From JERINI BIO TOOLS GMBH, Rudower Chaussee 5, 12489 Berlin, Germany, the § Institut für Medizinische
Immunologie, Universitätsklinikum Charite,
Humboldt-Universität zu Berlin, Schumannstraße 20-21, 10098 Berlin, Germany, and ¶ SCHERING AG Research Laboratories,
Müllerstra
e 170-178, 13342 Berlin, Germany
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ABSTRACT |
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Vascular endothelial growth factor (VEGF)
directly stimulates endothelial cell proliferation and migration via
tyrosine kinase receptors of the split kinase domain family. It
mediates vascular growth and angiogenesis in the embryo but also in the
adult in a variety of physiological and pathological conditions. The
potential binding site of VEGF with its receptor was identified using
cellulose-bound overlapping peptides of the extracytosolic part of the
human vascular endothelial growth factor receptor II (VEGFR II). Thus,
a peptide originating from the third globular domain of the VEGFR II
comprising residues 247RTELNVGIDFNWEYP261
was revealed as contiguous sequence stretch, which bound
125I-VEGF165. A systematic replacement with
L-amino acids within the peptide representing the putative VEGF-binding
site on VEGFR II indicates Asp255 as the hydrophilic key
residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding
with an IC50 of 0.5 µM on extracellular VEGFR
II fragments and 30 µM on human umbilical vein cells.
VEGF165-stimulated autophosphorylation of VEGFR II as well
as proliferation and migration of microvascular endothelial cells was
inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal
concentration of 3-10, 0.1, and 0.1 µM, respectively. We
conclude that transduction of the VEGF165 signal can be
interrupted with a peptide derived from the third Ig-like domain of
VEGFR II by blockade of VEGF165 binding to its receptor.
Angiogenesis, the formation of new blood vessels sprouting from
existing ones, plays an essential role in fetal and adult life. It is
important for proliferative processes in the reproductive tract, tissue
regeneration, and wound healing as well as for pathological conditions
such as solid tumor growth, rheumatoid arthritis, and retinopathies (1,
2).
Several putative angiogenic factors have been identified. Many of these
factors possess only very little or no direct mitogenicity on vascular
endothelial cells (3). Vascular endothelial growth factor
(VEGF)1 in contrast is a
potent endothelial cell-specific mitogen in vitro (4-6)
enhancing vascular permeability and stimulating angiogenesis in
vivo (7, 8). Its importance has been demonstrated by blockade of
tumor growth by neutralizing anti-VEGF monoclonal antibodies (9) and by
experiments where tumor growth was blocked by evoking the expression of
kinase truncated VEGFR II on cells in the vicinity of growing tumors
(10).
VEGF expression is induced by hypoxia in a variety of differentiated
cells (11, 12) whereas the two corresponding VEGF receptors denoted
fms-like tyrosine kinase (FLT-1, VEGFR I) and kinase insert
domain-containing receptor (KDR, VEGFR II), appear to be expressed
exclusively by endothelial and hematopoietic cells (13-16). VEGF is
encoded by a single gene yielding four isoforms containing 121, 165, 189, and 206 amino acids due to alternative splicing (17, 18). Isoforms
VEGF189 and VEGF206 remain bound to the
extracellular matrix. Isoforms VEGF121 and
VEGF165 are secreted (19). The most abundantly expressed
VEGF isoform is the homodimeric VEGF165 with an apparent
molecular weight of 43,000. VEGF is a member of the cysteine knot
family of growth factors and thus structurally related to the
platelet-derived growth factor (PDGF) and transforming growth factor
The two VEGF receptors I and II with apparent molecular weights of
about 220,000 consist of seven immunoglobulin-like extracellular domains, one transmembrane stretch, and intracellular split tyrosine kinase domains. These structural elements relate the VEGF receptors closely to the platelet-derived growth factor The VEGFR I and II bind most likely to different VEGF epitopes
dominated by basic and acidic residues, respectively, which appear to
be essential for ligand-receptor interaction (33). This is further
indicated by the finding that placental-like growth factor competes for
VEGF binding at VEGFR I but not at VEGFR II (34). When deletion mutants
of VEGFR I were constructed lacking extracellular Ig-like domains only
the deletion of domain 2 abolished VEGF binding completely. Binding was
restored by substituting with domain 2 of VEGFR II. However, the
specificity had changed and the placental-like growth factor did not
displace VEGF any longer (35). Truncation studies of both VEGF
receptors, VEGFR I (34) and VEGFR II (36), have mapped their
VEGF-binding site to the first three N-terminal globular domains.
Concomittant with the recognition of different binding sites, the VEGFR
I and II appear to exhibit different functions in angiogenesis. While
VEGFR II is mediating the stimulation of endothelial cell proliferation during angiogenesis and vasculogenesis (13), the VEGFR I seems to be
involved in the regulation of the assembly of the vascular endothelium,
which was demonstrated by studies with transgenic knockout mice (37).
