U.S. patent application number 10/662438 was filed with the patent office on 2004-07-15 for metalloprotease activation of myostatin, and methods of modulating myostatin activity.
Invention is credited to Tomkinson, Kathy, Wolfman, Neil.
Application Number | 20040138118 10/662438 |
Document ID | / |
Family ID | 31999236 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040138118 |
Kind Code |
A1 |
Wolfman, Neil ; et
al. |
July 15, 2004 |
Metalloprotease activation of myostatin, and methods of modulating
myostatin activity
Abstract
It has been determined that metalloprotease cleavage of a
myostatin pro peptide results in activation of a latent inactive
myostatin to an active form. Accordingly, methods of identifying
agents that modulate metalloprotease mediated activation of
myostatin are provided, as are agents identified using such
methods. Also provided are methods of modulating muscle growth in
an organism by increasing or decreasing metalloprotease mediated
cleavage of a myostatin pro peptide.
Inventors: |
Wolfman, Neil; (Dover,
MA) ; Tomkinson, Kathy; (Cambridge, MA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW,
GARRETT & DUNNER, L.L.P.
1300 I Street, N.W.
Washington
DC
20005
US
|
Family ID: |
31999236 |
Appl. No.: |
10/662438 |
Filed: |
September 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60486863 |
Jul 10, 2003 |
|
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60439164 |
Jan 9, 2003 |
|
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60411133 |
Sep 16, 2002 |
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Current U.S.
Class: |
435/6.11 ;
514/19.3; 514/3.8; 514/4.8; 514/6.9; 514/8.8 |
Current CPC
Class: |
C07K 14/475 20130101;
A61P 3/04 20180101; A61P 3/00 20180101; A61P 21/04 20180101; A61P
7/00 20180101; A61P 43/00 20180101; A61P 35/00 20180101; A61P 37/04
20180101; A61P 21/00 20180101; A61K 38/00 20130101; A61P 3/10
20180101; A61P 31/18 20180101; C07K 2319/00 20130101; A61P 37/02
20180101 |
Class at
Publication: |
514/012 ;
514/013; 514/014 |
International
Class: |
A61K 038/16 |
Goverment Interests
[0002] This invention was made in part with government support
under Grant Nos. HD35887, AR47746, and GM63471 awarded by the
National Institutes of Health. The United States government has
certain rights in this invention.
Claims
What is claimed is:
1. An agent that modulates metalloprotease-mediated activation of
latent myostatin, said agent comprising a peptide, wherein said
peptide comprises a peptide portion of a myostatin polypeptide, or
a derivative of said peptide portion, wherein the derivative of the
peptide portion of the myostatin polypeptide comprises a peptide
having a mutation of a cleavage site for the metalloprotease.
2. The agent of claim 1, wherein said agent reduces or inhibits
metalloprotease-mediated activation of latent myostatin.
3. The agent of claim 1, wherein said agent increases
metalloprotease-mediated activation of latent myostatin.
4. The agent of claim 1, wherein the peptide has an amino acid
sequence of: KDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETIITMPT
(SEQ ID NO:11); QLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETI (SEQ ID
NO:14); APPLRELIDQYDVQRADSSDGSLEDDDYHA (SEQ ID NO:17);
ELIDQYDVQRADSSDGSLED (SEQ ID NO:20); or YDVQRADSSD (SEQ ID
NO:23).
5. An agent of claim 1, wherein said agent is operatively linked to
a second molecule.
6. An agent of claim 5, wherein the second molecule comprises a
detectable label.
7. An agent of claim 5, wherein the second molecule comprises a
heterologous polypeptide.
8. An agent of claim 7, wherein the heterologous polypeptide
stabilizes the peptide.
9. An agent of claim 7, wherein the heterologous polypeptide
comprises an Fc domain of an antibody.
10. An agent of claim 7, comprising a fusion protein.
11. An agent of claim 10, wherein said fusion protein comprises a
peptide having an amino acid sequence as set forth in SEQ ID NO:11;
SEQ ID NO:14; SEQ ID NO:17; SEQ ID NO:20; or SEQ ID NO:23.
12. An agent of claim 11, wherein said fusion protein comprises an
operatively linked Fc domain of an antibody molecule.
13. An agent of claim 1, wherein said metalloprotease is a bone
morphogenetic protein-1/tolloid (BMP-1/TLD) family member.
14. An agent of claim 13, wherein the BMP-1/TLD family member is
BMP-1, TLD, tolloid-like protein-1 (TLL-1), or tolloid-like protein
2 (TLL-2).
15. An agent of claim 14, wherein the BMP-1/TLD family member is
BMP-1, mammalian TLD (mTLD), mammalian TLL-1 (mTLL-1), or mammalian
TLL-2 (m-TLL-2).
16. A method of increasing muscle mass in a subject, said method
comprising administration of the agent of claim 1.
17. A method of treating a metabolic disorder in a subject, said
method comprising administration of the agent of claim 1.
18. The method of claim 17, wherein said metabolic disorder is a
muscle wasting disorder.
19. The method of claim 18, wherein said muscle wasting disorder is
associated with muscular dystrophy, including Duchenne muscular
dystrophy; cachexia, including cachexia associated with cancer or
acquired immune deficiency syndrome; or sarcopenia, including
age-related sarcopenia.
20. The method of claim 17, wherein said metabolic disorder is
diabetes.
21. The method of claim 17, wherein said metabolic disorder is
associated with obesity.
Description
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of U.S. Ser. No. 60/486,863, filed Jul. 10,
2003; U.S. Ser. No. 60/439,164, filed Jan. 9, 2003; and U.S. Ser.
No. 60/411,133, filed Sep. 16, 2002; the entire content of each
which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The invention relates generally to metalloprotease
regulation of myostatin activity, and more specifically to methods
of using agonists or antagonists of the BMP-1/TLD family of
metalloproteases to modulate myostatin activity including, for
example, to regulate muscle development in an organism, to methods
of identifying agonists and antagonists of such metalloproteases,
and to agonists and antagonists so identified.
[0005] 2. Background Information
[0006] Myostatin is a transforming growth factor-.beta.
(TGF-.beta.) family member that is essential for proper regulation
of skeletal muscle growth. Myostatin is a secreted protein that is
expressed specifically by cells of the skeletal muscle lineage
during embryonic development and in adult animals; low levels of
myostatin mRNA also are present in fat cells in adults animals.
During early embryogenesis, myostatin mRNA is detectable in the
myotome compartment of developing somites. At later embryonic
stages and in postnatal life, myostatin is expressed widely in all
skeletal muscles that have been examined.
[0007] The function of myostatin was elucidated by gene targeting
studies in mice. Mice lacking myostatin demonstrated a dramatic and
widespread increase in skeletal muscle mass due to muscle fiber
hyperplasia and hypertrophy, indicating that myostatin is a
negative regulator of muscle growth. The myostatin gene is highly
conserved across evolution, with the predicted mature myostatin
protein sequence being identical among mice, rats, humans,
chickens, turkeys, and pigs, and highly homologous even with
respect to aquatic organisms. The function of myostatin also is
conserved, with mutations in the myostatin gene correlating to the
double muscling phenotype in cattle.
[0008] The role of myostatin in regulating muscle growth and
development indicates that methods and compositions that regulate
myostatin activity can have a broad variety of applications,
including, for example, for treating human diseases and for
improving livestock production. With respect to human therapeutic
applications, inhibitors of myostatin expression or function can
provide a clinical benefit in the treatment of muscle wasting
disorders such as muscular dystrophy, cachexia, and sarcopenia. In
addition, myostatin deficient animals have a significant reduction
in fat accumulation, and the loss of myostatin is protective
against the development of obesity and type II diabetes in genetic
models in mice. As such, modulation of myostatin activity also can
be useful in the treatment of metabolic disorders such as obesity
and type II diabetes. Further in this respect, inhibitors of
myostatin expression or function not only can be useful for
increasing the efficiency of livestock production, but also can
result in the production of meat with a lower fat content.
[0009] Various strategies for manipulating the biological
activities of myostatin have been described. Myostatin is
synthesized as a precursor protein that undergoes proteolytic
processing to generate an N-terminal fragment termed the "pro
peptide" and a C-terminal fragment, a disulfide-linked dimer of
which is the biologically active species. Currently described
strategies for inhibiting myostatin activity have utilized
molecules that can bind the myostatin C-terminal dimer and inhibit
its activity. For example, myostatin binds two activin type II
receptors, Act RIIA and Act RIIB, in vitro, and expression of a
truncated dominant negative form of Act RIIB in transgenic mice
resulted in the mice having increases in muscle mass comparable to
that of transgenic myostatin knock out mice.
[0010] The myostatin pro peptide also has been used to inhibit
myostatin activity. Following proteolytic processing, the myostatin
pro peptide remains non-covalently associated with the C-terminal
dimer and maintains the dimer in a latent, inactive state. The pro
peptide has been shown to block the activity of the purified
myostatin C-terminal dimer in various in vitro assays, and
overexpression of the pro peptide in transgenic mice resulted in a
phenotype characteristic of the myostatin null mutation.
Follistatin is another protein that acts as a myostatin inhibitor.
Follistatin can bind and inhibit the activity of a variety of
TGF-.beta. family members, including myostatin, and transgenic mice
overexpressing follistatin in muscle have dramatic increases in
muscle growth, consistent with inhibition of myostatin
activity.
[0011] The above described inhibitors of myostatin each
specifically interact with mature myostatin to inhibit its
activity. While inhibiting the activity of a protein such as
myostatin using an agent that directly interacts with the protein
provides great specificity, such a method can require that all or
most of the proteins be bound by the agent for the inhibitory
effect to be manifest. An alternative way to inhibit the activity
of a protein, particularly a protein that, itself must be activated
by a second protein such as an enzyme in order for the first
protein to be functional, is to target the second protein. Such a
method can be advantageous because activating proteins such as
enzymes generally are present at much lower levels than their
substrates. As such, there is a greater likelihood that all or most
of an activating protein such as an enzyme can be inhibited.
[0012] With respect to myostatin, at least two proteases are known
to be involved in processing promyostatin, the primary gene
product, into a signal peptide, a pro peptide and a C-terminal
fragment, the latter of which forms homodimers that have biological
myostatin activity. Unfortunately, these proteases also can act on
a variety of other proteins and, therefore, agents that target and
inhibit these proteases, for example, signal peptidase, likely
would have diverse and deleterious effects if administered to a
living organism. Thus, a need exists to identify biological
molecules that are more specifically involved in regulating
myostatin activation and activity. The present invention satisfies
this need and provides additional advantages.
SUMMARY OF THE INVENTION
[0013] The present invention is based on the identification of
proteases that cleave myostatin pro peptide, including when the
myostatin pro peptide is present in a complex with a myostatin
C-terminal dimer. As such, the proteases can convert a latent
inactive myostatin complex, which comprises a myostatin pro peptide
associated with a C-terminal myostatin polypeptide, to active
myostatin, which is a negative regulator of muscle growth and
development. Such proteases, which are exemplified by the
metalloprotease bone morphogenic protein-1/tolloid (BMP-1/TLD)
family of proteins, provide targets for drugs that can increase or
decrease the protease activity and, therefore, increase or decrease
myostatin activity. Accordingly, the present invention provides
agents that modulate metalloprotease mediated myostatin pro peptide
cleavage and activation of myostatin, as well as methods of using
such agents, for example, to modulate myostatin activity in an
organism. Methods of identifying such agents also are provided.
