Originally published In Press as doi:10.1074/jbc.M204107200 on July 31, 2002
J. Biol. Chem., Vol. 277, Issue 43, 40335-40341, October 25, 2002
Single-chain Vascular Endothelial Growth Factor Variant with
Antagonist Activity*
Thomas P.
Boesen
§,
Bobby
Soni,
Thue W.
Schwartz§, and
Torben
Halkier¶
From Maxygen ApS, Agern Alle 1, DK-2970 Hørsholm,
Denmark and § Laboratory for Molecular Pharmacology,
Department of Pharmacology, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark
Received for publication, April 29, 2002, and in revised form, July 23, 2002
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ABSTRACT |
Vascular endothelial growth factor is a specific
endothelial cell mitogen that is essential for the formation of
the vascular system but in the adult individual is
involved in several pathological conditions, including
cancer. It is a homodimeric protein that activates its receptor by
binding two receptor molecules and inducing dimerization. By mixing two
vascular endothelial growth factor monomers, each with different
substitutions, heterodimers with only one active receptor binding site
have previously been prepared. These heterodimers bind the receptor
molecule but are unable to induce dimerization and activation. However,
preparation of heterodimers is cumbersome, involving separate
expression of different monomers, refolding the mixture, and separating
heterodimers from homodimers. Here we show that a fully functional
ligand can efficiently be expressed as a single protein chain
containing two monomers. Single-chain vascular endothelial growth
factor is functionally equivalent to the wild-type protein. By
monomer-specific mutagenesis, one receptor binding site was altered.
This variant competitively and specifically antagonizes the mitogenic
effect of the wild-type protein on endothelial cells. The
results obtained with the single-chain antagonist show the feasibility
of the single-chain approach in directing alterations to single
specific regions in natural homodimeric proteins that would be
impossible to target in other ways.
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INTRODUCTION |
Angiogenesis, the sprouting of new blood vessels from existing
ones, is essential for the development of an organism. This is clearly
demonstrated by the early embryonic death of transgenic animals in
which angiogenesis is not regulated correctly (1-5). However,
beneficial angiogenesis is a rare event in the adult individual,
occurring only under special circumstances such as corpus luteum
formation and wound healing (6, 7). In contrast, several pathologies
are caused or promoted by excessive angiogenesis, including tumor
growth, rheumatoid arthritis, diabetic retinopathy, and age-related
macular degeneration (7). Therefore, angiogenesis has become a popular
target for drug development, and several antiangiogenic compounds based
on very different mechanisms of action are being developed (8).
Angiogenesis is regulated by a large number of both pro- and
antiangiogenic cytokines and growth factors. One of these, vascular endothelial growth factor
(VEGF),1 is highly specific
for endothelial cells, and it has been shown to be one of the key
inducers of angiogenesis (9). Many types of tumors produce VEGF to
create the vascular network necessary to support tumor growth, and
suppression of VEGF activity has been found to suppress tumor growth in
animal models (7).
VEGF is a dimeric protein that exists in several isoforms. The core
domain of VEGF that is involved in receptor binding and activation is
~110 amino acid residues/monomer (10). Two other domains of 24 and 44 amino acid residues are involved in binding of VEGF to the
extracellular matrix and to cell surface heparin (11, 12). Differential
RNA splicing leads to the formation of the isoforms VEGF121
(consisting of only the core domain) and VEGF165
(consisting of the core domain and the C-terminal heparin binding
domain) (12). Less common splice variants are VEGF145 and
VEGF189 that contain the 24-amino acid residue
extracellular matrix binding domain located C-terminal to the core
domain but N-terminal to the heparin binding domain (11, 12). Plasmin cleavage of VEGF leads to formation of VEGF110, which
contains only the core domain (10). VEGF165 has been found
to be a more potent inducer of angiogenesis than VEGF121,
possibly because binding of the molecule to cell surface heparin
presents the molecule to the receptor in a favorable way (10).
VEGF binds to and activates two tyrosine kinase receptors, kinase
insert domain receptor (KDR) and fms-like tyrosine kinase receptor 1 (13). Most of the functions of VEGF are found to be mediated
by KDR (14-16), and fms-like tyrosine kinase receptor 1 functions mainly as a decoy receptor, suppressing VEGF activity by
capturing VEGF and thereby making it unavailable to KDR (15, 17-19).
Activation of the VEGF receptors requires dimerization through binding
of one receptor molecule to each of the two receptor binding sites on
the VEGF dimer, located at the opposite ends of the VEGF dimer at the
interface between the monomers (13).
A number of reports describe investigation of VEGF by mutational
analysis (10, 14, 20-24), and a few describe heterodimeric VEGF
variants in which one but not the other receptor binding site has been
altered (21, 22). The three-dimensional structure of VEGF has been
determined by both NMR spectroscopy and x-ray crystallography (23,
25-29). Hence, extensive knowledge of VEGF structure-function
relationships is available, and it has been demonstrated that a VEGF
variant with only one functional receptor binding site is able to
antagonize the function of wild-type VEGF by binding to the receptor
without activating it (21, 22). However, due to the homodimeric nature
of VEGF, obtaining VEGF variants that have specifically been altered in
one but not the other receptor binding site is cumbersome, involving
the expression and purification of two separate protein chains and
refolding the two chains together, followed by a difficult isolation of correctly folded heterodimer (21, 22). This is not a procedure it is
possible to use in combination with medium- or high-throughput screening. Moreover, it is not well suited for larger-scale
manufacturing of heterodimeric VEGF variants. Expressing two VEGF
monomers as a single protein chain could be a solution to the problems
associated with creation of heterodimeric VEGF and heteromers of other
multimeric proteins, thereby allowing preparation of antagonists of a
number of receptor ligands (Fig. 1A). The present work
describes the construction and characterization of a single-chain VEGF
molecule that acts as a VEGF antagonist. Even though this protein is
not ideal, it represents an important step forward, and it shows the validity of the single-chain approach.
