LON-2/glypican is properly established to be a unfavorable regulator of BMP signaling in C. elegans [35]. We have now beforehand proven that the opposite glypican within the C. elegans genome, GPN-1/glypican, doesn’t perform in BMP signaling [36]. In distinction to LON-2, SMOC-1 can positively promote BMP signaling when overexpressed ([12], and this examine). Our co-IP outcomes and in silico structural modeling confirmed that SMOC-1 can bind each LON-2 and the mature area of DBL-1 and that SMOC-1 can mediate the formation of a LON-2-SMOC-1-DBL-1 tripartite complicated. Moreover, site-specific mutations within the endogenous smoc-1 (smoc-1(M1)) or the endogenous lon-2 (lon-2(mut)) loci that disrupt SMOC-1-LON-2 interplay give the worms a protracted phenotype, whereas compound mutations in smoc-1 (smoc-1(M1+M2)) that concurrently disrupt SMOC-1-LON-2 interplay and weaken SMOC-1-DBL-1 interplay considerably shorten the size of smoc-1(M1) mutants to close WT size. Based mostly on these information, we suggest the next mannequin for the way SMOC-1 features to manage BMP signaling in C. elegans (Fig 12). In our mannequin, SMOC-1 has each a unfavorable, LON-2/glypican-dependent position and a optimistic, DBL-1/BMP-dependent however LON-2-independent position in regulating BMP signaling. On the one hand, SMOC-1 concurrently binds to each the mature area of DBL-1/BMP and LON-2/glypican, ensuing within the sequestration of DBL-1/BMP to forestall DBL-1/BMP motion or interplay with the BMP receptors, thus inhibiting BMP signaling. Alternatively, secreted SMOC-1 can bind to the mature area of DBL-1/BMP, probably facilitating the motion of DBL-1/BMP by means of the extracellular house or the supply/presentation of DBL-1/BMP to its receptors, thus selling BMP signaling. The duality of SMOC-1 perform in our mannequin is according to earlier findings that smoc-1(0) mutants are solely barely (roughly 5%) smaller than WT worms and that smoc-1(0); lon-2(0) double mutants have an intermediate physique measurement between smoc-1(0) and lon-2(0) mutants [12]. It’s also according to overexpression research we offered in Fig 11, the place overexpression of SMOC-1 or DBL-1 can promote BMP signaling within the absence of LON-2.

Fig 12. A mannequin for the way SMOC-1 features to manage BMP signaling.

(A) In WT animals, SMOC-1 is proposed to each negatively and positively regulate BMP signaling. For its unfavorable position, SMOC-1 binds to LON-2/glypican and DBL-1/BMP to sequester DBL-1/BMP. Within the meantime, SMOC-1 may bind to DBL-1/BMP to advertise BMP signaling. The optimistic and unfavorable roles of SMOC-1 in WT animals steadiness one another out, resulting in a WT physique measurement. (B) In smoc-1(0) animals, each the optimistic and unfavorable roles of SMOC-1 are gone, resulting in a barely small physique measurement. (C–E) Eradicating LON-2 utterly [as in lon-2(0)] or disrupting SMOC-1-LON-2 interplay [as in smoc-1(M1) or lon-2(mut)] eliminates the unfavorable position of SMOC-1, resulting in a protracted physique measurement. (F) Weakening SMOC-1-DBL-1 interplay within the SMOC-1(M1) background [as in smoc-1(M1+M2)] considerably attenuates the optimistic position of SMOC-1, resulting in a non-long, WT-like physique measurement. BMP, bone morphogenetic protein; SMOC, secreted modular calcium-binding protein; WT, wild kind.

