Molecular Determinants of Arg-Gly-Asp Ligand Specificity for β3 Integrins

Abstract The Arg-Tyr-Asp (RYD) and Arg-Gly-Asp (RGD) sequences within the third complementarity-determining region of the heavy chain (H3) of murine recombinant Fab molecules OPG2 and AP7, respectively, are responsible for their specific binding to the platelet integrin αIIbβ3. In this study, we evaluated the influence of divalent cation composition and single amino acid substitutions at key positions within H3 on the selectivity of these Fab molecules for integrin αIIbβ3 versus the vitronectin receptor αVβ3. The parent Fab molecule OPG2 (H3 sequence, HPFYRYDGGN) binds selectively to αIIbβ3 and not at all to any other RGD-cognitive integrin, particularly αVβ3, under any divalent cation conditions. The binding of the AP7 Fab molecule (HPFYRGDGGN) to αIIbβ3 is not affected by the relative composition of calcium, magnesium or manganese. However, AP7 binding to αVβ3, either expressed by M21 cells or as the purified integrin, is supported by manganese and inhibited by calcium. If the flanking asparagine 108 residue within the AP7 H3 loop is replaced by alanine (HPFYRGDGGA), the resulting Fab molecule AP7.4 binds selectively to αVβ3 in a cation-dependent manner, but does not bind at all to αIIbβ3 under any conditions. AP7.4 binding to αVβ3 is supported by manganese, completely inhibited by calcium, and largely unaffected by magnesium. This behavior mimics that of the adhesive protein, osteopontin, another ligand that binds preferentially to αVβ3. Despite these differences in specificity for αIIbβ3 and αVβ3, AP7 and AP7.4 remain selective for the β3 integrins and do not bind to cell lines that express the RGD-cognitive integrins αVβ5 or α5β1. These results confirm that subtle changes in the amino acid composition immediately flanking the RGD or RYD motifs can have a profound effect on β3 integrin specificity, most likely because they influence the juxtaposition of the arginine and aspartate side chains within the extended RGD loop sequence.

The two members of the ␤ 3 integrin subgroup are ␣ IIb ␤ 3 , which is required for platelet cohesion mediated by the binding of fibrinogen or von Willebrand factor (1-3); and ␣ V ␤ 3 , the ubiquitous vitronectin receptor that mediates a variety of cel-lular processes, including migration, tumor cell metastasis, and angiogenesis (4 -7). The distinctive ␣ subunits, ␣ IIb and ␣ V , are relatively unique within the integrin family and exhibit only 36% sequence identity (8).
Although both ␤ 3 integrins recognize the RGD motif, each exhibits a preference for certain RGD-containing ligands. An example is the snake venom disintegrin barbourin (9), containing the KGD sequence, which binds with greater affinity to the platelet integrin ␣ IIb ␤ 3 . Smith et al. (10) have exploited this fact to alter the specificity of an engineered RGD-containing Fab molecule and increase its selectivity for ␣ IIb ␤ 3 over ␣ V ␤ 3 . In addition, this fundamental observation has led to the synthesis of a cyclic homoarginine-Gly-Asp (cHarGD) 1 peptide that has one of the highest differential affinities for ␣ IIb ␤ 3 versus ␣ V ␤ 3 (11). Nonetheless, each of these ligands, including this peptide, retain some affinity for ␣ V ␤ 3 , so that they still inhibit cell adhesion mediated by ␣ V ␤ 3 (11). The relative specificity of ligands for ␣ IIb ␤ 3 versus ␣ V ␤ 3 is also markedly influenced by divalent cations. For example, fibrinogen binds to ␣ V ␤ 3 in the presence of Mn 2ϩ but not in the presence of Ca 2ϩ (12).
To address these factors, Suehiro et al. (13) compared carefully the binding of various RGD ligands and peptides to the ␤ 3 integrins as a function of divalent cation composition, and grouped ligands into four classes. Class I, represented by RGD peptides and vitronectin, bind equivalently to ␣ IIb ␤ 3 and ␣ V ␤ 3 . Class II, represented by cHarGD, fibrinogen, or fibrinogen ␥-chain peptides, bind to both integrins in the presence of Mn 2ϩ , but only to ␣ IIb ␤ 3 in the presence of Ca 2ϩ . Class III, such as barbourin, bind exclusively to ␣ IIb ␤ 3 under any condition. Class IV, represented by osteopontin, bind primarily to ␣ V ␤ 3 .
