View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repository of the Academy's Library Influence of WFIKKN1 on BMP1-mediated activation of latent myostatin Gyorgy€ Szlama, Viktor Vas arhelyi, Maria Trexler and Laszl o Patthy Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary Keywords The NTR domain of WFIKKN1 protein has been shown to have signifi- BMP1; heparin; latent myostatin; myostatin; cant affinity for the prodomain regions of promyostatin and latent myo- WFIKKN1 statin but the biological significance of these interactions remained unclear. In view of its role as a myostatin antagonist, we tested the assumption that Correspondence L. Patthy, Institute of Enzymology, WFIKKN1 inhibits the release of myostatin from promyostatin and/or Research Centre for Natural Sciences, latent myostatin. WFIKKN1 was found to have no effect on processing of Hungarian Academy of Sciences, Budapest, promyostatin by furin, the rate of cleavage of latent myostatin by BMP1, P.O. Box 286, H-1519, Hungary however, was significantly enhanced in the presence of WFIKKN1 and Tel: +361 382 6751 this enhancer activity was superstimulated by heparin. Unexpectedly, E-mail: [email protected] WFIKKN1 was also cleaved by BMP1 and our studies have shown that the KKN1 fragment generated by BMP1-cleavage of WFIKKN1 con- (Received 26 May 2016, revised 19 September 2016, accepted 24 October tributes most significantly to the observed enhancer activity. Analysis of a 2016) pro-TGF-b -based homology model of homodimeric latent myostatin revealed that the BMP1-cleavage sites are buried and not readily accessible doi:10.1111/febs.13938 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 con- formational equilibrium of latent myostatin from the closed circular struc- ture 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 concentra- tions 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. Introduction Myostatin is a negative regulator of skeletal muscle formation of mature myostatin were also shown to growth: mice lacking myostatin are characterized by a cause hypermuscularity in mice, cattle, dog, and significant increase in skeletal muscle mass [1]. Muta- human [2–7]. In addition to its role in muscle develop- tions of the myostatin gene that interfere with the ment, myostatin is also involved in regulation of Abbreviations Act RIIB, activin receptor IIB; BMP1, bone morphogenetic protein 1; KKN1, The C-terminal half of WFIKKN1 protein, containing its Kunitz 1, Kunitz 2, and NTR domains; LM, latent myostatin; MSTN, myostatin gene; PCPE-1, procollagen C-proteinase enhancer-1; PMSF, phenylmethylsulfonyl fluoride; TGF, transforming growth factor; WFI1, The N-terminal half of WFIKKN1 protein, containing its WAP, follistatin, and immunoglobulin domains; WFIKKN1 and WFIKKN2, multidomain proteins containing WAP, follistatin, immunoglobulin, Kunitz 1, Kunitz 2, and NTR domains. The FEBS Journal 283 (2016) 4515–4527 ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of 4515 Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. WFIKKN1 and BMP1 activation of latent myostatin G. Szlama et al. adipose tissue: in MstnÀ/À mice, adipose tissue size is observation that delivery of WFIKKN2 gene into the significantly reduced [8,9]. muscles of mice resulted in an approximately 30% Myostatin is synthesized as an inactive precursor increase in muscle mass and that transgenic mice over- protein, containing an N-terminal prodomain and a expressing WFIKKN2 protein have larger muscles C-terminal growth factor domain [10]. In promyo- compared to wild-type animals [20,21]. statin, two molecules are linked through a single disul- WFIKKN1 and WFIKKN2 are closely related pro- fide bond present in the C-terminal growth factor teins that, in addition to a follistatin domain, contain domain. Active myostatin (corresponding to the cova- WAP domain, an immunoglobulin domain, two lently linked, homodimeric growth factor domain) is Kunitz domains, and an NTR domain [22,23]. The released from promyostatin by proteolytic processing. names of these proteins refer to the linear sequence of First, furin-type proprotein convertases cleave both their constituent WAP, follistatin, immunoglobulin, chains of the precursor at the boundary of the N-term- Kunitz, Kunitz, and NTR domains [22,23]. In Fig. 1, inal