VEGFR I-deficient embryos die at day 10.5 of gestation. Although
angioblasts are formed the assembly into functional blood vessels is
impaired. Similarly died homocygotic VEGFR II deficient embryos at day
8.5 with no vessels forming and defects in angioplastic and
hematopoietic lineages (38).
The molecular basis of the VEGF-VEGFR II interaction is poorly
understood. But recently the high resolution crystal structures of VEGF
(21, 39), VEGF complexed with domain 2 of the VEGFR I and VEGF mutation
data became available (21, 33). This VEGF mutation studies demonstrated
that high affinity binding of VEGF to VEGFR II is dependent on the
presence of three locally vicinal isoleucins (Ile43,
Ile46, and Ile83) and positively charged amino
acids Arg82, Lys84, and His86 in
VEGF. In addition, Asn63, Glu64, and
Glu67 are important especially for VEGFR II binding.
The aim of our investigations was to contribute to the topology of
VEGF-VEGFR II interaction and to use that knowledge to design novel
VEGF inhibitors. In the present study we report the mapping and
characterization of a putative VEGF165/VEGFR II contact site using cellulose-bound peptide libraries derived from the VEGFR II
sequence. The resulting peptide was characterized with respect to
VEGF165 binding, inhibition of VEGFR II
autophosphorylation, as well as growth and migration of microvascular
endothelial cells.
Synthesis of Peptides and Cellulose-bound Peptide
Libraries--
Cellulose-bound and cleavable sets of peptides
(PepSpotsTM and Cleavable PepSpotsTM, Jerini
Bio Tools GmbH, Berlin, Germany) were automatically prepared according
to standard spot synthesis protocols (40) using a spot synthesizer
(Abimed GmbH, Langenfeld, Germany) as described previously (41).
Cleavable peptides were released as amides from the cellulose support
using ammonia vapor (42). Subsequently, filter discs with adsorbed
peptides were punched out into microtiter plates. After desorption from
the cellulose matrix with buffer the peptide solution was directly used
in the test system. For generation of the sequence files, the in house
developed software DIGEN was applied. Soluble peptides in milligram
quantities were synthesized as amides on a multiple peptide synthesizer
AMS 422 (Abimed) according to the standard Fmoc machine protocol (43) using TentaGel S RAM resin (50 mmol/g; Rapp Polymere, Tübingen, Germany) and PyBOP activation. All peptides were analyzed by high performance liquid chromatography on a Vydac C18 column using a linear
gradient 5-60% acetonitrile/water (0.05% trifluoroacetic acid) for
20 min at 1.2 ml/min flow rate (detection at 214 nm) and by laser
desorption time-of-flight mass spectrometry using Solid Phase Binding Assay--
Peptide libraries bound
covalently to cellulose membranes were incubated in Tris-buffered
saline (50 mM Tris, pH 7.5, 150 mM NaCl)
containing 0.05% Tween 20 (TBS/Tween 20). After two changes of buffer
the paper was blocked by incubation for 60 min in TBS/Tween 20 containing 5% milk powder at 4° C. To this solution
125I-VEGF165 was added to a concentration of
0.1 µC/ml. The incubation was continued for 60 min before the
cellulose libraries were washed three times with TBS/Tween 20. Overnight the cellulose sheets were placed together with an
autoradiography film (Reflection NEF 496, DuPont, Bad Homburg, Germany)
and an intensifying screen NEF 491 (DuPont) before the film was
developed. Binding peptides were detected as black dots.
Human Umbilical Vein Endothelial Cell Culture--
Human
umbilical cords were canulated with a conical device, which was fixed
by a thread, and flushed with sterile phosphate-buffered saline (PBS)
containing calcium, magnesium (Biochrom KG, Berlin, Germany),
penicillin (500 units/ml), and streptomycin (1000 µg/ml) to remove
blood cells. The veins were then filled with PBS containing chymotrypsin (0.1% w/v) and incubated at room temperature for 25 min.
After slight manual massage of the umbilical cords the cell suspension
was brought into a tube containing 2 ml of fetal calf serum. After
dilution with buffer, cells were collected by centrifugation, washed
once in buffer, and seeded into two 25-cm2 flasks (coated
with 10 µg/ml Collagen) per cord in medium 199 (M199) containing 10%
fetal calf serum, 10% human serum, glutamin, or glutamax (2 mM), penicillin (100 units/ml), streptomycin (100 µg/ml),
ascorbic acid (1.27 mM), pyruvic acid (1 mM),
and 1% nonessential amino acids (Biochrom), 6 µg/ml endothelial
growth factor from bovine brain (Sigma), and 7.5 µg/ml heparin
(Sigma). After 2-3 h of plating we washed the adherent cells twice
with PBS and cells were cultured under standard conditions in medium
described above. Cells were passaged by trypsin digestion (0.02%
trypsin, 0.01% EDTA in phosphate-buffered saline without bivalent
ions) into microtiter plates at a density of 1.6 × 104 cells/well and cultured from 3 to 7 days.
