The absence of VEGFR-1 binding is surprising given that the structural predic-tions for viral VEGFs are very similar to that of Keywords Orf virus; Parapoxvirus; vascular endothelial gro
Trang 1factor-E partially blocks binding to VEGF receptor-1
Marie K Inder, Lyn M Wise, Stephen B Fleming and Andrew A Mercer
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
Members of the vascular endothelial growth factor
(VEGF) family of molecules have emerged as major
regulators of new blood vessel formation during
vascu-logenesis and angiogenesis [1–3] These proteins have
critical roles during embryogenesis and in normal adult
tissues, during wound healing and in pathological
con-ditions such as tumor formation Currently, the
mam-malian VEGF family includes VEGF-A, VEGF-B,
VEGF-C, VEGF-D and placenta growth factor
VEGF family members exert their biological activity
via a family of tyrosine kinase receptors, VEGF
recep-tor (VEGFR)-1, VEGFR-2 and VEGFR-3 [4–6]
VEGFR-1 is bound by VEGF-A, VEGF-B and
pla-centa growth factor and is primarily expressed on
endothelial and hematopoietic cells and may have a
role in monocyte recruitment and pro-inflammatory
gene expression VEGFR-2 is bound by VEGF-A,
VEGF-C and VEGF-D and is the primary signaling
receptor of VEGF-induced endothelial cell mitogenesis,
angiogenesis and vascular permeability VEGFR-3 regulates formation of the lymphatic vasculature via VEGF-C and VEGF-D
We, and others [7–16], have characterized a group of viral-derived VEGFs, collectively designated VEGF-E, which are encoded by members of the genus Parapox-virus, namely Orf virus (ORFV), Pseudocowpoxvirus (PCPV), Parapoxvirus of red deer in New Zealand (PVNZ) and bovine papular stomatitis virus (BPSV) These viruses infect specific ungulates and can readily infect humans [17,18], and the resulting lesions in the skin demonstrate extensive vascular dilation, dermal edema and endothelial cell proliferation [17,19–21] Viral VEGF proteins differ from mammalian VEGF family members in that they specifically bind and acti-vate VEGFR-2 and in general show little or no affinity for VEGFR-1 [8,9,11–16] The absence of VEGFR-1 binding is surprising given that the structural predic-tions for viral VEGFs are very similar to that of
Keywords
Orf virus; Parapoxvirus; vascular endothelial
growth factor; VEGF-E; vascular endothelial
growth factor receptor
Correspondence
L M Wise, Virus Research Unit,
Department of Microbiology and
Immunology, University of Otago,
PO Box 56, Dunedin, New Zealand
Fax: +64 3 479 7744
Tel: +64 3 479 7723
E-mail: lyn.wise@stonebow.otago.ac.nz
(Received 12 August 2007, revised
24 October 2007, accepted 12 November
2007)
doi:10.1111/j.1742-4658.2007.06189.x
Vascular endothelial growth factor (VEGF) family members play impor-tant roles in embryonic development and angiogenesis during wound healing and in pathological conditions such as tumor formation Parapox-viruses express a new member of the VEGF family which is a functional mitogen that specifically activates VEGF receptor (VEGFR)-2 but not VEGFR-1 In this study, we show that deletion from the viral VEGF of a unique C-terminal region increases both 1 binding and VEGFR-1-mediated monocyte migration Enzymatic removal of O-linked glycosyla-tion from the C-terminus also increased VEGFR-1 binding and migraglycosyla-tion
of THP-1 monocytes indicating that both the C-terminal residues and O-linked sugars contribute to blocking viral VEGF binding to VEGFR-1 The data suggest that conservation of the C-terminal residues throughout the viral VEGF subfamily may represent a means of reducing the immuno-stimulatory activities associated with VEGFR-1 activation while maintain-ing the ability to induce angiogenesis via VEGFR-2
Abbreviations
BPSV, bovine papular stomatitis virus; ORFV, Orf virus; PCPV, Pseudocowpoxvirus; PVNZ, Parapoxvirus of red deer in New Zealand; SA-HRP, streptavidin-peroxidase; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
Trang 2VEGF-A [10,22], and that viral VEGFs conserve many
residues shown to be vital for VEGF-A binding to
VEGFR-1 [23,24] Although domain-exchange studies
with a viral VEGF identified loop regions that are
essential for VEGFR-2 binding [25], a structural basis
for the inability of viral VEGF to bind VEGFR-1 has
not yet been determined There are, however, a
number of structural features that may influence the
specificity of viral VEGFs, including a C-terminal
Pro⁄ Thr-rich sequence, encoding potential O-linked
