alpha 2beta 1 Integrin Is Not Recognized by Rhodocytin but Is the Specific, High Affinity Target of Rhodocetin, an RGD-independent Disintegrin and Potent Inhibitor of Cell Adhesion to Collagen*

Johannes A. EbleDagger §, Bernd Beermann, Hans-Jürgen Hinz, and Alletta Schmidt-HederichDagger

From the Dagger  Institut für Physiologische Chemie und Pathobiochemie, Waldeyerstrasse 15 and the  Institut für Physikalische Chemie, Schlossplatz 7, Universität Münster, 48149 Münster, Germany

Received for publication, October 12, 2000, and in revised form, December 4, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have recombinantly expressed a soluble form of human alpha 2beta 1 integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble alpha 2beta 1 integrin binds to its collagen ligands the same way as the wild-type alpha 2beta 1 integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of alpha 2beta 1 integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombinantly expressed, soluble alpha 2beta 1 integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble alpha 2beta 1 integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble alpha 2beta 1 integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to alpha 2beta 1 integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with alpha 2beta 1 integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to alpha 2beta 1 integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking alpha 2beta 1 integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, alpha 2beta 1 integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, alpha 2beta 1 integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit alpha 2beta 1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Integrins are cell adhesion molecules that consist of two noncovalently associated subunits, alpha  and beta  (for review see Refs. 1 and 2). The subfamily of integrins sharing the beta 1 subunit are well known receptors for extracellular matrix molecules, such as collagens, laminins, and fibronectin. The subfamily of beta 3 subunit containing cytoadhesins comprise the platelet integrin, alpha IIbbeta 3 which binds fibrinogen/fibrin (3) and the vitronectin receptor alpha Vbeta 3. The latter ones, along with several beta 1 integrin, such as the fibronectin receptor alpha 5beta 1 integrin, recognize a linear arginyl-glycyl-aspartyl sequence within their respective ligands, such as fibrinogen, fibronectin, and vitronectin (4). In contrast, the collagen binding integrins alpha 1beta 1 and alpha 2beta 1 recognize arginine and aspartate/glutamate residues of different collagen chains, which are in close proximity to each other within the triple helical collagenous framework of the collagen (5-7), thus forming a completely different spatial structure than the linear RGD peptide.

Integrin-mediated cell adhesion not only anchors the cell mechanically within the extracellular matrix of the tissue but also elicits several cellular responses, such as cell spreading and migration, cell proliferation, and differentiation (for review see Ref. 2). A well studied example of cellular response triggered by integrin-ligand interaction is platelet activation and aggregation (8, 9). Thrombocytes abundantly possess the platelet integrin alpha IIbbeta 3 on their surface, which unless activated does not bind to fibrinogen/fibrin (3). Ablation of endothelial cells from the blood vessel wall or other injuries of blood vessels make type IV and type I collagen of the basement membrane and the underlying connective tissue, respectively, accessible to platelets. Once getting in contact with collagen, platelets avidly bind to collagen via their collagen receptors (9, 10), such as the alpha 2beta 1 integrin, GPVI, or indirectly via von Willebrand factor, which binds to both collagen and the vWF receptor on the platelet surface. Receptor-mediated adhesion to collagen elicits a cascade of signals within the platelets, which eventually results in secretion of platelet granula, in platelet aggregation and activation of platelet integrin alpha IIbbeta 3 which then binds to fibrin with high affinity. Insoluble fibrin, which has been produced by the enzymatic blood clotting cascade and provides a scaffold, which together with platelets form the blood clot as the first and essential step in hemostasis. The key role of the alpha 2beta 1 as the sole integrin collagen receptor on platelets is drastically manifested in patients with severe bleeding disorders, caused either by a genetic defect or lack of the integrin alpha 2 subunit (11) or by auto-antibodies against the integrin alpha 2 subunit (12).

Furthermore, snake, leeches, and ticks have developed natural inhibitors of integrin-ligand interactions, called disintegrins, that target at the integrin-mediated platelet adhesion to fibrinogen/fibrin and collagen (9). By inhibiting blood clotting, their venoms lead to severe bleeding, hemorrhages, or even death of their victims. Besides proteolytic enzymes, disintegrins are mainly responsible for these poisonous effects. Most of the known disintegrins contain a linear RGD sequence placed within a rather flexible loop, which prevents the RGD-dependent platelet integrin alpha IIbbeta 3 from binding to fibrin (13). However, very little is known about disintegrins that act on the interaction of alpha 2beta 1 integrin with collagen, the initial step of platelet activation and aggregation. From the venom of the Malayan pit viper (Calloselasma rhodostoma), rhodocytin/aggretin (14, 15), and, more recently, rhodocetin (16) have been shown to induce and inhibit, respectively, collagen-elicited platelet activation and aggretion. However, no direct proof was provided that rhodocytin/aggretin and rhodocetin are the agonist and antagonist, respectively, that interact directly and specifically with alpha 2beta 1 integrin among the different collagen receptors of blood platelets.

Using a recombinantly expressed, soluble human alpha 2beta 1 integrin, we could rule out that rhodocytin/aggretin binds directly to alpha 2beta 1 integrin. On the other hand, having established an inhibition assay with the purified soluble alpha 2beta 1 integrin apart from whole platelets, we could isolate an inhibitor of C. rhodostoma venom that inhibits the binding of soluble alpha 2beta 1 integrin to collagen on the molecular level. N-terminal sequencing identified this inhibitor to be the lately published rhodocetin (16). We could demonstrate that rhodocetin binds directly to the alpha 2beta 1 integrin. Rhodocetin efficiently competes with collagen for the alpha 2beta 1 integrin, even though it does not contain any collagenous triple helix domain, which has been surmised to be a prerequisite for high affinity binding to collagen-binding integrins. Nevertheless, the native conformation that is stabilized by disulfide bridges is essential for binding to alpha 2beta 1 integrin. In contrast to the majority of snake venom disintegrins, rhodocetin binds to alpha 2beta 1 integrin in an RGD-independent manner.

Of even more general importance, alpha 2beta 1 integrin is not only the integrin receptor for collagen on platelets but also abundantly expressed in various tissues (17), suggesting an important role of alpha 2beta 1 integrin within the organism. Having proved rhodocetin to be a very specific alpha 2beta 1-integrin antagonist, we have started to test rhodocetin as a tool in studying alpha 2beta 1-related functions on the cellular level and have demonstrated that rhodocetin can efficiently and entirely inhibit alpha 2beta 1 integrin-mediated adhesion of HT1080 fibrosarcoma cells to collagen.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of the cDNA Constructs of a Recombinant Human Soluble alpha 2beta 1 Integrin-- The transmembrane and cytoplasmic domain of the integrin alpha 2 subunit were substituted for a GGSTGGG spacer and the dimerizing motif of the transcription factor Fos. The cloning strategy started from the construct pUC-HygMT-alpha 3fos, which was described in a previous paper (18). Briefly, the cDNA sequence coding for the alpha 3 ectodomain within the pUC-HygMT-alpha 3fos was replaced by the cDNA sequence coding for the ectodomain of the integrin alpha 2 subunit. To this end, pUC-HygMT-alpha 3fos was cleaved by SalI and dephosporylated by calf intestine phosphatase. The 7.2-kilobase pair-long vector fragment still contains the Fos-coding sequence of the original construct pUC-HygMT-alpha 3fos, yet lacking the complete sequence coding for the integrin alpha 3 ectodomain. The human cDNA coding for the signal sequence and the N-terminal 912 amino acids of the mature alpha 2 ectodmain were excised from pFneo-alpha 2 construct (19) using SalI and BglII. The cDNA coding for the C-terminal 131 amino acids of the alpha 2 ectodomain and the first few amino acids of the GGSTGGG spacer, the latter one of which contains the SalI restriction site, were obtained by polymerase chain reaction using the alpha 2 cDNA of pFneo-alpha 2 as template, and the oligonucleotides ATGCTGAAATTCACTTAACAAGATCTACC with the BglII site underlined and GCCGCCCGTCGACCCTCCTGTTGGTACTTCGGCTTTCTC with the SalI site underlined as forward and reverse primer, respectively. In a triple ligation the SalI-cleaved vector fragment and the cDNA fragments for both the N- and C-terminal part of the alpha 2 ectodomain were ligated to the pUC-HygMT-alpha 2fos construct coding for the soluble alpha 2 ectodomain, which bears at its C terminus the short spacer sequence GGSTGGG and the dimerizing motif of Fos. The pUC-HygMT-beta 1jun construct was generated as described in a previous paper (18).

