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

Ohsawa Y, Takayama K, Nishimatsu S, Okada T, Fujino M, Fukai Y, Murakami T, Hagiwara H, Itoh F, Tsuchida K, Hayashi Y, Sunada Y - PLoS ONE (2015)

Bottom Line: We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Kawasaki Medical School, Kurashiki, Okayama, 701-0192, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

Identification of the inhibitory core of the myostatin prodomain.(A) Truncation and deletion constructs of human myostatin prodomain:human Fc fusion proteins (left). Percentage inhibitory effect of each construct on myostatin activity in comparison with the full-length prodomain (f-Pro, right). (B) Recombinant myostatin-induced transcriptional activity in HEK293 human embryonic kidney cells co-transfected with a pGL3-(CAGA)12-luciferase reporter gene, pCMV-β-Gal, and various prodomain region:Fc fusion constructs. Values are the mean ± SD (n = 6). RLU, relative luminescence units.
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pone.0133713.g001: Identification of the inhibitory core of the myostatin prodomain.(A) Truncation and deletion constructs of human myostatin prodomain:human Fc fusion proteins (left). Percentage inhibitory effect of each construct on myostatin activity in comparison with the full-length prodomain (f-Pro, right). (B) Recombinant myostatin-induced transcriptional activity in HEK293 human embryonic kidney cells co-transfected with a pGL3-(CAGA)12-luciferase reporter gene, pCMV-β-Gal, and various prodomain region:Fc fusion constructs. Values are the mean ± SD (n = 6). RLU, relative luminescence units.

Mentions: We designed a series of truncation and deletion constructs of the full-length human prodomain including 239 amino acid residues (24N–K262). We considered two known sites:, 54EAIKIQILSKL64, the putative binding site for TSP-1 [6], and 97QRD99, the cleavage site for BMP-1/tolloid family of metalloproteinases [4] (Fig 1A). Each construct was co-transfected with a TGF-β-sensitive Smad-responsive luciferase reporter gene into HEK293 human embryonic kidney cells. To evaluate in vitro myostatin activities, luciferase reporter assays were performed in triplicate, repeatedly at least twice. Stimulation of cells carrying an empty Fc vector with recombinant myostatin ligand significantly increased luciferase activity above the basal level (Fig 1B). Expression of the full-length prodomain:Fc fusion protein (f-Pro) resulted in a significant reduction of myostatin-induced transcriptional activity. Consistent with a previous report [4], a mutant (97QAD99) full-length prodomain:Fc fusion protein (f-ProD99A) was as effective at blocking myostatin-induced transcriptional activity as f-Pro. Additionally, f-ProD99A showed no proteolytic degradation products in immunoblot analysis (S1 Fig). We thus performed the following assays without considering the effect of cleavage by BMP-1/tolloid family of metalloproteinases.


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

Ohsawa Y, Takayama K, Nishimatsu S, Okada T, Fujino M, Fukai Y, Murakami T, Hagiwara H, Itoh F, Tsuchida K, Hayashi Y, Sunada Y - PLoS ONE (2015)

Identification of the inhibitory core of the myostatin prodomain.(A) Truncation and deletion constructs of human myostatin prodomain:human Fc fusion proteins (left). Percentage inhibitory effect of each construct on myostatin activity in comparison with the full-length prodomain (f-Pro, right). (B) Recombinant myostatin-induced transcriptional activity in HEK293 human embryonic kidney cells co-transfected with a pGL3-(CAGA)12-luciferase reporter gene, pCMV-β-Gal, and various prodomain region:Fc fusion constructs. Values are the mean ± SD (n = 6). RLU, relative luminescence units.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520684&req=5

pone.0133713.g001: Identification of the inhibitory core of the myostatin prodomain.(A) Truncation and deletion constructs of human myostatin prodomain:human Fc fusion proteins (left). Percentage inhibitory effect of each construct on myostatin activity in comparison with the full-length prodomain (f-Pro, right). (B) Recombinant myostatin-induced transcriptional activity in HEK293 human embryonic kidney cells co-transfected with a pGL3-(CAGA)12-luciferase reporter gene, pCMV-β-Gal, and various prodomain region:Fc fusion constructs. Values are the mean ± SD (n = 6). RLU, relative luminescence units.
Mentions: We designed a series of truncation and deletion constructs of the full-length human prodomain including 239 amino acid residues (24N–K262). We considered two known sites:, 54EAIKIQILSKL64, the putative binding site for TSP-1 [6], and 97QRD99, the cleavage site for BMP-1/tolloid family of metalloproteinases [4] (Fig 1A). Each construct was co-transfected with a TGF-β-sensitive Smad-responsive luciferase reporter gene into HEK293 human embryonic kidney cells. To evaluate in vitro myostatin activities, luciferase reporter assays were performed in triplicate, repeatedly at least twice. Stimulation of cells carrying an empty Fc vector with recombinant myostatin ligand significantly increased luciferase activity above the basal level (Fig 1B). Expression of the full-length prodomain:Fc fusion protein (f-Pro) resulted in a significant reduction of myostatin-induced transcriptional activity. Consistent with a previous report [4], a mutant (97QAD99) full-length prodomain:Fc fusion protein (f-ProD99A) was as effective at blocking myostatin-induced transcriptional activity as f-Pro. Additionally, f-ProD99A showed no proteolytic degradation products in immunoblot analysis (S1 Fig). We thus performed the following assays without considering the effect of cleavage by BMP-1/tolloid family of metalloproteinases.

Bottom Line: We identified a 29-amino acid region that inhibited myostatin-induced transcriptional activity by 79% compared with the full-length prodomain.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Kawasaki Medical School, Kurashiki, Okayama, 701-0192, Japan.

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.

No MeSH data available.


Related in: MedlinePlus