Use of phage display to probe the evolution of binding specificity and affinity in integrins

Renhao Li1, Ronald H. Hoess2, Joel S. Bennett3 and William F. DeGrado1,4

1 Department of Biochemistry and Biophysics and 2 Bristol-Myers-Squibb Pharmaceutical Company, Wilmington, DE 19880, USA 3 Hematology–Oncology Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 and

4 To whom correspondence should be addressed. E-mail: wdegrado{at}mail.med.upenn.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The specific binding of RGD-containing proteins to integrin is a function of both the conformation of and the local sequence surrounding the RGD motif. To study the effect of these factors on integrin binding affinity and specificity, we obtained RGD-containing ligands specific for different integrins presented on the same protein scaffold. The ß-turn region between two anti-parallel ß-strands on the loop I of tendamistat, an inhibitor of {alpha}-amylase, was extended by two residues and randomized in a phagemid library. This library and two subsequently constructed RGD-containing loop I libraries were biopanned with purified integrins {alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5 individually. The sequence analysis of selected tendamistat variants and characterization by phage ELISA revealed that phage adhesion is mediated exclusively by an RGD motif located at only two out of four possible positions on loop I. Further, sequences flanking the RGD motif were specific for different integrin targets. Interestingly, selected tendamistat variants mimic natural integrin ligands, both in sequence similarity and in integrin binding specificity, indicating that various ligand specificity patterns can be generated by driving towards maximum affinity in the integrin–ligand complexes.

Keywords: integrin/phage display/protein evolution/protein–ligand interaction/RGD motif


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Integrins are a family of heterodimeric cell surface receptors that interact with extracellular matrix proteins and are critical for biological processes such as cell adhesion and migration (Hynes, 1992Go). Many integrin ligands, such as fibronectin, vitronectin and osteopontin, contain an RGD sequence that is critical for integrin binding. Nonetheless, the three-dimensional presentation of this sequence and the surrounding residues account for the varied specificity and affinity to different integrins (Kopple et al., 1992Go; Pfaff et al., 1994Go; Bach et al., 1996Go; Haubner et al., 1997Go; Schumann et al., 2000Go).

It is interesting to consider what the amino acid sequences surrounding the RGD motif of these ligands can tell us about the evolutionary and biological processes that define a given integrin–ligand complex. One model might hold that a given integrin–ligand pair has evolved to maximize the specificity for this complex. For example, the RGD-containing protein osteopontin binds exclusively to {alpha}Vß3 on platelets, although the closely related RGD-binding integrin {alpha}IIbß3 is present at an ~100-fold greater level in platelet membranes (Bennett et al., 1997Go). Alternatively, other RGD-containing proteins might have evolved to have a broad specificity for a variety of integrins. Furthermore, it is not known whether the sequence of a natural RGD ligand has evolved to maximize overall affinity or whether affinity is fine-tuned to allow for an appropriate biological function.

Phage display provides an attractive tool to address these questions. In this method, randomized sequences are expressed on the phage surface and the peptide or protein with the highest affinity for a given receptor is selected through repeated rounds of biopanning. Here, we select for binding to three different integrins: {alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5. If the sequence of the phage-derived ligand for a given integrin closely resembles that of a natural ligand for this integrin, one can infer that the natural ligand has evolved to maximize its affinity (within the constraints of its overall three-dimensional structure). Furthermore, the specificity of the selected sequence can be compared with the specificities of the natural ligands. To the extent that they are similar, one can infer that natural ligand specificity is generated by maximizing affinity, without the need for negative selection against other integrins.

The RGD-containing sequences in integrin ligands are frequently flexible and they often occur in loops in many proteins (Leahy et al., 1992Go; Main et al., 1992Go; Krezel et al., 1994Go; Kodandapani et al., 1995Go). Nevertheless, a degree of conformational rigidity imposed on the RGD motif by, for instance, a disulfide bond (O’Neil et al., 1992Go) or cyclic backbone constraints (Kopple et al., 1992Go) can increase the binding affinity of the ligand. Moreover, studies on RGD-containing peptides or peptidomimetic compounds suggest that their integrin-binding specificities depend on (i) the relative orientation of the side chains of Arg and Asp, (ii) the specific turn conformation and (iii) the location of RGD in the turn (Pfaff et al., 1994Go; Bach et al., 1996Go; Haubner et al., 1997Go; Schumann et al., 2000Go).

