Regulation of Myostatin in Vivo by Growth and Differentiation Factor-Associated Serum Protein-1: A Novel Protein with Protease Inhibitor and Follistatin Domains

Jennifer J. Hill, Yongchang Qiu, Rodney M. Hewick and Neil M. Wolfman

Department of Protein Chemistry and Proteomics (J.J.H., Y.Q., R.M.H.), and Department of Musculoskeletal Sciences (N.M.W.), Wyeth Research, Cambridge, Massachusetts 02140

Address all correspondence and requests for reprints to: Jennifer J. Hill, Department of Protein Chemistry and Proteomics, Wyeth Research, 200 Cambridge Park Drive, Cambridge, Massachusetts 02140. E-mail: jhill{at}wyeth.com.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myostatin, a member of the TGFß superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFß superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFß binding proteins.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MYOSTATIN (ALSO CALLED growth and differentiation factor-8, GDF-8) was recently identified as a member of the TGFß superfamily that acts as a potent and specific negative regulator of skeletal muscle mass. Homozygous disruption of the myostatin gene in mice results in a significant increase in skeletal muscle mass and a reduction in body fat (1, 2, 3). Naturally occurring myostatin mutations in cattle display a similar double-muscled phenotype, confirming a role for myostatin in muscle development and regulation (4, 5, 6).

Myostatin appears to regulate muscle mass, at least in part, by controlling the proliferation of myoblasts, inducing cell cycle arrest in the G1 phase (7, 8). Some studies have suggested that myostatin may also negatively regulate the differentiation of myoblasts into myotubes by reducing the expression of the muscle-specific transcription factor, myogenic differentiation (MyoD) (9, 10). The regulation of muscle by myostatin is not merely a developmental phenomenon. In adult mice, systemic overexpression of myostatin leads to muscle wasting (11). Furthermore, changes in muscle mass due to disease or misuse are often reflected by changes in myostatin expression (12, 13, 14, 15).

Like other members of the TGFß superfamily, myostatin is a secreted protein that is produced from a precursor protein by proteolytic processing. Proteolysis at an internal dibasic site by a furin-type protease results in two polypeptide products, the N-terminal myostatin propeptide and the C-terminal fragment that is the mature myostatin ligand (1, 16, 17, 18). Like TGFß, dimers of the myostatin propeptide and mature myostatin remain noncovalently associated after cleavage, producing a latent complex in which the myostatin ligand is incapable of binding to its receptor (1, 18, 19). In fact, the majority of native myostatin circulating in serum is associated with its propeptide, forming what is known as the small latent complex (20, 21).

To produce a signal in vivo, the small latent myostatin complex must be activated by removal of the propeptide moiety. In the case of TGFß, which forms an analogous small latent complex, activation can occur via both physiochemical and proteolytic methods (reviewed in Ref. 21). In vitro, the small latent TGFß complex is activated by treatment with acid or heat, presumably reflecting a difference in the relative stability of mature TGFß and the TGFß propeptide (22, 23). Protease treatment with plasmin or cathepsin D also activates TGFß by cleaving the TGFß propeptide and unmasking mature active TGFß (24, 25). Despite the prevalence of TGFß research, the basis for TGFß activation in vivo remains unclear and may occur by multiple mechanisms depending on the cellular context.

Myostatin is very highly related to another TGFß superfamily member, bone morphogenetic protein-11 (BMP-11) (1, 26). Myostatin and BMP-11 differ by only 11 amino acids in the mature domain, but have different physiological roles, as highlighted by the phenotype of the BMP-11 knockout mouse which displays skeletal defects resulting from abnormal anterior-posterior patterning (27). Within the TGFß superfamily, myostatin and BMP-11 are closely related to the activin/inhibin-ß subfamily, displaying approximately 40% identity. Unlike myostatin, activin does not bind to its propeptide, but is instead negatively regulated by a family of proteins that contain follistatin (FS) domains (28, 29, 30). As the name implies, FS-domains were originally described as a 10-cysteine repeat found three times in follistatin, a protein that binds to activin and inhibits the activin-mediated release of FSH (31, 32, 33).

Like the myostatin propeptide, FS-domain proteins bind to certain TGFß family members and prevent association with cellular receptors (18, 28, 34, 35, 36). FS domain proteins have been categorized into two subfamilies, based on both sequence similarity and their ability to bind activin (37). The first subfamily contains follistatin and follistatin-related gene (FLRG), both of which bind and inhibit activin and myostatin (19, 20, 28, 29). Follistatin and FLRG show strong preferential binding for activin over other TGFß family members, including TGFß1, BMP-4, BMP-6, and BMP-7 (38, 39). However, the relative binding affinity of follistatin and FLRG for myostatin has not yet been determined. The second subfamily of FS-domain proteins contains proteins such as agrin, SPARC (secreted protein, acidic and rich in cysteine), hevin, and other proteins associated with the extracellular matrix. Members of this second subfamily have not yet been shown to bind activin or any other TGFß family protein, despite having as many as nine distinct FS domains (33).

