Modification of substrate inhibition of synaptosomal acetylcholinesterase by cardiotoxins

Different types of cardiotoxin (I-V and n) were isolated and purified from the venom of the Taiwan cobra (Naja naja atra). The effects of these cardiotoxins were studied on membrane-bound acetylcholinesterase, which was isolated from a sheep's brain cortex. The results showed that cardiotoxins I-III, V, and n activated the enzyme by modification of substrate inhibition, but cardiotoxin IV's reaction was different. The inhibition and activation of acetylcholinesterase were linked to the functions of the hydrophobicity index, presence of a cationic cluster, and the accessible arginine residue. Our results indicate that Cardiotoxins have neither a cationic cluster nor an arginine residue in their surface area of loop I; therefore, in contrast to fasciculin, cardiotoxins are attached by loop II to the peripheral site of the enzyme. As a result, fasciculin seems to stabilize nonfunctional conformation, but cardiotoxins seem to stabilize the functional conformation of the enzyme. Based on our experimental and theoretical findings, similar secondary and tertiary structures of cardiotoxins and fasciculin seem to have an opposite function once they interact with acetylcholinesterase.     

Lipid-protein interactions. A comparative study of the binding of cardiotoxins and neurotoxins to phospholipid vesicles

It has recently been shown that cardiotoxin II from Naja mossambica mossambica specifically interacts with negatively charged phospholipids (Dufourcq, J. and Faucon, J.F. (1978) Biochemistry 17, 1170–1176). In order to investigate whether or not short neurotoxins give rise to similar interactions, four techniques have been used, namely intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene, turbidity measurements and release of 6-carboxyfluorescein trapped inside single shelled vesicles.Neurotoxin III from Naja mossambica mossambica and neurotoxin I from the venom of the scorpion Androctonus australis Hector, specifically interact with negatively charged phospholipids leading to changes in tryptophan fluorescence and to a decrease of the fluidity of the bilayer. Cardiotoxin II from the same snake venom gives similar results. On the other hand, it seems that either a very weak or no interaction at all occurs in the case of neurotoxin I from the same Naja venom.There are important differences in the behaviour of cardiotoxin and neurotoxins: (i) neurotoxins lead to only weak release of 6-carboxyfluorescein from lipid vesicles, whereas cardiotoxin II induces fast and quantitative escape of the dye and then a general breakdown of the vesicular structure; (ii) binding of neurotoxins can be easily reversed by 100–200 mM NaCl or less than 1 mM Ca²⁺ and so it is essentially electrostatic, whereas binding of cardiotoxin II seems to involve some hydrophobic contribution.The short neurotoxins and cardiotoxins from snake venom having a great homology in sequence, their differences on binding properties are discussed in terms of changes in a particular area of the sequence.     

Cobra cardiotoxins. Purification, effects on skeletal muscle and structure/activity relationships [published errtum appears in Eur J Biochem 1988 Feb 1;171(3):727

A new preparative method for isolating homogeneous cardiotoxins from cobra venoms is described. The technique, based on reverse-phase high-pressure liquid chromatography, was used to isolate eight cardiotoxins of known sequence from four different venoms. In each case the method was found to be particularly efficient at removing trace quantities of contaminating phospholipase. Cardiotoxins isolated in this manner were found to retain their full biological activity. Without exception the purified cardiotoxins lacked powerful haemolytic activity at concentrations up to 0.01 mM (about 100 micrograms ml-1), although some lysis of human erythrocytes was induced at higher concentrations. The cardiotoxins displayed a wide range of depolarizing activity on cultured skeletal muscle, the lowest activity being associated with the highest LD50 value. Correlating variations in amino acid sequence and variations in depolarization potency revealed the importance of residues in the second and third loops, especially lysine-46, serine-48 and lysine-52, together with a number of hydrophobic residues. Further modifications of pharmacological activity were associated with the presence of additional basic residues in the first and second loops and to minor differences in secondary structure.     

Localization of the toxic site of Naja mossambica cardiotoxins: small synthetic peptides express an in vivo lethality

Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.     

A common cytolytic region in myotoxins, hemolysins, cardiotoxins and antibacterial peptides

Several proteins and polypeptides of reptilian, amphibian, insect, and microbial origin share a common cytolytic property. However, these cytolysins fulfill different objectives. They provide offensive armament in the case of toxins, but defensive systems in the case of antibacterial peptides. The sequences of several nonenzymatic cytolysins and their analogues were compared to identify the structural requirements for cytolytic activity. These cytolysins, although isolated from phylogenetically unrelated organisms, possess the common sequence features of a cationic site flanked by a hydrophobic surface. The presence of such a region apparently confers the cytolytic activity of various cytolysins. The concept of a cytolytic region is strongly supported by the existence of several natural and synthetic analogues of cytolysins and by chemical modification studies of these cytolysins. This prediction provides a new focus for cytolysin research. The understanding of this structure-function relationship should facilitate the design, synthesis, and development of better antibacterial and anticancer peptides.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Both aromatic and cationic residues contribute to the membrane-lytic and bactericidal activity of eosinophil cationic protein

Eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN) are proteins of the ribonuclease A (RNase A) superfamily that have developed biological properties related to the function of eosinophils. ECP is a potent cytotoxic molecule, and although the mechanism is still unknown this cytotoxic activity has been associated with its highly cationic character. Using liposome vesicles as a model, we have demonstrated that ECP tends to disrupt preferentially acidic membranes. On the basis of structure analysis, ECP variants modified at basic and hydrophobic residues have been constructed. Changes in the leakage of liposome vesicles by these ECP variants have indicated the role of both aromatic and basic specific amino acids in cellular membrane disruption. This is the case with the two tryptophans at positions 10 and 35, but not phenylalanine 76, and the two arginines 101 and 104. The bactericidal activity of both native ECP and point-mutated variants, tested against Escherichia coli and Staphylococcus aureus, suggests that basic amino acids play, in addition to the effect on the disruption of the cellular membrane, other roles such as specific binding on the surface of the bacteria cell.     

Molecular mechanism of cardiotoxin action on axonal membranes.

Cardiotoxin isolated from Naja mossambica mossambica selectively deactivates the sodium-potassium activated adenosine triphosphatase of axonal membranes. Tetrodotoxin binding and acetylcholinesterase activities are unaffected by cardiotoxin treatment. The details of association of cardiotoxin with the axonal membrane were studied by following the deactivation of the sodium-potassium activated adenosine triphosphatase and by direct binding measurements with a tritiated derivative of the native cardiotoxin. The maximal binding capacity of the membrane is 42-50 nmol of cardiotoxin/mg of membrane protein. The high amount of binding suggests association of the toxin with the lipid phase of the membrane. It has been shown that cardiotoxin first associates rapidly and reversibly to membrane lipids, then, in a second step, it induces a rearrangement of the membrane structure which produces and irreversible deactivation of the sodium-potassium activated adenosine triphosphatase. Solubilization of the membrane-bound ATPase with Lubrol WX gives an active enzyme species that is resistant to cardiotoxin-induced deactivation. Cardiotoxin binding to the membrane is prevented by high concentrations of Ca 2+ and dibucaine. Although cardiotoxins and neurotoxins of cobra venom have large sequence homologies, their mode of action on membranes is very different. The cardiotoxin seems to bind to the lipid phase of the axonal membrane and inhibits the sodium-potassium activated adenosine triphosphatase, whereas the neurotoxin associates with a protein receptor in the post-synaptic membrane and blocks acetylcholine transmission.     

Modifications of cysteine residues in the solution and membrane-associated conformations of phosphatidylinositol transfer protein have differential effects on lipid transfer activity

The alpha isoforms of mammalian phosphatidylinositol transfer protein (PITP) contain four conserved Cys residues. In this investigation, a series of thiol-modifying reagents, both alkylating and mixed disulfide-forming, was employed to define the accessibility of these residues and to evaluate their role in protein-mediated intermembrane phospholipid transport. Isolation and analysis of chemically modified peptides and site-directed mutagenesis of each Cys residue to Ala were also performed. Soluble, membrane-associated, and denatured preparations of wild-type and mutant rat PITPs were studied. Under denaturing conditions, all four Cys residues could be detected spectrophotometrically by chemical reaction with 4,4'-dipyridyl disulfide or 5,5'-dithiobis(2-nitrobenzoate). In the native protein, two of the four Cys residues were sensitive to some but not all thiol-modifying reagents, with discrimination based on the charge and hydrophobicity of the reagent and the conformation of the protein. With the soluble conformation of PITP, achieved in the absence of phospholipid vesicles, the surface-exposed Cys(188) was chemically modified without consequence to lipid transfer activity. Cys(188) exhibited an apparent pK(a) of 7.6. The buried Cys(95), which constitutes part of the phospholipid substrate binding site, was covalently modified upon transient association of PITP with a membrane surface. The Cys-to-Ala mutations showed that neither Cys(95) nor Cys(188) was essential for lipid transfer activity. However, chemical modification of Cys(95) resulted in the loss of lipid transfer activity. These results demonstrate that the Cys residues of PITP can be assigned to several different classes of chemical reactivity. Of particular interest is Cys(95), whose sulfhydryl group becomes exposed to modification in the membrane-associated conformation of PITP. Furthermore, the inhibition of PITP activity by thiol-modifying reagents is a result of steric hindrance of phospholipid substrate binding.     

Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.     

