Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.By using sensitive homology search and gene finding programs, we have previously identified two closely related multidomain proteins of unknown function: WFIKKN and WFIKKNRP (1, 2). Both proteins (recently renamed as WFIKKN1 and WFIKKN2) contain a WAP domain, a follistatin/Kazal domain, an immunoglobulin domain, two Kunitztype protease inhibitor domains, and an NTR domain. Because WAP-, Kazal-, and Kunitz-type protease inhibitor modules are frequently found in serine protease inhibitor proteins (3) and the inhibitory N-terminal domain of tissue inhibitors of metalloproteases belongs to the NTR domain family (4), we have suggested that WFIKKNs may be multivalent protease inhibitors that may control the action of different proteases.Although the second Kunitz-type protease inhibitor domain of human WFIKKN1 protein was indeed found to inhibit trypsin (and only trypsin) out of a panel of trypsin-related serine proteases (5), structural and evolutionary analyses suggest that trypsin may not be the prime target of this Kunitz domain (6).Studies on the expression characteristics of WFIKKN1 and WFIKKN2 did not provide obvious clues as to the biological function of these proteins as both genes were expressed in multiple tissues (1, 2). The WFIKKN1 gene was found to be expressed (in order of decreasing intensity) in pancreas, thymus, liver, kidney, lung, and testis with practically no expression in brain, heart, skeletal muscle, ovary, small intestine, colon, leukocyte, spleen, and prostate. The WFIKKN2 gene is expressed (in order of decreasing intensity) in ovary, testis, pancreas, brain, and lung but not in heart, liver, placenta, small intestine, colon, leukocyte, spleen and prostate. Thus the main difference between the two genes is that the WFIKKN2 gene is expressed in brain but not in liver, whereas the reverse is true for the WFIKKN1 gene.In the case of fetal tissues, the WFIKKN1 gene is expressed at the highest level in the lung with weaker expression in skeletal muscle and liver, whereas the WFIKKN2 gene is expressed primarily in fetal brain, skeletal muscle, thymus, and kidney. Thus, ...

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)β family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFβ1, bone morphogenetic protein (BMP)2 and BMP4 with relatively high affinity (Kd ∼ 10−6 m). To assess the biological significance of these interactions we studied the effect of WFIKKN proteins on the activity of GDF8, GDF11, TGFβ1, 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 TGFβ1, 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 TGFβ1, BMP2 and BMP4 they function as growth factor binding proteins. It is suggested that the physical association of WFIKKN proteins with these growth factors may localize their action and thus help to establish growth factor gradients in the extracellular space. Structured digital abstract  A list of the large number of protein‐protein interactions described in this article is available via the MINT article http://mint.bio.uniroma2.it/mint/search/publication.do?publicationAc=MINT-8044119

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.Structured digital abstractFurin cleaves Promyostatin by protease assay (View interaction)myostatin binds to PRO by surface plasmon resonance (View interaction)BMP-1 cleaves Promyostatin by protease assay (View interaction)ACR IIB physically interacts with Latent Myostatin by surface plasmon resonance (View interaction)Promyostatin and Promyostatin bind by comigration in gel electrophoresis (View interaction)WFIKKN1 binds to Latent Myostatin by pull down (View interaction)ACR IIB binds to Mature Myostatin by surface plasmon resonance (View Interaction: 1, 2, 3)WFIKKN1 binds to Myostatin Prodomain by surface plasmon resonance (View Interaction: 1, 2, 3)