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Reproductive Endocrine Unit (A.L.S., Y.S., A.G., J.L.S.), Massachusetts General Hospital, and Endocrine Unit (H.K.), Massachusetts General Hospital, Boston, Massachusetts 02114; and Pfizer Global Research and Development (P.A.K.), Groton, Connecticut 06340
Address all correspondence and requests for reprints to: Alan Schneyer, Ph.D., Pioneer Valley Life Science Institute, 3601 Main Street, Springfield, Massachusetts 01107. E-mail: alan.schneyer{at}bhs.org.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Follistatin (FST) and follistatin-like (FSTL)-3 (FLRG, FSRP) form a related group of follistatin domain (FSD)-containing proteins with overlapping biochemical, molecular, biosynthetic, and structure-function attributes, although there are also important differences (12). Neutralization of TGFβ superfamily ligands is their only known biological activity. Both proteins bind and neutralize activin irreversibly and bind myostatin with a 3- to 5-fold lower affinity (13, 14, 15). FST null mice die immediately after birth due to defects in muscle and bone formation (16), whereas FSTL3 null mice are viable but have altered glucose metabolism and enhanced pancreatic β-cell formation (17). FST overexpression in gonads leads to abnormal gonadal architecture and reduced fertility (18), whereas FSTL3 overexpression results in smaller testes and reduced male fertility (19), suggesting that regulation of activin activity by FST and/or FSTL3 is important for normal reproduction. In addition, transgenic overexpression of FST in muscle leads to increased musculature presumably due to decreased myostatin activity through antagonism by FST (20). These observations indicate that both in vitro and in vivo, FST and FSTL3 function to regulate the activity of activin and myostatin and that their absence or excess leads to abnormal development and/or function.
Experimental states of myostatin excess result in reduced body mass and cachexia (21), a state of body tissue wasting commonly associated with chronic diseases including cancer, AIDS, renal failure, and gastrointestinal disease and contributes to the morbidity and mortality of these conditions (22, 23). In addition, circulating myostatin levels were significantly elevated in AIDS patients with more than 10% weight loss (24). These observations have accelerated the search for myostatin inhibitors that might form the basis for novel therapeutic strategies to treat muscle-wasting conditions.
The crystal structure of FST bound to activin was recently reported in which FSD2 plays a prominent role in contact between each FST molecule and one subunit of the activin homodimer (25). Additional analysis of FST structure-function relationships using point and domain mutagenesis has identified numerous residues in FSD1, FSD2, and the N-terminal domain that have important effects on FST’s binding activity (26, 27). Moreover, these studies demonstrated that the order of the FST domains is also critical (26). Taken together, these studies demonstrate that specific residues in FST, especially within FSD2, are critical for the high-affinity interaction between FST and activin.
Although direct structural evidence is presently unavailable, it is theoretically possible that critical determinants for activin and myostatin binding to FST and FSTL3 do not completely overlap, suggesting that selective activin or myostatin antagonists might be derived through mutation of the natural protein. To begin to decipher the portions of FST critical for binding and neutralizing myostatin vs. activin, we tested previously prepared FST point and domain mutants with altered activin activity (26, 27) for their ability to inhibit myostatin activity in vitro. Our results indicate that mutations in FSD1 have the largest effect on myostatin binding, whereas FSD2 mutations that reduce or eliminate activin binding have little effect on myostatin binding. In addition, given the close structural relationship between myostatin and GDF11, we also examined binding and neutralization of GDF11 by wild-type (WT) and mutant FST. These results confirm the concept that the primary contacts of FST differ for activin and myostatin (28) and demonstrate that selectivity of FST mutants can be altered to reduce activin binding while preserving myostatin neutralization. Our results also indicate that such mutants will bind GDF11 and that further experimentation is required to separate myostatin and GDF11 binding determinants.
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Materials and Methods |
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Production of FST mutants
Mutations were introduced into the FST288 cDNA as previously discussed (26, 27), and mutant sequences were verified by bidirectional sequencing. The complete list of mutants analyzed is shown in Table 1
and were previously described in terms of activin binding and neutralization (26, 27). All mutant and WT FST constructs were cloned into pcDNA3.1-myc/his vector (Invitrogen, Carlsbad, CA) and prepared using NucleoBond Maxiprep kits (BD Biosciences CLONTECH, Palo Alto, CA).