glycosylation sites, that is unique to and highly
con-served among the viral VEGFs [8,10,13–16] In this
study, we examined the role of the C-terminal region
of the VEGF encoded by ORFV strain NZ2
(ORFVNZ2VEGF) in the receptor-binding specificity
and biological activities of the viral VEGFs
Results
Amino acid sequence comparisons
Comparison of the predicted amino acid sequences
of the viral VEGFs from ORFV strains NZ2
(ORFVNZ2VEGF) and NZ7 (ORFVNZ7VEGF), BPSV
strain V660 (BPSVV660VEGF), PCPV strain VR634
(PCPVVR634VEGF) and PVNZ strain RD86
(PVNZRD86VEGF) revealed a Thr⁄ Pro rich
C-termi-nus, containing putative O-linked glycosylation sites
(Fig 1A) that is not found in VEGF-A or other
VEGF family members [10] To examine the role of
this conserved C-terminus in the unique receptor
speci-ficity and biological activities of viral VEGFs, we
constructed a mutant of ORFVNZ2VEGF in which
the 16 C-terminal residues were deleted, designated
ORFVNZ2VEGF-DC (Fig 1B) We also constructed a
mutant in which the heparin-binding domain of
VEGF-A was replaced with the 16 C-terminal residues
of ORFVNZ2VEGF, designated VEGF-A-NZ2C
(Fig 1B)
Production and glycosylation state of VEGF
mutants
VEGF-A-NZ2C and ORFVNZ2VEGF-DC were
expressed with a FLAG octapeptide at the C-terminus
and then purified SDS⁄ PAGE under reducing
condi-tions followed by silver staining revealed bands at
26–28 and 19–20 kDa, for VEGF-A-NZ2C and
ORFVNZ2VEGF-DC, respectively (Fig 2)
Deglycosy-lation treatment with N-glycosidase reduced the
monomeric size of VEGF-A-NZ2C and ORFVNZ2
-VEGF-DC by 2–4 kDa (Fig 2), which was similar
to that seen for VEGF-A and ORFVNZ2VEGF,
indicating that each contained N-linked glycosylation Further treatment with sialidase and O-glycosidase reduced VEGF-A-NZ2C and ORFVNZ2VEGF by another 2–4 kDa, although no size shift was observed for VEGF-A and ORFVNZ2VEGF-DC (Fig 2) The absence of O-linked glycosylation on ORFVNZ2
VEGF-DC supports the prediction that ORFVNZ2VEGF contains an O-linked glycosylation site in the 16 C-terminal residues (Fig 1) The observation that the addition of the 16 C-terminal residues to VEGF-A-NZ2C are associated with the gain of O-linked glyco-sylation also supports this prediction (Fig 1)
Deletion of the C-terminus of ORFVNZ2VEGF increases its affinity for VEGFR-1 and VEGFR-2
To investigate the role of the ORFVNZ2VEGF C-ter-minus in receptor specificity, we tested the ability of the VEGF mutants to bind immobilized dimeric Ig fusion proteins containing the extracellular domains of human VEGFR-1 or VEGFR-2 using a receptor-bind-ing ELISA
VEGF-A and VEGF-A-NZ2C demonstrated signifi-cant levels of binding to VEGFR-1 compared with
A
B
Fig 1 Sequence comparison of the parapoxviral VEGF C-termini and schematic representation of ORFV NZ2 VEGF, VEGF-A and mutants (A) The C-terminal amino acid sequences of ORFV NZ2-VEGF, BPSV V660 VEGF, PCPV VR364 VEGF, ORFV NZ7 VEGF and PVNZ RD86 VEGF are shown [8,10,13,14] Predicted O-linked glyco-sylation sites are shaded gray [37] (B) Schematic representation of ORFVNZ2VEGF, VEGF-A and mutants ORFVNZ2VEGF-DC is a mutant of ORFV NZ2 VEGF in which the 16 C-terminal (C-term) amino acids have been deleted VEGF-A-NZ2C is a mutant of VEGF-A in which the heparin-binding domain (HBD) has been replaced with the 16 C-terminal amino acids from ORFV NZ2 VEGF All proteins are FLAG (F)-tagged at the C-terminus for detection and purification The locations of the conserved N-linked glycosylation site (N) and the variable loop regions (L1–3) are indicated by dashes and light gray boxes, respectively.
Trang 3mock-purified protein at all of the concentrations
tested (‡ 0.1 lgÆmL)1, P £ 0.05; Fig 3A) As reported
previously [15,16], ORFVNZ2VEGF did not bind
VEGFR-1 at any concentration tested (Fig 3A) By
contrast, ORFVNZ2VEGF-DC showed significant levels
of binding to VEGFR-1 from a concentration of
3.3 lgÆmL)1(P£ 0.05; Fig 3A)
VEGF-A and VEGF-A-NZ2C demonstrated
signifi-cant levels of binding to VEGFR-2 compared with
mock-purified protein at all of the concentrations tested
(‡ 0.1 lgÆmL)1, P£ 0.05; Fig 3B) ORFVNZ2VEGF
and ORFVNZ2VEGF-DC also showed significant
bind-ing to VEGFR-2 from a concentration of 0.4 lgÆmL)1
(P£ 0.05; Fig 3B) No significant differences in
binding to VEGFR-2 were observed at any
concen-tration between VEGF-A and VEGF-A-NZ2C or
ORFVNZ2VEGF and ORFVNZ2VEGF-DC (P £ 0.05;
Fig 3B)
To further examine the receptor specificity of the
