Latent myostatin has significant activity and this activity is controlled more efficiently by WFIKKN1 than by WFIKKN2

Abstract

Myostatin, a negative regulator of skeletal muscle growth, is produced from myostatin precursor by multiple steps of proteolytic processing. After cleavage by a furin-type protease, the propeptide and growth factor domains remain associated, forming a noncovalent complex, the latent myostatin complex. Mature myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases. Here, we show that, in reporter assays, latent myostatin preparations have significant myostatin activity, as the noncovalent complex dissociates at an appreciable rate, and both mature and semilatent myostatin (a complex in which the dimeric growth factor domain interacts with only one molecule of myostatin propeptide) bind to myostatin receptor. The interaction of myostatin receptor with semilatent myostatin is efficiently blocked by WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 1 or growth and differentiation factor-associated serum protein 2 (WFIKKN1), a large extracellular multidomain protein that binds both mature myostatin and myostatin propeptide [Kondás et al. (2008) J Biol Chem 283, 23677-23684]. Interestingly, the paralogous protein WAP, Kazal, immunoglobulin, Kunitz and NTR domain-containing protein 2 or growth and differentiation factor-associated serum protein 1 (WFIKKN2) was less efficient than WFIKKN1 as an antagonist of the interactions of myostatin receptor with semilatent myostatin. Our studies have shown that this difference is attributable to the fact that only WFIKKN1 has affinity for the propeptide domain, and this interaction increases its potency in suppressing the receptor-binding activity of semilatent myostatin. As the interaction of WFIKKN1 with various forms of myostatin permits tighter control of myostatin activity until myostatin is liberated from latent myostatin by bone morphogenetic protein 1/tolloid proteases, WFIKKN1 may have greater potential as an antimyostatic agent than WFIKKN2.

Keywords: WFIKKN1; WFIKKN2; latent myostatin; myostatin; promyostatin.

Fig 1

Schematic representation of the domain structure of human prepromyostatin. The vertical dashed lines indicate the positions of the sites of cleavage by furin-type proteases and BMP-1, and S indicates the signal peptide. The bottom part of the figure illustrates the position of the various prodomain fragments used in the present work. The numbers refer to the residue numbering of human prepromyostatin.

Fig 2

Luciferase reporter assay of myostatin activity of promyostatin and its derivatives. (A) Rhabdomyosarcoma A204 cells were transiently transfected with the SMAD Luciferase Reporter vector and a Renilla luciferase vector, and incubated for 16 h with different forms of myostatin. Firefly luciferase units were normalized to Renilla luciferase units. A, control medium; B, 5 nm promyostatin; C, 5 nm latent myostatin; D, 5 nm BMP-1-digested latent complex; E, 5 nm latent myostatin incubated at 80 °C for 5 min. (B) A204 cells transiently transfected with the SMAD Luciferase Reporter vector and a Renilla luciferase vector were incubated for 6 h with different concentrations of latent complex (▴) or with different concentrations of latent complex incubated at 80 °C for 5 min (•). Firefly luciferase units were normalized to Renilla luciferase units. Note that latent myostatin had significant activity even in the absence of BMP1-cleavage or heat treatment. Values are means ± standard errors. *P < 0.05 versus control samples; **P < 0.01 versus control samples.

Fig 3

Comparison of the interactions of promyostatin, latent myostatin and mature myostatin with ECD_ACRIIB. Promyostatin (100, 500, and 1000 nm) (A), latent myostatin (25, 100, 200, 350, 500, and 1000 nm (B) or mature myostatin (10, 20, 35, 50, 100, and 200 nm (C) in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) were injected over the surface of CM5 sensorchips containing the ligand-binding extracellular domain of ACRIIB. The insert in (B) shows the apparent association rate constants k_obs as a function of latent myostatin concentration. The observation that the value of k_obs did not increase linearly with the increase in analyte concentration indicates that the proportion of receptor-binding species decreased with the increase in total latent myostatin concentration. RU - SPR Response Units.

