Isolation and characterization of basic myotoxic phospholipases A2 from Bothrops godmani (Godman's pit viper) snake venom.

Two basic myotoxic phospholipases A2 were purified to homogeneity from the venom of Bothrops godmani from Costa Rica by ion-exchange chromatography on CM-Sephadex. They have molecular weights of 14,300 (myotoxin I) and 13,400 (myotoxin II) and isoelectric points of 8.2 (myotoxin I) and 8.9 (myotoxin II). They behave as amphiphilic proteins in charge-shift electrophoresis and have similar amino acid compositions. Both toxins induce drastic myotoxic effects when injected in the gastrocnemius muscle of mice and induce release of peroxidase trapped in negatively charged liposomes. In addition, myotoxin I has high phospholipase A2 activity and is anticoagulant at doses higher than 0.3 microgram/ml, whereas myotoxin II has a very low phospholipase A2 activity and exerts anticoagulant effect only at concentrations higher than 50 micrograms/ml. Immunochemical data indicate that both toxins are immunologically related to Bothrops asper myotoxins, although B. godmani myotoxin II gives a stronger cross-reactivity when tested with antisera raised against B. asper myotoxins I and II.     

Cloning and cDNA sequence analysis of Lys(49) and Asp(49) basic phospholipase A(2) myotoxin isoforms from Bothrops asper

Snake venom myotoxic phospholipases A(2) contribute to much of the tissue damage observed during envenomation by Bothrops asper, the major cause of snake bites in Central America. Several myotoxic PLA(2)s have been identified, but their mechanism of myotoxicity is still unclear. To aid in the molecular characterization of these venom toxins, the complete open reading frames encoding two Lys(49) and one Asp(49) basic PLA(2) myotoxins from the Central American snake B. asper (terciopelo) were obtained by cDNA cloning from venom gland poly-adenylated RNA. The amino acid sequence deduced from the myotoxins II and III open reading frames corresponded in each case to one of the reported amino acid sequence isoforms. The sequence of a new myotoxin IV-like sequence (MT-IVa) contains conservative Val-->Leu(18) and Ala-->Val(23) substitutions when compared with the reported N-terminus of the native myotoxin IV, suggesting minor isoform variations among specimens of a single species. Sequence alignment studies indicated significant (>75% sequence identity) identities with other crotalid venom Lys(49) PLA(2)s, particularly bothropstoxin I/Ia isoforms of B. jararacussu and myotoxin II of B. asper.     

Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A2, except that it shows only 0.1% of the phospholipase activity against L-alpha-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2(+)-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.     

Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue

Myotoxic phospholipases A2 of class II are commonly found in the venoms of crotalid snakes. As an approach to understanding their structure-activity relationship, diverse natural variants have been characterized biochemically and pharmacologically. This study describes a new myotoxic phospholipase A2 homologue, isolated from the venom of Atropoides (Bothrops) nummifer from Costa Rica. A. nummifer myotoxin 1 is a basic protein, with an apparent subunit molecular mass of 16 kDa, which migrates as a dimer in sodium dodecylsulfate-polyacrylamide gel electrophoresis under nonreducing conditions. It is strongly recognized by antibodies generated against Bothrops asper myotoxin II, by enzyme-immunoassay. The toxin induces rapid myonecrosis upon intramuscular injection in mice (evidenced by an early increase in plasma creatine kinase activity), and significant edema in the footpad assay. It also displays cytolytic activity upon cultured murine endothelial cells. The toxin (up to 50 microg) has no detectable phospholipase A2 activity on egg yolk phospholipids, and does not show an anticoagulant effect on sheep platelet-poor plasma in vitro. N-terminal sequence determination (53 amino acid residues) demonstrated that A. nummifer myotoxin II is a new Lys49 variant of the family of myotoxic, class II phospholipases A2. Sequence comparison with other phospholipases A2 revealed Asn53 as a novel substitution. In addition, this myotoxin is the first Lys49 variant presenting Asn in its N-terminus. Consequently, these findings suggest that neither Ser1 or Lys53, usually found in this family of proteins, are essential amino acid residues for their myotoxic, cytolytic, or edema-inducing effects.     

Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A(2) homologue from Bothrops pirajai venom

The complete amino acid sequence of the 121 amino acid residues of piratoxin II, a phospholipase A(2) like myotoxin from Bothrops pirajai venom, is reported. PrTX-II is a basic protein with a molecular mass of 13740 Da, a calculated pI of 9.03, but an experimental pI of 8.4 +/- 0.2, showing sequence similarity with other bothropic (90-99%) or non-bothropic ( approximately 80%) Lys49 PLA(2)-like myotoxins. This similarity falls to approximately 70% when this sequence is aligned with that of Asp49 PLA(2). Due to the substitution of Asp49 by Lys49 and alterations in the calcium binding loop structure, as the replacement of Gly32 by Leu32, piratoxin-II shows no PLA(2) activity when assayed on egg yolk. Piratoxin-II showed the same primary structure as piratoxin-I, except that it has Lys116 for Leu116. Despite this slightly higher basicity at the C-terminal region, piratoxin-II was shown to be less myotoxic than piratoxin-I. The change Leu --> Lys induced an alteration of the molecule surface shape and probably of the environment charge high enough to slightly decrease the myotoxic activity. When aligned with B. jararacussu bothropstoxin-I and with B. asper Basp-II, piratoxin-II revealed a single (position 132) and a quintuple (positions 17, 90, 111, 120 and 132) amino acid substitution, respectively, suggesting a common evolutionary origin for these three myotoxins.     

The effect of myotoxins isolated from Bothrops snake venoms on multilamellar liposomes: relationship to phospholipase A2, anticoagulant and myotoxic activities.

The effect of four myotoxins isolated from Bothrops snake venoms on the release of peroxidase trapped in large multilamellar liposomes was studied and correlated to their phospholipase A2, myotoxic and anticoagulant activities. The four myotoxins affected negatively-charged liposomes in a dose-dependent way, having no effect on positively-charged liposomes. Conditions that inhibited phospholipase A2 activity, i.e., substitution of calcium by EDTA, reduced liposome-disrupting activity of Bothrops asper myotoxin I and Bothrops atrox myotoxin, both of which have high phospholipase A2 activity, but did not affect the action of B. asper myotoxin II and Bothrops moojeni myotoxin II, which have extremely low phospholipase A2 activity. However, all myotoxins disrupted to some extent negatively-charged liposomes under conditions where phospholipase A2 activity was abolished. Since these toxins behave as amphiphilic proteins in charge-shift electrophoresis, it is suggested that membrane-disorganization is at least partially due to a non-enzymatic penetration and alteration of bilayers. There was no strict correlation between liposome-disrupting activity and myotoxicity in vivo. Thus, although both effects probably depend on the toxins' ability to disturb membranes, it is likely that variation in complexity between skeletal muscle plasma membrane and liposome bilayers are the basis for this difference. The anticoagulant effect seems to depend on the ability of the toxins to enzymatically degrade phospholipids, since only B. asper myotoxin I and B. atrox myotoxin prolonged the plasma recalcification time.     

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization,isolation of a myotoxic phospholipase A2 homologue and neutralization by two antivenoms

A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A₂ activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A₂ homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A₂ variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A₂ activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.     

Structural characterization and phylogenetic relationships of myotoxin II from Atropoides (Bothrops) nummifer snake venom,a Lys49 phospholipase A(2) homologue

In order to analyze its structure-function relationships, the complete amino acid sequence of myotoxin II from Atropoides (Bothrops) nummifer from Costa Rica was determined. This toxin is a Lys49-type phospholipase A(2) (PLA(2)) homologue, devoid of catalytic activity, structurally belonging to class IIA. In addition to the Asp49 --> Lys change in the (inactive) catalytic center, substitutions in the calcium-binding loop suggest that its lack of enzymatic activity is due to the loss of ability to bind Ca(2+). The toxin occurs as a homodimer of basic subunits of 121 residues. Its sequence has highest similarity to Lys49 PLA(2)s from Cerrophidion, Trimeresurus, Bothrops and Agkistrodon species, which form a subfamily of proteins that diverged early from Asp49 PLA(2)s present in the same species, as shown by phylogenetic analysis. The tertiary structure of the toxin was modeled, based on the coordinates of Cerrophidion godmani myotoxin II. Its exposed C-terminal region 115-129 shows several differences in comparison to the homologous sequences of other Lys49 PLA(2)s, i.e. from Agkistrodon p. piscivorus and Bothrops asper. Region 115-129 of the latter two proteins has been implicated in myotoxic activity, on the basis of the direct membrane-damaging of their corresponding synthetic peptides. However, peptide 115-129 of A. nummifer myotoxin II did not exert toxicity upon cultured skeletal muscle cells or mature muscle in vivo. Differences in several amino acid residues, either critical for toxicity, or influencing the conformation of free peptide 115-129 from A. nummifer myotoxin II, may account for its lack of direct membrane-damaging properties.     

