Biochemical and Biophysical Characterization of RefoldedDrosophila DPP, a Homolog of Bone Morphogenetic Proteins 2 and 4

Abstract

The mature C-terminal signaling domain of the Drosophila Decapentaplegic proprotein (DPP) can be efficiently refolded from chaotrope-solubilized inclusion bodies with the aid of a membrane protein-solubilizing detergent, high concentrations (0.75-2 M) of NaCl, and low temperatures (5-15°C). The disulfide-linked homodimeric product contains N-terminal heparin-binding sites that were utilized as intrinsic affinity tags to obtain a highly enriched preparation in one chromatographic step. A subsequent C4 reverse phase high pressure liquid chromatography step provides high purity, salt-free protein that is amenable to biophysical and structural studies at a yield of approximately 3 mg/liter of bacterial culture. The dimeric protein is correctly folded as determined by electrophoretic, spectroscopic, chemical, and proteolytic analyses. Refolded DPP is also bioactive as shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell culture, the refolded Escherichia coliexpressed protein is not glycosylated at a conserved N-linked site and is therefore homogeneous. The C-terminal domain dimer is more hydrophobic and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding in vitro. Hence solubilization of the mature ligand may be one of the principal roles of the large (250 -400 amino acids) N-terminal prodomains of transforming growth factor-␤ superfamily members, shown to act as intramolecular chaperones in vivo.The decapentaplegic locus (dpp) encodes a signaling ligand homologous to human bone morphogenetic proteins BMP-2 1 and -4 (1, 2) and has been the object of intense study due to its importance in many fundamental processes during Drosophila development. The mature DPP ligand is secreted as a disulfidelinked homodimer of the C-terminal domain of a large proprotein precursor and has been shown to act as a morphogen required for the establishment of the dorsoventral axis of the embryo (3) and to impart positional information over long range to specify cell fate along the anterior/posterior axis of the limbs (4, 5). DPP signaling also occurs at short range between germ layers of the developing gut (6 -8) and is required to direct the dorsal and ventral migration of tracheal cells during embryogenesis (9) and to act as a relay signal triggering epithelial cell shape changes during the process of dorsal closure (10). In contrast to the developmental roles of DPP signaling, which have been the focus of extensive genetic studies, little is known about the folding, modification, and processing of the DPP proprotein, its distribution and state extracellularly, or the biochemical and biophysical properties of the mature homodimer and specific aspects of its structure.Characterization of the protein products of the dpp locus has been hindered by...

Functions of heparan sulfate proteoglycans in cell signaling during development

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The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo

“…The prodomains of BMP7 and BMP4 play distinct roles in modulating the activity of their respective mature homodimers. The BMP7 prodomain fragment assists in solubilizing BMP7 homodimers by shielding hydrophobic residues present in the mature domain (18), and the prodomain of DPP has been suggested to play a similar role (24). The BMP7 prodomain also competes with type II receptors for binding to the BMP7 ligand, but does not confer latency because it can be easily displaced from the ligand by receptor binding (25).…”

HtrA1 serine protease inhibits signaling mediated by Tgfβ family proteins

“…All GST fusion proteins were expressed in insoluble fractions. They were solubilized in 8 M urea containing buffer and then renatured, as previously described (Groppe et al, 1998). The renatured proteins were dialyzed against the GST pull-down buffer [20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 0.05% NP-40, 10% glycerol].…”