Platelet-derived Growth Factor (PDGF)-C, a PDGF
Family Member with a Vascular Endothelial Growth Factor-like
Structure*
Laila J.
Reigstad
§,
Hege M.
Sande§,
Øystein
Fluge
,
Ove
Bruland¶,
Arturo
Muga
,
Jan Erik
Varhaug
,
Aurora
Martínez**, and
Johan R.
Lillehaug§
From the § Department of Molecular Biology,
¶ Department of Pediatrics,
Department of Surgery,
and ** Department of Biochemistry and Molecular Biology,
University of Bergen, Bergen 5009, Norway, and
Unidad de
Biofísica (Consejo Superior de Investigaciones
Científicas UPV/EHU) y Departmento de
Bioquímica y Biología Molecular, Universidad del
País Vasco, 48080 Bilbao, Spain
Received for publication, February 19, 2003
 |
ABSTRACT |
Platelet-derived growth factor (PDGF)-C is a
novel member of the PDGF family that binds to PDGF 
and 
receptors. The growth factor domain of PDGF-C (GFD-PDGF-C) was
expressed in high yields in Escherichia coli and was
purified and refolded from inclusion bodies obtaining a biologically
active growth factor with dimeric structure. The GFD-PDGF-C contains 12 cysteine residues, and Ellman assay analysis indicates that it contains
three intramonomeric disulfide bonds, which is in accordance with
GFD-PDGF-C being a member of the cystine knot superfamily of growth
factors. The recombinant GFD-PDGF-C was characterized by CD,
fluorescence, NMR, and infrared spectroscopy. Together, our data
indicate that GFD-PDGF-C is a highly thermostable protein that contains
mostly
-sheet secondary structure and some (6%)
-helix
structure. The structural model of PDGF-C, obtained by homology-based
molecular modeling using the structural representatives of this family
of growth factors, shows that GFD-PDGF-C has a higher structural homology to the vascular endothelial growth factor than to
PDGF-B. The modeled structure can give further insights into the
function and specificity of this molecule.
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INTRODUCTION |
Platelet-derived growth factor
(PDGF)1 is a major mitogen
and stimulant of motility of mesenchymal cells, such as fibroblasts and
smooth muscle cells, but also acts on other cell types including capillary endothelial cells and neurons (reviewed in Ref. 1). PDGFs and
their receptors have been extensively studied in nearly two decades,
but unexpectedly, two new members of the PDGF family were recently
identified by data base mining (i.e. PDGF-C (2-4) and
PDGF-D (5)). The PDGFs are members of the cystine knot family of growth
factors, which presently consists of at least 10 members
(i.e. vascular endothelial growth factor (VEGF) (VEGF-A), VEGF-B, VEGF-C, VEGF-D, and VEGF-E from the VEGF family; PDGF-A, PDGF-B, PDGF-C, and PDGF-D from the PDGF family); and placenta growth
factors PlGF-1 and PlGF-2 (reviewed in Ref. 6). Common to these factors
is the cystine knot domain that contains at least eight cysteine
residues perfectly conserved between the two chains (7). Two or three
intramonomeric disulfide bonds exist in each monomer, and the factors
dimerize in an antiparallel side-by-side mode by using two
additional intermonomeric disulfide bonds (8, 9).
Tumor cells of many different human tumor types express PDGFs and PDGF
receptors (PDGFRs). An autocrine stimulation of tumor cell growth may
prevail (10). The VEGFs induce proliferation of endothelial cells and
are involved in the development of the vascular system (angiogenesis)
(11, 12) through binding to the kinase domain-containing receptor/fetal
liver kinase-1 and the Fms-like tyrosine kinase 1 receptor (13,
14).
The recently described PDGF-C (3, 4) is a 345-amino acid protein with a
two-domain structure not previously observed in this family of growth
factors. An N-terminal CUB domain extends from residue 46 to 163 (15)
and a C-terminal growth factor domain (GFD) showing homology with PDGFs
and VEGFs extending from residue 235 to 345. These two domains are
separated by a linker region that includes a predicted protease
cleavage site at the N-terminal side of the GFD. The CUB domain of
PDGF-C shows highest homology to the CUB domain of neuropilins and
BMP-1, which are coreceptors for certain VEGF and VEGF-B isoforms (16,
17). The CUB domain might bind to the pericellular matrix. It seems to
block for receptor binding and is proteolytically cleaved off, making
an active PDGF-C of the latent growth factor (3). It has been proposed
that the isolated CUB domain of PDGF-C exhibits a mitogenic activity on
human coronary artery smooth cells, suggesting a possible biological activity of the CUB domain in addition to its role in maintaining latency of the GFD (18). A dominant negative form of the full-length PDGF-C was recently shown to inhibit growth of cultured Ewing tumor
cell (19), indicating the PDGF-C to be an important growth factor for
these tumor cells. The GFD of PDGF-C (GFD-PDGF-C) shows 27-35%
similarity with corresponding regions of PDGFs and VEGFs containing the
conserved pattern of eight invariant cysteine residues involved in
inter- and intrachain disulfide bonds similarly to the other members of
the cystine knot family (3). In addition, GFD-PDGF-C has four extra
cysteines (Cys52, Cys59, Cys64, and
Cys107), three located between invariant cysteines 3 (Cys48) and 7 (Cys103) and one located two
residues C-terminal to the eighth conserved cysteine
(Cys105). The GFD-PDGF-C, but not the full-length PDGF-C
protein, activates both the PDGFR
-form (3) and the PDGFR
-form
of the receptor (4). Although there is a high degree of sequence
identity between PDGF-C and VEGF (25%), no evidence that PDGF-C
(either the full-length protein or the isolated GFD domain) binds to
VEGF receptors exists (3, 4). Although previous studies have failed to
show increased expression of PDGF-C in tumor cell lines (18), we have
shown elevated levels of PDGF-C mRNA in human thyroid papillary
carcinoma (2).