[0014] The present invention relates to a method of modulating
myostatin activation. Such a method can be performed, for example,
by contacting a latent myostatin complex, which includes a
myostatin pro peptide and a myostatin C-terminal fragment,
particularly a C-terminal fragment dimer, with a metalloprotease
that can cleave the myostatin pro peptide, and with an agent that
can increase or decrease proteolytic cleavage of the pro peptide by
the metalloprotease, thereby modulating myostatin activation. The
metalloprotease can be any metalloprotease that can cleave the
myostatin pro peptide, particularly when the pro peptide comprises
a latent myostatin complex, including, for example, a BMP-1/TLD
family member such as BMP-1, TLD, tolloid-like protein-1 (TLL-1),
or tolloid-like protein-2 (TLL-2), particularly mammalian BMP-1/TLD
family members such as mammalian (m) TLD (mTLD), mTLL-1, and
mTLL-2.
[0015] A method of the invention can be used to increase the level
of myostatin activation (i.e., above a baseline level of myostatin
activation in the absence of an agent), for example, by contacting
a latent myostatin complex and metalloprotease with an agent that
increases proteolytic cleavage of the pro peptide by the
metalloprotease; or can be used to decrease the level of myostatin
activation (below a baseline level), for example, by contacting a
latent myostatin complex and metalloprotease with an agent that
decreases proteolytic cleavage of the pro peptide by the
metalloprotease. The method can be performed in vitro, using, for
example, cells or a tissue in culture, a cell extract, or
substantially purified reagents, including substantially purified
metalloprotease and/or latent myostatin complex; or can be
performed in vivo, for example, in a cell or tissue, either of
which can be in situ in an organism or isolated from an organism
(e.g., a cell ex vivo, which can be in culture). Thus, the method
can be performed by contacting a sample comprising a latent
myostatin complex and metalloprotease (e.g., a tissue sample and/or
a biological fluid) with an agent in vitro, or the contacting can
be performed in vivo, for example, by administering the agent to a
subject.
[0016] Free myostatin pro peptide, latent myostatin complex, and a
metalloprotease that can cleave a myostatin pro peptide can be
present intracellularly or extracellularly. However, the pro
peptide or latent myostatin complex generally is not present in the
same cells or cell type as the metalloprotease and, therefore,
cleavage of myostatin pro peptide by the metalloprotease generally
occurs extracellularly upon contact of the metalloprotease with the
pro peptide. As such, contacting of an agent with the pro peptide,
complex, and/or metalloprotease will depend in part on how the
agent acts to modulate the cleavage. For example, where the agent
can bind to and alter the conformation of the metalloprotease so as
to inhibit its cleavage activity with respect to a myostatin pro
peptide, cells that produce the metalloprotease can be contacted
with the agent such that the secreted metalloprotease lacks such
activity, or the agent can be administered to a medium into which
the metalloprotease is secreted (e.g., into the bloodstream of a
living organism) such that, upon contact with the agent in the
medium, the cleavage of the pro peptide by the metalloprotease is
reduced or inhibited. In comparison, where the agent acts, for
example, to destabilize an interaction of the metalloprotease and
the pro peptide, or where the agent acts as a competitive or
non-competitive inhibitor of the metalloprotease with respect to
the pro peptide, the agent generally is contacted with the medium
in which the metalloprotease and pro peptide are likely to interact
(e.g., the blood).
[0017] In one embodiment, the agent decreases proteolytic activity
of a metalloprotease that cleaves myostatin pro peptide from a
latent myostatin complex, thereby reducing or inhibiting myostatin
activation below a level of myostatin activation that occurs or
would occur in the absence of the agent. Where such an agent is
administered to a subject, the agent can result in increased muscle
mass or decreased fat content or both in the subject. The subject
can be any subject in which myostatin is expressed, particularly a
vertebrate organism, for example, animals that are raised as a food
source, such as a mammalian species (e.g., an ovine, porcine
species, or bovine species), avian species (e.g, chickens or a
turkeys), or a piscine species (e.g., salmon, trout, or cod). The
subject also can be a human subject, for example, a subject
suffering from a muscular disorder (e.g., a dystonia or dystrophy),
a subject suffering from wasting disorder (e.g., cachexia), or a
subject suffering from clinical obesity or other metabolic disorder
such as type II diabetes. In another embodiment, the agent
increases proteolytic activity of a metalloprotease that cleaves
myostatin pro peptide from a latent myostatin complex, thereby
increasing myostatin activation above a level, if any, of myostatin
activation that occurs or would occur in the absence of the agent.
Where such an agent is administered to a subject, the agent can
result in decreased muscle mass or increased fat content or both in
the subject.
[0018] The present invention also relates to a method of increasing
muscle mass in a subject. Such a method can be performed, for
example, by administering to the subject an agent that reduces or
inhibits proteolytic cleavage of a myostatin pro peptide by a
protease that cleaves myostatin pro peptide, thereby preventing
activation of latent myostatin in the cell and increasing muscle
mass in the subject. The metalloprotease can be any
metalloprotease, particularly a BMP-1/TLD family member such as
BMP-1, TLD, TLL-1, or TLL-2, including mTLD, mTLL-1 and mTLL-2. The
subject in which muscle mass is to be increased generally is
vertebrate, for example, a domesticated or farm animal, including a
mammal such as an ovine species, a porcine species, or a bovine
species; an avian species such as a chicken or a turkey; or a
piscine species; or can be a human subject.
[0019] The present invention further relates to a method for
ameliorating a metabolic disorder in a subject. Such a method can
be performed, for example, by administering to the subject an agent
that reduces or inhibits the proteolytic cleavage of a myostatin
pro peptide by a protease that cleaves myostatin pro peptide,
thereby preventing activation of latent myostatin in the cell and
ameliorating the metabolic disorder. The metabolic disorder can be
any such disorder associated with increased or undesirable
myostatin activation or activity, including, for example, a muscle
wasting disorder such as is associated with muscular dystrophy,
cachexia (e.g., associated with a cancer or acquired
immunodeficiency disease), or sarcopenia; or a metabolic disorder
such as clinical obesity or type 2 diabetes. The subject in which
the metabolic disorder is ameliorated can be any subject, and
generally is a vertebrate subject, for example, a domesticated
animal such as a cat or dog, or an animal raised as a source of
food (e.g., cattle, sheep, pigs, or fish); or can be a human
subject. Amelioration of the disorder can be identified using any
assay generally used to monitor the particular metabolic disorder,
for example, a glucose tolerance test for diabetes, or a serum
leptin assay for body fat analysis.
[0020] The present invention also relates to a method of
identifying an agent that modulates metalloprotease mediated
myostatin pro peptide cleavage and activation of latent myostatin.
Such a screening method can be performed, for example, by
contacting a myostatin pro peptide, a metalloprotease that can
cleave the myostatin pro peptide, and a test agent, under
conditions sufficient for cleavage of the pro peptide by the
metalloprotease; and detecting a change in the amount of cleavage
of the pro peptide in the absence of the test agent as compared to
the presence of the test agent, thereby identifying the test agent
as an agent that modulates metalloprotease mediated activation of
the latent myostatin. The myostatin pro peptide can be in an
isolated form, or can be a component of a latent myostatin complex
that further contains a myostatin C-terminal fragment or a
myostatin C-terminal dimer.
[0021] Where a test agent is identified as having metalloprotease
mediated myostatin modulating activity, a screening assay of the
invention can further include a step of determining an amount by
which the agent increases or decreases myostatin pro peptide
cleavage or myostatin activation. For example, where an agent is
identified that increases the proteolytic activity of the
metalloprotease above a basal level in a cell, a method of the
invention can further include determining an amount by which the
agent increases myostatin activation above the basal level. As
such, a method of the invention provides a means to obtain agents
or panels of agents that variously modulate myostatin activation by
a metalloprotease. Such a method further provides a means to
determine amounts of a particular agent useful for providing a
desired level of myostatin activity.
[0022] A difference in the amount of cleavage of the pro peptide
due to contact with a test agent can be detected, for example, by
detecting the pro peptide or a cleavage product of the pro peptide
using a method such as electrophoresis, chromatography, or mass
spectrometry, which can detect a myostatin pro peptide or cleavage
product thereof based on its size, charge, or both; an
immunological based assay such as an immunoblot analysis, an
enzyme-linked immunosorption assay (ELISA), or the like, which
utilizes an antibody specific for the intact pro peptide or the
cleaved pro peptide, but not an antibody that binds both the intact
and the cleaved pro peptide; or a fluorescence based assay,
including, for example, a fluorescence resonance energy transfer
(FRET) assay, wherein fluorescence of the intact pro peptide is
quenched, and the quenching is relieved upon cleavage of the pro
peptide. Depending on the relative amount of intact myostatin pro
peptide, pro peptide cleavage product, or a combination thereof
that is detected, a test agent can be identified as an agent that
increases or decreases metalloprotease mediated myostatin pro
peptide cleavage and activation of the latent myostatin.
[0023] A difference in the amount of cleavage of the pro peptide
also can be detected by detecting a change in binding of myostatin
to a myostatin receptor in vitro or expressed on a cell surface, or
by detecting a change in a myostatin mediated signal transduction
in a cell expressing a myostatin receptor. Where the assay is a
cell based assay, the cell can be one that expresses an endogenous
myostatin receptor, for example, L6 myocytes, or can be a cell
expressing a transgene encoding the myostatin receptor, for
example, a cell transfected with a polynucleotide encoding an
activin receptor such as an activin type II receptor. Myostatin
mediated signal transduction can be detected at any level in the
signal transduction pathway, including from binding of myostatin to
a cell surface receptor to expression of a gene that is regulated
due to myostatin binding to a myostatin receptor, wherein, in a
screening assay of the invention, the signal transduction is
dependent on metalloprotease mediated cleavage of a myostatin pro
peptide and activation of a latent myostatin complex. As such,
myostatin mediated signal transduction can be detected by detecting
myostatin binding to a myostatin receptor using a receptor binding
assay, or by detecting expression of a myostatin regulated gene,
including, for example, a reporter gene, which can comprise, for
example, a TGF-.beta. regulatory element operatively linked to a
polynucleotide encoding a detectable polypeptide. Accordingly, the
present invention provides agents that modulate metalloprotease
mediated myostatin pro peptide cleavage and myostatin activation,
wherein the agents are identified using a screening assay of the
invention. The present methods also are useful for confirming that
an agent modulates metalloprotease mediated myostatin pro peptide
cleavage and myostatin activation, including, if desired, the
specific activity of the agent.
[0024] The present invention also relates to an agent that
modulates metalloprotease mediated activation of latent myostatin.
The agent can be an agonist or an antagonist of metalloprotease
mediated activation of latent myostatin, and can reduce or inhibit
metalloprotease mediated activation of latent myostatin, or can
increase metalloprotease mediated activation of latent myostatin.
An agent that modulates metalloprotease mediated activation of
latent myostatin can be any type of molecule, including, for
example, a peptide agent, a polynucleotide agent, an antibody
agent, or a small organic molecule agent.
[0025] An agent that modulates metalloprotease mediated activation
of latent myostatin is exemplified herein by a peptide agent. A
peptide agent can include, for example, a peptide portion of a
myostatin polypeptide, or a derivative of such a peptide portion of
myostatin. In one embodiment, a derivative of a peptide portion of
myostatin is a peptide that corresponds to a myostatin pro peptide.
