From the
The murine monoclonal antibody OPG2 is an excellent paradigm of
natural RGD ligands and binds specifically to
Several integrins, including
The precise structure of an RGD (RYD) (KGD)
sequence in any adhesive protein had not been determined until the
recent solution of the crystal structure of the Fab fragment of OPG2 at
2.0-Å resolution (21). In other ligands, such as the type
III
In contrast, the entire OPG2 Fab molecule is well
determined, including the spatial organization of the RYD side chains
at the critical site for cell adhesion. The H3 loop of OPG2 protrudes
from the surface of the molecule, and the RYD sequence is located at
the tip of this loop, at a distance of about 12 Å from the
location of the combining site in other antibodies. Lacking a prominent
antigen-combining cavity, OPG2 binds to
In the present study, we compare the binding of recombinant
OPG2 Fab, Fd, and
OPG2 Fd
Recombinant virus was
plaque-purified by visual inspection of infected Sf9 cells at limiting
virus dilution and amplified according to the manufacturer's
protocol (Invitrogen). Media containing recombinant virus was harvested
4-6 days after infection of Sf9 cells. The presence of
recombinant virus and absence of wild-type virus was confirmed by PCR
of viral DNA using oligonucleotide primers BACWILDFOR and BACWILDREV () specific for flanking AcMNPV
sequences(31) . A PCR product of 800 base pairs is obtained from
wild-type virus: 650 base pairs plus size of insert, from recombinant
virus. The sequence of each recombinant clone was confirmed prior to
its use, using Sequenase (Promega Biotech, Inc., Madison, WI).
For sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), 5 µg (7.5 µl) of each lysate or 2.5
µl of each media sample were mixed with an equal volume of 2
The
presence of antigen-binding heterodimers (Fab fragments) in media from
coinfected cells was confirmed by analysis of murine Ig proteins that
bind to and are eluted from purified
For the first time, we demonstrate that the sequences RGD and
RYD are mutually replaceable within the framework of a natural ligand
without detectable change in affinity or specificity for the integrin
The binding of the recombinant
Fab to platelets, as determined by flow cytometry, precisely mimics the
binding of native OPG2 Fab(19, 39) . Upon platelet
activation, the number of Fab bound increases, reflecting an increase
in surface
The ability to express individual Fd and
In its ``quiescent'' state,
We thank Dr. Brunhilde Felding-Habermann (Scripps
Research Institute) for providing purified human placental
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
integrin. A reactive
Arg
-Tyr
-Asp
(RYD) tripeptide
is located in an extended loop, the third complementarity-determining
region of the heavy chain (H3). When compared to other RGD ligands, the
RYD tripeptide of OPG2 is unique, in that the side chains are fixed in
a stable orientation that we have defined by x-ray crystallography. In
this study, we express OPG2 H chain segments (Fd) and
chains as
components of active, Fab heterodimers by coinfection of Spodoptera
frugiperda cell lines with recombinant baculoviruses containing
cDNA specific for each protein. Recombinant AP7 Fd segments are
generated from the parent OPG2 Fd segments by replacement of
Tyr
with Gly, while recombinant AP7E Fd segments are
produced from AP7 Fd segments, by exchange of Asp
with
Glu. Neither the free Fd segments nor the free
chains of OPG2 or
AP7 can bind to
. The AP7 Fab
fragment, like the parent OPG2 Fab, binds strongly to purified
but weakly, if at all, to purified
. The affinity of OPG2 and AP7 Fab
fragments for gel-filtered platelets, whether nonstimulated or
activated by 0.2 µM phorbol 12-myristate 13-acetate, is
identical. As with other natural RGD ligands, the binding of
recombinant OPG2 Fab or AP7 Fab fragments to purified
or to gel-filtered platelets is
completely inhibited by the peptide RGDW or by addition of EDTA. AP7E
Fab fragments do not bind at all to either purified
or platelets. Our results
demonstrate, for the first time within a natural protein ligand, that
the tripeptides RGD and RYD exhibit equivalent binding capacity and
specificity for the integrin
.
