WFIKKN1 and WFIKKN2 bind growth factors TGFb1, BMP2 and BMP4 but do not inhibit their signalling activity Gyo ¨ rgy Szla ´ ma, Katalin Konda ´ s, Ma ´ ria Trexler and La ´ szlo ´ Patthy Institute of Enzymology, Budapest, Hungary Introduction Growth factors of the transforming growth factor b (TGFb) family regulate many cellular processes, includ- ing cell proliferation, differentiation and lineage deter- mination. In humans, more than 30 structurally related proteins belong to this family [1]. Members of this protein family are usually assigned to three main subfamilies: activins, TGFbs and bone morphogenic proteins (BMPs) ⁄ growth and differentiation factors (GDFs). TGFb family members are secreted as large precursor proteins and the mature growth factors are released from these precursors through cleavage by furin-type prote- ases. In several cases, the prodomain and the mature Keywords BMP; GDF11; GDF8; TGFb; WFIKKN Correspondence L. Patthy, Institute of Enzymology, Budapest, Karolina ut 29, Hungary Fax: +361 466 5465 Tel: + 361 209 3537 E-mail: patthy@enzim.hu (Received 1 June 2010, revised 5 October 2010, accepted 8 October 2010) doi:10.1111/j.1742-4658.2010.07909.x WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP domain, a follistatin domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain. Recent experiments have shown that both proteins have high affinity for growth and differentiation factor (GDF)8 and GDF11. Here we study the interaction of WFIKKN proteins with several additional representatives of the transforming growth factor (TGF)b family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFb1, bone morphogenetic pro- tein (BMP)2 and BMP4 with relatively high affinity (K d  10 )6 M). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFb1, BMP2 and BMP4 using reporter assays. These studies revealed that WFIKKN1 and WFIKKN2 inhibited the biological activity of GDF8 and GDF11 in the nanomolar range, whereas they did not inhibit the activities of TGFb1, BMP2 and BMP4 even in the micromolar range. Our data indicate that WFIKKN proteins are antagonists of GDF8 and GDF11, but in the case of TGFb1, BMP2 and BMP4 they function as growth factor binding pro- teins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to estab- lish growth factor gradients in the extracellular space. Structured digital abstract l A list of the large number of protein-protein interactions described in this article is available via the MINT article MINT-8044119 Abbreviations ACRIIB, activin receptor IIB; BMP2, bone morphogenetic protein 2; BMP3, bone morphogenetic protein 3; BMP4, bone morphogenetic protein 4; BMP8b, bone morphogenetic protein 8b; BMP11, bone morphogenetic protein 11 or growth and differentiation factor 11; BMPRIA, bone morphogenetic protein receptor IA; ECD, extracellular domain; GDF11, growth and differentiation factor 11 or bone morphogenetic protein 11; GDF8, growth and differentiation factor 8 or myostatin; TGFb1, transforming growth factor b1; TGF-bsRII, recombinant protein corresponding to the extracellular domain of TGFb1 receptor TGF-bbRII; WFIKKN1 and WFIKKN2 – WAP, follistatin, immunoglobulin, kunitz and netrin domain containing protein 1 and 2. 5040 FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS disulfide-bonded homodimer growth factor remain asso- ciated after proteolytic cleavage [2–4]. The prodo- main ⁄ growth factor complexes confer latency on the growth factors and the active homodimeric growth fac- tors may be liberated from the latent complexes through degradation of the propeptides by proteases [4–6]. TGFb family proteins signal through type I and type II serine–threonine kinase receptors; in verte- brates, seven type I receptors and five type II receptors have been identified [1]. Homodimeric growth factors bind to two type I and two type II receptors to form a hexameric signalling complex. In these complexes, type II receptors phosphorylate a short segment of type I receptors, which in turn phosphorylate down- stream targets [7,8]. The number of growth factors available for signal- ling is tightly regulated by several, structurally differ- ent antagonists that, by interacting with the growth factors, alter or diminish their binding to the receptors. Similar to the prodomain in latent complexes, inhibi- tory proteins, like chordin, noggin, follistatin, follista- tin-related protein and gremlin bind various members of the TGFb family with high affinity and block their interaction with their receptors [9]. Recent studies have expanded the list of TGFb antagonists to include WFIKKN1 and WFIKKN2 proteins: these proteins bind GDF8 (myostatin) and GDF11 (BMP11) with high affinity [10]. WFIKKN