VEGF mutants, we tested their ability to inhibit
VEGF-A bind the dimerized Ig fusion proteins
con-taining the extracellular domains of human VEGFR-1
or VEGFR-2 under soluble binding conditions using a
competitive displacement ELISA [8,13]
Preincubation of soluble VEGF-A or VEGF-A-NZ2C with VEGFR-1 significantly inhibited the binding of the receptor to immobilized VEGF-A at all concentrations tested (‡ 0.4 lgÆmL)1, P£ 0.05), whereas ORFVNZ2VEGF did not significantly inhibit VEGFR-1 binding to VEGF-A at any con-centration tested (Fig 3C) ORFVNZ2VEGF-DC did, however, inhibit binding of VEGFR-1 to VEGF-A from a concentration of 1.1 lgÆmL)1 (P£ 0.05; Fig 3C)
Preincubation of soluble VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF or ORFVNZ2VEGF-DC significantly inhibited the binding of VEGFR-2 to immobilized VEGF-A at all concentrations tested (‡ 0.2 lgÆmL)1,
P £ 0.05; Fig 3D) ORFVNZ2VEGF-DC was, however, significantly more potent than ORFVNZ2VEGF from a concentration of 0.6 lgÆmL)1, whereas no significant differences were observed between VEGF-A-NZ2C and VEGF-A at any of the concentrations tested (P£ 0.05; Fig 3D)
The interactions of the VEGF mutants with VEG-FR-1 and VEGFR-2 were further tested in bioassays that detect receptor binding and cross-linking at the cell surface These assays made use of BaF3 cell lines expressing chimeric receptors consisting of the extracel-lular domain of either human VEGFR-1 or murine VEGFR-2 and the transmembrane and cytoplasmic domains of the erythropoietin receptor [26,27] Binding and cross-linking of the chimeric receptors induce cell proliferation
VEGF-A and VEGF-A-NZ2C stimulated significant proliferation of cells expressing VEGFR-1 from the lowest concentration tested (‡ 1.2 ngÆmL)1, P£ 0.05), although ORFVNZ2VEGF did not induce cellular pro-liferation (Fig 3E) ORFVNZ2VEGF-DC stimulated significant proliferation of cells expressing VEGFR-1 from a concentration of 11 ngÆmL)1 (P£ 0.05; Fig 3E)
VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFVNZ2VEGF-DC were each able to stimulate sig-nificant proliferation of cells expressing VEGFR-2, in the presence of heparin, from a concentration of 0.8 ngÆmL)1 (P£ 0.05; Fig 3F) ORFVNZ2VEGF-DC was, however, significantly more potent than ORFVNZ2VEGF, VEGF-A and VEGF-A-NZ2C at all concentrations tested (0.8–22 ngÆmL)1, P£ 0.05; Fig 3F)
In summary, ORFVNZ2VEGF-DC showed a signi-ficant increase in VEGFR-1 binding compared with ORFVNZ2VEGF in the three different assays (Fig 3A,C,E) In addition, a consistent, but not signifi-cant, decrease was observed in VEGFR-1 binding
by VEGF-A-NZ2C compared with VEGF-A No
Fig 2 The C-terminus of ORFVNZ2VEGF has O-linked
glycosyla-tion Purified VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFV
NZ2-VEGF-DC were analyzed before and after enzymatic removal of
N- and O-linked sugars The proteins were resolved under reducing
conditions following treatment with the indicated combinations of
enzymes as described in Experimental procedures Proteins were
visualized by western blotting using anti-FLAG Ig Molecular mass
markers are indicated.
Trang 4consistent differences were noted in VEGFR-2 binding
between VEGF-A-NZ2C and VEGF-A (Fig 3B,D,F)
ORFVNZ2VEGF-DC did, however, show a small, but
significant increase, in VEGFR-2 binding, compared
with ORFVNZ2VEGF, in two of the three assay
sys-tems (Fig 3D,F)
Enzymatic removal of the O-linked glycosylation
of ORFVNZ2VEGF increases its affinity for VEGFR-1 and VEGFR-2
To examine the role O-linked glycosylation might play
in the receptor specificity and biological activities of
Trang 5the viral VEGFs, ORFVNZ2VEGF was treated with
sialidase and O-glycosidase to remove the O-linked
glycosylation The enzyme-treated protein, ORFVNZ2
-VEGF-DOglyc was then repurified and analyzed by
SDS⁄ PAGE and western blotting which revealed
bands at 38–42 and 19–20 kDa, under nonreducing
and reducing conditions, respectively (Fig 4A)
N-Gly-cosidase treatment reduced the monomeric size of
ORFVNZ2VEGF-DOglyc by 2–3 kDa (Fig 4A), but
further treatment with sialidase and O-glycosidase did
not result in a size shift These results confirm that
ORFVNZ2VEGF-DOglyc has retained its N-linked
glycosylation and the majority of the O-linked
glyco-sylation has been removed
To investigate the role of that ORFVNZ2VEGF
O-linked glycosylation plays in receptor specificity we
tested the ability of ORFVNZ2VEGF-DOglyc to bind
and cross-link VEGFR-1 and VEGFR-2 in the BaF3
bioassays Consistent with our previous results,