,
, and
, mediate cell adhesion phenomena
through the recognition of the tripeptide sequence Arg-Gly-Asp
(RGD)(1, 2, 3, 4, 5, 6) .
This recognition motif was first identified in fibronectin (7, 8) but has now been found in numerous adhesive
proteins, including fibrinogen, von Willebrand factor, vitronectin, and
several viper venom
disintegrins(9, 10, 11, 12, 13, 14, 15, 16, 17) .
Other variations upon the RGD motif that exist within natural ligands
include the Arg-Tyr-Asp (RYD) sequence within the major glycoprotein
gp63 of Leishmania(18) or the third heavy chain
complementarity-determining site (H3)(
)
of three
murine monoclonal antibodies, including OPG2, (19) and the
Lys-Gly-Asp (KGD) sequence within the disintegrin
barbourin(20) .
cell adhesion module of fibronectin (22, 23) and several disintegrins (24-29), the RGD
sequence is located in a long flexible loop, such that the conformation
of the RGD site could not be determined, even within the crystal
structure(23) . In the leech
antagonist decorsin, the loop containing the RGD site was
stabilized and defined in the polypeptide backbone. However, the side
chains, particularly the arginine side chain, could not be assigned a
clear orientation.
much as a ligand. This was further validated by comparing the
structure of OPG2 to that of fibronectin. When 47
-carbons are
superimposed with a root mean square deviation of 1.88 Å, the
location of the RYD(RGD) recognition loops within the overall modular
scaffold of the two proteins is identical.(
)
chains to
,
, and gel-filtered platelets before
or after treatment with PMA. We also compare the binding to the same
integrins of OPG2 mutants in which Tyr
is replaced by Gly
to generate RGD (designated ``anti-platelet no. 7'' or AP7)
or Asp
is replaced by Glu to generate RGE (AP7E). In this
manner, we directly compare, within a single natural ligand, the
relative affinity and specificity of RGD, RGE, and RYD tripeptide
sequences.
Synthesis of Recombinant OPG2 Fd and
The term Fd is used according to accepted practice to
indicate that segment of the Ig heavy chain, normally produced by
papain digestion, that includes the V Chain
cDNA
domain, the C
1
domain, and a portion of the hinge region up to and including the
cysteine residue which participates in a disulfide bond with the
carboxyl-terminal cysteine residue of the light chain (). In selected cases, we have eliminated the
carboxyl-terminal cysteine of the heavy chain segment and refer to that
construct as Fd
. Likewise, the terms
and
refer to the
chain constructs with or without a
carboxyl-terminal cysteine residue, respectively. Fab represents
disulfide-linked heterodimers composed of Fd+
, while
Fab
are noncovalently associated heterodimers composed of
Fd
+
.
and
chain cDNA were prepared and amplified by reverse
transcriptase-PCR from total cellular RNA, as described
previously(19) . Heavy chain primers were HFOR and HREV;
chain primers were KFOR and KREV (). A BglII/XbaI digest of the Fd
product was
cloned in pVL1392 (Invitrogen Corp., San Diego, CA) and amplified to
generate the H chain transfer vector pVL.Fd
. The
chain product was digested with XmaI and XbaI, then cloned in pVL1393 (Invitrogen) and amplified to
generate the
chain transfer vector
pVL.
. Alternate constructs in which the 3` cysteine
codons were retained, designated pVL.Fd and pVL.
, were generated
and amplified by PCR using oligonucleotide primer pairs HFOR/HCREV for
the Fd and KFOR/KCREV for the
chain. In each ligation, the OPG2
heavy chain or
chain signal peptide preceded the mature protein
sequence and was fused immediately downstream from the polyhedrin
promoter.
