Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals

Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (MBP) fusion were expressed in E. coli and purified. MSTNpro regions consisting of residues 45–68, -69, and -70 with MBP fusion suppressed MSTN activity with a potency comparable to that of full-sequence flatfish MSTNpro in a pGL3-(CAGA)12-luciferase reporter assay. Even though the MSTN-inhibitory potency was about 1,000-fold lower, the flatfish MSTNpro region containing residues 45–65 (MBP-Pro45-65) showed MSTN-inhibitory capacity but not the MBP-Pro45-64, indicating that the region 45–65 is the minimum domain required for MSTN binding and suppression of its activity. To examine the in vivo effect of MBP-fused, truncated flatfish MSTNpro, MBP-Pro45-70-His6 (20 mg/kg body wt) was subcutaneously injected 5 times for 14 days in mice. Body wt gain and bone mass were not affected by the administration. Grip strength and swimming time were significantly enhanced at 7 d after the administration. At 14 d, the effect on grip strength disappeared, and the extent of the effect on swimming time significantly diminished. The presence of antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 d after the administration with the titer value at 14 d being much greater than that at 7 d, suggesting that antibodies against MBP-Pro45-70-His6 neutralized the MSTN-inhibitory effect of MBP-Pro45-70-His6. We, thus, examined the MSTN-inhibitory capacity and in vivo effect of flatfish MSTNpro region 45–65 peptide (Pep45-65-NH2), which was predicted to have no immunogenicity in silico analysis. Pep45-65-NH2 suppressed MSTN activity with a potency similar to that of MBP-Pro45-65 but did not suppress GDF11, or activin A. Pep45-65-NH2 blocked MSTN-induced Smad2 phosphorylation in HepG2 cells. The administration of Pep45-65 (20 mg/kg body wt, 5 times for 2 weeks) increased the body wt gain with a greater gain at 14 d than at 7 d and muscle wt. Grip strength and swimming time were also significantly enhanced by the administration. Antibody titer against Pep45-65 was not detected. In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45–65 (Pep45-65) can suppress mouse MSTN activity and increase muscle mass and function without invoking an immune response, implying that Pep45-65 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.

Introduction Myostatin (MSTN), a member of the TGF-β superfamily, is a negative regulator of skeletal muscle development and growth. Knockout of the MSTN gene resulted in a dramatic increase in skeletal muscle mass in mice with little effect on other organs [1], while systemic overexpression of MSTN by transgenesis or selective gene transfer in skeletal muscle induced profound muscle atrophy in mice [2][3][4]. Owing to the potent inhibitory role of MSTN on muscle growth, there has been much interest in MSTN-blocking as a strategy to treat muscle atrophy caused by chronic diseases such as cancer, kidney failure, obstructive pulmonary disease, cardiomyopathy, liver cirrhosis, and age-associated sarcopenia [5][6][7]. Approaches like the administration of MSTN-blocking proteins or peptides [8][9][10][11][12][13][14], delivery of MSTN-blocking genes using an adeno-associated viral vector [15][16][17], and micro or short hairpin RNA inhibition of MSTN [18,19] have been used to block MSTN activity. Building on the results in lab animals, some of the MSTN-inhibitors, such as antibodies against MSTN, MSTN peptibody and soluble form of ActRIIB, have been applied in clinical trials [20][21][22][23].
MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN, suppresses MSTN activity by the complex formation in a latent/inactive state [4,24,25]. Members of the bone morphogenetic proteins-1/tolloid (BMP-1/TLD) of metalloproteinases specifically cleaves MSTNpro, leading to activation of MSTN by separating it from the MSTNpro/MSTN latent complex [26,27]. A dramatic increase in skeletal muscle mass was observed in transgenic mice overexpressing MSTNpro [28][29][30]. MSTNpro abundance in vivo by various approaches, including plasmid-mediated delivery [31], adeno-associated virus vector [17], and protein administration [9,11,27,32], also resulted in an increase in muscle mass, enhancement of muscle repair or amelioration of dystrophic pathophysiology in mice. These results together indicate that MSTNpro would be a potential agent to treat muscle atrophy caused by various chronic diseases and conditions.
Studies have shown that mouse MSTNpro region containing amino acid residues within 42-115 is effective in suppressing MSTN activity in vitro [12,13,33,34]. Furthermore, intramuscular injection of the truncated MSTNpro peptides increased muscle mass in Duchenne and caveolin 3-deficient limb-girdle muscular dystrophy model mice [12,13]. In our study, maltose binding protein (MBP)-fused pig MSTNpro region containing residues 42-175 exhibited full MSTN-inhibitory potency [35]. Similarly, flatfish MSTNpro region containing residues 45-80 showed MSTN-inhibitory potency comparable to that of full sequence mouse MSTNpro [36], indicating that MBP fused, truncated MSTNpro peptides would be potential agents to treat muscle wasting conditions. According to Takayama et al. [13], mouse MSTNpro region containing residues 43-67 was effective in suppressing the MSTN activity. It was thus, hypothesized that the minimum region of flatfish MSTNpro for MSTN suppression would potentially be smaller than the region containing residues . The objective of this study was to determine the minimum size of flatfish MSTNpro for MSTN inhibition and examine its effects on muscle growth and performance in mice.