In this paper, we present results on the use of a prokaryote expression
system to express GFD-PDGF-C on a large scale in an active, soluble
form. In order to obtain information on the structure and the
conformational stability of this domain, we have studied the
recombinant protein by CD, fluorescence, NMR and infrared spectroscopy,
and molecular modeling. The biological activity of the GFD-PDGF-C was
verified by assaying the stimulation of phosphorylation of p44/42
mitogen-activated protein kinase (MAPK), a kinase downstream from the
PDGFRs, by a cell growth assay
([methyl-3H]thymidine incorporation) and by a
modified in vitro "wound assay."
 |
EXPERIMENTAL PROCEDURES |
Cloning, Expression, and Purification of the Growth Factor Domain
of PDGF-C--
The coding sequence for the growth factor domain of
PDGF-C (GFD-PDGF-C) encoding the amino acid residues 235-345 of the
full-length PDGF-C and a His10 sequence added to the
N-terminal was cloned into the pET 19b expression vector (Novagen)
containing a T7 promoter, and positive clones were verified by
sequencing. Large scale protein expression was performed using the
BL21-CodonPlus(DE3)-RIL Escherichia coli cells (Stratagene)
grown at 37 °C using the pET 19b expression vector. Expression was
induced with 1 mM
isopropyl-thio-
-D-galactoside at
A600 = 0.6 and cultured for an additional 3-6
h. Pelleted bacteria were resuspended in 10 mM Tris-HCl, pH
8.0, 1 mM EDTA, 0.1 M NaCl (TEN buffer) (3 ml/g
of bacteria cells), containing 0.8 mg of lysozyme and 4 mg of sodium
deoxycholate (per g of bacteria) and French pressed (3 × 900 p.s.i.) using a French® pressure cell press (SLM-AMINCO®).
Inclusion bodies were collected by centrifugation (3,000 × g, 15 min, 4 °C), and the pellet was washed twice with 6 ml of TEN buffer/g of cells and centrifuged again. The inclusion bodies
were then dissolved in 7 M urea in either TEN buffer or in
25 mM NaH2PO4, 0.15 M
NaCl, pH 4.0, as indicated, and incubated at 50 °C for 40 min.
Unsolubilized material was discarded by centrifugation, and the
solution was adjusted to either 1 mM DTT or 10 mM
-mercaptoethanol, as indicated. The protein was
refolded by dialysis and stepwise removal of urea followed by an
overnight dialysis step in the absence of urea. Aggregated protein was
removed by centrifugation (17,000 × g, 20 min at
4 °C). When indicated, the renatured protein sample was dialyzed
against and stored on 2.5 mM KAc, 0.1 M KCl, pH
4.3. Solubilization and purification of the protein was analyzed by
SDS-PAGE run at constant voltage of 200 V for 35 min in 14.5% (w/v).
The gels were stained with Coomassie Brilliant Blue. The oligomeric
state was analyzed by both SDS-PAGE in the absence of treatment of the
protein samples with
-mercaptoethanol and by size exclusion
chromatography on a HiLoad Superdex 200 HR (Amersham Biosciences)
column (1.6 × 60 cm) at a flow rate of 23 ml/h (20). Blotting was
performed as described using the Bio-Rad Mini Trans-Blot System (21,
22). Rabbit polyclonal anti-GFD-PDGF-C/VEGF (4544g) (BioGenes, Berlin,
Germany) or anti-His (sc-803) (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) were used to detect the GFD-PDGF-C. The blots were developed
using ECL (Amersham Biosciences).
Titration of Thiols--
The Ellman assay (23, 24) was used to
determine the concentration of free thiols in urea-denatured and
refolded GFD-PDGF-C by spectroscopically determining the formation of
nitrothiobenzoate (
412(nitrothiobenzoate) = 14,290 M
1·cm
1). The content of
disulfide bonds was determined after reduction with sodium borohydride
(NaBH4).
