From the Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
Received for publication, October 25, 2002
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ABSTRACT |
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The vascular endothelial growth factor
(VEGF) family plays important roles in angiogenesis and vascular
permeability. Novel members of the VEGF family encoded in the Orf virus
genome, VEGF-E, function as potent angiogenic factors by specifically
binding and activating VEGFR-2 (KDR). VEGF-E is about 45% homologous
to VEGF-A at amino acid levels, however, the amino acid residues in
VEGF-A crucial for the VEGFR-2-binding are not conserved in VEGF-E. To
understand the molecular basis of the biological activity of VEGF-E, we
have functionally mapped residues important for interaction of VEGF-E
with VEGFR-2 by exchanging the domains between VEGF-ENZ-7 and PlGF, which binds only to VEGFR-1
(Flt-1). Exchange on the amino- and carboxyl-terminal regions had no
suppressive effect on biological activity. However, exchange on either
the loop-1 or -3 region of VEGF-ENZ-7 significantly reduced
activities. On the other hand, introduction of the loop-1 and -3 of
VEGF-ENZ-7 to placenta growth factor rescued the biological
activities. The chimera between VEGF-A and VEGF-ENZ-7 gave
essentially the same results. These findings strongly suggest that a
common rule exists for VEGFR-2 ligands (VEGF-ENZ-7 and
VEGF-A) that they build up the binding structure for VEGFR-2 through
the appropriate interaction between loop-1 and -3 regions.
Vascular endothelial growth factor-A
(VEGF-A)1 plays a pivotal
role in vasculogenesis, angiogenesis, and differentiation of hemangioblasts to hematopoietic precursor cells in embryogenesis. VEGF-A is also known to be closely involved in a variety of
pathological conditions such as tumor angiogenesis and diabetic
retinopathy (1-3).
VEGF-A is a member of the PDGF superfamily because of their structural
similarities. VEGF is found to be a dimeric glycoprotein of
Mr 34,000-42,000, and have conserved eight
cysteine residues in each monomer. These cysteine residues construct a
particular folding consisting of two intermolecular and three
intramolecular disulfide bonds that generate three loop-like
structures, loop-1, -2, and -3. VEGF-A exerts its biological activity
by interacting with receptor-type tyrosine kinases, VEGFR-1 (Flt-1) and
VEGFR-2 (KDR/Flk-1) (4-7). Homozygous loss of the VEGFR-1
or VEGFR-2 genes resulted in embryonic lethality between
days 8.5 and 9.5, indicating that these VEGF receptors play important
roles in vasculogenesis and angiogenesis (8, 9). The different
phenotypes of these VEGFR-mice suggest that VEGFR-2
is the major positive signal transducer, whereas VEGFR-1 has a negative
regulatory role in angiogenesis at early embryogenesis.
The VEGF family in vertebrate genomes includes VEGF-A, PlGF (placenta
growth factor), VEGF-B, -C, and -D. PlGF and VEGF-B specifically bind
to VEGFR-1 (10-13), whereas VEGF-C and -D bind and activate VEGFR-3
(Flt-4), regulating lymphangiogenesis as well as angiogenesis in the
middle stage of embryogenesis (14-18). Amino acids in VEGF-A essential
for binding with VEGFR-2 have been studied by the alanine scanning
method. Keyt et al. (19) have reported that Arg-82, Lys-84,
and His-86 are indispensable for the interaction between VEGF-A and
VEGFR-2.
Recently, we have shown that the VEGF-ENZ-7 protein, a
novel member of the VEGF family could bind specifically to VEGFR-2, activate the receptor, and promote the growth of endothelial cells in vitro and in vivo at a transient condition
(20). VEGF-E genes were originally found as an open reading
frame in the genome of the NZ-7, NZ-2, and D1701 strains of
parapoxvirus, Orf virus (21).
These three genes were structurally very similar to each other compared
with VEGF family proteins, and were designated as VEGF-ENZ-7,
ORFV2-VEGF/VEGF-ENZ-7, and
VEGF-ED1701 (20, 22, 23). Orf virus causes
contagious pustular dermatitis in sheep, goats and, sometimes, humans.
Histologically, the lesions are highly vascularized and edematous
with proliferation of endothelial cells and inflammatory cells. In
addition to VEGF-ENZ-7, other two VEGF-E members were also
shown to specifically bind to VEGFR-2 but not to other receptors.
VEGF-ENZ-7 has a high affinity to VEGFR-2 at similar levels
as VEGF-A, and efficiently competes to VEGF-A (20). This indicates that
the binding pocket on VEGFR-2 for VEGF-A and VEGF-ENZ-7 is significantly overlapped to each other. Interestingly, however, three
basic amino acids on VEGF-A essential for the VEGFR-2-binding are not
conserved in VEGF-ENZ-7, and these basic amino acids were changed to hydrophobic or non-charged ones, Val, Gly, and Ser, respectively. These results indicate that the local structure built up
by these basic amino acids in VEGF-A are not always required for the
ligands that bind to VEGFR-2.
To elucidate a novel rule for the ligand-binding to VEGFR-2, we carried
out a series of domain-exchange analysis between VEGF-E and PlGF, or
VEGF-E and VEGF-A. Our results clearly indicate that an intimate
relationship between the loop-1 and -3 of VEGF-ENZ-7 as
well as VEGF-A is crucial for the formation of the three-dimensional structure important for the high-affinity binding to VEGFR-2.
