J Biol Chem, Vol. 274, Issue 53, 37809-37814, December 31, 1999

Structural Requirements of Echistatin for the Recognition of _v3 and ₅₁ Integrins^*

Iwona Wierzbicka-Patynowski^§¶, Stefan Niewiarowski^§, Cezary Marcinkiewicz^§, Juan J. Calvete^**, Mariola M. Marcinkiewicz^§, and Mary Ann McLane^¶

From the  Physiology Department, Temple University and ^§ Sol Sherry Thrombosis Research Center, Philadelphia, Pennsylvania 19140, the ^¶ Medical Technology Department, University of Delaware, Newark, Delaware 19716, and the ^** Instituto de Biomedicina, Consejo Superior de Investigaciones Científicas, Valencia 46010, Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of _IIb3, _v3 and ₅₁, and eristostatin, a similar disintegrin selectively inhibiting _IIb3. In order to identify echistatin motifs required for selective recognition of _v3 and ₅₁ integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp²⁷ and Met²⁸ support recognition of both _v3 and ₅₁. Replacement of Met²⁸ with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met²⁸ with Leu selectively decreased echistatin's ability to recognize ₅₁ only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with ₅₁, which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on ₁ and ₃ integrin molecules.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The integrins are a family of cell surface glycoproteins that act as receptors for extracellular matrix (ECM)¹ proteins, or for membrane-bound counter-receptors on other cells. Each integrin is a heterodimer that contains an  and a  subunit, the pairing of which specifies the ligand binding abilities of integrins (1). Integrins can bind adhesive ligands and upon this binding undergo conformational changes leading to the exposure of neoepitopes recognized by specific monoclonal antibodies called anti-ligand-induced binding site (LIBS) antibodies (2).

The Arg-Gly-Asp (RGD) sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins, and nearly half of the over 20 known integrins recognize this sequence in their adhesion protein ligands (3). In addition to the RGD motif, many adhesive receptors recognize other integrin-binding domains, such as the KQAGDV sequence (4) of fibrinogen -chain, ILDV sequence of the CS1 region of fibronectin (5, 6), and RRETAWA sequence identified from a phage display library (7). It is known that the Arg and Asp residues are necessary but not sufficient to ensure binding activity. The RGD sequence is generally found to be associated with a series of probable -bends, which result in a highly ordered structure. This highly ordered conformation might form the basis of the specific binding of many proteins containing this cell surface recognition sequence. Additional determinants of integrin specificity and the high affinity of RGD-containing adhesive proteins for integrins may be the result of the specific conformation of the RGD sequence or by the amino acids immediately adjacent to the RGD site, creating an extended RGD locus.

Disintegrins are snake venom proteins capable of binding to integrins and interfering with integrin function (8-10). Disintegrins typically have an RGD sequence as their active site, except for barbourin containing a KGD sequence (11), and a new class of heterodimeric disintegrins such as EC3 and EMF10, containing MLD, VGD, and other recognition motifs (12). It appears that low molecular weight disintegrins, binding to integrins with an affinity approaching that of monoclonal antibodies, may represent a useful probe to identify functionally important motifs in the cell adhesion receptors. Scarborough et al. (13) postulated that the amino acid residue C-terminal to the RGD sequence determines selectivity of disintegrin for an integrin receptor. Pfaff et al. (14), however, demonstrated that disintegrins that have identical amino acid residues C-terminal to the RGD sequence do not necessarily show the same integrin's selectivity. They proposed that other regions of the disintegrins and the alignment of disulfide bridges are more likely to contribute to disintegrins' affinity and selectivity.

The purpose of this study was to identify structural motifs of echistatin responsible for its biological activity. We selected echistatin because this short disintegrin, occurring in Echis carinatus venom, has been extensively characterized by NMR spectroscopy (15-18). Moreover, eristostatin, another short disintegrin from Eristocophis macmahoni venom (8), shares a high degree of amino acid sequence similarity with echistatin, however both disintegrins are functionally different. Eristostatin binds with much higher affinity to _IIb3 integrin on platelet surface and is a much stronger inhibitor of ADP-induced platelet aggregation (14, 19). Eristostatin interactions with _v3 and ₅₁ expressed on cell surfaces are minimal to none, whereas echistatin strongly inhibits ligand binding to all three integrins. Marcinkiewicz et al. (20), using synthetic echistatin and its analogs, demonstrated that the C terminus of this disintegrin supports its binding to _v3 and _IIb3, and is critical for the expression of LIBS epitope on ₃ integrins. They proposed that the shape of the RGD loop determines the selectivity of disintegrins for integrin receptor while the C terminus is involved in the induction of conformational changes of integrins.