In one aspect of this embodiment, the derivative is a pro peptide
having a mutation of the metalloprotease cleavage site, for
example, a substitution, deletion, or insertion of an amino acid at
or in sufficient proximity to the cleavage site such that the
metalloprotease has increased or decreased cleavage activity with
respect to the peptide agent. In another aspect of this embodiment,
the derivative of a peptide portion of myostatin is a peptide agent
that reduces or inhibits metalloprotease mediated activation of
latent myostatin. The agent that modulates metalloprotease mediated
activation of latent myostatin can be operatively linked to a
second molecule, which facilitates the action or activity of the
agent, or increases or decreases the stability of the agent in a
particular environment. For example, a peptide agent can be
stabilized by operatively linking the peptide agent to a
polypeptide such as an Fc domain of an antibody molecule, thereby
increasing the half-life of the peptide agent in vivo.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 demonstrates that incubation of the myostatin complex
(MSTN; C-terminal myostatin dimer and pro peptide) with mTLL-1
resulted in a dramatic increase in expression of a luciferase
reporter gene (stippled bar; see Example 2), the expression of
which is regulated in transfected rhabdomyosarcoma cells upon
contact of the cells with active myostatin. Only background
expression was observed in cells contacted with myostatin complex,
alone (solid bar), or with mTLL-1, alone (hatched bar).
[0027] FIG. 2 shows a standard curve generated using the luciferase
reporter assay, wherein the transfected cells (see FIG. 1, above)
were contacted with the specified amounts of active purified
C-terminal myostatin dimer (diamonds). Control luciferase activity
(no myostatin) is shown by the circles.
[0028] FIGS. 3A to 3E show determination of cleavage of the
myostatin pro peptide by BMP-1/TLD family of proteinases.
[0029] FIGS. 3A and 3B show detection of a pro peptide degradation
product in CHO cell conditioned media. Conditioned media prepared
from CHO cells expressing the pro peptide (FIG. 3A) or wild type
and mutant forms of pro peptide/Fc fusion proteins (FIG. 3B) were
analyzed by SDS-PAGE followed by western blot analysis using
antibodies directed against either the myostatin pro peptide (FIG.
3A) or IgG (FIG. 3B). Note that mutation of D76 to A resulted in
loss of the degradation product.
[0030] FIG. 3C shows purification of wild type and mutant pro
peptide/C-terminal dimer complexes. Protein complexes were analyzed
by SDS-PAGE in the presence or absence of .beta.-mercaptoethanol
followed by western blot analysis, as indicated. Note that like the
wild type pro peptide, the D76A mutant pro peptide purified in a
complex with the C-terminal dimer. The pro peptide degradation
product did not co-purify with and was thus not part of the
complex. Bands denoted by the asterisk indicate misfolded myostatin
species, which were evident under non-reducing conditions.
[0031] FIGS. 3D and 3E show cleavage of the pro peptide by
BMP-1/TLD proteinases. Wild type and mutant complexes were
incubated with purified proteinases and analyzed by SDS-PAGE
followed by western blotting using antibodies directed against the
pro peptide. Incubations were carried out with 1 .mu.g latent
complex and 250 ng proteinase for 16 hours at 37.degree. C., except
that in FIG. 3D, the samples were incubated with an additional 250
ng BMP-1 for 4 more hours. In FIG. 3E, lanes labeled "no enzyme"
indicate samples incubated for 16 hours at 37.degree. C. in the
absence of enzyme. Note that all enzymes were capable of generating
the cleavage product and that the D76A mutant protein was
completely resistant to cleavage.
[0032] FIGS. 4A to 4D show activation of latent myostatin activity
by BMP-1/TLD proteinases. In FIGS. 4B to 4D, black bars represent
wild type, and gray bars represent D76A mutant complexes. Note that
although heat treatment activated both the wild type and mutant
complexes (FIG. 4B), each proteinase was capable of activating only
the wild type complex (FIGS. 4C and 4D). *p<0.05,
**p<0.01.
[0033] FIG. 4A shows activation of pGL3-(CAGA).sub.12-luciferase
reporter gene activity by purified myostatin C-terminal dimer.
[0034] FIG. 4B shows activation of the myostatin pro
peptide/C-terminal dimer latent complex by heat treatment. Control
(no myostatin (MSTN)) is indicated.
[0035] FIGS. 4C and 4D show activation of the myostatin pro
peptide/C-terminal dimer latent complex by BMP-1/TLD proteinases.
The samples used for the reporter assays in FIGS. 4C and 4D are the
same samples shown in FIGS. 3D and 3E, respectively.
[0036] FIG. 5 shows inhibition of reporter gene activity by wild
type and mutant pro peptide/Fc fusion proteins in vitro. A204 cells
transfected with the reporter construct were incubated with 10
ng/ml of purified myostatin C-terminal dimer and various
concentrations of wild type (dark) or D76A mutant (light) pro
peptide/Fc fusion protein. Note that the wild type and mutant
proteins were equally effective in blocking myostatin activity.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The present invention is based on the identification of
proteases that cleave myostatin pro peptide, including when the pro
peptide is present in a complex with a myostatin C-terminal dimer,
thereby converting latent inactive myostatin complex to active
myostatin. Proteases having such myostatin pro peptide cleaving
activity are exemplified by the metalloprotease bone morphogenic
protein-1/tolloid (BMP-1/TLD) family of proteins. As such, the
proteases provide targets and reagents for identifying drugs that
can increase or decrease the protease activity, or can increase or
decrease myostatin pro peptide cleavage mediated by the proteases,
and, therefore, increase or decrease myostatin activity.
[0038] Myostatin (growth differentiation factor-8; GDF-8) is
expressed as a pre-proprotein, promyostatin, which includes a
signal peptide (amino acid residues about 1 to 20), the myostatin
pro peptide domain (amino acid residues about 20 to 262 or 263) and
the myostatin C-terminal domain (amino acid residues about 267 or
268 to 375). Promyostatin polypeptides and encoding polynucleotides
are highly conserved evolutionarily (see McPherron and Lee, Proc.
Natl. Acad. Sci., USA 94:12457, 1997; GenBankAcc. Nos. AF019619,
AF019620, AF019621, AF019622, AF019623,AF019624, AF019625,AF019626,
and AF019627; U.S. Pat. No. 5,994,618, each of which is
incorporated herein by reference). Promyostatin polynucleotides and
encoded polypeptides are exemplified herein by human promyostatin
(SEQ ID NOS:1 and 2; pro peptide is amino acid residues about 20 to
263), bovine promyostatin (SEQ ID NOS:3 and 4; pro peptide is amino
acid residues about 20 to 262), chicken promyostatin (SEQ ID NOS:5
and 6; pro peptide is amino acid residues about 20 to 262), and
zebrafish promyostatin (SEQ ID NOS:7 and 8; pro peptide is amino
acid residues about 20 to 262).
[0039] Myostatin is activated by two proteolytic cleavage events--a
first removing the signal sequence (approximately the first 20
N-terminal amino acid residues of promyostatin; see, for example,
SEQ ID NO:2), and a second at a tetrabasic processing site (at
about amino acid residues 263 to 266 of promyostatin)--resulting in
the generation of a 26 kDa N-terminal pro peptide (approximately
amino acid residues 20 to 262 or 263) and a 12.5 kDa C-terminal
peptide (approximately amino acid residue 266 or 267 to the
C-terminus); a dimer of the C-terminal peptide is biologically
active. Upon secretion from cells, the myostatin C-terminal dimer
is maintained in a latent, inactive state due to its remaining
bound to the myostatin pro peptide (Lee and McPherron, Proc. Natl.
Acad. Sci., USA 98:9306-9311, 2001, which is incorporated herein by
reference). The latent myostatin complex that circulates in the
blood of adult mice can be activated in vitro by treatment with
acid (Zimmers et al., Science 296:1486-1488, 2002, which is
incorporated herein by reference).
[0040] Mice in which the myostatin gene has been knocked out show
increased muscle mass, and further exhibit a significant reduction
in fat accumulation with increasing age as compared to wild type
littermates (McPherron and Lee, J. Clin. Invest. 109:595-601, 2002,
which is incorporated herein by reference). Conversely,
over-expression of myostatin in vivo produces the signs and
symptoms characteristic of the muscle wasting syndrome, cachexia
(Zimmers et al., supra, 2002). The muscle wasting observed in mice
having increased levels of circulating myostatin can be partially
reversed by introducing myostatin binding agents such as the
myostatin pro peptide and follistatin to the mice (Zimmers et al.,
supra, 2002). These results confirmed that the observed muscle
wasting was due to increased myostatin, and indicate that methods
for decreasing the level of active myostatin or otherwise reducing
or inhibiting myostatin activity can be useful for ameliorating
muscle wasting. In view of the highly conserved nature of myostatin
among species as diverse as fish and humans, these results indicate
that myostatin also can be involved in the cachexia associated with
various disorders in humans, including, for example, cancer,
acquired immunodeficiency syndrome (AIDS), and sepsis, as well as
in neuromuscular disorders such as muscular dystrophy (see
Gonzalez-Kadavid et al., Proc. Natl. Acad. Med., USA
95:14938-14943, 1998, which is incorporated herein by
reference).
[0041] Proper skeletal muscle function also is involved in
maintaining normal glucose metabolism, and skeletal muscle
resistance to insulin stimulated glucose uptake is the earliest
manifestation of non-insulin dependent (type 2) diabetes (see
McPherron and Lee, supra, 2002). In two mouse models of obesity and
diabetes, loss of myostatin prevented an increase in adipose tissue
mass with age and attenuated the obese and diabetic phenotype in
the mouse models (McPherron and Lee, supra, 2002). As such, methods
that modulate myostatin activity also can be useful for reducing
body fat in an individual, and for treating disorders associated
with abnormal muscle function or obesity, for example, type 2
diabetes.
[0042] As disclosed herein, the myostatin pro peptide, either in a
free form or when part of a complex with the myostatin C-terminal
dimer, can be cleaved by members of the BMP-1/TLD family of
metalloproteases, and such cleavage releases the myostatin
C-terminal dimer from the inhibitory effects of the pro peptide,
thus generating active myostatin. As such, the BMP-1/TLD proteases
provide a target for drugs that can modulate myostatin activity
and, therefore, increase or decrease muscle mass or reduce or
prevent obesity in an organism. Accordingly, the invention provides
methods of identifying agents that modulate metalloprotease
mediated myostatin pro peptide cleavage, and that modulate
metalloprotease mediated activation of latent myostatin.
[0043] A screening method of the invention can be performed, for
example, by contacting a myostatin pro peptide, a metalloprotease
that can cleave the myostatin pro peptide, and a test agent, under
conditions sufficient for cleavage of the pro peptide by the
metalloprotease; and detecting a change in the amount of cleavage
of the pro peptide in the absence of the test agent as compared to
the presence of the test agent, thereby identifying the test agent
as an agent that modulates metalloprotease mediated myostatin pro
peptide cleavage. The myostatin pro peptide can be in an isolated
form, or can be a component of a latent myostatin complex that
further contains a myostatin C-terminal fragment or a myostatin
C-terminal dimer.
[0044] A metalloprotease examined according to a screening assay of
the invention can be any protease that cleaves a myostatin pro
peptide, particularly a metalloprotease that cleaves the pro
peptide when it is in a latent myostatin complex with a C-terminal
myostatin fragment or dimer thereof, such that active myostatin is
generated from the latent myostatin complex. Such metalloproteases
are exemplified by the BMP-1/TLD family of metalloproteases, which
includes four mammalian proteins, BMP-1 (Wozney et al., Science
242:1528-1534, 1988), mammalian Tolloid (mTLD; Takahara et al., J.