VEGF-A and ORFVNZ2VEGF-DC were able to
stimu-late significant proliferation of cells expressing
VEG-FR-1 from 1.2 and 11 ngÆmL)1, respectively (P£ 0.05),
whereas ORFVNZ2VEGF did not induce cellular
pro-liferation (Fig 4B) ORFVNZ2VEGF-DOglyc was less
potent than ORFVNZ2VEGF-DC but did stimulate
significant proliferation of cells expressing VEGFR-1
at the highest concentration tested (100 ngÆmL)1,
P£ 0.05; Fig 4B)
VEGF-A, ORFVNZ2VEGF, ORFVNZ2VEGF-DC
and ORFVNZ2VEGF-DOglyc were each able to
sti-mulate significant proliferation of cells expressing
VEGFR-2, in the presence of heparin, from a
concen-tration of 0.3 ngÆmL)1 (P£ 0.05; Fig 4C) ORFVNZ2
-VEGF-DC and ORFVNZ2VEGF-DOglyc were both
significantly more potent than VEGF-A and
ORFVNZ2VEGF at all concentrations tested (0.3–
7.4 ngÆmL)1, P£ 0.05; Fig 4C)
Removal of the C-terminal residues or O-linked glycosylation of ORFVNZ2VEGF increases its recruitment of THP-1 monocytes
Previous studies have shown that mammalian VEGF family members induce monocyte chemotaxis via their interaction with VEGFR-1 [28–30] Thus, using a Trans-well assay (Corning Costar, Corning, NY, USA), we examined the chemotactic response of THP-1 mono-cytes to treatment with the VEGF mutants The human THP-1 monocytic cell line has previously been shown to respond to VEGF family members in a manner similar
to human peripheral blood monocytes [29]
VEGF-A and VEGF-A-NZ2C were able to induce significant migration of cells from a concentration of
4 ngÆmL)1 (P£ 0.05), whereas ORFVNZ2VEGF did not induce cell migration (Fig 5) ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc were also able to induce significant migration of cells from a concentration of
20 ngÆmL)1 (P£ 0.05; Fig 5) Migration of THP-1 monocytes induced by VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc was significantly inhibited by preincubation of the cells with neutralizing antibody against VEGFR-1 (P£ 0.05) (Fig 5)
The increase in monocyte migration induced by ORFVNZ2VEGF-DC, compared with ORFVNZ2VEGF, was consistent with its increase in VEGFR-1 binding
in the receptor-binding assays (Figs 3 and 4) In addi-tion, the slight decrease in monocyte migration by VEGF-A-NZ2C, compared with VEGF-A, was consis-tent with its decrease in VEGFR-1 binding in the receptor-binding assays (Fig 3) By contrast, the increase in monocyte migration induced by ORFVNZ2 -VEGF-DOglyc, compared with ORFVNZ2VEGF, was greater than the increase in VEGFR-1 binding observed in the receptor-binding assay (Fig 4)
Fig 3 Deletion of the C-terminus of ORFV NZ2 VEGF increases binding to VEGFR-1 and VEGFR-2 Immobilized VEGFR-1–Ig (A) or VEGFR-2–
Ig (B) fusion protein was incubated for 2 h with increasing concentrations of soluble VEGF-A, VEGF-A-NZ2C, ORFV NZ2 VEGF, ORFV NZ2
VEGF-DC or with medium alone Bound VEGF protein was detected with horseradish peroxidase-conjugated M2 anti-FLAG Ig Values are expressed as a binding index, defined as the mean increase in absorbance ± SEM at 450 nm over the background (n = 2) and are represen-tative of three separate experiments Soluble VEGFR-1–Ig (C) or VEGFR-2–Ig (D) fusion protein was incubated for 2 h with increasing concentrations of VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFVNZ2VEGF-DC or with medium alone The mixture was then added to VEGF-A-coated wells to capture free VEGFR–Ig, which was detected with a biotinylated sheep anti-human Ig SA-HRP conjugate The results are presented as the percentage of the maximal absorbance of VEGFR–Ig bound Values are expressed as mean ± SEM (n = 2) and are rep-resentative of three separate experiments The abilities of the VEGF mutants to bind and cross-link VEGFR-1 (E) or VEGFR-2 (F) were tested using specific bioassay cell lines Bioassay cells were washed and resuspended in dilutions of VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF and ORFV NZ2 VEGF-DC, or with medium alone for 48 h at 37 C DNA synthesis was quantified by [ 3 H]thymidine incorporation and b-counting Values were expressed as a proliferation index, defined as the mean increase in cell proliferation ± SEM over the background (n = 2) and are representative of two separate experiments An asterisk indicates a significant (P £ 0.05) difference from the background level recorded when no growth factor was added A cross indicates a significant (P £ 0.05) difference in receptor binding between ORFV NZ2 VEGF-DC and the equivalent concentration of ORFV NZ2 VEGF.
