From the ¶ Laboratoire d'Ingénierie des
Macromolécules, the Laboratoire de Cristallographie
et Cristallogenèse des Protéines, the ** Laboratoire de
Biophysique Moléculaire, the
Laboratoire de Microscopie Electronique
Structurale, the §§ Laboratoire de
Résonance Magnétique Nucléaire, and the
Laboratoire de Spectrométrie de Masse des Protéines,
Commissariat à l'Energie Atomique/CNRS, Institut de Biologie
Structurale Jean-Pierre Ebel, 41, rue Jules Horowitz,
38027 Grenoble Cedex, France
Received for publication, March 28, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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Vascular endothelial cadherin (VE-cadherin) is a
transmembrane protein essential for endothelial cell monolayer
integrity (Gulino, D., Delachanal, E., Concord, E., Genoux, Y., Morand, B., Valiron, M. O., Sulpice, E., Scaife, R., Alemany, M., and Vernet, T. (1998) J. Biol. Chem. 273, 29786-29793).
This molecule belongs to the cadherin family of cell-cell adhesion
receptors, for which molecular details of homotypic interactions are
still lacking. In this study, a recombinant fragment encompassing the four N-terminal modules of VE-cadherin (VE-EC1-4) was shown to associate, in solution, as a stable
Ca2+-dependent oligomeric structure.
Cross-linking experiments combined with mass spectrometry demonstrated
that this oligomer is a hexamer. Gel filtration chromatography
experiments and analytical ultracentrifugation analyses revealed the
existence of an equilibrium between the hexameric and monomeric species
of VE-EC1-4. The concentration at which 50% of VE-EC1-4 is in its
hexameric form was estimated as 1 µM. The dimensions of
the hexamer, measured by cryoelectron microscopy to be 233 ± 10 × 77 ± 7 Å, are comparable to the thickness of adherens
endothelial cell-cell junctions. Altogether, the results allow us to
propose a novel homotypic interaction model for the class II
VE-cadherin, in which six molecules of cadherin form a hexamer.
The endothelium is a semipermeable barrier that controls the
passage of solutes and circulating cells between the bloodstream and
the neighboring tissues. The junctions between endothelial cells play a
crucial role in controlling this traffic. An adhesion receptor
belonging to the cadherin superfamily, vascular endothelial cadherin
(VE-cadherin)1 (1) is
specifically localized at these cell-cell junctions.
VE-cadherin is involved in the maintenance and restoration of
endothelium integrity (1-4). In fact, opening of VE-cadherin-mediated endothelial cell contacts was suggested to be a crucial step in neutrophil extravasation (5-7). VE-cadherin is also involved in
determining the vascular architecture since recent studies demonstrated
that targeted inactivation of VE-cadherin in transgenic mice leads to
embryonic mortality due to severe vasculogenic defects (8, 9).
Cadherins are Ca2+-dependent cell-cell adhesion
receptors that are able to bind cells together by means of homotypic
interactions. In these interactions, only identical cadherin types
interact. Consequently, cells expressing various cadherins segregate
into like groups when mixed. Due to this selective cell-cell
recognition property, cadherins are important regulators of
morphogenesis (10) by promoting clustering of cells with identical phenotypes.
The extracellular part of cadherins consists of five homologous protein
modules designated EC1-EC5, numbered from the N terminus to the C
terminus. Various lines of evidence have implicated the N-terminal
module EC1 in determining homotypic binding specificity (11, 12).
Fixation of Ca2+ on sites generally located between two
consecutive extracellular modules (13, 14) reduces cadherin
sensibility to proteolytic degradation, rigidifies the elongated
structure of cadherins (15, 16), and is required for homotypic
interactions (17). The cytoplasmic domains of cadherins are homologous
and interact either with Comparison of cadherin sequences provides evidence that VE-cadherin is
structurally different from class I cadherins, which include N-
and E-cadherins. For example, the EC1 module of VE-cadherin lacks the
sequence motif HAV (1) involved in the adhesion activity of class I
cadherins (11, 12). Consequently, VE-cadherin was classified in the
class II cadherin group (19), inferring that it might have particular
adhesive properties. The elucidation of how the extracellular region of
VE-cadherin assembles is fundamental to understanding the mechanism of
cell-cell adhesion in the endothelium and to clarifying the process by
which VE-cadherin modulates the transmigration of leukocytes. In this
study, we focus on the behavior in solution of VE-EC1-4, a fragment
consisting of the four N-terminal modules of VE-cadherin.
The molecular determinants at the basis of homotypic interactions and
organization of cadherins in the adherent structures at cell-cell
junctions have been studied by analyzing the behavior in solution of
cadherin fragments and by determining their high resolution structures.
Some of the results are conflicting. For example, the E-cadherin
N-terminal module (E-EC1) remains monomeric in solution (20, 21),
whereas the equivalent fragment of N-cadherin (N-EC1) self-associates
as dimers (14), suggesting that the homoassociation mechanism is
different according to the cadherin type. Comparison of the crystal
structures from various cadherin fragments is also ambiguous. Thus, the
N-EC1 crystal structure revealed the presence of parallel dimer
interfaces (cis-dimers) and antiparallel alignments
(trans-dimers) (14). It was suggested that both types of
association reflect the interactions occurring between cadherins at the
cell surface. cis-Dimers were proposed to mimic the
alignment of two molecules emerging from the same cell surface, whereas
trans-dimers may correspond to molecules protruding from
opposing cell surfaces. The presence of these parallel and antiparallel
dimers inspired Shapiro et al. to build a zipper
model for the molecular organization of cadherins at the cell surface,
in which each extracellular module contributes to lateral dimerization
(14, 22). Until now, no direct evidence for lateral dimerization
of the EC2-EC5 modules has been presented.