proteins are large extracellular multidomain proteins that contain a WAP domain, a Follistatin ⁄ Kazal domain, an immunoglobulin domain, two Kunitz-type protease inhibitor domains and an NTR domain [11,12]. The fact that, in luciferase reporter assays, WFIKKN2 inhibited the activity of myostatin and GDF11 [13] suggests that WFIKKNs may play crucial roles in the regulation of processes (muscle growth, anterior ⁄ posterior patterning of the axial skeleton, etc.) that are under the control of these growth factors. WFIKKN proteins are, however, expressed in numerous tissues other than those controlled by GDF8 or GDF11. For example, the WFIKKN1 gene is expressed in pancreas, thymus, liver, kidney, lung, tes- tis and inner ear, and the WFIKKN2 gene is expressed in ovary, testis, pancreas, brain and lung [11,12,14], raising the possibility that the proteins may have addi- tional functions. To investigate this possibility, we used SPR and luciferase reporter assays to study the inter- action of WFIKKN1 and WFIKKN2 proteins with several representatives of the TGFb family. Analyses of SPR sensorgrams have shown that both WFIKKN proteins bind TGFb1, BMP2 and BMP4 with relatively high affinity (K d  10 )6 m), but in reporter assays they do not inhibit their activities, even in the micromolar range. Our data suggest that WFIKKN proteins may function not only as antago- nists of GDF8 and GDF11, but also as proteins that localize the action of growth factors. Results Characterization of the interaction of WFIKKN1 and WFIKKN2 with BMP2, BMP3, BMP4, BMP8b and TGFb1 by SPR SPR analyses suggested that both WFIKKN proteins may bind BMP2, BMP3, BMP4, BMP8b and TGFb1 (Fig. 1), although the affinities of WFIKKN1 and WFIKKN2 for these growth factors are significantly lower than those determined for GDF8 and GDF11 (Table 1). No interaction was detected with activin A, even when high (up to 4 lm) concentrations of WFIKKNs were injected on the surface of immobi- lized activin A. The K d values calculated for the interactions of WFIKKN1 with BMP2 (7.2 · 10 )7 m), BMP3 (3.3 · 10 )6 m), BMP4 (8.2 · 10 )7 m), TGFb1 (4.5 · 10 )7 m) or for the interactions of WFIKKN2 with BMP2 (4.3 · 10 )8 m), BMP3 (1.8 · 10 )7 m), BMP4 (6.5 · 10 )8 m), TGFb1 (2.8 · 10 )8 m) were suggestive of relatively high affinities, raising the possibility that these interac- tions may have biological importance. It should be noted, however, that there was a major additional difference between sensorgrams obtained with GDF8 ⁄ GDF11 and TGFb1 ⁄ BMP2 ⁄ BMP3 ⁄ BMP4 ⁄ BMP8b: the former gave good fits with the simple model of a 1 : 1 Langmuir interaction [10], whereas the association and dissociation curves of the interaction of WFIKKN proteins with the various BMPs gave accept- able fits only with the model of ‘two state reaction with conformational change’. The association and dissocia- tion curves of the interaction of WFIKKN proteins with TGFb1 could be fitted to the model of ‘heteroge- neous ligand parallel reaction’ (see Experimental proce- dures). Because it has been pointed out recently in a critical review of the biosensor literature that parame- ters calculated with the 1 : 1 interaction model are most likely to give reliable estimates of binding constants [15], it may be doubtful whether the K d values calcu- lated for the interaction of WFIKKN proteins with TGFb1, BMP2, BMP3, BMP4, BMP8b are valid, and whether WFIKKNs are efficient inhibitors of the bind- ing of these growth factors to their cognate receptors. To answer these questions, we studied the ability of WFIKKN proteins to block the binding of growth fac- tors to their receptors using SPR in a solution-competi- tion format, as well as luciferase reporter assays. G. Szla ´ ma et al. WFIKKNs bind several members of the TGFb family FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS 5041 20 60 100 0 200 400 600 800 C Time (s) 40 100 160 220 0 200 400 600 800 D Time (s) 60 220 380 0 200 400 600 800 E Time (s) Time (s) 220 460 700 0 200 400 600 800 F Time (s) 140 300 0 200 400 600 800 G 150 550 950 0 200 400 600 800 H Time (s ) 300 620 0 200 400 600 800 I Time (s) 0 40 80 0 200 400 600 800 J Time (s) 20 100 0 200 400 600 800 A Time (s) RURURURURU RU RU RU RU RU WFIKKN1-BMP4 60 220 380 0 200 400 600 800 B Time (s) WFIKKN2-BMP4 WFIKKN1-TGFβ1WFIKKN2-TGF 1 WFIKKN1-BMP2 WFIKKN2-BMP2 WFIKKN1-BMP3 WFIKKN2-BMP3 WFIKKN1-BMP8b WFIKKN2-BMP8b Fig. 1. Characterization of the interaction of WFIKKN1 and WFIKKN2 with members of the TGFb family using SPR assays. Sensorgrams of the interactions of: (A) WFIKKN1 (500 n M, 1, 1.25, 1.5 and 2 lM) with BMP4; (B) WFIKKN2 (25, 50, 100, 250, 500 and 1000 nM) with BMP4; (C) WFIKKN1 (1 , 2, 3, 4 