https://doi.org/10.1371/journal.pbio.3002272.g012

The idea of SMOCs appearing as each antagonists and expanders of BMP signaling has been proposed by Thomas and colleagues [17]. Xenopus XSMOC-1 and the Drosophila SMOC homolog Pent have been proven to advertise the spreading of the BMP ligand, thus increasing the vary of BMP signaling, each in vitro and in vivo [10,11,17,37]. Each XSMOC-1 and human SMOC-1 can bind to heparin and heparan sulfate (HS), and within the case of Drosophila Pent, the BMP co-receptors Dally/glypican and Dally-like/glypican [10,17,27,28]. Research in Drosophila additional confirmed that the flexibility of Pent to increase the vary of BMP signaling depends on Dally/glypican in wing imaginal discs [10]. As a result of neither XSMOC-1 nor Pent have been discovered to bind BMPs, however BMPs can bind HSPGs [10,17,19,28], a mannequin was proposed the place the binding of SMOCs to HSPGs competitively reduces the interplay between HSPGs and BMPs, thus selling the spreading of BMPs [17]. Our information, combining in silico structural modeling, in vitro biochemical assays, and in vivo site-directed mutagenesis, strongly recommend that the optimistic position for CeSMOC-1 in regulating BMP signaling is impartial of LON-2/glypican, however depending on DBL-1/BMP. That is according to our earlier discovering that smoc-1(OE); dbl-1(0) double mutants are as quick as dbl-1(0) null mutants [12], suggesting that CeSMOC-1 acts by means of DBL-1/BMP to advertise BMP signaling.

At current, there isn’t a unified mannequin on how SMOCs perform to inhibit BMP signaling. Utilizing a Xenopus animal cap assay, Thomas and colleagues [16,17] confirmed that each XSMOC-1 and Pent can inhibit BMP signaling downstream of the BMP receptor, at the least for XSMOC-1, by activating the MAPK pathway. In distinction, Drosophila Pent has been proven to perform upstream of receptor activation to inhibit BMP signaling in zebrafish [10]. A more moderen examine confirmed that mouse SMOC2 can inhibit BMP signaling by competitively binding to BMPR1B [13]. Our outcomes are according to CeSMOC-1 functioning in a LON-2/glypican-dependent method to negatively regulate BMP signaling.

Utilizing each IP-MS of worm extracts and coimmunoprecipitation of proteins expressed in Drosophila S2 cells, now we have discovered that CeSMOC-1, particularly the EC area of CeSMOC-1, can bind to LON-2/glypican. Our findings are according to earlier findings exhibiting that Drosophila Pent can co-IP with Dally/glypican and Dally-like/glypican, whereas XSMOC-1, hSMOC2, and Pent can all bind to heparin and HS in vitro [10,17,27,28]. The EC domains of SMOCs all have a stretch of positively charged residues that was hypothesized to mediate its interplay with HSPGs [27]. Fascinatingly, our protein structural modeling means that the SMOC EC area might immediately work together with the protein core of HSPGs. As proven in Fig 8, the important thing residues recognized to mediate SMOC-1-LON-2 interplay are conserved amongst nematode SMOC proteins, in addition to vertebrate SMOCs. Additional, our ends in C. elegans present further readability to some beforehand perplexing findings by Taneja-Bageshwar and Gumienny [38,39]. These authors confirmed that overexpressing the LON-2/glypican protein core is ample to rescue the lengthy (Lon) physique measurement phenotype of lon-2(0) null mutants, suggesting that the LON-2/glypican protein core is ample to inhibit BMP signaling. Their earlier findings additional confirmed that the N-terminal area of LON-2, LON-2(1–368), is ample to inhibit BMP signaling. Notably, this area incorporates all the important thing residues in LON-2 that mediate LON-2-SMOC-1 interplay, as recognized by our structural modeling (between amino acids 100 and 325, S4 Fig). Mutating the three amino acids in LON-2 predicted to mediate LON-2-SMOC-1 interplay, as in LON-2(mut) [S311D A315D F319D], resulted in mutant worms exhibiting a protracted physique measurement phenotype indistinguishable from that of lon-2(0) null mutants (Fig 10). These outcomes strongly argue that the most important, if not the only, perform of LON-2 in regulating physique measurement is mediated by its interplay with SMOC-1 by way of the LON-2 protein core.

Within the C. elegans examine talked about above, Taneja-Bageshwar and Gumienny [38,39] additionally confirmed that the HS attachment websites are necessary for a truncated type of LON-2 (aa423-508) to perform in inhibiting BMP signaling when overexpressed. Whether or not related outcomes maintain true within the endogenous lon-2 locus stays to be decided. Kirkpatrick and colleagues discovered that in Drosophila, the protein core of Dally/glypican is partially practical, whereas its overexpression really limits Dpp/BMP signaling within the wing imaginal discs [40]. Whether or not Pent is concerned in mediating this perform of Dally/glypican is unclear.

