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4ß1: VCAM-1, Mucosal Addressin Cell Adhesion Molecule-1, and Fibronectin Induce Distinct Conformational Changes1
Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, M13 9PT, United Kindgom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 is a
receptor for both cell surface ligands (VCAM-1 and mucosal addressin
cell adhesion molecule-1 (MAdCAM-1)) and extracellular matrix
components (fibronectin). Through regulated interactions with these
molecules, 4ß1 mediates leukocyte
migration from the vasculature at sites of inflammation. Regulation of
integrin activity plays a key role in controlling leukocyte-adhesive
events and appears to be partly determined by changes in integrin
conformation. Several mAbs that recognize ligand-induced binding site
epitopes on integrins have been characterized, and a subset of these
mAbs are capable of stimulating integrin-ligand binding. Conversely,
some mAbs recognize epitopes that are attenuated by ligand engagement
and allosterically inhibit ligand binding. To gain insight into
ligand-specific effects on integrin conformation, we have examined the
ability of different ligands to modulate the binding of four distinct
classes (A, B1, B2, and C) of anti-4 Abs to
4ß1. VCAM-1 attenuated B (antifunctional)
class epitopes via an allosteric mechanism and also allosterically
inhibited the binding of the function-blocking
anti-ß1 mAb 13. Additional
4ß1 ligands (fibronectin fragments,
MAdCAM-1, and the CS1 peptide) also inhibited mAb 13-integrin binding;
however, the epitopes of the class B anti-4 mAbs
were attenuated by the fibronectin fragments, but not by MAdCAM-1 or
the CS1 peptide. Of the two anti-4 class A mAbs
examined, one recognized an epitope that was induced uniquely by
VCAM-1. Taken together, these data suggest that overlapping but
distinct binding mechanisms exist for different
4ß1 ligands and that distinct
conformational changes are induced upon integrin engagement by
different ligands. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The integrin 4ß1 (VLA-4, CD49d/CD29)
is expressed by a restricted set of cell types, including neural crest
cells, lymphocytes, monocytes, eosinophils, and myoblasts and has also
been reported to be present at low levels on polymorphonuclear cells
(6, 7, 8, 9, 10). 4ß1 is a key receptor for the
extracellular matrix macromolecule fibronectin and the cell surface Ig
superfamily member VCAM-1 (CD106); it has also been reported to bind
mucosal addressin cell adhesion molecule-1
(MAdCAM-1)4 (11, 12, 13, 14). In
vivo, 4ß1 has been implicated in a diverse
range of functions, including leukocyte interactions with activated
endothelia, anchorage of hematopoietic progenitor cells in the bone
marrow, and myoblast fusion during myogenesis (8, 15, 16).
The recognition of short peptide motifs by integrins is a
characteristic feature of integrin-ligand interactions (17). The main
4ß1 binding site in fibronectin is located
in the type III connecting segment (IIICS), a region distinct from that
recognized by other integrins (11). Analysis of the IIICS region of
fibronectin has identified a key adhesive motif, LDV, encoded in the
first alternatively spliced segment (represented by the CS1 peptide)
(18). Interestingly, mutagenesis studies performed on VCAM-1 and
MAdCAM-1 identified two homologous sequences (IDSPL and LDTSL,
respectively) that were responsible for
4ß1 interactions (14, 19, 20, 21, 22).
Furthermore, all 4ß1-ligand interactions
can be blocked by the CS1 peptide, suggesting common or overlapping
ligand binding sites on 4ß1, and
importantly, that the LDV motif of fibronectin represents the prototype
of a common 4ß1-binding motif found in all
of its ligands (23).
Despite advances in our understanding of ligand active sites, the
binding sites for ligands on integrins are less well characterized. A
number of approaches have been used to localize regions involved in the
ligand binding process. Peptide cross-linking, epitope mapping of
antifunctional mAbs, expression of recombinant integrin fragments, and
site-directed mutagenesis have localized active sites to several
regions in the N-terminal portions of both the and ß subunits
(see Refs. 1 and 3–5 for reviews). Putative ligand-binding sites in
the subunit map to a region that is predicted to be an array of
seven homologous modules organized into a ß-propeller structure (24).
A module homologous to the von Willebrand factor A domain, found in a
subset of integrin subunits, also represents a major ligand-binding
site, but this is absent in 4ß1 (25, 26, 27, 28, 29).
However, there is also strong evidence to suggest that a region
resembling a von Willebrand factor A domain is present in all ß
subunits and that this domain is a key module for ligand binding (Refs.
30 and 31; reviewed in Refs. 3 and 4).
