Tài liệu Báo cáo khoa học: The pro-form of BMP-2 interferes with BMP-2 signalling by competing with BMP-2 for IA receptor binding pptx

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
l
MINT-7261817:BMPR-IA (uniprotkb:P36894) and proBMP2 (uniprotkb:P12643) physically
interact (
MI:0915)bycross-linking studies (MI:0030)
l

MINT-7261681, MINT-7261693: BMP2 (uniprotkb:P12643) binds (MI:0407)toBMPR-IA
(uniprotkb:
P36894)byenzyme linked immunosorbent assay (MI:0411)
l
MINT-7261751, MINT-7261794: proBMP2 (uniprotkb:P12643) binds (MI:0407)toBMPR-IA
(uniprotkb:
P36894)bycompetition binding (MI:0405)
l
MINT-7261806, MINT-7261846: BMPR-IA (uniprotkb:P36894) physically interacts
(
MI:0915) with BMP2 (uniprotkb:P12643)byanti bait coimmunoprecipitation (MI:0006)
l
MINT-7261628, MINT-7261642: noggin (uniprotkb:Q13253) binds (MI:0407)toproBMP2
(uniprotkb:
P12643)bysurface plasmon resonance (MI:0107)
Abbreviations
AP, alkalinephosphatase; BMP-2,bone morphogeneticprotein-2; ECD,extracellular domain;GDF-8, growthand differentiationfactor-8; HA,
haemagglutinin; MBP,maltose bindingprotein; PFC,pre-formed receptorcomplex; Smad,small mothersagainst decapentaplegic;
TGFb, transforminggrowth factor b.
6386 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 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 TGFb has 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
TGFb superfamily, 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
l
MINT-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 ª 2009 The Authors Journal compilation ª 2009 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 nm was
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 nm BMP-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
A
B
C
BMP-2 [nM]
1 10 100
0
10
20
30
40
50
AP activity (%)

(ΔE × min
–1
× µg
–1
)
proBMP-2 [nM]
0 200 400 600
10
20
30
40
50
AP activity (%)
(ΔE × 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 n
M BMP-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 n
M. The lower amplitudes of the AP signals are due to the fact that, in this experiment, the signals
obtained using 10 n
M BMP-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 ª 2009 The Authors Journal compilation ª 2009 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 ª 2009 The Authors Journal compilation ª 2009 FEBS 6389
Next, competition experiments were performed. The
ECD at a concentration of 500 nm was 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,
a K
D
of 28 ± 16 nm for 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 n
M
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 n
M
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 n
M ECD 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 ª 2009 The Authors Journal compilation ª 2009 FEBS
antibody indicates that this signal represents the
complex of proBMP-2 with BMPR-IA. Complexes
of BMP-2 or proBMP-2 with BMPR-IA were also
immunoprecipitated using BMPR-IA ECD antibody
(Fig. 5C,D). After immuno-precipitation, for both
ligands, signals corresponding to the positions of the
ligand-receptor complexes were detected (Fig. 5C,D).
ProBMP-2 differs from BMP-2 in its ability to
induce Smad and p38 phosphorylation
BMP-2 can induce two signalling pathways depend-
ing on its mode of interaction with surface receptors
[5,6,33]: upon BMP-2 binding to PFCs, the Smad
pathway is induced, or BMP-2-induced receptor olig-
omerization triggers phosphorylation of p38, resulting
in AP induction [33,34], unless Smad proteins are
overexpressed [35,36]. The inability of proBMP-2
to induce AP prompted the question of whether