This model is in conflict with conclusions drawn from the crystal
structures of the two module fragments of E-cadherin (E-EC1-2) (23,
24) and N-cadherin (N-EC1-2) (25). In fact, both EC1-2 fragments
adopt twisted X structures maintained by a network of calcium-mediated
interactions that fixes the orientation between the EC1 and EC2 modules
(26). Formation of the antiparallel dimers was not observed in the
X-shaped E-EC1-2 and N-EC1-2 structures.
Studies to elucidate mechanisms involved in homotypic interactions have
essentially been performed on class I cadherins and more specifically
on murine E- and N-cadherins or on Xenopus C-cadherin. In
this study, we present results concerning the mechanism of homotypic
adhesion for the class II VE-cadherin. We show that the recombinant
fragment including the four N-terminal modules of VE-cadherin
self-associates in a Ca2+-dependent hexameric
structure. Its visualization by electron microscopy allows us to
constrain models for homotypic VE-cadherin associations.
Construction of the Recombinant VE-EC1-4 Expression
Vector--
Two oligonucleotides (oligonucleotide A, 5'-TATACAT
ATG GAT TGG ATT TGG AAC CAG ATG CAC-3'; and B, 5'-CTC
GAA TTC TCA CTC CGG GGC ATT GTC ATT
CTC ATC-3') were synthesized and used as primers to produce, by
polymerase chain reaction technology, a cDNA fragment containing
the sequence from nucleotides 166 to 1458 and encoding VE-cadherin
(Asp1-Glu431). The DNA template was isolated
from Escherichia coli strain XL1-Blue transformed with a
full-length VE-cadherin cDNA inserted into the pBluescript vector
(27).
Oligonucleotides A and B were designed to generate a 5'-NdeI
restriction site containing an ATG start codon (boldface in sequence above) and a 3'-EcoRI restriction site (underlined)
positioned immediately after an in-frame TGA stop codon (italic). The
polymerase chain reaction fragments were ligated with plasmid
pCR-Script (pCR-ScriptTM Amp SK(+) cloning kit, Stratagene,
La Jolla, CA) and then inserted, after digestion with NdeI
and EcoRI, into the pET-30b+ expression vector
(Novagen, Madison, WI). The resulting pET-VE-cadherin vector contained
the DNA sequences coding for VE-cadherin (amino acids 1-431) fused to
an N-terminal methionine. Prior to the expression of the protein, the
cDNA constructs were sequenced to verify that mutations had not
arisen during the polymerase chain reaction.
Purification of the Recombinant Fragments--
To obtain the
fragment designated VE-EC1-4 (Asp1-Glu431),
E. coli host strain BL21(DE3) was transformed with the
pET-VE-cadherin plasmid and grown in Lennox broth base containing 30 mg/liter kanamycin. Production of the recombinant fragment was
performed according to the supplier's instructions (Novagen,
Madison, WI) using a 100 µM
isopropyl- Determination of the Protein Concentrations--
The absorbance
coefficients for the VE-EC1-4 fragments, estimated from the amino acid
composition using molar absorption coefficients of 5540 M Analytical Gel Filtration Chromatography--
Multimers of
VE-EC1-4 were fractionated on the Superdex S200 analytical gel
filtration column (10,000-500,000 fractionation range) using a
biologic chromatography system (Bio-Rad, Ivry/Seine, France).
A molecular mass calibration curve was established using the following
standard globular proteins: ribonuclease A (13.7 kDa), chymotrypsinogen A (25 kDa; hydrodynamic radius
(Rh) = 20.9 Å), ovalbumin (43 kDa;
Rh = 30.5 Å), albumin (67 kDa;
Rh = 35.5 Å), aldolase (158 kDa;
Rh = 48.1 Å), catalase (232 kDa;
Rh = 52.2 Å), and ferritin (440 kDa;
Rh = 61 Å) (29).
A plot of (
The time required to reach equilibrium between the monomeric and
hexameric forms, after dilution of VE-EC1-4 from 45 to 2 µM, was evaluated at 20 and 4 °C. The hexameric form
rapidly decreased initially and stabilized after 30 h at 20 °C
and after >2 weeks at 4 °C.
The equilibrium between the monomeric (M) and hexameric (H) forms of
VE-EC1-4 can be described as 6M
The unit µM+5 for
KD(H) is dictated by the equation
KD(H) = [M]6/[H]. This
unusual unit of measurement for KD(H)
does not allow direct comparison with previous results established for
the monomer Matrix-assisted Laser Desorption Ionization (MALDI) Mass
Spectrometry--
Mass spectra of the recombinant fragment VE-EC1-4,
cross-linked or not, were obtained with a time-of-flight mass
spectrometer (VoyagerElite Xl, Perseptive Biosystems, Framingham, MA)
equipped with a 337-nm nitrogen laser. Aliquots of 0.5 µl of the
protein solution and 0.5 µl of the matrix solution were mixed on a
stainless steel sample plate and dried in the air prior to mass
spectrometry analysis. External calibration was performed with bovine
serum albumin (m/z 66452; Sigma). All experiments were
performed using a saturated solution of 2,5-dihydroxybenzoic acid
prepared in a 50% (v/v) solution of acetonitrile and aqueous 0.1%
trifluoroacetic acid. The accuracy of MALDI molecular mass
determinations is between 0.01 and 0.05%.