Circular Dichroism--
CD measurements were performed on a
Jasco J-810 spectropolarimeter equipped with a Jasco 423S Peltier
element for temperature control. Protein samples (25 µM)
were prepared in different buffers as indicated. The data were analyzed
using the Standard Analysis program provided with the instrument. The
amount of secondary structure elements was estimated by the CDNN
program that applies a neural network procedure (25).
Fluorescence Measurements--
Measurements of intrinsic
tryptophan fluorescence (excitation at 295 nm) were performed on a
PerkinElmer Life Sciences LS-50 luminescence spectrometer with a
constant temperature cell holder and 1-cm path length quartz cells. The
excitation and emission slits were 3 and 5, respectively. All spectra
were corrected for blank emission. The samples were prepared on 2.5 mM KAc, 0.1 M KCl, pH 4.3.
Infrared Spectroscopy--
Spectra were recorded in a Nicolet
Magna II 550 spectrometer equipped with a MCT detector, using a
demountable liquid cell (Harrick Scientific, Ossining, NY) with calcium
fluoride windows and 50-µm spacers. Samples (4 mg/ml) were
lyophilized in 2.5 mM KAc, 0.1 M KCl, pH 4.3, and prior to IR measurements they were resuspended in deuterium oxide
and centrifuged to remove any insoluble material (20 min at 15,000 × g). Deconvolution of the original amide I band has been
described previously (26). Thermal analysis was performed in the rapid
scan mode within the temperature interval 20-80 °C, using a scan
rate of 1 °C/min and recording one spectrum every 1 °C. A
tungsten-copper thermocouple was placed directly onto the window, and
the cell was placed into a thermostated cell mount.
Nuclear Magnetic Resonance--
Lyophilized samples of
GFD-PDGF-C for 1H NMR experiments were dissolved in 25 mM NaH2PO4, pH 4.0, containing 50 mM NaCl, 10% D2O (1.2 mM protein;
0.5 ml). NMR spectra were also taken of protein samples containing
0.2-0.4 mM dimer at pH 7.5. The pH of the samples was
checked with a glass microelectrode and was not corrected for isotope
effects. NMR experiments were acquired on a Bruker AMX-600 pulse
spectrometer operating at 600.13 MHz for the proton at a probe
temperature of 298 K. One-dimensional 1H NMR spectra were
run with water suppression by Pulse Sculpting (27) and "W5" pulse
train (28). Following parameters were set: 2-s delay time, 150-µs
"W5" pulse train parameter, 7183.908 Hz spectral width. 32 scans
were acquired. Data were processed using XWIN-NMR (Bruker) on an SGI
work station.
Molecular Modeling--
Structural models for GFD-PDGF-C were
prepared by sequence alignment modeling using the Homology module of
InsightII 2000 (Accelrys).
Assay of Biological Activity--
The biological activity of
GFD-PDGF-C was assayed both by a cell growth assay
([methyl-3H]thymidine incorporation) and by
mediation of phosphorylation of p44/22 MAPK downstream of the PDGF
receptors, as well as by an in vitro "wound assay." In
the cell growth assay of [methyl-3H]thymidine
(Amersham Biosciences) incorporation, confluent cultures of mouse 3T3
fibroblasts were starved in serum-free DCCM-1 medium (Biological
Industries, Kibbutz, Israel) for 24 h upon stimulation with equal
amounts of either the recombinant GFD-PDGF-C or PDGF-AA (Sigma).
Triplicate wells were used for each agent tested and also for the wells
not stimulated with any agents.
[methyl-3H]Thymidine (1 µCi/ml) and cold
thymidine (1 µM final concentration) were added to each
well 8, 10, and 24 h before termination of each experiment. At the
end of the labeling period, DNA was collected by trichloroacetic acid
precipitation (29), and the radioactivity was determined using a
scintillation counter. The biological activity of the recombinant
GFD-PDGF-C was then tested for mediation of phosphorylation of p44/42,
MAPK. Serum-starved mouse 3T3 fibroblasts were stimulated with the
recombinant refolded GFD-PGDF-C and with PDGF-AA and epidermal growth
factor (both from Sigma) in comparable amounts. Stimulation was
carried out for 15-60 min. Total cell protein was then isolated,
separated on a 12.5% SDS-PAGE, and immunoblotted. The effect of
stimulation was verified by assaying induction of MAPK phosphorylation
using anti-phospho-p44/42 MAPK monoclonal antibody (New England
Biolabs, Inc., Beverly, MA) and anti-Erk1 polyclonal antibody (Santa
Cruz Biotechnology). The anti-Erk1 polyclonal antibody was used as a
control for equal amounts of protein loaded in each well. In the
in vitro wound assay (30), cell monolayers were wounded by
scratching a line with a plastic scriber, and after washing with
phosphate-buffered saline, the cells were incubated in serum-free RPMI
1640 medium with 0.1% bovine serum albumin and with or without
refolded GFD-PDGF-C. The cells were fixed and stained with Giemsa stain.
 |
RESULTS |
Expression, Purification, and Renaturation--
Several small
scale experiments were performed in order to achieve an optimum
expression level and to obtain maximal GFD-PDGF-C solubility. In the
absence of denaturant, there was no soluble recombinant GFD-PDGF-C in
the supernatant of French press-treated bacterial suspensions (cleared
cell lysate) (Fig. 1A).
