The EMBO Journal Vol.16 No.13 pp.3898–3911, 1997
Proteolytic processing regulates receptor specificity
and activity of VEGF-C
D’Amore, 1996; Ferrara, 1997). Another growth factor ofVladimir Joukov, Tarja Sorsa, Vijay Kumar,
the VEGF family, placenta growth factor (PlGF), isMichael Jeltsch, Lena Claesson-Welsh1,
expressed predominantly in the placenta; it has minimalYihai Cao2, Olli Saksela3, Nisse Kalkkinen4
angiogenic activity, but is able to heterodimerize with andand Kari Alitalo5
to modulate the effects of VEGF (Maglione et al., 1991;
Molecular/Cancer Biology Laboratory and 3Department of Virology, Park et al., 1994; DiSalvo, 1995; Cao et al., 1996).
Haartman Institute, PL21 (Haartmaninkatu 3) and 4Biotechnology VEGF binds to and induces biological responses via two
Institute, PL52, University of Helsinki, 00014 Helsinki, Finland, tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-21Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, (Flk1/KDR), expressed mainly in endothelial cells (seeand 2Department of Cell and Molecular Biology, Karolinska Institutet,
Mustonen and Alitalo, 1995; Shibuya, 1995 for references).S-171 77, Stockholm, Sweden
PlGF is exclusively a ligand for VEGFR-1 (Park et al.,5Corresponding author
1994). VEGFR-1 and VEGFR-2 have seven immuno-
globulin (Ig)-like loops in the extracellular domain (EC),The recently identified vascular endothelial growth
a single transmembrane region and a tyrosine kinasefactor C (VEGF-C) belongs to the platelet-derived
domain, interrupted by an insert of 60–70 amno acidgrowth factor (PDGF)/VEGF family of growth factors
residues (de Vries et al., 1992; Terman et al., 1992;and is a ligand for the endothelial-specific receptor
Shibuya, 1995).tyrosine kinases VEGFR-3 and VEGFR-2. The VEGF
Three novel growth factors strikingly similar to VEGFhomology domain spans only about one-third of the
and PlGF have been identified recently. These factorscysteine-rich VEGF-C precursor. Here we have
are the VEGF-B/VEGF-related factor (VRF) (Grimmondanalysed the role of post-translational processing in
et al., 1996; Olofsson et al., 1996a), VEGF-C/VEGF-VEGF-C secretion and function, as well as the structure
related protein (VRP) (Joukov et al., 1996; Lee et al.,of the mature VEGF-C. The stepwise proteolytic pro-
1996) and c-fos-induced growth factor (FIGF) (Orlandinicessing of VEGF-C generated several VEGF-C forms
et al., 1996). VEGF-B is most closely related to VEGFwith increased activity towards VEGFR-3, but only
and is able to form heterodimers with it (Olofsson et al.,the fully processed VEGF-C could activate VEGFR-2.
1996a,b). VEGF-C and FIGF are similar in that both haveRecombinant ‘mature’ VEGF-C made in yeast bound
N- and C-terminal extensions flanking a VEGF homologyVEGFR-3 (KD J 135 pM) and VEGFR-2 (KD J 410
domain. Their C-terminal propeptides contain tandemlypM) and activated these receptors. Like VEGF, mature
repeated motifs with a spacing of cysteine residues typicalVEGF-C increased vascular permeability, as well as
of Balbiani ring 3 protein (BR3P) (Joukov et al., 1996;the migration and proliferation of endothelial cells.
Kukk et al., 1996; Lee et al., 1996; Orlandini et al., 1996).Unlike other members of the PDGF/VEGF family,
Thus, VEGF-C and FIGF comprise a novel subgroup ofmature VEGF-C formed mostly non-covalent homo-
the PDGF/VEGF family.dimers. These data implicate proteolytic processing as
The receptors for VEGF-B and FIGF have not yet beena regulator of VEGF-C activity, and reveal novel
identified, while VEGF-C is a ligand for two receptors,structure–function relationships in the PDGF/VEGF
VEGFR-3 (Flt4) (Joukov et al., 1996; Lee et al., 1996)family.
and VEGFR-2 (Joukov et al., 1996). VEGFR-3 differsKeywords: angiogenesis/growth factor/proteolytic
from the two other VEGFRs by being proteolyticallyprocessing/VEGF/VEGF-C
cleaved in the extracellular domain into two disulfide-
linked polypeptides (Aprelikova et al., 1992; Pajusola
et al., 1992, 1993; Galland et al., 1993) and by beingIntroduction
expressed in angioblasts of the head mesenchyme and in
the veins of embryos, and selectively in lymphatic endo-Angiogenesis, the formation of blood vessels by sprouting
thelia thereafter (Kaipainen et al., 1995). The paracrinefrom pre-existing ones, is regulated by a balance between
expression patterns of VEGF-C and VEGFR-3 in manypositive and negative regulators (Hanahan and Folkman,
tissues suggest that VEGF-C may function in angiogenesis1996). Vascular endothelial growth factor (VEGF) belongs
of the lymphatic vasculature (Kaipainen et al., 1995; Kukkto the platelet-derived growth factor (PDGF)/VEGF family
et al., 1996). On the other hand, the ability of VEGF-Cand is a major inducer of angiogenesis in normal and
to activate VEGFR-2 points to its possible functionalpathological conditions (Dvorak et al., 1995; Carmeliet
redundancy with VEGF.et al., 1996; Ferrara et al., 1996; Ferrara, 1997). The
The VEGF-C precursor is more than twice as large asbiological effects of VEGF are largely specific for endo-
the mature polypeptide, initially isolated from PC-3 cellthelial cells and include stimulation of their proliferation,
culture media (Joukov et al., 1996). This, combined withmigration and tube formation, and regulation of vascular
permeability (Dvorak et al., 1995; Klagsbrun and the unusual structure of the precursor, raised questions
3898 © Oxford University Press

Vascular endothelial growth factor C
about the role of its proteolytic processing, possibly
affecting receptor specificity, affinity and biological
activity. These questions have been addressed in the
present study.
