Date Published: April 18, 2019
Publisher: Public Library of Science
Author(s): Jeong Hwan Kim, Jeong Han Kim, Lisa Andriani Sutikno, Sang Beum Lee, Deuk-Hee Jin, Yong-Ki Hong, Yong Soo Kim, Hyung-Joo Jin, Stephen E Alway.
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.
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 , while systemic overexpression of MSTN by transgenesis or selective gene transfer in skeletal muscle induced profound muscle atrophy in mice [2–4]. 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 [5–7]. Approaches like the administration of MSTN-blocking proteins or peptides [8–14], delivery of MSTN-blocking genes using an adeno-associated viral vector [15–17], 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 [20–23].
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 smaller than that of pig (residues 42–175) for full MSTN-inhibitory capacity, suggesting that the large sequence variation between fish and mammalian MSTNpro affects the stability of MSTNpro and MSTN complex.