Bernard Saliou

COnvulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets

Convulxin, a very potent aggregating protein from rattlesnake venom, was purified by a new procedure and its heterodimeric structure alpha 3 beta 3 was confirmed. The polypeptide N-terminal sequences of convulxin subunits were determined by Edman degradation. They are very similar and appear homologous to botrocetin from Bothrops jararaca venom and to rattlesnake lectin from Crotalus atrox venom, both being classified among the C-type lectin family. The binding of 125I-labelled convulxin to blood platelets has also been analysed under equilibrium conditions. These studies indicated that convulxin binds to platelets with a high affinity (Kd = 30 pM) on a small number of binding sites (1000 binding sites per cell). The high-affinity binding of convulxin appears specific to platelets, since it is not observed on other cell types such as neutrophils and erythrocytes. Also, the high-affinity binding of convulxin to membranes platelet is not inhibited by alpha-thrombin, fibrinogen, collagen, laminin binding inhibitor, RGDS peptide, adenosine diphosphate, platelet-activating factor-acether, serotonin or epinephrine. This, together with the recent observation that platelet activation by convulxin is partially mediated by phospholipase C and involves other mechanisms as well, indicates that convulxin may interact with a specific platelet acceptor (receptor) protein which has yet to be characterized.

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non‐amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N‐glycans of the ‘complex’ and ‘hybrid’ types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non‐denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

The carboxy-terminal C2-like domain of the alpha-toxin from Clostridium perfringens mediates calcium-dependent membrane recognition

The lethal, cytolytic α‐toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N‐terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C‐terminal domain. Calcium ions are essential for the binding of α‐toxin to lipid films. Sixteen α‐toxin variants with single amino acid substitutions in the C‐terminal region were obtained using site‐directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were considerably less active than native α‐toxin under calcium‐limiting conditions. Replacement of Thr‐272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface‐exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C‐terminal region probably belongs to the growing family of C2β‐barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. The combined findings suggest that the C‐terminal region of α‐toxin mediates interactions with membrane phospholipids in a calcium‐dependent manner. Mutations to this domain may account for the natural lack of toxicity of the α‐toxin homologue, phospholipase C of Clostridium bifermentans.

Ceruleotoxin: Identification in the venom of Bungarus fasciatus, molecular properties and importance of phospholipase A2 activity for neurotoxicity

Ceruleotoxin is a potent neurotoxin which was originally purified from a batch of venom labelled Bungarus caeruleus, from the Pasteur Institute. Since NOBLE et al. have shown that this batch differs in its protein composition from that of B. caeruleus provided by Miami Serpentarium, we decided to clarify this point by comparing the composition of venoms from various Bungarus species of several origins. Although individual variations exist between samples of the same species, the venom from B. multicinctus, B. caeruleus and B. fasciatus possess characteristic protein compositions which allowed us to identify the batch used to purify ceruleotoxin as a B. fasciatus venom. We identified and purified ceruleotoxin from each of the five samples of B. fasciatus venoms tested. We failed to find this neurotoxin in either B. multicinctus or B. caeruleus venoms. Purified ceruleotoxin is a slightly basic protein with an isoelectric point of 7.4 which possesses a significant phospholipase A2 activity (200 mumoles lecithin hydrolyzed per min per mg) and a high lethal potency (i.v. LD50 in mice 0.03-0.07 mg/kg). It is composed of two identical subunits of 13,000 mol. wt. which resemble pancreas and snake venom phospholipases in their amino acid composition. Like crotoxin, ceruleotoxin irreversibly blocks the postsynaptic response of Torpedo and Electrophorus electroplaques to cholinergic agonists without preventing the binding of acetylcholine to its receptor. By hydrolyzing critical lipids of the postsynaptic membrane, it stabilizes the acetylcholine receptor - ionophore assembly in a desensitized state.

Binding of Crotoxin, a Presynaptic Phospholipase A2Neurotoxin, to Negatively Charged Phospholipid Vesicles

Abstract: Crotoxin, isolated from the venom of Crotalus durissus terrificus, is a potent neurotoxin consisting of a basic and weakly toxic phospholipase A2 subunit (component B) and an acidic nonenzymatic subunit (component A). The nontoxic component A enhances the toxicity of the phospholipase subunit by preventing its nonspecific adsorption. The binding of crotoxin and of its subunits to small unilamellar phospholipid vesicles was examined under experimental conditions that prevented any phospholipid hydrolysis. Isolated component B rapidly bound with a low affinity (Kapp in the millimolar range) to zwitterionic phospholipid vesicles and with a high affinity (Kapp of < 1 μM) to negatively charged phospholipid vesicles. On the other hand, the crotoxin complex did not interact with zwitterionic phospholipid vesicles but dissociated in the presence of negatively charged phospholipid vesicles; the noncatalytic component A was released into solution, whereas component B remained tightly bound to lipid vesicles, with apparent affinity constants from 100 to <1 μM, according to the chemical composition of the phospholipids. On binding, crotoxin or its component B caused the leakage of a dye entrapped in vesicles of negatively charged but not of zwitterionic phospholipids. The selective binding of crotoxin suggests that negatively charged phospholipids may constitute a component of the acceptor site of crotoxin on the presynaptic plasma membrane.

