Correspondence to: Erkki Koivunen, Department of Biosciences, Division of Biochemistry, University of Helsinki, Viikinkaari 5, FIN-00014 Helsinki, Finland. Tel:(358) 9-191-59023 Fax:(358) 9-191-59068 E-mail:erkki.koivunen{at}helsinki.fi.
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Abstract |
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Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginineglycineaspartic acid and leucineaspartatevaline. Using phage display, we have now found that the leukocyte-specific ß2 integrins bind sequences containing a leucineleucineglycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major ß2 integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel ß2 integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the ß2 integrinmediated leukocyte adhesion, but not ß1 or ß3 integrinmediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.
Key Words: cell adhesion, extracellular matrix, leukocyte, phage display, peptides
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Introduction |
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The migration of leukocytes through the body and the various lymphoid organs is an essential element of the immune system. While circulating in blood or lymphatic vessels, leukocytes are in a resting and low adhesive state. However, when leukocytes are stimulated by signals from the immune system, such as exposure to an immune complex or a chemokine gradient, their integrin adhesion receptors become activated (
Leukocytes express a specific subset of the integrin family, the ß2 integrins, of which four members are known. They have a common ß2 chain (CD18), but different subunits (
L or CD11a,
M or CD11b,
X or CD11c,
D or CD11d) (
subunits contain a conserved 200-residue A or I domain, which is essential for binding of most ligands. The crystal structures of I domains from the
L and
M subunits indicate the presence of a cation binding site called the metal-dependent adhesion site (
The major ligands of these integrins, the intercellular adhesion molecules (ICAMs),1 belong to the Ig superfamily, and five ICAMs with slightly different binding specificities have been described (Mß2 (Mac-1).
Because of the importance of the ß2 integrins for leukocyte function, antagonists of them are potential antiinflammatory agents. Antibodies to ß2 integrins or ICAMs have a therapeutic effect in animal models of immune system disorders (
To develop smaller peptide ligand-leads to the ß2 integrins, we have screened random peptide libraries displayed on filamentous phage. The phage display technique has previously yielded selective peptide ligands to the integrin species 5ß1 (
Vß3/ß5 (
Vß6 (
4ß1 and
4ß7 are known to have a specificity for peptides containing another type of tripeptide sequence, leucineaspartatevaline (
Mß2 integrin also shares the ability to recognize a motif comprising three amino acids, thus showing a functional similarity to other integrins. The tripeptide favored by
Mß2 turned out to be a previously unknown adhesion motif, leucineleucineglycine (LLG). Interestingly, such sequences are present on several adhesion proteins, such as ICAM-1 and von Willebrand factor. We developed a nonapeptide ligand LLG-C4, which has a compact disulfide-restrained structure as determined by nuclear magnetic resonance (NMR). This biscyclic peptide is a potent inhibitor of leukocyte cell adhesion and migration, and is a novel lead compound for development of antiinflammatory agents.
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Materials and Methods |
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Monoclonal Antibodies
Antibodies against the integrin ß2 subunit were 7E4, 11D3, 3F9, 2E7, 1D10, and 2F3 (L subunit antibodies were TS2/4 and MEM-83 (Monosan). The antibodies OKM1, OKM10, and MEM-170 were used against the anti-
M subunit, and the antibody 3.9 was used against the
X subunit (
IIbß3 integrin antibody P2 was purchased from Immunotech, and the
Vß3 integrin antibody LM609 and the ß1 subunit antibody 6S6 were from Chemicon.