Cloning of Recombinant Baculoviruses
Sf9 cells
(Invitrogen) (2 10
in 2 ml of complete
Grace's media) were seeded in T25 culture flasks and allowed to
attach for 30 min at ambient temperature. This results in an ideal
monolayer of cells that covers roughly 60% of the bottom surface of the
flask. Cells were transfected with a mixture prepared by emulsifying 20
µl of Cationic Liposome Solution (Invitrogen) into 1 ml of
Grace's media containing 1 µg of linearized Autographa
californica nuclear polyhedrosis virus (AcMNPV) DNA (Invitrogen)
and 3 µg of plasmid DNA (one of the pVL transfer vectors).
Transfection was confirmed by visual inspection of cells using an
inverted phase microscope. Cells were incubated at 27 °C. Media
from the transfected cells was harvested after 5-6 days and
stored at 4 °C until needed.
AP7 and AP7E
PCR-based mutagenesis was employed to
construct pVL.Fd, containing a Y104G substitution (Fig. 1). This mutation was inserted together with a SacII restriction site in stages. First, the 5` fragment A was
generated by PCR using primer pair HFOR + RGDREV. Next, the 3`
fragment B was produced by PCR using primer pair RGDFOR + HREV.
Fragments A and B were digested with SacII and ligated. The
ligated DNA was then amplified by PCR using primer pair HFOR +
HREV. A BglII/XbaI digest of the DNA product was then
ligated into pVL1392. By the same approach, pVL.Fd
,
containing the Y104G and D105E substitutions, was also constructed,
using the primer pair HFOR + RGDREV in stage 1 to generate
fragment A and primer pair RGEFOR + HREV in stage 2 to generate
fragment B. PCR products were cloned into PVL1392. The complete
sequence of both mutants was confirmed to be 100% accurate using
Sequenase (Promega).
Figure 1:
Strategy for the mutagenesis of
pVL.Fd to create
pVL.Fd
.
Analysis of Recombinant Protein
T25 flasks seeded
with 2.5 10
Sf9 cells in complete Grace's
media were infected with recombinant virus at a multiplicity of
infection of 5. Cells and media were collected 3 days post-infection.
Alternatively, T25 flasks were seeded with 2.5
10
High Five insect cells (Invitrogen, Inc.) in Ex-Cell 400
serum-free media. After allowing High Five cells to grow for 2 days at
27 °C, media were removed and replaced with fresh media, and cells
were then infected with recombinant virus at an multiplicity of
infection of 5 and incubated with the virus at 27 °C for 2 h. An
additional 7 ml of media was then added. Cells and media were collected
3 days later. In the case of either cell line, cells plus media were
removed from the flasks and separated by centrifugation for 5 min at
ambient temperature at 800
g. The media were
aspirated, and the cell pellet was resuspended in 0.1 M Tris/HCl, 0.15 M NaCl, pH 8.0 (TBS). The cells were
repelleted, resuspended in 1 ml of TBS, and repelleted at 1500
g for 5 min at 4 °C. The supernatant was completely
aspirated, and the cell pellet was lysed by resuspension in 0.25 ml of
ice-cold 0.1 M Tris/HCl, 0.1 M NaCl, 0.005 M CaCl
, 0.0002 M phenylmethylsulfonyl fluoride
containing 0.5% (v/v) Triton X-100 (lysis buffer). The lysate was
chilled on ice for 10 min. Nonsoluble, particulate matter, including
nuclei, were removed by centrifugation at 4 °C for 10 min at 10,000
g. Lysates were stored at 4 °C until used. Media
and lysates were separately analyzed as sources of secreted and
cellular protein, respectively. Protein concentration was determined by
the method of Markwell(32) . Controls included samples from
non-infected cells or cells infected for an equivalent time period with
nonrecombinant virus.
