1 Animal Genomics, AgResearch, Hamilton, New Zealand; and 2 Department of Biological Sciences, University of Waikato, Hamilton, New Zealand
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ABSTRACT |
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Myostatin, a
member of the transforming growth factor- superfamily, is a secreted
growth factor that is proteolytically processed to give COOH-terminal
mature myostatin and NH2-terminal latency-associated peptide in myoblasts. Piedmontese cattle are a heavy-muscled breed that
express a mutated form of myostatin in which cysteine
(313) is substituted with tyrosine. Here we have
characterized the biology of this mutated Piedmontese myostatin.
Northern and Western analyses indicate that there is increased
expression of myostatin mRNA and precursor myostatin protein in the
skeletal muscle of Piedmontese cattle. In contrast, a decrease in
mature myostatin was observed in Piedmontese skeletal muscle. However,
there is no detectable change in the circulatory levels of mature
myostatin in Piedmontese cattle. Myoblast proliferation assay performed
with normal and Piedmontese myostatin indicated that mature wild-type
myostatin protein inhibited the proliferation of
C2C12 myoblasts. Piedmontese myostatin, by
contrast, failed to inhibit myoblast proliferation. In addition, when
added in molar excess, Piedmontese myostatin acted as a potent
"competitive inhibitor" molecule. These results indicate that, in
Piedmontese myostatin, substitution of cysteine with tyrosine
results in the distortion of the "cystine knot" structure and a
loss of biological activity of the myostatin. This mutation also
appears to affect either processing or stability of mature myostatin
without altering the secretion of myostatin.
growth and differentiation factor 8; transforming growth
factor-
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INTRODUCTION |
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MYOSTATIN, ALSO KNOWN
AS growth and differentiation factor 8 (GDF-8), is a member of
the transforming growth factor (TGF)- superfamily. The targeted
deletion of the entire COOH-terminus of myostatin in mice
(8) and several naturally occurring mutations in the
myostatin gene in cattle (3) have been shown to
cause heavy muscling (also referred to as double muscling), mainly
resulting from hyperplasia. In the Belgian Blue cattle breed, for
instance, there is an 11-bp deletion causing a frame shift and
premature translational termination of myostatin (5). In
another heavy-muscled cattle breed, Piedmontese, there is a
guanine-to-adenine transition at position 938 (G938A) of
myostatin, causing a substitution of a critical cysteine to
a tyrosine in the signaling portion of myostatin (5). More
recently, Grobet et al. (3) have identified seven DNA
sequence polymorphisms of which five were predicted to disrupt
the function of myostatin in various European breeds of double-muscled
cattle. These studies have been used to clearly establish
myostatin as a negative regulator of skeletal muscle growth
in mammals.
Myostatin expression is first detected in the muscle precursor cells of the dermomyotome of somites, and the expression continues in the adult muscle (8). In addition to skeletal muscle, low levels of myostatin expression have also been detected in the mammary gland (4), heart (12), and brain (11). Despite the detection of myostatin in tissues other than skeletal muscle, the prominent phenotype observed in myostatin-null mice is one of increased skeletal muscle mass.
Function and structure studies of myostatin and other TGF- family
members have revealed insights into the biology of myostatin. Myostatin
is synthesized in myoblasts as a 52-kDa propeptide that appears to be
cleaved at RSRR (263-266), a furin protease site (9). This cleavage gives rise to a 26-kDa mature myostatin of 109 amino acids in length that is secreted to elicit its biological function. We have previously shown that a recombinant mature myostatin can regulate the proliferation of myoblasts (13). Mature
myostatin has also recently been suggested to function by binding to
the activin type II B receptor and possibly signals through the TGF-
type I receptor ALK5 (6).
Previous studies have also established that the mature portion of
TGF- members adopt a cystine knot structure (14), are secreted, and function as homodimers. Similarly, out of the nine cysteine residues in mature myostatin, six cysteine residues are positioned in a perfect spacing to form a cystine knot structure that
is seen in TGF-
(Fig. 1). Like
TGF-
, it is also established that mature myostatin forms a homodimer
(6) and is secreted (2).
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In the Piedmontese cattle, the third cysteine (C3; amino
acid 313) of myostatin is substituted with a tyrosine (Fig. 1). In this
manuscript, we have characterized the biology of Piedmontese myostatin.
