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• W4234496710 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The matricellular protein SMOC (Secreted Modular Calcium binding protein) is conserved phylogenetically from vertebrates to arthropods. We showed previously that SMOC inhibits bone morphogenetic protein (BMP) signaling downstream of its receptor via activation of mitogen-activated protein kinase (MAPK) signaling. In contrast, the most prominent effect of the Drosophila orthologue, pentagone (pent), is expanding the range of BMP signaling during wing patterning. Using SMOC deletion constructs we found that SMOC-∆EC, lacking the extracellular calcium binding (EC) domain, inhibited BMP2 signaling, whereas SMOC-EC (EC domain only) enhanced BMP2 signaling. The SMOC-EC domain bound HSPGs with a similar affinity to BMP2 and could expand the range of BMP signaling in an in vitro assay by competition for HSPG-binding. Together with data from studies in vivo we propose a model to explain how these two activities contribute to the function of Pent in Drosophila wing development and SMOC in mammalian joint formation. https://doi.org/10.7554/eLife.17935.001 eLife digest During the development of an embryo, a group of proteins known as growth factors stimulate cells to divide and direct how organs and limbs form. One family of growth factors called bone morphogenetic proteins (BMPs) regulate the formation of bone and many other tissues in the embryo. BMPs are released from cells, diffuse away and are then detected by other cells. When BMPs attach to docking station-like structures on the cell surface, called receptors, they stimulate a signaling process inside the cell. In 2009, researchers found that a protein called SMOC blocks BMP activity in animals with backbones by triggering an interfering signal inside the cell. In flies, however, the equivalent protein can make BMP diffuse further from the cell that releases it. To find out how SMOC can do both of these things, Thomas et al. – including some of the researchers involved in the 2009 study – conducted experiments to see which parts of SMOC are required to either block BMP signaling or encourage the diffusion of BMP. These experiments revealed that one end of SMOC can stick to molecules on the cell surface that are not receptors but are molecules where BMP can also bind. When this end of SMOC attaches to these sites, BMPs cannot bind and so diffuse further away. Thomas et al. then produced complete or shortened versions of SMOC proteins to see how this affected BMP activity in frogs. These experiments indicated that the opposite end of SMOC is required for short-circuiting the BMP signal. The results also showed that, at lower concentrations, SMOC stimulates BMPs to diffuse, and that higher concentrations are required to block BMP signaling. These findings suggest that similar to flies, SMOC can also stimulate BMPs to diffuse from the cell in animals with backbones. The next step will be to identify the cell surface receptor for SMOC to better understand the molecular mechanisms that inhibit BMP. The SMOC pathway could be targeted for therapeutic strategies to combat diseases associated with errors in BMP signaling like osteoarthritis, or in cell-based therapies where BMP signaling must be inhibited to produce cells needed to repair damaged tissues. https://doi.org/10.7554/eLife.17935.002 Introduction During development bone morphogenetic proteins (BMPs), comprising at least twenty structurally-related members of the transforming growth factor β (TGF-β) superfamily, are involved in many growth and differentiation events essential for determining body structure (Wu and Hill, 2009). Establishing temporospatial gradients or restricted distributions of BMP signaling is important for many of these processes, which are regulated by a number of mechanisms: ligand binding by extracellular BMP antagonists (Brazil et al., 2015), intracellular feedback inhibition downstream of the BMP receptor (Massagué et al., 2005), spatially restricted proteolytic processing (Thomas et al., 2006), and promotion or restriction of diffusion by interactions with extracellular matrix proteins such as collagen type IV (Umulis