From the
Changes in ligand binding ability of the integrin
Several of the integrins have been shown to undergo
activation-related changes in affinity for ligands that are thought to
reflect distinct structural transitions within one or both subunits
(1-8). The platelet integrin
Ligand binding to
We have developed a murine IgG
Since they are sensitive to conformational
constraints, most LIBS are difficult to identify by conventional
peptide mapping approaches or by Western blot assay. Not surprisingly,
most anti-LIBS mAb (with the exception of AP5) fail to bind to
denatured, reduced
One of the
LIBS, that recognized by mAb AP5, is precisely localized in this report
to the amino terminus,
The first unique
attribute of the AP5 epitope is its location. Most of the previously
defined LIBS, with the exception of D3
(22) , are clustered
within the carboxyl-terminal region of the
AP5 is also one of those anti-LIBS
antibodies
(19, 22, 25, 37) , all of
which bind to
A third attribute of the AP5 epitope that is certainly
relevant to a recently proposed model of ligand binding
(26) is
its sensitivity to divalent cations. We show here that AP5 LIBS
expression is inversely proportional to divalent cation concentration.
AP5, like other anti-LIBS specific for
It has been proposed that ligand and
divalent cation binding to the integrin
Since RGDW can
overcome the inhibition of AP5 binding that is observed in the presence
of divalent cations, it is likely that the conformational constraints
placed upon
The AP5 epitope is a simple
linear sequence GPNICT that is readily available on denatured
This study would not have been possible without the
generous collaboration of Dr. Douglas DeSimone (University of Virginia,
Charlottesville, VA) from whom we received the Xenopus
can be monitored by the
concomitant expression of ligand-inducible binding sites (LIBS). A new
LIBS, the hexapeptide sequence GPNICT (residues 1-6) at the amino
terminus of
recognized by the murine monoclonal
antibody (mAb) AP5, is sensitive both to the binding of ligand and to
micromolar differences in divalent cation levels. Calcium or magnesium
can completely inhibit the binding of AP5 to
on platelets, with ID
values of 80 and 1500 µM, respectively. The
inhibitory effect of calcium plus magnesium is cumulative. In the
presence of 1 mM calcium plus 1 mM magnesium, the
peptide RGDW overcomes this inhibition and induces maximal binding of
AP5. Maximal AP5 binding is also induced by a molar excess of EDTA. The
unique location of the AP5 LIBS was determined by comparing the binding
of LIBS-specific mAb to recombinant human-Xenopus
chimeras produced in a baculovirus expression system. AP5 defines
one region at the amino terminus
1-6. A second
region, defined by mAb D3GP3, is probably located within
422-490, confirming the finding of Kouns et
al. (Kouns, W. C., Newman, P. J., Puckett, K. J., Miller, A. A.,
Wall, C. D., Fox, C. F., Seyer, J. M., and Jennings, L. K.(1991)
Blood 78, 3215-3223). The third region, encompassing at
most residues 490-690, and perhaps more precisely located within
602-690 (Du X., Gu, M., Weise, J. W., Nagaswami, C., Bennett, J.
S., Bowditch, R., and Ginsberg, M. H.(1993) J. Biol. Chem. 268, 23087-23092), is recognized by the four mAb, anti-LIBS2,
anti-LIBS3, ant-LIBS6, and P41. Since its exposure is uniquely
regulated by both divalent cations and ligand, the amino terminus of
may be involved in control of ligand binding by
divalent cation mobilization.
perhaps best exemplifies the extent to which such
activation-related, conformational changes can impact upon integrin
function. In its ``quiescent'' state,
mediates platelet attachment
selectively to surfaces coated with fibrinogen
(9) . Once
activated, however, it mediates attachment to surfaces coated with von
Willebrand factor, fibronectin, or vitronectin, as well as
fibrinogen
(10, 11, 12) . This change in ligand
specificity likely results from changes in the accessibility and
affinity of multiple binding sequences on the ligand, including the RGD
tripeptide and the carboxyl-terminal decapeptide sequence of the
fibrinogen
chain
(13, 14) , as well as changes in
the availability of recognition sites on
(13, 15, 16, 17) .
itself alters
the conformation of this integrin, exposing neo-epitopes, known as
ligand-induced binding sites (LIBS)
(18-22), and
leading to ``post-receptor occupancy'' events that are
required for full adhesive function of this
receptor
(23, 24) . Certain antibodies, by binding to a
particular LIBS, can in turn increase the affinity of
for its ligands. Anti-LIBS1 or
anti-LIBS2 thus induce fibrinogen binding to platelets (25).
