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Regulation of Bone Morphogenetic Protein 9 (BMP9) by
Redox-dependent Proteolysis*
Received for publication, May 7, 2014, and in revised form, September 10, 2014 Published, JBC Papers in Press, September 18, 2014, DOI 10.1074/jbc.M114.579771
Zhenquan Wei1, Richard M. Salmon, Paul D. Upton, Nicholas W. Morrell2,3, and Wei Li2,4
From the Department of Medicine, University of Cambridge, School of Clinical Medicine, Box 157, Addenbrooke’s Hospital,
Hills Road, Cambridge CB2 0QQ, United Kingdom
Background: Bone morphogenetic protein 9 (BMP9) circulates at low concentrations maintaining endothelial integrity.
BMP9 has potent bone-forming activity at high concentrations.
Results:The intermolecular disulfide bond in BMP9 is regulated by redox potential; the loss of which renders BMP9 susceptible
to degradation by proteases present in serum.
Conclusion: BMP9 is regulated by redox-dependent proteolysis.
Significance:Optimum circulating BMP9 levels are essential for endothelium-specific activity.
BMP9, a member of the TGF superfamily, is a homodimer
that forms a signaling complex with two type I and two type II
receptors. Signaling through high-affinity activin receptor-like
kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a
vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic
signaling in mesenchymal stem cells in vitro and promoting
bone formation in vivo. This activity requires ALK1, the lower
affinity type I receptor ALK2, and higher concentrations of
BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential tomaintain the highly specific endothelial-protective function. Factors regulating BMP9 stability
and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers
could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were
capable of binding to the prodomain and ALK1, the M-form
demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by
changing the redox potential, and this redox switch caused a
major alteration in BMP9 stability. The M-form displayed
greater susceptibility to redox-dependent cleavage by proteases
present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide
new rationales for approaches to modify BMP9 levels for therapeutic purposes.
BMP9 is a circulating vascular quiescence factor (1), one of
only two BMP5 ligands that specifically activate the endothelial
ALK1/bonemorphogenetic protein receptor type II (BMPR-II)
pathway (2). ALK1 is an endothelial-specific type I receptor (3),
and BMPR-II is a type II receptor for the large family of BMP
ligands (4). ALK1 and BMPR-II play essential roles in early
developmental processes. Homozygous knock-out ALK1 or
BMPR-II in mice are embryonic lethal due to defects in early
heart and vessel development (5, 6). Humanmutations inALK1
lead to type II hereditary hemorrhagic telangiectasia (3), a vascular dysplasia of multiple telangiectasias and arteriovenous
malformations in internal organs typically affecting the lung,
brain, gastrointestinal tract, and liver, which can cause lifethreatening hemorrhage (7). Mutations in BMP9 have been
identified in patients with a vascular disorder phenotypically
overlapping with hereditary hemorrhagic telangiectasia (8).
Human mutations in BMPR-II are the commonest genetic
cause of pulmonary arterial hypertension (PAH), characterized
by increased pressure in the pulmonary circulation due to narrowing of the lung blood vessels, which leads to right ventricular hypertrophy and death within a few years of diagnosis (9,
10). ALK1 mutations were found in occasional PAH patients
(11, 12), and ALK1/ mice spontaneously develop PAH (13).
Endothelial dysfunction, characterized by endothelial cell
apoptosis and increased endothelium permeability, is a recognized trigger for PAH, and the ALK1/BMPR-II pathway plays
an essential role in maintaining the endothelium integrity (14,
15). Loss of BMPR-II predisposes human pulmonary artery
endothelial cells (hPAECs) to apoptosis and BMPs can inhibit
hPAEC apoptosis induced by serum starvation (16). BMPR-II
loss leads to the increased permeability of the hPAEC monolayer, reduced endothelial barrier function (14), and PAH (15).
Moreover, targeted gene delivery of BMPR-II to the pulmonary
endothelium attenuates PAH in rodent models (17).
* This work was supported by the British Heart Foundation Grants PG/12/54/
29734 (to W. L.) and CH/09/001/25945 (to N. W. M.).
Author’s Choice—Final version full access.
The atomic coordinates and structure factors (code 4MPL) have been deposited in
the Protein Data Bank (http://wwpdb.org/).
1 Present address: Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China.
2 Joint senior authors.
3 To whom correspondence may be addressed. Tel.: 44-1223-331666; E-mail:
nwm23@cam.ac.uk.
4 To whom correspondence may be addressed. Tel.: 44-1223-736862; Fax:
44-1223-336846; E-mail: wl225@cam.ac.uk.
