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Originally published In Press as doi:10.1074/jbc.M414413200 on February 4, 2005

J. Biol. Chem., Vol. 280, Issue 15, 14974-14980, April 15, 2005
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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{ddagger}§, Gerhard Herr¶, Rainer Rudolph{ddagger}, and Elisabeth Schwarz{ddagger}||

From the {ddagger}Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany and Advanced Tissue Regeneration GmbH, Turmstrasse 16, 35578 Wetzlar, Germany

Received for publication, December 22, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 motif has been discovered in both inhibitory peptides and growth factors (1, 2). The majority of bone morphogenetic proteins (BMPs)1 belong to the cystine knot structure family. With an antiparallel head to head arrangement of two monomers in the functional dimer, they belong to the TGF-{beta} subgroup. BMP-2 is one of the most intensively characterized BMP representatives, finding clinical application by its ability to induce bone formation (3). Biological activity is mediated by binding to cell membrane receptors that homo- and/or hetero-oligomerize upon ligand binding or to pre-formed dimeric receptor complexes (4–6). Crystal structures of BMP-2 alone (7) and in complex with either the extracellular domain of the BMP-2 receptor IA or the activin receptor II extracellular domain have been elucidated in atomic detail by x-ray crystallography (8, 9).

As with all other currently known cystine knot proteins, BMP-2 is synthesized in vivo as a pre-pro-protein. The pre-sequence mediates translocation into the lumen of the endoplasmic reticulum and thus secretion. The function of the 263-residue pro-peptide is hitherto unknown.

We and others could demonstrate for NGF/proNGF that the pro-peptide assists maturation in vivo and oxidative folding in vitro, probably assisting the latter process by facilitating formation of the correct disulfide connectivities (10–12). Recently, pro-forms of growth factors have received intensive scientific attention because it has been shown in some cases that the pro-peptides have functions in addition to those of maturation and/or folding of the mature growth factors. The presence of the pro-peptide can modulate or even change the biological activity of the respective cytokine. Several lines of evidence indicate that the pro-form of NGF induces pro-apoptotic signals (13, 14), in contrast to NGF, which acts as a mitogen (11). It has recently been shown that the pro-peptide targets NGF to a newly described receptor protein, sortilin, that specifically binds the pro-form (15). In the case of TGF-{beta}, the pro-peptide has been demonstrated to delay the function of the mature growth factor and thus has been termed latency-associated peptide {beta}-1-LAP (16–18). The pro-peptide of TFG-{beta} has a similar size to that of BMP-2; the homology of the two pro-peptides with 25% identical amino acids is moderate, however. A stabilizing role has been found in cell culture for a closely related pro-peptide, that of BMP-4 (19). Sequential cleavage at two distinct sites within the pro-domain by furin or furin-like proteases is required for the activation of the BMP-4 precursor (20, 21). Furthermore, proBMP-2 and proBMP-6 have been detected in arthritic synovial tissue (22). Whereas these results suggest a function of the pro-peptides in modulation of BMP activities, no specific function of the pro-peptide of BMP-2 could be assigned as yet. 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. Our results document that, similarly to NGF and proNGF, the pro-peptide part is stabilized by the mature part. Because oxidative folding in vitro is not stimulated by the pro-peptide, an in vivo role beyond structure formation can be anticipated for the pro-peptide moiety.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Biology—DNA encoding pre-proBMP-2 was provided by Roche Diagnostics. Cloning of coding sequences for proBMP-2, BMP-2, and the pro-peptide was achieved by PCR. The start of the coding sequence for the pro-peptide was determined by the program PSORT (psort.nibb.ac.jp.), which predicted cleavage of the signal sequence between Gly–263 and Gly–264. Gene expression with DNA for the pro-peptide and proBMP-2 was only successful in vector pET15b (Novagen), which codes for an N-terminal histidine tag. Expression of the BMP-2 cDNA was performed in pET11a (Novagen). Escherichia coli BL21(DE3) was used as bacterial host strain. Before transformation with plasmids encoding BMP-2 proteins, the cells were transformed with plasmid pUBS520 (23). The vector carries the gene for dnaY encoding the tRNAAGA/AGG for L-arginine codons. These codons are extremely rare in E. coli but are frequent in eukaryotic genes and thus often limit heterologous expression. Additional copies of dnaY overcome the lack of that otherwise rare tRNA. For recombinant production, cultivation in either shake flasks with 2YT medium or a Biostat ED fermenter was performed. For fermentation, the following medium was used: 54 g/liter yeast extract (OHLY KAV; DHW, Cologne, Germany), 0.54 g/liter NH4Cl, and 12 g/liter glycerol. The pH was adjusted with 1 M K2HPO4 to 7.0–7.4. Addition of 2.8 mM MgSO4, 0.01% thiamine, 0.1% ampicillin, and 0.05% kanamycin occurred by sterile filters. Feeding was started at A600 = ~20. At A600 = 60, induction of gene expression was started by addition of 1 mM isopropyl-1-thio-{beta}-D-galactopyranoside. After an additional 3 h of cultivation, cells were harvested by centrifugaion. Pellets were shock-frozen and stored at –80 °C.