Cross-linking of VE-EC1-4--
The recombinant fragment
VE-EC1-4 was partially cross-linked using
N-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC; Sigma) or ethylene glycol bis(succinimidyl succinate) (EGS). The molar ratio
between the recombinant fragment and the cross-linker reagent was
adjusted by varying the concentrations of the cross-linking reagents
from 1 to 3 mM and from 0.1 to 3 mM for EDAC
and EGS, respectively. In fact, these experiments were performed at
20 °C in MES (pH 7.0) for 2 h and in 2 mM Tris (pH
8.5) for 15 min for EDAC and EGS, respectively; and the concentration
of VE-EC1-4 was maintained at 110 µM. Electrophoresis
analyses were then performed on 4-15% gradient Phast gels (Amersham
Pharmacia Biotech).
To determine the oligomeric state of VE-EC1-4, the fragment was
cross-linked with EGS used at a 3 mM final concentration in 50 mM Tris-HCl, 100 mM NaCl, and 5 mM CaCl2 (pH 7.2) for 30 min at room
temperature under gentle stirring. Prior to the reaction, the
cross-linker was solubilized in Me2SO at a concentration of 50 mM to minimize pH and calcium concentration changes and
to avoid addition of a large volume. To maintain the oligomeric state of the fragment, the concentrations of VE-EC1-4 and calcium were adjusted to 20 µM and 5 mM, respectively. The
cross-linking reaction was terminated by adding 100 mM
glycine and cooling the reaction flask in ice.
Analytical Ultracentrifugation--
Sedimentation velocity and
sedimentation equilibrium experiments were performed using a Beckman
Model XL-A analytical centrifuge. Sedimentation velocity experiments
were carried out at 20 °C in 1.2- or 0.3-cm path length
double-sector cells with quartz windows and run at 42,000 rpm. The
recombinant fragment was diluted at 1.2 and 26.2 µM in 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and
5 mM CaCl2 5 days before the sedimentation
velocity experiments. Sample volumes of 400 µl were centrifuged, and
radial scans were taken at 12-min intervals. Data analysis was
performed using the computer programs SVEDBERG (30) and DCDT (31),
provided by J. S. Philo and W. F. Stafford, respectively. The
partial specific volume (v) for VE-EC1-4 (0.733 ml/g), the
density (
Sedimentation equilibrium experiments were performed in 1.2-cm double-
and six-channel centerpieces at 4500, 6000, and 12,000 rpm at VE-EC1-4
concentrations of 3, 12.5, 25, and 40 µM. After 24-48 h
of centrifugation, scans were compared at 2-h intervals to ensure that
equilibrium had been reached. An experimental base line was determined
at 42,000 rpm. Data of the absorbance (A) at 277 nm as a
function of radial position (r) were analyzed considering a
reversible self-associating system between monomers and hexamers using
the Multifit analysis program (Version 4.01, Beckman Instruments). An
apparent association constant (Kapp; in
absorbance units) was obtained from the set of 12 profiles. For each
profile, A1(r0), the
absorbance for monomers at the radial position
r0 (the first point of each data set), and
E, the residual base line, were fitted as follows:
A = A1(r0)exp(m1(1 Electron Microscopy--
Negatively stained specimens of 0.05 mg/ml VE-EC1-4 were prepared using uranyl acetate by the mica
floatation technique and observed with a Philips CM200 electron
microscope operating at 120 kV. Frozen hydrated specimens of 0.4 mg/ml
VE-EC1-4 were prepared on a holey carbon film supported on a
400-mesh copper grid. A 4-µl sample of the specimen was deposited
onto the grid; the excess was blotted with filter paper for 1-2 s; and
the grid was then rapidly plunged into liquid ethane at
Micrographs were digitized using an Optronics microdensitometer with a
step size of 25 mm, which corresponds to a pixel size of 3.69 Å for
the specimen, taking into account the corrected microscope
magnification of ×67,700. The length and width of the images of 100 particles were measured on a Silicon Graphics computer using the Semper
6 Plus image analysis package.
Individual particles were selected, masked, and normalized by
subtracting the mean and any density gradient present and normalizing the S.D. The particles were centered and aligned by cross-correlation procedures prior to averaging. The variation between particle images
suggests that they are in different orientations about their long axis,
so internal details are lost in the averaged image, but the overall
shape is clear.
Expression and Purification--
A recombinant fragment of human
VE-cadherin encompassing the four extracellular N-terminal modules
(EC1-EC4) was expressed in E. coli in sufficient amounts
for biophysical and biochemical studies (Fig.
1). The boundary of this fragment,
designated VE-EC1-4, was essentially defined according to the domain
organization proposed by Tanihara et al. (19). To avoid
cross-linking and the associated problems of insolubility, the fifth
module of VE-cadherin (EC5), the only module that may contain disulfide
bridges, was excluded (Fig. 1). Moreover, to increase solubility of the
fragment, the hydrophobic amino acid Phe432 in the
C-terminal region of the EC4 module was also deleted. Consequently, the
fragment starts immediately after the 47-amino acid long signal peptide
at Asp1 and ends at Glu431 of the mature
VE-cadherin molecule. A foreign methionine was added as a start signal
at the N terminus of the fragment.
