The pro-form of BMP-2 interferes with BMP-2 signalling by
competing with BMP-2 for IA receptor binding
Anja Hauburger
1
, Sabrina von Einem
1
, Gerburg K. Schwaerzer
2
, Anja Buttstedt
1
, Matthias Zebisch
3
,
Michael Schra
¨ml
4,
, Peter Hortschansky
5
, Petra Knaus
2
and Elisabeth Schwarz
1
1 Institut fu
¨r Biochemie und Biotechnologie, Martin-Luther-Universita
¨t Halle-Wittenberg, Germany
2 Institut fu
¨r Chemie ⁄Biochemie, Freie Universita
¨t Berlin, Germany
3 Biotechnologisch-Biomedizinisches Zentrum, Universita
¨t Leipzig, Germany
4 Scil Proteins GmbH, Halle, Germany
5 Leibniz-Institut fu
¨r Naturstoffforschung und Infektionsbiologie, Hans-Kno
¨ll-Institut (HKI), Jena, Germany
Keywords
alkaline phosphatase; BMPR-IA; BMPR-II;
bone morphogenetic protein-2; pro-domain
Correspondence
E. Schwarz, Institut fu
¨r Biochemie and
Biotechnologie, Martin-Luther-Universita
¨t
Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120
Halle, Germany
Fax: +49 345 55 27 013
Tel: +49 345 55 24 856
E-mail: elisabeth.schwarz@biochemtech.
uni-halle.de
Present address
Roche Diagnostics GmbH, Nonnenwald 2,
82372 Penzberg, Germany
(Received 2 April 2009, revised 18 August
2009, accepted 3 September 2009)
doi:10.1111/j.1742-4658.2009.07361.x
Pro-forms of growth factors have received increasing attention since it was
shown that they can affect both the maturation and functions of mature
growth factors. Here, we assessed the biological function of the pro-form of
bone morphogenetic protein-2 (BMP-2), a member of the transforming
growth factor b(TGFb)⁄BLP superfamily. The role of the 263 amino acids
of the pro-peptide is currently unclear. In order to obtain an insight into the
function of the pro-form (proBMP-2), the ability of proBMP-2 to induce
alkaline phosphatase (AP), a marker enzyme for cells differentiating into os-
teoblasts, was tested. Interestingly, in contrast to mature BMP-2, proBMP-2
did not lead to induction of AP. Instead, proBMP-2 inhibited the induction
of AP by BMP-2. This result raised the question of whether proBMP-2 may
compete with mature BMP-2 for receptor binding. ProBMP-2 was found to
bind to the purified extracellular ligand binding domain (ECD) of BMPR-
IA, a high-affinity receptor for mature BMP-2, with a similar affinity as
mature BMP-2. Binding of proBMP-2 to BMPR-IA was confirmed in cell
culture by cross-linking proBMP-2 to BMPR-IA presented on the cell sur-
face. In contrast to this finding, proBMP-2 did not bind to the ECD
of BMPR-II. ProBMP-2 also differed from BMP-2 in its capacity to induce
p38 and Smad phosphorylation. The data presented here suggest that the
pro-domain of BMP-2 can alter the signalling properties of the growth factor
by modulating the ability of the mature part to interact with the receptors.
Structured digital abstract
lMINT-7261817:BMPR-IA (uniprotkb:P36894) and proBMP2 (uniprotkb:P12643)physically
interact (MI:0915)bycross-linking studies (MI:0030)
lMINT-7261681,MINT-7261693:BMP2 (uniprotkb:P12643)binds (MI:0407)toBMPR-IA
(uniprotkb:P36894)byenzyme linked immunosorbent assay (MI:0411)
lMINT-7261751,MINT-7261794:proBMP2 (uniprotkb:P12643)binds (MI:0407)toBMPR-IA
(uniprotkb:P36894)bycompetition binding (MI:0405)
lMINT-7261806,MINT-7261846:BMPR-IA (uniprotkb:P36894)physically interacts
(MI:0915) with BMP2 (uniprotkb:P12643)byanti bait coimmunoprecipitation (MI:0006)
lMINT-7261628,MINT-7261642:noggin (uniprotkb:Q13253)binds (MI:0407)toproBMP2
(uniprotkb:P12643)bysurface plasmon resonance (MI:0107)
Abbreviations
AP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; ECD, extracellular domain; GDF-8, growth and differentiation factor-8; HA,
haemagglutinin; MBP, maltose binding protein; PFC, pre-formed receptor complex; Smad, small mothers against decapentaplegic;
TGFb, transforming growth factor b.