Protein Refolding and Purification—Inclusion body (IB) isolation and solubilization were performed according to Rudolph et al. (24). Renaturation of proBMP-2 and BMP-2 was carried out by 1:100 dilutions of IB protein solubilized in 5 M guanidinium chloride into refolding buffer (0.1 M Tris/Cl, pH 8.0, 1 M L-arginine, 5 mM EDTA, 5 mM oxidized glutathione, and 2 mM reduced glutathione). Prior to use, the buffer was degassed and pre-chilled to 10 °C. Protein concentration during renaturation was 3 µM. After renaturation (3–14 days), both proBMP-2 and BMP-2 were concentrated using a Filtron Minisette (PallGelman) cross-flow device. pH was adjusted to 5.5, and the material was dialyzed against 0.1 M Tris, 75 mM acetic acid, 0.2 M KH2PO4, 5 mM EDTA, and 6 M urea and then filtered. Subsequently, ~150 mg of protein was loaded onto a 5-ml HiTrapTM heparin-Sepharose HP (Amersham Biosciences) column that had been pre-equilibrated with 0.1 M Tris, 125 mM acetic acid, 5 mM EDTA, and 6 M urea (buffer A) containing 0.3 M NaCl. Loading was performed at a flow rate of 4 ml/min. The column was washed with 20 column volumes of buffer A containing 0.6 M NaCl. Elution of dimeric proBMP-2 was finally achieved by application of a linear gradient from 0.6 to 0.8 M NaCl in 5 column volumes.

Purification of renatured BMP-2 was identical to that of proBMP-2 with the following exceptions: after dialysis against buffer A, the column was loaded with a maximum of 30 mg of dialyzed protein. Dimeric BMP-2 species was eluted at a NaCl concentration of 0.7 M. Homogenous fractions were pooled, dialyzed against 10 mM NH4-acetate, pH 4.0, and lyophilized.

ProBMP-2 was further purified by hydroxylapatite chromatography (see below). Lyophilized protein was dissolved at a concentration of 2 mg/ml in 10 mM K-acetate, pH 4.5. The solution was then mixed with an equal volume of 0.1 M K-HEPES, pH 7.0, and incubated on ice overnight. Subsequently, the solution was loaded at a flow rate of 7.5 ml/min onto a self-packed 30-ml Macro-Prep CHT-I column (Bio-Rad) that had been equilibrated with 0.1 M K-HEPES, pH 7.0 (buffer M). For elution, 0.1 M K-HEPES, pH 7.0, and 1 M K-phosphate (buffer N) was used. Washing was performed with 4 column volumes of 10% and 20% buffer N. Elution was started by a gradient from 20% to 40% buffer N. ProBMP-2 eluted at 40% buffer N. Homogeneous fractions were pooled, concentrated, and dialyzed against 0.1 M K-HEPES, pH 7.0.

For renaturation and purification of the pro-peptide, IB protein was solubilized at a concentration of 2 mg/ml in buffer A containing 0.1 M NaCl. An equilibrated 5-ml HiTrapTM heparin-Sepharose HP column was loaded with ~40 mg of pro-peptide at a flow rate of 3 ml/min. Due to the high DNA content of the pro-peptide IB preparation, the column was washed extensively with buffer A containing 0.2 M NaCl until a baseline of the UV absorption was reached. Elution was performed by a linear gradient from 0.2 to 0.7 M NaCl in buffer A (20 column volumes). Pro-peptide-containing fractions were pooled and, for refolding by dialysis, diluted 1:2 with 2 M L-arginine/HCl, 2 M Tris/HCl, pH 8.0. A first dialysis was performed against 1 M Tris/HCl, pH 8.0, and a second dialysis was performed against 25 mM K-HEPES, pH 7.0. Renatured protein was then further purified by hydroxylapatite chromatography. Here, the same buffer system used for proBMP-2 was applied (buffers M and N). The column was loaded with 75 mg of refolded protein (0.5 mg/ml). Washing was performed at a flow rate of 5 ml/min with 1.5 column volumes of buffer M and then 3 column volumes of 7% buffer N. The pro-peptide eluted at 25% buffer N that was reached after 4 column volumes. Homogenous fractions were pooled, concentrated with polyethylene glycol 35000, and then dialyzed against 0.1 M K-HEPES, pH 7.0.