LON-2/glypican is a well-established unfavorable regulator of BMP signaling in C. elegans [35]. Earlier research confirmed that when expressed in mammalian HEK293T cells, membrane-tethered LON-2 can bind to mammalian BMP2 after chemical crosslinking [35]. Since Drosophila and vertebrate BMPs have been discovered to bind to HSPGs [19,41,42], a mannequin was proposed the place LON-2/glypican negatively regulates BMP signaling by sequestering the DBL-1/BMP ligand in C. elegans [35]. We didn’t detect any interplay between LON-2 and any type of DBL-1, both by co-IP assays utilizing the Drosophila S2 cell system, or by way of in silico structural modeling (Figs 5, S2 and S3). Though we can not rule out the chance that posttranslational modifications taking place in Drosophila S2 cells are totally different from these present in C. elegans, we consider that the absence of LON-2 and DBL-1 interplay in our S2 cell expression system is unlikely as a result of lack of glycosaminoglycan (GAG) modifications of LON-2. As proven in Figs 2, 5 and S2, we detected a number of increased molecular weight bands on western blots that seemingly correspond to GAG-decorated LON-2.

Our outcomes strongly argue that LON-2/glypican binds DBL-1/BMP not directly by way of CeSMOC-1 as a bridging molecule. We detected sturdy interplay between CeSMOC-1 and the mature area of DBL-1/BMP (Fig 5) and confirmed that CeSMOC-1 can mediate the formation of a tripartite complicated between LON-2/glypican and DBL-1/BMP (Fig 8). In silico structural modeling utilizing the ColabFold [34] implementation of AlphaFold2 [30] supported the interactions between LON-2/glypican, CeSMOC-1, and mature DBL-1/BMP (Figs 6–8). Importantly, key amino acids recognized by structural modeling in every of the three proteins to mediate pair-wise protein–protein interactions look like extremely conserved (Figs 6, S3 and S4). Lastly, mutations in CeSMOC-1 or LON-2/glypican that disrupt SMOC-1-LON-2 interplay (as in SMOC-1(M1) or LON-2(mut)) resulted in a protracted physique measurement phenotype, a manifestation of elevated BMP signaling, whereas mutations that considerably weaken CeSMOC-1-DBL-1/BMP interplay within the SMOC-1(M1) background, as in SMOC-1(M1+M2), abolished the lengthy phenotype of SMOC-1(M1) mutant worms (Figs 9 and 10). Our information recommend that LON-2/glypican acts as a unfavorable regulator of BMP signaling in C. elegans by sequestering the DBL-1/BMP ligand by way of CeSMOC-1 and that CeSMOC-1 facilitates the unfavorable regulation of BMP signaling by forming a tripartite complicated with LON-2/glypican and DBL-1/BMP. Since SMOC homologs from C. elegans, Drosophila, and Xenopus all functionally work together with HSPGs, we suggest that the SMOC-HSPG axis is an evolutionarily conserved module necessary in regulating BMP signaling.

Our earlier work [12] and this work confirmed that when overexpressed, CeSMOC-1 can act by means of DBL-1/BMP to positively promote BMP signaling. We argue that this optimistic position of CeSMOC-1 is LON-2/glypican-independent and primarily mediated by direct interactions between CeSMOC-1 and DBL-1/BMP. That is strongly supported by outcomes from our in vitro biochemical assays, in vivo mutagenesis research, and predictions from in silico structural modeling. Direct interplay between SMOCs and BMPs has not been beforehand reported in any organisms. SMOC proteins from totally different organisms additionally include totally different numbers of the TY or EC domains in several association (Fig 7A). However, our structural modeling predicted with excessive confidence direct interactions between SMOC and BMP homologs in H. sapiens, X. laevis, and D. melanogaster (Fig 7B). The expected interactions additionally share similarities with the predictions for CeSMOC-1 and DBL-1/BMP. We due to this fact suggest that the interplay between SMOCs and BMPs is evolutionarily conserved.

