(Received for publication, November 22, 1996)
From the Research and Development Center, Nippon Meat
Packers, Inc., 3-3 Midorigahara, Tsukuba, Ibaraki 300-26, Japan,
¶ Department of Insect Genetics and Breeding, National
Institute of Sericultural and Entomological Science, 1-2 Ohwashi,
Tsukuba, Ibaraki 305, Japan,
Department of Oral
Pathology, School of Dentistry, Showa University, Shinagawa-ku, Tokyo
142, Japan, ** Department of Orthopedic Surgery, School of
Medicine, Niigata University, Niigata 951, Japan, and
Faculty of Pharmaceutical Sciences, Hokkaido
University, Sapporo 060, Japan
Bone morphogenetic proteins (BMPs) are
multifunctional proteins that comprise the largest subfamily of the
transforming growth factor-. These proteins bind to types I and II
serine/threonine kinase receptors. Ligand-induced heteromeric
dimerization of these receptors is the key event in initiation of
biological responses. We report here large-scale expression and
purification of extracellular domain of the type I receptor for
BMP-2/4, using a silkworm expression system. This soluble form of BMP
receptor (sBMPR) was in monomer form in solution and bound to BMP-4 but
not to activin or transforming growth factor-
1. Surface plasmon
resonance studies showed that kinetic parameters of sBMPR for BMP-4
consisted of a relatively rapid association rate constant
(ka = 3.81 ± 0.19 × 104
s
1 M
1) and an extremely slow dissociation
rate constant (kd = 3.69 ± 0.26 × 10
4 s
1). From these two kinetic parameters,
affinity was determined to be similar to that of the intact
membrane-associated receptor expressed on COS cells. sBMPR inhibited
the alkaline phosphatase activity in BMP responsive cell lines such as
mouse osteoblastic cell MC3T3-E1 and bone marrow stromal cell ST2.
These data indicate that the extracellular domain of type I receptor
for BMP-2 and BMP-4 is sufficient for high-affinity binding to its
ligands and should prove useful in understanding the role of BMP-2/4
in vivo, because a suitable high-affinity anti-BMP antibody
has yet to be developed.
Bone morphogenetic proteins (BMPs),1
originally identified as proteins to induce endochondral bone formation
in ectopic extraskeletal sites in vivo (1), are the largest
subfamily of the transforming growth factor- (TGF-
). Of over a
dozen of these BMP members (2, 3), BMP-2 and BMP-4 (vertebrate ortholog
of Drosophila decapentaplegic) appear to play important
roles in embryogenesis and body patterning (4). We reported that a
Xenopus homologue of BMP-2 and BMP-4 present in developing
Xenopus embryos (5-8) regulates dorsal-ventral patterning
during mesoderm induction (9). In addition to the dorsal-ventral
specification, BMP-2 and BMP-4 are also involved in later stages of
development, e.g. differentiation of neural cells
(10-13), regulation of patterning in the limb bud (14, 15), and
epithelial-mesenchymal interactions during organogenesis (16).
Receptors for BMPs are a family of transmembrane serine/threonine
kinases (17). These receptors are divided into two distinct classes,
type I (18-20) and type II receptors (21, 22). Like receptors for
other TGF--related proteins, heteromeric dimerization of these
receptors, induced by binding to their ligands, is responsible for
initiating biological responses (22-25). However, a cross-linking study showed that when transiently expressed, these type I receptors are capable of binding to BMP-2 and BMP-4, without co-expression of the
type II receptor (18-20, 26). The type II receptor for BMPs bound to
ligands weakly, but the binding was facilitated by the presence of type
I receptors for BMPs (21, 22, 24). When the dominant-negative form of
type I receptor, lacking the serine/threonine kinase domain, was
expressed in ventral blastomeres of Xenopus embryos, the
BMP-4 signal was blocked, resulting in formation of a secondary body
axis (19, 26). These findings indicate that the extracellular domain
(ECD) of type I receptor is sufficient to mediate stable binding to
BMPs and subsequent formation of a heteromeric complex with the intact
(endogenous) type II receptor. Thus, a dominant-negative experiment
would be pertinent to study the biological function of BMP-2 and BMP-4 by specifically disrupting the BMP signal. However, properties of the
dominant-negative truncated type I receptor, its binding affinity,
specificity, and requirement of other components for binding to ligands
remain to be clarified.
