GDF5^W414R shows impaired Bmpr1a signaling in a SBE-Luciferase reporter gene assay.

<p>NIH/3T3 cells were transfected with the BMP type I receptors, <i>Bmpr1a</i> or <i>Bmpr1b,</i> as well as with wild type <i>GDF5</i> and the GDF5 variants <i>GDF5^W414R</i>, <i>GDF5^R399C</i> and <i>GDF5^E491K</i>. As reporter, the SMAD binding element (SBE) was used and firely luciferase was normalized against TK-Renilla luciferase. <b>A</b>: No Bmp type I receptor was co-expressed which resulted in a weak SBE reporter activation for wild type GDF5 and GDF5^E491K, whereas in case of GDF5^W414R and GDF5^R399C signaling activity was absent. <b>B</b>: <i>Bmpr1a</i> co-expression increased the signaling activity of wild type GDF5 and GDF5^E491K; however, GDF5^W414R and GDF5^R399C were not able to induce reporter gene expression. <b>C</b>: Co-expression of <i>Bmpr1b</i> further increased the signaling activity of wild type GDF5 and GDF5^E491K compared to co-expression with <i>Bmpr1a</i>. In case of GDF5^W414R and GDF5^R399C, <i>Bmpr1b</i> co-expression rescued their signaling activity. The means of triplicate measurements are shown, error bars indicate standard deviation and a represent experiment is shown. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; **p≤0.01). Significances are related to the respective wild type GDF5 value.</p

GDF5^W414R is positioned within the NOG and BMPR1A/B binding interface of the GDF5 dimer.

<p><b>A</b>: 3D presentation of the human GDF5 homodimer (PDB 1waq). The topology of the GDF5 monomer comprises two ß-sheets forming the fingers as well as the four-turn α-helix with the preceding pre-helix loop. The mutations are highlighted in pink (GDF5^W414R), violet (GDF5^R399C) and orange (GDF5^E491K). The image of the GDF5 structure was visualized using PyMol (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). <b>B</b>: Protein sequence alignment of human, mouse and chicken GDF5 comprising the seven cysteine residues (bold) of the mature domain. Numbering is referred to the pro-protein sequence. Amino acids predicted to form the NOG binding interface are depicted as framed white boxes and based on the BMP7:NOG complex (PDB 1m4u). Residues predicted to be involved in BMPR1A binding are shown as grey boxes and refer to the BMP2:BMPR1A structure (PDB 1rew). Black boxes mark amino acids that bind to BMPR1B (PDB 3evs). Arrows indicate the mutated sites for GDF5^W414R, GDF5^R399C and GDF5^E491K. Note that GDF5^W414R and GDF5^E491K are located within the NOG binding site. Moreover, all three mutations interfere with the BMP type I receptor (BMPR1A and BMPR1B) binding interface.</p

Binding affinities of GDF5^W414R to immobilized receptor ectodomains.

<p>GDF5^W414R shows altered NOG and BMP receptor type I binding affinities in the Biacore assay.</p><p>Mean values from two experiments using at least six different analyte concentrations are shown.</p

Clinical features of the affected family members with mutations in <i>GDF5</i>.

<p>The features are coded using terms from the Human Phenotype Ontology <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003846#pgen.1003846-Robinson1" target="_blank">[47]</a>. + present; − absent. <i>GDF5</i> mutations are presented with either features of brachydactyly (GDF5 p.R399C) or features of synostosis (GDF5 p.E491K) or a combination of multiple synostosis with additional brachydactyly (GDF5 p.W414R).</p

<i>Gdf5</i>, <i>Nog</i> and <i>Bmpr1b</i> are co-expressed during murine limb development.

<p>Mouse embryo<i>s</i> with the C57BL/6 genetic background at embryonic stages 11.5 (A–H), 12.5 (A′–H′) and 13.5 (A″–H″) were labeled with probes of <i>Gdf5</i> (A and E), <i>Nog</i> (B and F), <i>Bmpr1a</i> (C and G) or <i>Bmpr1b</i> (D and H) and signals are shown in red. Representatively, two sections of the coronal dorsal axis (A–D) and the autopod transversal axis (E–H) are depicted. The signal for <i>Gdf5</i> strongly co-localizes with the <i>Nog</i> and <i>Bmpr1b</i> expression pattern, whereas <i>Bmpr1a</i> expression is in direct proximity in the surrounding epithelium and underlying mesenchyme.</p

GDF5^W414R is resistant towards inhibition by NOG in chicken micromass cultures.