Structure and Function Relationship of Crotoxin, a Heterodimeric Neurotoxic Phospholipase A2 from the Venom of a South-American Rattlesnake

Snake neurotoxins that interfere with the release of transmitter at the neuromuscular junction have a clinical and a scientific interest and, therefore, their structure and their mode of action have been extensively studied. Phospholipase A2 s-neurotoxins have been found in the venom of Elapidae and Viperidae snakes. They are potent neurotoxins which specifically inhibit the release of acetylcholine from peripheral cholinergic synapses. Although they all possess phospholipase A2 activity, their quaternary structure is rather heterogeneous. Some of them consist of a single chain phospholipase A2 polypeptide chain, as ammodytoxin A from the venom of Vipera ammodytes ammodytes (Lee et al, 1984, Ritonja and Gubensek, 1985) or agkistrodotoxin from the venom of Agkistrodon blomhoffii brevicaudus (Chen et al, 1981). Multichain s-neurotoxins, such as crotoxin, from the venom of Crotalus durissus terrificus, are made of the non covalent association of several subunits homologous to phospholipases A2, at least one of which possessing enzymatic activity. Finally, s-bungarotoxins, which are only found in Bungarus venoms, covalently associate a phospholipase A2 subunit with a non-enzymatic polypeptide homologous to Kunitz-type protease inhibitors (Kondo et al, 1982a; 1982b). The mechanism of action of s-neurotoxins has been extensively studied in vivo and in vitro. This brief review focusses on crotoxin, the structure-function relationship of which was examined using the complementary approaches of biochemistry, pharmacology, immunochemistry and molecular biology.

Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis.

Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.

Crotoxin: A Biochemical Analysis of Its Mode of Action

AbstractCrotoxin, the major toxic component of the South American Rattlesnake, Crotalus durissus terrificus, is a potent neurotoxin which possesses a phospholipase A2 activity and blocks neuromuscular transmission primarily at the presynaptic level, although at higher doses it also reduces the postsynaptic response to acetylcholine by stabilizing the cholinergic receptor in an inactive conformational state.Crotoxin, which is in fact a mixture of very similar isoforms, consists of two non identical subunits. The basic component-B carries the phospholipase A2 activity of the toxin and possesses a low toxicity and the acidic component-A has no enzymatic activity although it resembles a phospholipase A2 in its primary structure. Component-A, is not toxic by itself but considerably enhances the lethal potency of the phospholipase ccmponent-B. Upon interaction with biological or artificial membranes, the two subunits dissociate: component-A is released free in solution and component-B is bound. The isolated pho...

Antipeptide antibodies directed to the C-terminal part of ammodytoxin A react with the PLA2 subunit of crotoxin and neutralize its pharmacological activity.

Crotoxin and ammodytoxin A are snake venom neurotoxic phospholipases A2. Polyclonal antibodies against three synthetic peptides selected from the C-terminal part of the primary structure of ammodytoxin A were tested by ELISA for their interaction with crotoxin and its subunits, CA and CB. All three antipeptide antibodies reacted specifically with corresponding parts of ammodytoxin A and CB, either native or reduced. Conversely, polyclonal antibodies produced against ammodytoxin A and CB reacted strongly with all three peptides, suggesting that they constitute at least a part of natural epitopes in both proteins. All antipeptide antibodies reacted also with the corresponding peptides derived from CB by cyanogen bromide cleavage. The biological activity of the immune complexes was tested. No significant change in the enzymatic activity of CB, ammodytoxin A or crotoxin was observed with any of the three antipeptide antibodies. These antibodies were, however, able to protect mice against the lethal potency of CB and to prolong survival time of mice injected with crotoxin. These antipeptide antibodies were assayed in vitro for their protective effect against the action of CB or crotoxin on synaptosomes from Torpedo marmorata electric organ. They partly inhibited the acetylcholine release induced by both proteins. These results indicate that the C-terminal part of CB is likely to be involved in the pharmacological action of crotoxin.

Partial purification of α-glycerotoxin, a presynaptic neurotoxin from the venom glands of the polychaete annelid glycera convoluta

The venom secreted from glands appended to the jaws of Glycera convoluta, a Polychaete Annelid, increases the spontaneous quantal release of transmitter from nerve terminals. The component that is biologically active on vertebrate cholinergic nerve terminals has recently been shown to be a high molecular weight protein. In the present work, the crude extract from the venom apparatus was shown to be toxic for mammals and crustaceans. It was fractionated by gel filtrations and ion exchange chromatographies. The biologically active component at frog neuromuscular junctions, ?-glycerotoxin, was purified more than 1,000-fold. It is distinct from the components that are toxic for crustaceans. Purified ?-glycerotoxin is a globular protein of 300,000 +/- 20,000 mol wt. It has a Stokes radius of 65 A and a sedimentation coefficient of 11 S. By its molecular properties, ?-glycerotoxin appears distinct from other neurotoxins such as ?-latrotoxin, which also trigger transmitter release.