To gain further insight into the molecular basis for differences in ligand specificity, we exploited our well characterized, recombinant RGD-containing Fab molecule AP7, and its RYDcontaining progenitor OPG2 (14). By the criteria of Suehiro et al. (13), OPG2 is a Class III ligand, binding only to ␣ IIb ␤ 3 under any condition, while AP7 is a Class II ligand, binding to ␣ V ␤ 3 in Mn 2ϩ but not Ca 2ϩ and to ␣ IIb ␤ 3 in either cation. The single amino acid replacement Asn 3 Ala within the RGD-containing loop of AP7 further changes the specificity of the mutagenized Fab molecule. The new Fab molecule, which we designate AP7.4, binds exclusively to ␣ V ␤ 3 and belongs to the Class IV RGD ligand group. These findings confirm that selectivity for the ␤ 3 integrins is determined by precise juxtapositions of the Arg and Asp side chains that are significantly affected by flanking amino acid sequences. 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 (14). Fab molecules represent disulfide-linked heterodimers composed of Fd plus chains. Hexahistidine-tagged Fd cDNAs and chain cDNA were prepared as described previously (14 -16). In each Fd construct, the oligonucleotide sequence (CATCAC-) 3 was inserted upstream from a TGA stop codon so as to encode a carboxyl-terminal (His) 6 sequence used to purify the Fab or Fd molecules by nickel affinity matrix chromatography, as described (15,16).

Synthesis of
The Fd construct AP7.4 was generated by splice overlap extension PCR (14,16), using AP7 Fd cDNA (14) as a template. The 5Ј cDNA fragment of AP7.4 Fd was obtained as a BglII/SacII fragment from AP7 Fd cDNA. The primer 5Ј-CTTCTACCGCGGCGACGGGGGAGCTTAC-TATGCTATGG-3Ј (AP74FOR) changes Asn 108 within AP7 H3 to Ala, while retaining a SacII site. AP74FOR was used in combination with the oligonucleotide 5Ј-CTATCTAGATCAATCCCTGGGCAC-3Ј (HREV) to produce the 3Ј fragment of AP7.4 Fd cDNA. Both fragments were then digested with SacII and ligated. Ligated cDNA served as template for subsequent PCR reactions using the oligonucleotide 5Ј-CTCAGATC-TACAATGGACTTCGGGCTC-3Ј (HFOR) and HREV to amplify AP7.4 Fd cDNA. A BglII-NcoI digest of the cDNA product was then ligated into pVL1392 containing a portion of the murine constant region, the carboxyl-terminal cysteine, and the (CATCAC-) 3 sequence followed by the TGA stop codon. The cloned, recombinant virus containing this new Fd construct is designated pVL7.4Fd.
The Fd construct of AP7.7 was generated by PCR-based overlap extension using FdOPG2 as template. The 5Ј fragment was produced with the primer HFOR and the oligonucleotide 5Ј-GGTCCATAGCAT-AGTAAGCTCCCCCGTCGTACC-3Ј (AP77REV). The 3Ј fragment was generated using the oligonucleotide 5Ј-GGTACGACGGGGGAGCTTAC-TATGCTATGGACC-3Ј (AP77FOR) and the primer HREV. The cDNA products were gel-purified and added together in a PCR reaction with no additional oligonucleotide primers. The DNA fragments were allowed to anneal and complimentary sequences were produced by extension from the overlapping sequences for 10 thermocycles. Subsequently, the primers HFOR and HREV were added, and an additional 25 cycles were carried out. The product was then digested with BglII and NcoI, purified, and ligated into the transfer vector employed for AP7.4 cDNA, as described above. The cloned, recombinant virus containing this insert is designated pVL7.7Fd.
