Model of interaction between GASP1 and Myostatin.

<p>(after Lee, 2004) A) Myostatin signalling through ActRIIB receptor is crucial for the regulation of muscle growth. Myostatin is synthesized as a precursor protein (pro-myostatin) that undergoes proteolytic processing (furin cleavage) to generate an N-terminal propeptide and a disulfide-linked C-terminal dimer, which is the biologically active molecule. Myostatin is in circulation as an inactive latent complex (propeptide and mature myostatin) which is activated by BMP/Tolloid family of metalloproteinases. Active myostatin, mostly binds to the ActRIIB receptor and engages the signalling cascade leading to the inhibition of myoblast differentiation and proliferation. B) Specific inhibitors can prevent binding of myostatin to ActRIIB. One of them is GASP1 which also binds to the propeptide. We hypothezise that GASP1 could interact with the inactive latent complex preventing propeptide cleavage by BMP/Tolloid through antiprotease activity of the second kunitz domain.</p

Synteny and paralogy of <i>Gasp</i>.

<p>A) Paralogons situated on human chromosomes 16 and 17 (HSA16; HSA17) including both <i>Gasp</i> genes. B) Orthology between each paralogon and chromosome 10q of <i>Ciona</i> and the contig 48 of <i>Branchiostoma</i>.</p

<i>Gasp1</i> expression patterns.

<p>A and B: <i>in situ</i> hybridization with a <i>Gasp1</i> probe on mouse embryos at 11.5 dpc and 12.5 dpc reveals expression in neural tube (nt), limb bud (lb), genital bud (gb) and caudal part of the embryo (cd). Enlargement of the limb bud (C) shows an expression in the precartilaginous condensations that give rise to fingers. D and E: <i>in situ</i> hybridization with <i>Gasp1</i> probes on zebrafish embryos at 2 dpf showing expression patterns in tail bud (tb), fin buds (fb) and angioblasts (ag).</p

Bootstrap values supporting that lamprey sequences belong to the GASP2 branch.

<p>ML: Maximum likelihood, ME: Minimum Evolution.</p

Substitution rate of GASP proteins in the different branches of the evolutionary tree.

<p>For each site the substitution rate was calculated from a moving average using a window size of 20 amino acids. The site position from start methionine is on the abscissa and the mean substitution rate on the ordinate. The black curves correspond to the ancestor of GASP1 and GASP2 proteins. The red and blue curves correspond to GASP1 and GASP2 respectively. Red asterisks represent gaps in sequence alignment due to one or two species. In the last two parts, the dotted lines correspond to fish and the solid lines to tetrapods. Abbreviations: SP: signal peptide, W: WAP, F: follistatin, K: kazal, Ku: kunitz, NTR: netrin domains.</p

Global view of GASP/Papilin evolution.

<p>This representation derived from the phylogenies conducted on the separate modules (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s001" target="_blank">Fig. S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s003" target="_blank">S3</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043710#pone.0043710.s004" target="_blank">S4</a>). From a protein close to the actual <i>N. vectensis</i> 292 aa protein (bottom right), an intermediate protein probably arose through a duplication of the kunitz domain. This form would represent a common ancestor to papilin (PPN, arrow 1) and GASP (arrow 2). The schematized papilin modular organization corresponds to that found in <i>Drosophila melanogaster</i> isoforms. The phylogeny of the IGc2 modules allowed us to situate ADAMTS protein close to papilin. The GASP kazal domain came from a donor polypeptide which could be agrin, FSTL1, 4, or 5 proteins. The FST and FSTL3 kazal domains are unlikely related to GASP module but very close to the units present in <i>N. vectensis</i> 4887 aa protein. Eppin, EXPI, and WAP8C are examples of proteins that probably evolved from the intermediate form. Domains related to <i>N. vectensis 292</i> aa protein are outlined in red, the new kunitz domain B (intermediate step) is outlined in yellow. The closest kazal domain between agrin, FSTL1, 4, 5 proteins and GASP is outlined in orange.</p

Recombinant BMP15 inhibition of progesterone secretion.

<p>Granulosa cells from small antral (1–3 mm in diameter) ovine follicles were cultured for 96 h in serum-free conditions. Cultures were performed in the absence (control) or in the presence of increasing dose (10, 50, 200ng/ml) of wild type recombinant human BMP15^wt or BMP15^Y235C mutant. Each treatment was tested in triplicate in each of 5 independent experiments. The results represent the amount of progesterone secreted by 50 000 cells between 48 h and 96 h of culture (mean ±SEM). Dose effect was analyzed by one-way ANOVA by comparing each dose to control (**, p<0.01; ***, p<0.001). Mutation effect was analyzed by Student t-test within each dose ($, p<0.05).</p

BMP15 evolution among TGFß/BMP family members.

<p>Phylogenetic tree of thirteen members of the TGFβ/BMP superfamily expressed by the mammalian ovary reconstructed using maximum likelihood. Bootstrap values are given when nodes are strongly supported (>80%). The scale represents the substitution rate.</p

BMP15 positive selection pressure among mammals.

<p>Phylogenetic tree of BMP15 from 24 mammals species reconstructed using TreeBeST and rooted by minimizing with the number of duplications and losses. Bootstrap values are given when nodes are strongly supported (>80%). Branches or species that have a significant LRT for positive selection calculation are indicated in bold (threshold of <i>q</i>= 5% of false positives). The scale represents the substitution rate. </p

Functional analysis of amino acids under positive selection in human BMP15.

<p>A, Human BMP15 full sequence showing prodomain, cleavage site (underlined) and mature peptide (framed sequence); known human variants for BMP15 found in POI patients (framed grey highligthed); and identified amino acids under positive selection (framed colored highlighted): BMP15^F146, BMP15^L189, BMP15^Y235. B, <i>In </i><i>vitro</i> reporter luciferase assay from COV434 granulosa cells transiently transfected with empty vector +/- 100ng recombinant human BMP15 (mock +/- BMP15) or wild type human BMP15 expressing vector (WT) or the different BMP15 variants vectors obtained by directed-mutagenesis of residues under positive selection. Results are expressed as the mean (±SD) of 4 independent experiments. Differences between means were analyzed by one-way ANOVA by comparing each condition to WT (*, p<0.05; **, p<0;01; ***, p<0.001).</p