From the Institute of Molecular Medicine, Tumor Biology Center, D-79106 Freiburg, Germany
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ABSTRACT |
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Vascular endothelial growth factor (VEGF) is an
endothelial cell-specific mitogen and a key mediator of aberrant
endothelial cell proliferation and vascular permeability in a variety
of human pathological situations such as tumor angiogenesis, diabetic
retinopathy, or psoriasis. By amino-terminal deletion analysis and by
site-directed mutagenesis we have identified a new domain within the
amino-terminal -helix that is essential for dimerization of VEGF.
VEGF121 variants containing amino acids 8 to 121 or
14 to 121, respectively, either expressed in Escherichia
coli and refolded in vitro, or expressed in Chinese
hamster ovary cells, were in a dimeric conformation and showed full
binding activity to VEGF receptors and stimulation of endothelial cell
proliferation as compared with wild-type VEGF. In contrast, a
VEGF121 variant covering amino acids 18 to 121, as well as
a variant in which the hydrophobic amino acids Val14,
Val15, Phe17, and Met18 within the
amphipathic
-helix near the amino terminus were replaced by serine,
failed to form biological active VEGF dimers. From these data we
conclude that a domain between amino acids His12 and
Asp19 within the amino-terminal
-helix is essential for
formation of VEGF dimers, and we propose hydrophobic interactions
between VEGF monomers to stabilize or favor dimerization.
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INTRODUCTION |
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Vascular endothelial growth factor (VEGF),1 also known as vascular permeability factor, is a mitogen that specifically regulates endothelial cell function. The biological activities of VEGF (for recent reviews, see Refs. 1-4) include stimulation of endothelial cell growth and migration, rapid enhancement of microvascular permeability in vivo, promotion of vasculogenesis and angiogenesis, and induction of differentiation of embryonic stem cells to hematopoietic precursors (5). Recent experiments of targeted disruption of the VEGF gene have demonstrated its essential role for vascular development in the embryo (6, 7). Even inactivation of a single VEGF allele results in defective development of large vessels, defective capillary sprouting, and embryonic lethality. Aberrant elevated expression of VEGF has been observed in a variety of human pathological situations such as tumor angiogenesis (8), diabetic retinopathy (9), rheumatoid arthritis (10), or psoriasis (11). Neutralizing of VEGF by antibodies or recombinant soluble receptor domains have shown therapeutic potential as agents capable of suppressing tumor growth (12) and retinal neovascularization (13). Two homologous cell-surface receptors of the tyrosine kinase family, Flt-1 (VEGFR-1) and KDR (VEGFR-2), bind VEGF with high affinity (14, 15).
VEGF is a homodimeric glycosylated protein that exists in five different isoforms of 121, 145, 165, 189, and 206 amino acids of which the amino-terminal 114 amino acids are identical. Together with placenta growth factor (PlGF) (16) and the recently described VEGF-B (17) and VEGF-C (18) VEGF builds a family of related growth factors which show structural homology to PDGF. In particular, the cysteines building up the structural fold of the proteins consisting of three intramolecular disulfide brigdes, and two intermolecular disulfide brigdes cross-linking the polypeptide chains, are conserved for these growth factors (19, 20). The very recently solved crystal structure of VEGF (21) confirmed the overall structural similarity of VEGF and PDGF. Alignment of VEGF121 and PDGF-B/v-sis amino acid sequences showed a 25% identity of the region Cys26 to Cys104 of VEGF and Cys16 to Cys99 of mature PDGF-B. This region of PDGF-B/v-sis, the minimal v-sis transforming domain, was described to retain biological activity of the growth factor (22, 23). In case of VEGF, it had previously been shown that a plasmin digested 110-amino acid amino-terminal fragment, which was cleaved between Arg110 and Ala111, retains full biological activity as compared with the VEGF121 isoform (24).
To analyze the contribution of amino acids Ala1 to Tyr25, which are located amino-terminal with respect to the homology region of VEGF to the minimal v-sis transforming domain, to structure and/or biological activity of VEGF, we generated amino-terminal truncated VEGF121 variants. Here were show that an amino-terminal domain between amino acids His12 and Asp19 is essential for in vitro dimerization of VEGF and for functional expression of VEGF in vivo. As the conversion of hydrophobic amino acids within this domain to serine impairs formation of VEGF dimers we propose hydrophobic interactions between VEGF monomers to stabilize or favor formation of dimers.
