Vipera lebetina Venom Contains Two Disintegrins Inhibiting Laminin-binding {beta}1 Integrins*

Johannes A. Eble {ddagger} §, Peter Bruckner {ddagger} and Ulrike Mayer ¶

From the {ddagger}Institute for Physiological Chemistry, Münster University Hospital, 48149 Münster, Germany and the Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Received for publication, February 21, 2003 , and in revised form, April 22, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To explain the myotoxic effects of snake venoms, we searched for inhibitors of {alpha}7{beta}1 integrin, the major laminin-binding integrin in skeletal muscle. We discovered two inhibitors in the venom of Vipera lebetina. One of them, lebein-1 (known as lebein), has already been proposed to be a disintegrin because of its RGD-containing primary sequence. The other, lebein-2, is a novel protein that also interacts firmly with {alpha}3{beta}1, {alpha}6{beta}1, and {alpha}7{beta}1 integrins, but not with the collagen-binding {alpha}1{beta}1 and {alpha}2{beta}1 integrins. Ligand binding of laminin-recognizing {beta}1 integrins was efficiently blocked by both lebein-1 and lebein-2. In cell attachment assays, lebein-1 and lebein-2 inhibited myoblast attachment not only to laminin, but also to fibronectin. However, neither lebein-1 nor lebein-2 interacted with {alpha}7{beta}1 integrin in an RGD-dependent manner, similar to the interaction of the laminin with {alpha}7{beta}1 integrin. Identical divalent cation dependence of integrin binding to laminin and to either of the two inhibitors and their mutually exclusive binding suggest that both lebein-1 and lebein-2 interact with the ligand-binding site of laminin-binding {beta}1 integrins by mimicking the yet unknown integrin-binding structure of laminins. Like lebein-1, lebein-2 is a soluble heterodimeric disintegrin of low molecular mass. Together with membrane-bound ADAM-2 and ADAM-9, the two inhibitors seem to form a small group of disintegrins that can bind to laminin-binding {beta}1 integrins. Because of their inhibitory capability both in vitro and in vivo, lebein-1 and lebein-2 may be valuable tools in influencing laminin-induced, integrin-mediated cell functions such as cell anchorage, migration, and mechanical force transduction on laminin-rich basement membranes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are cell adhesion molecules consisting of two genetically non-related {alpha} and {beta} subunits (reviewed in Refs. 1 and 2). Different combinations of these subunits give rise to 24 known integrin receptors with distinct ligand-binding specificities. The {beta}1 subunit-containing integrins are receptors mainly for extracellular matrix (ECM)1 proteins such as collagen, laminin, and fibronectin and are responsible for cell anchorage and motility. Moreover, by transducing mechanical forces between cells and the ECM, integrins maintain the tension within tissue and, consequently, tissue shape and structure. At the myotendinous junction, {beta}1 integrins, especially {alpha}7{beta}1 integrin, are thought to play a pivotal role (3, 4). Disruption of the {alpha}7 integrin gene leads to progressive muscle dystrophy, whereas the conditional knockout of the {beta}1 integrin subunit in cardiac muscle results in lethal cardiac failure (4). However, other integrins such as {alpha}3{beta}1, {alpha}6{beta}1, and {alpha}6{beta}4 (5) and non-integrin receptors such as {alpha}-dystroglycan (6) also bind to laminins, albeit with different preferences for various laminin isoforms (7, 8).

Laminins are major constituents of basement membranes (9, 10), and their isoform compositions vary with the tissue. The basement membrane of the myotendinous junction is enriched in laminin-2 (11). Merely differing in its {alpha} chain, laminin-2 has the {beta}1 and {gamma}1 chains in common with laminin-1. The networks of both laminin and type IV collagen, together with smaller proteins such as nidogen and proteoglycans, form the molecular basis of the basement membrane. The laminin meshwork of the basement membrane allows integrin-mediated anchorage of muscle fibers and other cells (9, 10). In addition, basement membranes separate the connective tissue from any other tissue. Only certain cells such as leukocytes and invasively growing tumor cells are able to penetrate through the basement membrane. Other processes such as wound healing require cell movement along the basement membrane. Cell migration through and along the basement membrane is mediated by laminin-binding integrins (5, 12).

Some snake venoms contain components that are selectively directed against integrins. These so-called disintegrins interfere with the ability of integrins to bind to their cognate ECM ligands. Hence, cell/matrix interactions are disrupted, and tissue integrity is destroyed. However, snake venoms are known to exert several different effects on snakebite victims, among which are failure of the cardiovascular system and damages to muscle and neural tissue. In addition to disintegrins, these pleiotropic effects are caused by a variety of venom proteins. Being abundant in most snake venoms, phospholipase A2 is mainly responsible for myotoxic effects (13, 14). Various proteases of snake venoms specifically cleave blood-clotting factors or components of the ECM, thus resulting in bleeding dysfunctions and tissue necrosis (1517).

Most disintegrins known to date contain an RGD sequence mimicking the ligands for the platelet {alpha}IIb{beta}3 and other RGD-dependent integrins (18), which cause failure of {alpha}IIb{beta}3 integrin-mediated platelet aggregation and blood clotting. Moreover, disintegrins are not limited to snake venoms, but are also found as domains in ADAM (a disintegrin and a metalloproteinase) family members, multidomain proteins that are abundant in a wide range of animal species (15, 19). ADAM proteins play important roles in various biological processes such as fertilization and cell/cell interactions and proteolytic processing (19). Whereas few ADAM proteins interact with integrins (1921), snake venom disintegrins interfering with integrin/laminin interaction have not been described so far.

From the venom of Vipera lebetina, we have purified and characterized two inhibitors that target laminin-binding integrins. One of them is the recently described lebein. Although it has been proposed to be a disintegrin for RGD-dependent integrins (22), we show in this work that lebein or lebein-1, as we refer to it, also avidly binds to the laminin-binding {beta}1 integrins in an RGD-independent manner. In addition, we isolated a new disintegrin (lebein-2) with homology to lebein-1 and similar binding specificity. The interaction of both inhibitors with laminin-binding {beta}1 integrins both in vitro and in in vivo cell attachment assays demonstrates their potential to manipulate integrin-dependent cell/laminin interactions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Production of Soluble Integrins—For the construction of pUC-hygMT-{alpha}7X2-Fos, the {alpha}7 integrin cDNA was amplified by reverse transcription-PCR from RNA isolated from mouse myotubes and verified by sequencing. The {alpha}7X2 integrin ectodomain cDNA encoding amino acids 1–1036 was generated by PCR, introducing an additional SalI site at the 3'-end. The {alpha}3 integrin ectodomain-coding cDNA of the previously described vector pUC-hygMT-{alpha}3-Fos (7) was replaced by the {alpha}7X2 integrin ectodomain.