In order to identify echistatin motifs required for selective recognition of _v3 and ₅₁ integrins, we expressed recombinant echistatin and eristostatin and a number of hybrid molecules. Based on previous studies (20-22), the introduced alterations were limited to the RGD loop and the C termini of the molecules. The major conclusions of our study are that echistatin uses two motifs, the RGD loop and the C terminus, to interact presumably with two different sites on ₅₁ integrin. In addition, conservative hydrophobic replacement of Met²⁸ with Leu did not significantly change the interaction of echistatin with _v3, but it was critical for the interaction of this disintegrin with ₅₁.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Lyophilized crude viper venoms were obtained from Latoxan (Valence, France) or Sigma. ADP, -thrombin, human fibronectin, and standard laboratory chemicals were obtained from Sigma. Pfu polymerase was obtained from Stratagene (La Jolla, CA). Hanks' balanced salt solution was acquired from Life Technologies, Inc. BL21(DE3) competent cells were from Novagen (Madison, WI). Restriction endonucleases, T4 DNA ligase, goat anti-rabbit Ig conjugated to alkaline phosphatase, trifluoroacetic acid, and acetonitrile were purchased from Fisher Scientific (King of Prussia, PA). The C-18 reverse phase columns were from Vydac (Hesperia, CA). Glutathione-Sepharose and 5-chloromethylfluorescein diacetate (CMFDA) were obtained from Amersham Pharmacia Biotech and Molecular Probes (Eugene, OR), respectively. The monoclonal antibody Ab62, which recognizes a LIBS2 epitope (23) in the C-terminal region of the extracellular domain of the ₃ subunit, was the gift of Dr. M. Ginsberg (Scripps Research Institute, La Jolla, CA). The monoclonal antibody 9EG7 recognizing LIBS epitope on the ₁ subunit was purchased from PharMingen (San Diego, CA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG was acquired from Jackson Immunoresearch (West Grove, PA). Polyclonal antibodies against native echistatin and eristostatin were raised in rabbits. Human von Willebrand factor (vWF) was the gift of Dr. M. Peng (Temple University, Philadelphia, PA). Synthetic echistatin D27W was the gift of Dr. V. Garsky (Merck Sharp and Dohme Research Laboratories, West Point, PA). All oligonucleotide primers were synthesized by Genosys Biotechnologies, Inc. (The Woodlands, TX). The C-terminal peptide PRNPHKGPAT was synthesized and purified by Genosys Biotechnologies, Inc.

Preparation of Purified Native Disintegrins-- Echistatin and eristostatin were prepared from crude venom of E. carinatus and E. macmahoni, respectively, by reversed-phase high pressure liquid chromatography using a Vydac C-18 column and an acetonitrile gradient as described previously (19).

Cloning and Expression of Recombinant Echistatin, Eristostatin, and Their Mutants-- A synthetic gene of echistatin was kindly provided by Dr. Marlene Jacobson (Merck Research Laboratories). The echistatin M28L gene was ligated as a BamHI insert into the vector pGEX-KG (24). The pGEX-KG vector placed a thrombin cleavage site between the sequence for the glutathione S-transferase gene and the BamHI restriction site used for insertion of the desired echistatin gene, which added four N-terminal amino acids (Gly, Ser, Thr, Met) to the final expressed sequence of echistatin and all echistatin clones. The gene for eristostatin was designed using optimal codons for E. coli expression (25), and synthesized by Genosys Biotechnologies, Inc. The resulting gene was ligated as a BamHI/EcoRI insert (to permit directional cloning) into the vector pGEX-KG. In contrast to echistatin's DNA construct, the placement of the BamHI site at the 5' end of the DNA insert for eristostatin added two N-terminal amino acids (Gly, Ser) to the final expressed protein. Recombinant echistatin and eristostatin clones were sequence-verified (DNA Sequencing Facility, University of Pennsylvania, Philadelphia, PA), and transformed into E. coli strain BL21(DE3), noted for its lack of cytoplasmic proteases. The glutathione S-transferase fusion proteins were induced and isolated as described (26).