Individual expression patterns of myotoxin isoforms in the venom of the snake Bothrops asper.

1. In order to investigate the distribution of myotoxin isoforms in the snake Bothrops asper, venoms from individual specimens were analyzed by a cathodic electrophoretic system for basic proteins under native conditions. 2. The electrophoretic system resolved at least five bands with slight differences in mobility, corresponding to the fastest migrating proteins in the venom. The identity of the bands was confirmed by immunoblotting, using a rabbit anti-myotoxin serum. 3. There were clear differences in the individual patterns of myotoxin isoform expression, both in specimens from the Atlantic and Pacific regions of Costa Rica. Some individuals possessed all five variants. 4. In agreement with previous reports, the venoms of ten newborn (less than 10 days of age) specimens completely lacked myotoxin bands, indicating an ontogenetic regulation in the expression of these toxins in B. asper. 5. One of the bands, corresponding to the lysine-49 phospholipase myotoxin II, was the only isoform present in all individuals studied, suggesting a possible selective pressure for the conservation of this type of protein in the venom of B. asper.     

Amino acid and cDNA sequences of a neutral phospholipase A2 from the long-nosed viper (Vipera ammodytes ammodytes) venom.

The amino acid sequence of a non-toxic phospholipase A2, ammodytin I2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) and its cDNA sequence have been determined. The protein sequence was elucidated by sequencing the peptides generated by CNBr cleavage, mild acid hydrolysis and tryptic digestion of maleylated and non-maleylated protein. Sequencing of the cDNA showed that the protein is synthesized as an 137-amino-acid-residue precursor molecule consisting of a 16-residue signal peptide, followed by a 121-residue mature enzyme. Ammodytin I2 cDNA shows 73% nucleotide and 59% amino acid identities in the mature protein region in comparison to that of ammodytoxin A, the most presynaptically neurotoxic phospholipase A2 from the long-nosed viper. Identities in the signal-peptide region are considerably higher, 96% and 100%, respectively.     

Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.     

The C-terminal and beta-wing regions of ammodytoxin A, a neurotoxic phospholipase A2 from Vipera ammodytes ammodytes, are critical for binding to factor Xa and for anticoagulant effect

Ammodytoxin A (AtxA) from the venom of Vipera ammodytes ammodytes belongs to group IIA secreted phospholipase A2 (sPLA2), for which the major pathologic activity is presynaptic neurotoxicity. We show here that this toxin also affects hemostasis because it exhibits strong anticoagulant activity. AtxA binds directly to human coagulation factor Xa (FXa) with Kdapp of 32 nM, thus inhibiting the activity of the prothrombinase complex with an IC50 of 20 nM. To map the FXa-interaction site on AtxA, various mutants of AtxA produced by site-directed mutagenesis and expressed in Escherichia coli were tested in the study. In surface plasmon resonance (SPR) measurements, with FXa covalently attached to the sensor chip, we show that the FXa-binding site on AtxA includes several basic amino acid residues at the C-terminal and beta-wing regions of the molecule. Applying an in vitro biological test for inhibition of prothrombinase activity, we further demonstrate that the same residues are also very important for the anticoagulant activity of AtxA. We conclude that the anticoagulant site of AtxA is located in the C-terminal and beta-wing regions of this phospholipase A2. Synthetic peptides comprising residues of the deduced anticoagulant site of AtxA provide a basis to synthesize novel anticoagulant drugs.     

The amino acid region 115-119 of ammodytoxins plays an important role in neurotoxicity

Quadruple (Y115K/I116K/R118M/N119L) and double (Y115K/I116K) mutants of ammodytoxin A, a presynaptically toxic phospholipase A(2) from Vipera ammodytes ammodytes venom, were prepared and characterized. The enzymatic activity of the quadruple mutant on phosphatidylcholine micelles was threefold higher than that of AtxA, presumably due to higher phospholipid-binding affinity, whereas the activity of the double mutant was twofold lower. The substantial decrease by more than two orders of magnitude in the lethal potency of both mutants, together with their decreased binding affinity for neuronal receptors, indicates involvement of the amino acid region 115-119 in neurotoxicity. The similar decrease of toxicity for the two mutants points to the importance of the residues Y115 and I116.     

Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production

Bothrops moojeni crude venom (MjCV) and its two major toxins, namely myotoxin I (MjTX-I) and myotoxin II (MjTX-II) were alkylated by p-bromophenacyl bromide (BPB). After alkylation the i.p. LD(50) (mice) of MjCV and MjTX-I/II increased from 6.0 to 15.7mg/kg and from 8.0 to 45.0mg/kg, respectively. In addition, doses of 5x LD(50) of alkylated MjTX-I did not cause a single death in mice and no myonecrosis was detected for the alkylated toxins, although both proteins still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A(2) found in snake venoms of the genus Bothrops. Myotoxic PLA(2)s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA(2)s, despite differences in their amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were apparently more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies raised against the native or modified toxins inhibited their PLA(2) and myotoxic activities. Therefore, alkylation of His48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while retaining antigenicity, which suggests a promising procedure for an enhanced antiophidian serum production for practical purposes.     

Structural and functional characterization of BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom

BnSP-7, a Lys49 myotoxic phospholipase A(2) homologue from Bothrops neuwiedi pauloensis venom, was structurally and functionally characterized. Several biological activities were assayed and compared with those of the chemically modified toxin involving specific amino acid residues. The cDNA produced from the total RNA by RT-PCR contained approximately 400 bp which codified its 121 amino acid residues with a calculated pI and molecular weight of 8.9 and 13,727, respectively. Its amino acid sequence showed strong similarities with several Lys49 phospholipase A(2) homologues from other Bothrops sp. venoms. By affinity chromatography and gel diffusion, it was demonstrated that heparin formed a complex with BnSP-7, held at least in part by electrostatic interactions. BnSP-7 displayed bactericidal activity and promoted the blockage of the neuromuscular contraction of the chick biventer cervicis muscle. In addition to its in vivo myotoxic and edema-inducing activity, it disrupted artificial membranes. Both BnSP-7 and the crude venom released creatine kinase from the mouse gastrocnemius muscle and induced the development of a dose-dependent edema. His, Tyr, and Lys residues of the toxin were chemically modified by 4-bromophenacyl bromide (BPB), 2-nitrobenzenesulfonyl fluoride (NBSF), and acetic anhydride (AA), respectively. Cleavage of its N-terminal octapeptide was achieved with cyanogen bromide (CNBr). The bactericidal action of BnSP-7 on Escherichia coli was almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The neuromuscular effect induced by BnSP-7 was completely inhibited by heparin, BPB, acetylation, and CNBr treatment. The creatine kinase releasing and edema-inducing effects were partially inhibited by heparin or modification by BPB and almost completely abolished by acetylation or cleavage of the N-terminal octapeptide. The rupture of liposomes by BnSP-7 and crude venom was dose and temperature dependent. Incubation of BnSP-7 with EDTA did not change this effect, suggesting a Ca(2+)-independent membrane lytic activity. BnSP-7 cross-reacted with antibodies raised against B. moojeni (MjTX-II), B. jararacussu (BthTX-I), and B. asper (Basp-II) myotoxins as well as against the C-terminal peptide (residues 115-129) from Basp-II.     

Isolation, characterization and molecular cloning of AnMIP, a new alpha-type phospholipase A2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae)

A new phospholipase A(2) (PLA(2))-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA(2) myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the alpha group of phospholipase A(2) inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247-22,301 and an isoelectric point of 4.1-4.7. This trimeric structure is the first observed in a PLIalpha from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIalphas isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA(2)s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper myotoxin I and A. nummifer myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.     

Tyr-->Trp-substituted peptide 115-129 of a Lys49 phospholipase A(2) expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect.

Myotoxin II is a group II Lys49 phospholipase A(2) (PLA(2)) isolated from the venom of the snake Bothrops asper. Previous studies on a synthetic peptide derived from its heparin-binding, cationic/hydrophobic sequence 115-129 demonstrated a direct functional role of this particular region in the in vitro cytolytic and bactericidal actions of the protein. Nevertheless, no significant myonecrosis has been observed after local intramuscular injection of peptide 115-129 (p115-129) in mice. Since the membrane-damaging action of p115-129 was proposed to depend on its amphiphilic character, the present study examined the effects of substituting its cluster of three tyrosine residues by tryptophan residues, on its toxic/pharmacological activities in vitro and in vivo. This substitution resulted in a drastic enhancement of the membrane-damaging activities of the peptide (p115-W3), together with the clear expression of myotoxic activity in vivo. Both the heparin-binding and antigenic characteristics of p115-129 were essentially conserved in p115-W3, suggesting that the modification did not lead to radical structural alterations. In addition to myotoxicity, cytotoxicity, and bactericidal action, p115-W3 exerted edema-forming activity in the mouse footpad assay. Thus, the synthetic 13-mer p115-W3 reproduced all the known toxic effects of myotoxin II. In spite of its potent membrane-damaging actions, p115-W3 did not acquire direct hemolytic activity upon mouse erythrocytes, an effect which is not present in myotoxin II, but that has been ascribed to the presence of tryptophan in other cationic, membrane-damaging peptides such as mellitin from bee venom. The myotoxic activity of p115-W3 herein described constitutes the first example of a short, PLA(2)-based linear synthetic peptide with the ability to reproduce this effect of a parent protein in vivo. This finding is in clear support of the proposed relevance of the C-terminal region 115-129 in all the membrane-damaging mechanisms exerted by myotoxin II, including the myotoxic mechanism.     