The construct pUC-hygMT-{alpha}6X1-Fos, coding for the {alpha}6 integrin ectodomain splice variant X1 (amino acids 1–989 of the published sequence) (23), was generated by isolating the HindIII- and XbaI-flanked cDNA fragment encoding amino acids 1–938 of the {alpha}6 integrin ectodomain from pRC-CMV-{alpha}6X1 (kindly provided by Dr. A. Sonnenberg, The Netherlands Cancer Institute, Amsterdam, The Netherlands) and by synthesizing an XbaI- and SalI-flanked cDNA fragment encoding amino acids 939–989 of the {alpha}6 integrin ectodomain by standard PCR. Taking advantage of the SalI site, the two cDNA fragments were directionally inserted into the pUC-hygMT-{alpha}3-Fos vector (7) from which the {alpha}3 integrin ectodomain-coding cDNA had been removed.

The vector pUC-hygMT-{beta}1-Jun was generated previously (7). Together with the pUC-hygMT-{beta}1-Jun vector, the vectors pUC-hygMT-{alpha}7X2-Fos and pUC-hygMT-{alpha}6X1-Fos were cotransfected into Drosophila Schneider's cells, and single cell clones were established as described previously (7, 24). Briefly, positive clones secreting {alpha}6{beta}1 and {alpha}7{beta}1 integrins were detected in a sandwich ELISA using monoclonal antibody GoH3 (kindly provided by Dr. A. Sonnenberg) and affinity-purified anti-Fos peptide antiserum, respectively, as capturing antibodies.

Clones G42 and ASH6, producing soluble {alpha}7X2{beta}1 and {alpha}6X1{beta}1 integrins, respectively, were grown as described previously (7). Both integrins were isolated by affinity chromatography with the immobilized cell-binding domain of invasin (7). Protein concentration and purity were determined by the BCA assay (Pierce) and SDS-PAGE, respectively.

Soluble {alpha}2{beta}1 and {alpha}3{beta}1 integrins were generated and isolated as described previously (7, 24). The generation and isolation of soluble {alpha}1{beta}1 integrin will be described elsewhere.2

Integrin Inhibition ELISA—To screen for inhibition of integrin-mediated laminin binding, microtiter plates were coated with 6 µg/ml murine laminin-1 (kindly provided by Dr. R. Timpl, Max Planck Institute for Biochemistry, Martinsried, Germany) or 10 µg/ml human laminin-2/4 (Merosin, Invitrogen, Karlsruhe, Germany) in Tris-buffered saline (TBS), pH 7.4, containing 1 mM MgCl2 (TBS/MgCl2 buffer) overnight at 4 °C. Nonspecific protein-binding sites were blocked with 1% heat-denatured bovine serum albumin (BSA) in TBS/MgCl2 buffer for 2 h at room temperature. Together with protease inhibitors, phenylmethylsulfonyl fluoride, and 1,10-phenanthroline (each at 1 mM) and aprotinin, leupeptin, and pepstatin (each at 3 µg/ml), soluble {alpha}7{beta}1 integrin was added at a concentration of 15 nM in 1% heat-denatured BSA in TBS, pH 7.4, containing 2 mM MgCl2 and 1 mM MnCl2, either without any supplements (positive control) or with 10 mM EDTA (nonspecific binding, negative control) or with snake venom solutions of 2 mg/ml. Lyophilized snake venoms were purchased from the Berchtesgadner Schlangenfarm (Berchtesgaden, Germany) and Sigma. After 2 h of incubation at room temperature, wells were washed twice with 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, and 1 mM MnCl2. Bound {alpha}7{beta}1 integrin was fixed with 2.5% glutaraldehyde in the same buffer for 10 min at room temperature and detected in an ELISA procedure using rabbit antiserum directed against the human {beta}1 integrin subunit (1: 400; kind gift of Dr. K. Kühn, Max Planck Institute for Biochemistry) and an alkaline phosphatase-conjugated anti-rabbit IgG antibody (1: 600; Sigma) as described previously (7). Nonspecific binding was measured in the presence of 10 mm EDTA and subtracted from all values. For the calculation of the relative inhibitory activity, binding signals in the presence of inhibitor were normalized to the non-inhibited control.

Isolation of Lebein-1 and Lebein-2—Lyophilized venom of V. lebetina was dissolved in 20 mM sodium phosphate, pH 6.5, 50 mM sodium chloride, and 1 mM EDTA and separated by gel filtration on a Superose 6 column (Amersham Biosciences AB, Uppsala, Sweden) in the same buffer. The fractions containing the {alpha}7{beta}1 integrin-inhibiting fractions were pooled; diluted with 20 mM MES, pH 6.0; and loaded onto a Mono S column (Amersham Biosciences AB). The {alpha}7{beta}1 integrin inhibitory activities of lebein-2 and lebein-1 were eluted in linear sodium chloride gradients at 80 and 140 mM, respectively. The eluate fractions were individually pooled and separated on a C8 reversed-phase column (Nucleosil, Macherey Nagel) in a linear gradient from 0.1% trifluoroacetic acid in water to 80% acetonitrile in 0.08% trifluoroacetic acid/water. Lebein-1 and lebein-2 were eluted as individual peaks, lyophilized, and dissolved in water. Protein concentration was determined by the BCA assay. Purity was proven by SDS-PAGE.

Far-Western Blotting—After separation by SDS-PAGE, snake venom proteins were blotted onto nitrocellulose membrane (Schleicher & Schüll). After nonspecific binding had been blocked with 1% heat-denatured BSA in TBS/MgCl2 buffer, 50 nM soluble {alpha}7{beta}1 integrin was added in the same solution supplemented with 1 mM MnCl2 for 2 h at room temperature. The membrane was washed twice with 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM MgCl2, and 1 mM MnCl2. Bound {alpha}7{beta}1 integrin was fixed with 2.5% glutaraldehyde for 10 min and detected by rabbit antiserum against the human {beta}1 integrin subunit (1:400) and subsequently by an alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody (1:700). Both antibodies were applied in 1% heat-denatured BSA in TBS/MgCl2 buffer, followed by three washes with TBS/MgCl2 buffer. The overlay blot was developed with 5-bromo-4-chloro-3-indolyl phosphate /nitro blue tetrazolium solution (Sigma).

Binding of Integrins to Lebein-1 and Lebein-2—Lebein-1 and lebein-2 (each at 3 µg/ml in 20 mM sodium phosphate buffer, pH 7.0), the CB4 fragment of human type IV collagen (10 µg/ml in TBS/MgCl2 buffer), murine laminin-1 (10 µg/ml in TBS/MgCl2 buffer), human laminin-5 (10 µg/ml in TBS/MgCl2 buffer), and bovine type I collagen (20 µg/ml in 0.1 M acetic acid) were immobilized onto microtiter plates overnight at 4 °C. After a blocking step, soluble integrins were added at 50 nM in 1% heat-denatured BSA in TBS/MgCl2 buffer in the presence of either 1 mM MnCl2 or 10 mM EDTA. {alpha}2{beta}1 integrin was further activated by addition of the {beta}1 integrin-activating antibody 9EG7 (kindly provided by Dr. D. Vestweber, Center for Molecular Biology of Inflammation, Münster, Germany) (25). Bound integrin was fixed and detected in an ELISA-type procedure as described before.