et al., 2009) and heparan sulfate proteoglycans (HSPGs) (Matsumoto et al., 2010; Belenkaya et al., 2004). In addition, BMP signaling is also influenced via communication with other signaling pathways, particularly those that act through mitogen-activated protein kinases (MAPKs). MAPK-directed phosphorylation of BMP receptor-regulated Smad 1/5/8 in the linker region inhibits BMP signaling by blocking Smad translocation to the nucleus (Alarcón et al., 2009; Sapkota et al., 2007). We showed previously that SMOC, a matricellular protein associated with basement membranes (Vannahme et al., 2002) and expressed in developing brain, branchial arches, eye, pronephros, limb bud cartilage condensations, and joint interzones (Okada et al., 2011; Rainger et al., 2011; Thomas et al., 2009), inhibits BMP signaling (Thomas et al., 2009); following the addition of BMP2 to NIH3T3 fibroblasts transfected with SMOC, downstream phosphorylation of Smad 1/5/8 is blocked (Thomas et al., 2009). In Xenopus ectodermal explants (animal caps), SMOC was shown to activate MAPK signaling and inhibit Smad 1/5/8-mediated BMP signaling downstream of the constitutively active BMP receptor, BMPR1B (Thomas et al., 2009). Although the exact mechanism is not known, the activity was lost in the presence of non-phosphorylatable linker-mutant Smad, suggesting that BMP inhibition results from activation of MAPK signaling and subsequent ubiquitination and degradation of Smad following linker Smad phosphorylation (Sapkota et al., 2007). In Drosophila, the SMOC orthologue pentagone (pent) is expressed in developing wing imaginal discs and has also been shown to inhibit BMP signaling (Vuilleumier et al., 2010). However, Pent did not appear to inhibit BMP signaling in the presence of the constitutively active zebrafish BMPR1 receptor, Alk8 (Vuilleumier et al., 2010). Structurally, SMOC and Pent are similar, containing an N-terminal follistatin-like domain (FS), followed by two thyroglobulin-like domains separated by a non-homologous domain, and a C-terminal extracellular calcium binding (EC) domain (Vannahme et al., 2002; Vuilleumier et al., 2010). Although Pent contains an additional EC domain located between the two thyroglobulin-like domains, their phylogenetic conservation would predict similar functions. In Drosophila, Pent binds to the cell surface HSPG, Dally, which is required for Pent to extend the range of BMP signaling during Drosophila wing patterning (Vuilleumier et al., 2010). While SMOC has also been shown to bind to HSPGs (Klemenčič et al., 2013), there have been no reports of SMOC promoting BMP signaling at a distance from its source. Indeed, the dual function of SMOC/Pent as a BMP inhibitor and an expander of BMP signaling have not been reconciled. Here, we present the first evidence showing how different domains within SMOC function either to inhibit BMP signaling locally or expand its range of effect. Results and discussion Drosophila Pent can inhibit BMP signaling in Xenopus downstream of the BMP receptor To support the applicability of functional information from Pent to the vertebrate SMOC (Figure 1A), we first confirmed that a pent cDNA construct was biologically active in Drosophila. As reported previously (Vuilleumier et al., 2010), compared to controls, flies homozygous for pent mutations display a characteristic truncation of the L5 longitudinal vein of the adult wing (Figure 1—figure supplement 1). When pent was expressed in its normal location during wing development, using the Gal4/UAS system, the mutant phenotype was rescued completely, demonstrating that the pent construct had full biological activity (Figure 1—figure supplement 1). Initial injections of pent mRNA into Xenopus embryos produced no apparent effects (not shown); however overexpression of a synthetic pent mRNA (co-pent) optimized for codon usage and translation efficiency in Xenopus (Villegas and Kropinski, 2008) (Figure 1—figure supplement 2) produced a dorsalized phenotype indistinguishable from that observed following overexpression of Xenopus SMOC-1 (XSMOC-1) (Figure 1B). The ability of Pent to inhibit BMP signaling downstream of the BMP receptor was analyzed in Xenopus ectodermal explants (animal