Approximately 60-70% of the recombinant protein present in inclusion
bodies was soluble in 7 M urea, and the solubilized protein
solution was almost free from proteins other than GFD-PDGF-C (Fig.
1A). For large scale purification, two equally effective
protocols were used to generate refolded GFD-PDGF-C from solubilized
inclusion bodies. First, the recombinant His10-tagged
GFD-PDGF-C was purified and refolded under denaturing conditions by
affinity chromatography on nickel-nitrilotriacetic acid silica resin
(Qiagen). The solubilized protein in TEN buffer, containing 7 M urea and 10 mM
-mercaptoethanol, was bound
to the nickel-nitrilotriacetic acid column, and refolding was performed
by gradient dilution of urea (7 to 1 M gradient) and
-mercaptoethanol (10 to 0 mM gradient). After extensive
washing at pH 6.2, the refolded protein was eluted with 250 mM imidazole in TEN buffer. The purified, refolded
GFD-PDGF-C was soluble, as determined by centrifugation (13,000 × g, 20 min) (Fig. 1B). Alternatively, the
GFD-PDGF-C was solubilized in NaH2PO4, pH 4.0, buffer supplemented with 7 M urea and 1 mM DTT
(see "Experimental Procedures") and refolded by dialysis at room
temperature with the stepwise removal of urea followed by an overnight
dialysis in the absence of urea. As seen by electrophoresis performed
at nonreducing conditions (Fig. 1C) and Western blot
analysis (data not shown), the refolded protein is mainly dimeric, with
a monomeric apparent molecular mass of 17 kDa due to the additional
N-terminal His10 tag. GFD-PDGF-C contains only one
methionine residue (Met1), and the N-terminal
His10 tag was easily removed by CNBr cleavage (31) (data
not shown). At nonreducing conditions, the monomer contained 3.52 (~4) free cysteines, and 8.48 (~8) -SH groups were in an
oxidized state. Thus, our results are compatible with a protein
homodimer having two intersubunit disulfide bonds, four free
sulfhydryls, and three intrasubunit S-S bonds (see below). Additional
proof for the dimeric structure of the protein and the absence of
aggregated or misfolded forms in the refolded preparation was obtained
by gel filtration chromatography analysis on a HiLoad Superdex
200TM column. Recombinant GFD-PDGF-C shows an elution
volume of 92 ml in runs performed on 25 mM
NaH2PO4, 0.15 M NaCl, pH 4.0-6.0, which corresponds to the elution volume of carbonic anhydrase (29 kDa)
in the calibrating runs of the column (data not shown).

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Fig. 1.
SDS-PAGE and Western blot analysis of the
expression, renaturation, and purification of recombinant
GFD-PDGF-C. A, SDS-PAGE under reducing conditions (0.33 M -mercaptoethanol in the sample buffer). Shown are
BenchMark prestained protein ladder (lane 1),
whole extract of noninduced (lane 2) and induced
(lane 3) bacteria, and cleared supernatant of the
cell lysate (soluble fraction) (lane 4) and
pellet (insoluble fraction) (lane 5).
B, SDS-PAGE under reducing conditions after solubilization
in 7 M urea, purification on a nickel-nitrilotriacetic acid
column at pH 8.0, and renaturation by dialysis to remove urea. Shown is
the renatured, refolded GFD-PDGF-C after centrifugation (13,000 × g, 20 min): supernatant (lane 1) and
pellet (insoluble fraction) (lane 2).
C, effect of reducing conditions on the oligomeric structure
of GFD-PDGF-C. Cleared supernatant of the cell lysate was treated under
reducing (10 mM -mercaptoethanol) (lane
1) and nonreducing conditions (no -mercaptoethanol in the
sample buffer) (lane 3). Unsoluble fraction
(pellet) was treated under reducing (10 mM
-mercaptoethanol) (lane 2) and nonreducing
conditions (no -mercaptoethanol in the sample buffer)
(lane 4).
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Biological Activity--
The biological activity of refolded
GFD-PDGF-C was determined by assaying GFD-PDGF-C-mediated
phosphorylation of p44/42 MAPK, a kinase downstream from the PDGFRs.
Serum-starved 3T3 cells were treated with recombinant renatured
GFD-PDGF-C or with commercially available PDGF-AA and epidermal growth
factor, and a strong increase in p44/42 phosphorylation was obtained
with each of the three factors (Fig. 2),
thus identifying a biological signal transduction activity of the
expressed and renatured GFD-PDGF-C preparation. Also, when GFD-PDGF-C
activity was assayed by a modified in vitro "wound
assay" (30), a well defined migratory activity into the cell-free
space was observed. Similarly, a significant increase in
[methyl-3H]thymidine incorporation (5-8-fold
over untreated controls, compared with 7-10-fold for PDGF-AA added at
equal amounts) into DNA was observed in 3T3 cells stimulated with the
recombinant protein (data not shown), indicating cell proliferation,
increased motility, and DNA synthesis and providing evidence of
biological active GFD-PDGF-C.

