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J Biol Chem, Vol. 273, Issue 44, 29052-29065, October 30, 1998
From the The mature C-terminal signaling domain of the
Drosophila Decapentaplegic proprotein (DPP) can be
efficiently refolded from chaotrope-solubilized inclusion bodies with
the aid of a membrane protein-solubilizing detergent, high
concentrations (0.75-2 M) of NaCl, and low temperatures
(5-15 °C). The disulfide-linked homodimeric product contains
N-terminal heparin-binding sites that were utilized as intrinsic
affinity tags to obtain a highly enriched preparation in one
chromatographic step. A subsequent C4 reverse phase high pressure
liquid chromatography step provides high purity, salt-free protein that
is amenable to biophysical and structural studies at a yield of
approximately 3 mg/liter of bacterial culture. The dimeric protein is
correctly folded as determined by electrophoretic, spectroscopic,
chemical, and proteolytic analyses. Refolded DPP is also bioactive as
shown by induction of chondrogenesis in embryonic chick limb bud cells and by high affinity binding to Noggin, an antagonist of bone morphogenetic protein signaling. In contrast to bone morphogenetic proteins extracted from demineralized bone or overexpressed in cell
culture, the refolded Escherichia coli-expressed protein is
not glycosylated at a conserved N-linked site and is
therefore homogeneous. The C-terminal domain dimer is more hydrophobic
and thus less soluble than its unfolded or partially folded forms, necessitating highly solubilizing conditions for recovery after folding
in vitro. Hence solubilization of the mature ligand may be
one of the principal roles of the large (250-400 amino acids) N-terminal prodomains of transforming growth factor- The decapentaplegic locus (dpp) encodes a
signaling ligand homologous to human bone morphogenetic proteins
BMP-21 and -4 (1, 2) and has been
the object of intense study due to its importance in many fundamental
processes during Drosophila development. The mature DPP
ligand is secreted as a disulfide-linked homodimer of the C-terminal
domain of a large proprotein precursor and has been shown to act as a
morphogen required for the establishment of the dorsoventral axis of
the embryo (3) and to impart positional information over long range to
specify cell fate along the anterior/posterior axis of the limbs (4,
5). DPP signaling also occurs at short range between germ layers of the
developing gut (6-8) and is required to direct the dorsal and ventral
migration of tracheal cells during embryogenesis (9) and to act as a
relay signal triggering epithelial cell shape changes during the
process of dorsal closure (10). In contrast to the developmental roles of DPP signaling, which have been the focus of extensive genetic studies, little is known about the folding, modification, and processing of the DPP proprotein, its distribution and state
extracellularly, or the biochemical and biophysical properties of the
mature homodimer and specific aspects of its structure.
Characterization of the protein products of the dpp locus
has been hindered by a lack of appreciable amounts of material for analysis. DPP is a relatively insoluble signaling ligand present in low
concentrations and cannot be isolated preparatively from Drosophila tissue. Attempts to express the DPP proprotein by
transient transfection in mammalian cell culture did not produce
detectable levels of the mature protein (11). To date the most
successful expression system has been a Drosophila embryonic
cell line (S2 cells) stably transfected with an inducible
metallothionein promoter-dpp cDNA fusion construct (12).
Although mature dimeric protein can be recovered from the conditioned
media, the bulk of the ligand is adsorbed to the surface of the tissue
culture plate and can be recovered in enriched form by washing with 0.5 M NaCl, 1% Tween 20. The S2 cell expression system has
provided DPP homodimer for the determination of its N terminus and
isoelectric point (12) and demonstration of ectopic bone-forming
activity in mammals (13). However, further studies to characterize the
signaling activity of the ligand have been hampered by the
unavailability of high purity preparations. For example, the affinity
of DPP for the type I receptors could not be quantitated due to the
inability to produce 125I-DPP for binding assays,
necessitating replacement with human recombinant BMP-2 (14, 15).
Similarly, human recombinant BMP-4 was substituted for the bona fide
Drosophila ligand to assay for Noggin function in the DPP
signaling pathway (16) and recombinant BMP-2 utilized in lieu of DPP to
stimulate ligand-dependent phosphorylation of MAD proteins
in Drosophila imaginal disk cell lines (17).
In this report, we show that the mature C-terminal signaling domain of
Drosophila DPP can be efficiently refolded in
vitro from chaotrope-solubilized inclusion bodies and purified to
near-homogeneity by heparin affinity chromatography followed by reverse
phase HPLC. Approximately 3 mg of high purity protein can be produced
per liter of bacterial culture, enabling biochemical studies and assays of signaling activity to be performed with DPP ligand rather than human
BMP homologs and opening the way for biophysical and structural analyses of the C-terminal domain. Our studies also show that, in
addition to the extracellular domains of heterodimeric receptor complexes, DPP binds with high affinity to heparin, a
glycosaminoglycan related to heparan sulfate of extracellular
matrix and cell-surface proteoglycans, and with high specificity to
Noggin, a protein antagonist of BMP activity in vertebrates. Because
the E. coli-expressed, refolded protein migrated
reproducibly faster in SDS-PAGE than homodimer secreted by insect
cells, DPP appears to be glycosylated like all BMPs at a conserved
N-linked site. Inspection of the crystal structure of BMP-7
(18) and a model of the DPP homodimer reveals that the modified
asparagine side chains reside in the bottom of a large cleft on one
side of the dimers, opposite the pair of highly basic, disordered
N-terminal peptides that extend from the folded core of the mature
domains. The surface of the DPP dimer appears to be predominantly
hydrophobic, comprised of two large symmetry-related pairs of
hydrophobic patches. As shown by reverse phase HPLC, the folded dimer
is more hydrophobic and thus less soluble than its unfolded or
partially folded forms. The highly solubilizing conditions found to be
crucial for the production of TGF- Expression Vector Construct--
The mature C-terminal domain of
DPP (DPPC) was overexpressed in Escherichia coli with the
bacteriophage T7 RNA polymerase/ Protein Expression--
For large scale production
(approximately 160 mg) of DPPC inclusion bodies, four 3-liter flasks
containing 1 liter each of medium (LB or NZY, pH 7.0; 200 µg/ml
ampicillin) were inoculated with 10 ml (1:100 final dilution) of an
overnight culture of the host expression strain BL21(DE3) transformed
with the pJC10/DPPC construct. The cultures were grown at 37 °C to
OD 0.6 at 600 nm (about 3 h), induced with 1 mM
isopropyl-1-thio-b-D-galactopyranoside, and harvested after
3 h. Overnight cultures were inoculated with a single colony from
a recent (1-2-day) transformation of Ca2+-treated, frozen
competent cells. Erlenmeyer flasks with baffles (Schikane-type) to
promote aeration and media pre-equilibrated to 37 °C prior to
inoculation appeared to improve yields of protein. Induction at OD
1.0-1.5 resulted in significantly lower levels of protein
expression.
Inclusion Body Isolation--
Inclusion bodies were isolated by
a modification of the method of Dekker et al. (22). Induced
cultures were transferred to polypropylene bottles, chilled on ice, and
harvested by centrifugation in a Sorvall GS3 rotor at 4 °C for 20 min at 4000 rpm (2700 × g). Cell pellets were
thoroughly resuspended in 30 ml of ice-cold 50 mM Tris-HCl,
40 mM EDTA, pH 8.0 (per liter of culture) followed by the
addition of 7.5 g of sucrose, 6 mg of lysozyme, and
phenylmethylsulfonyl fluoride at 100 µg/ml. After incubation on ice
for 30 min, the cells were lysed by osmotic shock by the addition of 30 ml of ice-cold 50 mM Tris-HCl, 40 mM EDTA, pH
8.0, and 100 µg/ml phenylmethylsulfonyl fluoride and incubated
another 30 min. The lysates were transferred to 250 ml of polypropylene
centrifuge bottles and chromosomal DNA sheared by repeated rounds
(3-5) of sonication with a Branson Sonifier 250 equipped with a 1-cm
diameter tip. Lysates were maintained on ice during each round
consisting of several minutes of sonication (50% duty cycle, output
60) followed by several minutes to cool down. Triton X-100 (0.1% final
concentration) was added to the sonicate prior to centrifugation in a
Sorvall GSA rotor at 4 °C for 30 min at 5500 rpm (4500 × g). The large, pinkish-brown pellets were resuspended in an
equal volume (60 ml) of ice-cold 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, 1 mM DTT (TED) by sonication,
recentrifuged, and washed again with TED buffer. The twice-washed
inclusion body pellets were resuspended in 2.0 ml of TED (per liter of
initial culture) and transferred to Eppendorf tubes for storage at
In Vitro Folding--
Inclusion bodies suspended in TED buffer
were centrifuged briefly (~2 min) in a microcentrifuge and
solubilized at an estimated concentration of 2.5 mg/ml in buffered
chaotrope. The final molarity of chaotrope is not significantly lowered
by addition of the inclusion bodies, which were diluted more than
70-fold from an estimated initial concentration of 180 mg of protein/ml
pellet. 8 M urea or 6 M GdmCl, buffered with 50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 1 mM DTT, gave equivalent results. After incubation for 1-2
h at ambient temperature, insoluble debris was removed by a second brief centrifugation, and the solubilized protein was diluted 50-fold
(final concentration 50 µg/ml), rapidly with stirring, into
pre-chilled folding buffer (50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 33 mM CHAPS detergent (1.8%
w/v), 1.25 M NaCl, 2 mM reduced glutathione,
and 1 mM oxidized glutathione) and incubated for at least
72 h at 4 or 15 °C.