To gain insight into the mechanism of myostatin regulation in vivo, we set out to define the binding partners of endogenous circulating myostatin. To accomplish this, we have isolated native myostatin from normal serum and analyzed proteins that copurified by mass spectrometry. Using this approach, we have previously reported that myostatin propeptide and FLRG bind and inhibit myostatin in vivo (20). Here, we report the identification and characterization of an additional myostatin binding protein identified in normal mouse and human serum. This protein, referred to here as GDF-associated serum protein-1 (GASP-1), is a novel member of the FS-domain protein family that also contains multiple protease inhibitor domains.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Myostatin Binds to a Novel Protein in Mouse and Human Serum
To characterize the major components of the circulating myostatin complex in vivo, we have isolated native myostatin and its associated proteins from wild-type mouse serum by affinity purification with an agarose-conjugated antimyostatin monoclonal antibody, JA16. JA16-bound proteins were subjected to subsequent elution steps with PBS buffer alone (mock elution), a peptide that could compete with myostatin for JA16 binding, and sodium dodecyl sulfate (SDS) detergent to remove all remaining proteins. These samples were concentrated, run on a one-dimensional SDS-PAGE gel, and visualized by silver stain (Fig. 1AGo). As previously reported, two bands unique to the JA16-purified samples are visible: a 12-kDa band identified as myostatin, and a 36-kDa band containing both myostatin propeptide and FLRG (20).



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Figure 1. A Novel Protein with Multiple Protease Inhibitor Domains Is Bound to Myostatin in Serum

A, Proteins were isolated from normal mouse serum using mock-conjugated (labeled "0") or JA16-conjugated agarose beads. Subsequent elutions with buffer ("mock elute"), a competing peptide, and sodium dodecyl sulfate (SDS) were separated by SDS-PAGE and visualized by silver stain. The gel was dissected into 15 slices from the peptide-eluted sample of both negative control and JA16 immunoprecipitates. The proteins in each slice were digested with trypsin and identified using nanoflow-LC-MS/MS. Proteins unique to the JA16 sample included mature myostatin (lower arrow), myostatin propeptide (upper arrow), FLRG (upper arrow), and a novel multivalent protease inhibitor named here as GASP-1 (boxed). B and C, Representative MS/MS spectra from two GASP-1 peptides, showing both N-terminal (b) and C- terminal (y) fragment ions.

 
To identify any additional myostatin binding proteins that are not visible by silver stain, the area of the silver-stained gel corresponding to molecular masses between 10 and 200 kDa was excised into 15 gel slices for both the mock and JA16 peptide-eluted samples (see Fig. 1AGo). Each gel slice was then subjected to in-gel trypsin digestion, and the resulting peptides were identified by nanoflow LC-MS/MS and database searching. These data were analyzed to provide a list of proteins that were found in each of the two samples, based on the presence of at least one sequenced peptide with a high quality MS/MS spectra. Many proteins were identified in both the negative control and JA16-purified samples, thus constituting background binding in the experiment. As expected, these proteins consisted of abundant serum proteins, such as albumin, immunoglobulins, and complement proteins.

We then looked for proteins that were found specifically in the JA16-immunopurified samples and not in the mock-purified negative control. Searches against the National Center for Biotechnology Information nonredundant (NCBI nr) database from June 2001 identified myostatin, myostatin propeptide, and FLRG, as previously reported, but did not reveal any additional proteins specific to the JA16 samples. There was no evidence of other TGFß superfamily members, including the highly related protein BMP-11/GDF-11, in the JA16 samples. Thus, the JA16 antibody specifically purified myostatin in these experiments.

These data were analyzed further by comparing the MS/MS spectra collected from the 15 samples to a database of theoretical proteins predicted from the Celera mouse genomic sequence (dated June 2001). This analysis identified a novel protein specific to the JA16-purified sample, hereby referred to as GASP-1. Since the initial identification of this protein, this sequence has been added to the NCBI nr database by the public genome sequencing effort as a predicted open reading frame under the accession number gi 20914039. Five peptides derived from the sequence of GASP-1 were identified on the basis of corresponding high-quality MS/MS spectra (Fig. 1Go, B and C). Table 1Go shows the peptide sequence, charge state (z), and Sequest correlation coefficient (Xcorr) for each identified peptide. Sequest Xcorr scores above 2.3 are generally considered significant for 2+ ions. Fortuitously, one of the peptides identified in our experiments (sequence = ECETDQECETYEK) spans the junction between the two exons that code for this protein, verifying the accuracy of the gene prediction algorithm in this instance. The GASP-1 peptides were found in the boxed region of Fig. 1AGo, which contains proteins between 70 and 80 kDa. However, a specific band corresponding to this protein is not visible on the silver-stained gel, probably due to the abundance of background immunoglobulins and albumin at this molecular mass. GASP-1 peptides were identified in the JA16 samples in four repetitions of this experiment, but were never found in the negative control samples. No additional myostatin-binding proteins were identified in these samples.


