From Biogen, Inc., Cambridge, Massachusetts 02142
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ABSTRACT |
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We have used the highly specific
Integrins comprise a large family of cell-surface receptors that
mediate cell-cell and cell-matrix interactions in diverse biological
settings (see Refs. 1 and 2 for reviews). Each integrin is a two-chain
heterodimer containing an Although all integrins require divalent cations to bind ligand, the
regulation of function by metal binding is complex and is not fully
understood (21, 22). Integrin Recently, we developed a series of highly selective
Synthesis of
[3H]BIO1211--
[3H]BIO1211 (50 Ci/mmol)
was synthesized by NEN Life Science Products using
[4,5-3H]leucine as a precursor. The radiochemical purity
of the compound was >95% as measured by reverse phase HPLC on a
C18 column, and the compound yielded the predicted spectrum
by 3H NMR. [3H]BIO1211 was dissolved in
Me2SO, diluted with 20 volumes of water, and sodium
phosphate, pH 8.8, was added to 20 mM (3.66 mCi/ml). The
solution was then aliquoted and stored at Binding of [3H]BIO1211 to
For kinetic on rate measurements, Jurkat cells were treated with 2 nM [3H]BIO1211 at room temperature for the
times indicated and then treated with a 500-fold excess of unlabeled
BIO1211 to quench further binding by the [3H]BIO1211.
Cells were collected by centrifugation and subjected to scintillation
counting. For kinetic off rate measurements, Jurkat cells were treated
with 5 nM [3H]BIO1211 at room temperature for
1 h. A 500-fold excess of unlabeled BIO1211 was added, and the
cells were further incubated for the times indicated. Cells were
pelleted at each time point, and cell-associated [3H]BIO1211 was measured by scintillation counting.
Binding and dissociation data are represented as a percent of the
maximum specific counts bound as a function of time. The data were
fitted to an exponential curve by nonlinear regression. For
koff, the exponential rate constant is the off
rate. For kon, the observed rate constant is the
true rate constant multiplied by the [3H]BIO1211
concentration. The effect of added unlabeled BIO1211 on dissociation
rates was tested over a wide range of concentrations from 100 nM (20-fold excess) to 50 µM (10,000-fold
excess). The dissociation curves were superimposable over this range of
concentrations, indicating that the excess unlabeled ligand was
exerting no allosteric effect on the rate of dissociation (data not shown).
Assessing Analysis of [3H]BIO1211 by Reverse Phase
HPLC--
Samples were analyzed by reverse phase HPLC on a
C18 column (Vydac, catalog number 218TP54, 0.46 × 25 cm) at 23 °C. The column was developed at 1 ml/min with the
following gradient of acetonitrile in 50 mM sodium acetate,
pH 4.5: 0-32 min 17.5-21.5%, 32-45 min 21.5-34.5%, 45-45.1 min
34.5-50%, 45.1-48 min 50%, 48-48.1 min 50-17.5%, and 48.1-53
min 17.5%, conditions that maximize the resolution of BIO1211 from
potential proteolytic and hydrolytic degradation products (data not
shown). The column effluent was monitored at 254 nm, and 0.5-ml
fractions were collected. The fractions were mixed with scintillation
mixture and analyzed by scintillation counting. Immediately prior to
injection on the HPLC, test samples were spiked with 2.5 µg each of
cold BIO1211 and cold BIO-1588, an analog of BIO1211 with the
C-terminal Val-Pro deleted, and a likely proteolysis product. The
elution profiles of the cold inhibitors were used to monitor column performance.
Development of a BIO1211-
Specific counts bound at saturation provided a direct measure of
A series of studies were performed to assess selected variables that
might affect the performance of the assay. First, no impact of
temperature on BIO1211 binding was observed under any of the activating
conditions tested when binding was compared at room temperature and at
37 °C (data not shown). Second, the presence of human serum or
plasma at concentrations up to Assessing the KD of BIO1211 for
Kinetics for Binding of BIO1211 to
To understand better the role metal ions have on activation, we
performed the study shown in Fig. 4,
where binding was tested as a function of changing Mg2+
concentrations. The dissociation rates were highly dependent on the
Mg2+ concentration and changed from 8.3 × 10
While the maximal affinity for binding of BIO1211 to
In the non-activated 1 mM Ca2+, 1 mM Mg2+ state, the kinetically determined
affinity of BIO1211 for
Since
An unexpected feature of the dissociation curve for the release of
BIO1211 from the divalent cation plus TS2/16-activated state was that
it required a double exponential fit to account for the data. A similar
biphasic curve for the release of BIO1211 was observed for
Independent evidence for the biphasic nature of BIO1211 release after
TS2/16 treatment was obtained by repeating the analysis on purified
The effects of Ca2+ on CS1 and VCAM-Ig Detect the Mn2+ + TS2/16 High Affinity
State--
As a result of its high affinity and slow rate of
dissociation, the [3H]BIO1211 can also be used as a probe
for ligand-integrin interactions through competition measurements in
which [3H]BIO1211 binding is used as a reporter for
receptor occupancy. In this format, Jurkat cells are first incubated
with test compound and then subjected to a brief treatment with
[3H]BIO1211 and counted. Counts bound under these
conditions measure integrin that is not occupied by the test compound
and is therefore free to bind the [3H]BIO1211. Typical
results from this type of analysis are shown in Fig.
6. Competition for
[3H]BIO1211 binding was seen with BIO1211, VCAM-Ig, and
CS1 at concentrations that are consistent with their known binding
constants for
To understand better whether the difference in affinities between the 2 mM Mn2+ alone and 2 mM
Mn2+ + TS2/16 states is specific to BIO1211 or whether it
reflects a property of We have used [3H]BIO1211 as a model LDV-containing
ligand to study A striking feature of the activated In the presence of 10 mM Mg2+ + TS2/16 or 2 mM Mn2+ + TS2/16, a further increase in
affinity was seen. The additive effect of activation by divalent
cations and by TS2/16 on the KD for
[3H]BIO1211 binding indicates that the mechanisms of
activation by divalent metals and by antibody are distinct. The kinetic
data are less informative about the transition between the "2
mM Mn2+ state" and the higher affinity state
observed with Mg2+ or Mn2+ plus TS2/16.