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EXPERIMENTAL PROCEDURES |
Materials--
Primary human umbilical vein endothelial cells
(HUVECs) from PromoCell were maintained in Endothelial Cell Growth
Medium from PromoCell for two to five passages before being used in
proliferation assays. WST-1 cell proliferation reagent for HUVEC
proliferation assay and Pfx polymerase for PCR were
from Roche Molecular Biochemicals. Other cell cultures reagents were
from Invitrogen. Human VEGF121, VEGF165, bFGF,
and antibodies against these proteins for use in Western blotting and
KDR/Fc chimera were from R&D Systems. The Pichia pastoris
protein expression system, including vectors, P. pastoris
strains, and media components, was from Invitrogen, as were precast
gels for SDS-PAGE. ToyoPerl SP550C cation-exchange resin was from Tosoh
BioSep. Resource S and NAP5 columns were from Amersham Biosciences. The
Jupiter C18 column was from Phenomenex. DNA
oligonucleotides used for gene synthesis, cloning, and mutagenesis were
from TAG Copenhagen or DNA Technology. Modified trypsin was from Promega.
Gene Synthesis, Cloning, and Mutagenesis--
Genes were
synthesized by mixing equimolar amounts of alternating sense and
antisense 70-base oligonucleotides covering the DNA sequence to be
synthesized with 20-bp overlaps and running 35 cycles of PCR using
Pfx polymerase and varying concentrations of the
oligonucleotide mixture. Different volumes of the primary PCR were used
as template in a secondary, 35-cycle PCR using end primers. The
resulting PCR reactions were run on agarose gels, and the reaction with
the strongest band of the right size and the least background was
selected. The correctly sized band was excised from the gel and cloned
into the pPICZ
A vector using the BamHI/XbaI
restriction sites, and several clones were sequenced to find one
without errors introduced during synthesis.
Subsequent mutagenesis was done by PCR using DNA oligonucleotides
containing the mutations of interest.
Protein Expression in P. pastoris--
Plasmid encoding the
protein of interest was transformed into the P. pastoris
X-33 strain. Expression was induced by growing the cells in buffered,
rich medium with methanol as the sole carbon source, as described in
the Invitrogen protocols. Supernatants were harvested by
centrifugation, and protein expression was checked by Western blot.
Protein Purification--
P. pastoris culture
supernatants (up to 500 ml) containing the protein of interest were
adjusted to pH 4.4 using acetic acid and diluted to a conductivity of
less than 15 millisiemens/cm in 100 mM sodium acetate, pH
4.4. The sample was applied to a 10 ml ToyoPerl SP550C cation-exchange
column, and bound protein was eluted in fractions with a linear
gradient from 0 to 2 M NaCl in 100 mM sodium
acetate, pH 4.4, using either an Äkta Basic or Äkta FPLC
chromatography system from Amersham Biosciences. Fractions
containing VEGF or VEGF variants were identified by Western blotting
and pooled. For initial screening, the pooled fractions were
concentrated, and buffer was changed to phosphate-buffered saline using VivaSpin concentration columns. For detailed
characterization of selected variants and wild-type VEGF, the pooled
fractions were diluted to a conductivity of less than 15 millisiemens/cm and applied to a Resource S cation-exchange column. The
bound protein was eluted in fractions with a linear gradient from 0 to
1 M NaCl in 100 mM sodium acetate, pH 4.4, using an Äkta Basic chromatography system. Fractions containing
VEGF or VEGF variants were identified by SDS-PAGE, pooled,
and concentrated, and buffer was changed to phosphate-buffered saline
using VivaSpin concentration columns. Protein preparations were stored
in aliquots at 
20 °C.
Amino Acid Sequence Analysis--
N-terminal amino acid sequence
analyses of protein samples were carried out following SDS-PAGE and
electroblotting onto polyvinylidene difluoride membranes using an
Applied Biosystems 494 Protein Sequencer equipped with a blot cartridge
and operated according to the manufacturer's instructions. N-terminal
amino acid sequence analyses of peptides were carried out on glass
fiber filters pretreated with Polybrene in the same protein sequencer
using a normal cartridge. All cysteine residues indicated were
positively identified as S-carboxamido-cysteines.
MALDI-TOF Mass Spectrometry--
MALDI-TOF mass spectrometry was
done using an Applied Biosystems Voyager DE-PRO mass spectrometer
operated in linear mode for protein and in reflector mode for peptides.
Sample preparation for MALDI-TOF mass spectrometry consisted of mixing
1 µl of sample with 1 µl of matrix solution. The matrix solution
was a saturated solution of -cyano-4-hydroxy cinnamic acid in
49.95% H2O:49.95% acetonitrile:0.1% trifluoroacetic
acid. Mass spectra were calibrated externally with proteins and
peptides of known masses.
Amino Acid Analysis--
Lyophilized protein samples were
hydrolyzed for 16 h at 110 °C in 6 M HCl containing
1% phenol in N2-blanketed glass vials before
quantification of the liberated amino acids by amino acid analysis
using the AccQTag system from Waters.
Enzymatic Degradation and Peptide Purification--
Before
degradation with modified trypsin, 460 µg of single-chain VEGF
variant 1:E64R/2:I46R was denatured, reduced, alkylated, and desalted.