Mutations within the RGD loop and the C termini of echistatin or eristostatin were performed by a polymerase chain reaction megaprimer mutagenesis method (Fig. 1 and Table I) (27). Purity of all recombinant proteins was assessed and molecular weight confirmed in mass spectrometric analysis (Wistar Institute, Philadelphia, PA). Moreover, the primary structure of eristostatin mutants Er (1-46)-HKGPAT and ErW30D/N31M (1-46)-HKGPAT was confirmed by amino acid sequencing (Edman degradation).

Cell Culture-- VNRC3 cells, Chinese hamster ovary (CHO) cells expressing human _v3 were kindly provided by Dr. M. Ginsberg (Scripps Research Institute). Nontransfected CHO-K1 cells, K562 erythroleukemic cell line, and ECV304 cell line were purchased from ATCC (Rockville, MD). CHO-K1 cells showed insignificant adhesion to vWF and fibronectin. K562 cells express predominantly ₅₁ receptor and do not express _IIb3 and _v3. ECV304 were considered previously to be an immortalized clone of human endothelial cells and used as such in the study on the disruption of angiogenesis (28). Using a number of monoclonal antibodies (SAM1 for ₅₁, LM609 for _v3, 7E3 for ₃, Lia1/2 for ₁, HP2/1 for ₄₁), we determined that ECV304 and human umbilical endothelial cells express the same pattern of integrins. According to the newest information from ATCC, chromosomal analysis of ECV304 suggests that they may be derived from human bladder cancer cells. CHO cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS), nonessential amino acids, glutamine, penicillin, and streptomycin. K562 cells were cultured in RPMI 1640 medium containing 10% FCS, glutamine, penicillin, and streptomycin. ECV304 cells were cultured in 199 medium containing 10% FCS, glutamine, penicillin, and streptomycin.

Preparation of Human Platelet Suspensions-- Aspirin-free blood was collected from healthy donors in 3.8% (w/v) sodium citrate (1:9 ratio), centrifuged at 1300 × g for 10 min, with the platelet-rich plasma separated from the cells within 30 min of collection for platelet aggregation studies.

Platelet Aggregation-- The concentration of each recombinant or native disintegrin that caused inhibition of platelet aggregation induced by 20 µM ADP was determined as described previously (29).

Adhesion Studies-- Each well of the Costar microplates was coated with human vWF (0.5 µg) or fibronectin (1 µg) in PBS buffer, pH 7.5, and incubated overnight at 4 °C. The plate was blocked with 1% (w/v) bovine serum albumin in Hanks' balanced salt solution and stored at 4 °C until needed. K562 or ECV304 cells were incubated with 12.5 µM CMFDA, and then free CMFDA was washed out. CMFDA-labeled K562 or ECV304 cells (1 × 10⁵/well) were incubated in the presence or absence of disintegrin inhibitor with appropriate substrata immobilized in the microtiter plate wells. After washing to remove unbound cells, the bound cells were lysed and their fluorescence read in a microplate reader (Cytofluor 2350, Millipore, Bedford, MA). Alternatively, we followed the same procedure with unlabeled VNRC3 cells (1 × 10⁵/well). After washing, the bound cells were fixed with 1% (w/v) formalin and stained with methylene blue. Cells were then solubilized with 50% (v/v) ethanol/HCl, and the resulting absorbance was read at 630 nm. The readings correlated with the number of adhering cells.

Expression of LIBS Epitope-- Expression of LIBS epitope on ₃ subunit was measured by means of flow cytometry. VNRC3 cultured cells (7 × 10⁵/well) were incubated in the presence or absence of recombinant disintegrins followed by the addition of mAb62 (23), an anti-LIBS antibody recognizing neoepitopes on ₃ subunit (1 µg/sample). The binding of this antibody was measured in flow cytometry using FITC-conjugated goat anti-mouse IgG (1 µg/sample). After each incubation step, the cells were washed with Hanks' balanced salt solution containing 1% (w/v) bovine serum albumin. Cells were fixed with 1% (w/v) formalin, and analysis was done with a Coulter Epics Elite flow cytometer (Miami, FL). Light scatter and fluorescence signals were analyzed for 10,000 cells per sample. Results were expressed as mean cell fluorescent intensity in arbitrary units.