Establishing a Stable, alpha 2beta 1 Secreting Schneider Cell Clone-- Both constructs were transfected in an equimolar ratio into Drosophila Schneider cells, using TransFastTM Transfection Reagents (Promega, Madison, WI) according to the manufacturer's instructions. Transfected cells were selected under 0.1 mg/ml hygromycin B. After two rounds of subcloning by limited dilution and after screening for positive clones by a sandwich ELISA1 described below, the stable clone alpha 2beta 1-G1.2 was established, which after induction of the metallothionine promoters upstream of both integrin alpha 2 and beta 1 ectodomain cDNAs secreted soluble alpha 2beta 1 integrin into the cell supernatant in concentrations of about 40 µg/liter.

To screen hygromycin B-resistant clones for their ability to secrete soluble alpha 2beta 1 integrin, supernatants of transfectant clones were tested in a sandwich ELISA 4-5 days after induction by copper sulfate. For the sandwich ELISA, the mouse monoclonal anti-integrin alpha 2 antibody JA218 (kindly provided by Danny Tuckwell, University of Manchester, UK) (20) was immobilized to the plastic surface of a microtiter plate at 8 µg/ml in TBS (50 mM Tris/HCl, pH 7.4, 150 mM NaCl ) with MgCl2 (TBS/MgCl2). After blockage of nonspecific binding sites on the microtiter plate with 1% (w/v) heat denatured BSA in TBS/MgCl2 (BSA/TBS/MgCl2), the cell supernatants were added into the coated wells. The antibody JA218 captured the soluble alpha 2beta 1 integrin, which was then detected by an rabbit anti-human beta 1 integrin-antiserum as primary antibody and goat anti-rabbit IgG-antibodies coupled to alkaline phosphatase (Sigma) as secondary antibody, diluted 1:300 and 1:600, respectively, in BSA/TBS/MgCl2. Before each antibody incubation and the final enzymatic detection reaction, wells were washed three times with TBS/MgCl2. As substrate of alkaline phosphatase, p-nitrophenylphosphate tablets were used according to the manufacturer's instructions (Sigma). Absorbance was measured at 405 nm using an ELISA-reader (Dynatech, Burlington, MA).

Isolation of Recombinant Human Soluble alpha 2beta 1 Integrin-- In spinner flasks, alpha 2beta 1 G1.2 cells were grown in Sf900 Medium (Life Technologies, Inc.) containing 0.1 mg/ml hygromycin B and 10% fetal calf serum. Once they had reached a density of about 12 million cells/ml, they were induced by addition of copper sulfate at 0.6 mM. Simultanously, glucose was added to 0.1% (v/w) and glutamine was added to 0.8 mM. Cell supernatant was harvested 5 days after induction and concentrated by ultrafiltration in a YM30 membrane cartridge (Amicon, Witten, Germany). Protease inhibitors aprotinin, leupeptin, and pepstatin were added at 1 µg/ml. Mn2+ ions that increase integrin affinity to ligands were added to a final concentration of 1 mM. The concentration of dithiothreitol (DTT) was adjusted to 2 mM, before the concentrated cell supernatant was loaded onto the collagen I column. The collagen I column had been generated by covalently coupling bovine type I collagen to cyanogen bromide-activated Sepharose 4B CL according to the manufacturer's instruction (Amersham Pharmacia Biotech). The loaded collagen I column was washed with TBS containing 2 mM MgCl2, 1 mM MnCl2, and 2 mM DTT (wash buffer A). After a stringent wash with buffer A with a NaCl concentration of 300 mM, the collagen I column was washed with buffer A, before the soluble alpha 2beta 1 integrin was eluted with TBS containing 20 mM EDTA. Immediately after elution, MgCl2 was added to 30 mM, and the eluate fraction was neutralized with M Tris/HCl, pH 8.0. The alpha 2beta 1 containing eluate fractions were concentrated by ultrafiltration.

Diluted with Mono Q buffer A (20 mM Tris/HCl, pH 8.0, 1 mM MgCl2), the alpha 2beta 1 containing solution was loaded onto a Mono Q column and eluted with a linear gradient of 0 to 50% Mono Q buffer B (1 M NaCl in Mono Q buffer A) within 60 min. The alpha 2beta 1 containing eluate fractions were concentrated by centrifugational ultrafiltration using a Centricon 50 tube (Amicon, Witten, Germany). Protein concentration was determined using the bichinonic acid assay according to the manufacturer's instructions (Pierce). Purity was assessed by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie staining.

Binding of Soluble alpha 2beta 1 Integrin to Various Extracellular Matrix Molecules-- Bovine type I collagen and chicken type II collagen was kindly provided by Peter Bruckner (University of Münster, Germany). Type IV collagen, the type IV collagen fragment CB3[IV], type V collagen, and murine Laminin-1 (Engelbreth-Holm-Swarm-Laminin) were gratefully obtained from Klaus Kühn, Rupert Timpl, and Albert Ries (Max-Planck-Institute for Biochemistry, Martinsried, Germany). Collagens were plated in 0.1 M acetic acid, except for CB3[IV], which like laminin was coated in TBS/MgCl2 onto the microtiter plate. After the wells were blocked with a BSA/TBS/MgCl2, the integrin dissolved in the same solution was allowed to bind to the immobilized substratum. MnCl2, activating antibody 9EG7 or EDTA were added as indicated. The activating monoclonal anti beta 1 integrin antibody 9EG7 (21) was isolated from cell supernatant according to standard protocols. The 9EG7 hybridoma was kindly provided by Dieter Vestweber (University of Münster, Münster, Germany). After a 2-h incubation at room temperature, nonbound integrin was washed away with HEPES wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM MnCl2) twice. Then collagen-bound alpha 2beta 1 integrin was covalently cross-linked to the substratum with 2.5% glutaraldehyde solution in HEPES wash buffer for 10 min at room temperature. After washing the plate three times with TBS/MgCl2, the amount of bound alpha 2beta 1 was measured in an ELISA-like procedure with a rabbit anti-human integrin beta 1 subunit antiserum as primary antibody and an anti-rabbit IgG-antibody conjugated to alkaline phosphatase as secondary antibody, diluted 1:300 and 1:600, respectively, in BSA/TBS/MgCl2. Each antibody incubation, all of which lasted for 1.5 h, was followed by washing the plate with TBS/MgCl2 three times. For detection, p-nitrophenylphosphate tablets (Sigma) were used as substrate for the alkaline phosphatase according to the manufacturer's recommendations. The yellow reaction product was measured at 405 nm in an ELISA reader.

Separation of the Snake Venom Proteins from C. rhodostoma-- Snake venom lyophilizate from C. rhodostoma (Sigma) was dissolved in TBS, pH 7.4, containing 1 mM EDTA (TBS/EDTA) at a protein concentration of about 200 mg/ml. The proteins were separated by gel filtration on a Superose 6 column HR30/30 (Amersham Pharmacia Biotech) using TBS/EDTA at 0.3 ml/min. Two distinct pools of fractions were able to inhibit the binding of soluble alpha 2beta 1 integrin to immobilized type I collagen. The fractions containing the Low molecular weight Calloselasma inhibitor (LMW-CI) was diluted in 20 mM MES/NaOH, pH 6.5 (Mono S buffer A) and passed through a Mono S HR5/5 column (Amersham Pharmacia Biotech). The retained proteins were eluted with a linear gradient of 0-20% Mono S-buffer B (1 M NaCl in Mono S-buffer A) within 60 min. In the third purification step, the LMW-CI containing solution was adjusted to pH 8.5 by diluting into 20 mM Tris/HCl, pH 8.5 (Mono Q-buffer A). The LMW-CI was eluted from the Mono Q column using a linear gradient of 0-50% Mono Q-buffer B (1 M NaCl in Mono Q-buffer A). The elute fractions containing LMW-CI were concentrated in a Centricon 10 tube by centrifugal ultrafiltration. To reduce contaminating proteins any further, a final gel filtration on a TSK G3000SWXL column (TosoHaas, Stuttgart, Germany) was performed at 0.4 ml/min. N-terminal sequencing by Edman degradation identified LMW-CI to be identical to rhodocetin (16).

Protein concentration was determined by bichinonic acid. Purity of LMW-CI and the apparent molecular masses of its subunits were assessed by SDS-PAGE and Coomassie staining.