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Fig. 2.
Induction of p44/42 MAPK
phosphorylation. Incubation of confluent, starved fibroblast (3T3)
cells with recombinant GFD-PDGF-C. Following the incubation, total
protein was harvested, separated on SDS-PAGE, immunoblotted, and
stained with anti-phospho-p44/42 MAPK monoclonal Ab (upper
panel) and anti-Erk1 polyclonal Ab (lower
panel), the last one used as control for equal amounts of
protein added to each well. Lane 1, no GFD-PDGF-C
added to the culture. Lanes 2 and 3,
recombinant refolded GFD-PDGF-C (about 20 ng) was given to the cultures
for 60 min and 15 min, respectively. Lane 4, 25 ng of PDGF-AA (Sigma) were given the culture for 15 min.
Lane 5, 20 ng of epidermal growth factor
(EGF) were given the culture for 15 min.
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Circular Dichroism--
The purified and urea-solubilized
GFD-PDGF-C shows a far UV CD spectrum with a minimum at around 215 nm,
and after renaturation of the protein by elimination of the urea, the
spectrum shows an even broader minimum with slightly decreased
ellipticity (Fig. 3A). Similar
spectra are obtained in the pH range 4.0-7.5. As estimated by the CDNN
neural network procedure (25), this spectrum corresponds to a content
of secondary structure of ~6 ± 1%
-helix, 52 ± 4%
-sheet, 17 ± 1%
-turn, and 25 ± 2% random coil. The near-UV CD spectrum of the renatured protein shows a broad positive transition centered at about 260 nm in Fig. 3B, which most
probably corresponds to disulfide bonds in a strained conformation,
i.e. with a C
SS torsion angle deviating significantly
from 90° (32). The positive signal around 260 nm diminishes on
incubation with 5 mM DTT at 25 °C and almost disappeared
when the GFD-PDGF-C was reduced with 5 mM DTT for 5 min at
95 °C (Fig. 3B). Upon heating the protein at 95 °C in
the absence of DTT, the strained orientation of the S-S bonds is
preserved in the protein, indicating a high conformational stability.
The high stability of GFD-PDGF-C is also manifested by the elevated
midpoint melting temperature (>100 °C) of the renatured protein in
the pH range 4.0-7.5 (Fig. 3C). Indeed, both the far-UV and
the near-UV spectra of the heat-treated (95 °C) protein at
nonreducing conditions were similar to the spectra of the renatured
protein at 25 °C (Fig. 3, A and B). The presence of urea in the protein samples decreases its thermal stability, and, in fact, the renaturation of the protein achieved by
the removal of urea parallels the increase in thermal stability of the
protein (Fig. 3C).

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Fig. 3.
CD spectra of GFD-PDGF-C: Effect
of urea and reducing conditions. A, far UV CD spectra
at 25 °C of isolated GFD-PDGF-C in the presence of 7 M
urea (dotted line) and after renaturation by
removal of the urea (solid line). B,
near UV CD spectra of the renatured protein taken at 25 °C
(solid line) and of the heat-treated (95 °C,
1 h) at nonreducing (dotted and
dashed line) and reducing (5 mM DTT
during heating) (dotted line) conditions, taken
at 95 °C. C, CD-monitored thermal scans following the
changes in ellipticity at 210 nm in samples of GFD-PDGF-C at different
stages of the renaturation (i.e. in the presence of 1.3 M (dotted line), 0.7 (dotted and dashed line),
0.1 (dashed line), and 0 M
(solid line) urea. The scan rate was 0.7 K/min.
In A and B, representative spectra are shown for
samples (0.6 mg/ml) prepared in 2.5 mM KAc, containing 100 mM KF, pH 4.3. Similar spectra are obtained at pH 7.5. [ ], mean residue molar ellipticity.
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Fluorescence Emission Spectroscopy--
GFD-PDGF-C contains one
Trp residue at position 39, which is conserved in PDGF-B
(Trp40). The tryptophan fluorescence emission spectrum of
the recombinant renatured GFD-PDGF-C (excitation at 295 nm) shows a
maximum at 342 nm (Fig. 4A)
and a quantum yield of 0.09, indicative of a solvent-exposed residue.
Fluorescence studies also showed that this residue is solvent-exposed
in PDGF-B (emission maximum at 347 nm) and that denaturation of the
protein results in a large red shift (the emission maximum increases to
356 nm) (33).

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Fig. 4.
Spectroscopic characterization of GFD-PDGF-C
by fluorescence emission (A) and one-dimensional
1H NMR (C) spectroscopy.
A, intrinsic fluorescence emission spectrum of GFD-PDGF-C, 1 µM dimer, pH 4.3. The excitation wavelength was 295 nm.