5 The abbreviations used are: BMP, bone morphogenetic protein; ALK1,
activin receptor-like kinase 1; BMPR-II, bone morphogenetic protein receptor type II; ECD, extracellular domain; pBMP9 (pHBMP9), prodomain bound
BMP9 (HBMP9) complex; hPAEC, human pulmonary artery endothelial cell;
PAH, pulmonary arterial hypertension; pSmad1/5/8, phosphorylated
Smad1/5/8; EGM, endothelial growth medium; D-form (M-form), dimeric
(monomeric) form on the non-reducing SDS-PAGE.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 45, pp. 31150 –31159, November 7, 2014
Author’s Choice © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
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The endothelial ALK1/BMPR-II pathway is constitutively activated by circulating BMP9, the only confirmed BMP that circulates at active concentrations (18, 19). BMP9 is produced by hepatocytes as the prepro-form and is processed by proprotein
convertase in the trans-Golginetwork intoprodomainandmature
BMP9 ligand (20). Theprodomain of BMP9 forms a complexwith
mature BMP9 in the circulation but does not affect BMP9 signaling activity (18). BMPR-II on the surface of hPAECs undergoes
rapid turnover (21), and BMP9 induces BMPR-II expression in
endothelial cells in an ALK1-dependent manner (22).
In addition to its role in the vascular endothelium, BMP9
signaling activity has also been demonstrated in mesenchymal
stem cells and C2C12 myoblasts. Among 14 BMPs tested,
BMP9 has the highest osteogenic signaling activity in vitro and
potently induces bone formation in vivo (23, 24). No defects in
bone or cartilage have been reported in the BMP9 knock-out
mice (25, 26), and the role of BMP9-induced osteogenic activity
in human physiology is not fully understood, but such potent
osteogenic potential of BMP9 raises the question why circulating BMP9 does not show osteogenic activity in blood vessels.
One possible reason for this is the concentration of BMP9.
Circulating levels of BMP9 are between 2–10 ng/ml measured
by activity (18, 19) and 300 pg/ml by ELISA (27). BMP9 signaling in endothelial cells is mediated by the high-affinity
receptor, ALK1 (28), whereas BMP9 osteogenic signaling activity requires both ALK1 and the low-affinity receptor, ALK2
(29). Whereas the EC50 of BMPs activating ALK2, ALK3, and
ALK6 is in the range of 50 ng/ml, BMP9 is particularly potent in
activating ALK1, with an EC50 of 50 pg/ml (18). Whereas other
unknown factors may contribute to the non-osteogenic, endothelial-specific ALK1-mediated signaling by BMP9, optimum
concentrations of circulating BMP9maybe an important factor
essential for maintaining endothelial specificity.
BMP9 is constitutively produced by the liver and secreted
into the circulation. We hypothesized that there may be mechanisms in place to regulate circulating BMP9 at the optimum
levels and activities for ALK1-specific signaling. We addressed
this question by characterizing recombinant BMP9 produced
in mammalian cells and purified under native, non-denaturing
conditions. We demonstrated that BMP9 stability and activity
was regulated by redox potential as well as proteolysis. The
redox-dependent cleavage of the non-covalently linked BMP9
dimer would provide a controlled natural degradation pathway
for BMP9. Such redox-dependent cleavage would suggest that
although there is a constant degradation of the BMP9 from the
circulation, a fraction of BMP9 (covalently linked BMP9 dimer)
remains stable and resistant to proteolysis, ensuring the constitutive activation of the endothelial ALK1/BMPRII pathway to
maintain the homeostasis of the vascular endothelium.
EXPERIMENTAL PROCEDURES
Materials—Anti-BMP9 antibody (MAB3209 and AF3209),
anti-BMP9 prodomain antibody (AF3879), and control BMP9
were purchased from R&D Systems, Inc. Anti-His tag antibody
(37–2900) was purchased from Invitrogen. Prodomain-bound
BMP9(pBMP9)andBMP6werekindgifts fromPfizer.Anti-phospho-Smad1/5/8 antibody was purchased from Cell Signaling
Technology.HiTrapnickel-nitrilotriacetic acid,HiTrapQFF, and
Superdex 10/30 columns were purchased from GE Healthcare.
hPAECs and endothelial growth medium (EGM-2) were purchased from Lonza, UK. All other tissue culture media were purchased from Invitrogen. Crystallization reagents were purchased
from Hampton Research, Inc. All plasmid purification kits were
purchased fromQiagen.
BMP9 Expression and Purification—Human full-length proBMP9 cDNA was cloned into pCEP4 between the HindIII and
XhoI sites and verified by DNA sequencing. To facilitate the
purification of mature BMP9, a His6 tag was introduced
immediately after the putative furin cleavage site to generate
pro-HBMP9. Plasmids containing pro-HBMP9 were transfected into HEK-EBNA cells using polyethylenimine in
DMEM medium containing 5% FBS. Cells were changed into
CDCHO expression medium without serum the following day,
and conditioned media were harvested after 3–4 days. To
purifyHBMP9, 5 liters of conditionedmediawere concentrated
using aVivaflow 200 concentrator (SartoriusAG) to 200ml and
dialyzed against 4 liters of 20mMTrisHCl, pH7.4, 250mMNaCl
at 4 °C overnight. Samples were loaded onto a 5-ml nickel-nitrilotriacetic acid column in 1 binding buffer (5 mM TrisHCl,
pH 7.4, 500 mM NaCl, 5 mM imidazole). After extensive washing
with 1binding buffer, bound fractionswere elutedwith an imidazole gradient (5–250 mM) over 10 column volumes. Fractions
were analyzed by non-reducing SDS-PAGE, and those containing
a mixture of partially processed pro-HBMP9, prodomain, and
matureHBMP9werepooledanddialyzedagainst20mMTrisHCl,
pH 7.4, and loaded onto a 1-ml Q Sepharose column. HBMP9
eluted in theunbound fraction at95%purity andwasused in the
characterization and crystallization assays.