Cloning and Analysis of Recombinant Baculoviruses-Recombinant viruses were cloned by infection of Sf9 cells (Invitrogen) (2 ϫ 10 6 in 2 ml of complete Grace's medium) seeded in T25 culture flasks, as described (14 -16). The sequence of each recombinant clone was confirmed prior to its use, using Sequenase version 2.0 (U.S. Biochemicals, Inc.). Recombinant viruses were used to coinfect High Five insect cells (Invitrogen, Inc.), and Fab molecules were harvested from the media, normally after 72-h cultures, as described (14 -16). Recombinant Fd and chains were detected by a quantitative Western blot assay using rabbit polyclonal anti-murine Fdϩ antibody, developed in our laboratory (14,16). Fab molecules were purified by adsorption to Ni-NTA resin (QIAGEN, Chatsworth, CA) and elution with imidazole buffer, as described (16). The purity of Fab molecules was assessed by silver staining of eluted proteins separated by electrophoresis on 10% SDS-polyacrylamide gel electrophoresis slab gels (16). Purified Fab molecule concentration was determined by optical density at 280 nm using an extinction coefficient of 1.4.
Purified Integrin ELISA-Integrin ␣ IIb ␤ 3 was purified as a functional heterodimer from human platelets as described by Fitzgerald et al. (17), except that protease inhibitors were included in the final buffer, namely 0.4 mM phenylmethylsulfonyl fluoride, 100 g/ml leupeptin, 0.02 g/ml pepstatin A, and 10 mM benzamidine. The vitronectin receptor ␣ V ␤ 3 was purified as a functional heterodimer from human placentas by immunoaffinity chromatography using the murine monoclonal antibody LM609, as described (18). Purified integrin heterodimers were adsorbed onto the wells of Immulon II microtiter plates (Dynatech, Inc., Chantilly, VA), and the ability of murine monoclonal Fab molecules or recombinant proteins to bind to each integrin was assessed by ELISA (19).
Flow Cytometry Analysis of Platelets and M21 Cells-Platelet-rich plasma was obtained by differential centrifugation of ACD(A)-anticoagulated whole blood. Platelet-rich plasma was harvested, and platelets were gently pelleted by centrifugation at 950 ϫ 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 re-combinant Fab molecules in Tyrode's buffer were added to 5 ϫ 10 5 platelets in the presence of either 20 ng/ml prostaglandin E 1 or 0.2 M phorbol myristate. After a 15 min incubation at ambient temperature, fluorescein isothiocyanate-labeled goat anti-mouse IgG F(abЈ) 2 (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) was added. After an additional 15-min incubation in the dark, samples were diluted 10-fold with Tyrode's buffer and analyzed on a Becton Dickinson FAC-Scan apparatus, as described (14,15).

RESULTS
The RGD-containing Fab ligand AP7 binds the integrin ␣ IIb ␤ 3 equivalently on both resting or activated platelets (14,16). On the premise that flanking sequences can influence integrin specificity, our goal was to determine the smallest possible mutation within the AP7 H3 loop that would change its specificity from ␣ IIb ␤ 3 to ␣ V ␤ 3 .
Binding of Recombinant Fab Molecules to Platelets-The binding of AP7, AP7.4, and AP7.7 Fab molecules to gel-filtered platelets was compared by flow cytometry as a function of extracellular divalent cation composition and platelet activation with phorbol myristate. Equivalent results were obtained in the presence of either 10 M Mn 2ϩ (Fig. 1) or 1 mM Ca 2ϩ plus 1 mM Mg 2ϩ (data not shown). As we have shown in the past (14,16), the binding of AP7 Fab molecules to platelets was saturable and could be completely inhibited by RGDW (Ն10 M) or EDTA (5 mM) (data not shown). AP7.4 and AP7.7, on the other hand, do not bind to platelets in the presence of any combination of divalent cations and regardless of the extent of platelet activation (Fig. 1).
Binding of Recombinant Fab Molecules to Human Cell Lines-The parent Fab molecule OPG2 fails to bind to ␣ V ␤ 3 expressed by the melanoma cell M21 under any conditions ( Fig.  2A), a finding that is consistent with our previous reports that this antibody is selective for the integrin ␣ IIb ␤ 3 (27,28). However, the comparative binding of AP7 and its derivatives to M21 cells is more complex and obviously influenced by divalent cation composition. While AP7.7 Fab molecules also fail to bind to M21 cells under any conditions (data not shown), differential binding of both AP7 and AP7.4 Fab molecules is observed. In general, we found that 1 mM Ca 2ϩ exerts an inhibitory effect on the binding of either AP7 or AP7.4 Fab molecules, that the binding of each is markedly augmented by the presence of Ն100 M Mn 2ϩ , and that 1 mM Mg 2ϩ has neither a supportive nor inhibitory influence ( Fig. 2A). In the case of AP7, a syner-gistic effect is observed in the presence of both 1 mM Mg 2ϩ and 100 M Mn 2ϩ , so that maximal binding equivalent to that seen with AP7.4 is observed. This is not true of AP7.4 itself, which shows maximal binding in the presence of either 100 M Mn 2ϩ alone or both 1 mM Mg 2ϩ and 100 M Mn 2ϩ . In any case, binding that would otherwise be supported by Mn 2ϩ is significantly attenuated in the presence of 1 mM Ca 2ϩ . Under optimum divalent cation conditions, the binding of either AP7 or AP7.4 Fab molecules to M21 melanoma cells is completely inhibited in a dose-dependent manner by GRGDSPK but not GRGESPK (Fig. 2B).