Trang 6In this study, we demonstrated a role for the highly
conserved O-glycosylated C-terminus in determining
the unique receptor-recognition profile and biological
activities of viral VEGFs Complete removal of the
C-terminus of ORFVNZ2VEGF increased its binding
to VEGFR-1 In addition, enzymatic removal of the
O-linked glycosylation within the C-terminal region
improved the ability of ORFVNZ2VEGF to activate
VEGFR-1, but not to the same level as that of the
deletion mutant These results indicate that the
C-ter-minal residues play a dominant role in preventing
ORFVNZ2VEGF recognition of VEGFR-1 and that the O-linked sugars associated with the C-terminus play a contributing role Interestingly, a previous study using Escherichia coli-expressed viral VEGF (strain D1701), which would not have contained O-linked glycosylation, reported minimal VEGFR-1 binding [11,12] The assay employed in this study directly mea-sures binding and cross-linking of chimeric VEGFR-1 and may be more sensitive than the endothelial cell-based assays used in the previous study
Removal of the C-terminal residues from ORFVNZ2 -VEGF also appeared to slightly enhance signaling via VEGFR-2, as measured in the BaF3 cell proliferation assay (Fig 3F) This suggests that the C-terminal region of viral VEGF interferes with both VEGFR-1 and VEGFR-2 binding This is consistent with previ-ous studies, which have shown that the binding sites for VEGFR-1 and VEGFR-2 overlap [23,31,32] A slight increase in VEGFR-2 binding by ORFVNZ2 -VEGF-DC was also seen in the competitive receptor-binding ELISA (Fig 3D) However, no difference in VEGFR-2 binding was observed in the direct receptor-binding ELISA (Fig 3B) This latter assay relies on immunodetection of the FLAG peptide fused to the VEGF protein Because the precise location of the FLAG peptide, in relation to the VEGF homology domain, varies between proteins, the accessibility of the FLAG peptide within the VEGF⁄ VEGFR complex may also differ This may influence the sensitivity of the assay and mask the slight differences in VEGFR-2 observed in other assays Interestingly, removal of the
Fig 4 Removal of the C-terminal O-linked glycosylation from ORFV NZ2 VEGF increases binding to VEGFR-1 and VEGFR-2 (A) Purified ORFVNZ2VEGF and ORFVNZ2VEGF-DOglyc were analyzed before and after enzymatic removal of N- and O-linked sugars The proteins were resolved under reducing conditions following treat-ment with the indicated combinations of enzymes as described in Experimental procedures Proteins were visualized by silver staining Molecular mass markers are indicated The ability of ORFVNZ2VEGF-DOglyc to bind and cross-link VEGFR-1 (B) or VEGFR-2 (C) was tested using specific bioassay cell lines Bioassay cells were washed and resuspended in dilutions of VEGF-A, ORFV NZ2 VEGF, ORFV NZ2 VEGF-DC and ORFV NZ2 VEGF-DOglyc, or with medium alone for 48 h at 37 C DNA synthesis was quanti-fied by [ 3 H]thymidine incorporation and b-counting Values were expressed as a proliferation index, defined as the mean increase in cell proliferation ± SEM over the background (n = 2) and are repre-sentative of two separate experiments An asterisk indicates a sig-nificant (P £ 0.05) difference from the background level recorded when no growth factor was added A cross indicates a significant (P £ 0.05) difference in receptor binding between ORFV NZ2
VEGF-DC or ORFV NZ2 VEGF-DOglyc and the equivalent concentration of ORFVNZ2VEGF.
Trang 7O-linked glycosylation from ORFVNZ2VEGF also
slightly increased the response seen in the VEGFR-2
BaF3 assay, to the level seen with the deletion mutant
(Fig 4C) This indicates that steric hindrance by the
O-linked sugars, and not the C-terminal residues, may
reduce the viral VEGF’s affinity for VEGFR-2
Recently the crystal structure of ORFVNZ2VEGF
was solved revealing a high similarity to the known
structures of other VEGF family members [22,25] The
C-terminal region, however, appeared to be highly
flex-ible and could not be resolved by crystallography Its
location adjacent to the receptor-binding face, as
illus-trated in Fig 6, suggests it is well placed to influence
the receptor-recognition profile of viral VEGF The
C-terminal residues may therefore form a direct
physi-cal block, preventing access of the VEGFRs to the
receptor-binding face Curiously, replacement of the
heparin-binding domain of VEGF-A with the
C-termi-nal residues of the viral VEGF did not significantly
alter the affinity of VEGF-A for VEGFR-1 or
VEG-FR-2 A simple explanation for this finding would be
that the C-terminal residues of the VEGF-A mutant,
due to the influence of adjacent residues or other
struc-tural features of VEGF-A, are not orientated towards
the receptor-binding face and are therefore unable
to influence receptor recognition Alternatively, the
inhibitory effects of the C-terminal residues on VEGFR binding may be dependent on their interac-tions with other structural features of the viral VEGF Removal of the C-terminal region from ORFVNZ2 -VEGF did not elevate -VEGFR binding to the level seen for VEGF-A, which suggests that other structural elements play a role in determining VEGFR specificity
of the viral VEGFs It has been hypothesized that the inability of viral VEGF to bind VEGFR-1 may be due
to the absence of a functional groove on the receptor-binding face of the homodimer [10,22] The lack of this groove may prevent viral VEGFs effectively binding the domain 2–3 linker region of VEGFR-1 In support
of this model, changing Arg46 to Iso within loop 1 of the groove region of ORFVNZ2VEGF increased its affinity for both VEGF receptors [22] In addition, replacement of both loop 1 and loop 3 of ORFVNZ2 -VEGF with those of -VEGF-A partially restored bind-ing to VEGFR-1 [22] Neither mutation, however, completely restored VEGFR-1 binding to the levels seen for VEGF-A [22] These findings in conjunction with the results of our study suggest that the groove region of the receptor binding face and the C-terminal residues make separate but additive contributions to the inability of viral VEGF to bind VEGFR-1 It would therefore be interesting to construct a viral
Fig 5 Removal of the C-terminal residues or O-linked glycosylation increases ORFV NZ2 VEGF-induced VEGFR-1-dependent chemotaxis of THP-1 monocytes THP-1 monocytes (1 · 10 5 cells) were added to the upper chamber of a Transwell insert The indicated concentrations
of VEGF-A, VEGF-A-NZ2C, ORFVNZ2VEGF, ORFVNZ2VEGF-DC and ORFVNZ2VEGF-DOglyc, or medium alone were added to the lower com-partments, the inserts were incubated for 6 h at 37 C and migrated cells that remained attached to the insert membrane were stained and counted as described in Experimental procedures Where indicated, THP-1 monocytes were preincubated with a neutralizing antibody against VEGFR-1 for 16 h and then assayed as described Results were expressed as a migration index, defined as the mean increase in cell migration, ± SEM, over background (n = 8) and are representative of three experiments Migration indexes that were significantly above that
of medium only are indicated by an asterisk (P £ 0.05) A cross indicates a significant (P £ 0.05) difference between cell migration induced in the presence and absence of antibody.