Fluorescence and CD Spectroscopy—Spectroscopic analyses were carried out at 20 °C. Dialysis buffer was used as a reference. Fluorescence spectroscopy was performed with a FluoroMax-3 spectrophotometer. Slit widths for excitation and emission were set to 2 and 5 nm, respectively. Excitation was at 295 nm. Measurements were recorded in 0.5-nm intervals in quartz cuvettes. CD spectroscopy was performed with either an AVIV 62A DS or a Jasco J710. Recordings were done in 1-nm steps, and 10 recordings were averaged. Ellipticities were calculated according to Schmid (25).

Preparation of Digit Removed BMP-2 (drBMP-2)—drBMP-2 was obtained via limited proteolysis from proBMP-2. Refolded protein was dialyzed in two steps against 0.1 M Tris, 0.125 M acetic acid, 5 mM EDTA, and 6 M urea and 1 M Tris/HCl, pH 7.5, 4 M urea, and 1 mM EDTA. For proteolysis, dialyzed samples were incubated at a concentration of 2 mg/ml with 1% (w/w) trypsin for 4 h at 4 °C. Proteolysis was stopped by the addition of a 10-fold molar excess of soybean trypsin inhibitor. drBMP-2 was purified by hydrophobic interaction chromatography. Fractogel EMD Phenyl S (Merck) was added directly to trypsin-cleaved protein at a ratio of 1:4 (v/v) followed by dialysis against 0.1 M Tris/HCl, pH 7.0. The loaded gel material was transferred to an empty column and thoroughly washed with (a) dialysis buffer, (b) 50 mM sodium acetate, pH 5.0, (c) 50 mM sodium acetate, pH 5, and 4 M urea, and (d) the same buffer in which urea was replaced by 1 M L-arginine. Elution was performed with 6 M guanidinium chloride and 0.2 M acetic acid. drBMP-2-containing fractions were pooled, dialyzed against 0.1% trifluoroacetic acid, and then sterile-filtered.

Denaturant-induced Unfolding—Proteins were incubated at the indicated concentrations of urea or guanidinium chloride for 24 h to ensure equilibrium conditions. The exact concentrations of the denaturants were controlled by refractrometry. Thermodynamic stabilities and m values were determined according to Refs. 26 and 27.

Biological Activity—Biological activity was tested with eight adult male Wistar rats (body weight, ~400 g; Charles River, Germany). The study was performed according to the current legal regulations of animal care and protection and the guidelines of the local authorities. Implant preparations were performed under laminar flow hoods. drBMP-2 and proBMP-2 were dissolved in 50% acetonitrile and 0.1% trifluoroacetic acid at protein concentrations of 0.125 and 0.65 mg/ml, respectively. Solutions were sterile-filtered and applied to ceramic {beta}-tri-calcium cubes (5 x 5 x 5 mm; ChronOs, Mathys, Switzerland). In total, 0.25 µmole (calculated for the dimeric species) of each protein was adsorbed. Cubes were implanted into abdominal wall pouches of anesthetized rats. Each rat received a total of four implants. 30 days after implantation, animals were sacrificed by drug overdose, and implants were excised. One half of the implants was examined histochemically, whereas the other half was deep-frozen for subsequent determination of alkaline phosphatase activity. For histochemical analysis, the implants were dehydrated and embedded in methylmethacrylate resins. 100–150-µm sections were cut with a diamond band saw and ground with a grinding machine to ~50 µm. Surface staining was done with Toluidine O. Determination of alkaline phosphatase was performed after homogenization of the samples with a Potter S (B. Braun Biotech) in 0.1 M Tris/HCl, pH 7.4, and 1% Triton X-100. Tissue debris were removed by centrifugation. The supernatant was tested for activity of alkaline phosphatase by the Ecoline test kit (Merck).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Pro-peptide Plays No Significant Role during in Vitro Oxidative Folding—Because our previous results had clearly indicated that oxidative folding of NGF is stimulated in vitro by the pro-peptide (10, 11), we investigated whether this is also the case for BMP-2/proBMP-2. The cDNAs for BMP-2, proBMP-2, and the BMP-2 pro-peptide were cloned into pET vectors to allow efficient expression. All three proteins were deposited in IBs upon recombinant production in E. coli. IB formation of the pro-peptide was unexpected because this protein fragment does not possess any cysteines that could be linked in disulfide bonds. Protocols were developed that enabled efficient recovery of native proteins from the IB pellets. After renaturation, the proteins were further purified as detailed under "Experimental Procedures" to ensure a single, homogenous, native species for further analysis. In Fig. 1, A–C, representative purification steps and the results of the final purification steps are illustrated. After purification, yields of mature BMP-2 and proBMP-2 were 25% and 10%, respectively, as calculated from the amount of protein originally present in the IB pellets. Differences in final yields were mainly due to losses during purification of renatured protein because SDS-PAGE analysis under non-reducing conditions revealed that after renaturation, disulfide-bonded dimeric species were obtained with yields of ~50%. Thus, in contrast to NGF, at least for in vitro structure formation, the pro-peptide of BMP-2 does not appear to be required.