In an attempt to address these problems and to determine the
physiological role of type I receptors for BMP signaling, we produced a
soluble form of the ECD of type I receptor fused to the
myc-epitope sequence (sBMPR), using a silkworm expression system. This system, first reported by Maeda et al. (27),
facilitates a high level of expression of the active form of
recombinant proteins that often become insolubilized in the
Escherichia coli expression system as an inclusion bodies
form. Using this system, a milligram quantity of the sBMPR was
expressed, and the purified sBMPR showed a high affinity for
recombinant Xenopus BMP-4 (rxBMP-4). Using a surface plasmon
resonance biosensor, sBMPR bound specifically to rxBMP-4 immobilized on
the sensor chip surface but not to activin or the TGF- immobilized
surface. Kinetic analysis showed that binding of sBMPR to rxBMP-4
consisted of a rapid association rate constant (ka = 3.81 ± 0.19 × 104 s
1
M
1) and an extremely slow dissociation rate
constant (kd = 3.69 ± 0.26 × 10
4 s
1). We also asked if sBMPR would
compete with membrane-associated BMP receptors in culture cells and act
as an antagonist of BMPs. The sBMPR inhibited ALP activity in
BMP-responsive cell lines such as MC3T3-E1 cells and ST2 cells.
To express
the soluble form of the BMP receptor (sBMPR), a truncated mutant of
cDNA that lacks both transmembrane and serine/threonine kinase
domains (coding sequences for amino acids 1-152) was amplified from
mTFR11 cDNA cloned in BlueScript SK() (19) by polymerase chain
reaction (PCR), using a universal forward primer and a
TFR11myc reverse primer. The TFR11myc primer,
3
-GAAACTACCGTCGTAGGCTCTCGTCTTCGACTAGAGGCTCCTCCTGGACATC, was designed
so that the myc epitope sequence (EQKLISEEDL) would be added
at the carboxyl terminus of the mutant protein (28). The PCR reaction
was performed in a volume of 100 µl containing 10 µl of the reverse
transcriptase reaction, 10 mM KCl, 20 mM Tris-HCl, pH 8.0, 10 mM (NH4)SO4, 6 mM MgSO4, 0.1% Triton X-100, 1 unit of Pfu DNA
polymerase (Stratagene), 40 ng of template DNA, and 50 pmol each of the
forward and the reverse primers. After 30 cycles of denaturation
(95 °C, 1 min), annealing (55 °C, 45 s), and extension
(72 °C, 2 min), the PCR product was recovered from low melting
temperature agarose gels and subcloned into a unique NurI
site of baculovirus transfer vector pBm4 (29). DNA sequencing was done
using an automated DNA sequencer (373A, Applied Biosystems, Inc.). The
full-length xBMP-4 cDNA was isolated from pUC19/xBMP-4 by digesting
with restriction enzyme EcoRI (5). The fragment was blunted
with T4 DNA polymerase and ligated into the NurI site of the
pBm4 vector.
The resulting transfer vectors, pBm4-sBMPR and
pBm4-rxBMP-4, were independently cotransfected with genomic DNA of
wild-type BmNPV into BmN4 insect cells (Nosannkoh, Japan), using a
Lipofectin technique (30) (Life Technologies, Inc.). Four days after
cotransfection, the culture supernatant was collected, and recombinant
viruses, in which the polyhedrin gene was replaced with sBMPR and
rxBMP-4 cDNA, were cloned by the end-point dilution method on
96-well plates (27). End point dilution was performed three times to clone the viruses genetically. The recombination of viruses was confirmed by extracting the viral genomic DNAs and by PCR with primers
designed to anneal the insertions, sBMPR and rxBMP-4 (data not shown).
The recombinant viruses were propagated and stored at 80 °C for
subsequent use. The fifth instar larvae of the silkworms (Kinshu X
Showa) fed artificial diets (Nohsannkoh, Japan) were obtained from
Kanebo (Aichi, Japan). About 105 plaque-forming units of
the recombinant viruses (50 µl) were injected subcutaneously into the
larva. Four days after the infection, abdominal legs of the larva were
pierced and the hemolymph recovered as described (31) was stored at
80 °C.
Immunodetection of sBMPR and rxBMP-4 was performed by Western blotting. After electrophoresis under reducing conditions on a 15% SDS acrylamide gel, proteins were blotted onto polyvinylidene difluoride membrane using a semidry Western transfer apparatus (Bio-Rad). The membrane was blocked in TBS-T (150 mM NaCl, 50 mM Bis-Tris, pH 7.5, and 0.05% Triton X-100) with 0.25% BSA for 1 h at room temperature and reacted with either anti-myc monoclonal antibody 9E10 (28) or anti BMP-4 polyclonal antibody Ab383 overnight at 4 °C (8). The filter was then incubated with a horseradish peroxidase-conjugated antibody for 1 h at room temperature. Western blot was developed by a chemiluminescent detection system described by the manufacturer (Amersham Corp.)