Consequently, a LIBS represents a sensitive, conformationally
constrained epitope on
whose
exposure is intimately related to ligand-binding capacity of this
integrin. Ligand binding to
is now
thought to be regulated by divalent cations
(26) , and it follows
that the expression of a LIBS might also be modulated by divalent
cations. Only one LIBS, localized to
844-859
(20) and recognized by the monoclonal antibody PMI-1, is known
to be inhibited by divalent cations
(27) , and comparable cation
sensitivity has not been reported for any LIBS associated with
.
monoclonal antibody (mAb) AP5 that binds to a linear sequence of
that is stable to denaturation and disulfide bond
reduction
(28) . Because RGD-containing peptides or the mAb OPG2
induce the expression of the AP5 epitope, and AP5 itself can induce
platelet aggregation, the epitope recognize by AP5 is properly
classified as a LIBS.
. To circumvent these
obstacles, we employ interspecies, recombinant
chimeras, in which antigenic sequences of human
are expressed in the context of antigenically silent Xenopus
. Our recombinant
molecules are
synthesized and secreted by Spodoptera frugiperda cell lines
employing baculovirus-based
vectors
(29, 30, 31) . In this report, we show
that this novel approach can be used to delimit regions of
that are bound by six different anti-LIBS mAb.
1-6. Within the
nondenatured
complex, the
availability of the AP5 LIBS, unlike the remaining
LIBS, is sensitive to the presence of divalent cations.
Antibodies
Several of the monoclonal
anti- antibodies used in this study
() were previously
described
(28, 32, 33, 34, 35) .
Additional antibodies were obtained from other investigators.
Anti-LIBS1, anti-LIBS2, anti-LIBS3, and anti-LIBS6 were generous gifts
from Dr. M. Ginsberg (Scripps Research Institute, La Jolla,
CA)
(19, 21, 25, 36, 37) . D3GP3
was kindly supplied by Dr. L. Jennings (The University of Tennessee,
Memphis, TN)
(22) . SZ21 was a gift from Dr. C. Ruan (Szuchou,
China), while P3-13 was obtained through the CD Workshop, Boston,
MA
(38) . Monoclonal IgG was purified from ascites fluid by
affinity chromatography on Protein A-Sepharose CL-4B (Pharmacia LKB
Biotechnol.). Fab fragments were produced by papain digestion of
purified IgG
(33) and separated from Fc fragments and undigested
IgG by chromatography on Protein A-Sepharose CL-4B. The control mAb
HP20 (IgG
,
), a gift from Dr. M. Fougereau (University
of Marseille, Marseille, France), is an anti-idiotypic antibody
specific for murine antibodies that recognize the GAT antigen (39) and
does not bind to platelet antigens. Fluorescein isothiocyanate
(FITC)-conjugated HP20 is used as a negative control in flow cytometry
analyses.
Peptides
Arg-Gly-Asp-Trp (RGDW), peptide
1-13 (GPNICTTRGVSSC), and peptide
722-735 (HDRKEFAKFEEERAR) were synthesized using Fmoc
chemistry and a Milligen/BioResearch Laboratories 9050 Automated
Pepsynthesizer (San Rafael, CA) and purified by preparative reverse
phase high pressure liquid chromatography (HPLC), as described
previously
(40) . All peptide preparations were judged at least
95% pure, as determined by analytical reverse phase HPLC, and peptide
composition is routinely confirmed by mass spectrometry.
Flow Cytometry
Platelet-rich plasma (PRP) was
obtained from acid-citrate-dextrose (NIH formula A)-anticoagulated
blood by differential centrifugation as described
previously
(33) . Prostaglandin E (PGE
,
Sigma) was added to the PRP to a final concentration of 20 ng/ml. After
a 15-min incubation, the PRP was centrifuged at 750
g for 10 min to sediment platelets. After three washes with
Ringer's citrate-dextrose containing 20 ng/ml PGE
, pH
6.5, the platelet pellet was resuspended in an appropriate buffer. Flow
cytometry was performed according to the method of Ginsberg et
al.(41) with slight modifications. Five-µl aliquots of
the washed platelets (3
10
/ml) suspended in 12
mM NaHCO
, 1 mM CaCl
, 1
mM MgCl
, 137.5 mM NaCl, 2.6 mM
KCl, pH 7.4, plus 1% bovine serum albumin (Cohn Fraction V) and 20
ng/ml PGE
(test buffer) were added to tubes containing:
peptides; 2 mM EDTA; or Fab fragments of OPG2 or AP2 in test
buffer. FITC-conjugated test mAb were added, the total volume was
adjusted to 50 µl with test buffer, and the mixtures were incubated
for 60 min at ambient temperature. The mixtures were then diluted to
0.5 ml with test buffer and analyzed in a flow cytometer
(Becton-Dickinson FACS Star
, Mountain View, CA). When it
was desired to activate platelets with ADP or thrombin, the washed
platelets were resuspended in test buffer without PGE
.