Inhibition ELISA: Inhibition of alpha 2beta 1 Binding to Immobilized Monomeric Type I Collagen-- Dissolved in 0.1 M acetic acid at 40 µg/ml, type I collagen was coated as monomeric molecule onto the plastic surface of a microtiter plate at 4 °C overnight. After washing with TBS/MgCl2, nonspecific binding sites on the plastic surface were blocked with BSA/TBS/MgCl2 for 2 h at room temperature. Then soluble alpha 2beta 1 integrin was added as a 6 µg/ml solution in BSA/TBS/MgCl2 either without any inhibitor (positive control of 100% binding), in the presence of a snake venom fraction, or with 10 mM EDTA (nonspecific binding; negative control with 0% binding). To increase the binding signal of alpha 2beta 1 integrin, both 1 mM MnCl2 and a 3-fold molar surplus of integrin-activating antibody 9EG7 was added. To prevent any protease activity of the snake venom that could degrade the alpha 2beta 1 integrin or the collagen substratum, resulting in a likewise decrease of binding signals, the following protease inhibitors were added to final concentrations as follows: 2 µg/ml of each aprotinin, leupeptin, and pepstatin, and 2 mM of each 1,10-phenanthroline and phenylmethylsulfonyl fluoride. After having bound to the immobilized collagen ligand in either the presence or the absence of inhibitor for 2 h at room temperature, nonbound alpha 2beta 1 integrin was washed off the plate with HEPES wash buffer. After chemical fixation, the bound integrin was measured in the ELISA-like procedure described above. As blank value, the binding signal obtained in the presence of EDTA was subtracted from all other values. To calculate relative binding values, the binding signal of alpha 2beta 1 integrin to type I collagen without any inhibitor was taken as 100%.

Titration of Immobilized Rhodocetin with Soluble alpha 2beta 1 Integrin-- Both native and inactive rhodocetin were coated onto a microtiter plate at 50 µg/ml in TBS/MgCl2 at 4 °C overnight. Rhodocetin had been inactivated by heat denaturation at 95 °C for 20 min in the presence of 40 mM DTT, followed by blockage of free thiol groups with 120 mM iodacetamide for 10 min at room temperature. After the microtiter plate was blocked with BSA/TBS/MgCl2, soluble alpha 2beta 1 integrin at different concentrations was incubated with the immobilized rhodocetin. Soluble alpha 2beta 1 integrin was dissolved in BSA/TBS/MgCl2 containing 1 µg/ml of each aprotinin, leupeptin, and pepstatin, as well as 0.5 mM phenylmethylsulfonyl fluoride and 1,10-phenanthroline. After a 2-h incubation at room temperature, wells were washed twice with HEPES wash buffer. Bound alpha 2beta 1 integrin was fixed, and its amount was determined by ELISA as described above. Nonspecific binding signals measured as alpha 2beta 1 binding to the blocking agent BSA were subtracted from the binding values for alpha 2beta 1 binding to native and denatured rhodocetin, respectively. The titration curves were linearized, and a Kd value was determined according to the algorithm given by Heyn and Weischet (22).

RGD Peptide Inhibition Assay of alpha 2beta 1 Binding to Rhodocetin-- Inhibition of alpha 2beta 1 binding to immobilized rhodocetin by RGD peptide was performed similarly to the titration experiments. After the microtiter plate was coated with rhodocetin at 50 µg/ml overnight at 4 °C and blocked with BSA/TBS/MgCl2 at room temperature for 2 h, soluble alpha 2beta 1 at 15 µg/ml was added either in the absence or presence of various concentrations of the linear GRGDSP peptide (Bachem, Heidelberg, Germany) for 2 h at room temperature. Then unbound alpha 2beta 1 integrin was removed by washing with HEPES wash buffer twice. Bound alpha 2beta 1 integrin was fixed with 2.5% glutaraldehyde in HEPES wash buffer. Its amount was determined by ELISA as described above. The binding signals were corrected for the blank values measured as alpha 2beta 1 binding to BSA and afterward normalized to the noninhibited binding of alpha 2beta 1 to rhodocetin in the absence of GRGDSP peptide (positive control, 100% binding).

Circular Dichroism Spectroscopy of Rhodocetin-- The buffer of the rhodocetin solution was changed to 20 mM sodium phosphate, pH 7.0, 50 mM NaCl by gel filtration on a TSK G3000SWXL column (TosoHaas, Stuttgart, Germany). The rhodocetin containing eluate fractions were concentrated in a Centricon 10 tube by centrifugal ultrafiltration to reach a concentration of about 0.3 mg/ml. The CD spectrum was recorded from 190 to 260 nm in a 0.1-mm cuvette in a CD spectrophotometer type CD6 (Jobin Yvon, Paris, France). Temperature was controlled by a self-constructed Peltier element cuvette holder. The relative amount of secondary structures (alpha  helix, parallel, and anti-parallel beta  strands, random coil) were calculated with the deconvolution program of CDNN by Gerhard Böhm (23).

Inhibition of Cell Adhesion to Collagen by Rhodocetin-- Monomeric bovine type I collagen at a concentration of 0.2 µg/ml in 0.1 M acetic acid was immobilized onto a microtiter plate at 4 °C overnight. After being washed with TBS/MgCl2 for three times, the plate was blocked with BSA/TBS/MgCl2 for 2 h at room temperature. HT1080 fibrosarcoma cells at a density of 500,000 cells/ml in Dulbecco's modified Eagle's medium were plated onto the plate for 35 min in a tissue culture incubator at 37 °C in both absence and presence of various concentrations of rhodocetin. Adherent cells were detected by staining with crystal violet (24). Briefly, adherent cells were fixed with 70% (v/v) solution of ethanol for 7 min and stained with a 0.1% (w/v) solution of crystal violet in destilled water. After washing the wells, cell bound dye was extracted with a 0.2% (v/v) Triton X-100 solution, and its amount was measured in an ELISA reader at 560 nm. Experiments with cells were done in triplicates. The adhesion signal of HT1080 cells measured on BSA was considered nonspecific background and subtracted from the adhesion signals of cells on type I collagen. Adhesion signals in the presence of rhodocetin were normalized to the adhesion signal of the noninhibited cell adhesion to type I collagen without any inhibitor.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production and Isolation of Recombinant Soluble Human alpha 2beta 1 Integrin-- A recombinant soluble human alpha 2beta 1 integrin that consists of the ectodomains of both alpha 2 and beta 1 integrin subunits being noncovalently associated by the dimerizing motif of Fos and Jun, respectively, was secreted by transfected Drosophila Schneider cells. Affinity purification of the cell supernatant on a type I collagen column yielded not only the soluble alpha 2beta 1 integrin but also a protein of 45 kDa as determined by SDS-PAGE under reducing conditions (Fig. 1, lane 5). Edman degradation of the latter one revealed its N-terminal sequence as STEFSEDLLDEDLDLDIDE and, thus, identified the 45-kDa protein as Drosophila BM40 (GenBankTM accession number AJ1333736). Interestingly, BM40 was abundantly expressed by Schneider cells. Like the soluble alpha 2beta 1 integrin, it bound to type I collagen column in a divalent cation-dependent manner. About 10 times more BM40 than soluble alpha 2beta 1 integrin was eluted from the type I collagen column by EDTA. However, binding of BM40 to type I collagen did not interfere with alpha 2beta 1 integrin binding to its collagen ligand. Being a less acidic protein than BM40, the soluble alpha 2beta 1 integrin was further purified by anion exchange chromatography on a Mono Q column, from which the soluble alpha 2beta 1 integrin was eluted at lower ion strength than the highly acidic BM40. Yields of soluble alpha 2beta 1 integrin ranged from 30 to 40 µg/liter of cell supernatant.



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Fig. 1.   SDS-PAGE of soluble human alpha 2beta 1 integrin (lanes 1 and 4, without and with prior reduction, respectively) and Drosophila BM40 (lanes 2 and 5, without and with prior reduction, respectively) in a 7.5-15% polyacrylamide gradient gel. Broad range molecular mass markers (Bio-Rad) were used as standard proteins (lane 3) with their molecular masses indicated on the far right side. Proteins in the gel were stained with Coomassie dye. Protein bands under both nonreduced and reduced conditions are labeled on the left and right side, respectively, of the gel.

Characterization of the Recombinant Soluble alpha 2beta 1 Integrin-- In SDS-PAGE, the soluble alpha 2beta 1 integrin heterodimer was separated into the Fos zipper containing alpha 2-ectodomain, alpha 2-Fos, and the Jun zipper containing beta 1-ectodomain, beta 1-Jun, which run at 150 and 95 kDa, respectively, under nonreducing conditions and at 140 and 100 kDa, respectively, after reduction (Fig. 1, lanes 1 and 4). The identity of the alpha 2 band was proven by N-terminal sequencing. Edman degradation revealed the sequence YNVGLPEAKI in agreement with the mature human integrin alpha 2 subunit (19), demonstrating that the human alpha 2 subunit was correctly processed proteolytically within the insect cells. Like the wild-type form on human cells, the human beta 1-Jun chain expressed by the insect cells was N-terminally blocked and thus inaccessible to Edman degradation. However, it was identified in Western blot by a polyclonal antiserum against the human integrin beta 1 subunit (data not shown). Unlike other integrin alpha  subunits, the alpha 2-ectodomain is not proteolytically processed into a heavy and light chain. Neither was the human soluble alpha 2beta 1 integrin cleaved in the heterologous expression system of the Drosophila Schneider cells. Having very similar isoelectric points, the alpha 2beta 1 integrin and BSA could not efficiently be separated by anion exchange chromatography leading to a slight contamination of BSA in the alpha 2beta 1 integrin preparation.