and 6 lM) with TGFb1; (D) WFIKKN2 (250 AND 500 nM, and 1, 2 and 4 lM) with TGFb1; (E) WFIKKN1 (400 and 750 n M, 2 and 3 lM) with BMP2; (F) WFIKKN2 (50, 100, 250 and 500 nM) with BMP2; (G) WFIKKN1 (500 and 750 nM, 1 and 1.5 lM) with BMP3; (H) WFIKKN2 (100 , 250 and 500 nM, 1 and 2 l M) with BMP3; (I) WFIKKN1 (250, 500 and 750 nM, 1, 1.25, 1.5 and 2 lM) with BMP8b; (J) WFIKKN2 (250 , 500 and 750 nM, 1 and 1.5 lM) with BMP8b. Various concentrations of WFIKKN1 or WFIKKN2 in 20 mM Hepes buffer, pH 7.5, containing 150 m M NaCl, 5 mM EDTA, 0.005% Tween 20 were injected over CM5 sensorchips containing the immobilized growth factors. For each type of experiment, one set of representative data from three parallel experiments are shown. For the sake of clarity, the concentrations of WFIKKN proteins are not indicated in the panels; in each case, the SPR response increased parallel to the increase in WFIKKN concentration. WFIKKNs bind several members of the TGFb family G. Szla ´ ma et al. 5042 FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS Effect of WFIKKN1 and WFIKKN2 on binding of GDF8, GDF11, BMP2, BMP4 and TGFb1 to the extracellular domains of their cognate receptors As shown in Fig. 2A, GDF8 binds tightly to the extra- cellular domain (ECD) of its receptor, activin recep- tor IIB (ACRIIB), with a K d value of 1.6 · 10 )9 m, comparable with that determined for intact receptors present on cells (Table 2). Preincubation of GDF8 with increasing concentrations of WFIKKN1 (Fig. 2B) or WFIKKN2 (Fig. 2C) efficiently decreased the recorded SPR response and association rate, indicating that GDF8–WFIKKN1 and GDF8–WFIKKN2 com- plexes formed are unable to bind to the ECD of the receptor protein. Analysis of the data revealed that WFIKKN1 and WFIKKN2 caused a 50% decrease in the rate of association of GDF8 to the ECD of ACRI- IB at  20 and 12 nm, respectively. GDF11 also binds tightly to the ECD of its receptor (Fig. 2D), with a K d value of 4.8 · 10 )10 m (Table 2). Preincubation of GDF11 with increasing concentrations of WFIKKN1 (Fig. 2E) or WFIKKN2 (Fig. 2F) efficiently decreased the recorded SPR response and association rate: WFIKKN1 and WFIKKN2 caused a 50% decrease in the rate of association of GDF11 to the ECD of ACRIIB at  40 and  5nm, respectively. In similar experiments, we investigated the effect of WFIKKN1 and WFIKKN2 on the binding of BMP2 and BMP4 to the ECD of their high-affinity receptor bone morphogenetic protein receptor IA (BMPRIA). As shown in Fig. 3, both BMP2 (Fig. 3A) and BMP4 (Fig. 3B) had high affinity for the ECD of BMPRIA, with K d values comparable with those determined by others (Table 2). Preincubation of BMP2 or BMP4 with increasing concentrations of WFIKKN1 resulted only in weak inhibition even at the highest (4 lm) concentration of WFIKKN1 (Fig. 3C,D). WFIKKN2 proved to be a slightly more efficient inhibitor of both BMP2 and BMP4 than WFIKKN1. As shown in Fig. 3E,F, WFIKKN2 decreased both the recorded SPR response and the association rate; WFIKKN2 caused a 50% decrease in the rate of association of BMP2 and BMP4 to the ECD of BMPRIA at  2 and 3 lm, respectively. TGFb1 had high affinity for the immobilized ECD of TGFbRII; based on analyses of sensorgrams, the interaction is characterized by a K d value of 5 · 10 )11 m (Table 2). Preincubation of TGF b1 with increasing concentrations of WFIKKN2 caused a 50% decrease in the rate of association to its receptor at  1 lm. In the case of WFIKKN1, even the highest concentration used (2 lm) caused only a 20% decrease in the rate of association of the growth factor to its receptor (data not shown). Effect of WFIKKN1 and WFIKKN2 on growth factor activity of GDF8, GDF11, BMP2, BMP4 and TGFb1 As shown in Fig. 4A, both WFIKKN1 and WFIKKN2 efficiently inhibited the activity of GDF8 in luciferase reporter assays, half-maximal inhibition being achieved by  6nm WFIKKN1 and by  3nm WFIKKN2. Similarly, WFIKKN1 and WFIKKN2 inhibited the activity of GDF11; 50 nm WFIKKN1 and WFIKKN2 caused 80% and 90% inhibition, respectively (data not shown). By contrast with GDF8 and GDF11, in the case of TGFb1, BMP2 and BMP4, neither WFIKKN1 nor WFIKKN2 was able to cause inhibition even at the highest concentrations (1 lm) used (Fig. 4B–D). Discussion SPR studies on the interaction of WFIKKN1 and WFIKKN2 proteins have raised the possibility that both proteins may bind several members of the TGFb family: their affinities for ligands were found to decrease in the order GDF8 ⁄ GDF11 >> TGFb1 ⁄ BMP2 ⁄ BMP4 > BMP3 > BMP8b, with no detectable affinity for activin A (Fig. 1 and Table 1). The lack of affinity of WFIKKNs for activin A is in harmony with the observation that, in luciferase reporter