Thrombin activation of platelets was performed as described
previously
(33) , namely, washed platelets (3
10
/ml) were incubated with 1 unit/ml of human
-thrombin (Sigma) at 37 °C for 15 min, and the reaction was
stopped by addition of 2 units/ml of hirudin (Sigma). The platelets
were washed once in Ringer's citrate-dextrose, pH 6.5,
resuspended in test buffer, and analyzed by flow cytometry.
Antibody Binding Assay
Platelets were isolated and
washed as described above, and mAb binding assays were performed as
described previously
(33) in buffer containing either 1
mM CaCl, 1 mM MgCl
, or 1
mM EDTA. The data obtained from the binding experiments were
analyzed by computerized, nonlinear, least-square regression analysis
in a main frame computer ACOS 1000 (NEC, Japan) with the SIMPLEX
program library of Osaka University.
Fibrinogen Binding Assay
Human plasma fibrinogen
was purified and labeled with I, and fibrinogen binding
was assayed as described previously
(11) . PRP was prepared from
acid-citrate-dextrose-anticoagulated blood, as described above, then
platelets were purified by gel filtration using a Sepharose 2B column
(Pharmacia), as described by Ginsberg and Plow
(42) , and
collected into 10 mM HEPES, 12 mM NaHCO
,
2 mM MgCl
(or 2 mM CaCl
),
137.5 mM NaCl, 2.6 mM KCl, pH 7.4, containing 0.1%
dextrose. One-ml aliquots of the suspension (10
platelets)
were incubated for 30 min at ambient temperature in the presence of 60
µg/ml AP5 IgG or 8 µM thrombin receptor peptide
SFLLRNPNDKY
(43) , in the presence or absence of 10
µM PGE
and in the presence of 5, 20, 100, or
200 µg/ml of purified, human
I-fibrinogen.
Platelet Aggregometry
Platelet aggregation was
monitored using a model PAP-4 NKK platelet aggregation tracer (Nikou
Bioscience Inc, Tokyo, Japan) at 37 °C with a stirring rate of
1,000 revolutions/min as described previously
(33) .
Construction of
In this paper, cDNA sequences are numbered beginning
with the ATG start codon. An XbaI-KpnI fragment
containing human Recombinant
Baculovirus
bp 1-485 was isolated from a
Bluescript cDNA clone (a gift from Dr. David R. Phillips, COR
Therapeutics, Inc., South San Francisco, CA) and ligated to a
3.1-kilobase KpnI-EcoRI fragment isolated from a
human Dami cell cDNA library (obtained from Dr. Robert I. Handin,
Dana-Farber Cancer Institute, Boston, MA) to generate full-length human
cDNA (H
). Full-length Xenopus
cDNA (X
) as a 4.0-kilobase
EcoRI fragment cloned in Bluescript was generously provided by
Dr. Douglas W. DeSimone (The University of Virginia, Charlottesville,
VA). Truncated H
(H) was constructed by insertion of a
pair of adjacent termination codons (TAATGA) 3` to bp 2155 followed by
a downstream BglII restriction site at bp 2161. Truncated
X
(X) was constructed by insertion of the identical
sequence 3` to bp 2148 of X
. To generate each chimera,
we used polymerase chain reaction-based mutagenesis to insert a silent
mutation(s) that creates a restriction site in one of the
subunits to complement the same site present within the native
sequence of the other
subunit (Fig. 1).
The following restriction sites were used for each chimera:
SacI for H6X, EcoRI for H53X and X53H, NcoI
for H287X, and PstI for H490X and X490H. cDNA was subcloned
into the transfer vector pVL1393 (Invitrogen, Inc., San Diego, CA).
After cotransfection of Sf9 cells with linearized Autographa
californica nuclear polyhedrosis virus DNA and the recombinant
transfer vector, recombinant virus was isolated by plaque purification
based on visual inspection. The absence of wild-type virus in
recombinant virus clones was confirmed by polymerase chain reacton.
Figure 1:
Schematic representation of
human-Xenopus constructs. The approximate
region of individual
domains (amino-terminal,
ligand-binding, cysteine-rich, and transmembrane) is depicted at the
top of the figure. The position of restriction sites, ATG start codons,
and inserted stop codons in each of the human (solid bar) and
Xenopus (open bar)
cDNAs is shown
at the top and bottom of the figure, respectively. Oligonucleotide
positions are numbered beginning with the start codon ATG. Restriction
sites introduced by silent mutations are indicated in
parentheses, while those without parentheses are
inherent in the corresponding native sequence. The human-Xenopus chimeras are depicted with the point of ligation designated by the
corresponding amino acid residue. Human
segments are
indicated by solid bars; segments from Xenopus
, by open
bars.