B, 1H NMR spectrum of GFD-PDGF-C, 0.2 mM dimer, pH 7.5, acquired at 25 °C.
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NMR Studies of GFD-PDGF-C--
In an attempt to obtain structural
information on the GFD-PDGF-C in solution, preliminary NMR experiments
were performed. At concentrations of about 1 mM GFD-PDGF-C,
pH 3.0-4.0, and temperatures between 25-50 °C, the line widths of
the NMR resonances were too broad, indicating some aggregation of the
protein at these conditions. A significant improvement of the spectral
quality was obtained when the pH of the sample was changed to pH 7.5 by
dialysis, lyophilization, and resuspension in 50 mM NaCl,
25 mM NaH2PO4, 100%
D2O, pH 7.5, and the protein concentration was adjusted to
0.2-0.5 mM (Fig. 4B). Two-dimensional TOCSY and
NOESY spectra could be recorded at this slightly alkaline pH (7.5), but
they were not of enough quality to attempt a complete assignment of the
signals. Future NMR investigations of the three-dimensional structure
of GFD-PDGF-C will require isotopic labeling of the protein.
Nevertheless, some structural information can be inferred from the NMR
spectrum, since most of the signals appearing between 6.0 and 5.0 ppm
probably correspond to low field shifted H
protons, indicating a
positive differential shift (i.e.
H
sample
H
coil > 0), characteristic of proteins with high
-sheet content.
Fourier Transform Infrared Spectroscopy--
The original and the
deconvolved spectra of GFD-PDGF-C are shown in Fig.
5A. The conformationally
sensitive amide I band, 1700-1600 cm
1, displays an
intense band component at 1625 cm
1 that has been assigned
to
-structures (34, 35) and weaker signals at 1673 and 1683 cm
1 that contain contributions from
-turns and the
high frequency component of the
-structure. The amide II spectral
region, 1600-1500 cm
1, is dominated by a strong band at
1587 cm
1 coming from ionized acidic side chains and
residual acetate buffer, that hampers the estimation of the relative
intensity of the amide I band components. Nevertheless, the spectrum
clearly indicates that the protein folds into a
-structure. There is
no spectral evidence for the existence of
-helical conformations,
although it cannot be ruled out that a weak helical signal (1653-1643
cm
1) may hide below the intense
-component.
Interestingly, the intensity of the residual amide II band at around
1545 cm
1 is negligible, indicating that the NH groups of
the protein readily exchange with the solvent and therefore that the
protein core is highly accessible. This characteristic, together with
the width of the amide I band argue against the presence of protein
aggregates. If this were the case, the amide I band would be narrower,
as expected from immobilized protein segments forming part of the aggregates, and these segments would, at least partially, be shielded from the solvent. The temperature dependence of the amide I band absorption maximum supports this interpretation (Fig. 5B).
Intermolecular aggregates usually give rise to a component at around
1620 cm
1 that slightly shifts downwards with increasing
temperatures (36, 37). The observed temperature-induced upward
displacement of the absorption maximum of the amide I band (from 1625 to 1633 cm
1) reinforces the interpretation that
GFD-PDGF-C folds into a
-structure and does not aggregate under
these experimental conditions.

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Fig. 5.
Fourier transform-infrared spectroscopy.
A, original (broken line) and
deconvolved (solid line) spectra of GFD-PDGF-C,
13 µM dimer, recorded in deuterated buffer at pD 4.3. The
deconvolution parameters are half-width = 18 and K = 2. B, temperature dependence of the absorption maximum of
the amide I band of GFD-PDGF-C.
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Structure Prediction of GFD-PDGF-C by Molecular Modeling--
The
structures of VEGF (VEGF-A) (Protein Data Bank accession code 1VPF)
(38) and PDGF-B (Protein Data Bank accession code 1PDG) (39) were used
as templates to construct a structural model of GFD-PDGF-C. In the
alignment of these three proteins, the sequence identity was 26% for
PDGF-B and 23% for VEGF, whereas the sequence similarity was 40 and
51%, respectively (Fig. 6). The model of
GFD-PDGF-C obtained using the Homology module of InsightII 2000 (MSI)
was similar to that obtained by the Swiss-Pdb Viewer in conjunction
with SWISS-MODEL (40). Both methods rendered three-dimensional models
of acceptable quality, as evaluated by PROCHECK (41) and Whatcheck
(42). The incorporation of the structure of human placenta growth
factor-1 (Protein Data Bank accession code 1FZV) (43) as template (16%
sequence identity with GFD-PDGF-C) did not have any influence on the
final predicted structure. The model of GFD-PDGF-C showed three
intramonomeric cystine bonds (Fig.
7A), and, as seen by the
overall structural alignments of GFD-PDGF-C with both VEGF and PDGF-B
(Fig. 7, B and C), the similarity between VEGF
and GFD-PDGF-C is higher than between GFD-PDGF-C and PDGF-B.

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Fig. 6.
