The C313Y Piedmontese mutation decreases myostatin covalent dimerisation and stability
© Sutherland-Smith et al; licensee BioMed Central Ltd. 2011
Received: 2 August 2011
Accepted: 24 October 2011
Published: 24 October 2011
Myostatin is a key negative regulator of muscle growth and development, whose activity has important implications for the treatment of muscle wastage disorders. Piedmontese cattle display a double-muscled phenotype associated with the expression of C313Y mutant myostatin. In vivo, C313Y myostatin is proteolytically processed, exported and circulated extracellularly but fails to correctly regulate muscle growth. The C313Y mutation removes the C313-containing disulphide bond, an integral part of the characteristic TGF-β cystine-knot structural motif.
Here we present in vitro analysis of the structure and stability of the C313Y myostatin protein that reveals significantly decreased covalent dimerisation for C313Y myostatin accompanied by a loss of structural stability compared to wild type. The C313Y myostatin growth factor, processed from full length precursor protein, fails to inhibit C2C12 myoblast proliferation in contrast to wild type myostatin. Although structural modeling shows the substitution of tyrosine causes structural perturbation, biochemical analysis of additional disulphide mutants, C313A and C374A, indicates that an intact cystine-knot motif is a major determinant in myostatin growth factor stability and covalent dimerisation.
This research shows that the cystine-knot structure is important for myostatin dimerisation and stability, and that disruption of this structural motif perturbs myostatin signaling.
Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors, acting as a primary negative regulator of muscle development and growth [1, 2]. Myostatin over expression in animal models induces profound muscle and fat loss analogous to that seen in human cachexia syndromes [3, 4]. Myostatin signaling can have negative consequences in a diseased background such as the muscular dystrophies  and may contribute to cachexia associated with many chronic disease states  including HIV  and cancer . Hence myostatin has been suggested to hold exciting potential for inhibitory targeting in a wide range of muscle wastage diseases [8, 9].
Similar to other TGF-β family members myostatin is translated as a precursor protein (MstnPP) consisting of an N-terminal signal sequence, a propeptide domain (residues 21-266) and a growth factor domain (MstnGF, residues 267-374) that contains the characteristic cystine-knot motif [10, 11] and dimerises at the C-terminus via an intermolecular disulfide bond [12–14]. The propeptide region plays a chaperone role assisting folding of the growth factor region [15, 16] before furin proteolysis at a conserved RSRR sequence [1, 14]. The propeptide remains non-covalently associated with the mature dimer regulating its activity and targeting in the latent complex [14, 17]. Myostatin remains latent until a second activating cleavage event in the propeptide region that disrupts the association [18, 19].
A number of myostatin null mutations that result in a double-muscled phenotype have been documented. In one human case a child has increased muscle mass, is unusually strong and does not show any negative effects from the mutation . Myostatin null mutations have also been identified in Texel sheep  and racing whippets . Double-muscled cattle breeds such as the Belgian Blue have been recognized for almost 200 years . The majority of myostatin null phenotypes result from premature stop codons and the ensuing absence of myostatin protein [22, 23]. In contrast, Piedmontese cattle have the myostatin missense mutation G938A that translates to C313Y myostatin protein with the consequent loss of one of the disulphide bonds (C313-C374) involved in the characteristic TGF-β family cystine-knot structural motif. Compared to wild type, Piedmontese cattle skeletal muscle C313Y myostatin precursor protein (C313Y-MstnPP) is translated at greatly elevated levels (> 10-fold) but the C313Y mature growth factor (C313Y-MstnGF) is detected at significantly reduced levels in skeletal muscle while circulating levels are similar . Refolded E. coli expressed C313Y-MstnGF failed to inhibit muscle cell proliferation and acted as a dominant negative inhibitor of wild type (WT) myostatin .
C313 mutations decrease myostatin disulphide-linked covalent dimerisation
C313 mutant myostatin precursor protein structures are similar to WT but show decreased stability
Structural modeling and tryptic digestion of C313Y-myostatin precursor protein suggest structural perturbation
Furin processing and secondary structure of C313Y-myostatin are maintained
C313Y myostatin growth factor proteolytically processed from C313Y-MstnPP fails to inhibit myoblast proliferation
In summary, in vitro characterization of C313Y myostatin shows that removal of the C313-C374 disulphide bond by site-directed mutagenesis decreases myostatin covalent dimerisation with subsequent loss of activity and lowered stability. An intact cystine-knot structural motif is essential for myostatin dimerisation and function.
Production of C313Y, C313A and C374A myostatin proteins
Mutant MstnPP expression constructs were prepared by PCR from the WT myostatin construct described previously . PCR fragments were cloned into a modified pET vector via BamHI and XhoI restriction sites and constructs confirmed by sequencing. The E. coli BL21 (DE3) expression, refolding and purification of the mutant proteins was conducted using the procedure previously described for WT myostatin .