The soluble alpha 2beta 1 integrin was able to bind to collagen types I, II, IV, and V and to laminin-1 (Engelbreth-Holm-Swarm-Laminin) (Fig. 2). The highest binding signals were observed to type I and II collagen, which is in good agreement with results of wild-type alpha 2beta 1 integrin (25). Like the wild-type form, the soluble alpha 2beta 1 integrin gave a smaller binding signal on the basal membrane collagen, type IV collagen, and likewise to its triple helical fragment CB3[IV], which comprise the binding sites for both alpha 1beta 1 and alpha 2beta 1 integrin (25). A significantly lower binding signal was measured to type V collagen, which together with type I collagen forms the collagen fibrils of the connective tissue. As a ligand without any collagenous triple helix, laminin-1 was bound by the soluble alpha 2beta 1 integrin, albeit with a much lower binding signal than the collagenous ligands. The latter finding corroborated studies of wild-type alpha 2beta 1 integrin binding to laminin-1 (26). Identical to cell membrane-anchored wild-type alpha 2beta 1 integrin, soluble alpha 2beta 1 required divalent cations to recognize its ligands. Therefore, EDTA abolished alpha 2beta 1 binding (Fig. 2). The soluble alpha 2beta 1 integrin seemed to be regulated by extracellular factors in a manner similar to that of the the wild-type alpha 2beta 1 integrin on the cell surface, because integrin-activating Mn2+ ions and the activating monoclonal antibody 9EG7 increase the binding signal of soluble alpha 2beta 1 integrin to its ligands (Fig. 2). Taken together, the soluble alpha 2beta 1 integrin showed ligand binding properties similar to the membrane-anchored wild-type alpha 2beta 1 integrin. However, no detergent was needed to extract the soluble alpha 2beta 1 integrin or to keep it in solution. Furthermore, unlike the detergent-extracted wild-type alpha 2beta 1 integrin, soluble alpha 2beta 1 integrin remained active even after a longer storage period of several months.



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Fig. 2.   Binding of soluble alpha 2beta 1 integrin to bovine type I collagen (bCol-1), chicken type II collagen (chCol-2), human type IV collagen (hCol-4) and its triple helical cyanogen bromidederived fragment (CB3[IV]), human type V collagen (hCol-5), and murine Engelbreth-Holm-Swarm-Laminin (mLam-1). The collagen molecules were immobilized onto the microtiter plate in 0.1 M acetic acid, except for CB3, which like laminin is coated in TBS containing Mg2+ ions. Binding of soluble alpha 2beta 1 integrin (12 µg/ml) was tested in the presence of 1 mM Mn2+ ions (lightly hatched bars), in the presence of 1 mM Mn2+ and a 3-fold molar surplus of activating antibody 9EG7 (densely hatched bars), or in the presence of 10 mM EDTA (open bars). Duplicate measurements were performed for every binding condition. Mean values and standard deviations of a representative experiment are shown.

Whole Snake Venom of C. rhodostoma Inhibits Binding of Soluble alpha 2beta 1 Integrin to Immobilized Type I Collagen-- The strong binding signal of soluble alpha 2beta 1 integrin to immobilized type I collagen (Fig. 2) was diminished and completely inhibited by the crude snake venom of C. rhodostoma in a dose-dependent manner with an IC50 value of about 50 µg/ml (data not shown). Like other snake venoms, C. rhodostoma venom contains several proteases that could be detected by zymogram developed with gelatin. Rhodostoxin (kistomin and major hemorrhagin), a metalloprotease (27, 28), and ancrod, a serine protease (29), could be detected in the zymogram among other proteolytic activities (data not shown). To rule out the possibility that any snake venom protease diminishes the alpha 2beta 1 binding signal to immobilized collagen, protease inhibitors directed against all four classes of proteases, such as aprotinin, leupeptin, phenylmethylsulfonyl fluoride, pepstatin, and 1,10-phenanthroline, were added to the venom protein fraction when applied in the inhibition ELISA to test its capability to inhibit binding of soluble alpha 2beta 1 to immobilized type I collagen by a nonproteolytic interaction.

Rhodocytin/Aggretin Does Not Inhibit Binding of Soluble alpha 2beta 1 Integrin to Type I Collagen-- Rhodocytin or aggretin are the two names of a 29-kDa protein of C. rhodostoma venom, which induces activation and aggregation of thrombocytes (15, 30). It was isolated from the snake venom according to Shin and Morita (31). In SDS-PAGE (Fig. 3, lane 1), the purified rhodocytin/aggretin showed a molecular mass of about 29 kDa under nonreducing conditions. Being a disulfide cross-linked heterodimer, it was cleaved under reducing conditions into two subunits of 19 and 15 kDa (Fig. 3, lane 4). The N-terminal sequences of both subunits, GLEDDFGWSPYDQ[H/(Q)]2 and DPSGWSSYEG[H/(G)](H)YK, proved their identities as alpha  and beta  chains, respectively, of rhodocytin/aggretin (14, 31). To test the postulated interaction of soluble alpha 2beta 1 integrin with rhodocytin/aggretin, the latter one was immobilized on a microtiter plate, and the binding of soluble alpha 2beta 1 was tested. Whereas the soluble alpha 2beta 1 binds to immobilized monomeric type I collagen in a divalent cation-dependent manner, no binding to immobilized rhodocytin/aggretin was observed (Fig. 4A). A similar result was obtained when wild-type alpha 2beta 1 integrin, which had been purified from platelets (kindly provided by Albert Ries and Rupert Timpl, Max-Planck-Institute for Biochemistry, Martinsried, Germany), was used (data not shown).



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Fig. 3.   SDS-PAGE of rhodocytin/aggretin (lanes 1 and 4, without and with prior reduction, respectively) and LMM-CI/rhodocetin (lanes 2 and 5, without and with prior reduction, respectively) in a 12-18% polyacrylamide gradient gel. Broad range molecular mass marker (Bio-Rad) was used as standard proteins (lane 3) with their molecular masses indicated on the far right side. Proteins in the gel were stained with Coomassie dye. Protein bands under nonreduced and reduced conditions are labeled on the left and right side, respectively, of the gel.



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Fig. 4.   Rhodocytin/aggretin from C. rhodostoma snake venom is not recognized by soluble alpha 2beta 1 integrin (A) and does not inhibit binding of alpha 2beta 1 integrin to type I collagen (B). In A, bovine type I collagen at 20 µg/ml in 0.1 M acetic acid and rhodocytin/aggretin at 50 µg/ml in TBS/MgCl2 were coated onto a microtiter plate. After blockage with heat denatured BSA, soluble alpha 2beta 1 integrin was added for 2 h at room temperature. After the wells had been washed twice, the bound alpha 2beta 1 integrin was chemically fixed to the immobilized substratum and its amount measured by ELISA. In B, bovine type I collagen was immobilized on the microtiter plate at 40 µg/ml in 0.1 M acetic acid. After blockage with heat denatured BSA, the soluble alpha 2beta 1 integrin was allowed to bind to the immobilized collagen in both absence and presence of various concentrations of soluble rhodocytin/aggretin for 2 h at room temperature. After the wells had been washed twice, the collagen-bound alpha 2beta 1 integrin was chemically fixed, and its amount was determined by ELISA. Blank values measured in BSA-coated wells were subtracted from the measured absorbance values. In B, the absorbance values were normalized to the noninhibited binding value, taken as 100%. Each value was measured in duplicate. Standard deviations are indicated.

Because immobilization may have caused inactivation of rhodocytin/aggretin, binding of soluble alpha 2beta 1 to soluble rhodocytin/aggretin was tested by measuring the capability of the snake venom component to inhibit alpha 2beta 1 integrin binding to immobilized type I collagen. However, rhodocytin/aggretin does not prevent alpha 2beta 1 integrin from binding to collagen (Fig. 4B). Both the binding test and the inhibition test rule out any direct interaction between rhodocytin/aggretin and soluble alpha 2beta 1 integrin on the molecular level.

