Bone morphogenetic protein-2 and -6 heterodimer illustrates the nature of ligand-receptor assembly.
ABSTRACT TGF-beta superfamily ligands are homo- or heterodimeric and recruit two type I and two type II Ser/Thr kinase receptors to initiate a transmembrane signaling cascade. Even with the known structure of the homodimer ligands in complex with extracellular domains of both receptor types, the sequential assembly of the signaling complex with its cognate receptors in the cell membrane remains elusive. We generated a bone morphogenetic protein-2/-6 heterodimer carrying two asymmetric interfaces for each receptor type. We demonstrate that the heterodimer possesses high affinity to both receptor types and increased Smad1-dependent signaling activity by both cell-based and chondrogenesis assays. Furthermore, we find that the minimal signaling complex consists of two type II receptors and one type I receptor per dimer. Our study reveals how the engineered heterodimers may use their independent binding interfaces to differentially recruit the different receptors for each receptor type to create new biological properties.
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ABSTRACT: The development of chemical refolding of transforming growth factor-beta (TGF-beta) superfamily ligands has been instrumental to produce the recombinant proteins for biochemical studies and exploring the potential of protein therapeutics. The osteogenic human bone morphogenetic protein-2 (hBMP-2) and its Drosophila DPP homolog were the early successful cases of refolding into functional form. Despite the similarity in their three dimensional structure and amino acid sequences, several other TGF-beta superfamily ligands could not be refolded readily by the same methods. Here, we report a comprehensive study on the variables of a rapid-dilution refolding method, including the concentrations of protein, salt, detergent and redox agents, pH, refolding duration and the presence of aggregation suppressors and host-cell contaminants, in order to identify the optimal condition to refold human BMP-9 (hBMP-9). To produce a recombinant form of hBMP-9 in E. coli cells, a synthetic codon-optimized gene was designed to encode the mature domain of hBMP-9 (Ser320 - Arg429) directly behind the first methionine, which we herein referred to as MB109. An effective purification scheme was also developed to purify the refolded MB109 to homogeneity with a final yield of 7.8 mg from 100 mg of chromatography-purified inclusion bodies as a starting material. The chemically refolded MB109 binds to ALK1, ActRIIb and BMPRII receptors with relatively high affinity as compared to other Type I and Type II receptors based on surface plasmon resonance analysis. Smad1-dependent luciferase assay in C2C12 cells shows that the MB109 has an EC50 of 0.61 ng/mL (25 pM), which is nearly the same as hBMP-9. MB109 is prone to be refolded as non-functional dimer and higher order multimers in most of the conditions tested, but bioactive MB109 dimer can be refolded with high efficiency in a narrow window, which is strongly dependent on the pH, refolding duration, the presence of aggregation suppressors and the concentrations of protein, salt and detegent. These results add to the current understanding of producing recombinant TGF-beta superfamily ligands in the microbial E. coli system. An application of the technique to produce a large number of synthetic TGF-beta chimeras for activity screen is also discussed.Microbial Cell Factories 02/2014; 13(1):29. DOI:10.1186/1475-2859-13-29 · 4.25 Impact Factor
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ABSTRACT: BMP- and Wnt-signalling play crucial roles in cartilage homeostasis. Our objective was to investigate whether activation of the BMP-pathway or stimulation of Wnt-signalling cascades effectively enhances cartilage-specific extracellular matrix (ECM) accumulation and functional biomechanical parameters of chondrocyte-seeded TE-constructs. Articular chondrocytes were cultured in collagen-type-I/III-matrices over 6 weeks to create a biomechanical standard curve. Effects of stimulation with 100 ng/mL BMP-4/-7 heterodimer or 10 mM lithium chloride (LiCl) on ECM-deposition was quantified and characterized histologically. Biomechanical parameters were determined by the Very Low Rubber Hardness (VLRH) method and under confined compression stress relaxation. BMP-4/-7 treatment resulted in stronger collagen type-II staining and significantly enhanced glycosaminoglycan deposition (3.2-fold; *p < 0.01) correlating with improved hardness (∼1.7-fold; *p = 0.001) reaching 83 % of native cartilage values after 28 days, a value not reached before nine weeks without stimulation. LiCl treatment enhanced VLRH slightly, but significantly (∼1.3-fold; *p = 0.016) with a trend to more ECM-deposition. BMP-4/-7 treatment significantly enhanced the E Modulus (105.7 ± 34.1 kPa; *p = 0.000001) compared to controls (8.0 ± 4.2 kPa). Poisson's ratio was significantly improved by BMP-4/-7 treatment (0.0703 ± 0.0409; *p = 0.013) versus controls (0.0432 ± 0.0284) and a significantly lower permeability (5.8 ± 2.1056 x 10(-14) mˆ4/N.s; *p = 0.00001) was detected compared to untreated scaffolds (4.4 ± 3.1289 x 10(-13) mˆ4/N.s). While Wnt-activation is less effective, BMP-4/-7 heterodimer stimulation approximated native cartilage features in less than 50 % of standard culture time representing a promising strategy for functional cartilage TE to improve biomechanical parameters of engineered cartilage.Osteoarthritis and Cartilage 11/2013; DOI:10.1016/j.joca.2013.11.011 · 4.66 Impact Factor
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ABSTRACT: BMP9, a member of the TGFβ superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower-affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations, and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.Journal of Biological Chemistry 09/2014; DOI:10.1074/jbc.M114.579771 · 4.60 Impact Factor