SDS/sample buffer (0.125 M Tris, pH 6.8, containing
20% (v/v) glycerol, 4% (w/v) SDS, and 0.002% (w/v) bromphenol blue, and
incubated at 100 °C for 3 min. To reduce disulfide bonds,
2-mercaptoethanol (final concentration = 5% (v/v)) was added to
samples prior to boiling. When samples were to be analyzed under
nonreducing conditions, and to then avoid disulfide rearrangement,
prior to boiling, N-ethylmaleimide (NEM) was added to a final
concentration of 5 mM by adding an appropriate volume of
freshly prepared 100 mM NEM in distilled water. SDS-PAGE was
performed as described(33) , using 10% acrylamide resolving slab
gels. Proteins resolved by SDS-PAGE were transferred to nitrocellulose
membranes(34) , and immunoglobulin protein was detected by
consecutive binding of: 1) rabbit anti-mouse IgG (specific for
and
chains, prepared and affinity-purified in our laboratory); 2)
biotin-conjugated, affinity-purified, goat anti-rabbit IgG H+L
(Jackson Immunoresearch Laboratories, Inc., West Grove, PA); 3)
alkaline phosphatase-conjugated streptavidin (Zymed, Inc., South San
Francisco, CA); and 4) color-generating substrate solution.
Quantitation by Western Blot
Purified native OPG2
Fab fragments at a range of concentrations from 0.025 to 0.25 µg
were subjected to SDS-PAGE under reduced conditions. In the same 10%
acrylamide slab gels, known volumes of recombinant media were loaded.
Following electrophoresis, separated proteins were transferred to
nitrocellulose membranes. Fd and bands were visualized as
described above. Membrane images were scanned and digitized using a
LACIE Limited Silverscanner II. Image data were analyzed using the
software NIH Image 1.52. The average gray value of pixels (mean) was
determined for each of the Fd bands. A standard curve was constructed
by plotting mean gray value versus amount of added protein for
aliquot of native OPG2 Fab. The quantity of recombinant Fd was
determined from this standard curve.
Purified Integrin ELISA
The integrin
was purified as a functional
heterodimer from human platelets as described by Fitzgerald et
al.(35) , except that protease inhibitors were included in
the final buffer, namely, 0.4 mM phenylmethylsulfonyl
fluoride, 100 µg/ml leupeptin, 0.02 µg/ml pepstatin A, and 10
mM benzamidine. The vitronectin receptor
, a gift from Dr. Brunhilde
Felding-Habermann (Scripps Research Institute), was purified as a
functional heterodimer from human placentae by immunoaffinity
chromatography using the murine monoclonal antibody LM609, as described
(36). Purified integrin heterodimers were adsorbed onto the wells of
Immulon II microtiter plates (Dynatech, Inc., Chantilly, VA), and the
ability of murine monoclonal Fab or recombinant proteins to bind to
each integrin was assessed by ELISA, as described(37) .
Flow Cytometry
Platelet-rich plasma was obtained
by differential centrifugation of whole blood anticoagulated with
acid-citrate-dextrose, formula A. Platelet-rich plasma was harvested,
and platelets were gently pelleted by centrifugation at 950 g for 11 min at ambient temperature. The pellet was
immediately resuspended in HEPES-modified, Tyrode's buffer, pH
6.5, containing 0.1% bovine serum albumin and 0.1% dextrose. The
platelet suspension was applied to a Sepharose 2B column, and fractions
containing platelets were collected. The recombinant Fab fragments in
Tyrode's buffer were added to 5
10
platelets
in the presence of either 20 ng/ml prostaglandin E
or 0.2
µM PMA. After a 15-min incubation at ambient temperature,
fluorescein isothiocyanate-labeled goat anti-mouse IgG
(F(ab`)
-specific; Jackson Immunoresearch Laboratories,
Inc., West Grove, PA) was added. After an additional 15-min incubation,
samples were diluted 10-fold with Tyrode's buffer and analyzed on
a Becton Dickinson FACScan apparatus.