assays, WFIKKN2 had no effect on the activity of activin [13]. Our observation, however, that both WFIKKN1 and WFIKKN2 appeared to bind TGFb1, BMP2 and BMP4 with relatively high affinity in SPR experiments does not necessarily mean that they are efficient inhibi- tors of these growth factors. The use of SPR to Table 1. Kinetic parameters of the interaction of BMP2, BMP3 BMP4, BMP8b, TGFb1, GDF8 and GDF11 with WFIKKN1 and WFIKKN2. The equilibrium dissociation constants of the interactions were determined from SPR measurements with BIAEVALUATION soft- ware 4.0. The K d values of the interaction of GDF8 and GDF11 with WFIKKN proteins are taken from Konda ´ s et al. [10]. Interacting proteins K d (M) Interacting proteins K d (M) BMP2 a –WFIKKN1 7.2 · 10 )7 BMP2 a –WFIKKN2 4.3 · 10 )8 BMP3 a –WFIKKN1 3.3 · 10 )6 BMP3 a –WFIKKN2 1.8 · 10 )7 BMP4 a –WFIKKN1 8.2 · 10 )7 BMP4 a –WFIKKN2 6.5 · 10 )8 BMP8b a –WFIKKN1 3.0 · 10 )5 BMP8b a –WFIKKN2 5.3 · 10 )5 TGFb1 a –WFIKKN1 4.5 · 10 )7 TGFb1 a –WFIKKN2 2.8 · 10 )8 8.9 · 10 )5 3.3 · 10 )5 GDF8 a –WFIKKN1 3.3 · 10 )8 GDF8 a –WFIKKN2 2.8 · 10 )10 GDF11 a –WFIKKN1 2.2 · 10 )9 GDF11 a –WFIKKN2 1.6 · 10 )10 a These proteins were immobilized on the sensorchips. G. Szla ´ ma et al. WFIKKNs bind several members of the TGFb family FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS 5043 determine dissociation constants of protein–protein interactions has numerous pitfalls, including problems associated with the immobilization of one of the inter- acting partners. As pointed out by Rich and Myszka [15], parameters calculated with the 1 : 1 Langmuir interaction model are most likely to give reliable esti- mates of binding constants, therefore that the interac- tion of WFIKKN proteins with TGFb1, BMP2, BMP3, BMP4, BMP8b gave acceptable fits only with alterna- tive models raised doubts about whether the dissocia- tion constants are valid and, consequently, whether the interactions detected by SPR have physiological rele- vance. To overcome this problem, we studied the ability of WFIKKN proteins to block the binding of growth factors to their receptors using SPR solution-competi- tion assay formats as well as reporter assays. These studies have shown that the conclusions drawn from the different types of assays are in agree- ment in the case of GDF8 and GDF11. For example, the high affinity of WFIKKN1 for GDF8 determined with SPR (K d  33 nm) is in agreement with its efficiency in inhibiting the binding of GDF8 to its receptor in vitro (IC 50  20 nm) and its ability to block the activity of GDF8 in reporter assays (IC 50  6nm). Similarly, the efficiency of WFIKKN2 to inhibit the binding of GDF8 to its receptor in vitro (IC 50  12 nm) is in agreement with its ability to block the activity of GDF8 in reporter assays (IC 50  3nm). 0 5 10 15 50 150 250 350 450 550 650 750 Time (s) RURURU RURURU A GDF8 0 5 10 15 50 150 250 350 450 550 650 Time (s) D GDF11 B –10 0 –10 5 15 50 150 250 350 450 550 650 750 Time (s) GDF8-WFIKKN1 0 5 10 15 50 150 250 350 450 550 650 Time (s) E GDF11-WFIKKN1 C 0 5 15 50 150 250 350 450 550 650 750 850 Time (s) GDF8-WFIKKN2 0 5 10 15 50 150 250 350 450 550 650 Time (s) F GDF11-WFIKKN2 Fig. 2. Effect of WFIKKN1 and WFIKKN2 on the binding of GDF8 and GDF11 with the ECD of ACRIIB monitored using SPR. Sensorgrams of the interactions of immobilized ECD of ACRIIB with: (A) GDF8, 5 , 10, 20 and 50 nM; (B) 50 nM GDF8 preincubated with 0, 25, 50, 100 and 250 n M WFIKKN1; (C) 50 nM GDF8 preincubated with 0, 12.5, 25, 30 and 50 nM WFIKKN2; (D) GDF11, 2, 2.5, 5, 7.5 and 10 nM; (E) 10 nM GDF11 preincubated with 0, 5, 10, 50, 100 and 200 nM WFIKKN1; (F) 10 nM GDF11 preincubated with 0, 0.5, 1, 2, 4, 6 and 8 nM WFIKKN2. Various concentrations of WFIKKN1 or WFIKKN2 and 50 n M GDF8 or 10 nM GDF11 were preincubated in 20 mM Hepes buffer, pH 7.5, con- taining 150 m M NaCl, 5 mM EDTA, 0.005% Tween 20 for 30 min at room temperature and were injected over CM5 sensorchips containing immobilized ECD of ACRIIB. For the sake of clarity, the concentrations of the proteins injected over the sensorchips are not indicated; in (A) and (D) the SPR response increased parallel to the increase in GDF8 and GDF11 concentration, respectively. In (B), (C), (E) and (F) the SPR response decreased parallel to the increase in WFIKKN concentration. WFIKKNs bind several members of the TGFb family G. Szla ´ ma et al. 5044 FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS The differences in the parameters in the different assays are likely to reflect true variations in the assay formats: in interactions with immobilized partner versus competition in the solution phase, immobilized ECD may not properly represent the