Cells and Culture Conditions--
Spodoptera
frugiperda (Sf9) cells and baculovirus transfer
vectors (pVL-1393) were purchased from Invitrogen (San Diego, CA) and
cultured in EX-CELL 400 medium (JRH Biosciences, Lenexa, KS). A
linearized DNA of a mutant Autographa californica nuclear
polyhedrosis viral DNA (BaculoGold) was from BD Pharmingen (San Diego,
CA). 125I-VEGF-A165 was the product of Amersham
Biosciences (Buckinghamshire, UK). NIH3T3 cell lines overexpressing
human KDR/VEGFR-2 (NIH3T3-KDR) were previously established and used for
signal transduction studies (13). NIH3T3-KDR cells were maintained in
Dulbecco's modified Eagle medium (Nissui, Tokyo) supplemented
with 10% calf serum, 2 mM L-glutamine, 40 µg/ml kanamycin, and 200 µg/ml G418 sulfate (Geneticin;
Invitrogen). Recombinant human VEGF-A165 was
prepared as described (13). The enhanced chemiluminescence detection kit was purchased from Amersham Biosciences.
Polyclonal and Monoclonal Antibodies--
Polyclonal antisera
against VEGF-ENZ-7 were raised in rabbits using a 20-amino
acid sequence of the carboxyl terminus as antigen (20). Anti-human
VEGFR-2 antiserum (B2) was prepared previously (24). A monoclonal
antibody specific to phosphotyrosine (PY20) was obtained from ICN
Biochemicals (Costa Mesa, CA). Secondary antibodies conjugated to
horseradish peroxidase were purchased from Amersham Biosciences.
Monoclonal neutralizing antibody to VEGF-A165 was purchased
from R&D Systems (Minneapolis, MN).
Construction of VEGF-ENZ-7 Chimeric Mutant--
The
synthetic cDNA encoding VEGF-ENZ-7 was cloned to the
BamHI and EcoRI restriction sites of pUC18 (20),
and PlGF cDNA was cloned to pUC18 (13). At first, sequences
encoding six histidines were introduced by using a double stranded
oligonucleotide encoding amino acid residues Cys-130 to Arg-148
of VEGF-ENZ-7 and six histidines followed by stop codon.
This oligomer also had the cohesive end for DraIII at the
NH2 terminus and for EcoRI at the COOH terminus, and it was ligated to the 3-kb DraIII-EcoRI
fragment of the plasmid that contains cDNA of
VEGF-ENZ-7 cloned to pUC18 (pUCE-his). With the same
manner, histidine residues were introduced into the COOH terminus of
PlGF-1 cDNA. The double stranded synthetic oligonucleotide encoding
Arg-131 to Arg-149 of PlGF-1 was followed by six histidine residues and
a stop codon. This oligomer had cohesive ends for the BsmI
site at the NH2 terminus and EcoRI site at the
COOH terminus. This oligomer was cloned to a 3-kb
BsmI-EcoRI fragment of the plasmid (pUCP-his)
with full-length cDNA of PlGF-I cloned to BamHI and the
EcoRI site of pUC18.
The constructs of chimera proteins were produced by exchanging variable
regions among VEGF-ENZ-7, VEGF-A, and PlGF. They were achieved by ligating a series of digested fragments of pUCP-his and/or
pUCE-his with synthetic double stranded oligonucleotides and/or the
fragments produced by the PCR technique. The details of each plasmid
construction are available on request.
Each construct contains the following amino acid sequence:
1, Met-1 to Val-40 of PlGF and Asn-35 to the stop codon of
VEGF-ENZ-7; 2, Met-1 to Tyr-51 of PlGF and Cys-46 to the
stop codon of VEGF-ENZ-7; 3, Met-1 to Glu-64 of PlGF and
Tyr-59 to the stop codon of VEGF-ENZ-7; 4, Met-1 to Arg-82
of PlGF and Cys-77 to the stop codon of VEGF-ENZ-7; 5, Met-1 to Glu-98 of PlGF and Thr-93 to the stop codon of
VEGF-ENZ-7; 6, Met-1 to Cys-130 of
VEGF-ENZ-7 and Glu-129 to the stop codon of PlGF; 7, Met-1
to Cys-88 of VEGF-ENZ-7 and Val-95 to the stop codon of
PlGF; 8, Met-1 to Cys-77 of VEGF-ENZ-7 and Thr-84 to the
stop codon of PlGF; 9, Met-1 to Val-40 of PlGF, Asn-35 to Cys-130 of
VEGF-ENZ-7 and Glu-129 to the stop codon of PlGF; numbers 10 to 27, shown in Figs. 1 and 4; number 28, Met-1 to Gly-45 of VEGF-ENZ-7, Cys-52 to Cys-77 of VEGF-A, and Val-72 to the
stop codon of VEGF-ENZ-7; number 29, Met-1 to Ile-87 of
VEGF-ENZ-7, Cys-95 to Cys-128 of VEGF-A, and Asp-131 to the
stop codon of VEGF-ENZ-7; number 30, Met-1 to Gly-45 of
VEGF-ENZ-7, Cys-52 to Cys-77 of VEGF-A, Val-72 to Ile-87 of
VEGF-ENZ-7, Cys-95 to Cys-128 of VEGF-A, and Asp-131 to the
stop codon of VEGF-ENZ-7; number 31, Met-1 to Tyr-51 of
PlGF, Cys-46 to Cys-72 of VEGF-ENZ-7, and Val-78 to the
stop codon of PlGF; number 32, Met-1 to His-93 of PlGF, Cys-88 to
Cys-130 of VEGF-ENZ-7, and Glu-129 to Arg-131 of PlGF;
number 33, Met-1 to Tyr-51 of PlGF, Cys-46 to Cys-72 of
VEGF-ENZ-7, Val-78 to His-93 of PlGF, Cys-88 to Cys-130 of VEGF-ENZ-7, and Glu-129 to the stop codon of PlGF.