Analysis of disulphide-linked dimerisation by reducing and non-reducing SDS-PAGE
Intermolecular disulphide formation was analysed by visualising purified myostatin (1 mg/ml) on reducing (R) versus non-reducing (NR) SDS-PAGE. All conditions were identical except for the presence (R) or absence (NR) of reducing agent (β-mercaptoethanol, 1.4 M) in the SDS sample buffer. Band densities were quantified with a correction for background staining using gel densitometry. The Student's t-test was performed using GraphPad Prism (GraphPad Software, Inc) using triplicate gels.
Circular dichroism spectroscopy and thermal denaturation
CD spectra in the far-UV region (195-240 nm) were measured for myostatin (1 mg/mL) with a Chirascan CD spectrometer (Applied Biophysics) using a 0.1 mm cell at 4°C. For each sample 20 spectra of 1 nm interval were collected every 2.5 seconds, followed by baseline subtraction, averaging and smoothing. For CD thermal denaturation, 1 nm/2.5 second readings were taken at every 5°C (precursor protein) or 10°C (latent complex) increase in temperature from 5 - 90°C with a 30 second equilibration time at each temperature and a tolerance level of 0.2°C. The change in C313Y-MstnPP CD signal was normalized by the decreased initial proportion of β-sheet for, relative to MstnPP, before thermally-induced denaturation (Figure 3A).
Fluorescence-based thermal shift assays
Sypro Orange (Sigma) was diluted 100x according to manufacturer's instructions in milliQ H2O with 2 μL then added to 18 μL myostatin (1 mg/ml in 50 mM Tris-HCl, 150 mM NaCl, pH 8.5). Negative controls contained buffer and dye only. A Rotor-Gene 6000 thermocycler (Corbett) was used for analysis with excitation at 470 nm and fluorescence emission measured at 555 nm over increasing temperature from 30 to 95°C in 1°C increments. Melting temperatures were calculated with the Rotor-Gene 6000 software.
The myostatin coordinates from the myostatin/follistatin complex structure (PDB code: 3HH2) were used to model the C313Y, C313A and C374A mutations with the amino acid substitution and rotamer tools in Coot . Structural figures were prepared with PyMol .
Protease resistance analysis
Protease resistance was assayed by incubating C313Y-MstnPP at a w/w ratio of 100:1 trypsin (bovine pancreas, Sigma) at either 4°C, room temperature or 37°C. Samples were taken after 0.5, 1, 2, 3, 4 and 18 hours (overnight), denatured, and then analysed by reducing and non-reducing SDS-PAGE. The decrease in concentration of full-length C313Y-MstnPP compared to MstnPP  as a function of time, normalised to the concentration at time zero, was quantified by gel densitometry using Image-J software  Owing to the different relative proportions of MstnPP and C313Y-MstnPP monomer and dimer under non-reducing conditions, the reduced SDS-PAGE monomer bands were quantified enabling direct comparison between the two proteins.
Purified myostatin precursor protein dimer, in HEPES pH 7.5, 150 mM NaCl buffer, was concentrated to 10 mg/mL and furin cleavage buffer (50 mM HEPES pH7.5 and 1 mM CaCl2) was added to a final volume of 250 μL per 1 mg of protein. Human furin convertase (Sigma, 2 U/μL) was added at a ratio of 1 μL furin to 100 μg protein. The reaction was incubated at 30°C for 64 hours and subsequently centrifuged at 17,000 × g, 4°C for 5 minutes to remove precipitated protein.
C2C12 myoblast proliferation myostatin activity assay
C2C12 mouse myoblasts were cultured in Advanced Dulbecco's Modified Eagle's Medium (4.5 g/L D-glucose and 110 mg/L sodium pyruvate; Gibco, Invitrogen) supplemented with 10% foetal calf serum, 4 mM L-glutamine and penicillin/streptomycin. Cells were incubated at 37°C in a 5% CO2 humidified environment and passaged at 80-90% confluency. Cells were plated in fresh medium in optical bottom 96-well plates at a density of 1,000 cells/well. After 24 hours, media was removed and 100 μL/well fresh media containing myostatin (10 μg/mL) was added. Equivalent concentrations of the untreated furin digest and MstnPP were used. Activation of the MstnGF was performed by acid treatment as described previously . Cells were incubated with protein for 72 hours and cell growth was measured using the WST-1 Cell Proliferation Assay (Roche). WST-1 (10 μL) was added to each well and incubated for a further 3 hours. Absorbance at 450 nm and 630 nm was measured in a PowerWave XS 96-well plate reader (BioTek Instruments, Inc). Cells incubated in media only and media containing furin cleavage buffer only were used as negative controls. Three independent experiments each with triplicate wells were used for each condition for WT; two independent experiments were conducted in triplicate for C313Y. The Student's t-test was performed using GraphPad Prism (v5.04, GraphPad Software, San Diego, California, USA).
We acknowledge New Zealand Tertiary Education Commission Top Achiever Doctoral and New Zealand Neuromuscular Alliance Henry Kelsey scholarships awarded to CSS, and grants from the Sir Thomas and Lady Duncan Trust, Massey University Research Fund and the Palmerston North Medical Research Foundation.
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