structure of the intact receptor complex present on the cell-surface. The three types of experiments suggest that WFIKKN1 and WFIKKN2 are potent inhibitors of GDF8 and GDF11 and may play a significant role in the regulation of biological processes controlled by these growth factors. In the case of BMP2 and BMP4, solution-competi- tion assays revealed that WFIKKNs inhibit the bind- ing of these growth factors to immobilized ECDs of their cognate receptors at higher concentrations (IC 50 values of 2–3 lm) than expected from the affinities of WFIKKNs for immobilized BMP2 and BMP4 (Table 1). In reporter assays, WFIKKNs failed to inhi- bit the activity of BMP2 and BMP4. In the case of TGFb1, solution-competition assays also indicated that WFIKKNs inhibit the binding of this growth factor to its receptor at higher concentra- tions than expected from the affinities of WFIKKNs for immobilized TGFb1 (Table 1). In reporter gene assays, neither WFIKKN1 nor WFIKKN2 inhibited the biological activity of this growth factor, even at 1 lm (Fig. 4B). This observation is in agreement with the finding of Hill et al. [13] that WFIKKN2 did not inhibit the activity of TGFb1. Although WFIKKN proteins do not inhibit the sig- nalling activites of BMP2, BMP4 and TGFb1, this does not necessarily mean that the interactions of WFIKKNs with BMP2, BMP4 and TGFb1 do not have physiological relevance. Growth factor binding proteins may control the action of growth factors not only by inhibiting their action, but also, by serving as a reservoir for growth factors, may localize their action in the vicinity of the binding proteins and thus help to establish growth factor gradients in the extracellular space through physical association. As a result, the same growth factor binding protein may serve as either an agonist or antagonist of a given growth factor in a context-dependent manner [16–20]. It may be relevant in this respect that WFIKKN1 was shown to be preferentially expressed in the developing inner ear, mainly in the BMP4-positive presumptive cristae, and it was suggested that WFIKKN1 may be involved in the early development of the inner ear sensory organ by controlling the action of BMP4 [14]. Because BMP4 not only specifies the sensory epithe- lium of the inner ear, but also regulates its structural development [21,22] we suggest that WFIKKN1 may influence this process by acting as a short-range diffus- ible protein that binds BMP4. Experimental procedures Reagents, enzymes, PCR primers, proteins, bacterial strains, cell lines and media Restriction enzymes, T4 DNA Ligase and Klenow polymer- ase were from New England Biolabs (Beverly, MA, USA). PCR primers were obtained from Integrated DNA Technol- ogies (Coralville, IA, USA). For amplification reactions, we used Taq DNA polymerase from Fermentas (Vilnius, Lith- uania) or the proofreading thermostable polymerase Accu- zyme (Bioline, London, UK). DNA purification was performed with Nucleospin Extract PCR purification kit (Macherey-Nagel, Duren, Germany). Escherichia coli JM109 bacterial strain was used for DNA propagation during DNA manipulation steps. Mature mouse GDF8 (O08689, GDF8_MOUSE), human GDF11 (O95390, GDF11_HUMAN), activin A (A4D1W7, A4D1W7_HU- MAN), BMP2 (P12643, BMP2_HUMAN), BMP3 (P12645, BMP3_HUMAN), BMP4 (P12644, BMP4_HUMAN), BMP8b (P34820, BMP8B_HUMAN), TGFb1 (P01137, TGFB1_HUMAN) and TGF-bsRII (P37173, TGFR2_HU- MAN) (corresponding to the ECD of TGF-bRII) were purchased from R&D Systems (Wiesbaden, Germany). CM5 sensorchips and the reagents for protein coupling to the chips were from Biacore AB (Uppsala, Sweden). Recombinant WFIKKN1 (Q96NZ8, WFKN1_HUMAN) and WFIKKN2 (Q8TEU8, WFKN2_HUMAN) were produced as described previously [10]. The Cignal SMAD Reporter Kit was purchased from SaBiosciences (Frederick, MD, USA), the firefly and Renilla luciferase kits were from Biotium (Hayward, CA, USA). Rhabdomyosarcoma A204 cells were from the German Collection of Microorganisms Table 2. Interaction of GDF8, BMP2 and BMP4 with immobilized extracellular domains of their receptors. The equilibrium dissocia- tion constants of the interactions were determined from SPR mea- surements with BIAEVALUATION software 4.0. For comparison K d values determined by others for the interaction of the growth factors with ECDs of receptors or intact receptors present on cell surfaces are also shown. Interacting proteins K d (M) References GDF8–cell surface 1.4 · 10 )10 3 GDF8–ECD ACRIIB 1.6 · 10 )9 GDF11–ECD ACRIIB 4.8 · 10 )10 TGFb1–ECD TbRII 6 · 10 )12 26 TGFb1–cell surface 25 · 10 )12 27 TGFb1–ECD TbRII 5 · 10 )11 BMP4–ECD BMPRIA 9.6 · 10 )9 28 BMP4–cell surface 2.5 · 10 )10 29 BMP4–ECD BMPRIA 1.2 · 10 )9 BMP2–ECD