Expression of Mutant Proteins and Purification--
The
full-length coding regions of chimera mutants were subcloned into the
BamHI-EcoRI site on the multicloning region of
pVL1393. These transfer vector DNAs were used for co-transfection into Sf9 cells along with the linearized baculovirus DNA
"BaculoGold" by liposome transfection. The recombinant viruses were
amplified at 3-day intervals. Sf9 cells grown in
serum-free medium EX-CELL 400 were used to produce chimera proteins.
For a large scale preparation of proteins, Sf9 cells
were infected with viruses at a multiplicity of infection of about 10. Three days after infection, the culture media were harvested and
centrifuged to remove debris. The supernatants were resolved by
SDS-PAGE on a 15% gel followed by Western blotting.
One-hundred ml of the supernatant of cells infected with chimera
recombinant virus was collected, concentrated, and dialyzed to 22.5 mM sodium phosphate buffer, pH 8.0, containing 375 mM NaCl and 62.5 mM imidazole. After dialysis
and filtration, glycerol was added for a final concentration at 20%,
giving rise to the final buffer with 20 mM sodium
phosphate, pH 8.0, 300 mM NaCl, and 50 mM
imidazole. The nickel-nitrilotriacetic acid beads (Qiagen, Germany)
were applied to the dialyzed sample and rotated for 3 h at
4 °C. The nickel-nitrilotriacetic acid containing sample was loaded
onto a 5-ml column, and washed with washing buffer (50 mM
imidazole, 20 mM sodium phosphate buffer, 300 mM NaCl, and 20% glycerol). The chimera proteins were
eluted with elution buffer (250 mM imidazole, 20 mM sodium phosphate buffer, pH 8.0, 300 mM
NaCl, and 20% glycerol). The positive fractions were collected and
further concentrated by Microcon (Millipore). For detection of VEGF-E
protein, aliquots of fractions were analyzed by Western blotting using
anti-His antibody (Qiagen, Germany) and Coomassie staining. The purity
of chimera proteins was above 80%.
Binding Competition Assays--
Binding competition assay was
performed as previously described (25, 26) using receptor protein
immobilized to solid phase. Preparation of the receptor protein
consisting of the extracellular region of VEGFR-2 tagged with the Fc
portion of IgG was described previously (25). Aliquots (50 µl) of the
Fc-tagged receptor (0.3 µg/ml) in phosphate-buffered saline were
attached to 96-well plates, Immunon 2 (Dynex Technologies, Inc.,
Chantilly, VA), overnight at 4 °C. The plates were washed twice and
blocked with binding buffer (1% bovine serum albumin in
phosphate-buffered saline) for 30 min at 25 °C. Competition activity
was examined by incubating the KDR-coated plates with a fixed
concentration of 125I-VEGF-A and increasing concentrations
of non-radiolabeled chimera mutant proteins for 3 h at 25 °C.
The wells were washed three times with binding buffer, then the bound
125I-labeled proteins were quantified in a Human Endothelial Cell Proliferation Assay--
Human umbilical
vein endothelial cells (HUVEC) (Morinaga, Tokyo) were grown in
HuMedia-EG2 (Kurabo, Tokyo) and used for endothelial cell growth assay.
HUVEC were seeded at 4,000 cells/well on 24-well collagen-coated plates
(CELLTIGHT, Sumitomo Bakelite Co., Ltd., Tokyo) in medium with
free-growth factor and low concentration serum (0.2%). Four h after
plating, mutant proteins containing medium were added. Two days after,
medium and mutant proteins were replaced with fresh sample. At day 4, cells were stained with the crystal violet. The cell numbers
were determined by averaging the counting of five spots randomly chosen
at each well using a Coulter counter.
VEGFR-2 Autophosphorylation Assay--
For in vivo
phosphorylation, NIH3T3-KDR (VEGFR-2) cells were grown to
semiconfluence and stimulated with a variety of ligands at 37 °C for
5 min. The cells were washed in ice-cold phosphate-buffered saline with
0.1 mM Na3VO4 twice and lysed in
1% Triton X-100 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 2% aprotinin, 1 mM
phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 10 mM Na4P2O7, and 2 mM Na3VO4). The lysates were
clarified by centrifugation (15,000 rpm for 10 min). Protein concentrations were measured using a Bio-Rad protein assay and the same
amounts of protein of each sample were used for analysis. For
immunoblotting, the cell lysates were subjected to 7.5% SDS-PAGE and
transferred to a nitrocellulose sheet. The blots were incubated with a
blocking solution (5% bovine serum albumin containing washing buffer
(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3%
Tween 20)) and probed with the primary antibody diluted in blocking
solution. The signal was visualized using peroxidase-conjugated
secondary antibodies and exchanged chemiluminescence (ECL, Amersham)
according to the manufacturer's instructions.
Tubular Formation Assay--
The angiogenesis kit was purchased
from Kurabo (Tokyo) and the tubular formation assay was demonstrated as
per the manufacturer's instructions. In this system, human endothelial
cells and fibroblasts were co-cultured in 24-well plates at 37 °C,
5% CO2. At days 1, 4, 7, and 9, medium was exchanged with
fresh medium, which contains chimera proteins and the excess amount of
neutralizing antibody for VEGF-A. At day 11, incubation was terminated,
fixed with 70% ethanol, and immunostained for CD31/PECAM-1. For
immunostaining, mouse anti-human CD31, alkaline
phosphatase-conjugated goat anti-mouse IgG and
5-bromo-4-chloro-3-indolyl phosphate were purchased from Kurabo
(Tokyo). The cells were incubated with mouse anti-human CD31 for 60 min
at 37 °C in blocking buffer (1% bovine serum albumin in
phosphate-buffered saline) and washed with blocking buffer. Secondary,
alkaline phosphatase-conjugated goat anti-mouse IgG was added to
the cells followed with incubation for 60 min at 37 °C. After
washing, 5-bromo-4-chloro-3-indolyl phosphate was used to develop a
color to visualize endothelial cells. Tubules were analyzed under a
bright-field microscope and total length of branching in a fixed area
(mm/mm2) of the randomly chosen 5 spots per well.