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  		FIG. 1.
Recombinant protein production. Proteins were analyzed after separation on 12% SDS-PAGE and staining with Coomassie Blue. A, production of histidine-tagged proBMP-2. Lane 1, molecular weight marker; lane 2, protein extract from induced cells; lane 3, inclusion body preparation; lane 4, protein after renaturation (not reduced); lane 5, protein after heparin-Sepharose; lane 6, protein after hydroxylapatite. B, BMP-2. Lanes 1–5 were as defined in A. Lane 6, protein after reverse phase high pressure liquid chromatography. In A and B, samples were not reduced in lanes 4–6. C, histidine-tagged pro-peptide. Lanes 1–3 were as defined in A. Lane 4, protein after heparin-Sepharose; lane 5, protein after hydroxylapatite.

 
ProBMP-2 Can Be Processed in Vitro to drBMP-2—In analogy to proBMP-4, it is assumed that BMP-2 becomes converted to mature BMP-2 by pro-hormone convertases of the furin type that cleave at a dibasic sequence motif (20, 28, 29). Because we had shown previously that the pro-form of NGF can also be cleaved in vitro by trypsin to obtain mature NGF (11), this protease was used to test maturation of proBMP-2 to BMP-2. Proteolysis with trypsin resulted in N-terminally truncated BMP-2 (Fig. 2). Peptide sequence analysis revealed that the ratio of proteolysis products was 2:1:3 corresponding to truncated species that started with Arg7, Lys8, and Arg9. N-terminally truncated forms of BMP-2 have been described previously and referred to as drBMP-2 (30). drBMP-2 lacks the heparin binding site. A non-natural, N-terminally modified form of BMP-2 lacking this site displayed increased biological activity in a limb bud cell assay when compared with full-length BMP-2, an observation that had been explained by sequestration of the full-length protein by heparin via the heparin binding site (31). The fact that the N-terminal truncation products were trypsin-resistant (30, 32) was taken as an indication that the native structures had been obtained by the in vitro folding protocol applied here.



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  		FIG. 2.
Processing of proBMP-2 and BMP-2 to drBMP-2. Proteolysis with trypsin was performed as indicated under "Experimental Procedures." Analysis of processing was performed by SDS-PAGE and subsequent silver staining (proBMP-2) or Coomassie Blue staining (BMP-2).

 
Both ProBMP-2 and drBMP-2 Induce Ectopic Bone Formation—The next question was whether drBMP-2 generated in vitro from proBMP-2 would be biologically active. Biological activity of drBMP-2 was analyzed in an animal model. Both drBMP-2 and proBMP-2 were applied to ceramic cubes and implanted into rats. Upon explantation after 30 days, all implants appeared to be well tolerated, and no signs of adverse effects were detectable. In both the drBMP-2 and proBMP-2 groups, clearly visible bone formation and hematopoietic bone marrow were observed in most cases on the outer surface and within the ceramic implant cubes (Fig. 3, A and B). In contrast, control implants were covered by thin layers of surrounding muscle with some nutrient vessels and appeared otherwise unchanged (Fig. 3C). No obvious differences in the basic histological results between proBMP-2 and drBMP-2 were detected. Thus, drBMP-2 derived from refolded proBMP-2 exhibited at least very similar, if not identical, osteoinductive activity as proBMP-2.



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  		FIG. 3.
Bone inducing activity of drBMP-2 (A) and proBMP-2 (B). Ceramic cubes were analyzed histochemically as described under "Experimental Procedures." Bright blue areas represent bone tissue, and dark blue cells represent osteoblasts. C, control implant.