6386 FEBS Journal 276 (2009) 6386–6398 ª2009 The Authors Journal compilation ª2009 FEBS
Introduction
Bone morphogenetic protein-2 (BMP-2) belongs to the
transforming growth factor b(TGFb) superfamily.
Structural features of proteins in this family include
the arrangement of disulfide bridges in a cystine knot
and the anti-parallel association of the two monomers,
which are linked by an intermolecular disulfide bond
[1,2]. The capacity of BMP-2 to induce bone formation
has been exploited for therapeutic application [3].
Signal transduction involves a BMP-2 dimer in asso-
ciation with two type I and two type II receptors. Two
binding modes for BMP-2 have been reported, which
indicate the existence of different signalling pathways
[4–7]. For the sequential mode of binding, two type I
receptor molecules are bound by the dimeric ligand.
Subsequently, two type II receptor molecules are
recruited by the ligand–type I receptor complex. This
association initiates the p38–MAPK pathway, which
finally leads to the induction of alkaline phosphatase.
The second binding mode is characterized by ligand
binding to pre-formed receptor complexes (PFC),
which consist of two type I and two type II receptors.
By binding of the ligand to PFCs, the Smad signalling
pathway is activated.
BMP-2 is translated as a prepro-protein in vivo. The
pre-sequence mediates translocation into the endoplas-
mic reticulum. However, the function of the pro-pep-
tide is presently unknown. We showed previously that
the pro-peptide is not required for in vitro oxidative
folding of the mature part [8]. Furthermore, recombi-
nant proBMP-2 induced ectopic bone formation in
rats, indicating that the pro-peptide does not signifi-
cantly impair the bone-inducing activity of mature
BMP-2 [8]. We are interested in the role of the 263
amino acid pro-peptide of BMP-2, because evidence
accumulated over recent years has shown that the
pro-forms of growth factors can modulate the activities
of the mature domains. In case of pro-neurotrophins,
for example, they can even elicit completely opposite
effects to those of the mature growth factors by bind-
ing to pro-form-specific receptors [9–11].
The pro-peptide of the related TGFbhas been
shown to retard the function of the mature protein by
non-covalent association with the mature part upon
proteolytic processing. This retarding role of the
pro-peptide led to it being named latency-associated
polypeptide [12]. In addition to regulating activity, at
least in the case of inhibins, which also belong to the
TGFbsuperfamily, the pro-domains appear to play a
role in assembly and secretion [13]. Similarly, an inhib-
itory role of the non-covalently attached pro-peptide
has been demonstrated for growth and differentiation
factor-8 (GDF-8) [14,15]. Furthermore, the pro-peptide
of GDF-8 impairs interaction of the mature part with
its receptors [16]. In the case of BMP-9, the pro-pep-
tide appears not to alter significantly the activity of the
mature part [17]. For the pro-peptide of BMP-7, a tar-
geting role to the extracellular matrix [18] has been
shown. The pro-peptide of BMP-4 is responsible for
stabilization of the mature part, intracellular traffick-
ing and folding in the endoplasmic reticulum [19–21].
Thus, the roles of the pro-peptides appear to be diver-
gent within the TGFb⁄BMP family, and appear to
modulate the function of the mature part by non-
covalent association after proteolytic cleavage by
pro-hormone convertases (for review, see [22]). The
biological relevance of pro-domains within the TGFb
family is highlighted by reports showing that muta-
tions in pro-domains lead to abnormal dorsoventral
patterning [23] and skeletal malformations [24,25]. In
the case of BMP-2, no published information is avail-
able on the physiological function of the pro-domain.
Increased levels of unprocessed proBMP-2 have been
shown to be present in synovial tissue from patients
suffering from rheumatoid arthritis and spondyloarthr-
opathies [26]. However, the relevance of this finding
for disease development is so far unclear.
In this work, we attempted to obtain an insight into
the function of proBMP-2. In order to obtain more
information about the role of the pro-form, proBMP-2,
i.e. BMP-2 with the covalently attached pro-peptide,
was recombinantly produced and compared to the
mature form. We show that proBMP-2 can compete
with mature BMP-2 for binding to BMP receptor IA
(BMPR-IA), one of the main receptors of mature
BMP-2 [6,27,28]. In contrast, the ECD of BMPR-II was
not bound by proBMP-2. Furthermore, the free pro-
peptide formed a non-covalent complex with mature
BMP-2 in vitro, thereby blocking binding of BMP-2 to
BMPR-II. The finding that proBMP-2 did not induce
alkaline phosphatase is consistent with the finding that
proBMP-2 does not lead to p38 phosphorylation. We
conclude that the pro-peptide of BMP-2, although it
lMINT-7261597,MINT-7261613:BMPR-IA (uniprotkb:P36894)binds (MI:0407)toBMP2
(uniprotkb:P12643)bysurface plasmon resonance (MI:0107)
A. Hauburger et al. The pro form of BMP-2 interferes with BMP-2 signalling
FEBS Journal 276 (2009) 6386–6398 ª2009 The Authors Journal compilation ª2009 FEBS 6387
does not disturb interaction of the mature part with
BMPR-IA, may nonetheless interfere with signal
induction, possibly at the level of receptor interaction.