BMP Response Element Luciferase Assays in C2C12 Cells—
C2C12 cells were seeded at 4  104 cells/well in 24-well plates.
The next day, cells were washed once and incubated in
OptiMEM I for 2 h. All wells were then co-transfected with
plasmids containing BMP response element luciferase (courtesy of Professor P. ten Dijke, Leiden University Medical Center, Leiden, Netherlands) and Renilla luciferase. In the wells for
testing BMP9 binding to ALK1, pcDNA3-hALK1 (courtesy of
ProfessorR. C.Trembath, King’sCollege London, London,UK)
was also added. After 4 h, the transfection mixtures were
removed, and DMEM containing 10% FBS and antibiotic-antimycotic (Invitrogen) added for 24 h. Cells were washed twice
with serum-free DMEM containing antibiotic, incubated in
serum-free DMEM for 10 h and then treated for 18 h with
dilutions of recombinant BMP9 proteins. At the end of the
treatment, cells were lysed and assayed for firefly and Renilla
luciferase activities using the Dual-Glo luciferase assay kit
(Promega) according to themanufacturer’s instructions. Firefly
luciferase activities were normalized to the Renilla control.
BMP9 Crystallization and Structure Determination—Crystallization trials were set up with a complex containingHBMP9
and ALK1 extracellular domain (ECD) (1:1 ratio, 4 mg/ml in 20
mMTrisHCl, pH7.4, 50mMNaCl), and diffraction quality crystals were obtained in 0.12 M Mg(NO3)2, 12% PEG3350 over 3
days. Analysis of the washed crystals by non-reducing SDSPAGE revealed that the crystals contained equal amounts of DandM-forms of BMP9, but not ALK1 ECD. Crystals were cryoprotected in 0.12MMg(NO3)2, 22%PEG3350, 20%glycerol, and
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data were collected at 100 K at Diamond Light Source I04 from
a single crystal. Data were processed using Mosflm, Scala, and
Truncate (30) to 1.90 Å, and the structure was solved bymolecular replacement using 1ZKZ as search model (31, 32). Model
building was carried out using Coot (33) and refinement with
REFMAC5 (34) and Phenix refine (35). Figures were produced
using Coot and PyMOL (The PyMOLMolecular Graphics System, version 1.2r3pre, Schrödinger, LLC.). Crystallographic
data and refinement statistics are given in Table 1, and the
coordinate files were deposited in the Protein Data Bank under
the accession code 4MPL.
BMP9 Signaling in hPAEC—HBMP9 C73S and control
HBMP9 were expressed in HEK-EBNA cells. To evaluate the
concentrations of BMP9 in the conditioned medium, we used
Western blot of the conditioned media against an anti-BMP9
prodomain antibody. This is because BMP9 C73S cannot be
detected with commercially available anti-BMP9 antibodies,
and the BMP9 prodomain is produced in the same peptide
chain and processed in a 1:1 ratio with mature BMP9 upon
secretion. The ratio of prodomain of C73S to wild type in the
transfected medium should be identical to the ratio of mature
ligands. The concentration of wild type BMP9 was determined
by ELISA using R&DBMP9 as standards and the concentration
of theC73Smutantwas calculated from the ratio obtained from
the prodomainWestern blot. For signaling assays, hPAECwere
seeded in 60-mm dishes at 3  105 cells/dish and cultured for
24 h in EGM-2 medium with 10% FBS. Cells were quiesced in
EGM-2 medium with 0.1% FBS for 16 h before treatment with
BMP9. At the designated time points, the medium was aspirated, and dishes were placed on dry ice to stop the signaling
reaction before lysis buffer (125 mM TrisHCl, pH 6.8, 2% SDS,
and 10% glycerol) was added. The protein concentration in the
total cell lysate was determined usingDCTM protein assay (BioRad), and 25–35 g of total cell protein was used for immunoblotting. BMP9 signaling was monitored by the phosphorylation of Smad1/5/8 detected by anti-pSmad1/5/8 antibody.
-Tubulin was used as a loading control.
BMP9 Redox Assay and Redox-dependent Cleavage—BMP9
redox assays were carried out by redox titration with glutathione (GSH)/glutathione disulfides (GSSG) adapted from Zhou
et al. (36). HBMP9 or pBMP9 were incubated with PBS or PBS
containing 0.1 mM GSSG with 0 to 20 mM GSH at room temperature overnight in the presence of protease inhibitor (Complete-EDTA, RocheDiagnostics). Samples were fractionated by
12% SDS-PAGE under non-reducing conditions and stained
with Coomassie Blue (Simply Blue, Invitrogen).
For redox-dependent proteolysis, HBMP9 or pBMP9 were
treated as above, but in the absence of protease inhibitor. The
following day, trypsin was added at the indicated concentrations (w/w), and samples were incubated for 3 h for HBMP9 or
overnight for pBMP9. All samples were fractionated on a 12%
non-reducing SDS-PAGE and stained with Coomassie Blue to
detect the cleavage products.