Because cysteine mutations in TGF- superfamily members can lead to
aberrant signaling (14), we have characterized the synthesis, processing, secretion, and biological activity of
Piedmontese myostatin. Here we report that, although the circulatory
levels of myostatin in Piedmontese cattle are unaffected, mature
Piedmontese myostatin fails to inhibit the growth of myoblasts in
vitro. Given that C3 is crucial for intramolecular
disulfide linkage in the cystine knot structure, it is likely that the
substitution of this cysteine to a tyrosine inactivates Piedmontese
myostatin by distorting the cystine knot structure required for
receptor binding.
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MATERIALS AND METHODS |
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Expression and purification of mature myostatin. Expression and purification of wild-type mature bovine myostatin (amino acids 267-375) has been described previously (13).
The pET protein expression system (Novagen, Madison, WI) was used to express and purify recombinant Piedmontese myostatin. Piedmontese myostatin coding sequencing, spanning amino acids 267-375, was PCR amplified using genomic DNA and PCR primers as described previously (13). Amplicons were cloned into pET 16-B vector, in-frame with the 10-histidine tag according to the manufacturer's protocol (Novagen). An overnight BL 21 Escherichia coli culture transformed with the Piedmontese myostatin expression vector was diluted and grown to an optical density of 0.8 (600 nm) in 1 liter of Lennox L broth medium containing ampicillin (50 mg/l). Expression of the myostatin fusion protein was induced by adding 0.5 mM isopropyl thio-C2C12 myoblast proliferation assay. C2C12 myoblasts (15) were grown, unless otherwise stated, in DMEM (Life Technologies, Grand Island, NY) buffered with 41.9 mM NaHCO3 (Sigma Cell Culture, St. Louis, MO) and 6% gaseous CO2, resulting in an initial media pH of ~7.4. Phenol red (7.22 nM; Sigma) was used as a pH indicator. Penicillin (1 × 105 IU/l; Sigma), 100 mg/l streptomycin (Sigma), and 10% FBS (Life Technologies) were routinely added to media.
Cell proliferation assays were carried out in uncoated 96-well Nunc microtitre plates (Nunc, Roskilde, Denmark). C2C12 cultures were seeded at 1,000 cells/well. After a 16-h attachment period, myostatin test media (either recombinant wild-type myostatin or Piedmontese myostatin) was added, and cells were incubated for a further 72 h. The test wells in the plate were randomly assigned, and all tests were run in replicates of eight. Although such proliferation assays were repeated at the very least two times for reproducibility, the results presented in this paper are SE of eight replicates obtained from one experiment. After the incubation period, proliferation was assessed using a methylene blue photometric end-point assay, as previously described (10). In this assay, absorbance at 655 nm is directly proportional to final cell number. To test whether Piedmontese myostatin could compete with wild-type myostatin, myoblasts were incubated with either wild-type myostatin alone or with increasing molar excess concentrations of Piedmontese myostatin. Proliferation of myoblasts was assessed as described above.Western analysis. Fifteen micrograms of muscle (musculus semitendinosus) extracts or plasma proteins were separated by SDS-PAGE (4-12% gradient gels) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in 50 mM Tris (pH 7.4), 100 mM NaCl, and 0.1% Tween 20 (TBST) and incubated with purified rabbit anti-myostatin antibodies (0.5 mg/ml; see Ref. 12) in 5% nonfat milk in TBST at room temperature for 3 h. The blot was washed (5 × 5 min) with TBST buffer and further incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (HRP; DAKO, Carpinteria, CA) for 1 h at room temperature. The membrane was washed as before, and HRP activity was detected using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) according to the manufacturer's protocol.
Northern analysis.
Total RNA (15 µg) from musculus semitendinosus was separated on a 1%
agarose-formaldehyde gel and transferred to a Hybond N+
membrane (Amersham). The membrane was autocross-linked and
prehybridized in 5× saline-sodium citrate (SSC), 50% formamide, 5×
Denhardt's solution, 1% SDS, and 0.25 mg/ml salmon sperm DNA for
2 h. Hybridization was carried out in the same solution with
bovine myostatin cDNA probe overnight at 42°C. The membrane was
washed at 50°C for 15 min with 2× SSC and 0.5% SDS and then with
1× SSC and 0.5% SDS. The bovine myostatin cDNA was obtained as
previously described (5) and radioactively labeled using
[-32P]dCTP (Amersham Pharmacia Biotech) and a
Rediprime II labeling kit (Amersham Pharmacia Biotech) according to the
manufacturer's protocol.