Expression and purification of MBP-fused truncated MSTNpros
The expression plasmids were transformed into E. coli strain K12TB1 (NEB, USA) separately by the heat-shock method. Selected colonies were inoculated in 8 ml of LB medium containing 100 μg/mL ampicillin plus 10 μg/mL streptomycin and incubated at 37˚C for overnight. For the expression of recombinant proteins, the 8 mL overnight-cultures were inoculated in 500 ml of fresh LB medium containing 100 μg/mL ampicillin plus 10 μg/mL streptomycin and incubated at 37˚C. When OD 600 reached 0.3~0.4, IPTG was added to the cultures to a final concentration of 0.3 mM, and the cultures were incubated at 37˚C for 3 h. Cells were harvested by centrifugation at 4,000 rpm, 4˚C for 20 min. Pellets were resuspended in 20 mL of column buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA) containing 1 mM PMSF and were disrupted by sonication for 10 min in an ice water bath. The lysate was centrifuged at 12,000 rpm for 20 min at 4˚C, and the supernatant was collected. MBP-fused truncated MSTNpro proteins were affinity-purified using amylose resin (NEB, MA, USA) as described previously (Lee et al., 2012). Protein concentration was determined by the BCA assay (Thermo Scientific, MA, USA) using BSA as a standard.

Expression and two-step affinity purification of MBP-Pro45-70-His6
A His6 tag was inserted at the C-terminus of pMALc5x-Pro45-70 by site-directed mutagenesis with primer sets in S1 Table. Sequencing analysis confirmed the insertion of the His6 tag.
Protein expression and preparation of cell lysates were carried out as described previously. E. coli lysate was centrifuged at 12,000 rpm for 20 min at 4˚C, and the supernatant was affinitypurified using Ni-NTA agarose (Qiagen, MD, USA). After loading, the column was washed with binding buffer and eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 40 mM imidazole). Elution fractions were subjected to an amylose resin affinity column. Eluted fractions containing the MBP-Pro45-70-His6 were pooled and dialyzed extensively against ultra-pure water using dialysis tubing (Spectrum Inc, Seoul, Korea). Protein concentration was determined by the BCA assay (Thermo Scientific. MA, USA), and then was dried by a speed vacuum concentrator and stored at -20˚C until use.

SDS-PAGE
SDS-PAGE (10% polyacrylamide gel) was performed according to the method of Laemmli [37]. Samples were mixed with loading buffer in the presence of 1.5% β-mercaptoethanol and incubated at 95˚C for 5 min before loading on the gel. Bands were stained with Coomassie brilliant blue solution.

pGL3-(CAGA) 12 luciferase assay
The anti-MSTN activity was measured using a pGL3-(CAGA) 12 -luciferase reporter assay in HEK293 cells stably expressing (CAGA) 12 -luciferase gene construct [38]. HEK293 cells were seeded on 96 well plates at 2×10 4 cells//well and grown in DMEM containing 10% FBS, 1% penicillin/streptomycin and 1% geneticin at 37˚C with 5% CO 2 for 24hr. After 24 hr, the medium was replaced with 100 μL serum-free DMEM. Then 1 nM MSTN, Activin A or Growth and Differentiation factor 11 (GDF11) (R&D systems, MN, USA) plus various concentrations of truncated flatfish MSTNpro were added to each well and incubated for 24 h. After incubation, luciferase activity was measured by a microplate luminometer using the Bright-Glo luciferase assay system (Promega, WI, USA) following the manufacturer's instruction. The percentage inhibition of MSTN activity was calculated by the following formula: Percentage of inhibition capability = (luminescence at ligands (1 nM MSTN, Activin A or GDF11)-luminescence at each truncated propeptide concentration) × 100 / (luminescence at ligands (1 nM MSTN, Activin A or GDF11)-luminescence at 0 nM ligands (MSTN, Activin A or GDF11). IC 50 (ligand concentration inhibiting 50% of MSTN, GDF11 or Activin A activity) values were estimated by a non-linear regression model defining a dose-response curve as described previously [39] using a Prism6 program (GraphPad, CA, USA). The equation for the model was Y = Bottom + (Top-Bottom)/[1+10^(X-LogIC 50 )], where Y is % inhibition, Bottom is the lowest value of % inhibition set at 0%, Top is the highest value of % inhibition set at 100%, and X is Log ligand concentration. IC 50 values were analyzed by ANOVA using the same program.