Integrins can exist in both active (ligand-competent) and inactive (ligand-incompetent) states. Physical data obtained from fluorescence resonance energy transfer experiments, the identification of enhanced ligand binding induced by mAbs, and the detection of neo epitopes expressed upon integrin-ligand engagement strongly suggest that integrins undergo conformational changes in response to cation-binding, ligand-binding, and mAb-binding events (reviewed in 4 . Several mAbs recognize ligand-induced binding sites (LIBS) on integrins, and a subset of these mAbs are also capable of activating integrins to bind ligand (32, 33, 34, 35, 36). One interpretation of these data is that integrins exist in an equilibrium between conformationally distinct inactive and active states. According to this model, Ab-induced integrin activation would be achieved when anti-LIBS mAbs bind to and stabilize integrin in an active ligand-competent or ligand-bound conformation. Recently, the mode of action of an inhibitory anti-ß1 mAb, mAb 13, has been described (37). Intriguingly, ligand-occupied integrin demonstrated a reduced affinity for mAb 13, implying that the Ab epitope is attenuated by ligand (i.e., mAb 13 recognizes a ligand-attenuated binding site, or LABS). Surprisingly, kinetic analysis of the competition between mAb 13 and ligand for integrin binding revealed an allosteric mechanism of inhibition in contrast to the expected direct competitive, steric mechanism (37). Thus, anti-integrin mAbs can either activate or perturb ligand recognition by stabilizing or destabilizing integrin conformers by an allosteric mode of action.
A large number of anti-4 mAbs have been described,
and these can be classified into several groups depending on epitope
location and the functional properties of the Ab (38, 39, 40, 41). Group A mAbs
induce leukocyte homotypic aggregation and have been reported to be
weak inhibitors of 4ß1-fibronectin
interactions but to have no effect on
4ß1-VCAM-1 binding. Group B mAbs are
potent inhibitors of all 4ß1-ligand
interactions, but these can be further subdivided into B1 and B2 mAbs,
where B2 classes stimulate homotypic aggregation and B1 mAbs do not.
Group C mAbs neither stimulate homotypic aggregation nor possess
antifunctional activity; however, they block homotypic aggregation
induced by either A or B2 mAbs. Interestingly, B1 and B2 mAbs block
aggregation induced by type A mAbs, whereas group A and B1 mAbs block
aggregation induced by type B2 mAbs.
Recently, we have shown for 5ß1 that
inhibitory anti-integrin mAbs can be used as probes to map the
integrin-ligand binding interface and that different regions of ligand
can differentially affect the binding of anti- and anti-ß
mAbs (42). Here, we have examined the integrin-binding characteristics
and epitope expression pattern of a panel of anti-4
mAbs representing the different epitope classes and the
anti-ß1 mAb 13 in response to specific
4ß1 ligands. The data suggest that
different 4ß1 ligands have overlapping but
distinct binding mechanisms. Moreover, the effects on mAb epitope
expression observed suggest that distinct conformational changes may be
induced upon integrin-ligand engagement, implying that
4ß1 may be able to transduce
ligand-specific signals into the cell.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse anti-human 4 mAb HP2/1 (directed
against epitope B1) was purchased from Serotec (Oxford, U.K.). Mouse
anti-4 mAbs HP1/1 (anti-epitope A), HP1/2
(anti-epitope B1), and HP1/3 (anti-epitope A) were purified
from culture supernatants (generous gifts of Dr. Francisco
Sánchez-Madrid, Universidad Autónoma de Madrid, Madrid,
Spain) using protein G-Sepharose affinity chromatography. Mouse
anti-4 mAb 8F2 (epitope C) was a gift of Dr. Chikao
Morimoto (Dana-Farber Cancer Institute, Boston, MA). Mouse
anti-4 mAbs GG5/3 and TY21/6 (both vs epitope B2)
were gifts of Dr. Ted Yednock (Athena Neurosciences, San Francisco,
CA). Rat anti-human ß1 mAb 13 and rat anti-human
5 mAb 16 were gifts of Dr. Steven Akiyama (National
Institute for Environmental Health Sciences, Research Triangle
Park, NC).
Recombinant ligands and peptides
Truncated CAM-Fc chimeras consisting of Ig domains 1 and 2 of human VCAM-1 or murine MAdCAM-1 fused to the hinge and Fc region of human IgG1 were constructed and purified as described previously (14). Control protein MUC18-Fc was a gift of Dr. David Simmons (University of Oxford, Oxford, U.K.). Recombinant fragments of fibronectin HepII/IIICS splice variants were expressed and purified as described previously (43). Variant H/120 comprises fibronectin type III repeats 12 to 14, full-length IIICS, and fibronectin type III repeat 15. Variant H/89 comprises fibronectin type III repeats 12 to 14, IIICS region (minus the final 31 amino acids) and fibronectin type III repeat 15. The synthetic peptide CS1 (DELPQLVTLPHPNLHGPEILDVPST) was synthesized using Fastmoc chemistry on an Applied Biosystems (Foster City, CA) model 431A peptide synthesizer and purified as previously described (44, 45).
