
cDNA clone by the hybridization of the target DNA to an
oligonucleotide probe in the mixture that is the complete
complement of the target nucleic acid (Wallace et al., Nucl. Acid
Res., 9:879, 1981, which is incorporated herein by reference).
Alternatively, a subtractive library can be used, thereby
eliminating nonspecific cDNA clones.
[0189] When the entire amino acid sequence of a desired polypeptide
is not known, the direct synthesis of DNA sequences is not possible
and the method of choice is the synthesis of cDNA sequences. Among
the standard procedures for isolating cDNA sequences of interest is
the formation of cDNA libraries prepared in plasmids or phage,
wherein the libraries are derived from reverse transcription of
mRNA that is abundant in donor cells having a high level of genetic
expression. When used in combination with polymerase chain reaction
technology, even rare expression products can be cloned. Where
significant portions of the amino acid sequence of the polypeptide
are known, the production of labeled single stranded or double
stranded DNA or RNA probe sequences duplicating a sequence
putatively present in the target cDNA can be employed in
hybridization procedures carried out on cloned copies of the cDNA,
which have been denatured into a single stranded form (Jay et al.,
Nucl. Acid Res., 11:2325, 1983, which is incorporated herein by
reference).
[0190] A cDNA expression library, such as a lambda gt11 library,
can be screened for GDF receptor peptides using an antibody
specific for a GDF receptor, for example, an anti-Act RIIB
antibody. The antibody can be polyclonal or monoclonal, and can be
used to detect expression product indicative of the presence of a
GDF receptor cDNA. Such an expression library also can be screened
with a GDF peptide, for example, with myostatin, or a functional
peptide portion thereof, to identify a clone encoding at least a
portion of a myostatin binding domain of a myostatin receptor.
[0191] Polynucleotides encoding mutant GDF receptors and mutant GDF
receptor polypeptides are also encompassed within the invention. An
alteration in a polynucleotide encoding a GDF receptor can be an
intragenic mutation such as point mutation, nonsense (STOP)
mutation, missense mutation, splice site mutation or frameshift, or
can be a heterozygous or homozygous deletion, and can be a
naturally occurring mutation or can be engineered using recombinant
DNA methods, for example. Such alterations can be detected using
standard methods known to those of skill in the art, including, but
not limited to, nucleotide sequence analysis, Southern blot
analysis, a PCR based analysis such as multiplex PCR or sequence
tagged sites (STS) analysis, or in situ hybridization analysis. GDF
receptor polypeptides can be analyzed by standard SDS-PAGE,
immunoprecipitation analysis, western blot analysis, or the like.
Mutant GDF receptors are exemplified by truncated GDF receptors,
including a soluble extracellular domain, which can have the
ability to specifically bind its cognate GDF, but lacks a kinase
domain; an intracellular GDF receptor kinase domain, which can
exhibit constitutive kinase activity; as well as by GDF receptors
that contain a point mutation, which disrupts the kinase activity
of the receptor or the ligand binding ability of the receptor; and
the like. Such GDF receptor mutants are useful for modulating GDF
signal transduction and, therefore, for practicing various methods
of the invention.
[0192] A polynucleotide encoding a GDF receptor can be expressed in
vitro by introducing the polynucleotide into a suitable host cell.
"Host cells" can be any cells in which the particular vector can be
propagated, and, where appropriate, in which a polynucleotide
contained in the vector can be expressed. The term "host cells"
includes any progeny of an original host cell. It is understood
that all progeny of the host cell may not be identical to the
parental cell due, for example, to mutations that occur during
replication. However, such progeny are included when the term "host
cell" is used. Methods of obtaining a host cell that transiently or
stably contains an introduced polynucleotide of the invention are
well known in the art.
[0193] A GDF receptor polynucleotide of the invention can be
inserted into a vector, which can be a cloning vector or a
recombinant expression vector. The term "recombinant expression
vector" refers to a plasmid, virus or other vehicle known in the
art that has been manipulated by insertion or incorporation of a
polynucleotide, particularly, with respect to the present
invention, a polynucleotide encoding all or a peptide portion of a
GDF receptor. Such expression vectors contain a promoter sequence,
which facilitates the efficient transcription of the inserted
genetic sequence of the host. The expression vector generally
contains an origin of replication, a promoter, as well as specific
genes which allow phenotypic selection of the transformed cells.
Vectors suitable for use in the present invention include, but are
not limited to, the T7-based expression vector for expression in
bacteria (Rosenberg, et al., Gene 56:125, 1987), the pMSXND
expression vector for expression in mammalian cells (Lee and
Nathans, J. Biol. Chem. 263:3521, 1988) and baculovirus-derived
vectors for expression in insect cells. The DNA segment can be
present in the vector operably linked to regulatory elements, for
example, a promoter, which can be a T7 promoter, metallothionein I
promoter, polyhedrin promoter, or other promoter as desired,
particularly tissue specific promoters or inducible promoters.
[0194] A polynucleotide sequence encoding a GDF receptor can be
expressed in either prokaryotes or eukaryotes. Hosts can include
microbial, yeast, insect and mammalian organisms. Methods of
expressing polynucleotides having eukaryotic or viral sequences in
prokaryotes are well known in the art, as are biologically
functional viral and plasmid DNA vectors capable of expression and
replication in a host. Methods for constructing an expression
vector containing a polynucleotide of the invention are well known,
as are factors to be considered in selecting transcriptional or
translational control signals, including, for example, whether the
polynucleotide is to be expressed preferentially in a particular
cell type or under particular conditions (see, for example,
Sambrook et al., supra, 1989).
[0195] A variety of host cell/expression vector systems can be
utilized to express a GDF receptor coding sequence, including, but
not limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors; yeast cells transformed with recombinant yeast expression
vectors; plant cell systems infected with recombinant virus
expression vectors such as a cauliflower mosaic virus or tobacco
mosaic virus, or transformed with recombinant plasmid expression
vector such as a Ti plasmid; insect cells infected with recombinant
virus expression vectors such as a baculovirus; animal cell systems
infected with recombinant virus expression vectors such as a
retrovirus, adenovirus or vaccinia virus vector; and transformed
animal cell systems genetically engineered for stable expression.
Where the expressed GDF receptor is post-translationally modified,
for example, by glycosylation, it can be particularly advantageous
to select a host cell/expression vector system that can effect the
desired modification, for example, a mammalian host cell/expression
vector system.
[0196] Depending on the host cell/vector system utilized, any of a
number of suitable transcription and translation elements,
including constitutive and inducible promoters, transcription
enhancer elements, transcription terminators, and the like can be
used in the expression vector (Bitter et al., Meth. Enzymol.
153:516-544, 1987). For example, when cloning in bacterial systems,
inducible promoters such as pL of bacteriophage .SIGMA., plac,
ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used.
When cloning in mammalian cell systems, promoters derived from the
genome of mammalian cells, for example, a human or mouse
metallothionein promoter, or from mammalian viruses, for example, a
retrovirus long terminal repeat, an adenovirus late promoter or a
vaccinia virus 7.5K promoter, can be used. Promoters produced by
recombinant DNA or synthetic techniques can also be used to provide
for transcription of the inserted GDF receptors coding
sequence.
[0197] In yeast cells, a number of vectors containing constitutive
or inducible promoters can be used (see Ausubel et al., supra,
1987, see chapter 13; Grant et al., Meth. Enzymol. 153:516-544,
1987; Glover, DNA Cloning Vol. II (IRL Press, 1986), see chapter 3;
Bitter, Meth. Enzymol. 152:673-684, 1987; see, also, The Molecular
Biology of the Yeast Saccharomyces (Eds., Strathern et al., Cold
Spring Harbor Laboratory Press, 1982), Vols. I and II). A
constitutive yeast promoter such as ADH or LEU2 or an inducible
promoter such as GAL can be used (Rothstein, DNA Cloning Vol. II
(supra, 1986), chapter 3). Alternatively, vectors can be used which
promote integration of foreign DNA sequences into the yeast
chromosome.
[0198] Eukaryotic systems, particularly mammalian expression
systems, allow for proper post-translational modifications of
expressed mammalian proteins. Eukaryotic cells which possess the
cellular machinery for proper processing of the primary transcript,
glycosylation, phosphorylation, and advantageously, plasma membrane
insertion of the gene product can be used as host cells for the
expression of a GDF receptor polypeptide, or functional peptide
portion thereof.
[0199] Mammalian cell systems which utilize recombinant viruses or
viral elements to direct expression can be engineered. For example,
when using adenovirus expression vectors, the GDF receptors coding
sequence can be ligated to an adenovirus transcription/translation
control complex, e.g., the late promoter and tripartite leader
sequence. Alternatively, the vaccinia virus 7.5K promoter can be
used (Mackett et al., Proc. Natl. Acad. Sci., USA 79:7415-7419,
1982; Mackett et al., J. Virol. 49:857-864, 1984; Panicali et al.,
Proc. Natl. Acad. Sci., USA 79:4927-4931, 1982). Particularly
useful are bovine papilloma virus vectors, which can replicate as
extrachromosomal elements (Sarver et al., Mol. Cell. Biol. 1:486,
1981). Shortly after entry of this DNA into mouse cells, the
plasmid replicates to about 100 to 200 copies per cell.
Transcription of the inserted cDNA does not require integration of
the plasmid into the host cell chromosome, thereby yielding a high
level of expression. These vectors can be used for stable
expression by including a selectable marker in the plasmid, such
as, for example, the neo gene. Alternatively, the retroviral genome
can be modified for use as a vector capable of introducing and
directing the expression of the GDF receptors gene in host cells
(Cone and Mulligan, Proc. Natl. Acad. Sci., USA 81:6349-6353,
1984). High level expression can also be achieved using inducible
promoters, including, but not limited to, the metallothionein IIA
promoter and heat shock promoters.
[0200] For long term, high yield production of recombinant
proteins, stable expression is preferred. Rather than using
expression vectors which contain viral origins of replication, host
cells can be transformed with the GDF receptors cDNA controlled by
appropriate expression control elements such as promoter, enhancer,
sequences, transcription terminators, and polyadenylation sites,
and a selectable marker. The selectable marker in the recombinant
plasmid can confer resistance to the selection, and allows cells to
stably integrate the plasmid into their chromosomes and grow to
form foci, which, in turn can be cloned and expanded into cell
lines. For example, following the introduction of foreign DNA,
engineered cells can be allowed to grow for 1 to 2 days in an
enriched media, and then are switched to a selective media. A
number of selection systems can be used, including, but not limited
to, the herpes simplex virus thymidine kinase (Wigler et al., Cell
11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase
(Szybalska and Szybalski, Proc. Natl. Acad. Sci., USA 48:2026,
1982), and adenine phosphoribosyltransferase (Lowy, et al., Cell
22:817, 1980) genes can be employed in tk-, hgprt- or aprt-cells
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA 77:3567,
1980; O'Hare et al., Proc. Natl. Acad. Sci., USA 78: 1527, 1981);
gpt, which confers resistance to mycophenolic acid (Mulligan and
Berg, Proc. Natl. Acad. Sci., USA 78:2072, 1981); neo, which
confers resistance to the aminoglycoside G-418 (Colberre-Garapin et
al., J. Mol. Biol. 150:1, 1981); and hygro, which confers
resistance to hygromycin (Santerre et al., Gene 30:147, 1984)
genes. Additional selectable genes, including trpB, which allows
cells to utilize indole in place of tryptophan; hisD, which allows
cells to utilize histinol in place of histidine (Hartman and
Mulligan, Proc. Natl. Acad. Sci., USA 85:8047, 1988); and ODC
(ornithine decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue, Curr. Comm. Mol. Biol. (Cold Spring Harbor Laboratory
Press, 1987), also have been described.
[0201] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors can be used.
Eukaryotic cells can also be cotransformed with DNA sequences
encoding the GDF receptors of the invention, and a second foreign
DNA molecule encoding a selectable phenotype, such as the herpes
simplex thymidine kinase gene. Another method is to use a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papilloma virus, to transiently infect or transform eukaryotic
cells and express the protein. (Gluzman, Eukaryotic Viral Vectors
(Cold Spring Harbor Laboratory Press, 1982)).
[0202] The invention also provides stable recombinant cell lines,
the cells of which express GDF receptor polypeptides and contain
DNA that encodes GDF receptors. Suitable cell types include, but
are not limited to, NIH 3T3 cells (murine), C2C12 cells, L6 cells,
and P19 cells. C2C12 and L6 myoblasts differentiate spontaneously
in culture and form myotubes depending on the particular growth
conditions (Yaffe and Saxel, Nature 270:725-727, 1977; Yaffe, Proc.
Natl. Acad. Sci., USA 61:477-483, 1968). P19 is an embryonal
carcinoma cell line. Such cells are described, for example, in the
Cell Line Catalog of the American Type Culture Collection (ATCC).
These cells can be stably transformed using well known methods
(see, for example, Ausubel et al., supra, 1995, see sections
9.5.1-9.5.6).
[0203] A GDF receptor can be expressed from a recombinant
polynucleotide of the invention using inducible or constitutive
regulatory elements, as described herein. The desired protein
encoding sequence and an operably linked promoter can be introduced
into a recipient cell either as a non-replicating DNA (or RNA)
molecule, which can either be a linear molecule or a covalently
closed circular molecule. Expression of the desired molecule can
occur due to transient expression of the introduced sequence, or
the polynucleotide can be stably maintained in the cell, for
example, by integration into a host cell chromosome, thus allowing
a more permanent expression. Accordingly, the cells can be stably
or transiently transformed (transfected) cells.
[0204] An example of a vector that can be employed is one which is
capable of integrating the desired gene sequences into the host
cell chromosome. Cells which have stably integrated the introduced
DNA into their chromosomes can be selected by also introducing one
or more markers which allow for selection of host cells which
contain the expression vector. The marker can complement an
auxotrophy in the host such as leu2, or ura3, which are common
yeast auxotrophic markers; can confer a biocide resistance, for
example, to an antibiotic or to heavy metal ions such as copper, or
the like. The selectable marker gene can either be directly linked
to the DNA gene sequences to be expressed, or can be introduced
into the same cell by cotransfection.
[0205] The introduced sequence can be incorporated into a plasmid
or viral vector capable of autonomous replication in the recipient
host. Any of a variety of vectors can be employed for this purpose.
Factors of importance in selecting a particular plasmid or viral
vector include the ease with which recipient cells that contain the
vector can be recognized and selected from those cells that do not
contain the vector; the number of copies of the vector desired in a
particular host cell; and whether it is desirable to be able to
"shuttle" the vector between host cells of different species.
[0206] For a mammalian host, several vector systems are available
for expression. One class of vectors utilizes DNA elements that
provide autonomously replicating extra-chromosomal plasmids derived
from animal viruses, for example, a bovine papilloma virus, polyoma
virus, adenovirus, or SV40 virus. A second class of vectors
includes vaccinia virus expression vectors. A third class of
vectors relies upon the integration of the desired gene sequences
into the host chromosome. Cells that have stably integrated the
introduced DNA into their chromosomes can be selected by also
introducing one or more markers genes (as described above), which
allow selection of host cells that contain the expression vector.
The selectable marker gene can be directly linked to the DNA
sequences to be expressed, or introduced into the same cell by
cotransfection. Additional elements can be included to provide for
optimal synthesis of an encoded mRNA or peptide, including, for
example, splice signals, transcription promoters or enhancers, and
transcription or translation termination signals. cDNA expression
vectors incorporating appropriate regulatory elements are well
known in the art (see, for example, Okayama, Mol. Cell. Biol.
3:280, 1983).
[0207] Once the vector or DNA sequence containing the construct has
been prepared for expression, the DNA construct can be introduced
into an appropriate host. Various methods can be used for
introducing the polynucleotide into a cell, including, for example,
methods of transfection or transformation such as protoplast
fusion, calcium phosphate precipitation, and electroporation or
other conventional techniques, for example, infection where the
vector is a viral vector.
[0208] The invention also provides transgenic animals, which have
cells that express a recombinant GDF receptor. Such transgenic
animals can be selected to have decreased fat content or increased
muscle mass, or both, and, therefore, can be useful as a source of
food products with high muscle and protein content, and reduced fat
and cholesterol content. The animals have been altered
chromosomally in their germ cells and somatic cells such that the
production of a GDF, particularly myostatin, is maintained at a
normal" level, but the myostatin receptor is produced in reduced
amounts, or is completely disrupted, resulting in the cells in the
animals having a decreased ability to bind myostatin and,
consequently, having greater than normal levels of muscle tissue,
preferably without increased fat or cholesterol levels.
Accordingly, the present invention also includes food products
provided by the animals. Such food products have increased
nutritional value because of the increase in muscle tissue. The
transgenic non-human animals of the invention include bovine,
porcine, ovine and avian animals, as well as other vertebrates, and
further includes transgenic invertebrates.
[0209] The invention also provides a method of producing animal
food products having increased muscle content. Such a method can
include modifying the genetic makeup of the germ cells of a
pronuclear embryo of the animal, implanting the embryo into the
oviduct of a pseudopregnant female, thereby allowing the embryo to
mature to full term progeny, testing the progeny for presence of
the transgene to identify transgene positive progeny, crossbreeding
transgene positive progeny to obtain further transgene positive
progeny, and processing the progeny to obtain a foodstuff. The
modification of the germ cell comprises altering the genetic
composition so as to reduce or inhibit the expression of the
naturally occurring gene encoding for production of a myostatin
receptor protein. For example, the transgene can comprise an
antisense molecule that is specific for a polynucleotide encoding a
myostatin receptor; can comprise a non-functional sequence that
replaces or intervenes in the endogenous myostatin receptor gene or
the transgene; or can encode a myostatin receptor antagonist, for
example, a dominant negative myostatin receptor such as a dominant
negative Act RIIB.
[0210] As used herein, the term "animal" refers to any bird, fish
or mammal, except a human, and includes any stage of development,
including embryonic and fetal stages. Farm animals such as pigs,
goats, sheep, cows, horses, rabbits and the like; rodents such as
mice; and domestic pets such as cats and dogs are included within
the meaning of the term "animal." In addition, the term "organism"
is used herein to include animals as described above, as well as
other eukaryotes, including, for example, other vertebrates such as
reptiles and amphibians, as well as invertebrates as described
above.
[0211] As used herein, the term "transgenic," when used in
reference to an animal or an organism, means that cells of the
animal or organism have been genetically manipulated to contain an
exogenous polynucleotide sequence that is stably maintained with
the cells. The manipulation can be, for example, microinjection of
a polynucleotide or infection with a recombinant virus containing
the polynucleotide. Thus, the term "transgenic" is used herein to
refer to animals (organisms) in which one or more cells receive a
recombinant polynucleotide, which can be integrated into a
chromosome in the cell, or can be maintained as an
extrachromosomally replicating polynucleotide, such as might be
engineered into a yeast artificial chromosome. The term "transgenic
animal" also includes a "germ cell line" transgenic animal. A germ
cell line transgenic animal is a transgenic animal in which the
genetic information has been taken up and incorporated into a germ
line cell, therefore conferring the ability to transfer the
information to offspring. If such offspring in fact possess some or
all of that information, the offspring also are considered to be
transgenic animals. The invention further encompasses transgenic
organisms.
[0212] A transgenic organism can be any organism whose genome has
been altered by in vitro manipulation of an early stage embryo or a
fertilized egg, or by any transgenic technology to induce a
specific gene knock-out. The term "gene knock-out" refers to the
targeted disruption of a gene in a cell or in vivo that results in
complete loss of function. A target gene in a transgenic animal can
be rendered nonfunctional by an insertion targeted to the gene to
be rendered nonfunctional, for example, by homologous
recombination, or by any other method for disrupting the function
of a gene in a cell.
[0213] The transgene to be used in the practice of the subject
invention can be a DNA sequence comprising a modified GDF receptors
coding sequence. Preferably, the modified GDF receptor gene is one
that is disrupted by homologous targeting in embryonic stem cells.
For example, the entire mature C-terminal region of the GDF
receptors gene can be deleted (see Example 13). Optionally, the
disruption (or deletion) can be accompanied by insertion of or
replacement with another polynucleotide, for example, a
nonfunctional GDF receptor sequence. A "knock-out" phenotype also
can be conferred by introducing or expressing an antisense GDF
receptor polynucleotide in a cell in the organism, or by expressing
an antibody or a dominant negative GDF receptor in the cells. Where
appropriate, polynucleotides that encode proteins having GDF
receptor activity, but that differ in nucleotide sequence from a
naturally occurring GDF gene sequence due to the degeneracy of the
genetic code, can be used herein, as can truncated forms, allelic
variants and interspecies homologs.
[0214] The present invention also provides antibodies that
specifically bind a GDF receptor, and that block GDF binding to the
receptor. Such antibodies can be useful, for example, for
ameliorating a pathologic condition such as a cell proliferative
disorder associated with muscle tissue.
[0215] A monoclonal antibody that binds specifically to a GDF
receptor, particularly to a myostatin receptor, can increase the
development of skeletal muscles. In preferred embodiments of the
claimed methods, a GDF receptor monoclonal antibody, polypeptide,
or polynucleotide is administered to a patient suffering from a
pathologic condition such as a muscle wasting disease, a
neuromuscular disorder, muscle atrophy, aging, or the like. The GDF
receptor antibody, particularly an anti-myostatin receptor
antibody, can also be administered to a patient suffering from a
pathologic condition such as a muscular dystrophy, spinal cord
injury, traumatic injury, congestive obstructive pulmonary disease
(COPD), AIDS or cachexia.
[0216] In a preferred embodiment, the anti-myostatin receptor
antibody is administered to a patient with muscle wasting disease
or disorder by intravenous, intramuscular or subcutaneous
injection; preferably, a monoclonal antibody is administered within
a dose range between about 0.1 Tg/kg to about 100 mg/kg; more
preferably between about 1 Tg/kg to 75 mg/kg; most preferably from
about 10 mg/kg to 50 mg/kg. The antibody can be administered, for
example, by bolus injunction or by slow infusion. Slow infusion
over a period of 30 minutes to 2 hours is preferred. The
anti-myostatin receptor antibody, or other anti-GDF receptor
antibody, can be formulated in a formulation suitable for
administration to a patient. Such formulations are known in the
art.
[0217] The dosage regimen will be determined by the attending
physician considering various factors which modify the action of
the myostatin receptor protein, for example, amount of tissue
desired to be formed, the site of tissue damage, the condition of
the damaged tissue, the size of a wound, type of damaged tissue,
the patient's age, sex, and diet, the severity of any infection,
time of administration and other clinical factors. The dosage can
vary with the type of matrix used in the reconstitution and the
types of agent, such as anti-myostatin receptor antibodies, to be
used in the composition. Generally, systemic or injectable
administration, such as intravenous, intramuscular or subcutaneous
injection. Administration generally is initiated at a dose which is
minimally effective, and the dose is increased over a preselected
time course until a positive effect is observed. Subsequently,
incremental increases in dosage are made limiting such incremental
increases to such levels that produce a corresponding increase in
effect, while taking into account any adverse affects that can
appear. The addition of other known growth factors, such as IGF I
(insulin like growth factor I), human, bovine, or chicken growth
hormone, which can aid in increasing muscle mass, to the final
composition, can also affect the dosage. In the embodiment where an
anti-myostatin receptor antibody is administered, the antibody is
generally administered within a dose range of about 0.1 Tg/kg to
about 100 mg/kg.; more preferably between about 10 mg/kg to 50
mg/kg.
[0218] As used herein, the term "antibody" is used in its broadest
sense to include polyclonal and monoclonal antibodies, as well as
antigen binding fragments of such antibodies. An antibody useful in
a method of the invention, or an antigen binding fragment thereof,
is characterized, for example, by having specific binding activity
for an epitope of a GDF receptor, for example, a myostatin
receptor. In addition, as discussed above, an antibody of the
invention can be an antibody that specifically binds a peptide
portion of a promyostatin polypeptide, particularly a myostatin
prodomain or functional peptide portion thereof. It will be
recognized that the following methods, which exemplify the
preparation and characterization of GDF receptor antibodies,
further are applicable to the preparation and characterization of
additional antibodies of the invention, including antibodies that
specifically bind a myostatin prodomain, antibodies that
specifically bind a promyostatin polypeptide and reduce or inhibit
proteolytic cleavage of the promyostatin to myostatin, and the
like
[0219] The term "binds specifically" or "specific binding
activity," when used in reference to an antibody means that an
interaction of the antibody and a particular epitope has a
dissociation constant of at least about 1.times.10-6, generally at
least about 1.times.10-7, usually at least about 1.times.10-8, and
particularly at least about 1.times.10-9 or 1.times.10-10 or less.
As such, Fab, F(ab')2, Fd and Fv fragments of an antibody that
retain specific binding activity for an epitope of a GDF receptor,
are included within the definition of an antibody. For purposes of
the present invention, an antibody that reacts specifically with an
epitope of a myostatin receptor, for example, is considered to not
substantially react with a TGF-.theta. receptor or a BMP receptor
if the antibody has at least a two-fold greater binding affinity,
generally at least a five-fold greater binding affinity, and
particularly at least a ten-fold greater binding affinity for the
myostatin receptor as compared to the TGF-.theta. or BMP
receptor.
[0220] The term "antibody" as used herein includes naturally
occurring antibodies as well as non-naturally occurring antibodies,
including, for example, single chain antibodies, chimeric,
bifunctional and humanized antibodies, as well as antigen-binding
fragments thereof. Such non-naturally occurring antibodies can be
constructed using solid phase peptide synthesis, can be produced
recombinantly or can be obtained, for example, by screening
combinatorial libraries consisting of variable heavy chains and
variable light chains (see Huse et al., Science 246:1275-1281
(1989), which is incorporated herein by reference). These and other
methods of making, for example, chimeric, humanized, CDR-grafted,
single chain, and bifunctional antibodies are well known to those
skilled in the art (Winter and Harris, Immunol. Today 14:243-246,
1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane,
Antibodies: A laboratory manual (Cold Spring Harbor Laboratory
Press, 1988); Hilyard et al., Protein Engineering: A practical
approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed.
(Oxford University Press 1995); each of which is incorporated
herein by reference).
[0221] Antibodies that bind specifically with a GDF receptor can be
raised using the receptor as an immunogen and removing antibodies
that crossreact, for example, with a TGF-.theta. type I or type II
receptor, with an activin receptor such as Act RIB, Act RIIA or Act
RIIB, or a BMP receptors such as BMP RII, BMP RIA and BMP RIB (see
Massague, supra, 1998). An antibody of the invention conveniently
can be raised using a peptide portion of a myostatin receptor that
is not present in a TGF-.theta., activin, or BMP receptor.