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RESULTS |
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Expression of myostatin mRNA in normal and Piedmontese cattle.
Piedmontese cattle, which are characterized by double muscling, express
a mutant form of myostatin as a result of a guanine-to-adenine substitution at nucleotide position 938. To determine if this mutation
leads to unstable myostatin mRNA and hence the double-muscling phenotype, we performed Northern blot analysis on the total RNA isolated from the skeletal muscle of normal and Piedmontese cattle. As
shown in Fig. 2, the 2.9-kb myostatin
mRNA is detected in the muscle of both normal and Piedmontese cattle.
Interestingly, more myostatin mRNA is detected in the skeletal muscle
of Piedmontese compared with normal cattle (Fig. 2B). This
result suggests that myostatin may, in part, regulate its own
expression by a "feedback inhibition" mechanism.
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Processing of myostatin in cattle.
Because the cysteine-to-tyrosine mutation may also affect the
translation efficiency, stability, or cleavage of the protein, we next
assessed the synthesis and processing of myostatin in Piedmontese
cattle. Total protein was extracted from the musculus semitendinosus of
normal and Piedmontese cattle and subjected to Western blot analysis.
As shown in Fig. 3A,
myostatin-specific antibodies recognized a 52-kDa precursor, a 40-kDa
latency-associated peptide, and a 26-kDa processed mature myostatin in
both normal and Piedmontese cattle muscle extracts. Densitometry
analysis of the Western blot indicates that there is relatively more
precursor myostatin protein in Piedmontese skeletal muscle, confirming
that increased myostatin mRNA results in the increased translation of
precursor myostatin (Fig. 3C). However, Western analysis
also indicated that there was less mature myostatin protein detected in
Piedmontese skeletal muscle (Fig. 3C). This result indicates that the cysteine-to-tyrosine mutation in Piedmontese myostatin decreases either the processing or stability of myostatin.
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Mature myostatin is secreted in Piedmontese cattle.
Processed mature myostatin has been previously shown to be secreted
(2) and biologically active (13). Because
mutations in the mature TGF- affecting the cysteine residues have
been shown to affect secretion (1), we investigated if the
double muscling seen in Piedmontese cattle is the result of abnormal myostatin secretion. To quantify the circulatory levels of myostatin in
normal and Piedmontese cattle, we collected blood and performed Western
blot analysis on plasma proteins. The Western analysis showed that the
26-kDa myostatin protein is detected in the plasma of both normal and
Piedmontese cattle (Fig. 3B). Moreover, there appears to be
no change in the circulatory levels of myostatin between normal and
Piedmontese cattle (Fig. 3D).
Expression and purification of mutant myostatin protein.
Because the cysteine-to-tyrosine mutation is predicted to disrupt the
cystine knot structure of Piedmontese myostatin, the biological
activity of Piedmontese mature myostatin was evaluated. To test the
biological activity, we expressed both wild-type and Piedmontese mature
myostatin as His-tagged proteins in Escherichia coli. The
expressed histidine fusion proteins were purified on a Ni-Agarose
column and separated by SDS-PAGE to check for purity. As shown on a
Coomassie blue-stained gel (Fig. 4), a
15-kDa myostatin fusion protein was purified to a high degree in a
single step. A typical yield of 6 mg of myostatin protein was purified
per liter of induced bacterial culture. Furthermore, there appears to
be no degradation of the recombinant myostatin during purification (Fig. 4).
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Piedmontese myostatin does not inhibit myoblast proliferation.