BMPRIA 2.6 · 10 )9 30 BMP2–ECD BMPRIA 3.3 · 10 )10 G. Szla ´ ma et al. WFIKKNs bind several members of the TGFb family FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS 5045 and Cell Cultures (DSMZ, Braunschweig, Germany). Mink lung epithelial cells stably transfected with a truncated PAI- 1 promoter ⁄ firefly luciferase construct (MLEC-clone32) [23] and HepG2-BRA cells stably transfected with the BRE–luc reporter construct [24] were generously provided by Profes- sor Daniel Rifkin (New York University). Culture media Dulbecco’s modified Eagle’s medium, McCoy’s 5A and heat-inactivated fetal bovine serum were obtained from Sigma-Aldrich (St Louis, MO, USA). Expression of the ECD of human BMPR1a and AVRIIB in Pichia pastoris The cDNA fragment coding for the extracellular domain of BMPR1A (P36894, BMR1A_HUMAN) was amplified from a human prostate first-strand cDNA library (Clontech, Mountain View, CA,USA) using 5¢-GAGGAATTCCAG AATCTGGATAGTATGCTT-3¢ sense and 5¢-GAGGTCG ACTCGAATGCTGCCATCAAAAAACGG-3¢ antisense 0 20 40 60 80 100 120 0 100 200 300 400 500 600 700 800 Time (s) B Time (s) 0 50 100 150 200 250 0 100 200 300 400 500 600 700 800 RURURU RURURU A 0 50 100 150 200 0 100 200 300 400 500 600 700 800 Time (s) C 0 20 40 60 80 100 120 0 100 200 300 400 500 600 700 800 Time (s) D 0 50 100 150 200 0 100 200 300 400 500 600 700 800 Time (s) E 0 20 40 60 80 100 120 0 100 200 300 400 500 600 700 800 Time (s) F BMP2 BMP4 BMP2-WFIKKN1 BMP4-WFIKKN1 BMP2-WFIKKN2 BMP4-WFIKKN2 Fig. 3. Effect of WFIKKN1 and WFIKKN2 on the binding of BMP2 and BMP4 to the ECD of BMPRIA monitored using SPR. Sensorgrams of the interactions of immobilized ECD of BMPRIA with: (A) BMP2, 5 , 10, 15 and 25 nM; (B) BMP4, 10, 15, 20 and 25 nM; (C) 15 nM BMP2 preincubated with 0 , 750, 2250 and 3900 nM WFIKKN1; (D) 25 nM BMP4 preincubated with 0, 500, 2500 and 4000 nM WFIKKN1; (E) 15 nM BMP2 preincubated with 0, 750, 2250 and 3900 nM WFIKKN2; and (F) 25 nM BMP4 preincubated with 0, 500, 2500 and 4000 nM WFIKKN2. Various concentrations of WFIKKN1 or WFIKKN2 and 50 n M GDF8 were preincubated in 20 mM Hepes buffer, pH 7.5, containing 150 mM NaCl, 5 mM EDTA, 0.005% Tween 20 for 30 min at room temperature and were injected over CM5 sensorchips containing immobilized ECD of BMPRIA. For the sake of clarity, the concentrations of the proteins injected over the sensorchips are not indicated; in (A) and (B) the SPR response increased parallel to the increase in BMP concentration, in (C), (D), (E) and (F) the SPR response decreased parallel to the increase in WFIKKN concentration. WFIKKNs bind several members of the TGFb family G. Szla ´ ma et al. 5046 FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS primers. The cDNA of the ECD of human AVRIIB (Q13705, AVR2B_HUMAN) was amplified with 5¢-GAGG AATTCTCTGGGCGTGGGGAGGCTGAG-3¢ sense and 5¢-GAGGTCGACCGTGAGCAGGGTGGGGGCTGT-3¢ antisense primers from a skeletal muscle cDNA library. Both reactions were performed with Accuzyme proofreading Taq DNA polymerase over 35 reaction cycles. The annealing temperature was 60 °C for amplification of the ECD of BMPRIA and 63.5 °C in the case of the ECD of AVRIIB. In both cases, amplified DNAs were digested with EcoRI and SalI restriction enzymes and ligated into pPiczalphaA Pichia pastoris expression vector digested with the same enzymes. The introduction of the linearized pPICZal- phaA_BMPR1A and pPICZalphaA_AVRIIB plasmids into Pichia pastoris GS115 cells and the expression of the recom- binant protein was performed according to the protocol described for WFIKKN proteins [10]. The calculated molecular mass of the ECDs of AVRIIB and BMPRIA are 14 971 and 15 499 Da, respectively. SDS ⁄ PAGE analysis of the proteins purified from the induction media showed diffuse bands with molecular masses higher than the expected, suggesting that the recom- binant proteins may be glycosylated. Deglycosylation of the proteins by EndoH digestion decreased the molecular mass of the proteins to  15–16 kDa. The N-terminal sequence of the recombinant ECD of BMPRIA was EFQNLDSMLHGT and that of the recombinant ECD of AVRIIB was EFSGRGEAETRE (residues in bold corre- 0 200 400 600 800 1000 20 40 60 80 100 120 140 160 B 0 200 400 600 800 1000 0 20 40 60 80 100 120 D 0 1020304050 0 20 40 60 80 100 RLU%RLU% RLU%RLU% WFIKKN (nM) WFIKKN2 WFIKKN1 WFIKKN2 WFIKKN1 WFIKKN2 WFIKKN1 0 200 400 600 800 1000 0 20 40 60 80 100 120 140 WFIKKN (nM) WFIKKN (n M) WFIKKN (n M) C WFIKKN2 WFIKKN1 A Fig. 4. Effect of WFIKKN1 and WFIKKN2 on the growth factor activities. (A) Rhabdomyosarcoma A204 cells transiently transfected with Cig- nal SMAD Luciferase Reporter vector and a Renilla luciferase vector were incubated for 16 h with 0.8 n M