RGD Peptide Inhibition Assay—Lebein-1 and lebein-2 (each at 3 µg/ml in 20 mM sodium phosphate buffer, pH 7.0) and murine laminin-1 (at 6 µg/ml in TBS/MgCl2 buffer) were coated onto microtiter plates overnight at 4 °C. After washing and blockage with 1% BSA in TBS/MgCl2 buffer, soluble {alpha}7{beta}1 integrin at 10.4 µg/ml (for lebein-1 and lebein-2 binding) and 1.3 µg/ml (for laminin binding) was added to the wells in the presence of peptides GRGDS, GRGES, and GRDGS (Bachem Biochemica GmbH, Heidelberg, Germany) for 2 h. Bound integrin was detected as described above.

Cell Adhesion Inhibition Tests—Microtiter plates were coated with fibronectin (gift of Dr. M. Humphries, University of Manchester) and murine laminin-1 at 10 and 40 µg/ml, respectively, overnight at 4 °C. After blocking for 6 h with 1% BSA in water, 100 µl of a C2C12 cell suspension (0.8 x 106 cells/ml) in Dulbecco's modified Eagle's medium without fetal calf serum and containing a 1:3 serial dilution of lebein-1 and lebein-2 were added to the wells and incubated for 45 min at 37 °C in a humidified 5% CO2 incubator. After the supernatant had carefully been discarded, attached cells were washed with phosphate-buffered saline and fixed for 10 min at room temperature with 70% ethanol. After air-drying, cells were stained with 0.1% crystal violet. For 100% binding values, lebein-1 and lebein-2 were replaced by an equal volume of Dulbecco's modified Eagle's medium. Three experiments with duplicate measurements were carried out.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant Production of a Soluble {alpha}7{beta}1 Integrin—{alpha}7X2{beta}1 integrin is the major laminin receptor in adult skeletal muscle. To establish a protein interaction assay, we first generated soluble {alpha}7X2{beta}1 integrin, similar to the recombinant production of soluble {alpha}3{beta}1 and {alpha}2{beta}1 integrins (7, 24). The {alpha}7X2-Fos/{beta}1-Jun integrin ectodomain heterodimer, hereafter called soluble {alpha}7{beta}1 integrin, consists of the ectodomains of both integrin subunits, to the C termini of which the dimerizing motifs of the transcription factors Fos and Jun have been fused. Although the cDNAs of the {alpha}7X2 and {beta}1 chains were from murine and human origins, respectively, the integrin heterodimer is formed and secreted by the insect cells with an expression yield (~600 µg/liter) comparable to that of soluble {alpha}3{beta}1 integrin (7). Soluble {alpha}7{beta}1 integrin was purified by affinity chromatography on a resin to which the cell-binding domain of invasin had been immobilized (Fig. 1A).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 1.
Isolation and characterization of soluble {alpha}7-Fos/{beta}1-Jun integrin, alias soluble {alpha}7{beta}1 integrin. A, SDS-PAGE of purified soluble {alpha}7{beta}1 integrin. Purified soluble {alpha}7{beta}1 integrin (1 µg) was separated on a 7.5–15% gradient polyacrylamide gel without (lane 1) and with (lane 3) prior reduction. Lane 2 contains molecular mass standards. HC, heavy chain; LC, light chain. B, binding of soluble {alpha}7{beta}1 integrin to immobilized ligands. Murine laminin-1 (mLN-1), human laminin-2/4 (hLN-2/-4), and human laminin-5 (hLN-5) (each at 10 µg/ml) and the cell-binding domain of invasin (MBP-invasin) (at 2 µg/ml) were coated onto microtiter plates and incubated with 15 µg/ml soluble {alpha}7{beta}1 integrin in the presence of 1 mM MnCl2 (hatched bars), 1 mM MnCl2 and the {beta}1 integrin-activating antibody 9EG7 (cross-hatched bars), or 10 mM EDTA (open bars). Bound integrin was detected as described under "Materials and Methods." Means ± S.D. of duplicate determinations are shown. C, titration of immobilized laminin-1 (LN-1; open symbols) and laminin-2/4 (LN-2/-4; closed symbols), coated at 10 µg/ml, with soluble {alpha}7{beta}1 integrin in the presence of 1 mM MnCl2 (circles), 1 mM MnCl2 and 9EG7 (squares), or 2 mM CaCl2 (triangles). A titration curve representative of at least two experiments is depicted. Means ± S.D. of duplicate measurements are shown.

 

The biological activity of the soluble {alpha}7{beta}1 integrin was tested qualitatively and quantitatively on different ECM substrates. As shown in Fig. 1B, {alpha}7{beta}1 integrin showed a higher binding signal for laminin-1 than for laminin-2/4. Laminin-2 is the most abundant laminin isoform of basement membranes in muscle tissue. In contrast, laminin-5 was not recognized. As expected from the purification protocol, invasin, a surface protein of Yersinia bacteria, also interacted with soluble {alpha}7{beta}1 integrin (Fig. 1B). Like other integrins, {alpha}7{beta}1 integrin is activated for ligand binding by Mn2+ ions. However, unlike other integrins, this activation seems sufficient, as addition of the integrin-activating antibody 9EG7, directed against the {beta}1 integrin subunit (25), did not increase the binding further. In the presence of EDTA, binding to any ligand was completely abolished, thus confirming the dependence of {alpha}7{beta}1 integrin on divalent cations (Fig. 1B).

Binding of soluble {alpha}7X2{beta}1 integrin to immobilized laminin-1 and laminin-2/4 was further studied by an ELISA-type titration assay, from which apparent Kd values could be calculated according to Heyn and Weischet (26). As shown in Fig. 1C, specific binding of soluble {alpha}7{beta}1 integrin to the immobilized laminin isoforms reached saturation. In the presence of Mn2+ ions, {alpha}7{beta}1 integrin bound more avidly to laminin-1 (Kd = 0.2 nM) than to laminin-2/4 (Kd = 2.3 nM). Addition of the activating antibody 9EG7 did not shift the titration curves to lower {alpha}7{beta}1 integrin concentrations and did not alter its apparent affinity constants significantly. However, Ca2+ ions reduced {alpha}7{beta}1 integrin binding to both laminin-1 and laminin-2/4, thus increasing the apparent Kd values by ~15-fold (Fig. 1C).

Screening Various Snake Venoms for Their Capability to Interfere with {alpha}7{beta}1 Integrin Binding to Laminin-1—Playing a key role in skeletal muscle, {alpha}7{beta}1 integrin conceivably is a target for myotoxic snake venoms. Because of its high affinity for {alpha}7{beta}1 integrin, we chose laminin-1 as its interaction partner to search for a snake venom inhibitor to this interaction (Table I). Crude snake venoms and soluble {alpha}7{beta}1 integrin were added to laminin-1-coated microtiter plates. Protease inhibitors were also supplemented to avoid proteolytic degradation of integrin or its ligand by the numerous proteases that are abundant in snake venoms. Of 33 snake venoms tested that may cause severe muscular dysfunctions, such as venoms of the Elapidae, Viperidae, and Crotalidae families, only the venom of V. lebetina showed a drastic abolition of {alpha}7{beta}1 integrin binding to laminin-1 (Table I). Titration of the V. lebetina venom demonstrated that it suppressed {alpha}7{beta}1 integrin binding entirely to both laminin-1 and laminin-2/4 in a dose-dependent manner (Fig. 2).