After purification of VE-EC1-4, a single 49-kDa band was observed by
SDS electrophoresis. N-terminal sequencing (Fig. 1) gave a unique
signal, confirming the purity and integrity of the extremity of the
molecule. Moreover, the molecular mass of the purified fragment (48,948 Da), obtained by MALDI mass spectrometry, matches the calculated one
(48,941 Da), indicating the absence of any internal cleavage within the
molecule. Circular dichroic measurements attested that VE-EC1-4
possesses a large fraction of Self-association of VE-EC1-4--
The capacity of the recombinant
fragment to self-associate was evaluated by gel filtration
chromatography (Fig. 2). Purified VE-EC1-4 was first injected at a concentration of 1 µM
onto an analytical gel filtration column (Fig. 2A). Two
peaks with elution volumes equal to 12.4 ± 0.1 ml (peak I;
Rh = 68 ± 1 Å) and 15.74 ± 0.05 ml
(peak II; Rh = 38.6 ± 0.8 Å) were detected on
the chromatograms. SDS electrophoresis analysis of the chromatographic
fractions corresponding to elution peaks I and II showed the presence
of a unique 49-kDa band (Fig. 2B). This attested that both
elution peaks correspond to the VE-EC1-4 fragment, thus excluding the
possibility that peak II was due to a contaminating protein. It can be
predicted that peaks I and II contain the oligomeric and monomeric
VE-EC1-4 forms, respectively.
Oligomeric State of VE-EC1-4--
To determine the oligomeric
state of VE-EC1-4 in solution, it was cross-linked using the
homobifunctional cross-linking reagents EGS (which reacts covalently
with amino groups) and EDAC (which reacts covalently with amino and
carboxylic groups). Before adding EGS or EDAC, the concentrations of
both VE-EC1-4 and Ca2+ were adjusted so that the fragment
was mostly in its multimeric form. Electrophoresis analysis of the
cross-linked product revealed the formation of a multiple band pattern
that varied with the concentration of the cross-linking reagent. In the
presence of 0.1 mM EGS, six bands clearly appeared on the
gel (Fig. 3A, lane 1). As the concentration of EGS was increased, the intensity of the five lower molecular mass bands decreased, whereas that of the
upper band increased (Fig. 3A, lane 2). At 3 mM EGS, only the higher molecular mass band was detected
(Fig. 3A, lane 3). Cross-linking with EDAC was
comparatively less efficient since the six previously mentioned bands
were observed at an EDAC concentration of 3 mM instead of
0.1 mM for EGS (Fig. 3A, lane 5),
whereas three bands were observed with 1 mM EDAC
(lane 4). The five lower molecular mass bands are partially
cross-linked VE-EC1-4 products, and the highest one reflects the
oligomeric association found in solution. By attributing a degree of
oligomerization to each band (Fig. 3A), it was possible to
relate the theoretical molecular mass to the electrophoretic mobility.
Indeed, a linear curve was obtained by plotting the log theoretical
molecular mass against the corresponding electrophoretic mobility for
each band detected on the gel (data not shown). This suggests that the
six discrete bands correspond to multimers containing one, two, three,
four, five, or six covalently cross-linked molecules from the bottom to
the top of the gel.
Using MALDI mass spectrometry, the molecular mass of the fragment
cross-linked with 3 mM EGS was measured to be 315 kDa.
There are 26 lysines on VE-EC1-4, all susceptible to coupling with
EGS. Assuming saturating cross-linking, the molecular mass of the
VE-EC1-4 multimer can be calculated as (49 + 26 × 0.224) × n, where 49 corresponds to the mass of a VE-EC1-4 monomer,
n is the degree of oligomerization, and 0.224 corresponds to
the mass of an EGS molecule coupled to the fragment. Accordingly, the
molecular masses were theoretically evaluated as 274 and 329 kDa for
pentameric and hexameric associations, respectively. Due to the lack of
accessibility to EGS of some lysines buried inside the core of the
protein, these calculated values are likely overestimated. This
suggested that the 315-kDa experimental value corresponds to a
hexameric association.
The oligomeric state of VE-EC1-4 was determined independently by
analytical centrifugation. Sedimentation velocity experiments were
performed using VE-EC1-4 at 1.2 and 26 µM. At 1.2 µM, the concentration profiles exhibited bimodal
boundaries (Fig. 3B). Consistent with this observation,
g(s*) versus s*, generated
from this set of concentration profiles with the software DCDT, showed a double distribution (Fig. 3C). This reflected the
heterogeneity existing within the molecular masses of the VE-EC1-4
protein at 1.2 µM. The curve fitting returned values of
s = 2.9 ± 0.1 S
(s20,w = 3.0 S) and 9.8 ± 0.1 S
(s20,w = 10.2 S) for the left-hand and
right-hand peaks, respectively. A good agreement was observed between
the s values generated according to the programs SVEDBERG and DCDT. At 26 µM VE-EC1-4, the lightest species
represented <5%, whereas the heaviest species migrated with a
sedimentation coefficient of 10.2 S
(s20,w = 10.6 S) (Fig.
3C).
The molar masses for each distribution were then calculated from the
sedimentation coefficients combined with the hydrodynamic radius
determined by gel filtration chromatography. Under these conditions,
the molar masses were estimated to 49 and 301 kDa for the 2.9 S and
10.2 S distributions, respectively. These results indicate that the 2.9 S peak distribution corresponds to the monomeric form of VE-EC1-4,
whereas the 10.2 S peak distribution corresponds to the hexameric form
of VE-EC1-4.
To determine the molecular mass by a method that does not depend on the
shape of the molecule, sedimentation equilibrium experiments were
performed. VE-EC1-4 at various initial concentrations was equilibrated
at 4500, 6000, and 12,000 rpm (Fig. 4,
A and B).The mean molecular mass increased with
increasing protein concentration and reached a plateau value of 285 kDa, compatible with a hexameric form (Fig. 4C).
Altogether, the results from gel filtration chromatography, analytical
ultracentrifugation, and MALDI spectrometry demonstrate that the
multimer of VE-EC1-4 is a hexamer. Moreover, variations in the
relative amounts of VE-EC1-4 hexamer over monomer as a function of
protein concentration show that both species are at equilibrium in solution.