Production and Dimer Formation of VEGF-ENZ-7
Mutants--
Alanine scanning analysis of VEGF-A has shown that basic
amino acids at 82, 84, and 86 as well as other amino acids in loop-3 are essential for VEGFR-2 binding, however, the VEGF-E family that
binds to VEGFR-2 at a similar affinity as VEGF-A does not conserve
these amino acids (Fig. 1). To examine
which region(s) in VEGF-E is crucial for the binding and activation of
VEGFR-2, we constructed a series of chimeric mutants by exchanging the variable region of VEGF-ENZ-7 with that of PlGF and/or
VEGF-A. The carboxyl terminus of all chimeric mutants had an insertion of six histidines as a tag sequence for the convenience of protein purification. This His tag did not suppress the activity of
VEGF-ENZ-7.
VEGF-ENZ-7 was exchanged with PlGF from the
NH2-terminal, and the
exchanging region was extended toward the COOH-terminal, serially
(Figs. 2 and 3). Secondary, exchanging
was performed from the COOH terminus toward serially. Loop-wise
exchanging on loop-1, -2, and -3 was produced by both PlGF and VEGF-A
amino acid sequences (Fig.
4A). The site-directed
mutagenesis was also performed by exchanging only 4 amino acid residues
(Fig. 4B). Finally, loop-1 and -3 regions of
VEGF-ENZ-7 were introduced to PlGF as the gain of function
mutants (Fig. 5) (see "Materials and
Methods").
The molecular size of mutant proteins in non-reducing conditions were
in the range of Mr 40,000 to 45,000, and 20,000 to 25,000 in reducing conditions, consistent with wild type
VEGF-ENZ-7 and PlGF (Fig. 3, data not shown). These results
indicate that all mutants did not show any disruption in dimerization.
NH2- and COOH-terminal Sequences in
VEGF-ENZ-7 Are Replaceable with the Corresponding Sequence
in PlGF without a Significant Loss of Biological Functions--
To
evaluate the affinity of each mutant to receptor, a binding assay was
carried out by using the extracellular domain of VEGFR-2 tagged with
the Fc portion of IgG (see "Materials and Methods"). Because
VEGF-ENZ-7 could compete with VEGF-A in binding to VEGFR-2,
chimeric proteins were tested for their ability to compete with
125I-labeled VEGF-A165 for interaction to
VEGFR-2-Fc immobilized onto 96-well plates.
When VEGF-ENZ-7 was exchanged with PlGF from the
NH2-terminal toward COOH-terminal serially (Fig.
2A), 34 amino acids of the NH2-terminal region
in VEGF-ENZ-7 were replaceable without any reduction of
affinity to VEGFR-2 (Fig. 2, chimera 1). An extension of the PlGF
region to the first conserved cysteine residue resulted in a minor
reduction of the affinity (Fig. 2B, 2). However,
as shown by chimeras 3, 4, and 5, the PlGF amino acid sequence close to
and over loop-1 resulted in the loss of affinity similar to intact PlGF
(Fig. 2B).
Secondary exchanging was performed from the COOH terminus toward the
NH2 terminus serially (Fig. 2A). Among the
mutants 6, 7, and 8, chimera 6 with 18 amino acids of the COOH-terminal
exchange from the 8th conserved cysteine to the COOH-terminal end
remained its affinity. However, the replacement over loop-3 such as
chimeras 7 and 8 had lost their binding ability. Taken together,
NH2 and COOH termini were not critical for
VEGF-ENZ-7 to interact with VEGFR-2.
To confirm this result, a chimera with simultaneous exchanging of both
NH2 and COOH termini were evaluated (Fig. 2A,
chimera 9), and showed no significant suppression in its affinity.
These results may imply that at least loop-1 and -3 are very critical for VEGF-ENZ-7, whereas the importance of loop-2 remained unclear.
Loop-2 but Not Loop-1 and -3 in VEGF-ENZ-7 Is
Exchangeable to Either PlGF or VEGF-A Sequence--
Keyt et
al. (27) reported that several amino acids in loop-3 are essential
for the binding of VEGF-A to VEGFR-2 (27). This result suggests that
loop-3 is a direct binding site for VEGFR-2 in VEGF-A. To analyze the
importance of each loop region of VEGF-ENZ-7, a variety of
chimera mutants with loop region-specific replacement were tested
(Figs. 4 and 5). These exchanged regions do not contain
As shown in Fig. 4A, chimeras 10 and 11 had an exchange on
loop-1 (Tyr-54 to Asn-68 between
We found that the loop-2 region in VEGF-ENZ-7 is
exchangeable to that of PlGF or VEGF-A (Fig. 4, chimera 12 and 13), whereas loop-1 and -3 specific exchanges to PlGF
caused a significant defect (Fig. 4, chimera 11, 15, and 17). These
results indicate that the important regions for VEGF-ENZ-7
to interact to VEGFR-2 are not only loop-3 but also loop-1.
Along with these PlGF replacements, the VEGF-A region was also
introduced into loop regions of VEGF-ENZ-7 as a reference. Surprisingly, independent introduction of loops of VEGF-A, which is a
strong ligand for VEGFR-2, also resulted in significant reduction in
biochemical and biological activities (Fig. 4, chimeras 10, 14, and 16). Therefore, both loop-1 and -3 regions are equally important in VEGF-A as well as in
VEGF-ENZ-7. This result implies that VEGF-ENZ-7
and VEGF-A may have a common mechanism in the interaction to VEGFR-2,
by having critical regions in loop-1 and -3.
