©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Topography of Ligand-induced Binding Sites, Including a Novel Cation-sensitive Epitope (AP5) at the Amino Terminus, of the Human Integrin Subunit (*)

Shigenori Honda , Yoshiaki Tomiyama , Anthony J. Pelletier , Doug Annis , Yumiko Honda , Randal Orchekowski , Zaverio Ruggeri , Thomas J. Kunicki (§)

From the (1) From The Roon Research Center for Arteriosclerosis and Thrombosis, Division of Experimental Hemostasis and Thrombosis of the Departments of Molecular and Experimental Medicine and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, the Blood Research Institute of the Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233, and The Second Department of Internal Medicine, Osaka University Medical School, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Changes in ligand binding ability of the integrin 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.


INTRODUCTION

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 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) .

Ligand binding to 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 .

We have developed a murine IgG 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.

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 . 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.

One of the LIBS, that recognized by mAb AP5, is precisely localized in this report to the amino terminus, 1-6. Within the nondenatured complex, the availability of the AP5 LIBS, unlike the remaining LIBS, is sensitive to the presence of divalent cations.


MATERIALS AND METHODS

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 Recombinant Baculovirus

In this paper, cDNA sequences are numbered beginning with the ATG start codon. An XbaI-KpnI fragment containing human 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 Kof 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 FcRII-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 FcRII 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 NaHPO, 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 .

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 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.

AP5 is also one of those anti-LIBS antibodies (19, 22, 25, 37) , all of which bind to , 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.

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 , 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.

It has been proposed that ligand and divalent cation binding to the integrin 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.

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 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.

The AP5 epitope is a simple linear sequence GPNICT that is readily available on denatured (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)



FOOTNOTES

*
This study was supported by National Heart, Lung, and Blood Institute Grants HL-46979 (to T. J. K.) and HL-31950 (to Z. M. R.). This is manuscript number 8196-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 correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The 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: LIBS, ligand-inducible binding sites; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; HPLC, high performance liquid chromatography; PRP, platelet-rich plasma; PGE, prostaglandin E; bp, base pair(s); ELISA, enzyme-linked immunosorbant assay; MFI, mean fluorescence intensity.

A. J. Pelletier, manuscript in preparation.


ACKNOWLEDGEMENTS

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 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.


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