Biol. Chem. 269:32572-32578, 1994), mammalian Tolloid-like-1
(mTLL-1; Takahara et al., Genomics 34:157-165, 1996), and mammalian
Tolloid-like-2 (mTLL-2; Scott et al., Devel. Biol. 213:283-300,
1999). The BMP-1/TLD family of metalloproteases, in turn, are
members of a larger family of proteins, the astacin family, which
includes proteases that are expressed in various vertebrate and
invertebrate organisms, including, for example, Xenopus (Xolloid;
UVS.2), fish (choriolysin H and L; zebrafish Tolloid), sea urchin
(BP-10 and SpAN), and hydra (HMP-1; see, for example, Li et al.,
Proc. Natl. Acad. Sci., USA 93:5127-5130, 1996, which is
incorporated herein by reference). As such, the screening assays of
the invention can be practiced using any of various
metalloproteases and, therefore, allow an identification of agents
that can be useful, for example, for modulating myostatin
activation in a variety of different organisms.
[0045] BMP-1 and mTLD are encoded by alternatively spliced mRNAs
from a single gene (Takahara et al., supra, 1994), whereas mTLL-1
and mTLL-2 are encoded by distinct genes. The BMP-1/TLD family of
proteases is known to have a role in regulating the activity of at
least three classes of substrates. First, BMP-1, mTLD, and mTLL-1
are capable of processing procollagen precursors into the mature
monomers required for assembly into the multimeric fibers that are
normally present in the extracellular matrix (Kessler et al.,
Science 271:360-362, 1996; Li et al., supra, 1996). Second, BMP-1,
mTLD, mTLL-1 and mTLL-2 each can process pro-lysyl oxidase into the
mature, biologically active enzyme (Uzel et al., J. Biol. Chem.
276:22537-22543, 2001). Third, BMP-1 and mTLL-1 can cleave chordin
(Scott et al., supra, 1999), which normally binds various members
of the BMP subgroup of the TGF-.beta. superfamily and maintains
them in a latent state (Blader et al., Science. 278:1937-1940,
1997; Marques et al., Cell 91:417-26, 1997; Piccolo et al., Cell
91:407-416, 1997). Cleavage of chordin by these metalloproteases
releases the BMP from the inhibitory effect of chordin. As such,
BMP-1 and TLL-1 are believed have a role in modulating the effects
of the BMPs during a variety of morphogenic processes. As disclosed
herein, BMP-1/TLD family members, including BMP-1, mTLD, mTLL-1 and
mTLL-2 also can cleave the myostatin pro peptide, either in its
free form or when bound to the myostatin C-terminal dimer (latent
myostatin complex), wherein cleavage of the pro peptide results in
activation of the myostatin C-terminal dimer (see Examples 1 and
2).
[0046] A test agent that can be examined according to a method of
the invention can be any type of molecule, including, for example,
a peptide, peptide derivative such as a peptide hydroxamate or a
phosphinic peptide, peptoid, polynucleotide, or small organic
molecule (see Example 3). Thus, the term "test agent" is used
broadly herein to mean any compound that is being examined for
agonist or antagonist activity with respect to metalloprotease
mediated myostatin pro peptide cleavage or myostatin activation.
Although the method generally is used as a screening assay to
identify previously unknown molecules (test agents) that can act as
agonist or antagonist agents, the method also can be used to
confirm that an agent known to have a particular activity in fact
has the activity, for example, in standardizing the activity of the
agent; and can be used to screen derivatives or other modified
forms or mimics of such known agents.
[0047] A screening method of the invention conveniently can be
adapted to high throughput analysis and, therefore, can be used to
screen combinatorial libraries of test agents, which can be a
library of random test agents, biased test agents, or variegated
test agents (see, for example, U.S. Pat. No. 5,571,698, which is
incorporated herein by reference), in order to identify those
agents that can modulate metalloprotease mediated cleavage of a
myostatin pro peptide and, therefore, myostatin activity. 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 library of peptide derivative compounds such as a
hydroxamate compound library, reverse hydroxamate compound library,
a carboxylate compound library, thiol compound library, a
phosphinic peptide library, or phosphonate compound library (see,
for example, Dive et al., Biochem. Soc. Trans. 28:455-460, 2000; Ye
and Marshall, Peptides: The Wave of the Future (Lebl and Houghten,
ed.; American Peptide Society, 2001), each of which is incorporated
herein by reference); a peptidomimetic library (Blondelle et al.,
Trends Anal. Chem. 14:83-92, 1995, which is incorporated herein by
reference); a nucleic acid library (O'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); 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, BioTechnology 13:351-360, 1995; each of
which is incorporated herein by reference).
[0048] Polynucleotides can be particularly useful as agents that
can modulate metalloprotease mediated myostatin pro peptide
cleavage or myostatin activation 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). 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. A
polynucleotide can be a naturally occurring nucleic acid molecule,
which can be isolated from a cell, or a synthetic molecule, which
can be prepared, for example, by methods of chemical synthesis or
by enzymatic methods such as by the polymerase chain reaction
(PCR).
[0049] A polynucleotide agent (or test agent) can contain
nucleoside or nucleotide analogs, or a backbone bond other than a
phosphodiester bond. 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).
[0050] 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.
[0051] 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).
[0052] Similarly, peptides, as exemplified herein (see Examples 3
and 4) can be useful as agents for modulating metalloprotease
mediated myostatin activation, or as test agents to screen for such
activity. Peptide agents (or test peptides) can contain one or more
D-amino acids and/or L-amino acids; and/or one or more amino acid
analogs, for example, an amino acid that has been derivatized or
otherwise modified at its reactive side chain. In addition, one or
more peptide bonds in the peptide can be modified, and a reactive
group at the amino terminus or the carboxy terminus or both can be
modified. Peptides containing D-amino acids, or L-amino acid
analogs, or the like, can 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 modulating
metalloprotease mediated myostatin activation as disclosed herein.
As disclosed herein, the stability of a peptide agent (or test
agent) also can be improved by generating (or linking) a fusion
protein comprising the peptide and a second polypeptide (e.g., an
Fc domain of an antibody) that increases the half-life of the
peptide agent in vivo (see Example 4; see, also, U.S. patent
application Publication No. 2003/0104406 A1, which is incorporated
herein by reference). Peptides also can be modified to have
decreased stability in a biological environment, if desired, such
that the period of time the peptide is active in the environment is
reduced.
[0053] Test agents also can be antibodies that are raised against
and specifically bind one or more epitopes of a metalloprotease
that cleaves a myostatin pro peptide; or against an epitope of the
pro peptide, which can be an isolated pro peptide or a pro peptide
component of a latent myostatin complex; or a complex of the
metalloprotease and pro peptide. 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. The term "binds specifically" or "specific binding
activity" or the like, 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.sup.-6 M,
generally at least about 1.times.10.sup.-7 M, usually at least
about 1.times.10.sup.-8 M, and particularly at least about
1.times.10.sup.-9 M or 1.times.10.sup.-10 M or less. As such, Fab,
F(ab').sub.2, Fd and Fv fragments of an antibody that retain
specific binding activity are included within the definition of an
antibody. In addition to specifically binding a particular epitope,
an antibody agent modulates the protease cleavage activity of a
metalloprotease for a myostatin pro peptide, including increasing
or decreasing such activity.
[0054] 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 (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, 1999); 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).
[0055] A panel of test agent antibodies conveniently can be
obtained by immunizing an animal using a peptide portion of a
myostatin pro peptide or of a metalloprotease, particularly a
BMP-1/TLD family member. Where such a peptide portion of the pro
peptide or metalloprotease 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, 1999). 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 (see, for example, Kohler and
Milstein, Nature 256:495, 1975, which is incorporated herein by
reference; see, also, Harlow and Lane, supra, 1999). 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. A
recombinant phage that expresses, for example, a single chain
antibody that modulates metalloprotease mediated cleavage of
myostatin pro peptide also provides an antibody that can used for
preparing standardized kits.
[0056] 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 gel, 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 are well known 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.
[0057] Therapeutic applications for antibody agents identified
according to a screening assay of the invention also are provided.
Where the therapeutic procedure is for treating a human subject,
the antibodies can be derived from a subhuman primate antibody
(see, for example, Goldenberg et al., Intl. Publ. WO 91/11465,
1991; and Losman et al., Intl. J. Cancer 46:310, 1990, each of
which is incorporated herein by reference). A therapeutically
useful antibody for human treatment 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). Alternatively, the antibodies 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).
[0058] The antibodies also can be derived from human monoclonal
antibodies, which, for example, can be obtained from transgenic
mice that have been genetically modified 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 well known (see, for example,
by Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature
368:856, 1994; and Taylor et al., Intl. Immunol. 6:579, 1994; each
of which is incorporated herein by reference), and commercial
sources of human antibodies are available (Abgenix, Inc.; Fremont
Calif.).
[0059] Antigen binding fragments of an antibody 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, F(ab').sub.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. Nos.
4,036,945 and 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).
[0060] 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
V.sub.H and V.sub.L 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 V.sub.H and V.sub.L 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 V.sub.H and V.sub.L 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., BioTechnology 11: 1271-1277, 1993; each of which is
incorporated herein by reference; see, also Sandhu, supra, 1992.
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).
[0061] A difference in the amount of cleavage of the pro peptide
due to contact with a test agent can be detected, for example, by
detecting the pro peptide and/or a cleavage product of the pro
peptide using a method such as electrophoresis, chromatography, or
mass spectrometry (see, for example, Thies et al., Growth Factors
18:251-259, 2001, which is incorporated herein by reference), which
can detect a myostatin pro peptide or cleavage product thereof
based on its size, charge, or both; an immunological based assay
such as an immunoblot analysis, an enzyme-linked immunosorption
assay (ELISA), or the like, which utilizes an antibody specific for
the intact pro peptide or the cleaved pro peptide, but not both; or
a fluorescence based assay, including, for example, a fluorescence
resonance energy transfer (FRET) assay, wherein fluorescence of the
intact pro peptide is quenched, and the quenching is relieved upon
cleavage of the pro peptide. Where an increased amount of a
cleavage product of the pro peptide is detected in the presence of
(or following contact with) the test agent as compared to an amount
of cleavage product in the absence of the test agent, the test
agent is identified as an agent that can increase metalloprotease
mediated activation of the latent myostatin. Similarly, where a
decreased amount of the pro peptide is detected in the presence of
(or following contact with) the test agent as compared to an amount
of pro peptide in the absence of the test agent, the test agent is
identified as an agent that can increase metalloprotease mediated
activation of the latent myostatin. Conversely, where a decreased
amount of a cleavage product of the pro peptide is detected in the
presence of (or following contact with) the test agent as compared
to an amount of cleavage product in the absence of the test agent,
the test agent is identified as an agent that can decrease
metalloprotease mediated activation of the latent myostatin. Where
a greater amount of the pro peptide is detected in the presence of
(or following contact with) the test agent as compared to an amount
of pro peptide in the absence of the test agent, the test agent is
identified as an agent that can decrease metalloprotease mediated
activation of the latent myostatin. Such activity can be confirmed
using a cell based or animal assay by detecting, for example, a
change in myostatin mediated signal transduction activity due to
the agent.
[0062] A difference in the amount of cleavage of the pro peptide
also can be detected by detecting a change in binding of myostatin
to a myostatin receptor, or by detecting a change in a myostatin
mediated signal transduction in a cell expressing a myostatin
receptor. Cells useful for performing a screening assay of the
invention include, for example, cells from mammals, birds, fish,
yeast, or Drosophila. Such functional assays can directly indicate
that a test agent modulates metalloprotease mediated myostatin
activation. A cell useful for such a method can be one that
expresses an endogenous myostatin receptor, for example, L6
myocytes, or can be a cell genetically modified, transiently or
stably, to express a transgene encoding the myostatin receptor, for
example, an activin receptor such as an activin type II receptor
(Thies et al., supra, 2001). Myostatin mediated signal transduction
can be detected at any level in the signal transduction pathway,
including from binding of myostatin to a cell surface receptor to
expression of a gene that is regulated by myostatin, which, in a
screening assay of the invention, is dependent on metalloprotease
mediated cleavage of a myostatin pro peptide and myostatin
activation.