Dissociation Rate Constants of the Hexameric Form--
To quantify
the equilibrium between the monomer and hexamer, the stability of the
hexamer was first analyzed by gel filtration chromatography. It was
noticed that, following dilution of the VE-EC1-4 stock solution, the
level of hexamer relative to monomer slowly decreased with time.
Equilibrium between the monomeric and hexameric forms required at least
30 h at 20 °C (Fig.
5A). At 4 °C, the
dissociation rate was considerably slowed down since equilibrium was
reached 2 weeks after the dilution (data not shown). From this kinetic
study, the hexamer dissociation rate constants (kd) at 4 and 20 °C were determined from a
plot of ln[H]0/[H] versus t,
assuming that the concentration of the monomer is negligible at the
beginning of the time course (where t,
[H]0, and [H] are the dissociation time and the hexamer
concentrations at time 0 and t, respectively). The
dissociation rate constants of the hexamer (kd) were
calculated to be 6 × 10 Apparent Dissociation Constant of the Hexamer--
The parameters
of the equilibrium between the monomeric and hexameric forms were
further established by loading VE-EC1-4 at a range of initial
concentrations onto the gel filtration column. The chromatographic runs
were carried out at 4 °C, taking advantage of the low hexamer
dissociation rate constant at this temperature. Indeed, we verified
that, at 4 °C, the hexamer was not dissociated during the
chromatographic runs despite the occurring dilution.
The chromatographic profiles showed that, as loading concentrations of
VE-EC1-4 were increased, a simultaneous increase in the intensity of
peak I and a decrease in that of peak II were observed (Fig.
2A). Based on the previous peak attribution, it was deduced
that increases in VE-EC1-4 concentration favor formation of the
hexamer, thus confirming results generated from sedimentation velocity experiments.
The monomer/multimer composition could be estimated from the areas of
peaks I and II seen on the chromatographic profiles. The relationship
between the percentage of multimer in solution and the initial
concentration of VE-EC1-4 was then established (Fig. 5B).
At VE-EC1-4 concentrations of 0.5 and 37.5 µM, 50 and 96% of the fragment was in the hexameric form, respectively. The apparent dissociation constant KD(H), as
defined under "Materials and Methods," was estimated as 0.3 µM5.
Sedimentation equilibrium experiments were also used to determine the
apparent dissociation constants (KD(H))
independently of the determination of Rh. As shown
in Fig. 4, our data were successfully described by the simplest model
considering an equilibrium between the monomeric and hexameric forms of
VE-EC1-4. A KD(H) value of 0.5 µM5, similar to that determined from gel
filtration chromatography experiments, was deduced. From this
KD(H) value,
C50% was estimated as 1 µM
(see "Materials and Methods"). This value is comparable to that
experimentally determined by gel filtration chromatography (0.5 µM).
Ca2+ Stabilization of the Hexamer--
To study the
Ca2+ dependence of the monomer Electron Microscopy of VE-EC1-4--
In the presence of 5 mM Ca2+ at a protein concentration of 2 µM, conditions for which the hexameric form is the
preponderant species, the electron micrographs of negatively stained
VE-EC1-4 show a quite homogeneous population of particles with an
elongated ellipsoidal shape (Fig.
6A). In the presence of EGTA,
smaller objects with various ill defined shapes replaced these
structures, indicating that formation of the hexamer is dependent on
the presence of Ca2+ (data not shown).
A more precise picture of the shape of the hexamer was obtained by
VE-EC1-4 in the frozen hydrated state (Fig. 6, B and
C). It has a hollow cigar shape when viewed from the
side (Fig. 6C) and appears as an annulus when viewed
from the end (Fig. 6B). There is apparently a specific
interaction between VE-EC1-4 and the air/water interface, so there are
only two views of the molecule. In each view, the protein density of
the hexamer is subdivided into smaller domains as expected for
VE-EC1-4, but it is too variable from particle to particle to allow
determination of the multimeric state from these images. The average
dimensions of 100 hexameric VE-EC1-4 particles, visualized by
cryoelectron microscopy, are 233 ± 10 × 77 ± 7 Å.
A recombinant fragment encompassing the four extracellular modules
of VE-cadherin was produced in E. coli. Recently, this fragment was demonstrated to be functional since it is able to inhibit
the aggregation of Chinese hamster ovary cells transfected with
VE-cadherin in a concentration-dependent manner (27). Here, we demonstrated that this fragment spontaneously forms a unique well
organized multimer. This study provides the first in vitro evidence that the extracellular part of cadherins can homoassociate in
solution in a structure possessing an oligomeric order higher than 2. Furthermore, this calcium-dependent oligomer self-assembles at lower concentrations than those previously published for cadherin dimeric associations (20).
Analysis of the oligomeric state of the VE-cadherin VE-EC1-4 fragment
both by analytical centrifugation and, after cross-linkage, by mass
spectrometry revealed a hexameric association. This is the first
description of such an organization in solution for a cadherin. Until
now, the extracellular recombinant fragments of other cadherins, such
as E-cadherin, were demonstrated either to stay monomeric (15, 33) or
to form dimers, as for Xenopus C-cadherin (34). Oligomeric
self-associations were observed for the extracellular region of
E-cadherin; but in this case, the molecule was fused to the assembly
domain of the cartilage oligomeric matrix protein to force
oligomerization (24, 33). Removal of the fifth module (EC5) to
the extracellular part of VE-cadherin does not change the properties of
the molecule since we recently verified that the VE-EC1-5 fragment
(Asp1-Asp542), which corresponds to the whole
extracellular domain of the receptor, was also able to form a hexameric structure.