However, the observed differences in the affinities and dissociation
rates between 2 mM Mn2+ + TS2/16 and 10 mM Mg2+ + TS2/16 suggested that the higher
affinity state can likewise lead to the observation of a continuum of
affinities. This notion was further supported by studies in which
dissociation rates were measured in the presence of 2 mM
Mg2+ + TS2/16 (koff = 0.76 × 104
1 inhibitor
4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic
acid-valine-proline (BIO1211) as a model LDV-containing ligand to study
4
1 integrin-ligand interactions on Jurkat
cells under diverse conditions that affect the activation state of
4
1. Observed KD
values for BIO1211 binding ranged from a value of 20-40 nM
in the non-activated state of the integrin that exists in 1 mM Mg2+, 1 mM Ca2+ to
100 pM in the activated state seen in 2 mM
Mn2+ to 18 pM when binding was measured after
co-activation by 2 mM Mn2+ plus 10 µg/ml of
the integrin-activating monoclonal antibody TS2/16. The large range in
KD values was governed almost exclusively by
differences in the dissociation rates of the integrin-BIO1211 complex,
which ranged from 0.17 × 10
4 s
1 to
>140 × 10
4 s
1. Association rate
constants varied only slightly under the same conditions, all falling
in the narrow range from 0.9 to 2.7 × 106
M
1 s
1. The further increase in
affinity observed upon co-activation by divalent cations and TS2/16
compared with that observed at saturating concentrations of metal ions
or TS2/16 alone indicates that the mechanism by which these factors
bring about activation are distinct and identified a previously
unrecognized high affinity state on
4
1
that had not been detected by conventional assay methods. Similar
changes in affinity were observed when the binding properties of
vascular cell adhesion molecule-1 and CS1 to
4
1 were studied, indicating that the
different affinity states detected with BIO1211 are an inherent
property of the integrin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-chain and a
-chain. The leukocyte
integrin
4
1 regulates cell migration into
tissues during inflammatory responses and normal lymphocyte trafficking
(3-5) and provides a key co-stimulatory signal supporting cell
activation (6-10). In vivo studies using blocking
monoclonal antibodies (4) and inhibitor peptides (11-13) have
demonstrated a critical role for
4 integrins in
leukocyte-mediated inflammation.
4
1
mediates cell adhesion by binding to either of two protein ligands,
vascular cell adhesion molecule-1
(VCAM-1)1 or the
alternatively spliced CS1-containing fibronectin variant (14-17).
Whereas expression of
4
1 is constitutive,
its interaction with ligands is strongly enhanced in an activated state
that can be induced by various stimuli including antigen, anti-T cell
receptor mAbs, phorbol esters, the divalent cation Mn2+,
and certain
1-specific antibodies (18-20). These
changes in affinity and/or avidity ultimately determine whether the
interaction is productive and stabilizes the ligand-integrin complex or
is nonproductive.
-subunits contain multiple
EF-hand-like Ca2+ binding loops (1, 23), which are in close
proximity to ligand-binding sites (24, 25). This region of the
-chain is made up of seven sequence repeats of about 60 amino acids
each, which are presumed to be organized in a
-propeller fold motif
found in various enzymes (24, 25). The
-chain contains a second type
of metal binding/ligand binding motif that shares homology with the
A-domain of von Willebrand's factor (26, 27). A homologous structure
is present in some
-subunits as well. This 200-residue protein
module has proven to be surprisingly tractable for biochemical
evaluation, and the corresponding regions from
M,
L, and
2, have been successfully crystallized (28-30). The crystal structures revealed a
Mg2+ or Mn2+ bound at the apex of a
dinucleotide binding motif (28-30). The observed structure defined by
the coordination of the cation with the peptide backbone has been
postulated to mimic the ligand-occupied structure in what is commonly
referred to as the metal ion-dependent adhesion site or
MIDAS (see Refs. 21 and 27 for references). Whereas the role of
cation-binding sites in regulating integrin function is well
established, how the sites are coordinated is unclear. Regulation of
ligand binding by cations is further complicated by the fact that
submillimolar concentrations of Ca2+ can non-competitively
inhibit ligand binding, indicating that certain of the metal-binding
sites can play an inhibitory role (22, 31).
4
1 inhibitors using the tetrapeptide ILDV
ligand binding sequence from the CS1 region of fibronectin as the
starting point for inhibitor design (32). This sequence is homologous
to the tetrapeptide QIDS, which comprises the
4
1-binding site in VCAM-1 (33). Whereas
both ILDV and QIDS peptides weakly inhibit ligand binding and cell
adhesion (33, 34), a compound that was 106 times more
potent, BIO1211, was generated by substituting isoleucine with a
4-((N'-2-methylphenyl)ureido)-phenylacetyl N-terminal cap (35). Here we used a tritiated version of BIO1211 as a probe to assess
4
1 function under various states of
activation. The data provide new information on the effect that
different activation conditions have on the affinity of
4
1 for its ligands and establish that
these differences in affinity are regulated by changes in the
dissociation rate of the ligand-integrin complex.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C. The binding affinity of the labeled compound was indistinguishable from unlabeled BIO1211 in the
4
1-direct binding assay
with VCAM-Ig-alkaline phosphatase as a reporter (36).
4
1 Expressing Cells--
Jurkat cells
that had been enriched for
4
1 expression
by FACS sorting were maintained in RPMI 1640 medium plus 10% fetal bovine serum at 37 °C in a tissue culture incubator. K562 cells that
had been transfected with either the human
4, human
2, or human
1 gene and selected for high
levels of
4
1,
2
1, and
1
1,
respectively, by FACS were grown in the same medium supplemented with 1 mg/ml G418, 10 µg/ml gentamicin sulfate, and 50 µg/ml streptomycin.
