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• W2022170395 abstract "SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1Δcub/Δcub) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like repeats 7–9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1Δcub/Δcub embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function. SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1Δcub/Δcub) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like repeats 7–9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1Δcub/Δcub embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function. SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is the founding member of an evolutionarily conserved SCUBE gene family (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 2Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (69) Google Scholar). To date, three distinct isoforms have been cloned and named SCUBE1 to SCUBE3 according to their order of discovery in mammals (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 2Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (69) Google Scholar, 3Grimmond S. Larder R. Van Hateren N. Siggers P. Morse S. Hacker T. Arkell R. Greenfield A. Mech. Dev. 2001; 102: 209-211Crossref PubMed Scopus (42) Google Scholar, 4Wu B.T. Su Y.H. Tsai M.T. Wasserman S.M. Topper J.N. Yang R.B. J. Biol. Chem. 2004; 279: 37485-37490Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These genes coding for polypeptide molecules of about 1000 amino acids share an organized protein domain structure of at least 5 recognizable motifs: an N-terminal signal peptide sequence, 9 tandem repeats of epidermal growth factor (EGF) 3The abbreviations used are: EGF, epidermal growth factor; BMP, bone morphogenetic protein; HA, hemagglutinin; GST, glutathione S-transferase; proBMP, prepro precursor of BMP; E, embryonic day; NTD, neural tube closure defect. -like modules, a large N-glycosylated spacer region followed by three repeated stretches of 6-cysteine residues with unique and regular spacing, and one CUB domain at the C terminus (see Fig. 1A). Our previous study demonstrated that the signal peptide sequence is sufficient to direct the expressed SCUBE1 protein into the secretory pathway and results in a secreted and surface-associated protein (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). However, little is known about the functional significance of the other four domains in SCUBE1. SCUBE genes have been shown to be expressed predominantly in a variety of developing tissues, including gonads, the central nervous system, dermomyotome, digital mesenchyme, and limb buds during mouse embryogenesis (2Grimmond S. Larder R. Van Hateren N. Siggers P. Hulsebos T.J. Arkell R. Greenfield A. Genomics. 2000; 70: 74-81Crossref PubMed Scopus (69) Google Scholar, 3Grimmond S. Larder R. Van Hateren N. Siggers P. Morse S. Hacker T. Arkell R. Greenfield A. Mech. Dev. 2001; 102: 209-211Crossref PubMed Scopus (42) Google Scholar, 5Haworth K. Smith F. Zoupa M. Seppala M. Sharpe P.T. Cobourne M.T. Gene. Expr. Patterns. 2007; 7: 630-634Crossref PubMed Scopus (19) Google Scholar), which implies that the proteins may play important roles in development. However, direct functional studies of the roles of the SCUBE genes during mammalian development are still lacking. In addition to its embryonic expression, SCUBE1 was found to be expressed in the endothelium and platelets (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 6Tu C.F. Su Y.H. Huang Y.N. Tsai M.H. Li L.T. Chen Y.L. Cheng C.J. Dai D.F. Yang R.B. Cardiovasc. Res. 2006; 71: 486-495Crossref PubMed Scopus (77) Google Scholar). Our recent study showed that SCUBE1 is stored in the platelet α-granules and exposed to the cell surface upon platelet stimulation and activation (6Tu C.F. Su Y.H. Huang Y.N. Tsai M.H. Li L.T. Chen Y.L. Cheng C.J. Dai D.F. Yang R.B. Cardiovasc. Res. 2006; 71: 486-495Crossref PubMed Scopus (77) Google Scholar). Yet, the molecular function for such targeted exposure of SCUBE1 on the cell surface has not been addressed. Here, we investigated the domain and biological functions of SCUBE1 by utilizing a series of SCUBE1 domain-specific deletion constructs and employing a new genetic mouse model with targeted disruption of the Scube1 gene. Our results demonstrated, for the first time, that the N-terminal