Loop-1 but Not the Adjacent Short Sequences Are Important for
Biological Function--
More detailed mutations were introduced to
identify important amino acid residues. The region for loop-1 exchange
was divided into 3 parts, which were composed of YLGE, ESTN, and LQYN.
These residues were changed to PlGF counterparts, DVVS, SEVE, and HMFS (Figs. 1A and 4B). Among these mutants, a chimera
with ESTN exchanged with SEVE severely lost the affinity, and exchange
of YLGE to DVVS had minor loss in affinity, whereas no effect in
another exchange mutant (chimeras 20, 19, and 21, respectively). These results suggest that the affinity reduction conferred by the exchange of the loop-1 region in chimera 11 was mainly caused by the replacement of ESTN on VEGF-ENZ-7 to SEVE in PlGF. In the loop-1 of
VEGF-ENZ-7, accumulation of negatively charged residues
might have disrupted the binding action of VEGF-ENZ-7. In
addition, regions upstream from loop-1 (chimera 22 and 23) and a region
downstream of loop-3 (number 26) were replaceable to the corresponding
regions of PlGF, however, short stretches upstream from loop-3 were not
(numbers 24 and 25).
VEGF-ENZ-7 have its specific insertion-like stretch with 8 amino acid residues in the loop-3 region (Fig. 1A). This
stretch was not found in other VEGF-E members or in the VEGF family.
Interestingly, the flanking region of this stretch showed similarities
in amino acid sequence to other VEGF-E genes. This
stretch was deleted to test its significance for the
VEGF-ENZ-7 protein. The deletion resulted in a complete
loss of biological activities, indicating that this short stretch is
indispensable for VEGF-ENZ-7 (Fig. 4B, chimera
27).
Cooperation between a Proper Set of Large Loop-1 and Large Loop-3
Is Crucial for Restoration of Biological Activity in
VEGF-ENZ-7--
To examine the regions in
VEGF-ENZ-7 necessary and sufficient for the activation of
KDR/VEGFR-2, we further constructed chimeric molecules between
VEGF-ENZ-7 and PlGF. Under the background of the PlGF
sequence, the loop-1-containing sequence and the loop-3-containing sequence in VEGF-ENZ-7 were introduced to the corresponding
regions (Fig. 5A, 31-33). For a control
experiment, under the background of VEGF-ENZ-7, the loop-1
and -3 regions in VEGF-A were replaced to the corresponding regions
(Fig. 5A, 28-30). As shown in chimera 33, both
loop-1 and -3-containing regions were found to be required for the
activation of VEGFR-2 kinase.
Although VEGF-ENZ-7 and VEGF-A are able to stimulate
VEGFR-2, to our surprise, a single replacement of either loop-1 or -3 in VEGF-A to the corresponding region in VEGF-ENZ-7
strongly suppressed the biological activity (Fig. 5, chimeras
28 and 29). Both loop-1 and -3 of VEGF-A are
required for a partial recovery of the activation of VEGFR-2 (chimera
30). Taken together, these results strongly suggest that an appropriate
pair of loop-1 and -3 is essential for the construction of
three-dimensional structure for the binding and activation of
VEGFR-2.
The Binding Ability of Mutants to VEGFR-2 Correlates Well with the
Activity of Receptor Autophosphorylation Assay and HUVEC Proliferation
Assay--
These mutant proteins were tested for their ability to
induce autophosphorylation of VEGFR-2 using a cell line, NIH3T3-KDR (see "Materials and Methods") (Fig.
6, data not shown). The ability was
highly correlated with the affinity of mutant proteins to the receptor
(Figs. 2, 4, and 5). The mutants with high affinity, as the wild type
VEGF-ENZ-7 protein to receptor, demonstrated a strong
activity in induction of autophosphorylation of KDR/VEGFR-2, whereas
the mutants with no affinity did not in a concentration range up to 150 ng/ml in the final medium. These results indicate that the critical
regions of VEGF-ENZ-7 for binding to VEGFR-2 are also
important for inducing autophosphorylation of VEGFR-2.
VEGF-A-induced signal transduction for the proliferation of endothelial
cells is mainly mediated by VEGFR-2. To evaluate the relationship of
the abilities to induce autophosphorylation of VEGFR-2 and to induce a
proliferation of endothelial cells, mutant proteins were tested for
their activity to induce proliferation of HUVEC. As expected, mutant
chimeras 1, 2, 6, 9, 12, 13, 19, 21-23, 26, 30 (weak), and 33 with
high affinity for VEGFR-2 could lead the proliferation of HUVEC,
whereas mutants with low receptor affinity facilitated weak mitogenesis
(Figs. 2, 4, and 5). Those mutants with no affinity failed to show such
activity, with one exception, number 18. Mutant number 18 did not show
detectable affinity to VEGFR-2, but it partially induced mitogenesis of
endothelial cells. These results suggest that the affinity strength of
mutant proteins mostly correlates with their activity to induce
mitogenesis of HUVEC.
Evaluation of Mutants in Tubular Formation Assay--
VEGF-A is
known to stimulate the endothelial cells to form a tubule-like
structure in vitro and in vivo. The chimeric
mutants of VEGF-ENZ-7 were tested for their activity for
tubular formation in a recently developed co-culture system between
HUVEC and human diploid fibroblasts (see "Materials and Methods")
(Fig. 7). To decrease a background tube
formation in this system detectable without any exogenous angiogenic
factors, we added anti-VEGF-A neutralizing antibody into culture
medium.