Similarly, an antibody that specifically binds a myostatin
prodomain can be raised using the prodomain, or a functional
peptide portion thereof as the immunogen. Where such a peptide is
non-immunogenic, it can be made immunogenic by coupling the hapten
to a carrier molecule such as bovine serum albumin (BSA) or keyhole
limpet hemocyanin (KLH), or by expressing the peptide portion as a
fusion protein. Various other carrier molecules and methods for
coupling a hapten to a carrier molecule are well known in the art
(see, for example, by Harlow and Lane, supra, 1988).
[0222] If desired, a kit incorporating an antibody or other agent
useful in a method of the invention can be prepared. Such a kit can
contain, in addition to the agent, a pharmaceutical composition in
which the agent can be reconstituted for administration to a
subject. The kit also can contain, for example, reagents for
detecting the antibody, or for detecting specific binding of the
antibody to a GDF receptor. Such detectable reagents useful for
labeling or otherwise identifying the antibody are described herein
and known in the art.
[0223] Methods for raising polyclonal antibodies, for example, in a
rabbit, goat, mouse or other mammal, are well known in the art
(see, for example, Green et al., "Production of Polyclonal
Antisera," in Immunochemical Protocols (Manson, ed., Humana Press
1992), pages 1-5; Coligan et al., "Production of Polyclonal
Antisera in Rabbits, Rats, Mice and Hamsters," in Curr. Protocols
Immunol. (1992), section 2.4.1; each or which is incorporated
herein by reference). In addition, monoclonal antibodies can be
obtained using methods that are well known and routine in the art
(Harlow and Lane, supra, 1988). For example, spleen cells from a
mouse immunized with a myostatin receptor, or an epitopic fragment
thereof, can be fused to an appropriate myeloma cell line such as
SP/02 myeloma cells to produce hybridoma cells. Cloned hybridoma
cell lines can be screened using labeled antigen to identify clones
that secrete monoclonal antibodies having the appropriate
specificity, and hybridomas expressing antibodies having a
desirable specificity and affinity can be isolated and utilized as
a continuous source of the antibodies. The antibodies can be
further screened for the inability to bind specifically with the
myostatin receptor. Such antibodies are useful, for example, for
preparing standardized kits for clinical use. A recombinant phage
that expresses, for example, a single chain anti-myostatin receptor
antibody also provides an antibody that can used for preparing
standardized kits.
[0224] Methods of preparing monoclonal antibodies well known (see,
for example, Kohler and Milstein, Nature 256:495, 1975, which is
incorporated herein by reference; see, also, Coligan et al., supra,
1992, see sections 2.5.1-2.6.7; Harlow and Lane, supra, 1988).
Briefly, monoclonal antibodies can be obtained by injecting mice
with a composition comprising an antigen, verifying the presence of
antibody production by removing a serum sample, removing the spleen
to obtain B lymphocytes, fusing the B lymphocytes with myeloma
cells to produce hybridomas, cloning the hybridomas, selecting
positive clones that produce antibodies to the antigen, and
isolating the antibodies from the hybridoma cultures.
[0225] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well established techniques,
including, for example, affinity chromatography with Protein-A
SEPHAROSE, size exclusion chromatography, and ion exchange
chromatography (Coligan et al., supra, 1992, see sections
2.7.1-2.7.12 and sections 2.9.1-2.9.3; see, also, Barnes et al.,
"Purification of Immunoglobulin G (IgG)," in Meth.: Molec. Biol.
10:79-104 (Humana Press 1992), which is incorporated herein by
reference). Methods of in vitro and in vivo multiplication of
monoclonal antibodies is well known to those skilled in the art.
Multiplication in vitro can be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo can be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, for example, syngeneic mice, to cause growth of antibody
producing tumors. Optionally, the animals are primed with a
hydrocarbon, especially oils such as pristane
(tetramethylpentadecane) prior to injection. After one to three
weeks, the desired monoclonal antibody is recovered from the body
fluid of the animal.
[0226] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention can also be derived from subhuman primate
antibody. General techniques for raising therapeutically useful
antibodies in baboons can be found, for example, in Goldenberg et
al., International Patent Publication WO 91/11465 (1991); and
Losman et al., Int. J. Cancer 46:310, 1990, each of which is
incorporated herein by reference.
[0227] A therapeutically useful anti-GDF receptor antibody also can
be derived from a "humanized" monoclonal antibody. Humanized
monoclonal antibodies are produced by transferring mouse
complementarity determining regions from heavy and light variable
chains of the mouse immunoglobulin into a human variable domain,
and then substituting human residues in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are known (see, for example, Orlandi et al., Proc. Natl.
Acad. Sci., USA 86:3833, 1989, which is hereby incorporated in its
entirety by reference). Techniques for producing humanized
monoclonal antibodies also are known (see, for example, Jones et
al., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988;
Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc.
Natl. Acad. Sci., USA 89:4285, 1992; Sandhu, Crit. Rev. Biotechnol.
12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993; each
of which is incorporated herein by reference).
[0228] Antibodies of the invention also can be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library (see, for example, Barbas et al., METHODS: A Companion to
Methods in Immunology 2:119, 1991; Winter et al., Ann. Rev.
Immunol. 12:433, 1994; each of which is incorporated herein by
reference). Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from STRATAGENE Cloning Systems (La Jolla, Calif.).
[0229] An antibody of the invention also can be derived from a
human monoclonal antibody. Such antibodies are obtained from
transgenic mice that have been "engineered" to produce specific
human antibodies in response to antigenic challenge. In this
technique, elements of the human heavy and light chain loci are
introduced into strains of mice derived from embryonic stem cell
lines that contain targeted disruptions of the endogenous heavy and
light chain loci. The transgenic mice can synthesize human
antibodies specific for human antigens, and the mice can be used to
produce human antibody-secreting hybridomas. Methods for obtaining
human antibodies from transgenic mice are described, for example,
by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature
368:856, 1994; and Taylor et al., Int. Immunol. 6:579, 1994; each
of which is incorporated herein by reference.
[0230] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of the antibody or by expression in E.
coli of DNA encoding the fragment. Antibody fragments can be
obtained by pepsin or papain digestion of whole antibodies by
conventional methods. For example, antibody fragments can be
produced by enzymatic cleavage of antibodies with pepsin to provide
a 5S fragment denoted F(ab')2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly (see, for example,
Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647,
each of which is incorporated by reference, and references
contained therein; Nisonhoff et al., Arch. Biochem. Biophys.
89:230. 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al.,
Meth. Enzymol., 1:422 (Academic Press 1967), each of which is
incorporated herein by reference; see, also, Coligan et al., supra,
1992, see sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
[0231] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light/heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques can also be used, provided the fragments
specifically bind to the antigen that is recognized by the intact
antibody. For example, Fv fragments comprise an association of VH
and VL chains. This association can be noncovalent (Inbar et al.,
Proc. Natl. Acad. Sci., USA 69:2659, 1972). Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as glutaraldehyde (Sandhu, supra,
1992). Preferably, the Fv fragments comprise VH and VL chains
connected by a peptide linker. These single-chain antigen binding
proteins (sFv) are prepared by constructing a structural gene
comprising DNA sequences encoding the VH and VL domains connected
by an oligonucleotide. The structural gene is inserted into an
expression vector, which is subsequently introduced into a host
cell such as E. coli. The recombinant host cells synthesize a
single polypeptide chain with a linker peptide bridging the two V
domains. Methods for producing sFvs are described, for example, by
Whitlow et al., Methods: A Companion to Methods in Enzymology 2:97,
1991; Bird et al., Science 242:423-426, 1988; Ladner et al., U.S.
Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271-1277, 1993;
each of which is incorporated herein by reference; see, also
Sandhu, supra, 1992.
[0232] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106, 1991, which is incorporated herein by
reference).
[0233] The invention also provides a method for identifying a GDF
receptor polypeptide. Such a method can be performed, for example,
by incubating components comprising GDF polypeptide and a cell
expressing a full length receptor or truncated receptor under
conditions sufficient to allow the GDF to bind to the receptor;
measuring the binding of the GDF polypeptide to the receptor; and
isolating the receptor. The GDF can be any of the known GDFs (e.g.,
GDF-1-16), and preferably is GDF-8 (myostatin) or GDF-11. Methods
of isolating the receptors are described in more detail in the
Examples section below. Accordingly, the invention also provides a
substantially purified GDF receptor, as well as peptides and
peptide derivatives of a GDF receptor that have fewer amino acid
residues than a naturally occurring GDF receptor. Such peptides and
peptide derivatives are useful as research and diagnostic tools in
the study of muscle wasting diseases and the development of more
effective therapeutics.
[0234] The invention further provides GDF receptor variants. As
used herein, the term "GDF receptors variant" means a molecule that
simulates at least part of the structure of GDF receptors. GDF
receptor variants can be useful in reducing or inhibiting GDF
binding, thereby ameliorating a pathologic condition as disclosed
herein. Examples of GDF receptor variants include, but are not
limited to, truncated GDF receptors such as a soluble extracellular
domain of a GDF receptor; a dominant negative GDF receptor such as
a dominant negative Act RIIB receptor, which lacks kinase activity;
or other truncated or mutant GDF receptors.
[0235] The invention relates not only to peptides and peptide
derivatives of a naturally-occurring GDF receptor, but also to GDF
receptor variants, including mutants GDF receptors, and chemically
synthesized derivatives of GDF receptors that specifically bind a
GDF, for example, myostatin. For example, changes in the amino acid
sequence of a GDF receptor are contemplated in the present
invention. GDF receptors can be altered by changing the DNA
encoding the protein. Preferably, only conservative amino acid
alterations are undertaken, using amino acids that have the same or
similar properties. Illustrative amino acid substitutions include
the changes of alanine to serine; arginine to lysine; asparagine to
glutamine or histidine; aspartate to glutamate; cysteine to serine;
glutamine to asparagine; glutamate to aspartate; glycine to
proline; histidine to asparagine or glutamine; isoleucine to
leucine or valine; leucine to valine or isoleucine; lysine to
arginine, glutamine, or glutamate; methionine to leucine or
isoleucine; phenylalanine to tyrosine, leucine or methionine;
serine to threonine; threonine to serine; tryptophan to tyrosine;
tyrosine to tryptophan or phenylalanine; valine to isoleucine or
leucine.
[0236] Variants useful for the present invention comprise analogs,
homologs, muteins and mimetics of a GDF receptor that retain the
ability to specifically bind to their respective GDFs. In another
embodiment, variant GDF receptors that have dominant negative
activity also are contemplated, regardless of whether the variant
also interacts specifically with its GDF. Peptides of the GDF
receptors refer to portions of the amino acid sequence of GDF
receptors that have these abilities. The variants can be generated
directly from GDF receptors itself by chemical modification, by
proteolytic enzyme digestion, or by combinations thereof.
Additionally, genetic engineering techniques, as well as methods of
synthesizing polypeptides directly from amino acid residues, can be
employed.
[0237] Peptides can be synthesized by such commonly used methods as
t-BOC or FMOC protection of alpha-amino groups. Both methods
involve stepwise syntheses whereby a single amino acid is added at
each step starting from the C terminus of the peptide (Coligan, et
al., Current Protocols in Immunology (Wiley Interscience, 1991),
Unit 9, which is incorporated herein by reference). Peptides of the
invention can also be synthesized by the well known solid phase
peptide synthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149,
1962; Stewart and Young, Solid Phase Peptides Synthesis (Freeman,
San Francisco, 1969), see pages 27-62, each of which is
incorporated herein by reference), using a
copoly(styrene-divinylbenze- ne) containing 0.1-1.0 mMol amines/g
polymer. On completion of chemical synthesis, the peptides can be
deprotected and cleaved from the polymer by treatment with liquid
HF-10% anisole for about 1/4-1 hours at 0.degree. C. After
evaporation of the reagents, the peptides are extracted from the
polymer with 1% acetic acid solution which is then lyophilized to
yield the crude material. This can normally be purified by such
techniques as gel filtration on Sephadex G-15 using 5% acetic acid
as a solvent. Lyophilization of appropriate fractions of the column
will yield the homogeneous peptide or peptide derivatives, which
can then be characterized by such standard techniques as amino acid
analysis, thin layer chromatography, high performance liquid
chromatography, ultraviolet absorption spectroscopy, molar
rotation, solubility, and quantitated by the solid phase Edman
degradation.
[0238] Non-peptide compounds that mimic the binding and function of
GDF receptors ("mimetics") can be produced by the approach outlined
by Saragovi et al. (Science 253: 792-95, 1991, which is
incorporated herein by reference). Mimetics are molecules which
mimic elements of protein secondary structure (Johnson et al.,
"Peptide Turn Mimetics," in Biotechnology and Pharmacy (Pezzuto et
al., Eds.; Chapman and Hall, New York 1993), which is incorporated
herein by reference). The underlying rationale behind the use of
peptide mimetics is that the peptide backbone of proteins exists
chiefly to orient amino acid side chains in such a way as to
facilitate molecular interactions. For the purposes of the present
invention, an appropriate mimetic can be considered to be the
equivalent of a GDF receptor.
[0239] Longer peptides can be produced by the "native chemical"
ligation technique which links together peptides (Dawson et al.,
Science 266:776, 1994, which is incorporated herein by reference).
Variants can be created by recombinant techniques employing genomic
or cDNA cloning methods. Site specific and region directed
mutagenesis techniques can be employed (Ausubel et al., supra, 1989
and 1990 to 1993 supplements), see volume 1, chapter 8; Protein
Engineering (Oxender and Fox eds., A. Liss, Inc., 1987)). In
addition, linker scanning and PCR mediated techniques can be
employed for mutagenesis (Erlich, PCR Technology (Stockton Press
1989); Ausubel et al., supra, 1989 to 1993). Protein sequencing,
structure and modeling approaches for use with any of the above
techniques are disclosed in the above cited references.
[0240] The present invention also provides GDF receptor-binding
agents that block the specific binding of a GDF to its receptor.
Such agents are useful, for example, as research and diagnostic
tools in the study of muscle wasting disorder as described above
and as effective therapeutics, and can be identified using the
methods as disclosed herein, for example, a molecular modeling
method. In addition, pharmaceutical compositions comprising GDF
receptor-binding agents can represent effective therapeutics. In
the context of the invention, the phrase "GDF receptor-binding
agent" denotes a naturally occurring ligand of a GDF receptor, for
example, GDF-1 to GDF-16; a synthetic ligand of GDF receptors, or
an appropriate derivative of the natural or synthetic ligands. The
determination and isolation of ligands is well known in the art
(Lerner, Trends Neurosci. 17:142-146, 1994, which is incorporated
herein by reference).
[0241] In yet another embodiment, the present invention relates to
GDF receptor-binding agents that interfere with binding between a
GDF receptor and a GDF. Such binding agents can interfere by
competitive inhibition, by non-competitive inhibition or by
uncompetitive inhibition. Interference with normal binding between
GDF receptors and one or more GDF can result in a useful
pharmacological effect.
[0242] The invention also provides a method for identifying a
composition that binds to a GDF receptor. The method includes
incubating components comprising the composition and a GDF receptor
under conditions sufficient to allow the components to interact
specifically, and measuring the binding of the composition to GDF
receptors. Compositions that bind to GDF receptors include
peptides, peptidomimetics, polypeptides, chemical compounds and
biologic agents as described above. Incubating includes exposing
the reactants to conditions that allow contact between the test
composition and GDF receptors, and provide conditions suitable for
a specific interaction as would occur in vivo. Contacting can be in
solution or in solid phase. The test ligand/composition can
optionally be a combinatorial library for screening a plurality of
compositions, as described above. Compositions identified in the
method of the invention can be further evaluated, detected, cloned,
sequenced, and the like, either in solution or after binding to a
solid support, by any method usually applied to the detection of a
specific DNA sequence such as PCR, oligomer restriction (Saiki et
al., Bio/Technology 3:1008-1012, 1985, which is incorporated herein
by reference), allele-specific oligonucleotide (ASO) probe analysis
(Conner et al., Proc. Natl. Acad. Sci., USA 80:278, 1983, which is
incorporated herein by reference), oligonucleotide ligation assays
(OLAs) (Landegren et al., Science 241:1077 1988, which is
incorporated herein by reference), and the like (see Landegren et
al., Science 242:229-237, 1988. which is incorporated herein by
reference).
[0243] To determine if a composition can functionally complex with
the receptor protein, induction of an exogenous gene can be
monitored by monitoring changes in the protein level of a protein
encoded for by the exogenous gene, or any other method as disclosed
herein. When a compositions is identified that can induce
transcription of the exogenous gene, it is concluded that this
composition can specifically bind to the receptor protein coded for
by the nucleic acid encoding the initial sample test
composition.
[0244] Expression of the exogenous gene can be monitored by a
functional assay or assay for a protein product, for example. The
exogenous gene is therefore a gene that provides an
assayable/measurable expression product in order to allow detection
of expression of the exogenous gene. Such exogenous genes include,
but are not limited to, reporter genes such as chloramphenicol
acetyltransferase gene, an alkaline phosphatase gene,
.theta.-galactosidase, a luciferase gene, a green fluorescent
protein gene, guanine xanthine phosphoribosyltransferase, alkaline
phosphatase, and antibiotic resistance genes such as neomycin
phosphotransferase (see above).
[0245] Expression of the exogenous gene is indicative of a specific
interaction of a composition and a GDF receptor; thus, the binding
or blocking composition can be identified and isolated. The
compositions of the present invention can be extracted and purified
from the culture media or a cell by using known protein
purification techniques commonly employed, including, for example,
extraction, precipitation, ion exchange chromatography, affinity
chromatography, gel filtration and the like. Compositions can be
isolated by affinity chromatography using the modified receptor
protein extracellular domain bound to a column matrix or by heparin
chromatography.
[0246] Also included in the screening method of the invention is
combinatorial chemistry methods for identifying chemical compounds
that bind to GDF receptors, as described above. Thus, the screening
method is also useful for identifying variants, binding or blocking
agents, etc., which functionally, if not physically (e.g.,
sterically) act as antagonists or agonists, as desired.
[0247] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE 1
Myostatin Acts in a Dose Dependent Manner
[0248] This example demonstrates that the activity of myostatin in
inhibiting muscle growth is dependent on the level of myostatin
expression in vivo.
[0249] Myostatin is a negative regulator of skeletal muscle mass
(McPherron et al., supra, 1997; McPherron and Lee, supra, 1997).
Myostatin knock-out mice that were homozygous for a deletion of the
myostatin gene had a 25-30% increase in total body mass. An
examination of the homozygous knock-out mice revealed that the
increased muscle mass was due to about a 100-200% increase in
skeletal muscle mass throughout the body.
[0250] Mice that were heterozygous for the myostatin mutation also
had an increase in total body mass. However, the increase mass of
the heterozygotes was less than that of the homozygotes, and was
statistically significant in only one age and sex group among the
many examined. In order to determine whether heterozygous mice have
an intermediate phenotype between that of wild type mice and
homozygous myostatin knock-out mice, the analysis of muscle weights
was extended to the heterozygous mice. Individual muscles sampled
from heterozygous mice weighed approximately 25-50% more than those
of wild type mice. These results demonstrate that mice that are
heterozygous for deletion of a myostatin gene have a phenotype that
is intermediate between that of wild type mice and homozygous
myostatin knock-out mice, and demonstrate that myostatin produces a
dose-dependent effect in vivo.
[0251] These results indicate that the manipulation of myostatin
activity can be useful in treating muscle wasting diseases and
other metabolic disorders associated with myostatin activity.
Furthermore, the dose-dependent effect of myostatin indicates that
a therapeutic effect can be obtained without achieving complete
inhibition of myostatin activity, thereby allowing for an
adjustment of myostatin activity if, for example, a certain level
of activity produces undesirable effects in a subject.
EXAMPLE 2
Myostatin Effect Decreases with Age in Knock-Out Mice
[0252] This example demonstrates that a decreased difference in
body weight between wild type mice and homozygous myostatin
knock-out mice is associated with a decline in muscle weight of the
mutant mice.
[0253] Myostatin knock-out mice weighed approximately 25-30% more
than wild type mice at five months of age (McPherron et al., supra,
1997). However, this difference in total body weights became
significantly smaller or disappeared altogether as the animals
aged. In order to determine whether this effect was due to a
relative loss of weight in the knock-out mice due, for example, to
muscle degeneration, or to a relatively greater weight gain by wild
type mice, a detailed analysis of muscle weights was made as a
function of age.
[0254] At all ages examined from 2 months to 17 months, the
pectoralis muscle weighed significantly more in homozygous mutant
mice than in wild type littermates. The most dramatic difference
was observed at 5 months, at which time the pectoralis weight was
approximately 200% greater in the mutant mice. Although the
pectoralis weight declined slightly at older ages, the weight of
this muscle in mutant mice remained greater than twice that of wild
type mice. This same basic trend was observed in all of the other
muscles examined, including the triceps brachii, the quadriceps,
the gastrocnemius and plantaris, and the tibialis anterior. Similar
trends were observed in both male and female mice. These results
demonstrate that the decreased difference in total body weights
between mutant and wild type mice observed with aging is due to a
slight decline in muscle weights in the mutant mice.
EXAMPLE 3
Myostatin Affects Fat Accumulation in a Dose Dependent Manner
[0255] This example demonstrates that myostatin knock-out mice fail
to accumulate fat, and that the decrease in fat accumulation is
associated with the level of myostatin expression in vivo.
[0256] Since the decline in muscle weights in myostatin mutants, as
demonstrated in Example 2, did not fully account for the
observation that the wild type animals eventually weighed about the
same as the mutant mice, the amount of fat accumulation in wild
type and mutant mice was examined. The inguinal, epididymal and
retroperitoneal fat pads in male mice were examined. There was no
difference in the weights of any of these fat pads between wild
type and mutant mice at two months of age. By 5 to 6 months of age,
wild type and heterozygous knock-out mice both exhibited a large
range of fat pad weights, and, on average, fad pad weights
increased by approximately 3-fold to 5-fold by the time the animals
reached 9 to 10 months of age. Due to the large range of fat pad
weights observed in these animals, some animals showed a much
larger increase (up to 10-fold) than others.
[0257] In contrast to the wild type and heterozygous knock-out
mice, the fat pad weights of myostatin homozygous mutant mice were
in a relatively narrow range and were virtually identical in 2
month old mice and in 9 to 10 month old mice. Thus, the increased
fat accumulation that occurred with aging in the wild type mice was
not observed in the homozygous myostatin knock-out mice. This
difference in fat accumulation, together with the slight decline in
muscle weights, as a function of age in the homozygous mutant mice
fully accounted for the observation that the wild type animals
eventually have the same total body weight as the mutants.
[0258] The mean fat pad weights of heterozygous knock-out mice at 9
to 10 months of age was intermediate between that of the wild type
mice and the homozygous mutant mice. Although this difference was
not statistically significant, due to the wide range of fat pad
weights in these and the wild type mice, these results nevertheless
indicate that myostatin has a dose-dependent effect on the
accumulation of fat, similar to its effect on muscle growth.
EXAMPLE 4
Effect of Myostatin on Metabolism
[0259] This example demonstrates that serum insulin and glucose
levels, as well as metabolic activity, are affected by the level of
myostatin expression.
[0260] In order to determine whether the skeletal muscle
hypertrophy and the lack of fat accumulation in the myostatin
mutant mice is due to an effect on overall metabolism, the
metabolic profile of the mutant mice was examined. Serum
triglyceride and serum cholesterol levels were significantly lower
in myostatin mutant mice as compared to wild type control mice
(Table 1). Serum insulin levels also appeared to be lower in the
myostatin mutant mice. However, the fed and fasting glucose levels
both were indistinguishable among homozygous mutant mice and wild
type mice (Table 1), and both groups of mice had a normal response
in a glucose tolerance test. The results demonstrate that the
homozygous myostatin knock-out mice can maintain normal levels of
serum glucose even though their serum insulin level is lower than
that of wild type animals.
1TABLE 1 SERUM PARAMETERS +/+ -/- triglycerides (mg/dl) 131.5 +/-
16.5 66.8 +/- 11.4 p = 0.012 cholesterol (mg/dl) 138.3 +/- 8.1 94.5
+/- 6.8 p = 0.0034 fed glucose (mg/dl) 114.0 +/- 4.8 119.3 +/- 5.2
n.s. (p = 0.43) fasting glucose (mg/dl) 86.5 +/- 3.8 103.3 + 9.3
n.s. (p = 0.13) +/+ indicates wild type mice; -/- indicate
homozygous knock-out mice
[0261] In order to determine whether differences in metabolic rates
could explain the lack of fat accumulation in the mutant mice, the
rate of oxygen consumption of wild type and mutant mice was
compared using a calorimeter. Mutant mice had a lower basal
metabolic rate and a lower overall metabolic rate than wild type
control mice. These results indicate that the lack of fat
accumulation in the myostatin mutant mice is not due to a higher
rate of metabolic activity.
EXAMPLE 5
Myostatin Affects Fat Accumulation in Genetically Obese Mice
[0262] This example demonstrates that a lack of myostatin
expression suppresses fat accumulation in mice that are a genetic
model for obesity.
[0263] In order to determine whether the loss of myostatin activity
could suppress fat accumulation not only in normal mice but also in
obese mice, the effect of the myostatin mutation in agouti lethal
yellow (Ay) mice, which represent a genetic model of obesity (Yen
et al., FASEB J. 8:479-488, 1994), was examined. Mice that were
doubly heterozygous for the lethal yellow and myostatin mutations
were generated, and offspring from crosses of these doubly
heterozygous mice were examined.
[0264] The total body weight of the Ay/a, myostatin -/- double
mutant mouse was dramatically reduced (approximately 9 grams)
compared to that of the Ay/a, myostatin +/+ mouse. This reduction
in total body weight was even more dramatic considering that the
Ay/a, myostatin -/- double mutant had about 2 to 3 times more
skeletal muscle than did the AY/a, myostatin +/+ mouse. The double
mutant had approximately 10 grams more muscle than the Ay/a,
myostatin +/+ mouse and, therefore, the total weight reduction in
the rest of the tissues was about 19 grams.
[0265] The reduction in total body weight resulted from a reduction
in overall fat content. As shown in Table 2, the weights of the
parametrial and retroperitoneal fat pads were reduced 5-fold to
6-fold in the Ay/a, myostatin -/- double mutant as compared to the
Ay/a, myostatin +/+ mouse. These results indicate that the presence
of the myostatin mutation dramatically suppresses fat accumulation
in obesity.
[0266] The presence of the myostatin mutation also dramatically
affected glucose metabolism. Agouti lethal mice lacking the
myostatin mutation had grossly abnormal glucose tolerance test
results, with serum glucose levels often reaching 450 to 600 mg/dl
and only slowly recovering to baseline levels over a period of 4
hours. Female agouti lethal mice were affected less than male mice,
and some females responded almost normally in this test, as
previously described (see Yen et al., supra, 1994). In contrast,
although the Ay/a, myostatin -/- mice had slightly abnormal glucose
tolerance tests, but none of these animals had the gross
abnormalities observed in the Ay/a myostatin +/+ mice.
