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. 2008 Feb 20;3(2):e1628.
doi: 10.1371/journal.pone.0001628.

Genetic analysis of the role of proteolysis in the activation of latent myostatin

Se-Jin Lee  1
Affiliations

Genetic analysis of the role of proteolysis in the activation of latent myostatin

Se-Jin Lee. PLoS One. .

Abstract

Myostatin is a secreted protein that normally acts to limit skeletal muscle growth. As a result, there is considerable interest in developing agents capable of blocking myostatin activity, as such agents could have widespread applications for the treatment of muscle degenerative and wasting conditions. Myostatin normally exists in an inactive state in which the mature C-terminal portion of the molecule is bound non-covalently to its N-terminal propeptide. We previously showed that this latent complex can be activated in vitro by cleavage of the propeptide with members of the bone morphogenetic protein-1/tolloid (BMP-1/TLD) family of metalloproteases. Here, I show that mice engineered to carry a germline point mutation rendering the propeptide protease-resistant exhibit increases in muscle mass approaching those seen in mice completely lacking myostatin. Mice homozygous for the point mutation have increased muscling even though their circulating levels of myostatin protein are dramatically increased, consistent with an inability of myostatin to be activated from its latent state. Furthermore, I show that a loss-of-function mutation in Tll2, which encodes one member of this protease family, has a small, but significant, effect on muscle mass, implying that its function is likely redundant with those of other family members. These findings provide genetic support for the hypothesis that proteolytic cleavage of the propeptide by BMP-1/TLD proteases plays a critical role in the activation of latent myostatin in vivo and suggest that targeting the activities of these proteases may be an effective therapeutic strategy for enhancing muscle growth in clinical settings of muscle loss and degeneration.

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Conflict of interest statement

Competing Interests: Under a licensing agreement between MetaMorphix, Inc. (MMI) and the Johns Hopkins University, the author is entitled to a share of royalty received by the University on sales of the factor described in this paper. Both the author, who is the scientific founder of MMI, and the University own MMI stock, which is subject to certain restrictions under University policy. The author is a paid consultant to MMI and to Merck on research areas related to the study described in this paper. The terms of these arrangements are being managed by the University in accordance with its conflict of interest policies.

Figures

Figure 1
Figure 1. Generation of mice carrying a Mstn point mutation rendering the propeptide resistant to cleavage by BMP-1/TLD proteases.
(a) Gene targeting strategy. Black and stippled boxes represent coding exons for the propeptide and C-terminal domain, respectively. The location of the point mutation (D76A) is denoted by an asterisk, and LoxP sites are denoted by triangles. Removal of the neo cassette using EIIa-cre mice resulted in Mstn alleles containing a single LoxP site (in intron 1) either with (MstnD76A) or without (MstnLoxP) the point mutation. (b) Northern analysis of Mstn RNA expression in mutant mice. Muscle RNA isolated from Mstn−/−, wild type, and MstnD76A/D76A mice was electrophoresed, blotted, and hybridized with a Mstn probe. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Analysis of myostatin protein in mutant mice. Hydroxylapatite-bound serum samples isolated from wild type, Mstn−/−, and MstnD76A/D76A mice were electrophoresed, blotted, and probed with antiserum directed against either the C-terminal domain or the propeptide . In each gel, the first lane contains purified myostatin latent complex isolated from Chinese hamster ovary cells .
Figure 2
Figure 2. Analysis of muscles of mutant mice.
(a) Muscle weight increases in MstnD76A/D76A mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green). (b) Distribution of fiber diameters. Gray bars represent muscle fibers from wild type mice, and red bars represent muscle fibers from Mstn−/− and MstnD76A/D76A.
Figure 3
Figure 3. Generation and analysis of mice carrying a loss-of-function mutation in the Tll2 gene.
(a) Gene targeting strategy. Locations of exons 6–9 are shown as black boxes, and LoxP sites are denoted by triangles. (b) Northern analysis of Tll2 expression levels. Twenty micrograms of poly A-selected brain RNA isolated from either wild type or Tll2−/− mice were electrophoresed, blotted, and hybridized with a Tll2 probe corresponding to exons 1–3. The blot was re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Muscle weight increases in Tll2−/− mice. Numbers represent percent increases relative to wild type mice and were calculated from the data shown in Table 1. Muscles analyzed were: pectoralis (red), triceps (gray), quadriceps (blue), and gastrocnemius (green).
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References

    1. Lee S-J. Regulation of muscle mass by myostatin. Annu Rev Cell Dev Biol. 2004;20:61–86. - PubMed
    1. McPherron AC, Lawler AM, Lee S-J. Regulation of skeletal muscle mass in mice by a new TGF-ß superfamily member. Nature. 1997;387:83–90. - PubMed
    1. Grobet L, Martin LJR, Poncelet D, Pirottin D, Brouwers B, et al. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nature Genet. 1997;17:71–74. - PubMed
    1. Kambadur R, Sharma M, Smith TPL, Bass JJ. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res. 1997;7:910–915. - PubMed
    1. McPherron AC, Lee S-J. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA. 1997;94:12457–12461. - PMC - PubMed

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