Preparation of Microvascular Endothelial Cells from Human
Foreskin--
Preparation of magnetic beads according to Jackson
et al. (Ref. 44, 10 million Dynabeads (anti-mouse Ig coated
by means of an DNA linker)) were washed with PBS/bovine serum albumin
(BSA) three times using the magnetic device. Anti-CD31 antibody
(Immunotech, Westbrook, ME) was added at a concentration of 3.5 µg/10
million beads and shaken for 30 min. The suspension was washed five
times with PBS/BSA and stored in 1.25 ml of 0.01% azide containing
PBS/BSA at 4° C. Beads were washed directly before use to remove azide.
Human Foreskin was washed with PBS containing penicillin (1000 units/ml) and streptomycin (1000 µg/ml) to remove blood. The skin was
cut in 1 ml of PBS into 2 × 2-mm pieces and left for 60 min in
0.3% trypsin/EDTA in PBS in the incubator at 37° C. Trypsin was
removed and the skin fragments were incubated for 4 h with dispase
grade II (2.4 units/ml; Boehringer Mannheim GmbH, Mannheim, Germany) at
37° C. The dermis of the skin was scraped with a spatula and the
resulting cell suspension was diluted in PBS and centrifuged at
200 × g for 5 min. The pellet was resuspended in
complete medium and filtered over a 100-µm net. The cell suspension was distributed in one or two culture wells of 10-cm2 area.
After 16 h in the incubator adherent cells were washed several
times with PBS and culture was continued until 50% confluence is
observed. Cell mixtures originating from 10 cm2 surface
were incubated at 4° C with 300,000 washed beads of the preparation
for 15 min and then washed cautiously five times with cold PBS/BSA. To
the pellet 50 µl of M199 containing 1% fetal calf serum and 200 units of DNase was added and incubation was continued for 15 min at
37° C. Then the cells were plated into a collagen-coated tissue
culture flask in the same full medium as HUVEC.
Receptor Binding Assay--
VEGF165 binding to
soluble extracellular fragment of VEGFR II (sVEGFR II) using microtiter
plates (Maxisorb, Nunc, Roskilde, Denmark) shaken for 30 min with 50 µl of sVEGFR II solution in PBS (Biochrom), in concentrations of
about 0.4 µg depending on the preparation. Then 200 µl of 4% BSA
in PBS was added for another 30 min. The solution was removed and the
plates were washed with 0.1% BSA in PBS. 10 µl of compounds in PBS,
0.1% BSA were added and 40 µl of
125I-VEGF165 (approximately 15,000 cpm or
300-600 pM) was added in PBS, 0.1% BSA. The incubation
mixture was shaken for 60 min and radioactivity was removed. After
three washes with 0.1% BSA in PBS, 100 µl of 0.5% sodium dodecyl
sulfate (SDS) was added and the plate was shaken for 30 min. 80 µl of
the solution was utilized for VEGF165 Binding to HUVEC--
300,000 HUVECs were seeded
per well (24-well plates) and cells were cultured for 2 days in
complete medium. Cells were washed with PBS containing calcium and
magnesium chloride (Biochrom). At 4° C 100 µl of medium M199
without additives but containing 0.1% BSA was added followed by 10 µl of peptide solution, that had been dissolved in dimethyl sulfoxide
and diluted appropriately in advance, and 40 µl of
125I-VEGF165. After 2 h of incubation at
4° C the medium was aspirated and washed three times with 500 µl
of PBS. The cells were lysed with 100 µl of 0.5% SDS solution by
vigorous shaking. The solution was transferred to a counting vial and
VEGFR II Autophosphorylation Assay--
100,000 MVECs were
plated into a 12-well plate. After 48 h the cells were put on ice
and fresh medium containing the solvent control, VEGF165 or
VEGF165 plus peptides in ice-cold medium M199 with 0.1%
BSA was added. Incubation was continued for 1 h. The medium was
removed and ice-cold RIPA buffer was added (50 mM HEPES, pH
7.2, 10 mM EDTA, 0.1% SDS, 1% Nonidet P-40, 0.5%
deoxycholate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 2 mM orthovanadate, 1 mM zinc acetate, 1.25 mM phenylmethylsulfonyl
fluoride, 10 mg/ml aprotinin). The DNA was removed by filtering through
a Millipore filter (0.65 µm). 50 µl of wheat germ
agglutinin-Sepharose was added to the filtrate and the mixture was
incubated under rolling for 1 h at 4° C. Sepharose was
separated by centrifugation, the supernatant was discarded and 25 µl
of twice concentrated SDS-electrophoresis buffer according to Laemmli
was added and boiled for 5 min. Proteins were separated on a 6% SDS
gel according to size. The proteins were transferred to a
polyvinylidene difluoride membrane by semidry electroblotting. The
membrane was blocked by 0.25% Tween 20 in PBS containing magnesium and
calcium chloride. After three washes for 15 min with 0.05% Tween 20 in
the same buffer, horseradish peroxidase-conjugated anti-phosphotyrosine
monoclonal antibody (PY-20, Transduction Laboratories, Inc., Lexington,
Ky) was added at a final concentration of 250 ng/ml. After three washes
for 15 min each, the blot was developed with an enhanced
chemiluminescence system (Amersham, Braunschweig, Germany).
