Isolation and Characterization of EMS16, a C-Lectin Type Protein from Echis multisquamatus Venom, a Potent and Selective Inhibitor of the α2β1 Integrin † (original) (raw)
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Toxicon, 2013
A domain C-type lectin-related protein a b s t r a c t Integrins are receptors of the extracellular matrix (ECM), playing a vital role in pathophysiological processes. They bind to ECM ligands like collagens and can mediate wound healing as well as tumor metastasis and thrombosis, thus being a part of cell adhesion and migration as well as platelet aggregation. For this reason, identifying a2b1 integrin-specific antagonists can assist in the development of drugs to treat tumor progression, angiogenesis, and cardiovascular diseases. Snake venoms have been shown to contain antagonists which target collagen-binding integrins. EMS16, rhodocetin, and VP12 are three toxins belonging to the C-type lectin-related protein family and have been proven to inhibit the a2b1 integrin, specifically the a2 integrin A domain. To specifically isolate antagonists targeting the a2b1 integrin A domain, we developed a protocol based on affinity chromatography. Using this novel approach, the toxin VP-i was isolated from Vipera palaestinae venom. We show that VP-i binds to the a2 integrin A domain and that it successfully inhibits adhesion of various cells to type I collagen as well as cell migration. Moreover, our results indicate that VP-i differs structurally from the previously purified VP12, although not functionally, and therefore is a further venom compound which can be utilized for drug development.
Toxicology and Applied Pharmacology, 2013
Snake venom antagonists of α2β1 integrin have been identified as members of a C-lectin type family of proteins (CLP). In the present study, we characterized three new CLPs isolated from Echis sochureki venom, which interact with this integrin. These proteins were purified using a combination of gel filtration, ion exchange chromatography and reverse phase HPLC. Sochicetin-A and sochicetin-B potently inhibited adhesion of cells expressing α2β1 integrin and binding of isolated α2β1 ectodomain to collagen I, as well as bound to recombinant GST-α2A domain in ELISA, whereas activity of sochicetin-C in these assays was approximately two orders of magnitude lower. Structurally, sochicetin-B and sochicetin-C are typical heterodimeric αβ CLPs, whereas sochicetin-A exhibits a trimer of its subunits (αβ) 3 in the quaternary structure. Immobilized sochicetins supported adhesion of glioma cell lines, LN18 and LBC3, whereas in a soluble form they partially inhibited adhesion of these cells to collagen I. Glioma cells spread very poorly on sochicetin-A, showing no cytoskeleton rearrangement typical for adhesion to collagen I or fibronectin. Adhesion on CLP does not involve focal adhesion elements, such as vinculin. Sochicetin-A also inhibited collagen-induced platelet aggregation, similar to other CLPs' action on the blood coagulation system.
The Biochemical journal, 1994
The inhibitory properties of a panel of snake-venom-derived RGD (Arg-Gly-Asp) proteins, including the disintegrins kistrin, elegantin and albolabrin, and the neurotoxin homologue dendroaspin, were investigated in a platelet-adhesion assay using three immobilized ligands of the glycoprotein IIb-IIIa complex (alpha IIb beta 3), namely fibrinogen, fibronectin and von Willebrand factor (vWF). The snake-venom proteins preferentially inhibited the adhesion of ADP-treated platelets to one or more of the immobilized ligands. Kistrin and dendroaspin exhibited similar inhibitory characteristics, abrogating platelet adhesion to fibrinogen and vWF at nanomolar concentrations, but poorly inhibiting adhesion to fibronectin. Kistrin and dendroaspin share little overall amino-acid-sequence identity, but a considerable level of sequence similarity exists around the RGD tripeptide. Synthetic cyclic peptides corresponding to these regions of kistrin and dendroaspin inhibited platelet adhesion to both ...
Thrombosis Research, 2002
Trimucytin is a powerful platelet aggregation inducer isolated from the venom of Taiwan habu snake (Trimeresurus mucrosquamatus). In this study, we found that the snake venom protein, crovidisin, which prevents collagen-platelet interaction through its high-affinity binding to collagen, inhibited competitively trimucytin-induced aggregation of washed human platelets with a pA 2 value of 6.65. The ability of trimucytin in triggering platelet aggregation was suppressed by a monoclonal antibody (A2-IIE10) raised against the a2 subunit of a2b1 integrin (glycoprotein Ia/IIa), indicating that platelet a2b1 integrin plays a central role in trimucytin's platelet reactivity. Many studies have localized the major reactive site of a2b1 integrin to the I-domain of a2 subunit. However, Escherichia coli-produced recombinant a2 I-domain (GST-a2I fusion protein) blocking collagen-induced platelet aggregation failed to inhibit aggregation of platelets in response to trimucytin. Based on these findings, it is concluded that the platelet reactivity of trimucytin is a2b1 integrin-dependent, while the I-domain present in the a2 subunit is not involved. This novel snake venom protein would be useful for mapping the functional domain of a2b1 integrin.
Journal of Organometallic Chemistry, 2011
Insularin (INS) was obtained from Bothrops insularis venom by reversed-phase highperformance liquid chromatography using a C18 column and characterized as a disintegrin by peptide mass fingerprint and inhibition of ADP-induced platelet aggregation. A cDNA coding for P-II a metalloproteinase/disintegrin was cloned from a cDNA library from B. insularis venom glands. The deduced protein sequence possesses 73 amino acid residues, including the N-terminal, internal peptides of native insularin, the ARGDNP-sequence and 12 cysteines in a conserved alignment. This cDNA fragment was subcloned in the pGEX-4T-1 vector and expressed in a prokaryotic expression system as a fusion protein with glutathione S-transferase (GST-INS). Both native and recombinant insularin inhibited ADP-induced platelet aggregation and endothelial cells (HUVEC) adhesion with similar activities indicating that GST-INS folded correctly and preserved the integrin-binding loop. Insularin may be a tool in studies that involve platelets and endothelial cell adhesion dependent on alphaIIbeta3 and alphavbeta3 integrins.