Trang 8VEGF mutant in which, loops 1 and 3 are replaced
with those from VEGF-A, and the C-terminal residues
are deleted, to ascertain whether these regions are the
determinants of VEGFR recognition or if additional
structural features are involved
The C-terminal residues are conserved among all
members of the viral VEGF family Recently, a variant
viral VEGF from BPSV has been reported that shows
greater recognition of VEGFR-1 and VEGFR-2 than
the other viral VEGFs [8] BPSVV660VEGF varies from
the other viral VEGFs in its C-terminal residues, yet
conserves the O-linked glycosylation sites This
sup-ports our finding that the specific amino acids within
the C-terminus influence the receptor-recognition pro-file of viral VEGFs It was also proposed that BPSVV660VEGF residues adjacent to the receptor-bind-ing face influence the orientation of loop 3 and the width of the groove, thereby affecting its ability to bind the VEGFRs [8] The orientation of the receptor-bind-ing face of BPSVV660VEGF may therefore be such that the C-terminal residues have less interaction with the loop regions Alternatively, this region of the other viral VEGFs, which differ from BPSVV660VEGF, maybe act
as a site of interaction with the C-terminal residues VEGFR-1 activation has been shown to mediate VEGF-induced immunostimulatory activities such as dendritic cell activation, monocyte migration, inflam-matory cytokine induction and other important anti-viral immune defenses [29,30,33,34] Removal of either the C-terminal residues or O-linked glycosylation from the viral VEGF resulted in an increase in VEGFR-1-mediated migration of THP-1 monocytes Conserva-tion of the C-terminal residues throughout the viral VEGF family may therefore represent a means of viral immune evasion The viral VEGFs, however, retain sufficient binding of VEGFR-2 to potently induce vas-cular dilation, dermal edema and proliferation of endothelial cells, thereby contributing to the prolifera-tive and highly vascularized nature of parapoxvirus lesions [15,35,36]
Experimental procedures
Expression vectors
Expression vectors for VEGF-A (murine VEGF iso-form 164) and ORFVNZ2VEGF were derived from pAPEX-3 and have been described previously [15] A DNA fragment containing nucleotides 4–368 of ORFVNZ2VEGF was amplified by PCR from viral DNA with the following primers: NZ2-DC 5¢ (5¢-AGCGCCCGGCGCGCCAGA AGTTGCTCGTCGGCATAC-3¢, AscI site underlined) and NZ2-DC 3¢ (5¢-ACTCGAACGCGTTCGTGGTCTA CAATCGCA-3¢, MluI site underlined) A DNA fragment containing nucleotides 4–458 of VEGF-A and 55 nucleo-tides from the C-terminus of ORFVNZ2VEGF was ampli-fied from pAPEX–mVEGF-A [15] with the following primers: mV-for 5¢ (5¢-CATGGCGCGCCTGATGAAC TTTCTGCTGTCTTGG-3¢) and mV-NZ2C 3¢ (5¢-CTGAC GCGTGCGGCGTCTTCTGGGCGGCCTTGTGGTCGT CGGTGGCGTGGTTGTGAACTTTGGTCTGCATTCA CATCGGCT-3¢) The PCR products were digested with AscI and MluI and ligated to pAPEX–mVEGF-A [15], from which the DNA sequence encoding VEGF-A but not the FLAG octapeptide (IBI⁄ Kodak, Rochester, NY, USA) had been removed by digestion with AscI
A
B
Fig 6 Structural elements of ORFV NZ2 VEGF involved in VEGFR
binding Ribbon representation of the structure of (A)
VEGF-A:Flt-1-D2 (VEGFR-1 domain 2) (PDB identifier 1QTY) [32] and (B)
ORFV NZ2 VEGF (PDB identifier 2GNN) [22] The VEGF dimers are
shaded gray with one monomer in a darker shade, and with
loops 1, 2 and 3, shaded blue, green and red, respectively
VEGFR-1 domain 2 is shaded purple Residues Ser94–Asn99 within loop 3
of one of the ORFV NZ2 VEGF monomers are missing, as they were
disordered due to their intrinsic flexibility The residues of VEGF-A
that form the groove implicated in VEGFR-1 binding, and the
resi-dues of ORFV NZ2 VEGF in the equivalent positions, are shaded
black The flexible C-terminus of each ORFVNZ2VEGF monomer is
labeled and shaded orange, with the disordered residues Thr120–
Arg133 drawn schematically as dashes The putative O-linked
glycosylation sites within the C-termini are shaded yellow.