Purification of 4ß1
MOLT4 cells (ECACC, Porton Down, U.K.) were cultured in RPMI
1640 supplemented with 2 mM glutamine and 10% (v/v) fetal bovine serum
with antibiotics (all from Life Technologies, Paisley, U.K.). Confluent
cultures (20 liters) were centrifuged at 2,000 x g for
5 min, and the cell pellet was washed in PBS (Life Technologies). The
cell pellet (approximately 10–15 ml) was then extracted for 30 min at
room temperature in 2% (v/v) Triton X-100, 150 mM NaCl, 2 mM PMSF, 10
µg/ml leupeptin, 5 mg/ml BSA, and 25 mM Tris-HCl, pH 7.4. Lysed cells
were then centrifuged at 1,600 x g for 5 min, and the
supernatant was recentrifuged at 30,000 x g for 30
min. The supernatant was absorbed first by filtration through Sepharose
4B and then by mixing with rat IgG-Sepharose (2 mg IgG/ml beads) for
1 h at room temperature. IgG-Sepharose was removed by filtration,
and a 2-ml wash with 0.1% (v/v) Triton X-100, 150 mM NaCl, 1 mM
MgCl2, 1 mM CaCl2, and 25 mM Tris-HCl, pH 7.4
(wash buffer) was pooled with the cleared filtrate. The supernatant was
then mixed with 3 ml mAb 13-Sepharose (2 mg IgG/ml beads) for 2 h
at room temperature. The suspension was then packed into a 1.6-cm
diameter column (Pharmacia) and washed with wash buffer at 4 to 8
ml/min for 16 h at 4°C. Material retained on the column was
eluted with 1 mM CaCl2, 1 mM MgCl2, 0.1%(v/v)
Triton X-100, and 10 mM sodium acetate, pH 3.5, at 0.5 ml/min at 4°C,
and 0.5-ml fractions were collected into a neutralizing volume of 1 M
Tris-HCl, pH 8.2. Aliquots of fractions were then analyzed by
electrophoresis on a 6% nonreducing SDS-polyacrylamide gel. After
Coomassie blue staining, bands corresponding to the expected positions
for 4, 5, ß1, and
4 cleavage products were detected. Peak integrin
fractions were pooled and mixed for 2 h at room temperature with
50 µl of mAb 16 (anti-5)-Sepharose (5 mg IgG/ml
beads) to remove 5ß1 from the preparation.
A second 5ß1 depletion was performed and
mAb 16-Sepharose removed by centrifugation. 4 and
ß1 were the only integrin subunits detected in the
cleared fractions by enhanced chemiluminescence ELISA.
Effect of ligand on mAb-4ß1
binding
Purified 4ß1 (
100 µg/ml)
was diluted 1:100 in PBS (containing divalent cations; Life
Technologies), and 100-µl aliquots were added to a 96-well ELISA
plate (Dynatech Immunulon-3; Dynatech Laboratories, Chantilly, VA).
Protein was allowed to coat wells for 16 h at 4°C, and then
nonspecific sites on the plate were blocked for 2 h at room
temperature with 200 µl 5% (w/v) BSA, 150 mM NaCl, 0.05% (w/v)
NaN3, and 25 mM Tris-HCl, pH 7.4. Wells were then washed
three times with 200-µl aliquots of binding buffer (0.1% (w/v) BSA,
150 mM NaCl, 1 mM MnCl2, and 25 mM Tris-HCl, pH 7.4),
before adding aliquots of mAb diluted to appropriate concentrations in
binding buffer (with or without varying concentrations of ligand) to
triplicate wells. HP1/1, HP1/3 (approximate concentration, 50 µg/ml),
and HP1/2 were diluted 1:300, 1:50, and 1:1000 respectively; HP2/1,
TY21/6, GG5/3, 8F2, and mAb 13 were used at 0.3 µg/ml. Plates were
then incubated for 2 to 2.5 h at 37°C. Unbound material was
removed with three 200-µl washes with binding buffer and bound Ab
detected with species-specific secondary Ab (IgG-specific, peroxidase
conjugate; Dako, High Wycombe, Bucks, U.K.) diluted 1:1000 in binding
buffer. After a 25-min incubation at room temperature, wells were
washed three times with 200-µl aliquots of binding buffer, and color
was developed using 0.55 mg/ml
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.0026% (v/v)
H2O2, 0.1 M sodium acetate, and 50 mM
NaH2PO4, pH 5. The effect of ligand on
mAb-4ß1 binding affinity was analyzed as
above, except that the level of mAb binding over a range of Ab
concentrations in the presence or absence of ligand (25 µg/ml) was
detected. The mode of ligand-mAb competition was determined by
monitoring the effect on mAb binding to
4ß1 at three different concentrations
(10-fold range) over a dose range of ligand.
