Gdf-8 Propeptide Binds to GDF-8 and Antagonizes Biological Activity by Inhibiting GDF-8 Receptor Binding

Thies, R. Scott; Chen, T; Davies, Monique V.; Tomkinson, Kathy N.; Pearson, Adele A.; Shakey, Q A; Wolfman, Neil M.

doi:10.3109/08977190109029114

Cited by 237 publications

(227 citation statements)

References 36 publications

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“…The ACVR2B͞Fc fusion protein was purified by using a protein A Sepharose column. Myostatin, GDF-11͞BMP-11, and activin activities were measured by using the pGL3-(CAGA) 12 -luciferase reporter assay in A204 rhabdomyosarcoma cells as described in ref. 12.…”

Section: Methodsmentioning

confidence: 99%

“…Various myostatin-binding proteins have been identified that are capable of inhibiting myostatin activity in vitro (8,(11)(12)(13)(14)(15)(16). Two of these proteins, the JA16 neutralizing monoclonal antibody (Ab) directed against myostatin (8,15) and a mutant form of the myostatin propeptide resistant to members of the BMP-1͞tolloid family of metalloproteases (16), have been shown to be capable of increasing muscle mass by Ϸ25% when administered to wild-type (WT) mice.…”

mentioning

confidence: 99%

“…These findings have raised the possibility that agents capable of targeting the myostatin signaling pathway may be useful for increasing muscle mass for both agricultural and human therapeutic applications. In this regard, loss of myostatin signaling has been shown to have beneficial effects in mouse models of muscle degenerative (8, 9) and metabolic (10) diseases.Various myostatin-binding proteins have been identified that are capable of inhibiting myostatin activity in vitro (8,(11)(12)(13)(14)(15)(16). Two of these proteins, the JA16 neutralizing monoclonal antibody (Ab) directed against myostatin (8, 15) and a mutant form of the myostatin propeptide resistant to members of the BMP-1͞tolloid family of metalloproteases (16), have been shown to be capable of increasing muscle mass by Ϸ25% when administered to wild-type (WT) mice.…”

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confidence: 99%

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Regulation of muscle growth by multiple ligands signaling through activin type II receptors

Lee

Reed

Davies

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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447

431

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Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn ؊/؊ mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo. Mice carrying a targeted mutation in the myostatin gene have muscles that are about twice the normal size as a result of a combination of muscle fiber hyperplasia and hypertrophy (2). Myostatin appears to play a similar role in other species as well; naturally occurring mutations in the myostatin gene have been shown to be responsible for the double-muscling phenotype in cattle (3-6), and recent studies have demonstrated that a human baby with approximately twice the normal muscle mass is also homozygous for a loss-of-function mutation in the MSTN gene (7). These findings have raised the possibility that agents capable of targeting the myostatin signaling pathway may be useful for increasing muscle mass for both agricultural and human therapeutic applications. In this regard, loss of myostatin signaling has been shown to have beneficial effects in mouse models of muscle degenerative (8, 9) and metabolic (10) diseases.Various myostatin-binding proteins have been identified that are capable of inhibiting myostatin activity in vitro (8,(11)(12)(13)(14)(15)(16). Two of these proteins, the JA16 neutralizing monoclonal antibody (Ab) directed against myostatin (8, 15) and a mutant form of the myostatin propeptide resistant to members of the BMP-1͞tolloid family of metalloproteases (16), have been shown to be capable of increasing muscle mass by Ϸ25% when administered to wild-type (WT) mice. To determine whether these increases in muscle growth are the maximal achievable by targeting this signaling pathway, we sought additional myostatin inhibitors that might have a broader specificity in their ability to target additional members of the TGF-␤ superfamily. Previous studies have demonstrated that myostatin is capable of binding the two activin type II receptors, ACVR2B and, to a lesser extent, ACVR2, in transfected COS cells (11,17). Moreover, transgenic mice in which a myosin light chain promoter͞ enhancer was used to express a truncated form of ACVR2B in skeletal muscle were found to have dramatic increases in muscle mass (11). Because the activin type II receptors have been shown to be capable of binding a number of other TGF-␤ family members in addition to ...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Regulation of muscle growth by multiple ligands signaling through activin type II receptors

Lee

Reed

Davies

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

447

431

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“…Another feature making myostatin potentially useful is its specificity. Like other TGF-b family members, the mature C-terminal protein binds to the N-terminal remnant of the original precursor molecule (LAP) resulting in a latent complex (Lee and McPherron, 2001;Thies et al, 2001;Yang et al, 2001). Myostatin then has to be released from this latent complex to be active.…”

Section: Rd Cell Growth Is Inhibited By Myostatinmentioning

confidence: 99%

Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway

et al. 2004

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Rhabdomyosarcoma (RMS) tumors are the most common soft-tissue sarcomas in childhood. In this investigation, we show that myostatin, a skeletal muscle-specific inhibitor of growth and differentiation is expressed and translated in the cultured RMS cell line, RD. The addition of exogenous recombinant myostatin inhibits the proliferation of RD cells cultured in growth media, consistent with the role of myostatin in normal myoblast proliferation inhibition. However, unlike normal myoblasts, upregulation of p21 was not observed. Rather, myostatin signalling resulted in the specific downregulation of both Cdk2 and its cognate partner, cyclin-E. The analysis of Rb reveals that there was no change in its phosphorylation status with myostatin treatment, consistent with D-type-cyclinCdk4/6 complexes being active in the absence of p21. Moreover, the activity of Rb appeared to be unchanged between treated and nontreated RD cells, as determined by the ability of Rb to bind E2F1. The examination of NPAT, a substrate of cyclin-E-Cdk2 involved in the transcriptional activation of replication-dependent histone gene expression, revealed that it undergoes a loss of phosphorylation with myostatin treatment. Supporting this, a downregulation in H4-histone gene expression was observed. These results suggest that myostatin could potentially be used as an inhibitor of RMS proliferation and define a previously uncharacterized, Rb-independent mechanism for the inhibition of muscle precursor cell proliferation by myostatin.

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“…Myostatin was expressed from a Chinese hamster ovary (CHO) cell line and purified similar to the method described in Thies et al 78 Crystallization of the RK35 and myostatin complex Chimeric RK35 was cleaved with immobilized papain for about 16 hours. Following cleavage, papain was removed using a disposable column.…”

Section: Cell Culture Expression and Purificationmentioning

confidence: 99%

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Apgar

Mader

Agostinelli

et al. 2016

mAbs

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Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted antimyostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

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Gdf-8 Propeptide Binds to GDF-8 and Antagonizes Biological Activity by Inhibiting GDF-8 Receptor Binding

Cited by 237 publications

References 36 publications

Regulation of muscle growth by multiple ligands signaling through activin type II receptors

Regulation of muscle growth by multiple ligands signaling through activin type II receptors

Myostatin inhibits rhabdomyosarcoma cell proliferation through an Rb-independent pathway

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

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