[0063] Metalloprotease mediated myostatin activation and consequent
myostatin mediated signal transduction can be detected by measuring
myostatin binding to a myostatin receptor using a receptor binding
assay, which can be an in vitro assay or cell based assay.
Metalloprotease mediated myostatin activation and consequent
myostatin mediated signal transduction also can be detected by
measuring expression of a myostatin regulated gene, which can be a
reporter gene comprising, for example, a TGF-.beta. regulatory
element operatively linked to a polynucleotide encoding a
detectable label. Expression of the reporter gene can be detected,
for example, by detecting an RNA transcript of the reporter gene
sequence, or by detecting a polypeptide encoded by the reporter
gene or an activity of the encoded polypeptide. A polypeptide
reporter can be, for example, .beta.-lactamase, chloramphenicol
acetyltransferase, adenosine deaminase, aminoglycoside
phosphotransferase, dihydrofolate reductase, hygromycin-B
phosphotransferase, thymidine kinase, .beta.-galactosidase,
luciferase, or xanthine guanine phosphoribosyltransferase, and can
be detected, for example, by detecting radioactivity, luminescence,
chemiluminescence, fluorescence, enzymatic activity, or specific
binding due to the reporter polypeptide, or survival in a selective
medium of cells expressing the reporter polypeptide. Methods for
introducing a transgene such as a polynucleotide encoding a
myostatin receptor or a reporter gene under conditions such that a
polypeptide encoded by the transgene can be expressed are disclosed
herein or otherwise known in the art.
[0064] Generally, a reporter gene includes a coding sequence, which
encodes the reporter polynucleotide or polypeptide, operatively
linked to one or more transcription and, as appropriate,
translation regulatory elements, and can be contained in a vector,
particularly an expression vector. If desired, the coding sequence
can further encode an operatively linked peptide tag such as a
His-6 tag, which can be detected using a divalent cation such as
nickel ion, cobalt ion, or the like; 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; glutathione
S-transferase, which can be detected using glutathione; or an Fc
domain of an antibody, which can be detected using Protein A or an
anti-Fc antibody, either of which, can, but need not, be detectably
labeled or attached to a solid support or, in turn, detected using
a second antibody. As such, it will be recognized that various
means for detecting a particular tagged molecule also can be used
to isolate the tagged molecule.
[0065] As used herein, the term "operatively linked" 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 reporter polypeptide 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 (see, e.g.,
Example 4), i.e., are covalently bound through a peptide bond; or
can be translated as two discrete peptides that, upon translation,
can associate with each other to form a stable complex.
[0066] A polynucleotide such as a reporter gene 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 polypeptide, 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.
[0067] An expression vector (or the polynucleotide) generally
contains or encodes a promoter sequence, which can provide
constitutive or, if desired, inducible, 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).
[0068] A polynucleotide encoding a reporter polypeptide can be
operatively linked, for example, to a tissue specific regulatory
element, for example, a muscle cell specific regulatory element,
wherein expression of the reporter polypeptide 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 include, 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).
[0069] Viral expression vectors can be particularly useful for
introducing a polynucleotide into a cell, including, if desired,
into 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. 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).
[0070] A polynucleotide such as a reporter gene or a polynucleotide
agent, which can be contained in a vector, can be introduced into a
cell by any of a variety of methods (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,
biolistic methods, 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.
[0071] Where a test agent is identified as having myostatin
modulating activity, the screening assay can further include a step
of determining an amount by which the agent increases or decreases
myostatin activation. For example, where an agent is identified
that increases the proteolytic activity of the metalloprotease for
the myostatin pro peptide above a baseline level of activity in a
particular system, for example, in an in vitro assay using purified
reagents or in vivo in a subject, the method can further include
determining an amount by which the agent increases myostatin
activation above the basal level. As such, different agents or
panels of agents can be obtained that increase or decrease
myostatin activation by a metalloprotease in a relatively defined
amount. Such a method further provides a means to determine amounts
of a particular agent useful for providing a desired level of
myostatin activity. As such, the present invention provides agents
and panels of agents that modulate metalloprotease mediated
myostatin activation, such agents being useful as medicaments to
modulate myostatin activation in a subject, for example, in a
subject having a metabolic disorder such as muscular dystrophy,
muscle wasting, obesity, or type 2 diabetes.
[0072] Accordingly, the invention provides methods of modulating
metalloprotease mediated myostatin activation. As used herein, the
term "modulate," when used in reference to an effect on
metalloprotease mediated cleavage of myostatin pro peptide or
metalloprotease mediated myostatin activation, means that the
amount of pro peptide cleavage or myostatin activation either is
increased or is reduced or inhibited. The terms "increase" and
"reduce or inhibit" are used in reference to the effect of an agent
on a baseline level of metalloprotease mediated myostatin pro
peptide cleavage or myostatin activation. The baseline level of
activity can be a level of cleavage or activation that is
identified as occurring in an in vitro assay using purified pro
peptide and metalloprotease under defined conditions, or using a
biological sample such as a cell or tissue extract obtained from a
subject, which can, but need not, be a normal healthy individual;
or a level of cleavage or activation that occurs in vivo in a
subject. The terms "reduce or inhibit" are used together herein
because it is recognized that, in some cases, the level of
metalloprotease mediated myostatin pro peptide cleavage or
myostatin activation 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, for example, a low level of
myostatin pro peptide cleavage remains, or whether such cleavage is
completely inhibited.
[0073] A method of modulating metalloprotease mediated myostatin
pro peptide cleavage or myostatin activation can be performed, for
example, by contacting a latent myostatin complex, which includes a
myostatin pro peptide and a myostatin C-terminal fragment,
particularly a C-terminal fragment dimer, with a metalloprotease
that can cleave the myostatin pro peptide, and with an agent that
can increase or decrease proteolytic cleavage of the pro peptide
mediated by the metalloprotease. The metalloprotease can be any
metalloprotease that can cleave the myostatin pro peptide,
particularly when the pro peptide comprises a latent myostatin
complex, including, for example, a BMP-1/TLD family member such as
BMP-1, mTLD, mTLL-1, or mTLL-2. The agent can act in any way to
modulate metalloprotease mediated cleavage of the myostatin pro
peptide, including, for example, by increasing or decreasing the
proteolytic activity of the metalloprotease, by competing with the
pro peptide for the metalloprotease, by facilitating contact of the
metalloprotease and a latent myostatin complex comprising the pro
peptide, or by inducing a conformational change in the latent
myostatin complex such that it is a less fit (or more fit)
substrate for the metalloprotease.
[0074] A method of modulating metalloprotease mediated myostatin
activation can be practiced with respect to any subject that
expresses myostatin, including vertebrates and invertebrates. For
example, the subject can be a human, mouse, cow, pig, sheep, goat,
dog, cat, 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.
[0075] A method of modulating metalloprotease mediated myostatin
activity can be performed in vitro or ex vivo using cells or a
tissue in culture, a cell or tissue extract, a biological fluid
such as a serum or plasma sample, or substantially purified
reagents, including substantially purified metalloprotease and/or
latent myostatin complex (see, for example, Thies et al., supra,
2001). Where the method is performed in vitro, the agent can be
contacted with sample comprising the metalloprotease and latent
myostatin complex by adding the agent to the sample, which
generally is in a culture medium or other buffered solution. For
example, where the method is performed using cells in culture, the
agent can be added to the culture medium such that it contacts the
metalloprotease and/or pro peptide, one or both of which can be
present in cells in the culture or secreted into the medium. The
agent can be selected such that it is soluble in the sample medium,
or can be formulated to increase solubility, if desired.
[0076] A method of modulating myostatin activation also can be
performed in vivo, including in a living subject, including with
respect to cells or a tissue in situ in a subject. In general, such
a method is performed by administering the agent to the subject
and, therefore, the agent generally is formulated in a composition
suitable for administration to the subject. As such, compositions
containing an agent that can modulate metalloprotease mediated
myostatin activation are provided, such compositions including the
agent in a pharmaceutically acceptable carrier. Such compositions
are useful as medicaments for treating a subject suffering from a
muscular and/or metabolic disorder as disclosed herein, and are
useful for administration to animals such as farm animals used for
labor or as food products.
[0077] A composition for administration to a living subject
generally includes the agent in a pharmaceutically acceptable
carrier. 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 agent. 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 agent to be administered, 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 composition
also can contain one or more additional reagent, including, for
example, nutrients or vitamins or, where the composition is
administered for a therapeutic purpose, a diagnostic reagent or
therapeutic agent relevant to the disorder being treated.
[0078] 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 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 remains 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).
[0079] The route of administration of a pharmaceutical composition
containing an agent that modulates metalloprotease mediated
myostatin activation 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
structures of peptide domains; or based on a peptoid such as a
vinylogous peptoid.
[0080] A composition as disclosed herein can be administered to a
subject by various routes including, for example, orally or
parenterally, such as intravenously, intramuscularly,
subcutaneously, intraorbitally, intracapsularly, intraperitoneally,
intrarectally, intracisternally or by passive or facilitated
absorption through the skin using, for example, a skin patch or
transdermal iontophoresis, respectively. Furthermore, the
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.
[0081] The pharmaceutical composition can be formulated as an 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).
[0082] 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. It will be recognized
that the amount of the pharmaceutical composition, for example, to
treat obesity 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
composition and the routes and frequency of administration are
determined, initially, using Phase I and Phase II clinical
trials.
[0083] A method of the invention can be used to increase the level
of myostatin activation (i.e., above a baseline level of myostatin
activation in the absence of an agent), for example, by contacting
a latent myostatin complex and/or metalloprotease with an agent
that increases proteolytic activity of the metalloprotease; or can
be used to decrease the level of myostatin activation (below a
baseline level), for example, by contacting a latent myostatin
complex and/or metalloprotease with an agent that decreases
metalloprotease mediated proteolytic activity of myostatin pro
peptide. The agent can be one that decreases proteolytic activity
of a metalloprotease that cleaves myostatin pro peptide of a latent
myostatin complex, thereby reducing or inhibiting myostatin
activation below a level of myostatin activation that occurs or
would occur in the absence of the agent. Where such an agent is
administered to a subject, the agent can result in increased muscle
mass or decreased fat content or both in the subject. For example,
the subject can be a human subject suffering from a muscle wasting
disorder, wherein increased muscle mass can ameliorate the signs
and symptoms of the disorder. Alternatively, the agent can be one
that increases metalloprotease mediated proteolytic cleavage of
myostatin pro peptide from a latent myostatin complex, thereby
increasing myostatin activation above a level, if any, of myostatin
activation that occurs or would occur in the absence of the agent.
Where such an agent is administered to a subject, the agent can
result in decreased muscle mass or increased fat content or both in
the subject. Such a subject can be, for example, an undesirable
organism such as an invasive fish species or rodents, wherein
decreased muscle mass and/or increased fat content places the
invasive species at a competitive disadvantage in the
environment.