caps) following co-injection of mRNAs for co-pent and constitutively active BMP receptor1B (caBMPR1B); the caBMPR1B, containing an intracellular activating mutation (Q203D), promotes phosphorylation of Smad 1/5/8 and subsequent BMP signaling events independent of ligand binding (Zou et al., 1997). As expected, XVent, a direct downstream target of BMP signaling (Gawantka et al., 1995), was strongly expressed in animal caps from embryos injected with caBMPR1B alone (Figure 1C). In contrast, XVent was markedly reduced in caps from embryos co-injected with caBMPR1B and either co-pent or XSMOC-1 (Figure 1C); these results suggested that, as we had shown previously for SMOC (Thomas et al., 2009), Pent can inhibit BMP signaling downstream of the BMPR1B receptor. It is unclear why, in a previous report (Vuilleumier et al., 2010), Pent was able to rescue ventralization caused by overexpression of Bmp2b in Zebrafish, but not following overexpression of the Zebrafish caBMPR1, Alk8. As both BMPR1B (Alk6) and Alk8 are type I BMP receptors that activate Smad 1/5/8, a possible reason could be the amounts of mRNA injected and/or the amounts of protein produced. In our previous study (Thomas et al., 2009), SMOC transfected NIH3T3 cells were able to inhibit BMP signaling in the presence of excess BMP2 (100 ng/ml); where the limiting factor would be the number of BMP receptors on the cells. Conversely, in order for SMOC/Pent to inhibit BMP signaling in the presence of caBMPR1B, SMOC/pent needed to be overexpressed at a 2:1 ratio (Figure 1C); SMOC was not able to inhibit BMP signaling in the presence of an excess of caBMPR1B (not shown). Figure 1 with 2 supplements see all Download asset Open asset Xenopus SMOC-1 and Drosophila pent are orthologues that inhibit BMP signaling downstream of the BMP receptor. (A) Schematic representation of vertebrate SMOC and Drosophila Pent: SP- signal peptide, FS – Follistatin-like domain, Tg1 – Thyroglobulin type I-like domain, EC- Extracellular calcium binding domain (B) Dorsalized phenotypes of stage 35 Xenopus embryos following overexpression of mRNAs for XSMOC-1 or codon-optimized pent (co-pent): Xenopus embryos were injected bilaterally at the two-cell stage with 200 pg of mRNA for either GFP (control), XSMOC-1, or co-pent. The exaggerated dorsal/anterior structures and diminished posterior structures observed following XSMOC-1 or co-pent overexpression were observed in 95% of embryos in four independent experiments (n = 130). (C) RT-PCR analysis of animal cap (AC) explants removed from stage 8/9 embryos injected bilaterally at the two-cell stage with either 450 pg of GFP (Control), 150 pg of constitutively active BMP receptor IB (caBMPR-IB) plus GFP (300 pg), or 150 pg of caBMPR-1B plus XSMOC-1 or co-pent (300 pg) mRNAs. The AC explants were incubated until stage 20 before RNA extraction. Induction of the BMP signaling target gene, XVent, by caBMPR-1B was blocked by co-expression of either XSMOC-1 or co-pent. –RT control, without reverse transcriptase. https://doi.org/10.7554/eLife.17935.003 Having established that Pent can function as a BMP antagonist in Xenopus assays, we wanted to determine whether SMOC could function as an expander of BMP signaling in Drosophila. However, attempts to express XSMOC-1 in Drosophila using a synthetic construct optimized for codon usage in Drosophila were unsuccessful; immunoblot analysis of two Drosophila lines generated to overexpress XSMOC-1 demonstrated that XSMOC-1 was below the level of detection (5 ng) for the assay (Figure 1—figure supplement 1), suggesting that despite codon optimization, XSMOC-1 was not translated in amounts sufficient to be effective. SMOC expressed in bacteria and refolded is biologically active in Xenopus and in mammalian cell lines As SMOC and Pent are structurally similar, SMOC may function as an expander of BMP signaling in vertebrates. To address this possibility we developed assays using SMOC expressed in bacteria and refolded together with two SMOC deletion mutant constructs; XSMOC-1∆EC lacking the EC domain, and XSMOC-1EC containing the EC domain only. The EC domain was of interest as hSMOC-1 binds to heparan sulphate proteoglycans (HSPGs) via the EC domain (Klemenčič et