LIBS epitope expression on ₁ subunit was assayed by means of an adhesion assay. Anti-LIBS antibody 9EG7 (30), recognizing neoepitopes on ₁ subunit, is sensitive to Ca²⁺ concentrations that interfere more in flow cytometry than in this adhesion assay. CMFDA-labeled K562 cells (1 × 10⁵ cells/well) were incubated in the presence or absence of recombinant disintegrins and then allowed to adhere to anti-LIBS antibody 9EG7 immobilized on the Costar microplate (1 µg/well). Adherent cells were lysed with 1% Triton X-100, and the fluorescence was measured as described above for the adhesion studies. K562 cells did not adhere to immobilized 9EG7 antibody in the absence of disintegrins.

For each experiment, the expression of LIBS in the presence of native echistatin was accepted as 100%. Nonspecific fluorescence was evaluated by measuring the binding of FITC-conjugated goat anti-mouse IgG in the presence of a primary monoclonal antibody in the absence of disintegrin and was subtracted from each test result.

Statistical Analysis-- Data were analyzed for statistical significance using Student's t test (two-tailed).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of Recombinant Mutants of Echistatin and Eristostatin-- There are significant differences between echistatin's and eristostatin's RGD loop and their C termini (Fig. 1). Echistatin and eristostatin differ by four amino acid residues within the 13-amino acid residue RGD loop. At the C termini, echistatin contains 10 and eristostatin 7 amino acid residues. The PRNP sequence is common for both disintegrins while the remaining residues are completely different. To hybridize both disintegrins, eight different mutants of echistatin and seven of eristostatin were made. Native and recombinant echistatin and eristostatin wild types were compared for their inhibitory effect on ADP-induced platelet aggregation. There was no significant difference between the IC₅₀ values of native and recombinant echistatin (136 ± 29 nM versus 124 ± 18 nM) and native and recombinant eristostatin (59 ± 22 nM versus 58 ± 15 nM). Fig. 2 demonstrates that the recombinant wild type echistatin and eristostatin show similar effect to the native proteins in inhibition of adhesion and expression of LIBS epitopes in cells expressing _v3 and ₅₁ receptors. It can be concluded that the additional amino acid residues at the N termini of recombinant echistatin and eristostatin did not affect their biological activity.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   The amino acid sequence of echistatin and eristostatin. The differences between the amino acid residues of the RGD loops and C termini of echistatin and eristostatin are underlined.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   The effect of disintegrins on cell adhesion and expression of LIBS epitope. The effect of native (circle) and recombinant (triangle) echistatin or native (square) and recombinant (diamond) eristostatin on the adhesion of VNRC3 cells to immobilized vWF (panel A) or K562 cells to immobilized fibronectin (panel B) was checked by the absorbance or fluorescence measurement of methylene blue or FITC-stained cells, respectively. The effect of disintegrins on the expression of LIBS epitope on 3 (panel C) or 1 (panel D) integrins was checked using anti-LIBS Mab62 (anti-3) or 9EG7 (anti-1) monoclonal antibodies. The results represent mean ± S.E. from at least five independent experiments. For each experiment, the expression of LIBS in the presence of native echistatin was accepted as 100%. Nonspecific fluorescence was evaluated by measuring the binding of FITC-conjugated goat anti-mouse IgG in the presence of a primary monoclonal antibody in the absence of disintegrin and was subtracted from each test result. The percentage of expression of LIBS epitope for each sample was calculated using formula: (sample fluorescence/native echistatin fluorescence) × 100%.