Amino-acid sequence of ammodytoxin B partially reveals the location of the site of toxicity of ammodytoxins

The complete amino-acid sequence of ammodytoxin B, a presynaptically toxic phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined by manual and automated protein sequencing. Ammodytoxin B (i.v. LD50 = 0.58 mg/kg for white mice) is 30-fold less toxic than ammodytoxin A, the most toxic phospholipase isolated from the same venom. The two proteins (each 122 residues long) differ in only 3 residues located in positions 115, 118 and 119 (numbering according to R. Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634) suggesting that an exposed hydrophobic residue in position 115 and a basic residue in position 118 may be responsible for the increased toxicity of ammodytoxin A and should form at least one part of the site of toxicity in ammodytoxins.     

Binding to the high-affinity M-type receptor for secreted phospholipases A(2) is not obligatory for the presynaptic neurotoxicity of ammodytoxin A

R180, isolated from porcine brain cortex, is a high-affinity membrane receptor for ammodytoxin A (AtxA), a secreted phospholipase A(2) (sPLA(2)) and presynaptically active neurotoxin from venom of the long-nosed viper (Vipera ammodytes ammodytes). As a member of the M-type sPLA(2) receptors, present on the mammalian plasma membrane, R180 has been proposed to be responsible for one of the first events in the process of presynaptic neurotoxicity, the binding of the toxin to the nerve cell. To test this hypothesis, we prepared and analyzed three N-terminal fusion proteins of AtxA possessing a 12 or 5 amino acid residue peptide. The presence of such an additional "propeptide" prevented interaction of the toxin with the M-type receptor but not its lethality in mouse and neurotoxic effects on a mouse phrenic nerve-hemidiaphragm preparation. In addition, antibodies raised against the sPLA(2)-binding C-type lectin-like domain 5 of the M-type sPLA(2) receptor were unable to abolish the neurotoxic action of AtxA on the neuromuscular preparation. The specific enymatic activities of the fusion AtxAs were two to three orders of magnitude lower from that of the wild type, yet resulting in a similar but less pronounced neurotoxic profile on the neuromuscular junction. This is in accordance with other data showing that a minimal enzymatic activity suffices for presynaptic toxicity of sPLA(2)s to occur. Our results indicate that the interaction of AtxA with the M-type sPLA(2) receptor at the plasma membrane is not essential for presynaptic activity of the toxin. Interaction of AtxA with two intracellular proteins, calmodulin and the R25 receptor, was affected but not prevented by the presence of the N-terminal fusion peptides, implying that these proteins may play a role in the sPLA(2) neurotoxicity.     

Sequence homology between phospholipase and its inhibitor in snake venom. The primary structure of phospholipase A2 of vipoxin from the venom of the Bulgarian viper (Vipera ammodytes ammodytes, Serpentes)

The amino-acid sequence of phospholipase A2 from the neurotoxin vipoxin of the Bulgarian Viper (Vipera ammodytes ammodytes, Serpentes) is presented. The enzyme consists of 122 amino-acid residues including 7 disulfide bonds and thus belongs to phospholipases A2 group IIA. The sequence was determined by automatic Edman degradation of the intact chain and of the peptides obtained after tryptic hydrolysis of the oxidized chain. The short cleavage time of 30 min and another limited tryptic digestion of the oxidized and citraconylated chain provided overlapping peptides. Sequencing was done with liquid- and gas-phase sequenators. The complete alignment of all peptides was facilitated by the high degree of homology with known viperid venom phospholipases A2. In common with mammalian phospholipases, the tryptophan residue in position 30 (essential for enzymatic activity) as well as the histidine in position 47 in the active site are present. Vipoxin phospholipase A2 shows 53.3% homology with another phospholipase A2 from Vipera ammodytes ammodytes venom (Ammodytoxin B), whereas 62% homology was found between both subunits of vipoxin phospholipase A2 and its inhibitor. This high degree of identity can be accounted for in terms of a common origin by gene duplication.