GDF8 preincubated with different concentrations of WFIKKN1 ( ) and WFIKKN2 ( ). Firefly luciferase units were normalized to Renilla luciferase units and background values obtained from control cells were subtracted. (B) Mink lung epithelial cells stably transfected with a truncated PAI-1 promoter ⁄ firefly lucifer- ase construct (MLEC-clone32) were incubated for 15 h with 8 p M TGFb1 preincubated with different concentrations of WFIKKN1 ( ) and WFIKKN2 ( ). (C) HepG2 cells stably transfected with the BRE–luc reporter construct were incubated for 15 h with 250 pM BMP2 preincu- bated with different concentrations of WFIKKN1 ( ) and WFIKKN2 ( ). (D) HepG2 cells stably transfected with the BRE–luc reporter con- struct were incubated for 15 h with 250 p M BMP4 preincubated with different concentrations of WFIKKN1 ( ) and WFIKKN2 ( ). In the case of (B), (C) and (D), the luciferase activities were normalized to the protein content of the wells and background values obtained from control cells were subtracted. The figure shows the mean values of three parallel experiments. Error bars represent the SEM. G. Szla ´ ma et al. WFIKKNs bind several members of the TGFb family FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS 5047 spond to residues of the ECDs, the N-terminal residues EF originate from the expression constructs). The structural integrity and stability of recombinant ECDs was checked by CD spectroscopy. CD spectra were measured over the range 195–250 nm by using a JASCO J-720 spectropolarimeter thermostatted with a N eslab RT-111 water bath. The measurements were carried out in 1 mm pathlength cells and protein solutions of 0.1 mgÆmL )1 in 10 mm Tris ⁄ HCl buffer, pH 8.0. Spectra were measured at 25 °C with a 16 s time constant and 20 nmÆmin )1 scan rate. The spectral slit width was 1.0 nm. The spectra of the recombinant proteins were also recorded at different temper- atures from 25 to 95 °Cat10°C intervals. The thermal unfolding of the recombinant ECD of BMPRIA and the ECD of AVRIIB were monitored at 213 and 230 nm, respectively, where the difference of the CD spectra recorded at different temperatures was the largest. The heating rate was 60 °CÆh )1 . The native fold of both recombinant ECDs collapsed with single melting temperatures: T m =72°C for the ECD of BMPRIA and 67 °C for the ECD of AVRIIB. Protein analyses The composition of protein samples was analysed by SDS ⁄ PAGE under both reducing and nonreducing condi- tions. The gels were stained with Coomassie Brilliant Blue G-250. The concentration of the recombinant proteins was determined using the following extinction coefficients: WFIKKN1, 64 440 m )1 Æcm )1 ; WFIKKN2, 57 470 m )1 Æcm )1 ; ECD of BMPRIA, 5095 m )1 Æcm )1 ; ECD of AVRIIB, 26 065 m )1 Æcm )1 . The extinction coefficients were calculated with the online protein analysis tool protparam. SPR analysis SPR measurements were performed on a BIACORE X (GE Healthcare, Stockholm, Sweden) instrument. Proteins to be immobilized were dissolved in 50 mm sodium acetate, pH 4.5, and 100 lL of 0.7 lm activin A or 50 lL of 0.8 lm BMP2 or 100 lL of 0.83 lm BMP3 or 75 lL of 0.7 lm BMP8b or 50 lL of 0.8 lm BMP4 solutions were injected with a 5 lLÆmin )1 flow rate on a CM5 sensor chip activated by the amine coupling method, according to the manufac- turer’s instructions. TGFb1 was dissolved in 50 mm sodium acetate, pH 4.1, and 42 lL of a 0.8 lm solution was injected with a 3 lLÆmin )1 flow rate. The ECDs of ACRIIB and BMPRIA were dissolved in 50 mm sodium acetate, pH 4.0 or 4.2 respectively, and 100 lL of 17.5 l m solutions were injected with 5 lLÆmin )1 flow rate. TGFbsRII was dis- solved in 50 mm sodium acetate, pH 4.2, and 100 lLof 6.6 lm solution was injected with 5 lLÆmin )1 flow rate. For interaction measurements, 80-lL aliquots of pro- tein solutions were injected over the sensor chips with a 20 lLÆmin )1 flow rate, followed by 10 min flow of buffer. Binding and washes were performed in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA, 0.005% Tween 20 pH 7.5 buffer. After each cycle the chips were regenerated with 20 mm Hepes, 150 m m NaCl, 5 mm EDTA, 0.005% Tween 20, pH 7.5 buffer containing 8 m urea. In solution-competition assays, constant concentrations of growth factors were incubated with increasing concentra- tions of WFIKKN1 or WFIKKN2 in 20 mm Hepes, 150 mm NaCl, 5 mm EDTA, 0.005% Tween 20 pH 7.5 buf- fer for 30 min at room temperature prior to injection on chips with immobilized ECDs of growth factor receptors. Control flow cells were prepared by executing the cou- pling reaction in the presence of coupling buffer alone. Control flow cells were used to obtain control sensorgrams showing nonspecific