Affinity Purification of Recombinant Fab
Purified
was adsorbed onto the wells of
Immulon II microtiter plates as described(37) . To each well was
added 300 µl of 1% (w/v) powdered milk (Carnation, Los Angeles, CA)
in 0.02 M Tris, 0.15 M NaCl, 0.002 M CaCl
, 0.02% NaN
, pH 7.2, containing 0.05%
(v/v) Tween 20 (TBS-TW), and plates were incubated at ambient
temperature for 1 h. The wells were then rinsed four times with TBS-TW.
Media containing recombinant Fab were diluted 1:10 in TBS-TW, 50 µl
of the diluted media were added to each well, and the plates were
incubated for 2.5 h at ambient temperature. The wells were again rinsed
four times with TBS-TW. Recombinant Fabs were eluted by consecutively
rinsing each set of four identical wells with a single 20-µl volume
of SDS/sample buffer, containing either 5 mM NEM (for
nonreduced samples) or 5% (v/v) 2-mercaptoethanol (to reduce proteins
in samples). The 20-µl sample solution was then analyzed by the
procedure described above for SDS-PAGE.
Quantitation of Secreted Fd+
Insect cells were infected with recombinant viruses, and
media and cell lysates were analyzed after 72 h. Native OPG2 Fab
fragments, prepared by papain digestion, were used as the standard in a
quantitative Western blot assay (Fig. 2) to determine the
amount of recombinant Fab secreted into the media within 72 h by
infected cells. A comparison is made between cells coinfected with AP7E
Fd Heterodimers
(Fab)
+
(AP7E Fab
; lanes
1 and 2), AP7 Fd
+
(AP7
Fab
, lanes 3 and 4), OPG2
Fd
+
(OPG2 Fab
; lanes
4 and 5), and OPG2 Fd+
(OPG2 Fab, lanes 7 and 8). Known concentrations of purified, native OPG2 Fab ranging from 0.025 to 0.25 µg/lane established the
standard curve. One- or 5-µl aliquots of each media containing
recombinant Fab were analyzed in the experiment depicted. The
calculated levels of each recombinant Fab are listed in I. In cells coinfected with Fd+
, Fab
heterodimers (mass = 48 kDa) are detected under nonreduced
conditions. On the other hand, in cells coinfected with
Fd
+
, functional Fab, lacking an
interchain disulfide, are secreted (Fig. 3). Because the
disulfide is absent, however, individual
Fd
+
chains are resolved whether or
not disulfide bond reducing agents are included during SDS-PAGE. The
apparent molecular mass of the recombinant
or
chain in the media is
23 kDa ( Fig. 2and Fig. 3), while that of the Fd
or Fd segment is
27
kDa. In cells coinfected with either Fd+
or
Fd
+
, Ig protein bands of 23 and 27
kDa are detected in both cell lysates and media.
Figure 2:
Quantitative Western blot of recombinant
Fab fragments in media from coinfected High Five cells. In the lefthand portion of the gel, 1- or 5-µl aliquots of media
were added to each lane containing: AP7E Fab (lanes1 and 2), AP7 Fab
(lanes3 and 4), OPG2 Fab
(lanes5 and 6), and OPG2 Fab (lanes7 and 8). In the righthalf of the gel,
known quantities of purified, native OPG2 Fab were added (µg/lane).
The positions of molecular mass standards are indicated at the right edge of the gel: a, lysozyme (14.3
kDa); b, trypsin inhibitor (21.5 kDa); c, carbonic
anhydrase (30 kDa); d, ovalbumin (46 kDa); e, bovine serum albumin (69 kDa); f, phosphorylase b (97.4 kDa); g,
myosin heavy chain (200 kDa). The position of Fd segments and
chains are indicated.
Figure 3:
Western blot of recombinant murine
immunoglobulin protein affinity-purified from insect cell media by
adsorption to human . Proteins were
separated under nonreduced conditions. Ig protein from 72 h media were
analyzed in each case from cells infected with: OPG2 Fd+
(lane 1), OPG2 Fd
+
(lane
2), Fd
+
(lane 3), or
Fd
+
(lane 4).