A lyophilized sample was denatured in 400 µl of 6 M
guanidinium-HCl, 0.3 M Tris-HCl, pH 8.3, and incubated
overnight at 37 °C before the addition of 50 µl of 0.2 M dithiothreitol in the same buffer. After a 2-h incubation
at ambient temperature to reduce the disulfide bonds, 50 µl of 1.0 M iodoacetamide in denaturation buffer was added, and the
free thiol groups were alkylated for 30 min at ambient temperature.
Finally, the sample was buffer-changed into freshly made 50 mM NH4HCO3, 0.4 M urea, pH 8.3, on a NAP5 column. Enzymatic degradations of reduced and alkylated single-chain VEGF variant 1:E64R/2:I46R with modified trypsin
were carried out for 16 h at 37 °C in a thermomixer using 2.3 µg of enzyme. The peptides generated by the enzymatic degradation were separated using a Jupiter C18 column (2 × 50 mm)
and eluted with a linear gradient of acetonitrile in 0.1% aqueous
trifluoroacetic acid. Collected fractions were analyzed using MALDI-TOF
mass spectrometry and amino acid sequence analysis.
Endothelial Cell Proliferation Assay--
HUVECs were seeded at
a density of 4,000-5,000 cells/well in 96-well plates in 100 µl of
Dulbecco's modified Eagle's medium/F-12 containing 0.5% fetal bovine
serum and varying concentrations of VEGF121,
VEGF165, bFGF, and/or single-chain variants. The cells were
incubated for 2 days at 37 °C in an incubator with 5%
CO2. To quantify viable cells, 10 µl of WST-1 cell
proliferation reagent was added to each well, the plates were incubated
at 37 °C for 1-1.5 h, and then A450 was read
in an enzyme-linked immunosorbent assay reader. Relevant standard
curves of wild-type growth factors (VEGF121,
VEGF165, or bFGF) were included on each plate. For
antagonist assays, a background stimulation with 1 nM of
either VEGF121, VEGF165 or bFGF was included.
All curves displayed are representative of at least three independent experiments.
BiaCore Receptor Binding Assay--
The interaction of
single-chain VEGF proteins and a VEGF-Fc receptor dimer (R&D Systems)
was analyzed using a BIAcore 3000 analyzer (BIAcore). VEGF-Fc was
coupled to a BIAcore CM5 chip, and equivalent molar amounts (133 nM) of a VEGF single-chain wild-type and an antagonist
mutant were injected over the receptor. Sensograms of association (in
the presence of ligand) and dissociation (subsequently, in the absence
of ligand) were recorded.
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RESULTS |
Creating a Single-chain Form of VEGF121--
A
synthetic single-chain gene consisting of coding sequences for two
VEGF121 monomers connected by a 14-residue (42-bp; amino acid sequence, GSTSGSGKSSEGKG) linker was prepared as described under
"Experimental Procedures" (Fig.
1B). The codon usage was optimized for expression in yeast systems (such as Saccharomyces cerevisiae or P. pastoris) while maximizing the
difference in codon usage between the two monomers to reduce the risk
of recombination events between the two monomers in vivo and
facilitate monomer-specific PCR on the plasmid constructs. The
synthesized gene was cloned into the pPICZA P. pastoris
expression vector after the KEX2 signal cleavage site, and several
clones were sequenced to find one with no errors in the
sequence.
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Fig. 1.
Single-chain antagonist concept.
A shows the basic idea behind the single-chain concept. When
the dimeric VEGF variant is formed from separate monomers, alterations
of the molecule (represented by the white dot) will affect
both of the receptor binding sites (located at the left and
right poles of the dimer, as shown) due to the symmetry of
the molecule. However, when the dimer is formed from a
single-chain with a single alteration, only one of the receptor binding
sites is affected. B schematically shows the gene that was
used to express single-chain VEGF. The gene encodes a prepro-peptide
that is cleaved off before secretion from the yeast, and two monomers
of VEGF121 separated by a short linker peptide. The
difference in codon usage between the two VEGF monomers is maximized to
avoid recombination events and enable monomer-specific PCR.
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Subsequently, the plasmid encoding single-chain VEGF was transformed
into P. pastoris strain X-33, and the protein was expressed by growing X-33 clones in rich medium with methanol as carbon source.
The protein was purified to >90% purity (as evaluated by SDS-PAGE) by
two cation-exchange steps.
SDS-PAGE and MALDI-TOF mass spectrometry of single-chain VEGF both show
the presence of two components (Fig. 2).
N-terminal amino acid sequence analysis of the two components after
SDS-PAGE and electroblotting gave the same N-terminal sequence
(EGGGQNHHEVVKFMD) corresponding to amino acid residues 5-19 of
wild-type VEGF121.
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Fig. 2.
Characterization of protein
preparations. A shows Coomassie Blue-stained SDS-PAGE
analysis of three different preparations of single-chain VEGF variants.
Only in the highly overloaded lanes are impurities observed (two very
weak low molecular weight bands and a little VEGF tetramer and hexamer;
the identity of the VEGF tetramers and hexamers was confirmed by
Western blotting). B shows a MALDI-TOF mass spectrum of
purified single-chain VEGF. The three sets of twin peaks correspond to
the single-charged ((M + H)+), double-charged ((M + 2H)2+), and triple-charged ((M + 3H)3+)
species. The two peaks for each species correspond to the
monoglycosylated and diglycosylated form. In some cases, a
nonglycosylated form was observed on the ascending slope of the first
peak. No other significant peaks were observed, indicating that the
protein is highly pure. In the inset, the mass heterogeneity
caused by glycosylation is clearly seen on the zoomed peak for the
single-charged species.