Preparation of Recombinant Protein
Sf9 cell
monolayers in a 25-cm tissue culture flask were each
infected with 15-20 multiplicity of infection units of
recombinant virus. Recombinant protein was assayed by dot-blot and
Western blot assays each 24 h interval following infection to determine
the time course of recombinant protein production. Cells and culture
media were harvested as soon as maximum synthesis was attained
(nominally, 48 h) to obtain maximum yield and avoid potential
proteolytic degradation that can occur at later time intervals. Cells
were pelleted, and the media were used as a source of secreted
recombinant protein. Cells were lysed by resuspension in 100
mM Tris-HCl, 100 mM NaCl, 0.2 mM PMSF, pH
8.0, containing 0.5% Triton X-100. The mixture was centrifuged at
10,000
g for 10 min in a microcentrifuge to pellet
particulate matter, and the supernatant was used as a source of
recombinant protein (lysate). Protein concentration was determined by
the method of Markwell et al.(44) .
Additional Procedures
Purification of human
platelet , Western blot assays,
dot-blot assays, and ELISA were performed as described
previously
(40, 45) .
RESULTS
The anti-LIBS mAbs used in this study have already been
characterized
(19, 21, 22, 25, 36, 37) with the exception of AP5. Consequently, preliminary
studies were performed to establish the nature of the epitope
recognized by AP5 as a LIBS.
AP5 Recognizes a Calcium-sensitive LIBS on
The binding characteristics of AP5 to intact
platelets were initially analyzed by flow cytometry using nonlabeled
AP5 and FITC-conjugated secondary antibody. In the presence of 1
mM Ca (or 1 mM Ca
+ 1 mM Mg
) and 20 ng/ml
PGE
, as an inhibitor of platelet activation, the level of
AP5 that binds to platelets (Fig. 2, panel B) is
comparable to that obtained with the control mAb, HP20 (Fig. 2,
panel A). Addition of
50 µM RGDW to
the platelet suspension results in a marked increase in the expression
of the AP5 epitope (Fig. 2, panel C), while the
control peptide, RGEW (
1 mM), fails to induce its
expression (Fig. 2, panel D). Fab
fragments of another mAb, OPG2, which contains the sequence RYD within
its antigen-binding site and binds to
as an RGD-containing ligand
(34, 46) , also induce
maximum expression of AP5 epitopes (Fig. 2, panel
E). Fab fragments of the isotype identical mAb AP2, which
binds to an identical number of
sites, are without effect (Fig. 2, panel
F).
Figure 2:
Expression of AP5-binding sites on
platelets, as determined by flow cytometry. The binding of FITC-HP20
(20 µg/ml) (A) or FITC-AP5 (20 µg/ml) (B) to
nonactivated, washed platelets after 60 min at ambient temperature in
the presence of divalent cations (1 mM Ca, 1
mM Mg
) is depicted. Also shown is the
binding of FITC-AP5 (20 µg/ml) to washed platelets following
pretreatment (30 min at ambient temperature) with: RGDW (500
µM) (C), RGEW (1 mM) (D), OPG2
Fab fragments (80 µg per ml; 1.6 µM) (E), or
AP2 Fab fragments (80 µg/ml) (F). Mean fluorescence
intensity is indicated on the abscissa; the percentage of
fluorescence-positive cells is depicted on the
ordinate.
The effect of divalent cations on AP5 binding to
intact platelets was analyzed using FITC-conjugated AP5. The binding of
FITC-AP5 (60 µg/ml) to intact platelets is inhibited by divalent
cations (Fig. 3A). In these studies, FITC-AP5
binding is measured at room temperature in the presence of 20 ng/ml
PGE and in the absence of RGD peptides. Maximal binding is
observed in the presence of 1 mM EDTA (MFI = 16.5
± 1.43, mean ± 2S.D., n = 3). A
significant reduction in binding to roughly half-maximal levels is
attained when neither chelator nor divalent cations, i.e. Ca
and Mg
, are added to the
buffer (MFI = 7.98 ± 2, p < 0.001). Relative
to Ca
- and Mg
-free buffer, a
marginal decrease in binding in the presence of 1 mM
Mg
(MFI = 6.36 ± 1.04) is not
significant (p > 0.05), whereas binding is significantly
depressed in the presence of 2 mM Mg
(MFI
= 1.83 ± 0.77, p < 0.001), 1 mM
Ca
(MFI = 0.58 ± 0.55, p <
0.0001) or 1 mM Mg
+ 1 mM
Ca
(MFI = -0.51 ± 0.2, p < 0.0001).