Purification--
The folding solution was concentrated 10-fold
by ultrafiltration at 4 °C with a stirred cell device fitted with a
YM10 membrane (Amicon), diluted 12.5-fold with 6 M urea, 50 mM Tris-HCl, 2 mM EDTA, pH 8.0, to yield a
final NaCl concentration of 0.1 M and reconcentrated to
approximately 50 ml. The turbid solution was cleared of precipitate by
centrifugation at 5000 × g for 5 min. Residual
unfolded monomer, folded dimeric product, and higher order multimers
were separated by heparin affinity chromatography on a 5-ml Heparin
HiTrap column by fast protein liquid chromatography (Amersham Pharmacia
Biotech) at ambient temperature through combined step and gradient
elution with NaCl. The column was equilibrated with 6 M
urea, 50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 0.1 M NaCl at a flow rate of 1.0 ml/min, and the concentrated,
buffer-exchanged folding reaction was pumped onto the column with a
50-ml Superloop. After washing with 5 column volumes, the bulk of the
unfolded monomer was eluted with a step gradient of 0.25 M
NaCl as a single peak. The folded dimeric product eluted as a broad
peak with a 25-ml gradient from 0.25 to 0.4 M NaCl and the
multimeric forms with a 5-ml gradient from 0.4 to 1.0 M
NaCl. Fractions containing the folded dimer were identified by SDS-PAGE
and pooled and then dialyzed exhaustively against 0.1% trifluoroacetic
acid (two times, 2 h each, followed by a third dialysis
overnight), concentrated by ultrafiltration with a Centriplus device
(Amicon) in a tabletop centrifuge to approximately 1.5-2.5 mg/ml, and
stored at 4 °C. The folded dimer was purified to near-homogeneity by
reverse phase HPLC on a semi-preparative, 10 × 250 mm
C4 column (Vydac, 214TP510). Aliquots of the protein were
adjusted to 60% solvent A (0.1% trifluoroacetic acid), 40% solvent B
(80% acetonitrile, 0.08% trifluoroacetic acid) in a total volume of
1.0 ml, injected onto the column at a flow rate of 2.5 ml/min, and
eluted with a gradient from 40 to 60% B over 60 min. Injections of
approximately 1.3, 1.7, 2.3, and 3.4 mg provided similar recovery
efficiencies with no apparent decrease in resolution (larger loadings
were not tested). The peak of folded dimer, which reproducibly eluted
with a retention time of 43.5 min, was collected manually. Peak
fractions from multiple injections were pooled, the UV absorption
spectrum measured, and aliquots of 15-20 ml frozen in 50 ml
polypropylene tubes, lyophilized, and stored at SDS-PAGE--
Proteins were analyzed on denaturing 15%
polyacrylamide gels by discontinuous SDS-PAGE with glycine as the
trailing ion (24), or after digestion with trypsin, on denaturing 15%
polyacrylamide gels by discontinuous SDS-PAGE with Tricine as the
trailing ion (25). Samples containing folded, oxidized proteins were
denatured under non-reducing conditions prior to electrophoresis by
boiling in SDS-PAGE sample buffer in the presence of 2% SDS. For
denaturation and reduction of proteins, the sample buffer contained 2%
SDS and 0.2 M DTT. To quench folding reactions, concentrate
proteins, and remove salt, detergent, or chaotrope, trichloroacetic
acid was added to a final concentration of 15-20%, and the sample was incubated on ice for 20 min. Proteins were collected by centrifugation in a tabletop microcentrifuge for 10 min at 4 °C, washed with ice-cold 10% trichloroacetic acid, and recentrifuged. The protein pellet was rinsed with ice-cold 100% ethanol and allowed to dry prior
to boiling in SDS-PAGE sample buffer as described above. After
electrophoresis, proteins were visualized by staining with 0.25%
Coomassie Brillant Blue R-250 in acidic methanol. In contrast to
unfolded or misfolded forms of DPPC which reacted positively with
silver stain, the folded dimer stained negatively leaving a clear zone
or halo, which if initially subjected to staining with Coomassie
remained blue after staining with silver.
Activated Thiol-Sepharose Chromatography--
100 mg of
freeze-dried activated thiol-Sepharose 4B powder (Amersham Pharmacia
Biotech) was swollen in deaerated 1 mM EDTA for 15 min, and
a portion was poured into a disposable polypropylene column to yield a
bed volume of 0.2 ml. The gel was washed extensively with 1 mM EDTA (deaerated) to remove preservatives added prior to
freeze-drying and equilibrated with 3 column volumes of Tris-buffered GdmCl (6 M GdmCl, 50 mM Tris-HCl, 2 mM EDTA, pH 8.0). A standard folding reaction (1.3 ml) was
concentrated, and the buffer was exchanged by 3 cycles of concentration
and dilution with a Centricon C-10 (Amicon) ultrafiltration unit and
0.5% CHAPS, 50 mM Tris-HCl, pH 8.0, 1 mM GSSG
as dilution buffer, resulting in a final dilution of approximately
80-fold. One-half of the sample (50 µl) was diluted 10-fold with 450 µl of buffered GdmCl and passed over the column 5 times to ensure
quantitative coupling of free thiols. The flow-through was collected,
and the column was washed with 3 column volumes of buffered GdmCl.
Covalently coupled protein was eluted with 3 column volumes of buffered
GdmCl containing 50 mM DTT. The two pools of proteins,
bound and unbound, were precipitated with trichloroacetic acid (15%),
collected by centrifugation, and washed with ice-cold ethanol. The
samples were boiled in SDS-PAGE loading buffer (without reducing agent)
and analyzed on denaturing 15% polyacrylamide gels.
Trypsin Cleavage--
Chemically modified sequencing grade
trypsin (Boehringer Mannheim), which is more resistant to autolysis and
thus more stable during prolonged incubations, was dissolved in 1 mM HCl at a concentration of 1 mg/ml. For analytical
reactions, 2.5 µl of DPPC monomer or dimer (1 mg/ml in 0.1%
trifluoroacetic acid) was incubated with 1 µl of trypsin in a total
volume of 15 µl made 0.1 M in Tris-HCl, pH 8.5. The
solubility of the proteins, and hence the extent of the reaction, was
enhanced by addition of zwitterionic detergent (Zwittergent 3-12, Boehringer Mannheim) to 10 mM or urea to 4 M.
Digit-removed DPPC2 (DR-DPPC2) was produced
preparatively by a modification of the method of Koenig et
al. (26) for limited hydrolysis of recombinant BMP-2. 500 µg of
DPPC2 was solubilized in 1.0 ml of 4 M urea,
0.1 M NaCl, 0.1 M Tris-HCl, pH 8.5, 10 mM Zwittergent 3-12. Trypsin was added at a mass-wise ratio
of 1:20, trypsin:DPPC2, and the reaction was incubated at
37 °C for 5 h. The reaction mixture was dialyzed exhaustively
against 0.1% trifluoroacetic acid and DR-DPPC2 purified by
reverse phase HPLC on a semi-preparative C4 column (Vydac 214TP510,
10 × 250 mm) with a gradient of acetonitrile as described above
for the preparation of DPPC2. The digit-removed form of the
dimer eluted as a single symmetrical peak with a retention time of 48.6 min.
Protein Sequence Determination--
The N termini of
DPPC2 and DR-DPPC2 were determined by automated
cycles (9 and 5, respectively) of Edman degradation performed with an
Applied Biosystems 473A Protein Sequencer and analyzed on line with a
phenylthiohydantoin-amino acid analyzer.
UV Absorption Spectroscopy--
To assess the yield, purity, and
solubility of the protein, UV absorption spectra (240-340 nm) of the
dimer preparations were determined routinely following enrichment by
heparin affinity chromatography, dialysis, and concentration and
purification by reverse phase HPLC. For the determination of difference
spectra, 70 µg of protein in 0.1% trifluoroacetic acid was diluted
10-fold into 6.67 M GdmCl (AA-Grade, NIGU Chemie GMBH,
Waldkraiburg, Germany) buffered with 50 mM Tris-HCl, pH
8.0. Final protein concentration was 100 µg/ml or approximately 7 µM. After establishment of a base line with buffer only,
the absorption spectra of the monomer from 240-310 nm was recorded. A
second base line was then established with the monomer sample, the
spectrum of the dimer measured, and the difference spectrum obtained
from the derivative. Concentrations of the reduced monomer and the
folded, oxidized dimer were estimated from absorbance at 280 nm with
molar absorption coefficients of 19,940 and 40,755 M Noggin Binding Assay--
Anti-myc monoclonal antibody (9E10)
was coated on ELISA plates (Corning) in phosphate-buffered saline at 2 µg/ml by passive binding. Unbound 9E10 was removed by washing with
phosphate-buffered saline, and nonspecific binding was blocked with 1%
bovine serum albumin in phosphate-buffered saline. Xenopus
BMP-4 tagged with a myc epitope (xBMP-4myc) was bound to the
9E10-coated ELISA plates by incubation for 1 h and unbound
xBMP-4myc removed by washing. Increasing amounts of hNG-Fc were added
to the plate coated with xBMP-4myc and incubated for 1 h. Unbound
hNG-Fc was removed by washing, and 0.5 µg/ml alkaline
phosphatase-conjugated anti-human IgG (anti-Fc AP) was added to each
well. After 1 h, unbound anti-Fc AP was removed by washing, and
alkaline phosphatase substrate was added, and the reactions were
allowed to proceed for approximately 15 min. The extent of the
reactions was determined by measuring the absorbance at 405 nm with an
automatic plate reader, and the data in tabular form were converted to
a graphic format with the program Cricket Graph (Computer Associates).
