Naziha Marrakchi

Lebectin, a Macrovipera lebetina venom‐derived C‐type lectin, inhibits angiogenesis both in vitro and in vivo

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti‐angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin–ligand interactions. Recently, the C‐type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti‐angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express αvβ3, αvβ5, and α5β1 integrins, as well as the α2, α3, α6, and β4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC50 ≈ 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the α5β1 and αV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel™ (IC50 = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel™ Plug assay in nude mice, our results show that lebectin displays potent anti‐angiogenic activity in vivo. Lebectin thus represents a new C‐type lectin with anti‐angiogenic properties with great potential for the treatment of angiogenesis‐related diseases. J. Cell. Physiol. 211: 307–315, 2007.

Molecular cloning of disintegrins from Cerastes vipera and Macrovipera lebetina transmediterranea venom gland cDNA libraries: insight into the evolution of the snake venom integrin-inhibition system

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 alpha3beta1, alpha6beta1 and alpha7beta1 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 alpha-chains, providing novel insights into the evolutionary adaptation of the snake venom antagonists to the ligand-binding sites of their target integrin receptors.

Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes.

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.

Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom.

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi=9.9 and migrates in SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of 15015 and 16296 Da for alpha and beta subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A, B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds to platelet GPIb.

Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration

Two non-toxic PLA2s were purified to homogeneity from Cerastes cerastes Tunisian snake venom. The purification process employed gel filtration on Sephadex G-75 followed by C18 reverse phase high-pressure liquid chromatography. These two acidic enzymes, namely CC-PLA2-1 and CC-PLA2-2, have a molecular weight of 13,737.52 and 13,705.63 Da, respectively. These two PLA2 are the first reported glycosylated phospholipases A2 purified from snake venom. The rates of glycosylation are 2.5% and 0.5% (w/w), respectively. Specific activities of 1800 U/mg and 2400 U/mg for CC-PLA2-1 and CC-PLA2-2, respectively, were measured at optimal conditions. CC-PLA2-1 and CC-PLA2-2 strongly inhibited coagulation. They also exhibited a marked dose-dependent inhibitory effect on platelet aggregation induced by ADP and arachidonic acid in platelet-rich plasma. Interestingly, CC-PLA2-1 and CC-PLA2-2 inhibited in a dose-dependent manner adhesion of IGR39 melanoma and HT1080 fibrosarcoma cells to fibrinogen and fibronectin. Furthermore, both CC-PLA2-1 and CC-PLA2-2 abolished HT1080 cell migration towards fibrinogen and fibronectin. This activity is reported for the first time for PLA2 enzymes.

PIVL, a new serine protease inhibitor from Macrovipera lebetina transmediterranea venom, impairs motility of human glioblastoma cells

A novel Kunitz-type serine proteinase inhibitor, termed PIVL, was purified to homogeneity from the venom of the Tunisian snake Macrovipera lebetina transmediterranea. It is a monomeric polypeptide chain cross-linked by three disulfide linkages with an isotope-averaged molecular mass of 7691.7 Da. The 67-residue full-length PIVL sequence was deduced from a venom gland cDNA clone. Structurally, PIVL is built by a single Kunitz/BPTI-like domain. Functionally, it is able to specifically inhibit trypsin activity. Interestingly, PIVL exhibits an anti-tumor effect and displays integrin inhibitory activity without being cytotoxic. Here we show that PIVL is able to dose-dependently inhibit the adhesion, migration and invasion of human glioblastoma U87 cells. Our results also show that PIVL impairs the function of αvβ3 and to a lesser extent, the activity of αvβ6, αvβ5, α1β1 and α5β1 integrins. Interestingly, we demonstrate that the (41)RGN(43) motif of PIVL is likely responsible for its anti-cancer effect. By using time lapse videomicroscopy, we found that PIVL significantly reduced U87 cells motility and affected cell directionality persistence by 68%. These findings reveal novel pharmacological effects for a Kunitz-type serine proteinase inhibitor.

MVL-PLA2, a Snake Venom Phospholipase A2, Inhibits Angiogenesis through an Increase in Microtubule Dynamics and Disorganization of Focal Adhesions