Figure 3:
Effect of divalent cations on the binding
of AP5 to platelets, as determined by flow cytometry. A,
FITC-AP5 was added at a final concentration of 60 µg/ml. Mean
fluorescence intensity (MFI) is depicted on the
ordinate. Binding was measured after 60 min of incubation at
room temeprature in the presence of (left to right on the
abscissa): 1 mM EDTA, divalent-cation free buffer
(0), 1 mM Mg, 2 mM
Mg
, 1 mM Ca
, or 1
mM Ca
+ 1 mM
Mg
. Values represent mean ± 2 S.D. (n = 3). B, FITC-AP5 binding to platelets
(ordinate) as a function of divalent cation concentration
(abscissa). Binding was measured in the presence of a range of
Mg
alone (
), a range of Ca
alone (
), or a range of Ca
in the
presence of a constant (1 mM) Mg
concentration (
). Mean values of triplicate
determinations are depicted.
Binding of FITC-AP5 (60 µg/ml) to washed
platelets as a function of divalent cation concentration is depicted in
Fig. 3B. Half-maximal inhibition of AP5 binding
is observed in the presence of 80 µM Ca or 1.5 mM Mg
. At a constant 1
mM Mg
, half-maximal inhibition is obtained
at 20 µM Ca
. The results indicate that
calcium is a more efficient inhibitor of the binding of AP5 but that
the inhibitory effects of calcium and magnesium are cumulative.
Scatchard Analysis
The binding of
I-AP5 IgG to nonactivated platelets in the presence of 1
mM Ca
and 1 mM Mg
is negligible. With careful platelet handling, the presence of
activation inhibitors, such as PGE
, and the absence of
RGD-containing ligands, there is no significant binding. However,
addition of
100 µM RGDW leads to a marked increase in
the number of AP5 molecules bound by platelets (B
= 48, 620 ± 6,810, n = 3), and a
single class of binding sites with an apparent
K
of 9.9 ± 3.5 nM (n = 3) is observed. When divalent cations in the platelet
resuspension buffer are replaced by 1 mM EDTA, the number of
AP5-binding sites/platelet is again maximal (B
= 44, 122 ± 8,870, n = 3).
Fibrinogen Binding and Platelet Aggregation
The
ability of AP5 to induce binding of I-fibrinogen to
gel-filtered platelets was measured by direct assay. As shown in
Fig. 4, even in the presence of 1 mM
Mg
and 10 µM PGE
, AP5 IgG
(final concentration = 60 µg/ml) induces saturable binding
of fibrinogen to platelets. In the depicted experiment, at 300
µg/ml added fibrinogen, AP5 induces the binding of 19,601 ±
149 (mean ± S.D.) molecules of fibrinogen/platelet in the
absence of PGE
, and 14,056 ± 183 molecules/platelet
in the presence of PGE
. Under the same conditions, thrombin
receptor peptide induces the binding of 17,854 ± 1,658
molecules/platelet in the absence of PGE1, but only 3,393 ± 47
molecules/platelet in the presence of this inhibitor. While thrombin
receptor peptide induced binding is maximal at
300 µg/ml of
added fibrinogen, AP5-induced binding is already 75% maximal at 25
µg/ml added fibrinogen, and saturated binding occurs at
100
µg/ml added fibrinogen.
Figure 4:
Direct assay of I-fibrinogen
binding to washed platelets. The concentration of labeled fibrinogen
added is indicated on the abscissa, while the amount of
fibrinogen bound to platelets is depicted on the ordinate.
Values represent the mean of duplicate determinations. Error bars indicate one standard deviation. Represented is fibrinogen
binding:
, induced by AP5;
, induced by AP5 in the presence
of PGE
;
, induced by thrombin receptor peptide;
, induced by thrombin receptor peptide plus PGE
;
, in the absence of agonist; and
, in the absence of
agonist plus PGE
.
Addition of AP5 IgG or Fab to PRP
induces modest and gradual platelet aggregation
(Fig. 5A) similar to that previously reported for
anti-LIBS1
(25) . AP5-induced aggregation reaches a maximum
within 5-10 min. While the expected platelet shape change and
release of dense granule ATP is observed in PRP after addition of 10
µM ADP (Fig. 5B), aggregation
induced by AP5 is neither preceded by platelet shape change nor
associated with release of dense granules
(Fig. 5C). Aggregation occurs independently of
platelet FcRII activity since: 1) Fab fragments of AP5, on an
equimolar basis, are as effective as intact IgG in the induction of
platelet aggregation (Fig. 5A); 2) the
Fc
RII-specific mAb IV.3, which blocks Fc receptor-mediated
platelet activation
(47) , does not inhibit AP5-induced platelet
aggregation (not shown); and 3) the extent or rate of platelet
aggregation does not correlate with the Fc
RII phenotype (active or
inactive)
(48) of the platelet donor.
Figure 5:
Platelet aggregation induced by AP5. In
each panel, percent light transmission is shown on the
ordinate, and the horizontal bar represents 1 min.
