Abstract

Myostatin (MSTN) is a potent negative regulator of skeletal muscle growth, and its activity is suppressed by MSTN propeptide (MSTNpro), the N-terminal part of MSTN precursor cleaved during post-translational MSTN processing. The current study examined which region of flatfish (Paralichthys olivaceus) MSTN-1 propeptide (MSTN1pro) is critical for MSTN inhibition. Six different truncated forms of MSTN1pro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in Escherichia coli, and partially purified by an affinity chromatography for MSTN-inhibitory activity examination. Peptides covering different regions of flatfish MSTN1pro were also synthesized for MSTN-inhibitory activity examination. A MBP-fused MSTN1pro region consisting of residues 45–100 had the same MSTN-inhibitory potency as the full sequence flatfish MSTN1pro (residues 23–265), indicating that the region of flatfish MSTN1pro consisting of residues 45–100 is sufficient to maintain the full MSTN-inhibitory capacity. A MBP-fused MSTN1pro region consisting of residues 45–80 (Pro45–80) also showed MSTN-inhibitory activity with a lower potency, and the Pro45–80 demonstrated its MSTN binding capacity in a pull-down assay, indicating that the MSTN-inhibitory capacity of Pro45–80 is due to its binding to MSTN. Flatfish MSTN1pro synthetic peptides covering residues 45–65, 45–70, and 45–80 demonstrated MSTN-inhibitory activities, but not the synthetic peptide covering residues 45–54, indicating that residues 45–65 of flatfish MSTN1pro are essential for MSTN inhibition. In conclusion, current study show that like the mammalian MSTNpro, the MSTN-inhibitory region of flatfish MSTN1pro resides near its N-terminus, and imply that smaller sizes of MSTNpro can be effectively used in various applications designed for MSTN inhibition.