Interaction of Echistatin, Eristostatin, and Their Mutants with _v3 Receptor-- Echistatin, eristostatin, and their mutants were tested for their inhibitory effect on adhesion of VNRC3 cells to immobilized vWF and for their effect on expression of LIBS epitope on _v3 receptor (Table I). Substitution of Met²⁸ with Leu did not significantly change echistatin's interaction with _v3, whereas substitution with Asn completely abolished echistatin's effect on _v3 receptor. Echistatin mutant EcM28L also showed an inhibitory effect similar to that for echistatin wild type on the adhesion of ECV304 cells, expressing both _v3 and ₅₁ receptors, to immobilized vWF (data not shown). Furthermore, eristostatin in which Asn³¹ has been replaced with Met showed significantly increased inhibitory effect on adhesion of VNRC3 cells to vWF and on expression of LIBS in _v3 as compared with the wild type eristostatin. Replacement of Asp²⁷, an amino acid residue adjacent to the RGD sequence in echistatin, with Trp present at the same position in eristostatin, significantly reduced echistatin's inhibitory effect in VNRC3 cells but it did not significantly change echistatin's effect on the expression of LIBS in _v3. Parallel substitution of eristostatin's Trp³⁰ with Asp significantly increased both eristostatin's inhibitory effect on adhesion of VNRC3 cells to immobilized vWF as well as expression of LIBS epitope in _v3. Substitution of three residues within the RGD loop of echistatin (R22V/D27W/M28N) and substitution of two residues within the RGD loop of eristostatin (W30D/N31M) made echistatin mutant resemble eristostatin and eristostatin mutant resemble echistatin in their interaction with _v3 receptor. Truncation, full or partial, of the C terminus of echistatin significantly reduced its inhibitory effect and ability to express LIBS. Full or partial truncation of eristostatin showed the same trend; however, it should be noted that the level of wild type eristostatin interaction with both integrins was very low. Echistatin M28L, in which six C-terminal amino acid residues HKGPAT have been replaced with the three amino acid residues from the C-terminal eristostatin (WNG), showed the same inhibitory effect as echistatin M28L on the adhesion of VNRC3 cells and on expression of LIBS on _v3. A hybrid molecule of echistatin with the R22V/D27W/M28N mutation of the RGD loop and the WNG sequence at the C terminus resembled eristostatin in its interaction with _v3 integrin. Furthermore, a hybrid molecule of eristostatin with the HKGPAT sequence at the C terminus had a similar inhibitory effect on VNRC3 cell adhesion and the same LIBS-promoting effect as echistatin wild type. Similar observation was made for the eristostatin hybrid ErW30D/N31M (1-46)-HKGPAT.

View this table: [in this window] [in a new window]   Table I The effect of recombinant echistatin, eristostatin, and their mutants on cell adhesion and expression of LIBS epitopes The effect of recombinant echistatin and eristostatin and their mutants on the adhesion of VNRC3 cells to immobilized vWF (column 1) or K562 cells to immobilized fibronectin (column 3), and on the expression of LIBS epitope on 3 (column 2) or 1 (column 4) integrins. The results represent mean ± S.E. from at least five independent experiments. Calculations were as described in Fig. 2. All echistatin mutants were statistically different (p < 0.05) from echistatin wild type except for those marked with *, while all eristostatin mutants were significantly different (p < 0.05) from eristostatin wild type except for those marked with . In addition, there was no statistical difference (p > 0.05) between echistatin wild type and eristostatin mutants marked with , and between eristostatin wild type and echistatin mutants marked with §. Ec stands for echistatin, Er for eristostatin, r for recombinant, wt for wild type, and s for synthetic.