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Protein production and purification
Human embryonic kidney FreeStyle 293-F cells were maintained in 30 ml of FreeStyle 293 expression medium (Life Technologies) to a density of 1 x 104 cells/ml with a minimum of 85% viability. Plasmid DNA (35 µg per 30 ml culture) was transfected using 40 µl of 293fectin reagent and 2 ml of Opti-MEM (Life Technologies) according to the manufacturer’s protocol. The culture was incubated for 72 h at 37 C, after which the supernatant was collected and circulated over a nickel-Sepharose affinity column (QIAGEN) using a peristaltic pump at 4 C overnight. Purified FST was eluted with imidazole [300 mM (pH 6.8)] and then concentrated and exchanged into Dulbecco’s PBS using an Ultra-15 centrifugal filter unit (Amicon, Bedford, MA).
Quantitation of purified protein
The protein concentration of WT and mutant FST preparations was determined using two immunoassays for FST or the myc tag as previously described (26) and adjusted for results from silver stained gels of the concentrated FST after electrophoresis, which was compared with a standard of known concentration (>90% pure). FST bands were identified by Western blot of SDS-PAGE gels using anti-myc (clone 4A6; Upstate Biologicals, Lake Placid, NY) and detection with goat antimouse IgG linked to horse radish peroxidase (1:15,000; Jackson ImmunoResearch, West Grove, PA).
Solid-phase direct binding assay
Myostatin or activin was plated onto 96-well Immulon-2 (Dynatech Laboratory, Chantilly, VA) strips in carbonate buffer overnight at 4 C at a concentration of 50 ng per 50 µl/well (24). Each well was aspirated and blocked with 200 µl of blocking buffer (10 mM PBS containing 3% BSA) for at least 2 h. Nonspecific binding wells were left in blocking solution, and all other wells were washed three times using Tween 20 and Tris-buffered saline (TTBS) (TBS/0.05% Tween 20). Increasing concentrations of FST WT or mutants, diluted in TBS, were added to the well for 1 h at room temperature. After washing, 100 µl of anti-myc antibody (clone 4A6; Upstate Biologicals) were added at a final dilution of 1:500 in TBS/0.1% BSA and incubated for 1 h at room temperature. After three washes of TTBS, goat antimouse IgG-alkaline phosphatase (Jackson ImmunoResearch) was used at a final dilution of 1:500 as the secondary antibody in TBS/0.1% BSA. The plate was incubated for 1 h at room temperature and washed three times with TTBS. 
-Nitrophenol phosphate (1 x 15 mg tablet; Sigma, St. Louis, MO) was dissolved in 15 ml of 0.1 M glycine buffer with 1 mM MgCl2 and 1 mM ZnCl2 (pH 10.4). Two hundred microliters were added to each well for 30 min in room temperature. The plate was analyzed on a microplate reader at 405 nm.
Solid-phase radioligand binding assay
Activin was iodinated as previously described (13). Purified WT FST was plated onto 96-well Immulon-2 plates (Dynatech Laboratories) in 0.1 M carbonate buffer (pH 9.6) overnight at 4 C at 25 ng/well (13). After blocking nonspecific sites with 200 µl of blocking buffer (0.01 M PBS/0.05% Tween 20/3% BSA) for 2 h, increasing concentrations of unlabeled activin or GDF11 were added to each well in 100 µl assay buffer (0.01 M PBS/0.05% Tween 20 + 0.1% gelatin). Radiolabeled activin was diluted to 50,000 cpm per 50 µl, and 50 µl were added to all wells. The plate was incubated for 2 h at room temperature. After three washes, the wells were aspirated and counted in a 
-counter.
Data analysis
Reporter activity results were expressed as percent of maximum (no FST) for each ligand. Each experiment also included WT FS 288 as a positive control. Mutants showing significant differences between activin and myostatin inhibition were tested at least three times.
For comparison of activin and myostatin binding activity of FST mutants at 200 ng DNA/well (Fig. 1), the activin and myostatin inhibition by each mutant was normalized to the activity of WT FST in that assay. For assays comparing activin and myostatin inhibition of increasing doses of WT or mutant FST (Fig. 2), the ED50 was estimated at the dose at which 50% of maximal stimulation was inhibited. This point was compared for mutant FST vs. WT FST and to compare myostatin with activin inhibition activity.
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Results |
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) containing single-amino acid substitutions in FSD1, FSD2, the N-terminal domain, or mutations that altered the number or order of FSDs were examined for myostatin and activin inhibitory activity. The N-terminal domain mutations that were previously shown to suppress activin inhibition (27) also reduced myostatin activity to a similar degree (not shown) and were thus not investigated further. The remaining 13 mutants retaining antagonistic activity to at least one ligand were screened by in vitro bioassay at a single, maximal dose to compare activin and myostatin antagonism relative to WT FST. We found that outright deletion of FSD1 diminished both myostatin and activin inhibition, indicating that this domain was required for both activities (Fig. 1). However, the remaining FST mutants revealed differential inhibition of activin vs. myostatin, with six mutants, primarily focused on FSD2, retaining substantial or complete myostatin antagonism with reduced activin inhibition (Fig. 1) and were investigated further.