[0267] These results indicate that the myostatin mutation at least
partially suppressed the development of abnormal glucose metabolism
in the agouti lethal mice. Significantly, mice that were
heterozygous for the myostatin mutation had an intermediate
response compared to myostatin +/+ and myostatin -/- mice, thus
confirming the dose-dependent effect of myostatin.
EXAMPLE 6
Purification of Recombinant Myostatin
[0268] This example provides a method for preparing and isolating
recombinant myostatin.
[0269] In order to elucidate the biological activity of myostatin,
large quantities of myostatin protein were purified for bioassays.
Stable Chinese hamster ovary (CHO) cell lines producing high levels
of myostatin protein were generated by co-amplifying a myostatin
expression cassette with a dihydrofolate reductase cassette using a
methotrexate selection scheme (McPherron et al., supra, 1997).
Myostatin was purified from the conditioned medium of the highest
producing line by successive fractionation on hydroxyapatite,
lentil lectin SEPHAROSE, DEAE agarose, and heparin SEPHAROSE.
Silver stain analysis revealed that the purified protein obtained
following these four column-chromatography steps (referred to as
"heparin eluate") consisted of two species with molecular masses of
approximately 35 kilodaltons (kDa) and 12 kDa.
[0270] The purified protein preparation was determined by various
criteria to represent a complex of two myostatin prodomain peptides
and a disulfide-linked dimer of mature C-terminal myostatin
peptides. First, western blot analysis, using antibodies raised
against specific portions of the promyostatin sequence, identified
the 35 kDa band as the prodomain and the 12 kDa band as the mature
C-terminal peptide. Second, under non-reducing conditions, the
species reacting with antibodies directed against the mature
C-terminal peptide had an electrophoretic mobility consistent with
a disulfide linked dimer. Third, the molar ratio of prodomain to
mature C-terminal peptide was approximately 1:1. Fourth, the
prodomain and mature C-terminal peptide copurified through the four
column chromatography steps. Finally, the mature C-terminal peptide
bound to the lentil lectin column even though the C-terminal region
does not contain consensus N-linked glycosylation signals,
indicating that the mature C-terminal peptide bound to the column
due to its interaction with the prodomain peptide, which contains a
potential N-linked glycosylation site.
[0271] These results indicate that myostatin produced by the
genetically modified CHO cells is secreted in a proteolytically
processed form, and that the resulting prodomain and mature
C-terminal region associate non-covalently to form a complex
containing two prodomain peptides and a disulfide-linked dimer of
C-terminal proteolytic fragments, similar to that described for
TGF-.beta.3. In the TGF-.theta. complex, the C-terminal dimer
exists in an inactive, latent form (Miyazono et al., J. Biol. Chem.
263:6407-6415, 1988), and the active species can be released from
this latent complex by treatment with acid, chaotropic agents,
reactive oxygen species, or plasmin, or by interactions with other
proteins, including thrombospondin and integrin Iv.beta.6 (Lawrence
et al., Biochem. Biophys. Res. Comm. 133:1026-1034, 1985; Lyons et
al., J. Cell Biol. 106:1659-1665, 1988; Schultz-Cherry and
Murphy-Ullrich, J. Cell Biol. 122:923-932, 1993; Barcellos-Hoff and
Dix, Mol. Endocrinol. 10:1077-1083, 1996; Munger et al., Cell
96:319-328, 1999). Furthermore, the addition of purified prodomain
peptide (also known as latency-associated peptide or LAP) to the
TGF-.theta. complex inhibits the biological activity of the
purified C-terminal dimer in vitro and in vivo (Gentry and Nash,
Biochemistry 29:6851-6857, 1990; Bottinger et al., Proc. Natl.
Acad. Sci., USA 93:5877-5882, 1996).
[0272] The heparin eluate, which consisted of a complex of
prodomain and mature C-terminal peptide, was further purified using
an HPLC C4 reversed phase column. The C-terminal dimer eluted from
the HPLC column earlier than the prodomain, thus allowing the
isolation of the C-terminal dimer free of prodomain. Fractions that
contained mostly prodomain also were obtained, although these
fractions contained small amounts of the C-terminal dimer. Some of
the protein also was present as higher molecular weight complexes.
The nature of the higher molecular weight complexes is unknown,
but, based on western blot analysis in the presence or absence of
reducing agents, these complexes can contain at least one prodomain
peptide and one C terminal mature myostatin peptide linked by one
or more disulfide bonds. In fact, most of the mature C-terminal
peptide present in the HPLC fraction enriched for the propeptide
(HPLC fractions 35-37) was present in these high molecular weight
complexes. These higher molecular weight complexes likely represent
improperly folded proteins that are secreted by the genetically
modified CHO cells.
EXAMPLE 7
Myostatin Specifically Interacts with an Activin Receptor
[0273] This example demonstrates that myostatin specifically binds
an activin type II receptor expressed on cells in culture, and that
this specific binding is inhibited by a myostatin prodomain.
[0274] The receptors for some TGF-.beta. family members have been
identified, and most are single membrane spanning serine/threonine
kinases (Massague and Weis-Garcia, Cancer Surveys 27:41-64, 1996).
The activin type II receptors (Act RIIA and/or Act RIIB), for
example, are known to bind members of the TGF-.beta. superfamily.
The phenotype of mice lacking the Act RIIB receptor showed
anterior/posterior axial patterning defects and kidney
abnormalities that were very similar to those observed in GDF-11
knock-out mice (McPherron et al., Nat. Genet. 22:260-264, 1999; Oh
and Li, Genes Devel. 11:1812-1826, 1997). Since the amino acid
sequences of GDF-11 and myostatin (GDF-8) are 90% identical in the
mature C-terminal region, the ability of myostatin to specifically
interact with a activin type II receptor was examined
[0275] Myostatin was labeled by radio-iodination, and binding
studies were performed using COS cells transfected with an Act RIIB
expression construct. Myostatin interacted specifically with the
transfected COS cells. Myostatin binding was competed in a dose
dependent manner by excess unlabeled myostatin, and was
significantly lower in control COS cells, which were transfected
with an empty vector. No significant binding occurred to cells
transfected with a BMP RII or TGF-.beta. RII expression construct.
Myostatin binding to Act RIIB-transfected cells was saturable, and
the binding affinity was approximately 5 nM as determined by
Scatchard analysis.
[0276] The receptor binding assay also was used to examine the
ability of the myostatin prodomain to inhibit the ability of the
mature C-terminal dimer to interact specifically with Act RIIB in
this system. The addition of purified prodomain peptide blocked the
ability of the C-terminal dimer to bind the Act RIIB transfected
COS cells in a dose-dependent manner. These results indicate that
the myostatin prodomain is a natural inhibitor of myostatin.
EXAMPLE 8
Increased Myostatin Levels Induce Weight Loss
[0277] This example demonstrates that elevated levels of myostatin
can lead to substantial weight loss in vivo.
[0278] In one set of experiments, CHO cells that express myostatin
were injected into nude mice. The nude mice that had myostatin
expressing CHO cell tumors showed severe wasting over the course of
approximately 12 to 16 days following injection of the cells. This
wasting syndrome was not observed in nude mice injected with any of
a variety of control CHO lines that had undergone a similar
selection process but did not express myostatin. Furthermore, the
myostatin coding sequence in the construct used to transfect the
CHO cells was under the control of a metallothionein promoter, and
the wasting syndrome was exacerbated when mice bearing the
myostatin expressing tumors were maintained on water containing
zinc sulfate. Western blot analysis revealed high levels of
myostatin protein in the serum of the nude mice that bore the
myostatin-expressing CHO cells. These results indicate that the
wasting syndrome was induced in response to the elevated level of
myostatin in the nude mice and, as discussed below, this result was
confirmed by observing similar effects in mice injected with
purified myostatin.
[0279] The dramatic weight loss observed in the nude mice bearing
myostatin expressing CHO cells was due primarily to a
disproportionate loss of both fat and muscle weight. White fat pad
weights (intrascapular white, uterine, and retroperitoneal fat)
were reduced by greater than 90% compared to mice bearing control
CHO cell tumors. Muscle weights were also severely reduced, with
individual muscles weighing approximately half as much in myostatin
expressing mice as in control mice by day 16. This loss in muscle
weight was reflected by a corresponding decrease in fiber sizes and
protein content.
[0280] Mice bearing the myostatin expressing CHO cell tumors also
became severely hypoglycemic. However, the weight loss and
hypoglycemia were not due to a difference in food consumption, as
all of the mice consumed equivalent amounts of food at each time
interval examined during the 16 day course of the study. These
results indicate that myostatin overexpression induces a dramatic
weight loss, which resembles the cachectic wasting syndrome that
occurs in patients suffering from chronic diseases such as cancer
or AIDS.
[0281] With more chronic administrations using lower doses of
myostatin, changes in fat weight were observed. For example; twice
daily injections of 1 .mu.g of myostatin protein for 7 days
resulted in an approximately 50% decrease in the weights of a
number of different white fat pads (intrascapular white, uterine,
and retroperitoneal fat pads) with no significant effect on brown
fat (intrascapular brown). These results confirm that myostatin can
induce weight loss and, in extreme cases, a wasting syndrome in
vivo.
EXAMPLE 9
Characterization of Myostatin Binding to an Activin Receptor
[0282] This example describes a method for characterizing the
relationship of myostatin binding to an activin receptor with the
biological effects produced by myostatin in vivo.
[0283] Act RIIA or Act RIIB knock-out mice can be used to confirm
that Act RIIA or Act RIIB is a receptor for myostatin in vivo. A
detailed muscle analysis of these mice can determine whether
knock-out of an activin receptors is associated with a change in
muscle fiber number or size. Since Act RIIA/Act RIIB double
homozygous mutant die early during embryogenesis (Song et al.,
Devel. Biol. 213:157-169, 1999), only the various
homozygous/heterozygous combinations can be examined. However,
tissue-specific or conditional knock-out mice can be generated such
that both genes can be "deleted" only in muscle tissue, thus
allowing postnatal examination of the double homozygous knock-out
mice.
[0284] The effect on adipose tissue can be examined with aging of
the mice to determine whether the number of adipocytes or the
accumulation of lipid by these adipocytes is altered in the
knock-out mice. Adipocyte number and size is determined by
preparing cell suspensions from collagenase treated tissue
(Rodbell, J. Biol. Chem. 239:375-380, 1964; Hirsch and Gallian, J.
Lipid Res. 9:110-119, 1968). Total lipid content in the animals is
determined by measuring dry carcass weights and then the residual
dry carcass weights after lipid extraction (Folch et al., J. Biol.
Chem. 226:497-509, 1957).
[0285] A variety of serum parameters also can be examined,
including fed and fasting glucose and insulin, triglycerides,
cholesterol, and leptin. As disclosed above, serum triglycerides
and serum insulin are decreased in the myostatin mutant animals.
The ability of the activin receptor knock-out mice to respond to an
exogenous glucose load also can be examined using glucose tolerance
tests. As disclosed above, the response to a glucose load
essentially was identical in wild type and myostatin mutant mice at
5 months of age. This observation can be extended by measuring
these parameters in the mice as they age. Serum insulin levels also
can be measured at various times during the glucose tolerance
tests.
[0286] Basal metabolic rates also can be monitored using a
calorimeter (Columbus Instruments). As disclosed above, myostatin
mutant mice have a lower metabolic rate at 3 months of age than
their wild type counterparts. This analysis can be extended to
older mice, and the respiratory quotient also can be measured in
these animals. The ability to maintain normal thermogenesis can be
determined by measuring the basal body temperatures as well as
their ability to maintain body temperature when placed at 4.degree.
C. Brown fat weights and expression levels of UCP1, UCP2, and UCP3
in brown fat, white fat, muscle, and other tissues also can be
examined (Schrauwen et al., 1999).
[0287] Food intake relative to weight gain can be monitored, and
feed efficiency can be calculated. In addition, the weight gain of
the animals placed on high fat diets can be monitored. Wild type
mice maintained on a high fat diet accumulate fat rapidly, whereas
the results disclosed herein indicate the activin receptor mutant
animals will remain relatively lean.
[0288] The results of these studies can provide a more complete
profile of the effect of myostatin mice, particularly with respect
to their overall metabolic state, thereby providing insight as to
whether the ability of the myostatin knock-out mice to suppress the
accumulation of fat is an anabolic effect of the myostatin mutation
in muscle that leads to a shift in energy utilization such that
little energy is available for storage in the form of fat. For
example, the decreased fat accumulation can be due to an increased
rate of thermogenesis. These results also will provide a baseline
for comparison of the effect of the myostatin activity in the
context of different genetic models of obesity and type II
diabetes.
EXAMPLE 10
Characterization of Myostatin Effects in Genetic Models of Obesity
and Type II Diabetes
[0289] This example describes methods for determining the effect of
myostatin in treating obesity or type II diabetes.
[0290] The dramatic reduction in overall fat accumulation in
myostatin mutant mice as compared to wild type mice indicates that
myostatin activity can be manipulated to treat or prevent obesity
or type II diabetes. The effect of the myostatin mutation can be
examined in the context of several well characterized mouse models
of these metabolic diseases, including, for example, "obese" mice
(ob/ob), "diabetic" mice (db/db), and agouti lethal yellow (Ay)
mutant strains. Each of these strains is abnormal for virtually
every parameter and test described above (see, for example, Yen et
al., supra, 1994, Friedman and Halaas, Nature 395:763-770, 1998).
The ability of the myostatin mutation to slow or suppress the
development of these abnormalities in mice carrying these other
mutations can be examined by constructing double mutants, then
subjecting the double mutant animals, along with appropriate
control littermates carrying only the ob/ob, db/db, or agouti
lethal yellow mutations, to the various tests.
[0291] As disclosed above, the myostatin mutation in Ay mice was
associated with about a 5-fold suppression of fat accumulation in
the myostatin mutant Ay mice, and with a partial suppression of the
development of abnormal glucose metabolism as assessed by glucose
tolerance tests. These results can be extended to include
additional animals at various ages, and similar studies can be
performed with the ob/ob and db/db mutants. As both the these
mutations are recessive, mice that are doubly homozygous for the
myostatin mutation and either the ob or db mutation can be
generated. In order to examine the effects of partial loss of
myostatin function in these genetic model systems, mice that are
homozygous for the ob or db mutation and heterozygous for the
myostatin mutation also are examined. Mice that are doubly
heterozygous for the myostatin and ob mutations have been
generated, and the offspring from matings of these doubly
heterozygous mice can be examined, particularly with respect to fat
accumulation and glucose metabolism. Partial suppression of either
or both of these abnormalities in the obese mutants can indicate
that myostatin is a target for the treatment of obesity and type II
diabetes.
EXAMPLE 11
Characterization of Transgenic Mice Expressing Dominant Negative
Polypeptides that can Affect Myostatin Activity
[0292] This example describes methods for characterizing the effect
of myostatin postnatally by expressing dominant negative
polypeptides that can block myostatin expression or myostatin
signal transduction.
[0293] Myostatin Inhibitors
[0294] The modulation of myostatin activity postnatally can be used
to determine the effect of myostatin on muscle fiber number
(hyperplasia) and muscle fiber size (hypertrophy). Conditional
myostatin knock-out mice, in which the myostatin gene is deleted at
defined times during the life of the animal, can be used for these
studies. The tet regulator in combination with the cre recombinase
provides a system for generating such mice. In this system, the
expression of cre is induced by administration of doxycycline.
[0295] Transgenic mice expressing an inhibitor of myostatin from an
inducible promoter also can be generated such that myostatin
activity can be reduced or inhibited at defined times during the
life of the animal. The tetracycline regulators are useful for
generating such transgenic mice, in which the myostatin expression
is induced by doxycycline.
[0296] A modification of the tet system, which utilizes
co-expression of a hybrid reverse tet-transactivator (fusion
protein of the activation domain of VP16 with the mutant reverse
tet repressor) and a hybrid tet-transrepressor (fusion protein of
the KRAB repressor domain of mammalian Kox1 with the native tet
repressor), can be particularly useful for producing the transgenic
mice (Rossi et al., Nat. Genet. 20:389-393, 1998; Forster et al.,
Nucl. Acids Res. 27:708-710, 1999). In this system, the hybrid
reverse tet-transactivator binds tet operator sequences, and
activates transcription only in the presence of tetracycline; the
hybrid tet-transrepressor binds tet operator sequences and
represses transcription only in the absence of tetracycline. By
co-expressing these two fusion proteins, the basal activity of the
target promoter is silenced by the tet-transrepressor in the
absence of tetracycline, and is activated by the reverse
tet-transactivator upon administration of tetracycline.
[0297] Two types of transgenic lines can be generated. In the first
type, the transgene encodes a myostatin inhibitor polypeptide under
the control of a muscle specific promoter, for example, the muscle
creatine kinase promoter (Stemberg et al., supra, 1988) or the
myosin light chain enhancer/promoter (Donoghue et al., supra,
1991). Individual transgenic lines are screened for specific
expression of the tet regulators in skeletal muscle, and several
independent lines for each of the two promoters are selected and
examined to confirm that any effects observed are not due, for
example, to integration site-specific effects. A construct
containing the two tet regulators under the control of the myosin
light chain promoter/enhancer has been constructed, and can be used
for pronuclear injections. In the second type of line. the
transgene contains a myostatin inhibitor polypeptide under the
control of a minimal CMV promoter that further contains tet
operator sequences.
[0298] The myostatin inhibitor can be a dominant negative form of
myostatin or a myostatin prodomain, which, as disclosed herein, can
inhibit myostatin activity. Dominant negative forms of TGF-.beta.
family members have been described (see, for example, Lopez et al.,
Mol. Cell Biol. 12:1674-1679, 1992; Wittbrodt and Rosa, Genes
Devel. 8:1448-1462, 1994), and contain, for example, a mutant
proteolytic cleavage site, thereby preventing the protein from
being processed into the biologically active species. When
co-expressed in a cell with the endogenous wild type gene, the
mutant protein forms non-functional heterodimers with the wild type
protein, thus acting as a dominant negative. A mutant myostatin
polypeptide containing a mutation in the promyostatin cleavage site
has been constructed, and can be examined for a dominant negative
effect by co-expressing the mutant with wild type myostatin in
varying ratios in 293 cells. Conditioned medium from 293 cells
transiently transfected with the constructs can be examined by
western blot analysis and the ability of the mutant to block the
formation of mature C-terminal dimers can be examined.
[0299] An expression construct encoding only the myostatin
prodomain also can be utilized. As disclosed above, the prodomain
forms a tight complex with the mature C-terminal dimer and blocks
the ability of the mature C-terminal myostatin dimer to bind Act
RIIB in cells expressing the receptor in culture. By analogy to
TGF-.beta., the myostatin prodomain also can maintain the mature
C-terminal dimer in an inactive latent complex in vivo.
[0300] These transgenic animals can be bred with those expressing
the tet regulators to generate doubly transgenic lines containing
both the tet regulators and the inhibitor target construct. These
doubly transgenic lines can be screened for those in which all of
the different components are expressed appropriately. Northern blot
analysis using RNA obtained from various muscles and control
tissues from representative mice in each line, before and after
administration of doxycycline in the drinking water, can be used to
identify such transgenic lines. Transgenic lines will be selected
that do not express the transgene in any tissue in the absence of
doxycycline, and that express the transgene only in muscle in the
presence of doxycycline.
[0301] Doxycycline is administered to the selected transgenic
animals and the effect on muscle mass is examined. Doxycycline can
be administered to pregnant mothers to induce the expression of the
inhibitor during embryogenesis. The effect of blocking myostatin
activity during development of the transgenic animals can be
compared to the effects observed in myostatin knock-out mice. Since
the promoters for driving expression of the tet regulators can be
induced at a later time during development than the time when
myostatin is first expressed, the effect on muscle mass in the
transgenic mice can be compared to the effect that occurs in the
myostatin knock-out mice.
[0302] The effect of inhibiting myostatin activity postnatally can
be examined by administering doxycycline to the doubly transgenic
mice at various times after birth. Doxycycline treatment can begin,
for example, at 3 weeks of age, and the animals can be analyzed at
5 months of age, which is the age at which the difference in muscle
weights were at a maximum in the myostatin knock-out mice versus
wild type mice. The animals are examined for the effects of the
inhibitor on muscle mass. Muscles also can be examined
histologically to determine effects on fiber number and fiber size.
In addition, a fiber type analysis of various muscles in the
transgenic mice can be performed to determine whether there is a
selective effect on type I or type II fibers.
[0303] Doxycycline can be administered in different doses and at
different times to characterize the effect of the myostatin
inhibitors. Doubly transgenic mice also can be maintained
chronically on doxycycline, then examined for effects on fat pad
weights and other relevant metabolic parameters as described above.
The results of these studies can confirm that modulating myostatin
activity postnatally can increase muscle mass or decrease fat
accumulation, thus indicating that targeting myostatin can be
useful for the treatment of a variety of muscle wasting and
metabolic diseases clinically.
[0304] Myostatin
[0305] Transgenic mice containing a myostatin transgene also can be
examined and the effects produced upon expression of myostatin can
be compared with those observed in the nude mice containing the
myostatin-expressing CHO cells. Similarly as described above,
myostatin can be placed under control of conditional (tet) and
tissue specific regulatory elements, and expression of myostatin in
the transgenic mice can be examined to determine whether a wasting
syndrome occurs similar to that observed in the nude mice. The
myostatin transgene can include, for example, processing signals
derived from SV40, such that transgene can be distinguished from
the endogenous myostatin gene.
[0306] Serum samples can be isolated from the myostatin transgenic
mice at various times following the administration of doxycycline
and the level of myostatin transgene product in the serum can be
determined. Total body weights of the animals are monitored over
time to determine whether the animals show significant weight loss.
In addition, individual muscles and fat pads are isolated and
weighed, and the number, size and type of muscle fibers are
determined in selected muscle samples.
[0307] The level of myostatin transgene expression can be varied by
varying the dose of doxycycline administered to the animals.
Transgene expression can be monitored using, for example, northern
blot analysis of transgene RNA levels in muscle, or myostatin
protein levels in serum. The identification of specific levels of
myostatin transgene expression allows a correlation of the extent
of wasting induced by myostatin. The transgenic lines also can be
crossed with the myostatin knock-out mice to generate mice in which
the only source of myostatin is expression from the transgene.
Expression of myostatin at various times during development can be
examined and the effect of myostatin on fiber number, fiber size,
and fiber type can be determined. The availability of mice in which
the expression of myostatin can be precisely and rapidly controlled
provides a powerful tool for further characterizing the myostatin
signal transduction pathway and for examining the effects of
various agents that potentially can be useful for modulating
myostatin signal transduction.
[0308] Effectors of Myostatin Signal Transduction
[0309] Transgenic mice containing either dominant negative forms of
a myostatin signal transduction pathway, which can include
components of a TGF-.theta. signal transduction pathway, that is
expressed specifically in skeletal muscle can be generated. As
disclosed herein, the Smad proteins, which mediate signal
transduction through a pathway induced by activin type II
receptors, can be involved in myostatin signal transduction.
[0310] Act RIIB can bind GDF-11, which is highly related to
myostatin (McPherron et al., supra, 1997; Gamer et al., supra,
1999; Nakashima et al., Mech. Devel. 80:185-189, 1999), and
expression of c-ski, which can bind an inhibit Smad 2, Smad 3, and
Smad 4, dramatically affects muscle growth (Suprave et al., supra,
1990; Berk et al., supra, 1997; see, also, Luo et al., supra, 1999;
Stroschein et al., supra, 1999; Sun et al., supra, 1999a and b;
Akiyoshi et al., supra, 1999). As disclosed herein, myostatin
interacts specifically with Act RIIB and, therefore, can exert its
biological effect, at least in part, by binding to activin type II
receptors in vivo and activating the Smad signaling pathway.
[0311] The role of the Smad signaling pathway in regulating muscle
growth can be examined using transgenic mouse lines that are
blocked, or capable of being blocked, at specific points in the Act
RIIB/Smad signal transduction pathway. The muscle creatine kinase
promoter or myosin light chain enhancer/promoter can be used to
drive expression of various inhibitors of the Smad signal
transduction pathway.
[0312] An inhibitor useful in this system can include, for example,
follistatin; a dominant negative Act RIIB receptor; a dominant
negative Smad polypeptide such as Smad 3; c-ski; or an inhibitory
Smad polypeptide such as Smad 7. Follistatin can bind and inhibit
the activity of certain TGF-.beta. family members, including GDF-11
(Gamer et al., supra, 1999). Dominant negative forms of an activin
type II receptor can be obtained by expressing a truncated GDF
receptor, for example, by expressing the extracellular domain,
particularly a soluble form of an Act RIIB extracellular domain, or
by expressing a truncated Act RIIB receptor that lacks the kinase
domain or contains a mutation such that the mutant receptor lacks
kinase activity. Smad 7 functions as an inhibitory Smad that can
block the signaling pathway induced by activin, TGF-.beta., and
BMP. A dominant negative form of Smad 3, for example, can be
constructed by mutating the Smad 3 C-terminal phosphorylation
sites, thereby blocking Smad 3 function (Liu et al., supra, 1997).
c-ski overexpression has been correlated to muscle hypertrophy in
transgenic mice (Suprave et al., supra, 1990).
[0313] Transgenic mice can be prepared and each founder line
examined for proper, muscle-specific expression of the transgene.
The selected mice are examined for total body weights, individual
muscle weights, and muscle fiber sizes, numbers and types. Those
lines demonstrating a clear effect on muscle mass can be examined
further for fat accumulation and other relevant metabolic
parameters as described above. The use of these different agents to
target specific steps in the activin receptor/Smad signal
transduction pathway is particularly informative because the
signaling pathways for the different agents overlap at different
steps. For example, follistatin binds to and inhibits activin and
GDF-11 activity, but not TGF-.beta., whereas a dominant negative
Smad 3 can block signaling through both activin and TGF-.beta.
receptors. Smad 7 can have an even more pleotropic because it
blocks signaling through BMP receptors as well. The studies can
allow the identification of specific targets for modulating
myostatin activity, thus providing various strategies for
developing drugs or other agents that modulate myostatin signal
transduction and, therefore, myostatin activity.
[0314] In particular, the transgenic lines described herein can be
used to determine the effect of blocking myostatin function or the
Smad signaling pathway postnatally on the development of obesity or
type II diabetes. For example, the inhibitory transgenes can e
crossed into the ob/ob, db/db, and Ay mutant mice. In the absence
of doxycycline, an inhibitor transgene is not expressed and,
therefore, the animals are indistinguishable from each of the
parental mutant mice. In the presence of doxycycline, the inhibitor
is expressed and can block myostatin activity. The effect of
blocking myostatin activity on development of the metabolic
abnormalities in these mutant animals can be examined.