Results
Characterization of VEGF-C antibodies and
mapping of peptide epitopes in reduced and
alkylated VEGF-C polypeptides
To study VEGF-C processing, we first generated antisera
recognizing two different regions of the VEGF-C pre-
cursor. Antiserum 882 was obtained by immunization with
a synthetic peptide corresponding to amino acid residues
2–18 of the N-terminus of mature secreted human VEGF-C
[residues 104–120 of the VEGF-C prepropeptide (Joukov
et al., 1996); EMBL, GenBank and DDBJ entry X94216].
Antiserum 905 was raised against the N-terminus of
the putative VEGF-C propeptide (residues 33–54) (see
Figure 3). These antisera and the extracellular domain of
VEGFR-3 (R-3EC) were then compared for their ability
to bind metabolically labelled recombinant VEGF-C from
the conditioned media (CM) of transfected 293-EBNA
cells. Both antibodies precipitated VEGF-C forms with
molecular masses of 15, 21, as well as a doublet of
29/31 kDa (Figure 1A, lanes 3 and 5, arrows). At higher
levels of VEGF-C expression, polypeptides of 43 and
58 kDa were also detected in the immunoprecipitates
(Figures 1B and 2). Importantly, both antibodies immuno-
precipitated the VEGF-C forms which were able to bind
Fig. 1. Recognition of VEGF-C polypeptides by antibodies andVEGFR-3 (Figure 1A, lane 2). The doublet of 29/31 kDa
VEGFR-3. 293-EBNA cells were transfected with VEGF-C,was the major component of the immunoprecipitates. The
metabolically labelled, and secreted polypeptides were isolated from21 kDa band was precipitated by antiserum 905 less the medium with subsequent analysis by SDS–PAGE and
efficiently than by antiserum 882, suggesting that a fraction autoradiography. (A) Wild-type VEGF-C was precipitated from CM
using protein A–Sepharose (PAS) only (lane 1), PAS and R-3ECof this form is bound to (a) polypeptide(s) containing also
(lane 2), antiserum 882 (lanes 3 and 4) or antiserum 905 (lanes 5 andthe N-terminal VEGF-C sequence recognized by antiserum
6). Lanes 4 and 6 show immunoprecipitation using the antisera905. Pre-treatment of the antisera with the corresponding pre-treated with the corresponding peptides used for immunizations.
peptides used for immunizations abolished their ability R-3EC means recombinant soluble extracellular domain of VEGFR-3.
(B) The antisera 882 and 905 were used to immunoprecipitate wtto immunoprecipitate the above-mentioned polypeptides
(lanes 1–4) or ∆N VEGF-C (lanes 5–8) from non-treated CM (lanes 1,(Figure 1A, lanes 4 and 6), indicating that they were
3, 5 and 7) or from CM treated with dithiothreitol and iodoacetamidespecific for VEGF-C.
to reduce and alkylate disulfide bonds (lanes 2, 4, 6 and 8).In order to explore the structure of the VEGF-C peptides
further, we compared the abilities of the antisera to bind other hand, the 21, 29 and 43 kDa forms were not affected
VEGF-C after reduction and alkylation of disulfide bonds. by the R102S mutation, suggesting that these polypeptides
This treatment prevented the precipitation of the 29 and contain peptide sequences located C-terminally of R102.
43 kDa polypeptides by both antisera and of the 21 kDa The specificity of antiserum 905 was demonstrated further
form by antiserum 905 (Figure 1B, lanes 1–4). Reduction by its inability to immunoprecipitate a VEGF-C mutant
and alkylation slowed down the migration of the VEGF- in which the N-terminal propeptide (residues 32–102) was
C polypeptides in SDS–PAGE, presumably by dissociating deleted (∆N, see Figures 1B and 3). The ∆N polypeptide,
intrachain bonds. Therefore, the absence of the 29 kDa immunoprecipitated with the 882 antiserum, migrated in
form in these conditions could have been due to its co- SDS–PAGE with a mobility corresponding to the size of
migration with the 31 kDa component of the doublet. To the deletion (~8 kDa) and it was co-precipitated with an
show that this is not the case, we generated an artificial equal amount of another pair of polypeptides of
N-glycosylation site in the N-terminal part of VEGF-C by 29–32 kDa, which were not recognized by antiserum
replacing Arg102 with a serine residue, resulting in the 882 upon reduction/alkylation of disulfide bonds. These
NSS(102) peptide (see Figure 3). This mutation slowed polypeptides were considered to represent heterogenously
down the mobility of the polypeptide normally migrating cleaved/glycosylated C-terminal fragments of the ∆N
at 31 kDa and therefore improved the separation of the precursor.
doublet, thus confirming the above conclusion (data not
shown). The mobilities of the 58 and 15 kDa forms were Biosynthesis, dimerization and proteolytic
also reduced to 64 and 21 kDa respectively, indicating processing of VEGF-C
that these VEGF-C polypeptides contained the appropriate To analyse the kinetics of VEGF-C biosynthesis and
processing, we performed metabolic pulse–chase labellingN-terminal peptide of VEGF-C (data not shown). On the
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