proBMP-2 might induce cellular signals by preferen-
tially interacting with PFCs, and thus predominantly
activate the Smad pathway. When 10 nM BMP-2
was added to C2C12 cells, Smad proteins 1, 5 and 8
were phosphorylated at their C-termini after 15 min.
In contrast, the same concentration of proBMP-2
did not lead to significant phosphorylation even after
120 min (Fig. 6A,B). Only at high concentrations
(200 nm) did proBMP-2 led to instantaneous Smad
phosphorylation (Fig. S3). When the effect of
BMP-2 and proBMP-2 on the phosphorylation status
of p38 was tested, addition of BMP-2 resulted in
maximal phosphorylation of p38 after 60 min, while
proBMP2 showed no induction of p38 phosphoryla-
tion (Fig. 6A,C).
To measure Smad activation in long-term experi-
ments, a BRE–luciferase assay was used, in which
luciferase (as reporter gene) is under the control of a
Smad-responsive element [5]. Figure 7 shows that both
ligands induce luciferase in a concentration-dependent
manner; however, proBMP-2 was less effective than
BMP-2.
Discussion
BMP-7, BMP-9, GDF-8 and TGFb are highly homol-
ogous to BMP-2. For all these growth factors, non-
covalent association of the pro-peptides with the
mature domains after proteolytic processing has been
demonstrated [12,15,17,18,37]. In case of BMP-2, it is
not clear whether the pro-peptide moiety remains
associated with the mature part of BMP-2 after pro-

hormone processing because precise and detailed infor-
mation on the levels of BMP-2 and proBMP-2 in the
C
A
B
Time (s)
050
1
00 150 200
RU
0
100
200
300
400
500
200 nM BMP-2
800 n
M proBMP-2
Pro-peptide in pre-incubation [µM]
R
eq
(RU)
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
50
100
150
200
250

300
D
Time (s)
050
1
00 150 200
RU
0
100
200
300
400
500
600
700
400 nM
200 nM
100 nM
50 nM
25 nM
12.5 nM
Time (s)
050
1
00 150 200
RU
0
100
200
300

400
500
600
700
400 nM
200 nM
100 nM
50 nM
25 nM
12.5 nM
0 nM
Fig. 4. ProBMP-2 does not bind to the immobilized ECD of BMPR-
II. (A) Interaction of BMP-2 with immoblilized BMPR-II–Fc. (B) Com-
parison of proBMP-2- and BMP-2 interaction with immobilized
BMPR-II–Fc. (C) Inhibition of BMP-2 binding to BMPR-II–Fc by the
isolated pro-peptide. Signal reduction is dependent on the
concentrations of pro-peptide in the pre-incubation. (D) Complex for-
mation of BMP-2 and the free pro-peptide was tested by BIAcore.
For the experiments, the pro-peptide was immobilized on a CM5 chip.
A. Hauburger et al. The pro form of BMP-2 interferes with BMP-2 signalling
FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6391
extracellular space is not available. In fact, only one
such study has been performed, which found that a
small amount of unprocessed proBMP-2 is secreted
upon recombinant expression in CHO cells [38]. Lories
et al. [26] described accumulation of the pro-form of
BMP-2 in human tissue, with high levels of proBMP-2
being found in tissue from patients with rheumatoid
arthritis or spondyloarthropathy. Interestingly, BMPR-
IA-positive cells have been detected in synovial tissue

from arthritic patients [39], and a role of the receptor
in the development of the arthritis has been discussed
[26].
The data presented here provide clear evidence that
the pro-form of BMP-2 can interact with BMPR-IA.
However, receptor binding of pro-forms of the
TGFb ⁄ BMP family appears to be complex, depending
on both the receptor type and the individual pro-form.
While indirect evidence has been obtained that a
97
116
kDa
proBMP-2
DSS
160
proBMP-2-
BMPR-IA-
complex
+
–
+
+
kDa
BMP-2
DSS
+
–
+
+
97

116
kDa
proBMP-2
DSS
160
proBMP-2-
BMPR-IA-
complex
+
–
+
+
116
97
66
97
116
kDa
proBMP-2
DSS
160
proBMP-2 -
BMPR-IA-
complex
proBMP-2
–
+
+
+
proBMP-2

WB: anti-BMP-2AB
CD
WB: anti-BMP-2 WB: anti-BMP-2
WB: anti-HA
BMP-2 -
BMPR-IA-
dimer
complex
BMP-2 -
BMPR-IA-
complex
Fig. 5. ProBMP-2 can be cross-linked to
recombinantly expressed BMPR-IA. COS-7
cells expressing BMPR-IA were incubated
with proBMP-2 (A,B,D) or BMP-2 (C). After
blotting, cell extracts (A,B) were decorated
with BMP-2 antibody (A) or with HA
antibody (B). Cross-linked ligand–receptor
complexes were immunoprecipitated using
BMPR-IA ECD antibody (C,D) and analysed
with BMP-2 antibody after blotting.
proBMP-2A
BC
BMP-2
P-Smad 1/5/8
Smad 1
P-p38
Actin
0 15 30 60 120 15 30 60 120 min
Time (min)