At first, we demonstrated that VEGF-ENZ-7 could induce the
tubular formation in vitro (Fig. 7A). Next we
examined tubular formation by chimera mutants. Representative results
and quantitative analysis are shown in Fig. 7, B and
C. The strength of chimera proteins for tubular-forming
activity was correlated with the affinity to the receptor, and with the
abilities to autophosphorylate the receptor and induce the
proliferation of endothelial cells. Interestingly, the tubules induced
by VEGF-ENZ-7 were morphologically distinguished from that
of VEGF-A, where VEGF-ENZ-7-induced tubules were slightly
thicker. Chimera number 18, which showed only weak HUVEC proliferation
without the significant affinity to receptor and the receptor
autophosphorylation ability in vitro could induce the
tubular formation as effectively as VEGF-A and VEGF-ENZ-7. The morphology induced by number 18 was closely related to that of
tubules induced rather by VEGF-A than VEGF-ENZ-7. This
result suggests that this tubular formation system is more sensitive than other assays such as the binding assay, receptor
autophosphorylation assay, and HUVEC proliferation assay, so that the
activity of chimera 18 was detected. In conclusion, both loop-1
and -3-containing regions in VEGF-ENZ-7 were found to be
required for the binding and activation of VEGFR-2 in a cooperative
manner (Fig. 8).
In this study, the important regions of VEGF-ENZ-7 for
binding to VEGFR-2 were analyzed by using domain exchanging with PlGF and site-directed mutagenesis. PlGF does not bind to VEGFR-2 but only
to VEGFR-1 (12, 13, 28). VEGF-ENZ-7 and PlGF have a similar
amino acid composition, and they also completely conserve the critical
cysteine-knot motif that was composed of eight cysteine residues (see
Fig. 1B). Recently, the crystal structure study for PlGF
(29) revealed that PlGF actually conserves the tertially structure with
VEGF (30, 31) and PDGF (32). Thus, the VEGF-ENZ-7/PlGF chimera mutants were assumed to fold appropriately in a similar manner
and build up tertiary structure. We confirmed that these chimeric
mutants conserve the dimerization property in post-translational modification (Fig. 3).
In previous reports, the receptor interacting amino acids for PDGF-B
and VEGF-A were identified. For PDGF, site-directed mutagenesis revealed that Arg-27 and Ile-30 were critical amino acid residues in
interacting to PDGF receptor (33). These amino acid residues were found
in the loop-1 region of PDGF-B. The alanine scanning mutagenesis of
VEGF-A demonstrated important amino acid residues for binding to
receptors. For VEGFR-1 binding, negatively charged residues Asp-63,
Glu-64, and Glu-67 were critical, and they were found in the loop-2
region (27). For VEGFR-2 binding, mutation in positively charged
residues, Arg-82, Lys-84, and His-86 resulted in severe reduction in
receptor binding, and these basic amino acid residues are located in
the loop-3 region (27). Because these three basic amino acids are not
conserved and changed to hydrophobic ones in the VEGF-E family, we
suggest that these basic amino acids in VEGF-A are not directly
interacting with VEGFR-2 but are important for keeping the proper
tertially structure within the VEGF-A molecule.
In another report (31), supporting the previous result, critical
amino acids for binding VEGFR-2 were found clustered at We found that PlGF could replace the amino and carboxyl terminus of
VEGF-ENZ-7 without any changes in biochemical and
biological activities. This result indicates that the amino and
carboxyl termini in VEGF-ENZ-7 are not involved directly in
binding with VEGFR-2. As shown in mutants 3-5, 7, and 8 (Fig. 2),
extending the exchange region into the core region and over loop-1 and
-3 regions resulted in a significant defect in biological and
biochemical activities. These results suggest that the regions
containing loop-1 and -3 are critical for VEGF-ENZ-7 to be
functionally intact. As expected, exchanging the narrowed regions of
VEGF-ENZ-7 only containing loops-1 and -3 (mutant 10 and 11 for loop-1, 14-17 for loop-3) also produced functionally defective
proteins, whereas the loop-2 exchange did not reduce activity (mutants
12 and 13). Mutant proteins with low affinity to VEGFR-2 could not
induce autophosphorylation of VEGFR-2, proliferation of HUVEC, or
tubular formation by endothelial cells except for chimera 18. These
results suggest that the affinity strength to the receptor basically
reflect their effectiveness in biological response.
In addition to the replacement by PlGF, VEGF-A regions were also
introduced to VEGF-ENZ-7 in a loop-specific manner, as a reference (Figs. 4A and 5A). Surprisingly, these
mutants with VEGF-A loops also had significant reduction in biochemical
and biological activities. It could suggest that a single loop of VEGF-A is not enough to be biologically functional. A pair of loop-1
and -3 of VEGF-ENZ-7 or VEGF-A may be required to build up
the receptor-binding determinant for VEGFR-2. Mutants 10 and 14 did not
bind to VEGFR-2 and lost biological activity. Mutant 18, which is a
combination of numbers 10 and 14 with regions of loops-1 and -3 of
VEGF-A onto VEGF-ENZ-7, had a tubular formation activity,
whereas it did not show activities in other VEGFR-2-associated analysis
at detectable levels. Chimera 18 molecule might interact with VEGFR-2
better in a two-dimensional co-culture system that seems closer to the
in vivo situation, compared with the one-dimensional culture
of HUVEC.
As reported previously, regional exchange of the VEGF-A loop-3 region
with that of PlGF resulted with significant reduction in VEGFR-2
binding and proliferation of endothelial cells (36, 37). However, its
activity in inducing vascular permeability was still functional (37).