 
For quantification of the observed bone inducing activity of proBMP-2 and drBMP-2, the activity of alkaline phosphatase was determined for each implant (Fig. 4). The activities were analyzed using the Friedman rank test and pairwise comparison according to Wilcoxon and Wilcox (33). Enzyme activities of drBMP-2- and proBMP-2-coated implants were significantly increased compared with the control implants but did not differ significantly from each other ({alpha} = 5%). In summary, both drBMP-2 and proBMP induced ectopic bone formation to the same extent, at least in the animal test performed here.



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  		FIG. 4.
Quantification of biological activities. Alkaline phosphatase activity was determined as described under "Experimental Procedures." Units indicate total enzyme activity of half an implant block.

 
The Mature Part of BMP-2 Influences the Spectroscopic Properties of the Pro-peptide Moiety in ProBMP-2—Besides the biological activity in the case of proBMP-2 and drBMP-2, the correctly folded state of the three proteins (proBMP-2, BMP-2, and the pro-peptide) was confirmed by spectroscopic analyses. First, fluorescence spectra of the native proteins were compared with those of the denatured samples. These studies were complicated by the low solubility of BMP-2 at neutral pH. Improved solubility of BMP-2 at pH values of <6 had been reported previously (31, 34). Therefore, in order to obtain comparable data, fluorescence spectra of proBMP-2 and the pro-peptide were also recorded at acidic pH. A clear shift of the emission maxima from 339.5 to 349 nm (proBMP-2) and from 340 to 351 nm (pro-peptide) indicated that incubation at pH 4.7 resulted in an at least partial denaturation of the pro-peptide (Fig. 5, A and C). Incubation of proBMP-2 in 6 M guanidinium chloride and of the pro-peptide in 5.25 M urea at pH 7.0 caused additional shifts of the emission maxima to 359 nm. This additional shift was interpreted as a final complete solvent exposure of the tryptophans in strong denaturants at high concentrations and a partial solvent exposure of either some or all of the three tryptophan residues in the pro-peptide at pH 4.7. Fluorescence analyses were complemented by UV spectroscopy under identical conditions (data not shown). The UV difference spectra of native and denatured proBMP-2 clearly differed depending on whether the protein was incubated at acidic pH or in guanidinium chloride, again supporting the hypothesis that acidic pH partially denatures the pro-peptide moiety. This interpretation is supported by the observation that the UV difference spectra of the native pro-peptide and the pro-peptide that had been incubated at low pH or in urea were almost superimposable.



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  		FIG. 5.
Fluorescence spectra of proBMP-2 (A), BMP-2 (B), and pro-peptide (C). Protein concentrations were 0.4 µM (proBMP-2), 1.9 µM (BMP-2), and 1.3 µM (pro-peptide). A, fluorescence of proBMP-2 was recorded in 0.1 M K-HEPES, pH 7.0 (solid line); 10 mM K-acetate, pH 4.7 (dashed-dotted line); 5.25 M urea, 0.1 M K-HEPES, pH 7.0 (dotted line); and 6 M guanidinium chloride, 0.1 M Tris/Cl, pH 7.0 (dashed line). B, BMP-2 in 50 mM sodium acetate, pH 4.7 (solid line), and in the same buffer with 5.25 M urea (dotted line) or 6 M guanidinium chloride (dashed line). C, pro-peptide was recorded in 0.1 M K-HEPES, pH 7.0 (solid line); 10 mM K-acetate, pH 4.7 (dashed-dotted line); 10 mM K-HEPES with 5.25 M urea (dotted line); and Tris/Cl, pH 7.0, 6 M guanidinium chloride (dashed line). D, pH dependence of the fluorescence properties of the pro-peptide. Fluorescence intensity at 320 nm is indicated by filled circles; the position of the maxima is indicated by open circles. Measurements were performed in 50 mM K-HEPES/acetate buffer at the indicated pH values. Protein concentration was 0.94 µM.

 
Titration of the pH sensitivity of the pro-peptide structure by fluorescence analysis revealed that loss of structure occurs already under mildly acidic conditions such as pH 6.5 (Fig. 5D). This finding reveals an interesting correlation with in vivo studies performed by Christian and co-workers (21), who demonstrated a second proteolytic cleavage within the pro-peptide of BMP-4 that is enhanced by slightly acidic pH and necessary for activation of BMP-4.