EGF-like repeats participate in the formation of adhesions in a Ca2+-dependent fashion and the EGF-like repeats 7–9 are sufficient for reciprocal and lateral interactions between SCUBE1 in homophilic adhesions. Moreover, gene-targeting study indicated that SCUBE1 is required during the early stages of central nervous system development, possibly through modulating the activity of members of the bone morphogenetic protein (BMP) family of proteins via its C-terminal 6-cysteine repeat motifs and the CUB domain. Cell Culture, Construction of Expression Plasmids, and Transfection—A2058 melanoma cells, human embryonic kidney (HEK)-293T and GP2–293 retrovirus packaging cells were maintained in Dulbecco's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in an atmosphere of 5% CO2. The epitope-tagged version of the SCUBE1 mutant was constructed as described (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Cells were transfected by using Lipofectamine 2000 (Invitrogen). Immunoprecipitation and Western Blot Analysis—Two days after transfection, cell lysates were clarified by centrifugation at 10,000 × g for 20 min at 4 °C. Samples underwent immunoprecipitation, followed by Western blot analysis as described (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Cell Aggregation Assay—A2058 stable cells were detached by trypsin (0.01%)-EDTA (2.5 mm) treatment and suspended in Dulbecco's minimal essential medium, either containing 5 mm CaCl2 or Ca2+ free, at 1 × 106 cells/ml in polystyrene tubes. Then, tubes were incubated on a rotating platform (10 rpm) at 37 °C for 1 h. The extent of cell aggregation was viewed microscopically and photographed. For quantitation, cell clusters of more than four cells were counted as being aggregated. The data are shown as the means ± S.E. of three independent experiments in duplicate. Luciferase Activity Assays—Human HepG2 cells (3 × 105 cells per well) were seeded into 24-well plates and transfected on the following day with 0.4 μg of the BMP-inducible luciferase reporter I-BRE-Luc and 0.01 μg of the Renilla luciferase reporter vector used as an internal control. The transfected responding cells (HepG2) were stimulated with conditioned media derived from the signaling cells (HEK-293T) transfected with the BMP2 expression plasmid alone or in a combination of various SCUBE1 deletion constructs. Luciferase activity was measured following 24-h incubation by the use of the dual reporter system (Promega, Madison, WI). Data are expressed as relative luciferase activity (firefly luciferase activity divided by Renilla luciferase activity). Targeted Disruption of the Scube1 Gene in Mice—The targeting vector obtained from the MICER website was digested with ClaI (see Fig. 8A). The ES cell line R1, established from a hybrid of two 129 substrain (129X1/SvJ and 129S1) F1 male blastocysts (7Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar), was transfected with linearized targeting vector, and selection involved use of puromycin. Of a total of 251 ES cell clones, we obtained 13 correctly targeted ES clones verified by Southern blot and/or PCR analysis. The correctly targeted ES cell clones were injected into blastocysts of C57BL/6 mice. The targeted allele was backcrossed with C57BL/6 females for at least three generations before phenotyping experiments. Histological and Immunohistochemical Analysis—Embryos were dissected from pregnant mice at the indicated time, fixed, and embedded in paraffin. Sections were cut at 5 μm and stained with hematoxylin and eosin. For SCUBE1 immunohistology, sections were probed with a monoclonal antibody specifically against the CUB domain of SCUBE1 (6Tu C.F. Su Y.H. Huang Y.N. Tsai M.H. Li L.T. Chen Y.L. Cheng C.J. Dai D.F. Yang R.B. Cardiovasc. Res. 2006; 71: 486-495Crossref PubMed Scopus (77) Google Scholar). Proliferation and Apoptosis Assays—Sections were stained with rabbit anti-mouse Ki-67 antibody (DakoCytomation) for active cell proliferation and counterstained with hematoxylin to detect all nuclei. Detection of apoptotic cells involved the terminal dUTP nick-end labeling reaction kit (Roche Applied Science) and counterstaining by 4′,6-diamidino-2-phenylindole. Statistical Analysis—Data are expressed as mean ± S.E. Differences between groups were analyzed by unpaired Student's t test. p < 0.05 was considered significant. Molecular