The six differentially active mutants were examined at multiple doses to compare their relative activin and myostatin antagonist activities with WT FST in more detail. Interestingly, outright deletion of FSD2 (dFSD2) had little effect on myostatin inhibition (Fig. 2A; compare triangle, solid vs. dotted lines), whereas inhibition of activin activity was almost completely ablated (Fig. 2A; compare circle, solid vs. dotted lines). Altering the order of the FST domains so that FSD3 preceded FSD1 and -2 (Fig. 2B; FSD3/1/2) had a similar effect on activin activity but also reduced myostatin inhibition by nearly 10-fold. Placing FSD2 before FSD1 (FSD2/1/3) was not as effective at reducing activin inhibition, but it had no effect on myostatin inhibition (Fig. 2C). These results suggest that FSD2 is more critical for activin antagonism, whereas FSD1 appears to be more critical for myostatin inhibition.
We therefore replaced FSD2 with an extra copy of FSD1 (FSD1/1/3), which was more effective than WT FST in antagonizing myostatin but lost the majority of its activin antagonist activity (Fig. 2D). Conversely, replacing FSD1 with an extra copy of FSD2 (FSD2/2/3) reduced myostatin inhibition nearly 10-fold more than the reduction in activin inhibition (Fig. 2E), consistent with FSD2 having greater effect on activin bioactivity, compared with FSD1 as hypothesized. Point mutations in FSD2, such as Y185A, had little effect on myostatin inhibition but reduced activin antagonism more than 10-fold, consistent with this domain being more important for activin inhibition. Taken together, these results indicate that mutations affecting FSD2 have a greater effect on activin antagonism, whereas FSD1 appears to be more critical for inhibiting myostatin activity.
To determine whether the altered activity of the mutants was due to altered binding of the ligands, we investigated three mutants with the greatest difference in activity for direct binding to solid-phase ligand. Although WT FST bound well to solid-phase activin, none of the mutants bound activin, even at the maximal dose tested (Fig. 3A). In contrast, WT FST and all of the FST mutants bound to solid-phase myostatin in rank order, consistent with bioactivity (WT > dFSD2 > FSD3/1/2 > FST Y185A; Fig. 3B). Thus, the differential activity appears to be due to a difference in binding affinity with FSD2 mutants retaining myostatin binding but demonstrating vastly reduced activin binding. These results also indicate that by deleting FSD2, altering its position in the FST molecule, or introducing mutations into FSD2 selectively reduces activin relative to myostatin inhibition, selective myostatin antagonists were created.
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), equivalent to the difference in FST binding to myostatin relative to activin in a similar assay (15). We then examined whether dFSD2, the FST mutant with greatest differential antagonism between activin and myostatin, would similarly differentially antagonize GDF11. Whereas dFSD2 had vastly reduced activin inhibitory activity (Fig. 4B), this mutant had identical activity to WT FST in inhibiting GDF11 in the same in vitro bioassay (Fig. 4C). Moreover, this differential activity was identical with that seen with myostatin (Fig. 2A). These results indicate that like myostatin, FST mutants with domain 2 deletion are selective for GDF11 relative to activin. Furthermore, they demonstrate that such mutants will antagonize both myostatin and GDF11 similarly and thus not be selective with respect to these two ligands.
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Discussion |
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Consistent with the recently solved crystal structure of activin bound to FST that demonstrated extensive contacts between FSD2 and activin (25) and our previous mutational analyses that identified FSD2 as a critical activin binding region (26), the present results indicate that deletion of FSD2, alteration of its position in the FST molecule, or point mutations within FSD2 all reduce activin antagonism. Conversely, mutations that eliminate FSD1 or alter its position usually alter myostatin inhibition. Significantly, those FSD2 mutations that reduce activin antagonism have little effect on myostatin inhibition, demonstrating that indeed these ligands are differentially bound and antagonized by FST. This observation demonstrates that it is possible to create FST mutants that are selective for myostatin inhibition. The most selective mutant was dFSD2 in which the second FST domain was deleted, leaving the N domain and FSD1 followed by FSD3. This mutant antagonized myostatin similarly to WT FST, but its activin-inhibitory activity was reduced more than 250-fold. Moreover, direct binding to activin was undetectable, whereas myostatin binding was reduced only slightly. These results indicate that the dFSD2 mutant might act as a myostatin selective mutant in vivo and therefore constitute a starting point for development of a selective myostatin inhibitor of therapeutic value.