binding to the surface as well as refrac- tive index changes resulting from changes in the bulk prop- erties of the solution. Control sensorgrams were subtracted from sensorgrams obtained with immobilized ligand. To correct for differences between the reaction and reference surfaces, we also subtracted the average of sensorgrams obtained with blank running buffer injections. The kinetic parameters for each interaction were deter- mined by globally fitting the experimental data with bia- evaluation software 4.0 and the closeness of the fit was characterized by the chi-square values. Fits were accepted only if the v 2 values were < 5% of R max [25]. The associa- tion and dissociation curves of the interaction of GDF8, GDF11, BMP2 and BMP4 with the ECDs of their recep- tors gave good fits with the model of 1 : 1 Langmuir inter- action. The sensorgrams of the interaction of WFIKKN proteins with BMPs, however, gave acceptable fits only with the model of ‘two state reaction with conformational change’. In the case of the interaction of WFIKKN pro- teins with TGFb1, the data gave acceptable fits with the model ‘of heterogeneous ligand parallel reaction’. Cell culture Rhabdomyosarcoma A204 cells were cultured in McCoy’s 5A medium supplemented with 10% fetal bovine serum, penicillin (100 UÆmL )1 ) and streptomycin (100 lgÆmL )1 )at 37 °C, 5% CO 2 . Mink lung epithelial cells and HepG2- BRA cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, peni- cillin (100 UÆmL )1 ) streptomycin (100 lgÆmL )1 ) and geneti- cin at a concentration of 200 lgÆmL )1 (MLEC-clone32) or 700 lgÆmL )1 (HEPG2-BRA) at 37 °C, 5%CO 2 . Reporter assays TGFb1 activity was measured with MLEC-clone32 cells, whereas the activities of BMP2 and BMP4 were monitored with HEPG2-BRA cells, using 96-well tissue culture dishes. In these reporter assays MLEC-clone32 cells (1.6 · 10 4 cellsÆwell )1 ) or HEPG2-BRA cells (5 · 10 3 cellsÆwell )1 ) were allowed to attach for 3 or 24 h respectively, then the WFIKKNs bind several members of the TGFb family G. Szla ´ ma et al. 5048 FEBS Journal 277 (2010) 5040–5050 ª 2010 The Authors Journal compilation ª 2010 FEBS medium was changed to Dulbecco’s modified Eagle’s med- ium supplemented with 0.1% BSA, penicillin (100 UÆmL )1 ) streptomycin (100 lgÆmL )1 ) containing 8 pm TGFb1, 250 pm BMP2 or 250 pm BMP4, preincubated for 30 min with different concentrations of WFIKKN1 or WFIKKN2. Control experiments were performed similarly, except that no growth factor was added. After incubation for 15 h at 37 °C, 5% CO 2 , cells were lysed in 100 lL lysis buffer and the luciferase activity of the samples was determined using the firefly luciferase assay kit of Biotium on an Appliskan luminometer (Thermo Electron Corp., Beverly, MA, USA). The protein content of the samples was determined with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) and the luciferase activity was normalized to the protein content of the wells. The activities of GDF8 and GDF11 were studied on Rhabdomyosarcoma A204 cells. 2.5–3 · 10 4 cells were pla- ted in wells of a 96-well plate and were allowed to attach for 24 h, then transiently transfected with 150 ng Cignal SMAD Luciferase Reporter vector mixture with 0.4 lL Lipofectamin 2000 reagent per well, according to the manu- facturer’s instructions. Transfections were performed in serum-free McCoy’s 5A medium containing 1 mgÆmL )1 BSA without antibiotics. The plasmid preparation used for transfections was a 40 : 1 ratio mixture of an inducible, TGFb-responsive firefly lucif- erase construct and a constitutively expressing Renilla lucif- erase construct. Sixteen hours later, the transfection medium was changed to McCoy’s 5A containing 1mgÆmL )1 BSA and conditioned for 5 h. WFIKKN1 and WFIKKN2 were incubated with 0.8 nm growth factors for 30 min at 37 °C and were added to the cells. After 16 h, the cells were washed with NaCl ⁄ P i and lysed using 50 lL passive lysis buffer from the Biotium luciferase kit, and fire- fly and Renilla luciferase activities were measured. The firefly luciferase units obtained were normalized to the Renilla luciferase units and background values obtained from cells grown in McCoy’s 5A medium–1 mgÆmL )1 BSA alone were subtracted to generate relative luciferase units. Three parallel experiments were performed in all cases and were repeated at least twice. 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WFIKKN1 and WFIKKN2 bind growth factors TGFb1, BMP2 and BMP4 but do not inhibit their signalling activity Gyo ¨ rgy Szla ´ ma,. 800 Time (s) F BMP2 BMP4 BMP2 -WFIKKN1 BMP4 -WFIKKN1 BMP2 -WFIKKN2 BMP4 -WFIKKN2 Fig. 3. Effect of WFIKKN1 and WFIKKN2 on the binding of BMP2 and BMP4 to the