Peptide Synthesis
Peptide synthesis was carried out using Fmoc chemistry (model 433A; Applied Biosystems). Disulfides were formed by oxidation in 10 mM ammonium bicarbonate buffer, pH 9, overnight. Peptides were then purified by HPLC on an acetonitrile gradient. Generation of disulfides was confirmed by mass spectrometry analysis. The C(1-8;3-9) and C(1-9;3-8) peptides with the guided disulfide bridges were custom-made by Anaspec. The ACDCRGDCFCG (RGD-4C) peptide (
Phage Display
The Mß2 integrin was purified by antibody affinity chromatography from buffy coats obtained from the Finnish Red Cross blood transfusion service (
Preparation of Glutathione S-transferase and Fc Fusion Proteins
The nucleotide sequence coding for LLG-C4 was PCR amplified from phage DNA with the primers containing a BamHI 5'-AGGCTCGAGGATCCTCGGCCGACGGGGCT-3' and an EcoRI site 5'-AGGTCTAGAATTCGCCCCAGCGGCCCC-3'. The PCR product was purified on an agarose gel, digested with the two restriction enzymes, and ligated into the PGEX-2TK vector (Amersham Pharmacia Biotech). Recombinants expressing LLG-C4-Glutathione S-transferase (GST) were verified by DNA sequencing. LLG-C4-GST was produced in E. coli strain BL 21 and purified by glutathione affinity chromatography followed by dialysis. ICAM-1-Fc fusion protein containing the five ICAM-1 Ig domains was produced in CHO cells and purified by protein A affinity chromatography (M I domain was expressed as a GST fusion protein in E. coli and purified by affinity chromatography on glutathione-coupled beads followed by cleavage with thrombin to release the recombinant I domain (
Integrin Binding Assays
Integrins were immunocaptured on microtiter wells that were coated with nonspecific IgG or the subunit antibodies OKM1, MEM170, TS2/4, 2E7, or 7E4. A 200-µl aliquot of the buffy coat lysate in 1% octylglucoside/1 mM MnCl2/TBS was allowed to incubate for 2 h at 4°C. The wells were then washed five times with the octylglucoside-containing buffer. LLG-C4-GST or GST (10 µg/ml) was incubated in the integrin-coated or the M I domaincoated wells in 25 mM octylglucoside/TBS/1 mM MnCl2 for 1 h. After washing of the wells, the bound GST was determined with anti-GST antibodies (Amersham Pharmacia Biotech), which were labeled with an Eu3+ chelate according to the instructions of the manufacturer (Wallac). The Eu3+ fluorescence was measured with a fluorometer (1230 Arcus; Wallac).
Cell Culture
The leukocytic cell lines THP-1, Jurkat, U-937, and K562 were maintained as described (Xß2 integrintransfected L cell line were obtained from Dr. Y. van Kooyk (University Hospital, Nijmegen, Netherlands).
Cell Adhesion
Fibrinogen (Calbiochem), fibronectin (Boehringer), von Willebrand factor (Calbiochem), GST fusion proteins, Fc fusion proteins, or synthetic peptides were coated on microtiter wells at a concentration of 2 µg in 50 µl TBS unless otherwise indicated. The wild-type and A2 domaindeleted recombinant von Willebrand factors ( (10 ng/ml; Roche). T cells (1.5 x 105 per well) were allowed to bind to Eahy926 cells for 30 min at 4°C, and then 15 min at 37°C. The unbound T cells were removed by immersing the microtiter plate upside down in PBS. The bound cells were determined by the phosphatase assay.
Cell Migration
Cell migration was studied using 8-µm pore size Transwell filters (Costar). Both the upper and lower filter surfaces were coated with fibrinogen, LLG-C4-GST, or GST at a concentration of 40 µg/ml. Free binding sites were blocked with 5% BSA. THP-1 cells (5 x 104 in 100 µl) were plated on the upper compartment in 10% serum-containing medium in the absence or presence of C(1-8;3-9) or C(1-9;3-8) (200 µM). The lower compartment was filled with 750 µl of the same medium. After a culture for 18 h at 37°C, the filters were immersed in methanol for 15 min, in water for 10 s, and in 0.1% toluidine blue (Sigma-Aldrich) for 5 min. The filters were then washed three to five times with water until cell staining was clear. Cells were removed from the upper surface of the filter with a cotton swab, and cells migrated on the lower surface were counted microscopically. A Student's t test was used for statistical analysis.