0 20 40 60 80 100 120 140
Time (min)
0 20406080100120140
0
20
40
60
80
100
120
Relative intensity (%)
Relative intensity (%)
0
20
40
60
80
100
120
P-Smad 1/5/8 signal intensity P-p38 signal intensity
Fig. 6. BMP-2 and proBMP-2 differ in their
ability to lead to Smad1 ⁄ 5 ⁄ 8 or p38 phos-
phorylation. C2C12 cells were treated with
10 n
M ligand for the indicated time periods.
(A) After blotting, whole-cell lysates were
analysed using antibodies against phosphor-
ylated (P-Smad1 ⁄ 5 ⁄ 8) and total Smad1 ⁄ 5 ⁄ 8,
phosphorylated p38 (P-p38), or actin as a
loading control. (B) Quantification of C-termi-

nally phosphorylated Smad1 ⁄ 5 ⁄ 8 in relation
to total Smad1 ⁄ 5 ⁄ 8. (C) Quantification of
phosphorylated p38 in relation to actin.
Circles, BMP-2; squares, proBMP-2.
The pro form of BMP-2 interferes with BMP-2 signalling A. Hauburger et al.
6392 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS
non-covalent complex of the pro-peptide and BMP-9
can bind to the type I receptor Alk1 [17], the latency-
associated polypeptide of TGFb inhibits the interac-
tion of TGFb isoforms with type II and III receptors
[40]. An even more complex situation has very recently
been described for the non-covalent complex of BMP-
7 and pro-peptides, which binds to type I receptors
and type II receptors, depending on the experimental
set-up [41]. Upon binding to the complex of mature
part and pro-domain(s), type II receptors are able to
dissociate the pro-domains from the complex [41]. As
we showed here that the ECD of BMPR-II is not
bound by proBMP-2, displacement of the covalently
bound pro-domain is unlikely. Moreover, our results
on the inhibitory effect of the free pro-peptide on com-
plex formation between BMPR-II and BMP-2 indicate
that, in this case, BMPR-II per se cannot rearrange
the pro-peptide. Generally, however, displacement of
the pro-peptide cannot be excluded although in cova-
lent peptide linkage, as the peptide region between the
pro-peptide and the mature part may be flexible and
thus allow sufficient conformational freedom to dis-
charge the pro-peptide part from a receptor binding
interface. Thus, the slower association kinetics of

proBMP-2 with noggin could be due to such a
displacement, induced by noggin.
In addition to the potential physiological implica-
tions of our findings, the interaction of proBMP-2 with
BMPR-IA and noggin allows indirect conclusions
about the position of the pro-peptide with respect to
the mature part. As neither the interaction with
BMPR-IA nor with noggin was affected in quantita-
tive terms, the pro-peptide moiety probably does not
obstruct the key residues of BMP-2 that mediate bind-
ing to either the receptor or noggin. This observation
is consistent with crystallographic results, which
revealed that the BMPR-IA binding site of the related
BMP-7 largely coincides with the noggin-binding area
[32,42]. Thus, the proBMP-2 binding to noggin
detected here indirectly confirms the interaction of
proBMP-2 with BMPR-IA. Conversely, as noggin cov-
ers part of the BMPR-II binding site of BMP-2, an
interaction of noggin with proBMP-2 is counter-intui-
tive given that proBMP-2 did not bind to BMPR-II.
Consequently, the pro-peptide masks at least the inter-
action site in BMP-2 for BMPR-II binding. On the
other hand, a recent NMR study showed that both the
ECD of BMPR-IA and BMP-2 undergo structural
transitions upon association [43], indicating that
discussions on ligand–receptor interactions have to
consider the high inherent flexibility of probably all
the partners involved. In what way BMPR-IA binding
by proBMP-2 may rearrange the pro-peptide moiety as
discussed above remains to be tested.