Dissection of the SCUBE1 Domain Responsible for Secretion and Cell Surface Association—We previously showed that overexpression of SCUBE1 in HEK-293T cells resulted in a secreted glycoprotein tethered on the cell surface (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). However, the structure responsible for the surface targeting and membrane association was not precisely defined. To delineate the domain required for the secretion and cell surface anchorage, we then generated a comprehensive panel of mutated SCUBE1 expression constructs (D1–D7) with specific deletions of the EGF-like repeats, spacer region, cysteine-rich repeats, or CUB domain (Fig. 1A). The FLAG epitope tag was added at the N terminus for easy detection of these mutated SCUBE1 forms. The SCUBE1 deletion proteins were expressed by means of transient expression in HEK-293T cells. Two days after transfection, the conditioned media and cell lysates were collected and subjected to Western blot analysis with anti-FLAG antibody. As shown in Fig. 1B, all SCUBE1 deletion constructs produced corresponding recombinant proteins in HEK-293T cells (bottom panel). Furthermore, immunoblotting of the conditioned media from transfected cells showed an effective secretion of all expression products, except for the SCUBE1-D2 mutant protein, which lacks the spacer region (Fig. 1B, top panel). Adding back this region restored the secretion of the D2 mutant into the conditioned culture medium (data not shown). Together, these data suggest that the spacer region, located between the EGF-like domains and the cysteine-rich repeats, is capable of directing the expressed SCUBE1 protein through the secretory pathway for secretion, at least when overexpressed in HEK-293T cells. The same set of transfected cells was detached and stained with anti-FLAG antibody to determine their surface expression by flow cytometry (Fig. 1C). Flow cytometric analysis showed SCUBE1-D2, -D4, -D5, and -D6 deletion mutants defective in membrane association, but SCUBE1-FL, -D1, -D3, and -D7 mutant protein, which all contain the spacer region, was expressed on the cell surface. Together, our results support the concept that the spacer region has at least two functions: to target SCUBE1 for secretion and anchor the molecule on the cell surface. Establishment of Cell Lines Expressing a Series of SCUBE1 EGF-like Repeat Deletion Mutants—Our previous study showed that the preformed SCUBE1 protein stored in the platelet α-granules could be translocated to the surface upon platelet activation (6Tu C.F. Su Y.H. Huang Y.N. Tsai M.H. Li L.T. Chen Y.L. Cheng C.J. Dai D.F. Yang R.B. Cardiovasc. Res. 2006; 71: 486-495Crossref PubMed Scopus (77) Google Scholar). However, the functional significance of such targeted exposure of SCUBE1 on the cell surface remained elusive. Because certain EGF-like repeats mediate homophilic or heterophilic protein-protein interactions (8Ishii J. Adachi H. Aoki J. Koizumi H. Tomita S. Suzuki T. Tsujimoto M. Inoue K. Arai H. J. Biol. Chem. 2002; 277: 39696-39702Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 9Balzar M. Briaire-de Bruijn I.H. Rees-Bakker H.A. Prins F.A. Helfrich W. de Leij L. Riethmuller G. Alberti S. Warnaar S.O. Fleuren G.J. Litvinov S.V. Mol. Cell Biol. 2001; 21: 2570-2580Crossref PubMed Scopus (143) Google Scholar), we next examined by suspension cell aggregation assay whether the N-terminal EGF-like repeats of SCUBE1 play a role on cell-cell interaction. To further evaluate the relative contribution of the EGF-like repeats in the adhesion function of SCUBE1, we first constructed four deletion mutants consisting of the EGF-like modules 1–9 (E1–9), 4–9 (E4–9), 7–9 (E7–9), or only E7–9 deletion (ΔE7–9) into a retrovirus vector, pLNCX-eB7 (10Su Y.C. Chuang K.H. Wang Y.M. Cheng C.M. Lin S.R. Wang J.Y. Hwang J.J. Chen B.M. Chen K.C. Roffler S. Cheng T.L. Gene Ther. 2007; 14: 565-574Crossref PubMed Scopus (26) Google Scholar). In addition, an HA epitope tag was added at the N terminus for easy detection, and the mutant constructs were further fused to the B7 extracellular and transmembrane domain (B7TM) to effectively target the chimeric protein on the plasma membrane (Fig. 2A). The melanoma A2058 cells lacking endogenous SCUBE1 expression were infected with recombinant retroviral particles and selected in G418 to obtain four