Earlier research on SMOC homologs have proven that the EC area of SMOC proteins can bind to HSPGs. On this examine, we confirmed that the EC-mediated interplay between CeSMOC-1 and LON-2/HSPG is important for the inhibitory features of CeSMOC-1 and LON-2/glypican in regulating BMP signaling. We additional confirmed that each the TY and the EC domains are required for full perform of CeSMOC-1 on the endogenous locus. The requirement for each the TY and the EC domains for full CeSMOC-1 perform is according to each of our co-IP and structural modeling outcomes, which demonstrated that residues in each the TY and the EC domains are concerned within the interplay between CeSMOC-1 and DBL-1/BMP (Figs 5, 6 and 9). Nonetheless, paradoxically, overexpression of SMOC-1(EC) is ample to advertise BMP signaling in vivo (Fig 3), regardless that SMOC-1(EC) didn’t bind DBL-1/BMP in our in vitro co-IP assays (Fig 2). It’s doable that the stringent IP situation that we used precludes doable low-affinity interactions between SMOC-1(EC) and DBL-1/BMP that may happen in C. elegans, particularly when SMOC-1(EC) is drastically overexpressed. In line with this notion, now we have beforehand proven that 2 single amino acid substitution mutations within the respective TY area and EC area of SMOC-1, jj85(E105K) and jj65(C210Y), induced partial loss-of-function phenotypes on the native setting [12]. But, like overexpressing SMOC-1(EC), overexpressing both SMOC-1(E105K) or SMOC-1(C210Y) was additionally ample to advertise BMP signaling by rising the physique measurement (S8 Fig). Since many practical assays in C. elegans contain the utilization of repetitive transgenic arrays that may trigger overexpression of the transgene, our outcomes additionally spotlight the significance of finishing up practical research of proteins on the endogenous expression ranges.

On this examine, we confirmed that key residues within the TY area of C. elegans SMOC-1 mediate CeSMOC-1-DBL-1/BMP interplay, as mutating these key residues (as in SMOC-1(M3) [Y72A Y90A Y95A W97A]) considerably compromised the flexibility of CeSMOC-1 to bind DBL-1/BMP in vitro (Fig 9). Our structural modeling prompt that the TY domains in SMOC proteins from C. elegans to people are additionally concerned in mediating SMOC-BMP interplay (Fig 7). Apparently, Thomas and colleagues discovered that one of many TY domains (TY1) in XSMOC-1 is required for XSMOC-1’s perform in inhibiting BMP signaling [17]. Based mostly on our findings, we speculate that maybe this position of XSMOC-1 TY1 in BMP signaling is mediated by way of its direct interplay with BMP.

The TY area is evolutionarily conserved throughout metazoans and seen in quite a lot of functionally various proteins, together with thyroglobulin, SMOCs, nidogens, and IGFBPs [43,44]. The TY (kind 1a) area is characterised by 6 conserved cysteine residues, together with “QC” and “CWCV” residue motifs, that type intramolecular disulfide bridges [45]. Exterior of those conserved areas, the TY area has extremely variable loops which will contribute to the big variety of related features. TY domain-containing proteins might perform to inhibit cysteine cathepsins, whereas others inhibit aspartate peptidases, papain-like proteases, or metalloproteases [43,44]. Testicans, which include a TY area in addition to an EC area, can work as a aggressive inhibitor of cathepsin L as present in human cell tradition. Alternatively, human testicans have additionally been discovered to bind to cathepsin L to behave as a chaperone, stopping degradation and inspiring cathepsin exercise throughout the ECM [46,47]. Future analysis will decide whether or not the TY domains in these different proteins can perform analogously to the TY area in CeSMOC-1 by binding to members of the TGFβ tremendous household of development elements and whether or not SMOC TY domains have further features past TGFβ signaling.

There are 2 SMOC homologs in mammals, SMOC1 and SMOC2. Each mice and human people with mutations that severely cut back SMOC1 expression exhibit irregular tooth, eye, and limb improvement [14,15,48–50]. Mutations in hSMOC2 have been discovered to be related to defects in tooth improvement [51,52] and vitiligo [14,53,54]. Irregular expression of SMOCs has additionally been related to a number of cancers in addition to kidney and pulmonary fibrosis [55–61]. Our work described on this examine recognized evolutionarily conserved mechanisms on how SMOC proteins regulate BMP signaling. We have now beforehand proven that each hSMOC1 and hSMOC2 can partially rescue the BMP signaling defect of smoc-1(0) null mutants in C. elegans [12]. Future analysis on the mechanistic particulars of SMOC perform to manage BMP signaling in an in vivo system akin to C. elegans might have vital implications for human well being.

All plasmids and oligonucleotides used on this examine are listed in S4 and S5 Tables, respectively. Plasmids used for expression in Drosophila S2 cells have been generated from pCB313 (present from Dr. Claire Bénard, [62]).