Alternatively the blot was developed for control purposes with a poly-
or monoclonal antibody against VEGFR II (Dr. Towbin, Basel; Dr.
Martiny-Baron, Freiburg, Germany).
c-kit Autophosphorylation Assay--
TF-1 cells (CRL-2003, ATCC,
Manassas, VA) that had been cultured in the presence of 2 ng/ml
granulocyte macrophage-colony stimulating factor were centrifuged.
Three million cells in 50 µl of PBS containing 0.1% BSA were
transferred into wells of a C96 white microtiter plates (Maxisorb,
Nunc) that had been coated previously with 1 µg/ml anti-c-kit = CD 117 antibody (Research Diagnostics Inc., Flanders, NJ) overnight at
pH 9.6 and then blocked with BSA. 10 µl of inhibitor solution was
added 5 min before stem cell factor (40 µl, 1 nM) was
added. Incubation time was 60 min at 4° C. Cells were lysed in the
original plate with 50 µl of lysis buffer (150 mM HEPES
pH 7.1, 450 mM sodium chloride, 3 mM magnesium
chloride, 30 mM sodium diphosphate
(Na4P2O7), 300 mM sodium flouride, 3 mM sodium vanadate
(Na3VO4), 30% glycerol, 4.5% Triton X-100, 3 tablets/50 ml of protease inhibitor mixture (B#1836145, Boehringer
Mannheim GmbH). After washing several times (50 mM Tris pH
7.4, 150 mM NaCl, Tween 0.1%), the wells were incubated with 1:20,000-fold diluted anti-phosphotyrosine antibody horseradish peroxidase-conjugated (4G10, Upstate Ltd., Lake Placid, NY) and after
repeated washings developed using chemiluminescence substrate (Boehringer Mannheim GmbH), according to the manufacturer.
Endothelial Cell Migration--
Endothelial cells were passaged
and maintained overnight in medium 199, 10% fetal calf serum, 10%
human serum containing glutamin, penicillin, and streptomycin but no
growth factors. The next day cells were detached from the culture
plates with trypsin. Ten thousand cells in 100 µl of M199 containing
2% human serum and glutamin were given into a culture well insert with
a porous filter bottom that had been washed with PBS and coated with
collagen (10 µg/ml) previously. In the lower chamber are 600 µl of
medium (M199 + 2% human serum). After 2 h at standard culture
conditions VEGF165 (250 pM final concentration)
and 10 µl of the inhibitors were added. The medium was mixed by
cautious swirling and cells were incubated for an additional 18 h.
The inserts were rinsed with PBS and stained by immersion into a PBS
solution containing 1% rose bengal in 30% of ethanol. Excess rose
bengal was removed by washing with PBS. Cells from the upper side of
the insert were removed by cotton swaps. For quantification randomized
inserts were evaluated under the microscope counting three field each of three independent experiments. Quantitative image scan was performed
with Densitometer SI (Molecular Dynamics, Sunnyvale, CA).
Proliferation of Microvascular Endothelial Cells--
50,000
cells were plated into a 24-well plate in full medium based on M199.
After 2 h the medium was changed to 3% human serum. The next
morning various amounts of VEGF165 and inhibitors were added. After 3 days in culture the cells were washed and 1% rose bengal in 30% ethanol was added. After 5 min the dye solution was
removed, and the cells were washed three times thoroughly with PBS and
lysed with 0.1% sodium dodecyl sulfate in PBS. Optical density of the
resulting solutions was determined at 560 nm.
Mapping of the Potential VEGF/VEGFR II Contact Site--
Two scans
of overlapping peptides (13-mers, shifted by 2 amino acids) derived
from the entire soluble VEGFR II (sVEGFR II) sequence were
independently synthesized (Fig. 1). In
the second scan (Fig. 1B) all cysteine residues in the
peptides were substituted by serine to prevent unspecific disulfide
bridging of the target molecule (125I-VEGF165).
Incubation of both scans with radiolabeled
125I-VEGF165 was performed utilizing only short
washing times in order to permit kinetically rapid binding processes.
The peptide scan with cysteine containing peptides probed with soluble
125I-VEGF165 showed eight strong spots (Fig.
1A), whereas the peptide scan with the serine substitutions
(Fig. 1B) displayed only two spots (124 and 125)
corresponding to the peptide sequences
247RTELNVGIDFNWE259 and
249ELNVGIDFNWEYP261 of VEGFR II. The activity
bound to spots 124 and 125 could be easily stripped with routinely used
buffers (Tween 20, Tris-buffered saline, data not shown). In contrast,
the activity displayed on spots 227-229 and 236-239 (Fig.
1A), where all corresponding peptides contain a cystein
residue, could only be removed under reducing conditions indicating
nonspecific disulfide bridging of the ligand with the matrix-bound
peptides. The specific signals of the spots 124 and 125, which thus
were reproduced in two independent experiments (Fig. 1), represent an
amino acid sequence located in the third globular domain of the
extracellular part of the VEGFR II.