A standard curve of hNG-Fc (human Noggin fused to the Fc domain of
human IgG1) was performed to demonstrate dose-dependent
binding of hNG-Fc to xBMP-4myc and to allow correlation between
A405 units and the fraction of hNG-Fc bound.
Competitive binding of DPPC2, human BMP-4, and human Noggin against
hNG-Fc was determined as described above, except that the concentration
of hNG-Fc was held constant (110 ng/ml) and the concentration of the
competitive ligands varied (1-1000 ng/ml). The buffer components of
either the DPPC2 or the hNG preparations used in this assay
have no effect on the binding of hNG-Fc to xBMP-4myc at the levels
employed. Similar results are obtained when blocking antibodies to hNG
are used and no binding of hNG-Fc to the plates is observed if
xBMP-4myc is omitted.
Chick Limb Bud Cell Assay--
BMP-induced synthesis of
proteoglycan in chicken embryonic limb bud cells was assayed as
described previously (28). Briefly, limb buds were isolated from day 7 chicken embryos, disintegrated with protease, and filtered cells
aliquoted into 24-well plates. After incubation in BMP-containing
medium for 4 days, 20 µl of 30 µCi/ml
Na235SO4 was added to each well,
and the cultures were incubated for an additional 6 h. Cells were
lysed with GdmCl and proteoglycans precipitated with Alcian blue and
collected on glass paper with a Skatron cell harvester.
35SO4 incorporation was determined with a
radio-TLC analyzer, and the data in tabular form were converted to a
graphic format with the program GraFit (Erithacus Software). Curves
were fit with a four-parameter equation for EC50 or
IC50 determination without providing initial estimates for
the iterative calculations.
Model of the DPP Homodimer--
The three-dimensional structure
of the DPP homodimer was modeled with ProMod (29, 30), an automated,
homology-based method.2 The
polypeptide backbone of the DPPC monomer was produced by sequence
alignment and three-dimensional superposition with the four
representatives of the TGF- In Vitro Folding Reaction
Ruppert et al. (28) have shown that the human homolog
of DPP, BMP-2, can be refolded in vitro in the presence of a
zwitterionic detergent and high salt, conditions determined empirically
for the refolding of TGF- Cerletti (33) has shown that supplementation of the folding reaction
developed for TGF- The absolute requirement for high salt was examined in detail by
varying the concentration of NaCl in the standard reaction buffer from
0 to 2.0 M NaCl in increments of 0.25 M (Fig.
2C). As initially determined (cf. Fig.
2A), omission of NaCl from the reaction had a severe effect
on the yield or recovery of all forms of the protein. Residual unfolded
monomer and the intermediate could be recovered from reactions
containing moderate concentrations of NaCl (0.25-0.5 M);
however, yields and/or recoveries of the dimeric product were
negligible in this range. Concentrations from 0.75-2.0 M
were found to be suitable for folding and/or recovery of the product,
with an apparent optimum around 1.25 M NaCl. A pronounced
increase between 0.5 and 0.75 M NaCl was observed relative to the gradual decrease in yield attained from 1.25 to 2.0 M. Recovery of monomeric forms was essentially independent
of NaCl concentration at 0.25 M and above. The decrease in
dimeric product at 1.0 M NaCl may have been an artifact of
sample manipulation rather than an actual minimum in the efficiency of
folding or recovery at this concentration.
The absolute requirement for low temperature was also examined in
detail by performing reactions between 5 and 35 °C in increments of
10 °C (Fig. 2D). As initially observed (cf.
Fig. 2A), incubation at low temperature (5 °C) yielded
both monomeric intermediate and folded dimeric forms of the protein and
incubation at 25 °C almost undetectable levels of these forms.
However, folding and/or recovery of the dimeric product was found to be
optimal at the intermediate temperature of 15 °C. Incubation at
35 °C was equally as inefficient as near-ambient temperature
(25 °C).
Purification
A variety of chromatography matrices (cation exchange, anion
exchange, and chromatofocusing) and buffer combinations (sodium acetate, Tris-HCl, HEPES, MES, 30% isopropyl alcohol, and 6 M urea) were screened for their ability to separate
residual unfolded and misfolded forms from the folded dimeric product.
However, all forms of the protein failed to bind these matrices and
co-eluted in the flow-through. Because the DPP dimer was expected to
contain two N-terminal heparin-binding sites (28), heparin-Sepharose was tested and found to provide efficient separation of the folded dimer from non-native forms (Fig. 3,
A and B). All forms of the protein were bound to
heparin quantitatively in low salt (6 M urea, 50 mM Tris-HCl, 2 mM EDTA, pH 8.0, 0.1 M NaCl). Dimers, both folded and misfolded, bound with
affinity intermediate between the low affinity of the monomer and the
high affinity of the larger multimeric misfolded forms. Gradient
elution alone resulted in partially overlapping peaks of monomer and
dimer which could not be resolved by alteration of the shape of the
gradient.3 However, an initial step gradient resulted in
the quantitative elution of the bound monomer in a single well formed
peak. A subsequent linear gradient of NaCl yielded a broad peak of
dimeric protein containing the folded product in highly enriched
form.
Biochemical and Biophysical Characterization of Refolded
Drosophila DPP, a Homolog of Bone Morphogenetic
Proteins 2 and 4*
§,
,, and§§
Department of Cell Biology and
¶ Department of Biophysical Chemistry Biozentrum, University of
Basel, CH-4056 Basel, Switzerland, ** Regeneron Pharmaceuticals, Inc.,
Tarrytown, New York 10591, and
Theodor-Boveri-Institut (Biozentrum),
Physiologische Chemie II, Am Hubland,
D-97074 Würzburg, Germany
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
superfamily members, shown to act as intramolecular chaperones in
vivo.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
superfamily signal ligands
in vitro, a membrane protein-solubilizing detergent, high
concentrations of NaCl, and low temperatures, appear to be required for
recovery of the folded product rather than to promote the process of
folding per se, as is more commonly observed. Thus
solubilization of the folded C-terminal domain of TGF- superfamily
members may be one of the principal roles of the large (250-400 amino
acids) N-terminal prodomains, which are required as intramolecular
chaperones in vivo to provide for secretion of the mature
signal ligands (19).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
10 promoter system (20). A PCR
product encoding the mature protein was synthesized with Taq
DNA polymerase (Stehelin) from a cDNA template and introduced into
pJC10 (21), a high copy number derivative of pET3c, by cleavages with
NdeI and BamHI. These sites were incorporated
into the DNA product by oligonucleotide primers encoding N- and
C-terminal sequences, respectively. The NdeI site provided a
methionine initiation codon downstream of the 10 promoter and
ribosome-binding site of pJC10. The BamHI site is preceded by a stop codon and flanked by a terminator. Analysis of the reverse phase HPLC-purified folded dimer prepared from the E. coli-expressed protein by electrospray ionization mass
spectrometry using direct infusion indicated a molecular mass of
29,576.9 ± 2 Da, which was not in agreement with the value
(29,591.6 Da) calculated from the cDNA-encoded amino acid sequence
in the data base (accession number P07713). The nucleotide sequence of the PCR product was determined by automated
fluorescent-dideoxynucleotide sequence analysis, and three variant
positions were identified, two of which were in accordance with the
encoded genomic sequence (see legend to Fig. 1). The third variation,
apparently introduced by the low fidelity thermostable polymerase
employed, was a valine to alanine substitution (Val80 
Ala; numbering as in Fig. 1). A PCR product encoding the wild-type protein was synthesized with a high fidelity enzyme, Pfu DNA
polymerase (Stratagene), and the nucleotide sequence of the PCR product
was verified by dideoxynucleotide sequence analysis. Wild-type DPPC protein (Val80) was analyzed in experiments depicted in
Fig. 2, C and D, and Fig. 7, variant protein
(Ala80) in all other experiments shown.
20 °C. Protein concentration (approximately 18 mg/ml) was
estimated by the method of Bradford (23) by solubilizing microliter
volumes of a 10-fold dilution of resuspended inclusion bodies directly in the phosphoric acid/methanol reagent which was then diluted with
water. HPLC-purified DPPC protein and bovine serum albumin gave similar
responses in the dye-binding assay, indicating that bovine serum
albumin standard curves can be used to estimate DPPC protein
concentration, as has been shown previously to be feasible for DPP
dimer from S2 cells and for the BMP family of proteins in general (13).
Insufficient sonication resulted in loose, jelly-like pellets of
inclusion bodies due to contamination with high molecular weight DNA
which could be eliminated by additional cycles. As an alternative to
sonication, a French press could also be employed and may be more
practical with larger preparations.
20 °C.
1 cm1, respectively.
Coefficients were calculated by the method of Pace et al.