Transgenic strains have been generated utilizing pCFJ90[myo-2p::mCherry::unc-54 3’UTR] (present from Erik Jorgensen) or LiuFD290[ttx-3p::mCherry] (present from Oliver Hobert) as co-injection markers. Two transgenic traces with one of the best transmission effectivity throughout a number of generations have been analyzed with every plasmid. Built-in transgenic traces have been generated utilizing gamma irradiation, after which outcrossed with WT N2 worms at the least 2 instances. S6 Desk lists all of the strains generated on this examine.

For IP/MS experiments, built-in transgenic traces overexpressing untagged SMOC-1 or SMOC-1::2xFLAG have been used (see S6 Desk). Transgenic strains overexpressing both SMOC-1(TY)::2xFLAG or SMOC-1(EC)::2xFLAG have been generated utilizing an embryonic deadly temperature-sensitive pha-1(e2123) background, by together with a pha-1 expressing plasmid (pC1) and a florescent co-injection marker throughout the array. Solely transgenic worms are viable on the restrictive temperature (25°C) [63]. The florescent co-injection marker allowed for visible evaluation of multi-generational transgene transmission effectivity. The utilization of those 2 markers allowed us to develop giant populations of solely transgenic worms carrying the transgenes at excessive transmission effectivity.

All sgRNA information plasmids have been generated utilizing the technique described in [64,65]. To generate every particular modifications, the precise sgRNA plasmids (see S4 Desk) have been injected with the Cas9-encoding plasmid, pDD162 (Dickinson and colleagues), and the single-strand oligodeoxynucleotide homologous restore template, into WT N2 worms. pRF4(rol-6(d)) was used as a co-injection marker. Injected P0 animals have been singled. F1 progeny have been picked from plates that gave probably the most curler progeny, allowed to put eggs and screened for profitable CRISPR occasions by way of PCR (see S5 Desk for oligo data). The ensuing secure strains have been confirmed by genotyping and Sanger sequencing. The smoc-1(TY)::2xflag and the smoc-1(EC)::2xflag strains have been generated within the smoc-1(jj276[smoc-1::2xflag]) background utilizing both single-strand oligodeoxynucleotide or plasmid as homologous restore template (see S4 and S5 Tables).

CRISPR–Cas9-mediated mutations within the smoc-1 or lon-2 locus have been generated utilizing ribonucleoprotein (RNP) complexes by following the protocol described in [66]. RNP mixes containing 0.25 mg/ml Cas9 (IDT 1081059), 0.1 mg/ml tracrRNA (IDT 1072534), 56 ng/ml gene-specific crRNA (IDT), 25 ng/ml single-stranded oligodeoxynucleotide restore template (IDT ultramers), and 40 ng/ml of pRF4(rol-6(d)) plasmid have been injected into both the smoc-1(jj276[smoc-1::2xflag]) (for smoc-1 edits) or PD1074 WT (for lon-2 edits) worms. Injected P0 animals have been singled. F1 progeny have been picked from plates that gave probably the most curler progeny, allowed to put eggs and screened for profitable CRISPR occasions by way of PCR. See S5 Desk for oligo data. The ensuing secure strains have been confirmed by genotyping and Sanger sequencing of your entire smoc-1 or lon-2 coding area.

Physique measurement measurements have been carried out as beforehand described [12,67]. Gravid adults have been bleach synchronized, with ensuing embryos incubated in M9 buffer rotating at 15°C till hatched (24 to 48 h). Synchronized L1s have been plated and grown at 20°C till the L4.3 vulval stage was seen in a majority of worms. To measure the physique size of heterozygous jj276/+ or jj499 jj276/+ hermaphrodites, jjIs3900/+ males that specific mCherry within the pharynx (S6 Desk) have been mated with jj276 or jj499 jj276 hermaphrodites, pink cross progeny on the L4.3 vulval stage have been collected for measurement. For imaging, worms have been washed from plates, handled with 0.3% sodium azide till straightened, after which mounted onto 2% agarose pads. Pictures have been taken utilizing a Hamamatsu Orca-ER digicam utilizing the iVision software program (Biovision Know-how). Utilizing Fiji, worm physique lengths have been measured from photos utilizing the segmented line device. An ANOVA and Tukey’s sincere vital distinction (HSD) was used to check for variations in physique measurement between genotypes utilizing R (https://www.R-project.org/).