al., 2013) and the expander function of Pent is associated with binding to the cell surface-associated HSPG, Dally (Vuilleumier et al., 2010). For expression of XSMOC-1 in bacteria, the predicted signal peptide (2-24) was omitted and a C-terminal hexahistidine-tag added. When first expressed, two predominant induced proteins were observed on SDS-PAGE (Figure 2A); one migrating at 49 kDa, the other at approximately 24 kDa. Protein sequencing revealed the 49 kDa protein to be XSMOC-1, whereas the 24 kDa protein was a partial XSMOC-1 sequence beginning at V235. The base sequence (GTG) was consistent with an alternative start codon (Villegas and Kropinski, 2008); when changed to GTA by site-directed mutagenesis only the expected 49 kDa product was produced (Figure 2A). All subsequent XSMOC-1 and XSMOC-1∆EC constructs contained GTA at V235. Figure 2 with 1 supplement see all Download asset Open asset Expression and refolding of recombinant mature XSMOC-1, XSMOC-1∆EC, and XSMOC-1EC. (A) Coomassie stained SDS-PAGE of wild type (V235-GTG) and silent mutant (V235-GTA) recombinant mature XSMOC-1 following size exclusion chromatography (SEC). The product migrating at 24 kDa is a partial XSMOC-1 sequence beginning at the cryptic start site encoded by GTG at V235. (B–E) Solid lines: SEC profiles obtained following refolding of XSMOC-1 (B, C), XSMOC-1∆EC (D) and XSMOC-1 EC (E) either in the absence (B) or presence of 2 mM Calcium Chloride (C–E). Dashed line: SEC profile (C) obtained for human SMOC-1 refolded in the presence of calcium. Asterisk symbols indicate the peaks corresponding to each schematic diagram. https://doi.org/10.7554/eLife.17935.006 Initial XSMOC-1 refolding studies were conducted using the protocol described previously, where calcium is absent, and produces hSMOC-1 that is monomeric (Novinec et al., 2008). Analysis by S-200 size-exclusion chromatography (SEC) showed a mixture of poorly separated peaks, one of which had a calculated molecular weight of 45.5 kDa, approximate to the predicted 49.6 kDa of monomeric XSMOC-1 (Figure 2B). However, when tested in the Xenopus animal cap assay the protein was inactive (not shown). As occupancy of the calcium binding sites may be necessary for biological activity, we modified the refolding buffer to include 2 mM CaCl2. With this change, in addition to poorly-separated higher molecular weight material, both XSMOC-1 (Figure 2C) and XSMOC-1∆EC (Figure 2D) migrated as single, symmetrical peaks. Their calculated molecular weights of 95.4 kDa and 53 kDa, respectively, were approximately twice the predicted monomeric sizes (49.6 kDa and 32.3 kDa). This suggested that XSMOC-1 and XSMOC-∆EC refolded in the presence of calcium form dimers. Furthermore, hSMOC-1, shown previously to elute as a monomer (Novinec et al., 2008), also migrated as an apparent dimer under these conditions with a calculated molecular weight of 90.3 kDa (Figure 2C); no peak was observed at the expected monomeric size of 46.6 kDa. In contrast, XSMOC-1EC eluted at the calculated molecular weight of 23.7 kDa (Figure 2E), consistent with that predicted for a monomer (18.4 kDa). Dimeric XSMOC-1 could not be dissociated by chelation of Ca++ ions and continued to elute as a dimer following dialysis in the presence of 10.5 mM EDTA (Figure 2—figure supplement 1). Indeed, a dimer was still observed in the presence of 50 mM EDTA and 1 mM nitriloacetic acid (not shown). Analysis by SDS-PAGE under non-reducing and reducing conditions demonstrated that dimeric XSMOC-1 and XSMOC-∆EC were not formed through disulfide linkages (Figure 2—figure supplement 1). BMP inhibition and neural induction by SMOC does not require the EC domain The biological activity of the SMOC proteins was assessed using the Xenopus animal cap explant assay in which overexpression of XSMOC-1 mRNA was shown previously to induce anterior neural markers (Thomas et al., 2009). XSMOC-1, XSMOC-1∆EC, or XSMOC-1EC proteins were incubated with stage 9 (Nieuwkoop and Faber, 1994) late blastula animal caps, at equimolar concentrations, until wild type embryos reached the late neurula stage (stage 20–21). RT-PCR demonstrated the induction of anterior neural markers in the presence of XSMOC-1 and XSMOC-1∆EC, but