Biophysical Comparison of BMP-2, ProBMP-2, and the Free Pro-peptide Reveals Stabilization of the Pro-peptide by the Mature Growth Factor

Abstract

Pro-forms of growth factors have received intensive scientific attention recently because in some cases different biological activities have been ascribed compared with the mature growth factors. Examples are the pro-apoptotic role of the nerve growth factor (NGF) proform (proNGF) or the latency of the transforming growth factor (TGF)-␤ pro-form (proTGF-␤). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-␤ family, mature BMP-2, proBMP-2, and the isolated pro-peptide were recombinantly produced in Escherichia coli cells, and a biophysical comparison was performed. Protocols were developed that allowed efficient refolding and subsequent purification of the proteins. ProBMP-2 could be processed to an N-terminally truncated form of BMP-2, digit removed BMP-2 (drBMP-2), that possessed biological activity, i.e. it induced ectopic bone formation. Bone inducing activity was also displayed by proBMP-2. The three proteins were characterized both by fluorescence and CD spectroscopy. From these analyses, predominant ␤-sheet secondary structural elements in the pro-peptide were deduced. The thermodynamic stability of the pro-peptide was determined by chemical unfolding assays. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. However, in contrast to NGF/proNGF, the pro-peptide did not stimulate oxidative folding of the mature part in vitro.

The combined mechanism of bone morphogenetic protein- and calcium phosphate-induced skeletal tissue formation by human periosteum derived cells

“…Interestingly, clustering was found between total bone, percentage of: bone marrow, trabeculae, cortex and total bone to elevated phosphorylation levels J Bolander et al BMP-and CaP-induced -release to cause a decrease in pH, providing a more soluble environment for the BMP (Hillger et al, 2005;Kamp et al, 1998). Alternatively, the release of Ca 2+ may mediate the subsequent release and biological availability of BMPs bound to the CaP-granules through hydrogen/ionic/electrostatic/hydrophobic interactions (Wang et al, 2013).…”

“…For 3D analysis of in vivo formed bone, explants were analysed using the Phoenix NanoTom S nanoCT system, (GE Measurement and Control Solutions, Wunstorf, DE) with a 180 kV/15 W high-performance nanofocus X-ray tube as previously described (Kerckhofs et al, 2013). Briefly, the system was equipped with a tungsten target and was operated at a voltage of 60 kV and a current of 200 µA.…”

Biophysical Characterization of Refolded Drosophila Spätzle, a Cystine Knot Protein, Reveals Distinct Properties of Three Isoforms

“…These interactions are supplemented by a network of charged/polar interactions. The side chain carboxylate of Glu 45 is in contact with Arg 14 and Lys B100 and His B71 of the second monomer; Arg 14 in turn contacts the side chain of Gln 43 and the main chain carbonyl oxygen of Tyr…”

The pro‐peptide of proNGF: Structure formation and intramolecular association with NGF

“…It has also been reported that propeptides guide the assembly of subunits into active protein complexes as in the case of the von Willebrand factor or caspase-3 (Wise et al 1988;Feeney and Clark 2005). The pro-peptides of TGF-b and very likely also bone morphogenetic proteins retard the biological activities of the mature growth factors (Böttinger et al 1996;Degnin et al 2004;Hillger et al 2005). While for some pro-peptides their biological activities are displayed even when they are not covalently linked to their mature parts (Gray and Mason 1990), in the case of NGF, the propeptide has to be covalently linked to the mature part to promote oxidative structure formation (Rattenholl et al 2001a).…”