2 and
4 K562 cells were a gift of Dr.
Martin Hemler. For binding studies, the cells were pelleted by
centrifugation, washed two times with TBS (50 mM Tris HCl,
150 mM NaCl, 0.1% bovine serum albumin, 2 mM
glucose, 10 mM HEPES, pH 7.4), suspended at approximately 2 × 106 cells/ml in TBS, and counted using a Neubauer
hemocytometer. The cells were further diluted with TBS to the
concentration indicated and treated with [3H]BIO1211 at
room temperature. The cells were then pelleted by centrifugation,
resuspended in 100 µl of TBS plus Mn2+, and transferred
to a scintillation vial containing 2.9 ml of ScintiVerse II (Fisher).
Cell-associated radioactivity was quantified by scintillation counting.
All studies were performed in siliconized 1.5-ml Eppendorf tubes with a
standard 1-ml sample volume. Each condition was tested in at least two
independent studies. In the studies indicated, a 100-1000-fold excess
of unlabeled BIO1211 was added to samples after the incubation with
[3H]BIO1211 to prevent further binding. Binding studies
testing the effects of cell number, incubation time, and
[3H]BIO1211 concentration were performed in TBS plus 2 mM MnCl2 as described. Nonspecific binding of
[3H]BIO1211 to cells was assessed at each cell density
and [3H]BIO1211 concentration in TBS but in the absence
of added metal ion. Specific counts bound were calculated by
subtracting nonspecific counts from total counts bound. Other studies
testing the effects of activation on binding were performed as
indicated. In the 1 mM Ca2+, 1 mM
Mg2+ state where KD = 20-40
nM for binding of BIO1211 to
4
1, a high background at the higher
BIO1211 concentrations (3,000 cpm at 10 nM) in the standard
assay format limited the concentration of [3H]BIO1211
that could be tested to ~10 nM; however, by diluting the
specific activity of the label from 50 to 5 Ci/mmol, binding could be
evaluated at BIO1211 concentrations up to 100 nM, albeit with reduced precision.
4
1-Ligand Interactions by
Competition--
[3H]BIO1211 was also used to study
4
1 function by competition, using the
radioactivity as a reporter for
4
1
occupancy. In this format, Jurkat cells (1 × 106/ml)
in the buffers indicated were treated with serial dilutions of test
compound for 1 h, and then 5 nM
[3H]BIO1211, an amount sufficient to bind all unoccupied
receptors, was added for 10 min before measuring the bound counts. The
cells were then pelleted by centrifugation and subjected to
scintillation counting. Counts bound under these conditions measure
integrin that is not occupied by the test compound and is therefore
free to bind the [3H]BIO1211. The competition format was
also used for kinetic binding studies. Binding and dissociation
constants were calculated from
4
1 that
after treatment with test compound was free to bind the
[3H]BIO1211.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1 Binding
Assay Using [3H]BIO1211--
The ability of
[3H]BIO1211 to bind Jurkat
(
4
1 positive) cells and
1-transfected K562 (
4
1
negative) cells was examined under diverse conditions known to alter
the activation state of
4
1 (Fig.
1). [3H]BIO1211 binding to
Jurkat cells was greatest in the presence of 2 mM
Mn2+ alone, 10 mM Mg2+ alone, and 2 mM Mn2+ + 10 µg/ml mAb TS2/16, treatments
that activate
4
1. Binding was greatly
reduced under non-activating conditions exemplified by treatment with
Ca2+, Mg2+ (1 mM each) (see Fig. 1,
A and B). No binding was observed to the
1-transfected K562
(
4
1-negative) control cell line. The ~2000 cpm background seen in Fig. 1A is residual free
[3H]BIO1211, which was removed if samples were washed
prior to analysis. In the studies presented below, specific binding was
calculated by subtracting background counts from total counts bound.
Background binding was determined for each test sample using a control
sample that was subjected to the same treatment but in the absence of any divalent cation.
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Fig. 1.
Binding of [3H]BIO1211 to
Jurkat and 1-K562 cells under
various states of
4
1
activation. Jurkat cells were incubated with
[3H]BIO1211 in the indicated buffers and conditions. The
cells were then pelleted by centrifugation, resuspended in 100 µl of
TBS plus Mn2+, transferred to a scintillation vial
containing 2.9 ml of ScintiVerse II, and cell-associated radioactivity
was quantified by scintillation counting. A, a series of
studies were performed in which 1-ml samples (2 × 106
cells) were incubated with 5 nM [3H]BIO1211
in the indicated buffers for 2 h at room temperature.
B, Jurkat cells at 0.75 × 106/ml were
incubated at room temperature for 40 min with the indicated
concentrations of [3H]BIO1211 in the presence of 1 mM Ca2+, 1 mM Mg2+
(
); Ca2+, Mg2+, Mn2+ (1 mM each) (
); 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16 (
); 2 mM Mn2+ (
); 2 mM
Mn2+ plus 10 µg/ml TS2/16 (+). Background
counts were determined at each concentration of
[3H]BIO1211 tested by incubating identical samples in TBS
buffer in the absence of divalent cations. Specific counts bound were
calculated by subtracting background counts from total counts bound.
C, Jurkat cells were treated with mAb B5G10 under the
conditions indicated, then with phycoerythrin-labeled anti-mouse Ig
antibody, and analyzed by FACS.
4
1 expression levels, and based on this
number, we estimate that the Jurkat cells used in these studies have
approximately 80,000 copies of
4
1 per
cell. To rule out the possibility that
4
1
levels changed under the different treatments,
4
1 levels were compared under the 1 mM Ca2+, 1 mM Mg2+
(non-activating), and 2 mM Mn2+ (activating)
conditions by FACS, using the non-neutralizing anti-
4 mAb B5G10 as a reporter (Fig. 1C). The
4
1 levels in the activated and
non-activated samples were indistinguishable.
100% of the total assay volume had
no effect on the binding assay (data not shown). Third, bound
[3H]BIO1211 could be quantitatively released from Jurkat
cells with EDTA, indicating that the
BIO1211·
4
1 complex remained on the cell
surface. The released BIO1211 was further characterized by reverse
phase HPLC for the presence of potential degradation products that
might impact our interpretation of binding. As shown in Fig. 2, over 95% of the bound
[3H]BIO1211 was released by the EDTA treatment unchanged,
indicating that only minimal degradation had occurred. Previously, we
showed that the kinetically determined KD values for
binding of [3H]BIO1211 to Mn2+-activated
cells of various types (peripheral blood lymphocytes, ~10,000 copies
of
4
1/cell; Jurkat cells, ~80,000
copies/cell;
4-K562 cells ~250,000 copies/cell) were
similar, showing that BIO1211 binding was not affected by differences
in surface expression of
4
1 (35).