VCAM-Fc-4ß1 binding
Binding experiments were performed (with or without mAb at 3
µg/ml) as before, except that VCAM-Fc (1 µg/ml) was detected using
an alkaline-phosphatase-conjugated goat anti-human, IgG Fc-specific
secondary Ab (Sigma, Poole, U.K.). Color was developed using soluble
phosphatase substrate (p-nitrophenyl phosphate;
Sigma) in 0.1 M glycine, 1 mM MgCl2, and 1 mM
ZnCl2, pH 10.4. Maximum
4ß1-VCAM-Fc binding was observed at 10
µg/ml, with half-maximal binding of VCAM-Fc at 1 µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 binding
Recent work from this laboratory has investigated the mechanism
underlying the antifunctional activity of mAbs that block adhesion
mediated by the integrin 5ß1. These
studies showed that both anti-5 and
anti-ß1 Abs are allosteric inhibitors that work by
preventing conformational changes within the integrin subunits that are
necessary for ligand engagement. This finding implies that
anti-integrin Abs can be used as probes for dissecting the sites
within integrin ligands that contact specific receptor active sites.
Because the integrin 4ß1 has the
interesting property of recognizing both the alternatively spliced
extracellular matrix protein fibronectin and the cell surface Igs
VCAM-1 and MAdCAM-1, we decided to investigate the mechanism of action
of anti-4 Abs. We hypothesized that these studies
would inform us about the molecular basis of differential ligand
recognition by 4ß1 and may ultimately feed
into the process of rational design of 4ß1
antagonists. To determine the nature of such protein-protein
interactions, it is necessary to accurately measure the apparent
binding affinity over a range of conditions; to do this we employed a
cell-free solid-phase binding assay using purified integrin and
recombinant ligands.
Anti-4 mAbs can be grouped into four classes according
to activity, and for this study we used the following representative
panel: HP1/1 and HP1/3 (class A); HP1/2, HP2/1, and TY21/6 (class B1);
GG5/3 (class B2); and 8F2 (class C). We first monitored the effect of
the mAb panel on 4ß1 binding to VCAM-Fc.
The results confirm previous reports (40), in that only mAbs directed
against either B1 or B2 class epitopes inhibited integrin-ligand
binding (Fig. 1
).
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4-specific was investigated. To maximize ligand binding,
experiments were performed at saturating ligand concentrations (50
µg/ml VCAM-Fc) and in the presence of 2 mM Mn2+. The
effect of VCAM-Fc occupancy of 4ß1
produced contrasting effects on mAb-4ß1
binding (Fig. 2). The antifunctional
mAbs, HP1/2, HP2/1, TY21/6 (class B1), GG5/3 (class B2), and mAb 13
(anti-ß1) were all substantially inhibited by
VCAM-Fc, whereas the epitope A mAb HP1/1 and the epitope C mAb 8F2 were
unperturbed. Intriguingly, binding of one epitope A mAb (HP1/3) to
4ß1 was markedly enhanced by VCAM-Fc
(192%). The control protein MUC18-Fc had no significant effect on any
of the mAbs tested.
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4 and anti-ß1 mAbs
Analysis of the mode of competition between mAb 13 and fibronectin
for 5ß1 integrin binding revealed an
allosteric mode of inhibition in contrast to a direct competitive
steric mechanism (37). To determine the type of inhibition observed
with the anti-4 B1 class, B2 class, and
anti-ß1-mAbs recognizing
4ß1, we examined the effect of a constant
concentration of VCAM-Fc (25 µg/ml) on mAb binding to
4ß1 over a range of mAb concentrations.
The results obtained with HP2/1 (B1 class), and GG5/3 (B2 class) were
essentially identical (Fig. 3,
A and B). Although the maximum level of
mAb-4ß1 binding appeared to be unchanged
in the presence of VCAM-Fc, a marked shift in the apparent affinity of
mAb for integrin was observed. Double-reciprocal plots of these data
revealed that the apparent affinities of each anti-4
mAb for 4ß1 were similar (HP2/1, 0.29 nM;
GG5/3, 0.23 nM; Fig. 3, D and E). VCAM-Fc,
however, had marked effects on their apparent affinities for
4ß1 (HP2/1, 0.9 nM; GG5/3, 1.0 nM). Thus,
occupancy of integrin by ligand resulted in a more than threefold
decrease in the apparent affinity of both mAbs.
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4ß1 (Fig. 3
C). A
double-reciprocal plot of the mAb 13 data revealed that Ab binding was
biphasic (Fig. 3F), in contrast to the monophasic
binding obtained with both of the anti-4 mAbs. By
separating the mAb 13 data into two populations and performing linear
regression analysis, the apparent affinity of mAb 13 for both of the
integrin populations could be determined. This analysis revealed that
20% of the total integrin could bind mAb 13 with identical affinity
with that in the absence of ligand (0.8 nM), suggesting that this
population of integrin represented integrin not occupied by ligand.
However, the second population of integrin was found to bind mAb 13
with much lower affinity (28 nM) and could only be detected with high
concentrations of mAb 13 (Fig. 3F, dashed line). Thus, these
results suggest that mAb 13 has a 35-fold lower affinity for integrin
occupied by VCAM-1.