RESULTS
Expression and Purification of Human BMP9 fromMammalian Cells—BMP9 is synthesized as the prepro-form and processed into the mature ligand and the prodomain during secretion (Fig. 1A). To ensure the proper folding and processing of
BMP9, we used a mammalian transient transfection system to
overexpress human BMP9. Full-length pro-BMP9 and proHBMP9 (Fig. 1B) were overexpressed in HEK-EBNA cells, and
transfection supernatants were blotted against anti-BMP9 antibody (Fig. 1C). Similar to the pulse-chase experiment from
CHO cells transfected with pro-BMP7 (37), both dimeric
(D-form) and monomeric (M-form) forms of BMP9, together
with the partially processed pro-BMP9, could be readily
detected (Fig. 1C). Activity assays using BMP response element
luciferase-transfected C2C12 cells and BMP9 transfection
media showed that pHBMP9 has the same signaling activity as
pBMP9 and BMP9 from R&D Systems (Fig. 1D).
The mature ligand HBMP9 was purified to 95% purity and
both D- and M-forms were co-purified (Fig. 1E). We also
obtained pBMP9, a purified protein complex of prodomain
bound human BMP9 (a kind gift from Pfizer), representing the
circulating form of BMP9 in human (Fig. 1E). The identities of
the bands of pBMP9 and HBMP9 in Fig. 1E were confirmed by
Western blot using antibodies against BMP9, prodomain, and
His tag, respectively (Fig. 1F). Both forms of BMP9 andHBMP9
reacted with anti-BMP9 antibody, and only prodomain reacted
with the anti-prodomain antibody. Anti-His antibody reacted
with both D- andM-forms ofHBMP9, butmuchmore potent in
detecting M-form BMP9.
Although pBMP9 was generated from a totally different
source and by different purification methods, a similar ratio of
D- and M-forms of BMP9 was present in the pBMP9 as in
HBMP9. Because the presence of stable BMP9 in the absence of
an intermolecular disulfide bond and its co-existence with the
disulfide-linked form has not been reported for the TGF
superfamily ligands, we investigatedwhether the disulfide bond
plays a role in regulating BMP9 activity and stability. In the
following sections, pBMP9 represented the circulating form
andHBMP9 representedmatureBMP9, akin to the commercial
BMP9 (R&D Systems) that has been used in most of the literature to date.
The M-form BMP9 Is a Non-covalently Linked Dimer—
Forming a stable dimer is essential for BMP signaling activity
because the minimum signaling unit for BMP comprises one
BMP dimer, two copies of the type II receptor, and one copy
of the type I receptor (38, 39). To determine whetherM-form
BMP9 is a monomer or dimer in solution, semi-purified
HBMP9 was passed through a gel filtration column. The Dand M-forms of BMP9 were co-eluted under a single peak,
which was confirmed by analyzing the fractions (B3 to B5) on a
non-reducing SDS-PAGE (Fig. 2A). To confirm the presence of
the intermolecular disulfide bond in the D-form and to further
delineate any local conformational differences between the two
forms of BMP9, crystallization trialswere set up, anddiffraction
quality crystals were grown within a week. A single crystal contained both D- and M-forms of HBMP9 (Fig. 2B). Because two
previously published BMP9 structures were either in the
M-formor theD-form and crystallized under different proteinprotein interaction contexts (32, 40), our crystal is unique in
addressing the local differences between D- and M-forms of
BMP9 under identical conditions. We solved the structure to
1.9 Å with a space group I4122 (Table 1). Similar to the pubRedox-dependent Proteolysis of BMP9
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FIGURE 1. Expression, activity, and purification of recombinant BMP9. A, schematic diagram of BMP9 production and processing. B, schematic diagram of
the generation of HBMP9. A His tag (filled star) is introduced at the beginning of the mature BMP9. C, conditioned medium from HEK-EBNA cells transfected with
prepro-BMP9 (lane 1) or prepro-HBMP9 (lane 2) were separated on a non-reducing SDS-PAGE and probed with anti-BMP9 antibody (MAB3209). The diagram on
the right shows the schematic drawing of the BMP9 molecules. D denotes dimer on non-reducing SDS-PAGE, and M denotes monomer on SDS-PAGE. D- and
M-forms migrate slower in the HBMP9 due to the addition of the His6 tag at the N terminus of the mature ligand as depicted in B. D, HEK cell-produced BMP9
and HBMP9 have comparable activity with BMP9 from R&D Systems. Conditioned media containing pBMP9 (because it is very likely to be present as a
prodomain bound complex in the conditioned media) or pHBMP9 were quantified by ELISA using R&D BMP9 as a standard and subjected to signaling assay
using C2C12 cells transfected with ALK1. Plasmid containing Renilla was co-transfected with reporter plasmid containing BMP response element-luciferase,
and the luciferase activity induced by BMP9 signaling was read using the Promega Dual-Luciferase system. E, purified pBMP9 and HBMP9 were fractionated on
a 12% non-reducing SDS-PAGE and stained by Coomassie Blue. F, identical samples of R&D Systems BMP9, pBMP9, and HBMP9 were run in parallel on three
12% non-reducing SDS-PAGE, then blotted separately against the following: anti-BMP9 antibody (left), anti-BMP9 prodomain antibody (middle), and anti-His
tag antibody (right). A single asterisk indicates a minor band that could not be seen on the SDS-PAGE in E but reacted very strongly with anti-BMP9 antibody.