Results
ProBMP-2 inhibits AP induction by BMP-2
In order to determine whether proBMP-2 elicits biologi-
cal responses similar those elicited by mature BMP-2,
induction of alkaline phosphatase (AP) was investi-
gated. AP represents a marker enzyme for differentia-
tion into osteoblasts, thus AP activity is usually
measured to test the response to mature BMP-2 [29,30].
Using BMP-2 as a control, an EC
50
of 18 ± 4 nmwas
calculated (Fig. 1A), which corresponds well with the
published EC
50
of 19 nm[30]. AP activity induced by
BMP-2 was blocked by noggin (Fig. 1D). When AP
activity was tested upon addition of the isolated pro-
peptide as a negative control, no signal increase was
observed (Fig. S1). Similarly, using proBMP-2 under
identical assay conditions, only a low AP signal increase
and no concentration dependence was recorded
(Fig. 1B). Even when cells were stimulated with 1 lm
proBMP-2, the AP signal was in a similar range to that
obtained after induction with 2 nmBMP-2 (data not
shown). This very small signal increase upon addition of
proBMP-2 may be due to slow cleavage of proBMP-2 to
BMP-2 over time, possibly by proteases secreted from
the C2C12 cells, rather than an AP-inducing activity of
proBMP-2. Contamination of the proBMP-2 protein
sample with traces of mature BMP-2 could be excluded
as neither staining of SDS–PAGE gels nor western blot
analysis using a rhBMP-2 antibody yielded any evidence
for the presence of mature BMP-2 during the first 48 h
of incubation (Fig. S2).
Next, we tested whether proBMP-2 suppresses
induction of AP by mature BMP-2. For the competi-
tion experiments, cells were incubated with BMP-2 at
a concentration of 200 nm, which had been proven to
elicit the maximal AP response (Fig. 1A), and increas-
ing concentrations of proBMP-2. A proBMP-2 concen-
tration-dependent inhibition of the BMP-2-induced AP
activity was observed (Fig. 1C). The possibility of con-
tamination of the proBMP-2 preparation with endo-
toxins was excluded by using a chromogenic limulus
amoebocyte lysate (LAL) detection kit (Charles River,
Wilmington, MA, USA), which showed that endotoxin
AB
C
BMP-2 [nM]
1 10 100
0
10
20
30
40
50
AP activity (%)
(∆E × min–1 × µg–1)
proBMP-2 [nM]
0 200 400 600
10
20
30
40
50
AP activity (%)
(∆E × min–1 × µg–1)
proBMP-2 [nM]
0 200 400 600 800 1000
AP inhibition (%)
20
40
60
80 D
AP activity (%)
I II III IV
0
20
40
60
80
100
Fig. 1. Mature BMP-2 but not proBMP-2 leads to induction of AP. Effects of BMP-2 (A) and proBMP-2 (B) on the induction of alkaline phos-
phatase in C2C12 cells. AP activity was measured by determination of the change in extinction (DE) per minute and microgram protein. In
(C), 200 nMBMP-2 and the indicated concentrations of proBMP-2 were added simultaneously to the cells; the maximal AP activity in the
absence of proBMP-2 was set to 100%. (D) The AP assay was controlled by endpoint determinations of substrate turnover in the presence
of an equimolar amount (III) or fivefold molar excess (IV) of noggin over BMP-2 (black) or proBMP-2 (grey); (I) no ligand; (II) absence of nog-
gin. Ligand concentrations were 10 nM. The lower amplitudes of the AP signals are due to the fact that, in this experiment, the signals
obtained using 10 nMBMP-2 were set to 100%. Data represent means and standard deviations from four independent measurements.
The pro form of BMP-2 interferes with BMP-2 signalling A. Hauburger et al.
6388 FEBS Journal 276 (2009) 6386–6398 ª2009 The Authors Journal compilation ª2009 FEBS
levels were below the determination threshold. Thus,
based on these data, we conclude that the observed
reversal of BMP-2-elicited AP induction by proBMP-2
may reflect a biological mechanism.