<p>Chicken micromass cells were infected with RCASBP(A) containing the coding sequence (cds) of either wild type <i>GDF5</i> or the GDF5 variants <i>GDF5^W414R</i>, <i>GDF5^R399C</i> or <i>GDF5^E491K</i>. RCASBP(B) contained the cds of <i>NOG</i> and was used for co-transfection. Chicken micromass cultures and quantification of Alcian blue incorporation at 595 nm into the extracellular matrix (ECM) are shown for day 5. In the chicken micromass system, wild type GDF5 strongly induced chondrogenesis compared to the untransfected control. Chondrogenic differentiation was completely blocked in both, the control and wild type GDF5 cultures, when <i>NOG</i> is co-transfected. A similar pattern was observed for GDF5^R399C. Contrary, GDF5^W414R and GDF5^E491K exhibited insensitivity towards the antagonist. Values represent the mean of triplicates and error bars indicate standard deviation. Statistical analysis was performed using a two-tailed Student's t test (n.s.: not significant; *p≤0.05; ***p≤0.001).</p

NOG insensitivity of rhN445T GDF5 in vitro.

<p>(A) ALP activity induced in C2C12 myoblastic mouse cells overexpressing the Bmpr1b receptor (C2C12-Bmpr1b) after three days of stimulation. All BMPs show dose-dependent induction of ALP activity. (B) Inhibition of rhBMP-induced ALP activity in the C2C12-Bmpr1b cell line by rhNOG. ALP activity was measured after co-stimulation of 5 nM rhGDF5, rhN445T GDF5, rhBMP2, and 0.05 nM rhBMP9 with up to 40 nM rhNOG (rhBMP9 up to 0.4 nM). RhBMP2 is strongly inhibited by rhNOG. In contrast to rhGDF5 the mutant rhN445T GDF5 shows no response to rhNOG inhibition.</p

Bmp9 and Bmp10 are not inhibited by Nog.

<p>Chicken limb bud micromass cultures were retrovirally infected to express the indicated proteins and stained with Alcian blue after five days of cultivation. (A) Schematic overview of the mature domain of mouse Bmp9. Conserved cysteines marked in black. (B) 3D surface model of a BMP9 dimer (light grey and dark grey; PDB∶1ZKZ) linked via a disulfide bridge. Mut2 (K371N) represents the homologous mutation site to GDF5 N445T and is indicated in red. Mut1 (K348L, yellow) and mut3 (YH415Q, blue) are additional sites, which were predicted to be important for the interaction of GDF5 with NOG. (C) Expression of wtBmp9 and wtBmp10 in chicken micromass strongly induce chondrogenesis. This effect cannot be inhibited by co-expression of Nog. The Bmp9 mutants mut1 (K348L), mut2 (K371N), mut3 (YH415Q), mut1+2, and mut2+3 are similar to wtBmp9. In contrast, the combination of K371N and YH415Q (mut2+3) results in partial inhibition by Nog. The combination of all three mutations (mut1+2+3) leads to a complete inhibition by Nog. (D) Quantification of Alcian blue staining after extraction and photometric measurement at OD₅₉₅ (n = 4).</p

Gdf5 mutants N445T and N445K as well as Bmp9 cause strong chondrogenic differentiation when overexpressed in chicken limbs.

<p>Retroviral overexpression of indicated proteins after injection into chicken hind limbs at stage HH10. The contralateral hind limb was used as control. Embryos were harvested at day 7.5 and skeletal preparation stained with Alcian blue and Alizarin red. WtGdf5 lead to fusion of joints in the phalanges and metatarsals. In contrast, N445T and N455K Gdf5 as well as Bmp9 overexpression in vivo caused a severe phenotype with massive cartilage production leading to complete fusion of all skeletal elements of the limb and absence of interdigital mesenchyme. Scale: 1 mm.</p

Biacore interaction analysis (n = 6) showed no binding differences for rhGDF5 and rhN445T GDF5 with BMPR1Aecd and BMPR1Becd.

<p>Biacore interaction analysis (n = 6) showed no binding differences for rhGDF5 and rhN445T GDF5 with BMPR1Aecd and BMPR1Becd.</p