To test the biological activity of the normal and mutant myostatin, we
incubated actively growing myoblast cultures with either wild-type or
Piedmontese myostatin protein for 72 h and determined the myoblast
number by methylene blue assay. As shown in Fig. 5A, increasing concentrations
of wild-type myostatin inhibited the growth of myoblasts, with
half-maximal inhibition occurring at a myostatin concentration of
~3.5 µg/ml. Moreover, myostatin treatment did not cause any
morphological changes to the myoblasts (Fig. 5C). This is
consistent with our previous observation (13). In contrast
to wild-type myostatin, the Piedmontese myostatin protein failed to
inhibit the proliferation of myoblasts, even at the concentration of 10 µg/ml (Fig. 5B). Furthermore, not only did Piedmontese
myostatin fail to inhibit myoblast proliferation, high concentrations
significantly increased the proliferation of
C2C12 cells compared with nontreated control
myoblasts (Fig. 5B). These results suggest the
cysteine-to-tyrosine substitution at amino acid position 313 results in
a biologically inactive myostatin molecule. In addition, the inactive
Piedmontese myostatin appears to be acting as a "competitive
inhibitor" protein over the wild-type myostatin already present in
the media.
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Piedmontese myostatin functions as a competitive inhibitor.
To experimentally prove that the mutant myostatin can compete with the
wild-type myostatin, myoblasts were incubated with either wild-type
myostatin alone or with wild-type myostatin and increasing molar
concentrations of Piedmontese myostatin. As shown in Fig.
6, a 1.5 µg/ml concentration of
wild-type myostatin inhibited the growth of myoblasts
(P = 0.018). Increased molar ratios of Piedmontese
protein over the wild-type protein rescued the myoblasts from this
growth inhibitory effect, with a one-to-one molar ratio of Piedmontese
myostatin ablating the observed wild-type myostatin inhibition. These
results are consistent with the Piedmontese myostatin competing with
the wild-type myostatin for receptor binding, thereby neutralizing
wild-type myostatin function.
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DISCUSSION |
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Myostatin, a member of the TGF- superfamily, is a key regulator
of skeletal muscle growth. The targeted deletion of
myostatin in mice (8) and natural mutations in
bovine myostatin (5) lead to hyperplasia of
skeletal muscle. Of the myostatin mutations that are
described in double-muscled cattle, the Piedmontese allele is very
interesting, since a single-point mutation (which results in
replacement of cysteine 313 with tyrosine) causes heavy muscling (5). In this communication, we have characterized the
molecular basis for the double-muscle phenotype caused by the
Piedmontese myostatin. The results indicate that the
cysteine-to-tyrosine transition does not seem to affect the circulatory
levels of myostatin. Rather, the mutation appears to interfere with
receptor binding ability, possibly through distortion of the cystine
knot structure. Furthermore, we show that Piedmontese myostatin acts as
a potent competitive inhibitor molecule.
TGF- family member proteins are proteolytically processed at the
site of synthesis, and mature biologically active peptides are secreted
in blood (9). Therefore, the biological function of mature
myostatin may be affected at the level of synthesis, processing, or
secretion. To test if the Piedmontese mutation in myostatin
affects mRNA transcription or stability, we performed Northern blot
analysis on total RNA from skeletal muscle of Piedmontese and normal
cattle. The results indicated that there is an increased expression of
myostatin mRNA in Piedmontese skeletal muscle compared with the normal
skeletal muscle (Fig. 2). The increased myostatin expression seen in
double-muscled Piedmontese cattle may be attributed to lack of feedback
inhibition of myostatin expression by nonfunctional myostatin. Indeed,
unpublished results from our laboratory using the myostatin
promoter and luciferase reporter gene support this argument, since
mature recombinant myostatin negatively regulates the myostatin promoter.
Experiments altering cysteine residues in the mature portion of TGF-
resulted in aberrant cleavage, suggesting that disulfide bonding may be
required for proteolytic processing (1). To test if this
may be the case for Piedmontese myostatin, we performed Western blot
analysis to determine the processing of myostatin protein in
Piedmontese cattle. In agreement with the Northern blot results, we
observed a severalfold increase in the levels of Precursor myostatin in
the Piedmontese relative to normal-muscled cattle. However, there
appears to be a decrease in the levels of mature myostatin protein
(Fig. 3, A and C). This paradox could be because
of 1) inefficient processing of the mutant myostatin protein
or 2) decreased stability of the mutant myostatin.
Systematic mutations of most cysteine residues within the mature
TGF- protein have also been shown to result in the loss of secretion
(1). To test this possibility, we performed Western blot
analysis on normal-muscled and Piedmontese cattle plasma to quantify
the levels of mature myostatin in the circulation. Although there are
decreased levels of mature myostatin at the site of synthesis, the
circulatory levels of mature myostatin do not appear to be different
between normal-muscled and Piedmontese cattle (Fig. 3, B and
C).