independent A2058/SCUBE1-E1–9, -E4–9, -E7–9, or -ΔE7–9 stable transfectant cell lines. Western blot analysis with the anti-HA antibody demonstrated the SCUBE1 EGF-like repeat deletion mutant proteins to be produced at comparable levels with the expected molecular masses (Fig. 2B). Most importantly, flow cytometry using the anti-HA or ant-SCUBE1 antibody confirmed that all of these SCUBE1 EGF-like repeat mutants expressed by A2058 cells were transported to the cell surface (Fig. 2C). The EGF-like Repeats 7–9 Are Sufficient, but Not Necessary, for Reciprocal (trans) or Lateral (cis) Interactions between SCUBE1 Molecules—To examine the involvement of SCUBE1 EGF-like repeats in intercellular adhesion, we evaluated the aggregating properties of the SCUBE1 EGF-like repeat mutant stable transfectant cell lines by cell aggregation assay. Monolayer cultures of the parental (control) or transfected stable cell lines were dissociated into single cells and allowed to aggregate in suspension culture. When the suspensions were gently shaken, A2058 cells expressing SCUBE1 EGF-like repeat mutants tended to aggregate (Fig. 3). The parental A2058 cells aggregated poorly in suspension (<3%), but a good proportion (∼8–20%) of the SCUBE1 EGF-like repeat mutant-transfected clones aggregated after 1 h of shaking (Fig. 3). The chelating of calcium by Ca2+-binding EGF-like domains is important for the structural integrity and function of several other EGF-like domain-containing proteins (11Downing A.K. Knott V. Werner J.M. Cardy C.M. Campbell I.D. Handford P.A. Cell. 1996; 85: 597-605Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar, 12Hambleton S. Valeyev N.V. Muranyi A. Knott V. Werner J.M. McMichael A.J. Handford P.A. Downing A.K. Structure. 2004; 12: 2173-2183Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Because six of nine EGF-like modules (i.e. repeats 1–3 and 7–9) contain Ca2+-binding consensus sequences (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 6Tu C.F. Su Y.H. Huang Y.N. Tsai M.H. Li L.T. Chen Y.L. Cheng C.J. Dai D.F. Yang R.B. Cardiovasc. Res. 2006; 71: 486-495Crossref PubMed Scopus (77) Google Scholar), we hypothesized that these EGF-like repeats may function in a Ca2+-dependent manner. When the same cell lines were dissociated with gentle pipetting in the presence of 5 mm EDTA or allowed to aggregate in the absence of Ca2+, no apparent aggregation was observed during 1 h of incubation for either the parental or EGF-like repeat deletion transfectants (Fig. 3A). To further address the specificity of the EGF-like repeats, the recombinant GST fusion protein fragment containing the EGF-like repeats (GST-E4–9) or spacer region (GST-Spacer) was added into the cell aggregation assay to block the cell-cell interactions. Soluble EGF-like repeat protein GST-E4–9 clearly blocked A2058-SCUBE1-E7–9-mediated cell aggregation assay, whereas GST alone or recombinant spacer region protein (GST-Spacer) could not block SCUBE1-induced cell aggregation (Fig. 4). We previously showed that human SCUBE1 proteins are capable of forming oligomeric complexes when overexpressed in HEK-293T cells (1Yang R.B. Ng C.K. Wasserman S.M. Colman S.D. Shenoy S. Mehraban F. Komuves L.G. Tomlinson J.E. Topper J.N. J. Biol. Chem. 2002; 277: 46364-46373Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). To further determine the relative contribution of which EGF-like repeat modules involved in lateral (cis) intracellular oligomerization, we transiently co-expressed three deletion constructs, HA-tagged SCUBE1-E1–9, -E4–9, -E7–9, or -ΔE7–9 with FLAG. SCUBE1-D2 containing only the EGF-like repeats 1–9 in HEK-293T cells. Immunoprecipitation of HA.SCUBE1-E1–9, -E4–9, -E7–9, or -ΔE7–9 resulted in the co-precipitation of FLAG.SCUBE1-D2 (Fig. 5). Therefore, the EGF-like repeats 7–9 are also sufficient, but not necessary, for homophilic lateral SCUBE1 interactions within the same cells. Molecular Analysis of the C-terminal Six-cysteine Repeat Motif and CUB Domain—Recent genetic analysis identified that the zebrafish orthologue of the mammalian SCUBE2 gene acts upstream of hedgehog ligands or through a parallel pathway (13Hollway G.E. Maule J. Gautier P. Evans T.M. Keenan D.G. Lohs C. Fischer D. Wicking C. Currie P.D. Dev. Biol. 2006; 294: 104-118Crossref PubMed Scopus (81) Google Scholar, 14Kawakami A. Nojima Y. Toyoda A. Takahoko