Despite the observed changes in selectivity for ␣ IIb ␤ 3 versus ␣ V ␤ 3 , the AP7 and AP7.4 Fab molecules remained selective for only these ␤ 3 integrins and did not bind to either M21-L cells, which express ␣ 5 ␤ 1 but not ␣ V ␤ 3 , or UCLA-P3 cells, which express ␣ V ␤ 5 but not ␣ V ␤ 3 (data not shown).
Binding of Recombinant Fab Molecules to Purified ␣ IIb ␤ 3 or ␣ V ␤ 3 -The selectivity of each Fab molecule for ␣ IIb ␤ 3 or ␣ V ␤ 3 and the dependence of binding on the RGD sequence were further investigated using the purified integrins in an ELISA (Fig. 3).
In the presence of 1 mM each of Ca 2ϩ and Mg 2ϩ , AP7 or OPG2 Fab molecules exhibit a strong affinity for ␣ IIb ␤ 3 (Fig. 3A) but fail to bind to ␣ V ␤ 3 (Fig. 3B). Binding of either Fab molecule is completely inhibited by 1 mM EDTA or Ն10 M RGDW but not by Յ2 mM RGEW (data not shown). Under the same divalent cation conditions, both AP7.4 and AP7.7 Fab molecules fail to bind to either ␣ IIb ␤ 3 (Fig. 3A) or ␣ V ␤ 3 (Fig. 3B).
In the presence of Ն10 M Mn 2ϩ , the binding of each Fab molecule to purified ␣ IIb ␤ 3 is unchanged (Fig. 3C). However, AP7 and AP7.4 Fab molecules now bind strongly to ␣ V ␤ 3 in the presence of Mn 2ϩ , while OPG2 Fab molecules fail to bind (Fig.  3D). The binding of AP7 or AP7.4 Fab molecules to ␣ V ␤ 3 in this cell-free system is completely inhibited by 1 mM EDTA or Ն 10 M RGDW, but not at all by up to 2 mM RGEW (data not shown). As a negative control, AP7.7 Fab molecules (50 g/ml) fail to bind to either integrin under any conditions (Fig. 3, A-D). DISCUSSION Using the recombinant murine Fab molecule OPG2 as a versatile framework, our results validate the hypothesis that the amino acid composition immediately flanking an RGD tripeptide can profoundly influence the specificity and divalent cation modulation of ligand binding to ␤ 3 integrins. The relevant sequences of the recombinant Fab molecules, OPG2, AP7, AP7.4, and AP7.7, and their comparative specificities, as determined by this study, are summarized in Table I.
The model that we have developed using OPG2 and the AP7 series of recombinant Fab molecules provides a unique opportunity to compare both RGD and RYD analogs of the same ligand and to predict the impact of single amino acid substitutions on specificity based upon known side chain interactions defined by x-ray crystallography of the parent Fab molecule (28). In the case of each Fab molecule in our series, a single amino acid substitution within the third complementarity-determining region of the heavy chain results in a profound change in specificity. For example, there is the complete loss of binding to either ␤ 3 integrin that is characteristic of AP7.7 created by the replacement of Asn 108 by an Alanine within the OPG2 H3 sequence. The major reason for our selection of Asn 108 as a target for substitution is the fact that, in the crystal structure of the OPG2 Fab molecule (28), the side chain of Asn 108 is in a position to form a hydrogen bond with that of Asp 105 , i.e. the distance between Asp 105 -OD1 and Asn 108 -ND1 is 2.8 or 3.2 Å in each of two alternate conformers of the H3 loop. We reasoned that disruption of such a side chain interaction would likely influence the juxtaposition of the remaining side chains, particularly those of Arg 103 and Asp 105 . This hypothesis is borne out by our experimental evidence, and our results provide strong support for the presence of a hydrogen bond between these side chains. It follows that this side chain interaction probably holds the Asp 105 carboxyl group in a unique orientation with respect to the amino group of Arg 103 such that OPG2 is recognized exclusively by ␣ IIb ␤ 3 .