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Table 1. GASP-1 Peptides Identified in JA16 Immunoprecipitates from Mouse and Human Serum

 
GASP-1 Contains an FS Domain and Multiple Protease-Inhibitory Domains
Based on the Celera predicted sequence, we used PCR to amplify GASP-1 from mouse heart cDNA and cloned the resulting product into a mammalian expression vector encoding a C-terminal V5-His tag. Sequencing confirmed that this clone matched the predicted Celera sequence, with the exception of some base substitutions in wobble codons that did not change the translated amino acid sequence. The cDNA and associated protein sequence of our mouse GASP-1 clone are shown in Fig. 2AGo.



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Figure 2. GASP-1 Is a Multivalent Protease Inhibitor

A, cDNA and protein sequence of mouse GASP-1. The peptides identified by mass spectrometry in JA16 affinity-purified samples are underlined. B, The domain structure of GASP-1. GASP-1 has a signal sequence/cleavage site after amino acid (aa) 29. In addition, GASP-1 contains two Kunitz/BPTI serine protease inhibitor domains, an FS-domain (including a Kazal serine protease inhibitor motif), and a netrin domain. C, Phylogenetic tree of GASP-1 and -2 predicted from the mouse (m) and human (h) genomic sequences. Mouse and human GASP-1 are 90% identical. GASP-1 and GASP-2 are 54% identical.

 
The GASP-1 cDNA encodes a 571-amino-acid protein with a predicted molecular mass of 63 kDa. It has a putative signal sequence/cleavage site at its N terminus and two possible sites for N glycosylation at amino acids 314 and 514. Analysis of the GASP-1 protein sequence by Pfam and BLAST (40, 41) revealed that GASP-1 contains many conserved domains, including a WAP domain, a follistatin/Kazal domain, an immunoglobulin domain, two tandem Kunitz domains, and a netrin domain (Fig. 2BGo). WAP domains, originally identified in whey acidic protein, contain eight cysteines that form a four-disulfide core and are often found in proteins with antiprotease activity (42, 43). The C-terminal region of FS domains contains a similarity to Kazal serine protease inhibitor domains. Kunitz domains, originally identified in bovine pancreatic trypsin inhibitor, also inhibit serine proteases, thus establishing a likely role for GASP-1 in the regulation of this class of proteins. Furthermore, netrin domains have been implicated in the inhibition of metalloproteases (44, 45). Thus, based on the presence of these conserved regions, GASP-1 is likely to inhibit the activity of proteases.

BLAST searches against the mouse Celera transcript database revealed a protein that has more than 50% identity with GASP-1, referred to here as GASP-2. GASP-2 contains the same domain structure as GASP-1, suggesting that these proteins define a two-member family of multivalent protease inhibitors (Fig. 2CGo). Interestingly, we found only peptides corresponding to GASP-1, not GASP-2, in our JA16-purified samples. This result suggests that GASP-1 and GASP-2 likely have different biological specificity. Alternatively, GASP-2 may not be present in serum.

GASP-1 and GASP-2 appear to be the mouse homologs of two human proteins (WFIKKNRP and WFIKKN, respectively) that also contain multiple protease inhibitory domains (46, 47). Although, the concentration of myostatin in human serum is considerably lower than that found in mouse serum (20), the sensitivity of mass spectrometric analysis of proteins allowed us to identify three peptides corresponding to the human homolog of GASP-1 from JA16 immunoprecipitations from human serum (Table 1Go). None of these peptides was found in the corresponding negative control. Again, there was no evidence of human GASP-2 in these experiments. Thus, the interaction between GASP-1 and myostatin is present in both mouse and human.

Characterization of GASP-1
Myostatin RNA is produced nearly exclusively in skeletal muscle (1). To determine the tissue distribution of GASP-1 mRNA, a 551-bp fragment of GASP-1 was amplified from first-strand cDNA produced from a variety of mouse tissues and staged embryos (Fig. 3Go). GASP-1 appears to be fairly widely expressed, with particularly high expression in skeletal muscle and heart. Significant expression is also seen in brain, lung, and testis. In contrast, liver and kidney express relatively low levels of GASP-1 mRNA. Developmentally, the level of GASP-1 mRNA remains fairly constant, perhaps increasing slightly between d 7 and d 11 of mouse embryogenesis.