To test the biological function of Piedmontese myostatin, we used the
myoblast proliferation assay (13). We show that increasing concentrations of wild-type mature myostatin inhibited the
proliferation of C2C12 myoblasts in culture
(Fig. 5A), which is consistent with the negative regulatory
function of myostatin (13). By contrast, the Piedmontese
myostatin could not inhibit the proliferation of actively growing
C2C12 cells, even at a very high concentration (Fig. 5B). TGF- family members function by binding to
their respective receptors, initiating a downstream signaling cascade
(7). For receptor binding, mature TGF-
family members
attain a cystine knot structure mediated by six cysteine residues in
the mature portion of the molecule (14). These cysteine
residues are thus highly conserved in TGF-
family members and are
classified as 10-membered cystine knot proteins (14).
Myostatin has nine cysteine residues within the mature portion of the
peptide, six of which are appropriately spaced to form a cystine
knot-forming domain identical to that in TGF-
(Fig. 1). It is also
noteworthy that a recent scanning of a human cDNA sequence database for
10-membered cystine knot proteins has identified GDF-8 (myostatin) as
one of the proteins that contains a potential cystine knot structure (14). The six cysteines involved in knot formation are
spaced out with intervening amino acids. Cysteines 2 and 3 form
intrachain disulfide bonds with cysteines 5 and 6, respectively, thus
forming a ring. The third disulfide bond formed between cysteines 1 and 4 penetrates the ring. With Piedmontese myostatin, the cysteine at
position 313 (which is C3 in the cystine knot) is
substituted with tyrosine and thus would not be able to form the
required intrachain disulfide linkage, thereby distorting the cystine
knot structure. This would probably affect receptor binding or receptor interaction. It has recently been confirmed that mature myostatin preferentially binds to activin type IIB receptor (6).
When a high concentration of Piedmontese myostatin was used in the bioassay, we observed that C2C12 myoblasts actually proliferated significantly faster than the myoblasts incubated in the control media (Fig. 5B). This indicates that the mutant myostatin added to the actively growing media is acting as a "competitor" to the wild-type myostatin secreted in the media by the C2C12 cells. Furthermore, in a competition experiment in which wild-type myostatin protein was incubated with increasing concentrations of Piedmontese protein, a decrease in the inhibitory effect of wild-type protein was seen (Fig. 6). This experiment confirmed that the Piedmontese myostatin indeed acts as a competitor over the wild-type myostatin. Two scenarios can be used to explain this neutralizing effect of Piedmontese myostatin. When added in excess, Piedmontese myostatin may bind to the receptor, albeit improperly, thereby outcompeting wild-type myostatin and attenuating its growth inhibitory effect. Alternatively, Piedmontese myostatin may be heterodimerizing with wild-type mature myostatin, resulting in a receptor binding-incompetent myostatin complex. The first scenario is the most plausible, since wild-type myostatin is secreted in a homodimerized form, which would be unlikely to disassociate, allowing Piedmontese myostatin to dimerize with it. However, because the cysteine mutation affects the required cystine knot structure, in either scenario high concentrations of Piedmontese myostatin would act as a myostatin mimetic and possibly disrupt the further downstream signaling by mature wild-type myostatin. Thus Piedmontese myostatin presents itself as a potential inhibitor of myostatin. Experiments regarding the in vivo efficacy of Piedmontese myostatin to inhibit the wild-type myostatin function and myostatin-receptor cross-linking are underway.
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ACKNOWLEDGEMENTS |
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We are indebted to the Animal Breeding Services, Hamilton, New Zealand for providing Piedmontese muscle samples. We also thank Dr. John Bass and the rest of the Functional Muscle genomics group for their continued support.
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FOOTNOTES |
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Funding was provided by the Foundation for Research and Technology (New Zealand).
Address for reprint requests and other correspondence: R. Kambadur, AgResearch, East St., Hamilton, New Zealand (E-mail: Ravi.Kambadur{at}agresearch.co.nz).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published March 6, 2002;10.1152/ajpcell.00458.2001
Received 25 September 2001; accepted in final form 17 February 2002.
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