VCAM-1 is an allosteric inhibitor of antifunctional
anti-4 and anti-ß1
mAb-4ß1 binding
The above data indicate that VCAM-Fc is capable of inhibiting the
binding of all classes of antifunctional 4 mAbs and
the antifunctional anti-ß1 mAb, mAb 13 to
4ß1. The shifts in apparent integrin
binding affinities indicate a competitive mode of inhibition. To
determine whether ligand was acting as a direct competitor of mAbs or
an allosteric inhibitor, we performed binding experiments with several
different mAb concentrations and a dose range of VCAM-Fc (Fig. 4, A–C). All three
mAb-4ß1 interactions were inhibited by
VCAM-Fc in a dose-dependent fashion. Importantly, the maximum extent of
inhibition decreased with increasing mAb concentration. These data are
inconsistent with the behavior of a direct-competitive inhibitor, in
which the maximum level of inhibition observed would be unchanged with
increasing mAb concentration. Single-reciprocal plots of these data
reveal that in all cases, curves of
mAb-4ß1 binding with increasing dose
ligand were hyperbolic, indicative of an allosteric mechanism of
inhibition (46) (Fig. 4, D–F).
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4ß1
binding, yet HP1/3 weakly inhibits
VCAM-Fc-4ß1 binding
We next further analyzed the increase in
HP1/3-4ß1 binding detected in the
presence of VCAM-Fc (Fig. 1). Inclusion of VCAM-Fc in the binding assay
did not change the apparent affinity of the mAb for integrin; however,
the maximum level of HP1/3 binding was markedly enhanced, suggesting
induction of HP1/3 epitope expression by VCAM-Fc (Fig. 5A). HP1/3 has
previously been reported to be a weak inhibitor of
4ß1-fibronectin binding but to have no
effect on 4ß1-VCAM-1 interactions (40).
Because VCAM-Fc altered HP1/3-4ß1 binding
characteristics, we examined the effect of HP1/3 on
4ß1-VCAM-Fc binding. The results showed
that in contrast to the effect of VCAM-Fc on
4ß1-HP1/3 binding, HP1/3 increased the
concentration for half-maximal VCAM-Fc binding and slightly reduced the
maximal level of 4ß1-VCAM-Fc binding (Fig. 5B). Although integrin was activated in these
experiments by the inclusion of 2 mM Mn2+, VCAM-Fc binding
could be increased when the activating mAb TS2/16 was coincubated with
ligand (Fig. 5C). Thus, these results show that
despite recognizing a LIBS, HP1/3 is not an activator of
4ß1-VCAM-Fc binding.
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4ß1 binding observed with
MAdCAM-Fc and fibronectin variants
Because different biologic properties and different binding
affinities are associated with each
4ß1 ligand, they might be expected to
trigger different conformational changes when they occupy
4ß1. We therefore examined the effect of
an array of 4ß1 ligands including
recombinant fibronectin (IIICS region) splice variants and MAdCAM-Fc on
the binding of the anti-4 mAbs. Two fibronectin
variants were tested: H/120, encompassing fibronectin type III repeats
12 to 14, full-length IIICS (120 amino acids), and fibronectin type III
repeat 15, and H/89, comprising fibronectin type III repeats 12 to 14,
IIICS region (minus the CS5 and CS6 peptides; 89 amino acids), and
fibronectin type III repeat 15. The control protein, MUC18-Fc, had a
negligible effect on mAb-4ß1 binding,
whereas the effects of MAdCAM-Fc and the fibronectin variants differed
from VCAM-Fc in a number of respects (Fig. 6). MAdCAM-Fc did not attenuate any of
the anti-4 mAbs, whereas fibronectin variants H/120
and H/89, which both possess a potent LDV motif, attenuated the binding
of all antifunctional anti-4 mAbs. Fibronectin
variants that lacked the LDV motif did not affect any mAb-integrin
interactions at the concentrations tested (data not shown). In
addition, MAdCAM-Fc as well as H/120 and H/89 markedly reduced the
level of mAb 13-4ß1 binding. However, none
of the alternative 4ß1 ligands were
capable of enhancing the binding of HP1/3 to
4ß1.
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4ß1 binding
Despite variations in their effects on the
anti-4 mAbs, all of the ligands tested here
were capable of perturbing mAb 13-4ß1
binding. Because all physiologic ligands for
4ß1 possess an adhesion motif closely
related to LDVP, we compared the abilities of the LDV-peptide CS1
(derived from the IIICS of fibronectin), H/120, and MAdCAM-Fc to
attenuate mAb13-4ß1 binding. The results
obtained indicate that MAdCAM-Fc, H/120, and CS1 are dose-dependent
inhibitors of mAb 13-4ß1 binding (Fig. 7, A–C). Although CS1
is a weaker inhibitor than either MAdCAM-Fc or H/120, a significant
level of mAb attenuation was observed. However, CS1 did not perturb the
binding of any of the anti-4 mAbs to
4ß1 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 mAbs and identified
differential responses of the epitopes of these mAbs to occupancy of
the integrin by a range of 4ß1 ligands.