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Figure 3. GASP-1 mRNA Is Expressed in Many Tissues and throughout Development

Tissue expression profiles of mouse GASP-1. A 551-bp fragment of GASP-1 was amplified from normalized first-strand cDNA panels from CLONTECH Laboratories, Inc. A portion of G3PDH was amplified as a control. G3PDH expression is known to be high in skeletal muscle and low in testis. The cDNA panels were normalized against ß-actin, phospholipase A2, and ribosomal protein S29, in addition to G3PDH.

 
To determine the N-terminal sequence of the secreted GASP-1 protein, we transfected COS cells with a mammalian expression vector encoding GASP-1 with a C-terminal V5-His tag (GASP1-V5-His). Serum-free conditioned media was harvested 48 h later and analyzed by Western blot analysis with an anti-V5 polyclonal antibody. A single band, running at approximately 80 kDa was seen, confirming that recombinant GASP-1 is secreted into the conditioned media (data not shown). Approximately 10 ml of this conditioned media were run over a His-affinity column and further purified by reverse phase chromatography. This purification scheme yielded a band corresponding to the expected size of full-length GASP-1 on a Coomassie-stained SDS-PAGE gel. Edman sequencing of this band determined an N-terminal sequence of L-P-P-I-R-Y-S-H-A-G-I. Thus, amino acids 1–29 of GASP-1 constitute the signal sequence that is removed during processing and secretion.

GASP-1 Binds Directly to Both Mature Myostatin and Myostatin Propeptide
To confirm and further characterize the interaction between myostatin and GASP-1, we incubated purified recombinant myostatin and purified recombinant myostatin propeptide with conditioned media from COS1 cells transfected with either a vector control or GASP1-V5-His. We then immunoprecipitated myostatin with JA16-conjugated agarose beads and looked for copurification of GASP-1 and myostatin propeptide using Western blots (Fig. 4Go, left panel). Both GASP-1 (lane 3) and myostatin propeptide (lane 1) coimmunoprecipitated with myostatin, demonstrating that myostatin can interact with both of these proteins. Neither GASP-1 nor propeptide was detected in JA16 immunoprecipitates in the absence of myostatin (lane 4), eliminating the possibility of nonspecific binding in these experiments. When all three proteins were present, both GASP-1 and myostatin propeptide were pulled down with myostatin, suggesting the possibility that these proteins may form a ternary complex (lane 5). However, this experiment does not eliminate the possibility that GASP-1 and propeptide are bound to the same epitope on separate myostatin molecules.



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Figure 4. GASP-1 Binds Separately to Both Myostatin and Myostatin Propeptide

Left panel, JA16 was used to immunoprecipitate myostatin from mock- or GASP-1-V5-His-transfected COS cell-conditioned media supplemented with recombinant purified myostatin and/or propeptide. Western blots with anti-V5 (top panel), antimyostatin (middle panel), or antimyostatin propeptide polyclonal antibodies were used to determine whether these proteins were present in the immunoprecipitate. Right panel, GASP-1 protein was immunoprecipited by anti-V5 tag antibodies from mock- or GASP-1-V5-His conditioned media supplemented with recombinant purified myostatin and/or propeptide. The immunoprecipitate was analyzed by Western blotting as above.

 
To further confirm the interaction between GASP-1 and myostatin, we performed the reverse immunoprecipitation by pulling down GASP-1 from conditioned media supplemented with myostatin and/or myostatin propeptide recombinant protein. To achieve this, we used an agarose-conjugated monoclonal antibody directed against the V5 epitope of the C-terminal V5-His tag on GASP-1. As expected, myostatin coimmunoprecipitated with GASP-1 (Fig. 4Go, right panel, lanes 3 and 5), further confirming a direct interaction between these proteins. Surprisingly, myostatin propeptide also copurified with GASP-1, even in the absence of myostatin (lane 4), suggesting that myostatin propeptide can bind directly to GASP-1. Thus, GASP-1 binds to both myostatin and myostatin propeptide independently. Addition of both myostatin and propeptide consistently showed less propeptide binding to GASP-1 than when propeptide was added alone, suggesting the possibility that mature myostatin may compete with myostatin propeptide for GASP-1 binding. Because both propeptide and GASP-1 are pulled down by antimyostatin antibodies, further experiments will be necessary to determine whether GASP-1 binds effectively to the myostatin small latent complex.