[0084] Accordingly, in one embodiment, the invention provides a
method of increasing muscle mass or reducing the fat content or
both of a subject by modulating proteolytic cleavage of a myostatin
pro peptide by a metalloprotease such as a BMP-1/TLD family
metalloprotease. Such a method can be performed, for example, by
administering to the subject an agent that reduces or inhibits the
proteolytic activity of a protease that cleaves myostatin pro
peptide, thereby preventing activation of latent myostatin and
increasing muscle mass in the subject. The subject in which muscle
mass is to be increased can be any subject in which myostatin is
expressed, particularly a vertebrate organism, including
domesticated animals (e.g., a feline or canine species), farm
animals or animals that are raised as a food source, including
mammalian species (e.g., an ovine, porcine, or bovine species),
avian species (e.g., chickens or turkeys), and piscine species
(e.g., salmon, trout, or cod). 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.
Thus, a method of the invention can be performed on any eukaryotic
organism that expresses myostatin and relies on metalloprotease
mediated cleavage of myostatin pro peptide to activate myostatin,
including a vertebrate organism, for example, mammalian, avian or
piscine organism, or an invertebrate organism, for example, a
mollusk, echinoderm, gastropod or cephalopod. In one embodiment,
the subject is a human subject, for example, a subject suffering
from a metabolic disorder such as a muscular disorder (e.g., a
dystonia or dystrophy), a wasting disorder (e.g., cachexia),
clinical obesity, or type 2 diabetes.
[0085] As such, the invention also provides a method for
ameliorating a metabolic disorder in a subject by administering an
agent that modulates metalloprotease mediated myostatin activation
in the subject. As used herein, the term "ameliorate," when used in
reference to a metabolic disorder, means that signs or symptoms
associated with the disorder are lessened. Amelioration of the
disorder can be identified using any assay generally used by the
skilled clinician to monitor the particular metabolic disorder, for
example, a glucose tolerance test for monitoring diabetes, or a
serum leptin assay for body fat analysis (McPherron and Lee, supra,
2002). Amelioration of a metabolic disorder such as obesity or
cachexia can be monitored simply by measuring the subject's body
weight.
[0086] Heterozygous myostatin knock-out mice have increased
skeletal muscle mass, although to a lesser extent than that
observed in homozygous mutant mice, indicating that myostatin acts
in a dose-dependent manner in vivo. Furthermore, overexpression of
myostatin in animals has 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, and resembling cachexia as occurs in
patients with chronic diseases such as cancer or AIDS. In addition,
the serum levels of myostatin immunoreactive material have been
correlated with the status of patients with respect to muscle
wasting (Gonzalez-Kadavid et al., supra, 1998). 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. 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. 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 (A.sup.y) mice), which
suppressed fat accumulation by five-fold. Abnormal glucose
metabolism also was partially suppressed in agouti mice containing
the myostatin mutation.
[0087] As such, the agents and methods of the present invention,
which reduce or inhibit metalloprotease mediated myostatin
activation, can be used to treat or prevent metabolic diseases such
as obesity and type 2 diabetes. The methods of the invention are
useful, for example, for ameliorating various metabolic disorders,
including, for example, the cachexia associated with chronic
diseases such as cancer (see Norton et al., Crit. Rev. Oncol.
Hematol. 7:289-327, 1987, which is incorporated herein by
reference), as well as conditions such as type 2 diabetes, obesity,
and other metabolic disorders. As used herein, the term "metabolic
disorder" refers to a condition that is characterized, at least in
part, by an abnormal amount, development or metabolic activity of
muscle and/or adipose tissue. Such metabolic disorders include, for
example, obesity; muscle wasting disorders such as muscular
dystrophy, neuromuscular diseases, cachexia, and anorexia; and
disorders such as type 2 diabetes, which generally, but not
necessarily, is associated with obesity. The term "abnormal," when
used in reference to the amount, development or metabolic activity
of muscle and/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.
[0088] A method for ameliorating a metabolic disorder in a subject
can be performed, for example, by administering to the subject an
agent that reduces or inhibits the proteolytic activity of a
protease that cleaves myostatin pro peptide, thereby preventing
activation of latent myostatin in the cell and ameliorating the
metabolic disorder. As indicated above, the metabolic disorder can
be any disorder associated with increased or undesirably high
myostatin activation or activity, including, for example, a muscle
wasting disorder such as is associated with muscular dystrophy,
cachexia (e.g., associated with a cancer or acquired
immunodeficiency disease), or sarcopenia; or a metabolic disorder
such as clinical obesity or type 2 diabetes. By way of example,
sarcopenia is a metabolic disorder that is characterized by a loss
of skeletal muscle mass, quality, and strength, and can lead to
frailty in the elderly. Examples of skeletal muscle properties that
contribute to its overall quality include contractility, fiber size
and type, and glucose uptake and metabolism. Sarcopenia has
important consequences because the loss of lean body mass reduces
function, and because a loss of approximately 40% of lean body mass
generally is fatal (see, for example, Roubenoff and Castaneda, J.
Amer. Med. Assn. 286, 2001). A method of the invention provides a
means to ameliorate sarcopenia by reducing or inhibiting
metalloprotease mediated myostatin activation, thereby allowing
increased muscle growth and development in the subject.
[0089] The following examples are intended to illustrate but not
limit the invention.
EXAMPLE 1
BMP-1/TLD Metalloprotease Family Members Cleave Myostatin Pro
Peptide
[0090] This example demonstrates that the members of the bone
morphogenic protein-1/Tolloid (BMP-1/TLD) family of
metalloproteases cleave the myostatin pro peptide.
[0091] Five hundred ng of purified myostatin pro peptide or of
purified latent myostatin complex comprising the pro peptide and
C-terminal dimer (Lee and McPherron, supra, 2001) was incubated
overnight at 37.degree. C. with 100 ng purified BMP-1, mTLD,
mTLL-1, or mTLL-2 (Scott et al., Devel. Biol. 213:283-300, 1999,
which is incorporated herein by reference). Reaction products were
analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) followed by western blot analysis using
antiserum raised against the myostatin pro peptide (Lee and
McPherron, supra, 2001).
[0092] A discrete proteolytic cleavage product of the pro peptide
was detected in each reaction containing one of the four proteases,
but not in control reactions that did not contain a protease.
Moreover, each of the proteases cleaved the pro peptide whether it
was in a purified form or in a complex with the myostatin
C-terminal dimer. These results demonstrate that the BMP-1/TLD
metalloproteases cleave the myostatin pro peptide.
EXAMPLE 2
Metalloprotease Cleavage of Myostatin Pro Peptide Activates Latent
Myostatin
[0093] This example demonstrates that cleavage of the myostatin pro
peptide by a BMP-1/TLD metalloprotease activates latent
myostatin.
[0094] Purified myostatin pro peptide and C-terminal dimer complex
was incubated with mTLL-1, then examined using a reporter gene
assay that specifically detects myostatin activity. A204
rhabdomyosarcoma cells were transfected with the pGL3-(CAGA).sub.12
luciferase reporter gene construct, which comprises the luciferase
coding sequence linked to the TGF-.beta. responsive CAGA sequence
from the promoter of the TGF-.beta. inducible PAI-1 gene (Thies et
al., supra, 2001). The transfected cells were contacted with either
untreated pro peptide/C-terminal dimer complex or complex that had
been pre-incubated with mTLL-1. Incubation of the complex with
mTLL-1 dramatically increased the amount of luciferase activity
detected in the reporter cell assay, whereas no change was observed
in cells treated with mTLL-1 alone or with the myostatin complex
alone (FIG. 1).
[0095] In order to determine the extent of myostatin activation by
mTLL-1, a standard curve was generated using purified myostatin
C-terminal dimer in the reporter gene assay (FIG. 2), then the
amount of luciferase activity in cells treated with the mTLL-1
treated complex was compared to the standard curve. A comparison of
the amount of myostatin activity present in the mTLL-1-treated
sample and the degree of proteolytic processing of the pro peptide
by mTLL-1 in this sample revealed that at least about 50% of the
proteolytically-cleaved myostatin complex was active in the
reporter assay. These results demonstrate that cleavage of the
myostatin pro peptide in a complex of the pro peptide and myostatin
C-terminal dimer by the BMP-1/TLD metalloprotease, mTLL-1,
activates myostatin.
EXAMPLE 3
Peptide Substrates for Tolloid Family Members
[0096] A series of three peptides each of 10, 20, 30, 40, or 50
amino acid residues was synthesized based on the sequence of the
myostatin pro peptide, and encompassing the BMP-1/TLD
metalloprotease cleavage site (amino acid residues "RD" as shown in
bold, below, in wild type peptides; SEQ ID NOS:9, 12, 15, 18, and
21). Peptides in which the arginine residue at the P1 position just
upstream of the cleavage site was changed to a glutamine residue
(SEQ ID NOS:10, 13, 16, 19, and 22; see bold), and peptides in
which the aspartic acid at the P1' position just downstream of the
cleavage site was changed to an alanine (SEQ ID NOS:11, 14, 17, 20,
and 23; see bold), also were synthesized. The sequences of the
peptides are shown below:
1 50-mer KDVIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMP- T (SEQ
ID NO:9) KDVIRQLLPKAPPLRELIDQYDVQQDDSSDGSLEDDDYHATT- ETIITMPT (SEQ
ID NO:10) KDVIRQLLPKAPPLRELIDQYDVQRADSSDGSLE- DDDYHATTETIITMPT (SEQ
ID NO:11) 40-mer: QLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETI; (SEQ ID
NO:12) QLLPKAPPLRELIDQYDVQQDDSSDGSLEDDDYHATTETI; (SEQ ID NO:13) and
QLLPKAPPLRELIDQYDVQRADSSDGSLEDDDYHATTETI. (SEQ ID NO:14) 30-mer:
APPLRELIDQYDVQRDDSSDGSLEDDDYHA; (SEQ ID NO:15)
APPLRELIDQYDVQQDDSSDGSLEDDDYHA; (SEQ ID NO:16) and
APPLRELIDQYDVQRADSSDGSLEDDDYHA- . (SEQ ID NO:17) 20-mer:
ELIDQYDVQRDDSSDGSLED; (SEQ ID NO:18) ELIDQYDVQQDDSSDGSLED; (SEQ ID
NO:19) and ELIDQYDVQRADSSDGSLED. (SEQ ID NO:20) 10-mer: YDVQRDDSSD;
(SEQ ID NO:21) YDVQQDDSSD; (SEQ ID NO:22) and YDVQRADSSD. (SEQ ID
NO:23)
[0097] Peptides were supplied as lyophilized powders and stock
solutions of 1.0 mM were prepared in 60% acetonitrile--0.1%
trifluoroacetic acid (TFA) and 40% water. Activity of the enzymes
on the peptide substrates was assessed by combining 70 .mu.l of
water, 20 .mu.l of either mock conditioned medium or conditioned
medium containing the protein of interest, and 10 .mu.l of
synthetic peptide. Samples were incubated overnight at either room
temperature or 37.degree. C., then reactions were quenched by
reducing the pH through the addition of 1.0 .mu.l of 0.1% TFA. Each
aliquot was applied to a 2 cm C18 guard column cartridge (Supelco)
and peptides were eluted using an acetonitrile gradient (0-40% over
20 minutes) in 0.1% TFA. Peaks corresponding to cleaved peptide
fragments were identified and confirmed using mass spectrometry.
The 40-mer, 30-mer, and 20-mer wild type and R.fwdarw.Q mutant
peptides were cleaved by conditioned media containing TLL-2,
whereas the peptides containing the D.fwdarw.A mutation at the P1'
position were not cleaved; the 50-mer was insoluble under the
conditions used, and the cleavage products of the 10-mer were
difficult to detect due to their small size (i.e., 5-mers).
EXAMPLE 4
Activation of Latent Myostatin by BMP-1/Tolloid Family
Metalloproteases
[0098] This example demonstrates that BMP-1/TLL family members can
cleave and activate latent myostatin.