Integrins are essential protagonists of the complex multi-step process of angiogenesis that has now become a major target for the development of anticancer therapies. We recently reported and characterized that MVL-PLA2, a novel phospholipase A2 from Macrovipera lebetina venom, exhibited anti-integrin activity. In this study, we show that MVL-PLA2 also displays potent anti-angiogenic properties. This phospholipase A2 inhibited adhesion and migration of human microvascular-endothelial cells (HMEC-1) in a dose-dependent manner without being cytotoxic. Using Matrigel™ and chick chorioallantoic membrane assays, we demonstrated that MVL-PLA2, as well as its catalytically inactivated form, significantly inhibited angiogenesis both in vitro and in vivo. We have also found that the actin cytoskeleton and the distribution of αvβ3 integrin, a critical regulator of angiogenesis and a major component of focal adhesions, were disturbed after MVL-PLA2 treatment. In order to further investigate the mechanism of action of this protein on endothelial cells, we analyzed the dynamic instability behavior of microtubules in living endothelial cells. Interestingly, we showed that MVL-PLA2 significantly increased microtubule dynamicity in HMEC-1 cells by 40%. We propose that the enhancement of microtubule dynamics may explain the alterations in the formation of focal adhesions, leading to inhibition of cell adhesion and migration.

Lebectin, a novel C-type lectin from Macrovipera lebetina venom, inhibits integrin-mediated adhesion, migration and invasion of human tumour cells

The adhesion receptors of the integrin family play an essential role during tumour progression and thus represent interesting potential targets for the development of new therapeutic agents. The snake venom contains natural inhibitors of integrin–ligand interactions called disintegrins. It also contains C-type lectin proteins mainly known as modulators of platelet aggregation. In this study, we demonstrate that lebectin, a novel C-type lectin isolated from Macrovipera lebetina venom, displayed an anti-integrin activity. Lebectin inhibited the integrin-mediated attachment of various tumour cell lines to different adhesion substrata. The C-type lectin also completely blocked cell migration towards fibronectin in haptotaxis assays and prevented invasion of fibrin gels by tumour cells. In addition, lebectin proved to be a potent inhibitor of tumour cell proliferation. Although the specific integrins affected by lebectin are not identified in this study, the integrin α5β1 might be involved.

CC-PLA2-1 and CC-PLA2-2, two Cerastes cerastes venom-derived phospholipases A2, inhibit angiogenesis both in vitro and in vivo

Integrins are essential in the complex multistep process of angiogenesis and are thus attractive targets for the development of antiangiogenic therapies. Integrins are antagonized by disintegrins and C-type lectin-like proteins, two protein families from snake venom. Here, we report that CC-PLA2-1 and CC-PLA2-2, two novel secreted phospholipases A2 (PLA2) isolated from Cerastes cerastes venom, also showed anti-integrin activity. Indeed, both PLA2s efficiently inhibited human brain microvascular endothelial cell adhesion and migration to fibrinogen and fibronectin in a dose-dependent manner. Interestingly, we show that this anti-adhesive effect was mediated by α5β1 and αv-containing integrins. CC-PLA2s also impaired in vitro human brain microvascular endothelial cell tubulogenesis on Matrigel and showed antiangiogenic activity in vivo in chicken chorioallantoic membrane assay. The complete PLA2 cDNAs were cloned from a venom gland cDNA library. Mature CC-PLA2-1 and CC-PLA2-2 contain 121 and 120 amino acids, respectively, including 14 cysteines each and showed 83% identity. Tertiary model structures of CC-PLA2-1 and CC-PLA2-2 were generated by homology modeling. This is thus the first study describing an antiangiogenic effect for snake venom PLA2s and reporting first clues to their mechanism of action on endothelial cells.

αvβ5/β6 integrin suppression leads to a stimulation of α2β1 dependent cell migration resistant to PI3K/Akt inhibition

Crosstalk between integrins is involved in the regulation of various cell functions including cell migration. Here we identify the interplay between the integrins alphavbeta5/beta6 and alpha2beta1 during cell migration toward type I collagen. Human colon cancer cell lines HT29-D4 and SW480 were used as cell models. To improve our understanding of the consequences of alphavbeta5/beta6 function on alpha2beta1, we decreased the expression of alphav integrins by either siRNA or lysosomal targeting strategies or inhibited their function using, as antagonists, blocking antibodies or disintegrins. In all cases, we observed a greatly enhanced alpha2beta1 integrin-dependent cell migration associated with focal adhesion rearrangements and increased outside-in signaling as demonstrated by elevated phosphorylation of focal adhesion kinase and MAPKinase (ERK1 and ERK2). The alphavbeta5/beta6-dependent limitation of alpha2beta1 function could be overridden by TS2/16, an activating anti-beta1 antibody. Interestingly, compared to control cells, the pharmacological inhibition of PI3Kinase or the siRNA-mediated knockdown of AKT had little effect on the high alpha2beta1-mediated cell migration observed in the absence of alphav integrins or following activation of alpha2beta1 integrins by the TS2/16. These results suggest that integrins alphavbeta5/beta6 repress alpha2beta1 possibly by interfering with their activation process and thereby modify the cell signaling regulation of alpha2beta1-mediated migration.