GASP-1 Inhibits the Activity of Myostatin and BMP-11, But Not Activin or TGFß1
Two FS-domain proteins, follistatin and FLRG, have been previously shown to inhibit myostatin activity in a (CAGA)12 luciferase transcriptional reporter assay (11, 20). Both of these proteins also inhibit the biological activity of the related proteins, activin and BMP-11 (20, 26, 28, 29). Because GASP-1 contains an FS-domain, we tested the ability of GASP-1 to inhibit myostatin, BMP-11, activin, and TGFß1 activity in the (CAGA)12 reporter assay.

Various dilutions of conditioned media from COS cells transfected with V5-His tagged GASP-1 or a vector control were incubated with purified recombinant myostatin, BMP-11, activin, or TGFß1 and assayed for growth factor activity in rhabdomyosarcoma cells expressing the (CAGA)12 reporter construct. GASP-1 inhibited myostatin activity in a concentration-dependent manner (Fig. 5AGo). GASP-1 similarly inhibited the activity of BMP-11 in this assay (Fig. 5BGo), as might be expected because mature myostatin and BMP-11 are highly conserved and differ by only 11 amino acids. Surprisingly, GASP-1 did not inhibit the activity of activin or TGFß1 (Fig. 5Go, C and D), suggesting a level of specificity not yet seen in other FS-domain proteins. Thus, GASP-1 exhibits specificity in its inhibition of myostatin and BMP-11. To estimate the potency of GASP-1 inhibition of myostatin activity, we purified a small amount of GASP-1 and determined the IC50 for the inhibition of myostatin in the reporter gene assay. As can be seen in Fig. 6Go, GASP-1 inhibits 20 ng/ml myostatin with an IC50 of approximately 3 nM.



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Figure 5. GASP-1 Inhibits the Biological Activity of Myostatin and the Highly Related BMP-11, But Not Activin or TGFß

Various dilutions of conditioned media from mock (open circles) or GASP-1-V5-His (solid squares) were incubated with (A) 10 ng/ml myostatin, (B) 10 ng/ml BMP-11, (C) 10 ng/ml activin, or (D) 0.5 ng/ml TGFß. These samples were then subjected to a luciferase reporter activity assay in A204 (A–C) or RD (D) cells to determine the activity of the added growth factors. Luciferase activity is shown in relative luciferase units (RLU). The activity resulting from each of the growth factors alone is shown by the solid diamonds and short dashed line. Without addition of any growth factor, the background activity in the assay is low, as shown by the long dashed line with error bars but no symbol.

 


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Figure 6. GASP-1 Inhibits Myostatin with an IC50 of 3 nM

Purified GASP-1 was tested for its ability to inhibit 20 ng/ml myostatin in the (CAGA)12 luciferase reporter assay in RD cells (solid squares). The activity resulting from myostatin alone is shown by the solid diamonds and short dashed line. The activity present when no growth factors are added is shown by the long dashed line.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this paper, we have used affinity purification and mass spectrometry to identify a novel endogenous myostatin-binding protein that we have named GASP-1. GASP-1 is associated with myostatin in normal mouse and human serum and can inhibit activity resulting from 20 ng/ml myostatin in a reporter gene assay with an IC50 of approximately 3 nM. The myostatin/GASP-1 interaction was identified in a pool of normal mouse and human serum, suggesting that this interaction occurs under physiological conditions. Previously, we have shown that myostatin propeptide and FLRG also bind and inhibit myostatin in serum (20). Thus, nature has provided three structurally distinct proteins to regulate circulating myostatin, emphasizing the biological importance of tight and specific regulation of this growth factor. These three proteins may form distinct multicomponent myostatin complexes that play unique roles in the regulation of skeletal muscle.

The possibility remains that additional proteins bind and regulate myostatin in serum. Although we did not identify any additional myostatin-binding proteins in these experiments, we are limited to interacting proteins that do not mask the JA16 epitope, that are present in the protein databases, and that bind myostatin with a sufficiently low dissociation rate to withstand the washing steps. In addition, our experiments are biased against extremely high abundance serum proteins, since these proteins tend to be identified in the negative controls. Because mass spectrometry is not inherently quantitative, we are not able to determine whether a given protein is present at a higher level in the JA16 samples than in the control. Rather, we are limited to determining whether a protein is present or absent. Intriguingly, {alpha}2-macroglobulin has been shown to bind activin and inhibin, possibly playing a role in delivery or clearance (48, 49). In fact, we did identify {alpha}2-macroglobulin in both the mock-conjugated and JA16-conjugated peptide-eluted samples by mass spectrometry. However, because this protein was present in the negative control, we are unable to determine whether {alpha}2-macroglobulin binds myostatin by these experiments.