Studies with phage-displayed peptide libraries indicate that peptides with different sequences flanking the RGD motif show varying degrees of specificity for different integrins (O’Neil et al., 1992Go; Koivunen et al., 1993Go, 1994Go; Healy et al., 1995Go; Kraft et al., 1999Go). However, well-defined integrin-specific motifs are not obvious in these peptide ligands. Also, mutational studies on several protein ligands have demonstrated that residues flanking the RGD motif play a critical role in their integrin binding specificity (Kunicki et al., 1997Go; Wierzbicka-Patynowski et al., 1999Go). Nevertheless, it appeared desirable to conduct a more comprehensive evaluation of the role of the RGD-flanking sequence in defining specificity and affinity of a conformationally defined protein ligand for a variety of integrins. To accomplish this objective, we constrained the RGD motif in loop I of tendamistat. Tendamistat is a small protein inhibitor of {alpha}-amylase. Its 75 amino acids form an immunoglobulin-like fold with three CDR-like loops projecting from the hydrophobic core formed by two ß-sheets (Pflugrath et al., 1986Go). Its stability, folding and dynamics have been thoroughly investigated (Renner et al., 1992Go; Schönbrunner et al., 1997Go; Bonvin and van Gunsteren, 2000Go; Bachmann and Kiefhaber, 2001Go). Thus, any perturbation in structural and/or dynamic property can be subsequently analyzed in detail. Since tendamistat does not bind to integrin or extracellular matrix proteins, its impact on integrin–ligand interaction should be minimal. Therefore, tendamistat can provide a non-discriminating structural ‘platform’ on which various integrin ligands can be generated and compared.

Tendamistat has been previously used as a protein scaffold for display of random peptides on phage (McConnell and Hoess, 1995Go). In tendamistat, loop I, the loop with the lowest B-factors, is the major contributor to {alpha}-amylase binding (Wiegand et al., 1995Go). Most of the side chains comprising this loop point in the same direction and are exposed to solvent (Figure 1AGo). One of the two disulfide bonds in tendamistat connects the bases of the antiparallel ß-strands flanking loop I. Hence loop I should be ideal for presentation of an RGD motif. In this paper, we describe the construction of RGD-containing tendamistat loop I phagemid libraries and the selection of ligands specific for integrins {alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5. The sequences of identified ligands resemble those found in natural integrin ligands. The initial examination of these ligands by phage ELISA revealed a pattern of integrin-binding specificity consistent with that observed in nature. Thus, nature would appear to generate various integrin-specific patterns by driving towards maximum affinity in these integrin–ligand complexes.



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Fig. 1. Tendamistat loop I libraries. (A) A ribbon diagram of tendamistat highlighting the loop I. Side chains of Q16 and S21 (gray) point inside the protein, whereas side chains of the other residues on the loop (black) point outside to form the {alpha}-amylase binding site. The structure used here is 1HOE in the PDB databank (Pflugrath et al., 1986Go). (B) Illustrations of various loop I libraries made in this study.

 

    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction of the phagemid for the tendamistat loop I library

Tendamistat has been cloned into a phagemid pRH704 in an earlier study (McConnell and Hoess, 1995Go). In the present study, four synthetic oligonucleotides were designed to carry out the following changes in the tendamistat gene in pRH704 using PCR. First, the KpnI site near the 3'-end of the tendamistat gene was deleted, leaving the remaining KpnI site in the signal sequence region upstream of tendamistat gene as a unique site. Second, a FLAG tag was placed at the N-terminus of tendamistat for immunological detection and a unique restriction site (MfeI) was inserted between the tag and the tendamistat sequence, permitting the insertion of a cassette containing the randomized loop sequence. Because inspection of the crystal and NMR structures of tendamistat (Pflugrath et al., 1986Go; Kline et al., 1988Go) revealed that the N-terminus of tendamistat is flexible and does not form extensive contacts with the rest of the protein, the first five residues were deleted to minimize the length of the cassette inserted to create the peptide library.