View this table:
[in this window]
[in a new window]
 
TABLE I
Inhibition of {alpha}7{beta}1 integrin binding to laminin-1 by Elapidae, Viperidae, and Crotalidae snake venoms

Murine laminin-1 was coated at 10 µg/ml onto microtiter plates. Soluble {alpha}7{beta}1 integrin was added at 3.9 µg/ml in the presence of 2 mg/ml snake venom and protease inhibitors. Bound integrin was quantified as described under "Materials and Methods." Means ± S.D. of duplicate determinations are shown. Reduction of integrin binding to <50% indicated a strong inhibition by the venom.

 


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 2.
Dose-dependent inhibition of {alpha}7{beta}1 integrin binding to laminin-1 and laminin-2/4 by V. lebetina venom. Murine laminin-1 (LN-1; {square}) and human laminin-2/4 (LN-2/-4; {circ}) (each at 10 µg/ml) were immobilized and incubated with soluble {alpha}7{beta}1 integrin (3.9 µg/ml) in the presence of crude venom of V. lebetina at the indicated concentrations. Bound integrin was detected as described under "Materials and Methods." Means ± S.D. of duplicate measurements are shown.

 

Purification of the {alpha}7{beta}1 Integrin Inhibitory Activity of the V. lebetina Venom—To purify the {alpha}7{beta}1 integrin inhibitor, the venom was separated by gel filtration, ion-exchange chromatography, and reversed-phase chromatography. From the gel filtration column, the {alpha}7{beta}1 integrin inhibitory activity was eluted in a single peak (Fig. 3A, gray bar). However, further purification on a Mono S column separated the inhibitory activity into two different fractions, called MS-I and MS-II (Fig. 3B). Each of these two inhibitory peaks, MS-I and MS-II, showed a characteristic elution profile upon reversed-phase chromatography (Fig. 4, A and B). Fraction MS-I could not further be separated by reversed-phase chromatography and contained only one inhibitor that eluted as a single peak (Fig. 4A).



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 3.
Purification of the {alpha}7{beta}1 integrin-inhibiting factor from V. lebetina venom by gel filtration (A) and cation-exchange chromatography on a Mono S cation-exchange column (B). The absorbance of the eluate was measured at 280 nm (solid lines). Eluate fractions were tested for their capacity to inhibit {alpha}7{beta}1 integrin binding to immobilized laminin-1 ({circ}). The {alpha}7{beta}1 integrin-blocking inhibitors were eluted from the gel filtration column as a single peak, marked by the gray bar in A. The two inhibitors were separated by Mono S ion-exchange chromatography into two distinct peaks, marked by the gray bars labeled MS-I and MS-II in B. Fraction MS-I contained lebein-2, whereas fraction MS-II was highly enriched in lebein-1.

 


View larger version (35K):
[in this window]
[in a new window]
 
FIG. 4.
Purification of lebein-2 and lebein-1 from the two Mono S fractions (MS-I and MS-II) by reversed-phase chromatography on a C8 reversed-phase column. The absorbance of the eluate was monitored at 280 nm (solid lines). Eluate fractions were tested for inhibition of {alpha}7{beta}1 integrin binding to immobilized laminin-1 ({circ}). A, the Mono S fraction MS-I contains one inhibitory activity, which is the novel disintegrin lebein-2. B, the Mono S fraction MS-II contains two peaks of inhibitory activities. At lower acetonitrile concentrations, lebein-1 was eluted, whereas lebein-2 required higher acetonitrile concentrations to be eluted.

 

After reversed-phase chromatography, the major inhibitory peak of fraction MS-II (Fig. 4B) was identified as lebein, a recently discovered disintegrin (22), which we refer to as lebein-1. Edman degradation of lebein-1 revealed two staggered N-terminal sequences, MNGSNPXXD and NGSNPXXD, at a 2:1 ratio. Except for the initial methionine residues, this sequence is identical to the published primary sequence of lebein (Swiss-Prot accession number P83253 [GenBank] ) (22). Furthermore, mass spectrometry determined its mass to be 14,083 Da and proved its identity to lebein. Upon SDS-PAGE, the heterodimeric lebein-1 showed an apparent molecular mass of 18 kDa. After reduction, it was separated by SDS-PAGE into two highly homologous subunits, {alpha} and {beta}, with apparent molecular masses of ~14 kDa (Fig. 5A). The difference in molecular mass determined by SDS-PAGE and mass spectrometry is likely due to the fact that the molecular masses of proteins below 20 kDa show a nonlinear behavior on polyacrylamide gels. Furthermore, the nonlinearity of the apparent molecular masses of the heterodimeric protein and its subunits upon SDS-PAGE can be explained by the fact that the Stoke radius of the two closely associated subunits within the nonreduced heterodimeric disintegrin is likely to be smaller than the sum of the Stoke radii of the individual unfolded subunits after reductive cleavage of disulfide bridges.



View larger version (36K):
[in this window]
[in a new window]
 
FIG. 5.
SDS-PAGE (A) and {alpha}7{beta}1 integrin overlay assay (B) of purified V. lebetina toxins. Equal volumes of fractions MS-I and MS-II and purified lebein-2 and lebein-1 (2 µg of each) were separated under nonreducing (without {beta}-mercaptoethanol (w/o {beta}-ME)) and reducing (with {beta}-mercaptoethanol) conditions. The gel was either stained with Coomassie (A) or blotted onto a nitrocellulose membrane to detect {alpha}7{beta}1 integrin-binding proteins on the far-Western blot (B). The molecular masses of standard proteins as well as the bands of lebein-1 and lebein-2 are indicated. The labels refer to both panels.

 

The inhibitory activity of fraction MS-I (Fig. 4A) was identified as a novel protein from V. lebetina venom. Despite its high sequence identity, its N-terminal sequence (MNSANPXXDDI), especially residues alanine and isoleucine in positions 4 and 11, respectively, clearly discriminated this inhibitor from lebein-1. Furthermore, its molecular mass of 14,735 Da, as determined by mass spectrometry, differed from that of lebein-1. The different molecular mass of lebein-2 was also observed upon SDS-PAGE (Fig. 5A). Upon reduction, the novel V. lebetina inhibitor with an apparent molecular mass of 20 kDa dissociated into two subunit chains ({alpha} and {beta}) with apparent molecular masses of 15 and 7 kDa, respectively (Fig. 5A). Edman degradation revealed that its {alpha} subunit was N-terminally blocked, whereas the N-terminal amino acid sequence of the {beta} subunit was proven to be identical to the N terminus of the nonreduced protein. This amino acid sequence has not been published yet. Because of its sequence homology to lebein and similar binding affinities, we propose the name lebein-2 for it. Thus, lebein-2 is a 14,735-Da heterodimeric protein consisting of two subunits, {alpha} and {beta}, the latter one of which has the N-terminal amino acid sequence MNSANPXXDDI.