M. Satoh M. Tanaka H. Wada H. Masai I. Terasaki H. Sakaki Y. Takeda H. Okamoto H. Curr. Biol. 2005; 15: 480-488Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Woods I.G. Talbot W.S. PLoS Biol. 2005; 3: e66Crossref PubMed Scopus (87) Google Scholar). In addition, a nonsense mutation in the ty97 allele encodes a truncated null protein lacking the cysteine-rich repeat motif and the CUB domain, implying that this C-terminal region is essential for SCUBE2 function (13Hollway G.E. Maule J. Gautier P. Evans T.M. Keenan D.G. Lohs C. Fischer D. Wicking C. Currie P.D. Dev. Biol. 2006; 294: 104-118Crossref PubMed Scopus (81) Google Scholar, 14Kawakami A. Nojima Y. Toyoda A. Takahoko M. Satoh M. Tanaka H. Wada H. Masai I. Terasaki H. Sakaki Y. Takeda H. Okamoto H. Curr. Biol. 2005; 15: 480-488Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Woods I.G. Talbot W.S. PLoS Biol. 2005; 3: e66Crossref PubMed Scopus (87) Google Scholar). From this genetic evidence, the SCUBE2 protein is postulated to play a critical role for transport or stability of hedgehog signaling in the extracellular environment (15Woods I.G. Talbot W.S. PLoS Biol. 2005; 3: e66Crossref PubMed Scopus (87) Google Scholar), function as an endocytic receptor during reception of the hedgehog signal (13Hollway G.E. Maule J. Gautier P. Evans T.M. Keenan D.G. Lohs C. Fischer D. Wicking C. Currie P.D. Dev. Biol. 2006; 294: 104-118Crossref PubMed Scopus (81) Google Scholar), or modulate the long range action of BMP-dependent signaling in the neural tube and somites (14Kawakami A. Nojima Y. Toyoda A. Takahoko M. Satoh M. Tanaka H. Wada H. Masai I. Terasaki H. Sakaki Y. Takeda H. Okamoto H. Curr. Biol. 2005; 15: 480-488Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), but the exact biochemical and molecular mechanism underlying the SCUBE2 action remains unclear. To further explore the molecular function of the C-terminal region of SCUBE1, we generated one additional deletion mutant, SCUBE1-ty97 (Q623 to stop codon), mimicking the ty97 mutant allele in the zebrafish Scube2 gene by removing the 6-cysteine repeat motif and the CUB domain (Fig. 6A). Since one previous report demonstrated that BMP activity can be attenuated by the co-expression of SCUBE2 in the zebrafish (14Kawakami A. Nojima Y. Toyoda A. Takahoko M. Satoh M. Tanaka H. Wada H. Masai I. Terasaki H. Sakaki Y. Takeda H. Okamoto H. Curr. Biol. 2005; 15: 480-488Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), we next investigated whether the SCUBE1 C-terminal domain could directly interact with BMP2 protein and form a complex. HEK-293T cells were transfected with a Myc-tagged BMP2 expression plasmid alone or in combination with a series of FLAG-tagged SCUBE1 domain deletion constructs (Fig. 6A). Two days after transfection, cell lysates underwent immunoprecipitation with the anti-Myc monoclonal antibody, and the precipitates were analyzed by immunoblotting with anti-FLAG monoclonal antibody to determine the protein association. Immunoprecipitation with anti-Myc antibody resulted in a specific coimmunoprecipitation of the SCUBE1-D4 or -D5 deletion protein (Fig. 6B), which suggests that SCUBE1 could form a complex with BMP2 through its C-terminal CUB domain. To further evaluate whether the interaction between SCUBE1-D4/-D5 and BMP2 affects the long range signaling of BMP2, we co-cultured the conditioned media derived from HEK-293T cells co-transfected with BMP2 alone or in combination with SCUBE1 deletion constructs (signaling cells) added to the responding cells, HepG2 cells containing the BMP-inducible promoter luciferase reporter construct I-BRE-Luc (16Benchabane H. Wrana J.L. Mol. Cell Biol. 2003; 23: 6646-6661Crossref PubMed Scopus (76) Google Scholar) (Fig. 7A). As expected, BMP2 alone produced by the signaling cells could act as a long range signaling molecule by inducing an increase of ∼10-fold in luciferase activity (Fig. 7B). Although the BMP2 proteins co-expressed with the SCUBE1-FL, -ty97, -D5, or -D6 triggered the BMP-mediated transcriptional activation equally well, co-expression with the SCUBE1-D4 mutant resulted in a marked attenuation of the BMP response (Fig. 7B). Because the proteolytic processing of the large prepro precursor of BMP (proBMP) and its subsequent secretion into the extracellular environment are the required steps in the formation