The amplified tendamistat gene fragment was subsequently digested by EcoRI and StyI and inserted back into the pRH704 vector (McConnell and Hoess, 1995Go). The resulting phagemid, designated pRL101, was transformed into XL1-Blue cells (Stratagene, La Jolla, CA). After the library was made, a pool of mixed colonies was selected randomly for sequencing. The signal readouts of four bases at each randomized position are very similar, confirming the randomness of the library. To generate the phage particles, M13-CVS helper phage (Stratagene) was added to the bacteria culture at the MOI of 20:1. The purified phage particles were titered by counting the number of colonies that resulted from phage transformation to Escherichia coli cells. Phage ELISA and western blot were performed to confirm that pRL101 was capable of displaying the FLAG tag and consequently tendamistat, on the phage surface.

Construction of phagemid libraries based on the loop I of tendamistat

In the construction of the randomized loop I library, the oligo serving as the template for PCR carried an MfeI site at its 5'-end and eight randomized codons in the middle: GACAGACAATTGGAACCAGCGCCATCTTGCGTTACCCTGNNSCAGNNSNNSNNSNNSNNSNNSTCTNNSGCTGACAACGGTTGCGCA (N = A, T, C or G; S = C or G). The forward primer has the same 5'-end of the template: GACAGACAATTGGAACCAGC. The reverse primer covers the tendamistat sequence between the randomized region and the BstEII site: CTTTTACGGTAACCGTTTCTGCGCAACCGTTGTCAGC. Five cycles of PCR were carried out with Deep Vent polymerase (New England Biolabs, Beverly, MA). The resulting DNA fragment was digested by MfeI and BstEII and ligated with the 4 kb MfeI–BstEII fragment of pRL101. Following ligation, the DNA was electroporated into XL1-Blue cells and the phagemid library was amplified as described (Kay et al., 1996Go).

The construction of two RGD-containing loop I libraries was carried out following the procedures described above. For the RGDX library, the template oligo was GACAGACAATTGGAACCAGCGCCATCTTGCGTTACCCTGNNSNN-SCGTGGTGACNNSNNSNNSNNSNNSGCTGACAACGG-TTGCGCA and for the XRGD library, the template was GACAGACAATTGGAACCAGCGCCATCTTGCGTTACC-CTGNNSNNSNNSCGTGGTGACNNSNNSNNSNNSGCTGACAACGGTTGCGCA.

Selection of integrin binding phages

Purified {alpha}Vß3 and {alpha}Vß5 were obtained from CHEMICON (Temecula, CA) and integrin {alpha}IIbß3 from Enzyme Research Laboratories (South Bend, IN). Integrins (2 µg per well) in 200 µl of 0.05% Tween20–TBS buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2) were coated on the wells of Maxisorp microtiter plates (NUNC, Denmark) at 4°C overnight. On the following day, coated wells were washed four times with TBS buffer and blocked with 5% non-fat milk in TBS buffer for 2 h, followed by six washes with TBS buffer, all at room temperature. The phage library of 1011 cfu in 100 µl of TBS buffer was mixed with 100 µl of 5% non-fat milk in microcentrifuge tubes for 2 h prior to addition to the integrin-coated wells. After incubation for 1 h with integrin, unbound phages were removed and the wells were washed five times with TBS–Tween buffer, followed by five washes with TBS buffer. Bound phages were then eluted with 200 µl of 100 mM glycine, 1 mg/ml BSA, pH 2.0 for 10 min and the pH was neutralized with 100 µl of 1 M Tris–HCl, pH 7.4. In each ensuing round of selection, the wells were coated with 0.5 µg of integrin and phage input was 109–1010 cfu. The incubation time of each washing step was increased to 5 min for the second round and 10 min for the third round. After each round, eluted phages were titered and the recovery efficiency was calculated as the ratio of the output versus input phage titers.