A complete L-substitution analysis of the peptide
249ELNVGIDFNWEYP261 (Fig. 1, A and
B), spot 125, which exhibits the stronger signal in the
peptide scan in comparison to spot 124) in which all residues were
replaced by all other 19 L-amino acids (Fig.
2) was performed to determine the
residues involved in binding of VEGF165. The results
indicate that Asp255 is the essential residue for peptide
binding since no other amino acid substitution is allowed at this
position. Glu249 can only be replaced by Asp and Glu with
comparable signal intensity favoring a negatively charged amino acid
residue at position 249. Important hydrophobic residues are located in
positions Leu250, Val252, Ile254,
Phe256, and Trp258. These five hydrophobic
residues can only be exchanged by physicochemically similar amino acids
comprising aliphatic and aromatic residues. Interestingly, amino acids
Val252 and Ile254 can be replaced by Phe but
not by Tyr suggesting that the hydroxyl groups of Tyr are unfavorable
for binding. Ser and Thr can be substituted for Asn251 with
equal signal intensity favoring H-bonding of that amino acid residue,
which appears also to be the type of interaction at position
Glu259 since charged amino acids can be replaced with
comparable intensity by potential H-bond donors (Gln, Ser, and Thr).
Positively charged amino acids (Lys, Arg, and His) are strongly
disfavored for substitution since only C-terminal positioned amino
acids (Tyr260, Pro261, and Asn257)
can be replaced by Arg and His without eliminating VEGF165
binding. In addition, substitution of Pro is only allowed at the
C-terminal position suggesting that the secondary structure inducing
Pro interferes with the binding conformation of the peptide. A similar situation is found for Gly, which also can only be exchanged with comparable signal intensities for the C-terminal amino acids and additionally for Gly253.
Inhibition of Peptide Binding to VEGFR II--
The peptides
obtained from this analysis (Table I)
were synthesized according to standard methods (44). The resulting
purity of the peptides was >95%, which was analyzed by high
performance liquid chromatography and mass spectrometry. To overcome
solubility problems the peptides were prolongated with hydrophilic
amino acids at the C termini according to the primary sequence of VEGFR II. The elongated peptides exhibit in the solid-phase VEGF binding assay similar signal intensities (data not shown) as compared with the
peptides obtained in the initial peptide scan (Fig. 1). The peptide
representing the potential VEGFR II-binding site was synthesized as
monomer and dimer. The dimeric peptides were synthesized as branched
peptides with one common C-terminal lysine.
To find out whether the peptides bind to VEGF165 at a site
relevant for the interaction of VEGF165 with its receptor
the peptides were studied as competitors in a binding test utilizing
either the extracellular part of a VEGFR II bound to microtiter plates or endothelial cells as receptor source and
125I-VEGF165 as ligand. In Fig.
3 the competition of peptide
RTELNVGIDFNWEYPASK with 125I-VEGF165 is
compared with that of VEGF165. Unlabeled
VEGF165 reduces binding of
125I-VEGF165 with an IC50 of about
1 nM from its receptor. Peptides RTELNVGIDFNWEYPASK
(Je-7) and (RTELNVGIDFNWEYPAS)2K (Je-11)
inhibit binding of 125I-VEGF165 at 10 and 0.5 µM, respectively.
Inhibition of VEGF165 Signal Transduction--
For
functional testing MVECs were incubated with VEGF165, or
VEGF165 in the presence of peptides. Receptor
autophosphorylation of lysed cells was quantified in a Western blot
using wheat germ agglutinin-Sepharose for precipitation and an
anti-phosphotyrosine monoclonal antibody coupled to horseradish
peroxidase for the detection. With increasing concentrations of
VEGF165 a band at 200 kDa stained increasingly at an
IC50 of 75 pM (Fig.
4). In control blots polyclonal
anti-VEGFR II antibody was used for identification of the receptor band
(Dr. Towbin, Basel, Switzerland). Compared with VEGF165
addition alone the intensity of this phosphotyrosine containing band
was suppressed in the presence of the peptide with an IC50
3-10 and 0.3-1 µM for peptide monomer (Je-7) and dimer
(Je-11), respectively, thus providing evidence of the antagonistic effect (Fig. 5). The peptides alone did
not activate receptor phosphorylation nor did they have any effect on
the morphology of the cells (data not shown).
To determine the specificity of the peptides inhibiting
autophosphorylation of VEGFR II we performed control experiments with TF-1 cells expressing the c-kit receptor. C-kit is a member of the
receptor tyrosine kinases class III comprising split kinase domains
comparable to VEGFR II differing only in the number of extracellular Ig
loops. VEGFR II possesses seven Ig loops whereas c-kit consists of five
Ig loops thus belonging to the platelet-derived growth factor-receptor
family. Using stem cell factor, the endogenous c-kit ligand, we found a
concentration-dependent increase in receptor autophosphorylation. The autophosphorylation was blocked by the kinase
inhibitor staurosporine, but not by peptides Je-7 and Je-11 (Table
II).
