©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Exchange of Arg-Gly-Asp (RGD) and Arg-Tyr-Asp (RYD) Binding Sequences in a Recombinant Murine Fab Fragment Specific for the Integrin Does Not Alter Integrin Recognition (*)

Thomas J. Kunicki (1)(§), Kathryn R. Ely (2), Thomas C. Kunicki (1), Yoshiaki Tomiyama (3), Douglas S. Annis (1)

From the (1)Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis, Departments of Molecular and Experimental Medicine and Vascular Biology, Scripps Research Institute, La Jolla, California 92037, the (2)La Jolla Cancer Research Foundation, La Jolla, California 92037, and the (3)Second Department of Internal Medicine, Osaka University Medical School, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The murine monoclonal antibody OPG2 is an excellent paradigm of natural RGD ligands and binds specifically to 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 .


INTRODUCTION

Several integrins, including , , 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) .

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 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.

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 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.()

In the present study, we compare the binding of recombinant OPG2 Fab, Fd, and 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.


MATERIALS AND METHODS

Synthesis of Recombinant OPG2 Fd and Chain cDNA

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 domain, the C1 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+.

OPG2 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.

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).

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.

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 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.


RESULTS

Quantitation of Secreted Fd+ Heterodimers (Fab)

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+ (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.

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 (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, Chains, and Fab to Purified

By ELISA, it was apparent that neither Fd alone nor 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 (, ▾).




DISCUSSION

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 . 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 .

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 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.

The ability to express individual Fd and 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.

In its ``quiescent'' state, 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



FOOTNOTES

*
This study was supported by R01 Grant HL-46979 from the NHBLI, National Institutes of Health. This is Manuscript 8754-MEM from the Scripps Research Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Dept. of Molecular and Experimental Medicine, Scripps Research Institute, 10666 N. Torrey Pines Rd., Maildrop SBR13, La Jolla, CA 92037. Tel.: 619-554-3668; Fax: 619-554-6679.

The abbreviations used are: H3, third complementarity determining region of the heavy chain; PMA, phorbol 12-myristate 13-acetate; AcMNPV, A. californica nuclear polyhedrosis virus; AP7, murine monoclonal antibody containing the RGD sequence in H3; AP7E, murine monoclonal antibody containing the RGE sequence in H3; ELISA, enzyme-linked immunosorbent assay; Fab, immunoglobulin light chain + Fd; Fd, V domain + C1 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.

Ely, K. R., Kunicki, T. J., and Kodandapani, R. (1995) Protein Eng. in press.


ACKNOWLEDGEMENTS

We thank Dr. Brunhilde Felding-Habermann (Scripps Research Institute) for providing purified human placental and Dr. Dan Salomon (Scripps Research Institute) for assistance in the computerized visualization and quantitation of proteins adsorbed to nitrocellulose membranes.


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