not XSMOC-1EC (Figure 3A); hSMOC-1 was also effective in this assay (not shown). For these studies, the optimal concentration of SMOC was found to be 100 μg/ml, which appears relatively high. However, SMOC is a matricellular protein and though it is difficult to estimate the effective concentration in vivo, its affinity for HSPGs suggests that its diffusion will be restricted unless HSPG sites are saturated. Consequently, it may remain concentrated near the site of secretion and thus achieve high levels locally. Temporal analyses showed that a two-hour pulse of XSMOC-1 or XSMOC-1∆EC protein was sufficient to commit the naïve ectoderm of the Xenopus animal cap to an anterior neural fate sixteen hours later (Figure 3B); a one-hour pulse was not. This suggests that, following a two hour exposure to SMOC, a duration of exposure (Rogers and Schier, 2011) is reached whereby sufficient changes in gene transcription occur in SMOC-responsive cells to convert their fate from epidermal to neural. While it is well established that inhibition of endogenous BMP activity in Xenopus ectodermal explants promotes a neural fate (Vonica and Brivanlou, 2006) and a number of genes have been implicated in neural fate specification (Kishi et al., 2000; Ueno et al., 2008; Milet et al., 2013; Green and Vetter, 2011; Gammill and Sive, 2001), the sequence of events resulting in commitment to the neural lineage is not known. It should now be feasible to design an unbiased genome-wide screen to identify the early transcriptional changes, following a two hour exposure to SMOC, that initiate neural differentiation. Figure 3 Download asset Open asset XSMOC-1 and XSMOC-1∆EC, but not XSMOC-1EC convert the fate of naïve Xenopus ectoderm explants (animal caps) to anterior neural tissue within two hours. (A) RT-PCR analysis of animal caps removed at stage 8/9 and incubated in 0.7X MMR/0.1% BSA (control) containing equimolar amounts of XSMOC-1 (100 μg/ml), XSMOC-1∆EC (75 μg/ml) or XSMOC-1EC (50 μg/ml) until sibling embryos reached the late neurula stage (20); anterior neural markers (N-CAM, Nrp-1, Otx2, Xag-1) were induced by both XSMOC-1 and XSMOC-1∆EC, but not by XSMOC-1EC. Expression of the ectodermal marker Keratin was suppressed by both XSMOC-1 and XSMOC-1∆EC, but not by XSMOC-1EC. (B) Animal caps removed at stage 8/9 were incubated in 0.7X MMR/0.1% BSA (control) in the presence of XSMOC-1 (100 μg/ml) for six minutes, one hour, or two hours before replacing with 0.7X MMR/0.1% BSA and incubating until sibling embryos reached stage 20. RT-PCR analysis shows that a two hour exposure to XSMOC-1was sufficient to induce the naïve ectoderm to express anterior neural markers 16 hr post-pulse; a one hour exposure was not. The continual presence of XSMOC-1 (16 hr) was used as a positive control. https://doi.org/10.7554/eLife.17935.008 The ability of the SMOC proteins to inhibit BMP signaling was assessed in mammalian cell lines. Serum-starved NIH-3T3 and HEK-293 cells were incubated with either XSMOC-1 (100 μg/ml) or equimolar amounts of XSMOC-1∆EC or XSMOC-1EC for thirty minutes, followed by the addition of BMP2 (50 ng/ml) for an additional thirty minutes. As expected, BMP2 treatment alone caused phosphorylation of Smad 1/5/8 (Figure 4A,B). This was blocked in the presence of XSMOC-1 and XSMOC-1∆EC, whereas XSMOC-1EC significantly enhanced BMP2-mediated Smad phosphorylation (Figure 4A,B). Potentiation of Smad phosphorylation by the EC domain was not due to an additive effect, as the addition of XSMOC-1EC alone to NIH-3T3 cells did not result in Smad1/5/8 phosphorylation (Figure 4C). Figure 4 Download asset Open asset The two thyroglobulin-like domains are necessary for BMP inhibition, whereas the EC domain promotes BMP signaling. (A) Immunoblot showing phosphorylation of Smad 1/5/8 (pSmad) by BMP2 (50 ng/ml) in HEK-293 cells is inhibited by the addition of XSMOC-1 (100 μg/ml) or XSMOC-1∆EC (75 μg/ml), but is enhanced by the addition of XSMOC-1EC (50 μg/ml). Total Smad is shown as a loading control. (B) Graph showing the relative fluorescence of pSmad 1/5/8 obtained on immunoblots from four separate experiments using both HEK293 and NIH3T3 cells; each treatment is displayed as the percent