Finally, because of the presence of two carboxylic acid groups in the
BIO1211 sequence, we tested whether it could function as a chelator.
Using fluorescent metal ion indicators that are sensitive to the
concentrations of free Mg2+, Mn2+, and
Ca2+ as probes for binding, we were unable to detect an
association between BIO1211 and these metal ions (data not shown).
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Fig. 2.
Analysis of [3H]BIO1211
stability by reverse phase HPLC. Jurkat cells (7.5 × 106 cells/ml) in TBS containing 2 mM
Mn2+ were incubated at room temperature for 30 min with 1 or 5 nM [3H]BIO1211 as indicated. The cells
were pelleted, and the supernatant was collected (supernatant 1). The
cells were washed with TBS containing 2 mM Mn2+
and treated with TBS + 2 mM Mn2+ plus 10 mM EDTA for 60 min, with each step followed by a
centrifugation step. Supernatants from the initial incubation medium,
wash, and EDTA extraction were analyzed by reverse phase HPLC. 0.5-ml
fractions were collected, mixed with scintillation mixture, and
analyzed by scintillation counting. A shows HPLC elution
profile for [3H]BIO1211 without treatment. B
shows the corresponding data from supernatant 1 of the 1 nM
BIO1211 treatment, and C shows the data from the EDTA
extract of the 5 nM treatment.
4
1 by Equilibrium Binding--
The low
level of occupancy seen in Fig. 1B, for binding of
[3H]BIO1211 to Jurkat cells under non-activating
conditions, suggested that the affinity of BIO1211 for
4
1 was lower for non-activated than for
the activated integrin. In order to confirm this possibility and to
obtain a more accurate measure of KD, the analysis was repeated using higher BIO1211 concentrations. The resulting curves
showed dose-dependent binding and, at 100 nM
[3H]BIO1211, specific counts bound were comparable for
the 1 mM Ca2+, 1 mM
Mg2+ state and the 2 mM Mn2+ state
(data not shown). Based on these binding data, a KD of 20-40 nM was calculated for the 1 mM
Ca2+, 1 mM Mg2+ (non-activated)
state. Attempts to estimate KD values for activated
states using equilibrium binding methods were unsuccessful because the
KD values were lower than the concentration of
4
1 in the assay. Thus, while we observed
dose-dependent and saturable binding (see Fig.
1B), the binding curves were in fact simply measuring a
titration of the receptor to full occupancy and could not be used to
accurately calculate affinity. A more accurate assessment of the
affinity of BIO1211 for activated
4
1 was
obtained using kinetic measurements (see below). For unknown reasons,
total counts bound were 20% lower in the 1 mM
Ca2+, 1 mM Mg2+ + TS2/16 state
under conditions that should have been saturating for
[3H]BIO1211 binding compared with other activated states
(Fig. 1B). It was not possible to perform FACS analysis in
the presence of TS2/16 to test if TS2/16 treatment had altered surface
levels of integrin, since the analysis would have required co-treatment with two murine anti-
4
1 mAbs.
4
1
in Different Activation States--
Kinetic data for the binding and
dissociation of [3H]BIO1211 to
4
1 were measured under the following
conditions, as shown in Fig. 3: 1 mM Ca2+, 1 mM Mg2+;
Ca2+, Mg2+, Mn2+ (1 mM
each); 1 mM Ca2+, 1 mM
Mg2+ + 10 µg/ml TS2/16; 2 mM
Mn2+; and 2 mM Mn2+ + 10 µg/ml
TS2/16. Binding reached a plateau level within 10 min of treatment for
all test conditions (Fig. 3A). Association rate constants
(kon) calculated from these time courses all
fell in the narrow range from 0.9 to 2.7 × 106
M
1 s
1 (Table
I). Differences in the percent
4
1 occupied after 10 min were identical
to those seen in the equilibrium experiments shown in Fig.
1B. Time courses for the rates of dissociation of BIO1211
from Jurkat cells are shown in Fig. 3B. Unlike the
association rates, which were similar across the different assay
conditions, the dissociation rates varied over a wide range and were
highly dependent on the activation state of the integrin. Values for koff ranged from 0.17 × 10
4
s
1 for the 2 mM Mn2+ + TS2/16
state to >140 × 10
4 s
1 for the 1 mM Ca2+, 1 mM Mg2+
state (Table I). The kinetic rate constants from these and other binding studies were used to calculate KD values,
which are summarized in Table I. The close correspondence between the variations in KD values and in dissociation rates
indicates that the affinity of BIO1211 for
4
1 is governed almost exclusively by off
rates. KD values observed under conditions commonly considered to be activating ranged from 470 pM in the
Ca2+, Mg2+, Mn2+ (1 mM
each) state to 18 pM in the 2 mM
Mn2+ + TS2/16 state. These differences in affinity that
resulted from activation were not apparent from the equilibrium binding
measurements because the KD values were lower than
the concentration of
4
1 in the assays and
therefore were masked by the format of the assay. Similar problems were
encountered when affinities were estimated by measuring the ability of
BIO1211 to block cell adhesion to CS1 or VCAM or to block direct
binding of VCAM-Ig to Jurkat cells with VCAM-Ig as the reporter (data
not shown).
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Fig. 3.