NMR Analysis of Peptides
For NMR structure determination, the C(1-8;3-9) peptide was dissolved in DMSO/H2O (90/10) and C(1-9;3-8) in H2O at the concentrations of 13 mM. Two-dimensional spectra, acquired with spectrometers operating at 600- and 800-MHz 1H frequency, allowed us to identify 114 nuclear Overhauser enhancements (nOes) for C(1-8;3-9) and 85 for C(1-9;3-8) peptide. 40 structures with no restraint violations above 0.2 Å were selected from families of 200 structures generated by simulated annealing (DYANA program;
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Results |
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The LLG Peptide Motif Binds to a ß2 Integrin
We used the CX7C and CX9C phage libraries to search for peptide ligands to purified Mß2 integrin. After the fifth round of selection, the CX7C library gave a 600-fold enrichment and CX9C a 1,000-fold enrichment of phage bound to the integrin in comparison to background. Sequencing of the bound phage revealed altogether only seven different sequences, indicating selection of specific peptides by the integrin (Table 1). Four of them contained the LLG tripeptide motif. The two sequences most strongly enriched were CPCFLLGCC (LLG-C4) and CWKLLGSEEEC, and these were the only clones remaining after searching for high affinity binders by using low integrin-coating concentrations. Screening protein databases indicated that the LLG tripeptide sequence is present on several adhesion proteins. Most interestingly, it is located on the first Ig domain of ICAM-1, just preceding the Glu-34 residue, which is critical for ICAM-1 binding to the
Lß2 integrin (
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We focused our studies on the LLG-C4 nonapeptide because it showed higher affinity to Mß2 in phage-binding experiments in comparison to the other clones (data not shown). Due to the presence of four cysteine residues, the peptide appeared to be structurally constrained by two disulfide bonds. We first examined whether an integrin-binding peptide could be obtained by bacterial expression of LLG-C4 tethered to GST. The LLG-C4-GST fusion protein, but not GST alone, had a potent activity and bound to the
Mß2 integrin in a divalent cationsensitive manner like a typical integrin ligand. The cation chelator EDTA inhibited the binding of LLG-C4-GST to the integrin, which was immunocaptured on microtiter wells with the
M subunit antibodies MEM170 or OKM1 (Fig 1 A). Similar EDTA-inhibitable binding of LLG-C4-GST was detected with the
Lß2 integrin, which was captured with the TS2/4 antibody. Surprisingly, EDTA only partially inhibited LLG-C4-GST binding when the ß2 subunit antibody 2E7 was used. We have found this antibody to stimulate leukocyte adhesion to various matrix proteins. LLG-C4-GST binding did not differ from GST control and was not inhibitable by EDTA, when a nonspecific IgG was used for immunocapture (not shown).
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We next studied whether the peptide can directly interact with the I domain of the Mß2 integrin, the known ligand binding site. LLG-C4-GST, examined at the concentrations of 0.01100 µg/ml, showed a concentration-dependent binding to the isolated I domain of the
M subunit (Fig 1 B). GST at the same concentrations did not bind. The ability of the I domain to bind LLG-C4-GST was dependent on the Mn2+ cations added to the binding medium, and chelating Mn2+ with EDTA blocked the binding (Fig 1 C). Initially, we encountered difficulties in chemical synthesis of an active and water-soluble LLG-C4 peptide, apparently because mixed disulfides easily formed during air oxidation. One LLG-C4 (1) preparation was highly active and blocked the ability of the I domain to bind the LLG-C4-GST (Fig 1 C). The same peptide was also active in cell culture experiments. Another preparation, LLG-C4 (2), was inactive apparently due to disadvantageous disulfide bonding and did not inhibit LLG-C4-GST binding to the I domain.
Immobilized LLG-C4 Nonapeptide Selectively Supports ß2 Integrinmediated Cell Adhesion
We examined the integrin-binding specificity of LLG-C4 in cell adhesion assays. Phorbol esteractivated THP-1 monocytic cells efficiently bound to LLG-C4-GST, but not to GST or peptide-GST controls (CLRSGRGC-GST, CPPWWSQC-GST) coated on microtiter wells (Fig 2 A). EDTA at a concentration of 2.5 mM abolished the binding. Screening with a panel of antiintegrin antibodies indicated that the cell adhesion on LLG-C4-GST was completely inhibited by the blocking antibody to the ß2 chain, 7E4 (Fig 2 B). Antibodies to the ß1 (6S6) and ß3 integrins (LM609, P2) had no effect. Partial inhibition was obtained with the ß2 chain antibodies 11D3 and 3F9. The order of the potency of the three ß2 antibodies is the same as that obtained previously in other assays (
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Studies with antibodies against the integrin subunits showed that the
X subunit antibody 3.9 effectively inhibited the THP-1 adhesion to LLG-C4-GST. The
M subunit antibodies OKM10, MEM170, and 60.1 were weakly inhibitory, whereas the
L-directed antibodies TS1/22 and TS2/4 had hardly any effect. Furthermore, we found that the
X antibody 3.9 and the
M antibody OKM10 had a synergistic effect when added together, causing a complete inhibition of the cell adhesion.