Although suppression of TGFb effects by latency-
associated polypeptide has been reported [44], the find-
ing that proBMP-2 inhibits AP induction by mature
BMP-2 was unexpected, as our previous results
had shown ectopic bone formation in response to
proBMP-2. In these studies, however, growth factor-
induced bone formation was assessed at a single time
point (30 days), and neither the pharmacokinetics of
proBMP-2 nor the kinetics of bone formation were anal-
ysed. Thus, the fate of administered proBMP-2 in the
animal over time is unclear and it cannot be excluded
that proBMP-2 becomes converted to mature BMP-2 by
extracellular proteases with time.
The fact that proBMP-2 elicited Smad-mediated
luciferase transcription in cell culture after 16 h is con-
sistent with a slow action of proBMP-2. The Smad
pathway is predominantly induced by ligand binding
to PFCs. Possibly, proBMP-2 signalling occurs pre-
dominantly via this binding mode. However, how
proBMP-2 transmits signals despite being unable to
bind BMPR-II is unclear, and is the next issue to be
addressed.
Experimental procedures
Recombinant proteins
Recombinant production and purification of BMP-2,
proBMP-2 and the pro-domain of BMP-2 were performed
as described previously [8]. The extracellular ligand binding
domain (ECD) of BMPR-IA was prepared and biotinylated
as described previously [31]. The BMPR-II ⁄ Fc chimera
(ECD) was purchased from R&D Systems (Minneapolis,

MN, USA). Noggin was produced in E. coli BL21 (DE3) as
a soluble maltose binding protein (MBP) fusion protein. To
Ligand concentration [nM]
100 0.1 0.5 1 20 50
RLU
0
2
4
6
8
10
12
14
16
18
Fig. 7. Activation of the Smad1 ⁄ 5 ⁄ 8 pathway by BMP2 and
proBMP2 as measured by reporter gene assay (BRE-luciferase).
C2C12 cells were stimulated with the indicated concentrations of
proBMP-2 and BMP-2 for 16 h. Black columns, BMP-2; grey
columns, proBMP-2.
A. Hauburger et al. The pro form of BMP-2 interferes with BMP-2 signalling
FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6393
overcome codon usage limitations, cells were additionally
transformed with plasmid pUBS520, which carries the gene
dnaY that encodes a rare tRNA for arginine codons in
E. coli [45]. Cultivation in LB medium was performed in
shake flasks at 37 °C. Noggin cDNA expression was
induced using 1 mm isopropyl thio-b-d-galactoside when the
cells had reached an attenuance at 600 nm of 0.5–0.8. Cells
were harvested 3 h after induction. A volume of 10 mL

buffer A (20 mm Tris ⁄ HCl pH 7.4, 200 mm NaCl, 1 mm
EDTA) was added per gram of cell pellet. Cells were dis-
rupted by high-pressure cell dispersion. After sedimentation
of cell debris at 9000 g for 30 min at 4 °C, the supernatant
was diluted at a ratio of 1 : 5 in buffer A, and loaded at a
flow rate of 1 mLÆmin
)1
on a 15 mL bed size amylose
column (New England Biolabs, Beverly, MA, USA). The
column was washed with 10 column volumes of buffer A.
Elution of the MBP–noggin fusion protein was achieved
using buffer A containing 10 mm maltose. Pooled elution
fractions were adjusted to a concentration of 8 m with solid
urea, and renaturation of the fusion protein was achieved by
fourfold dilution into refolding buffer (50 mm Tris ⁄ HCl pH
8.5, 1 m NaCl, 2 mm EDTA, 1 mm phenylmethanesulfonyl
fluoride, 25 mm Chaps, 2 mm oxidized glutathione and
0.2 mm reduced glutathione). The protein concentration
during renaturation was 100 lgÆmL
)1
. After 4 days of rena-
turation, the protein solution was concentrated using a
Vivaflow 200 ultrafiltration tube (Sartorius Vivascience,
Go
¨
ttingen, Germany), and was then dialysed against 20 mm
Tris ⁄ HCl pH 8.5, 150 mm NaCl. For proteolytic MBP
removal, the protein solution (0.35 mgÆml
)1
) was incubated