This may be a species of partially processed BMP9. Double asterisks indicate nonspecific carrier protein from R&D Systems BMP9. Pro, prodomain.
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lished BMP9 structure (Protein Data Bank code 1ZKZ), there
was only oneHBMP9monomer in an asymmetric unit, forming
a canonical BMP dimer with the symmetry-related molecule.
This is the highest resolution BMP9 structure reported so far,
showing an overall well ordered molecule with an average B
factor of 32.6. Indeed, Cys-73, the critical amino acid involved
in disulfide bond formation, was in two conformations as demonstrated by clear electron density (Fig. 2C), with an intermolecular
disulfide bond formed between the two monomers in one of the
conformations. Interestingly, no other major difference could be
observed for the remaining residues between theD- andM-forms.
Overlaying the HBMP9 structure with the previously published
BMP9 structures revealed that the core regions of BMP9 were
almost identical. Loops contacting the type I and type II receptors
had diverged conformations. These are also the regions with the
highest B factors in all BMP9 structures, indicating an intrinsically
higherdegreeof flexibility at these regions (Fig. 2D).This structure
confirms that stable BMP9 dimers can form with or without an
intermolecular disulfide bond.
Both D- and M-forms of BMP9 Can Bind to ALK1 and
Prodomain—We next investigated whether the M-form
BMP9 maintained protein-protein interactions known for the
D-form. Because BMP9 binds to ALK1 ECD with high affinity
and circulates as a complex with its prodomain, we examined
whether the M-form could form a complex with ALK1 ECD
and BMP9 prodomain using native PAGE followed by SDSPAGE (Fig. 3, A and B). HBMP9 migrates as a single band on a
native PAGE, containing both D- and M-forms (Fig. 3A, band
1). In the presence of excess ALK1 ECD (Fig. 3A, band 3), both
D- and M-forms of BMP9 formed complexes with ALK1 ECD
and shifted to a new band (Fig. 3A, band 2). To investigate the
prodomain-BMP9 interaction, pBMP9 was run on a native
PAGE. It fractionated into three bands on the native PAGE.
SDS-PAGE analysis of bands 4–6 revealed that both D- and
FIGURE 2. M-form BMP9 is a non-covalently linked dimer. A, HBMP9 was loaded onto a Superdex 75 10/30 gel filtration column pre-equilibrated in 50 mM
TrisHCl, pH 7.4, containing 150 mM NaCl. Non-reducing SDS-PAGE of the peak fractions (B3 to B5) revealed the D- and M-forms of HBMP9 co-elute under the
same peak. The doublet in the M-form on SDS-PAGE was probably due to a partial reduction of intramolecular disulfide bonds. B, a representative of the HBMP9
crystal (left) and two examples of washed single crystals ran on a non-reducing SDS-PAGE (right) demonstrated that each single crystal contains a mixture of Dand M-forms of HBMP9. C, crystal structure of HBMP9 (left), colored according to the B-factors (spectrum, blue to white to red, from 20 to 100) with Cys-73 in two
conformations shown in sticks. Electron density (2Fo  Fc map at 1) clearly shows two conformations of Cys-73 (right). D, HBMP9 was overlaid with the
published BMP9 structures (Protein Data Bank codes 1ZKZ and 4FAO, all colored as described in C). In the semi-transparent schematic, type I receptor ALK1 is
shown in yellow, and activin receptor type 2B is shown in cyan as in BMP9ALK1activin receptor type 2B complex (Protein Data Bank code 4FAO).
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M-forms of BMP9migrate as a single entity, either as free form
(band 4) or in the prodomain bound complex (band 5). These
data agree with the crystal structure that in solution, M-form
was indistinguishable from the disulfide-linked D-form. M-form
co-migrated with the D-form on the native PAGE, both in free
form, and in complexeswithALK1ECDorprodomain, indicating
M-form BMP9 can form complexes with ALK1 ECD and the
prodomain as the D-form.
The Intermolecular Disulfide Bond Is Not Required for BMP9
Signaling Activity—To investigate whether the intermolecular
disulfide bond is required for BMP9 signaling activity, a
HBMP9 C73S mutant was generated where Cys-73 was substituted with Ser, and it could only exist as the M-form. After
quantification of the mature BMP9 in the conditioned media
using the prodomain Western blot (Fig. 4A), a signaling assay
using Smad1/5/8 phosphorylation was carried out in hPAECs.
As shown in Fig. 4B, although slightly less active than wild type
at low concentrations, C73S mutant is active in inducing
Smad1/5/8 phosphorylation. However, a time course experiment revealed that at 1 ng/ml, whereas wild type BMP9 signal
lasted for at least 6 h, HBMP9 C73S almost completely lost its
signaling activity at 2 h (Fig. 4C). This indicates that although
the intermolecular disulfide bond did not affect BMP9 receptor
binding and the ability to signal, it could affect its stability and
hence the half-life of BMP9.