Assessing
[3H]BIO1211/ 4
1
binding on Jurkat cells under various states of activation using
kinetic measurements. Jurkat cells were incubated with
[3H]BIO1211 in the presence of 1 mM
Ca2+, 1 mM Mg2+ (
);
Ca2+, Mg2+, Mn2+ (1 mM
each) (
); 1 mM Ca2+, 1 mM
Mg2+ plus 10 µg/ml TS2/16 (
); 2 mM
Mn2+ (
); 2 mM Mn2+ plus 10 µg/ml TS2/16 (+). For kon measurements, 1-ml
samples (1.35 × 106 cells/ml) were treated with 2 nM [3H]BIO1211 for the times indicated. Cells
were collected by centrifugation and subjected to scintillation
counting. For koff measurements, 1-ml samples
(2 × 106 cells/ml) were treated with 5 nM
[3H]BIO1211 for 120 min at room temperature. 5 µM unlabeled BIO1211 was added, and the cells were
further incubated for the times indicated. Cells were pelleted at each
time point, and cell-associated [3H]BIO1211 was measured
by scintillation counting. The data in A (association) and
B (dissociation) were fitted to exponential curves by
nonlinear regression, and kon and
koff values were calculated from the curve fits
(Table I). For kon, 100% refers to maximum
[3H]BIO1211 bound in the Mn2+-activated
state, whereas for koff, 100% bound reflects
maximum binding under each test condition (i.e. specific
binding at t = 0).
Binding of [3H]BIO1211 to 4
1 on Jurkat
cells under various states of activation
4
1 were determined
from kinetic measurements as described in the legend to Fig. 3. mAbs
TS2/16 and HP1/2 were used at 10 µg/ml. On rates for the Mg2+
states were determined at 1, 10, and 100 mM Mg2+
and were indistinguishable. The value measured at those concentrations
has been assigned to the other Mg2+ alone states shown.
4 s
1 to 5.3 × 10
4
s
1 to 4.3 × 10
4 s
1 to
3.2 × 10
4 s
1 to 2.1 × 10
4 s
1 when dissociation rates were
measured in the presence of 2, 10, 50, 100, and 300 mM
Mg2+. When KD was plotted as a function
of Mg2+ concentration, the data fit a hyperbolic curve,
suggesting that the measurements were part of a continuum rather than
discrete points (Fig. 4B). At high concentrations of added
Mg2+, binding appears to be saturating out at an affinity
that approximates the value seen at 2 mM Mn2+
(Fig. 4B). Suboptimal concentrations of Mn2+
produced a similar titration of the dissociation rate to that seen with
Mg2+, although at a much lower concentration of the metal
ion (data not shown). Variations in the concentration of TS2/16 from
0.1 to 10 µg/ml had no effect on KD (data not
shown), indicating that 10 µg/ml was a saturating concentration.
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Fig. 4.
Dissociation curves for
[3H]BIO1211 from the Mg2+-activated
BIO1211· 4
1
complexes. One-ml aliquots of Jurkat cells (1 × 106 cells) under the conditions indicated were treated with
5 nM [3H]BIO1211 for 2 h at room
temperature. 5 µM of unlabeled BIO1211 was added, and the
cells were further incubated for 0, 5, 15, 30, 60, 90, and 120 min. The
cells were pelleted, and cell-associated [3H]BIO1211 was
measured by scintillation counting. The data were fitted to exponential
curves by nonlinear regression. 100% occupied was defined as specific
counts bound at t = 0. Over the range of
Mg2+ concentrations tested there was no change in this
number. 100% binding = 11,000 cpm in the Mg2+ alone
samples and 12,000 cpm in the Mg2+ + TS2/16 samples.
Background = 1500 cpm for both sets of data. A, 2 mM Mg2+ (
); 10 mM
Mg2+ (
); 100 mM Mg2+ (
); 2 mM Mg2+ plus 10 µg/ml TS2/16 (
); 10 mM Mg2+ plus 10 µg/ml TS2/16 (
); 100 mM Mg2+ plus 10 µg/ml TS2/16 (
).
B, hyperbolic dependence of KD on
[Mg2+] (solid line), saturating at high
[Mg2+] to the value of KD = 100 pM seen at 2 mM Mn2+ (dashed
line).
4
1 in the presence of divalent cations
was achieved with 2 mM Mn2+, a further increase
in affinity was observed if samples were co-activated with 2 mM Mn2+ + TS2/16, suggesting that the
mechanisms by which TS2/16 and divalent cations brought about
activation were distinct. Results from these analyses are summarized in
Table I. In the presence of 2 mM Mn2+ alone and
2 mM Mn2+ + TS2/16, KD
values of 100 and 18 pM, respectively, were observed. The
KD value resulting from activation by TS2/16 alone
(i.e. 1 mM Mg2+, 1 mM
Ca2+ + TS2/16) was 220 pM. A similar increase
in affinity was seen after treatment of the 10 mM
Mg2+ alone state with TS2/16 (KD = 440, 220, and 40 pM for the 10 mM Mg2+
alone, TS2/16 alone, and 10 mM Mg2+ + TS2/16
states, respectively). Interestingly, the final KD observed after co-activation by divalent cations plus TS2/16 was strongly influenced by the concentration and type of divalent cation.
KD values varied with metal ion concentrations in a
manner analogous to the data observed in the divalent cation alone
state, whereas the TS2/16 treatment appeared to produce a quantum step.
This distinction in effect is particularly apparent from the
dissociation data shown in Fig. 4A. In contrast to the additive effect seen after co-activation by TS2/16 plus divalent cations, the dissociation rate observed in the presence of 2 mM Mn2+ plus 10 mM Mg2+
was not significantly slower than that seen with 2 mM
Mn2+ alone, indicating that activation caused by
Mn2+ and Mg2+ was not additive (data not shown).
4
1 was
significantly lower (>5 nM) than the affinities determined
for the activated states. Whereas the on-rate for the Ca2+,
Mg2+ state was similar to that observed under activating
conditions (see Table I), dissociation was too rapid to allow an
accurate determination of koff, and so the
kinetic data gave only a lower limit to KD (see Fig.
3B). The limit of KD >5 nM
is consistent with the more accurate estimate of KD = 20-40 nM that was obtained from equilibrium measurements
described above. Together the data in Table I demonstrate that the
affinity of BIO1211 for
4
1 is highly
sensitive to the activation state of
4
1
and can vary by over 1000-fold depending on the assay conditions.