prodomain and the C-terminal growth factor we show a schematic representation of the domain domain. The importance of this step in the control of architecture of the WFIKKN1 protein. myostatin activity is underlined by the observation Our studies on these WFIKKN proteins have that the common K153R polymorphism of human revealed that their follistatin domains are primarily promyostatin that significantly increases the rate of its responsible for the myostatin antagonist activity proteolysis by furin is associated with obesity and [18,19,24], but relatively little is known about the func- lower muscle strength [11]. Furin-cleavage of promyo- tion of the other domain types. statin, however, does not lead to the release of active As WAP, Kunitz, and NTR domains have been myostatin: the myostatin prodomains and the disul- implicated in inhibition of various types of proteases, fide-bonded growth factor domains remain associated, we have hypothesized that the corresponding domains forming a noncovalent myostatin/prodomain complex of WFIKKN proteins might also function as protease [12]. As the myostatin/prodomain complex does not inhibitors [22–24]. Full-length WFIKKN proteins were bind to the myostatin receptor, Act RIIB, the nonco- found to inhibit the proteolytic activity of bovine tryp- valent myostatin/prodomain complex is referred to as sin but they had no effect on the peptidolytic activities latent myostatin [12,13]. of bovine elastase, chymotrypsin, tissue-type plasmino- Active myostatin is released from latent myostatin gen activator, urokinase-type plasminogen activator, by proteases of the BMP1/Tolloid family that cleave furin, and BMP1 [24]. At the domain level, the trypsin the prodomain of myostatin, thereby disrupting the inhibitory activity of WFIKKN proteins was assigned noncovalent myostatin/prodomain complex [14]. The to their second Kunitz-type domain [25]. key importance of BMP1-mediated cleavage of latent Interestingly, the NTR domain of WFIKKN1 (but myostatin for the release of mature myostatin is not of WFIKKN2) was found to have significant affin- supported by the observation that mice carrying a ity for myostatin prodomain [18,26]. This difference mutation that renders the myostatin prodomain between WFIKKN1 and WFIKKN2 is also reflected BMP1-resistant exhibit significant increase in muscle in their interactions with promyostatin and latent myo- mass [15]. statin; although WFIKKN1 has affinity for both Several myostatin antagonists exist that may control promyostatin and latent myostatin, WFIKKN2 had the activity of mature myostatin. Lee and McPherron no affinity for these proteins, suggesting that the were the first to show that follistatin, an activin antag- NTR/prodomain interaction mediates the interaction onist, is also a potent inhibitor of myostatin [12]. Fur- of WFIKKN1 with promyostatin and latent myostatin thermore, Hill et al. [16] have shown that myostatin is [18,26]. Detailed analysis of the WFIKKN1–prodomain bound to another inhibitory binding protein, the interaction revealed that WFIKKN1 binds the FSTL3/FLRG protein (the product of the follistatin- C-terminal subdomain of myostatin prodomain, but related gene, FLRG). not the N-terminal subdomain that plays a critical role Two other follistatin-related proteins, the products in the interaction of the prodomain with mature myo- of the WFIKKN1 and WFIKKN2 genes were also statin [26]. It thus appears that myostatin prodomain shown to have high affinity for myostatin [17,18]. Sig- consists of two functionally distinct subdomains: the nificantly, WFIKKN1 and WFIKKN2 have higher N-terminal subdomain binds mature myostatin, specificity for myostatin (and the closely related GDF- whereas the C-terminal subdomain binds WFIKKN1. 11) than follistatin or FLST3/FLRG protein [19]. The Very little is known about the biological significance physiological significance of WFIKKN2 protein as a of the interaction of WFIKKN1 with myostatin key regulator of myostatin activity is supported by the propeptide, promyostatin, and latent myostatin. In 4516 The FEBS Journal 283 (2016) 4515–4527 ª 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. G. Szlama et al. WFIKKN1 and BMP1 activation of latent myostatin SP WAP FS IG KU KU NTR Fig. 1. Domain architecture of WFIKKN1 protein. The name of this