GASP-1 is a fairly widely expressed protein that contains an FS-domain and multiple protease-inhibitory domains. The expression of GASP-1 is relatively high in skeletal muscle, suggesting that GASP-1 may bind to myostatin during, or shortly after, secretion. However, whereas myostatin expression is generally limited to the skeletal muscle (1), GASP-1 is also produced in many other tissues and throughout development. This pool of GASP-1 may be released into circulation where it can bind to myostatin. Alternatively, GASP-1 may also act as an inhibitor of other growth factors including BMP-11. In fact, we have shown that BMP-11, which is known to play an important role in embryonic development (26, 27), can be inhibited by GASP-1.

The FS-domain of GASP-1 may be responsible for mediating the interaction with myostatin. Two other FS-domain proteins, follistatin and FLRG, also bind and inhibit myostatin, presumably by preventing it from associating with its cellular receptor (11, 19, 20). FLRG has been shown to bind to circulating myostatin under physiological conditions, confirming a physiological role for FLRG in regulating myostatin activity (20).

Follistatin and FLRG comprise a subfamily of FS-domain proteins that share a common N-terminal domain and high binding affinity for activin (37). Follistatin requires this 63-amino-acid N-terminal domain, in particular some hydrophobic residues, to bind and inhibit activin (50, 51). Although GASP-1 has an N-terminal domain of the same size as follistatin, the two domains are not highly related, showing only 7% identity at the amino acid level. In contrast, the N-terminal domain of FLRG is more than 30% identical to that of follistatin. Intriguingly, the N-terminal domain of FLRG does not appear to be required for activin binding because a glutathione-S-transferase fusion protein containing the second FS-domain and the short acidic C-terminal tail is sufficient to bind activin (29). Because GASP-1 lacks significant similarity to follistatin and FLRG in this N-terminal domain, GASP-1 appears to fall within the second subfamily of FS-domain proteins. Members of this second subfamily are not generally thought to inhibit activin or other TGFß family members (37). Thus, GASP-1 is the first example of a member of this second subfamily that can inhibit a TGFß superfamily member. In addition, GASP-1 is the first example of an FS-domain protein that can bind and inhibit a specific member of the TGFß superfamily without also inhibiting activin. This selectivity may result from regions outside of the FS-domain of GASP-1 that facilitate binding to myostatin. Alternatively, GASP-1 may bind to myostatin in a manner independent of its FS-domain. It is interesting to note that GASP-1 shows the same selectivity as the myostatin propeptide, which does not contain an FS-domain, but also inhibits myostatin and BMP-11, not activin (18).

Based on our in vitro binding results, GASP-1 may also regulate myostatin activity by a mechanism independent of the FS-domain. Surprisingly, myostatin propeptide coimmunoprecipitates with GASP-1, even in the absence of mature myostatin. This is in contrast to the myostatin-FLRG interaction that is mediated entirely by mature myostatin (20). Furthermore, our data suggest that mature myostatin may compete with myostatin propeptide for binding to GASP-1. Thus, GASP-1 interacts independently with both mature myostatin and myostatin propeptide. We have shown that GASP-1 inhibits the biological activity of mature myostatin, but a role for GASP-1 in the regulation of myostatin propeptide is less clear.

Intriguingly, GASP-1 contains multiple protease-inhibitory domains. Two tandem Kunitz domains show a very high degree of similarity to inter-{alpha}-trypsin inhibitor, indicating that these are almost certainly functional serine protease inhibitor domains. GASP-1 may also inhibit metalloproteases, based on the presence of a netrin domain at the C terminus (44, 45). In addition, the FS-domain of GASP-1 is similar to Kazal serine protease-inhibitory domains. All FS-domains contain two subdomains that can be distinguished based on their pattern of disulfide bond linkages and three-dimensional structure (52). The N-terminal region of the FS domain encompasses the first four cysteine residues and displays structural similarity to epidermal growth factor. The C-terminal region, including cysteines 5–10, is tellingly similar to Kazal serine protease-inhibitory domains. Many FS-domain proteins, including the three FS-domains in follistatin and the single FS-domain in SPARC, contain a hydrophobic insertion into the region between cysteines 6 and 7, corresponding to the inhibitory loop of the Kazal protease inhibitor, ovomucoid (52). It has been proposed that this insertion may explain the lack of demonstrated protease activity in these proteins (52). Interestingly, GASP-1 does not contain an insertion in this region, opening the possibility that this domain may act as a functional Kazal-like serine protease inhibitor. Thus, based on the presence of conserved domains, GASP-1 may act as a multifunctional protease inhibitor with activity against both serine proteases and metalloproteases.