Our novel findings are that 1) the binding of function-blocking mAbs,
directed against either the 4 or ß1
subunit, are attenuated by ligand occupancy and that ligand-dependent
mAb attenuation is via an allosteric mechanism in all cases; 2) the
degenerate LDV motif, a version of which is present in all
4ß1 ligands, is the minimal sequence
required for attenuation of the mAb 13 binding site on the
ß1 subunit (MAdCAM-1 or CS1 peptide did not perturb any
4 epitopes); and 3) a non-antifunctional class A
4 mAb recognizes a LIBS, uniquely induced by VCAM-1.
Taken together, these data show that engagement of different
4ß1 ligands elicits different
conformational responses in the integrin.
Four distinct activities have been attributed to
anti-4 mAbs depending on their ability to perturb
integrin-ligand binding or induce homotypic cell aggregation and have
been classified as either A, B1, B2, or C mAbs (40). The epitopes of
these mAbs have since been localized on the 4 subunit
using human/mouse chimeras and site-directed mutagenesis. Class A mAbs
map to the extreme N-terminus of the -chain, whereas class B mAbs
locate to the third repeat; class C mAbs map to residues 422 to 606
(38, 39, 41). The mechanism of both antifunctional and homotypic
adhesion-inducing activities is presently poorly understood.
We first demonstrated that only mAbs of the B1 and B2 subsets blocked
the binding of VCAM-Fc to 4ß1 in a
cell-free solid-phase assay, confirming previous data (40). A recent
report has demonstrated that ligand (fibronectin-fragments) could
attenuate the antifunctional anti-ß1 mAb 13 binding
site on 5ß1 (37). Thus, we examined the
effect of VCAM-Fc on the ability of the anti-4 mAb
panel to bind 4ß1. We found that in the
presence of VCAM-Fc, the binding of representatives of both B1 and B2
(antifunctional class) mAbs was reduced by approximately 70%,
indicating competition between ligand and mAb for integrin binding. In
addition, mAb 13 binding was also reduced by VCAM-Fc. In contrast to
these observations, the binding of HP1/3 (class A) was dramatically
enhanced (twofold increase) by VCAM-Fc, suggesting that HP1/3
recognizes an epitope induced by ligand. Thus, HP1/3 represents a
member of the anti-LIBS family.
We further investigated the mechanism of ligand attenuation of
mAb-4ß1 binding and found that in the
presence of VCAM-Fc, the affinity of class B1 (HP2/1), class B2
(GG5/3), and anti-ß1 (mAb 13) mAbs was reduced. These
data closely resemble those obtained for mAb
13-5ß1 interactions in the presence of
fibronectin central cell-binding domain region fragments (37). Thus, it
would appear that mAb 13, HP2/1, and GG5/3 have high affinities for
unliganded integrin but low affinities for ligand-occupied
integrin.
Further data indicated that the mechanism of ligand-dependent mAb
epitope attenuation was not directly competitive but was allosteric in
nature. Thus, HP2/1 and GG5/3 can be grouped with mAb 13 as
anti-LABS Abs. These findings have important implications for our
understanding of integrin function and the mode of action of
antifunctional integrin mAbs. First, the concept that integrins are
allosteric proteins and undergo conformational changes when ligands or
mAbs engage is further reinforced. Second, the use of
anti-functional mAbs to identify ligand binding sites should be
interpreted cautiously, because mAb-dependent perturbation of integrin
function could potentially be distal from the ligand binding site.
Third, the fact that ligand-induced conformational changes, as detected
by modulation of mAb epitope recognition, indicates that both the
and ß integrin subunits are involved in integrin-ligand binding
events. These data also provide an insight into the mode of action of
A, B1, B2, and C class mAbs. Although the precise molecular basis of
anti-4 mAb (class A and class B2)-induced homotypic
cell aggregation is unclear, a possible explanation for mAb effects is
apparent. Inhibition of class B2 mAb-induced aggregation by class A
mAbs, the inhibition of class A mAb-induced aggregation by class B2
mAbs, and the blockade of both by B1 and C class mAbs have been
observed; however, the epitopes of class A, class B, and class C mAbs
are distinct according to competition assay and epitope mapping
experiments (38, 39, 41). Furthermore, the class C mAb, 8F2, which did
not block 4ß1-VCAM-Fc binding and was not
affected by VCAM-Fc occupancy of integrin, is capable of blocking some
ligand-dependent responses (47). A likely explanation for these effects
might be that integrin activity is modulated by conformational changes
induced when mAbs engage their epitopes.