Inhibition of Endothelial Cell Activity--
To further
substantiate the antagonistic activity of the peptides we studied the
migration of microvascular endothelial cells through nucleopore
filters. The number of cells migrating through the filters to the lower
compartment within 18 h can be increased in the presence of 250 pM VEGF165 by a factor of 2-4 depending on the
cell preparation. If in addition increasing concentrations of the
antagonistic peptide are added the number of transmigrating cells is
decreased (Fig. 6). Migration of MVECs
was inhibited at half-maximal concentrations of about 0.1 and 0.3 µM by RTELNVGIDFNWEYPASK (Je-7) and
(RTELNVGIDFNWEYPAS)2K (Je-11), respectively.
VEGF165 stimulates MVEC proliferation in a
concentration-dependent manner with an EC50 of
100 pM (Fig. 7). In the
presence of peptides Je-7 and Je-11, the cell number assayed as
described was reduced concentration dependently with an
IC50 of 0.1 and 0.5 µM, respectively (Fig.
8). The peptides did not inhibit cell proliferation in the absence of VEGF165, showing the lack
of toxicity of the agents for basal cell growth.
VEGFR II and VEGF dimers are relatively large proteins that
interact in a 2:1 stoichiometry at the extracellular part of the molecules. The cytoplasmic tails of the dimerizing receptors are autophosphorylated subsequently.
To get more information on the size of the interacting surface on both
molecules we have mapped a potential contact site of VEGF165/VEGFR II using cellulose-bound overlapping
peptides, which were already successfully applied for mapping different
types of protein-protein interactions (45-48). The mapped peptides
were further characterized by a complete L-substitutional analysis which reveals binding information on a resolution of one amino acid
side chain. A special feature of the peptide/VEGF165
contact site which may reflect partly the VEGFR II-VEGF-binding site is that only negatively charged and hydrophobic amino acids contribute to binding.
The VEGF crystal structure and mutation analysis (21) reveal a
hydrophobic groove in the VEGF surface which is accompanied by two
nearby hydrogen bond forming amino acids (Asp63,
Glu64, and Glu67) or basic residues
Arg82, Lys84, and His86, the basic
amino acids being relevant for VEGFR II binding, the acidic ones for
VEGFR I binding (21, 33).
As indicated through binding experiments using shortened extracellular
domains of the VEGFR I by Davis-Smyth et al. (35) and
Barleon et al. (34) only extracellular Ig-like domains 1-3 appear to be necessary for binding. These findings are in line with the
presumed VEGF contact site on the third globular domain of VEGFR II.
Recently it was shown that domains 2 and 3 of VEGFR I and VEGFR II were
sufficient to bind VEGF with comparable affinities to the wild type
receptors (39). If only the Ig-like domain 2 of both receptors was
tested for binding, a 60-fold decrease of binding affinity was observed
for the VEGFR I, whereas the binding of VEGFR II-domain 2 was reduced
by a factor of 1000. This clearly indicates that the Ig-like domain 3 of the VEGFR II contributes significantly to the binding of VEGF, which
is also strongly supported by our results since we only detect a contiguous peptide sequence on the third Ig-like domain of VEGFR II and
not in the second domain. High resolution data obtained from the
crystal structure analysis of VEGF8-109 complexed with the
second Ig-like domain of VEGFR I (FLT-1) (39) reveal similar
VEGF-binding sites for the VEGFR I and VEGFR II. Five of seven residues
of VEGF reported to be important for VEGFR II interaction are located
in the interface of the VEGF/VEGFR I-domain 2 complex (33, 39).
Interestingly, the residual 2 VEGF amino acids described to be involved
in VEGFR II binding (21, 33, 49) form a groove adjacent to the
C-terminal segment of VEGFR I-domain 2 suggesting that this groove is a
potential binding site of domain 3 of the native complex (39). It is
tempting to speculate that the discussed groove is the binding site for the identified peptide VEGFR II247-261 located on the
third Ig-like domain. This would partly explain why we could only map a
peptide binding VEGF165 with good affinity on the third
domain and not on the second domain of VEGFR II; because for peptide binding, a binding groove is much more favorable than a flat
discontinuous surface as demonstrated for the interface of VEGF and
VEGFR I-domain 2. This discussed mode of binding would also be in
agreement with the low solubility observed for the initially
synthesized hydrophobic peptides and the importance of 5 hydrophobic
residues in the VEGF-peptide interaction as deduced from the
substitutional analysis (Fig. 2). Although the data strongly suggest a
direct binding of the mapped peptide to the VEGFR II-binding site of
VEGF, we cannot rule out an allosteric mode of action or an
interference with VEGF dimerization, which causes the inhibitory effect
of the peptide.