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In MALDI-TOF mass spectrometry, both components are seen to be
heterogeneous with respect to their masses. The shapes of the two peaks
clearly indicate that glycosylation of the components is the basis for
the mass heterogeneity. The smaller component has an average mass of
30.8 kDa, with masses ranging from 30 to 31.5 kDa, whereas the larger
component has an average mass of 32.8 kDa, with masses ranging from 32 to 34 kDa. The theoretical mass of the polypeptide part of single-chain
VEGF starting at Glu5 is 28915 Da. Because
single-chain VEGF contains two potential N-glycosylation sites (one in
each monomer), incomplete glycosylation of either of these sites is the
likely explanation for the two components. In some mass spectra, the
nonglycosylated version of single-chain VEGF is sometimes observed in
minute amounts.
The ability of single-chain VEGF to induce proliferation of endothelial
cells in vitro was found to be indistinguishable from that
of wild-type VEGF121 in both potency and efficacy, as shown in Fig. 3. This indicates that connecting
the two monomers with a peptide linker does not affect the interaction
between VEGF and the KDR receptor; in other words, single-chain VEGF
functions as effectively as a wild-type VEGF homodimer.
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Fig. 3.
The dose-dependent stimulation of
endothelial cell proliferation by wild-type VEGF121 and
single-chain VEGF. The mitogenic effect on endothelial cells is
one of the main functions of VEGF, and the single-chain form ( ) is
functionally equivalent to the wild-type form ( ) in this
assay.
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Creating a Single-chain VEGF Antagonist--
Initially, nine
single-chain VEGF variants were prepared by site-directed mutagenesis
aimed at creating an antagonist by altering one of the receptor binding
sites of the single-chain VEGF to make that site unable to bind the
receptor (Fig. 4A). Positions are given relative to wild-type VEGF121 for each monomer,
so that 1:E64 is glutamic acid 64 in the first (N-terminal) monomer,
and 2:I46 is isoleucine 46 in the second (C-terminal) monomer. The 1:
and 2: tell whether the alteration is present in the N-terminal (1:) or
C-terminal (2:) monomer. 2:MRI(81-83)del means that the Met-Arg-Ile
sequence at position 81-83 in the C-terminal monomer was deleted.
2:I80IM means that a methionine was inserted after isoleucine 80 in the
C-terminal monomer.
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Fig. 4.
Induction of endothelial cell proliferation
by single-chain VEGF variants. A shows the average
maximum activity observed in three independent experiments on partially
purified single-chain VEGF variants relative to wild-type
VEGF121. Eight of the nine variants demonstrated a
dose-dependent ability to induce endothelial cell
proliferation that was indistinguishable from that of wild-type
VEGF121. However, the 1:E64R/2:I46R variant had only about
30% maximum activity and was able to antagonize itself at high
concentrations. B shows agonist assays run on two highly
purified single-chain VEGF variants and wild-type VEGF121
(, wild-type VEGF121; , 1:E64R/2:I46R variant; x,
1:E64R variant).
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These variants were expressed in P. pastoris as described
for the wild-type single-chain VEGF and partially purified by a single
cation-exchange step. This step removes most of the media components
and some proteins and allows analysis by mass spectrometry and
N-terminal amino acid sequencing. However, the purity is not sufficient
to allow quantification by amino acid analysis or
A280 absorption measurement. Thus, whereas it is
possible to evaluate the efficacy in HUVEC proliferation assay, it is
not possible to make an estimate of agonist or antagonist potency. Most
of these variants were able to stimulate HUVEC proliferation in a way
that was indistinguishable from wild-type VEGF121 (Fig.
4A). However, one single variant, 1:E64R/2:I46R,
demonstrated only a very modest induction of HUVEC proliferation and
was able to fully antagonize VEGF121-induced proliferation
(Figs. 4B and 5).
Protein Characterization--
SDS-PAGE and MALDI-TOF mass
spectrometry of the variants of single-chain VEGF always show the
presence of two components like those seen for wild-type single-chain
VEGF. N-terminal amino acid sequence analysis of the two components
after SDS-PAGE and electroblotting invariably gave the same N-terminal
sequence starting at amino acid residue Glu5 in
single-chain VEGF.
In MALDI-TOF mass spectrometry, results equivalent to those described
for wild-type single-chain VEGF were obtained, with minor variations in
the absolute amount of carbohydrate attached and the relative intensity
of the peaks for the two components.
After degradation of the highly purified antagonistic variant
single-chain VEGF with trypsin, confirmation of the two substitutions (1:E64R and 2:I46R) was obtained at the protein level through peptide
sequencing. The evidence for the 1:E64R substitution is indirect
because the peptide containing the substitution (amino acid residues
1:C57-1:E64) was not identified in the degradation. However, the
peptide resulting from trypsin-catalyzed cleavage following 1:R64
(amino acid residues 1:G65-1:R82) was purified in good yields, showing
that position 1:64 was indeed occupied by a residue after which trypsin
cleaves. The evidence for the 2:I46R substitution is direct because the
peptide containing the substitution (amino acid residue 2:S24-2:R46)
was purified and sequenced through the C-terminal R2:46 residue. In
addition, MALDI-TOF mass spectrometry showed that the peptide had the
expected mass of amino acid residues 2:24-2:46 with position 2:46
occupied by an arginine residue.