Heterodimers that bind to and can be eluted from
are detected in the case of OPG2
Fab (lane 1), OPG2 Fab
(lane 2), and AP7
Fab
(lane 3), but the negative control AP7E
Fab
does not bind to antigen (lane 4). The
apparent molecular masses of protein standards (cf. legend to
Fig. 2) are depicted to the left of the panel. The positions
of protein bands corresponding to Fab, Fd
segments, and
chains are indicated to the right of the
gel.
Additional analyses
of cell lysates and media (not shown) established that, in cells
transfected with alone, a dense Ig protein band at 46 kDa is
detected under nonreduced conditions. This protein band represents
-
homodimers. On the other hand, in transfections with Fd
alone, Fd are recovered in only trace levels in either the media or
cell lysates. The Fd segments are likely sequestered by interactions
with other cellular proteins and poorly secreted, if at all.
(Fig. 3). When media from cells coinfected with OPG2
Fd+
to generate OPG2 Fab are adsorbed to and eluted from
, a single major protein band with
an apparent molecular mass characteristic of Fab (
48 kDa) is
detected (lane1). Upon reduction of purified Fab,
individual Fd and
chains are resolved (not shown). On the other
hand, when media from cells coinfected with OPG2
Fd
+
to generate OPG2 Fab
are analyzed in an identical manner (lane2),
Fd
and
are resolved under both nonreduced (Fig. 3) and reduced (not shown) conditions. Identical
results are obtained with AP7 Fab
(Fig. 3, lane3), while the control AP7E Fab
(lane4) does not bind to the solid-phase
antigen,
, as expected.
These results confirm that, in the absence of an interchain disulfide,
Fd
and
chains still form Fab
heterodimers that bind specifically to
.
Binding of Fd,
By ELISA, it was
apparent that neither Fd alone nor Chains, and Fab to Purified
chains bind to purified
, notwithstanding the presence or
absence of the carboxyl-terminal cysteine (Fig. 4). On the
other hand, AP7 Fab
and OPG2 Fab
bind
specifically to
and not to
. Binding to
is completely inhibited by
10
µM RGDW or 1 mM EDTA. In this respect, the
recombinant Fab precisely mimic native OPG2 IgG or Fab. No difference
in binding is observed between recombinant OPG2 Fab
or OPG2
Fab. The control, AP7E Fab
, does not bind at all to
purified
. Saturable binding of the
remaining recombinant Fab fragments to purified
is observed (Fig. 5),
with half-maximal binding attained at 8 ng/ml for OPG2 Fab
,
15 ng/ml for OPG2 Fab, and 5 ng/ml for AP7 Fab
. The binding
of AP7 Fab
, OPG2 Fab
, or OPG2 Fab to purified
is completely inhibited by
10
µM RGDW ( Fig. 4and Fig. 5) or 1 mM EDTA (Fig. 5).
Figure 4:
Binding
to purified integrins of recombinant chains, Fd segments, and Fab
fragments. The ability of recombinant proteins in media from infected
cells to bind to
(solidbars) or
, in the
presence (diagonalstripedbars) or absence (openbars) of RGDW peptide (20 µM) was
measured by ELISA. The ordinate depicts the OD at 405 nm.
Recombinant proteins are indicated on the abscissa. Media from
cells infected with single Ig proteins were analyzed (left to right): OPG2
chains, OPG2
chains, OPG2
Fd
segments, OPG2 Fd segments, AP7 Fd
segments,
and AP7E Fd
segments. To the right side of the
figure are depicted the results obtained with media containing
recombinant Fab fragments (100 ng/ml) generated by cells coinfected
with Fd segment-
chain pairs (left to right):
OPG2 Fab
(Fd
+
), OPG2 Fab
(Fd+
), AP7 Fab
(Fd
+
), and AP7E Fab
(Fd
+
). Bars represent mean + S.D. (n =
3).