Fig 4

Myostatin prodomain blocks the interaction of mature myostatin with ECD_ACRIIB. SPR sensorgrams of the interactions of immobilized ECD_ACRIIB with 10 nm myostatin preincubated with 0, 1, 2, 5, 10, 20, 50 and 100 nm myostatin prodomain are shown. Various concentrations of myostatin prodomain and 10 nm myostatin were preincubated in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) for 30 min at room temperature, and were injected over CM5 sensorchips containing immobilized ECD_ACRIIB. For the sake of clarity, the concentrations of myostatin prodomain injected over the sensorchip are not indicated in the panels; the SPR response decreased in parallel with the increase in myostatin prodomain concentration. The insert shows that the value of the apparent association rate k_obs decreased with the increase in myostatin prodomain concentration. Note that 50 nm myostatin prodomain completely eliminated the interaction; half-maximal inhibition was achieved with ∼ 1 × 10⁻⁸ m myostatin prodomain. RU - SPR Response Units.

Fig 5

Myostatin prodomain binds to the myostatin–myostatin receptor complex. (A) The myostatin–ECD_ACRIIB complex was formed by injection of 100 nm myostatin over the surface of immobilized ECD_ACRIIB, and, after the completion of the injection, different concentrations of myostatin prodomain (0, 20, 50, 100, 200, and 500 nm) were injected over the receptor–myostatin complex. For the sake of clarity, the concentrations of prodomain injected over the sensorchip are not indicated in the panels; the SPR response increased with the increase in myostatin prodomain concentration. (B) Sensorgrams of the interaction of myostatin prodomain with the ACRIIB–myostatin complex fitted with the 1 : 1 interaction model of biaevaluation 4.1. The sensorgrams in (B) were calculated from those shown in (A) by subtracting the RU values observed at 0 nm myostatin prodomain. The equilibrium dissociation constant of the interaction of myostatin prodomain with the myostatin–ECD_ACRIIB complex was 3 × 10⁻⁸ m. RU - SPR Response Units.

Fig 6

Interaction of promyostatin with immobilized WFIKKN1 and WFIKKN2. (A) Sensorgrams of the interactions of promyostatin (50, 100, 250, 500, 1000, 1500, 2000, and 2500 nm) with WFIKKN1. (B) Sensorgrams of the interactions of promyostatin (100, 500, and 2000 nm) with WFIKKN2. Various concentrations of promyostatin in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) were injected over CM5 sensorchips containing immobilized WFIKKN1 or WFIKKN2. For the sake of clarity, the concentrations of promyostatin are not indicated in the panels; in (A), the SPR response increased in parallel with the increase of promyostatin concentration. In (A), the inset shows the equilibrium responses plotted against the concentration of injected promyostatin; the equilibrium dissociation constant was determined by fitting the curve with the general fitting model ‘Steady state affinity’ of biaevaluation 4.1. The equilibrium dissociation constant of the interaction of promyostatin with WFIKKN1 was ∼ 1 × 10⁻⁶ m. RU - SPR Response Units.

Fig 7

Interaction of myostatin prodomain with immobilized WFIKKN1 and WFIKKN2. (A) Sensorgrams of the interactions of myostatin prodomain (25, 50, 100, 200, 350, 500, 750, and 1000 nm) with WFIKKN1. (B) Sensorgrams of the interactions of myostatin prodomain (100, 500, and 1000 nm) with WFIKKN2. Various concentrations of myostatin prodomain in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) were injected over CM5 sensorchips containing immobilized WFIKKN1 or WFIKKN2. For the sake of clarity, the concentrations of myostatin prodomain are not indicated in these panels; in (A), the SPR response increased in parallel with the increase in myostatin prodomain concentration. The response curves were fitted with the 1 : 1 interaction model of biaevaluation 4.1, and the K_D for binding of WFIKKN1 to myostatin prodomain was calculated to be 2 × 10⁻⁸ m (A). Note that WFIKKN2 did not bind myostatin prodomain (B). (C) Sensorgrams of the interaction of immobilized WFIKKN1 with 200 nm myostatin prodomain preincubated with or without 1 μm WFIKKN1. (D) Sensorgrams of the interaction of immobilized WFIKKN1 with 200 nm myostatin prodomain preincubated with or without 1 μm WFIKKN2. Mixtures of WFIKKN1 or WFIKKN2 with myostatin prodomain were incubated for 30 min in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) before injection over CM5 sensorchips containing immobilized WFIKKN1. Note that soluble WFIKKN1 efficiently inhibited the interaction of myostatin prodomain with immobilized WFIKKN1 (C), whereas soluble WFIKKN2 had no effect on the interaction (D). RU - SPR Response Units.