Searching for the Component of C. rhodostoma Venom That Inhibits the Interaction of Soluble alpha 2beta 1 Integrin with Collagen-- Although rhodocytin/aggretin did not inhibit alpha 2beta 1 binding to collagen (Fig. 4), the whole snake venom hampered binding of soluble alpha 2beta 1 integrin to immobilized type I collagen. Taking advantage of the inhibition ELISA, the constituent of C. rhodostoma venom that is responsible for the inhibition of alpha 2beta 1 integrin binding to type I collagen was searched. In the first purification step, the venom proteins were separated according to their molecular masses by gel filtration on a Superose 6 column (Fig. 5A). When the eluate fractions were screened for their capability to inhibit alpha 2beta 1 binding to immobilized type I collagen, two peaks of inhibitory activity could be identified (Fig. 5B). Because of their different molecular masses, they were referred to as high molecular weight and low molecular weight Calloselasma inhibitor. Purification and identification of the LMW-CI activity was further pursued. Ion exchange chromatography both on Mono S and Mono Q could clearly separate rhodocytin/aggretin from the alpha 2beta 1 integrin inhibitory activity of LMW-CI. The Mono S column retained LMW-CI at pH 6.5 up to a ionic strength of 105 mM NaCl, whereas rhodocytin/aggretin barely bound to Mono S at pH 6.5 and was washed off the column at very low ionic strength. In the opposite elution order, LMW-CI was eluted from the Mono Q column at pH 8.5 at low ionic strength of about 100 mM NaCl, whereas rhodocytin/aggregetin remained bound to the Mono Q resin at NaCl concentrations of up to 300 mM NaCl. In conclusion, the isoelectric point of LMW-CI must be higher than the one of rhodocytin/aggretin, although the isoelectric points of both proteins must be in a pH range of 6.0-8.5. As final purification step of LMW-CI, another gel filtration chromatography on a TSK G3000SWXL was performed, resulting in a highly purified band at 27 kDa in SDS-PAGE. Furthermore, coprecipitation experiments with alpha 2beta 1 integrin showed that the 27-kDa protein binds to the alpha 2beta 1 integrin, suggesting that the 27-kDa protein is the LMW-CI (data not shown).



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Fig. 5.   Purification of LMM-CI from C. rhodostoma venom using gel filtration on a Superose 6 HR30/30 column (A). The eluate fractions were tested in the inhibition ELISA for their capability to inhibit binding of soluble alpha 2beta 1 to immobilized type I collagen (B). The inhibitory activity of the eluate fractions is shown as relative inhibition, which is defined as difference between noninhibited and inhibited binding signal normalized to the noninhibited binding signal. Note that two inhibitory peaks are separated on the size exclusion column that differ in their molecular masses. They are named high molecular mass Calloselasma inhibitor (HMW-CI) and low molecular mass Calloselasma inhibitor (LMW-CI), respectively.

Characterization of LMW-CI-- Under nonreducing conditions, LMW-CI shows an apparent molecular mass of 27 kDa in SDS-PAGE (Fig. 3, lane 2). LMW-CI is a heterodimer, which upon reduction falls apart in two subunits of 16 and 14 kDa (Fig. 3, lane 5). The N-terminal sequences of both LMW-CI subunits were identified by Edman degradation with the N terminus of the 16-kDa subunit being D[-/(F)]PD[G/S]WSSTKSYYR[P/(R)][F/(P)][K/(F)][E/(K)][K/(E)]3 and the N terminus of the 14 kDa subunit being DFRPTTWSMSKLY [-/(S)]YKPF(K). These N-terminal sequences clearly showed that the LMW-CI is distinct from the rhodocytin/aggretin. However, these sequences disclosed that LMW-CI is identical to rhodocetin, a recently published inhibitor of collagen-induced platelet aggregation (16).

On a molecular level, rhodocetin inhibited binding of soluble alpha 2beta 1 integrin to immobilized type I collagen in a dose-dependent manner (Fig. 6), thus proving that, in contrast to rhodocytin/aggretin, the effect of rhodocetin on whole platelets (16) can indeed be imitated on a molecular scale, i.e. on the interaction of isolated alpha 2beta 1 integrin to collagen. With increasing concentrations, LMW-CI/rhodocetin decreased the binding signal of the collagen receptor to its ligand and eventually abolished it entirely. From Fig. 6, an IC50 value of about 30 nM could be determined.



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Fig. 6.   LMM-CI/rhodocetin inhibits binding of soluble alpha 2beta 1 integrin to immobilized type I collagen in a dose-dependent manner. Monomeric bovine type I collagen was coated onto a microtiter plate at 40 µg/ml in 0.1 M acetic acid. After blockage of the wells with heat denatured BSA, soluble alpha 2beta 1 integrin at 6 µg/ml was allowed to bind to immobilized type I collagen in both absence and presence of various concentrations of LMM-CI for 2 h at room temperature. After the wells had been washed twice, the collagen-bound alpha 2beta 1 integrin was detected by ELISA. Nonspecific binding signal as measured in the presence of 10 mM EDTA was subtracted from all values. The binding signals were then normalized to the noninhibited binding signal. Each value was measured in duplicate. Relative standard deviations are shown.

Rhodocetin Is a Disintegrin That Directly and Specifically Binds to alpha 2beta 1 Integrin-- Addition of various protease inhibitors to the inhibition ELISA ruled out the possibility that the decrease of alpha 2beta 1 binding to collagen was caused by proteolytic digestion of either binding partner by a snake venom protease. Therefore, a direct, yet nonenzymatic binding interaction of LMW-CI/rhodocetin with either alpha 2beta 1 integrin or with the integrin-binding site on type I collagen must be responsible for its inhibitory effect. To test a direct interaction of rhodocetin with the soluble alpha 2beta 1 integrin, rhodocetin was immobilized onto a microtiter plate, and binding of soluble alpha 2beta 1 was measured. As shown in Fig. 7, the soluble alpha 2beta 1 integrin directly bound to rhodocetin, thereby qualifying it to be a disintegrin. The binding signal could be increased slightly by addition of 1 mM MnCl2 and the integrin-activating antibody 9EG7. However, in contrast to other integrin ligands, binding of alpha 2beta 1 to rhodocetin did not require any divalent cations, because addition of EDTA did not abolish alpha 2beta 1 binding to rhodocetin. A binding signal similar to the one of soluble alpha 2beta 1 integrin was obtained when detergent-extracted wild-type alpha 2beta 1 integrin from human platelets was applied (data not shown). Another soluble integrin, the laminin-5 receptor alpha 3beta 1 integrin (18), did not bind to immobilized, native rhodocetin, although it showed binding activity to laminin-5 (Fig. 7). The soluble alpha 3beta 1 integrin had been produced in our lab by insect cells similarly to the soluble alpha 2beta 1 integrin (18). Even more striking, another widespread collagen receptor, alpha 1beta 1 integrin, which had been isolated from human placenta according to Kern et al. (25) and was tested biologically active by its binding to type I and IV collagen, entirely fails to bind to rhodocetin (Fig. 7), proving the specificity of LMW-CI/rhodocetin to recognize alpha 2beta 1 integrin selectively.



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Fig. 7.   LMW-CI is a disintegrin which interacts directly and specifically with alpha 2beta 1 integrin. LMW-CI at 30 µg/ml, monomeric bovine type I collagen (bCol-1) at 30 µg/ml, monomeric human type IV collagen at 30 µg/ml (hCol-4), and human laminin-5 (hLam-5) at 10 µg/ml were coated onto the microtiter plate in TBS/Mg2+ ions, except for the collagen, which were dissolved in 0.1 M acetic acid. After blocking with heat denatured BSA, the wells were incubated with octylglucoside-solubilized alpha 1beta 1 integrin (open, filled, and gray bars), with soluble alpha 2beta 1 integrin (thinly, thickly, and densely striped bars), or with soluble alpha 3beta 1 integrin (thinly, thickly, and densely hatched bars) for 2 h at room temperature either in the presence of 1 mM Mn2+ ions (open, thinly striped, and hatched bars, respectively), or in the presence of both 1 mM Mn2+ and a 3-fold molar surplus of integrin-activating 9EG7 antibody (filled, thickly striped, and hatched bars, respectively) or in the presence of 10 mM EDTA (gray, densely striped, and hatched bars, respectively). Wild-type alpha 1beta 1 had been extracted and isolated from human placental tissue according to Kern et al. (25). Both soluble human alpha 2beta 1 and alpha 3beta 1 integrin had been recombinantly expressed in Drosophila Schneider cells and isolated as described "Experimental Procedures" and according to Eble et al. (18), respectively. After the wells had been washed twice, substratum-bound integrin was chemically fixed, and its amount was determined by ELISA. The binding signals onto heat denatured BSA were taken as blanks. Each value was measured in duplicate, and standard deviations are shown. Binding of alpha 1beta 1 and alpha 2beta 1 integrin to laminin-5 and binding of alpha 3beta 1 integrin to type I and IV collagen were not determined (n.d.).