[0315] Expression of the inhibitor can be induced at an early age,
for example, at 3 weeks of age, to maximize the effect. In
addition, myostatin activity can be blocked prior to the time that
the metabolic abnormalities become so severe as to be irreversible.
Animals can be maintained on doxycycline and assessed at various
ages using the tests described above, including those relating to
fat accumulation and glucose metabolism. Any delay in the age at
which one or more test results becomes abnormal in the ob/ob,
db/db, and Ay mutant animals can be identified. Similar studies can
be performed using older animals, which have developed some of the
signs of obesity or type II diabetes, and the effect of blocking
myostatin activity on various parameters, including fat weight and
glucose metabolism, can be determined. The results of these studies
can further identify specific targets that can be manipulated in an
effort to prevent or treat obesity or type II diabetes.
EXAMPLE 12
Characterization of Myostatin Effect on the Induction of
Cachexia
[0316] This example describes methods for determining the role of
myostatin signal transduction in the development and progression of
cachexia.
[0317] The activin receptor and Smad pathway can constitute at
least part of the signal transduction pathway involved in mediating
myostatin activity in normal individuals and, therefore, can be
involved in mediating the effects that occur in an individual due
to excess levels of myostatin. As disclosed herein, cachexia, for
example, can be mediated, at least in part, by abnormally high
levels of myostatin. As such, methods for manipulating signal
transduction through the Smad pathway can provide a new strategy
for developing drugs for the treatment of muscle wasting in general
and cachexia in particular.
[0318] The role of the Smad signaling pathway in cachexia can be
examined by examining the susceptibility of the various transgenic
lines described above to cachexia, which can be induced, for
example, by interleukin-6 (IL-6; Black et al., Endocrinology
128:2657-2659, 1991, which is incorporated herein by reference]),
tumor necrosis factor-I (TNF-I; Oliff et al., Cell 50:555-563,
1987, which is incorporated herein by reference), or certain tumor
cells. In the case of IL-6 and TNF-I, the inhibitor transgenes can
be crossed into a nude mouse background, then the animals can be
challenged with CHO cells that produce IL-6 or TNF-I, which induce
wasting in nude mice when overexpressed in this manner. CHO cells
that overproduce IL-6 or TNF-I can be prepared using the methods
described above for generating myostatin overproducing cells. For
example, TNF-I cDNA can be cloned into the pMSXND expression vector
(Lee and Nathans, J. Biol. Chem. 263:3521-3527, 1988), then cells
carrying amplified copies of the expression construct can be
selected stepwise in increasing concentrations of methotrexate.
[0319] Tumor cells such as Lewis lung carcinoma cells (Matthys et
al., Eur. J. Cancer 27:182-187, 1991, which is incorporated herein
by reference) or colon 26 adenocarcinoma cells (Tanaka et al., J.
Cancer Res. 50:2290-2295, 1990, which is incorporated herein by
reference), which can induce cachexia in mice, also can be utilized
for these studies. These cell lines cause severe wasting when grown
as tumors in mice. Thus, the effect of these tumors can be examined
in the various transgenic mice described herein. It is recognized
that the various tumor cells will only grow in certain genetic
backgrounds. For example, the Lewis lung carcinoma cells are
routinely grown in C57 BL/6 mice, and the colon 26 carcinoma cells
are routinely grown in BALB/c mice. Thus, the transgenes can be
backcrossed into these or other genetic backgrounds to allow growth
of the tumor cells.
[0320] Various parameters, including total body weight, individual
muscle weight, muscle fiber size and number, food intake and serum
parameters, including glucose levels, can be monitored. In
addition, serum myostatin levels and myostatin RNA levels in muscle
can be examined to confirm that increased myostatin expression is
correlated with cachexia. The results of these studies can confirm
that the action of myostatin is downstream of the cachexia-inducing
agents in these experimental models. The results also can confirm
that the Smad signaling pathway is essential for development of
cachexia in these models, and can demonstrate that a therapeutic
benefit can be obtained in the treatment of cachexia by modulating
the Smad signaling
EXAMPLE 13
Identification and Characterization of Growth Differentiation
Factor-8 (GDF-8) and GDF-11 Receptors
[0321] This example describes methods for identifying and
characterizing cell surface receptors for GDF-8 (myostatin) and
GDF-11.
[0322] The purified GDF-8 and GDF-11 proteins will be used
primarily to assay for biological activities. In order to identify
potential target cells for GDF-8 and GDF-11 action cells expressing
their receptors will be searched. For this purpose, the purified
protein will be radio-iodinated using the chloramine T method,
which has been used successfully to label other members of this
superfamily, like TGF-.beta. (Cheifetz et al., supra, 1987),
activins (Sugino et al., J. Biol. Chem. 263:15249-15252, 1988), and
BMPs (Paralkar et al., Proc. Natl. Acad. Sci., USA 88:3397-3401,
1991), for receptor binding studies. The mature processed forms of
GDF-8 and GDF-11 each contain multiple tyrosine residues. Two
different approaches will be taken to identify receptors for these
proteins.
[0323] One approach will determine the number, affinity, and
distribution of receptors. Either whole cells grown in culture,
frozen sections of embryos or adult tissues, or total membrane
fractions prepared from tissues or cultured cells will be incubated
with the labeled protein, and the amount or distribution of bound
protein will be determined. For experiments involving cell lines or
membranes, the amount of binding will be determined by measuring
either the amount of radioactivity bound to cells on the dish after
several washes or, in the case of membranes, the amount of
radioactivity sedimented with the membranes after centrifugation or
retained with the membranes on a filter. For experiments involving
primary cultures, where the number of cells can be more limited,
binding sites will be visualized directly by overlaying with
photographic emulsion. For experiments involving frozen sections,
sites of ligand binding will be visualized by exposing these
sections to high resolution Beta-max hyperfilm; if finer
localization is required, the sections will be dipped in
photographic emulsion. For all of these experiments, specific
binding will be determined by adding excess unlabeled protein as
competitor (for example, see Lee and Nathans, supra, 1988).
[0324] A second approach will be to characterize the receptor
biochemically. Membrane preparations or potential target cells
grown in culture will be incubated with labeled ligand, and
receptor/ligand complexes will be covalently cross-linked using
disuccinimidyl suberate, which has been commonly used to identify
receptors for a variety of ligands, including members of the
TGF-.beta. superfamily (Massague and Like, J. Biol. Chem.
260:2636-2645, 1985). Cross-linked complexes are separated by
electrophoresis on SDS polyacrylamide gels to look for bands
labeled in the absence, but not in the presence, of excess
unlabeled protein. The molecular weight of the putative receptor
will be estimated by subtracting the molecular weight of the
ligand. An important question that these experiments will address
is whether GDF-8 and GDF-11 signal through type I and type II
receptors like many other members of the TGF-.beta. superfamily
(Massague and Weis-Garcia, supra, 1996).
[0325] Once a method for detecting receptors for these molecules
has been achieved, more detailed analysis will be carried out to
determine the binding affinities and specificities. A Scatchard
analysis will be used to determine the number of binding sites and
dissociation constants. By carrying out cross-competition analyses
between GDF-8 and GDF-11, it will be possible to determine whether
they are capable of binding to the same receptor and their relative
affinities. These studies will give an indication as to whether the
molecules signal through the same or different receptors.
Competition experiments using other TGF-.beta. family members will
be performed to determine specificity. Some of these ligands are
available commercially, and some others are available from Genetics
Institute, Inc.
[0326] For these experiments, a variety of embryonic and adult
tissues and cell lines will be tested. Based on the specific
expression of GDF-8 in skeletal muscle and the phenotype of GDF-8
knock-out mice, initial studies focus on embryonic and adult muscle
tissue for membrane preparation and for receptor studies using
frozen sections. In addition, myoblasts will be isolated and
cultured from embryos at various days of gestation or satellite
cells from adult muscle as described (Vivarelli and Cossu, Devel.
Biol. 117:319-325, 1986; Cossu et al., Cell Diff. 9:357-368, 1980).
The binding studies on these primary cells after various days in
culture will be performed and binding sites localized by
autoradiography so that the binding sites can be c-o-localized with
various myogenic markers, such as muscle myosin (Vivarelli et al.,
J. Cell Biol. 107:2191-2197, 1988), and correlate binding with the
differentiation state of the cells, such as formation of
multinucleated myotubes. In addition to using primary cells, cell
lines will be utilized to look for receptors. In particular, the
initial focus will be on three cells lines, C2C12, L6, and P19.
C2C12 and L6 myoblasts differentiate spontaneously in culture and
form myotubes depending on the particular growth conditions (Yaffe
and Saxel, supra, 1977; Yaffe, supra, 1968). P19 embryonal
carcinoma cells can be induced to differentiate into various cell
types, including skeletal muscle cells in the presence of DMSO
(Rudnicki and McBurney, Teratocarcinomas and Embryonic Stem Cells:
A practical approach (E. J. Robertson, IRL Press, Cambridge 1987).
Receptor binding studies will be carried out on these cell lines
under various growth conditions and at various stages of
differentiation. Although the initial studies will focus on muscle
cells, other tissues and cell types will be examined for the
presence of GDF-8 and GDF-11 receptors.
[0327] Recombinant human GDF-8 (rhGDF-8) homodimer will be used in
these binding studies. RhGDF-8 was expressed using CHO cells and
purified to approximately 90% purity. The rhGDF-8 had the expected
25 kDa to 27 kDa molecular weight and, upon reduction, was reduced
to the 12 kDa monomer. Using I-125 labeled GDF-8 in a
receptor-ligand binding assay, two myoblast cell lines, L6 and G-8,
bound GDF-8. The binding was specific since non labeled GDF-8
effectively competed the binding of the labeled ligand. The
dissociation constant (Kd) was 370 pM, and L6 myoblasts have a high
number (5,000 receptors/cell) of cell surface binding proteins.
GDF-11 (BMP-11) is highly homologous (>90%) to GDF-8. Receptor
binding studies revealed that GDF-8 and GDF-11 bound to the same
binding proteins on L6 myoblasts. It is important to establish
whether or not GDF-8 binds to the known TGF-.theta. receptor.
TGF-.theta. did not compete the binding of GDF-8, indicating that
the GDF-8 receptor is distinct from the TGF-.theta. receptor. The
GDF-8 receptor was not expressed on all myoblast cell lines,
including four myoblast cell lines, C2C12, G7, MLB13MYC c14 and
BC3H1, which do not bind GDF-8.
[0328] The gene or genes encoding receptors for GDF-8 and GDF-11
can be obtained. As a first step towards understanding the
mechanism by which GDF-8 and GDF-11 exert their biological effects,
it is important to clone the genes encoding their receptors. From
the experiments above, it will be more clear as to whether GDF-8
and GDF-11 bind to the same receptor or to different receptors.
There will also be considerable information regarding the tissue
and cell type distribution of these receptors. Using this
information, two different approaches will be taken to clone the
receptor genes.
[0329] The first approach will be to use an expression cloning
strategy. In fact, this was the strategy that was originally used
by Mathews and Vale (Cell 65:973-982, 1991) and Lin et al. (Cell
68:775-785, 1992) to clone the first activin and TGF-.beta.
receptors. Poly A-selected RNA from the tissue or cell type that
expresses the highest relative number of high affinity binding
sites will be obtained, and used to prepare a cDNA library in the
mammalian expression vector pcDNA-1, which contains a CMV promoter
and an SV40 origin of replication. The library will be plated, and
cells from each plate will be pooled into broth and frozen.
Aliquots from each pool will be grown for preparation of DNA. Each
individual pool will be transiently transfected into COS cells in
chamber slides, and transfected cells will be incubated with
iodinated GDF-8 or GDF-11. After washing away the unbound protein,
the sites of ligand binding will be visualized by autoradiography.
Once a positive pool is identified, the cells from that pool will
be replated at lower density, and the process will be repeated.
Positive pools will then be plated, and individual colonies will be
picked into grids and re-analyzed as described (Wong et al.,
Science 228:810-815, 1985).
[0330] Initially, using pool sizes of 1500 colonies will be
screened. In order to be certain to identify a positive clone in a
mixture of this complexity, a control experiment using TGF-.beta.
and a cloned type II receptor will be performed. The coding
sequence for the TGF-.beta.3 type II receptor will be cloned into
the pcDNA-1 vector, and bacteria transformed with this construct
will be mixed with bacteria from our library at various ratios,
including 1:1500. The DNA prepared from this mixture then will be
transfected into COS cells, incubated with iodinated TGF-.beta.,
and visualized by autoradiography. If positive signals are observed
at a ratio of 1:1500, pools of 1500 clones will be screened.
Otherwise, smaller pool sizes corresponding to ratios at which the
procedure is sensitive enough to identify a positive signal in
control experiments will be used.
[0331] A second parallel strategy to attempt to clone the GDF-8 and
GDF-11 receptors also will be used, taking advantage of the fact
that most receptors for members of the TGF-.beta. superfamily that
have been identified belong to the membrane-spanning
serine/threonine kinase family (Massague and Weis-Garcia, supra,
1996). Because the cytoplasmic domains of these receptors are
related in sequence, degenerate PCR probes will be used to clone
members of this receptor family that are expressed in tissues that
contain binding sites for GDF-8 and GDF-11. In fact, this is the
approach that has been used to identify most of the members of this
receptor family. The general strategy will be to design degenerate
primers corresponding to conserved regions of the known receptors,
to use these primers for PCR on cDNA prepared from the appropriate
RNA samples (most likely from skeletal muscle), to subclone the PCR
products, and finally to sequence individual subclones. As
sequences are identified, they will be used as hybridization probes
to eliminate duplicate clones from further analysis. The receptors
that are identified then will be tested for their ability to bind
purified GDF-8 and GDF-11. Because this screen will yield only
small PCR products, full-length cDNA clones will be obtained for
each receptor from cDNA libraries prepared from the appropriate
tissue, inserted into the pcDNA-1 vector, transfected into COS
cells, and the transfected cells will be assayed for their ability
to bind iodinated GDF-8 or GDF-11. Ideally, every receptor that is
identified in this screen will be tested for the ability to bind
these ligands. However, the number of receptors that are identified
can be large, and isolating all of the full-length cDNAs and
testing them can require considerable effort. Almost certainly some
of the receptors that are identified will correspond to known
receptors, and for these, either obtaining full-length cDNA clones
from other investigators or amplifying the coding sequences by PCR
based on the published sequences should be straightforward. For
novel sequences, the tissue distribution will be determined by
northern blot analysis and the highest priority will be directed to
those receptors whose expression pattern most closely resembles the
distribution of GDF-8 and/or GDF-11 binding sites as determined
above.
[0332] In particular, it is known that these receptors fall into
two classes, type I and type II, which can be distinguished based
on the sequence and which are both required for full activity.
Certain ligands cannot bind type I receptors in the absence of type
II receptors while others are capable of binding both receptor
types (Massague and Weis-Garcia, supra, 1996). The cross-linking
experiments outlined above should give some indication as to
whether both type I and type II receptors are also involved in
signaling GDF-8 and GDF-11. If so, it will be important to clone
both of these receptor subtypes in order to fully understand how
GDF-8 and GDF-11 transmit their signals. Because it cannot be
predicted as to whether the type I receptor is capable of
interacting with GDF-8 and GDF-11 in the absence of the type II
receptor, type II receptor(s) will be cloned first. Only after at
least one type II receptor has been identified for these ligands,
will an attempt be made to identify the type I receptors for GDF-8
and GDF-11. The general strategy will be to cotransfect the type II
receptor with each of the type I receptors that are identified in
the PCR screen, then assay the transfected cells by crosslinking.
If the type I receptor is part of the receptor complex for GDF-8 or
GDF-11, two cross-linked receptor species should be detected in the
transfected cells, one corresponding to the type I receptor and the
other corresponding to the type II receptor.
[0333] The search for GDF-8 and GDF-11 receptors is further
complicated by the fact at least one member of the TGF-.beta.
superfamily, namely, GDNF, is capable of signaling through a
completely different type of receptor complex involving a
GPI-linked component (GDNFR-alpha) and a receptor tyrosine kinase
(c-ret; Trupp et al., Nature 381:785-789, 1996; Durbec et al.,
Nature 381:789-793, 1996; Treanor et al., Nature 382:80-83, 1996;
Jing et al., Cell 85:1113-1124, 1996). Although GDNF is the most
distantly-related member of the TGF-.beta. superfamily, it is
certainly possible that other TGF-.beta. family members can also
signal through an analogous receptor system. If GDF-8 and GDF-11 do
signal through a similar receptor complex, the expression screening
approach should be able to identify at least the GPI-linked
component (indeed GDNFR-alpha was identified using an expression
screening approach) of this complex. In the case of GDNF, the
similar phenotypes of GDNF- and c-ret-deficient mice suggested
c-ret as a potential receptor for GDNF.
EXAMPLE 14
Preparation and Characterization of GDF-11 Knock-Out Mice
[0334] The phenotype of GDF-11 knock-out mice in several respects
resembles the phenotype of mice carrying a deletion of a receptor
for some members of the TGF-.theta.superfamily, including the
activin type IIB receptor (Act RIIB). To determine the biological
function of GDF-11, the GDF-11 gene was disrupted by homologous
targeting in embryonic stem cells.
[0335] A murine 129 SvJ genomic library was prepared in lambda
FIXII according to the instructions provided by Stratagene (La
Jolla, Calif.). The structure of the GDF-11 gene was deduced from
restriction mapping and partial sequencing of phage clones isolated
from the library. Vectors for preparing the targeting construct
were kindly provided by Philip Soriano and Kirk Thomas. To ensure
that the resulting mice would be null for GDF-11 function, the
entire mature C-terminal region was deleted and replaced by a neo
cassette. R1 ES cells were transfected with the targeting
construct, selected with gancyclovir (2 TM) and G418 (250 Tg/ml),
and analyzed by Southern blot analysis.
[0336] Homologous targeting of the GDF-11 gene was observed in
8/155 gancyclovir/G418 doubly resistant ES cell clones. Following
injection of several targeted clones into C57BL/6J blastocysts,
chimeras were obtained from one ES clone that produced heterozygous
pups when crossed to both C57BL/6J and 129/SvJ females. Crosses of
C57BL/6J/129/SvJ hybrid F1 heterozygotes produced 49 wild type
(34%), 94 heterozygous (66%), and no homozygous mutant adult
offspring. Similarly, there were no adult homozygous null animals
seen in the 129/SvJ background (32 wild type (36%) and 56
heterozygous mutant (64%) animals).
[0337] To determine the age at which homozygous mutants were dying,
litters of embryos isolated at various gestational ages from
heterozygous females that had been mated to heterozygous males were
genotyped. At all embryonic stages examined, homozygous mutant
embryos were present at approximately the predicted frequency of
25%. Among hybrid newborn mice, the different genotypes were also
represented at the expected Mendelian ratio of 1:2:1 (34+/+(28%),
61+/-(50%), and 28-/- (23%)). Homozygous mutant mice were born
alive and were able to breath and nurse. All homozygous mutants
died, however, within the first 24 hours after birth. The precise
cause of death was unknown, but the lethality may have been related
to the fact that the kidneys in homozygous mutants were either
severely hypoplastic or completely absent.
[0338] Homozygous mutant animals were easily recognizable by their
severely shortened or absent tails. To further characterize the
tail defects in these homozygous mutant animals, their skeletons
were examined to determine the degree of disruption of the caudal
vertebrae. A comparison of wild type and mutant skeleton
preparations of late stage embryos and newborn mice, however,
revealed differences not only in the caudal region of the animals
but in many other regions as well. In nearly every case where
differences were noted, the abnormalities appeared to represent
homeotic transformations of vertebral segments in which particular
segments appeared to have a morphology typical of more anterior
segments. These transformations were evident throughout the axial
skeleton extending from the cervical region to the caudal region.
Except for the defects seen in the axial skeleton, the rest of the
skeleton, such as the cranium and limb bones, appeared normal.
[0339] Anterior transformations of the vertebrae in mutant newborn
animals were most readily apparent in the thoracic region, where
there was a dramatic increase in the number of thoracic (T)
segments. All wild type mice examined showed the typical pattern of
13 thoracic vertebrae each with its associated pair of ribs. In
contrast, homozygous mutant mice showed a striking increase in the
number of thoracic vertebrae. All homozygous mutants examined had 4
to 5 extra pairs of ribs for a total of 17 to 18, although in over
1/3 of these animals, the 18th rib appeared to be rudimentary.
Hence, segments that would normally correspond to lumbar (L)
segments L1 to L4 or L5 appeared to have been transformed into
thoracic segments in mutant animals.
[0340] Moreover, transformations within the thoracic region in
which one thoracic vertebra had a morphology characteristic of
another thoracic vertebra were also evident. For example, in wild
type mice, the first 7 pairs of ribs attach to the sternum, and the
remaining 6 are unattached or free. In homozygous mutants, there
was an increase in the number of both attached and free pairs of
ribs to 10-11 and 7-8, respectively. Therefore, thoracic segments
T8, T9, T10, and in some cases even T11, which all have free ribs
in wild type animals, were transformed in mutant animals to have a
characteristic typical of more anterior thoracic segments, namely,
the presence of ribs attached to the sternum. Consistent with this
finding, the transitional spinous process and transitional
articular processes which are normally found on T10 in wild type
animals were instead found on T13 in homozygous mutants. Additional
transformations within the thoracic region were also noted in
certain mutant animals. For example, in wild type mice, the ribs
derived from T1 normally touch the top of the sternum. However, in
2/23 hybrid and 2/3 129/SvJ homozygous mutant mice examined, T2
appeared to have been transformed to have a morphology resembling
that of T1; that is, in these animals, the ribs derived from T2
extended to touch the top of the sternum. In these cases, the ribs
derived from T1 appeared to fuse to the second pair of ribs.
Finally, in 82% of homozygous mutants, the long spinous process
normally present on T2 was shifted to the position of T3. In
certain other homozygous mutants, asymmetric fusion of a pair of
vertebrosternal ribs was seen at other thoracic levels.
[0341] The anterior transformations were not restricted to the
thoracic region. The anterior most transformation that we observed
was at the level of the 6th cervical vertebra (C6). In wild type
mice, C6 is readily identifiable by the presence of two anterior
tuberculi on the ventral side. In several homozygous mutant mice,
although one of these two anterior tuberculi was present on C6, the
other was present at the position of C7 instead. Hence, in these
mice, C7 appeared to have been partially transformed to have a
morphology resembling that of C6. One other homozygous mutant had 2
anterior tuberculi on C7 but retained one on C6 for a complete C7
to C6 transformation but a partial C6 to C5 transformation.
[0342] Transformations of the axial skeleton also extended into the
lumbar region. Whereas wild type animals normally have only 6
lumbar vertebrae, homozygous mutants had 8 to 9. At least 6 of the
lumbar vertebrae in the mutants must have derived from segments
that would normally have given rise to sacral and caudal vertebrae
as the data described above suggest that 4 to 5 lumbar segments
were transformed into thoracic segments. Hence, homozygous mutant
mice had a total of 33-34 presacral vertebrae compared to 26
presacral vertebrae normally present in wild type mice. The most
common presacral vertebral patterns were C7/T18/L8 and C7/T18/L9
for mutant mice compared to C7/T13/L6 for wild type mice. The
presence of additional presacral vertebrae in mutant animals was
obvious even without detailed examination of the skeletons as the
position of the hind limbs relative to the forelimbs was displaced
posteriorly by 7 to 8 segments.
[0343] Although the sacral and caudal vertebrae were also affected
in homozygous mutant mice, the exact nature of each transformation
was not as readily identifiable. In wild type mice, sacral segments
S1 and S2 typically have broad transverse processes compared to S3
and S4. In the mutants, there did not appear to be an identifiable
S1 or S2 vertebra. Instead, mutant animals had several vertebrae
that appeared to have morphology similar to S3. In addition, the
transverse processes of all 4 sacral vertebrae are normally fused
to each other although in newborns often only fusions of the first
3 vertebrae are seen. In homozygous mutants, however, the
transverse processes of the sacral vertebrae were usually unfused.
In the caudal-most region, all mutant animals also had severely
malformed vertebrae with extensive fusions of cartilage. Although
the severity of the fusions made it difficult to count the total
number of vertebrae in the caudal region, up to 15 transverse
processes were counted in several animals. It could not be
determined whether these represented sacral or caudal vertebrae in
the mutants because morphologic criteria for distinguishing S4 from
caudal vertebrae even in wild type newborn animals could not be
established. Regardless of their identities, the total number of
vertebrae in this region was significantly reduced from the normal
number of approximately 30. Hence, although the mutants had
significantly more thoracic and lumber vertebrae than wild type
mice, the total number of segments was reduced in the mutants due
to the truncation of the tails.
[0344] Heterozygous mice also showed abnormalities in the axial
skeleton although the phenotype was much milder than in homozygous
mice. The most obvious abnormality in heterozygous mice was the
presence of an additional thoracic segment with an associated pair
of ribs. This transformation was present in every heterozygous
animal examined, and in every case, the additional pair of ribs was
attached to the sternum. Hence, T8, whose associated rib normally
does not touch the sternum, appeared to have been transformed to a
morphology characteristic of a more anterior thoracic vertebra, and
L1 appeared to have been transformed to a morphology characteristic
of a posterior thoracic vertebra. Other abnormalities indicative of
anterior transformations were also seen to varying degrees in
heterozygous mice. These included a shift of the long spinous
process characteristic of T2 by one segment to T3, a shift of the
articular and spinous processes from T10 to T1, a shift of the
anterior tuberculus on C6 to C7, and transformation of T2 to T1
where the rib associated with T2 touched the top of the
sternum.
[0345] In order to understand the basis for the abnormalities in
axial patterning seen in GDF-11 mutant mice, mutant embryos
isolated at various stages of development were examined, and
compared o wild type embryos. By gross morphological examination,
homozygous mutant embryos isolated up to day 9.5 of gestation were
not readily distinguishable from corresponding wild type embryos.
In particular, the number of somites present at any given
developmental age was identical between mutant and wild type
embryos, suggesting that the rate of somite formation was unaltered
in the mutants. By day 10.5-11.5 p.c., mutant embryos could be
easily distinguished from wild type embryos by the posterior
displacement of the hind limb by 7-8 somites. The abnormalities in
tail development were also readily apparent at this stage. Taken
together, these data suggest that the abnormalities observed in the
mutant skeletons represented true transformations of segment
identities rather than the insertion of additional segments, for
example, by an enhanced rate of somitogenesis.