BMP9 Is Regulated by Redox Potential—To investigate the
stability of BMP9, we first asked whether D- and M-forms of
BMP9 could be converted into each other. Incubating HBMP9
with 0.5mM thiol-oxidizing agent diamide (41) overnight could
not promote intermolecular disulfide bond formation (data not
shown), indicating that theSH groups from twoCys-73 in the
non-covalently linked dimer are not in a distance readily forming a disulfide bond, consistent with our and previous observations that they are 4.8 Å apart (32). Redox buffer comprising 0.1
mM GSSG and 0 to 20 mM GSH can provide an oxidized to
reduced redox range allows disulfide bond exchange and has
been previously used to demonstrate a redox switch in circulating angiotensinogen. It has been shown that the angiotensinogen redox switch is reduced in plasma from healthy individuals
and oxidized in plasma from preeclampsia patients (36). When
HBMP9 was incubated in such redox buffer, the M-form was
very sensitive to the presence of the redox buffer, readily converted into the D-form under oxidizing conditions (0.1/1 mM
GSSG/GSH), and the totally reduced form (M*-form, with all
three intramolecular disulfide bonds reduced) under all other
conditions. The D-form BMP9 was more stable and gradually
converted into M*-form under more reducing conditions (Fig.
5A). Because BMP9 circulates in blood as the prodomainbound form, we investigated whether the presence of the
prodomain would protect BMP9 from redox changes (Fig. 5B).
Similar to HBMP9, in the presence of prodomain, M-form
BMP9was readily converted into theD-form andM*-form under
the oxidizing and reducing buffers, respectively, although there
were still some M-form left in all conditions. The D-form BMP9
could be reduced to the M-form in the reducing buffer (Fig. 5B,
0.1/10 mMGSSG/GSH), but there were still significant amounts
of the D-form remaining even at the most reducing condition
tested, supporting a role for the prodomain in stabilizing the
BMP9 structure. To investigate whether this redox sensitivity
was unique for BMP9, we incubated BMP6 in the same redox
buffer. Purified BMP6 was a disulfide-linked dimer without the
TABLE 1
Crystals, data processing, refinement, and models
Crystals HBMP9 (PDB code 4MPL)a
Space group I4122
Cell dimensions a  b  71.27, and c  145.89 Å,
      90°
Solvent content (%) 65.66
Data processing statistics
Wavelength (Å) 0.9795
Resolution (Å) 40.17-1.90 (2.00-1.90)
Total reflections 117,427
Unique reflections 15,096
Mn (I/sd) 15.6 (2.7)
Completeness (%) 99.3 (98.6)
Multiplicity 7.8 (8.0)
Rpim 0.029 (0.279)
Model
No. of protein atoms 930
No. of water molecules 95
Average B-factor (Å2) 32.6
Refinement statistics
Reflections in working/free set 14,327/769
Rfactor/Rfree 19.8/22.6
r.m.s.d. of bonds (Å)/angles from
ideality
0.008/1.198°
Ramachandran plotb
Favored (%) 94.44
Outlier (%) 0
a PDB, Protein Data Bank; r.m.s.d., root mean square deviation.
b Calculated using Molprobity (52).
FIGURE 3. M-form BMP9 can bind to ALK1 and prodomain as the D-form.
A, M- and D- forms of HBMP9 co-migrate on native PAGE, and both can form
complexes with ALK1 ECD. B, M- and D-forms of BMP9 co-migrate, both as
free form and as prodomain-bound form. In A and B, bands 1 to 6 from native
PAGE were cut out, boiled in 2 SDS-loading buffer for 20 min before loading
onto a non-reducing SDS-PAGE to confirm the identities. For SDS-PAGE in A,
two parts of the same gel are shown.
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prodomain (Fig. 5C). Although there was a trend toward faster
migration with increasing concentration of GSH, probably due
to the reduction of intramolecular disulfide bonds, BMP6
remained as disulfide-linked dimer at the redox range tested
and no monomeric form of BMP6 could be detected either in
M- or M*-form. This indicates that the BMP6 intermolecular
disulfide bond is likely to bemore stable than that of BMP9 and
may require the intramolecular disulfide bonds to be reduced
before the intermolecular disulfide bond can be reduced.
Hence, labile intermolecular disulfide bond and redox regulation is not a general feature for BMPs but may be unique to
BMP9.