A sequence comparison of the mapped VEGFR II-derived epitope with the
corresponding sequences of VEGFR I and VEGFR III of different species
(Fig. 9) reveal significant differences
among the VEGF receptors. No negative charge is present in the compared sequence of the human VEGFR I. Moreover, the VEGFR I contains an Arg
and a Met at positions 253 and 256 (human VEGFR II numbering) instead
of Gly and Phe in all other VEGF receptors. Both residues specific for
VEGFR I would abolish binding of the VEGFR II-derived peptide (Fig. 2).
Asp255, the key residue for peptide binding (Fig. 2), is
the only residue specific for VEGFR II in different species suggesting
that it might also play a role in VEGFR II binding. Besides two
strictly conserved residues (Trp258 and Pro261)
the potential VEGFR II-binding site comprise several residues differing
strongly from the VEGFR I which may contribute to the different binding
modes of the two major VEGF receptors.
INTRODUCTION
Top
Abstract
Introduction
References
(TGF
) (20, 21). It contains a heparin-binding site and
glycosylation sites, which appear not to be involved in binding (22,
23). Recently three VEGF homologues, placental-like growth factor
(PlGF), VEGF-B, and VEGF-C, have been identified (24-26).
Placental-like growth factor originally found in placenta is binding to
VEGFR I but not to VEGFR II (26). VEGF-B heterodimerizes with
VEGF165 in vitro and is particularly expressed
in muscle. VEGF-C is binding to the FLT-4 receptor (VEGFR III), which
is mainly expressed in the lymphatic system, and after complete
processing it also binds to VEGFR II (25). Formation of heterodimers
among the various VEGF homologues in addition may govern the diverse
physiological functions of VEGF (27-29).
/
receptors having five extracellular immunoglobin-like domains and pertaining to the
class III tyrosine kinase receptors (30-32).
EXPERIMENTAL PROCEDURES
-cyano-4-hydroxycinnamic acid as matrix (LaserTec fOCUS mass spectrometer; VESTEC/Perseptive Biosystems, Wiesbaden-Nordenstadt, Germany).
-counting.
-radiation was measured. The result is given as the concentration
that reduces specific binding to 50%.
RESULTS
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Fig. 1.
Autoradiographs of the cellulose-bound VEGFR
II-derived peptides probed with
125I-VEGF165. The primary sequence of
VEGFR II is fragmented in 376 13-mer peptides overlapping 11 amino
acids. A, original sequence of VEGFR II. The signals
represent the following sequences: spot 124, RTELNVGIDFNWE; spot 125, ELNVGIDFNWEYP; spot 227, HIHWYWQLEEECA; spot 228, HWYWQLEEECANE; spot 229, YWQLEEECANEPS; spot 236, AVSVTNPYPCEEW;
spot 237, SVTNPYPCEEWRS; spot 238, TNPYPCEEWRSVE; spot 239, PYPCEEWRSVEDF. B, the cysteine residues of the original
sequence are replaced by serine residues. The signals represent
the following sequences: spot 124, RTELNVGIDFNWE; spot 125, ELNVGIDFNWEYP.
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Fig. 2.
Autoradiograph of the cellulose-bound
L-substitutional analysis of VEGFR II249-261-peptide
probed with 125I-VEGF165. Every amino acid
in the wild type peptide (wt, left column) is exchanged
against the 20 L-amino acids (rows) resulting in a complete set of the
possible point substitutions.
Summary of results
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Fig. 3.
Binding competition of the peptides and VEGF
for the VEGFR II. Competition curves for the monomeric peptide
RTELNVGIDFNWEYPASK (Je-7), the dimeric peptide
(RTELNVGIDFNWEYPAS)2K (Je-11), and
VEGF165 were obtained according to "Experimental
Procedures" by adding increasing concentrations of peptides and
VEGF165 to microtiter plates, coated previously with
soluble VEGFR II preparations before adding
125I-VEGF165.
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Fig. 4.
Autophosphorylation of VEGFR II in MVECs
induced by VEGF165. Cells were treated as described
under "Experimental Procedures." The experiments were performed as
duplicates of each concentration. A, quantitative
densitometric scan of the Western blot of lysed cells after stimulation
with VEGF165. B, 200-kDa band of the Western
blot stained with an anti-phosphotyrosine antibody. C, as a
control the stripped membrane was reprobed with a polyclonal antibody
against VEGFR II (obtained from Dr. Towbin).
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Fig. 5.
Inhibition of VEGF165-stimulated
VEGFR II autophosphorylation in MVECs. Cells were incubated with
75 pM VEGF165 and increasing concentrations of
peptide RTELNVGIDFNWEYPASK (Je-7). Each concentration was tested twice.
The preparation of the blots was performed as described under
"Experimental Procedures" (right lane marker protein
myosin). A, quantitative densitometric scan of the Western
blot of lysed cells after stimulation with VEGF and competition with
Je-7. B, 200-kDa band of the Western blot stained with an
anti-phosphotyrosine antibody. C, as a control the stripped
membrane was reprobed with a polyclonal antibody against VEGFR II
(obtained from Dr. Towbin).
C-kit autophosphorylation in TF-1 cells
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Fig. 6.