Trang 9Protein synthesis from the expression vectors described
above gave rise to secreted polypeptides tagged with the
FLAG octapeptide at their C-termini The C-terminal
deletion mutant of ORFVNZ2VEGF was designated
ORFVNZ2VEGF-DC, and the domain exchange mutant of
VEGF-A with the ORFVNZ2VEGF O-glycosylated
C-ter-minus was designated VEGF-A-NZ2C
Recombinant protein production
Recombinant FLAG-tagged proteins were expressed in
293-EBNA cells, purified and quantitated as previously
described [15] A mock elution sample was obtained from
conditioned medium of pAPEX-3-transfected 293-EBNA
cells that underwent the same purification process
Protein deglycosylation
Proteins were treated with N-glycosidase F (Roche,
Mann-heim, Germany), sialidase (neuraminidase, Roche), and
O-glycosidase (Roche), and resolved by SDS⁄ PAGE and
visualized by silver staining or western blotting, as
previ-ously described [14]
ELISA receptor-binding assay
Maxisorp 96-well immunoplates (Nunc, Roskilde,
Den-mark) were coated with 500 ngÆmL)1 VEGFR-1–Ig or
VEGFR-2–Ig fusion proteins (R&D Systems, Minneapolis,
MN, USA) in coating buffer (15 mm Na2CO3, 35 mm
NaHCO3, pH 9.6) at 4C for 16 h and blocked with
0.5% BSA and 0.02% Tween 20 at room temperature for
1 h Plates were washed between steps with wash buffer
(NaCl⁄ Piand 0.02% Tween 20) Immobilized VEGFR–Igs
were then incubated with a titration of purified VEGFs at
room temperature for 2 h Captured VEGF was detected
by horseradish peroxidase-conjugated M2 anti-FLAG Ig
(Sigma, St Louis, MO, USA) and developed with
tetra-methylbenzidine substrate reagent (B&D Biosciences, San
Diego, CA, USA) and quantified by measuring absorbance
at 450 nm
ELISA competitive displacement receptor
binding assay
Maxisorp 96-well immunoplates were incubated with
400 ngÆmL)1 VEGF-A in coating buffer at 4C for 16 h
and blocked with 1% BSA and 0.02% Tween 20 at 37C
for 45 min Plates were washed between steps with wash
buffer Samples of purified growth factors, serially diluted
in binding buffer (NaCl⁄ Pi with 0.4% BSA, 0.02%
Tween 20 and 2 lgÆmL)1heparin in VEGFR-2 assays
only), were incubated with 300 ngÆmL)1human VEGFR-1–
Ig or VEGFR-2–Ig in non-absorbent plates at 25C for
1 h The mixture was then transferred to plates coated with VEGF-A and incubated at 25C for 1 h to capture the unbound VEGFR–Ig fusion protein The captured VEGFR–Ig fusion protein was detected by biotinylated anti-human Ig (Dako, Glostrup, Denmark) and streptavi-din-peroxidase (SA-HRP; Sigma) and tetramethylbenzidine substrate reagent and quantified by measuring the absor-bance at 450 nm
Bioassays for the binding and cross-linking of the extracellular domains of VEGFR-1 and VEGFR-2
Bioassays for monitoring the binding and cross-linking of VEGFR-1 and VEGFR-2, using BaF3-derived cell lines expressing chimeric receptors consisting of the extracellular, ligand-binding domains of human VEGFR-1 or mouse VEGFR-2 and the transmembrane and cytoplasmic domains of the erythropoietin receptor, were carried out as previously described [26,27] Briefly, the bioassay cell lines were incubated with various concentrations of purified growth factors for 48 h at 37C DNA synthesis was quan-tified by measuring [3H]thymidine incorporation during a further 16 h incubation
Chemotaxis assay
Chemotaxis assays using THP-1 monocytes were carried out in 24-well plates containing Transwell inserts of 5 lm pore size (Corning Costar, Corning, NY, USA), as previ-ously described [8] Briefly, monocytes were loaded into inserts with various concentrations of purified growth fac-tor in the bottom compartment and then incubated for 6 h
at 37C Nonmigrated cells were removed from the upper side of the filter membrane and the adherent cells on the lower side were fixed in gluteraldehyde then stained using Gill’s hemotoxylin For a quantitative assessment of migrated cells, a total of four fields of ·40 magnification from two different wells was counted
Enzymatic removal of O-linked glycosylation from ORFVNZ2VEGF
Purified protein (150 lg) was diluted in 0.05 m sodium phosphate buffer (pH 7) containing 0.1% SDS Twenty mU
of sialidase and 25 mU of O-glycosidase was added and the mixture was incubated at 37C for 3 h The protein was then re-purified and quantitated, as previously described [15]
Statistical analysis
Statistical analysis was performed using analysis of variance (single factor anova) with significant points of difference (P£ 0.05) determined using Tukey’s test
Trang 10This study was partially supported by the Health
Research Council of New Zealand Lyn Wise was
sup-ported in part by the University of Otago Health