The fluorescence properties of native BMP-2 were compared with those of BMP-2 that had been incubated in either 5.25 M urea or 6 M guanidinium chloride. Here, a shift of the fluorescence maxima upon denaturation from 346 to 352 (urea) and 356 nm (guanidinium chloride) was observed (Fig. 5B). In addition, an increase of fluorescence intensity upon unfolding was observed that could reflect tryptophan quenching of BMP-2 in the native state. Another noteworthy observation was that the maximum fluorescence emission of proBMP-2 was found at a lower wavelength than the emission maxima of the pro-peptide and BMP-2, indicating that the tryptophan residues of BMP-2 are less solvent-exposed in proBMP-2 than in the mature protein. The fact that denaturation of BMP-2 in guanidinium chloride resulted in a shift of the emission maximum to 356 nm, whereas shifts to 359 nm were observed with the pro-peptide-containing proteins (Fig. 5, A and C), is probably due to the cystine knot. It may prevent complete solvent exposure of the tryptophan residues in the mature part, even under strongly denaturing conditions.

The three proteins were also analyzed using far-UV CD spectroscopy. The spectrum recorded from proBMP-2 at pH 7.0 exhibited two minima at 212 and 218 nm. Beyond 212 nm toward shorter wavelengths, signal intensity was reduced (Fig. 6A). This shape of the spectrum indicates prevailing {beta}-sheet structures. In contrast, the CD spectra recorded for proBMP-2 at pH 4.7 showed weaker signal intensities in the wavelength range from 212 to 225 nm than the spectrum obtained from protein at neutral pH. However, an increased signal intensity was observed below 210 nm. These pH-dependent spectroscopic properties confirmed the conclusions drawn from fluorescence analyses: incubation at acidic pH is likely to cause loss of secondary structural elements that are located in the pro-peptide moiety.



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  		FIG. 6.
Far-UV-CD analysis. A, proBMP (14.3 µM) was analyzed in 0.1 M K-HEPES, pH 7.0 (•); 10 mM sodium acetate, pH 4.7 (15 µM; gray diamonds); and 6 M guanidinium chloride (6.0 µM; {triangleup}). B, BMP-2 was measured in 50 mM sodium acetate, pH 4.7 (25 µM; gray diamonds); 6 M guanidinium chloride, 50 mM sodium actetate (27 µM; {triangleup}); and the same buffer after reduction (48 µM; {blacktriangledown}). C, pro-peptide was recorded in 0.1 M K-HEPES, pH 7.0 (•); 10 mM K-acetate, pH 4.7 (gray diamonds); 5.25 M urea, 0.1 M K-HEPES, pH 7.0 ({circ}); and 6 M guanidinium chloride, 0.1 M Tris/HCl, pH 7.0 ({triangleup}). Protein concentration was between 30 and 33 µM. D, comparison of the spectrum of proBMP-2 (15 µM; •) and the calculated additive spectrum of BMP-2 and the pro-peptide ({circ}).

 
A corresponding but less pronounced effect of low pH was detected with the isolated pro-peptide (Fig. 6C). The increased signal intensity at low pH was interpreted as a moderate increase of random coil structures in the overall fold. Considering the large pH dependence of the fluorescence signals (Fig. 5C), in contrast to the rather weak changes in ellipticities upon pH shift, we conclude that mainly tertiary structures of the pro-peptide are affected by incubation at pH 4.7. Because the CD spectrum of the pro-peptide at pH 7 was found to be almost superimposable with the spectrum of {beta}-1-LAP from TGF-{beta}, we conclude that our in vitro refolding protocol indeed leads to the native fold of the BMP-2 pro-peptide (35).

CD analysis of BMP-2 was performed under native, denaturing, and denaturing plus reducing conditions. Whereas denaturation with 6 M guanidinium chloride resulted in a loss of signal intensity, a further loss of signal was observed upon additional reduction of disulfide bonds (Fig. 6B). The spectrum of BMP-2 under native conditions resembles that of NGF (11) and is typical for a protein that contains predominantly {beta}-sheet structures.

An additive spectrum was calculated from the spectra of BMP-2 and the pro-peptide in order to detect structural differences between the BMP-2-bound peptide and the isolated pro-peptide (Fig. 6D). Due to the extremely low solubility of BMP-2 at pH 7, the measurements were carried out at pH 4.7, where the pro-peptide moiety of proBMP-2 is at least partially unfolded (see above). Although the calculated additive spectrum resembles that of proBMP-2, small differences indicate changes in secondary structural elements that are likely to take place in the pro-peptide moiety.

