Abstract
Signaling by bone morphogenetic proteins (BMPs) plays a central role in early embryonic patterning, organogenesis, and homeostasis in a broad range of species. Chordin, an extracellular antagonist of BMP signaling, is thought to readily diffuse in tissues, thus forming gradients of BMP inhibition that result in reciprocal gradients of BMP signaling. The latter determine cell fates along the embryonic dorsoventral axis. The secreted protein Twisted Gastrulation (TSG) is thought to help shape BMP signaling gradients by acting as a cofactor that enhances Chordin inhibition of BMP signaling. Here, we demonstrate that mammalian Chordin binds heparin with an affinity similar to that of factors known to functionally interact with heparan sulfate proteoglycans (HSPGs) in tissues. We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cell-surface HSPGs and that Chordin bound to cell-surface HSPGs (e.g. syndecans), but not to basement membranes containing the HSPG perlecan. Surprisingly, mammalian TSG did not bind heparin unless prebound to Chordin and/or BMP-4, although Drosophila TSG has been reported to bind heparin on its own. Results are also presented that indicate that Chordin-HSPG interactions strongly potentiate the antagonism of BMP signaling by Chordin and are necessary for the retention and uptake of Chordin by cells. These data and others regarding Chordin diffusion have implications for the paradigm of how Chordin is thought to regulate BMP signaling in the extracellular space and how gradients of BMP signaling are formed.
Highlights
In Drosophila, the gene products Tolloid (TLD) and Twisted Gastrulation (TSG) help shape the gradient of DPP signaling that forms the dorsoventral axis through interactions with Short Gastrulation (SOG) and DPP
We further demonstrate that Chordin binding in mouse embryonic tissues was dependent upon its interaction with cellsurface Heparan sulfate proteoglycans (HSPGs) and that Chordin bound to cell-surface HSPGs, but not to basement membranes containing the HSPG perlecan
Chordin and bone morphogenetic proteins (BMPs)-4, but Not Mouse TSG, Bind Heparin In Vitro—To gain insight into the potential of Chordin to bind heparan sulfate (HS) in vivo, purified recombinant murine Chordin was incubated with heparin-Sepharose, and the mixture was placed in a column and eluted with a step gradient of 0.15–2.0 M NaCl
Summary
Production of Recombinant Proteins—FLAG-tagged mouse Chordin, FLAG-tagged Chordin fragments, and protein C-tagged mouse TSG were expressed and purified as described previously [19, 25]. Three 5-min washes with PBS were followed by incubation with anti-Chordin antibody (described above) at 1:200, with rat anti-syndecan-1 monoclonal antibody 281.2 at a concentration of 1 g/ml, or with rabbit anti-syndecan-4 polyclonal antibody (see below) at 1:300 for 1 h. Transfected M2-10B4 cells were fixed as described above, but were not incubated for 1 h at room temperature prior to addition of anti-Chordin antibody. Characterization of the distribution of total HS in tissues was performed with monoclonal antibody (mAb) 3G10 (Seikagaku America, Inc.), which detects unsaturated glucuronate residues remaining on core proteins following heparinase III treatment [46]. Sections were incubated with mAb 3G10 at 1:200 in RPMI 1640 medium and 10% fetal calf serum, followed by Cy3-conjugated donkey anti-mouse secondary antibody (Molecular Probes, Inc.) at 1:300. AP activity was measured by absorbance readings taken at 405 nm using a Universal Microplate Reader (Bio-Tek Instruments, Winooski, VT)
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