Immunoblotting

Freshly prepared phage particles (1011 cfu) were resuspended in standard SDS sample loading buffer and electrophoresed in precast SDS–polyacrylamide gels (4–12% NuPAGE Bis-Tris gel with MES running buffer; Invitrogen, Carlsbad, CA). Separated proteins were transferred to a nitrocellulose membrane (0.45 µm, Schleicher & Schuell, Keene, NH) and immunoblotted with anti-FLAG M2 mAb followed by HRP-labeled Fc-specific anti-mouse IgG (Sigma, St. Louis, MO). Proteins were visualized by treating the blot with chemiluminescence reagent (NEN Life Science Products, Boston, MA) and exposed to Kodak X-0mat AR film.

Phage ELISA

Maxisorp plates (200 ng integrin per well) were prepared as described above. Phage particles were serially diluted with 0.3% BSA blocking buffer and added to the wells. After incubation for 1 h at room temperature, the plates were washed five times each with TBS–Tween buffer and TBS buffer. HRP–anti-M13 Ab conjugate (Amersham-Pharmacia, Piscataway, NJ), 1:5000 diluted in BSA blocking buffer, was then added for 1 h. Subsequently, the plates were washed eight times with TBS buffer, followed by addition of the substrate ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] with H2O2 and incubated for a further 20 min. The absorbance at 405 nm was measured with a Bio-Tek EL800 microplate reader.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Construction and panning of a randomized phagemid library in loop I of tendamistat

Loop I of tendamistat contains a type I ß-turn flanked by an anti-parallel ß-sheet. The side chains of Q16 and S21 are directed into the interior of the protein as part of its core, whereas the side chains of residues 17–20 point outwards and constitute the binding site for {alpha}-amylase (Figure 1AGo). To attempt to conserve the fold of the protein, we chose to keep residues Q16 and S21 constant in the initial library, while randomizing the rest of the loop. To introduce additional variation, two residues were inserted at the turn (Figure 1BGo). A DNA fragment containing the randomized region was generated and cloned into the vector pRL101 as a MfeI–BstEII fragment. The diversity of the resulting phagemid library was 3.9x108.

To determine whether the randomized loop I library was capable of generating integrin-binding ligands, the phagemid library was biopanned with integrin {alpha}Vß3. After three rounds of selection, the phage recovery efficiency increased by two orders of magnitude to 2x10-6, indicating significant enrichment for phages that bind to {alpha}Vß3. Individual clones were then selected for DNA sequencing. Twelve out of the 15 sequenced clones contained an RGD sequence (Table IGo). The other three contained a slightly altered sequence, RSD and a pair of cysteine residues. No other motifs, in particular the sequence NGR previously selected from other peptide libraries (Healy et al., 1995Go; Koivunen et al., 1995Go), were identified. Interestingly, the RGD motif was only observed in the N-terminal half of the loop, in two out of the four possible registers within the randomized region. Further, there was no apparent consensus residues flanking the RGD motif.


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Table I. Deduced tendamistat loop I sequences of phages binding to integrin {alpha}Vß3 from the randomized library, xQxxxxxxSx
 
Construction and panning of RGD-containing loop I libraries

Based on these results, we constructed two position-specific RGD-containing loop I libraries. In each library, the RGD motif was fixed at a particular position of loop I, with the libraries being designated as RGDX and XRGD (Figure 1BGo). In each of these libraries, seven residues flanking the motif, including the conserved residues Q16 and S21, were randomized. The diversities of the RGDX and XRGD libraries were 3.2x108 and 1.4x108, respectively.

To study the effects of the flanking residues on integrin binding specificity and affinity, both libraries were biopanned using immobilized integrins {alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5. To identify the optimum flanking sequence, panning stringency was increased by gradually lengthening the incubation time of the washing steps in each round. When phage recovery efficiency increased significantly to 10-5 after three rounds of panning (Figure 2Go), individual clones were selected for DNA sequencing. Table IIGo summarizes the sequences selected for binding individual integrins. In some instances, a fourth round of panning was carried out, but the sequences detected were as diverse as those obtained after the third round (data not shown).