For the suppression of sma-9(0) M-lineage defect (Susm) assay, worms have been grown at 20°C, and the variety of grownup animals with 4 CCs and 5 to six CCs have been tallied throughout a number of plates [68]. The reported Susm penetrance refers back to the p.c of animals with 1 or 2 M-derived CCs as scored utilizing the CC::GFP reporter. For every genotype, 2 impartial isolates have been generated (as proven within the pressure record in S6 Desk), 3 to 7 plates of worms from every isolate have been scored for the Susm phenotype, and the Susm information from the two isolates have been mixed, and the technique of the Susm penetrance have been offered. The shortage of M-derived CCs phenotype will not be absolutely penetrant in sma-9(cc604) mutants [28]. For the Susm rescue experiments, we used R to generate a common linear mannequin with binomial error and a logit hyperlink perform designating transgenic state because the explanatory perform. The Wald statistic take a look at was used to find out if transgenic state (transgenic versus non-transgenic worms throughout the similar line) is related to CC quantity.

C. elegans strains have been repeatedly bleach synchronized and grown on 90 mm NGM plates seeded with NA22 micro organism, till desired inhabitants measurement was reached. Roughly 25 NA22 plates containing about 10,000 synchronized gravid adults have been bleached to get a goal inhabitants of about 2 million or extra synchronized L1s in M9. Roughly 4,000,000 synchronized L1s have been plated on 15 cm egg plates (NGM strep with OP50-1 and hen egg; [66] and grown till inhabitants reached the L4 stage (48 h at 25°C)). Worms have been washed from plates and picked up with H150 (50 mM HEPES (pH 7.6), 150 mM KCl). Successive pelleting and washing have been accomplished to take away any extra meals or particles. Lastly, worms have been pelleted and an equal quantity of H150g10 (H150 with 10% glycerol) with protease inhibitor (Pierce, A32965) was added to make a worm slurry. “Worm popcorn” was made by including the slurry dropwise immediately into liquid nitrogen. Ensuing popcorn was saved in 50 mL conical tubes at −80°C.

Eluates have been submitted to Biotechnology Useful resource Heart (BRC) at Cornell College for evaluation by MS. Briefly, samples have been ready by in-solution trypsin digestion earlier than conducting nanoLC-ESI-MS/MS evaluation utilizing an Orbitrap Fusion Tribrid (Thermo Fisher Scientific, San Jose, California, United States of America) mass spectrometer geared up with a nanospray Flex Ion Supply and paired with a Dionex UltiMate 3000 RSLCnano system (Thermo, Sunnyvale, California, USA) [69,70]. Processing workflow used SequestHT and MS Amanda with Percolator validation. Database search was carried out towards a C. elegans database downloaded from NCBI in June 2021. Solely excessive confidence peptides outlined by Sequest HT with a 1% FDR by Percolator have been thought of for assured peptide identification. Abundance ratios relative to untagged (e.g., smoc-1::2xflag/smoc-1) have been assessed to establish candidate interplay companions, with values over 2.0 being thought of enriched. Solely hits with 2 or extra mapped peptides have been thought of right here.

The ColabFold [34] implementation of AlphaFold ([30] was used to foretell constructions of complexes involving totally different combos of LON-2, SMOC-1, and the mature type of DBL-1. Default parameters have been used besides 3 fashions have been predicted for every question. Pairwise combos resulted in predictions for an interplay between LON-2 and SMOC-1, with an interface predicted template modeling (“iptm”) rating of 0.59 and an interplay between SMOC-1 and mature DBL-1 with an iptm rating of 0.76. In distinction, no interplay was predicted between mature DBL-1 and LON-2. Given these predictions, we then tried a prediction with LON-2, SMOC-1, and a couple of copies of mature DBL-1 (mature DBL-1 is understood to homodimerize). The consequence was a structural prediction wherein SMOC-1 interacts with each mature DBL-1 and LON-2 (iptm rating of 0.5) by means of interfaces which might be the identical as predicted within the 2 corresponding pairwise combos. In every of those profitable predictions, the three fashions predicted for every case had similar predicted interfaces. For predictions of complexes fashioned between CeSMOC-1 and mature DBL-1 homologs in different species, the next iptm scores have been obtained for the highest hits: C. elegans SMOC-1:DBL-1:DBL-1, 0.76; H. sapiens SMOC1:BMP2:BMP2, 0.81; X. laevis XSMOC1:BMP2:BMP2, 0.78; D. melanogaster Pentagone:Dpp:DPP, 0.80. The iptm rating is a confidence rating generated by AlphaFold ([33]).