The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy

Abstract

Myostatin, a muscle-specific transforming growth factor-β (TGF-β), negatively regulates skeletal muscle mass. The N-terminal prodomain of myostatin noncovalently binds to and suppresses the C-terminal mature domain (ligand) as an inactive circulating complex. However, which region of the myostatin prodomain is required to inhibit the biological activity of myostatin has remained unknown. We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain. This inhibitory core resides near the N-terminus of the prodomain and includes an α-helix that is evolutionarily conserved among other TGF-β family members, but suppresses activation of myostatin and growth and differentiation factor 11 (GDF11) that share identical membrane receptors. Interestingly, the inhibitory core co-localized and co-immunoprecipitated with not only the ligand, but also its type I and type II membrane receptors. Deletion of the inhibitory core in the full-length prodomain removed all capacity for suppression of myostatin. A synthetic peptide corresponding to the inhibitory core (p29) ameliorates impaired myoblast differentiation induced by myostatin and GDF11, but not activin or TGF-β1. Moreover, intramuscular injection of p29 alleviated muscle atrophy and decreased the absolute force in caveolin 3-deficient limb-girdle muscular dystrophy 1C model mice. The injection suppressed activation of myostatin signaling and restored the decreased numbers of muscle precursor cells caused by caveolin 3 deficiency. Our findings indicate a novel concept for this newly identified inhibitory core of the prodomain of myostatin: that it not only suppresses the ligand, but also prevents two distinct membrane receptors from binding to the ligand. This study provides a strong rationale for the use of p29 in the amelioration of skeletal muscle atrophy in various clinical settings.

Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli

“…The result of Jiang et al[16] in which a GST-fused human MSTNpro consisting of residues 42–98 showed no MSTN-inhibitory capacity supports the mechanism, as well as our current result. On the other hand, it has been reported that synthetic MSTNpro peptides containing residues in the N-terminal fragment (below Arg-98) of BMP-1-digested MSTNpro have MSTN-inhibitory capacity in vitro [17–19], as well as enhancement of muscle growth upon administration of the peptides[17, 18]. The apparent contradictory results can be related to the administration dose of peptides.…”

“…However, the MSTN-binding affinity of the BMP-1-digested N-terminal fragment of MSTNpro appears to be very weak, it is thus speculated that C-terminal region from the BMP-1-digested point stabilizes the binding to MSTN. In support of this speculation, the MSTNpro peptides containing residues in the BMP-1-digested N-terminal fragment required micromolar concentration to suppress MSTN activity [17–19]. …”

“…Similarly, a region of human MSTNpro containing residues 42–99 fully suppressed MSTN activity in co-transfection experiments [17], and a synthetic mouse MSTNpro peptide covering residues 45–68 was effective in suppressing MSTN activity [18]. We have also shown that maltose binding protein (MBP)-fused flatfish MSTN1pro region consisting of residues 45–100 had the same MSTN inhibitory potency as the MBP-fused full sequence flatfish MSTN1pro [19].…”

Effect of N‐Terminal Acylation on the Activity of Myostatin Inhibitory Peptides

“…[10] It is important to note that all strategies to inhibit myostatin are not equal in their action. [17] Moreover,i no ur synthetic peptide-based study, amouse-derived peptide consisting of 23 aa residues (position: 21-43, peptide A; Scheme 1) showed effective inhibitory activity against myostatin, while ah uman-derived peptides equence did not. To the best of authors' knowledge,n os mall peptides inhibiting myostatin have been describedt od ate in the literature.…”

“…[2,14] In the structural study of inactivated TGF-b1, the N-terminal a-helical region of the prodomain,w hich is conserved among superfamily proteins, interacts with the type Ir eceptor binding site of mature TGF-b1. [17] Moreover,i no ur synthetic peptide-based study, amouse-derived peptide consisting of 23 aa residues (position: 21-43, peptide A; Scheme 1) showed effective inhibitory activity against myostatin, while ah uman-derived peptides equence did not. [16] In recent investigations on the key peptide sequence responsible for myostatin inhibition from the same prodomain, using as eries of fusion proteins with the antibody Fc fragment, Ohsawa et al successfully found as horter 29 aa residue fragment located at positions 19-47.…”