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

Frank Hillger,Gerhard Herr,Rainer Rudolph,Elisabeth Schwarz

doi:10.1074/jbc.m414413200

Frank Hillger, Gerhard Herr + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m414413200

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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)-beta pro-form (proTGF-beta). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-beta 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 beta-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.

Highlights

The cystine knot is a structural signature that describes disulfide connectivities in which two pairs of disulfide bridges form a ring through which a third disulfide bridge is threaded
The Pro-peptide Plays No Significant Role during in Vitro Oxidative Folding—Because our previous results had clearly indicated that oxidative folding of nerve growth factor (NGF) is stimulated in vitro by the pro-peptide [10, 11], we investigated whether this is the case for bone morphogenetic protein (BMP)-2/proBMP-2
BMP-2 belongs to the family of cystine knot proteins, a family that has received attention from the protein folding community and from the pharmaceutical industry

Summary

Introduction

The cystine knot is a structural signature that describes disulfide connectivities in which two pairs of disulfide bridges form a ring through which a third disulfide bridge is threaded. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. In the present study, refolding and purification protocols for proBMP-2, BMP-2, and the pro-peptide have been established to allow a biophysical comparison of the three proteins.

Results

Conclusion