Gel filtration chromatography and ultracentrifugation experiments
showed evidence of a concentration-dependent monomer Our results also indicate that the hexamer is able to dissociate after
dilution, with a very slow kd dissociation rate
constant that increases from 10 Formation of the hexameric structure observed for the VE-cadherin
fragment is strictly dependent on the presence of Ca2+.
This result reflects the behavior of cadherins expressed at the cell
surface and is in agreement with studies done on various cadherin-derived recombinant fragments. Indeed, except for the earliest
works of Shapiro et al. (14) and Brieher et al.
(34), multimers of cadherins were formed only in the presence of
Ca2+ (20, 23-25, 35).
Cryoelectron microscopy allows us to visualize the hexameric structures
as elongated cigar-shaped particles. From these images, the average
dimensions of the hexamers were evaluated as 233 ± 10 × 77 ± 7 Å (Fig. 6, B and C). The length of
the hexameric structure is comparable to the distance separating two
adjacent endothelial cells, estimated by electron microscopy to be
~200 Å (36). It is not clear from the cryoelectron microscope images
whether the six cadherin molecules adopt a parallel or an antiparallel
orientation within the hexamer. Determination of the relative
orientation of the six molecules of VE-cadherin within the hexamer is a
key step in understanding how adherent junctions are formed between endothelial cells. A parallel orientation would indicate formation of
hexamers with molecules emanating from a single cell surface, whereas
an antiparallel orientation would indicate formation of adhesive
hexamers consisting of molecules protruding from neighboring cells. The
crystal structure of the hexameric VE-EC1-4 complex should resolve
this question. Nevertheless, the fact that VE-cadherin was
demonstrated, by atomic force microscopy, to be able to constitute multiple trans-interactions is compatible with an
antiparallel orientation of the hexamer (37). This
trans-orientation is also in agreement with results recently
published demonstrating that formation of antiparallel dimers requires
Ca2+, in contrast to parallel dimers (38, 39). Moreover, as
attested by the determination of the N-EC1 structure (14) and as
deduced from the electron microscopy measurement for the E-EC1-5
cadherin construct (i.e. 220 Å) (33), the average dimension
of a cadherin module is 45 ± 1 Å. Consequently, the length of
the VE-EC1-4 hexamer (233 ± 10 Å) corresponds
approximately to the length of five cadherin modules. Thus, this is
compatible with a model in which the six monomers are disposed in an
antiparallel fashion to form a barrel with three overlapping modules
along its length.
Due to its relatively high affinity constant, the hexamer appears at
low concentrations. Placed in a cellular context, this result indicates
that this hexameric structure may be physiologically relevant at the
cell-cell junctions where VE-cadherin is concentrated. In fact, this
hexameric assembly may constitute a basic association motif whose
assembly may promote formation of the continuous sealing region between
endothelial cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
- or
-catenins in a mutually exclusive
fashion. By connecting cadherins to the actin-based cytoskeleton,
binding of
-catenin to
- or
-catenins (18) strengthens
cadherin-mediated cell adhesion.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside induction for 4 h. The cells were then harvested and resuspended in 40 mM Tris (pH 7.4) containing 5 mM EDTA and a
protease inhibitor mixture (Complete tablets, Roche Molecular
Biochemicals, Meylan, France). After sonication, the insoluble proteins
were collected by centrifugation and washed four times with 0.5 M urea and 0.5% Triton X-100 dissolved in Tris-HCl (pH
7.5). The insoluble proteins were then dissolved in Tris-HCl containing
8 M urea and chromatographed on an anion-exchange Mono
Q column (Amersham Pharmacia Biotech, Orsay, France) using a NaCl
gradient (0-300 mM). Fractions, eluted at a NaCl
concentration of 120 mM, were pooled and concentrated to 3 mg/ml prior to the refolding according to the following method.
Purified urea-containing fractions were diluted in renaturing buffer
(50 mM Tris-HCl (pH 8) at 4 °C) so that the final
concentrations of protein and urea were in the ranges of 20-50 µg/ml
and 0.1-0.2 M, respectively. To facilitate the refolding
of the protein, CaCl2 was then added at a 5 mM
final concentration, and the solution was incubated overnight without
stirring at 4 °C before addition of NaCl (150 mM final
concentration). After concentration to 2 mg/ml, the protein solution
was filtered (0.22 µm) and eluted on a Superdex S200 column (Amersham
Pharmacia Biotech) to eliminate aspecific aggregates.
1 cm
1
for tryptophan residues and 1280 M
1 cm
1
for tyrosine residues (20, 28), were evaluated to 45,800 M
1 cm
1
.
log Kav)1/2 versus
Rh for the globular proteins previously mentioned
allowed us to determine the hydrodynamic radius of the various
VE-EC1-4 forms observed on chromatograms (Kav = (Ve
Vo)/(Vt
Vo), where Ve is the elution
volume for the protein, Vo is the column void
volume, and Vt is the total bed volume (24 ml)). A
knowledge of the Rh associated with the
sedimentation coefficient allowed an accurate determination of the
VE-EC1-4 monomer and multimer molecular masses (see
"Analytical Ultracentrifugation").