The value of such an antagonist was recently demonstrated by Nakatani et al. (28) in a transgenic model. Overexpression of a FST mutant containing the N domain and two copies of FSD1 (FS I-I) in muscle led to increased muscle mass and strength resulting from both hyperplasia and hypertrophy (28). Moreover, overexpression of this mutant in muscle of mdx mice (a model for Duchene muscular dystrophy) resulted in at least partial rescue from this disease (28). Although this study clearly demonstrated the potential of one selective FST mutant for reversing muscle degeneration, the only other FSD1 or -2 mutant that was investigated was a construct containing two copies of FSD1 and FSD3 (FS I-I-3). This protein was found to bind activin and myostatin similarly to the FS I-I mutation and was not investigated further (28). Interestingly, the FS 1–1-3 mutant appears identical with our FSD 1/1/3 mutant in structure, but in our assay, the FSD 1/1/3 mutant was less selective than our dFSD2 mutant because substantial activin inhibitory activity was retained in FSD1/1/3. Although we used the same reporter, we used human embryonic kidney 293 cells as opposed to A204 cells used by Nakatani et al., which may be one source for the disparate results. Nevertheless, both studies clearly demonstrate that FST mutants can be constructed allowing selective inhibition of myostatin activity with respect to activin binding.
GDF11 was recently shown to be a critical regulator of pancreatic β-cell development and differentiation (11). Given its extensive structural identity with myostatin, we investigated whether FST could neutralize its bioactivity and whether FST mutants selective for myostatin were similarly selective for GDF11. We found that GDF11 inhibited activin binding to FST with approximately 6-fold lower potency, a difference that is comparable with myostatin’s binding relative to activin (15, 28). Moreover, WT FST antagonized the bioactivity of both activin (Fig. 4B) and GDF11 (Fig. 4C), and this antagonism was equivalent to that of the mutant dFSD2 (Fig 4C), which also antagonized myostatin (Fig. 2A). However, whereas the binding results suggest greater potency for inhibiting activin relative to GDF11, the bioassay results suggest greater inhibition of GDF11 relative to activin. This discrepancy is likely due to the GDF11 preparation being less than 100% bioactive, thereby decreasing its effective concentration relative to FST or dFSD2 in the bioassay, which would shift the binding curve to the right and the bioassay curve to the left. Nevertheless, these results demonstrate that FST is a binding and neutralization protein for all members of the activin branch of the TGFβ superfamily and mutants selective for myostatin over activin are similarly selective for GDF11. This observation suggests that whereas FST mutants relatively deficient for activin neutralization can be created, they will likely inhibit both myostatin and GDF11. Because the activity of GDF11 in adults is unknown at present, our findings indicate that potential side effects of myostatin-selective FST mutants need further investigation.
In this study, we have demonstrated that the surfaces of FST necessary for binding and neutralizing activin and myostatin are at least partially distinct, with FSD1 being more critical for myostatin binding and FSD2 more important for activin neutralization. The mutant with the greatest decrease in activin antagonism relative to myostatin, dFSD2, had at least a 250-fold greater inhibition of myostatin relative to activin. We also showed that both WT FST and the dFSD2 mutant bind GDF11. Taken together, our results confirm the presence of differential binding sites for activin and myostatin/GDF11 in FST and support the development of selective antagonists with reduced activin inhibition. Although such antagonists may still not be able to differentially neutralize myostatin and GDF11, further mutational analysis of FST antagonists may identify residues or regions that permit their functional discrimination.
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Acknowledgments |
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Footnotes |
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Current address for A.L.S.: Pioneer Valley Life Science Institute, 3601 Main Street, Springfield, Massachusetts 01107.
Disclosure Statement: P.A.K. is currently employed by and has equity interest in Pfizer. A.L.S., Y.S., H.K., and P.A.K. are inventors on a U.S. patent application. A.G. and J.L.S. have nothing to declare.
First Published Online June 5, 2008
Abbreviations: dFSD2, Deletion of FSD2; FSD, follistatin domain; FST, follistatin; FSTL, FST-like; GDF11, growth and differentiation factor 11; TBS, Tris-buffered saline; TTBS, Tween 20 and TBS; WT, wild type.
Received February 25, 2008.
Accepted for publication May 23, 2008.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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