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EXPERIMENTAL PROCEDURES |
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Construction and Bacterial Expression of VEGF Mutant Proteins-- cDNAs encoding amino-terminal truncated VEGF proteins were generated by PCR technique using the 5'-primers VEGF-8 (5'-GAGCCATGGAGAATCATCACGAAGTGGTGAAG-3'), VEGF-14 (5'-CACGCCATGGTGAAGTTCATGGATGTCT-3'), and VEGF-18 (5'-AAGTCCATGGATGTCTATCAGCGCAGC-3'), respectively, the 3'-primer VEGF-Bam (5'-AGTGGATCCCCCCCTGGTGA-3'), and cloned human VEGF121 cDNA (25) as template. Mutant VEGF-V14S/V15S/F17S/M18S was generated by PCR using 5'-primer 207 (5'-TCGGGCCTCCGAAACCATGA-3') and 3'-primer VEGF-N1 (5'-GGCTCCTGAAGCTGCTAAGCACTACTAAGACGGGAGG-3') for amplification of the 5'-fragment and 5'-primer VEGF-N2 (5'-AATCGTCGAAGTCCTCGGATGTCTATCAGCGCAGCTA-3') and 3'-primer VEGF-Bam for amplification of the 3'-fragment. The fragments were purified by agarose gel electrophoresis and used in an equimolar ratio as template for fusion-PCR using 5'-primer 207 and 3'-primer VEGF-Bam. VEGF variant C61S was generated by amplification of the VEGF121 cDNA cloned in pBluescript vector using the 5'-primer C61S (5'-GGCTGCTCCAATGACGAGGGC-3') and the 3'-primer 252c (5'-CCCGCATCGCATCAGGGGCAC-3'). The resulting PCR fragment containing mutant VEGF sequence and vector sequence was phosphorylated by T4 polynucelotide kinase (Pharmacia, Freiburg, Germany), gel purified, religated, and transformed into Escherichia coli XL1-blue. The mutant VEGF cDNAs were cloned into the His-pET vector (26) via NcoI and BamHI sites, and transformed into E. coli BL21DE3 (27). All constructs were verified by DNA sequencing. Solubilization of VEGF proteins from inclusion bodies, refolding, and purification was performed essentially as described for wild-type VEGF121 (26).
Transfection Analysis--
For the construction of eukaryotic
expression plasmids NcoI/BamHI fragments encoding
mutant VEGF variants were fused to the VEGF signal sequence by
substitution of the NcoI/BamHI restriction fragment of wild-type VEGF121 in a plasmid, which contains
the entire VEGF121 coding region cloned into the
SmaI site of pBluescript (25). The cDNA fragments were
released by EcoRI/XbaI restriction endonuclease
digestion and ligated into pCI-neo expression vector (Promega,
Heidelberg, Germany) which provides a cytomegalovirus promoter and
enhancer. Transient transfection of chinese hamster ovary (CHO) cells
was performed in six-well plates containing approximately 2 × 105 cells/well, which were incubated at 37 °C overnight
in the presence of 2 µg/well of calcium phosphate-precipitated
VEGF-variant/pCI-neo DNA. For determination of transfection efficiency
and protein secretion 1 µg/well pSBC-2/SEAP expression vector DNA
(28) encoding secreted placental alkaline phosphatase (SEAP) was
cotransfected. Cell culture supernatant was replaced with serum-free
medium, and cells were incubated for 48 h at 37 °C. Conditioned
media (2 ml) were harvested, centrifuged, and stored at 80 °C.
Total RNA was prepared from transfected cells using an RNeasy kit
(Quiagen, Hilden, Germany). Relative SEAP activity was determined as
optical density at 405 nm of heat-inactivated (5 min at 65 °C)
aliquots (100 µl) of conditioned media, which were incubated for
30-60 min at room temperature with 100 µl of SEAP-buffer (1 M diethanolamine, 10 mM homoarginine, 1.5 mM MgCl2, 23 mM
p-nitrophenyl phosphate). Aliquots (30 µl) of conditioned
media were electrophoresed on 15% SDS-polyacrylamide gels under
nonreducing conditions, electrotransferred to nitrocellulose Hybond-N
membranes (Amersham, Braunschweig, Germany), probed with the polyclonal
antiserum K7.16 (29) raised against human VEGF, and detected using the
ECL detection system (Amersham, Braunschweig, Germany).