Animal experiments
ICR male mice were obtained from Hyochang Science (Seoul, Korea). We followed the current regulations for the care and use of laboratory animals under the guidance of the Animal Ethics Committee of Pukyong National University, Busan, Korea. The ethics committee approved this study under protocol AEC-201405. The mice were maintained in a room at a constant temperature of 24±1˚C under a 12 h light/12 h dark cycle at 65% humidity. Animal pellet (Formula M07; FeedLab, Korea) and water were provided ad libitum. For the MBP-Pro45-70-His6 administration study, mice (5~6 week old weighing 24-26 g) were randomly divided into two groups (n = 12 per group). Animals were injected subcutaneously with 0 or 24 mg/kg body wt of MBP-Pro45-70-His6 five times (3 days apart) for 14 days. MBP-Pro45-70-His6 was dissolved in PBS (2 μg/μL) for mice in the treatment group, and PBS was injected for mice in the control group. Body wt was measured at the time of injection.
For the Pep45-65-NH2 administration study, mice (6~8 week old weighing 27-30 g) were randomly divided into two groups (n = 12 per group). Pep45-65-NH2 was dissolved in PBS (2 μg/μL), and subcutaneously injected (10 μL/g body wt) five times (3 days apart) for 14 days for mice in the treatment group. Mice in the control group were injected with PBS. Body wt was measured at the time of injection.

Forelimb grip strength measurement
Forelimb grip strength was measured 1 h after each injection. The tensile force generated by each mouse was measured using a force transducer with a rectangular 4×5 cm 2 metal net (Model-RX-10 Aikoh Engineering Co., Osaka, Japan). Briefly, the mouse was slowly pulled by the tail in the opposite direction while its two forelimbs gripped the metal net. The grip strength meter was recorded in Newton (N). Each mouse was subjected to three times grip test with at least a 1 min rest between trials. The maximum grip force was recorded as the forelimb grip strength. Grip strength (N) was measured on day 0 (before the first injection), day 7, and day 14.

Swimming time measurement
The forced swimming test was performed 1 h after the grip strength measurement. For the forced swimming test, mice were dropped individually in a 1,000 mL beaker (25 cm in height, 10 cm in diameter) filled with water at 24±1˚C. After a 2 min adaptation period, the swimming time (s) was measured following the procedure described previously [40]. Another swimming time was measured at 4 h after the 1 st measurement.

Analysis of blood glucose, cholesterol, triglyceride, and fatty acids
On day 14, the mice were anesthetized with Zoletil 50 (Virbac, Carros, France; 10 mg/kg, intramuscular), and blood was collected by cardiac puncture [41]. The blood, after clotting for 10 min on ice, was centrifuged at 3,000 rpm for 15 min to obtain serum and the serum was stored at -70˚C until use. The serum levels of total cholesterol, triglyceride, and glucose were measured by SD LipidoCare system-analyzer (SD Biosensor, Seoul, Korea). The levels of free fatty acid (FFA, #K612-100) in serum were measured by colorimetric assay kits (Biovision, CA, USA).

Skeletal muscle and bone mass measurement
After peeling the skin off the euthanized mice, forelimbs and hindlimbs were disarticulated from scapula to carpus and from ilium to medial malleolus, respectively. The wt of forelimbs and hindlimbs were immediately measured, as well as the bone wt. The skeletal muscle mass was calculated by subtracting the wt of the bone from those of the forelimbs and hindlimbs, respectively.