of the biologically active BMP ligand, we next investigated whether the inhibition of BMP2 signaling by SCUBE1-D4 occurs in the intracellular or extracellular space. HEK-293T cells were transfected with plasmids expressing proBMP2 alone or in combination with various SCUBE1 deletion constructs. Western blot analysis of the cell lysates and condition medium from these cultures revealed all deletion mutants with no effect on total proBMP2 synthesis (Fig. 7C), whereas only the SCUBE1-D4 mutant potently inhibited the secretion of mature BMP2 into the culture medium. Targeted Disruption of the Scube1 Gene in Mice Produces a Neural Tube Closure Defect over the Midbrain—To further evaluate the biological function of the C-terminal domain of SCUBE1 in vivo, we disrupted the mouse Scube1 gene at the end of exon 16 by use of an insertional targeting vector obtained from a public resource (17Adams D.J. Biggs P.J. Cox T. Davies R. van der Weyden L. Jonkers J. Smith J. Plumb B. Taylor R. Nishijima I. Yu Y. Rogers J. Bradley A. Nat. Genet. 2004; 36: 867-871Crossref PubMed Scopus (122) Google Scholar). This vector was expected to result in the duplication of exons 11–16 and disruption of splicing to produce a mutant transcript coding for a truncated protein lacking the functional 6-cysteine repeat motifs (exons 16–19) and the CUB domain (exons 20 and 21) (Fig. 8A). The correctly targeted embryonic stem cell clones and chimeric mice carrying germ line transmission of the mutant allele of Scube1 were confirmed by Southern blot (Fig. 8B) and genomic PCR analyses with one primer pair designed from the vector backbone and the other designed from outside the targeting construct (Fig. 8A). In addition, the expression of the mutant transcript in hetero- or homozygous mutant mice was confirmed by reverse transcriptase PCR analysis and direct sequencing of the PCR product (data not shown). Most importantly, the Scube1Δcub/Δcub mutant mice lacked the expression of the functional C-terminal region, because SCUBE1 immunoreactivity was not seen in the mutant brain sections stained with a monoclonal antibody specific against the CUB domain (6Tu C.F. Su Y.H. Huang Y.N. Tsai M.H. Li L.T. Chen Y.L. Cheng C.J. Dai D.F. Yang R.B. Cardiovasc. Res. 2006; 71: 486-495Crossref PubMed Scopus (77) Google Scholar) (Fig. 8C). However, immunohistochemistry stained with the N terminus-specific monoclonal antibody could still detected the immunoreactivity in the mutant neural tissues, including forebrain (f), hindbrain (h), and cranial ganglia (asterisks), suggesting the targeted allele is not a true null allele (see supplemental Fig. S1). Instead, a truncated form of SCUBE1 containing the N-terminal fragment is expressed in the mutant animal, as the protein fragment can be detected using an antibody specific for the N terminus. Thus, this allele is designated as Scube1Δcub/Δcub on the basis of the lack of the CUB immunoreactivity (Fig. 8C). Mice heterozygous for the Scube1 mutation have a wild-type phenotype and normal fertility. However, a fraction of the Scube1Δcub/Δcub newborn pups died shortly after birth and manifested a phenotypic defect showing acrania and lack of brain tissue (Fig. 9A). In addition, mutant embryos recovered from E12.5 to E17.5 were alive (determined by active heart beating and/or limb movement) with exposed and progressively degenerating brain tissue (Fig. 10B and data not shown).FIGURE 10Exencephaly in Scube1-mutant mice as a result of an early neural tube closure defect. A and B, lateral view of E12.5 wild-type (A) and Scube1Δcub/Δcub (B) embryos. The mutant shows an overproliferation of neural tissue and the everted cranial neural folds (between arrows) in the midbrain region. C and D, transversal sections stained with hematoxylin and eosin through the midbrain at E12.5, depicting the everted neuroepithelium (asterisks) and neural tube closure defects in the Scube1Δcub/Δcub animal. D, compared with wild-type control (C). m, midbrain. E and F, transverse forebrain sections of the Ki-67-stained wild-type (E) and Scube1Δcub/Δcub (F) E12.5 embryos. Brown nuclei are Ki-67-positive cells and indicate active cell" @default.
• W2022170395 created "2016-06-24" @default.
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• W2022170395 date "2008-05-01" @default.
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