at room temperature for 24 h with 0.5% w ⁄ w tobacco etch
virus (TEV) protease related to the fusion protein content.
Upon proteolysis, noggin precipitated and was recovered by
a 20 min centrifugation at 48 000 g. Precipitated protein was
further purified by reverse-phase high-performance liquid
chromatography. Protein resuspended in solvent A (0.1%
v ⁄ v trifluoroacetic acid in 20% v ⁄ v aqueous acetonitrile) was
loaded at a flow rate of 0.7 mLÆmin
)1
onto a SOURCE 15
reverse phase chromatography column (GE Healthcare,
Munich, Germany). The column was washed with three
column volumes of solvent A. Elution of noggin was
achieved using a non-linear gradient of four column volumes
of 0–37.5% solvent B (0.1% v ⁄ v trifluoroacetic acid in
aqueous acetonitrile) and 1.5 column volumes of 37.5–100%
solvent B. For some experiments, noggin from R&D Systems
was used as a reference, and was found to behave identically.
Cell culture
C2C12 cells (DSMZ, Braunschweig, Germany) were main-
tained in RPMI-1640 medium (PAA Laboratories GmbH,
Co
¨
lbe, Germany) supplemented with 10% fetal bovine
serum at 37 °Cin5%CO
2
. To allow differentiation and
induction of AP, the serum concentration was reduced
to 2%.
Cross-linking of ligands to transiently transfected

COS-7 cells
Transfection and cross-linking of ligand–receptor complexes
were performed as described previously [4]. COS-7 cells
were grown in six-well plates (10 cm
2
per well) in
Dulbecco’s modified Eagle’s medium with 10% fetal bovine
serum. Transfection with RotifectÔ (Roth, Karlsruhe, Ger-
many) was performed according to the supplier’s instruc-
tions. To each well, 2 lg pcDNA-3.1-BRIA-HA [46] was
added for expression of HA-tagged BMPR-IA. Forty-eight
hours after transfection, cells were washed twice in buf-
fer W (50 mm Hepes pH 7.5, 128 mm NaCl, 1.2 mm CaCl
2
,
5mm MgSO
4
,5mm KCl, 2 mg ÆmL
)1
BSA). Subsequently,
10 nm BMP-2 or proBMP-2 in buffer W were added and
allowed to interact with surface receptors for 3 h at 4 °C.
Cross-linking was performed as described previously [47].
After cross-linking, cells were solubilized in 100 lL lysis
buffer per well [NaCl ⁄ P
i
pH 7.4, 0.5% Triton X-100, 1 mm
EDTA and protease inhibitor cocktail (Roche Diagnostics,
Mannheim, Germany)] at 4 °C for 40 min. Cell extracts
were analysed by western blotting or after immunoprecipi-

tation. Receptor complexes were immunoprecipitated using
BMPR-IA ECD antibodies (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) at a ratio of 5 lgÆmL
)1
cell extract
for 2 h at 4 °C. Then 25 lL of the solution was added to
25 lL protein A–Sepharose slurry (GE Healthcare) in lysis
buffer for 1 h at 4 °C. After washing the beads three times
with NaCl ⁄ P
i
, SDS–PAGE sample buffer containing b-mer-
captoethanol was added, and the samples were heated for
3 min at 95 °C. All protein samples were run on 4–12%
Bis–Tris pre-cast gradient gels (Invitrogen, Karlsruhe,
Germany). Western blotting was performed according to
standard protocols. After blocking (10 mm Tris pH 7.9,
150 mm NaCl, 0.5% Tween-20, 3% BSA), blots were incu-
bated with 10 lgÆmL
)1
HA antibody (Covance, Emeryville,
CA, USA) or 0.4 lgÆmL
)1
BMP-2 antibody (Dianova,
Hamburg, Germany). Detection was achieved using horse-
radish peroxidase-coupled secondary antibody with the
ECL system (GE Healthcare).
Alkaline phosphatase (AP) assay and
luciferase-based reporter gene assay
C2C12 cells (2 · 10
3