Sciences Career Development Program Postdoctoral
Fellowship Award We thank Nicola Real for expert
technical assistance
References
1 Carmeliet P (2005) VEGF as a key mediator of
angio-genesis in cancer Oncology 69(Suppl 3), 4–10
2 Ferrara N (2004) Vascular endothelial growth factor:
basic science and clinical progress Endocrin Rev 25,
581–611
3 McColl BK, Stacker SA & Achen MG (2004)
Molecu-lar regulation of the VEGF family – inducers of
angio-genesis and lymphangioangio-genesis APMIS 112, 463–480
4 Olsson AK, Dimberg A, Kreuger J & Claesson-Welsh L
(2006) VEGF receptor signalling – in control of
vascu-lar function Nat Rev Mol Cell Biol 7, 359–371
5 Shibuya M & Claesson-Welsh L (2006) Signal
transduc-tion by VEGF receptors in regulatransduc-tion of angiogenesis
and lymphangiogenesis Exp Cell Res 312, 549–560
6 Zachary I (2003) VEGF signalling: integration and
multi-tasking in endothelial cell biology Biochem Soc
Trans 31, 1171–1177
7 Delhon G, Tulman ER, Afonso CL, Lu Z,
delaConcha-Bermejillo A, Lehmkuhl HD, Piccone ME, Kutish GF
& Rock DL (2004) Genomes of the parapoxvirus orf
virus and bovine papular stomatitis virus J Virol 78,
168–177
8 Inder MK, Ueda N, Mercer AA, Fleming SB & Wise
LM (2007) Bovine papular stomatitis virus encodes a
functionally distinct VEGF that binds both VEGFR-1
and VEGFR-2 J Gen Virol 88, 781–791
9 Lyttle DJ, Fraser KM, Fleming SB, Mercer AA &
Rob-inson AJ (1994) Homologs of vascular endothelial
growth factor are encoded by the poxvirus orf virus
J Virol 68, 84–92
10 Mercer AA, Wise LM, Scagliarini A, McInnes CJ,
Buettner M, Rhiza HJ, McCaughan CA, Fleming SB,
Ueda N & Nettleton PF (2002) Vascular endothelial
growth factors encoded by Orf virus show surprising
sequence variation but have a conserved, functionally
relevant structure J Gen Virol 83, 2845–2855
11 Meyer M, Clauss M, Lepple-Wienhues A, Waltenberger
J, Augustin HG, Ziche M, Lanz C, Buttner M, Rziha
HJ & Dehio C (1999) A novel vascular endothelial
growth factor encoded by Orf virus, VEGF-E, mediates
angiogenesis via signalling through VEGFR-2 (KDR)
but not VEGFR-1 (Flt-1) receptor tyrosine kinases
EMBO J 18, 363–374
12 Ogawa S, Oku A, Sawano A, Yamaguchi S, Yazaki Y
& Shibuya M (1998) A novel type of vascular endothe-lial growth factor, VEGF-E (NZ-7 VEGF), preferen-tially utilizes KDR⁄ Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain J Biol Chem 273, 31273–31282
13 Ueda N, Inder MK, Wise LM, Fleming SB & Mercer
AA (2007) Parapoxvirus of red deer in New Zealand encodes a variant of viral vascular endothelial growth factor Virus Res 124, 50–58
14 Ueda N, Wise LM, Stacker SA, Fleming SB & Mercer
AA (2003) Pseudocowpox virus encodes a homolog of vascular endothelial growth factor Virology 305, 298– 309
15 Wise LM, Ueda N, Dryden NH, Fleming SB, Caesar
C, Roufail S, Achen MG, Stacker SA & Mercer AA (2003) Viral vascular endothelial growth factors vary extensively in amino acid sequence, receptor-binding specificities and the ability to induce vascular permeabil-ity yet are uniformLy active mitogens J Biol Chem 278, 38004–38024
16 Wise LM, Veikkola T, Mercer AA, Savory LJ, Fleming
SB, Caesar C, Vitali A, Makinen T, Alitalo K & Stacker SA (1999) Vascular endothelial growth factor (VEGF)-like protein from orf virus NZ2 binds to VEGFR2 and neuropilin-1 Proc Natl Acad Sci USA
96, 3071–3076
17 Haig DM & Mercer AA (1998) Ovine diseases Orf Vet Res 29, 311–326
18 Mercer A, Fleming S, Robinson A, Nettleton P & Reid
H (1997) Molecular genetic analyses of parapoxviruses pathogenic for humans Arch Virol Suppl 13, 25–34
19 Groves RW, Wilson-Jones E & MacDonald DM (1991) Human orf and milkers’ nodule: a clinicopathologic study J Am Acad Dermatol 25, 706–711
20 Horner GW, Robinson AJ, Hunter R, Cox BT & Smith
R (1987) Parapoxvirus infections in New Zealand farmed red deer (Cervus elaphus) NZ Vet J 35, 41–45
21 Nagington J, Lauder IM & Smith JS (1967) Bovine papular stomatitis, pseudocowpox and milker’s nodules Vet Rec 81, 306–313
22 Pieren M, Prota A, Ruch C, Kostrewa D, Wagner A, Biedermann K, Winkler F & Ballmer-Hofer K (2006) Crystal structure of the Orf virus NZ2 variant of vascu-lar endothelial growth factor-E J Biol Chem 281, 19578–19587
23 Keyt B, Nguyen H, Berleau L, Duarte C, Park J, Chen
H & Ferrara N (1996) Identification of vascular endo-thelial growth factor determinants for binding KDR and Flt-1 receptors J Biol Chem 271, 5638–5646
24 Li B, Fuh G, Meng G, Xin X, Gerritsen M, Cunning-ham B & deVos A (2000) Receptor-selective variants of human vascular endothelial growth factor J Biol Chem
275, 29823–29828