Interaction of Echistatin, Eristostatin, and Their Mutants with ₅₁ Receptor-- Further experiments demonstrated that any mutations introduced into the RGD loop or truncation of the C terminus of echistatin completely abolished echistatin's ability to inhibit adhesion of K562 cells to immobilized fibronectin and its ability to induce expression of LIBS epitope in ₅₁ receptor (Table I). Echistatin mutants, EcM28L (1-43)-WNG and EcR22V/D27W/M28N (1-43)-WNG, both resembled eristostatin in its weak interaction with ₅₁ receptor. Replacement of Trp³⁰ with Asp significantly increased eristostatin's inhibitory effect on adhesion of K562 cells to fibronectin, although it did not increase its ability to express LIBS epitope. Other mutations of the RGD loop of eristostatin, N31M and W30D/N31M, did not alter the original effect of eristostatin on ₅₁ receptor. Interestingly, however, hybrid eristostatin Er (1-46)-HKGPAT and ErW30D/N31M (1-46)-HKGPAT, while not being as effective as wild type echistatin, clearly endowed this molecule with a new ability to block K562 cell adhesion and induce LIBS in the ₁ integrin. This observation was further confirmed with ECV304 cells expressing both _v3 and ₅₁ integrin, in which adhesion to immobilized fibronectin was completely blocked by both native echistatin and eristostatin mutant Er (1-46)-HKGPAT (data not shown). In contrast, the eristostatin mutant ErW30D/N31M (1-46)-HKGPAT showed increased inhibitory effect with this cell line as compared with eristostatin wild type, but not as significant as the effect of Er (1-46)-HKGPAT (data not shown). Furthermore, EcM28L and native eristostatin did not block adhesion of ECV304 cells to immobilized fibronectin. Synthetic peptide PRNPHKGPAT at 1 mM concentration was unable, however, to inhibit adhesion of K562 cells to immobilized fibronectin and, at concentration of 10 mM, adhesion of ECV304 to immobilized fibronectin (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we investigated the significance of various protein motifs in echistatin on the preferential recognition of _v3 and ₅₁ integrins. The data were obtained by studying the effect of various mutants of echistatin and another short disintegrin, eristostatin, on inhibition of the adhesion of cells transfected with _v3 and ₅₁ to vWF and fibronectin and expression of LIBS epitopes on the two types of cells. The results showed definitive trends that can be summarized as follows. We confirmed previous observations that the amino acid adjacent C-terminally to the RGD motif, Asp²⁷ in echistatin corresponding to Trp³⁰ in eristostatin, appears to be critical for selective recognition of integrins. Tryptophan favors the recognition of _IIb3 (20, 21), whereas aspartic acid favors recognition of _v3 (20, 21) and ₅₁. Met²⁸ in echistatin, adjacent to the RGDD motif, corresponds to Asn³¹ in eristostatin, adjacent to the RGDW motif. Our experiments suggest that methionine enhances interaction of echistatin with both integrins, as tested in cell adhesion and LIBS assays. It is especially critical for the interaction between echistatin and ₅₁ integrin. Substitution of Met²⁸ with Asn significantly decreased interaction of echistatin with both integrins. Substitution of Met²⁸ with Leu in echistatin abolished interaction with ₅₁, having no significant effect on _v3. Substitution of Asn³¹ in eristostatin with Met enhanced interaction with _v3 and ₅₁ in the cell adhesion assay and increased expression of LIBS epitope in _v3 integrin. C termini of both echistatin (RNPHKGPAT) and eristostatin (RNPWNG) are absolutely essential for each disintegrin's interaction with the two integrins. Mutation of R22V/D27W/M28N within the RGD loop of echistatin made echistatin mutant resemble eristostatin in its interaction with both _v3 and ₅₁ receptors. Mutation of W30D/N31M within eristostatin's RGD loop enhanced the interaction of this mutant with _v3, but not with ₅₁. A hybrid molecule of eristostatin, however, in which C-terminal WNG sequence was substituted with HKGPAT, caused an extensive expression (over 20-fold higher than that of eristostatin wild type) of LIBS epitope on ₁ integrin (Table I). This mutant also inhibited ECV304 cell adhesion to fibronectin with identical potency as native echistatin. These experiments suggest that the amino acids situated at the end of the C-terminal domain of echistatin also play a role in selective recognition of integrins.

This study demonstrates a novel observation, that the substitution of Met²⁸ in echistatin with Asn completely abolished echistatin's interaction with _v3 and ₅₁. It has been reported that labeling of echistatin with chloramine T results in the oxygenation of Met²⁸, which makes echistatin prone to dissociation from the _v3 receptor (22). Modeling and molecular dynamic simulation studies showed that the extra oxygen atom on the methionine residue could form hydrogen bonds with the glycine and aspartic acid residues of the RGD motif. The hydrogen bond formation constrains the conformational flexibility of the RGD loop and may contribute to the lower binding affinity of the molecule and to the dissociable nature of the interaction with _v3 receptor. These observations are in agreement with the studies done on accutin, another member of the short disintegrin family (31). Accutin, which naturally contains Leu at the position of Met²⁸ within echistatin, inhibits angiogenesis in vivo and in vitro by blocking integrin _v3 of endothelial cells and by inducing apoptosis. Accutin, however, did not inhibit the binding of the anti-5 monoclonal antibody to endothelial cells. This study, for the first time, presents evidence for the important role of Met²⁸ in echistatin for the selectivity for both ₅₁ and _v3 receptors. The hydrophobic character of methionine is especially important for disintegrin interactions with ₅₁.