4
1 affinity states are dependent on
metal ion binding, we investigated the effects of EDTA treatment on
dissociation of the BIO1211·
4
1 complex
under various activating conditions. Results from this analysis are
shown in Fig. 5 and Table
II. EDTA treatment of the 2 mM Mn2+ alone state resulted (Fig.
5A), as expected, in a more rapid release of
[3H]BIO1211 from the complex, although even after EDTA
treatment the rate of release was not as rapid as from the
non-activated state. EDTA treatment also resulted in a more rapid
release of BIO1211 from the 2 mM Mn2+ + TS2/16
state; however, the rate of release was clearly slower in the presence
of TS2/16 than when either the 10 mM Mg2+ alone
or 2 mM Mn2+ alone activated states were
treated with EDTA.
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Fig. 5.
Dissociation curves for
[3H]BIO1211 from the 2 mM Mn2+ + TS2/16-activated
BIO1211· 4
1
complex after treatment with EDTA and Ca2+. One-ml
aliquots of Jurkat cells (1 × 106 cells) in TBS plus
2 mM Mn2+ and 10 µg/ml TS2/16 or with 2 mM Mn2+ alone were treated with 5 nM [3H]BIO1211 for 2 h at room
temperature. 5 µM unlabeled BIO1211 was added in the same
buffer either alone or with the addition of 10 mM EDTA or 1 mM Ca2+, and the cells were further incubated
for 0, 5, 15, 30, 60, 90, and 120 min. The cells were pelleted, and
cell-associated [3H]BIO1211 was measured by scintillation
counting. The data were fitted to exponential curves by nonlinear
regression. A, 2 mM Mn2+ alone
(
); 2 mM Mn2+ plus EDTA (
); 2 mM Mn2+ plus 10 µg/ml TS2/16 (+);
2 mM Mn2+ plus 10 µg/ml TS2/16 plus EDTA
(
); 2 mM Mn2+ plus 10 µg/ml TS2/16 plus 1 mM Ca2+ (×). B, 1 mM Ca2+, 1 mM Mg2+ plus
10 µg/ml TS2/16 (
); 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16 plus EDTA (
).
Data sets for the 2 mM Mn2+ plus 10 µg/ml
TS2/16 plus EDTA and 2 mM Mn2+ plus 10 µg/ml
TS2/16 plus 1 mM Ca2+ conditions shown in
A and for both sets of conditions shown in B were
fit to double exponential curves.
Rate constants for the dissociation of BIO1211-integrin complexes under
various conditions
4
1·BIO1211 complexes formed in the
presence of 1 mM Ca2+, 1 mM
Mg2+ + TS2/16 and treated with EDTA (see Fig.
5B). Unlike the large effect of EDTA treatment on the
release of BIO1211 from the divalent cation alone and divalent cation
plus TS2/16-activated states, the effect of EDTA on the 1 mM Ca2+, 1 mM Mg2+ + TS2/16 state was modest, causing only a 2-3-fold increase in the rate
of the fast phase of release. Although we originally had assumed that
the dissociation of BIO1211 from
4
1·BIO1211 complexes would follow a
single exponential based on the release data that had been generated
for the Mn2+-activated integrin, a reevaluation of the data
summarized in Table I indicated that this was not necessarily true for
all methods of activation. Additional experiments with more data points added along the relevant regions of the dissociation curves supported our original finding that BIO1211 release from divalent cation alone
activated
4
1 follows a single exponential
and reconfirmed the observation that the dissociation of BIO1211 from
the 1 mM Ca2+, 1 mM
Mg2+ + TS2/16 state was biphasic. By extrapolating the line
defined by the slowly dissociating component back to the y
intercept, we can estimate that about 50% of the integrin-BIO1211
complex for the 1 mM Ca2+, 1 mM
Mg2+ + TS2/16 state was in this form (Fig. 5B).
For the tight binding states that were promoted by divalent cation plus
TS2/16 treatment, koff without EDTA treatment
was so slow that it was impossible to distinguish between a single and
double exponential due to the limited dissociation that occurred during
the 120-min duration of the experiment, so the potential biphasic
nature of these states remains unclear.
4
1 that had been immobilized on plastic
and assayed in an enzyme-linked immunosorbent assay-type format.
Dissociation rates for the release of BIO1211 from
BIO1211·
4
1 complexes that were formed
in the presence of 2 mM Mn2+ or 2 mM Mn2+ + TS2/16 were similar to values
observed on live cells, and when the same complexes were treated with
EDTA, TS2/16 treatment dramatically decreased the rate of release of
BIO1211 from the complex (data not shown). The effect of TS2/16 in the
plate format was even more pronounced than on cells. Whereas about 50%
of the BIO1211 was rapidly dissociable from cells after EDTA treatment,
in the plate format only about 10% was in the rapidly dissociable
form, and 90% of the
4
1·BIO1211
complexes were in the EDTA-resistant form.
4
1
activation are complex. For
4
1·BIO1211
complexes, formed in the presence of 2 mM Mn2+ + TS2/16, Ca2+ treatment was as effective as EDTA at
promoting release of BIO1211 and, like EDTA treatment, required a
double exponential fit to account for the data (Fig. 5A).
Ca2+ had less of an effect on the divalent cation alone or
TS2/16 states (see Table I). In particular for activation by
Mn2+, 1 mM Ca2+ treatment resulted
in only a modest 3-fold effect on koff
(koff = 5.3 and 1.4 × 10
4
s
1 for the Ca2+, Mg2+,
Mn2+ (1 mM each) and 2 mM
Mn2+ states, respectively). In the studies shown in Fig.
1A, we observed that increasing the Ca2+
concentration from 1 to 10 mM slightly stimulated BIO1211
binding. Although the ability of Ca2+ to induce the release
of bound BIO1211 from the 2 mM Mn2+ + TS2/16
state is consistent with the published model based on studies for
RGD-binding integrins in which high concentrations of Ca2+
can function as a non-competitive inhibitor of ligand binding (22, 31),
the results we observed for Ca2+ on other states show that
Ca2+ can have a wide range of effects on activation. Recent
findings (37) have further highlighted differences between
4
1 and other
1 integrins
in their responsiveness to calcium. A more thorough study is needed to
understand better the interplay between Ca2+ and other
metal ion binding and how these effects are connected to ligand binding.