Sequence alignment of GFD-PDGF-C with PDGF-B
and VEGF (VEGF-A). The signs above and the
histogram below the alignment indicate the degree
of similarity between the sequences. The stars (and
highest bars) indicate identity, whereas
homologue properties of the residues are indicated with two
dots or one dot, depending on
the degree of similarity. The first conserved cystine bond
(1) corresponds to Cys18-Cys62, the
second (2) corresponds to
Cys48-Cys103, and the third corresponds to
Cys55-Cys105, whereas the intermonomeric
cystine bonds (I) correspond to
Cys42-Cys54.
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|

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Fig. 7.
Model of the three-dimensional structure of
GFD-PDGF-C based on sequence alignment with PDGF-B and VEGF.
A, -helixes are shown in red, and -sheets
are shown in turquoise. Cysteine residues are numbered and
shown in a stick representation, sulfur is
colored in yellow, and nitrogen and oxygen are colored in
blue and red, respectively. The first
intramonomeric cystine bond is Cys18-Cys62,
the second is Cys48-Cys103, and the third is
Cys55-Cys105. The two intermonomeric cysteines
making the dimer are Cys42 and Cys54.
B, overall backbone structural alignment of GFD-PDGF-C
(turquoise) with PDGF-B (violet); root mean
square deviation = 4.42 Å for backbone atoms. C,
overall backbone structural alignment of GFD-PDGF-C
(turquoise) with VEGF (orange); root mean square
deviation = 0.23 Å for backbone atoms.
|
|
Based on the dimeric structure of both VEGF-AA (38) and PDGF-BB (39), a
structural model of the dimeric form of GFD-PDGF-C was also constructed
(Fig. 8). The two cysteine residues
Cys42 and Cys54 form the interdisulfide bonds
between the monomers, giving a dimer of the GFD-PDGF-C. The
-helix
of one monomer stretches over the other monomer, contributing to the
stability of the dimer. The areas most probably involved in the binding
to the Fms-like tyrosine kinase 1 receptor, based on the structure of
VEGF-AA complexed with domain 2 of the receptor (44), are indicated in
Fig. 8. The location of the solvent-exposed Trp39,
responsible for the intrinsic fluorescence of GFD-PDGF-C (Fig. 4A), is also shown in Fig. 8.

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Fig. 8.
Model structure of the dimeric GFD-PDGF-CC
based on the structural alignment with dimeric VEGF-AA. The two
monomers, colored red and yellow, are bound
together by the two inter disulfide bonds (yellow). The
indigo color indicates putative areas for
binding to the receptor. See "Structure Prediction of GFD-PDGF-C by
Molecular Modeling" for comments on the indicated residues.
|
|
 |
DISCUSSION |
Our main aim was to study the structure of the growth factor
domain of the novel PDGF-C protein. This protein domain has low solubility in bacterial expression systems, most likely due to its 12 cysteine residues. Therefore, we first established methods for large
scale (milligram amounts) expression, purification, and renaturation of
the recombinant GFD-PDGF-C protein. The system of choice was expression
in E. coli using the pET 19b vector with a His tag placed in
the N terminus. Although almost no soluble protein was obtained in the
cell lysates, it was possible to renature and purify the protein in one
step on a nickel-nitrilotriacetic acid column and in a stepwise
dialysis procedure. Both [methyl-3H]thymidine
incorporation and mediation of phosphorylation of MAPK downstream of
the PDGF receptors and the in vitro "wound assay" showed
biological activity of the recombinant GFD-PDGF-C protein, indicating
that it was refolded correctly into its native structure. To our
knowledge, to date no structural characterization has been performed on
GFD-PDGF-C, possibly because in previous studies only microgram amounts
have been purified (3, 4). Li et al. (3) grouped PDGF-C
within the PDGF family, since it bound to the PDGF 
-receptor
after proteolytic cleavage and separation from the N-terminal CUB
domain. Later it was shown that GFD-PDGF-C binds to the PDGF

-receptor as well (4, 45) but not to the VEGF receptors.
Nevertheless, recent studies suggest that PDGF-C can also possess
VEGF-like activity (18), and, in fact, the CUB domain of this factor
shows highest homology to the CUB domain of neuropilins and BMP-1,
which are coreceptors for certain VEGF isoforms (16, 17). Moreover, the
existence of an as yet unknown receptor specific for PDGF-C cannot be
ruled out.