Disintegrins are extremely potent inducers of LIBS epitopes on _IIb3 and _v3 integrins. Their LIBS inducing activity is 3-4 orders of magnitude higher than the LIBS inducing activity of short linear RGD-containing peptides or peptidomimetics (20). Marcinkiewicz et al. (20) hypothesized that the C-terminal domain of echistatin supports echistatin binding to the resting integrin and significantly contributes to the expression of LIBS epitope and to the conformational changes of the receptor. This study confirms the important role of the C terminus of disintegrins on their interaction with integrins. However, the C terminus appears to be important not only for the induction of conformational changes in the integrin receptor, but, together with the RGD loop, it determines disintegrin selectivity. The amino acid sequence at the C terminus seems to be less or at least equally important as the amino acid sequence of the RGD loop for the recognition of _v3. There are more spatial constraints regarding ligand binding to the ₅₁ than to the _v3 integrin. On the basis of the NMR structure of the disintegrin flavoridin, showing close association of the C terminus with the active site loop structure, it has been revealed that the C terminus can act as a secondary binding determinant for specific interaction with integrin receptors (32). Similar evidence has been presented for echistatin's C-terminal involvement in determining specificity of this disintegrin for integrin receptors (15).

It has been documented very well that Asp¹¹⁹ in the integrin's ₃ subunit represents an RGD recognition site. This is supported by identification of mutations in patients with thrombasthenia and by studies on Chinese hamster ovary cells transfected with _IIb3 and its mutants (33-36). Sequence ₃-(118-128) (MDLSYSMKDDL) is highly conserved in ₃ and ₁ integrins, and it corresponds to ₁-(153-163). Other sequences in the ₃ subunit, ₃-(214-218) (RNRDA) and ₃-(217-231) (DAPEGGFDAIMQATV), may represent additional binding sites (37). The cross-linking site of disintegrins echistatin and eristostatin is located within ₃-(214-302) (38). Using synthetic peptides, Steiner et al. (39) presented evidence that the RNRDA motif plays a role in the expression of LIBS epitope. Two patients with Glanzmann's thrombasthenia, in which Arg²¹⁴ is substituted with either Trp or Glu, show deficient platelet aggregation and ligand binding activity (34, 40). In one patient, deficient LIBS expression was observed. It can be proposed that echistatin's and eristostatin's RGD loops interact with Asp¹¹⁹ of ₃ integrin, while both PRNPHKGPAT and PRNPWNG may interact with other motifs located on both ₃ and ₁ integrins. Although our data suggest a separate binding site for HKGPAT in ₁ integrin, there is no indication where this site can be localized.

In conclusion, these studies have identified amino acid residues in the hairpin loops and in the C termini of short disintegrins that contribute to the selective recognition of integrin receptors: methionine, aspartic acid C-terminal in the RGD sequence, and the HKGPAT motif at the C terminus. The most important finding is the identification of the structural feature of echistatin required for interaction with ₅₁ integrin. This study confirms selective recognition of integrin receptors by disintegrins and suggests that the RGD loop and the C terminus of echistatin bind to different sites within integrins. Our study may facilitate synthesis of short peptides and peptidomimetics blocking _v3 and ₅₁, two integrins involved in angiogenesis, tissue repair, and cancer metastasis.

	ACKNOWLEDGEMENT

We are grateful for the technical skill of Tom Riggs.

	FOOTNOTES

^* This work was supported by National Institutes of Health Training Grant HL 0777 (to I. W. P.), American Heart Association Initial Investigatorship (to M. A. M. and C. M.), and grants-in-aid from the American Heart Association (to S. N.) and Barra Foundation (to S. N.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Temple University.

To whom correspondence should be addressed: Dept. of Medical Technology, University of Delaware, McKinly Laboratory 057, Newark, DE 19808. Tel.: 302-831-8737; Fax: 302-831-4180; E-mail: mclane@udel.edu.

	ABBREVIATIONS

The abbreviations used are: ECM, extracellular matrix; vWF, von Willebrand factor; FITC, fluorescein isothiocyanate; CHO, Chinese hamster ovary; FCS, fetal calf serum; LIBS, ligand-induced binding site; CMFDA, 5-chloromethylfluorescein diacetate.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES