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: 6.27). 10/2003; 130(17):4047-56. DOI: 10.1242/dev.00633
Source: PubMed

ABSTRACT 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, Aug 08, 2015
  • 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 · 3.50 Impact Factor
  • Source
    • "Noggin was selected for these studies rather than TWSG1 to simplify interpretation. While BMP-agonist activity has been reported for TWSG1 in some circumstances [Oelgeschlager et al., 2003; Ross et al., 2001], only BMP-antagonist activity has been described for noggin. RANKL-stimulated bone marrow derived cultures were treated with noggin for defined intervals, and stained for TRAP on day 5. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF-kappaB ligand (RANKL) to enhance in vitro differentiation of osteoclast-like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow-derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p-SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p-SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL-stimulated formation of multinuclear TRAP-positive cells. The BMP antagonist noggin inhibited RANKL-mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts-like cells.
    Journal of Cellular Biochemistry 11/2009; 109(4):672-82. DOI:10.1002/jcb.22462 · 3.37 Impact Factor
  • Source
    • "Given the significance of BMP levels, it is important to understand how BMP signaling is regulated during BA1 morphogenesis. Twisted gastrulation (Twsg1 in mice and humans or tsg in Drosophila) gene product is a secreted protein, which has been shown to either antagonize or promote BMP activity depending on the species, developmental timing, the organ being studied, and also its interactions with other proteins such as CHRD (Larrain et al., 2001; Nosaka et al., 2003; Oelgeschlager et al., 2003; Petryk et al., 2005; Ross et al., 2001; Wills et al., 2006). TWSG1 binds to several BMPs, including BMP2, BMP4, and BMP7. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The mandibular arch (BA1) is critical for craniofacial development. The distal region of BA1, which gives rise to most of the mandible, is dependent upon an optimal level of bone morphogenetic protein (BMP) signaling. BMP activity is modulated in the extracellular space by BMP-binding proteins such as Twisted gastrulation (TWSG1). Twsg1(-/-) mice have a spectrum of craniofacial phenotypes, including mandibular defects that range from micrognathia to agnathia. At E9.5, the distal region of the mutant BA1 was prematurely and variably fused with loss of distal markers eHand and Msx1. Expression of proximal markers Fgf8 and Barx1 was expanded across the fused BA1. The expression of Bmp4 and Msx2 was preserved in the distal region, but shifted ventrally. While wild type embryos showed a gradient of BMP signaling with higher activity in the distal region of BA1, this gradient was disrupted and shifted ventrally in the mutants. Thus, loss of TWSG1 results in disruption of the BMP4 gradient at the level of signaling activity as well as mRNA expression. Altered distribution of BMP signaling leads to a shift in gene expression and increase in apoptosis. The extent of apoptosis may account for the variable degree of mandibular defects in Twsg1 mutants.
    Developmental Biology 02/2009; 328(1):13-23. DOI:10.1016/j.ydbio.2008.12.041 · 3.64 Impact Factor
Show more