Interactions of lactoferricin-derived peptides with LPS and antimicrobial activity

Synthetic peptides derived from human and bovine lactoferricin, as well as tritrpticin sequences, were assayed for antimicrobial activity against wild-type Escherichia coli and LPS mutant strains. Antimicrobial activity was only obtained with peptides derived from the bovine lactoferricin sequence and peptides corresponding to chimeras of human and bovine sequences. None of the peptides corresponding to different regions of native human lactoferricin showed any antimicrobial activity. The results underline the importance of the content of tryptophan and arginine residues, and the relative location of these residues for antimicrobial activity. Results obtained for the same assays performed with LPS mutants suggest that lipid A is not the main binding site for lactoferricin which interacts first with the negative charges present in the inner core. Computer modelling of the most active peptides led to a model in which positively charged residues of the cationic peptide interact with negative charges carried by the LPS to disorganise the structure of the outer membrane and facilitate the approach of tryptophan residues to the lipid A in order to promote hydrophobic interactions.     

Characterization of elapidae snake venom components using optimized reverse-phase high-performance liquid chromatographic conditions and screening assays for alpha-neurotoxin and phospholipase A2 activities

The vast majority of Elapidae snake venoms, genus Naja, includes three classes of toxic polypeptides: alpha-neurotoxins, phospholipases A2, and cardiotoxins. A new experimental approach using reverse-phase high-performance liquid chromatography in particular has been developed, allowing their respective resolution, identification, and quantitation from milligram quantities of venom. First, definition of optimal chromatographic conditions for Naja mossambica mossambica toxins has been ascertained. Different column packing and solvent systems were compared for their efficiency, with particular attention to the ionic strength of the aqueous solvent. A medium-chain alkyl support (octyl) in conjunction with a volatile ammonium formate (0.15 M, pH 2.70)/acetonitrile solvent system was found to be particularly effective. All the components known until now from this venom could be resolved in a single step, and the elution order was alpha-neurotoxins, phospholipases A2, and cardiotoxins with a total recovery of absorbance and toxicity. Then, with these suitable conditions, we describe a new major cardiotoxin molecule in this venom by hydrophobic and not ionic-charge discrimination. Second, specific assays were designed to detect alpha-neurotoxin and phospholipase A2 activities in chromatographic fractions: alpha-neurotoxin activity was determined by competition for the binding of a radiolabeled alpha-neurotoxin to the acetylcholine receptor of the ray electric organ, and phospholipase A2 activity was defined by the enzymatic activity of these toxins with a fluorescent phospholipid as substrate. Finally, the applicability of these new methods to study other Naja snake venoms was demonstrated.     

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.     

The complete amino acid sequence of a cardiotoxin from the venom of Naja naja (Cambodian Cobra).

The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.     

Effects of Ca2+ and membrane surface charge on the direct lytic activity of cobra cardiotoxin - a membrane spin assay.

Direct membrane lytic activity of cobra (Naja Naja atra) cardiotoxin was studied by measuring the release of trapped spin label (2,2,6,6-tetramethyl-piperidinyloxyl)-choline chloride from liposomes. Results suggest that (a) presence of membrane negative charge and Ca²⁺ are required for optimal direct lytic activity of cardiotoxin, (b) direct Ca²⁺ binding to cardiotoxin may affect the equilibrium between its membrane active and inactive form.     

Structure and action of heteronemertine polypeptide toxins: importance of amphipathic helix for activity of Cerebratulus lacteus toxin A-III

The marine heteronemertine Cerebratulus lacteus produces a family of protein cytolysins designated as A-toxins. Limited subtilisin digests of the most abundant homolog, toxin A-III, yield two major products which may be purified by high-performance liquid chromatography. One product is shown to represent residues 1-86 and the other contains the entire toxin sequence (1-95). Both polypeptides are shown to lack internal protease nicks. The 1-95 polypeptide retains full cytolytic activity in comparison to native toxin, whereas 1-86 has an activity that is approximately four times lower. Extensive treatment of A-III with carboxypeptidase Y yields a polypeptide containing residues 1-75 which is totally devoid of hemolytic activity. Residues 63-95 of native A-III have been predicted to form a relatively hydrophobic alpha-helix which is potentially important for activity. The circular dichroism spectrum of 1-95 is in excellent agreement with both experimental and Chou-Fasman-predicted secondary structures of native A-III, while the spectra of 1-86 and 1-75 indicate a loss of helicity quantitatively consistent with the removal of residues 87-95 and 76-95, respectively. Combined with our earlier data on bilayer penetration by N-terminal sequences (K. M. Blumenthal (1982) Biochemistry 21, 4229-4233], the current results indicate a direct involvement of both ends of A-III in lytic activity. The C-terminal region may function by contributing a membrane binding site in the form of an amphipathic helix.     