The most dramatic and novel finding of our study is the change in specificity created by the engineering of the AP7.4 Fab molecule. The sole difference between the Fab molecule AP7, which binds preferentially to ␣ IIb ␤ 3 in the presence of calcium ions, and AP7.4, which binds solely to ␣ V ␤ 3 in the presence of manganese ions, is a single amino acid substitution adjacent to the RGD motif within the H3 loop (HPFYRGDGGN in AP7 versus HPFYRGDGGA in AP7.4). To our knowledge, this is the first published report of a complete change in specificity of an RGD ligand from ␣ IIb ␤ 3 to ␣ V ␤ 3 as a result of a single amino acid substitution. While others have shown that single amino acid differences in RGD peptides or an Fab mol- ecule can increase their relative affinities for ␣ IIb ␤ 3 (10,11,29,30), the engineered AP7.4 molecule represents the first instance in which a dramatic decrease in affinity for ␣ IIb ␤ 3 and reciprocal increase in affinity for ␣ V ␤ 3 has been produced. This change in relative affinities is so extreme that the binding of the Fab molecule to ␣ IIb ␤ 3 has fallen below the level of detection. Apparently, because Tyr 104 of OPG2 has been replaced by Gly in AP7, the subsequent replacement of Asn 108 with Ala no longer results in the dramatic loss of binding to either integrin that was observed with the creation of AP7.7 (see above). In the case of AP7.4, the elimination of the putative hydrogen bond and the accommodation of this change by the mutated H3 loop must increase the flexibility of the Arg 103 and Asp 105 side chains and facilitate the increased selectivity of AP7.4 for the integrin ␣ V ␤ 3 . Our results would argue that both ␣ IIb ␤ 3 and ␣ V ␤ 3 are highly restrictive with respect to the Arg and Asp side chain orientations that each will recognize. There are at least two mechanisms that may be involved in the divalent cation regulation of ligand binding to integrins. On one hand, integrin conformation is likely influenced by cations, particularly Mn 2ϩ . As an example, the monoclonal antibody 9EG7 binds to a Mn 2ϩ -induced epitope and stimulates ␤ 1 integrin functions (31). On the other hand, there is substantial evidence that divalent cations support an initial ternary complex with integrin and ligand. As the ligand-integrin binding becomes stabilized, the divalent cation is displaced (32). Replacement of Asn 108 with Ala may eliminate the ability of AP7.4 to disrupt cation coordination upon contact with an integrin. This would not explain, however, why AP7.4 then binds selectively to ␣ V ␤ 3 , an integrin whose recognition of RGD ligands is equally regulated by divalent cations.
Osteopontin was the first RGD ligand identified that has a substantial preference for ␣ V ␤ 3 relative to ␣ IIb ␤ 3 (33). It is particularly relevant that Ca 2ϩ is a strong inhibitor of osteopontin binding to ␣ V ␤ 3 , while Mn 2ϩ enhances this interaction (33). In the microenvironment of bone tissue, osteoclasts liberate Ca 2ϩ from demineralized bone during resorption such that levels of free Ca 2ϩ increase locally. This would then favor detachment of osteoclasts from bone by inhibition of the osteopontin binding to ␣ V ␤ 3 . Conversely, increases in the relative level of Mg 2ϩ compared to Ca 2ϩ in areas of bone growth would favor osteopontin-mediated cell attachment. Micromolar levels of Mn 2ϩ found in many tissues, including bone and liver, would support cell attachment via ␣ V ␤ 3 (12). Clearly, the engineered, recombinant Fab molecule AP7.4 behaves precisely as does osteopontin with respect to its selectivity for the integrin ␣ V ␤ 3 and the influence of divalent cations on its binding properties. Thus, AP7.4 is a powerful new tool to investigate the role of the integrin ␣ V ␤ 3 in various biological processes, including bone resorption.