Biological functions of the WAP domain-containing multidomain proteins WFIKKN1 and WFIKKN2.

WFIKKN1 and WFIKKN2 are two closely related multidomain proteins consisting of a WAP (whey acidic protein)-, a follistatin-, an immunoglobulin-, two Kunitz-type protease inhibitor-domains and an NTR domain (netrin domain). Recent experiments have shown that both WFIKKN1 and WFIKKN2 bind myostatin and GDF11 (growth and differentiation factor 11) with high affinity and are potent antagonists of these growth factors. Structure-function studies on WFIKKN proteins have revealed that their interactions with GDF8 and GDF11 are mediated primarily by the follistatin and NTR domains.

Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin.

The NTR domain of WFIKKN1 protein has been shown to have significant affinity for the prodomain regions of promyostatin and latent myostatin but the biological significance of these interactions remained unclear. In view of its role as a myostatin antagonist, we tested the assumption that WFIKKN1 inhibits the release of myostatin from promyostatin and/or latent myostatin. WFIKKN1 was found to have no effect on processing of promyostatin by furin, the rate of cleavage of latent myostatin by BMP1, however, was significantly enhanced in the presence of WFIKKN1 and this enhancer activity was superstimulated by heparin. Unexpectedly, WFIKKN1 was also cleaved by BMP1 and our studies have shown that the KKN1 fragment generated by BMP1-cleavage of WFIKKN1 contributes most significantly to the observed enhancer activity. Analysis of a pro-TGF-ß -based homology model of homodimeric latent myostatin revealed that the BMP1-cleavage sites are buried and not readily accessible to BMP1. In view of this observation, the most plausible explanation for the BMP1-enhancer activity of the KKN1 fragment is that it shifts a conformational equilibrium of latent myostatin from the closed circular structure of the homodimer to a more open form, making the cleavage sites more accessible to BMP1. On the other hand, the observation that the enhancer activity of KKN1 is superstimulated in the presence of heparin is explained by the fact KKN1, latent myostatin, and BMP1 have affinity for heparin and these interactions with heparin increase the local concentrations of the reactants thereby facilitating the action of BMP1. ENZYMES: Furin: EC 3.4.21.75; BMP1, bone morphogentic protein 1 or procollagen C-endopeptidase: EC 3.4.24.19.

K153R polymorphism in myostatin gene increases the rate of promyostatin activation by furin.

Recent studies demonstrated an association between the K153R polymorphism in the myostatin gene with extreme longevity, lower muscle strength and obesity but the molecular basis of these associations has not been clarified. Here, we show that the K153R mutation significantly increases the rate of proteolysis of promyostatin by furin, but has no effect on the activity of the latent complex or the cleavage of the latent complex by bone morphogenetic protein 1 (BMP-1). The increased rate of activation of K153R mutant promyostatin may explain why this polymorphism is associated with obesity, lower muscle strength and extension of lifespan.

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

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.

Correction: Nagy, A., et al. Reassessing Domain Architecture Evolution of Metazoan Proteins: Major Impact of Gene Prediction Errors. Genes 2011, 2, 449-501.

We found some errors in the published versions of Figure S2, Figure S3 and Figure S8 of our paper [1]. The correct Figures are presented below. [...].

Reassessing domain architecture evolution of metazoan proteins: major impact of gene prediction errors.

In view of the fact that appearance of novel protein domain architectures (DA) is closely associated with biological innovations, there is a growing interest in the genome-scale reconstruction of the evolutionary history of the domain architectures of multidomain proteins. In such analyses, however, it is usually ignored that a significant proportion of Metazoan sequences analyzed is mispredicted and that this may seriously affect the validity of the conclusions. To estimate the contribution of errors in gene prediction to differences in DA of predicted proteins, we have used the high quality manually curated UniProtKB/Swiss-Prot database as a reference. For genome-scale analysis of domain architectures of predicted proteins we focused on RefSeq, EnsEMBL and NCBI's GNOMON predicted sequences of Metazoan species with completely sequenced genomes. Comparison of the DA of UniProtKB/Swiss-Prot sequences of worm, fly, zebrafish, frog, chick, mouse, rat and orangutan with those of human Swiss-Prot entries have identified relatively few cases where orthologs had different DA, although the percentage with different DA increased with evolutionary distance. In contrast with this, comparison of the DA of human, orangutan, rat, mouse, chicken, frog, zebrafish, worm and fly RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with those of the corresponding/orthologous human Swiss-Prot entries identified a significantly higher proportion of domain architecture differences than in the case of the comparison of Swiss-Prot entries. Analysis of RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences with DAs different from those of their Swiss-Prot orthologs confirmed that the higher rate of domain architecture differences is due to errors in gene prediction, the majority of which could be corrected with our FixPred protocol. We have also demonstrated that contamination of databases with incomplete, abnormal or mispredicted sequences introduces a bias in DA differences in as much as it increases the proportion of terminal over internal DA differences. Here we have shown that in the case of RefSeq, EnsEMBL and NCBI's GNOMON predicted protein sequences of Metazoan species, the contribution of gene prediction errors to domain architecture differences of orthologs is comparable to or greater than those due to true gene rearrangements. We have also demonstrated that domain architecture comparison may serve as a useful tool for the quality control of gene predictions and may thus guide the correction of sequence errors. Our findings caution that earlier genome-scale studies based on comparison of predicted (frequently mispredicted) protein sequences may have led to some erroneous conclusions about the evolution of novel domain architectures of multidomain proteins. A reassessment of the DA evolution of orthologous and paralogous proteins is presented in an accompanying paper [1].

WFIKKN1 and WFIKKN2 bind growth factors TGFß1, BMP2 and BMP4 but do not inhibit their signalling activity.

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)ß family using SPR measurements. Analyses of SPR sensorgrams suggested that, in addition to GDF8 and GDF11, both WFIKKN proteins bind TGFß1, bone morphogenetic protein (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, TGFß1, 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 TGFß1, 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 TGFß1, 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.

Both WFIKKN1 and WFIKKN2 have high affinity for growth and differentiation factors 8 and 11.

WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP, a follistatin, an immunoglobulin, two Kunitz-type protease inhibitor domains, and an NTR domain. Recent experiments have shown that WFIKKN2 protein binds mature GDF8/myostatin and myostatin propeptide and inhibits the biological activity of myostatin (Hill, J. J., Qiu, Y., Hewick, R. M., and Wolfman, N. M. (2003) Mol. Endocrinol. 17, 1144-1154). 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.