THP-1 cells similarly bound strongly to the synthetic air-oxidized LLG-C4 nonapeptide coated on plastic, and the antibodies against the Mß2 and
Xß2 integrins (3.9, OKM10, and 7E4) prevented the binding (data not shown). To determine the arrangement of the disulfide bonds in the active form of LLG-C4, we prepared synthetic peptides with different disulfide configurations. The most active peptide, C(1-8;3-9), was obtained by directing one disulfide bond between the C1 and C8 cysteines and a second one between the C3 and C9 cysteines. Cells bound to the C(1-8;3-9) disulfidecontaining peptide but failed to bind to the conformer with C(1-9;3-8) disulfides (Fig 2 C). Cross-linking of the C(1-8;3-9) peptide with glutaraldehyde further enhanced cell binding, apparently due to better coating of the multimeric peptide. C(1-9;3-8) was inactive even after the cross-linking. In general, the C(1-8;3-9) peptide specifically supported the binding of ß2 integrinexpressing cells lines such ß2 integrintransfected L cells and the leukocytic cell lines THP-1, U-937, and Jurkat. The binding of
Xß2-transfected L cells to LLG-C4-GST was inhibited by EDTA and the ß2 integrinblocking antibody 7E4 (Fig 2 D). Nonleukocytic cell lines L929, K562, SKOV-3, KS6717, and Eahy96, which do not express ß2 integrins, showed no binding to the peptide or LLG-C4-GST, whether the cells were pretreated with phorbol ester or not (data not shown).
LLG-C4 Nonapeptide Specifically Blocks ß2 Integrinmediated Adhesion of Leukocytes
We examined the ability of LLG-containing peptides to block leukocyte binding to adhesion proteins containing or lacking an LLG tripeptide sequence. THP-1 cell adhesion on LLG-C4-GST was inhibited by the C(1-8;3-9) peptide with an IC50 of 20 µM (Fig 3 A). The other conformer, C(1-9;3-8), was 20-fold less active than C(1-8;3-9). To study whether the LLG tripeptide sequence is sufficient for recognition by the ß2 integrins, we prepared the minimal cyclic CLLGC peptide. In a control peptide the leucines were replaced by alanines. THP-1 cell adhesion experiments using the LLG-C4-GST substratum indicated that CAAGC was only a weak competitor of cell adhesion, whereas CLLGC readily inhibited cell adhesion at concentrations of 1 mM, indicating a specific recognition of the LLG motif by the ß2 integrins (Fig 3 B).
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We next examined the ability of LLG-containing peptides to inhibit the Lß2 integrinmediated binding of Jurkat cells to ICAM-1-Fc recombinant protein, which contains the LLG sequence of the first Ig domain. ICAM-1-Fc was directly coated on microtiter wells or captured via protein A. In both cases we found concentration-dependent inhibition by C(1-8;3-9) on Jurkat cell adhesion and the IC50 was
80 µM (Fig 4 A). The C(1-9;3-8) conformer was severalfold less active and had hardly any effect. C(1-8;3-9) similarly inhibited the binding of freshly isolated T cells to cultured endothelial cells which were stimulated to express ICAM-1 by treatment with TNF-
(Fig 4 B). T cells did not bind to unstimulated endothelial cells. As a control, the RGD-C4 peptide had no effect on T cell binding to endothelial ICAM-1.
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As von Willebrand factor contains LLG peptide motifs, we were interested in the capability of the protein to function as a substratum for leukocytes. We found that phorbol esteractivated THP-1 cells strongly bound. The ß2 integrin antibody 7E4 blocked the THP-1 cell binding to von Willebrand factor (Fig 5 A) and was nearly as efficient an inhibitor as the cation chelator EDTA (data not shown). The ß3 integrin antibodies LM609 and P2 were without effect. C(1-8;3-9) was a potent inhibitor of THP-1 cell binding to von Willebrand factor. The peptide inhibited with an IC50 of 20 µM (Fig 5 B). In addition, CLLGC but not CAAGC inhibited at a 500 µM concentration (data not shown). Similar C(1-8;3-9) peptidemediated inhibition was observed on Jurkat cell binding to von Willebrand factor (not shown). Importantly, THP-1 showed weaker binding (35% of wild-type) to a mutated von Willebrand factor, from which the A2 domain, including the LLG sequence, was deleted (Fig 5 C). Furthermore, THP-1 adhesion to the A2-deleted von Willebrand factor was not blocked by C(1-8;3-9), but by the RGD-4C peptide. To further study the specificity of the LLG peptides, we examined THP-1 adhesion to fibronectin, a known ligand of several ß1 and ß3 integrins. C(1-8;3-9) showed no significant inhibition of fibronectin binding by THP-1 cells. C(1-8;3-9) also had no effect on binding of nonleukocytic cell lines such as HT1080 on fibronectin or fibrinogen (data not shown).