In single-chain VEGF, amino acid residues 1:N75 and 2:N75 are potential
N-glycosylation sites. After degradation of the antagonistic single-chain VEGF with trypsin, the peptides containing these two amino
acid residues (amino acid residues 1:G65-1:R82 and 2:C57-2:R82) were
purified and characterized. Both 1N:75 and 2:N75 were found to be
partially glycosylated because both a glycosylated and a nonglycosylated version of each peptide were purified. Based upon the
relative amounts of the nonglycosylated and glycosylated versions of
each peptide in the high pressure liquid chromatography
purification, it was clear that 1:N75 was almost fully glycosylated,
whereas 2:N75 was glycosylated to a lesser extent.
A very interesting observation was that both the nonglycosylated and
the glycosylated version of the peptide containing 2:N75 were purified
in two forms distinguished by a mass difference of 17 Da. Because the
N-terminal amino acid residue in the peptide (amino acid residue 2:57)
is an S-carboxamidomethyl-Cys residue, we propose that
the mass difference is caused by cyclization of the
S-carboxamidomethyl-Cys residue with concomitant loss of
NH3 (mass, 17 Da) in a reaction analogous to the
cyclization of N-terminal glutamine residues. N-terminal amino acid
sequence determination of the peptide with the highest mass gave the
expected amino acid sequence (CGGCCNDEGLECVPTEES[N]ITMQIMR; [N] is
found either glycosylated or nonglycosylated) with the 4 Cys residues
identified as S-carboxyamidomethyl-Cys residues. A peptide
with cyclized N-terminal S-carboxamidomethyl-Cys residue
should be inaccessible to N-terminal amino acid sequencing. However,
upon N-terminal amino acid sequencing of the peptides with the
supposedly cyclized S-carboxamidomethyl-Cys residue at the N
terminus, it was possible to obtain sequence information for the
peptide, but with residue 1 identified as an
S-carboxymethyl-Cys residue. An N-terminal
S-carboxymethyl-Cys residue can only be explained by
in-sequencer hydrolysis of a cyclized N-terminal S-carboxamidomethyl-Cys residue to an
S-carboxymethyl-Cys residue. This specific observation
clearly supports the interpretation that the N-terminal
S-carboxamidomethyl-Cys residue can undergo cyclization just
like N-terminal Gln residues. It should be stressed that all other Cys
residues covered by amino acid sequencing were clearly identified as
S-carboxamidomethyl-Cys residues without any presence of
S-carboxymethyl-Cys residues, ruling out the possibility for
contamination of the iodoacetamide used with iodoacetic acid. Such a
contamination also would not explain the observation of the loss of 17 Da in the peptide mass.
MALDI-TOF mass spectrometry of the glycosylated peptides showed that
the glycan structures attached to 1:N75 and 2:N75 were almost identical
and heterogeneous in accordance with the data obtained from intact
single-chain VEGF. The masses of the glycan structures are in
accordance with glycan structures composed of 2 N-acetylglucosamine residues and 6-14 hexose residues.
Taking into account that the single-chain VEGF is produced in P. pastoris, it is highly likely that the hexose residues are mannose residues.
Further Characterization of the 1:E64R/2:I46R
Variant--
To verify that the antagonist activity of the
1:E64R/2:I46R variant is specific and not caused by cytotoxic
impurities in the protein preparations, antagonist assays were run
against variable doses of VEGF121 and against bFGF. As
shown in Fig. 5A, the
single-chain VEGF variant is able to fully antagonize the mitogenic
effects of VEGF121 in a dose-dependent manner.
In contrast, the mitogenic effect of bFGF is not affected,
demonstrating that the apparent antagonism is VEGF-specific and not due
to cytotoxic impurities in the protein preparation. As shown in Fig.
5B, the antagonist potency of the single-chain VEGF variant
is reduced (the IC50 is increased) when the concentration
of VEGF121 is increased. Together, these assays demonstrate
that the single-chain VEGF variant is a specific, competitive VEGF
antagonist, in accordance with the proposed model for how a VEGF
molecule with only one active receptor binding site would affect the
interaction between VEGF and its receptor.
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Fig. 5.
VEGF antagonism by single-chain VEGF
1:E64R/2:I46R. A shows the ability of single-chain VEGF
1:E64R/2:I46R to antagonize the mitogenic effect of VEGF121
() and bFGF () on endothelial cells. The curves have been
normalized to wild-type VEGF121 and bFGF standard curves,
respectively. This panel demonstrates that the apparent antagonist
effect of the variant is VEGF-specific and is not due to the presence
of unspecific, cytotoxic impurities in the protein preparation.
Furthermore, it demonstrates that the variant is a full antagonist
because the level of endothelial proliferation is reduced to the level
observed without stimulation. B shows the antagonist
activity of the single-chain VEGF variant against low (1 nM, ) or high (200 nM, ) concentrations
of wild-type VEGF121. The reduced potency of the antagonist
when the concentration of agonist is increased (as seen from the right
shift of the curve) demonstrates that the single-chain VEGF variant has
competitive antagonist behavior. Furthermore, it shows that the
apparent antagonist behavior is not due to a bell-shaped agonist
behavior of the wild-type VEGF protein or another type of high-dose
inhibition, in which case an increase in the concentration of agonist
would not have led to a right shift of the antagonist curve.
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To further characterize the interaction between VEGF and the KDR
receptor, BiaCore analyses were run in which single-chain VEGF and the
1:E64R/2:I46R variant were floated over immobilized KDR/Fc chimera,
which is dimeric. As shown in Fig. 6,
wild-type single-chain VEGF displays a biphasic dissociation with
half-lives of ~55 s and 43 min, respectively, corresponding to VEGF
dimer bound with one or two sites. The 1:E64R/2:I46R variant also
displays biphasic dissociation. However, whereas the half-life of the
fast dissociation is indistinguishable from that of wild-type
single-chain VEGF, the half-life of the slow dissociation is 23 min,
i.e. only half of that of the wild-type single-chain VEGF.