BMP9 Is Susceptible to Redox-dependent Proteolysis—We
next applied limited trypsin proteolysis to probe whether there
is any difference in the stability between D-,M-, andM*- forms
of BMP9. HBMP9 (Fig. 6A) or pBMP9 (Fig. 6B) were incubated
in PBS alone, oxidizing redox buffer (0.1/1 mM GSSG/GSH) or
slightly reducing redox buffer (0.1/4mMGSSG/GSH) overnight
FIGURE 4. Intermolecular disulfide bond is not required for BMP9 signaling activity. A, conditioned media (6 l) from HEK-EBNA cells transfected with
transfection reagent alone (lane 1), empty vector (lane 2), pro-BMP9 (lane 3), pro-HBMP9 (lane 4), or pro-HBMP9 C73S (lane 5) were fractionated on a 12%
SDS-PAGE and probed with anti-BMP9 prodomain antibody. The intensities of bands were quantified using ImageJ, and the ratio of HBMP9 C73S to HBMP9
obtained. HBMP9 C73S concentration in the conditioned medium was normalized to HBMP9 using the above ratio. B, left: hPAECs were serum-restricted in
EGM-2, 0.1% FBS overnight, and stimulated with HBMP9 or HBMP9 C73S (both 0.25–3 ng/ml) for 1 h. Cells were harvested, and total cell protein was
immunoblotted with anti-pSmad1/5/8 antibody. Band intensities of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the
-tubulin blot, and normalized to a 0.25 ng/ml wild type sample. Data of the mean  S.E. from three repeats are shown on the right. C, after quiescence
overnight in EGM-2, 0.1% FBS, hPAECs were treated with 1 ng/ml of HBMP9 or HBMP9 C73S. Samples were harvested at 1, 2, 6, and 24 h, and immunoblotting
was carried out as described in B. Band intensity of pSmad1/5/8 blots were analyzed using ImageJ, corrected by ratios obtained from the -tubulin blot and
normalized to a wild type 1-h treatment sample. Data of the mean  S.E. from three repeats are shown on the right. NS, not significant.
FIGURE 5. BMP9 is regulated by redox potential. Purified HBMP9 (A), pBMP9 (B), or BMP6 (C) were incubated at room temperature overnight with PBS alone
or redox buffer containing 0.1 mM GSSG and 0 –20 mM GSH. Proteins were then run on a non-reducing SDS-PAGE and detected by Coomassie Blue. M* is the fully
reduced form of BMP9.
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before limited proteolysis by trypsin.More reducing conditions
containing higher concentrations of GSH were not included
due to the inactivation of trypsin, probably due to the reduction
of the disulfide bonds in trypsin (data not shown). As shown in
Fig. 6A, BMP9 is unusually stable without any redox buffer and
resistant to 2% trypsin digestion at 37 °C for 3 h. After overnight
incubation with oxidizing redox buffer, M-form BMP9 had
almost completely disappeared, mostly converted into the
D-form,whichwashighly resistant to trypsindigestion, althougha
small amount of M*-form could be seen and was fully degraded
evenby0.5% trypsin.Under 0.1/4mMGSSG/GSH, the level of the
D-form BMP9 had not changed compared with PBS control,
but almost all the M-form BMP9 was converted into M*-form
and readily cleaved by 0.5% trypsin. In the presence of the
prodomain, BMP9 was slightly more resistant to trypsin. But
when higher trypsin concentrations and overnight incubation
were used, similar redox-dependent cleavage of the M-form
could be observed (Fig. 6B).
M-form BMP9 Is Preferentially Cleaved in Serum from Control Subjects—We next probed whether there is evidence of
redox-dependent proteolysis in vivo because there is GSSG/
GSH redox buffer in serum (42). As the levels of circulating
BMP9 are reported to be 0.07–10 ng/ml (18, 19, 27), too low to
be detected by Western blot, we reconstituted pBMP9 into 10
control human serum samples and investigated whether there
were any protease activity in serum that could cleave pBMP9.
Aliquots were taken at 0 h and after overnight incubation at
37 °C, and the BMP9 levels weremeasured byWestern blotting
for BMP9 (AF3209, control experiments showed that this
antibody can detect the D- and M-forms of BMP9 equally
well, data not shown). As shown in Fig. 7, after overnight
incubation at 37 °C, although there was an overall small
reduction in the D-form BMP9, the amount of M-form had
decreased significantly.
DISCUSSION
TGF family ligands are powerful pleiotropic cytokines that
function at very low concentrations.Withmore than 30 ligands
signaling through seven type I and five type II receptors in
human, there is a large degree of promiscuity in ligand-receptor
recognition. Thus, their activities need to be tightly regulated
both temporally and spatially. Dysregulated BMP signaling has
been associated with a number of diseases characterized by
bone and tissue remodeling.Whereas TGF is stored in a latent
complex with its prodomain and activated upon binding to
integrin (43), BMPs are secreted in the active forms andmostly
regulated by BMP antagonists, including noggin and chordin
(44). Although noggin inhibits most of the BMPs, it does not
inhibit BMP9 or BMP10 (45). Among the many known BMP
antagonists, only crossveinless 2 (CV2, also called BMPER) has
been shown to bind and inhibit BMP9 activity (46). However,
the mechanism of crossveinless 2 function is not fully understood as it has been shown that it can act as both an activator
and an inhibitor of BMP signaling depending on the concentrations (47). Other factors regulating the stability and degradation of BMPs have not been well documented. Limited number
of reports include the cleavage of the two sites within the BMP4
prodomain directing the intracellular trafficking and degradation of mature BMP4 (48) and megalin, a low-density lipoprotein receptor-related protein, mediating the clearance of BMP4
in the neuroepithelium (49).