H, with an apparent dissociation
constant KD(H) = [M]6/[H], where [M] and [H] correspond to the
concentrations of the monomer and hexamer, respectively. To estimate
KD(H), serial dilutions of VE-EC1-4
were prepared from the 37.8 µM stock solution and
equilibrated at 20 °C for periods longer than 48 h. 100 µl of
each VE-EC1-4 dilution were injected onto the Superdex S200 column
using a flow rate of 0.2 ml/min and 20 mM Tris (pH 8.5)
running buffer containing 200 mM NaCl and 5 mM
CaCl2. To minimize hexamer dissociation during
chromatographic runs, experiments were performed at 4 °C. To verify
the stability of the hexamer during chromatographic runs, fractions
containing the multimer were reinjected onto the column immediately
after the first separation. No significant dissociation was noticed for
VE-EC1-4 loading concentrations ranging 0.4 from 45 µM,
despite dilution occurring during the chromatographic process. Thus,
the very slow dissociation rate at 4 °C allowed us to estimate the
percentage of the monomeric (% M) and hexameric (% H) forms from
chromatographic profiles. Practically, they were calculated, from their
respective chromatographic peaks, after fitting and integration with
two gaussian curves. From these percentages, [M] and [H] were
deduced using the following equations: [H] = (% H)Ci/6) × 100 and [M] = (% M)Ci/100, where Ci corresponds to
the total concentration of VE-EC1-4. Then, from the straight line
ln[H] = f(ln[M]), KD(H) could be deduced.
dimer equilibrium of E-cadherin. For comparison, we
calculated the C50% values for each
equilibrium. (C50% corresponds to the
concentration at which 50% of the fragments by weight are in the
multimeric form.) C50% can be deduced from
KD using the following equation:
C50% = (2/n1/n
1)(KD)1/n
1,
where n corresponds to the degree of association. When
n is equal to 2, the C50% value
corresponds to the KD for a monomer
dimer
equilibrium. In contrast, when n is equal to 6, the
C50% value becomes equal to 1.4 ×
for a
monomer
hexamer equilibrium.
, 1.0059 g/ml) and the viscosity (
, 1.022 centipoise) of
the buffer were calculated according to the program Sedinterp, supplied
by D. B. Hayes, T. Laue, and J. S. Philo. By combining
the sedimentation coefficient (S) and the hydrodynamic
radius (Rh) of VE-EC1-4 determined by
ultracentrifugation and gel filtration chromatography experiments, the
molar mass (m) of the multimer could be calculated using the following equation: S = m(1
v)/6
N
Rh, where
N is Avogadro's number.
v)
2(r2
r02)/2RT) + A1(r0)6Kappexp(6m1(1
v)
2(r2
r02)/2RT) + E,
where m1 is the molecular mass of the monomer,
is the angular velocity in s
1,
R is 8.31 J·K
1·mol
1
(the gas constant), and T is the temperature in Kelvin. The
dissociation constant KD(H) was
calculated from Kapp using an extinction coefficient of 48,843 cm
1
M
1.
175 °C.
Specimens were observed at a temperature of approximately
180 °C
using a Gatan 626 cryo-holder with a Phillips CM200 electron microscope
operating at 200 kV. Images were obtained under low dose conditions
(<10 electrons/Å2) at a nominal magnification of
×66,000. The images were recorded on Kodak SO163 electron image films
and developed in full-strength D19 developer for 12 min at room
temperature. The magnification was calibrated in independent
experiments using the 23-Å pitch of tobacco mosaic virus.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (9K):
[in a new window]
Fig. 1.
Schematic representation of VE-cadherin and
its derived recombinant fragment VE-EC1-4. The
extracellular part of VE-cadherin (VE Cad) consists of five
modules designated EC1-EC5. The 48.9-kDa recombinant fragment
VE-EC1-4 overlaps the four N-terminal modules of VE-cadherin
(EC1-EC4). The N-terminal sequence of VE-EC1-4 is marked.
CYT, cytoplasmic tail of VE-cadherin; signal peptide of
VE-cadherin.
-sheets and indicated that it is
correctly folded (data not shown).
View larger version (31K):
[in a new window]
Fig. 2.
Gel filtration analysis of VE-EC1-4
self-association. A, fast pressure liquid
chromatography molecular exclusion profile for the VE-EC1-4 fragment
at an initial loading concentration of 1 µM. Two distinct
peaks (designated peaks I and II) are clearly detected on the
chromatograms performed at 4 °C. B, SDS-polyacrylamide
gel electrophoresis analysis of the fractions corresponding to peaks I
and II. 40 µl of each fraction were set at the top of a 12.5%
polyacrylamide gel. Following migration, the protein was detected by
Coomassie Blue staining.
View larger version (35K):
[in a new window]
Fig. 3.
Determination of the oligomeric state for
VE-EC1-4. A, cross-linking experiments. VE-EC1-4 at
110 µM was cross-linked using EGS (0.1 (lane
1), 0.300 (lane 2), and 3 (lane 3)
mM) and EDAC (1 (lane 4) and 3 (lane
5) mM) at the indicated concentrations. Lane
6 shows the molecular mass markers. Cross-linked products were
analyzed by SDS electrophoresis using a 4-15% gradient Phast gel. The
oligomeric states of each cross-linked product are indicated on the
left. B and C, analytical ultracentrifugation.
Shown in B are the sedimentation velocity profiles of
VE-EC1-4 at 1.2 (upper) and 26.2 (lower)
µM. The first and last profiles
presented here for each VE-EC1-4 concentration were obtained at
20 °C after 50 and 120 min of sedimentation at 42,000 rpm. The path
length was 1.2 cm for the 1.2 µM sample and 0.3 cm for
the 26.2 µM sample. Shown in C are the
sedimentation coefficient distribution functions
(g(s*)) for VE-EC1-4 at 1.2 (- - -) and
26.2 (- - - - -) µM. The apparent sedimentation
coefficient distribution functions (g(s*),
i.e. normalized for loading concentrations, but without
correction for diffusion effect) were evaluated using DCDT (32),
considering six profiles of absorbance obtained for
2t between 1.1 × 1011
and 1.4 × 1011 s
1.