The biological significance of the protease-inhibitory domains in GASP-1 remains unclear, but we can speculate that they may play a role in one of the regulatory processes thought to involve proteases: 1) the activation of latent myostatin complex, or 2) the internal processing of the myostatin proprotein to release myostatin propeptide and mature myostatin.

TGFß is produced by proteolytic cleavage of a proprotein at a furin cleavage site. This processing results in the production of two polypeptide chains, the TGFß propeptide and the mature TGFß ligand. After processing, these two protein products remain noncovalently associated and TGFß is secreted as a small latent complex consisting of two mature TGFß molecules and two TGFß propeptide molecules. Although there is considerable interest in the regulation of TGFß during development and homeostasis, it remains unclear how the small latent complex is activated in vivo. In vitro, the serine protease plasmin can cleave the propeptide of active TGFß at an internal site, resulting in the release of the noncovalently bound propeptide moiety and the subsequent activation of the small latent complex (25). If the small latent myostatin complex is also activated by proteolysis of the propeptide, GASP-1 may inhibit this process and serve to keep myostatin in a latent, inactive form.

Alternatively, GASP-1 could prevent the processing of the myostatin proprotein through inhibition of a furin-like protease, presumably resulting in an inactive unprocessed myostatin molecule. Treatment of C2C12 myoblasts with hydroxamate-based chemical inhibitors of metalloproteases induces an increase in cell fusion leading to hypertrophy (53). This treatment also leads to an increase in unprocessed myostatin in C2C12 lysates of these cells, suggesting that the hypertrophy may be the result of inhibiting the proteolytic processing of myostatin. In fact, we see evidence of unprocessed myostatin in our JA16 immunoprecipitations from normal serum (Hill, J. J., unpublished observations; see 48-kDa band in Fig. 1Go), suggesting that regulation of secreted myostatin activity may occur partially through the regulation of furin-mediated myostatin processing.

In this paper, we have identified a novel protein, GASP-1, that interacts with myostatin in normal mouse and human serum. GASP-1 inhibits myostatin activity in a reporter gene assay, which suggests that it acts as a negative regulator of myostatin action. Surprisingly, GASP-1 binds independently to both mature myostatin and myostatin propeptide. This finding, in addition to the observation that GASP-1 contains numerous putative protease-inhibitory domains, suggests that GASP-1 may regulate myostatin activity by an additional mechanism, not associated with the presence of a FS-domain and thus may constitute a novel class of inhibitory TGFß-binding proteins.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Antibodies and Conditioned Media
JA16 monoclonal antimyostatin antibody and the procedure used to produce JA-16-conjugated agarose beads have been previously described, as have the polyclonal antimyostatin and antimyostatin propeptide antibodies used for Western blots (20). Anti-V5 antibodies were purchased from Sigma (St. Louis, MO). Immunoprecipitations were performed with clone V5–10 agarose conjugate and Western blots used an anti-V5 polyclonal rabbit antibody (catalog no. V8137). Conditioned media were collected 48 h after transfection of COS1 cells with GASP1-V5-His/pcDNA3.1D-V5-His-TOPO or empty vector using the FuGENE 6 reagent (Roche, Indianapolis, IN) in serum-free DMEM.

Immunoprecipitations and Mass Spectrometry
JA16 immunoprecipitations from mouse and human serum were performed essentially as described (20), but were scaled up approximately 5-fold by adding 8 µl of 4x lithium dodecyl sulfate sample buffer to the 100 µl eluant and reducing this volume to less than 30 µl by evaporation. All samples were supplemented with water up to a final volume of 35 µl and analyzed as previously described. Briefly, reduced and iodoacetamide-alkylated samples were separated by SDS-PAGE (NuPage, 10% Bis-Tris, Invitrogen, San Diego, CA) and silver stained. Each gel lane was sliced into 13–15 pieces and subjected to in-gel trypsin digestion. The resulting peptides were analyzed by nanoflow reverse phase LC-MS/MS on an LCQ Deca XP ion trap mass spectrometer as previously described (20). Peptide sequences were determined from MS/MS spectra by database searching using the Sequest algorithm and confirmed by manual inspection.

For immunoprecipitations with recombinant proteins, 400 µl conditioned media from vector- or GASP1-transfected cells were combined with 1.2 µg of recombinant purified myostatin and/or myostatin propeptide protein (18). JA16 (10 µl packed volume) or anti-V5 (30 µl) conjugated agarose beads were incubated with the supplemented conditioned media for 2 h at 4 C and washed twice in cold 1% Triton in PBS and twice in PBS. Beads were resuspended in 50 µl 1x lithium dodecyl sulfate buffer with dithiothreitol. Western blots were performed as previously described (20).