difference from control. The inhibition of Smad 1/5/8 phosphorylation by XSMOC-1 or XSMOC-1 ∆EC and the potentiation of BMP signaling by XSMOC-1EC are both significant (p=≤0.01). (C) Immunoblot of HEK293 cell extracts showing that XSMOC-1 EC alone does not promote Smad phosphorylation. (D) RT-PCR analysis of Xenopus animal cap (AC) explants removed at stage nine from embryos injected bilaterally at the two-cell stage with mRNAs for GFP (Control), caBMPR1B (120 pg), or caBMPR1B and equimolar amounts of XSMOC-1(600 pg), XSMOC-1 ∆FS (540 pg), XSMOC-1 ∆EC (420 pg), XSMOC-1 Tg1 (330 pg), XSMOC-1 ∆Tg1 (360 pg) orXSMOC-1 EC (240 pg). The AC explants were incubated until stage 20 (late neurula) before RNA extraction and analysis. The induction of the direct BMP signaling target gene, XVent, by caBMPR1B was blocked by co-expression with XSMOC-1, XSMOC-1 ∆FS, or XSMOC-1 ∆EC, but not by XSMOC-1 Tg1, XSMOC-1 ∆Tg1, or XSMOC-1 EC. H4: Histone loading control, –RT: Negative control. https://doi.org/10.7554/eLife.17935.009 Figure 4—source data 1 Source data file for generating Figure 4B. Absorbance values obtained from pSmad immunoblots in four separate experiments. https://doi.org/10.7554/eLife.17935.010 Download elife-17935-fig4-data1-v1.docx Having established that the SMOC EC domain is not required for the inhibition of BMP signaling we designed deletion constructs for use in mRNA overexpression studies to determine which domain(s) of SMOC are required for BMP inhibition; XSMOC-1∆FS contained a 49 amino acid deletion of the N-terminal FS-like domain (∆Q43 to A91); XSMOC-1∆Tg1 contained only the follistatin-like domain and EC domain (∆K95 to S304); XSMOC-1∆FS∆EC (referred to as XSMOC-1Tg1) contained deletions of the FS-like and EC domains (∆Q43 to A91 and ∆N310 to end), leaving only the two Tg1-like domains. The effect on BMP inhibition was analyzed in Xenopus embryos following overexpression of mRNAs for each deletion construct and caBMPRIB. Analysis of animal caps following co-injection of Xenopus embryos with caBMPRIB and XSMOC-1, XSMOC-1∆FS, or XSMOC-1∆EC showed inhibition of BMP signaling, indicated by the suppression of XVent expression (Figure 4D). Whereas these data suggest that the FS and EC domains are not required for BMP inhibition overexpression of XSMOC-1Tg1, containing the Tg1-like domains only, did not inhibit BMP signaling in this assay (Figure 4D); deletion of the Tg1-like domains (XSMOC-1∆Tg1) produced a similar result. While we believe the Tg1-like domains to be important for BMP inhibition, by process of elimination, the effects of large deletions on protein folding are difficult to predict; in experiments of this type, where protein function is lost, improper folding is common (Valastyan and Lindquist, 2014). Consequently, we consider misfolding of the protein produced by XSMOC-1∆FS∆EC as the most likely reason for the inability of this construct to block BMP signaling. Additional evidence for the importance of the Tg1-like domains in SMOC comes from studies of human Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome, an autosomal disorder caused by mutations in SMOC-1 (Rainger et al., 2011). The phenotype of anopthalmia, oligodactyly, and joint abnormalities was found to be the same in patients with nonsense or frameshift mutations and those with missense mutations in the second Tg1-like domain (Rainger et al., 2011). As the nonsense and frameshift mutations were predicted to result in a complete loss of SMOC-1 function, the two pedigrees harboring two different single amino acid missense mutations in the Tg1-like domain suggests this domain is indeed essential for SMOC-1 function. Alternative approaches will be required to elucidate the exact role of the Tg1-like domains in BMP inhibition. Many proteins contain Tg1-like domains, including thyroglobulin, insulin-like growth factor binding proteins (IGFBPs) 1–6, the proteoglycan testican, and the basement membrane associated protein nidogen/entactin (Novinec et al., 2006). However, there have been no reports of any of these proteins inhibiting BMP signaling. The SMOC EC domain can expand the range of BMP signaling in vitro by competitive binding to HSPGs The potentiation of BMP signaling by the