Lebein-1 and Lebein-2 are the {alpha}7{beta}1 Integrin-binding Components of V. lebetina Snake Venom—A far-Western blot was established to test the fractions of V. lebetina snake venom for {alpha}7{beta}1 integrin-binding components (Fig. 5B). After electrophoretic separation, snake venom components were transferred onto a nitrocellulose membrane. Soluble {alpha}7{beta}1 integrin was allowed to bind to the blotted snake venom proteins and detected immunologically. The strongest signal was observed with purified lebein-2 (Fig. 5B). Remarkably, an intense signal was detected not only with the lebein-2 monomer at 20 kDa, but also with a band at ~30 kDa, which may be the aggregate of two lebein-2 molecules. In contrast, this band with an apparent molecular mass of 30 kDa was hardly visible on the Coomassie-stained SDS-polyacrylamide (Fig. 5A). The additional band at ~43 kDa, which was seen only on the far-Western blot, could not be identified yet, but might be an even higher aggregate of lebein-2. The far-Western blot (Fig. 5B) provided the first evidence that lebein-2 binds {alpha}7{beta}1 integrin directly. This interaction seemed to be specific and depended on the disulfide bridge-stabilized quaternary and/or tertiary structure of lebein-2 because reduction of lebein-2 entirely abolished the binding signal on the far-Western blot (data not shown).

Lebein-1 also bound {alpha}7{beta}1 integrin in the far-Western assay only under nonreducing conditions (Fig. 5B), albeit with a weaker binding signal than lebein-2. However, far-Western blot assays can be assessed only qualitatively, as protein interaction may be weakened because of only partial renaturation of the blotted proteins after SDS treatment. The Mono S fraction MS-II, which contains lebein-1 (Fig. 5B, MS-II lane), included an additional band at an apparent molecular mass of 44 kDa, which was also recognized by {alpha}7{beta}1 integrin. This may be the precursor of lebetase, a snake venom metalloproteinase that also possesses a disintegrin domain highly homologous to lebein-1 (22, 27). Indeed, this band showed proteolytic activity in a gelatin zymogram (data not shown). Our data suggest that the lebetase precursor is also able to interact with {alpha}7{beta}1 integrin presumably via its disintegrin domain.

Lebein-2 and Lebein-1 Are Disintegrins That Bind to the Laminin-binding {beta}1 Integrins—To further analyze whether binding of lebein-2 and lebein-1 is specific for {alpha}7{beta}1 integrin, we investigated their integrin-binding spectra. To this end, lebein-2 and lebein-1 were immobilized, and the soluble integrins ({alpha}1{beta}1, {alpha}2{beta}1, {alpha}3{beta}1, {alpha}6{beta}1, and {alpha}7{beta}1) were tested for their binding. Although all integrins showed binding activity for their cognate ECM ligands such as the CB3 fragment of type IV collagen, type I collagen, laminin-5, and laminin-1, only the laminin-binding integrins ({alpha}3{beta}1, {alpha}6{beta}1, and {alpha}7{beta}1) showed a clear binding signal for immobilized lebein-2 and lebein-1, which depended on the presence of divalent cations (Fig. 6). Soluble {alpha}1{beta}1 integrin also showed a slight binding signal for both disintegrins, which, however, was considered nonspecific due to its persistence in the presence of EDTA (Fig. 6, open bars). Antibody 9EG7 increased the binding signals of {alpha}3{beta}1 and {alpha}6{beta}1 (but not {alpha}7{beta}1) integrins for both disintegrins (Fig. 6). These binding analyses demonstrated that lebein-2 and lebein-1 are disintegrins that recognize the laminin-binding {beta}1 integrins, but not the collagen-binding integrins ({alpha}1{beta}1 and {alpha}2{beta}1), in a divalent cation-dependent manner.



View larger version (47K):
[in this window]
[in a new window]
 
FIG. 6.
Lebein-2 and lebein-1 are disintegrins that bind to laminin-binding integrins in a divalent cation-dependent manner. Lebein-2 and lebein-1 (each at 5 µg/ml) were immobilized and incubated with various soluble integrins ({alpha}1{beta}1 (no-pattern bars), {alpha}2{beta}1 (horizontally striped bars), {alpha}3{beta}1 (vertically striped bars), {alpha}6{beta}1 (hatched bars), and {alpha}7{beta}1 (cross-hatched bars)) in the presence of 1 mM MnCl2 (light-gray bars), 1 mM MnCl2 and 9EG7 (dark-gray bars), or 10 mM EDTA (white bars). The activity of each individual integrin was proven by binding to its cognate ligand as a positive control: the type IV collagen fragment CB3 for {alpha}1{beta}1, type I collagen for {alpha}2{beta}1, laminin-5 for {alpha}3{beta}1, and laminin-1 for {alpha}6{beta}1 and {alpha}7{beta}1. Means ± S.D. of duplicate determinations are shown.

 

Titration of immobilized lebein-2 and lebein-1 with soluble {alpha}7{beta}1 and {alpha}3{beta}1 integrins in the presence of divalent cations yielded titration curves that showed saturation (data not shown). Furthermore, linearization of these titration curves according to Heyn and Weischet (26) provided apparent affinity constants in the nanomolar range (Table II). Identical to laminin-1, both lebein-2 and lebein-1 bound avidly to soluble {alpha}7{beta}1 integrin in the presence of 1 mM Mn2+ ions without any marked alteration of affinities after addition of the activating antibody 9EG7. Ca2+ ions increased the apparent dissociation constants significantly. In addition, the presence of Ca2+ showed more clearly that lebein-1 bound more avidly to {alpha}7{beta}1 integrin compared with lebein-2. Although the affinities of {alpha}3{beta}1 integrin for both disintegrins are lower, the tendency of Mn2+ and Ca2+ ions to increase and to decrease, respectively, the affinity of soluble {alpha}3{beta}1 integrin for lebein-2 and lebein-1 resembled the effects on soluble {alpha}7{beta}1 integrin. In contrast to soluble {alpha}7{beta}1 integrin, binding of soluble {alpha}3{beta}1 integrin to both disintegrins could be substantially improved by the activating antibody 9EG7 (Table II).


View this table:
[in this window]
[in a new window]
 
TABLE II
Apparent Kd values for the interaction of soluble {alpha}7{beta}1 and {alpha}3{beta}1 integrins with lebein-2 and lebein-1

Lebein-2 and lebein-1 were coated at 3 µg/ml onto microtiter plates and titrated with either soluble {alpha}7{beta}1 or soluble {alpha}3{beta}1 integrin at a concentration range of 300-0.3 nM in the presence of 1 mM MnCl2, 1 mM MnCl2 and 9EG7, 2 mM CaCl2, or 10 mM EDTA. Bound integrins were quantified as described under "Materials and Methods." From these titration curves, apparent Kd values were calculated using a linearization algorithm according to Heyn and Weischet (26). Titration curves of at least two independent experiments were evaluated. Mean ± S.D. are shown.

 

Inhibition of Binding of {alpha}7{beta}1 and {alpha}3{beta}1 Integrins to Their Cognate Laminin Isoform Ligands—Both lebein-2 and lebein-1 bound to the laminin-binding {beta}1 integrins with the same divalent cation dependences as to the laminin ligands (Fig. 1C and Table II). Furthermore, both disintegrins completely and efficiently blocked binding of {alpha}7{beta}1 and {alpha}3{beta}1 integrins to their respective laminin isoform ligands in a dose-dependent manner (Fig. 7). Because of its higher affinity for both integrins, lebein-1 showed half-maximal inhibition at up to 3-fold lower concentrations than lebein-2 (Fig. 7 and Table III).