Purification of Recombinant ProteinsrxBMP-4 was purified by affinity chromatography on a heparin column using documented procedures (31), with the following modifications. The hemolymph recovered from infected larvae was precipitated with 50% ammonium sulfate, resolved in Tris-buffer (20 mM Tris-HCl, pH 7.4), and desalted on a phenyl-Sepharose column (Pharmacia Biotech Inc.). The column was preequilibrated with the same buffer and then eluted with a linear gradient of a decreasing concentration of ammonium sulfate concentration from 0.5 to 0 M. The fraction recovered from 0.5 M ammonium sulfate concentration elution, containing immunoreactivity of the anti-BMP4 antibody analyzed by Western blot, was pooled and dialyzed against Tris buffer (20 mM Tris, 4 M urea, pH 8.0). The active fraction was loaded on a heparin-Sepharose CL-6B column (Pharmacia), which was eluted by 0.25 M sodium chloride containing 4 M urea. The active fraction was further separated on the ResourceS column (Pharmacia) in the same buffer, developed with a gradient from 0 to 1.0 M sodium chloride. The active fraction in 0.5 M NaCl elution was pooled, acidified to pH 3.0 with CF3COOH (TFA), and concentrated on a µRPC C2/C18 column (Pharmacia, SMARTsystem). The column was preequilibrated with 0.1% TFA and was eluted with a linear gradient from 0 to 80% acetonitrile containing 0.1% TFA. The main peak eluted at 60% acetonitrile was pooled and stored at 4 °C until use. The purity of the protein was >98%, determined by silver staining of SDS-PAGE (data not shown).
The initial two purification steps of sBMPR were the same as that of rxBMP-4, i.e. 50% ammonium sulfate precipitation and phenyl-Sepharose column chromatography. Following these steps, the fraction, containing immunoreactivity of anti-myc antibody 9E10 analyzed by Western blot, was dialyzed against Tris-buffer (20 mM Tris-HCl, pH 7.4) and loaded on a ResourceQ column (Pharmacia) preequilibrated with the same buffer. The column was eluted with a linear gradient from 0 M to 1.0 M sodium chloride. The immunoreactive fraction in 0.5 M NaCl elution was further separated by gel filtration performed on Superdex 75 (Pharmacia) equilibrated in Tris-buffered saline (50 mM Tris-HCl, 100 mM KCl, pH 7.4). The immunoreactive sBMPR was eluted as the main peak of low molecular mass (<20 kDa). The protein concentration of purified sBMPR was determined by measuring the absorbance in a 1-cm-path length cell at 280 nm.
Peptide Growth Factors and AntibodiesPurified recombinant
human activin produced in insect cells was a gift from Dr. I. Eto
(Ajinomoto, Inc., Kawasaki, Japan). Recombinant human TGF-1 was
purchased form King Jyohzo (Tokyo, Japan). The 9E10 cell line, which
produces anti-myc antibody, was provided by Dr. H. Brivanlou
(Rockefeller University, New York, NY). The anti-myc
monoclonal antibody was purified from the growth medium of 9E10,
using a protein A affinity column.
Binding experiments and
kinetic analysis were performed using the BIAcore2000 (Biacore). The
basic principles and its use have been documented (32, 33). Purified
rxBMP-4 described above, recombinant human activin, and recombinant
human TGF-1 were prepared in immobilization buffer (10 mM acetate buffer, pH 4.8) at a concentration of 10 µg/ml
and were immobilized on sensor chips (CM5, certified grade, Biacore) at
a flow rate of 20 µl/min at 25 °C for 150 s by the amine
coupling method (32). The immobilization levels for rxBMP-4, activin,
and TGF-
1 were 1031, 1543, and 1012 RUs, respectively. For binding
studies, sBMPR was injected over a range of concentrations between 10 nM and 12.5 µM at 25 °C for 60-150 s.
Before the analysis, the flow rate of the analyte injection was
optimized to minimize mass transport limitations (33). A flow rate of
30 µl/min was found to be sufficient to overcome the mass transport
limitation (data not shown). The kinetic parameters, association rate
constant (ka) and dissociation rate constant
(kd), were determined from three independent
experiments, using BIAevaluation software version 2.1 (Biacore).
The buffer for sample dilution and running buffer for BIAcore2000 was
HBS (50 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, and 0.005% Nonidet P-40, pH 7.4). Prior to data
collection, several methods for surface regeneration after ligand
binding were evaluated. Injection of 100 mM HCl (20 µl)
efficiently removed the bound proteins and preserved the binding
capacity of the sensor chip surface. rxBMP-4 surfaces were stable
for over 100 binding and regeneration cycles (data not shown).