This supports the proposed model for how the antagonist should work:
the 1:E64R/2:I46R variant has one intact receptor binding site, and
hence variant ligand bound with only one (intact) site dissociates with
the same kinetics as the wild-type ligand. However, the variant ligand bound with two sites dissociates much faster than the wild-type ligand
because one of the sites has reduced affinity for the receptor.
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Fig. 6.
Interaction between the KDR receptor and
single-chain VEGF. The interaction was analyzed using a BiaCore
system as described under "Experimental Procedures." The two curves
represent the dissociation of single-chain VEGF and the 1:E64R/2:I46R
variant from predimerized receptor (KDR/Fc chimera). Both molecules
show biphasic dissociation corresponding to a mix of ligands bound with
either one or two receptor binding sites. The two molecules show an
indistinguishable fast dissociation (half-life, ~55 s), in accordance
with the expectation that they both have at least one intact
high-affinity receptor binding site. However, when both sites are used
for binding, the dissociation of the 1:E64R/2:I46R variant is much
faster (half-life, 23 min) than that of the wild-type single-chain
(half-life, 43 min), demonstrating that the arginine substitutions
reduce the affinity for the receptor.
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1:E64R Variant--
The 2:I46R substitution is present in three
single-chain VEGF variants that have full mitogenic activity, so it is
not clear from the above-mentioned results whether the change in
activity of the 1:E64R/2:I46R variant is due solely to the 1:E64R
substitution or to a combined effect of the two substitutions. To
investigate this, a single-chain VEGF variant with only the 1:E64R
substitution was created. This variant was expressed and purified by
the two cation-exchange steps described above. The ability of this
variant to stimulate endothelial cell proliferation was compared with that of the 1:E64R/2:I46R variant (Fig. 4B). It is clearly
seen that the mitogenic effect of the 1:E64R variant is much higher than that of the 1:E64R/2:I46R variant.
| |
DISCUSSION |
Single-chain VEGF--
Application of the single-chain antagonist
generation approach to VEGF requires that single-chain VEGF be
expressed as a molecule that is functionally equivalent to wild-type
VEGF. Therefore, the initial experiments were aimed at creating a
single-chain form of wild-type VEGF with functional properties
identical to those of normal dimeric VEGF.
Several parameters can be varied in the design of a single-chain VEGF,
including the selection of which monomer to use (e.g. VEGF110, VEGF121, or VEGF165) and
the choice of peptide (if any) to link the two monomers. Based on the
crystal structure of the core domain of VEGF (i.e. most of
VEGF121), in which the C terminus of one monomer is located
in close proximity to the N terminus of the other monomer, it was
anticipated that two VEGF121 monomers could be expressed as
a single-chain dimer connected by a short linker peptide. In contrast,
it is difficult to contemplate the feasibility of preparing
single-chain dimers of VEGF165 because the structures of
the two domains of this molecule have not been determined together. Of
course, because the difference between the VEGF121 and
VEGF165 monomers is a domain located at the C terminus of
the monomer, a single-chain dimer in which the N-terminal monomer is
VEGF121 and the C-terminal monomer is VEGF165
would have the same requirements for the connection between the
monomers as would a single-chain dimer of VEGF121 monomers.
A described above, the first single-chain VEGF that was tested
consisted of two VEGF121 monomers connected by a 14-residue linker peptide. This protein was successfully expressed in P. pastoris and found to stimulate endothelial cell proliferation in
a manner indistinguishable from that of wild-type VEGF121, indicating that this construct would provide a suitable scaffold for
the preparation of single-chain VEGF121 heterodimers. The lack of 4 N-terminal residues of wild-type VEGF121 that
were removed from the protein during posttranslational processing was
not found to influence the activity of the molecule, in good accordance with previous studies on VEGF (30). Mass spectrometry of the expressed
protein revealed two main forms, a monoglycosylated form and a
diglycosylated form. The nonglycosylated form was only observed in
minute amounts. Analysis of the glycosylation at the glycopeptide
level identified the glycosylation as being well-known P. pastoris N-glycan structures. No observation of large
mannan structures that are sometimes observed on proteins expressed in P. pastoris was made. Similar patterns of glycosylation were
observed for all of the variants prepared. However, because previous
studies have shown that glycosylation does not affect VEGF activity
(31-33), no attempts were made to obtain homogenously glycosylated
protein preparations. Later, peptide map studies on the 1:E64R/2:I46R variant revealed a difference in the frequency of glycosylation of the
two monomers. The most N-terminal glycosylation site had a higher
frequency of glycosylation than the most C-terminal site, a property
that is likely to apply to wild-type single-chain VEGF too. Taking the
closeness of 2:N75 to the C terminus of the protein into consideration,
this is hardly surprising.
Preparing a Single-chain Antagonist--
As mentioned above,
extensive structure-function information regarding the interaction
between VEGF and its receptors is available in the literature. Based on
this information, two strategies were used to make one of the receptor
binding sites nonfunctional. One strategy was to substitute arginine
residues into one of the receptor binding sites on positions known from
previously published studies to be important for the VEGF-receptor
interaction (mainly from alanine scanning studies). A different
strategy was to insert or delete one or more residues in the receptor
binding site to try to disrupt the structure of the entire region.