(27), which includes contributions from disulfide bonds
(
280M or
cm
1 = Trp5500 + Tyr1450+ Cys125). The calculated molecular
weights of the reduced monomer and the oxidized dimer, including the
N-terminal methionine residues, are 14,819.8 Da and 29,625.6 Da.
Monomer and dimer at 1 mg/ml therefore correspond to absorbances at 280 nm of 1.35 and 1.38, respectively. All spectra were determined with a
single-beam, microprocessor-controlled UV/VIS spectrophotometer (Hewlett-Packard 8452A Diode Array) with data storage capacity.
superfamily (TGF-1, TGF-2, TGF-3, and BMP-7) currently in the Brookhaven Protein Data Bank. As
the C-terminal domains of BMP-7 and DPP were aligned without the
introduction of gaps, no additional loops were constructed de
novo and only non-conserved side chains had to be introduced and
existing side chains corrected by referencing a library of allowed side
chain rotamers. The model was subjected to several cycles of energy
minimization with CHARMm and refined by optimizing intramolecular
contacts and relieving steric strain. A symmetry transformation was
employed to generate a dimeric structure from the final model of the
monomer. Template structures and models were visualized with RASMOL
(31). The model of the monomer, when transformed to a dimer, does not
result in any apparent large gaps or steric conflicts within the
structure. Because the disordered N-terminal arm is unresolved in the
electron density map and therefore lacking from the crystal structure
of BMP-7, the structure of this polypeptide could not be incorporated
into the model of the DPP homodimer.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2 (32). We found that the C-terminal domain of DPP (DPPC; Fig. 1) also can be efficiently
refolded in vitro under these conditions. As is common for
many secreted disulfide-containing proteins, the DPP homodimer contains
no unpaired cysteines and thus the oxidized dimeric folding product can
be denatured under non-reducing conditions and analyzed by SDS-PAGE
without introducing rearrangements due to scrambling of disulfide bonds
by thiol-disulfide exchange (Fig.
2A). A single major dimeric
product was observed (DPPC2 ox) migrating within a well
defined zone, suggestive of a single species or conformer. The dimeric
product also migrated with increased mobility compared with aggregates
of unfolded monomer (2 DPPC red), indicative of reduced hydrodynamic
volume and consistent with an extensively disulfide-linked form. A
partially oxidized, monomeric intermediate was also observed in
reactions that yielded folded product. Moderate alteration of the
protein concentration, pH, ratio of the redox pair, or supplementation
with EDTA to chelate oxidation-catalyzing metals had negligible effects
on the overall efficiency of the reaction, although minor influences on
the rate of the reaction were observed with respect to pH and redox
pair.3 In contrast, low
temperature, high salt, and zwitterionic detergent were each found to
be absolutely necessary for refolding the C-terminal domain of DPP
in vitro.
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Fig. 1.
Structural features of the C-terminal domain
of DPP. Maturation of the DPP proprotein in vivo
produces a homodimeric signaling ligand composed of two
disulfide-linked, 132-amino acid monomers of the C-terminal domain
(12). The N-formylmethionine residue, required for bacterial
overexpression, was not cleaved post-translationally by the E. coli host. Like other members of the BMP family, each monomer of
the secreted form of the DPP homodimer is expected to be glycosylated
at a conserved N-linked site (*) contained by the consensus
motif, NX(S/T). The cystine-knot which is the structural
hallmark of the mature domain of all TGF- superfamily members is
composed of a pair of linkages (Cys61-Cys130;
Cys65-Cys132) through which a third
(Cys32-Cys98) is formed. The homodimer is
covalently linked via an intermolecular disulfide bond between residue
97 of each monomer. The N-terminal polypeptide arm, which is not
conserved among TGF- superfamily members, is a flexible extension
from the folded, disulfide-linked core (shaded) and contains
clusters of basic residues (bold) which have been implicated
in mediating high affinity binding to heparin (28). Arginine and lysine
residues within the core which are potential substrates for tryptic
cleavage are also indicated. The boundary between the disordered
N-terminal arm and the folded core is predicted from the electron
density map of BMP-7 (18). Arrows indicate N termini of the
folded dimer after limited proteolysis with trypsin, their lengths
proportional to the relative frequency of cleavage. The amino acid
sequence encoded by the expression construct is not in agreement with
the cDNA-derived sequence (1), and its data base entry at residues
18 and 19 (Gln, Pro in place of His, Ala), however, is in accordance
with the encoded genomic sequence (2).
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Fig. 2.
Characterization of the in vitro
folding reaction. A, parameters of the reaction.
Chaotrope-solubilized inclusion bodies of the C-terminal domain of DPP
(DPPC) were rapidly diluted 50-fold in standard reaction buffer (2 M NaCl, 1.8% w/v CHAPS, 2 mM GSH, 1 mM GSSG, 50 mM Tris-HCl, pH 8.0) to a final
concentration of approximately 50 µg/ml and incubated at 4 °C for
3 days (std rxn). The effects of supplementation with 5 mM EDTA, incubation at ambient temperature, reduced protein
concentration, varied pH, an equimolar (1 mM) ratio of
redox pair, and elimination of either NaCl or CHAPS from the reaction
were analyzed. The reactions were terminated and the protein
precipitated by the addition of trichloroacetic acid to 20% (see
"Experimental Procedures") and analyzed by non-reducing SDS-PAGE.
An aliquot of the unfolded inclusion body protein was subjected to
boiling in the presence of 2% SDS and 0.2 M DTT and
co-electrophoresed (reduced monomer). B, effect of organic co-solvent and
folding trials with other Drosophila BMP homologs. Folding
reactions containing the C-terminal domains of DPP, 60A, and SCREW were
performed in standard reaction buffer (std rxn) or in
standard reactions supplemented with 20% Me2SO
(+DMSO) and analyzed as described above. Products in the
folding reactions with the C-terminal domain of DPP are indicated with
arrows as follows: upper arrow, oxidized dimer
and lower arrow, monomeric intermediate. C, NaCl
dependence. Reactions containing the C-terminal domain of DPP were
performed under standard conditions except that the concentration of
NaCl was varied from 0 to 2.0 M NaCl in increments of 0.25 M. The reactions were quenched by the addition of
trichloroacetic acid, adjusted to an equivalent concentration of NaCl
(2.0 M) without altering the composition of CHAPS (1.8%),
and then incubated on ice and collected by centrifugation.
D, temperature dependence. Standard reactions containing
DPPC were incubated between 5 and 35 °C in increments of 10 °C
for 3 days. An aliquot of the solubilized inclusion body protein was
stored in 6 M GdmCl at 5 °C during the course of the
folding reaction and then diluted into trichloroacetic acid-quenched
folding buffer for precipitation and analysis by non-reducing SDS-PAGE
( ). Inclusion body protein analyzed in C and D
was prepared under reducing conditions as described under
"Experimental Procedures," whereas the preparations shown in
A and B were washed in TE buffer without DTT and
subjected to a final wash with 10% acetic acid, which allowed for
removal of residual lysozyme (migrates slightly faster than DPPC
monomer) and other more high molecular weight contaminants. DPPC
red, reduced monomer; DPPC int, a monomeric folding
intermediate; DPPC2 ox, oxidized
dimer; 2 DPPC reduced, non-covalently linked, dimeric
aggregate.
2 with an organic co-solvent (20-30% dimethyl
sulfoxide or 10% dimethylformamide) provided for efficient refolding
of TGF-3. Addition of organic co-solvent (20% dimethyl sulfoxide)
to the standard folding reaction described above had a marked
inhibitory effect on the refolding of DPPC, reducing the yield of
folded dimer relative to monomeric or misfolded forms (Fig.
2B). Folding trials with the other Drosophila BMP
family members, 60A (34) and SCREW (35), under both conditions
(±dimethyl sulfoxide) failed to produce detectable levels of folded
dimeric product, i.e. a single species or conformer
migrating as a compact form ahead of the population of dimeric
aggregate. As well, no monomeric folding intermediate indicative of
disulfide bond formation was observed in either reaction with the
C-terminal domains of 60A and SCREW.
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Fig. 3.