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Finally, we examined THP-1 adhesion to fibrinogen, which is predominantly mediated via the Mß2 and
Xß2 integrins (
Mß2 and
Xß2 integrins (data not shown). As RGD-directed integrins can also mediate cell attachment on fibrinogen, we compared C(1-8;3-9) to the RGD-4C peptide, the selective ligand of
Vß3/ß5 integrins. We prestimulated THP-1 cells with low concentrations of C(1-8;3-9) and RGD-4C to fully activate both the ß2 and RGD-dependent integrins. After the peptide prestimulation, RGD-4C inhibited THP-1 cell adhesion on fibrinogen more effectively than C(1-8;3-9) (Fig 6 B). To study whether C(1-8;3-9) and RGD-4C target different integrins, the peptides were given together to cells. The effects of C(1-8;3-9) and RGD-4C were additive and the peptide combination blocked cell adhesion efficiently.
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As a model of monocyte rolling and extravasation, we examined in vitro migration of THP-1 cells on fibrinogen immobilized on Transwell filters. Cells effectively migrated in the presence of 10% serum. C(1-8;3-9) at a concentration of 200 µM completely abolished the ability of the cells to traverse the filter and bind to its lower surface (Fig 6 C; P = 0.005, n = 6). The C(1-9;3-8) conformer was less active than C(1-8;3-9) and inhibited only partially (P = 0.01, n = 6). The activity difference between C(1-8;3-9) and C(1-9;3-8) was significant (P = 0.003). In a reverse strategy, when the filter was coated with LLG-C4, cell migration was strongly enhanced. Approximately 10-fold more cells migrated on the LLG-C4-GST substratum than on control GST substratum (Fig 6 D). Cell migration on LLG-C4-GST was also more efficient when compared with fibronectin and fibrinogen coatings. C(1-8;3-9) at the 200 µM concentration completely suppressed the cell migration on LLG-C4-GST (P = 0.0026, n = 6; data not shown).
NMR Structures of Nonapeptide Conformers
We analyzed the C(1-8;3-9) and C(1-9;3-8) peptides by NMR spectroscopy to determine whether there are differences in peptide conformations due to the directed arrangement of the disulfide bonds. The structure determinations resulted in well-defined backbone conformations. The root mean square of deviation of the main chain atoms was 0.4 ± 0.2 Å for C(1-8;3-9) and 0.3 ± 0.2 Å for C(1-9;3-8) calculated from ensembles of 40 structures. For both peptides, all main chain dihedrals and
are in the favorable and allowed regions of Ramachandran plot. There are only a few nOes to define the side chain orientation, and therefore the side chain dihedrals of F4, L5, and L6, in particular, are dispersed (Fig 7 A).
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The pairing of the disulfides in the two ways influenced the structure of the nonapeptide considerably. The "crossing arrangement of disulfides" of C(1-8;3-9) constrains the overall structure tighter than the "parallel arrangement of disulfides" of C(1-9;3-8). This is reflected by the larger number of nOes observed for C(1-8;3-9) (114) than for C(1-9;3-8) (85). There is no bias towards shorter distance restraints in C(1-8;3-9) compared with those of C(1-9;3-8). As a result of the different disulfide configurations, there are interresidue nOes found exclusively in one of the structures, 37 in C(1-8;3-9) and 20 in C(1-9;3-8). The crossing arrangement of disulfides in C(1-8;3-9) is topologically more complicated than the parallel bridging in C(1-9;3-8). In the short nonapeptide the adjacent disulfides with large van der Waals radii of sulphur atoms give rise to numerous steric restraints. The residue P2 also limits conformational freedom, whereas G7 contributes to it. The impact of mere topology on the steric restraints is apparent from the representative structures (Fig 7 B). C(1-8;3-9) is more compact than C(1-9;3-8). Furthermore, there is a continuous hydrophobic surface patch composed of aliphatic groups of P2, F4, and L5 in the C(1-8;3-9) peptide. Overall, the disulfide bridges and the F4-L6 strand are buckled in C(1-8;3-9), whereas in C(1-9;3-8) they are extended. This likely accounts for the poorer water solubility of C(1-8;3-9) and may contribute to its higher activity.