per well) were seeded into 96-well
plates. Cells were allowed to attach overnight, and then
complete medium (10% fetal bovine serum) was replaced
by 200 lL differentiation medium (2% fetal bovine serum)
supplemented with the growth factors. After 4 days, the
medium was removed, cells were washed with NaCl ⁄ P
i
and
lysed in 100 lL lysis buffer [100 mm glycine ⁄ Na
+
,1mm
MgCl
2
,1mm ZnCl
2
pH 9.6, 1% Nonidet P-40 (Applichem,
Darmstadt, Germany)] by gentle shaking at room tempera-
ture for 2–3 h. 20 lL lysate was transferred to a new
96-well plate. Then 200 lL substrate solution (9 mm
The pro form of BMP-2 interferes with BMP-2 signalling A. Hauburger et al.
6394 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS
p-nitrophenyl phosphate in lysis buffer) was added per well
[29]. Changes in absorption at 405 nm were followed over
30 min using an ELISA plate reader. For determination of
inhibition by noggin, the AP assay was slightly modified:
1 · 10
4
cells were seeded and tests were performed after
3 days with 100 lL substrate solution per well. For the
luciferase-based reporter gene assay, C2C12 cells were

seeded into 48-well plates at a density of 10
4
cells per well
under normal growth conditions. After 24 h, cells were
transfected with a construct containing firefly luciferase dri-
ven by the BMP response element (pBRE-Luc) [48] and a
constitutive active Renilla luciferase (pRLTK) as an internal
control using Lipofectamine 2000 (Invitrogen, Life Technol-
ogies, Carlsbad, CA, USA) according to the manufacturer’s
instructions. The next day, cells were starved for 5 h and
stimulated with ligand in medium containing 0.5% fetal
bovine serum for 16 h. Luciferase activity was measured
using the dual luciferase reporter assay system (Promega,
Madison, WI, USA) and a Mithras LB 940 luminometer
(Berthold Detection Systems, Pforzheim, Germany).
Test for phosphorylated p38 and Smad
The test was performed as described previously [49]. C2C12
cells were seeded in six-well plates (2 · 10
5
cells per well).
After 24 h, cells were starved for 3 h and then incubated in
the absence or presence of BMP-2 (10 nm) or proBMP-2
(10 nm) for the indicated time. Cells were washed with
NaCl ⁄ P
i
and lysed in 200 lL SDS–PAGE sample buffer
(60 mm Tris ⁄ HCl pH 6.8, 2% SDS, 10% glycerol, 15%
b-mercaptoethanol, 0.01% bromophenol blue) per well. After
boiling at 95 °C for 5 min, protein was separated by SDS–
PAGE and blotted to poly(vinylidene difluoride) membranes.

Membranes were blocked with TBST (TBS containing 0.1%
Tween-20) containing 5% BSA at room temperature for
60 min. Membranes were washed three times for 10–15 min
in TBST. For antibody staining, the primary antibodies (anti-
phospho-p38 and anti-phospho-Smad1 ⁄ 5 ⁄ 8, Cell Signaling
Technology Inc., Beverly, MA, USA) were diluted 1 : 1000 in
TBST containing 5% BSA. Detection was performed using a
secondary horseradish peroxidase-coupled antibody using the
ECL system and ChemiSmart 5000 (Peqlab, Erlangen,
Germany). Phosphorylation was quantified relative to total
Smad (anti-Smad, Cell Signaling Technology Inc.) or actin
(anti-b-actin, Sigma-Aldrich, St Louis, MO, USA). For quanti-
fication, the maximum phosphorylation level was set to 100%.
Surface plasmon resonance
Binding of BMP-2 and proBMP-2 to the immobilized pro-
teins was examined using the BIA2000 system (BIAcore,
Uppsala, Sweden). Biotinylated BMPR-IA ECD or noggin
(sulfo-NHS-LC-biotin, Pierce ⁄ Thermo Fisher, Rockford, IL,
USA) were immobilized on streptavidin-coated chips at a
density of approximately 300 resonance units for the ECD or
64 resonance units for noggin. For BMPR-II–Fc, 1000 reso-
nance units were immobilized on a CM5 sensorchip (GE
Healthcare) by amine coupling. BMP-2 or proBMP-2 (in
10 mm Hepes pH 7.4, 500 mm NaCl, 3.4 mm EDTA,
0.005% Tween-20) were injected at the indicated concen-
trations. Sensorgrams were recorded at a flow rate of
30 lLÆmin
)1
at 25 °C. Regeneration of the ECD-coated chip
surface was achieved by perfusion with 10 mm glycine pH 2,