Figure 5:
Titration
of Fab binding to purified by
ELISA. The final concentration of recombinant Fab (ng/ml), based on the
calculated amount of recombinant Fd segments in the media, is depicted
on the abscissa. The amount of Fab binding to
is proportional to the OD at 405
nm depicted on the ordinate. Recombinant Fab analyzed here
are: OPG2 Fab
(
), OPG2 Fab (
), and AP7
Fab
(
). Also depicted is the binding of 100 ng/ml AP7
Fab
in the presence of the inhibitors 10 µM RGDW (
) or 1 mM EDTA (▾). Values represent
mean of two determinations in a single representative
assay.
Binding of Recombinant Fab to Platelets
The
behavior of each recombinant Fab against platelets, as determined by
flow cytometry, was equivalent to that observed in the purified antigen
ELISA. In addition, the binding of each Fab fragment as a function of
platelet activation was assessed (Fig. 6). To nonactivated
platelets, saturable binding was obtained with each Fab fragment at
concentrations 50 µg/ml, although half-saturation with AP7
Fab
is attained at about 3 µg/ml. When platelets are
activated with 0.2 µM PMA, an increase in the binding of
each Fab is noted, with half-saturation attained at levels ranging from
about 1 µg/ml for AP7 Fab
to 2-3 µg/ml for
OPG2 Fab
or OPG2 Fab. Moreover, the number of Fab molecules
bound in each case increases significantly, presumably because of the
exposure of additional
on the
platelet surface. Either RGDW (
10 µM) or EDTA (1
mM) completely inhibits the binding of AP7 Fab
(Fig. 6) as well as OPG2 Fab
or OPG2 Fab
(not shown) to either nonactivated or PMA-treated platelets.
Figure 6:
Titration of recombinant Fab binding to
gel-filtered platelets by flow cytometry. Nonactivated platelets (whitesymbols) were assayed in the presence of the
activation inhibitor prostaglandin E (50 ng/ml). Activated
platelets (blacksymbols) were preincubated for 15
min with 0.2 µM PMA. The final concentration of added Fab
fragments (µg/ml) is depicted on the abscissa. The amount
of bound Fab fragments is proportional to the mean fluorescence
intensity (MFI) shown on the ordinate. Values
represent the mean of duplicate determinations in one representative
experiment. Recombinant Fab fragments are: OPG2 Fab
(
,
), OPG2 Fab (
,
), and AP7 Fab
(
,
). Also shown is the binding of AP7 Fab
at two
concentrations (10 or 50 µg/ml) in the presence of inhibitors: 10
µM RGDW (
,
) or 1 mM EDTA (
,
▾).
. In the crystal structure of OPG2
(21), the Tyr
side chain is oriented away from the
Arg
and Asp
side chains on the opposite
side of the RYD loop (Fig. 7). In OPG2, within the
framework of the antibody binding face, the substitution Tyr
Gly to create AP7 has no effect on specificity or affinity
of the recombinant Fab. As is the case with native and recombinant OPG2
Fab, AP7 Fab
bind to
but not to
. Thus, the
replacement of RYD by RGD to generate AP7 renders this recombinant
derivative an even more faithful molecular mimic of other
RGD-containing ligands. Leishmania gp63 is another natural
ligand whose binding site for integrins contains the RYD
sequence(18) . However, it is not possible to compare the
structure of the OPG2 H3 loop with the structure of the RYD-containing
sequence in gp63, since the latter glycoprotein has not yet been
crystallized.
Figure 7:
Structure of the OPG2 H3 loop from x-ray
diffraction data (left). The
Arg-Tyr
-Asp
side chains are
labeled. The
-carbon backbone of the H3 loop is in the plane of
the paper; Arg
and Asp
side chains project
outward from the plane of the paper, while the Tyr
side
chain projects into the paper. The structure is replicated in the image
on the right, without alteration of side chain orientations,
except that the sequence of AP7 is shown, wherein Tyr
is
replaced by Gly.