[0346] Alterations in expression of homeobox containing genes are
known to cause transformations in Drosophila and in vertebrates. To
see if the expression patterns of Hox genes (the vertebrate
homeobox containing genes) were altered in GDF-11 null mutants, the
expression pattern of 3 representative Hox genes, Hoxc-6, Hoxc-8
and Hoxc-11, was determined in day 12.5 p.c. wild type,
heterozygous and homozygous mutant embryos by whole mount in situ
hybridization. The expression pattern of Hoxc-6 in wild type
embryos spanned prevertebrae 8-15 which correspond to thoracic
segments T1-T8. In homozygous mutants, however, the Hoxc-6
expression pattern was shifted posteriorly and expanded to
prevertebrae 9-18 (T2-T11). A similar shift was seen with the
Hoxc-8 probe. In wild type embryos, Hoxc-8 was expressed in
prevertebrae 13-18 (T6-T11) but, in homozygous mutant embryos,
Hoxc-8 was expressed in prevertebrae 14-22 (T7-T15). Finally,
Hoxc-11 expression was also shifted posteriorly in that the
anterior boundary of expression changed from prevertebrae 28 tin
wild type embryos to prevertebrae 36 in mutant embryos. (Note that
because the position of the hind limb is also shifted posteriorly
in mutant embryos, the Hoxc-11 expression patterns in wild type and
mutant appeared similar relative to the hind limbs). These data
provide further evidence that the skeletal abnormalities seen in
mutant animals represent homeotic transformations.
[0347] The phenotype of GDF-11 mice suggested that GDF-11 acts
early during embryogenesis as a global regulator of axial
patterning. To begin to examine the mechanism by which GDF-11
exerts its effects, the expression pattern of GDF-11 in early mouse
embryos was examined by whole mount in situ hybridization. At these
stages, the primary sites of GDF-11 expression correlated precisely
with the known sites at which mesodermal cells are generated.
Expression of GDF-11 was first detected at day 8.25-8.5 p.c. (8-10
somites) in the primitive streak region, which is the site at which
ingressing cells form the mesoderm of the developing embryo.
Expression was maintained in the primitive streak at day 8.75, but
by day 9.5 p.c., when the tail bud replaces the primitive streak as
the source of new mesodermal cells, expression of GDF-11 shifted to
the tail bud. Hence, at these early stages, GDF-11 appears to be
synthesized in the region of the developing embryo where new
mesodermal cells arise and presumably acquire their positional
identity.
[0348] The phenotype of GDF-11 knock-out mice in several respects
resembled the phenotype of mice carrying a deletion of a receptor
for some members of the TGF-.theta. superfamily, the activin type
IIB receptor (Act RIIB). As in the case of GDF-11 knock-out mice,
the Act RIIB knock-out mice have extra pairs of ribs and a spectrum
of kidney defects ranging from hypoplastic kidneys to complete
absence of kidneys. The similarity in the phenotypes of these mice
raises the possibility that Act RIIB can be a receptor for GDF-11.
However, Act RIIB cannot be the sole receptor for GDF-11 because
the phenotype of GDF-11 knock-out mice is more severe than the
phenotype of Act RIIB mice. For example, whereas the GDF-11
knock-out animals have 4-5 extra pairs of ribs and show homeotic
transformations throughout the axial skeleton, the Act RIIB
knock-out animals have only 3 extra pairs of ribs and do not show
transformations at other axial levels. In addition, the data
indicate that the kidney defects in the GDF-11 knock-out mice are
also more severe than those in Act RIIB knock-out mice. The Act
RIIB knock-out mice show defects in left/right axis formation, such
as lung isomerism and a range of heart defects that we have not yet
observed in GDF-11 knock-out mice. Act RIIB can bind the activins
and certain BMPs, although none of the knock-out mice generated for
these ligands show defects in left/right axis formation.
[0349] If GDF-11 does act directly on mesodermal cells to establish
positional identity, the data presented here would be consistent
with either short range or morphogen models for GDF-11 action. That
is, GDF-11 can act on mesodermal precursors to establish patterns
of Hox gene expression as these cells are being generated at the
site of GDF-11 expression, or alternatively, GDF-11 produced at the
posterior end of the embryo can diffuse to form a morphogen
gradient. Whatever the mechanism of action of GDF-11 may be, the
fact that gross anterior/posterior patterning still does occur in
GDF-11 knock-out animals suggests that GDF-11 may not be the sole
regulator of anterior/posterior specification. Nevertheless, it is
clear that GDF-11 plays an important role as a global regulator of
axial patterning and that further study of this molecule will lead
to important new insights into how positional identity along the
anterior/posterior axis is established in the vertebrate
embryo.
[0350] Similar phenotypes are expected in GDF-8 knock-out animals.
For example, GDF-8 knock-out animals are expected to have increased
number of ribs, kidney defects and anatomical differences when
compared to wild type.
EXAMPLE 15
Production of Transgenic Mice Expressing Myostatin Pro-Peptide,
Follistatin or a Dominant Negative Act RIIB
[0351] Purification of myostatin. A Chinese hamster ovary (CHO)
cell line carrying amplified copies of a myostatin expression
construct was transfected with an expression construct for the
furin protease PACE (kindly provided by Monique Davies) in order to
improve processing of the precursor protein. Conditioned medium
(prepared by Cell Trends, Middletown, Md.) was passed successively
over hydroxylapatite (eluted with 200 mM sodium phosphate pH 7.2),
lentil lectin Sepharose (eluted with 50 mM Tris pH 7.4, 500 mM
NaCl, 500 mM methyl mannose), DEAE agarose (collected material that
flowed through the column in 50 mM Tris pH 7.4, 50 mM NaCl), and
heparin Sepharose (eluted with 50 mM Tris pH 7.4, 200 mM NaCl). The
eluate from the heparin column was then bound to a reverse phase C4
HPLC column and eluted with an acetonitrile gradient in 0.1%
trifluoroacetic acid. Antibodies directed against the mature
C-terminal protein were described previously (see U.S. Pat. No.
5,827,733, herein incorporated by reference). In order to raise
antibodies against the pro peptide, the portion of the human
myostatin protein spanning amino acids 122-261 was expressed in
bacteria using the RSET vector (Invitrogen, San Diego, Calif.),
purified by nickel chelate chromatography, and injected into
rabbits. Immunizations were carried out by Spring Valley Labs
(Woodbine, Md.).
[0352] Receptor binding. Purified myostatin was radioiodinated
using the chloramine T method (Frolik, C. A., Wakefield, L. M.,
Smith, D. M. & Sporn, M. B. (1984) J Biol Chem 259,
10995-11000). COS-7 cells grown in 6 or 12 well plates were
transfected with 1-2 .mu.g pCMV5 or pCMV5/receptor construct using
lipofectamine (Gibco, Rockville, Md.). Crosslinking experiments
were carried out 2 days post transfection as described (Franzen,
P., ten Dijke, P., Ichijo, H., Yamashita, H., Schultz, P., Heldin,
C.-H. & Miyazono, K. (1993) Cell 75, 681-692). For quantitative
receptor binding assays, cell monolayers were washed twice with PBS
containing 1 mg/ml BSA and incubated with labeled myostatin in the
presence or absence of various concentrations of unlabeled
myostatin, pro peptide, or follistatin at 4.degree. C. Cells were
then washed 3 times with the same buffer, lysed in 0.5N NaOH, and
counted in a gamma counter. Specific binding was calculated as the
difference in bound myostatin between cells transfected with Act
RIIB and cells transfected with vector. This method of calculating
specific binding was especially important in assessing the effect
of the pro peptide as the addition of the pro peptide also reduced
non-specific binding in a concentration-dependent manner.
[0353] Transgenic mice. DNAs encoding a truncated form of murine
Act RIIB spanning amino acids 1-174, the murine myostatin pro
peptide spanning amino acids 1-267, and the human follistatin short
form were cloned into the MDAF2 vector containing the myosin light
chain promoter and 1/3 enhancer (McPherron, A. C. & Lee, S.-J.
(1993) J Biol Chem 268, 3444-3449). Purified transgenes including
the myosin light chain regulatory sequences and SV40 processing
sites were used for microinjections. All microinjections and embryo
transfers were carried out by the Johns Hopkins School of Medicine
Transgenic Core Facility. Transgenic founders in a hybrid
SJL/C57BL/6 background were mated to wild type C57BL/6 mice, and
all studies were carried out using F1 offspring. For analysis of
muscle weights, individual muscles were dissected from both sides
of nearly all animals, and the average of the left and right muscle
weights was used. Analysis of fiber numbers and sizes was carried
out as described (McPherron, A. C., Lawler, A. M. & Lee, S.-J.
(1997) Nature 387, 83-90). RNA isolation and Northern analysis were
carried out as described (McPherron, A. C. & Lee, S.-J. (1993)
J Biol Chem 268, 3444-3449).
[0354] In order to overproduce myostatin protein, a CHO cell line
carrying amplified copies of a myostatin expression construct was
produced (McPherron, A. C., Lawler, A. M. & Lee, S.-J. (1997)
Nature 387, 83-90). Myostatin was purified from the conditioned
medium of this cell line by successive fractionation on
hydroxylapatite, lentil lectin Sepharose, DEAE agarose, and heparin
Sepharose. Silver stain analysis of the purified protein
preparation revealed the presence of two protein species of 29 kd
and 12.5 kd. A variety of data suggested that this purified protein
consisted of a non-covalent complex of two pro peptide molecules
bound to a disulfide-linked C-terminal dimer. First, by Western
analysis, the 29 kd and 12.5 kd species were immunoreactive with
antibodies raised against bacterially-expressed fragments of
myostatin spanning the pro peptide and C-terminal mature region,
respectively. Second, in the absence of reducing agents, the
C-terminal region had an electrophoretic mobility consistent with
that of a dimer. Third, the two species were present in a molar
ratio of approximately 1:1. And fourth, the C-terminal dimer was
retained on the lectin column and could be eluted with methyl
mannose even though this portion of the protein contains no
potential N-linked glycosylation sites; the simplest interpretation
of these data is that the C-terminal region bound the lectin
indirectly by being present in a tight complex with the pro
peptide, which does have a glycosylation signal.
[0355] Because the C-terminal dimer is known to be the biologically
active molecule for other TGF-.beta. family members, the C-terminal
dimer of myostatin was purified away from its pro peptide by
reverse phase HPLC. The fractions containing the purified
C-terminal dimer (32-34) appeared to be homogeneous. However, the
fractions most enriched for the pro peptide (35-37) were
contaminated with small amounts of C-terminal dimer and with high
molecular weight complexes that most likely represented misfolded
proteins.
[0356] Most members of the TGF-.beta. superfamily have been shown
to signal by binding serine/threonine kinase receptors followed by
activation of Smad proteins (Heldin, C.-H., Miyazono, K. & ten
Dijke, P. (1997) Nature 390, 465-471; Massagu, J., Blain, S. W.
& Lo, R. S. (2000) Cell 103, 295-309). The initial event in
triggering the signaling pathway is the binding of the ligand to a
type II receptor. In order to determine whether myostatin is
capable of binding any of the known type II receptors for related
ligands, cross-linking studies were carried out with
radio-iodinated myostatin C-terminal dimer on COS-7 cells
transfected with expression constructs for either TGF-.beta., BMP,
or activin type II receptors. Cross-linked complexes of the
predicted size (full length receptor bound to myostatin) were
detected for cells expressing either Act RIIA or Act RIIB. Higher
levels of binding to Act RIIB than to Act RIIA were observed in
both cross-linking and standard receptor binding assays, therefore
receptor binding studies were focussed on Act RIIB. Binding of
myostatin to Act RIIB was specific (binding could be competed by
excess unlabeled myostatin) and saturable, and assuming that all of
the myostatin protein was bioactive, we estimated the dissociation
constant by Scatchard analysis to be approximately 10 nM. It is
known in the case of TGF-.beta. that the affinity for the type II
receptor is significantly higher in the presence of the appropriate
type I receptor and that other molecules are involved in presenting
the ligand to the receptor.
[0357] In order to determine whether activin type II receptors may
be involved in myostatin signaling in vivo, the effect of
expressing a dominant negative form of Act RIIB in mice was
investigated. For this purpose, we generated a construct in which a
truncated form of Act RIIB lacking the kinase domain was placed
downstream of a skeletal muscle-specific myosin light chain
promoter/enhancer. From pronuclear injections of this construct, a
total of 7 founder animals positive for the transgene were
identified. Analysis of these founder animals at 7 months of age
revealed that all seven had significant increases in skeletal
muscle mass with individual muscles of these founder animals
weighing up to 125% more than those of control non-transgenic
animals derived from similar injections (Table 2).
[0358] Three lines of evidence suggested that the increases in
muscle weights in these founder animals resulted from the
expression of the transgene. First, analysis of offspring derived
from matings of three founder animals (the other four founder
animals did not generate sufficient numbers of offspring for
analysis) with wild type C57BL/6 mice showed that the increases in
muscle weights correlated with the presence of the transgenes
(Table 3). Second, although muscle weights varied among the
different transgenic lines, the magnitude of the increase was
highly consistent among animals in any given line for all muscles
examined and for both males and females (Table 3). For example, all
muscles of both male and female mice from the C5 line weighed
approximately 30-60% more than those of control animals, whereas
all muscles from C11 mice weighed approximately 110-180% more.
Third, Northern analysis of RNA samples prepared from transgenic
animals showed that the expression of the transgene was restricted
to skeletal muscle and that the relative levels of transgene
expression correlated with the relative magnitude of the increase
in muscle weights (Table 3). For example, animals from the C11
line, which had the greatest increases in muscle weights, also had
the highest levels of transgene expression.
[0359] These data showed that expression of a dominant negative
form of Act RIIB can cause increases in muscle mass similar to
those seen in myostatin knockout mice. In myostatin knockout mice,
the increase in muscle mass has been shown to result from increases
in both fiber number and fiber size. In order to determine whether
expression of dominant negative Act RIIB also causes both
hyperplasia and hypertrophy, sections of the gastrocnemius and
plantaris muscles of animals from the C27 line were analyzed.
Compared to control muscles, the muscles of the C27 animals showed
a clear increase in overall cross-sectional area. This increase in
area resulted partially from an increase in fiber number. At the
widest point, the gastrocnemius and plantaris muscles had a total
of 10015+1143 fibers in animals from the C27 line (n=3) compared to
7871+364 fibers in control animals (n=3). However, muscle fiber
hypertrophy also contributed to the increase in total area. The
mean fiber diameter was 51 .mu.m in animals of the C27 line
compared to 43 .mu.m in control animals. Hence, the increase in
muscle mass appeared to result from an approximately 27% increase
in the number of fibers and 19% increase in fiber diameter
(assuming the fibers to be roughly cylindrical, this increase in
diameter result in an approximately 40% increase in cross sectional
area). Except for the increase in fiber number and size, however,
the muscles from the transgenic animals looked grossly normal. In
particular, there were no obvious signs of degeneration, such as
widely varying fiber sizes (the standard deviation of fiber sizes
was similar between control and transgenic animals) or extensive
fibrosis or fat infiltration.
[0360] These approaches were used to explore other possible
strategies for inhibiting myostatin. First, we investigated the
effect of the myostatin pro peptide. In the case of TGF-.beta., it
is known that the C-terminal dimer is held in an inactive, latent
complex with other proteins, including its pro peptide, and that
the pro peptide of TGF-.beta. can have an inhibitory effect on
TGF-.beta. activity both in vitro and in vivo (Miyazono, K.,
Hellman, U., Wernstedt, C. & Heldin, C.-H. (1988) J Biol Chem
263, 6407-6415; Gentry, L. E. & Nash, B. W. (1990) Biochem. 29,
6851-6857; Bottinger, E. P., Factor, V. M., Tsang, M.L.-S.,
Weatherbee, J. A., Kopp, J. B., Qian, S. W., Wakefield, L. M.,
Roberts, A. B., Thorgeirsson, S. S. & Sporn, M. B. (1996) Proc
Natl Acad Sci, USA 93, 5877-5882). The observation that the
myostatin C-terminal dimer and pro peptide co-purified raised the
possibility that myostatin may normally exist in a similar latent
complex and that the myostatin pro peptide may have inhibitory
activity. Second, we examined the effect of follistatin, which has
been shown to be capable of binding and inhibiting the activity of
several TGF-.beta. family members. In particular, follistatin can
block the activity of GDF-11, which is highly related to myostatin,
and follistatin knockout mice have been shown to have reduced
muscle mass at birth, which would be consistent with over-activity
of myostatin (Gamer, L., Wolfman, N., Celeste, A., Hattersley, G.,
Hewick, R. & Rosen, V. (1999) Dev Biol 208, 222-232; Matzuk, M.
M., Lu, N., Vogel, H., Sellheyer, K., Roop, D. R. & Bradley, A.
(1995) Nature 374, 360-363).
[0361] The effect of the pro peptide and follistatin in vitro was
then studied. Both the myostatin pro peptide and follistatin were
capable of blocking the binding of the C-terminal dimer to Act
RIIB. The Ki of follistatin was estimated to be approximately 470
pM and that of the pro peptide to be at least 50-fold higher. The
calculation of the Ki for the pro peptide, however, assumes that
all of the protein in the final preparation represented
biologically active pro peptide and therefore is likely to be an
overestimate. As discussed above, the pro peptide preparation was
contaminated both with small amounts of C-terminal dimer and with
misfolded high molecular weight species.
[0362] In order to determine whether these molecules are also
capable of blocking myostatin activity in vivo, transgenic mice
were generated in which the myosin light chain promoter/enhancer
was used to drive expression of either the myostatin pro peptide or
follistatin. From pronuclear injections of the pro peptide
construct, three transgenic mouse lines (two of these, B32A and
B32B, represented independently segregating transgene insertion
sites in one original founder animal) were identified that showed
increased muscling. As shown in Table 3, muscle weights of animals
from each line were increased by approximately 20-110% compared to
those of non-transgenic control animals. Northern analysis of RNA
samples prepared from representative animals of each of these lines
showed that the expression levels of the transgene correlated with
the magnitude of the increase in muscle weights. Specifically,
animals from the B32A line, which had only an approximately 20-40%
increase in muscle mass, had the lowest levels of transgene
expression, and animals from the B32B and B53 lines, which had an
approximately 70-110% increase in muscle mass, had the highest
levels of transgene expression. Perhaps significantly, muscle
weights in animals that were doubly transgenic for the B32A and
B32B insertion sites were similar to those observed in animals
transgenic only for the B32B insertion site (Table 3) despite the
fact that the doubly transgenic animals appeared to have higher
levels of transgene expression. These findings suggest that the
effects seen in the B32B line (and B53 line) were the maximal
achievable from overexpressing the pro peptide. As in the case of
animals expressing the dominant negative form of Act RIIB, animals
expressing the pro peptide showed increases in both muscle fiber
number and size. Analysis of the gastrocnemius and plantaris
muscles from two animals that were doubly transgenic for the B32A
and B32B insertion sites showed that fiber numbers were increased
by approximately 40% (the two animals had 11940 and 10420 fibers),
and fiber diameters were increased by approximately 21% (to 52
.mu.m) compared to control animals.
[0363] The most dramatic effects on skeletal muscle were obtained
using the follistatin construct. Two founder animals (F3 and F66)
showed increased muscling (Table 2). In one of these animals (F3),
muscle weights were increased by 194-327% relative to control
animals, resulting from a combination of hyperplasia (66% increase
in fiber number to 13051 in the gastrocnemius/plantaris) and
hypertrophy (28% increase in fiber diameter to 55 .mu.m). Although
we have not analyzed muscle weights of myostatin knockout mice in a
hybrid SJL/C57BL/6 background, the increases in muscle mass
observed in the F3 founder animal were significantly greater than
the increases we have seen in myostatin null animals in other
genetic backgrounds. These results suggest that at least part of
the effect of follistatin may result from inhibition of another
ligand besides myostatin. Clearly, analysis of additional
follistatin transgenic lines will be essential in determining
whether other ligands may also be involved in negatively regulating
muscle growth.
[0364] Following proteolytic processing, the myostatin C-terminal
dimer is likely maintained in a latent complex with its pro peptide
and perhaps other proteins as well. Myostatin is also negatively
regulated by follistatin, which binds the C-terminal dimer and
inhibits its ability to bind to receptors. Release of the
C-terminal dimer from these inhibitory proteins by unknown
mechanisms allows myostatin to bind to activin type II receptors.
By analogy with other family members, we presume that activation of
these receptors then leads to activation of a type I receptor and
Smad proteins.
[0365] This overall model for myostatin regulation and signaling is
consistent not only with the data presented here but also with
other genetic data. As discussed earlier, follistatin knockout mice
have been shown to have reduced muscle mass at birth, which is what
one might expect for uninhibited myostatin activity. A similar
muscle phenotype has been reported for mice lacking ski, which has
been shown to inhibit the activity of Smad2 and 3, and the opposite
phenotype, namely excess skeletal muscle, has been observed in mice
overexpressing ski. Based on the present findings, one hypothesis
is that these observed phenotypes reflect the over-activity and
under-activity, respectively, of myostatin in these mice.
[0366] Although all of the in vitro and genetic data are consistent
with the overall model that we have put forth here, these data
would also be consistent with alternative models involving other
receptors and ligands. For example, we do not know the mechanism by
which the truncated form of Act RIIB enhances muscle growth in our
transgenic mice. It is possible that the truncated receptor is not
acting to block signaling in the target cell but is rather merely
acting as a sink to deplete extracellular concentrations of
myostatin. It is also possible that the truncated receptor is
blocking signaling of other ligands besides myostatin. In this
regard, it has been shown that dominant negative forms of type II
activin receptors can block signaling of a variety of different
TGF-.beta. related ligands in other species. Similarly, our data do
not show definitively that follistatin is blocking myostatin
activity in vivo to promote muscle growth. In this regard, the
extraordinary degree of muscling seen in one of the follistatin
expressing founder animals suggests that other
follistatin-sensitive ligands may be involved in regulating muscle
growth.
[0367] To date, however, myostatin is the only secreted protein
that has demonstrated to play a negative role in regulating muscle
mass in vivo. Although additional experiments will be required to
prove aspects of this overall model and to identify the other
signaling components, our data suggest that myostatin antagonists,
such as follistatin and the myostatin pro peptide, or activin type
II receptor antagonists may be effective muscle enhancing agents
for both human and agricultural applications.
2TABLE 2 Muscle weights (mg) transgenic animals pectoralis triceps
quadriceps gastroc./plantaris male controls (7 mo., n = 10) 100.8
.+-. 5.4 115.6 .+-. 5.5 243.8 .+-. 12.5 168.1 .+-. 7.6 dom. neg.
Act RIIB (7 mo.) C5 male founder 148 155 318 252 C11 male founder
227 250 454 338 C33 male founder 158 176 352 244 C42 male founder
196 212 309 269 female controls (7 mo., n = 10) .sup. 68.9 .+-. 2.7
96.9 .+-. 3.5 208.3 .+-. 7.1 140.3 .+-. 4.3 dom. neg. Act RIIB (7
mo.) C2 female founder 104 163 352 263 C4 female founder 103 139
303 194 C27 female founder 135 117 181 256 male controls (4 mo., n
= 12) .sup. 98.3 .+-. 3.3 110.9 .+-. 2.9 251.7 .+-. 8.5 169.3 .+-.
4.7 follistatin (4 mo.) F3 male founder 296 494 736 568 F66 male
founder 169 263 421 409 All animals (including controls) represent
hybrid SJL/C57BL/6 F.sub.0 mice born from injected embryos.
[0368]
3TABLE 3 Muscle weights (mg) transgenic line pectoralis triceps
quadriceps gastroc./plantaris 1. Males controls (n = 50) 104.6 .+-.
1.5 113.9 .+-. 1.6 246.2 .+-. 3.0 167.7 .+-. 2.1 dom. neg. Act RIIB
C5 (n = 11) 153.7 .+-. 6.0*** 177.5 .+-. 6.0*** 322.7 .+-. 9.3***
247.1 .+-. 8.1*** C27 (n = 5) 190.4 .+-. 7.1*** 230.8 .+-. 13.0***
406.8 .+-. 11.6*** 283.8 .+-. 6.9*** C11 (n = 2) 278.0 .+-. 18.4*
244.5 .+-. 4.9** 515.5 .+-. 7.8** 366.0 .+-. 21.2* pro peptide B32A
(n = 8) 139.9 .+-. 7.1*** 160.6 .+-. 7.8*** 322.5 .+-. 10.3***
222.6 .+-. 7.1*** B32B (n = 4) 214.0 .+-. 19.9** 206.5 .+-. 6.7***
435.8 .+-. 15.0*** 289.5 .+-. 8.6*** B32A + B (n = 8) 212.4 .+-.
8.4*** 220.3 .+-. 6.4*** 429.1 .+-. 11.1*** 288.3 .+-. 8.5*** B53
(n = 8) 215.1 .+-. 6.4*** 229.6 .+-. 8.1*** 413.3 .+-. 13.2***
293.5 .+-. 10.5*** 2. Females controls (n = 50) 64.7 .+-. 1.4 75.7
.+-. 1.1 164.5 .+-. 2.0 109.6 .+-. 1.4 dom. neg. Act RIIB C5 (n =
15) 89.7 .+-. 2.8*** 115.9 .+-. 4.0*** 229.3 .+-. 6.5*** 161.8 .+-.
4.7*** C27 (n = 5) 117.6 .+-. 10.9*** 138.6 .+-. 12.3*** 314.0 .+-.
27.7*** 207.6 .+-. 18.3*** C11 (n = 3) 180.3 .+-. 38.9 208.7 .+-.
45.7 430.3 .+-. 72.2* 291.7 .+-. 48.8* pro peptide B32A (n = 9)
78.8 .+-. 2.9*** 100.1 .+-. 3.7*** 206.0 .+-. 2.7*** 138.9 .+-.
3.1*** B32B (n = 2) 131.0 .+-. 18.4 151.5 .+-. 23.3 315.5 .+-. 58.7
199.5 .+-. 24.7 B32A + B (n = 4) 109.3 .+-. 9.5* 132.8 .+-. 6.0**
270.8 .+-. 6.9*** 177.0 .+-. 2.4*** B53 (n = 6) 134.7 .+-. 7.7***
148.2 .+-. 12.1*** 303.8 .+-. 18.5*** 212.8 .+-. 12.9*** *p <
0.05, **p < 0.01, ***p < 0.001. All animals (including
controls) represent 4 month old offspring of transgenic founders
(SJL/C57BL/6) mated with wild type C57Bl/6 mice.