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Fig. 2. Phage recovery efficiency of RGD-containing tendamistat loop I libraries, panned against integrins {alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5. (A) RGDX library; (B) XRGD library. The number of round is indicated below each bar column.

 

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Table II. Deduced tendamistat loop I sequences of phages binding to different integrins from RGD-containing librariesa
 
Figure 3Go and Table IIGo illustrate the sequences of 15 clones selected randomly from each category. There are major differences, in addition to some similarities, in the degrees of conservation of specific positions within each experiment, depending on the library (XRGD or RGDX) and the type of integrin used in biopanning. In particular, the RGDX library gave the following consensus sequences:



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Fig. 3. Residue distribution histograms of the loop I sequences in identified integrin-binding tendamistat ligands. The histograms are grouped by biopanning categories (in columns, indicated on the top) and by residue positions relative to the RGD motif (in rows, indicated on the right). In each histogram, the number of times for one residue to appear at a certain position in 15 sequenced clones (Table IIGo) is plotted for all amino acids excluding cysteine. The dashed line indicates the location of the RGD motif. The preferred residues in each category and position are labeled by their residue type.

 

-2 -1 +1 +2 +3 +4 +5
{alpha}IIbß3 R A RGD N P S N L
{alpha}Vß3 A/V S/T RGD X P/Y X X X
{alpha}Vß5 V/A T/S RGD T F/Y X X S

The {alpha}V integrins tend to prefer a small hydrophobe (A or V) at the -2 position. Residues T and S are preferred at the -1 position for {alpha}Vß5, whereas S or other small side chain residues are preferred over T when the library is panned against {alpha}Vß3. There was a clear preference for residue P at the +2 position for both ß3 integrins, but C-terminal to the RGD site there was a substantial difference between the preferred side chains in all three cases. The very strong preference for T at position +1 was particularly striking for {alpha}Vß5.

The consensus sequences arising from the XRGD library were


-3 -2 -1 +1 +2 +3 +4
{alpha}IIbß3 R R/K S/T RGD M/L G L/I Y
{alpha}Vß3 R P/H A/G RGD S D H/L X
{alpha}Vß5 H L A RGD D L T Y

The ß3 integrins show a strong preference for residue R at the -3 position. The {alpha}V integrins prefer mostly A at the -1 position, whereas {alpha}IIbß3 prefers either S or T at this position. Beyond these similarities, the sequences were quite distinct. For example, G was absolutely conserved at the +2 position for {alpha}IIbß3, whereas D is strongly preferred at the same position for {alpha}Vß3 (14 out of 15 clones).

Phage ELISA for characterization of the binding affinity

We chose to study the integrin-binding specificities of the tendamistat-based variants by comparing their affinities to individual integrins, which can be carried out in parallel by phage ELISA. Earlier studies of RGD-containing peptides identified by phage display have demonstrated a correlation between peptide inhibition of attachment of integrin-expressing cells and peptide-displaying phage binding to isolated integrins (Koivunen et al., 1993Go, 1995Go; Healy et al., 1995Go). Representative sequences from each category, underlined in Table IIGo, were selected for phage ELISA studies. Control experiments described below demonstrated that the phage ELISA signal can only be attributed to the conformation and sequence of the RGD-containing loop I of tendamistat. To insure that the ELISA signal reflected only the intrinsic binding activity of the ligands, not the ligand concentration, we confirmed that the expression level of each variant protein displayed on the phage surface was very similar by immunoblotting (Figure 4Go) and ELISA (Figure 5Go, top plot) probed with an anti-FLAG monoclonal antibody. Further, the addition of 5 mM EDTA to the ELISA mixture abolished the absorbance at 405 nm, confirming that divalent cations were required for phage binding to integrins (data not shown). Finally, phages displaying wild-type tendamistat failed to bind to integrins (Figure 5Go), demonstrating the inability of the protein scaffold itself to bind integrins and the necessity of an RGD motif for integrin binding. It should be noted that the comparison of phage ELISA data among variants are made on a semi-quantitative basis.