The Mature Part Stabilizes the Pro-peptide of BMP-2—Spectroscopic analyses of proBMP-2 and the pro-peptide had indicated that the mature part influences both secondary and tertiary structures of the pro-peptide moiety. In order to investigate whether the mature part stabilizes the pro-peptide moiety, the thermodynamic stability of proBMP-2 was compared with that of the pro-peptide and BMP-2. Equilibrium unfolding studies were complicated by the low solubility of BMP-2 at neutral pH and the aggregation of proBMP-2 and the pro-peptide upon denaturation. Furthermore, as described above, at pH 4.7, where BMP-2 is soluble, the pro-peptide moiety appears to be at least partially unfolded. Because L-arginine suppresses aggregation and oligomerization of proBMP-2 and the pro-peptide (data not shown), unfolding and refolding studies were performed in the presence of 1 M L-arginine (36). Unfolding of proBMP-2 and BMP-2 by increasing concentrations of guanidinium chloride was monitored by fluorescence spectroscopy (Fig. 7A). A biphasic unfolding curve was recorded with proBMP-2. Transition midpoints were at 0.8 and 3.2 M guanidinium chloride. Because the transition midpoint of BMP-2 corresponded to the second transition in proBMP-2, the latter transition very likely reflects unfolding of the BMP-2 part. Consequently, the first transition should mirror unfolding of the pro-peptide moiety. Thermodynamic stabilities were quantified by non-linear regression (Table I). The calculated {Delta}G values indicate that the thermodynamic stability of BMP-2 is independent of the presence of the pro-peptide.



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  		FIG. 7.
Equilibrium unfolding studies. A, proBMP-2 and BMP-2 were unfolded by guanidinium chloride in the presence of 1 M L-arginine, 0.1 M K-HEPES, pH 7.0. For simplicity, refolding curves were omitted. Protein concentrations were 0.45 µM (proBMP-2) and 12 µM (BMP-2). Unfolding was monitored by fluorescence emission at 320 (first transition) and 360 nm (second transition). Excitation was at 295 nm. To ensure complete unfolding, samples were incubated at the indicated guanidinium chloride concentrations for 24 h at 24 °C before spectroscopy. {diamondsuit}, transition of the pro-peptide domain of proBMP-2; •, transition of the BMP-2 domain of proBMP-2; {square}, transition of BMP-2. B, unfolding of the isolated pro-peptide ({diamondsuit}, unfolding; {diamond}, refolding) and the pro-peptide attached to mature BMP-2 (•, unfolding; {circ}, refolding).

 


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  		TABLE I
Thermodynamic stabilities and m values as derived from experiments illustrated in Fig. 7

 
In order to test whether the BMP-2 part stabilizes the pro-peptide as in case of NGF/proNGF (37), unfolding of the pro-peptide was tested. Because preliminary experiments had indicated that the pro-peptide is only marginally stable under native conditions and in the presence of L-arginine, urea was used as a weaker denaturant than guanidinium chloride. (Fig. 7B). Unfolding was tested under identical conditions with proBMP-2. It is clearly visible that in proBMP-2, the pro-peptide undergoes a cooperative transition, whereas unfolding of the isolated pro-peptide is clearly less cooperative (Fig. 7B). The calculated m values for unfolding of the isolated pro-peptide and the pro-peptide moiety of proBMP-2 differ by a factor of 2. From this it is reasonable to conclude that the solvent-exposed surface area of native, free pro-peptide is approximately twice as large as that fused to BMP-2. Thus, a significant stabilization of the pro-peptide in proBMP-2 at the level of tertiary contacts results from extensive interactions with the mature part. These results support the conclusions derived from the spectroscopic analysis, which indicated interactions between both moieties of proBMP-2 leading to structural rearrangements.

With the pro-peptide, no plateau at low urea concentrations was detected. Thus, a small but significant proportion of the pro-peptide was non-native in the absence of denaturant under the conditions applied. This fact rendered {Delta}G calculations for the pro-peptide problematical. Despite this complication, we assumed that the change in fluorescence of the native pro-peptide is identical to the change in fluorescence of native proBMP-2. The calculation of the thermodynamic parameters of unfolding then led to a value of 90% native pro-peptide in the absence of urea under the given conditions. The values in Table I show a significant contribution of the mature part to the overall stability of the pro-peptide with a {Delta}{Delta}G between the pro-peptide linked to the mature part and the isolated pro-peptide of 13 kJ/mol. Thus, as with NGF/proNGF, the mature part has a strong effect on the stability of the pro-peptide, likely caused by a structure induction.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
BMP-2 belongs to the family of cystine knot proteins, a family that has received attention not only from the protein folding community but also from the pharmaceutical industry. Although BMP-2 has prospects as an important therapeutic protein due to its bone inducing activity, virtually nothing is known about the structure and functions of the pro-form and/or the isolated pro-peptide. In case of NGF, the pro-form has recently been attributed pro-apoptotic functions (13, 15). In addition, we have previously demonstrated that the pro-peptide significantly contributes to in vitro oxidative folding (10, 11). The original objective of the presented work was to show a similar role for the pro-peptide of BMP-2. However, as folding yields have shown, both BMP-2 and proBMP-2 can be refolded with comparable yields from the denatured state. Thus, at least under the conditions employed here, oxidative structure formation of BMP-2 is independent of the presence of the pro-peptide. This result still does not exclude a folding assisting role of the pro-peptide in the cell.