ProBMP-2 binds to the extracellular ligand
binding domain of BMPR-IA, but not that of
BMPR-II
To investigate whether inhibition of BMP-2-induced
AP activity by proBMP-2 results from competition of
proBMP-2 with BMP-2 for binding to the main
receptor BMPR-IA, BIAcore experiments were per-
formed. For these studies, the ECD of the receptor
was recombinantly produced in Escherichia coli cells,
refolded and purified [31]. The ECD was biotinylated
and immobilized on streptavidin-coated BIAcore chips.
Ligand binding was first analysed using the mature
growth factor. The fast association rate and the very
slow dissociation rate are in accordance with published
results (K
D
= 0.9 ± 0.8 ·10
)9
m) (Fig. 2A and
Table 1) [29]. When proBMP-2 was tested as an ana-
lyte, a comparable K
D
(4 ± 1.8 ·10
)9
m) was
obtained (Fig. 2B and Table 1). This result shows that
the pro-peptide moiety does not interfere with binding
of the mature part to BMPR-IA. As the BMPR-IA
binding site for BMP-2 partially overlaps with the area
bound by the antagonist noggin [32], we attempted
to verify these findings by testing the binding of
proBMP-2 to noggin. Biotinylated noggin was immo-
bilized on streptavidin-coated chips, and proBMP-2 or
BMP-2 were injected at various concentrations
(Fig. 2C,D). The sensorgrams reveal that proBMP-2
binds to noggin with a comparable affinity to that for
mature BMP-2, a result that confirms indirectly that
the pro-peptide moiety does not interfere with binding
of the mature part to the BMPR-IA ECD. Due to the
very slow release of both analytes from the immobi-
lized ligand, K
D
values based on the association and
dissociation rates could not be determined for the
interaction with noggin.
The BIAcore experiments that revealed binding of
proBMP-2 were corroborated by ELISA studies. For
these experiments, BMP-2 or proBMP-2 was adsorbed
on to the well surfaces of microtitre plates. After
blocking free binding sites of the wells, the BMPR-IA
ECD was added at various concentrations. Growth
factor-bound ECD was detected by incubation with
BMPR-IA ECD antibody and subsequent detection
via a horseradish peroxidase-conjugated antibody.
The BMPR-IA ECD bound to both immobilized
BMP-2 and proBMP-2 (Fig. 3A,B). The final signal
for proBMP-2 was approximately twice as high as that
for BMP-2. Presumably, this effect is due to more
efficient coating of proBMP-2 to the well surface than
with BMP-2, as has also been observed in other experi-
ments (data not shown).
A
D
C
B
Time (s)
0 100 200 300 400 500
RU
0
100
200
300
400
400 nM
200 nM
100 nM
50 nM
25 nM
12.5 nM
6.2 nM
3.1 nM
1.6 nM
Time (s)
0 100 200 300 400 500
RU
0
50
100
150
200
250
400 nM
200 nM
100 nM
50 nM
25 nM
12.5 nM
6.2 nM
3.1 nM
Time (s)
0 100 200 300 400
RU
0
10
20
30
40
600 nM
300 nM
200 nM
100 nM
50 nM
25 nM
12.5 nM
6.2 nM
0 nM
Time (s)
0 100 200 300 400 500
RU
0
10
20
30
40
400 nM
200 nM
100 nM
50 nM
25 nM
12.5 nM
6.2 nM
3.1 nM
0 nM
Fig. 2. Surface plasmon resonance experi-
ments demonstrate binding of proBMP-2 to
the ECD of BMPR-IA. Interaction of BMP-2
(A) and proBMP-2 (B) with immobilized ECD
of BMPR-IA. Interaction of BMP-2 (C) and
proBMP-2 (D) with immobilized noggin are
indicated. The higher scattering of the sen-
sorgrams in (C) and (D) is due to the fact
that only 64 resonance units of noggin were
immobilized in these experiments.
Table 1. Kinetic constants for interaction of the ECD of BMPR-IA
with the growth factors. Association (k
a
) and dissociation (k
d
) rates
for the ligands with the ECD and the apparent dissociation con-
stants K
D
as determined using BIAcore are shown.
k
a
(M
)1
Æs
)1
)k
d
(s
)1
)K
D
(nM)
BMP-2 3.1 ± 1.8 ·10
5
2.8 ± 1.0 ·10
)4
0.9 ± 0.8
proBMP-2 7.4 ± 2.9 ·10
4
3.0 ± 0.2 ·10
)4
4.0 ± 1.8
A. Hauburger et al. The pro form of BMP-2 interferes with BMP-2 signalling
FEBS Journal 276 (2009) 6386–6398 ª2009 The Authors Journal compilation ª2009 FEBS 6389
Next, competition experiments were performed. The
ECD at a concentration of 500 nmwas pre-incubated
for 30 min with increasing concentrations of BMP-2 to
allow complex formation. Subsequently, the pre-incu-
bated samples were added to wells that had been
coated with BMP-2 or proBMP-2. ECD binding to
immobilized BMP-2 or proBMP-2 decreased with
increasing concentrations of the growth factors in the
pre-incubations (Fig. 3C,D). These data confirmed that
proBMP-2 binds specifically to the ECD of BMPR-IA.