Migration assay with MVEC through micropore
filters. Inhibition of VEGF165-stimulated migration of
MVECs with increasing concentrations of peptides. Three independent
filters, three visual fields each. A, peptide Je-7
(RTELNVGIDFNWEYPASK). B, peptide Je-11
((RTELNVGIDFNWEYPAS)2K).
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Fig. 7.
Proliferation assay with MVEC. MVEC were
cultured overnight in a serum-reduced medium before increasing
concentrations of VEGF165 were added. After 1 (blue
curve), 2 (green curve), or 3 (red curve)
days in culture, cell mass was determined by colorimetric stain.
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Fig. 8.
Peptide inhibition of
VEGF165-stimulated MVEC proliferation. A
and C, inhibitory peptides were present in increasing
concentrations. B and D, for control of peptide
toxicity the cells were cultured with peptides in the absence of
VEGF165.
DISCUSSION
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Fig. 9.
Sequence comparison of different VEGF
receptors. The bar and the asterisks
indicate the mapped VEGFR II-derived peptide sequence and
physicochemically similar amino acids, respectively. The
underlined residues, SSK, were used to improve
the solubility of the VEGFR II-derived peptides. Conserved amino acids
are shown in green, the negatively charged amino acids are
stained in blue. Amino acid residues differing significantly
from the sequence VEGFR II247-261 are colored in
red.
The identified contiguous peptide from the sequence of Ig-like domain 3 binds to VEGF165 and competes with VEGFR II for VEGF165 binding. Utilizing this peptide in a monomeric and dimeric form, the signal transduction pathway of VEGF165 in endothelial cells could be interrupted in cellular test systems measuring receptor autophosphorylation, proliferation, and migration. The inhibition of the VEGF/VEGF-receptor function is specific for VEGF signaling since the ligand-receptor interaction of stem cell factor/c-kit, belonging together with VEGFR II to the same tyrosine kinase receptor class III, is not affected.
The concentrations observed for inhibition of VEGF are slightly different for the different assays which might be due to the different duration, variation in protein content of the media, and sensitivity of the experiments (1 h at 4° C for receptor autophosphorylation, 18 h at 37° C for migration, and 3 days at 37° C in the proliferation assays). Especially the duration of the experiment could have an influence on the effective concentration of the peptides showing a limited solubility. This is in line with the fact that the monomeric peptide (Je-7) possesses a lower solubility compared with the dimeric peptide (Je-11), which does not show relevant differences in the IC50 values in the endothelial cell migration assay and proliferation assay in contrast to the competition with sVEGFR and the receptor autophosphorylation assay. In addition, the different sensitivity of the respective assays might contribute to the different IC50 values. Experiments with other tested compounds indicate that the migration and proliferation seem to be more sensitive assays compared with the binding and phosphorylation assays.
The binding constant of the monomeric peptide to VEGF165 is
considerably lower than the VEGF/VEGFR II binding constant which might
also be due to an avidity effect of the homodimeric
VEGF165, which possesses two identical receptor-binding
sites. This is supported by the fact that the dimeric peptide has an
order of magnitude higher binding constant than the monomeric peptide. We assume that the binding between the peptide dimer and VEGF occurs in
a 1:2 stoichiometry since the length of the peptide length does not
allow for the simultaneous occupation of the two VEGF-binding sites,
which are most likely located on the poles of the oval VEGF molecule
(21, 39). Our findings open the opportunity to search for nonpeptidic
small molecules that block the interaction of VEGF with VEGFR II for
possible use for pharmaceutical purposes.
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ACKNOWLEDGEMENTS |
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We thank Dr. Towbin and Dr. Reusch for the VEGFR II antibodies. G. Fenten and M. Seeman for expert technical help, Dr. Martiny-Baron for baculovirus, expressing VEGFR II protein, and VEGF, Dr. Holger Wenschuh for peptide synthesis, and Dr. Ulrich Reineke for critical reading of the manuscript.
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Note Added in Proof |
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The importance of the third Ig-like domain of VEGFRII in VEGF binding is supported by studies of Shinkai, A., Ito, M., Anazawa, H., Yamaguchi, S., Shitara, K., and Shibuya, M. (1998) J. Biol. Chem. 273, 31283-31288, published after submission of this manuscript.
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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.
To whom correspondence may be addressed. Tel.:
49-30-6392-6392; Fax: 49-30-6392-6395; E-mail: biotools{at}jerini.de.
** To whom correspondence may be addressed. Tel.: 49-30-4681-5798; Fax: 49-30-4681-8069; E-mail: karlheinz.thierauch{at}schering.de.
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor/vascular permeability factor; VEGFR II, VEGF receptor II or KDR (kinase domain insert receptor); VEGFR I, VEGF receptor I or Flt-1 (fms like tyrosine kinase); FLT-4, VEGF receptor III or VEGFR III; HUVEC, human umbilical vein cell; MVEC, microvascular endothelial cell; sVEGFR II, soluble VEGF receptor II; c-kit, stem cell growth factor receptor; BSA, bovine serum albumin; PBS, phosphate-buffered saline.
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REFERENCES |
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