In order to compare the three proteins on a biochemical level, protocols were developed that enabled efficient renaturation of BMP-2, proBMP-2, and the pro-peptide from inclusion body material. Besides biological activities of BMP-2 and proBMP-2, spectroscopic characterization indicated that native species were obtained with all three proteins. As observed for {beta}-1-LAP of TGF-{beta} (38) and the BMP-4 pro-domain (21), the pro-peptide of BMP-2 loses structural integrity at low pH and likely dissociates from the mature part. Whether dissociation causes or is the result of unfolding is currently not clear. Strikingly, CD analysis of the pro-peptide indicated that secondary structure is mainly dictated by {beta}-sheets. A comparable predominance of {beta}-structure has been demonstrated for {beta}-1-LAP (35). The authors had employed CD analyses and unfolding experiments that suggested a stabilization of the pro-peptide by the mature part. In addition, evidence is provided that the differences between isolated pro-peptide and that linked to the mature part are mainly secondary structural elements. The authors reported structural rearrangements upon association of {beta}-1-LAP from the mature growth factor. In the case of NGF/proNGF, we could demonstrate that the mature part induces the structure of the pro-peptide, which is is barely structured in its isolated form (37). Thus, the pro-peptide of BMP-2 resembles that of both NGF and TGF-{beta} with regard to its interaction with the mature part. As in NGF, stabilization of the pro-peptide is conferred by the mature part. However, in contrast to that of NGF, the pro-peptide of BMP-2 appears to possess at least some tertiary contacts in the absence of the mature part because a weakly cooperative unfolding curve was recorded. The comparison of native and denatured proBMP-2 by fluorescence measurements indicated restricted solvent accessibility of the tryptophan residues in the mature domain. Because both tryptophan residues of the mature part (Trp28 and Trp31) contribute to the interaction of the cytokine with the type I receptor (9), BMP-2 in the pro-form may not be competent for type I receptor binding.

ProBMP-2 can be processed in vitro to yield drBMP-2 that displays biological activity as tested by its bone inducing activity and induction of alkaline phosphatase. To a comparable extent, proBMP-2 also induces ectopic bone formation in vivo. It is presently unclear whether the bone inducing activity of proBMP-2 results from mature BMP-2 that could be the product of proteolytic processing within the animal. Alternatively, biological activity could be delayed by the pro-peptide. If this were true, the pro-peptide would exert a role comparable with that of {beta}-1-LAP (16–18). Besides the limited sequence homology, the pro-peptides also differ in other aspects: first, whereas {beta}-1-LAP is disulfide-linked, the pro-peptide of BMP-2 lacks cysteines; second, {beta}-1-LAP possesses the integrin binding motif, RGD (39), which is absent in the pro-peptide of BMP-2. Consequently, if a delaying function for the pro-peptide of BMP-2 can be demonstrated, then it would be independent of a pro-peptide-mediated adhesion to integrins. Thus, the biological role of the BMP-2 pro-peptide remains to be analyzed and will require comprehensive cell biological approaches combined with biochemical approaches.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant SCHW375/3-1-3 (to E. S. and R. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Present address: Biochemisches Institut, Winterthurer-Strasse 190, 8057 Zürich, Switzerland. Back

|| To whom correspondence should be addressed. Tel.: 49-345-55-24-856; Fax: 49-345-55-01-327; E-mail: Elisabeth.Schwarz{at}biochemtech.uni-halle.de.

1 The abbreviations used are: BMP, bone morphogenetic protein; TGF, transforming growth factor; NGF, nerve growth factor; drBMP-2, digit removed BMP-2; LAP, latency-associated peptide; IB, inclusion body. Back


   ACKNOWLEDGMENTS
 
We thank Milton Stubbs and Marco Kliemannel for critical reading of the manuscript and fruitful suggestions.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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