ELISA for antibody titer against MBP-Pro45-70-His6
Blood for ELISA was collected on day 7 using the facial vein technique [42] and on day 14 by cardiac puncture. ELISA plates were coated with 100 μL of an MBP-pro45-70-His6 solution (1 μg/mL PBS) per well overnight at 4˚C. The plates were washed three times with PBS, followed by blocking of the plate with 150 μL PBS containing 1% bovine serum albumin at room temperature for 3 h. The plates were washed three times with PBS and added with 100 μL diluted sera (1:1000 with blocking solution) in each well, then incubated at room temperature for 3 h. The plates were washed four times with PBS, then, 100 μL alkaline-phosphate conjugated anti-mouse IgG (Sigma, USA) diluted 1:5,000 with blocking solution was added to each well and incubated at room temperature for 3 h. After washing four times with PBS, 100 μL of p-Nitrophenyl Phosphate (Sigma, USA) liquid substrate solution was added to each well and incubated for 30 min at 37˚C in the dark. The absorbance was measured at 450 nm with a microplate reader (Biotek, Seoul, Korea). All samples were tested in triplicate. The His6 tag was inserted at the C-terminus of Pro45-70 to facilitate further purification and to examine the effect of the presence of His6 tag on MSTN-inhibitory capacity and its administration on muscle growth and performance in mice. SDS-PAGE analysis of the MBP-Pro45-70-His6 during the two-step purification revealed the production of MBP-Pro45-70-His6 with very high purity (S1 Fig). The IC 50 value of MBP-Pro45-70-His6 was 1.43 nM for 1 nM MSTN inhibition (Fig 1), which is very close to that of MBP-Pro45-70 (1.18 nM). To examine the extent of specificity of MBP-Pro45-70-His6's binding to MSTN, the inhibitory activities of MBP-Pro45-70-His6 against GDF11, structurally very similar member to MSTN, and Activin A, another TGF-beta family member, were measured. IC 50 values of MBP-pro45-70-His6 for 1 nM GDF11 (12.5 nM) and Activin A (52.8 nM) were about 8-fold and 37-fold higher, respectively than for 1 nM MSTN (Fig 1), indicating that MBP-Pro45-70-His6 is highly specific in suppressing MSTN activity.

MBP-Pro45-70-His6 blocks Smad2/3 phosphorylation induced by MSTN
MSTN binds to specific serine/threonine kinase transmembrane receptor type IIB (ActRIIB) and induces phosphorylation of activin receptor-like kinase4 (ALK4) or activin receptor-like kinase5 (ALK5) type I receptor [43]. The MSTN/receptors complex allows intracellular signal propagation via phosphorylation of the Smad transcription factor proteins. Thus, we examined the phosphorylation level of Smad2 and Smad3 in HepG2 cells treated with MSTN plus MBP-Pro45-70-His6. We used MBP-Pro45-70 in this experiment because it showed the highest MSTN-inhibitory potency, indicating that less amount will be required in animal studies for MSTN inhibition among the truncated MSTN propeptides. In the control group without any treatment, the phosphorylation of Smad2 and Smad3 were not detected, while MSTN treatment induced phosphorylation of Smad2 and Smad3. Both MBP-Pro45-70-His6 and SB431542, an ALK5 inhibitor, blocked the MSTN-induced phosphorylation of Smad2 and Smad3, indicating that MBP-Pro45-70-His6 suppresses the Smad signaling pathway induced by MSTN (Fig 2).

MBP-Pro45-70-His6 administration does not affect body wt growth but increases muscle wt, forelimb grip strength, and swimming time
The administration of MBP-Pro45-70-His6 tended to increase the body wt gain during 7 days administration period as compared to the control, but no difference in body wt gain was observed after 14 days administration (Fig 3A). The muscle wts of forelimb (1.40 g) and hindlimb (3.00 g) of treated mice were significantly heavier than those (1.34 g and 2.69 g, respectively) of control mice after 14 days administration (Fig 3B). The bone wts of both forelimb and hindlimb were not affected by the MBP-pro45-70-His6 administration (Fig 3C). Apparently, the increase in muscle wt did not affect the body wt gain. Since suppression of MSTN is known to decrease the accumulation of adipose tissue [44], it is speculated that fat mass of the treated group was less than that of the control group, resulting in no difference in body wt gain. The effect of MBP-Pro45-70-His6 on forelimb grip strength was examined by measuring the change in grip strength from day 0 to day 7 and from day 0 to day 14 ( Fig 3D). The mice treated with MBP-pro45-70-His6 had a significantly greater increase in grip strength from day 0 to day 7 than the control mice (0.090 N vs 0.0325 N). However, the increase in grip strength of the mice administered with MBP-pro45-70-His6 from day 0 to day 14 (0.0442 N) was not different from that of control mice (0.0525 N). The effect of MBP-pro45-70-His6 on swimming time was also examined by measuring the change in swimming time from day 0 to day 7 and from day 0 to day 14 ( Fig 3E). The mice treated with MBP-pro45-70-His6 had 8-fold greater increase in swimming time from day 0 to