In addition to these observations, we show that a class A mAb, HP1/3,
recognizes a site induced by VCAM-Fc, further reinforcing the
perception that ligand engagement results in a conformational response
by ligand. Analysis of HP1/3 binding data in the presence of VCAM-Fc
reveals an increase in the maximal level of
HP1/3-4ß1 binding (but not an increase in
binding affinity), indicating that HP1/3 represents an anti-LIBS
mAb as opposed to the anti-LABS mAbs described above. Several
anti-LIBS mAbs have been previously described, including a number
that map to the ß1 subunit (e.g., 15/7, 9EG7, and 12G10);
however, HP1/3 represents the first anti-4
anti-LIBS mAb (35, 36, 37, 48). Interestingly, data obtained here with
VCAM-Fc and elsewhere with fibronectin fragments demonstrate that HP1/3
is capable of inhibiting integrin-ligand interactions. Published
findings have suggested that HP1/3 is an inhibitor of
4ß1-fibronectin binding but not
4ß1-VCAM-1 binding (40). Here, we
demonstrate that HP1/3 is a weak inhibitor of
4ß1-VCAM-1 interactions. A possible
explanation to reconcile these data may reside in the higher apparent
affinity of 4ß1 for VCAM-1 as opposed to
fibronectin and the more sensitive cell-free assay system used here. In
the cases examined so far, all anti-LIBS mAbs are also activators
of integrin-ligand binding activity; however, for reasons that are
unclear, HP1/3 does not fall into this category.
Integrin 4ß1 is also a weak receptor for
the HepII/IIICS region of fibronectin and MAdCAM-1 (13, 14). When we
analyzed the effect of fibronectin splice variants and MAdCAM-Fc on the
binding of the anti-4 mAb panel to
4ß1, a set of epitope expression profiles
were obtained that were ligand-specific. Only fibronectin variants and
VCAM-Fc were capable of perturbing B1 class, B2 class, and mAb 13
epitopes, whereas MAdCAM-Fc only affected mAb
13-4ß1 binding. In addition, only VCAM-Fc
was capable of enhancing the HP1/3 epitope. Thus, ligand occupancy of
integrin appears to result in the modulation of integrin epitopes, with
an epitope profile unique to each ligand. These data therefore imply
that 4ß1 undergoes distinct conformational
changes, depending upon the ligand engaged. Interestingly, mutagenesis
of the 4 subunit class A epitope region has been shown
to abolish homotypic cell aggregation and
4ß1-fibronectin binding but not
4ß1-VCAM-1 binding, further reinforcing
the unique effects of mAb epitope expression induced by VCAM-Fc (49).
On the basis of these findings, 4ß1 might
be expected to respond differently to distinct
4ß1 ligands, with important implications
for integrin signaling and cell phenotypic changes.
4ß1 represents an attractive pharmacologic
target, given that it plays a key role in a variety of
adhesion-dependent disease states, including atherosclerosis, asthma,
and rheumatoid arthritis (5, 50). The data obtained with the
anti-4 mAb panel suggest that careful consideration
of 4ß1 antagonists needs to be made,
because potent anti-4ß1 agents may be
capable of inducing cellular responses that result in unwanted side
effects. Further characterization of the cellular responses associated
with anti-4 mAb binding activity might be useful in
identifying potent antifunctional compounds with true antagonistic
characteristics.
A propeller model for the N-terminus of the 4 subunit
has been proposed (24). In this model, the seven 60-amino acid repeats
found in all integrin subunits are organized into a large domain
with the overall appearance of a seven-bladed propeller with
pseudo-sevenfold symmetry. In this model, each repeat folds into a
four-stranded ß-sheet and constitutes a single blade of the
propeller. When mapped onto the model, the class A mAbs locate to blade
1, whereas B1 and B2 mAbs map to blades 3 and 4. Class C mAbs fall
outside the propeller structure. The data described here suggest that
the propeller structure would be capable of conformational changes in
response to ligand; indeed, HP1/3 binding is sensitive to mutations at
apparently distal sites (38, 39, 41). Moreover, ligands appear to
differentially affect the conformation of blades independently of other
parts of the propeller.
A related, but degenerate adhesion motif is a common feature of all
4ß1 ligands. A consensus sequence, based
upon amino acid homology, can be detected within
4ß1 ligands (LDV (fibronectin IIICS
region), IDS (VCAM-1), and LDT (MAdCAM-1)), with the first aliphatic
and aspartate residue of each sequence being crucial for biologic
activity (14). Interestingly, the peptide CS1, derived from the IIICS
of fibronectin and encompassing the LDV motif, is capable of inhibiting
4ß1-fibronectin and
4ß1-VCAM-1 interactions via a competitive
mechanism, indicating that the ligand binding site(s) on
4ß1 for fibronectin and VCAM-1 are closely
overlapping or identical (23). When we analyzed the effect of CS1 on
the binding of the anti-4 mAb panel to
4ß1, we found that no attenuation or
enhancement of epitopes was observed, a response identical with that of
MAdCAM-1. Interestingly, like MAdCAM-1, CS1 was able to perturb mAb
13-4ß1 binding. These data suggest that
the active LDV motif (present in CS1) interacts primarily with the
ß1 subunit of 4ß1, with no
detectable effect on the 4 subunit. These data are
comparable with the recent finding that the FnIII(10) of fibronectin
interacts with the ß1 subunit of
5ß1 via its RGD adhesion motif (42). Both
RGD and LDV represent important integrin adhesion motifs in a variety
of ligands, and it is intriguing that both perturb the same
ß1 epitope (mAb 13) in separate integrins. The
implication of these findings is that key adhesion sequences within
integrin ligands appear to engage integrin-active sites via similar
mechanisms with similar conformational responses and therefore that RGD
and LDV perform equivalent functions in the integrin-ligand binding
mechanism.
