SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans.

Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium-binding (EC) domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. We showed that CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. We provide both in vitro biochemical and in vivo functional evidence demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. We further showed that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling

GFP-tagged SMOC-1 is non-functional.

(A) Diagrams depicting various endogenously tagged SMOC-1 proteins, with the corresponding CRISPR alleles shown on the left of the diagrams. (B) Table showing the penetrance of the Susm phenotype of strains carrying specified smoc-1 allele in a sma-9(cc604) background. The Susm penetrance refers to the percent of animals with 1 or 2 M-derived CCs as scored using the arIs37(secreted CC::GFP) reporter. For each genotype, 2 independent isolates were generated (as shown in the strain list), 4 to 7 plates of worms from each isolate were scored for the Susm phenotype at 20°C, and the Susm data from the 2 isolates were combined and presented in the table. a The lack of M-derived CCs phenotype is not fully penetrant in sma-9(cc604) mutants [25]. b Data from [12]. Statistical analysis was conducted by comparing various double mutants with the sma-9(cc604) single mutant. **** P t test). (C) Relative body sizes of various strains at the same developmental stage (WT set to 1.0). Body sizes of 35 to 70 worms of each genotype were measured. A dbl-1 null allele (ok3749) and smoc-1 null allele (tm7125) were included as controls. Groups marked with distinct symbols are significantly different from each other (P P-value between tm7125 and jj271 is 0.0099), while groups with the same symbol are not. The exception is tested using an ANOVA with a Tukey HSD. Original data sets are in S1 Data. (PDF)</p

Oligonucleotides used in this study.

Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium (EC)-binding domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. In this study, we used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. We showed that CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. We provide both in vitro biochemical and in vivo functional evidence demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. We further showed that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. Our work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling.</div

SMOC-1, but not LON-2, binds to DBL-1 when expressed in S2 cells.

(A) Diagrams of LON-2 and DBL-1 expression constructs used in the Drosophila S2 cell expression system. (B) Results of co-IP experiments testing the interaction between LON-2::Myc and V5::DBL-1 prodomain::FLAG::DBL-1 mature domain. IP with anti-Myc beads, anti-V5 beads or anti-FLAG beads and IB with anti-Myc, anti-V5, or anti-FLAG antibodies, as indicated. Experiments were independently repeated in triplicate, with representative results shown in this figure, and C) Diagrams of SMOC-1 and DBL-1 expression constructs used in the Drosophila S2 cell expression system. (D) Results of co-IP experiments testing the interaction between HA::DBL-1 prodomain::FLAG::DBL-1 mature domain and different versions of SMOC-1::V5*. IP with anti-V5 beads or anti-FLAG beads and IB with anti-V5 or anti-FLAG antibodies, as indicated. The source of DBL-1 in these experiments was cell media, which does not contain full-length DBL-1, but only HA-tagged prodomain and FLAG-tagged mature domain. Experiments were independently repeated in triplicate, with representative results shown in this figure. Original images of western blots can be found in S1 Raw Images. co-IP, coimmunoprecipitation; EC, extracellular calcium; IB, immunoblot; IP, immunoprecipitation; SMOC, secreted modular calcium-binding protein; TY, thyroglobulin type-1.</p

The in vitro and in vivo consequences of mutations in LON-2 that affect SMOC-1 binding.

(A) Diagrams of SMOC-1, WT, and mutant LON-2 expression constructs used in the Drosophila S2 cell expression system. (B) Results of co-IP experiments testing the interaction between SMOC-1::V5 and Myc tagged WT and mutant LON-2 proteins. IP with anti-V5 beads or anti-Myc beads and IB with anti-V5 or anti-Myc antibodies, as indicated. Experiments were independently repeated in triplicate, with representative results shown in this figure. (C) DIC images of WT, lon-2(jj678) null, and lon-2(jj507) worms, showing their body sizes. Scale bar represents 50 μm. (D) Relative body sizes of worms at the same developmental stage (WT set to 1.0). ***P N = 21. e678: N = 31. jj507: N = 26. jj508: N = 27. Original data sets are in S1 Data. Original images of western blots can be found in S1 Raw Images. co-IP, coimmunoprecipitation; EC, extracellular calcium; IB, immunoblot; IP, immunoprecipitation; SMOC, secreted modular calcium-binding protein; TY, thyroglobulin type-1; WT, wild type.</p

Results of IP-MS experiment 2 showing proteins that are both enriched in the tagged (SMOC-1::2xFLAG(OE)) vs.

untagged (SMOC-1(OE)) samples, as well as in the (SMOC-1(EC)::2xFLAG(OE)) vs. untagged (SMOC-1(OE)) samples. (XLSX)</p

LON-2 does not interact with full-length DBL-1.

(A) Diagrams of LON-2 and DBL-1 expression constructs used in the Drosophila S2 cell expression system. (B) Results of co-IP experiments testing the interaction between LON-2::Myc and V5::DBL-1 prodomain::FLAG::DBL-1 mature domain. Immunoprecipitation (IP) with anti-Myc beads or anti-V5 beads and immunoblot (IB) with anti-Myc or anti-FLAG antibodies, as indicated. Full-length DBL-1 detected by anti-FLAG antibody runs at around 55KD. Experiments were independently repeated in triplicate, with representative results shown in this figure. Original images of western blots can be found in S1 Raw Images. (PDF)</p

Co-IP results testing the model for tripartite complex formation.

(A) Predicted structure of a complex formed between LON-2, SMOC-1, and a homodimer of the DBL-1 mature domain. The membrane-anchoring region of LON-2 lies at the bottom of the panel. (B) The same structure prediction as in A, but shown from the “top.” (C) Diagrams of LON-2, SMOC-1, and DBL-1 expression constructs used in the Drosophila S2 cell expression system. (D) Results of co-IP experiments testing the interaction between LON-2::Myc and HA::DBL-1 prodomain::FLAG::DBL-1 mature domain in the presence or absence of SMOC-1::V5. IP with anti-FLAG beads, or anti-Myc beads, and IB with anti-Myc, anti-V5 or anti-FLAG antibodies, as indicated. Note that immunoprecipitation of FLAG::DBL-1mature domain with anti-FLAG beads can pull down LON-2::Myc only in the presence of SMOC-1::V5. No FLAG::DBL-1mature protein was detected when IP was performed using anti-Myc antibodies, very likely due to the low amount of FLAG::DBL-1mature protein in the co-IP experiments, as FLAG::DBL-1mature protein was not detectable in the Input lanes where approximately 1% of materials used for the co-IP experiments were loaded. Experiments were independently repeated in triplicate, with representative results shown in this figure. Original images of western blots can be found in S1 Raw Images. co-IP, coimmunoprecipitation; EC, extracellular calcium; IB, immunoblot; IP, immunoprecipitation; SMOC, secreted modular calcium-binding protein; TY, thyroglobulin type-1.</p

Multiple sequence alignment of LON-2 homologs.

Clustal Omega (CLUSTAL O(1.2.4)) [71] of C. elegans (Ce) LON-2 with its homologs from other nematode species, including C. Japonica (Cjp), C. brenneri (Cbn), C. briggsae (Cbr), and C. remanei (Cre), as well as with Drosophila Dally and Glypican from M. musculus (mouse) and H. sapiens (human). Red $ marks the residues at the interface between LON-2::SMOC-1, as identified via ColabFold [34] and #### marks the glycosaminoglycan attachment site. Residues highlighted in yellow are those mutated to generate LON-2(mut) that cannot bind SMOC-1. (PDF)</p