MBP-Pro45-70-His6 administration suppresses blood cholesterol, triglyceride, and free fatty acids concentrations
The concentrations of serum cholesterol, triglyceride, and free fatty acid of mice administered with MBP-Pro45-65-His6 were significantly lower than those of control mice, while no difference in serum glucose concentration was observed between the two groups ( Table 2).

MBP-Pro45-70-His6 administration induced MBP-Pro45-70-His6 antibody production
MBP-Pro45-70-His6 is a heterologous protein to mice, thus the generation of antibody against MBP-Pro45-70-His6 was examined using ELISA (Fig 4). The presence of an antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 days after the administration with the titer value at day14 being significantly greater than that at day 7.

Pep45-65-NH2 administration enhances body wt growth, muscle wt, forelimb grip strength, and swimming time
The administration of Pep45-65-NH2 increased the body wt gain during the 7 and 14 days administration period as compared to the control (Fig 7A). The increase in gain at day 14 (0.809 g) was greater than that at day 7 after administration (0.55 g), suggesting that the increase in body wt gain by Pep45-65-NH2 continued during the 14 days administration. The muscle wts of forelimb (1.30 g) and hindlimb (2.67 g) of treated mice were significantly heavier than those (1.14 g and 2.16 g, respectively) of control mice after 14 days administration ( Fig  7B). Forelimb bone wt of the treatment group was significantly lower than that of the control group after 14 days administration, but no difference in hindlimb bone wt was observed between the two groups ( Fig 7C). The mice treated with Pep45-65-NH2 had a greater increase in grip strength from day 0 to day 7 than the control mice (0.0533 N vs 0.0208 N), as well as from day 0 to day 14 (0.0800 N vs 0.0325 N) (Fig 7D). The mice treated with Pep45-65-NH2 had a greater increase in swimming time from day 0 to day 7 than the control mice (7.17 s vs 2.42 s), as well as from day 0 to day 14 (8.08 s vs 3.25 s) (Fig 7E).

Pep-45-65-NH2 administration suppresses blood glucose, triglyceride, and free fatty acids concentrations
The concentrations of serum glucose, triglyceride, and free fatty acid of mice administered with Pep45-65-NH2 were significantly lower than those of control mice, while no difference in serum cholesterol concentration was observed between the two groups (Table 3).