Purification of the dimeric folding
product. A and B, separation of DPPC,
DPPC2, and multimeric forms by heparin affinity
chromatography. A, profile of HiTrap heparin affinity
column. Approximately 40 mg of chaotrope-solubilized inclusion body
protein was rapidly diluted into standard folding buffer (200 ml) and
incubated at 4 °C for 3.5 days. The reaction was concentrated and
the buffer exchanged to 6 M urea, 50 mM
Tris-HCl, pH 8.0, by ultrafiltration and pumped onto a
1-ml HiTrap heparin affinity column with a 10-ml Superloop
mounted on an fast protein liquid chromatography system (Amersham
Pharmacia Biotech). The bound protein was washed extensively with 6 M urea, 50 mM Tris-HCl, pH 8.0, 0.1 M NaCl until the absorbance at 280 nm of the elution
returned to base line. Residual monomeric protein was eluted with a
step gradient from 0.1 to 0.25 M, the folded dimeric
product with a linear gradient from 0.25 to 0.4 M, and
misfolded multimeric forms with a linear gradient from 0.4 to 1.0 M NaCl. B, non-reducing SDS-PAGE analysis of
fractionation depicted in A. 1-µl aliquots from the
indicated column fractions (1.0 ml each) were boiled in SDS-PAGE
loading buffer containing 2% SDS (without reducing agent) and analyzed
on a denaturing 15% polyacrylamide gel. Protein was visualized by
staining with Coomassie. An aliquot of the flow-through (FT)
peak, not depicted in A, was also analyzed. C and
D, C4 reverse phase HPLC purification and analysis of folded
DPPC2. C, purification of folded dimer from
residual monomer and misfolded forms. The folded dimer eluted late in
the acetonitrile gradient (retention time ~40 min) as a single,
uniform peak compared with unfolded or misfolded species that appeared
earlier as a broad shoulder (18-38 min). D, analysis of
purified DPPC2. An aliquot (~30%) of the peak material
collected from the preparative chromatographic run shown in
A was lyophilized, redissolved in 50% acetonitrile, 0.1%
trifluoroacetic acid, and rechromatographed. The purified,
re-solubilized protein eluted as a single sharp peak, equilibrating
with or contaminated by little or no detectable denatured forms. The
small peaks at approximately 16 and 28 min, which were not observed in
the preparative profile, appear to be solvent artifacts or ghost peaks
emanating from a less stable base line. The purified protein
reproducibly eluted slightly later (~42 min) in the gradient when
rechromatographed. HPLC depicted in C and D were
performed with an analytical C4 column (Vydac 214TP54, 4.6 × 250 mm). Milligram quantities were purified with a semi-preparative C4
column (Vydac 214TP510, 10 × 250 mm) from which the dimeric
product eluted in a peak from 41 to 46 min.
After enrichment, the dimeric product could be bound quantitatively to a cation exchange matrix (SP-Sepharose; 6 M urea, 50 mM HEPES, pH 7.5, 0.15 M NaCl), but the folded protein eluted from a linear gradient of NaCl in a broad peak contaminated by the trace non-native forms, perhaps due to shared ionic properties of the proteins.3 In contrast, the folded dimer was purified to near-homogeneity by reverse phase HPLC with a linear gradient of acetonitrile (Fig. 3C), which separates proteins primarily due to differences in surface hydrophobicity. Residual unfolded monomer and misfolded forms eluted in a broad shoulder preceding a single uniform peak of dimeric product, which was retained last by the C4 matrix.
Approximately 3 mg of HPLC-purified folded dimer was obtained per liter of bacterial culture (Table I). Differences in yield of the purified product arose predominantly from variability in the yield of inclusion body protein, which in turn depended on the efficiency of induction (cf. "Experimental Procedures"). The induced protein was found exclusively in the inclusion body fraction and quantitatively recovered in a highly purified form (cf. Fig. 2A). Around 15% of the chaotrope-solubilized protein could be refolded routinely under the standard conditions and obtained in highly enriched form in one step by heparin affinity chromatography. Approximately one-half of the enriched pool of dimeric product was recovered at near-homogeneity by C4 reverse phase HPLC.
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Evidence for a Folded Product
The inclusion body-purified protein appears to be correctly refolded and oxidized to a disulfide-linked homodimer, as evidenced by several independent observations.
Chromatographic-- Following lyophilization and resolubilization in 50% acetonitrile, 0.1% trifluoroacetic acid, the purified dimer was reanalyzed by C4 reverse phase HPLC and found to elute in a single sharp peak (Fig. 3D). Little or no unfolded or misfolded forms were observed, indicative of a stable disulfide-linked structure resistant to rearrangement or denaturation.
Electrophoretic-- Comparison of the mobilities of oxidized and reduced forms of denatured proteins in polyacrylamide gels provides a simple assay for disulfide bonds, which restrict the flexibility of the polypeptide and decrease its hydrodynamic volume (36). Analysis of HPLC-purified dimeric and monomeric forms of DPPC clearly shows the greater mobility of the folded dimeric product compared with that of aggregated monomer, indicative of a more compact, disulfide-linked structure (Fig. 4A). SDS-PAGE analysis of the purified proteins also clearly demonstrates that the dimeric product migrates as an apparent single species or conformer, in contrast to the aggregated monomer which migrates in a disperse zone indicative of a heterogeneous population of unfolded or misfolded polypeptides.
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Chemical-- Activated thiol-Sepharose 4B (Amersham Pharmacia Biotech) provides a means of separating thiol-containing from nonthiol-containing proteins. A mixed disulfide between Sepharose-linked glutathione and 2-pyridyl reacts through thiol-disulfide exchange with proteins containing free thiols, whereas nonthiol-containing proteins are unreactive and therefore not covalently bound. The extent of oxidation of the DPPC protein forms was examined by passing a standard folding mixture equilibrated in 6 M GdmCl repeatedly over a column of activated thiol-Sepharose 4B and analyzing the bound and unbound fractions by non-reducing SDS-PAGE (Fig. 4B). The dimeric product appeared to be quantitatively recovered in the unbound fraction, indicating that no reaction had occurred and consistent with an oxidized form containing no free or accessible thiols. The monomeric intermediate (DPPC int) was also found to be unreactive, indicating that the majority of the cysteines had formed disulfide linkages or were inaccessible to the mixed disulfide linked to the column matrix. Most of the unfolded monomer and misfolded forms reacted covalently, as little of these forms were detected in the flow-through fraction and could be decoupled with DTT-containing buffer. Also consistent with a completely oxidized native structure containing multiple disulfides and no unpaired cysteines is the observation that the dimeric product could be denatured prior to electrophoresis in the presence of SDS by boiling at neutral pH, conditions which would allow for the scrambling of disulfide bonds by free thiols, without any evidence of rearrangement to forms with altered migration rates (cf. Fig. 4A).
Proteolytic-- Proteolytic cleavage of proteins in the native conformation is usually observed only in disordered regions of the polypeptide backbone, such as segments linking separate domains, large flexible loops, or extended termini. In contrast, unfolded, misfolded, or intermediately folded proteins are much more sensitive to proteolysis at multiple sites along the polypeptide chain. Koenig et al. (26) have observed that the compact, disulfide-linked core of human BMP-2 is resistant to proteolysis, whereas the N-terminal polypeptide extending from the folded C-terminal domain is protease-sensitive. Incubation with trypsin yielded truncated products collectively designated DR-BMP-2, a "digit-removed" form. Similarly, incubation of DPPC2 with trypsin also yielded a truncated, digit-removed form (Fig. 4C). Solubilization of the dimer in aqueous buffer with either zwitterionic detergent or 4 M urea allowed for a quantitative conversion from full length to the truncated form, which consisted of two major products as revealed by amino-terminal sequence analysis. Approximately 35 and 65% of the DR-DPPC2 was found to have N-terminal extensions of four and five residues, respectively, from the domain core (cf. Fig. 1). A similar gradient of protease sensitivity with respect to the folded core was observed for BMP-2. Approximately 30 and 70% of the DR-BMP-2 had extensions of two and four residues, respectively (26). In comparison, treatment of the unfolded monomer under these conditions led to an apparent total proteolysis of the polypeptide at additional lysine and arginine residues within the polypeptide (cf. Fig. 1). The resistance of the disulfide-linked core of the folded dimer to proteolysis was significant. Digestion of the folded dimer under even more rigorous conditions (stoichiometric ratio of dimer and protease, 4 M urea, 24 h incubation with chemically modified, high stability trypsin) failed to yield additional detectable products, demonstrating that the core of the folded product did not contain any protease-accessible disordered regions expected of a partially folded or misfolded state.3
Spectroscopic-- Differences in conformation and solubility of the unfolded monomer and the folded dimer were analyzed by ultraviolet absorption spectroscopy (Fig. 5). Each DPPC monomer contains 3 phenylalanine, 6 tyrosine, and 2 tryptophan residues that serve as chromophores, allowing not only for estimation of protein concentration, but also for comparison of the molecular environment of these side chains and the relative solubility of the two forms. Because aromatic amino acids do not absorb at wavelengths greater than 310 nm, a sloping base line in the 310-400 nm region is generally due to light scattering by large particles or aggregates in solution (37). The spectrum of the unfolded monomer slopes strongly in this region, indicative of aggregation (Fig. 5A). In contrast, the spectrum of the folded dimer maintains a base-line value throughout this region, demonstrating that the preparation is more soluble than the unfolded monomer at low pH (~2) in this mildly chaotropic buffer (0.1% trifluoroacetic acid). Differences in the curvature of the descending slope (285-295 nm) of the spectra were also observed. Such differences are due to increases in absorbance of tyrosine (285-288 nm) and tryptophan (291-294 nm) residues upon burial in the hydrophobic core of the native, folded protein and are usually accompanied by a red shift in the absorbance maximum (37). Thus, in addition to the environment-specific differences in the descending slope, the absorbance maximum of the unfolded monomer (276 nm) is red-shifted by 2 nm relative to that of the folded dimer (278 nm). To minimize potential artifacts due to the aggregated state of the monomer and to compare the stability of the two forms, uv spectra were also measured under denaturing conditions (6 M GdmCl). Although the spectra were no longer shifted relative to one another, differences in the descending slope were again observed (Fig. 5B). The difference spectra of the two protein forms in 6 M GdmCl (Fig. 5C) reveals peaks at 285 and 293 nm resulting from differences in the solvent exposure of the tyrosine and tryptophan residues, respectively. Thus even under strongly denaturing conditions the folded dimer retains some native-like structure, presumably due to the constraints imposed by the extensive and intertwined disulfide linkages of the cystine knot (cf. Fig. 1).