It is noteworthy that the ability of LMW-CI/rhodocetin to interact with alpha 2beta 1 integrin depended on its disulfide bridges, which stabilize both its quartenary and tertiary structure. Preincubation of LMW-CI at DTT concentrations higher than 0.016 mM without any thermal denaturation resulted in a strong decrease of alpha 2beta 1 binding (Fig. 8A). However, when scrutinized by SGS-PAGE (Fig. 8B), the partially reduced LMW-CI/rhodocetin run as stable heterodimer even up to 10 mM DTT. Amazingly, rhodocetin does not possess any intercatenary disulfide bridges (16), but its subunits stayed together even under the harsh denaturating condition of the SDS-PAGE sample buffer containing 2% SDS. Reduction of the intracatenary disulfide bridges at DTT concentrations higher than 10 mM made the rhodocetin heterodimer dissociate. Although lacking an intercatenary disulfide bridge, quaternary structure of rhodocetin is very stable and depends on the tertiary structure of both subunits, which is stabilized by intracatenary disulfide bridges. As the binding signal of soluble alpha 2beta 1 integrin gradually decreased with increasing DTT concentrations higher than 0.016 mM and is entirely lost at 10 mM DTT, it can be envisioned that the intracatenary disulfide bridges are of paramount importance in maintaining the native tertiary structure of rhodocetin, which is essential for alpha 2beta 1 integrin binding. Furthermore, the tertiary structure of its subunits as evidenced by its integrin binding function seems to be even more sensitive to denaturation than its quaternary structure, i.e. dissociation into its two subunits.



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Fig. 8.   The native tertiary structure of LMW-CI/rhodocetin, as evidenced by its inhibitory activity toward alpha 2beta 1 integrin (A), is destroyed at much lower concentration of reducing agents than its quartenary structure, i.e. association of its two subunits, as detected by SDS-PAGE under nonreducing conditions (B). A, LMW-CI was incubated for an hour with increasing concentrations of DTT at 37 °C. After inactivation of DTT with a surplus of iodacetamide, LMW-CI was coated onto a microtiter plate at 40 µg/ml. Soluble alpha 2beta 1 integrin at 6 µg/ml was allowed to bind to the pretreated LMW-CI. After being chemically fixed, bound integrin was detected by ELISA. Binding signals were corrected for the nonspecific binding signal on heat denatured BSA and normalized to LMW-CI, which had been incubated without DTT and treated with iodacetamide. Values were measured in duplicate. Standard deviations are shown. Note that inibitory activity drops at DTT concentration higher than 0.016 mM, and is completely lost at 10 mM. B, DTT and iodacetamide-treated LMW-CI, which was used to coat the microtiter plate, was separated in a 12-18% polyacrylamide gradient gel under nonreducing conditions. Note that nonreduced LMW-CI heterodimer vanishes at DTT concentrations higher than 2 mM with a concomitant pronounced appearance of the two LMW-CI subunits.

To determine the binding affinity of rhodocetin to alpha 2beta 1 integrin, both native and denatured rhodocetin were immobilized onto a microtiter plate and titrated with soluble alpha 2beta 1 integrin (Fig. 9). Treatment of rhodocetin with 40 mM DTT in addition to thermal denaturation entirely abolished its binding activity to the integrin, again demonstrating that the specific interaction of rhodocetin with alpha 2beta 1 integrin requires the disulfide-stabilized native conformation of rhodocetin. For binding of soluble alpha 2beta 1 integrin to native rhodocetin, saturation was achieved at alpha 2beta 1 concentrations of about 100 nM. From such titration curves, an apparent Kd value of LMW-CI/rhodocetin binding to alpha 2beta 1 integrin was calculated to be 10.3 nM.



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Fig. 9.   Titration of native (closed symbols) and inactivated (open symbols) LMW-CI/rhodocetin with soluble alpha 2beta 1 integrin. Inactivation of LMW-CI was achieved by reduction of disulfide bridges with 40 mM DTT and heat denaturation at 95 °C for 20 min. Reduced thiol groups were then blocked with 120 mM iodacetamide. Both native and inactivated LMW-CI was coated onto the microtiter plate at 40 µg/ml and titrated with the indicated concentrations of soluble alpha 2beta 1. Wells coated with heat denatured BSA were taken as blanks. The bound alpha 2beta 1 integrin was chemically fixed, and its amount was determined by ELISA. The blank values were subtracted from the binding signals. Each value was measured in duplicate. Standard deviations are indicated.

Rhodocetin Does Not Contain a Triple Helical Collagen Domain-- Being essential for its inhibitory activity, the native conformation of LMW-CI/rhodocetin was further studied by CD. We were especially interested in whether or not LMW-CI contains any triple helical collagenous motifs, because high affinity ligands of alpha 2beta 1 integrin are mostly collagenous molecules. Laminin-1, which lacks any collagenous structure, is bound by alpha 2beta 1 integrin with much lower affinity. Although LMW-CI/rhodocetin competed with the high affinity binding of alpha 2beta 1 to type I collagen, the CD spectrum of LMW-CI (data not shown) did not reveal any collagenous triple helical structure within the disintegrin. Although lacking a collagen domain, rhodocetin possesses a distinct native structure, because deconvulation of the CD spectrum recorderd at 20 °C disclosed a high content of 59.5% beta -sheet and a minor amount of 10.3% alpha -helical secondary structure for rhodocetin. Heat denaturation abrogated any secondary structural signals in the CD spectrum, leaving a spectrum typical of random coil.

Rhodocetin Is an RGD-independent Integrin-- Many disintegrins bind to RGD-dependent integrins in an RGD peptides inhibitable manner. However, the linear GRGDSP peptide failed to inhibit the interaction of alpha 2beta 1 integrin with immobilized LMW-CI/rhodocetin (Fig. 10). Even at concentrations of 4 mM, which represented an 800,000-fold molar surplus to the soluble alpha 2beta 1 integrin in the inhibition experiment, the GRGDSP peptide did not affect the alpha 2beta 1 disintegrin interaction, thus showing that LMW-CI/rhodocetin belongs to the small group of RGD-independent disintegrins.



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Fig. 10.   Binding of the disintegrin LMW-CI/rhodocetin to alpha 2beta 1 integrin does not depend on an RGD peptide sequence. LMW-CI was coated onto a mircrotiter plate at 40 µg/ml. After being blocked with heat denatured BSA, wells were incubated with soluble alpha 2beta 1 integrin for 2 h in the absence and presence of the linear peptide GRGDSP at concentrations indicated in the plot. After wells had been washed twice, bound receptor was chemically fixed, and its amount was determined by ELISA. alpha 2beta 1 binding to heat denatured BSA was taken as blank and subtracted from the binding values. Binding signals were normalized to the noninhibited binding signal measured in the absence of peptide. Values were determined in duplicate. Standard deviations are indicated.

Effect of Rhodocetin on alpha 2beta 1-mediated Adhesion of Fibroblasts-- Whether LMW-CI/rhodocetin can be used in vivo, e.g. to inhibit alpha 2beta 1-mediated cell adhesion and migration or to influence other cellular reactions triggered by the alpha 2beta 1-collagen interaction, the effect of the isolated rhodocetin on adhesion of HT1080 cells onto immobilized type I collagen was examined. HT1080 is a human fibrosarcoma cell line that abundantly expresses alpha 2beta 1 integrin on its surface and that adheres to immobilized type I collagen mainly via its alpha 2beta 1 integrin (32). When HT1080 cells were plated onto monomeric type I collagen in the presence of soluble rhodocetin, cell adhesion declined with increasing concentrations of the snake venom disintegrin and was eventually abolished completely (Fig. 11). At a cell density of 500,000 cells/ml, plated onto 0.2 mg/ml type I collagen, an IC50 value for LMW-CI/rhodocetin of about 2 µg/ml = 60 nM was measured, showing that, even at low concentrations, this disintegrin is able to specifically affect alpha 2beta 1-collagen interaction on a cellular level.