View larger version (20K):
[in this window]
[in a new window]
 
FIG. 7.
Lebein-2 and lebein-1 inhibit both {alpha}3{beta}1 and {alpha}7{beta}1 integrin binding to laminin. Laminin-1 (LN-1; {circ}) and laminin-2/4 (LN-2/-4; {square}) (each at 6 µg/ml) and human laminin-5 (LN-5; {triangleup}) (at 10 µg/ml) were coated onto microtiter plates. Laminin-1 and laminin-2/4 were incubated with soluble {alpha}7{beta}1 integrin (3.9 µg/ml), and laminin-5 was incubated with soluble {alpha}3{beta}1 integrin (18.2 µg/ml) in the presence of lebein-2 (A) and lebein-1 (B) at the indicated concentrations. Bound integrins were quantified as described under "Materials and Methods." Means ± S.D. of duplicate determinations are shown.

 

View this table:
[in this window]
[in a new window]
 
TABLE III
IC50 values for the inhibition of binding of {alpha}7{beta}1 and {alpha}3{beta}1 integrins to their cognate laminin isoform ligands by lebein-2 and lebein-1

IC50 values were determined as the disintegrin concentration that is necessary to inhibit the integrin binding signal for its ligand by 50%. Inhibition curves of at least two independent experiments were evaluated. Mean ± S.D. are shown.

 

RGD Peptides Interfere Only Weakly with {alpha}3{beta}1 and {alpha}7{beta}1 Integrin Binding to Lebein-1 and Lebein-2—Because at least lebein-1 is known to contain RGD sequences in both chains, we investigated whether the inhibitory activities of lebein-1 and lebein-2 on {alpha}7{beta}1 integrin depend on an RGD peptide sequence. To this end, binding of soluble {alpha}7{beta}1 integrin to immobilized laminin-1, lebein-1, and lebein-2 was challenged by increasing concentrations of RGD peptides or their derivatives (Fig. 8). The {alpha}7{beta}1 integrin/laminin-1 interaction was not affected by the GRGDS peptide even at a 1.6 million-fold molar excess (8 mM) to the integrin. In contrast, the binding of {alpha}7{beta}1 integrin to both lebein-1 and lebein-2 decreased at GRGDS peptide concentrations above 1 mM. However, this decline in binding was incomplete even at the very high peptide concentration of 8 mM. Furthermore, control peptides such as GRGES and the scrambled sequence GRDGS showed a similar, yet less pronounced decline in integrin binding to both disintegrins at high peptide concentrations above 1 mM. At RGD peptide concentrations below 1 mM, lebein-2 and lebein-1 bound to soluble {alpha}7{beta}1 integrin in an RGD-independent manner, similar to the natural integrin ligand. Hence, both lebein-2 and lebein-1 must mechanistically be considered RGD-independent disintegrins when interacting with laminin-binding {beta}1 integrins.



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 8.
Binding of {alpha}7{beta}1 integrin to lebein-2 and lebein-1 is only slightly affected by an RGD peptide sequence. Immobilized laminin-1 (LN-1; {blacksquare}) (at 6 µg/ml) as well as lebein-2 (white symbols) and lebein-1 (gray symbols) (each at 3 µg/ml) were incubated with soluble {alpha}7{beta}1 integrin at concentrations of 1.3 and 10.4 µg/ml, respectively, in the presence of peptide GRGDS (squares), GRGES (triangles), or GRDGS (circles) at the indicated concentrations. Bound integrin was quantified as described under "Materials and Methods." Means ± S.D. of duplicate determinations are shown.

 

Lebein-2 and Lebein-1 Affect Muscle Cell Interactions Not Only with Laminin, but also with Fibronectin—To examine the efficiency of the purified V. lebetina venom components on muscle cells, attachment of myoblasts to laminin and fibronectin was assessed in the presence of either lebein-2 or lebein-1 (Fig. 9). Both disintegrins inhibited cell attachment to laminin-1, presumably by blocking the interaction of {alpha}7{beta}1 integrin, although complete inhibition could not be achieved even at high disintegrin concentrations. Interestingly, cell adhesion to immobilized fibronectin was even more severely compromised by both lebein-2 and lebein-1. Most likely, this was caused by RGD sequences that are located in both chains of lebein-1 (22). The complete primary structure of lebein-2 (and hence the presence of RGD sequences within lebein-2) is not known yet.



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 9.
Effect of lebein-2 and lebein-1 on myoblast adhesion to laminin-1 and fibronectin. C2C12 myoblastic cells were plated at 0.8 x 106 cells/ml onto microtiter plates coated with fibronectin ({blacksquare}) and laminin-1 ({circ}) at 10 and 40 µg/ml, respectively. Lebein-2 (A) and lebein-1 (B) were added at the indicated concentrations. Attached cells were quantified in a colorimetric assay.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Among the various laminin-binding integrins, {alpha}7{beta}1 integrin plays an important role in the anchorage of muscles to their extracellular matrix, especially at the myotendinous junctions and intercalating disks of skeletal and cardiac muscle (3, 4). Snake venoms may contain inhibitors directed against {alpha}7{beta}1 integrin, thus causing muscle dysfunction and paralysis. However, other venom components such as phospholipase A2, which leads to necrosis of muscle and other tissue (13, 14), may also exert myotoxic and cytotoxic effects. Their presence precluded screening of whole snake venoms in cell tests. Therefore, we have established a cell-free screening assay in which we used a recombinantly expressed soluble {alpha}7{beta}1 integrin heterodimer and its binding to laminin-1. Within the muscle, {alpha}7{beta}1 is highly concentrated at the myotendinous and neuromuscular junctions, which are essential for the transduction of mechanical forces and nerve signals, respectively (12). Because {alpha}7X2{beta}1 integrin seems to be the only isoform involved in laminin-induced acetylcholine receptor recruitment into neuromuscular junctions (12) and is the predominant isoform in adult skeletal muscle, the splice variant X2 was chosen for recombinant production. A high yield expression of soluble {alpha}7X2{beta}1 integrin was achieved by coexpression of the murine {alpha}7X2 integrin ectodomain with the human {beta}1 integrin ectodomain in Drosophila Schneider's cells. Similar to the results of von der Mark et al. (28), we showed that soluble {alpha}7X2{beta}1 integrin bound to laminin-1 and laminin-2/4, but failed to interact with laminin-5. Despite being the major laminin isoform in muscle tissue (11), laminin-2/4 was bound by {alpha}7X2{beta}1 integrin with a lower affinity than laminin-1. Compared with other integrins (7, 24), soluble {alpha}7{beta}1 integrin binds its ligands with very high affinity, emphasizing its important role in muscle tissue. We also demonstrated that {alpha}7{beta}1 integrin belongs to the group of {beta}1 integrins that are recognized by invasin, the surface protein of Yersinia pseudotuberculosis, and thus can be utilized by the pathogen to invade {alpha}7{beta}1 integrin-bearing host cells (29).