[0099] Myostatin purification and analysis. The generation of CHO
cell lines overexpressing myostatin was described
previously.sup.5,6 (numbered references listed at end of Example
4). Similar strategies were used to generate CHO lines expressing
mutant forms of full-length human myostatin and pro peptide/Fc
fusion proteins (see U.S. Publ. No. US 2003/0104406 A1). Mutant
human full-length myostatin sequences were based on SEQ ID NO:2,
and the mutant pro peptide sequences were based on amino acid
residues 24 to 266 of SEQ ID NO:2. Myostatin pro peptide/C-terminal
dimer complexes were purified from the conditioned medium of CHO
expressing cells as described.sup.5. Pro peptide/Fc fusion proteins
were purified using a Protein A-SEPHAROSE gel column. Antibodies
directed against bacterially-produced myostatin C-terminal domain
and pro peptide were as described.sup.1,5.
[0100] Proteinase and reporter gene assays. Purified BMP-1, mTLD,
mTLL-1, and mTLL-2 proteinases were prepared as described.sup.15.
Myostatin activity was measured using the
pGL3-(CAGA).sub.12-luciferase reporter assay in A204
rhabdomyosarcoma cells as described.sup.6. A standard curve using
purified myostatin C-terminal dimer was generated for each set of
assays in order to quantify myostatin activity.
[0101] Injection of mice. Female BALB/c mice (Charles River)
weighing 17 g to 19 g were injected intraperitoneally on days 1, 4,
8, 15, and 22 either with PBS alone or with various proteins
diluted in PBS; doses of proteins administered were as follows: pro
peptide/Fc fusion proteins--1 mg/kg or 10 mg/kg; IgG2am (control
antibody)--10 mg/kg; and JA16 (myostatin neutralizing antibody)--60
mg/kg. Mice were sacrificed on day 29 for muscle analysis. Muscles
from both sides of each animal were dissected and weighed; the
average weight was used for each muscle.
[0102] The generation of Chinese hamster ovary (CHO) cells
overexpressing myostatin has been described.sup.5,6. Like other
TGF-.beta. family members, myostatin produced by CHO cells is
cleaved at a dibasic site to generate an N-terminal pro peptide and
a disulfide-linked dimer of C-terminal fragments. In the course of
characterizing the secretion of myostatin by these cells, the
presence was noted of a discrete cleavage product of the pro
peptide (as detected by western blot analysis using antibodies
specific for the pro peptide). This cleavage product was detected
in the conditioned medium of CHO cells transfected with expression
constructs containing either the full-length myostatin precursor
protein (not shown) or the myostatin pro peptide alone in the
absence of the C-terminal domain (FIG. 3A). Because the myostatin
pro peptide can maintain the C-terminal dimer in a latent state
both in vitro.sup.5,6 and in vivo.sup.7,8, and because proteolytic
cleavage of the TGF-.beta. pro peptide is believed to be one
mechanism for activating latent TGF-.beta..sup.10-14, a role for
cleavage of the myostatin pro peptide in regulating myostatin
latency was investigated.
[0103] N-terminal sequencing revealed that the pro peptide
degradation product detected in CHO cell conditioned medium
resulted from proteolytic cleavage between arginine 75 and
aspartate 76. In order to determine whether either of these amino
acid residues is essential for proteolytic cleavage, CHO cell lines
expressing mutant versions of the pro peptide, in which either the
arginine or aspartate residue was changed to glutamine or alanine,
respectively, were generated. To enhance stability of these
proteins for in vivo studies (see below), the mutant pro peptides
were fused with an Fc domain. Although changing the arginine to
glutamine had no effect on proteolytic cleavage, no degradation
product could be detected in conditioned medium prepared from CHO
cells expressing the aspartate to alanine mutant pro peptide/Fc
fusion protein (FIG. 3B; see, also, Example 3). The requirement for
aspartate at the cleavage site suggested that members of the
BMP-1/TLD family of metalloproteinases were responsible for
generating this degradation product. A number of substrates have
been identified for mammalian members of the BMP-1/TLD family, and
in nearly every case, proteolytic cleavage has been shown to occur
immediately N-terminal to an aspartate residue.sup.15,16.
Furthermore, mutagenesis studies have documented the importance of
the aspartate residue in rendering these sites susceptible to
proteolytic cleavage.sup.17. As there were no apparent reports of
other proteinases with a similar specificity or requirement for an
aspartate residue just C-terminal to the scissile bond in protein
substrates, the ability of members of the BMP-1/TLD family to
cleave the myostatin pro peptide in vitro was investigated.
[0104] Myostatin was purified from the conditioned medium of
overproducing CHO cells.sup.5. After successive fractionation on
hydroxyapatite, lentil lectin SEPHAROSE gel, DEAE agarose, and
heparin SEPHAROSE gel, a purified preparation of the myostatin
latent complex was obtained that consisted of the N-terminal pro
peptide bound non-covalently to the C-terminal dimer (FIG. 3C). As
shown in FIG. 3D, incubation of the purified latent complex with
purified BMP-1 resulted in complete cleavage of the pro peptide to
generate a single product with an electrophoretic mobility
identical to that detected in conditioned medium prepared from CHO
cells engineered to overproduce myostatin. N-terminal sequencing of
BMP-1-treated pro peptide confirmed that cleavage occurred
immediately N-terminal to aspartate 76.
[0105] The ability of the other mammalian members of the BMP-1/TLD
family, including mTLD, mTLL-1, and mTLL-2, to cleave the pro
peptide also was tested. For these experiments, enzyme
concentrations were used that resulted in only partial cleavage,
thus allowing a comparison of the relative activities of the four
enzymes. As shown in FIG. 3E, incubation of the latent complex with
each of the four proteinases resulted in cleavage of the pro
peptide. Three of the proteinases, BMP-1, mTLL-1, and mTLL-2, were
approximately equally effective in cleaving the pro peptide, while
mTLD was consistently less active than the other three, even though
the same mTLD preparation was fully active against known substrates
such as procollagen. All four of these proteinases also cleaved pro
peptide that had been purified away from the C-terminal dimer.
[0106] In order to determine the effect of proteolytic cleavage of
the pro peptide on myostatin latency, myostatin biological activity
was measured in latent complexes treated with each of the four
proteinases. For this purpose, a reporter gene assay was used in
which A204 rhabdomyosarcoma cells were transfected with the
pGL3-(CAGA).sub.12-luciferase construct and incubated with
myostatin.sup.6. As described previously, the addition of purified
myostatin C-terminal dimer to these cells resulted in an increase
in luciferase activity above basal levels (FIG. 4A). In contrast,
purified myostatin latent complex was inactive in this assay, but
could be activated by incubation at 80.degree. C. for 5 minutes
(FIG. 4B). As shown in FIG. 4C, the latent complex was also
activated by pretreatment with BMP-1. Based on quantification of
myostatin activity relative to a standard curve, cleavage of the
pro peptide by BMP-1 was approximately as effective as heat
treatment in activating the latent complex. The latent complex was
also activated by pretreatment with the other proteinases, and the
extent of activation correlated roughly with the extent of
proteolytic cleavage by these enzymes (FIG. 4D).
[0107] The requirement for aspartate at the cleavage site also was
examined. A CHO cell line expressing high levels of a mutant form
of myostatin, in which aspartate 76 was changed to alanine, was
generated and the latent complex was purified from the conditioned
medium of these cells. As shown in FIG. 3C, the mutation had no
effect on the ability of the pro peptide to bind to the C-terminal
dimer; the mutant pro peptide and C-terminal dimer remained tightly
associated throughout the purification. Moreover, the mutant pro
peptide maintained the complex in a latent form that could be
activated by heating, as assessed by the luciferase reporter assay
(FIG. 4B). However, the mutant pro peptide in the latent complex
was completely resistant to proteolysis by each of the four
proteinases, BMP-1, mTLD, mTLL-1, and mTLL-2 (FIGS. 3D and E), and
was resistant to activation by these proteinases (FIGS. 4C and
4D).
[0108] Finally, the role of proteolytic cleavage of the pro peptide
in vivo was investigated by examining the effect of injecting wild
type and mutant versions of the pro peptide into mice. As
determined in previous experiments, the half-life of wild type pro
peptide after intraperitoneal injections into mice could be
increased from approximately 2 hours to 5 to 7 days by fusing the
pro peptide to an Fc domain. For this reason, CHO cell lines
expressing wild type or mutant (aspartate 76 to alanine) pro
peptide fused to an Fc domain were generated, and the fusion
proteins were purified using a Protein A SEPHAROSE gel column. The
aspartate to alanine mutation did not affect the activity of the
pro peptide in vitro, as the purified wild type and mutant pro
peptide/Fc fusion proteins were equally effective in inhibiting the
activity of the purified myostatin C-terminal dimer in the reporter
gene assay (FIG. 5).
[0109] In order to assess the activities of these proteins in vivo,
adult mice were given weekly injections of purified wild type or
mutant pro peptide/Fc fusion proteins and sacrificed after four
weeks for muscle analysis. For comparison, a set of mice also was
injected with the JA16 myostatin neutralizing monoclonal antibody,
which causes an approximately 25-30% increase in muscle mass after
12 weeks of treatment.sup.18. As shown in Table 1 (below),
injection of wild type pro peptide/Fc fusion protein had no effect
on muscle mass at doses of 1 and 10 mg/kg/week. Similarly, little
or no effect was seen following injection of the aspartate to
alanine mutant pro peptide/Fc fusion protein at a dose of 1
mg/kg/week. However, injection of the mutant pro peptide/Fc fusion
protein at 10 mg/kg/week led to a statistically significant
(p<0.0001) increase of 18-27% in the weight of each skeletal
muscle examined. This magnitude of increase in muscle weights
observed at the higher dose of the mutant pro peptide/Fc fusion
protein was approximately twice that seen following injection of
the JA16 myostatin neutralizing monoclonal antibody, which resulted
in muscle weight increases of 10-16%.
[0110] These results demonstrate that members of the BMP-1/TLD
family of metalloproteinases cleave myostatin pro peptide bound to
the C-terminal dimer and activate the latent complex. Furthermore,
a mutant form of the pro peptide that was resistant to cleavage by
BMP-1/TLD proteinases caused increases in muscle mass when injected
into adult mice, presumably by forming latent complexes incapable
of being activated by this group of proteinases. This general
mechanism for regulating the activity of the C-terminal dimer has
been described for certain other TGF-.beta. family members. In the
case of TGF-.beta., proteolytic cleavage of its associated pro
peptide by plasmin.sup.10,11 or by matrix
metalloproteinases.sup.12-14 is believed to be one mechanism for
activating latency in vivo. In the case of the BMPs, members of the
BMP-1/TLD family appear to play an important role in regulating the
activity of the C-terminal dimer by cleaving and inactivating the
BMP antagonist chordin.sup.15,19-22.
[0111] All four mammalian proteinases in the BMP-1/TLD family can
cleave the myostatin pro peptide in vitro, and one or more can be
involved in regulating myostatin activity in vivo. In this regard,
mTLL-2, unlike the other three proteinases, is expressed
specifically in skeletal muscle during embryonic
development.sup.15. The identification of the specific proteinase
or proteinases involved in regulating myostatin latency will
provide targets for identifying agents useful for modulating muscle
mass, and will allow targeting of these enzymes for the development
of novel muscle enhancing agents for both human therapeutic and
agricultural applications.