[0369] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
29 1 2743 DNA Homo sapiens CDS (59)...(1183) 1 aagaaaagta
aaaggaagaa acaagaacaa gaaaaaagat tatattgatt ttaaaatc 58 atg caa aaa
ctg caa ctc tgt gtt tat att tac ctg ttt atg ctg att 106 Met Gln Lys
Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15 gtt
gct ggt cca gtg gat cta aat gag aac agt gag caa aaa gaa aat 154 Val
Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25
30 gtg gaa aaa gag ggg ctg tgt aat gca tgt act tgg aga caa aac act
202 Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr
35 40 45 aaa tct tca aga ata gaa gcc att aag ata caa atc ctc agt
aaa ctt 250 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser
Lys Leu 50 55 60 cgt ctg gaa aca gct cct aac atc agc aaa gat gtt
ata aga caa ctt 298 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val
Ile Arg Gln Leu 65 70 75 80 tta ccc aaa gct cct cca ctc cgg gaa ctg
att gat cag tat gat gtc 346 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu
Ile Asp Gln Tyr Asp Val 85 90 95 cag agg gat gac agc agc gat ggc
tct ttg gaa gat gac gat tat cac 394 Gln Arg Asp Asp Ser Ser Asp Gly
Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 gct aca acg gaa aca atc
att acc atg cct aca gag tct gat ttt cta 442 Ala Thr Thr Glu Thr Ile
Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 atg caa gtg gat
gga aaa ccc aaa tgt tgc ttc ttt aaa ttt agc tct 490 Met Gln Val Asp
Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 aaa ata
caa tac aat aaa gta gta aag gcc caa cta tgg ata tat ttg 538 Lys Ile
Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155
160 aga ccc gtc gag act cct aca aca gtg ttt gtg caa atc ctg aga ctc
586 Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu
165 170 175 atc aaa cct atg aaa gac ggt aca agg tat act gga atc cga
tct ctg 634 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg
Ser Leu 180 185 190 aaa ctt gac atg aac cca ggc act ggt att tgg cag
agc att gat gtg 682 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln
Ser Ile Asp Val 195 200 205 aag aca gtg ttg caa aat tgg ctc aaa caa
cct gaa tcc aac tta ggc 730 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln
Pro Glu Ser Asn Leu Gly 210 215 220 att gaa ata aaa gct tta gat gag
aat ggt cat gat ctt gct gta acc 778 Ile Glu Ile Lys Ala Leu Asp Glu
Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 ttc cca gga cca gga
gaa gat ggg ctg aat ccg ttt tta gag gtc aag 826 Phe Pro Gly Pro Gly
Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 gta aca gac
aca cca aaa aga tcc aga agg gat ttt ggt ctt gac tgt 874 Val Thr Asp
Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 gat
gag cac tca aca gaa tca cga tgc tgt cgt tac cct cta act gtg 922 Asp
Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280
285 gat ttt gaa gct ttt gga tgg gat tgg att atc gct cct aaa aga tat
970 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr
290 295 300 aag gcc aat tac tgc tct gga gag tgt gaa ttt gta ttt tta
caa aaa 1018 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe
Leu Gln Lys 305 310 315 320 tat cct cat act cat ctg gta cac caa gca
aac ccc aga ggt tca gca 1066 Tyr Pro His Thr His Leu Val His Gln
Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc cct tgc tgt act ccc aca
aag atg tct cca att aat atg cta tat 1114 Gly Pro Cys Cys Thr Pro
Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 ttt aat ggc aaa
gaa caa ata ata tat ggg aaa att cca gcg atg gta 1162 Phe Asn Gly
Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 gta
gac cgc tgt ggg tgc tca tgagatttat attaagcgtt cataacttcc 1213 Val
Asp Arg Cys Gly Cys Ser 370 375 taaaacatgg aaggttttcc cctcaacaat
tttgaagctg tgaaattaag taccacaggc 1273 tataggccta gagtatgcta
cagtcactta agcataagct acagtatgta aactaaaagg 1333 gggaatatat
gcaatggttg gcatttaacc atccaaacaa atcatacaag aaagttttat 1393
gatttccaga gtttttgagc tagaaggaga tcaaattaca tttatgttcc tatatattac
1453 aacatcggcg aggaaatgaa agcgattctc cttgagttct gatgaattaa
aggagtatgc 1513 tttaaagtct atttctttaa agttttgttt aatatttaca
gaaaaatcca catacagtat 1573 tggtaaaatg caggattgtt atataccatc
attcgaatca tccttaaaca cttgaattta 1633 tattgtatgg tagtatactt
ggtaagataa aattccacaa aaatagggat ggtgcagcat 1693 atgcaatttc
cattcctatt ataattgaca cagtacatta acaatccatg ccaacggtgc 1753
taatacgata ggctgaatgt ctgaggctac caggtttatc acataaaaaa cattcagtaa
1813 aatagtaagt ttctcttttc ttcaggtgca ttttcctaca cctccaaatg
aggaatggat 1873 tttctttaat gtaagaagaa tcatttttct agaggttggc
tttcaattct gtagcatact 1933 tggagaaact gcattatctt aaaaggcagt
caaatggtgt ttgtttttat caaaatgtca 1993 aaataacata cttggagaag
tatgtaattt tgtctttgga aaattacaac actgcctttg 2053 caacactgca
gtttttatgg taaaataata gaaatgatcg actctatcaa tattgtataa 2113
aaagactgaa acaatgcatt tatataatat gtatacaata ttgttttgta aataagtgtc
2173 tcctttttta tttactttgg tatattttta cactaaggac atttcaaatt
aagtactaag 2233 gcacaaagac atgtcatgca tcacagaaaa gcaactactt
atatttcaga gcaaattagc 2293 agattaaata gtggtcttaa aactccatat
gttaatgatt agatggttat attacaatca 2353 ttttatattt ttttacatga
ttaacattca cttatggatt catgatggct gtataaagtg 2413 aatttgaaat
ttcaatggtt tactgtcatt gtgtttaaat ctcaacgttc cattatttta 2473
atacttgcaa aaacattact aagtatacca aaataattga ctctattatc tgaaatgaag
2533 aataaactga tgctatctca acaataactg ttacttttat tttataattt
gataatgaat 2593 atatttctgc atttatttac ttctgttttg taaattggga
ttttgttaat caaatttatt 2653 gtactatgac taaatgaaat tatttcttac
atctaatttg tagaaacagt ataagttata 2713 ttaaagtgtt ttcacatttt
tttgaaagac 2743 2 375 PRT Homo sapiens 2 Met Gln Lys Leu Gln Leu
Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15 Val Ala Gly Pro
Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 Val Glu
Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40 45
Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50
55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Val Ile Arg Gln
Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln
Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu
Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu Thr Ile Ile Thr Met
Pro Thr Glu Ser Asp Phe Leu 115 120 125 Met Gln Val Asp Gly Lys Pro
Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 Lys Ile Gln Tyr Asn
Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 Arg Pro
Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175
Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180
185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp
Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser
Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His
Asp Leu Ala Val Thr 225 230 235 240 Phe Pro Gly Pro Gly Glu Asp Gly
Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 Val Thr Asp Thr Pro Lys
Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 Asp Glu His Ser
Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 Asp Phe
Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300
Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305
310 315 320 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly
Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile
Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly
Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg Cys Gly Cys Ser 370
375 3 2676 DNA Mus musculus CDS (104)...(1231) 3 gtctctcgga
cggtacatgc actaatattt cacttggcat tactcaaaag caaaaagaag 60
aaataagaac aagggaaaaa aaaagattgt gctgattttt aaa atg atg caa aaa 115
Met Met Gln Lys 1 ctg caa atg tat gtt tat att tac ctg ttc atg ctg
att gct gct ggc 163 Leu Gln Met Tyr Val Tyr Ile Tyr Leu Phe Met Leu
Ile Ala Ala Gly 5 10 15 20 cca gtg gat cta aat gag ggc agt gag aga
gaa gaa aat gtg gaa aaa 211 Pro Val Asp Leu Asn Glu Gly Ser Glu Arg
Glu Glu Asn Val Glu Lys 25 30 35 gag ggg ctg tgt aat gca tgt gcg
tgg aga caa aac acg agg tac tcc 259 Glu Gly Leu Cys Asn Ala Cys Ala
Trp Arg Gln Asn Thr Arg Tyr Ser 40 45 50 aga ata gaa gcc ata aaa
att caa atc ctc agt aag ctg cgc ctg gaa 307 Arg Ile Glu Ala Ile Lys
Ile Gln Ile Leu Ser Lys Leu Arg Leu Glu 55 60 65 aca gct cct aac
atc agc aaa gat gct ata aga caa ctt ctg cca aga 355 Thr Ala Pro Asn
Ile Ser Lys Asp Ala Ile Arg Gln Leu Leu Pro Arg 70 75 80 gcg cct
cca ctc cgg gaa ctg atc gat cag tac gac gtc cag agg gat 403 Ala Pro
Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val Gln Arg Asp 85 90 95
100 gac agc agt gat ggc tct ttg gaa gat gac gat tat cac gct acc acg
451 Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His Ala Thr Thr
105 110 115 gaa aca atc att acc atg cct aca gag tct gac ttt cta atg
caa gcg 499 Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu Met
Gln Ala 120 125 130 gat ggc aag ccc aaa tgt tgc ttt ttt aaa ttt agc
tct aaa ata cag 547 Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser
Ser Lys Ile Gln 135 140 145 tac aac aaa gta gta aaa gcc caa ctg tgg
ata tat ctc aga ccc gtc 595 Tyr Asn Lys Val Val Lys Ala Gln Leu Trp
Ile Tyr Leu Arg Pro Val 150 155 160 aag act cct aca aca gtg ttt gtg
caa atc ctg aga ctc atc aaa ccc 643 Lys Thr Pro Thr Thr Val Phe Val
Gln Ile Leu Arg Leu Ile Lys Pro 165 170 175 180 atg aaa gac ggt aca
agg tat act gga atc cga tct ctg aaa ctt gac 691 Met Lys Asp Gly Thr
Arg Tyr Thr Gly Ile Arg Ser Leu Lys Leu Asp 185 190 195 atg agc cca
ggc act ggt att tgg cag agt att gat gtg aag aca gtg 739 Met Ser Pro
Gly Thr Gly Ile Trp Gln Ser Ile Asp Val Lys Thr Val 200 205 210 ttg
caa aat tgg ctc aaa cag cct gaa tcc aac tta ggc att gaa atc 787 Leu
Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly Ile Glu Ile 215 220
225 aaa gct ttg gat gag aat ggc cat gat ctt gct gta acc ttc cca gga
835 Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr Phe Pro Gly
230 235 240 cca gga gaa gat ggg ctg aat ccc ttt tta gaa gtc aag gtg
aca gac 883 Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Lys Val
Thr Asp 245 250 255 260 aca ccc aag agg tcc cgg aga gac ttt ggg ctt
gac tgc gat gag cac 931 Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu
Asp Cys Asp Glu His 265 270 275 tcc acg gaa tcc cgg tgc tgc cgc tac
ccc ctc acg gtc gat ttt gaa 979 Ser Thr Glu Ser Arg Cys Cys Arg Tyr
Pro Leu Thr Val Asp Phe Glu 280 285 290 gcc ttt gga tgg gac tgg att
atc gca ccc aaa aga tat aag gcc aat 1027 Ala Phe Gly Trp Asp Trp
Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn 295 300 305 tac tgc tca gga
gag tgt gaa ttt gtg ttt tta caa aaa tat ccg cat 1075 Tyr Cys Ser
Gly Glu Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His 310 315 320 act
cat ctt gtg cac caa gca aac ccc aga ggc tca gca ggc cct tgc 1123
Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys 325
330 335 340 tgc act ccg aca aaa atg tct ccc att aat atg cta tat ttt
aat ggc 1171 Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr
Phe Asn Gly 345 350 355 aaa gaa caa ata ata tat ggg aaa att cca gcc
atg gta gta gac cgc 1219 Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro
Ala Met Val Val Asp Arg 360 365 370 tgt ggg tgc tca tgagctttgc
attaggttag aaacttccca agtcatggaa 1271 Cys Gly Cys Ser 375
ggtcttcccc tcaatttcga aactgtgaat tcaagcacca caggctgtag gccttgagta
1331 tgctctagta acgtaagcac aagctacagt gtatgaacta aaagagagaa
tagatgcaat 1391 ggttggcatt caaccaccaa aataaaccat actataggat
gttgtatgat ttccagagtt 1451 tttgaaatag atggagatca aattacattt
atgtccatat atgtatatta caactacaat 1511 ctaggcaagg aagtgagagc
acatcttgtg gtctgctgag ttaggagggt atgattaaaa 1571 ggtaaagtct
tatttcctaa cagtttcact taatatttac agaagaatct atatgtagcc 1631
tttgtaaagt gtaggattgt tatcatttaa aaacatcatg tacacttata tttgtattgt
1691 atacttggta agataaaatt ccacaaagta ggaatggggc ctcacataca
cattgccatt 1751 cctattataa ttggacaatc caccacggtg ctaatgcagt
gctgaatggc tcctactgga 1811 cctctcgata gaacactcta caaagtacga
gtctctctct cccttccagg tgcatctcca 1871 cacacacagc actaagtgtt
caatgcattt tctttaagga aagaagaatc tttttttcta 1931 gaggtcaact
ttcagtcaac tctagcacag cgggagtgac tgctgcatct taaaaggcag 1991
ccaaacagta ttcatttttt aatctaaatt tcaaaatcac tgtctgcctt tatcacatgg
2051 caattttgtg gtaaaataat ggaaatgact ggttctatca atattgtata
aaagactctg 2111 aaacaattac atttatataa tatgtataca atattgtttt
gtaaataagt gtctcctttt 2171 atatttactt tggtatattt ttacactaat
gaaatttcaa atcattaaag tacaaagaca 2231 tgtcatgtat cacaaaaaag
gtgactgctt ctatttcaga gtgaattagc agattcaata 2291 gtggtcttaa
aactctgtat gttaagatta gaaggttata ttacaatcaa tttatgtatt 2351
ttttacatta tcaacttatg gtttcatggt ggctgtatct atgaatgtgg ctcccagtca
2411 aatttcaatg ccccaccatt ttaaaaatta caagcattac taaacatacc
aacatgtatc 2471 taaagaaata caaatatggt atctcaataa cagctacttt
tttattttat aatttgacaa 2531 tgaatacatt tcttttattt acttcagttt
tataaattgg aactttgttt atcaaatgta 2591 ttgtactcat agctaaatga
aattatttct tacataaaaa tgtgtagaaa ctataaatta 2651 aagtgttttc
acatttttga aaggc 2676 4 376 PRT Mus musculus 4 Met Met Gln Lys Leu
Gln Met Tyr Val Tyr Ile Tyr Leu Phe Met Leu 1 5 10 15 Ile Ala Ala
Gly Pro Val Asp Leu Asn Glu Gly Ser Glu Arg Glu Glu 20 25 30 Asn
Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn 35 40
45 Thr Arg Tyr Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys
50 55 60 Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile
Arg Gln 65 70 75 80 Leu Leu Pro Arg Ala Pro Pro Leu Arg Glu Leu Ile
Asp Gln Tyr Asp 85 90 95 Val Gln Arg Asp Asp Ser Ser Asp Gly Ser
Leu Glu Asp Asp Asp Tyr 100 105 110 His Ala Thr Thr Glu Thr Ile Ile
Thr Met Pro Thr Glu Ser Asp Phe 115 120 125 Leu Met Gln Ala Asp Gly
Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser 130 135 140 Ser Lys Ile Gln
Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145 150 155 160 Leu
Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg 165 170
175 Leu Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser
180 185 190 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln Ser
Ile Asp 195 200 205 Val Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro
Glu Ser Asn Leu 210 215 220 Gly Ile Glu Ile Lys Ala Leu Asp Glu Asn
Gly His Asp Leu Ala Val 225 230 235 240 Thr Phe Pro Gly Pro Gly Glu
Asp Gly Leu Asn Pro Phe Leu Glu Val 245 250 255 Lys Val Thr Asp Thr
Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp 260 265 270 Cys Asp Glu
His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 275 280 285 Val
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 290 295
300 Tyr
Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln 305 310
315 320 Lys Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly
Ser 325 330 335 Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile
Asn Met Leu 340 345 350 Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly
Lys Ile Pro Ala Met 355 360 365 Val Val Asp Arg Cys Gly Cys Ser 370
375 5 1131 DNA Rattus norvegicus CDS (1)...(1128) 5 atg att caa aaa
ccg caa atg tat gtt tat att tac ctg ttt gtg ctg 48 Met Ile Gln Lys
Pro Gln Met Tyr Val Tyr Ile Tyr Leu Phe Val Leu 1 5 10 15 att gct
gct ggc cca gtg gat cta aat gag gac agt gag aga gag gcg 96 Ile Ala
Ala Gly Pro Val Asp Leu Asn Glu Asp Ser Glu Arg Glu Ala 20 25 30
aat gtg gaa aaa gag ggg ctg tgt aat gcg tgt gcg tgg aga caa aac 144
Asn Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn 35
40 45 aca agg tac tcc aga ata gaa gcc ata aaa att caa atc ctc agt
aaa 192 Thr Arg Tyr Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser
Lys 50 55 60 ctc cgc ctg gaa aca gcg cct aac atc agc aaa gat gct
ata aga caa 240 Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala
Ile Arg Gln 65 70 75 80 ctt ctg ccc aga gcg cct cca ctc cgg gaa ctg
atc gat cag tac gac 288 Leu Leu Pro Arg Ala Pro Pro Leu Arg Glu Leu
Ile Asp Gln Tyr Asp 85 90 95 gtc cag agg gat gac agc agt gac ggc
tct ttg gaa gat gac gat tat 336 Val Gln Arg Asp Asp Ser Ser Asp Gly
Ser Leu Glu Asp Asp Asp Tyr 100 105 110 cac gct acc acg gaa aca atc
att acc atg cct acc gag tct gac ttt 384 His Ala Thr Thr Glu Thr Ile
Ile Thr Met Pro Thr Glu Ser Asp Phe 115 120 125 cta atg caa gcg gat
gga aag ccc aaa tgt tgc ttt ttt aaa ttt agc 432 Leu Met Gln Ala Asp
Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser 130 135 140 tct aaa ata
cag tac aac aaa gtg gta aag gcc cag ctg tgg ata tat 480 Ser Lys Ile
Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145 150 155 160
ctg aga gcc gtc aag act cct aca aca gtg ttt gtg caa atc ctg aga 528
Leu Arg Ala Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg 165
170 175 ctc atc aaa ccc atg aaa gac ggt aca agg tat acc gga atc cga
tct 576 Leu Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg
Ser 180 185 190 ctg aaa ctt gac atg agc cca ggc act ggt att tgg cag
agt att gat 624 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln
Ser Ile Asp 195 200 205 gtg aag aca gtg ttg caa aat tgg ctc aaa cag
cct gaa tcc aac tta 672 Val Lys Thr Val Leu Gln Asn Trp Leu Lys Gln
Pro Glu Ser Asn Leu 210 215 220 ggc att gaa atc aaa gct ttg gat gag
aat ggg cat gat ctt gct gta 720 Gly Ile Glu Ile Lys Ala Leu Asp Glu
Asn Gly His Asp Leu Ala Val 225 230 235 240 acc ttc cca gga cca gga
gaa gat ggg ctg aat ccc ttt tta gaa gtc 768 Thr Phe Pro Gly Pro Gly
Glu Asp Gly Leu Asn Pro Phe Leu Glu Val 245 250 255 aaa gta aca gac
aca ccc aag agg tcc cgg aga gac ttt ggg ctt gac 816 Lys Val Thr Asp
Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp 260 265 270 tgc gat
gaa cac tcc acg gaa tcg cgg tgc tgt cgc tac ccc ctc acg 864 Cys Asp
Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 275 280 285
gtc gat ttc gaa gcc ttt gga tgg gac tgg att att gca ccc aaa aga 912
Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 290
295 300 tat aag gct aat tac tgc tct gga gag tgt gaa ttt gtg ttc tta
caa 960 Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu
Gln 305 310 315 320 aaa tat ccg cat act cat ctt gtg cac caa gca aac
ccc aga ggc tcg 1008 Lys Tyr Pro His Thr His Leu Val His Gln Ala
Asn Pro Arg Gly Ser 325 330 335 gca ggc cct tgc tgc acg cca aca aaa
atg tct ccc att aat atg cta 1056 Ala Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu 340 345 350 tat ttt aat ggc aaa gaa
caa ata ata tat ggg aaa att cca gcc atg 1104 Tyr Phe Asn Gly Lys
Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met 355 360 365 gta gta gac
cgg tgt ggg tgc tcg tga 1131 Val Val Asp Arg Cys Gly Cys Ser 370
375 6 376 PRT Rattus norvegicus 6 Met Ile Gln Lys Pro Gln Met Tyr
Val Tyr Ile Tyr Leu Phe Val Leu 1 5 10 15 Ile Ala Ala Gly Pro Val
Asp Leu Asn Glu Asp Ser Glu Arg Glu Ala 20 25 30 Asn Val Glu Lys
Glu Gly Leu Cys Asn Ala Cys Ala Trp Arg Gln Asn 35 40 45 Thr Arg
Tyr Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys 50 55 60
Leu Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln 65
70 75 80 Leu Leu Pro Arg Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln
Tyr Asp 85 90 95 Val Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu
Asp Asp Asp Tyr 100 105 110 His Ala Thr Thr Glu Thr Ile Ile Thr Met
Pro Thr Glu Ser Asp Phe 115 120 125 Leu Met Gln Ala Asp Gly Lys Pro
Lys Cys Cys Phe Phe Lys Phe Ser 130 135 140 Ser Lys Ile Gln Tyr Asn
Lys Val Val Lys Ala Gln Leu Trp Ile Tyr 145 150 155 160 Leu Arg Ala
Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg 165 170 175 Leu
Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser 180 185
190 Leu Lys Leu Asp Met Ser Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp
195 200 205 Val Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser
Asn Leu 210 215 220 Gly Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His
Asp Leu Ala Val 225 230 235 240 Thr Phe Pro Gly Pro Gly Glu Asp Gly
Leu Asn Pro Phe Leu Glu Val 245 250 255 Lys Val Thr Asp Thr Pro Lys
Arg Ser Arg Arg Asp Phe Gly Leu Asp 260 265 270 Cys Asp Glu His Ser
Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 275 280 285 Val Asp Phe
Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 290 295 300 Tyr
Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln 305 310
315 320 Lys Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Arg Gly
Ser 325 330 335 Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile
Asn Met Leu 340 345 350 Tyr Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly
Lys Ile Pro Ala Met 355 360 365 Val Val Asp Arg Cys Gly Cys Ser 370
375 7 1128 DNA Gallus gallus CDS (1)...(1125) 7 atg caa aag ctg gca
gtc tat gtt tat att tac ctg ttc atg cag atc 48 Met Gln Lys Leu Ala
Val Tyr Val Tyr Ile Tyr Leu Phe Met Gln Ile 1 5 10 15 gcg gtt gat
ccg gtg gct ctg gat ggc agt agt cag ccc aca gag aac 96 Ala Val Asp
Pro Val Ala Leu Asp Gly Ser Ser Gln Pro Thr Glu Asn 20 25 30 gct
gaa aaa gac gga ctg tgc aat gct tgt acg tgg aga cag aat aca 144 Ala
Glu Lys Asp Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35 40
45 aaa tcc tcc aga ata gaa gcc ata aaa att caa atc ctc agc aaa ctg
192 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu
50 55 60 cgc ctg gaa caa gca cct aac att agc agg gac gtt att aag
cag ctt 240 Arg Leu Glu Gln Ala Pro Asn Ile Ser Arg Asp Val Ile Lys
Gln Leu 65 70 75 80 tta ccc aaa gct cct cca ctg cag gaa ctg att gat
cag tat gat gtc 288 Leu Pro Lys Ala Pro Pro Leu Gln Glu Leu Ile Asp
Gln Tyr Asp Val 85 90 95 cag agg gac gac agt agc gat ggc tct ttg
gaa gac gat gac tat cat 336 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu
Glu Asp Asp Asp Tyr His 100 105 110 gcc aca acc gag acg att atc aca
atg cct acg gag tct gat ttt ctt 384 Ala Thr Thr Glu Thr Ile Ile Thr
Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 gta caa atg gag gga aaa
cca aaa tgt tgc ttc ttt aag ttt agc tct 432 Val Gln Met Glu Gly Lys
Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 aaa ata caa tat
aac aaa gta gta aag gca caa tta tgg ata tac ttg 480 Lys Ile Gln Tyr
Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 agg
caa gtc caa aaa cct aca acg gtg ttt gtg cag atc ctg aga ctc 528 Arg
Gln Val Gln Lys Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170
175 att aag ccc atg aaa gac ggt aca aga tat act gga att cga tct ttg
576 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu
180 185 190 aaa ctt gac atg aac cca ggc act ggt atc tgg cag agt att
gat gtg 624 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile
Asp Val 195 200 205 aag aca gtg ctg caa aat tgg ctc aaa cag cct gaa
tcc aat tta ggc 672 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu
Ser Asn Leu Gly 210 215 220 atc gaa ata aaa gct ttt gat gag act gga
cga gat ctt gct gtc aca 720 Ile Glu Ile Lys Ala Phe Asp Glu Thr Gly
Arg Asp Leu Ala Val Thr 225 230 235 240 ttc cca gga cca gga gaa gat
gga ttg aac cca ttt tta gag gtc aga 768 Phe Pro Gly Pro Gly Glu Asp
Gly Leu Asn Pro Phe Leu Glu Val Arg 245 250 255 gtt aca gac aca ccg