BMP9 is constitutively secreted from the liver in an active
form into the circulation. Mechanisms controlling its bioavailability need to be in place to ensure the optimum signaling
activity and specificity. Based on our findings that BMP9 protein level can be regulated by redox-dependent proteolysis, we
propose the following model as one way to control the BMP9
levels in circulation (Fig. 7B). BMP9 is unique among the BMP
family in that stable dimers can form with or without the intermolecular disulfide bond and the equilibrium is determined by
the redox potential. The two forms of BMP9 dimer show minFIGURE 6. M-form BMP9 is more susceptible to redox-dependent proteolysis. HBMP9 (A) or pBMP9 (B) was incubated in PBS or redox buffer containing 0.1/1 mM GSSG/GSH (0.1/1) or 0.1/4 mM GSSG/GSH (0.1/4) at room temperature overnight. The following day, an aliquot of each treatment was
subjected to limited trypsin digestion at 37 °C, 3 h for HBMP9 or overnight for
pBMP9. Reactions were stopped by addition of SDS-loading buffer and boiling at 100 °C for 10 min. Cleavage was monitored by fractionating samples on
a 12% non-reducing SDS-PAGE and Coomassie Blue staining.
FIGURE 7. M-form BMP9 is preferentially cleaved in serum and model of
BMP9 regulation by redox-dependent proteolysis. A, pBMP9 (0.3 g) was
reconstituted into 10 l of serum (10 healthy human controls), and equal
aliquots of samples were taken at 0 h (lane 1) and after overnight incubation
at 37 °C (lane 2). Samples were then fractionated on a 12% SDS-PAGE under
non-reducing conditions and detected by anti-BMP9 antibody (AF3209). The
D- and M-forms of BMP9 at 0 h and overnight were quantified using ImageJ,
and the % BMP9 remaining after overnight incubation was calculated and
plotted using GraphPad Prism. Data are mean  S.E. (n  10). B, model of the
regulation of BMP9 concentration by redox-dependent proteolysis.
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imal differences in structure and protein-protein interactions.
But the M-form is highly sensitive to the redox buffer and can
be converted into the D-form under oxidizing environment or
the fully reduced M*-form that is readily cleaved by proteases in the normal sera from healthy subjects. This regulation by redox-dependent cleavage may provide a natural
degradation pathway in humans, in addition to the inhibition by CV2, to maintain BMP9 at low concentrations for
ALK1-specific signaling.
It is difficult to provide in vivo evidence for this model or the
presence of theM-form in nature: the circulating level of BMP9
is too low for detecting the M-form or BMP9 cleavage products, and our results would predict that theM-formwould be
most susceptible to degradation in the circulation. We have
attempted BMP9 immunoblotting using liver samples from
mice, but the multiple bands on the blots prevent the robust
interpretation of the results (data not shown). However, there
were several indirect lines of evidence supporting that our
observations are likely to be relevant to the in vivo setting. First,
using a pulse-chase assay, it has been shown that when proBMP7 was expressed and processed in CHO cells, the supernatant has the exactly same pattern of processed and partially
processed BMP7 as we observed for pro-BMP9, including the
monomeric forms of BMP7 (37). It was hypothesized that the
close spacing of the disulfide bonds in the BMP may promote
the disulfide bond exchange that resulted in themonomeric form
(37).Our observation that the thiol-oxidizing agent diamide could
not promote Cys-73 disulfide bond formation, but redox buffer
could, also supports a role for disulfide exchange in the D- and
M-form conversion. Second, in a previously published BMP9
structure inwhichBMP9was generated froma special CHOcells,
BMP9 was crystallized in the presence of the prodomain and
existed exclusively in theM-form (32).
Apart from the many recent reports on the role of BMP9
signaling in the endothelial cell biology and cardiovascular diseases, BMP9 is also a neurotrophic factor, potently inducing
andmaintaining the cholinergic phenotype in the central nervous system (50). Administration of BMP9 was effective in
reversing the A42 amyloid plaque burden and reversing cholinergic neuron abnormalities in a mouse model of Alzheimer
disease (51). BMP9 clearly shows therapeutic potential in cardiovascular diseases, neurodegenerative diseases, as well as the
widely explored bone and cartilage defects. This study has provided new insights into the structure and regulation of BMP9,
and these findings have broad implications for our understanding of the regulation of BMP9 in vivo and have provided essential information for developing BMP9 for therapeutic use.
Acknowledgments—We thank Prof. JimHuntington for critical review
of the structure. Infrastructure support was provided by the Cambridge National Institute for Health Research Biomedical Research
Centre.
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Redox-dependent Proteolysis of BMP9
NOVEMBER 7, 2014 • VOLUME 289 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 31159
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Zhenquan Wei, Richard M. Salmon, Paul D. Upton, Nicholas W. Morrell and Wei Li
Proteolysis
Regulation of Bone Morphogenetic Protein 9 (BMP9) by Redox-dependent
doi: 10.1074/jbc.M114.579771 originally published online September 18, 2014
2014, 289:31150-31159.J. Biol. Chem.
10.1074/jbc.M114.579771Access the most updated version of this article at doi:
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