4
1 (35, 36). An interesting
aspect of the binding data was that the VCAM-Ig titration required a
non-hyperbolic curve fit to account for the data, as had been
previously seen in FACS binding experiments using VCAM-Ig (38). The
binding equation used to fit the data for VCAM-Ig describes the data
expected for a bivalent ligand (39) and differs from the simple
hyperbolic competition curve seen for the monovalent CS1. VCAM-Ig
binding was also evaluated by competition using a kinetic readout.
Surprisingly, the t1/2 for dissociation of the
VCAM-Ig·
4
1 complex was only 8 min for the Mn2+ state, which identified a limitation in using
VCAM-Ig as a reporter of integrin function (t1/2 = 70 min for BIO1211 under these conditions). [3H]BIO1211
as a reporter type readout should have broad applications as a probe
for
4
1 structure-function.
View larger version (23K):
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Fig. 6.
Analysis of CS1 and VCAM-Ig binding through
competition. The apparent affinities of CS-1 and VCAM-Ig for
4
1 were assessed on Jurkat cells through
competition with [3H]BIO1211. Jurkat cells (2 × 106 cells/ml) in TBS plus 2 mM Mn2+
buffer were incubated for 1 h at room temperature with serial
dilutions of CS1, VCAM-Ig, or unlabeled BIO1211 at the concentrations
indicated. At the end of the incubation, 5 nM
[3H]BIO1211 was added, and the cells were further
incubated for 10 min. The cells were then pelleted, and the
bound [3H]BIO1211 was quantified by
scintillation counting. The titration data were plotted as a percent of
maximum counts bound with no added competitor.
, BIO1211;
,
VCAM-Ig;
, CS1.
4
1 common to its
interaction with other ligands, we tested VCAM-Ig and CS1 binding to
Jurkat cells under the same conditions described for BIO1211. As shown
in Table III, VCAM-Ig and CS1 were also
sensitive to differences between the 2 mM Mn2+
alone and 2 mM Mn2+ + TS2/16 states. These
observations support the notion that BIO1211 mimics many of the
properties of
4
1 ligands and that the
different activation states of
4
1 it
distinguishes are indeed relevant to the binding of more physiological
ligands.
The Mn2+ + TS2/16 high affinity state is a property of
4
1 that affects binding of VCAM-Ig and CS-1
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
1 integrin function.
When binding was tested under various conditions that affect the
activation state of
4
1,
KD values were observed that ranged from a value of
20-40 nM in the 1 mM Mg2+, 1 mM Ca2+ (non-activated) state to 100 pM in the 2 mM Mn2+ (activated)
state to 18 pM in a newly identified state detected when
binding was measured in the presence of 2 mM
Mn2+ + 10 µg/ml mAb TS2/16. The differences in affinity
were regulated almost exclusively by changes in the dissociation rate
of the complex. Although we had expected to see affinity differences between non-activated and activated
4
1,
the large variation in affinity among the various activated states was
surprising. Over a 25-fold difference in affinity for BIO1211 was
detected between the Ca2+, Mg2+,
Mn2+ (1 mM each) state and the 2 mM
Mn2+ + TS2/16 state. To verify that the variations in the
integrin affinity state are relevant to ligands other than BIO1211, we tested VCAM-1 and CS-1 binding under selected conditions and showed that similar trends were also seen for these more physiologically relevant ligands.
4
1
was that activation was not defined by a single high affinity state but
rather by a continuum of affinities that was easily manipulated by
changes in the assay conditions. This result is particularly apparent from the data shown in Fig. 4B where the affinity changed
from 700 to 440 to 360 to 220 to 170 pM by simply changing
Mg2+ concentrations. At high concentrations of added
Mg2+, BIO1211 affinity and dissociation rate converge to
the values seen at 2 mM Mn2+ (Fig.
4B). This observation suggests that the activating effects seen with Mg2+ and Mn2+ at saturation involve
formation of the same affinity state of the integrin. This conclusion
was supported by the observation that no further increase in affinity
was observed when samples that had been treated with 2 mM
Mn2+ were also treated with 10 mM
Mg2+. Suboptimal concentrations of Mn2+
produced a similar titration of the dissociation rate to that seen with
Mg2+, with the most dramatic effects seen at concentrations
of less than 50 µM Mn2+ (data not shown).
Presumably, this continuum represents the effects of metal
ion-dependent interconversion of a finite number of
discrete states of the integrin. If the interconversion between the
states is fast as few as two states could account for the data shown in
Fig. 4B. Further studies are needed to define the exact
number of affinity states involved.
4 s
1), 10 mM Mg2+ + TS2/16 (koff = 0.43 × 10
4
s
1), and 100 mM Mg2+ + TS2/16
(koff = 0.17 × 10
4
s
1). The relative changes in the dissociation rate of the
BIO1211·
4
1·TS2/16 complex that were
observed as Mg2+ was increased from 2 to 10 mM
and from 10 to 100 mM are strikingly similar to the
relative changes observed at the same Mg2+ concentrations
in the absence of TS2/16 (Table I). In fact, when
koff values for the Mg2+ + TS2/16
states were plotted against koff values for the
corresponding Mg2+ alone states, we observed a straight
line with a slope of 0.125. Key features of the binding data are
summarized in the schematic drawing shown in Fig.
7. In particular, Fig. 7 highlights the distinct and additive mechanisms by which divalent cations and TS2/16
promote
4
1 activation.
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Fig. 7.
Schematic summary of binding data.
Dissociation constants for BIO1211 binding are summarized using actual
koff measurements to define integrin activation
states as described in Table I. I = non-activated
4
1 observed in the presence of 1 mM Ca2+, 1 mM Mg2+.
IT = activated
4
1
observed in the presence of 1 mM Ca2+, 1 mM Mg2+ plus 10 µg/ml TS2/16.
IM2+ = activated
4
1 observed in the presence of divalent
cations. IM2+ = activated
4
1 observed after co-stimulation with
divalent cation plus TS2/16. Hatch marks indicate the
different affinity states observed as a result of changes in the type
and concentrations of divalent cations. The single
IT state was observed over a range of TS2/16
concentrations from 0.1 to 10 µg/ml.