2TABLE 1 pectoralis triceps quadriceps gastrocnemius tibialis PBS
(n = 10) 82.8 .+-. 2.8 85.5 .+-. 1.6 142.0 .+-. 2.6 95.5 .+-. 1.5
32.6 .+-. 0.8 IgG2am 87.7 .+-. 1.9 87.8 .+-. 1.6 148.4 .+-. 2.3
98.7 .+-. 2.1 33.8 .+-. 0.9 (10 mg/kg, n = 10) wild type 84.3 .+-.
1.6 85.3 .+-. 1.8 145.7 .+-. 2.2 96.0 .+-. 1.4 33.2 .+-. 0.4 (1
mg/kg, n = 10) D76A 89.4 .+-. 3.5 90.0 .+-. 2.0 150.7 .+-.
2.9.sup.a 97.7 .+-. 2.2 34.1 .+-. 0.6 (1 mg/kg, n = 9) wild type
87.5 .+-. 3.4 88.5 .+-. 2.7 147.1 .+-. 4.0 98.2 .+-. 2.2 33.3 .+-.
0.8 (10 mg/kg, n = 10) D76A 105.1 .+-. 1.2.sup.b,c 102.1 .+-.
1.2.sup.b,d 175.6 .+-. 1.2.sup.b,e 112.6 .+-. 1.1.sup.b,d 40.3 .+-.
1.2.sup.b,d (10 mg/kg, n = 10) JA16 96.0 .+-. 1.2.sup.f 94.8 .+-.
1.1.sup.f 160.3 .+-. 1.1.sup.b 104.9 .+-. 1.1.sup.f 37.5 .+-.
1.1.sup.f (60 mg/kg, n = 10) .sup.ap < 0.05 (vs. PBS) .sup.bp
< 0.0001 (vs. PBS) .sup.cp < 0.05 (vs. JA16) .sup.dp <
0.01 (vs. JA16) .sup.ep < 0.001 (vs. JA16) .sup.fp < 0.001
(vs. PBS)
[0112] Each of the following publications is incorporated herein by
reference:
[0113] 1. McPherron, A. C., Lawler, A. M. & Lee, S.-J.
Regulation of skeletal muscle mass in mice by a new TGF-.beta.
superfamily member. Nature 387, 83-90 (1997).
[0114] 2. Bogdanovich, S. et al. Functional improvement of
dystrophic muscle by myostatin blockade. Nature 420, 418-421
(2002).
[0115] 3. Wagner, K. R., McPherron, A. C., Winik, N. & Lee,
S.-J. Loss of myostatin attenuates severity of muscular dystrophy
in mdx mice. Ann Neurol 52, 832-836 (2002).
[0116] 4. McPherron, A. C. & Lee, S.-J. Suppression of body fat
accumulation in myostatin-deficient mice. J Clin Invest 109,
595-601 (2002).
[0117] 5. Lee, S.-J. & McPherron, A. Regulation of myostatin
activity and muscle growth. Proc Natl Acad Sci USA 98, 9306-9311
(2001).
[0118] 6. Thies, R. et al. GDF-8 propeptide binds to GDF-8 and
antagonizes biological activity by inhibiting GDF-8 receptor
binding. Growth Factors 18, 251-259 (2001).
[0119] 7. Zimmers, T. et al. Induction of cachexia in mice by
systemically administered myostatin. Science 296, 1486-1488
(2002).
[0120] 8. Hill, J. J. et al. The myostatin propeptide and the
follistatin-related gene are inhibitory binding proteins of
myostatin in normal serum. J Biol Chem 277, 40735-40741 (2002).
[0121] 9. Hill, J. J., Qiu, Y., Hewick, R. M. & Wolfman, N. M.
Regulation of myostatin in vivo by GASP-1: a novel protein with
protease inhibitor and follistatin domains. Mol Endocrin 17,
1144-1154 (2003).
[0122] 10. Lyons, R. M., Keski-Oja, J. & Moses, H. L.
Proteolytic activation of latent transforming growth factor-.beta.
from fibroblast-conditioned medium. J. Cell Biol. 106, 1659-1665
(1988).
[0123] 11. Sato, E. & Rifkin, D. Inhibition of endothelial cell
movement by pericytes and smooth muscle cells: activation of a
latent transforming growth factor-.beta.1-like molecule by plasmin
during co-culture. J Cell Biol 109, 309-315 (1989).
[0124] 12. Yu, Q. & Stamenkovic, I. Cell surface-localized
matrix metalloproteinase-9 proteolytically activates TGF-.beta. and
promotes tumor invasion and angiogenesis. Genes Dev 14, 163-176
(2000).
[0125] 13. D'Angelo, M., Billings, P., Pacifici, M., Leboy, P.
& Thorsten, K. Authentic matrix vesicles contain active
metalloproteases (MMP). J. Biol Chem 276, 11347-11353 (2001).
[0126] 14. Maeda, S., Dean, D., Gay, I., Schwartz, Z. & Boyan,
B. Activation of latent transforming growth factor .beta.1 by
stromelysin 1 in extracts of growth plate chondrocyte-derived
matrix vesicles. J Bone Min Res 16, 1281-1290 (2001).
[0127] 15. Scott, I. et al. Mammalian BMP-1/Tolloid-related
metalloproteinases, including novel family member mammalian
Tolloid-like 2, have differential enzymatic activities and
distributions of expression relevant to patterning and
skeletogenesis. Dev Biol 213, 283-300 (1999).
[0128] 16. Scott, I. C. et al. Bone morphogenetic protein-I
processes probiglycan. J Biol Chem 275, 30504-30511 (2000).
[0129] 17. Lee, S.-T., Kessler, E. & Greenspan, D. S. Analysis
of site-directed mutations in human pro-a2(I) collagen which block
cleavage by the C-proteinase. J Biol Chem 265, 21992-21996
(1990).
[0130] 18. Whittemore, L.-A. et al. Inhibition of myostatin in
adult mice increases skeletal muscle mass and strength. BBRC 300,
965-971 (2003).
[0131] 19. Blader, P., Rastegar, S., Fischer, N. & Strahle, U.
Cleavage of the BMP-4 antagonist chordin by zebrafish tolloid.
Science 278, 1937-1940 (1997).
[0132] 20. Piccolo, S. et al. Cleavage of chordin by Xolloid
metalloprotease suggests a role for proteolytic processing in the
regulation of Spemann organizer activity. Cell 91, 407-416
(1997).
[0133] 21. Marques, G. et al. Production of a DPP activity gradient
in the early Drosophila embryo through the opposing actions of the
SOG and TLD proteins. Cell 91, 417-426 (1997).
[0134] 22. Pappano, W., Steiglitz, B., Scott, I. C., Keene, D. R.
& Greenspan, D. S. Use of Bmp1/Tll1 doubly homozygous null mice
and proteomics to identify and validate in vivo substrates of BMP-1
tolloid-like metalloproteinases. Mol Cell Biol 23, 4428-4438
(2003).
[0135] 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
23 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 1128 DNA
Bovine CDS (1)..(1125) 3 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 4 375 PRT Bovine 4 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 5 1128 DNA Gallus gallus CDS (1)..(1125) 5 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 6 375 PRT Gallus gallus 6 Met Gln Lys Leu Ala Val Tyr
Val Tyr Ile Tyr Leu Phe Met Gln Ile 1 5 10 15 Ala Val Asp 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 Thr 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
7 1125 DNA Danio rerio CDS (1)..(1122) 7 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 8 374 PRT Danio rerio 8
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 9 50 PRT Homo sapiens 9 Lys Asp Val Ile Arg Gln
Leu Leu Pro Lys Ala Pro Pro Leu Arg Glu 1 5 10 15 Leu Ile Asp Gln
Tyr Asp Val Gln Arg Asp Asp Ser Ser Asp Gly Ser 20 25 30 Leu Glu
Asp Asp Asp Tyr His Ala Thr Thr Glu Thr Ile Ile Thr Met 35 40 45
Pro Thr 50 10 50 PRT Artificial sequence Mutant peptide portion of
human myostatin 10 Lys Asp Val Ile Arg Gln Leu Leu Pro Lys Ala Pro
Pro Leu Arg Glu 1 5 10 15 Leu Ile Asp Gln Tyr Asp Val Gln Gln Asp
Asp Ser Ser Asp Gly Ser 20 25 30 Leu Glu Asp Asp Asp Tyr His Ala
Thr Thr Glu Thr Ile Ile Thr Met 35 40 45 Pro Thr 50 11 50 PRT
Artificial sequence Mutant peptide portion of human myostatin 11
Lys Asp Val Ile Arg Gln Leu Leu Pro Lys Ala Pro Pro Leu Arg Glu 1 5
10 15 Leu Ile Asp Gln Tyr Asp Val Gln Arg Ala Asp Ser Ser Asp Gly
Ser 20 25 30 Leu Glu Asp Asp Asp Tyr His Ala Thr Thr Glu Thr Ile
Ile Thr Met 35 40 45 Pro Thr 50 12 40 PRT Homo sapiens 12 Gln Leu
Leu Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr 1 5 10 15
Asp Val Gln Arg Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp 20
25 30 Tyr His Ala Thr Thr Glu Thr Ile 35 40 13 40 PRT Artificial
sequence Mutant peptide portion of human myostatin 13 Gln Leu Leu
Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr 1 5 10 15 Asp
Val Gln Gln Asp Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp 20 25
30 Tyr His Ala Thr Thr Glu Thr Ile 35 40 14 40 PRT Artificial
sequence Mutant peptide portion of human myostatin 14 Gln Leu Leu
Pro Lys Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr 1 5 10 15 Asp
Val Gln Arg Ala Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp 20 25
30 Tyr His Ala Thr Thr Glu Thr Ile 35 40 15 30 PRT Homo sapiens 15
Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val Gln Arg Asp 1 5
10 15 Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp Tyr His Ala 20 25
30 16 30 PRT Artificial sequence Mutant peptide portion of human
myostatin 16 Ala Pro Pro Leu Arg Glu Leu Ile Asp Gln Tyr Asp Val
Gln Gln Asp 1 5 10 15 Asp Ser Ser Asp Gly Ser Leu Glu Asp Asp Asp
Tyr His Ala 20 25 30 17 30 PRT Artificial sequence Mutant peptide
portion of human myostatin 17 Ala Pro Pro Leu Arg Glu Leu Ile Asp
Gln Tyr Asp Val Gln Arg Ala 1 5 10 15 Asp Ser Ser Asp Gly Ser Leu
Glu Asp Asp Asp Tyr His Ala 20 25 30 18 20 PRT Homo sapiens 18 Glu
Leu Ile Asp Gln Tyr Asp Val Gln Arg Asp Asp Ser Ser Asp Gly 1 5 10
15 Ser Leu Glu Asp 20 19 20 PRT Artificial sequence Mutant peptide
portion of human myostatin 19 Glu Leu Ile Asp Gln Tyr Asp Val Gln
Gln Asp Asp Ser Ser Asp Gly 1 5 10 15 Ser Leu Glu Asp 20 20 20 PRT
Artificial sequence Mutant peptide portion of human myostatin 20
Glu Leu Ile Asp Gln Tyr Asp Val Gln Arg Ala Asp Ser Ser Asp Gly 1 5
10 15 Ser Leu Glu Asp 20 21 10 PRT Homo sapiens 21 Tyr Asp Val Gln
Arg Asp Asp Ser Ser Asp 1 5 10 22 10 PRT Artificial sequence Mutant
peptide portion of human myostatin 22 Tyr Asp Val Gln Gln Asp Asp
Ser Ser Asp 1 5 10 23 10 PRT Artificial sequence Mutant peptide
portion of human myostatin 23 Tyr Asp Val Gln Arg Ala Asp Ser Ser
Asp 1 5 10
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