It was proposed that the determinant of VEGF-A to facilitate vascular
permeability could be different from that to bind to VEGFR-2 and induce
endothelial cell proliferation. It is also possible that the Miles
assay, an assay for activity to facilitate vascular permeability could
be very sensitive compared with other assays, so that activity of that
mutant was detectable. The construction pattern of two chimera proteins
in this study, numbers 15 and 17, are similar to such loop-3 exchanged
mutants of VEGF-A. Interestingly, these VEGF-ENZ-7 chimeric
proteins lost the activity to facilitate vascular
permeability,2 indicating
that the loop-3 region of VEGF-ENZ-7 was critical in this
particular biological activity. This result suggests that some of the
biochemical properties of VEGF-A and VEGF-E could be different,
especially in the aspect of the mechanisms for facilitating vascular permeability.
The chimera proteins that could be clinically applied as
angiogenic stimulators include numbers 1, 6, 9, and 33. In keeping with
the strong activities as wild type VEGF-ENZ-7, they have human PlGF amino acid residues at the amino and/or carboxyl termini instead of viral amino acid residues of VEGF-ENZ-7. Amino-
or carboxyl-terminal peptide sequences are generally highly
immunogenic. Therefore, these "humanized VEGF-E family" are
expected to be less immunogenic compared with the wild type
VEGF-ENZ-7 and clinically more useful.
The important region of VEGF-ENZ-7 for interacting with
VEGFR-2 was identified as loop-1 and -3. Loop-1 and -3 may be playing an important role in presenting the binding determinant of
VEGF-ENZ-7 for VEGFR-2. According to these results, a
heterodimer in which one molecule binds only VEGFR-2 but the other does
not bind either VEGFR-1 or VEGFR-2 could be designed and synthesized.
Such peptides may be very useful as a VEGFR-2 antagonist for clinical
application to inhibit the pathological angiogenesis in which VEGFR-2
plays the major role as a signal transducer.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-counter. All
experiments were performed in duplicate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the amino acid sequence for the
VEGF family and three-dimensional structure of the dimeric
molecule of VEGF-A. A, the amino acid
sequences of VEGF-E (VEGF-ENZ-7,
VEGF-ENZ-2/VEGF-ORFVNZ-2,
VEGF-ED1701) and human VEGF-A, -B, -C, and -D are shown.
The amino acid residues of VEGF-A critical for interacting with VEGFR-2
are shaded in orange. The numbers in
red and blue boxes represent the amino acid
residues of VEGF-A and VEGF-ENZ-7, respectively. The
numbers and thin lines in black
represent the regions of VEGF-ENZ-7 that were replaced with
corresponding amino acid residues of human PlGF and/or VEGF-A. The
double black line with number 27 indicates a unique amino
acid stretch only found in VEGF-ENZ-7. Red
character, the eight cysteine residues conserved in all VEGF
family members. Blue character, the amino acid residues
identical to VEGF-ENZ-7. Black line, the core
region that is conserved in all VEGF family. Red bar, loop
regions 1, 2, and 3 of VEGF-A. Green and blue
bars, the -helix regions and
-strands of VEGF-A,
respectively. Signal peptide sequences at the NH2-terminal
region of the VEGF family were omitted in this figure.
, no amino
acids. B, dimer structure of VEGF-A. One monomer of the
VEGF-A dimer is shown in red and another in
green. The VEGF-A molecule forms three intramolecular and
two intermolecular disulfide bonds. The disulfide bonds and cysteine
residues of a homodimer are shown in yellow in one monomer
and in red in another. The numbers in
black indicate the loop structure. The amino acid residues
of VEGF-A important for interacting VEGFR-2 are shaded in
red. This figure was adapted from Muller et al.
(30, 31).
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Fig. 2.
Amino- and carboxyl-terminal regions of the
VEGF-E are not essential for the biological activity.
A, a variety of constructs for the replacement of amino- and
carboxyl-terminal regions in VEGF-ENZ-7 (orange)
with the corresponding regions of PlGF (green).
a, VEGFR-2 (KDR) autophosphorylation was measured by using
NIH3T3-KDR cells. The cells were stimulated with chimera mutant
proteins (4-120 ng/ml), lysed, and then subjected to SDS-PAGE for
Western blotting with an anti-phosphotyrosine antibody
( -PY) or with an anti-VEGFR-2 antibody (
-KDR
IK-5). The activity was indicated by +. +++, equal to wild type
VEGF-ENZ-7; ++, 2-3-fold weaker than wild type
VEGF-ENZ-7; +, 3-10-fold weaker than wild type
VEGF-ENZ-7;
, phosphorylation was not detected in this
concentration range. b, proliferation of HUVEC. Quiescent
HUVEC were stimulated with 1, 10, and 100 ng/ml chimera mutant
proteins. After 4 days, cells were stained and the cell number was
determined by averaging the counting of five spots to wild type
VEGF-ENZ-7. ++, 2-fold weaker than wild type
VEGF-ENZ-7 at 100 ng/ml; +, 4-fold weaker than wild type
VEGF-ENZ-7;
, HUVEC proliferation was not detected.
B, binding activities of the chimeric proteins between
VEGF-ENZ-7 and PlGF to VEGFR-2. The activities were
measured by competition experiments using 125I-VEGF-A and
VEGFR-2 binding systems (see "Materials and Methods").
C, the activities of chimeric proteins for stimulation of
HUVEC growth.
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Fig. 3.
Dimer formation of all chimera proteins are
not disrupted. Representative chimera proteins (1-9)
are demonstrated for their dimer formation ability. Each mutant protein
is analyzed in reducing and non-reducing conditions by Western
blotting. Note that the size of all mutant proteins are doubled in the
non-reducing condition compared with that in reducing condition.
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Fig. 4.
Functional analysis of
VEGF-ENZ-7/PlGF chimera mutant proteins, 10-27.
A, 10-18; and B, 19-27,
the construct maps of chimera mutants and their activities for VEGFR-2
autophosphorylation and HUVEC growth (see Fig. 2, legend).