In this study, we have searched for an inhibitor that interferes with integrin-mediated adhesion to laminin. With the help of soluble {alpha}7X2{beta}1 integrin and its high affinity binding to laminin-1, we could screen various snake venoms for their inhibitory capabilities in a protein interaction assay. Several venomous snakes from the Elapidae, Viperidae, and Crotalidae families are considered to exert myotoxic effects. However, in most of them, proteolytic enzymes, phospholipase activities, and other factors may account for their destructive effects on muscle tissue (13, 14), as only one of 33 tested venoms entirely inhibited interaction of {alpha}7{beta}1 integrin with its laminin ligands. In addition to abundant phospholipase activity and numerous proteases (14, 17), the venom of V. lebetina contains two disintegrins (lebein-2 and lebein-1) that inhibit {alpha}7{beta}1 integrin binding to its laminin ligands. In this study, we have established and optimized their purification from the crude toxin and have characterized their integrin-inhibiting function.

Lebein-1 and its primary structure have been published recently, as lebein (22). Because both subunits bear RGD sequences, lebein-1 was considered an RGD-dependent disintegrin. However, our study provides experimental evidence not only that lebein-1 interferes with the RGD-dependent cell attachment to fibronectin, but also that it additionally binds to laminin-binding {beta}1 integrins in an RGD-independent manner. Therefore, it efficiently inhibits the interaction of laminin-binding integrins with their respective laminin isoforms.

During the purification of lebein-1, we also found a 44-kDa protein that showed proteolytic activity. This protein might be a precursor of lebetase, a Zn2+ ion-containing metalloprotease that has been identified at the cDNA level (27). This precursor also contains a disintegrin domain (27), in good agreement with our observation that the 44-kDa protein bound {alpha}7{beta}1 integrin on the far-Western blot.

We isolated lebein-2 as a novel protein of the V. lebetina venom. It has a molecular mass of 14,735 Da, as determined by mass spectrometry, and consists of two subunits, {alpha} and {beta}. Whereas the {alpha} subunit of lebein-2 was N-terminally blocked, its {beta} subunit shows an N-terminal amino acid sequence similar to, yet distinct from, those of the lebein-1 subunits, thus proving that lebein-2 is an independent gene product. Lebein-2 and lebein-1 strongly bound to the laminin-binding {beta}1 integrins ({alpha}7{beta}1, {alpha}6{beta}1, and {alpha}3{beta}1), but did not recognize the collagen-binding integrins ({alpha}1{beta}1 and {alpha}2{beta}1) in a divalent cation-dependent manner. Although we have tested only the X1 splice variant of the {alpha}6 integrin subunit, it can be assumed that {alpha}6{beta}1 integrin is, in general, a target of lebein-2 and lebein-1, as all its splice variants have similar ligand-binding specificities (30). We also limited our experiments to the X2 splice variant of {alpha}7 integrin. However, this is the predominant {alpha}7{beta}1 integrin isoform in skeletal muscle and thus the potential target of the snake venom. Furthermore, because of their broad binding specificity for all laminin-binding {beta}1 integrins, we assume that both disintegrins are also functional against the X1 splice variant of {alpha}7{beta}1 integrin, which is mainly expressed during myogenesis.

Having identified lebein-2 and lebein-1 in a protein interaction assay, we also proved their inhibitory activities on myoblasts in cell attachment studies. Interestingly, both lebein-2 and lebein-1 inhibited integrin-mediated cell adhesion not only to laminin, but also to fibronectin. The inhibition mechanism for fibronectin is probably caused by RGD sequences, although so far, only lebein-1 is known to be an RGD-dependent disintegrin. Our in vitro binding data support the conclusion that inhibition of cell attachment to laminin-1 is caused by a direct inhibitory interaction of lebein-2 or lebein-1 with laminin-binding {beta}1 integrins on the cell surface that is independent of an RGD sequence.

Laminin-binding {beta}1 integrins avidly bound to both lebein-2 and lebein-1, with Kd values similar to those of the natural laminin ligands (Fig. 1C) (7). Like the binding to laminin, integrin binding affinities for the two V. lebetina disintegrins increased in the presence of Mn2+ ions and decreased in the presence of Ca2+ ions. Furthermore, EDTA completely abolished integrin binding to both laminin and disintegrins. Additionally, the fact that binding of laminin and that of lebein-1 or lebein-2 to integrins are mutually exclusive suggests that both disintegrins act as laminin mimetics that bind to or close to the ligand-binding site of laminin-binding integrins. The interaction of {alpha}7{beta}1 integrin with lebein-1 and lebein-2 did not depend on an RGD sequence, although at least lebein-1 bears two RGD sequences, nor did soluble {alpha}7{beta}1 integrin interact with laminin-1 in an RGD-dependent manner in our study, in agreement with earlier findings (31). However, binding of laminin-1 to its integrin receptors not only requires a yet undefined three-dimensional recognition site within the laminin G module, but also depends on the presence of the coiled-coil domain consisting of all three laminin chains, {alpha}1, {beta}1, and {gamma}1 (3235). Further mechanistic and structural studies will answer the question of how the small size disintegrins lebein-2 and lebein-1 can mimic the large laminin molecules and how they can achieve this inhibition.

Both lebein-1 and lebein-2 belong to the rare group of disintegrins that interact with laminin-binding {beta}1 integrins in an RGD-independent manner. To our knowledge, fertilin (ADAM-2) and meltrin-{gamma} (ADAM-9) are the only examples of disintegrins that interact with the laminin-binding {alpha}6{beta}1 integrin (20, 21, 36). Like lebein-1 and lebein-2, ADAM-2 is functional only as a heterodimeric molecule together with ADAM-1, thus taking an essential part in sperm-oocyte fusion during fertilization (20). Lebein-1 shows some sequence similarities to the disintegrin domains of these ADAM proteins. However, neither ADAM-2 nor ADAM-9 contains an RGD peptide sequence (21, 36). In contrast to ADAM-2 and ADAM-9, which are anchored in the plasmalemma, snake venom disintegrins are highly soluble and are able to inhibit cell/matrix interaction, thus leading to tissue dissipation.

In this study, we have identified and characterized two disintegrins from V. lebetina venom (lebein-1 and the novel lebein-2) that interact with laminin-binding {beta}1 integrins in a divalent cation-dependent and RGD-independent manner both in vitro and in vivo. Through their potential to inhibit integrin-mediated cell attachment to laminins, both lebein-1 and lebein-2 may be valuable tools to influence laminin-dependent cell functions. Among these are the attachment, mechanical force transduction, and migration of cells such as myoblasts (5, 12) and {alpha}7{beta}1 integrin-bearing melanoma cells (31) along or through the laminin-rich basal membranes. These cell functions are of paramount importance in complex physiological processes such as wound healing and tumor invasion.


    FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 492/B3 (to J. A. E.) and Wellcome Trust Grant 060549 (to U. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Münster University Hospital, Inst. for Physiological Chemistry, Waldeyerstr. 15, 48149 Münster, Germany. Tel.: 49-251-835-2289; Fax: 49-251-835-5596; E-mail: eble{at}uni-muenster.de.