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Fig. 4. Western blot of tendamistat phage particles. 1011 phage particles were loaded for each lane and the FLAG–tendamistat–PIII fusion protein was probed by anti-FLAG M2 mAb. The sequence of loop I for each tendamistat construct is labeled on the top. Molecular markers in kDa are shown on the left.

 


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Fig. 5. Phage ELISAs for display of tendamistat mutants. Serial dilutions of tendamistat–phage solutions were incubated in microtiter wells with immobilized anti-FLAG M2 mAb or integrins, indicated in each plot. After removal of the non-bound phage, bound phages were measured by absorbance at 405 nm using a reaction catalyzed by HRP–anti-M13 mAb conjugate. All four plots utilize the same plot symbols, which are identified by their corresponding loop I sequence (shown only in the top plot) and biopanning categories (shown on the right side of the bottom three plots). The data represent means from two separate experiments.

 
In the phage ELISA, binding of each variant to all three integrins ({alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5) was measured, regardless of its intended target during biopanning. Two major binders were identified for {alpha}IIbß3. Both were selected originally for {alpha}IIbß3: the tendamistat variant derived from the XRGD library (designated as ‘XRGD–{alpha}IIbß3') with higher affinity and the variant from the RGDX library with lower affinity based on ELISA measurements. Two other variants, selected from RGDX–{alpha}Vß3 and RGDX–{alpha}Vß5, bound detectably to {alpha}IIbß3.

Except for wild-type tendamistat, all proteins tested bound {alpha}Vß3 to various degrees. One variant selected from XRGD–{alpha}Vß3 is the best binder. Other variants showed slightly lower affinity, as judged by their ELISA signals. Proteins selected from RGDX–{alpha}Vß3 showed only modest affinity for {alpha}Vß3. It is noteworthy that the variant displaying the least binding affinity was from XRGD–{alpha}IIbß3, the same protein with the highest affinity for integrin {alpha}IIbß3.

In contrast to {alpha}Vß3, only one variant, the protein derived from XRGD–{alpha}Vß5, showed high affinity for {alpha}Vß5 based on ELISA measurements. Consistently, this clone was completely dominant after three rounds of panning. Two other proteins, one from RGDX–{alpha}Vß5 and one from RGDX–{alpha}IIbß3, also bound to {alpha}Vß5, albeit with lower affinity.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The conformation of the RGD motif in tendamistat

Although the randomized loop I library used in the initial biopanning had few sequence constraints, the majority of the identified ligands contained an RGD motif and the remainder contained an RGD-like RSD sequence (Table IGo). This emphasizes the importance and dominance of RGD in binding to integrin. In the randomized loop I library, the RGD motif could be placed on the loop at four different positions. However, only two positions yielded productive sequences. In both cases, the RGD motif resided on the N-terminal half of the loop, suggesting that the Asp residue of RGD must be present at the apex of the loop and that exposure of its side chain is critical to integrin binding.

The RGD motif has been inserted into a number of proteins and exhibited integrin-binding activity (Yamada et al., 1993Go; Smith et al., 1995Go; Hufton et al., 2000Go). The structural diversity of the loops in these proteins suggests that the RGD motif could undergo some conformational changes upon binding to integrins and there are no strict requirements for its initial conformation. In our study, displacement of the RGD motif by one residue in loop I almost certainly alters the conformation of the motif. Given the nature of the ß-sheet conformation, it is likely that the side chains of the RGD motif at these positions point in the opposite direction. However, variants from both libraries can bind to integrins. Nevertheless, the ligands selected from the XRGD library in general display better binding specificity and affinity than those from the RGDX library. This suggests that although the RGD motif on the tip of a loop can adopt different conformations to bind integrins, its initial conformation can affect the integrin binding affinity and specificity of the ligand.