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Noggin Binding
Holley et al. (16) have shown by injection of mRNAs in Drosophila embryos that noggin, an antagonist of BMP signaling in vertebrates, also blocks signaling by DPP. Because Noggin exerts its antagonistic effect by binding to BMP-2, -4, and -7 and blocking their ability to interact with their receptors (38), the potent biological activity of noggin may be mediated by direct binding to the dpp-encoded signal ligand. Thus we determined whether refolded DPPC2 could bind Noggin in a competition binding assay (Fig. 6). After determination of the dose dependence of epitope-tagged human Noggin (hNG) binding to antibody-immobilized Xenopus BMP-4, the ability of the refolded DPPC2, human BMP-4, and refolded hNG to compete against the binding of hNG to Xenopus BMP-4 was determined. hNG at fixed concentration was challenged with each ligand at a series of concentrations, and the fraction of epitope-tagged hNG remaining bound was determined by ELISA. The human BMP-4 standard was found to displace half of the bound hNG at a concentration of approximately 0.3 nM. Refolded DPPC2 competed slightly less efficiently, displacing half of the bound hNG at about 3-fold higher concentration or 1 nM. The refolded hNG control was only slightly more effective than refolded DPPC2, competing half of the bound Noggin at around 0.6 nM. Thus the refolded DPPC2 bound Noggin with an affinity comparable to that of vertebrate BMP-4 and was therefore bioactive with respect to regulation of activity by antagonist binding.
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Induction of Chondrogenesis
Recombinant DPP and 60A proteins produced in tissue culture have been shown to induce the formation of endochondral bone after subcutaneous implantation in rats (13), the principal and most definitive means of determining BMP activity (39, 40). However, for optimal results, insect BMP orthologs must be reconstituted with rat collagen carrier prepared from demineralized bone by GdmCl extraction, rather than with commercially available basement membrane-extracted matrices. In addition, analysis of extensive dose curves or direct comparison with other BMP activities is impractical due to the large number of implantations required. An alternative assay, induction of chondrogenesis in micromass cultures of mesenchymal cells from embryonic chicken limb buds (41-44), provides quantitative dose-dependent determination of activity as well as direct comparison between BMPs. In this assay, the EC50 (effector concentration at half-maximal response) for BMP-2 was 33 ± 13 nM and the maximum incorporation at saturation 20,600 cpm ± 4,200 cpm (Fig. 7). The DPP ligand exhibited approximately 5-fold reduced activity relative to BMP-2, as estimated by the calculated EC50 value (170 ± 60 nM). The maximum incorporation induced by DPPC2 at saturation was estimated at 14,900 ± 2,600 cpm, approximately 70% that achieved by BMP-2. However, due to the limited solubility of these ligands above 1 µM, saturation values could not be directly measured and required estimation from the fitted curves, the shapes of which were highly sensitive to small deviations in the measured response at the highest concentrations of ligand. Thus the difference in maximum response achieved by the two ligands did not appear to be significant due to the limitations imposed at higher concentrations. The activity of DPPC2 as a function of concentration, on the other hand, is indeed reduced relative to the mammalian protein but may reflect the heterologous nature of the assay. Mammalian fibroblasts such as C3H10T1/2 cells are markedly less responsive to BMP-2 than the avian mesenchyme cells (28). In keeping with this reduction in responsiveness, DPP has no measurable inductive effect on this cell line. In fact, none of the mammalian fibroblast cell lines commonly employed to assay BMP activity has been found to respond to insect BMPs. Thus the reduced activity of the insect ligand relative to the human ortholog at lower concentrations in the chick mesenchyme cell assay most likely reflects reduced complementarity between the insect ligand and the avian receptors rather than a non-native ligand structure. Despite the inherent limitations imposed by the insolubility of the ligands and the heterologous nature of the assay, the refolded DPP dimer induced a strong chondrogenic response in a dose-dependent manner and was therefore clearly bioactive as a signal ligand.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Although the genetic control of many key developmental processes by dpp has been intensively studied, little has been known about the biochemical and biophysical properties of the encoded signaling ligand and specific aspects of its structure due to the lack of appreciable amounts of highly purified protein for analysis. We have shown that the mature C-terminal signaling domain of DPP can be efficiently refolded from chaotrope-solubilized inclusion bodies, yielding a properly folded, bioactive ligand that can be purified to near-homogeneity in two chromatographic steps. Approximately 3 mg of high purity protein can be produced routinely per liter of bacterial culture, enabling not only biochemical but also biophysical and structural studies to be performed.
DPP C-terminal domain dimer was produced in high yield under conditions
determined empirically for TGF-2 (32) that also provided for the
production of the human homolog of DPP, BMP-2 (28). The solubilized,
denatured proteins are refolded in a zwitterionic detergent and high
concentrations of NaCl at low temperature in the presence of a redox
system. Glutathione, in its reduced and oxidized forms at a ratio and
in concentrations similar to those observed in vivo (45),
provides for thiol-disulfide exchange leading to a fully oxidized
dimeric product and is employed in many, if not most, in
vitro reactions for the production of disulfide-containing
proteins.
In contrast to the requirements for the glutathione pair, the roles of
the zwitterionic detergent, high concentrations of salt, and low
temperature are not as readily apparent. Cerletti (33) determined that
the optimal detergents for production of TGF- superfamily members
are the non-ionic detergent digitonin and the zwitterionic detergent
CHAPS, the latter and its derivative (CHAPSO) being most preferable.
These are mild detergents employed frequently and almost exclusively
for solubilization of membrane proteins without denaturation. An
absolute requirement for detergent to solubilize the monomer was
observed (cf. Fig. 2A); however, it remains to be
determined whether other detergents would suffice in this capacity, and
if so, whether the specific effect of the membrane protein-solubilizing
detergents is to promote folding, enhance recovery, or both.
In the absence of NaCl the monomer was only marginally soluble (cf. Fig. 2, A and C); however, at 0.25 M and above, residual unfolded monomer and the folding intermediate could be quantitatively recovered. Thus despite the limited amount of dimer observed in reactions containing low to moderate concentrations of NaCl (0.25-0.5 M), the potential for folding is present under these conditions, as opposed to reactions that lacked salt or detergent. The high concentrations of NaCl (0.75-2.0 M) found to allow for efficient production of the dimer are therefore required to promote folding and/or to provide for recovery of the native form.
Low to moderate concentrations of all salts have a salting-in effect by
screening the net charge of a protein in aqueous solution, which leads
to a reduction in electrostatic free energy and an increase in its
solubility (46). Thus addition of NaCl at low ionic strength to the
folding reaction appears to produce such a salting-in effect, allowing
for the recovery of the monomeric and intermediate forms. However, at
higher concentrations, NaCl, found to be the preferred salt for
production of TGF-2 (32) and TGF-3 (33) in vitro, can
have more complex effects on protein solubility and structure.
Consistent with their central positions in the Hofmeister series,
Na+ and Cl ions are relatively neutral with
respect to their effects on the properties of bulk water but
nonetheless can increase the solubility of proteins dramatically at
concentrations up to 2 M (46, 47). Several observations
suggest that in the case of the production of TGF- superfamily
members, high salt enhances the solubility and hence the recovery of
the dimeric product but is not required to promote its folding. First,
TGF-2 can be efficiently refolded in vitro in low salt
(250 mM NaCl) as a fusion protein containing a tripartite
N-terminal peptide composed of a hexahistidyl tag, a thrombin cleavage
site, and a collagen-binding site (48). The role of high salt can thus
be mimicked by the N-terminal peptide which most likely has a
solubilizing effect, as the efficiency of renaturation of other
inclusion body proteins can be significantly enhanced through fusion
with hydrophilic partners (49). For example, fusion of insulin-like
growth factor I with the IgG binding domain of staphylococcal protein A
allowed for refolding at relatively high concentrations without the
need for solubilizing agents (50), and a clear correlation between the
efficiency of folding and the hydrophilicity of short N-terminal fusion
peptides could be demonstrated for the renaturation of human
granulocyte-stimulating factor proteins (51). Thus high concentrations
of salt are not strictly required to promote folding, since the
TGF-2 fusion protein could be renatured in 250 mM NaCl,
but rather appear to have a solubilizing effect. As mentioned above,
the unfolded and intermediately folded forms of DPPC are soluble at low
concentrations of salt, without the aid of a fusion partner, suggesting
that high salt is required to enhance the solubility of the folded form. Second, DPPC seems competent to fold under conditions of low
salt, due to the following similarities with the productive reactions
in high salt: (i) the amount of residual monomer is equivalent,
indicative of consumption in the reaction, (ii) the folding
intermediate is formed, and to a similar extent, and (iii) the folded
dimer is indeed produced, albeit at significantly reduced efficiency.
Together these observations suggest that the dimer is refolded just as
efficiently at low to moderate salt concentrations as at high but is
insufficiently soluble under these conditions to be quantitatively
recovered. Third, the reduced DPPC monomer appears to adopt a
native-like, dimeric state in the absence of high salt, as determined
by fluorescence spectroscopy and sedimentation equilibrium
ultracentrifugation analyses in 0.1% trifluoroacetic acid, pH
2.3 Therefore, high concentrations of NaCl are not required
to promote folding by collapsing the monomer or inducing structure, in
keeping with the efficient folding of the TGF-2 fusion protein
observed in low salt and consistent with a singular role in
solubilizing the dimeric product.