Matrix Biology, 2007
Integrins are essential protagonists in the complex multistep process of cancer progression and metastasis. We recently reported that lebectin, a novel C-type lectin from Macrovipera lebetina venom, displays an anti-integrin activity. In this study, we extend this observation to lebecetin, a second C-type lectin isolated from the same venom and previously reported as a potent inhibitor of platelet aggregation. Both venom lectins appear to exert their effect on cell adhesion, migration, invasion and proliferation by inhibiting α5β1 and αv-containing integrins. Moreover, the inhibition of α5β1 and αv integrins is likely due to the binding of venom peptides, as both lebectin and lebecetin co-immunoprecipitate with these integrins. Lebectin and lebecetin are thus the first examples of venom C-type lectins inhibiting an integrin other than the collagen receptor α2β1.
Toxicon, 2010
SVMPs are multi-domain proteolytic enzymes in which disintegrin-like and cysteine-rich domains bind to cell receptors, plasma or ECM proteins. We have recently reported that jararhagin, a P-III class SVMP, binds to collagen with high affinity through an epitope located within the Da-disintegrin sub-domain. In this study, we evaluated the binding of jararhagin to a 2 b 1 integrin (collagen receptor) using monoclonal antibodies and recombinant jararhagin fragments. In solid phase assays, binding of jararhagin to a 2 b 1 integrin was detectable from concentrations of 20 nM. Using recombinant fragments of jararhagin, only fragment JC76 (residues 344-421), showed a significant binding to recombinant a 2 b 1 integrin. The anti-jararhagin monoclonal antibody MAJar 3 efficiently neutralised binding of jararhagin to collagen, but not to recombinant a 2 b 1 integrin nor to cell-surface-exposed a 2 b 1 integrin (a 2-K562 transfected cells and platelets). The same antibody neutralised collagen-induced platelet aggregation. Our data suggest that jararhagin binding to collagen and a 2 b 1 integrin occurs by two independent motifs, which are located on disintegrin-like and cysteine-rich domains, respectively. Moreover, toxin binding to collagen appears to be sufficient to inhibit collagen-induced platelet aggregation.
Laboratory Investigation, 2005
Lebestatin, a new member of the lysine-threonine-serine (KTS)-disintegrin family, was purified to homogeneity from Tunisian snake (Macrovipera lebetina) venom. It is a single-chain polypeptide composed of 41 amino acids. The amino-acid sequence of lebestatin shows that it displays a pattern of cysteines similar to other short disintegrins, but contains the sequence KTS rather than RGD in its integrin-binding loop. Lebestatin presents a high homology with obtustatin and viperistatin. Lebestatin interacts specifically with the a1b1 integrin. It was thus able to inhibit both adhesion and migration of PC12 and a1b1 integrin-expressing CHO cells (CHO-a1) to type I and IV collagens. This disintegrin also affected adhesion and migration of endothelial cells and exhibited an anti-angiogenic effect in vivo when using the 8-day-old embryo chick chorioallantoic membrane model.
Archives of Biochemistry and Biophysics, 2003
The a 5 b 1 integrin is one of the major fibronectin receptors which plays an essential role in the adhesion of normal and tumor cells to extracellular matrix. Here, we describe the isolation and characterization of a novel dimeric metalloproteinase/disintegrin, which is an inhibitor of fibronectin binding to the a 5 b 1 integrin. This protein (BaG) was isolated from the venom of the South American snake Bothrops alternatus by gelatin-Sepharose affinity and anion exchange chromatography. The molecular mass of BaG was approximately 130 kDa under non-reducing conditions and 55 kDa under reducing conditions by SDS-PAGE. BaG shows proteolytic activity on casein that was inhibited by EDTA. 1,10-phenanthroline-treated BaG (BaG-I) inhibits ADP-induced platelet aggregation with an IC 50 of 190 nM. BaG-I inhibits fibronectin-mediated K562 cell adhesion with an IC 50 of 3.75 lM. K562 cells bind to BaG-I probably through interaction with a 5 b 1 integrin, since anti-a 5 b 1 antibodies inhibited K562 cell adhesion to BaG-I. In addition, BaG-I induces the detachment of K562 cells that were bound to fibronectin. In summary, we have purified a novel, dimeric snake venom metalloproteinase/disintegrin that binds to the a 5 b 1 integrin.
Biochemical Journal, 2006
We report the cloning and sequence analysis of Cerastes vipera and Macrovipera lebetina transmediterranea cDNAs coding for short non-RGD (Arg-Gly-Asp) disintegrins and for dimeric disintegrin subunits. The mRNAs belong to the short-coding class, suggesting that these disintegrin mRNAs may be more widely distributed than previously thought. Our data also argue for a common ancestry of the mRNAs of short disintegrins and those coding for precursors of dimeric disintegrin chains. The Macrovipera lebetina transmediterranea dimeric disintegrin reported to inhibit the laminin-binding integrins α 3 β 1 , α 6 β 1 and α 7 β 1 was analysed using a proteomic approach and was shown to bear MLD (Met-Leu-Asp) and VGD (Val-Gly-Asp) motifs. The results highlight the fact that disintegrins have evolved a restricted panel of integrin-blocking sequences that segregate with defined branches of the phylogenetic tree of the integrin α-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.