When high affinity 4
1·BIO1211 complexes
were treated with EDTA, BIO1211 was released; however, the rate of
release differed depending on the degree of activation. Dissociation
data for cation-activated
4
1 could be
fitted to a single exponential, indicating that once the integrin is
treated with EDTA, BIO1211 is released at a single characteristic rate.
In contrast, the data for TS2/16-activated
4
1 did not fit a single exponential and
required a double exponential fit indicating that more than one
population of sites contributes to the release process. The rate
constant for the rapidly dissociating population was similar to that
for release from the divalent ion alone state after treatment with
EDTA, whereas the rate constant for the slowly dissociating population
was similar to that for the divalent cation plus TS2/16 state in the
absence of EDTA. Similar biphasic release data were obtained regardless
of whether samples were activated by TS2/16 alone or by TS2/16 plus
divalent cation, indicating that the complex dissociation curves were
not unique to the high affinity state. Thus, it appears either that the
divalent cations are not accessible to EDTA to the same degree in the
different activated states of
4
1 or that
a different step governs the release rate. Further studies are needed
to distinguish between these alternatives. It is not clear whether
activation with TS2/16 induces heterogeneity in the integrin or allows
preexisting heterogeneity to become visible. Nevertheless, the fact
that dissociation data after EDTA treatment can distinguish between
divalent cation and TS2/16-activated states provides additional support
for the idea that the two stimuli bring about activation of
4
1 in distinct ways.
Although the precise mechanism by which the affinity states of
integrins are regulated is unknown, a large body of data implicates the
cation-binding sites as a key component (21, 22, 31). Since
ligand-binding sites map to the same regions on the - and
-chains
as the cation-binding sites, various models have been proposed in which
the cations coordinate with ligands or compete with ligand for binding
(28-30, 32, 40), or in which cation binding regulates the opening of
the integrin dimer to expose the ligand-binding site (21). Our data
argue against the latter model since only the affinities and not the
association rate constants for ligand binding are altered by changes in
the activation state, indicating that the ligand-binding site is
equally accessible for binding under all states of activation. In one
published study where a peptide spanning the MIDAS site from the
3 subunit of
IIb
3 was used
as a model for the ligand-binding site, the authors suggested ligand
binding could directly compete for Mn2+ binding (40). We
see no evidence for this competition using [3H]BIO1211 as
a reporter for
4
1 function.
Although many models have been proposed for how integrin activation is
regulated, the simplest explanation for the independent and additive
effects of divalent cations and TS2/16 on
4
1 activation is that the metal-binding
sites on the
4-chain and the MIDAS site on
1-chain can be independently regulated. Thus, the
dependence of
4
1 affinity on divalent
cations could reflect regulation on the
-chain elements, whereas the
dependence of the affinity on TS2/16 binding could reflect regulation
on the
-chain. Because of the multiple putative metal-binding sites
on the
4-chain, variations in the type and concentration
of metal ions could affect occupancy of these sites and thereby change
the affinity of
4
1 for ligand. In
contrast, since TS2/16 would either be bound or not bound, one would
expect a quantum effect of the antibody on affinity through a
conformational change induced by antibody binding that can either
directly induce ligand binding or, alternatively, that affects metal
binding at the MIDAS which would indirectly affect ligand binding.
Although the current studies do not allow us to evaluate BIO1211
binding at a molecular level, the availability of
-chain mutants
(41) that are targeted at these key regulatory sites should allow us to
define more precisely the effects of these distinct elements on ligand binding.
Ligand binding and integrin activation induce a cascade of
conformational changes within the integrin that ultimately lead to the
activation of intracellular signaling pathways. These changes have been
studied in detail using mAbs whose epitopes are either exposed (termed
LIBS) or lost following ligand binding (21, 42). BIO1211 is a LIBS
inducer and therefore might be expected to exhibit the same profile of
effects that natural ligands exhibit (35). The small size of BIO1211
makes it particularly well suited for this type of analysis, since it
minimizes the chance of steric inhibition of antibody binding. This
notion is particularly apparent from the data for HP1/2 shown in Table
I. HP1/2 is a B1 class anti-4-antibody, which is defined
as a potent inhibitor of
4
1-ligand interactions that does not induce leukocyte homotypic aggregation (42).
Although HP1/2 is a potent inhibitor of VCAM-Ig binding (36), our data
clearly demonstrate that HP1/2 has no effect on BIO1211 binding and
therefore that it blocks ligand binding through steric effects rather
than through direct binding at the ligand-binding pocket. The exquisite
sensitivity of the [3H]BIO1211 to differences in
4
1 activation suggests that the labeled
probe will prove to be an extremely valuable readout for this type of analysis.
Although many assays have been used to study
4
1 function (36, 43, 44), the data we
generated with [3H]BIO1211 revealed various features
about ligand binding that were not evident from these conventional
assay methods. Most significant were the observations that activation
is not defined by a single state but rather by several distinct states
that give rise to a range of affinities and that the affinity
differences are tightly coupled to dissociation rates of the
integrin-ligand complex. As soluble, monovalent probes for
4
1 function, BIO1211, and related
inhibitors represent novel tools that should aid in further unraveling
the complexities associated with integrin activation.
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ACKNOWLEDGEMENTS |
---|
We thank Francisco Sanchez-Madrid for the
TS2/16 antibody; Martin Hemler for providing the 2- and
4-K562 transfectants and the B5G10 hybridoma; and Phil
Gotwels for providing the
1-transfected K562 cell line.
We also thank Diane Leone, Andrew Sprague, and William Delahunt for
providing data on the biochemical properties of BIO1211 in diverse
assays in which IC50 values were calculated using adhesion
and direct binding formats with VCAM-Ig as reporters.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Biogen, Inc., 14 Cambridge Center, Cambridge, MA 02142. Tel.: 617-679-3310; Fax: 617-679-2616.
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ABBREVIATIONS |
---|
The abbreviations used are: VCAM-1, vascular cell adhesion molecule-1; BIO1211, 4-((N'-2-methylphenyl)ureido)-phenylacetyl-leucine-aspartic acid-valine-proline; mAb, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; HPLC, high pressure liquid chromatography; FACS, fluorescence-activated cell sorter.
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REFERENCES |
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