Molecular Biology
The GASP-1 coding sequence was amplified from mouse heart QUICKCLONE cDNA (CLONTECH Laboratories, Inc., Palo Alto, CA) by PCR with PfuTurbo polymerase (Stratagene, La Jolla, CA) (forward primer: 5'-CACCATGTGTGCCCCAGGGTATCATCGGTTCTGG-3'; reverse primer: 5'-TTGCAAGCCCAGGAAGTCCTTGAGGAC-3'). The PCR product from this reaction ran as a single major band of approximately 1700 bp on a 1% agarose gel. The amplified cDNA was then cloned into the TOPO sites of the pcDNA3.1D/V5-His-TOPO vector (Invitrogen) so as to include an in-frame C-terminal V5-His tag according to manufacturer’s recommendations. The full-length cDNA insert was sequenced on both strands to produce the sequence given in Fig. 2Go.

Tissue expression profiling was performed using PCR to amplify a 551-bp fragment of GASP-1 from normalized mouse first strand cDNA panels (CLONTECH Laboratories, Inc.) using the Advantage cDNA PCR Kit (CLONTECH Laboratories, Inc.) according to the manufacturer’s recommendations (forward primer: 5'-TTGGCCACTGCCACCACAATCTCAACCACTT-3'; reverse primer: 5'-TCTCAGCATGGCCATGCCGCCGTCGA-3'). Aliquots of the PCR reaction were removed after 23 cycles [glyceraldehyde-3-phosphate dehydrogenase (G3PDH) control only], 34, and 38 cycles (GASP-1 samples) and visualized by ethidium bromide staining of a 1% agarose gel.

GASP-1-His Purification/N-Terminal Sequencing
GASP1-V5-His was purified from 10 ml conditioned media using a cobalt affinity column (CLONTECH Laboratories, Inc.). Proteins were eluted with a linear gradient from 0 to 500 mM imidazole in 50 mM Tris, 1 M NaCl (pH 8.0), followed by reverse-phase high-pressure liquid chromatography on a 2-cm LC-304 C4 cartridge (Supelco, Bellafonte, PA) using an acetonitrile gradient in 0.1% trifluoroacetic acid. Fractions containing GASP-1 were concentrated by evaporation, separated by SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and visualized with Coomassie blue. A major band corresponding to the expected size of full-length GASP-1 (~80 kDa) was excised and sequenced by Edman degradation on an ABI 492 sequencer (ABI, Foster City, CA). GASP1-V5-His protein used in the luciferase reporter gene assay (Fig. 6Go) was purified from 100 ml conditioned media on a cobalt affinity column and eluted as above. Fractions containing GASP-1 were further purified by size exclusion chromatography in PBS using a BioSepS3000 column (Phenomenex, Inc., Torrance, CA).

Reporter Gene Assay
A luciferase reporter construct, pGL3-(CAGA)12 (54) was transiently transfected into A204 or RD rhabdomyosarcoma cells. Dilutions of conditioned media from vector or GASP-1-transfected cells were incubated for 30 min at 37 C with 10 ng/ml myostatin, 10 ng/ml BMP-11, 10 ng/ml recombinant human activin A (R&D Systems, Minneapolis, MN), or 0.5 ng/ml recombinant human TGFß1 (R&D Systems). Luciferase activity was measured as described previously (11, 18). In this assay, A204 cells respond to myostatin, BMP-11, and activin, but do not respond well to TGFß1. RD cells respond to both myostatin and TGFß1. Thus, we used A204 cells to test for the ability of GASP-1 to inhibit myostatin, BMP-11, and activin and RD cells to monitor the activity of TGFß and myostatin. A standard curve measuring the concentration dependence of the luciferase activity induced by each of these growth factors was generated for each experiment (data not shown). The growth factor concentrations used fall in the linear region of this curve such that small changes in growth factor concentration result in measurable changes in luciferase activity.


    ACKNOWLEDGMENTS
 
We thank Amanda Bemis and Ying Huang for N-terminal sequencing and Adele Pearson for providing recombinant purified myostatin and myostatin propeptide. We also thank Ioannis Moutsatsos, Patrick Cody, and Gary Van Domselaar for the development and support of programs and databases to analyze mass spectrometric data.


    FOOTNOTES
 
Abbreviations: BMP, Bone morphogenetic protein; FLRG, follistatin-related gene; FS-domain, follistatin domain; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GASP,growth and differentiation factor-associated serum protein; GDF, growth and differentiation factor; SPARC, secreted protein, acidic and rich in cysteine.

Received for publication November 5, 2002. Accepted for publication February 17, 2003.


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