**, VEGFR-2 phosphorylation by mutant 18 was detected at a higher
concentration of chimera protein (500 ng/ml). The box
presents the amino acid sequences of VEGF-ENZ-7
(orange), PlGF (green), and VEGF-A
(blue) that are actually exchanged.
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Fig. 5.
Functional analysis of
VEGF-ENZ-7/PlGF chimera mutant proteins, 28-33.
A, the construct map of chimera mutants and their activities
for VEGFR-2 autophosphorylation and HUVEC growth (see Fig. 2,
legend). B, binding activities of the chimeric
proteins to VEGFR-2. C, the activities of chimeric proteins
for stimulation of HUVEC growth.
-strands
that flank the loop site. Therefore, the basic architecture of these
chimeric proteins would not be disrupted.
1 and
2 strands), 12 and 13 on
loop-2 (Gly-78 to Ile-87 between
3 and
4), 14 and 15 on the loop-3 containing region (Val-98 to Ser-123 between
5 and
8), and
16 and 17 on the loop-3 short region (Val-105 to Asn-118 between
6
and
7) with the corresponding regions of VEGF-A or PlGF (Fig. 1A).
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Fig. 6.
Analysis of VEGFR-2 autophosphorylation by
chimera mutant proteins. NIH3T3-KDR cells were stimulated with
chimera mutant proteins at variable concentrations. Representative
results of VEGFR-2 phosphorylation are shown. Total cell lysate
(A) or anti-VEGFR-2 immunoprecipitates (B) were
subjected to SDS-PAGE and Western blotting with anti-phosphotyrosine
( -PY) and anti-VEGFR-2 (
-KDR)
(arrows). The autophosphorylation of VEGFR-2 by mutants 1, 2, 9, 11, 12, and 13 are representatively shown. B,
autophosphorylation of VEGFR-2 by chimera mutants were demonstrated by
anti-VEGFR-2 immunoprecipitation. 9 and 13 could
induce VEGFR-2 autophosphorylation as effective as wild type
VEGF-ENZ-7.
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Fig. 7.
Analysis of tubular formation by chimera
mutant proteins. VEGF-E chimera proteins were tested for
stimulatory activity of endothelial tubular formation using the
HUVEC-fibroblast co-culture system (see "Materials and Methods").
At days 1, 4, 7, and 9, medium was replaced with fresh medium that
contains angiogenic factor and neutralizing antibody for VEGF-A (500 ng/ml). At day 11, incubation was terminated, fixed, and immunostained
with -CD31/PECAM-1 antibody for endothelial cells
(arrows). The co-cultured fibroblasts were not stained but
slightly giving rise to the yellow background. A,
VEGF-ENZ-7 (50 ng/ml) was tested for tubular formation
activity. a, the supplied medium induced tubular formation.
b, tubular formation was completely inhibited by an excess
amount of neutralizing antibody (500 ng/ml) for human VEGF-A.
c and d shows tubular formation by
VEGF-ENZ-7 without and with an excess amount of
neutralizing antibody for human VEGF-A, respectively. It indicates that
VEGFR-2 activation only by VEGF-ENZ-7 is sufficient for
tubular formation. B, chimera mutants (50 ng/ml) were tested
for tubular formation. The number in each panel indicates
the serial number of chimera mutants. 9, 12, and
18 show the tubular formation activity as effective as wild
type VEGF-ENZ-7. C, quantitative analysis of
tubular formation with mutant VEGF-ENZ-7 (see "Materials
and Methods"). Control, VEGF-A (50 ng/ml); Ab,
anti-VEGF-A antibody (500 ng/ml); E, VEGF-ENZ-7
(50 ng/ml); number, each chimera mutant
VEGF-ENZ-7 (50 ng/ml).
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Fig. 8.
Schematic representation of exchangeable
regions and non-exchangeable regions for VEGF-ENZ-7 to
interact with VEGFR-2. Exchangeable regions are shown in
red, unexchangeable regions obtained from the chimera study
in blue, and the regions conserved in VEGF-ENZ-7
and PlGF in green. Regions exchangeable with PlGF in a
detailed chimeric analysis (Fig. 4B) are shown in weak
red color. Exchangeable regions include the NH2
terminus, COOH terminus, loop-2, -helix, and part of loop-1 regions.
Non-exchangeable regions include the middle part of loop-1 and the
broad region associated with loop-3. By introducing these
"non-exchangeable regions" of PlGF to VEGF-ENZ-7
significantly reduces activities for receptor interaction and
endothelial proliferation. Note that the conserved regions were not
mutated. These results suggest that loop-1 and -3 play the important
roles in providing the binding determinant for VEGFR-2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helix,
loop-1, -2, and -3 regions. They were designated "hot spots." The
majority of these hot spot amino acid residues are located in loop-1
and -3 regions. In addition, VEGF-A neutralizing antibody and peptides,
which inhibit the interaction of VEGF-A to VEGFR-2, have been reported.
Interestingly, the epitopes for these inhibitors are located at loop-1
and -3, thus, they might modify the structure of loop-1 and/or loop-3
regions (34, 35).
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FOOTNOTES |
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* This work was supported by Special Project Research on Cancer-Bioscience Grant-in-aid 12215024 from the Ministry of Education, Culture, Sports, Science and Technology in Japan and the program "Research for the Future" of Japan Society for Promotion of Science.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. Tel.: 81-3-5449-5550;
Fax: 81-3-5449-5425; E-mail: shibuya@ims.u-tokyo.ac.jp.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M210931200
2 A. Kiba and M. Shibuya, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; HUVEC, human umbilical vein endothelial cells; PlGF, placenta growth factor; PDGF, platelet-derived growth factor.
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