Recovery of unfolded monomer from the folding reaction was independent
of temperature (cf. Fig. 2, A and D),
just as recovery of monomer was independent of the concentrations of
NaCl at 0.25 M and above. However, production of dimer was
strongly dependent on the temperature of the 3-day incubation. Low
temperature (5-15 °C) was strictly required for efficient
production of the dimer, which was present in only limited amounts in
reactions incubated at near ambient temperature and higher
(25-35 °C). However, the dimeric form is indeed produced at these
temperatures to some extent, as is the case for reactions containing
low concentrations of NaCl, again suggesting that recovery is
singularly affected rather than folding. In addition, the levels of
residual unfolded monomer after incubation at high temperature are
equivalent to those of productive reactions (low temperature and high
salt), consistent with the notion that the less productive conditions
allow the reactions to proceed nearly equally but have disparate
effects on the solubility of the product. The positive effect of
temperatures in this range (5-15 °C) on the solubility of many
proteins is well documented and has a physical basis due to the
increased tendency of water to form clathrate-like structures around
non-polar molecules. This water-ordering effect of lower temperatures
results in a decrease in the free energy of transfer of a non-polar
molecule into water, 
Gtr, a measure of the
magnitude of the hydrophobic interaction (47). Interestingly, the
optimum temperature of folding proteins from mesophiles in
vitro is 10-15 °C (52), which coincides well with the optimum
observed for the C-terminal domain of DPP. However, low temperature
presumably aids in the solubilization of intermediately folded forms of
most mesophilic proteins, reducing aggregation in the absence of
solubilizing agents, whereas low temperature appears to be required for
solubilization and recovery of the product, not the intermediate forms,
of TGF- superfamily members folded in the presence of high salt and
a membrane protein-solubilizing detergent. Thus two of the factors found to be crucial for production of C-terminal dimers in
vitro, high concentrations of NaCl and low temperature, appear to
influence the solubility of the folded form, not the unfolded or
partially folded forms as is commonly the case (53, 54).
The insolubility of non-native forms is generally attributed to
intermolecular interaction between hydrophobic side chains, inaccessible to solvent in the core of the native form, but transiently exposed to solvent during the folding process, leading to aggregation at high protein concentrations or in the absence of solubilizing factors. In this regard, it is particularly interesting that the folded
dimeric product DPPC2 is actually more hydrophobic than the
unfolded or misfolded forms, as shown by reverse phase HPLC (cf. Fig. 3C). The non-native forms elute in a
broad shoulder preceding the peak of folded dimer, which is released
last from the hydrophobic matrix by the gradient of organic solvent. In contrast, the native forms of the vast majority of proteins are more
hydrophilic than their partially folded forms and elute significantly earlier. This pronounced hydrophobicity appears to be responsible for
the insolubility of DPPC2 and TGF- superfamily members
in general. The DPP C-terminal dimer not only requires solubilizing conditions to be recovered from the folding reaction as described above, but as well must be maintained in chaotropic aqueous buffers or
in aqueous organic mixtures throughout purification and during storage
and handling (cf. Table I), a shared property of BMP (28,
55, 56) and TGF- (32, 33) ligands. At relatively high concentrations
(2.5 mg/ml) in a non-chaotropic, aqueous buffer (10 mM
sodium acetate, pH 3.5), the DPP dimer was soluble but highly
aggregated, so much so that the size of the particles could not be
estimated by dynamic light scattering.
Mittl et al. (57) have noted that the surface of TGF-3 is
mainly hydrophobic, which provides the structural basis for its low
solubility in aqueous solvents. A similar analysis of the structures of
the other TGF- superfamily members in the Brookhaven Protein Data
Bank, the refined crystal structures of TGF-2 (58, 59), the solution
structure of TGF-1 (60), and the crystal structure of BMP-7 (18),
demonstrates that this is a conserved structural feature and accounts
for the general tendency for these ligands to aggregate under
non-denaturing conditions. The hydrophobic surface is composed of two
symmetry-related pairs of patches that wrap around the dimeric
C-terminal domains in a nearly contiguous fashion, as demonstrated by
the crystal structure of BMP-7 and a homology-based model of the
DPPC2 (Fig. 8). Thus, both with respect to surface structure and solubility properties, the mature ligands of the TGF- superfamily are strikingly similar to membrane proteins, which typically are surrounded by a broad band of hydrophobic surface for insertion into the non-polar lipid bilayer and extremely insoluble in aqueous buffers without the aid of detergents. The preference for membrane protein-solubilizing detergents in the folding
reaction is consistent with the observed membrane protein-like properties of these ligands and a singular role for these detergents in
enhancing the solubility of the folded product, as hypothesized for the
roles of high concentrations of NaCl and low temperatures.
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and C' atoms of the backbone are shown in yellow. The two
N
-2 atoms of the asparagine residues of the conserved
N-linked glycosylation site common to all BMPs are indicated
in blue for each dimer. Views are perpendicular to the
2-fold axis in upper panels and down the 2-fold axis in
lower panels. The two disordered hydrophilic N-terminal
peptides (not depicted) extend from the folded core of each BMP
homodimer at central positions approximately 180° opposite the two
N-linked glycosylation sites (lower surface,
center, of dimers viewed in upper panels).
The conditions that appear to be required to solubilize the folded
C-terminal domain in vitro are clearly far from
physiological, thus a means for solubilizing the mature domain in
vivo must have evolved. Gray and Mason (19) have shown that
in vivo the prodomains of two TGF- superfamily members,
TGF-1 and activin A, are required as intramolecular chaperones to
provide for secretion of the mature domains, as well as to promote
folding and disulfide bond formation. Subsequently, Mason and
co-workers (61) showed that BMP-2 and BMP-4 also cannot be produced in
cell culture in the absence of the prodomain, which appears to serve a
similar role as a chaperone for this family of signal ligands. Because
aggregates of the folded C-terminal dimer would be degraded before
exiting the secretory organelles, the prodomain may allow for secretion
of the mature form by enhancing its solubility as a complex of the two
domains. After proteolytic processing in the trans-Golgi
network by members of the family of subtilisin-like prohormone
convertases (furins; see review by Nakayama (62)), two representatives
of the TGF- superfamily, TGF-1 (63, 64) and Müllerian
inhibiting substance (65), are secreted from cultured cells and
embryonic testes, respectively, as non-covalent complexes. The
prodomains of TGF-1 (66, 67) and Müllerian inhibiting
substance (68) have also been shown to reassociate with their
respective signaling ligands in vitro, demonstrating that
stable, non-covalent complexes between the N- and C-terminal domains
form both in vitro and in vivo. The interface
between the two domains almost certainly includes, and thus renders
solvent-inaccessible, the hydrophobic surface patches of the folded
dimer, since the largest or the second largest patch forms the
intersubunit interface in 90% of all multimeric proteins (69).
Moreover, the burial of exposed hydrophobic surface area due to
hydrophobic interaction is suggested to be the main driving force for
the formation of these multimeric complexes (70). Similarly, formation
of complexes of the N- and C-terminal domains of TGF- superfamily
members may be mediated by, and mask from solvent, hydrophobic surface
patches at domain interfaces. Hence, one of the principal roles of the
prodomains as intramolecular chaperones may be to enhance the
solubility of the folded dimers by masking the large hydrophobic
patches from the aqueous solvent, reducing kinetic partitioning into
off-pathway aggregates (53, 54) as has been shown for the molecular
chaperones of the hsp70 and chaperonin families, and thereby allowing
for secretion.
In addition to a requirement for secretion, the prodomain was also
shown to be required to promote the folding and the formation of
disulfide bonds of the C-terminal domain (19). In vitro
folding of the TGF- and BMP C-terminal domains is sluggish,
requiring 3-7 days to reach completion, and thus again far from the
physiological situation, since disulfide-containing proteins typically
fold and exit the endoplasmic reticulum within 0.5-1.5 h (71). The slow kinetics of TGF- superfamily members is reminiscent of the in vitro folding behavior of proteases that have also been
shown to require their prodomains as intramolecular chaperones,
however, not to allow for secretion but to enhance the rate of folding (reviewed by Eder and Fersht (72)). Because they promote folding in a
direct and positive fashion by providing essential steric information
to the C-terminal domain, Ellis (73) has categorized prodomains that
act as intramolecular chaperones as "steric chaperones," in
contrast to hsp70 and chaperonin proteins that are considered "non-steric" because they assist folding indirectly by preventing aggregation of newly synthesized polypeptides. In this context, the
prodomains of TGF- superfamily members appear to function non-sterically, but to prevent aggregation of the folded rather than
intermediate forms, and possibly also sterically, to reduce kinetic
barriers preventing conversion of reduced native-like states to the
oxidized native structure.