A, to citrated PRP (3 10
platelets/ml),
AP5 IgG (60 µg/ml) or Fab fragments (60 µg/ml) were added at
the point indicated by the vertical arrow. B,
platelets in PRP were induced to aggregate by addition of 10
µM ADP (vertical arrow). Release of dense granule
ATP was measured by the luciferin-luciferase reaction using a Lumi
aggregometer. In B and C, the amount of ATP is
depicted as arbitrary units. Maximum release is represented in
B. C, same as B except that AP5 IgG (60
µg/ml) was used as agonist. D, washed platelets (3
10
platelets/ml) were resuspended in 5 mM HEPES,
0.3 mM NaH
PO
, 12 mM
NaHCO
, 5.5 mM glucose, 1 mM
MgCl
, 2 mM CaCl
, 2 mM KCl,
137 mM NaCl, pH 7.4. AP5 IgG (60 µg/ml) (solid
vertical arrow) and fibrinogen (1 mg/ml) (open vertical
arrow) were added at the times
indicated.
AP5-induced platelet
aggregation is mediated by fibrinogen binding to
since washed platelets treated
with AP5 do not aggregate until exogenous fibrinogen is added
(Fig. 5D). Preincubation of PRP or washed
platelets with AP2 completely inhibits aggregation induced by AP5 (not
shown). Neither PGE
(1 µM) nor dibutyl cAMP (2
mM), which completely inhibit ADP (10 µM)-induced
platelet aggregation, have any effect on aggregation induced by AP5
(not shown).
Expression of Truncated Human
Culture media and cell lysates were both
tested at 1-5 days post-infection to evaluate levels of
recombinant human by immunoblot using mAb AP3
(Fig. 6). The electrophoretic mobility of the truncated
human
(approximate molecular mass = 90 kDa) is
noticeably faster than that of the native human
subunit (approximate molecular mass = 100 kDa), consistent
with the absence in the former of the transmembrane and cytoplasmic
sequences (residues 691-762) which represent a combined mass of
at least 8 kDa. The fact that there is only a difference of about 10
kDa in apparent molecular mass would suggest that the recombinant
protein is glycosylated to an extent equivalent to that of the native
protein.
Figure 6:
Time course of truncated human expression by Sf9 cells infected with recombinant virus. Thirty
µl of culture media (A) or 10 µg of cell lysate
protein (B) were electrophoresed in each lane of a 10%
polyacrylamide slab gel under nonreduced conditions. The separated
proteins were transferred to a polyvinylidene fluoride membrane, and
-related protein was detected by binding of mAb AP3
followed by alkaline-posphatase-conjugated goat anti-mouse IgG. Samples
were obtained 1-5 days post-infection. Additional controls
include: purified human platelet
(40 ng/lane), lysates of non-infected Sf9 cells (NIC)
obtained on day 1 (10 µg/lane), and lysates of Sf9 cells infected
with nonrecombinant virus (NRV) obtained on day 2 (10
µg/lane).
The 90-kDa recombinant was detected as
early as day 1 in both media and lysates. Although the amount of the
recombinant
in lysates peaks by day 2 and begins to
decline by day 3 post-infection, recombinant
in the
media is maximal by day 2 and remains at essentially the same level up
to day 5 post-infection. Essentially the same rates of synthesis and
levels of total recombinant protein were observed for truncated
Xenopus
and each of the human-Xenopus chimeras produced in this study, with the exception of H287X.
Levels of H287X in Sf9 cell lysates were consistently 3-fold lower than
truncated human or Xenopus
molecules.
Identification of the AP5 Epitope on
Using a dot-blot assay and Sf9 cell lysates as
the source of recombinant antigen, we compared the binding of AP5 and
10 other -specific mAb to recombinant human
(H), recombinant Xenopus
(X), and human-Xenopus chimeras (Fig. 7). These mAb include the six
anti-LIBS, D3, anti-LIBS1, anti-LIBS2, anti-LIBS3, anti-LIBS6, and P41.
All 11 mAb bind to H, while three mAb (AP6, anti-LIBS1 and P3-13)
bind equally well to both H and X. We were thus precluded from further
localizing the epitopes recognized by these three mAb using this
approach. Two mAb (AP5 and SZ21) bind to H53X, H287X, and H490X, but
only AP5 binds to H6X. As expected, neither AP5 nor SZ21 bind to X53H
or X490H. These data suggest that the AP5 epitope is located in the
amino-terminal hexapeptide GPNICT and confirm the localization of the
SZ21 epitope to residues 7-52, as predicted
(35) . Of the
remaining six mAb, AP3 and D3 bind to epitopes within residues
287-489, consistent with a previous localization
study
(22) , and four of the anti-LIBS, P41, anti-LIBS2,
anti-LIBS3, and anti-LIBS6, bind to epitopes within residues
490-690. Our results with anti-LIBS2 are consistent with a
previous report
(21) .
Figure 7:
Dot-blot to measure mAb binding to
truncated human (H), truncated Xenopus
(X) and human-Xenopus
chimeras (H-X or X-H). Lysates of
Sf9 cells producing recombinant
-related protein were
dialyzed against 10,000 volumes of lysis buffer and adsorbed onto
nitrocellulose membranes. After adsorbed protein had air-dried onto the
membrane, the binding of mAb was detected. nrv represents an
equivalent lysate of Sf9 cells infected with nonrecombinant virus. The
recombinant constructs are indicated from left to right at the top of
the figure; the mAb tested are listed from top to bottom at the right
of the figure.
The results of a peptide inhibition
assay confirm the localization of the AP5 epitope
(Fig. 8). The peptide 1-13, which
corresponds to residues 1-13 of human
, inhibits
the binding of AP5 to purified
(ID
800 µM) in an
antigen-specific ELISA. The control peptide
722-736 from the cytoplasmic tail of human
has no effect on AP5 binding. In comparison, purified
human
inhibits AP5 binding with an
ID
60 µM.
Figure 8:
Peptide inhibition of the binding of AP5
to solid-phase as determined by
ELISA. Purified human platelet
was
adsorbed to wells of microtiter plates. Inhibitor peptides in
phosphate-buffered saline + 10% bovine serum albumin at the
indicated concentrations (abscissa) were preincubated with 0.2
µg/ml of AP5 IgG. The mixtures were then added to the
-coated wells, and the extent of
binding of AP5 to coated
was
determined by subsequent binding of alkaline-phosphatase-conjugated
goat anti-mouse IgG. Inhibitors included: purified
(
),
pep-tide GPNICTTRGVSSC (residues 1-13) (
), and
peptide HDRKEFAKFEEERAR (residues 722-736)
(
).
Since the precise epitope
recognized by anti-LIBS1 could not be localized by dot-blot assay to
the chimeras that we tested, we employed a
competitive binding assay using flow cytometry to demonstrate that
anti-LIBS1 and AP5 do not compete for the same binding site on
(). It is apparent from these
competition assays that the LIBS1 epitope is distinct from the AP5
epitope.
DISCUSSION
The AP5 epitope, located at the amino terminus of the
integrin subunit, can be included in the group of
epitopes that are defined as LIBS. There are several attributes of AP5,
relative to the other LIBS defined to date, that are particularly
germane to our appreciation of the structure and function of the
integrin
.
subunit,
i.e. within residues
602-690
(19, 21, 25, 36, 37) .
The existence of a LIBS at the opposite extreme, the amino terminus, of
reinforces the hypothesis that activation-related
events induce global conformational changes within this subunit of the
integrin
. These global changes are
undoubtedly facilitated by long range disulfide bonds, and the putative
disulfide between Cys
and Cys
(49) likely
contributes to physical proximity between the AP5 epitope and the
carboxyl-terminal portion of
. The location of the D3
epitope within residues 422-490 is consistent with this physical
model, since this region of
that surrounds
Cys
is likely to be proximal to the AP5 epitope
surrounding Cys
. Furthermore, both the AP5 and D3 epitopes
could then be brought into physical proximity to the larger group of
LIBS localized within residues 602-690 by another long range
disulfide bond between Cys
and
Cys
(49) . We postulate that there is a
structurally constrained cluster of
LIBS, created by
the juxtaposition of three noncontiguous
segments,
(1-6),
(422-490), and
(602-690), that is sensitive to conformational
changes in the molecule coordinate with activation of this receptor. In
view of the findings of Frelinger et al.(19) , we can
likely include two regions of the
subunit within
this cluster, the PMI-1 site at the extreme carboxyl terminus of the
heavy chain of
, residues 844-859, and a
distinct, but undefined, site on
recognized by
PMI-2.
, that can activate the integrin
in the absence of an external
agonist. One proposed explanation for this phenomenon rests on the
supposition that there are equilibria between various conformations of
the integrin
(25, 50) . In this model, proposed by
Frelinger et al.(25) , a portion of the integrin
population always has the potential to exist transiently in an
activated conformational state. When this subpopulation is bound by an
anti-LIBS, like AP5, the equilibrium of the entire integrin pool is
shifted, more integrin undergoes a transition to that state, and more
AP5 binds. In practice, it is difficult to distinguish between this
model and one in which AP5 binds with low affinity to the nonactivated
conformation of the integrin causing the integrin within the
antibody-integrin complex to undergo a transition to the activated
state. All of the
-specific anti-LIBS which have been
reported thus far to activate
,
namely anti-LIBS1, D3GP3
(22) , anti-LIBS2 (mAb62)
(37) ,
anti-LIBS3, and anti-LIBS6
(19) , recognize epitopes that would
be physically clustered by long range disulfide bonds, as described
above.