One of the nine variants that were initially prepared, 1:E64R/2:I46R,
showed almost no agonist activity and was able to fully antagonize the
activity of wild-type VEGF121. Antagonist assays against
bFGF and elevated concentrations of VEGF121 demonstrated that the observed effect is due to a VEGF-specific, competitive antagonist. Subsequently, BiaCore analysis of dissociation of wild-type
single-chain VEGF and the 1:E64R/2:I46R variant from the KDR receptor
supported the notion that the antagonist variant contains one intact
receptor binding site and one site with reduced receptor binding
affinity. Together, these data clearly show that the 1:E64R/2:I46R
single-chain VEGF variant is effectively a VEGF variant with only a
single receptor binding site.
Interestingly, all eight other variants had full agonist activity.
Whereas effects such as receptor saturation may hide a difference
between some of the variants and wild-type VEGF, there is obviously a
huge difference between 1:E64R/2:I46R and the other variants. This is
in interesting contrast to earlier alanine scanning studies on VEGF, in
which Glu64 and Ile46 do not appear to be much
more important than the other residues that were substituted in this
study (Tyr45, Gln79, and Ile83).
The subsequent preparation of a single-chain VEGF with 1:E64R as the
only substitution revealed that both 1:E64 and 2:I46 must be
substituted to arginines at the same time to eliminate agonist activity.
The effect of arginine substitutions on receptor binding obviously
cannot be predicted from alanine scanning mutagenesis results, emphasizing that alanine scanning alone presents a biased picture of
the relative importance of the residues in a protein and that arginine
scanning, glutamate scanning, or tryptophan scanning mutagenesis would
provide complementary data for structure-function studies. A different
consequence of this observation is that to improve the antagonist
identified in this study by reducing its low agonist activity further,
a more complete arginine scanning is necessary. This is the case
because the results of previous alanine scanning studies cannot be used
to predict the positions in which arginine substitution will have the
greatest effects.
The single-chain VEGF antagonist variant prepared in this study is not
an optimal solution to the problem of preparing a VEGF variant with
antagonist properties. For example, the variant shows limited agonist
activity within a narrow concentration range, and the antagonist
potency is low. However, the construction of this variant demonstrates
the feasibility of using the single-chain approach for creating VEGF
antagonists, and the variant may serve as a scaffold for further
improvement of antagonistic properties.
| |
FOOTNOTES |
*
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.:
45-7020-5550; Fax: 45-7020-5530; E-mail: th@maxygen.dk.
Published, JBC Papers in Press, July 31, 2002, DOI 10.1074/jbc.M204107200
| |
ABBREVIATIONS |
The abbreviations used are:
VEGF, vascular
endothelial growth factor;
bFGF, basic fibroblast growth factor;
HUVEC, human umbilical vein endothelial cell;
KDR, kinase insert domain
receptor;
MALDI-TOF, matrix-assisted laser desorption ionization
time-of-flight.
| |
REFERENCES |
| 1.
|
Ferrara, N.,
Carver-Moore, K.,
Chen, H.,
Dowd, M., Lu, L.,
O'Shea, K. S.,
Powell-Braxton, L.,
Hillan, K. J.,
and Moore, M. W.
(1996)
Nature
380,
439-442[CrossRef][Medline]
[Order article via Infotrieve]
|
| 2.
|
Carmeliet, P.,
Ferreira, V.,
Breier, G.,
Pollefeyt, S.,
Kieckens, L.,
Gertsenstein, M.,
Fahrig, M.,
Vandenhoeck, A.,
Harpal, K.,
Eberhardt, C.,
Declercq, C.,
Pawling, J.,
Moons, L.,
Collen, D.,
Risau, W.,
and Nagy, A.
(1996)
Nature
380,
435-439[CrossRef][Medline]
[Order article via Infotrieve]
|
| 3.
|
Shalaby, F., Ho, J.,
Stanford, W. L.,
Fischer, K. D.,
Schuh, A. C.,
Schwartz, L.,
Bernstein, A.,
and Rossant, J.
(1997)
Cell
89,
981-990[CrossRef][Medline]
[Order article via Infotrieve]
|
| 4.
|
Fong, G. H.,
Rossant, J.,
Gertsenstein, M.,
and Breitman, M. L.
(1995)
Nature
376,
66-70[CrossRef][Medline]
[Order article via Infotrieve]
|
| 5.
|
Shalaby, F.,
Rossant, J.,
Yamaguchi, T. P.,
Gertsenstein, M., Wu, X. F.,
Breitman, M. L.,
and Schuh, A. C.
(1995)
Nature
376,
62-66[CrossRef][Medline]
[Order article via Infotrieve]
|
| 6.
|
Ferrara, N.,
Chen, H.,
Davis-Smyth, T.,
Gerber, H. P.,
Nguyen, T. N.,
Peers, D.,
Chisholm, V.,
Hillan, K. J.,
and Schwall, R. H.
(1998)
Nat. Med.
4,
336-340[CrossRef][Medline]
[Order article via Infotrieve]
|
| 7.
|
Ferrara, N.
(2001)
Am. J. Physiol. Cell Physiol.
280,
C1358-C1366[Abstract/Free Full Text]
|
| 8.
|
Brower, V.
(1999)
Nat. Biotechnol.
17,
963-968[CrossRef][Medline]
[Order article via Infotrieve]
|
| 9.
|
Ferrara, N.
(2000)
Curr. Opin. Biotechnol.
11,
617-624[CrossRef][Medline]
[Order article via Infotrieve]
|
| 10.
|
Keyt, B. A.,
Berleau, L. T.,
Nguyen, H. V.,
Chen, H.,
Heinsohn, H.,
Vandlen, R.,
and Ferrara, N.