Biological
activity of sBMPR was examined using two different cell lines. MC3T3-E1
cells were provided by Dr. M. Kumegawa. ST2 cells were obtained from
the RIKEN Cell Bank (Tsukuba, Japan). These cells were inoculated at a
density of 3 × 103 cells/well in 24-well plates and
cultured with -modified Minimum Essential Medium (Life Technologies,
Inc., Grand Island, NY) containing 10% fetal bovine serum (Life
Technologies, Inc.) and antibiotics (100 units/ml of penicillin-G and
100 µg of streptomycin). To examine the biological activity of sBMPR,
ALP activity of MC3T3-E1 cells was examined. Various concentrations of
sBMPR were added to the medium on day 1 of the culture, and ALP
activity was determined on day 6 of the culture, using an established
technique (34). ST2 cells were grown in the presence of rxBMP-4
(100 ng/ml) and various concentrations of sBMPR. ALP activity was
determined on day 3 of culture.
To confirm the synthesis of sBMPR in the silkworm expression
system we used, 0.5 µl of silkworm hemolymph was subjected to electrophoresis on a 15% SDS polyacrylamide gel. Under reducing conditions, a defuse band with a relative molecular mass of ~20 kDa
was observed for hemolymph recovered from larvae infected with
sBMPR-recombinant virus but not in noninfected or in wild virus-infected hemolymph (Fig. 1A, lane
2-4). Western blot analysis showed that this species reacted
strongly with the anti-myc monoclonal antibody 9E10 (Fig.
1A, lane 7), thereby suggesting the production of
myc-epitope tagged sBMPR in silkworm larvae. To obtain a
large quantity of sBMPR, 100 ml of hemolymph were collected from 200 larvae infected with the recombinant virus. The recovered hemolymph was
precipitated with 50% ammonium sulfate, and sBMPR was purified using
hydrophobic, ion-exchange, and size-exclusion chromatography, as
described under "Material and Methods." Using this expression system, a milligram quantity of sBMPR (~5 mg) was obtained.
The N-terminal amino acid sequence of purified sBMPR was analyzed using an automated pulse liquid-phase sequencer. A complete single amino acid sequence of 15 amino acid residues was determined to be "GMKSDLDQKKPENGV." This sequence was a perfect match with the coding sequence for amino acids 34-49 deduced from the cDNA sequence. However, the signal peptide of the TFR11 had been predicted to be cleaved after S19 (19). It cannot be ruled out that the observed N-terminal sequence is the original cleavage site in vivo; several recombinant proteins expressed in insect cells were found to be processed at different positions of natural proteins (31, 35).
In SDS-PAGE analysis, the purified sBMPR migrated on the gel at a molecular mass of ~16 and ~20 kDa, under nonreducing and reducing conditions, respectively (Fig. 1B). Molecular masses of the observed bands in SDS-PAGE were greater than that predicted by cDNA sequence (129 residues; 1,430 Da). This was presumably the result of glycosylation on the two potential N-glycosylation sites of the molecule (19). A molecular mass of 16 kDa was also determined by matrix-assisted laser desorption mass spectrometry, indicating that sBMPR was monomeric (data not shown), despite the presence of 10 conserved cysteine residues of ECD. This was further supported by a global analysis of equilibrium sedimentation, using an analytical ultracentrifuge. Data from this yielded a single molecular mass of 16 ± 0.5 kDa and indicated that sBMPR was a monomer in solution and did not aggregate further.2 Recently, we confirmed that all cysteines of ECD formed five intramolecular disulfide bridges and no unpaired cysteine residues.3 Differences in molecular mass under reducing conditions in SDS-PAGE analysis would be the result of disruption of these intramolecular disulfide bonds.
For binding assay, purified recombinant rxBMP-4 was immobilized
on sensor chips of BIAcore, and 5 µg/ml of sBMPR were passed over the
sensor chip. During the injection of sBMPR, a rising slope of the RU
was evident, and after the injection, the RU decreased slowly. However,
this change in sensorgram did not occur when the sBMPR was injected
over either an activin or a TGF-1 immobilized sensor chip surface.
This was identical to the control injection with mock-coupled sensor
chip surface in the absence of proteins (Fig.
2A). To detect the myc epitope tag
of sBMPR, injection of anti-myc monoclonal antibody 9E10
followed the injection of sBMPR over the xBMP-4 surface. After binding
of sBMPR to rxBMP-4 immobilized on the sensor chip, a second increase
of 1000 RU was observed by sequential injection of 9E10 (3 µg/ml).