Furthermore, the results indicate that both proteins
interact with the same epitope on the ECD because
proBMP-2 binding can be inhibited by BMP-2.
To assess binding of proBMP-2 to BMPR-II, BIA-
core experiments were performed using the ECD of
BMPR-II linked to a Fc domain of human IgG
(BMPR-II-Fc). After immobilization of the chimeric
protein on a CM5 chip, binding of BMP-2 as a
positive control (Fig. 4A) and of proBMP-2 (Fig. 4B)
were recorded. ProBMP-2 did not bind to the immo-
bilized ECD chimera of BMPR-II. When mature
BMP-2 was pre-incubated with increasing concentra-
tions of separately produced, free pro-peptide,
decreased signals were observed (Fig. 4C), which
confirms that the pro-peptide inhibits association of
mature BMP-2 with the ECD of BMPR-II, probably
by masking binding sites of BMP-2. Consistently,
maximal inhibition was observed by using equimolar
concentrations (0.4 lm) of both pro-peptide and
BMP-2. Furthermore, the ability of BMP-2 to inter-
act with the free pro-peptide could be proven by
BIAcore experiments (Fig. 4D). From these studies,
aK
D
of 28 ± 16 nmfor the non-covalent complex
of mature BMP-2 and the pro-peptide was calcu-
lated.
BMP-2 and proBMP-2 bind to BMPR-IA at the cell
surface
After demonstrating that proBMP-2 binds to the ECD
of BMPR-IA in vitro, we performed an in vivo experi-
ment to test binding of proBMP-2 to this receptor
presented at the cell surface. COS-7 cells were trans-
fected with an expression construct for BMPR-IA
carrying a haemagglutinin (HA) epitope [4]. Transient
expression of the receptor was first tested 2 days after
transfection by examination of whole-cell extracts
using SDS–PAGE, western blotting and decoration
with HA antibodies (data not shown).
COS-7 cells transiently expressing BMPR-IA were
incubated with BMP-2 or proBMP-2. After removal of
unbound ligands, cell-bound growth factors were
chemically cross-linked using disuccinimidylsuberate
(DSS). Ligand–receptor complexes were detected
directly in whole-cell lysates after western blotting.
Detection of the proBMP-2 moiety was done using a
BMP-2 antibody (Fig. 5A) and the BMPR-IA part by
a HA antibody (Fig. 5B). A band at the expected size
of approximately 140 kDa was detected using each
antibody, but was never observed in the negative con-
trols to which neither proBMP-2 nor DSS were added.
Detection of bands of comparable sizes with either
C D
A
B
BMPR-IA-ECD
[µM]
0.0 0.5 1.0 1.5 2.0 2.5 5.0
Slope at 405 nm
0
5
10
15
20
Immobilized BMP-2
BMPR-IA-ECD
[µM]
0.0 0.5 1.0 1.5 2.0 2.5 5.0
Slope at 405 nm
0
10
20
30
40
50
Immobilized proBMP-2
In the pre-incubation with 500 nM ECD
0
0
2
2
4
4
6
6
8
8
10
10 12 14
BMP-2
[µM]
Slope at 405 nm
Immobilized BMP-2
In the pre-incubation with 500 nM ECD
0 2 4 6 8 10 12 14
0
2
4
6
8
10
12
14
16
18
20
BMP-2
[µM]
Slope at 405 nm
Immobilized proBMP-2
Fig. 3. Binding of proBMP-2 to the ECD
was confirmed by ELISA. Binding of the
BMPR-IA ECD to immobilized BMP-2 (A)
and proBMP-2 (B). For the competition
experiments, 500 nMECD were pre-incu-
bated with the indicated concentrations of
BMP-2. Unbound ECD reacted with immobi-
lized BMP-2 (C) or proBMP-2 (D). Data
represent means and standard deviations
from three independent measurements.
The pro form of BMP-2 interferes with BMP-2 signalling A. Hauburger et al.
6390 FEBS Journal 276 (2009) 6386–6398 ª2009 The Authors Journal compilation ª2009 FEBS