Based on our CD experiments, the secondary structure of GFD-PDGF-C
includes about 52%
-sheet structure. This large percentage of
-sheet is also corroborated by Fourier transform infrared spectroscopy and by NMR studies in which signals corresponding to low
field shifted H
protons show a positive differential shift characteristic of proteins with high
-sheet content. This high content of
-sheet structure is also found in both PDGF-B (39) and
VEGF (44) (Fig. 7) and seems to be characteristic of members of the
cystine knot family (43). The
-sheet structure and the cystine knot
confer a high conformational stability to the proteins. Thus, as seen
with GFD-PDGF-C, the midpoint melting temperature was found to be
>95 °C, and the thermal treated protein (at 95 °C for 1 h)
still retains a high degree of secondary structure, even when the
disulfide bridges of the cystine knot motive have been cleaved by
reduction. This high stability could be suggestive of extensive
aggregated
-structure, but this does not seem to be the case for the
recombinant refolded GFD-PDGF-C, which shows a dimeric structure both
by size exclusion chromatography and by SDS-PAGE in the absence of
reducing agent. Moreover, its dispersed 1H NMR spectrum,
its intrinsic fluorescence spectrum, resultant from a solvent-exposed
Trp39, and its Fourier transform infrared spectroscopic
properties are also typical of a folded nondenatured and nonaggregated
protein. The results from all of these spectroscopic techniques support the homology-base modeled structure of GFD-PDGF-C
(Figs. 7 and 8). The modeled structure contains 50% of
-sheet
secondary structure, and about 6% of
-helical structure
localized at a short helix at the N-terminal of the protein. The
presence of this
-helical content is also indicated by the CD
measurements. Whereas PDGF-B is devoid of
-helical structure (Fig.
7B) (39), VEGF contains about 12% helix structure
distributed in two
-helixes per monomer (Fig. 7C) (44).
The good structural alignment between the modeled structure of
GFD-PDGF-C and VEGF (the root mean square deviation for the backbone
atoms between the modeled structure of GFD-PDGF-C is 0.23 and 4.42 Å when compared with the structure of VEGF and PDGF-B, respectively (Fig.
7)) also provides evidence for a large structural closeness between
GFD-PDGF-C and VEGF. Moreover, as seen in the sequence alignment (Fig.
6), the four proline residues in GFD-PDGF-C are conserved in VEGF (only
two with PDGF), and their position is coincident in the
three-dimensional structures. Prolines are important in defining the
overall conformation of the proteins and are usually found in the
flanking segments of protein-protein interaction sites (46). Although
the structures of the PDGFR-
and PDGFR-
receptors are not known,
there is evidence that the receptors of the PDGF family may all use
domain 2 for factor binding in a way analogous to the structure of
VEGF8-109 in complex with domain 2 of Fms-like tyrosine
kinase 1 receptor (47, 48). According to this complex structure, the
putative receptor-interacting regions in the modeled dimeric structure of GFD-PDGF-C (shown in Fig. 8) are flanked by the conserved proline residues. As seen in this structure, a region including
Trp39, Leu79, and Arg80
might be an important binding site for the PDGF
- or/and
-receptor. Amino acid sequence alignments (see, for instance, Fig. 1 in Ref. 3) show that PDGF-A and PDGF-B contain a similar sequence, Trp40, Val78, and Arg79,
that is not found in the members of the VEGF family. With these structure considerations in mind, it is possible that the PDGF-like specificity of PDGF-C is associated with the residues in regions supposed to interact with the receptor, despite the similarity in
overall structure to the growth factor domain to VEGF.
It is also interesting to note that an insertion of three amino acid
residues (Asn51-Cys52-Ala53,
numbering for the GFD) is specific for PDGF-C but is not present in the
other members of the PDGF/VEGF family. The insertion occurs close to
the loop-2 region that is thought to be involved in receptor binding.
Moreover, three out of the four extra nonconserved cysteine residues in
the GFD-PDGF-C sequence (i.e. Cys59,
Cys64, and Cys107) are positioned forming a
cluster close to the cystine knot, indicating their putative
involvement in further binding with coreceptors, with or without
intermolecular disulfide bonding. Only mutagenesis and binding
experiments may elucidate the receptor specificity of PDGF, and our
modeled structure of the growth factor domain may contribute to provide
the frame for a rational choice of the residues to be mutated.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Nils Åge Frøystein and Dr. M. Angeles Jiménez for valuable help with the NMR experiments.
 |
FOOTNOTES |
*
This work was supported by grants from the Norwegian Cancer
Society (to Ø. F., L. J. R., J. E. V., and J. R. L.), the Locus for Cancer Research, University of Bergen (to J. R. L.), and the Norwegian Research Council (to A. M.) and by Comisión
Interministerial de Ciencia y Tecnologiá BMC2001/1561 (to
A. M.).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.

To whom correspondence should be addressed: Dept. of Molecular
Biology, HIB, University of Bergen, Thormøhlensgate 55, N-5020 Bergen,
Norway. Tel.: 47 55 58 64 21; Fax: 47 55 58 96 83; E-mail: johan.lillehaug@mbi.uib.no.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M301728200
 |
ABBREVIATIONS |
The abbreviations used are:
PDGF, platelet-derived growth factor;
DTT, dithiothreitol;
GFD, growth factor
domain;
GFD-PDGF-C, growth factor domain of PDGF-C;
MAPK, mitogen-activated protein kinase;
PDGFR, PDGF receptor;
PDGF-C, PDGF
type C;
VEGF, vascular endothelial growth factor;
PlGF, placenta growth
factor.
 |
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