Semiquantitative reverse-transcriptase PCR for determination of VEGF
RNA in transfected cells was performed by converting 1 µg of total
RNA to cDNA using a first-strand cDNA synthesis kit (Pharmacia,
Freiburg, Germany) with random hexanucleotide primers followed by PCR
amplification of a 247-base pair VEGF cDNA fragment and a 397-base
pair GAPDH cDNA fragment simultaneously using the primers VEGF-E3
(5'-GGTGGACATCTTCCAGGAGTACCC-3'), VEGF-E5R (5'-TTCTTGTCTTGCTCTATCTTTCTTTG-3'), GAPDH1
(5'-AGCGAGACCCCACTAACATCAAA-3'), and GAPDH2
(5'-GTGGATGCAGGGATGATGTTCTG-3').
Binding Assays-- Recombinant extracellular domain of human VEGF receptor Flt-1 (30) and KDR,2 respectively, were coated onto Maxisorb plates (1 µg/well) and were incubated with biotinylated VEGF165 (10 ng/ml) in the presence of increased concentrations of VEGF121 proteins as described previously (30).
Thymidine Incorporation Assays-- Quiescent HUVE cells were stimulated with increased concentrations of VEGF121 proteins. After 18 h of VEGF-incubation [3H]thymidine (0.5 µCi) was added and the incubation was continued for additional 6 h. The cells were washed and the incorporation of radioactivity was determined by scintillation counting.
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RESULTS |
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Characterization of Amino-terminal VEGF121 Variants
Expressed in E. coli--
Amino-terminal truncated VEGF121
variants were expressed in E. coli using the T7
RNA-polymerase driven pET system (27). The proteins were solubilized
from inclusion body material, refolded, and finally purified by
Ni2+-affinity chromatography essentially as described
previously for wild-type VEGF121 (26). The E. coli derived "wild-type" VEGF121 in fact covered
amino acids Met3 to Arg121 of mature human
VEGF121 and showed virtually identical biological activity
as compared with human VEGF121 covering amino acids 1 to
121 produced by recombinant techniques in Sf9 insect cells (26).
The VEGF variants VEGF1211-7 contained amino acids 8 to
121 of mature human VEGF121 with Gly8 changed
to Met, and Gln9 changed to Glu due to insertion of an
NcoI restriction endonuclease site,
VEGF121
1-13 contained amino acids 14-121 with
Val14 changed to Met, and VEGF121
1-17
contained amino acids 18-121 (Fig.
1A). For control,
VEGF121-C61S was prepared, a monomeric VEGF mutant with
Cys61 replaced by serine which did not bind to endothelial
cells and showed no significant biological activity (19). An additional amino-terminal His6 tag facilitated affinity purification
of the recombinant proteins. On nonreducing SDS gels
VEGF121, VEGF121
1-7, and
VEGF121
1-13 migrated with apparent molecular masses of
approximately 34, 32, and 30 kDa, respectively, which correspond to the
molecular mass of VEGF121 dimers (Fig. 1B,
lanes 1, 3, and 4). In contrast, VEGF121
1-17 (Fig. 1B, lane 5), as
well as the control, VEGF121-C61S (Fig. 1B,
lane 2), migrated as monomers of approximately 16 kDa. Upon
electrophoresis on reducing SDS gels all of the VEGF variants migrated
as monomeric forms of approximately 16-17 kDa (not shown).