Further analogy between 5ß1-fibronectin
interactions and 4ß1-ligand interactions
is also apparent; in both cases, additional sequences outside the
RGD/LDV motif are required for further regulation of anti- mAb
epitopes. In the case of 5ß1,
anti-5 mAbs were sensitive to the presence of the
synergy sequence of FnIII(9), indicating that this region of
fibronectin contacts the 5 subunit (42). Recently,
additional sites important for integrin-binding activity in VCAM-1 Ig
domain 2 (adjacent to the IDS motif domain) have been identified;
however, none of the anti-4 mAbs tested here
demonstrated altered binding to 4ß1 in the
presence of VCAM-Fc when these sites were mutated (P. Newham and
M. J. Humphries, unpublished observations; 14 . Nevertheless,
for two ß1 integrins at least, there appears to be the
requirement for multiple, subunit-specific contact sites on ligands. It
is noteworthy that MAdCAM-1 is a relatively poor ligand for
4ß1 and is only capable of attenuating mAb
13-4ß1 binding and not the binding of
anti-4 mAbs. The lack of activity associated with
MAdCAM-1 may therefore be attributable to poor or no contact with the
4 subunit. However, it should be noted that
4ß1 and 4ß7-MAdCAM-1
interactions can be perturbed by antifunctional
anti-4 mAbs. Thus, although MAdCAM-1 contact sites
on 4ß1 are distinct from other
4ß1 ligands, they can be perturbed by
antifunctional mAbs, perhaps the result of a close functional
relationship between both of the integrin subunits.
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Acknowledgments |
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Footnotes |
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2 Current address: Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, SK10 4TG, U.K.
3 Address correspondence and reprint requests to Professor Martin J. Humphries, Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester, M13 9PT U.K. E-mail address:
4 Abbreviations used in this paper: MAdCAM-1, mucosal addressin cell adhesion molecule-1; IIICS, type III connecting segment; LIBS, ligand-induced binding site; LABS, ligand-attenuated binding site.
Received for publication September 8, 1997. Accepted for publication December 29, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 (LPAM-1), 4ß1 (VLA-4), and IELß7. Int. Immunol. 6:263.4 integrin binding interfaces on VCAM-1 and MAdCAM-1: integrin binding footprints identify accessory binding sites that play a role in integrin specificity. J. Biol. Chem. 272:19429.4ß1). J. Cell Biol. 124:601.4ß7: common themes among integrin-Ig family interactions. J. Immunol. 156:719.[Abstract]
4ß1. J. Biol. Chem. 269:4005.-subunits into a ß-propeller domain. Proc. Natl. Acad. Sci. USA 94:65.2ß1 (VLA-2, CD49b/CD29) in a divalent cation-independent manner. J. Biol. Chem. 269:26006.2 I-domain is a binding site for collagens. J. Cell Sci. 108:1629.[Abstract]
1 A domain is a ligand binding site for collagens and laminin. J. Biol. Chem. 272:12311. subunit of integrin CR3 (CD11b/CD18). Cell 80:631.[Medline]
IIbß3 (glycoprotein IIb-IIIa) alter receptor affinity, specificity, and function. J. Biol. Chem. 266:17106.4ß1 (VLA-4, CD49d/CD29). Biochem. J. 305:945.
4 and 5 subunits. EMBO J. 14:5550.[Medline]
4 epitopes. J. Biol. Chem. 266:10241.4 integrin. J. Biol. Chem. 270:14270.5ß1-fibronectin interactions using inhibitory anti-5 and ß1 monoclonal antibodies: evidence that the synergy sequence of fibronectin is recognized by the amino-terminal repeats of the 5 subunit. J. Biol. Chem. 272:17283.4ß1-mediated melanoma cell adhesion and migration on vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced IIICS region of fibronectin. J. Biol. Chem. 269:27224.4ß1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand. J. Biol. Chem. 270:28740.4 subunit involved in homotypic cell aggregation and in fibronectin but not vascular cell adhesion molecule-1 binding. J. Biol. Chem. 271:2696.4 integrins in vivo. J. Clin. Invest. 94:1722.
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signifies mAb-
(0.3 µg/ml), and 
(0.1
µg/ml); C, • (3 µg/ml), 