In contrast to BMP-2, -4, and DPP signal ligands, which can be efficiently produced in vitro under highly solubilizing conditions, the C-terminal domains of SCREW, 60A, and the vertebrate homolog of 60A, BMP-7, cannot. Thus structural features that are common to the BMP-2 and -4 class of proteins, yet not shared by the BMP-7 class and other groups, appear to engender competency to refold in vitro. One striking difference between these two classes is the nature of the N-terminal polypeptides that extend from the folded core of the C-terminal dimers. The polypeptides of BMP-2, -4 ligands are composed primarily of charged or polar residues and thus hydrophilic, whereas the polypeptides extending from the folded C-terminal domains of BMP-7, 60A, and SCREW are composed of a larger fraction of non-polar residues and thus appear to be less water-soluble in comparison. This observation suggests that perhaps by increasing their solubility, e.g. by fusion to hydrophilic N-terminal peptide partners, the core domains of BMP-7, 60A, and SCREW may be rendered competent to spontaneously refold in vitro.
Although the heparin-binding property of DPP has not been reported previously, high affinity for heparin has been observed for BMP-2, -3, -4, and -7 (28, 55, 56, 74) and is a functional consequence of the primary structure of the N-terminal polypeptide (cf. Fig. 1). Ruppert et al. (28) have shown that the N-terminal arm of human BMP-2 comprises a high affinity heparin-binding site. Clusters of basic amino acids are a common feature of these polypeptides of many BMPs (28), suggesting that high affinity binding is mediated through specific ionic interactions between positively charged protein side chains and anionic carboxylate and sulfate groups of heparin and heparan sulfate glycosaminoglycan chains (reviewed by Hileman et al. (75)). This is in contrast to complexes of low affinity which are presumed to arise from relatively nonspecific ionic interactions. Isolation of both the unfolded monomer and the folded dimer by heparin affinity chromatography in 6 M urea, which would diminish the contributions to binding primarily of hydrogen bonds and hydrophobic interactions, provides evidence that ionic interactions indeed play a significant role in formation of the protein-carbohydrate complex. The thermodynamic parameters of DPPC2 binding to low molecular weight heparin have been determined by isothermal titration calorimetry and have shown that the binding affinity is comparable to that of bovine fibroblast growth factor.4 Because heparan sulfate proteoglycan co-receptors play a key role in mediating the interaction between bovine fibroblast growth factor and its receptor, the in vitro binding studies have significant implications concerning the distribution and activity DPP in vivo during embryonic development and imaginal disk morphogenesis. dally, encoding a Drosophila glypican modified with heparan sulfate, has recently been shown to interact genetically with dpp and thus may serve such a co-receptor role (76).
The competition binding assays provide direct biochemical evidence for interaction between the mature DPP signal ligand and Noggin, which had been previously demonstrated indirectly by injection experiments with Drosophila embryos (16). As no Drosophila Noggin homolog has been identified to date, it remains to be determined whether this interaction is only a reflection of the apparently highly conserved structure of BMPs or whether in addition to the Drosophila Chordin homolog encoded by the short gastrulation gene, SOG (77), a second antagonist related to vertebrate Noggin participates in the modulation of DPP activity.
The E. coli-expressed, refolded protein migrated at a reproducibly faster rate through denaturing polyacrylamide gels than the homodimer secreted by the Drosophila S2 embryonic cell line (12, 13), consistent with the observation that all BMPs are post-translationally modified through N-linked glycosylation by insect as well as mammalian cell lines (55, 78). The measured difference in molecular mass of approximately 1.75 kDa per monomer is in good agreement with the calculated molecular mass of a mature, high mannose oligosaccharide (1.4 kDa), indicating that the secreted DPP dimer is also modified by N-linked glycosylation by the Drosophila embryonic cell line. The C-terminal domains of a family of BMPs, which includes DPP, share a consensus N-linked glycosylation site (Fig. 9), shown to be the single site of modification of mature BMP-7 (56). Inspection of the model of DPPC2 indicates that the modified asparagine side chains reside in the bottom of a large hydrophilic cleft (Fig. 8). The two N-linked oligosaccharides, branched polymers of 8-10 carbohydrate residues, would be expected to fill this cleft and render the secreted dimer somewhat more soluble in non-denaturing, aqueous buffers. However, S2-secreted DPP dimer behaves qualitatively like the non-glycosylated, refolded protein from E. coli, requiring chaotropic aqueous buffers or aqueous organic solvent mixtures for solubilization (13). These modifications also impart heterogeneity onto the population of protein molecules, unlike the bacterially expressed protein which is homogeneous. For many applications, especially biophysical, a homogeneous preparation may be advantageous. The availability of large amounts of highly purified, homogeneous DPP ligand may now allow for biochemical and biophysical analyses of receptor, antagonist, and heparin/heparan sulfate interactions and determination of the three-dimensional structure of these complexes.
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ACKNOWLEDGEMENTS |
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The following members of the Biozentrum der Universität Basel are acknowledged for their support and assistance: Prof. Kasper Kirschner, Dept. of Biophysical Chemistry, for unlimited access to the HPLC apparatus and UV/VIS spectrophotometer and to all of the members of the Kirschner laboratory for their courtesy and helpfulness; Dr. Thomas Kiefhaber, Dept. of Biophysical Chemistry, for critical comments on the manuscript and valuable discussion about protein folding theory; Dr. Paul Jenö and Thierry Mini, Protein Chemistry Facility, for mass spectrometry measurements and N-terminal sequence determinations, respectively; Guido Capitani, Dept. of Structural Biology, for generating a dimeric model by symmetry transformation; Georges Martin, Dept. of Cell Biology, for providing the pJC10 expression vector and technical expertise about bacterial overexpression and fast protein liquid chromatography; Thomas Marty, Dept. of Cell Biology, for automated fluorescent-dideoxynucleotide sequence analysis; Remo Amherd, Dept. of Pharmacology, for curve-fitting analysis of the chondrogenesis data; Dr. Stephania DiMarco performed the dynamic light scattering analysis while at Ciba-Geigy, Basel, and her effort and expertise are greatly appreciated. Special thanks go to Dr. John McCartney (Creative Biomolecules, Hopkinton, MA) for stimulating discussion about in vitro folding of BMPs and Kay Rashka (University of Wisconsin) for generously providing details about DPP expression in S2 cells.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Kantons Basel. To whom correspondence should be addressed: Dept. of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.: 41 61 267 2070; Fax: 41 61 267 2078; E-mail: groppe{at}ubaclu.unibas.ch.
Present address: Pfizer Central Research, Sandwich, Kent, CT13
9NJ UK.
§§ Supported by a START fellowship of the Swiss National Science Foundation and by the Kantons Basel.
The abbreviations used are: BMP, bone morphogenetic protein(s); TGF, transforming growth factor; DPPC, reduced, unfolded monomeric form of the mature C-terminal domain of DPP; MES, 4-morpholineethanesulfonic acid; hNG, human Noggin fused to the Fc domain of human IgG1; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; DPPC2, oxidized, folded dimeric form of the mature C-terminal domain of DPP.
2 Accessible through the Swiss model server on the World Wide Web at http://www.expasy.ch/swissmod/SWISS-MODEL.html.
3 J. Groppe and M. Affolter, unpublished observations.
4 R. Hileman, J. Groppe, M. Affolter, and R. Linhardt, unpublished data.
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Abstract
Introduction
Procedures
Results
Discussion
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 K. E. Gregory, R. N. Ono, N. L. Charbonneau, C.-L. Kuo, D. R. Keene, H. P. Bachinger, and L. Y. Sakai The Prodomain of BMP-7 Targets the BMP-7 Complex to the Extracellular Matrix J. Biol. Chem., July 29, 2005; 280(30): 27970 - 27980. [Abstract] [Full Text] [PDF]
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F. Hillger, G. Herr, R. Rudolph, and E. Schwarz Biophysical Comparison of BMP-2, ProBMP-2, and the Free Pro-peptide Reveals Stabilization of the Pro-peptide by the Mature Growth Factor J. Biol. Chem., April 15, 2005; 280(15): 14974 - 14980. [Abstract] [Full Text] [PDF]
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X. Lin Functions of heparan sulfate proteoglycans in cell signaling during development Development, December 15, 2004; 131(24): 6009 - 6021. [Abstract] [Full Text] [PDF]
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D. J. Bornemann, J. E. Duncan, W. Staatz, S. Selleck, and R. Warrior Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways Development, May 1, 2004; 131(9): 1927 - 1938. [Abstract] [Full Text] [PDF]
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C. Oka, R. Tsujimoto, M. Kajikawa, K. Koshiba-Takeuchi, J. Ina, M. Yano, A. Tsuchiya, Y. Ueta, A. Soma, H. Kanda, et al. HtrA1 serine protease inhibits signaling mediated by Tgf{beta} family proteins Development, March 1, 2004; 131(5): 1041 - 1053. [Abstract] [Full Text] [PDF]
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 J. Groppe, J. Greenwald, E. Wiater, J. Rodriguez-Leon, A. N. Economides, W. Kwiatkowski, K. Baban, M. Affolter, W. W. Vale, J. C. Belmonte, et al. Structural Basis of BMP Signaling Inhibition by Noggin, a Novel Twelve-Membered Cystine Knot Protein J. Bone Joint Surg. Am., August 1, 2003; 85(90003): 52 - 58. [Abstract] [Full Text] [PDF]
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M. Fujise, S. Takeo, K. Kamimura, T. Matsuo, T. Aigaki, S. Izumi, and H. Nakato Dally regulates Dpp morphogen gradient formation in the Drosophila wing Development, April 15, 2003; 130(8): 1515 - 1522. [Abstract] [Full Text] [PDF]
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