aaa cgg tcc cgc aga gat ttt ggc ctt gac tgt 816 Val Thr Asp Thr Pro
Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 gat gag cac
tca acg gaa tcc cga tgt tgt cgc tac ccg ctg aca gtg 864 Asp Glu His
Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 gat
ttc gaa gct ttt gga tgg gac tgg att ata gca cct aaa aga tac 912 Asp
Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295
300 aaa gcc aat tac tgc tcc gga gaa tgc gaa ttt gtg ttt cta cag aaa
960 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys
305 310 315 320 tac ccg cac act cac ctg gta cac caa gca aat ccc aga
ggc tca gca 1008 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro
Arg Gly Ser Ala 325 330 335 ggc cct tgc tgc aca ccc acc aag atg tcc
cct ata aac atg ctg tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys Met
Ser Pro Ile Asn Met Leu Tyr 340 345 350 ttc aat gga aaa gaa caa ata
ata tat gga aag ata cca gcc atg gtt 1104 Phe Asn Gly Lys Glu Gln
Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 gta gat cgt tgc
ggg tgc tca tga 1128 Val Asp Arg Cys Gly Cys Ser 370 375 8 374 PRT
Gallus gallus 8 Gln Lys Leu Ala Val Tyr Val Tyr Ile Tyr Leu Phe Met
Gln Ile Ala 1 5 10 15 Val Asp Pro Val Ala Leu Asp Gly Ser Ser Gln
Pro Thr Glu Asn Ala 20 25 30 Glu Lys Asp Gly Leu Cys Asn Ala Cys
Thr Trp Arg Gln Asn Thr Lys 35 40 45 Ser Ser Arg Ile Glu Ala Ile
Lys Ile Gln Ile Leu Ser Lys Leu Arg 50 55 60 Leu Glu Gln Ala Pro
Asn Ile Ser Arg Asp Val Ile Lys Gln Leu Leu 65 70 75 80 Pro Lys Ala
Pro Pro Leu Gln Glu Leu Ile Asp Gln Tyr Asp Val Gln 85 90 95 Arg
Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His Ala 100 105
110 Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu Val
115 120 125 Gln Met Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser
Ser Lys 130 135 140 Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp
Ile Tyr Leu Arg 145 150 155 160 Gln Val Gln Lys Pro Thr Thr Val Phe
Val Gln Ile Leu Arg Leu Ile 165 170 175 Lys Pro Met Lys Asp Gly Thr
Arg Tyr Thr Gly Ile Arg Ser Leu Lys 180 185 190 Leu Asp Met Asn Pro
Gly Thr Gly Ile Trp Gln Ser Ile Asp Val Lys 195 200 205 Thr Val Leu
Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly Ile 210 215 220 Glu
Ile Lys Ala Phe Asp Glu Thr Gly Arg Asp Leu Ala Val Thr Phe 225 230
235 240 Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Arg
Val 245 250 255 Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu
Asp Cys Asp 260 265 270 Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr
Pro Leu Thr Val Asp 275 280 285 Phe Glu Ala Phe Gly Trp Asp Trp Ile
Ile Ala Pro Lys Arg Tyr Lys 290 295 300 Ala Asn Tyr Cys Ser Gly Glu
Cys Glu Phe Val Phe Leu Gln Lys Tyr 305 310 315 320 Pro His Thr His
Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala Gly 325 330 335 Pro Cys
Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr Phe 340 345 350
Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val Val 355
360 365 Asp Arg Cys Gly Cys Ser 370 9 1128 DNA Baboon CDS
(1)...(1125) 9 atg caa aaa ctg caa ctc tgt gtt tat att tac ctg ttt
atg ctg att 48 Met Gln Lys Leu Gln Leu Cys Val Tyr Ile Tyr Leu Phe
Met Leu Ile 1 5 10 15 gtt gct ggt cca gtg gat cta aat gag aac agt
gag caa aaa gaa aat 96 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser
Glu Gln Lys Glu Asn 20 25 30 gtg gaa aaa gag ggg ctg tgt aat gca
tgt act tgg aga caa aac act 144 Val Glu Lys Glu Gly Leu Cys Asn Ala
Cys Thr Trp Arg Gln Asn Thr 35 40 45 aaa tct tca aga ata gaa gcc
att aaa ata caa atc ctc agt aaa ctt 192 Lys Ser Ser Arg Ile Glu Ala
Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 cgt ctg gaa aca gct
cct aac atc agc aaa gat gct ata aga caa ctt 240 Arg Leu Glu Thr Ala
Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80 tta ccc aaa
gcg cct cca ctc cgg gaa ctg att gat cag tat gat gtc 288 Leu Pro Lys
Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 cag
agg gat gac agc agc gat ggc tct ttg gaa gat gac gat tat cac 336 Gln
Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105
110 gct aca acg gaa aca atc att acc atg cct aca gag tct gat ttt tta
384 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu
115 120 125 atg caa gtg gat gga aaa ccc aaa tgt tgc ttc ttt aaa ttt
agc tct 432 Met Gln Val Asp Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe
Ser Ser 130 135 140 aaa ata caa tac aat aaa gtg gta aag gcc caa cta
tgg ata tat ttg 480 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu
Trp Ile Tyr Leu 145 150 155 160 aga ccc gtc gag act cct aca aca gtg
ttt gtg caa atc ctg aga ctc 528 Arg Pro Val Glu Thr Pro Thr Thr Val
Phe Val Gln Ile Leu Arg Leu 165 170 175 atc aaa cct atg aaa gac ggt
aca agg tat act gga atc cga tct ctg 576 Ile Lys Pro Met Lys Asp Gly
Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 aaa ctt gac atg aac
cca ggc act ggt att tgg cag agc att gat gtg 624 Lys Leu Asp Met Asn
Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 aag aca gtg
ttg caa aat tgg ctc aaa caa cct gaa tcc aac tta ggc 672 Lys Thr Val
Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 att
gaa ata aaa gct tta gat gag aat ggt cat gat ctt gct gta acc 720 Ile
Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 225
230
235 240 ttc cca gga cca gga gaa gat ggg ctg aat ccc ttt tta gag gtc
aag 768 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val
Lys 245 250 255 gta aca gac aca ccc aaa aga tcc aga agg gat ttt ggt
ctt gac tgt 816 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly
Leu Asp Cys 260 265 270 gat gag cac tca aca gaa tcg cga tgc tgt cgt
tac cct cta act gtg 864 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg
Tyr Pro Leu Thr Val 275 280 285 gat ttt gaa gct ctt gga tgg gat tgg
att atc gct cct aaa aga tat 912 Asp Phe Glu Ala Leu Gly Trp Asp Trp
Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aag gcc aat tac tgc tct gga
gag tgt gaa ttt gta ttt tta caa aaa 960 Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 tat cct cat act
cat ctg gta cac caa gca aac ccc aga ggt tca gca 1008 Tyr Pro His
Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc
cct tgc tgt act ccc aca aag atg tct cca att aat atg cta tat 1056
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340
345 350 ttt aat ggc aaa gaa caa ata ata tat ggg aaa att cca gcc atg
gta 1104 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala
Met Val 355 360 365 gta gac cgc tgc ggg tgc tca tga 1128 Val Asp
Arg Cys Gly Cys Ser 370 375 10 375 PRT Baboon 10 Met Gln Lys Leu
Gln Leu Cys Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15 Val Ala
Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30
Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln Asn Thr 35
40 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys
Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile
Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile
Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser
Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu Thr Ile Ile
Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 Met Gln Val Asp Gly
Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 Lys Ile Gln
Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160
Arg Pro Val Glu Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165
170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser
Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser
Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro
Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu Asp Glu Asn
Gly His Asp Leu Ala Val Thr 225 230 235 240 Phe Pro Gly Pro Gly Glu
Asp Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 Val Thr Asp Thr
Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 Asp Glu
His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285
Asp Phe Glu Ala Leu Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290
295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln
Lys 305 310 315 320 Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro
Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser
Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu Gln Ile Ile
Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg Cys Gly Cys
Ser 370 375 11 1128 DNA Bovine CDS (1)...(1125) 11 atg caa aaa ctg
caa atc tct gtt tat att tac cta ttt atg ctg att 48 Met Gln Lys Leu
Gln Ile Ser Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15 gtt gct
ggc cca gtg gat ctg aat gag aac agc gag cag aag gaa aat 96 Val Ala
Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30
gtg gaa aaa gag ggg ctg tgt aat gca tgt ttg tgg agg gaa aac act 144
Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Leu Trp Arg Glu Asn Thr 35
40 45 aca tcg tca aga cta gaa gcc ata aaa atc caa atc ctc agt aaa
ctt 192 Thr Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys
Leu 50 55 60 cgc ctg gaa aca gct cct aac atc agc aaa gat gct atc
aga caa ctt 240 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile
Arg Gln Leu 65 70 75 80 ttg ccc aag gct cct cca ctc ctg gaa ctg att
gat cag ttc gat gtc 288 Leu Pro Lys Ala Pro Pro Leu Leu Glu Leu Ile
Asp Gln Phe Asp Val 85 90 95 cag aga gat gcc agc agt gac ggc tcc
ttg gaa gac gat gac tac cac 336 Gln Arg Asp Ala Ser Ser Asp Gly Ser
Leu Glu Asp Asp Asp Tyr His 100 105 110 gcc agg acg gaa acg gtc att
acc atg ccc acg gag tct gat ctt cta 384 Ala Arg Thr Glu Thr Val Ile
Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 acg caa gtg gaa gga
aaa ccc aaa tgt tgc ttc ttt aaa ttt agc tct 432 Thr Gln Val Glu Gly
Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 aag ata caa
tac aat aaa cta gta aag gcc caa ctg tgg ata tat ctg 480 Lys Ile Gln
Tyr Asn Lys Leu Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160
agg cct gtc aag act cct gcg aca gtg ttt gtg caa atc ctg aga ctc 528
Arg Pro Val Lys Thr Pro Ala Thr Val Phe Val Gln Ile Leu Arg Leu 165
170 175 atc aaa ccc atg aaa gac ggt aca agg tat act gga atc cga tct
ctg 576 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser
Leu 180 185 190 aaa ctt gac atg aac cca ggc act ggt att tgg cag agc
att gat gtg 624 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser
Ile Asp Val 195 200 205 aag aca gtg ttg cag aac tgg ctc aaa caa cct
gaa tcc aac tta ggc 672 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro
Glu Ser Asn Leu Gly 210 215 220 att gaa atc aaa gct tta gat gag aat
ggc cat gat ctt gct gta acc 720 Ile Glu Ile Lys Ala Leu Asp Glu Asn
Gly His Asp Leu Ala Val Thr 225 230 235 240 ttc cca gaa cca gga gaa
gat gga ctg act ccc ttt tta gaa gtc aag 768 Phe Pro Glu Pro Gly Glu
Asp Gly Leu Thr Pro Phe Leu Glu Val Lys 245 250 255 gta aca gac aca
cca aaa aga tct agg aga gat ttt ggg ctt gat tgt 816 Val Thr Asp Thr
Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265 270 gat gaa
cac tcc aca gaa tct cga tgc tgt cgt tac cct cta act gtg 864 Asp Glu
His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285
gat ttt gaa gct ttt gga tgg gat tgg att att gca cct aaa aga tat 912
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290
295 300 aag gcc aat tac tgc tct gga gaa tgt gaa ttt gta ttt ttg caa
aag 960 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val Phe Leu Gln
Lys 305 310 315 320 tat cct cat acc cat ctt gtg cac caa gca aac ccc
aga ggt tca gcc 1008 Tyr Pro His Thr His Leu Val His Gln Ala Asn
Pro Arg Gly Ser Ala 325 330 335 ggc ccc tgc tgt act cct aca aag atg
tct cca att aat atg cta tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys
Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 ttt aat ggc gaa gga caa
ata ata tac ggg aag att cca gcc atg gta 1104 Phe Asn Gly Glu Gly
Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 gta gat cgc
tgt ggg tgt tca tga 1128 Val Asp Arg Cys Gly Cys Ser 370 375 12 375
PRT Bovine 12 Met Gln Lys Leu Gln Ile Ser Val Tyr Ile Tyr Leu Phe
Met Leu Ile 1 5 10 15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser
Glu Gln Lys Glu Asn 20 25 30 Val Glu Lys Glu Gly Leu Cys Asn Ala
Cys Leu Trp Arg Glu Asn Thr 35 40 45 Thr Ser Ser Arg Leu Glu Ala
Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala
Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys
Ala Pro Pro Leu Leu Glu Leu Ile Asp Gln Phe Asp Val 85 90 95 Gln
Arg Asp Ala Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105
110 Ala Arg Thr Glu Thr Val Ile Thr Met Pro Thr Glu Ser Asp Leu Leu
115 120 125 Thr Gln Val Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe
Ser Ser 130 135 140 Lys Ile Gln Tyr Asn Lys Leu Val Lys Ala Gln Leu
Trp Ile Tyr Leu 145 150 155 160 Arg Pro Val Lys Thr Pro Ala Thr Val
Phe Val Gln Ile Leu Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly
Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn
Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val
Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile
Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230
235 240 Phe Pro Glu Pro Gly Glu Asp Gly Leu Thr Pro Phe Leu Glu Val
Lys 245 250 255 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly
Leu Asp Cys 260 265 270 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg
Tyr Pro Leu Thr Val 275 280 285 Asp Phe Glu Ala Phe Gly Trp Asp Trp
Ile Ile Ala Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr
His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro
Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350
Phe Asn Gly Glu Gly Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355
360 365 Val Asp Arg Cys Gly Cys Ser 370 375 13 1128 DNA Porcine CDS
(1)...(1125) 13 atg caa aaa ctg caa atc tat gtt tat att tac ctg ttt
atg ctg att 48 Met Gln Lys Leu Gln Ile Tyr Val Tyr Ile Tyr Leu Phe
Met Leu Ile 1 5 10 15 gtt gct ggt ccc gtg gat ctg aat gag aac agc
gag caa aag gaa aat 96 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser
Glu Gln Lys Glu Asn 20 25 30 gtg gaa aaa gag ggg ctg tgt aat gca
tgt atg tgg aga caa aac act 144 Val Glu Lys Glu Gly Leu Cys Asn Ala
Cys Met Trp Arg Gln Asn Thr 35 40 45 aaa tct tca aga cta gaa gcc
ata aaa att caa atc ctc agt aaa ctt 192 Lys Ser Ser Arg Leu Glu Ala
Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 cgc ctg gaa aca gct
cct aac att agc aaa gat gct ata aga caa ctt 240 Arg Leu Glu Thr Ala
Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80 ttg ccc aaa
gct cct cca ctc cgg gaa ctg att gat cag tac gat gtc 288 Leu Pro Lys
Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 cag
aga gat gac agc agt gat ggc tcc ttg gaa gat gat gat tat cac 336 Gln
Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105
110 gct acg acg gaa acg atc att acc atg cct aca gag tct gat ctt cta
384 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Leu Leu
115 120 125 atg caa gtg gaa gga aaa ccc aaa tgc tgc ttc ttt aaa ttt
agc tct 432 Met Gln Val Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe
Ser Ser 130 135 140 aaa ata caa tac aat aaa gta gta aag gcc caa ctg
tgg ata tat ctg 480 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu
Trp Ile Tyr Leu 145 150 155 160 aga ccc gtc aag act cct aca aca gtg
ttt gtg caa atc ctg aga ctc 528 Arg Pro Val Lys Thr Pro Thr Thr Val
Phe Val Gln Ile Leu Arg Leu 165 170 175 atc aaa ccc atg aaa gac ggt
aca agg tat act gga atc cga tct ctg 576 Ile Lys Pro Met Lys Asp Gly
Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 aaa ctt gac atg aac
cca ggc act ggt att tgg cag agc att gat gtg 624 Lys Leu Asp Met Asn
Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 aag aca gtg
ttg caa aat tgg ctc aaa caa cct gaa tcc aac tta ggc 672 Lys Thr Val
Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 att
gaa atc aaa gct tta gat gag aat ggt cat gat ctt gct gta acc 720 Ile
Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230
235 240 ttc cca gga cca gga gaa gat ggg ctg aat ccc ttt tta gaa gtc
aag 768 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val
Lys 245 250 255 gta aca gac aca cca aaa aga tcc agg aga gat ttt gga
ctc gac tgt 816 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly
Leu Asp Cys 260 265 270 gat gag cac tca aca gaa tct cga tgc tgt cgt
tac cct cta act gtg 864 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg
Tyr Pro Leu Thr Val 275 280 285 gat ttt gaa gct ttt gga tgg gac tgg
att att gca ccc aaa aga tat 912 Asp Phe Glu Ala Phe Gly Trp Asp Trp
Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aag gcc aat tac tgc tct gga
gag tgt gaa ttt gta ttt tta caa aaa 960 Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 tac cct cac act
cat ctt gtg cac caa gca aac ccc aga ggt tca gca 1008 Tyr Pro His
Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc
ccc tgc tgt act ccc aca aag atg tct cca atc aat atg cta tat 1056
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340
345 350 ttt aat ggc aaa gaa caa ata ata tat ggg aaa att cca gcc atg
gta 1104 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala
Met Val 355 360 365 gta gat cgc tgt ggg tgc tca tga 1128 Val Asp
Arg Cys Gly Cys Ser 370 375 14 375 PRT Porcine 14 Met Gln Lys Leu
Gln Ile Tyr Val Tyr Ile Tyr Leu Phe Met Leu Ile 1 5 10 15 Val Ala
Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30
Val Glu Lys Glu Gly Leu Cys Asn Ala Cys Met Trp Arg Gln Asn Thr 35
40 45 Lys Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys
Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile
Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile
Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser Asp Gly Ser
Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu Thr Ile Ile
Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 Met Gln Val Glu Gly
Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140 Lys Ile Gln
Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160
Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165
170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser
Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser
Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro
Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu Asp Glu Asn
Gly His Asp Leu Ala Val Thr
225 230 235 240 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu
Glu Val Lys 245 250 255 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp
Phe Gly Leu Asp Cys 260 265 270 Asp Glu His Ser Thr Glu Ser Arg Cys
Cys Arg Tyr Pro Leu Thr Val 275 280 285 Asp Phe Glu Ala Phe Gly Trp
Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys
Ser Gly Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 Tyr Pro
His Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340
345 350 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met
Val 355 360 365 Val Asp Arg Cys Gly Cys Ser 370 375 15 1128 DNA
Ovine CDS (1)...(1125) 15 atg caa aaa ctg caa atc ttt gtt tat att
tac cta ttt atg ctg ctt 48 Met Gln Lys Leu Gln Ile Phe Val Tyr Ile
Tyr Leu Phe Met Leu Leu 1 5 10 15 gtt gct ggc cca gtg gat ctg aat
gag aac agc gag cag aag gaa aat 96 Val Ala Gly Pro Val Asp Leu Asn
Glu Asn Ser Glu Gln Lys Glu Asn 20 25 30 gtg gaa aaa aag ggg ctg
tgt aat gca tgc ttg tgg aga caa aac aat 144 Val Glu Lys Lys Gly Leu
Cys Asn Ala Cys Leu Trp Arg Gln Asn Asn 35 40 45 aaa tcc tca aga
cta gaa gcc ata aaa atc caa atc ctc agt aag ctt 192 Lys Ser Ser Arg
Leu Glu Ala Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 cgc ctg
gaa aca gct cct aac atc agc aaa gat gct ata aga caa ctt 240 Arg Leu
Glu Thr Ala Pro Asn Ile Ser Lys Asp Ala Ile Arg Gln Leu 65 70 75 80
ttg ccc aag gct cct cca ctc cgg gaa ctg att gat cag tac gat gtc 288
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val 85
90 95 cag aga gat gac agc agc gac ggc tcc ttg gaa gac gat gac tac
cac 336 Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr
His 100 105 110 gtt acg acg gaa acg gtc att acc atg ccc acg gag tct
gat ctt cta 384 Val Thr Thr Glu Thr Val Ile Thr Met Pro Thr Glu Ser
Asp Leu Leu 115 120 125 gca gaa gtg caa gaa aaa ccc aaa tgt tgc ttc
ttt aaa ttt agc tct 432 Ala Glu Val Gln Glu Lys Pro Lys Cys Cys Phe
Phe Lys Phe Ser Ser 130 135 140 aag ata caa cac aat aaa gta gta aag
gcc caa ctg tgg ata tat ctg 480 Lys Ile Gln His Asn Lys Val Val Lys
Ala Gln Leu Trp Ile Tyr Leu 145 150 155 160 aga cct gtc aag act cct
aca aca gtg ttt gtg caa atc ctg aga ctc 528 Arg Pro Val Lys Thr Pro
Thr Thr Val Phe Val Gln Ile Leu Arg Leu 165 170 175 atc aaa ccc atg
aaa gac ggt aca agg tat act gga atc cga tct ctg 576 Ile Lys Pro Met
Lys Asp Gly Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 aaa ctt
gac atg aac cca ggc act ggt att tgg cag agc att gat gtg 624 Lys Leu
Asp Met Asn Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205
aag aca gtg ttg caa aac tgg ctc aaa caa cct gaa tcc aac tta ggc 672
Lys Thr Val Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210
215 220 att gaa atc aaa gct tta gat gag aat ggt cat gat ctt gct gta
acc 720 Ile Glu Ile Lys Ala Leu Asp Glu Asn Gly His Asp Leu Ala Val
Thr 225 230 235 240 ttc cca gaa cca gga gaa gaa gga ctg aat cct ttt
tta gaa gtc aag 768 Phe Pro Glu Pro Gly Glu Glu Gly Leu Asn Pro Phe
Leu Glu Val Lys 245 250 255 gta aca gac aca cca aaa aga tct agg aga
gat ttt ggg ctt gat tgt 816 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg
Asp Phe Gly Leu Asp Cys 260 265 270 gat gag cac tcc aca gaa tct cga
tgc tgt cgt tac cct cta act gtg 864 Asp Glu His Ser Thr Glu Ser Arg
Cys Cys Arg Tyr Pro Leu Thr Val 275 280 285 gat ttt gaa gct ttt gga
tgg gat tgg att att gca cct aaa aga tat 912 Asp Phe Glu Ala Phe Gly
Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aag gcc aat tac
tgc tct gga gaa tgt gaa ttt tta ttt ttg caa aag 960 Lys Ala Asn Tyr
Cys Ser Gly Glu Cys Glu Phe Leu Phe Leu Gln Lys 305 310 315 320 tat
cct cat acc cat ctt gtg cac caa gca aac ccc aaa ggt tca gcc 1008
Tyr Pro His Thr His Leu Val His Gln Ala Asn Pro Lys Gly Ser Ala 325
330 335 ggc cct tgc tgt act cct aca aag atg tct cca att aat atg cta
tat 1056 Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met
Leu Tyr 340 345 350 ttt aat ggc aaa gaa caa ata ata tat ggg aag att
cca ggc atg gta 1104 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys
Ile Pro Gly Met Val 355 360 365 gta gat cgc tgt ggg tgc tca tga
1128 Val Asp Arg Cys Gly Cys Ser 370 375 16 375 PRT Ovine 16 Met
Gln Lys Leu Gln Ile Phe Val Tyr Ile Tyr Leu Phe Met Leu Leu 1 5 10
15 Val Ala Gly Pro Val Asp Leu Asn Glu Asn Ser Glu Gln Lys Glu Asn
20 25 30 Val Glu Lys Lys Gly Leu Cys Asn Ala Cys Leu Trp Arg Gln
Asn Asn 35 40 45 Lys Ser Ser Arg Leu Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys Leu 50 55 60 Arg Leu Glu Thr Ala Pro Asn Ile Ser Lys