When 9E10 was injected alone (blank surface) or injected with a 10-fold
molar excess of myc epitope peptide (EQKLISEEDL), this second response
during the injection of 9E10 was precluded (Fig. 2B),
indicating that the second response was specific binding of 9E10 to the
protein containing the myc epitope sequence, that is sBMPR. These
results clearly suggest that the ECD of TFR11 was sufficient for
binding to BMP-4 and did not bind either activin or TGF-
1. The
myc epitope sequence at the amino terminus of sBMPR was well
recognized by 9E10, forming a stable complex with sBMPR and rxBMP-4 on
the sensor chip.
To determine kinetic parameters, association rate constant and
dissociation rate constant, we next injected increasing concentrations of sBMPR over immobilized xBMP-4 on the sensor chip (0.1-12.5 µM). Sensorgrams consisted of a rapid increase in RU
during injections and an extremely slow decrease of dissociation phase
(Fig. 3A); curves were analyzed using
nonlinear least squares methods. Dissociation phase (140-200 s) gave a
good fit to a single exponential interaction model at all
concentrations, and the dissociation rate constant, kd, was calculated to be 3.69 ± 0.26 × 104 s
1 (Fig. 3C). However, the
association phase (80-130 s) was fitted to a single exponential
interaction model only at a low concentration range (<3
µM), and the association rate constant,
ka, was estimated to be 3.81 ± 0.19 × 104 s
1 M
1 (Fig.
3B). The apparent equilibrium dissociation constant
(KD) determined from these two rate constants
(kd/ka) was 9.6 nM.
The affinity determined by this method is similar to
KD values (0.5-3.5 nM) for intact
type I receptors for BMPs binding to their ligand (18, 19, 26).
Determination of kinetic parameters of the sBMPR/BMP-4 interaction was also done in reverse orientation, i.e. with sBMPR immobilized on the sensor chip surface and rxBMP-4 in solution. However, the injected BMP-4 was absorbed in the micro flow channel of the instrument and hardly reached flow cells of the sensor chip surface at neutral pH range (data not shown), probably because of the sticky nature of BMP-4 (1). Thus, using a sBMPR immobilized surface was not feasible for reproducible data collection and quantitative analysis.
We then measured ALP activity of MC3T3-E1 cells to determine if the
sBMPR could compete with the membrane-bound BMP receptors on cultured
cells and could act as an antagonist of BMPs. MC3T3-E1 cells are well
characterized as an osteoblastic cell line that differentiates into
mature osteoblasts responding to BMPs and shows high ALP activity. The
cells produce several families of BMPs and respond in an autocrine
manner. Reverse transcription-PCR blot analysis showed that this cell
line expresses BMP-2 and BMP-4 of
transcripts.4 Fig.
4A shows the dose-dependent
inhibition effect of sBMPR on ALP activity in MC3T3-E1 cells on day 6 of culture. Maximum inhibition was observed with a concentration of 3 µg/ml of sBMPR. Added sBMPR was considered to bind to BMPs secreted
into the culture medium and to act as a antagonist by competing with
wild membrane-bound receptors; the major type I receptor for BMP
expressed on the cell was TFR11 (ALK-3), which was cloned using a
cDNA library of MC3T3-E1 cells (19).
Next we asked if sBMPR could block the differentiation of bone marrow stromal cells ST2 into osteoblasts induced by BMP-4 (36). They do not exhibit osteoblastic features, such as increase in ALP activity, in control culture. However, when the cells are cultured in the presence of rxBMP-4 (100 ng/ml), the level of ALP activity increased 6-fold over that of control culture. This effect of BMP-4 on ALP activity was suppressed when sBMPR was added, dose-dependently (Fig. 4B). In the presence of 1 µg/ml of sBMPR, the ALP activity was reduced to basal level, and 50% inhibition was obtained at about 100 ng/ml of sBMPR (molar ratio of added sBMPR to rxBMP-4 was ~2:1).
sBMPR described here has facilitated structure-function analysis of the ECD of the type I receptor for BMP-2 and BMP-4. Our data show that sBMPR retains high-affinity binding and forms a stable complex with BMP-4 because of an extremely slow dissociation rate constant.
Because only the active form (mature form) of BMPs is capable of binding to its receptor (17), the sBMPR might be useful to detect active forms of BMP-2 and BMP-4 as well as for use as a BMP antagonist. An anti-BMP antibody that recognizes active BMP and neutralizes the BMP signal has yet to be developed.
We thank Mariko Ohara for editing the manuscript. We acknowledge the support of Minoru Hata and Dr. Katsuhiko Mikoshiba.