Our results provide definitive evidence that the
tyrosine residue in the RYD tripeptide motif within H3 of OPG2 is not
required for the specific binding of this antibody to the integrin
. The Asp
Glu
substitution to generate AP7E resulted in not just a marked decrease in
binding to
but a complete loss of
that binding, demonstrating that the stereochemical requirement of Asp
for
recognition previously seen in
peptides and synthetic ligands (19, 38) holds true in
the case of AP7. Thus, both AP7 and its progenitor OPG2 continue to
behave as macromolecular RGD ligands in assays that test RGD-mediated
binding to the integrins
and
.
as a result of both the
fusion of platelet
-granule membranes with the plasma membrane and
increased accessibility of sequestered plasma membrane from the
canalicular system(40) . Consistent with our findings using
native OPG2(19, 39) , the concomitant increase in
affinity of OPG2 (or AP7) Fab fragments results in a significant
decrease in the concentration of Fab at which half-saturable binding is
attained.
chain
segments permits us to evaluate the relative contribution of each
component of the active Fab. Free
chains do not bind to
and exhibit a tendency to form
homodimers. There is little, if any, antigen-binding by OPG2 or AP7 H
chain segments (Fd) when these are produced in the baculovirus
expression system in the absence of
chains. This is probably a
result of both a decreased secretion of free Fd and the inherent
inability of the Fd alone to bind with appreciable affinity to
. Decreased secretion is evidenced
by trace to undetectable quantities of free Fd in the media when
proteins are analyzed by SDS-PAGE after disulfide bond reduction.
However, even though secreted Fd levels are very low and no antigen
binding is observed in the absence of
chains, antigen recognition
can be rescued when media from cells infected with Fd
or Fd
is mixed with media from cells infected with
or
(not shown). This indicates that assembly of Fd and
chains into
active Fab can occur subsequent to secretion and in the absence of cell
processing mechanisms. This establishes that Fd, and for that matter
the H chain itself, is inherently unable to bind to antigen in the
absence of
chains. In the absence of
chains, it is possible
that Fd or H chains assume a conformation that masks the H3 loop or
that the Fd are rendered inactive by associations with other proteins
in the media. The latter explanation would be consistent with the
observation that more Fd protein is detectable at the expected
electrophoretic mobility in the same media sample after disulfide bond
reduction.
mediates platelet attachment
selectively to surfaces coated with fibrinogen(41) . Once
activated, however, it can also mediate attachment to surfaces coated
with von Willebrand factor, fibronectin, or
vitronectin(24, 25) . The ability of OPG2 to bind
selectively to
but not to other
RGD-cognitive integrins must depend on more than just the RYD
tripeptide. It is likely that other residues within H3 that flank the
tripeptide and/or other CDR sequences also contribute substantially to
specificity. The development of recombinant Fab will permit us to
address these questions. The molecular basis of specificity could prove
to be quite complex, however, since the binding of certain ligands,
including fibrinogen and fibronectin, to activated
can be mediated by more than one
recognition motif(30, 42) . Nonetheless, the inherent
specificity of OPG2 for
, the
similarities in binding between OPG2, AP7, and other natural ligands
that contain RGD sequences, and the fact that the structure of OPG2 has
been solved by x-ray crystallography make OPG2 and its derivative AP7
excellent tools for future studies of the molecular basis of RGD ligand
specificity.
Table: List of murine immunoglobulin products developed
and/or used in this study
Table: Oligonucleotide primers
Table: Secretion of recombinant Fab by
baculovirus-infected insect cells
domain +
C
1 domain; H chain, immunoglobulin heavy chain; NEM, N-ethylmaleimide; OPG2, murine monoclonal antibody containing
the RYD sequence in H3; PAGE, polyacrylamide gel electrophoresis; PCR,
polymerase chain reaction; TBS, Tris-buffered saline.
and Dr. Dan Salomon (Scripps
Research Institute) for assistance in the computerized visualization
and quantitation of proteins adsorbed to nitrocellulose membranes.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.