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Discussion |
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We have developed highly specific peptide antagonists of the leukocyte ß2 integrins using phage display. The most active antagonist, LLG-C4, is a biscyclic nonapeptide that is structurally restrained by two disulfide bonds and contains a novel LLG tripeptide adhesion motif. The LLG-C4 peptide specifically blocked the ß2 integrinmediated leukocyte adhesion and inhibited leukocyte binding to their major ligand ICAM-1. Furthermore, like a typical integrin ligand, the peptide supported cell adhesion when immobilized on plastic and bound leukocytic cell lines, but not cells lacking ß2 integrins. The effectiveness and leukocyte specificity of the peptide are explained by its ability to interact with the I domain, which is a known active site in the leukocyte integrins. Interestingly, not only ICAM-1 but also several other adhesion proteins, including von Willebrand factor, contain the consensus PP/XXLLG sequence identified by phage display.
The activity of the LLG-C4 nonapeptide was strictly dependent on the correct formation of two disulfide bridges. There was a 20-fold difference in the activities of two biscyclic conformers that differed only in the configuration of the disulfide bridges. The more active peptide had a very compact structure due to a "crossing" arrangement of the disulfide bonds as shown by NMR. Interestingly, the leucine side chains protrude from the cyclic structure like antennae, suggesting that they can directly interact with the integrin. The small glycine residue may adjust a correct distance between the leucine side chains. The biscyclic RGD-4C peptide can also exist in two different isomers, depending on internal disulfide bonding, and the two structures have clearly different integrin-binding activities (
LLG-C4-GST is a highly efficient adhesion substratum for phorbol esteractivated THP-1 leukemia cells. We also detected cell binding to the immobilized nonapeptide, but the overall binding was weaker, apparently because the short peptide coats less efficiently on microtiter plates. We were not able to detect a similar strong binding of the Xß2 integrintransfected L cells to LLG-C4-GST as with THP-1. This is likely due to the fact that the integrin expression was limited only to a subset of L cells as determined by FACS® analysis.
Immunocapture experiments with different ß2 integrin antibodies showed that LLG-C4 is able to bind to each of the three integrin species, Lß2,
Mß2, and
Xß2. EDTA inhibition showed that the binding of LLG-C4 to the integrins as well as to purified I domain is cation dependent. However, one of the antibodies used for integrin immunocapture gave an exceptional result in that EDTA could not completely inhibit the binding of LLG-C4-GST fusion protein. This antibody, 2E7, which recognizes the common ß2 subunit, shows an integrin-activating effect in cell culture and stimulates leukocyte cell adhesion to LLG-C4-GST and various matrix proteins. Thus, it is possible that this antibody changes the conformation of integrin, resulting in stronger binding. The antibody may expose secondary binding sites for ligands in integrins, and GST protein itself may then contribute to the cation-independent binding.
Previous studies have indicated that synthetic peptides spanning the LLG region of ICAM-1 (
von Willebrand factor contains two LLG sequences, but an ability of these sequences to interact with integrins has not been reported. von Willebrand factor is a multifunctional adhesive ligand binding several proteins, and it prevents bleeding during vascular injury by mediating platelet adhesion to exposed subendothelium (IIbß3 (
subunits of the ß2 integrins (
As the ß2 integrins exist in an inactive state and become activated only after physiologic stimuli, such as by chemokines or through contact with antigen-presenting cells, it would be desirable to develop compounds binding preferentially to cells bearing the activated integrins. We found that LLG-C4 exhibits such properties and reacts with cells after integrin activation. Furthermore, LLG-C4 is a promising ß2 integrintargeting agent, as the sequence can specifically direct phage binding to ß2 integrinexpressing cell lines, and low concentrations of the soluble peptide inhibit the binding (Koivunen, E., R. Pasqualini, and W. Arap, manuscript in preparation). Finally, the presence of LLG sequences in von Willebrand factor suggests a novel function for the protein in mediating not only platelet but also leukocyte adhesion.
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Footnotes |
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1 Abbreviations used in this paper: GST, glutathione S-transferase; ICAM, intercellular adhesion molecule; LLG, leucineleucineglycine; NMR, nuclear magnetic resonance; nOe, nuclear Overhauser enhancement; RGD, arginineglycineaspartic acid; TNF, tumor necrosis factor.
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Acknowledgements |
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We thank Minna Ekström, Marja Pietilä, Sanna Pesonen, and Kari Kaitila for technical assistance.
This work was supported by the Academy of Finland, the Finnish Cancer Society, the Sigrid Juselius Foundation, the Finnish Cultural Fund, and the Technology Development Centre of Finland. G. van Willigen was supported by the Netherlands Organization for Scientific Research (grant R91-266), the Catharijne Foundation, and the Dirk-Zwager Assink Foundation.
Submitted: 1 September 2000
Revised: 26 March 2001
Accepted: 11 April 2001
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References |
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