Selected tendamistat variants mimic natural integrin ligands

Using two RGD-containing loop I phage libraries, we have identified a family of tendamistat-based integrin ligands. These ligands resemble many natural integrin ligands in terms of the sequence surrounding the RGD motif (Table IIIGo). Remarkably, this resemblance is not limited to one particular variant or biopanning category. Rather, examples from many categories can be found to resemble the natural ligands. For instance, the RGDxP motif, identified from the RGDX library for {alpha}IIbß3 or {alpha}Vß3, shares identity with the sequences in fibronectin and several disintegrins. Similarly, the RGDxD motif, identified from the XRGD library for {alpha}Vß3, shares identity with the RGD sequence found in decorsin and fibronectin. Two sequences selected from the RGDX library for {alpha}Vß5, VTRGDTFTQS or VTRGDTFTIS, are nearly identical with the sequence VTRGDVFTMP from vitronectin, a natural ligand for {alpha}Vß5.


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Table III. Loop I sequence of tendamistat variants mimic many natural integrin ligandsa
 
In addition to sequence similarity, the integrin-binding specificity of our ligands also mirrors those of natural ligands. Highly expressed on the platelet surface, integrin {alpha}IIbß3 is essential to hemostasis and thrombosis. To avoid spontaneous clotting in the normal blood vessel, {alpha}IIbß3 must stay inactive and ligand-free. Therefore, it is critical for it not to interact with ligands targeted for other integrins. Consistently, in our experiments, two variants with the highest affinity for {alpha}IIbß3 were targeted for this integrin, while variants selected for binding to {alpha}Vß3 and {alpha}Vß5 showed little or no binding to {alpha}IIbß3. Many snakes and other animals have evolved disintegrins in their venoms that inhibit hemostasis by interacting with platelet integrins {alpha}IIbß3 and {alpha}Vß3 (Niewiarowski et al., 1994Go). Some of the disintegrins contain the residue P at the +2 position after the RGD motif (Table IIIGo) and they can bind to {alpha}IIbß3 as well as {alpha}Vß3. Consistently, with the sequence RARGDNPSNL, the ligand selected from RGDX–{alpha}IIbß3 also binds very strongly to {alpha}Vß3 (Figure 5Go). Conversely, the variant identified from RGDX–{alpha}Vß3 also contains the RGDxP motif and shows limited affinity for {alpha}IIbß3.

In nature, there have been very few ligands identified to bind {alpha}Vß5. The most prominent ligand is vitronectin. Such a limited supply of ligands was also reflected in our phage ELISA results, as all but one tendamistat ligands selected for other integrins do not bind {alpha}Vß5. On the other hand, the variants screened for {alpha}Vß5 also bound to {alpha}Vß3 with high affinity. This reflects the fact that all known natural {alpha}Vß5 ligands also bind to {alpha}Vß3.

In contrast to {alpha}IIbß3, {alpha}Vß3 is present in many types of cells and involved in various biological processes including viral entry and tumor development (Jackson et al., 1997Go; Parise et al., 2000Go). Therefore, it is conceivable for {alpha}Vß3 to interact with a variety of RGD-containing ligands, including those capable of binding other integrins. Our phage ELISA results reflected such a broad binding reactivity for {alpha}Vß3 in that all variants screened for the other integrins bound to {alpha}Vß3 (Figure 5Go).

In summary, we have screened a family of tendamistat-based conformation-restricted ligands against integrins {alpha}IIbß3, {alpha}Vß3 and {alpha}Vß5, respectively. With selection against one particular integrin for three rounds of panning, we maximized the binding affinity of these ligands towards individual integrins. The sequences of identified ligands resemble those found in natural integrin ligands. In addition, our results showed that the integrin-binding specificities of these ligands also resemble those of natural ligands. Therefore, it is possible for a ligand to achieve desirable binding specificity by evolving towards maximum affinity for one particular integrin.


    Acknowledgments
 
This work was supported partly by grants HL54500 and HL40387 from the National Institutes of Health. R.L. was supported by a postdoctoral fellowship from the Cancer Research Fund of the Damon Runyon–Walter Winchell Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 4, 2002; revised September 19, 2002; accepted October 16, 2002.





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