Asp Ala Ile Arg Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Arg
Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Val Thr Thr Glu
Thr Val Ile Thr Met Pro Thr Glu Ser Asp Leu Leu 115 120 125 Ala Glu
Val Gln Glu Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140
Lys Ile Gln His Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145
150 155 160 Arg Pro Val Lys Thr Pro Thr Thr Val Phe Val Gln Ile Leu
Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Leu
Asp Glu Asn Gly His Asp Leu Ala Val Thr 225 230 235 240 Phe Pro Glu
Pro Gly Glu Glu Gly Leu Asn Pro Phe Leu Glu Val Lys 245 250 255 Val
Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265
270 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
275 280 285 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys
Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Leu
Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val His Gln
Ala Asn Pro Lys Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu
Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val 355 360 365 Val Asp Arg
Cys Gly Cys Ser 370 375 17 1128 DNA Meleagris gallopavo CDS
(1)...(1125) 17 atg caa aag cta gca gtc tat gtt tat att tac ctg ttc
atg cag att 48 Met Gln Lys Leu Ala Val Tyr Val Tyr Ile Tyr Leu Phe
Met Gln Ile 1 5 10 15 tta gtt cat ccg gtg gct ctt gat ggc agt agt
cag ccc aca gag aac 96 Leu Val His Pro Val Ala Leu Asp Gly Ser Ser
Gln Pro Thr Glu Asn 20 25 30 gct gaa aaa gac gga ctg tgc aat gct
tgc acg tgg aga cag aat act 144 Ala Glu Lys Asp Gly Leu Cys Asn Ala
Cys Thr Trp Arg Gln Asn Thr 35 40 45 aaa tcc tcc aga ata gaa gcc
ata aaa att caa atc ctc agc aaa ctg 192 Lys Ser Ser Arg Ile Glu Ala
Ile Lys Ile Gln Ile Leu Ser Lys Leu 50 55 60 cgc ctg gaa caa gca
cct aac att agc agg gac gtt att aaa caa ctt 240 Arg Leu Glu Gln Ala
Pro Asn Ile Ser Arg Asp Val Ile Lys Gln Leu 65 70 75 80 tta ccc aaa
gct cct ccg ctg cag gaa ctg att gat cag tat gac gtc 288 Leu Pro Lys
Ala Pro Pro Leu Gln Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 cag
aga gac gac agt agc gat ggc tct ttg gaa gac gat gac tat cat 336 Gln
Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105
110 gcc aca acc gaa acg att atc aca atg cct acg gag tct gat ttt ctt
384 Ala Thr Thr Glu Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu
115 120 125 gta caa atg gag gga aaa cca aaa tgt tgc ttc ttt aag ttt
agc tct 432 Val Gln Met Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe
Ser Ser 130 135 140 aaa ata caa tat aac aaa gta gta aag gca caa tta
tgg ata tac ttg 480 Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu
Trp Ile Tyr Leu 145 150 155 160 agg caa gtc caa aaa cct aca acg gtg
ttt gtg cag atc ctg aga ctc 528 Arg Gln Val Gln Lys Pro Thr Thr Val
Phe Val Gln Ile Leu Arg Leu 165 170 175 att aaa ccc atg aaa gac ggt
aca aga tat act gga att cga tct ttg 576 Ile Lys Pro Met Lys Asp Gly
Thr Arg Tyr Thr Gly Ile Arg Ser Leu 180 185 190 aaa ctt gac atg aac
cca ggc act ggt atc tgg cag agt att gat gtg 624 Lys Leu Asp Met Asn
Pro Gly Thr Gly Ile Trp Gln Ser Ile Asp Val 195 200 205 aag aca gtg
ttg caa aat tgg ctc aaa cag cct gaa tcc aat tta ggc 672 Lys Thr Val
Leu Gln Asn Trp Leu Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 atc
gaa ata aaa gct ttt gat gag aat gga cga gat ctt gct gta aca 720 Ile
Glu Ile Lys Ala Phe Asp Glu Asn Gly Arg Asp Leu Ala Val Thr 225 230
235 240 ttc cca gga cca ggt gaa gat gga ctg aac cca ttt tta gag gtc
aga 768 Phe Pro Gly Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val
Arg 245 250 255 gtt aca gac aca cca aaa cgg tcc cgc aga gat ttt ggc
ctt gac tgc 816 Val Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly
Leu Asp Cys 260 265 270 gac gag cac tca acg gaa tct cga tgt tgt cgc
tac ccg ctg aca gtg 864 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg
Tyr Pro Leu Thr Val 275 280 285 gat ttt gaa gct ttt gga tgg gac tgg
att ata gca cct aaa aga tac 912 Asp Phe Glu Ala Phe Gly Trp Asp Trp
Ile Ile Ala Pro Lys Arg Tyr 290 295 300 aaa gcc aat tac tgc tct gga
gaa tgt gaa ttc gta ttt cta cag aaa 960 Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu Phe Val Phe Leu Gln Lys 305 310 315 320 tac ccg cac act
cac ctg gta cac caa gca aat cca aga ggc tca gca 1008 Tyr Pro His
Thr His Leu Val His Gln Ala Asn Pro Arg Gly Ser Ala 325 330 335 ggc
cct tgc tgc aca ccc acc aag atg tcc cct ata aac atg ctg tat 1056
Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 340
345 350 ttc aat gga aaa gaa caa ata ata tat gga aag ata cca gcc atg
gtt 1104 Phe Asn Gly Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ala
Met Val 355 360 365 gta gat cgt tgc ggg tgc tca tga 1128 Val Asp
Arg Cys Gly Cys Ser 370 375 18 375 PRT Meleagris gallopavo 18 Met
Gln Lys Leu Ala Val Tyr Val Tyr Ile Tyr Leu Phe Met Gln Ile 1 5 10
15 Leu Val His Pro Val Ala Leu Asp Gly Ser Ser Gln Pro Thr Glu Asn
20 25 30 Ala Glu Lys Asp Gly Leu Cys Asn Ala Cys Thr Trp Arg Gln
Asn Thr 35 40 45 Lys Ser Ser Arg Ile Glu Ala Ile Lys Ile Gln Ile
Leu Ser Lys Leu 50 55 60 Arg Leu Glu Gln Ala Pro Asn Ile Ser Arg
Asp Val Ile Lys Gln Leu 65 70 75 80 Leu Pro Lys Ala Pro Pro Leu Gln
Glu Leu Ile Asp Gln Tyr Asp Val 85 90 95 Gln Arg Asp Asp Ser Ser
Asp Gly Ser Leu Glu Asp Asp Asp Tyr His 100 105 110 Ala Thr Thr Glu
Thr Ile Ile Thr Met Pro Thr Glu Ser Asp Phe Leu 115 120 125 Val Gln
Met Glu Gly Lys Pro Lys Cys Cys Phe Phe Lys Phe Ser Ser 130 135 140
Lys Ile Gln Tyr Asn Lys Val Val Lys Ala Gln Leu Trp Ile Tyr Leu 145
150 155 160 Arg Gln Val Gln Lys Pro Thr Thr Val Phe Val Gln Ile Leu
Arg Leu 165 170 175 Ile Lys Pro Met Lys Asp Gly Thr Arg Tyr Thr Gly
Ile Arg Ser Leu 180 185 190 Lys Leu Asp Met Asn Pro Gly Thr Gly Ile
Trp Gln Ser Ile Asp Val 195 200 205 Lys Thr Val Leu Gln Asn Trp Leu
Lys Gln Pro Glu Ser Asn Leu Gly 210 215 220 Ile Glu Ile Lys Ala Phe
Asp Glu Asn Gly Arg Asp Leu Ala Val Thr 225 230 235 240 Phe Pro Gly
Pro Gly Glu Asp Gly Leu Asn Pro Phe Leu Glu Val Arg 245 250 255 Val
Thr Asp Thr Pro Lys Arg Ser Arg Arg Asp Phe Gly Leu Asp Cys 260 265
270 Asp Glu His Ser Thr Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val
275 280 285 Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys
Arg Tyr 290 295 300 Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu Phe Val
Phe Leu Gln Lys 305 310 315 320 Tyr Pro His Thr His Leu Val His Gln
Ala Asn Pro Arg Gly Ser Ala 325 330 335 Gly Pro Cys Cys Thr Pro Thr
Lys Met Ser Pro Ile Asn Met Leu Tyr 340 345 350 Phe Asn Gly Lys Glu
Gln Ile Ile Tyr Gly Lys Ile Pro Ala Met Val 355 360 365 Val Asp Arg
Cys Gly Cys Ser 370 375 19 1125 DNA Danio rerio CDS (1)...(1122) 19
atg cat ttt aca cag gtt tta att tct cta agt gta tta att gca tgt 48
Met His Phe Thr Gln Val Leu Ile Ser Leu Ser Val Leu Ile Ala Cys 1 5
10 15 ggt cca gtg ggt tat gga gat ata acg gcg cac cag cag cct tcc
aca 96 Gly Pro Val Gly Tyr Gly Asp Ile Thr Ala His Gln Gln Pro Ser
Thr 20 25 30 gcc acg gag gaa agc gag ctg tgt tcc aca tgt gag ttc
aga caa cac 144 Ala Thr Glu Glu Ser Glu Leu Cys Ser Thr Cys Glu Phe
Arg Gln His 35 40 45 agc aag ctg atg aga ctg cat gcc atc aag tcc
caa att ctt agc aaa 192 Ser Lys Leu Met Arg Leu His Ala Ile Lys Ser
Gln Ile Leu Ser Lys 50 55 60 ctc cga ctc aag cag gct cca aac atc
agc cgg gac gtg gtc aag cag 240 Leu Arg Leu Lys Gln Ala Pro Asn Ile
Ser Arg Asp Val Val Lys Gln 65 70 75 80 ctg tta ccc aaa gca ccg cct
ttg caa caa ctt ctg gat cag tac gat 288 Leu Leu Pro Lys Ala Pro Pro
Leu Gln Gln Leu Leu Asp Gln Tyr Asp 85 90 95 gtt tta gga gat gac
agt aag gat gga gct gtg gaa gag gac gat gaa 336 Val Leu Gly Asp Asp
Ser Lys Asp Gly Ala Val Glu Glu Asp Asp Glu 100 105 110 cat gcc acc
aca gag acc atc atg acc atg gcc aca gaa cct gac ccc 384 His Ala Thr
Thr Glu Thr Ile Met Thr Met Ala Thr Glu Pro Asp Pro 115 120 125 att
gtt caa gta gat cgg aaa ccg aag tgt tgc ttt ttc tcc ttc agt 432 Ile
Val Gln Val Asp Arg Lys Pro Lys Cys Cys Phe Phe Ser Phe Ser 130 135
140 ccg aag atc caa gcg aac cgg atc gta aga gcg cag ctc tgg gtt cat
480 Pro Lys Ile Gln Ala Asn Arg Ile Val Arg Ala Gln Leu Trp Val His
145 150 155 160 ctg aga ccg gcg gag gag gcg acc acc gtc ttc tta cag
ata tct cgg 528 Leu Arg Pro Ala Glu Glu Ala Thr Thr Val Phe Leu Gln
Ile Ser Arg 165 170 175 ctg atg ccc gtt aag gac gga gga aga cac cga
ata cga tcc ctg aaa 576 Leu Met Pro Val Lys Asp Gly Gly Arg His Arg
Ile Arg Ser Leu Lys 180 185
190 atc gac gtg aac gca gga gtc acg tct tgg cag agt ata gac gta aag
624 Ile Asp Val Asn Ala Gly Val Thr Ser Trp Gln Ser Ile Asp Val Lys
195 200 205 cag gtg ctc acg gtg tgg tta aaa caa ccg gag acc aac cga
ggc atc 672 Gln Val Leu Thr Val Trp Leu Lys Gln Pro Glu Thr Asn Arg
Gly Ile 210 215 220 gag att aac gca tat gac gcg aag gga aac gac ttg
gcc gtc act tca 720 Glu Ile Asn Ala Tyr Asp Ala Lys Gly Asn Asp Leu
Ala Val Thr Ser 225 230 235 240 acc gag act ggg gag gat gga ctg ctc
ccc ttt atg gag gtg aaa ata 768 Thr Glu Thr Gly Glu Asp Gly Leu Leu
Pro Phe Met Glu Val Lys Ile 245 250 255 tca gag ggc cca aaa cga atc
cgg agg gac tcc gga ctg gac tgc gat 816 Ser Glu Gly Pro Lys Arg Ile
Arg Arg Asp Ser Gly Leu Asp Cys Asp 260 265 270 gag aat tcc tca gag
tct cgc tgc tgc agg tac cct ctc act gtg gac 864 Glu Asn Ser Ser Glu
Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp 275 280 285 ttc gag gac
ttt ggc tgg gac tgg att att gct cca aaa cgc tat aag 912 Phe Glu Asp
Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys 290 295 300 gcg
aat tac tgt tca gga gaa tgc gac tac atg tac ctg cag aag tat 960 Ala
Asn Tyr Cys Ser Gly Glu Cys Asp Tyr Met Tyr Leu Gln Lys Tyr 305 310
315 320 ccc cac acc cat ctg gtg aac aag gcc agt ccg aga gga acg gct
ggg 1008 Pro His Thr His Leu Val Asn Lys Ala Ser Pro Arg Gly Thr
Ala Gly 325 330 335 ccc tgc tgc act ccc acc aag atg tct ccc atc aac
atg ctt tac ttt 1056 Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile
Asn Met Leu Tyr Phe 340 345 350 aac ggc aaa gag cag atc atc tac ggc
aag atc cct tcg atg gta gta 1104 Asn Gly Lys Glu Gln Ile Ile Tyr
Gly Lys Ile Pro Ser Met Val Val 355 360 365 gac cgc tgt ggc tgc tca
tga 1125 Asp Arg Cys Gly Cys Ser 370 20 374 PRT Danio rerio 20 Met
His Phe Thr Gln Val Leu Ile Ser Leu Ser Val Leu Ile Ala Cys 1 5 10
15 Gly Pro Val Gly Tyr Gly Asp Ile Thr Ala His Gln Gln Pro Ser Thr
20 25 30 Ala Thr Glu Glu Ser Glu Leu Cys Ser Thr Cys Glu Phe Arg
Gln His 35 40 45 Ser Lys Leu Met Arg Leu His Ala Ile Lys Ser Gln
Ile Leu Ser Lys 50 55 60 Leu Arg Leu Lys Gln Ala Pro Asn Ile Ser
Arg Asp Val Val Lys Gln 65 70 75 80 Leu Leu Pro Lys Ala Pro Pro Leu
Gln Gln Leu Leu Asp Gln Tyr Asp 85 90 95 Val Leu Gly Asp Asp Ser
Lys Asp Gly Ala Val Glu Glu Asp Asp Glu 100 105 110 His Ala Thr Thr
Glu Thr Ile Met Thr Met Ala Thr Glu Pro Asp Pro 115 120 125 Ile Val
Gln Val Asp Arg Lys Pro Lys Cys Cys Phe Phe Ser Phe Ser 130 135 140
Pro Lys Ile Gln Ala Asn Arg Ile Val Arg Ala Gln Leu Trp Val His 145
150 155 160 Leu Arg Pro Ala Glu Glu Ala Thr Thr Val Phe Leu Gln Ile
Ser Arg 165 170 175 Leu Met Pro Val Lys Asp Gly Gly Arg His Arg Ile
Arg Ser Leu Lys 180 185 190 Ile Asp Val Asn Ala Gly Val Thr Ser Trp
Gln Ser Ile Asp Val Lys 195 200 205 Gln Val Leu Thr Val Trp Leu Lys
Gln Pro Glu Thr Asn Arg Gly Ile 210 215 220 Glu Ile Asn Ala Tyr Asp
Ala Lys Gly Asn Asp Leu Ala Val Thr Ser 225 230 235 240 Thr Glu Thr
Gly Glu Asp Gly Leu Leu Pro Phe Met Glu Val Lys Ile 245 250 255 Ser
Glu Gly Pro Lys Arg Ile Arg Arg Asp Ser Gly Leu Asp Cys Asp 260 265
270 Glu Asn Ser Ser Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr Val Asp
275 280 285 Phe Glu Asp Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg
Tyr Lys 290 295 300 Ala Asn Tyr Cys Ser Gly Glu Cys Asp Tyr Met Tyr
Leu Gln Lys Tyr 305 310 315 320 Pro His Thr His Leu Val Asn Lys Ala
Ser Pro Arg Gly Thr Ala Gly 325 330 335 Pro Cys Cys Thr Pro Thr Lys
Met Ser Pro Ile Asn Met Leu Tyr Phe 340 345 350 Asn Gly Lys Glu Gln
Ile Ile Tyr Gly Lys Ile Pro Ser Met Val Val 355 360 365 Asp Arg Cys
Gly Cys Ser 370 21 4 PRT Artificial Sequence proteolytic cleavage
site 21 Arg Xaa Xaa Arg 1 22 4 PRT Eukaryotes SITE (0)...(0)
proteolytic processing site 22 Arg Ser Arg Arg 1 23 4 PRT
Eukaryotes SITE (0)...(0) proteolytic processing site 23 Arg Ile
Arg Arg 1 24 1393 DNA Homo sapiens CDS (54)...(1274) GDF-11 24
ccgcgggact ccggcgtccc cgccccccag tcctccctcc cctcccctcc agc atg 56
Met 1 gtg ctc gcg gcc ccg ctg ctg ctg ggc ttc ctg ctc ctc gcc ctg
gag 104 Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu
Glu 5 10 15 ctg cgg ccc cgg ggg gag gcg gcc gag ggc ccc gcg gcg gcg
gcg gcg 152 Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala Ala
Ala Ala 20 25 30 gcg gcg gcg gcg gcg gca gcg gcg ggg gtc ggg ggg
gag cgc tcc agc 200 Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly Gly
Glu Arg Ser Ser 35 40 45 cgg cca gcc ccg tcc gtg gcg ccc gag ccg
gac ggc tgc ccc gtg tgc 248 Arg Pro Ala Pro Ser Val Ala Pro Glu Pro
Asp Gly Cys Pro Val Cys 50 55 60 65 gtt tgg cgg cag cac agc cgc gag
ctg cgc cta gag agc atc aag tcg 296 Val Trp Arg Gln His Ser Arg Glu
Leu Arg Leu Glu Ser Ile Lys Ser 70 75 80 cag atc ttg agc aaa ctg
cgg ctc aag gag gcg ccc aac atc agc cgc 344 Gln Ile Leu Ser Lys Leu
Arg Leu Lys Glu Ala Pro Asn Ile Ser Arg 85 90 95 gag gtg gtg aag
cag ctg ctg ccc aag gcg ccg ccg ctg cag cag atc 392 Glu Val Val Lys
Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln Ile 100 105 110 ctg gac
cta cac gac ttc cag ggc gac gcg ctg cag ccc gag gac ttc 440 Leu Asp
Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp Phe 115 120 125
ctg gag gag gac gag tac cac gcc acc acc gag acc gtc att agc atg 488
Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser Met 130
135 140 145 gcc cag gag acg gac cca gca gta cag aca gat ggc agc cct
ctc tgc 536 Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro
Leu Cys 150 155 160 tgc cat ttt cac ttc agc ccc aag gtg atg ttc aca
aag gta ctg aag 584 Cys His Phe His Phe Ser Pro Lys Val Met Phe Thr
Lys Val Leu Lys 165 170 175 gcc cag ctg tgg gtg tac cta cgg cct gta
ccc cgc cca gcc aca gtc 632 Ala Gln Leu Trp Val Tyr Leu Arg Pro Val
Pro Arg Pro Ala Thr Val 180 185 190 tac ctg cag atc ttg cga cta aaa
ccc cta act ggg gaa ggg acc gca 680 Tyr Leu Gln Ile Leu Arg Leu Lys
Pro Leu Thr Gly Glu Gly Thr Ala 195 200 205 ggg gga ggg ggc gga ggc
cgg cgt cac atc cgt atc cgc tca ctg aag 728 Gly Gly Gly Gly Gly Gly
Arg Arg His Ile Arg Ile Arg Ser Leu Lys 210 215 220 225 att gag ctg
cac tca cgc tca ggc cat tgg cag agc atc gac ttc aag 776 Ile Glu Leu
His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe Lys 230 235 240 caa
gtg cta cac agc tgg ttc cgc cag cca cag agc aac tgg ggc atc 824 Gln
Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly Ile 245 250
255 gag atc aac gcc ttt gat ccc agt ggc aca gac ctg gct gtc acc tcc
872 Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr Ser
260 265 270 ctg ggg ccg gga gcc gag ggg ctg cat cca ttc atg gag ctt
cga gtc 920 Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu Leu
Arg Val 275 280 285 cta gag aac aca aaa cgt tcc cgg cgg aac ctg ggt
ctg gac tgc gac 968 Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu Gly
Leu Asp Cys Asp 290 295 300 305 gag cac tca agc gag tcc cgc tgc tgc
cga tat ccc ctc aca gtg gac 1016 Glu His Ser Ser Glu Ser Arg Cys
Cys Arg Tyr Pro Leu Thr Val Asp 310 315 320 ttt gag gct ttc ggc tgg
gac tgg atc atc gca cct aag cgc tac aag 1064 Phe Glu Ala Phe Gly
Trp Asp Trp Ile Ile Ala Pro Lys Arg Tyr Lys 325 330 335 gcc aac tac
tgc tcc ggc cag tgc gag tac atg ttc atg caa aaa tat 1112 Ala Asn
Tyr Cys Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys Tyr 340 345 350
ccg cat acc cat ttg gtg cag cag gcc aat cca aga ggc tct gct ggg
1160 Pro His Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala
Gly 355 360 365 ccc tgt tgt acc ccc acc aag atg tcc cca atc aac atg
ctc tac ttc 1208 Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn
Met Leu Tyr Phe 370 375 380 385 aat gac aag cag cag att atc tac ggc
aag atc cct ggc atg gtg gtg 1256 Asn Asp Lys Gln Gln Ile Ile Tyr
Gly Lys Ile Pro Gly Met Val Val 390 395 400 gat cgc tgt ggc tgc tct
taagtgggtc actacaagct gctggagcaa 1304 Asp Arg Cys Gly Cys Ser 405
agacttggtg ggtgggtaac ttaacctctt cacagaggat aaaaaatgct tgtgagtatg
1364 acagaaggga ataaacaggc ttaaagggt 1393 25 407 PRT Homo sapiens
25 Met Val Leu Ala Ala Pro Leu Leu Leu Gly Phe Leu Leu Leu Ala Leu
1 5 10 15 Glu Leu Arg Pro Arg Gly Glu Ala Ala Glu Gly Pro Ala Ala
Ala Ala 20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Val Gly
Gly Glu Arg Ser 35 40 45 Ser Arg Pro Ala Pro Ser Val Ala Pro Glu
Pro Asp Gly Cys Pro Val 50 55 60 Cys Val Trp Arg Gln His Ser Arg
Glu Leu Arg Leu Glu Ser Ile Lys 65 70 75 80 Ser Gln Ile Leu Ser Lys
Leu Arg Leu Lys Glu Ala Pro Asn Ile Ser 85 90 95 Arg Glu Val Val
Lys Gln Leu Leu Pro Lys Ala Pro Pro Leu Gln Gln 100 105 110 Ile Leu
Asp Leu His Asp Phe Gln Gly Asp Ala Leu Gln Pro Glu Asp 115 120 125
Phe Leu Glu Glu Asp Glu Tyr His Ala Thr Thr Glu Thr Val Ile Ser 130
135 140 Met Ala Gln Glu Thr Asp Pro Ala Val Gln Thr Asp Gly Ser Pro
Leu 145 150 155 160 Cys Cys His Phe His Phe Ser Pro Lys Val Met Phe
Thr Lys Val Leu 165 170 175 Lys Ala Gln Leu Trp Val Tyr Leu Arg Pro
Val Pro Arg Pro Ala Thr 180 185 190 Val Tyr Leu Gln Ile Leu Arg Leu
Lys Pro Leu Thr Gly Glu Gly Thr 195 200 205 Ala Gly Gly Gly Gly Gly
Gly Arg Arg His Ile Arg Ile Arg Ser Leu 210 215 220 Lys Ile Glu Leu
His Ser Arg Ser Gly His Trp Gln Ser Ile Asp Phe 225 230 235 240 Lys
Gln Val Leu His Ser Trp Phe Arg Gln Pro Gln Ser Asn Trp Gly 245 250
255 Ile Glu Ile Asn Ala Phe Asp Pro Ser Gly Thr Asp Leu Ala Val Thr
260 265 270 Ser Leu Gly Pro Gly Ala Glu Gly Leu His Pro Phe Met Glu
Leu Arg 275 280 285 Val Leu Glu Asn Thr Lys Arg Ser Arg Arg Asn Leu
Gly Leu Asp Cys 290 295 300 Asp Glu His Ser Ser Glu Ser Arg Cys Cys
Arg Tyr Pro Leu Thr Val 305 310 315 320 Asp Phe Glu Ala Phe Gly Trp
Asp Trp Ile Ile Ala Pro Lys Arg Tyr 325 330 335 Lys Ala Asn Tyr Cys
Ser Gly Gln Cys Glu Tyr Met Phe Met Gln Lys 340 345 350 Tyr Pro His
Thr His Leu Val Gln Gln Ala Asn Pro Arg Gly Ser Ala 355 360 365 Gly
Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu Tyr 370 375
380 Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly Lys Ile Pro Gly Met Val
385 390 395 400 Val Asp Arg Cys Gly Cys Ser 405 26 476 DNA Salmon-1
CDS (3)...(473) 26 gg cag ccg gag acg aat tgg ggg atc gag att aat
gcg ttc gac tcg 47 Gln Pro Glu Thr Asn Trp Gly Ile Glu Ile Asn Ala
Phe Asp Ser 1 5 10 15 aag gga aat gat ctg gcc gtt acc tca gca gaa
gcg gga gaa gga ctg 95 Lys Gly Asn Asp Leu Ala Val Thr Ser Ala Glu
Ala Gly Glu Gly Leu 20 25 30 caa ccc ttc atg gag gtg acg att tca
gag ggc ccg aag cgc tcc agg 143 Gln Pro Phe Met Glu Val Thr Ile Ser
Glu Gly Pro Lys Arg Ser Arg 35 40 45 aga gac tcg ggc ctg gac tgt
gac gag aac tcc ccc gag tcc cgc tgt 191 Arg Asp Ser Gly Leu Asp Cys
Asp Glu Asn Ser Pro Glu Ser Arg Cys 50 55 60 tgc cgc tac ccc ctc
acg gta gac ttt gaa gac ttt ggc tgg gac tgg 239 Cys Arg Tyr Pro Leu
Thr Val Asp Phe Glu Asp Phe Gly Trp Asp Trp 65 70 75 att att gcc
ccc aag cgc tac aag gcc aac tac tgc tct ggt gag tgt 287 Ile Ile Ala
Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys 80 85 90 95 gag
tac atg cac ctg cag aag tac ccc cac acc cac ctg gtg aac aag 335 Glu
Tyr Met His Leu Gln Lys Tyr Pro His Thr His Leu Val Asn Lys 100 105
110 gct aac cct cgc ggc acc gca ggg ccc tgc tgc acc ccc acc aag atg
383 Ala Asn Pro Arg Gly Thr Ala Gly Pro Cys Cys Thr Pro Thr Lys Met
115 120 125 tcc ccc atc aac atg ctc tac ttc aac cgc aaa gag cag atc
atc tac 431 Ser Pro Ile Asn Met Leu Tyr Phe Asn Arg Lys Glu Gln Ile
Ile Tyr 130 135 140 ggc aag atc ccc tcc atg gtg gtg gac cgt tgc gga
tgc tcg 473 Gly Lys Ile Pro Ser Met Val Val Asp Arg Cys Gly Cys Ser
145 150 155 tga 476 27 157 PRT Salmon-1 27 Gln Pro Glu Thr Asn Trp
Gly Ile Glu Ile Asn Ala Phe Asp Ser Lys 1 5 10 15 Gly Asn Asp Leu
Ala Val Thr Ser Ala Glu Ala Gly Glu Gly Leu Gln 20 25 30 Pro Phe
Met Glu Val Thr Ile Ser Glu Gly Pro Lys Arg Ser Arg Arg 35 40 45
Asp Ser Gly Leu Asp Cys Asp Glu Asn Ser Pro Glu Ser Arg Cys Cys 50
55 60 Arg Tyr Pro Leu Thr Val Asp Phe Glu Asp Phe Gly Trp Asp Trp
Ile 65 70 75 80 Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly
Glu Cys Glu 85 90 95 Tyr Met His Leu Gln Lys Tyr Pro His Thr His
Leu Val Asn Lys Ala 100 105 110 Asn Pro Arg Gly Thr Ala Gly Pro Cys
Cys Thr Pro Thr Lys Met Ser 115 120 125 Pro Ile Asn Met Leu Tyr Phe
Asn Arg Lys Glu Gln Ile Ile Tyr Gly 130 135 140 Lys Ile Pro Ser Met
Val Val Asp Arg Cys Gly Cys Ser 145 150 155 28 412 DNA Salmon-2 CDS
(2)...(409) 28 g gtt acc tca act gaa gcc gga gaa gga ctg caa ccc
ttc atg gag gtg 49 Val Thr Ser Thr Glu Ala Gly Glu Gly Leu Gln Pro
Phe Met Glu Val 1 5 10 15 aag att tcg gag ggc ccg aag cgc tcc agg
aga gat tcg ggc ctg gac 97 Lys Ile Ser Glu Gly Pro Lys Arg Ser Arg
Arg Asp Ser Gly Leu Asp 20 25 30 tgt gat gag aac tcc ccc gag tcc
cgc tgc tgc cgg tac ccc ctc acg 145 Cys Asp Glu Asn Ser Pro Glu Ser
Arg Cys Cys Arg Tyr Pro Leu Thr 35 40 45 gtg gac ttt gaa gac ttt
ggc tgg gac tgg att att gcc ccc aag cgc 193 Val Asp Phe Glu Asp Phe
Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 50 55 60 tac aag gcc aac
tac tgc tct ggt gag tgc gag tac atg cac ctg cag 241 Tyr Lys Ala Asn
Tyr Cys Ser Gly Glu Cys Glu Tyr Met His Leu Gln 65 70 75 80 aag tac
ccc cac acc cac ctg gtg aac aag gct aac cct cgc ggc acc 289 Lys Tyr
Pro His Thr His Leu Val Asn Lys Ala Asn Pro Arg Gly Thr 85 90 95
gcg ggg ccc tgc tgc acc ccc acc aag atg tcc ccc atc aac atg ctc 337
Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met Leu 100
105 110 tac ttc aac cgc
aaa gag cag atc atc tac ggc aag atc ccc tcc atg 385 Tyr Phe Asn Arg
Lys Glu Gln Ile Ile Tyr Gly Lys Ile Pro Ser Met 115 120 125 gtg gtg
gac cgc tgc ggc tgc tcg tga 412 Val Val Asp Arg Cys Gly Cys Ser 130
135 29 136 PRT Salmon-2 29 Val Thr Ser Thr Glu Ala Gly Glu Gly Leu
Gln Pro Phe Met Glu Val 1 5 10 15 Lys Ile Ser Glu Gly Pro Lys Arg
Ser Arg Arg Asp Ser Gly Leu Asp 20 25 30 Cys Asp Glu Asn Ser Pro
Glu Ser Arg Cys Cys Arg Tyr Pro Leu Thr 35 40 45 Val Asp Phe Glu
Asp Phe Gly Trp Asp Trp Ile Ile Ala Pro Lys Arg 50 55 60 Tyr Lys
Ala Asn Tyr Cys Ser Gly Glu Cys Glu Tyr Met His Leu Gln 65 70 75 80
Lys Tyr Pro His Thr His Leu Val Asn Lys Ala Asn Pro Arg Gly Thr 85
90 95 Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser Pro Ile Asn Met
Leu 100 105 110 Tyr Phe Asn Arg Lys Glu Gln Ile Ile Tyr Gly Lys Ile
Pro Ser Met 115 120 125 Val Val Asp Arg Cys Gly Cys Ser 130 135
* * * * *
References