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Fig. 11.   LMW-CI/rhodocetin efficiently and entirely inhibits alpha 2beta 1 integrin-mediated cell adhesion of HT1080 fibrosarcoma cells to type I collagen. Monomeric type I collagen was coated at 0.2 µg/ml in 0.1 M acetic acid. After the wells were washed and blocked with heat denatured BSA, HT1080 cells were plated onto the collagen substratum at a density of 50,000 cells/well for 35 min in the absence and presence of various concentrations of LMW-CI as indicated. Adhered cells were stained with crystal violet, which was solubilized from the cells after the wells had been destained. Absorbance was measured at 560 nm. Integrin-specific cell adhesion was completely abolished in the presence of 10 mM EDTA. This blank value was subtracted from the binding signals. The adhesion signals were normalized to the noninhibited adhesion of HT1080 cells. Each value was measured in triplicate. Standard deviations are shown.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here, we report that rhodocytin, an inducer of platelet aggregation from the hemorrhagic snake venom of C. rhodostoma, does not interact with alpha 2beta 1 integrin, one of the collagen receptors on the surface of blood platelets. However, from the same snake venom, we have isolated and characterized LMW-CI, which is identical to rhodocetin, a recently published inhibitor of collagen-induced platelet aggregation (16). We show that LMW-CI/rhodocetin is a disintegrin that specifically and avidly binds to alpha 2beta 1 integrin. Independently of Wang et al. (16), we have purified LMW-CI/rhodocetin as a component of C. rhodostoma venom, which inhibits the binding of alpha 2beta 1 integrin to immobilized collagen on the molecular level. We used recombinantly expressed and purified, soluble alpha 2beta 1 integrin in an inhibition ELISA to screen the snake venom for components that specifically and nonproteolytically inhibit the interaction of alpha 2beta 1 integrin with collagen. Although commonly used, the method of using whole platelets to test for integrin agonists and antagonists may be biased by the presence of various other collagen receptors on the platelet surface. Furthermore, here we describe our detailed studies on the interaction of the novel disintegrin LMW-CI/rhodocetin with alpha 2beta 1 integrin.

Identification of LMW-CI/rhodocetin as disintegrin, which specifically binds to alpha 2beta 1 integrin, was made feasible by the recombinant production of a soluble alpha 2beta 1 integrin and its purification in sufficient amounts. Soluble alpha 2beta 1 integrin was generated by replacing the transmembrane and cytoplasmic domain of both integrin subunits alpha 2 and beta 1 with the dimerizing motifs of the transcription factor Fos and Jun, respectively. Lately, a similar attempt to produce soluble alpha 3beta 1 had been successful (18). However, yields of soluble alpha 2beta 1 integrins were generally lower than with soluble alpha 3beta 1 integrin in compliance with the comparatively lower expression of membrane-bound alpha 2beta 1 on transfected mammalian cells, such as erythroleukemic K562 cells.4 Although the human integrin ectodomains were heterologously expressed by Drosophila cells, both sunbunits were correctly processed proteolytically, because the leader sequences were cleaved off to give the N termini of both mature human subunits. Whereas the N-terminal amino acid sequence of mature human alpha 2 subunit was accessible to Edman degradation, the beta 1 subunit was N-terminally blocked. However, loss of signal sequence of the beta 1 subunit and its subsequent N-terminal blockage reaction to a pyroglutamate residue also occurs in the homologously expressed human beta 1 subunit, indicating a correct processing of the soluble human beta 1 integrin subunit by the insect cells. Furthermore, the ectodomain heterodimer of the collagen receptor alpha 2beta 1 integrin binds avidly to monomeric type I collagen, suggesting that the integrin is not only correctly processed but also correctly folded. The binding signals of soluble alpha 2beta 1 to different types of collagen and laminin-1 demonstrated a ligand preference similar to the wild-type alpha 2beta 1 integrin, with decreasing binding signals in the order of type I, type II, type IV, and type V collagen and laminin-1 (25, 26). Binding affinities of soluble alpha 2beta 1 integrin could be increased with Mn2+ ions and an activating antibody 9EG7, an observation that resembles the activity regulation of wild-type alpha 2beta 1 integrin.

A great advantage of the soluble alpha 2beta 1 integrin in comparison with the membrane-bound wild-type alpha 2beta 1 integrin, which was isolated by extracting blood platelets with the mild detergent octylglucoside, is its stability and the comparatively high yield. Whereas the detergent-extracted wild-type alpha 2beta 1 integrin lost activity within days after preparation,2 the soluble alpha 2beta 1 integrin remained stable for several weeks. However, we have not found any explanation for this observation yet.

Having sufficient amounts of a stable, soluble alpha 2beta 1 integrin at hand, we could address the question of which component of the hemorrhagic snake venom of C. rhodostoma is responsible for inhibiting the interaction of alpha 2beta 1 integrin with type I collagen. This interaction is of major importance for platelet reactions to collagen. Collagen becomes accessible to platelets after damage of the blood vessels or tissue injury. It initiates the activation of platelets (8, 30, 33), which results in degranulation and an increase in number and/or affinity of other cell adhesion molecules on the platelet, such as the major platelet integrin alpha IIbbeta 3 (3), which eventually leads to platelet aggregation and blood clotting. RGD-containing disintegrins, such as rhodostomin (kistrin) from C. rhodostoma (27, 34), inhibit the interaction of the RGD-dependent alpha IIbbeta 3 integrin with fibrin, thereby impairing a later step in the blood clotting mechanism. Moreover, snake venoms contain several proteases, such as the metalloprotease rhodostoxin (kistomin and major hemorrhagin) (27, 28), and the serine protease ancrod (29) from C. rhodostoma, which cleave fibrinogen/fibrin or prothrombin, thus again interfering in the blood clotting cascade and resulting in bleeding and hemorrhages.

Being the initial step of collagen-triggered platelet activation and aggregation, alpha 2beta 1 integrin is of paramount importance in hemostasis (9, 10, 17). Previous studies with the hemorrhagic snake venom of C. rhodostoma were performed on whole platelets. However, platelets contain various collagen receptors on their surface with different characteristics, e.g. alpha 2beta 1 integrin (GPIa/IIa) and GPVI (9, 10, 17). Whereas GPVI mainly recognizes type I collagen molecules, which are bundled into collagen fibrils, in a divalent cation-independent manner, alpha 2beta 1 integrin mainly binds to monomeric type I collagen molecules in the presence of divalent cations (35-37). The importance of alpha 2beta 1 integrin in normal hemostasis is corroborated by severe bleeding disorders in patients, who either lack the alpha 2beta 1 integrin receptor on their platelets (11) or who have developed inhibiting autoantibodies against the alpha 2 integrin subunit (12).

Possessing various collagen receptors and being easily activated by several stimuli other than collagen, such as ADP, thrombin, etc., whole platelets are rather coarse targets to screen hemorrhagic snake venoms for specific inhibitors to the alpha 2beta 1 integrin-collagen interaction, inasmuch as snake venoms by themselves contain a whole battery of various agents interfering with platelet activation and blood clotting. Based on such studies with whole platelets, rhodocytin/aggretin have been published to be an activator of alpha 2beta 1 integrin-mediated platelet aggregation (15, 31). However, we have isolated rhodocytin/aggretin, proved its identity by N-terminal sequencing, and could not see any interaction of rhodocytin with alpha 2beta 1 integrin. Nor could we observe any influence of rhodocytin on the integrin-collagen interaction. Therefore, a direct interaction of rhodocytin/aggretin with the soluble alpha 2beta 1 integrin ectodomain on the protein level can be ruled out. Alternatively, its effects on platelets aggregation may be caused by protein-carbohydrate interactions. Wild-type alpha 2beta 1 integrin on the platelets surface may differ from recombinantly expressed soluble alpha 2beta 1 integrin in their carbohydrate side chains, because Drosophila Schneider cells are unable to process the N-linked carbohydrate side chains of proteins from high mannose type into complex type carbohydrate antennary structures (18). Because rhodocytin/aggretin belongs to the family of C-type lectins bearing homology to the carbohydrate recognition domains of Ca2+-dependent lectins (31), it can be surmised that rhodocytin cross-links several alpha 2beta 1 integrins on the platelet surface via their carbohydrate side chains, thereby imitating the recruitment of integrins into focal contact-like structures, which eventually leads to platelet activation and aggregation. However, because both recombinantly expressed soluble alpha 2beta 1 integrin and wild-type alpha 2beta 1 integrin extracted from platelets that are likely to bear high-mannose type and complex-type N-linked carbohydrate side chains, respectively, fail to interact with rhodocytin/aggretin in our binding tests, even in the presence of Ca2+ ions, a mechanism involving a direct interaction of rhodocytin/aggretin with alpha 2beta 1 integrin to explain platelet activation and aggregation by rhodocytin can be considered very unlikely.