Discussion
Myostatin propeptide (MSTNpro) is a well-known MSTN suppressor, and the administration of recombinant MSTNpro enhanced skeletal growth or muscle repair in mice [9,11,27,32], demonstrating its potential as a pharmaceutical agent to treat various muscle wasting conditions or diseases. Recent studies have shown that synthetic peptides containing 29 or 24 amino acids from the mouse MSTNpro were sufficient to suppress MSTN activity in vitro and to increase muscle mass in mice of muscular dystrophy model [12,13]. We also have shown that truncated forms of pig and flatfish MSTNpro with an N-terminal fusion of maltose binding protein (MBP) had the MSTN-inhibitory capacity not different from the full sequence mouse MSTNpro [35,36]. Notably, the size of MSTNpro region of flatfish (residues 45-80) was much  mouse MSTN region 44-67 (Fig 8), which was reported as a minimum domain for MSTN suppression [13]. The region is in the α-helix forming domain of MSTNpro (Fig 8). A recent study on a crystal structure of human unprocessed MSTN showed that the region from Arg45 to Leu 64 forms α-helical structure, and six aliphatic residues in the helical structure are buried within the hydrophobic groove of an MSTN domain that is known to interact with MSTN type I and II receptors [45], A peptide fragment bearing flatfish MSTNpro residues from 45-65 (Pep45-65), which suppressed MSTN activity, was predicted to induce little immunogenicity in silico assay. Pep45-65 suppressed the MSTN-induced Smad2/3 phosphorylation dose-dependently in HepG2 cells, suggesting its potential to suppress MSTN activity in vivo. In mice experiment, the administration of Pep45-65 significantly enhanced body wt gain, muscle mass, grip strength, and swimming time. Unlike the administration of MBP-MSTNpro45-70-His6, the extent of increase in body wt gain and grip strength at week 2 was greater than at week 1, indicating that the effect of Pep45-65 on muscle growth and performance was accumulating during the prolonged administration. No antibody titer was detected against Pep45-65 (S2 Fig), suggesting that the non-mammalian nature of the peptide would probably cause little side effects that may arise from the antigenic response to heterologus peptide administration in mammalian species. Furthermore, Pep45-65 did not suppress GDF11, and Activin A in the concentration range tested. Given that MSTN and GDF11 share 89% homology and signal through the same transcription factors [1], the administration of Pep45-65 is likely to invoke minimal non-targeted effects in vivo. These results together, thus, point to the importance of the immunogenicity of recombinant proteins or peptides as pharmaceutical agents and indicate that Pep45-65 has a great therapeutic potential to treat muscle atrophy caused by various conditions and diseases.
In addition to its effect on muscle mass, MSTN also regulates fat mass and energy metabolism. Studies have shown that the inhibition of MSTN decreases fat mass, improves insulin sensitivity and whole-body metabolism in mice [47][48][49][50][51][52], and pig [53]. MSTN-suppression increased the expression of PGC-1α, a key regulator of mitochondrial biogenesis, brown adipocyte differentiation, and fatty acid metabolism, in the muscle and adipose tissue [49,50,53], indicating that the changes in metabolic profiles affected by MSTN-suppression are associated with the upregulation of PGC-1α. Recent studies also have shown that the serum level of Irisin, a myokine inducing white adipocyte browning and enhancing energy expenditure [54], was increased by MSTN suppression [50,53,55], suggesting a presence of cross talk between muscle and adipocyte in improving insulin sensitivity and enhancing energy metabolism by MSTN suppression. According to Dong et al. [50], MSTN-suppression increases PGC-1α expression and Irisin production in skeletal muscle, leading to the stimulation of white adipocyte browning and consequently enhanced energy expenditure and improved insulin sensitivity. In this study, the administration of MSTN suppressors (MBP-MSTNpro45-70-His6 and Pep45-65) lowered blood concentrations of glucose, triglyceride, and free fatty acids. In agreement with this result, others also reported that MSTN suppression lowers blood glucose, triglyceride, or fatty acids levels [47,49,53]. It is, thus, likely that energy metabolism was enhanced by the administration of MBP-MSTNpro45-70-His6 or Pep45-65, and future studies need to examine the potentials of these compounds to improve insulin sensitivity.
The size of Pep45-65 is smaller than that of mouse MSTNpro peptide fragment consisting of residues from 44 to 66 (p23) that was reported to be the minimum peptide for MSTN inhibition [13]. In spite of the smaller size the MSTN-inhibitory capacity of flatfish Pep45-65 appears to be comparable to or better than that of p23. The IC 50 value of Pep45-65 for MSTN inhibition (12.5 ng/mL) was about 3 μM in our current and a previous study [36], while the IC 50 value of p23 for MSTN inhibition (10 ng/mL) was reported to be 4.1 μM in a (CAGA) 12 reporter gene assay [13]. The removal of residues from 64 to 66 (Leu-Arg-Leu) from p23 failed to show MSTN-inhibitory capacity [13], suggesting an important role of these sequences for effective inhibition of MSTN activity. However, our result shows that the region in flatfish MSTNpro may not be necessary for an effective MSTN suppression because in the absence of the corresponding region (67Leu-Arg-69Met), Pep45-65 was effective in suppressing MSTN activity. There are many other subtle differences in amino acid sequence between Pep45-65 and p23 (Fig 8), and future studies need to investigate the contributions of the differences to the interaction of MSTNpro peptides and MSTN and subsequence MSTN inhibition.
In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45-65 (Pep45-65-NH2) can suppress MSTN activity and increase muscle mass and function without invoking an immune response in the mouse. The Pep45-65-NH2 with its smaller size than the mouse propeptide (p23, aa 44-66) showed a comparable to or better MSTN-inhibitory potency than the mouse propeptide. Also, Pep45-65-NH2 did not interfere with the GDF11 and Activin A signaling, suggesting minimal off-target effect in vivo. These results, together, imply that Pep45-65-NH2 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.