The pro-BMP activity of Twisted gastrulation is independent of BMP binding

Howard Hughes Medical Institute and Department of Biological Chemistry, University of California-Los Angeles, Los Angeles, CA 90095-1662, USA.
Development (Impact Factor: 6.46). 10/2003; 130(17):4047-56. DOI: 10.1242/dev.00633
Source: PubMed


The determination of the vertebrate dorsoventral body axis is regulated in the extracellular space by a system of interacting secreted molecules consisting of BMP, Chordin, Tolloid and Twisted Gastrulation (Tsg). Tsg is a BMP-binding protein that forms ternary complexes with BMP and Chordin. We investigated the function of Tsg in embryonic patterning by generating point mutations in its two conserved cysteine-rich domains. Surprisingly, Tsg proteins with mutations in the N-terminal domain were unable to bind BMP, yet ventralized the embryo very effectively, indicating strong pro-BMP activity. This hyperventralizing Tsg activity required an intact C-terminal domain and could block the anti-BMP activity of isolated BMP-binding modules of Chordin (CRs) in embryonic assays. This activity was specific for CR-containing proteins as it did not affect the dorsalizing effects of Noggin or dominant-negative BMP receptor. The ventralizing effects of the xTsg mutants were stronger than the effect of Chordin loss-of-function in Xenopus or zebrafish. The results suggest that xTsg interacts with additional CR-containing proteins that regulate dorsoventral development in embryos.

Download full-text


Available from: Shawn C Little, Oct 07, 2015
7 Reads
  • Source
    • "Although there is some evidence that TWSG1 can promote BMP activity in some species, in mice it appears to act mostly as a BMP antagonist (Larraín et al., 2001; Nosaka et al., 2003; Oelgeschläger et al., 2003; Petryk et al., 2005; Ross et al., 2001; Sotillo Rodriguez et al., 2009; Wills et al., 2006). We examined the expression of several BMP pathway genes and BMP targets after BMP2 treatment alone, after ATRA treatment alone and after combined treatment (Fig. 4). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Holoprosencephaly (HPE) is a developmental anomaly characterized by inadequate or absent midline division of the embryonic forebrain and midline facial defects. It is believed that gene-environment interactions play a role in the widely variable penetrance and expressivity of HPE, although a direct investigation of such effects has been limited. The goal of this study was to examine if mice carrying a mutation in a gene encoding a BMP antagonist Twisted gastrulation (Twsg1) associated with a low penetrance of HPE are sensitized to retinoic acid (RA) teratogenesis. Pregnant Twsg1(+/-) dams were treated by gavage with a low dose of all-trans RA (3.75 mg/kg). Embryos were analyzed between E9.5 and E11.5 by microscopy and geometric morphometric analysis by microCT. P19 embryonal carcinoma cells were used to examine potential mechanisms mediating combined effects of increased BMP and retinoid signaling. While only 7% of wild type embryos exposed to RA showed overt HPE or neural tube defects (NTD), 100% of Twsg1 null mutants exposed to RA manifested severe HPE compared to 17% without RA. Remarkably, up to 30% of Twsg1+/- mutants also showed HPE (23%) or NTD (7%). The majority of shape variation among Twsg1+/- mutants was associated with narrowing of the midface. In P19 cells, RA induced the expression of Bmp2, acted in concert with BMP to increase p53 expression, caspase activation, and oxidative stress. This study provides direct evidence for modifying effects of the environment in a genetic mouse model carrying a predisposing mutation for HPE in the Twsg1 gene. Further study of the mechanisms underlying these gene-environment interactions in vivo will contribute to better understanding of the pathogenesis of birth defects and present an opportunity to explore potential preventive interventions.
    Disease Models and Mechanisms 12/2014; 8(2). DOI:10.1242/dmm.018275 · 4.97 Impact Factor
  • Source
    • "Based on data for xTsg in which glycosylation at Asn52 has been shown to occur (Oelgeschlager et al., 2003), we hypothesized that if the consensus serine at position 53 were restored in rodent TWSG1, Asn51 of mTWSG1 would also be a glycosylation site. As predicted, when Pro53 was mutated in the mouse sequence to serine, a decrease in electrophoretic mobility was observed, and a triplet of bands appeared on SDS-PAGE analysis at 27, 30, and 33 kD (Figure 2F). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to glycosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.
    Frontiers in Physiology 09/2011; 2:59. DOI:10.3389/fphys.2011.00059 · 3.53 Impact Factor
  • Source
    • "To examine Chordin proteolysis more directly, we also performed anti-Myc immunoblotting of extracts from zebrafish embryos injected with mRNA encoding Myc-tagged Chordin together with cv2, tolloid or tsgW67G mRNA. The latter encodes a mutant version of Xenopus Tsg that enhances BMP signaling by promoting Tolloid-dependent Chordin degradation [18]. As shown in Figure 4P, compared to embryos injected with chordin mRNA only, co-injection of cv2 mRNA led to a very moderate reduction in the levels of full-length Chordin and Chordin fragments, whereas much more strongly reduced Chordin levels were obtained after co-injection of chordin with tolloid or tsgW67G mRNA. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Crossveinless-2 (CV2) is an extracellular BMP modulator protein of the Chordin family, which can either enhance or inhibit BMP activity. CV2 binds to BMP2 via subdomain 1 of the first of its five N-terminal von Willebrand factor type C domains (VWC1). Previous studies showed that this BMP binding is required for the anti-, but not for the pro-BMP effect of CV2. More recently, it was shown that CV2 can also bind to the BMP inhibitor Chordin. However, it remained unclear which domains mediate this binding, and whether it accounts for an anti- or pro-BMP effect. Here we report that a composite interface of CV2 consisting of subdomain 2 of VWC1 and of VWC2-4, which are dispensable for BMP binding, binds to the VWC2 domain of Chordin. Functional data obtained in zebrafish embryos indicate that this binding of Chordin is required for CV2's pro-BMP effect, which actually is an anti-Chordin effect and, at least to a large extent, independent of Tolloid-mediated Chordin degradation. We further demonstrate that CV2 mutant versions that per se are incapable of BMP binding can attenuate the Chordin/BMP interaction. We have physically dissected the anti- and pro-BMP effects of CV2. Its anti-BMP effect is obtained by binding to BMP via subdomain1 of the VWC1 domain, a binding that occurs in competition with Chordin. In contrast, its pro-BMP effect is achieved by direct binding to Chordin via subdomain 2 of VWC1 and VWC2-4. This binding seems to induce conformational changes within the Chordin protein that weaken Chordin's affinity to BMP. We propose that in ternary Chordin-CV2-BMP complexes, both BMP and Chordin are directly associated with CV2, whereas Chordin is pushed away from BMP, ensuring that BMPs can be more easily delivered to their receptors.
    PLoS ONE 09/2010; 5(9):e12846. DOI:10.1371/journal.pone.0012846 · 3.23 Impact Factor
Show more