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Amino-terminal Domain His12 to Asp19 Is
Essential for Functional Expression of VEGF in Vivo--
To analyze
whether the amino-terminal domain between His12 and
Asp19 is also essential for in vivo dimerization
of VEGF the variant VEGF121 cDNAs were fused to the
VEGF signal sequence, ligated into pCI-neo expression vector providing
cytomegalovirus promoter/enhancer sequences, and transiently
transfected into CHO cells. Due to fusion to the signal sequence the
resulting expression plasmid pCI-neo/VEGF121 encoded the
entire human VEGF121 coding region, whereas in construct
pCI-neo/VEGF1213-13 amino acids Met3 to
Glu13 had been deleted and Val14 was replaced
by Met, and in construct pCI-neo/VEGF121
3-18 amino acids Met3 to Phe17 had been deleted. Construct
pCI-neo/VEGF121-
/S encoded the entire VEGF121 coding sequence in which the hydrophobic amino
acids Val14, Val15, Phe17, and
Met18 had been replaced by serine. Immunoblot analysis of
conditioned media of the transfected cells using the polyclonal
antiserum K7.16 raised against human VEGF protein showed that
VEGF121 and VEGF121
3-13 migrated under
nonreducing conditions as dimers (Fig. 3A, lanes 1 and
2). In contrast, in conditioned media of
pCI-neo/VEGF121
3-18 and
pCI-neo/VEGF121-
/S, as well as in pCI-neo vector control transfected CHO cells, no VEGF protein was detectable by Western blot
analysis (Fig. 3A, lanes 3-5). Western blot
analysis of VEGF variants expressed in E. coli showed that
the antiserum used was able to recognize all of the various VEGF
variants including the monomeric ones (data not shown). Measurement of
SEAP activity in conditioned media of the transfected cells revealed
efficient transfection of the cells and efficient secretion of proteins even in transfections in which VEGF protein was not detectable in the
conditioned medium (Fig. 3B). Using lysates of transfected cells for Western blot analysis neither wild-type VEGF nor mutant VEGF
variants were detectable demonstrating that wild-type VEGF and
VEGF121
3-13 were secreted efficiently by the cells, and
that the VEGF variants VEGF121
3-18 and
VEGF121-
/S were not accumulated within the cells (not
shown). Expression of transfected constructs for the VEGF variants at
the level of mRNA was shown by semiquantitative reverse-transcriptase PCR (Fig. 3C). Transfection of human
"293" embryonic kidney cells gave similar results (not shown).
Taken together these results show that the amino-terminal
His12 to Asp19 domain is essential for
functional expression of VEGF at least in transfected CHO and 293 cells. Truncation or mutation of this domain either impairs synthesis
of VEGF or the cells recognize these variants as aberrant proteins
which were apparently degraded.
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Binding of VEGF Variants to VEGF Receptors and Stimulation of
Proliferation of HUVE Cells--
A PDGF-B variant in which the two
cysteines involved in interchain disulfide bonds had been converted to
serine, migrated as a monomer on non-reducing SDS gels, but exists as a
noncovalent dimer at pH 4-7 in solution and shows similar mitogenic
activity as compared with wild-type PDGF-BB (31, 32). To analyze the biological activity of the amino-terminal VEGF121 variants
receptor binding assays and proliferation assays were performed.
Binding of the VEGF variants expressed in E. coli to
recombinant extracellular domain of human VEGF receptors Flt-1 (Fig.
4A) and KDR (Fig.
4B), respectively, was studied by competition assays with
biotinylated VEGF165. The dimeric VEGF variants
VEGF1211-7 and VEGF121
1-13 competed
with biotinylated VEGF for binding of both of the VEGF receptors in an
almost undistinguishable manner as compared with wild-type
VEGF121. Binding of the monomeric variant
VEGF121
1-17, as well as the monomeric control
VEGF121-C61S, to the receptors was strongly impaired.
Growth of HUVE cells was stimulated by the VEGF variants
VEGF121
1-7 and VEGF121
1-13 in a dose
dependent manner which was almost undistinguishable from wild-type
VEGF121 stimulated growth of the cells. The monomeric
variants VEGF121
1-17 and VEGF121-C61S
failed to induce proliferation of HUVE cells (Fig. 4C).
Taken together these results show that truncation or mutation of the
VEGF amino-terminal
-helical domain prevents the formation of stable
VEGF dimers although the cysteines involved in formation of the core
structure of VEGF had not been affected. Dimerization of VEGF had
previously been shown to be a prerequisite for biological activity
(19).
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DISCUSSION |
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The analysis of amino-terminal truncated VEGF121
variants revealed a domain between amino acids His12 and
Asp19 that is essential for in vitro
dimerization of VEGF and for functional expression of VEGF in
vivo. This domain showed an interspersed sequence of charged
and hydrophobic amino acids which may anticipate an amphipathic
-helical conformation. We postulate that the interaction of
hydrophobic interfaces stabilize or favor dimerization of VEGF. Conversion of the hydrophobic amino acids to serine by site-directed mutagenesis, which results in VEGF monomers, supports this model. The
very recently by Muller et al. (21) solved crystal structure of VEGF indeed revealed that the amino acids near the amino terminus anticipate an
-helical conformation. The crystal structure shows that hydrophobic amino acids from this
-helix together with residues from helix
2, loop regions
1-
3, and
5-
6, and sheet
6 of the
other monomer form a small hydrophobic core that presumably stabilizes
the central structure of the VEGF dimer. In addition, they found that a
Phe17 to alanine mutant VEGF displayed on a phage surface
lost KDR receptor binding implicating a contribution of
Phe17 to receptor binding. As the conformation of the
Phe17
Ala mutant displayed on the phage surface was not
investigated, our results implicate that loss of KDR binding is more
likely due to impaired dimerization of the mutant VEGF.