View larger version (26K):
[in a new window]
Fig. 4.
Global fitting of the sedimentation
equilibrium data for VE-EC1-4. A, the loading
concentrations/angular velocities for the nine profiles presented are
as follows: 40 µM/4500 rpm ( ), 40 µM/6000 rpm (
), 40 µM/12,000 rpm (+),
12.5 µM/4500 rpm (
), 12.5 µM/6000 rpm
(
), 12.5 µM/12,000 rpm (×), 3 µM/4500
rpm (
), 3 µM/6000 rpm (
), and 3 µM/12,000 rpm (
). The global fit also takes into
account data at a VE-EC1-4 concentration of 25 µM, which
are not shown here for clarity. The lines correspond to the fitted
curves, from which a dissociation constant of 0.5 µM5 was deduced. B, the
differences between experimental and calculated values of absorbance
(normalized by the statistical error of experimental absorbance
(dA)) are presented. The separation between the graduations
is 1; for clarity, the curves are displayed with respect to each other
by a value of 2. C, the apparent molar mass was calculated
from d ln A/dr2 as a
function of the total concentration of VE-EC1-4. A is the
absorbance.
6 and
1 × 10
6 s
1 at
20 and 4 °C, respectively.
View larger version (18K):
[in a new window]
Fig. 5.
Gel filtration analysis of the monomer
hexamer equilibrium. All the chromatographic
runs were performed at 4 °C to avoid dissociation of the hexamer.
A, kinetic study of the hexamer dissociation. VE-EC1-4 was
diluted from 45 to 2 µM and stored at room temperature.
The percentage of VE-EC1-4 present as hexamers was estimated from
chromatographic profiles performed at different time intervals after
dilution. B, relationship between hexamer formation and
total concentration of VE-EC1-4. Multimer/monomer molar ratios were
estimated from molecular exclusion profiles by integrating the area of
peaks I and II (see "Materials and Methods") for various loading
concentrations of VE-EC1-4. C,
Ca2+-dependent association of the hexameric
VE-EC1-4 form. VE-EC1-4 at 18 µM (equilibrated in 5 mM Ca2+) was injected onto a Superdex S200 gel
filtration column. Elutions were performed in Tris/saline (pH 7.4)
running buffer containing either 5 mM Ca2+
(------) or 10 mM EGTA (- - -). In the presence of 10 mM EGTA, the hexamer (peak I) dissociated into the monomer
(peak II).
hexamer
equilibrium by gel filtration chromatography, a modified elution buffer containing 10 mM EGTA was used to elute VE-EC1-4 loaded in
the presence of Ca2+. The resulting progressive removal of
free Ca2+ prevented the formation of aggregates that clog
the column. As illustrated in Fig. 5C, progressive
Ca2+ depletion induced a large increase in peak II at the
expense of peak I. This shows that the multimer dissociates into
monomers in the absence of Ca2+.
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Fig. 6.
Electron microscope images of VE-EC1-4.
A, electron microscope image of 2 µM VE-EC1-4
in the presence of 5 mM Ca2+ negatively stained
with uranyl acetate. B and C, cryoelectron
microscope images of frozen hydrated VE-EC1-4: end-on views in a thick
region of amorphous ice and side-on views of the hexamer in a thin
region of ice, respectively. Several hexamers are marked with
arrowheads in B and C. Below
C is a gallery of hexamers, and at the far right is an
averaged image of the side-on views of the VE-EC1-4 hexamer as in
C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
hexamer equilibrium with a dissociation constant
KD(H) = 0.3-0.5 µM+5. Although the detailed mechanism
resulting in the elaboration of the hexamer is not known, it can be
assumed that this occurs via intermediate species such as dimers or
trimers. These transient associations probably appear for VE-EC1-4
concentrations lower than those required for hexameric associations,
suggesting that VE-EC1-4 dimers may be formed at concentrations lower
than 0.5 µM. The unusual unit used for this
KD(H) value does not allow a direct
comparison between our value and those previously published for other
cadherins. To palliate this inconvenience, C50%
was evaluated for comparison with other cadherins. The VE-EC1-4
fragment associates as a hexamer with a C50% of
~0.5 µM, whereas the E-EC1-2 fragment, containing the
two N-terminal modules of E-cadherin, forms dimers with a
C50% ranging from 80 µM (20) to
170 µM (35). Differences in propensity to self-associate
observed for E- and VE-cadherin fragments suggest that class I and II
cadherins elaborate various types of homotypic interactions.
6 to
6.10
6 s
1 when the
temperature is raised from 4 to 20 °C. This reflects the stability
of the hexameric structure.
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ACKNOWLEDGEMENTS |
---|
We thank I. Arnal and Y. Petillot for help in electron microscopy and mass spectrometry, respectively.
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FOOTNOTES |
---|
* This work was supported in part by Association pour la Recherche sur le Cancer Grant 9463 and by a grant from the Groupement des Entreprises Françaises dans la Lutte contre le Cancer.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.
The on-line version of this article (available at
http://www.jbc.org) contains Figs. A-E and accompanying text.
§ Recipient of a fellowship from the Association pour la Recherche sur le Cancer.
¶¶ To whom correspondence should be addressed. Tel.: 33-4-7688-9204; Fax: 33-4-7688-5494; E-mail: gulino@ibs.fr and vernet{at}ibs.fr.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M002667200
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ABBREVIATIONS |
---|
The abbreviations used are: VE-cadherin, vascular endothelial cadherin; MALDI, matrix-assisted laser desorption ionization; EDAC, N-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EGS, ethylene glycol bis(succinimidyl succinate); MES, 2-(N-morpholino)ethanesulfonic acid.
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