Both WFIKKN1 and WFIKKN2 Have High Affinity for Growth and Differentiation Factors 8 and 11

Abstract

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, ...

Peroxisome Proliferator-activated Receptor β/δ Induces Myogenesis by Modulating Myostatin Activity

“…As mentioned earlier, Gasp-1 is a secreted protein that has been demonstrated previously to bind and inhibit myostatin activity (27,28). Therefore, if PPAR␤/␦-mediated induction of Gasp-1 is associated with inactivation of myostatin, we reasoned that treatment with L165041 would increase the levels of secreted Gasp-1, inhibit myostatin activity, and increase myoblast proliferation.…”

“…Subsequent expression analysis confirmed up-regulation of Gasp-1 following activation of PPAR␤/␦ and also revealed enhanced association of Gasp-1 with myostatin in response to PPAR␤/␦ activation. Importantly, Gasp-1 has been shown previously to be a potent antagonist of myostatin (27,28), which is a well characterized potent negative regulator of myoblast proliferation and differentiation (29,30) as well as muscle stem cell (satellite cell) activation and self-renewal (31). Therefore, we propose that PPAR␤/␦ positively regulates myogenesis through a mechanism that results in Gasp-1-mediated inhibition of myostatin activity.…”

“…3A, right panel). Gasp-1 is a secreted protein, which has been shown to interact directly with both the mature and Latency Associated Peptide forms of myostatin resulting in inhibition of myostatin signaling (27,28). Interestingly, loss of myostatin function, much like what we observed following L165041-mediated activation of PPAR␤/␦, results in enhanced myoblast proliferation and differentiation (25,29,30,46) therefore, we propose that PPAR␤/␦ activation enhances myogenesis through a mechanism that involves Gasp-1-mediated inhibition of myostatin activity.…”

Variation in the ovine WFIKKN2 gene

“…All the sequences identified shared high homology to the WFIKKN2 sequences from cattle and other animal species. These results suggest that these sequences represent variants of the ovine WFIKKN2 gene, and that sheep have a single WFIKKN2 gene, rather than the two WFIKKN2-related genes reported in fish genomes (Kondas et al, 2008). Five and two variants were identified in intron 1 and the 3′ UTR of WFIKKN2 respectively, suggesting that the gene is polymorphic in sheep.…”

“…In vitro experiments reveal that WFIKKN2 is a potent inhibitor of two TGF beta family members that are implicated in musculoskeletal development: GDF8 (growth and differentiation factor 8, also known as myostatin) and GDF11 (growth and differentiation factor 11, also known as bone morphogenetic protein 11) (Hill et al, 2003;Kondas et al, 2008). Specifically, 3 nM and 50 nM WFIKKN2 cause 50% and 90% inhibitions of GDF8 and GDF11 activities respectively, using a SPR (surface plasmon resonance) assay in a solution-competition format (Szlama et al, 2010).…”