Without using whole platelets, we have established a cell-free inhibition assay as a tool to search for an inhibitor of the integrin-collagen interaction on the molecular level without any interfering cellular reactions that occur on platelets during or after their activation. Furthermore, the use of soluble alpha 2beta 1 integrin instead of detergent-extracted wild-type alpha 2beta 1 integrin even allows us to work not only in a cell-free but also in a detergent-free test system. Therefore, we could identify the disintegrin LMW-CI from C. rhodostoma venom, which specifically binds to alpha 2beta 1 integrin in an RGD-independent manner, thereby inhibiting the interaction of alpha 2beta 1 integrin with immobilized, monomeric type I collagen. N-terminal sequencing of LMW-CI revealed its identity with rhodocetin (16). We called this inhibitor low molecular weight Calloselasma inhibitor to distinguish it from another alpha 2beta 1 integrin inhibiting activity of the C. rhodostoma venom that was found in an earlier eluate fraction, i.e. higher molecular mass fraction, of a size exclusion column. However, we have not yet characterized the latter one, which we referred to as high molecular weight Calloselasma inhibitor. Although binding with high affinity and specificity, the LMW-CI/rhodocetin does not need any divalent cations to be bound by the integrin. Its native three-dimensional structure, which is stabilized by disulfide bridges, is essential for alpha 2beta 1 bindung. Unlike the other high affinity collagen ligands of alpha 2beta 1 integrin, LMW-CI/rhodocetin lacks a collagenous triple helical conformation. Nevertheless, it binds to alpha 2beta 1 integrin avidly and even competes with collagen efficiently. Because we had included protease inhibitors into the test assay and proved absence of protease activities in the LMW-CI/rhodocetin preparation by zymogram, the inhibitory effect of LMW-CI/rhodocetin is not caused by proteolytic activity.

LMW-CI/rhodocetin is a heterodimer with an apparent molecular mass of 27 kDa consisting of two subunits of 16 and 14 kDa, which are firmly attached. Despite lacking any covalent, intercatenary disulfide bridges (16), the two subunits remained associated under the harsh denaturation conditions of the SDS-PAGE sample buffer, containing 2% SDS. However, reduction of intracatenary disulfide bridges leads to destruction of the tertiary structure and subsequentially to the dissociation of the two subunits. Judging the native tertiary structure by its capability to bind to alpha 2beta 1 integrin, we found that the native tertiary structure of rhodocetin, which is required for integrin binding, is lost at much lower concentrations of reducing agents than the quartenary structure, detected as dissociation of the two subunits in SDS-PAGE under nonreducing conditions.

Interestingly, both rhodocytin/aggretin and LMW-CI/rhodocetin belong to a family of snake venom proteins that bear similarity to the carbohydrate recognition domain of C-type lectins (16, 31). However, platelet-derived wild-type alpha 2beta 1 integrin and recombinantly expressed, soluble alpha 2beta 1 integrin bind equally well to immobilized LMW-CI/rhodocetin, although the two integrins may vary in their glycosylation pattern of N-linked carbohydrate side chains, suggesting that the interaction of the novel disintegrin LMW-CI/rhodocetin bases on a protein-protein interaction. This direct interaction then causes the inhibition of collagen binding to alpha 2beta 1 integrin.

It is noteworthy that LMW-CI/rhodocetin does not bind to alpha 1beta 1 integrin, the other collagen-binding integrin with a widespread tissue distribution. Nor does this disintegrin interact with alpha 3beta 1 integrin. Therefore, LMW-CI/rhodocetin differs from other, mainly RGD-dependent disintegrins in its unique specificity toward alpha 2beta 1 integrin. Interestingly, LMW-CI/rhodocetin does not require divalent cations to bind to alpha 2beta 1 integrin, because, in contrast to other integrin ligands, deprivation of divalent cations by EDTA does not abolish alpha 2beta 1 binding to LMW-CI/rhodocetin. This suggests a binding mechanism distinct from the integrin binding mechanism to collagen. Nevertheless, LMW-CI can completely abolish alpha 2beta 1 integrin binding to collagen. Either LMW-CI binds at a site within alpha 2beta 1 integrin distinct from the ligand binding site that leads to a conformational change and to an allosteric inactivation of the integrin, or LMW-CI binds to a site within alpha 2beta 1 integrin, which is overlapping or even identical to the collagen binding site, thereby inhibiting collagen binding sterically. Future structural studies will help to answer this question.

As a prerequisite for its binding activity to alpha 2beta 1 integrin, LMW-CI indispensibly needs its native conformation, which is stabilized by disulfide bridges. Further structural insights into LMW-CI were gained by CD spectroscopy. The CD spectrum of LMW-CI is in good agreement with the CD spectrum provided by in Refs. 16. It clearly demonstrated that LMW-CI/rhodocetin does not bear any structural resemblance to a collagenous triple helix, which is typical of high affinity ligands of alpha 2beta 1 integrin. Still, LMW-CI/rhodocetin avidly binds to alpha 2beta 1 integrin and efficiently competes with type I collagen.

Having found and characterized LMW-CI/rhodocetin as snake venom disintegrin that specifically recognizes alpha 2beta 1 integrin and preventing it from binding to its collagen ligand, we eventually returned to whole cells to study the alpha 2beta 1-related functions in the cellular context. alpha 2beta 1 integrin not only is a pivotal trigger in hemostasis, but its widespread distribution on other cell types also suggests a much broader biological role in the organism (17, 38). The presence of alpha 2beta 1 integrin on endothelial cells of newly grown blood capillaries suggests a potential role in angiogenesis (39). Fibroblasts also bear alpha 2beta 1 integrin and use it to exert mechanical forces to a surrounding collagen gel, which in vivo takes place in connective tissue to maintain the shape of tissues and organs, during wound contraction and scar formation (40). Furthermore, ligand occupancy of alpha 2beta 1 integrin on fibroblasts and epithelial tumor cells elicits expression of various matrix metalloproteases (MMPs) (40), such as interstitial collagenase (MMP-1) (41, 42), stromelysin-1 (MMP-3) (43), collagenase-3 (MMP13), and membrane-bound metallomatrixproteinase-1 (MT1-MMP, MMP14) (44). The latter one itself proteolytically activates gelatinase A (MMP-2) (40). alpha 2beta 1 integrin-triggered secretion of MMPs is a key point in tumor invasion and metastasis, because these proteases degrade extracellular matrix proteins, among them basal membrane proteins, thus opening the path for invading tumor cells. To manipulate such alpha 2beta 1-triggered effects, LMW-CI/rhodocetin may be a valuable tool because of its unique specificity and high affinity toward alpha 2beta 1 integrin. Another advantage of LMW-CI/rhodocetin is its high solubility under physiological conditions compared with the poor solubility of collagen, which because of its high tendency to aggregate and precipitate cannot be applied as soluble inhibitor. Furthermore, because of its independence of divalent cations, LMW-CI is likely to bind in vivo as effectively as in the cell-free test, whereas alpha 2beta 1 integrin binds less avidly to collagen in vivo because of the presence of beta 1-integrins attenuating Ca2+ ions under physiological conditions. With HT1080 fibrosarcoma cells, which adhere to type I collagen mainly via alpha 2beta 1 integrin, we demonstrated that LMW-CI indeed inhibits cell adhesion to type I collagen as the initial step of integrin-mediated cell migration, gene activation, and anchorage-dependent growth. LMW-CI/rhodocetin efficiently and completely inhibits alpha 2beta 1-mediated cell adhesion to type I collagen, proving its suitability as specific alpha 2beta 1 integrin inhibitor in vivo. Thus, LMW-CI may be a useful agent to study and influence alpha 2beta 1 integrin-triggered cell function, like cell adhesion, cell migration, or secretion of MMPs. Therefore, it may help not only in treating thrombosis but also in treatments aimed to prevent tumor invasion and metastasis.


    ACKNOWLEDGEMENTS

Type IV collagen and its fragment CB3[IV], as well as type V collagen and laminin-1 were kindly provided by Klaus Kühn, Rupert Timpl, and Albert Ries (Max-Planck-Institute for Biochemistry, Martinsried, Germany). Type II collagen was gratefully obtained by Peter Bruckner (Institute for Physiological Chemistry, Münster, Germany). We also appreciated the monoclonal antibody JA218, a kind gift of Danny S. Tuckwell (School of Biological Sciences, Manchester, UK).


    FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft Grant Eb177/3-1 (to J. A. E.).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.

§ To whom correspondence should be addressed: Inst. für Physiologische Chemie und Pathobiochemie, Universität Münster, Waldeyerstr. 15, 48149 Münster, Germany. Tel.: 49-251-835-2289; Fax: 49-251-835-5596; E-mail: eble@uni-muenster.de.

Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009338200

4 J. A. Eble, unpublished observations.

2 Brackets indicate two possible amino acids that could not be clearly identified in the sequencing cycle. Parentheses indicate a less likely amino acid when the Edman degradation cycle did not give a clear identification but an option of two or more possible amino acids.

3 See footnote 2. A hyphen indicates that no amino acid could be detected in the respective cycle of Edman degradation.


    ABBREVIATIONS

The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; MMP, matrix metalloprotease.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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