Fig 8

Myostatin and WFIKKN1 bind to different regions of myostatin prodomain. (A) Sensorgrams of the interaction of PRO_43–115 (500 nm, 1 μm, 2 μm, 5 μm, and 10 μm) with immobilized myostatin. (B) Sensorgram of the interaction of PRO_116–266 (1 μm) with immobilized myostatin. Note that myostatin bound to the N-terminal region but not the C-terminal region of myostatin prodomain. (C) Sensorgrams of the interaction of PRO_43–115 (400 nm, 1 μm, and 2.5 μm) with immobilized WFIKKN1. (D) Sensorgrams of the interaction of PRO_116–266 (50 nm, 100 nm, 200 nm, 500 nm, 1 μm, and 5 μm) with immobilized WFIKKN1. Note that WFIKKN1 bound to the C-terminal region but not the N-terminal region of myostatin prodomain. Various concentrations of prodomain fragments in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) were injected over the surface containing immobilized myostatin (A, B) or immobilized WFIKKN1 (C, D). The inset in (D) shows the equilibrium response plotted against the concentration of injected PRO_116–266. The equilibrium dissociation constant was determined by fitting the curve with the general fitting model ‘Steady state affinity’ of biaevaluation 4.1, and the K_D for binding of WFIKKN1 to PRO_116–266 was calculated to be 4.3 × 10⁻⁷ m. RU - SPR Response Units.

Fig 9

Latent myostatin binds WFIKKN1 but not WFIKKN2. In Ni²⁺-affinity pull-down assays, 1 μm latent myostatin was incubated for 1 h with 2 μm His-tagged WFIKKN1 or 2 μm His-tagged WFIKKN2 in NaCl/P_i containing 50 mm imidazole, 0.1% Tween-20 and 100 μm phenylmethanesulfonyl fluoride (pH 7.5), and the solutions were then mixed with 20 μL of Ni²⁺–nitrilotriacetic acid resin. After 15 min of agitation, the resin was washed with NaCl/P_i, 50 mm imidazole, 0.5% Tween-20 and 100 μm phenylmethanesulfonyl fluoride (pH 7.5), and the bound proteins were eluted with NaCl/P_i and 500 mm imidazole (pH 7.5). The eluted samples were analyzed by SDS/PAGE, and the proteins were visualized by staining with Coomassie Brilliant Blue and by western blotting with specific antibodies against myostatin prodomain (anti-prodomain) and against mature myostatin (anti-myostatin). LM, latent myostatin. The numbers indicate the molecular mass values of proteins of the Low Molecular Weight Calibration Kit. In the upper panel, the proteins were visualized by staining with Coomassie Brilliant Blue; in the lower panels, the proteins were visualized by western blotting.

Figure 10

WFIKKN1 and WFIKKN2 inhibit the binding of latent myostatin to ECD_ACRIIB. (A) Sensorgrams of the interactions of immobilized ECD_ACRIIB with 500 nm latent myostatin preincubated with WFIKKN1 (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 nm). (B) Sensorgrams of the interactions of immobilized ECD_ACRIIB with 500 nm latent myostatin preincubated with WFIKKN2 (0, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, and 100 nm). For the sake of clarity, the concentrations of WFIKKNs are not indicated in the panels; the SPR response decreased in parallel with the increase in WFIKKN concentration. (C) Values of the apparent association constant k_obs from (A) and (B) were plotted against WFIKKN1 (▼) and WFIKKN2 (•) concentrations. Note that k_obs values decreased with the increase in WFIKKN1 or WFIKKN2 concentration; half-maximal inhibition was achieved with ∼ 1 × 10⁻⁹ m WFIKKN1 or ∼ 5 × 10⁻⁹ m WFIKKN2. In these experiments, various concentrations of WFIKKN1 or WFIKKN2 were preincubated with latent myostatin in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA and 0.005% Tween-20 (pH 7.5) for 30 min at room temperature, and were injected over CM5 sensorchips containing immobilized ECD_ACRIIB. RU - SPR Response Units.