, exhibits a
significant increase in affinity for the
complex when that receptor is occupied by a protein ligand or an
RGD-containing peptide. Nonetheless, binding to
``nonactivated'' platelets in the absence of agonists or
ligands can occur, and the level of binding to nonactivated platelets
depends upon the composition of divalent cations in the buffer. Both
calcium and magnesium inhibit AP5 binding, but the inhibitory effect of
calcium (ID
80 µM) is greater than that
of magnesium (ID
1.5 mM). The effect of
these divalent cations is cumulative, and in the presence of 1
mM magnesium, inhibition by calcium is enhanced (ID
20 µM). Chelation of divalent cations with
millimolar EDTA results in a significantly higher level of AP5 binding
(roughly 2-fold) than that observed in the presence of divalent
cation-free buffer, confirming that the effect of divalent cations
occurs at micromolar or submicromolar concentrations. Only one other
anti-LIBS, PMI-1, specific for a sequence of the
subunit
(25) , is sensitive to the concentration of
divalent cations in the milieu. Interestingly, none of the other LIBS
associated with
are significantly affected by
divalent cation concentration.
are mutually exclusive (26), since RGD ligands displace two
cations from
and a single metal
ion from the active sequence
(118-131). Based on
these findings, a model is proposed wherein a ternary intermediate
complex is formed between ligand, receptor, and cations. The involved
cations are eventually displaced as the interaction between receptor
and ligand is stabilized. Receptor-bound cations are required to
maintain the receptor recognition pocket in a conformation that has the
potential to associate with ligand. Following initial capture of
ligand, however, cations are no longer required and are displaced. This
model is strongly supported by exposure of the LIBS recognized by AP5
by either ligand binding or chelation of cations. While D'Souza
et al.(26) argue that
(118-131)
is one of the sites of ternary complex formation, they also provide
presumptive evidence for at least one more site. Given its sensitivity
to divalent cations, the AP5 epitope at the amino terminus of
may represent a second sequence involved in the
stability of the proposed ternary complex. While the PMI-1 epitope is
another candidate for such a cation-binding site, binding by PMI-1 to
does not influence ligand binding to this receptor.
Thus, the AP5 epitope appears to be more intimately involved in the
physical relationships between the three components, receptor, ligand,
and cations. Moreover, the expression of the AP5 epitope on the related
integrin
is comparably influenced by
both ligand binding and divalent cation chelation.(
)
Consequently, any model of
activation, ligand
binding, and the requirement for divalent cations must include the
amino terminus of the
subunit.
by calcium or
magnesium are thus eliminated when RGD peptides bind to this receptor.
This interpretation is consistent with the recent findings of
D'Souza et al. (26). It is also likely that RGD peptides
free the AP5 epitope when they displace calcium from the vicinity of
the recognition pocket of this receptor.
(28) or on the individual native
subunit, expressed in the baculovirus system. However, the
integrins are particularly complex molecules whose function is
modulated by dynamic changes in subunit association and conformation of
the heterodimer. Thus, even simple contiguous epitopes, like the AP5
LIBS, can be influenced greatly by conformational constraints imparted
by changes in the tertiary structure of the receptor. The further study
of epitopes like that recognized by AP5 that are modulated by ligand
binding and/or divalent cations will lead to a better understanding of
the relationship between structure and function of integrins.
Table:
Murine monoclonal antibodies
Table:
Competitive binding assay (flow
cytometry)
,
prostaglandin E
; bp, base pair(s); ELISA, enzyme-linked
immunosorbant assay; MFI, mean fluorescence intensity.
cDNA clone. We obtained monoclonal antibodies and technical
support from several individuals: we thank Drs. M. H. Ginsberg and X.
Du (Scripps Research Institute, La Jolla, CA) for the mAb anti-LIBS1,
anti-LIBS2, anti-LIBS3, and anti-LIBS6; Dr. Lisa Jennings (University
of Tennessee-Memphis, Memphis, TN) for the mAb D3GP3; Dr. Changgeng
Ruan (University of Suzhou, Suzhou, China) for mAb SZ21; Dr. Peter
Newman (Blood Research Institute, Milwaukee, WI) for mAb AP3; Dr. Steve
Rosenfeld (University of Rochester, NY) for mAb IV.3; Dr. Shuji Uchida
(Osaka University Medical School, Osaka, Japan) for analyzing the
radiolabeled mAb binding data; Dr. Michel Fougereau (University of
Marseille, Marseille, France) for mAb HP20; and Dr. Fumihiro Ishida
(The Scripps Research Institute, La Jolla, CA) for advice and technical
support on screening the cDNA library.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.