Kinetics of erythrocyte lysis by snake venom cardiotoxins

Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 μg) could be conveniently and rapidly assayed with the high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated.Cardiotoxins from two different genera (Hemachatus haemachates and Naja mossambica mossambica) displayed virtually identical hemolytic properties. Hemolysis increased linearly with time, in contrast with a sigmoidal pattern when phospholipase was present as an impurity. Low concentrations of Ca²⁺ (<1 mM) stimulated cardiotoxin action. A limiting plateau rate of hemolysis reached during concentration dependence experiments in which the level of either cardiotoxin or of erythrocytes was varied, suggested that the interaction of cardiotoxin with erythrocyte membranes is a saturation phenomenon only at a high ratio of cardiotoxin: erythrocytes. No hemolysis was observed with an homologous neurotoxin of S-methylated cardiotoxin, providing evidence for specificity. The linear Arrhenius plots obtained for the temperature dependence of cardiotoxin-induced hemolysis strengthened the conclusion that its action involves more than a detergent-like effect on membrane phospholipids.     

The complete covalent structure of a cardiotoxin from the venom of Naja nigricollis (African black-necked spitting cobra).

The complete covalent structure of a small, basic protein with cardiotoxic activity is described. This has been isolated from the venom of Naja nigricollis by gel filtration on Sephadex G-75 and gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin, molecular weight 6806 from amino acid composition, consists of 60 amino acids, cross-linked by four disulfide bridges, connecting 3-21, 14-38, 42-53, and 54-59. The protein contains one residue of tryptophan, phenylalanine, and glutamic acid, two residues of arginine and tyrosine, four residues of methionine, and nine residues of lysine. Histidine is absent. The chymotryptic peptides of the oxidized and S-carboxymethylated protein were isolated by gel filtration on Sephadex G-25 and zone electrophoresis on a cellulose column. The sequence was determined by Edman degradation, using the (manual) direct phenylthiohydantoin method and with the use of carboxypeptidase A. Disulfide pairing was determined on thermolysin cleaved peptides from the native protein. The sequence is shown to be homologous to other cardiotoxins and a lytic factor from snake venoms and also shows homology, both in sequence and disulfide pairing to neurotoxins. A partial reduction experiment in the absence of denaturing agent using 14-C-labeled iodoacetic acid as S-carboxymethylating agent shows that disulfide bonds 14-38 and 42-53 were reduced fastest followed marginally by 54-59, and then bond 3-21.     

Delineation of the functional site of a snake venom cardiotoxin: preparation, structure, and function of monoacetylated derivatives

Toxin gamma, a cardiotoxin from the venom of the cobra Naja nigricollis, was modified with acetic anhydride, and the derivatives were separated by cation-exchange and reverse-phase chromatography. Nine monoacetylated derivatives were obtained, and those modified at positions 1, 2, 12, 23, and 35 were readily identified by automated sequencing. The overall structure of toxin gamma, composed of three adjacent loops (I, II, and III) rich in beta-sheet, was not affected by monoacetylation as revealed by circular dichroic analysis. Trp-11, Tyr-22, and Tyr-51 fluorescence intensities were not affected by modifications at Lys-12 and Lys-35, whereas Trp-11 fluorescence intensity slightly increased when Lys-1 and Lys-23 were modified. The cytotoxic activity of toxin gamma to FL cells in culture was unchanged after modification at positions 1 and 2, whereas it was 3-fold lower after modification at Lys-23 and Lys-35. The derivative modified at Lys-12 was 10-fold less active than native toxin. Using two isotoxins, we found that substitutions at positions 28, 30, 31, and 57 did not change the cytotoxic potency of toxin gamma. A good correlation between cytotoxicity, lethality, and, to some extent, depolarizing activity on cultured skeletal muscle cells was found. In particular, the derivative modified at Lys-12 always had the lowest potency. Our data show that the site responsible for cytotoxicity, lethality, and depolarizing activity is not diffuse but is well localized on loop I and perhaps at the base of loop II. This site is topographically different from the AcChoR binding site of the structurally similar snake neurotoxins.     

Relationship between antimicrobial activity and amphiphilic property of basic model peptides

Several cationic model peptides of the prepiece moieties of mitochondrial protein precursors were found to be active against Gram-positive bacteria, but inactive against Gram-negative bacteria. The CD spectra of the model peptides in the presence of phospholipid liposomes demonstrated that antimicrobial activity was generally in parallel with the content of the alpha-helical amphiphilicity. The results indicate that appropriate positioning of cationic and hydrophobic groups in the stable alpha-helical structure of the peptides is important to exhibit antimicrobial activity. These peptides also have an ability to leak carboxyfluorescein from acidic and neutral phospholipid vesicles, suggesting that the peptides interact with the bacterial membrane to perturb it.