1 The abbreviations used are: ECM, extracellular matrix; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; BSA, bovine serum albumin; MES, 4-morpholineethanesulfonic acid. Back

2 S. Niland and J. A. Eble, manuscript in preparation. Back


    ACKNOWLEDGMENTS
 
We acknowledge the excellent technical assistance of Alletta Schmidt-Hederich and I. Jannetti. We also thank Dr. Gottfried Pohlentz for the molecular mass determination of both lebein-1 and lebein-2 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Humphries, M. J. (2000) Biochem. Soc. Trans. 28, 311–339[Medline] [Order article via Infotrieve]
  2. Hynes, R. O. (2002) Cell 110, 673–687[Medline] [Order article via Infotrieve]
  3. Mayer, U., Saher, G., Fässler, R., Bornemann, A., Echtermeyer, F., von der Mark, H., Miosge, N., Pöschl, E., and von der Mark, K. (1997) Nat. Genet. 17, 318–323[Medline] [Order article via Infotrieve]
  4. Shai, S.-Y., Harpf, A. E., Babitt, C. J., Jordan, M. C., Fishbein, M. C., Chen, J., Umura, M., Leil, T. A., Becker, D., Jiang, M., Smith, D. J., Cherry, S. R., Loftus, J. C., and Ross, R. S. (2002) Circ. Res. 90, 458–464[Abstract/Free Full Text]
  5. Belkin, A. M., and Stepp, M. A. (2000) Microsc. Res. Tech. 51, 280–301[CrossRef][Medline] [Order article via Infotrieve]
  6. Hemler, M. E. (1999) Cell 97, 543–546[Medline] [Order article via Infotrieve]
  7. Eble, J. A., Wucherpfennig, K. W., Gauthier, L., Dersch, P., Krukonis, E., Isberg, R. R., and Hemler, M. E. (1998) Biochemistry 37, 10945–10955[CrossRef][Medline] [Order article via Infotrieve]
  8. Hogervorst, F., Admiraal, L. G., Niessen, C., Kuikman, I., Janssen, H., Daams, H., and Sonnenberg, A. (1993) J. Cell Biol. 121, 179–191[Abstract]
  9. Timpl, R., and Brown, J. C. (1996) Bioessays 18, 123–132[Medline] [Order article via Infotrieve]
  10. Tunggal, P., Smyth, N., Paulsson, M., and Ott, M.-C. (2000) Microsc. Res. Tech. 51, 214–227[CrossRef][Medline] [Order article via Infotrieve]
  11. Schuler, F., and Sorokin, L. M. (1995) J. Cell Sci. 108, 3795–3805[Abstract/Free Full Text]
  12. Burkin, D. J., and Kaufman, S. J. (1999) Cell Tissue Res. 296, 183–190[CrossRef][Medline] [Order article via Infotrieve]
  13. Mebs, D., and Ownby, C. L. (1990) Pharmacol. Ther. 48, 223–236[CrossRef][Medline] [Order article via Infotrieve]
  14. Harris, J. B. (1985) Pharmacol. Ther. 31, 79–102[CrossRef][Medline] [Order article via Infotrieve]
  15. Bjarnason, J. B., and Fox, J. W. (1995) Methods Enzymol. 248, 345–368[Medline] [Order article via Infotrieve]
  16. Matsui, T., Fujimura, Y., and Titani, K. (2000) Biochim. Biophys. Acta 1477, 146–156[Medline] [Order article via Infotrieve]
  17. Siigur, J., Aaspóllu, A., Tónismägi, K., Trummal, K., Samel, M., Vija, H., Subbi, J., and Siigur, E. (2001) Haemostasis 31, 123–132[Medline] [Order article via Infotrieve]
  18. Calvete, J. J. (1997) in Integrin-Ligand Interaction (Eble, J. A., and Kühn, K., eds) pp. 157–174, Springer-Verlag New York Inc., New York
  19. White, J. M., Bigler, D., Chem, M., Takahashi, Y., and Wolfsberg, T. G. (2001) in Cell Adhesion (Beckerle, M. C., ed) Vol. 39, pp. 189–216, Oxford University Press, Oxford
  20. Almeida, E. A. C., Huovila, A.-P. J., Sutherland, A. E., Stephens, L. E., Calarco, P. G., Shaw, L. M., Mercurio, A. M., Sonnenberg, A., Primakoff, P., Myles, D. G., and White, J. M. (1995) Cell 81, 1095–1104[Medline] [Order article via Infotrieve]
  21. Nath, D., Slocombe, P. M., Webster, A., Stephens, P. E., Docherty, A. J. P., and Murphy, G. (2000) J. Cell Sci. 113, 2319–2328[Abstract/Free Full Text]
  22. Gasmi, A., Srairi, N., Guermazi, S., Dkhil, H., Karoui, H., and El Ayeb, M. (2001) Biochim. Biophys. Acta 1547, 51–56[Medline] [Order article via Infotrieve]
  23. Hogervorst, F., Kuikman, I., Kessel, V., and Geurts, A. (1991) Eur. J. Biochem. 199, 425–433[Abstract]
  24. Eble, J. A., Beermann, B., Hinz, H.-J., and Schmidt-Hederich, A. (2001) J. Biol. Chem. 276, 12274–12284[Abstract/Free Full Text]
  25. Lenter, M., Uhlig, H., Hamann, A., Jenö, P., Imhof, B., and Vestweber, D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9051–9055[Abstract]
  26. Heyn, M. P., and Weischet, W. O. (1975) Biochemistry 14, 2447–2453[Medline] [Order article via Infotrieve]
  27. Siigur, E., Aaspóllu, A., Tu, A. T., and Siigur, J. (1996) Biochem. Biophys. Res. Comm. 224, 229–236[CrossRef][Medline] [Order article via Infotrieve]
  28. von der Mark, H., Williams, I., Wendler, O., Sorokin, L., von der Mark, K., and Pöschl, E. (2002) J. Biol. Chem. 277, 6012–6016[Abstract/Free Full Text]
  29. Isberg, R. R., and Leong, J. M. (1990) Cell 60, 861–871[Medline] [Order article via Infotrieve]
  30. Delwel, G. O., Kuikman, I., and Sonnenberg, A. (1995) Cell Adhes. Commun. 3, 143–161[Medline] [Order article via Infotrieve]
  31. Kramer, R. H., McDonald, K. A., and Vu, M. P. (1989) J. Biol. Chem. 264, 15642–15649[Abstract/Free Full Text]
  32. von der Mark, H., Dürr, J., Sonnenberg, A., von der Mark, K., Deutzmann, R., and Goodman, S. L. (1991) J. Biol. Chem. 266, 23593–23601[Abstract/Free Full Text]
  33. Deutzmann, R., Aumailley, M., Wiedemann, H., Pysny, W., Timpl, R., and Edgar, D. (1990) Eur. J. Biochem. 191, 513–522[Abstract]
  34. Yurchenco, P., Sung, U., Ward, M. D., Yoshihiko, Y., and O'Rear, J. J. (1993) J. Biol. Chem. 268, 8356–8365[Abstract/Free Full Text]
  35. Sung, U., O'Rear, J. J., and Yurchenco, P. D. (1993) J. Cell Biol. 123, 1255–1268[Abstract]
  36. Bigler, D., Takahashi, Y., Chen, M. S., Almeida, E. A. C., Osbourne, L., and White, J. M. (2000) J. Biol. Chem. 275, 11576–11584[Abstract/Free Full Text]