The His12 to Asp19 domain is highly conserved
between human, sheep (34), porcine (35), bovine (36), and mouse (37)
VEGF (Fig. 5). Similar interspersed
sequences of charged and hydrophobic amino acids are located
amino-terminal with respect to the v-sis homology regions of
the VEGF-related growth factors PlGF (16), VEGF-B (17), and VEGF-C (18,
38). Heterodimerization of VEGF and PlGF has shown to occur in
vitro (39, 40) and in vivo (41), as well as
heterodimerization of VEGF and VEGF-B was reported for cells expressing
both growth factors (17). Heterodimerization of the members of the
family of VEGF-related growth factors is thought to contribute to a
fine tuning of angiogenic stimuli (41). Although the involvement of the
amino-terminal domain in the heterodimerization between various
VEGF-related growth factor monomers has not been investigated so far,
the similarity of the amino-terminal domains of the VEGF-related
proteins suggest that heterodimerization would be supported by these
domains. Heterodimerization of VEGF and PDGF-B has not been observed so
far although the eight cysteines building the core structure are
perfectly conserved. One of the most significant differences in the
crystal structure of VEGF (21) and PDGF-B (42) is the structure of the
amino terminus which is extended in PDGF rather than alpha-helical as in VEGF, whereas the monomer topology and side-by-side dimer
association is highly similar for both proteins. In the PDGF-BB dimer
the extended amino terminus of one chain makes contact to the other PDGF chain, but in contrast to VEGF, the amino terminus is not essential for dimerization of PDGF as it was shown by mutagenic analyses which resulted in the identification of the minimal
v-sis transforming domain (22, 23). Analysis of the crystal
structure of transforming growth factor (TGF)-2 (43),
which is besides PDGF, VEGF, and nerve growth factor, another member of
the cystine knot family of growth factors, revealed a close contact of
-helix H3 of one TGF-
2 chain to
-strand structures of the other chain in the TGF-
2
dimer, which presumably stabilizes the dimeric structure.
-Helix
H3 is located within TGF-
2 at a position
that correlates to loop/turn II in PDGF-B and VEGF.
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Interference with the VEGF/VEGF-receptor system is generally viewed as an attractive target for therapeutical intervention in a variety of human pathological situations which involve elevated VEGF expression and aberrant endothelial cell proliferation such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. Enhanced VEGF expression is induced by various factors and growth conditions including growth factors (see Finkenzeller et al. (44), and references therein), activated oncogenes (28, 45, 46), inactivated tumor suppressor genes (47, 48), and hypoxia (49). Diverse mechanisms acting at the level of promoter activation (44), mRNA stabilization (50, 51), and translational regulation (52) have been shown to be involved in up-regulation of VEGF expression. The multiplicity of factors and mechanisms involved in VEGF expression hamper the development of therapeutical approaches directed to reduce enhanced expression of the growth factor in pathological settings. As VEGF dimerization is an event that is necessary for biological activity of the growth factor, but is independent from the diverse mechanisms of regulation of gene expression and translation, prevention of dimerization by interference with the amino-terminal domain may be a promising strategy for therapeutical down-regulation of VEGF expression.
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ACKNOWLEDGEMENTS |
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We thank Steffi Koidl, Gabi Bader, and Katja Mohrs for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Kirstins Weg.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: Institute of Molecular
Medicine, Tumor Biology Center, Breisacher Str. 117, D-79106 Freiburg,
Germany. Tel.: 49-761-206-1711; Fax: 49-761-206-1705.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; CHO, chinese hamster ovary; Flt-1, fms-like tyrosine kinase-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HUVE, human umbilical vein endothelial; KDR, kinase domain receptor; PCR, polymerase chain reaction; PDGF, platelet-derived growth factor; PlGF, placenta growth factor; SEAP, secreted placental alkaline phosphatase; TGF, transforming growth factor.
2 G. Martiny-Baron, B. Barleon, F. Totzke, D. Marmé, and G. Siemeister, unpublished result.
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REFERENCES |
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