EC domain was examined further by investigating the relative affinities of XSMOC-1EC and BMP2 for each other and for HSPGs. The binding of SMOC/Pent and BMP2/4 to HSPGs is known, as evidenced by the co-purification of SMOC and BMPs following heparin affinity chromatography of bovine cartilage extracts (Chang et al., 1994), and the binding of BMP2/4 and the EC domain of hSMOC/Pent to heparin/HSPGs (Vuilleumier et al., 2010; Klemenčič et al., 2013; Ruppert et al., 1996). In addition, the basic amino acid-rich putative heparin-binding region identified within the EC domain of SMOC (Klemenčič et al., 2013) is highly conserved in Pent (Figure 5—figure supplement 1). Using the Protein Homology/analogY Recognition Engine (PHYRE), an unsupervised homology model for XSMOC-1EC was constructed based on the structure of the EC domain of the related family member BM-40 (Hohenester et al., 1996). XSMOC-1EC aligned well with the BM-40-EC model (Figure 5—figure supplement 1) and the electrostatic surface potential map predicted an area of positive charge similar to that reported in the EC domain of hSMOC1 (Klemenčič et al., 2013) (Figure 5—figure supplement 1). As monomeric hSMOC-1, refolded in the absence of calcium, was used in previous heparin-binding studies (Klemenčič et al., 2013), we first determined whether dimeric XSMOC-1 can bind heparin. XSMOC-1 and XSMOC-1EC bound to heparin Sepharose in the presence of 0.5M NaCl, whereas XSMOC-1∆EC did not (Figure 5A), confirming the EC domain to be the site of HSPG binding. Comparison of the heparin-binding affinities of XSMOC-1EC and BMP2 showed a striking similarity, with both eluting between 0.65M and 0.7M NaCl (Figure 5B). The possibility that XSMOC-1 and BMP2 bind to each other was discounted (Figure 5C); when BMP2 was incubated with XSMOC-1 or XSMOC-1 EC, pre-bound to heparin-Sepharose, BMP2 was only present in the unbound fraction (Figure 5C; lanes 2 and 5) and did not co-elute with XSMOC-1 or XSMOC-1 EC (Figure 5C; lanes 3 and 6). The lack of interaction of SMOC with BMP2 agrees with an analogous finding observed for Pent and the Drosophila BMP, decapentaplegic (Dpp) (Vuilleumier et al., 2010). This, combined with SMOC and BMP2 having similar heparin-binding affinities, suggests that XSMOC-1EC could compete with BMPs for HSPG binding on HEK293 cells and thereby increase BMP bioavailability. Figure 5 with 1 supplement see all Download asset Open asset XSMOC-1EC and BMP2 have similar binding affinities for heparin sepharose (HS), but do not bind to each other. SDS-PAGE analysis (Coomassie staining) of HS elution profiles showing (A) binding of XSMOC-1, XSMOC-1∆EC, and XSMOC-1EC in PBS or PBS/0.5M NaCl; binding of XSMOC-1 to HS requires the EC domain. (B) XSMOC-1EC and mature BMP2 (A284-R396) in a NaCl gradient (400–700 mM) have equivalent HS binding affinities. (C) BMP2 does not bind directly to XSMOC-1 (lanes 1–3) or XSMOC-1EC (lanes 4–6) at physiological ionic strength (PBS); BMP2 (4 μg) incubated with HS (0.3 μl) saturated with XSMOC-1 or XSMOC-1EC (6 μg), did not co-elute with XSMOC-1 or XSMOC-1EC (lanes 3 and 6) and was only present in the unbound fraction (lanes 2 and 5). Saturation of HS by XSMOC-1 and XSMOC-1EC was confirmed by their presence in the unbound fractions (lanes 1 and 4) prior to incubation with BMP2. https://doi.org/10.7554/eLife.17935.011 We designed an in vitro assay to test the hypothesis that SMOC can expand the range of BMP signaling by competing with BMP2/4 for HSPG-binding. BMP4-soaked beads represented a cellular source of BMPs and agarose gels (0.7%) containing heparan sulfate (HS) (10 μg/ml) represented an extracellular matrix (ECM) capable of binding SMOC and BMPs. Chamber slides containing BMP4-soaked beads embedded in the agarose/HS/XSMOC-1EC matrices were seeded with the stable reporter cell line C33A-2D2-09, harboring luciferase under the control of a BMP response element (BRE). After 24 hr, immunohistochemical analysis of cells in fields of view adjac" @default.
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• W4234496710 title "Decision letter: SMOC can act as both an antagonist and an expander of BMP signaling" @default.
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