(1996)
J. Biol. Chem.
271,
7788-7795[Abstract/Free Full Text]
|
| 11.
|
Poltorak, Z.,
Cohen, T.,
Sivan, R.,
Kandelis, Y.,
Spira, G.,
Vlodavsky, I.,
Keshet, E.,
and Neufeld, G.
(1997)
J. Biol. Chem.
272,
7151-7158[Abstract/Free Full Text]
|
| 12.
|
Robinson, C. J.,
and Stringer, S. E.
(2001)
J. Cell Sci.
114,
853-865[Abstract]
|
| 13.
|
Shibuya, M.
(2001)
Cell Struct. Funct.
26,
25-35[CrossRef][Medline]
[Order article via Infotrieve]
|
| 14.
|
Gille, H.,
Kowalski, J., Li, B.,
LeCouter, J.,
Moffat, B.,
Zioncheck, T. F.,
Pelletier, N.,
and Ferrara, N.
(2001)
J. Biol. Chem.
276,
3222-3230[Abstract/Free Full Text]
|
| 15.
|
Rahimi, N.,
Dayanir, V.,
and Lashkari, K.
(2000)
J. Biol. Chem.
275,
16986-16992[Abstract/Free Full Text]
|
| 16.
|
Bernatchez, P. N.,
Soker, S.,
and Sirois, M. G.
(1999)
J. Biol. Chem.
274,
31047-31054[Abstract/Free Full Text]
|
| 17.
|
Hiratsuka, S.,
Minowa, O.,
Kuno, J.,
Noda, T.,
and Shibuya, M.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9349-9354[Abstract/Free Full Text]
|
| 18.
|
Fong, G. H.,
Zhang, L.,
Bryce, D. M.,
and Peng, J.
(1999)
Development
126,
3015-3025[Abstract]
|
| 19.
|
Dunk, C.,
and Ahmed, A.
(2001)
Am. J. Pathol.
158,
265-273[Abstract/Free Full Text]
|
| 20.
|
Li, B.,
Fuh, G.,
Meng, G.,
Xin, X.,
Gerritsen, M. E.,
Cunningham, B.,
and de Vos, A. M.
(2000)
J. Biol. Chem.
275,
29823-29828[Abstract/Free Full Text]
|
| 21.
|
Fuh, G., Li, B.,
Crowley, C.,
Cunningham, B.,
and Wells, J. A.
(1998)
J. Biol. Chem.
273,
11197-11204[Abstract/Free Full Text]
|
| 22.
|
Siemeister, G.,
Schirner, M.,
Reusch, P.,
Barleon, B.,
Marme, D.,
and Martiny-Baron, G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4625-4629[Abstract/Free Full Text]
|
| 23.
|
Muller, Y. A., Li, B.,
Christinger, H. W.,
Wells, J. A.,
Cunningham, B. C.,
and de Vos, A. M.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7192-7197[Abstract/Free Full Text]
|
| 24.
|
Keyt, B. A.,
Nguyen, H. V.,
Berleau, L. T.,
Duarte, C. M.,
Park, J.,
Chen, H.,
and Ferrara, N.
(1996)
J. Biol. Chem.
271,
5638-5646[Abstract/Free Full Text]
|
| 25.
|
Wiesmann, C.,
Christinger, H. W.,
Cochran, A. G.,
Cunningham, B. C.,
Fairbrother, W. J.,
Keenan, C. J.,
Meng, G.,
and de Vos, A. M.
(1998)
Biochemistry
37,
17765-17772[CrossRef][Medline]
[Order article via Infotrieve]
|
| 26.
|
Muller, Y. A.,
Chen, Y.,
Christinger, H. W., Li, B.,
Cunningham, B. C.,
Lowman, H. B.,
and de Vos, A. M.
(1998)
Structure
6,
1153-1167[Medline]
[Order article via Infotrieve]
|
| 27.
|
Fairbrother, W. J.,
Champe, M. A.,
Christinger, H. W.,
Keyt, B. A.,
and Starovasnik, M. A.
(1998)
Structure
6,
637-648[Medline]
[Order article via Infotrieve]
|
| 28.
|
Wiesmann, C.,
Fuh, G.,
Christinger, H. W.,
Eigenbrot, C.,
Wells, J. A.,
and de Vos, A. M.
(1997)
Cell
91,
695-704[CrossRef][Medline]
[Order article via Infotrieve]
|
| 29.
|
Muller, Y. A.,
Christinger, H. W.,
Keyt, B. A.,
and de Vos, A. M.
(1997)
Structure
5,
1325-1338[Medline]
[Order article via Infotrieve]
|
| 30.
|
Siemeister, G.,
Marme, D.,
and Martiny-Baron, G.
(1998)
J. Biol. Chem.
273,
11115-11120[Abstract/Free Full Text]
|
| 31.
|
Claffey, K. P.,
Senger, D. R.,
and Spiegelman, B. M.
(1995)
Biochim. Biophys. Acta
1246,
1-9[CrossRef][Medline]
[Order article via Infotrieve]
|
| 32.
|
Peretz, D.,
Gitay-Goren, H.,
Safran, M.,
Kimmel, N.,
Gospodarowicz, D.,
and Neufeld, G.
(1992)
Biochem. Biophys. Res. Commun.
182,
1340-1347[CrossRef][Medline]
[Order article via Infotrieve]
|
| 33.
|
Yeo, T. K.,
Senger, D. R.,
Dvorak, H. F.,
Freter, L.,
and Yeo, K. T.
(1991)
Biochem. Biophys. Res. Commun.
179,
1568-1575[CrossRef][Medline]
[Order article via Infotrieve]
|
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ABSTRACT
INTRODUCTION