and regeneration of the noggin-coupled chip surface by 6 m
guanidinium chloride ⁄ acetic acid pH 2.0, 1 m NaCl. Correc-
tion for background signals was performed by subtraction of
the signals from the control flow cell. Non-specific binding
was negligible. The sensorgrams were evaluated using the
software BIAevaluation version 2.0 (biacore) assuming a
1 : 1 interaction. Although one BMP-2 dimer is known to
interact with two ECD monomers [42], a 1 : 1 interaction
has been used previously for the assessment of relative bind-
ing affinities [29]. Association and dissociation rate constants
were obtained from 7–9 analyte concentrations. Mean values
with a standard deviation were deduced from five indepen-
dent measurements. Apparent dissociation constants, K
D
,
were calculated as K
D
= k
d
⁄ k
a
.
The influence of the free pro-peptide on BMP-2 binding
to BMPR-II was tested using 400 nm BMP-2 that had been
pre-incubated for 2 h at room temperature with increasing
pro-peptide concentrations. Binding to BMPR-II was then
detected by recording equilibrium binding via BIAcore. To
this end, the time interval for the association was prolonged
until a constant maximal response representing an equilib-
rium was observed. Signals were then plotted against the

pro-peptide concentrations.
ELISA
For the assays, a previously published protocol [50] was
modified. Tests were performed in 96-well microtitre plates
(Nunc-ImmunoÔ Maxisorp, Nunc, Wiesbaden, Germany)
at room temperature. Washing was performed with 200 lL
10 mm NaCl ⁄ P
i
pH 7.4. Wells were coated with 100 lLof
either 100 nm BMP-2 or 100 nm proBMP-2 in 50 mm car-
bonate buffer, pH 8, at 4 °C overnight. Free sites were
blocked for 2 h at room temperature using 200 lL blocking
buffer per well [5% BSA (Sigma), 10 mm NaCl ⁄ P
i
pH 7.4,
0.05% Tween-20]. After washing, the BMPR-IA ECD was
added at the indicated concentrations in blocking buffer
and incubated for 1 h. Tests for non-specific binding were
performed using blocking buffer alone. After three washing
steps, goat human BMPR-IA ECD antibody (Santa Cruz
Biotechnology) was added to a final concentration of
0.2 lgÆmL
)1
. Detection was performed using goat IgG
horseradish peroxidase-conjugated antibody (Perbio Sci-
ence, Bonn, Germany) at a concentration of 0.4 lgÆmL
)1
in
blocking buffer. Photometric detection was achieved using
0.5 mgÆmL

)1
2,2¢-azino-bis(3-ethylbenzthiazoline-6-sulpho-
nic acid (ABTS; Roche Diagnostics) in ABTS buffer.
A. Hauburger et al. The pro form of BMP-2 interferes with BMP-2 signalling
FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS 6395
Changes of absorption at 405 nm were followed every 30 s
for 1 h using a microtitre plate reader (MR 7000, Dynex
Technologies, Denkendorf, Germany), and the slopes were
calculated.
Acknowledgements
This work was funded by the Land Sachsen-Anhalt
through the program ‘Structures and Mechanisms
of Biological Information Processing’ and a grant
from the Deutsche Forschungsgemeinschaft (DFG
SCHW375 ⁄ 5-1) to E.S.
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Supporting information
The following supplementary material is available:
Fig. S1. The pro-peptide does not cause AP induction.
Fig. S2. ProBMP-2 is maintained stably in cell culture
over 49 h.
Fig. S3. At high concentrations, proBMP-2 induces
immediate Smad phosphorylation.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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should be addressed to the authors.
The pro form of BMP-2 interferes with BMP-2 signalling A. Hauburger et al.
6398 FEBS Journal 276 (2009) 6386–6398 ª 2009 The Authors Journal compilation ª 2009 FEBS