Asper Myotoxin Research Articles

Articular inflammation induced by an enzymatically-inactive Lys49 phospholipase A2: activation of endogenous phospholipases contributes to the pronociceptive effect

BackgroundArthritis is a set of inflammatory conditions that induce aching, stiffness, swelling, pain and may cause functional disability with severe consequences to the patient’s lives. These are multi-mediated pathologies that cannot be effectively protected and/or treated. Therefore, the aim of this study was to establish a new model of acute arthritis, using a Lys49-PLA2 (Bothrops asper myotoxin II; MT-II) to induce articular inflammation.MethodsThe articular inflammation was induced by MT-II (10 μg/joint) injection into the left tibio-tarsal or femoral-tibial-patellar joints. Cellular influx was evaluated counting total and differential cells that migrated to the joint. The plasma extravasation was determined using Evans blue dye. The edematogenic response was evaluated measuring the joint thickness using a caliper. The articular hypernociception was determined by a dorsal flexion of the tibio-tarsal joint using an electronic pressure-meter test. The mediators involved in the articular hypernociception were evaluated using receptor antagonists and enzymatic inhibitors.ResultsPlasma extravasation in the knee joints was observed 5 and 15 min after MT-II (10 μg/joint) injection. MT-II also induced a polymorphonuclear cell influx into the femoral-tibial-patellar joints observed 8 h after its injection, a period that coincided with the peak of the hyperalgesic effect. Hyperalgesia was inhibited by the pretreatment of the animals with cyclooxygenase inhibitor indomethacin, with type-2 cyclooxygenase inhibitor celecoxib, with AACOCF3 and PACOCF3, inhibitors of cytosolic and Ca2+-independent PLA2s, respectively, with bradykinin B2 receptor antagonist HOE 140, with antibodies against TNFα, IL-1β, IL-6 and CINC-1 and with selective ET-A (BQ-123) and ET-B (BQ-788) endothelin receptors antagonists. The MT-II-induced hyperalgesia was not altered by the lipoxygenase inhibitor zileuton, by the bradykinin B1 receptor antagonist Lys-(Des-Arg9,Leu8)-bradykinin, by the histamine and serotonin antagonists promethazine and methysergide, respectively, by the nitric oxide inhibitor LNMMA and by the inhibitor of matrix 1-, 2-, 3-, 8- and 9- metalloproteinases GM6001 (Ilomastat).ConclusionThese results demonstrated the multi-mediated characteristic of the articular inflammation induced by MT-II, which demonstrates its relevance as a model for arthritis mechanisms and treatment evaluation.

Isolation, characterization and molecular cloning of AnMIP, a new α-type phospholipase A 2 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 α 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 PLIα 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 PLIαs isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA 2s, 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.

A Cellular Deficiency of Gangliosides Causes Hypersensitivity to Clostridium perfringens Phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.

Inhibition of Myotoxic Activity of Bothrops asper Myotoxin II by the Anti-trypanosomal Drug Suramin

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity.

Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A 2 from Bothrops snake venoms: Isolation of 4-nerolidylcatechol as active principle

Phospholipases A 2 (PLA 2) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA 2 from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA 2s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of ∼1 mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA 2s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA 2 activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA 2s studied.

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A 2 homologue from the venom of the snake Bothrops atrox

A new myotoxin was isolated from the venom of Bothrops atrox from Colombia. B. atrox myotoxin I is a homodimer, with a subunit molecular mass of 13,826, and a p I of 8.9. Its complete nucleotide sequence was obtained by cDNA cloning, indicating a mature product of 122 residues that belongs to the family of Lys49 phospholipase A 2 (PLA 2) homologues, a subgroup of catalytically inactive proteins within the group IIA. Accordingly, the toxin was devoid of phospholipase and anticoagulant activities, in vitro. In mice, it induced conspicuous local myonecrosis, edema, and a systemic interleukin-6 response. In vitro, it was cytolytic upon myoblasts, and weakly bactericidal. The toxin showed highest homology with other Lys49 PLA 2s, both in its primary and three-dimensional modeled structure, although with an evident difference in the C-terminal region. Unlike Lys49 proteins of American crotalids having 121 residues, this toxin presents an insertion (Asn) between positions 118 and 119. Despite several substitutions within the C-terminal region 115–129 between B. atrox myotoxin I and B. asper myotoxin II, antibodies against synthetic peptide 115–129 of the latter were strongly cross-reactive to the former, indicating the antigenic conservation of this site, known to be critical for the membrane-damaging activities of Lys49 myotoxins.

Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A 2

Myotoxic phospholipases A 2 account for most of the muscle necrosis that results from envenenomation by crotaline snakes. In this study, we investigated the protective effect of fucoidan, a natural sulfated polysaccharide obtained from the brown seaweed Fucus vesiculosus, against the cytotoxic and myotoxic activities of a group of phospholipase A 2 myotoxins from crotaline snake venoms: Bothrops asper myotoxins I, II, III, and IV, Cerrophidion godmani myotoxins I and II, Atropoides nummifer myotoxins I and II, and Bothriechis schlegelii myotoxin I. All of the toxins tested were efficiently inhibited by fucoidan, in both their cytotoxic and myotoxic effects. The basis for this inhibition appears to be the rapid formation of complexes between fucoidan and myotoxins, as evidenced by turbidimetric analysis. The possible binding site of fucoidan on the myotoxins was investigated using short synthetic peptides that represent the membrane-damaging region (residues 115–129) for three of these toxins. Fucoidan clearly inhibited the cytolytic activity of the peptides, indicating its ability to interact with the C-terminal myotoxic region of these phospholipases A 2. Fucoidan significantly inhibited muscle damage in mice, when administered locally, immediately after experimental envenomation with crude venom from B. asper. These results encourage further studies of sulfated fucans as compounds of potential use to improve the treatment of envenomations by crotaline snakes.

Neutrophils do not contribute to local tissue damage, but play a key role in skeletal muscle regeneration, in mice injected with Bothrops asper snake venom.

Local tissue damage induced by crotaline snake venoms includes edema, myonecrosis, hemorrhage, and an inflammatory response associated with a prominent cellular infiltrate. The role of neutrophils in the local tissue damage induced by Bothrops asper snake venom and by myotoxin I, a phospholipase A2 isolated from this venom, was investigated. Male Swiss mice were pretreated with either an antimouse granulocyte rat monoclonal immunoglobulin G (IgG) antibody or with isotype-matched control antibody. No significant differences in these local effects were observed between mice pretreated with antigranulocyte antibodies and those receiving control IgG. Moreover, myotoxicity induced by B. asper myotoxin I was similar in neutrophil-depleted and control mice. The role of neutrophils in the process of skeletal muscle regeneration was also assessed. Muscle regeneration was assessed by quantifying the muscle levels of creatine kinase and by morphometric histological analysis of the area comprised by regenerating cells in damaged regions of skeletal muscle. Mice depleted of neutrophils and then injected with B. asper venom showed a more deficient regenerative response than mice pretreated with control IgG. Moreover, a drastic difference in the regenerative response was observed in mice injected with myotoxin I, because animals pretreated with control IgG showed a successful regeneration, whereas those depleted of neutrophils had abundant areas of necrotic tissue that had not been removed 7 days after injection, associated with reduced contents of creatine kinase. It is concluded that (1) neutrophils do not play a significant role in the acute local pathological alterations induced by the venom of B. asper, and (2) neutrophils play a prominent role in the process of skeletal muscle regeneration after injection of B. asper venom and myotoxin I, probably related to the phagocytosis of necrotic material and the recruitment of other inflammatory cells, two events directly associated with a successful muscle regenerative response.

Biochemical and biological properties of phospholipases A 2 from Bothrops atrox snake venom

Phospholipases A 2 (PLA 2s), of molecular mass 13–15 kDa, are commonly isolated from snake venom. Two myotoxins with PLA 2 activity, BaPLA 2I and BaPLA 2III, with estimated molecular masses of 15 kDa were isolated from the venom of Bothrops atrox using Sephacryl S-100-HR and reverse-phase chromatography. BaPLA 2I was basic, with a pI of 9.1, while BaPLA 2III was neutral with a pI of 6.9. On a molecular basis, BaPLA 2III exhibited higher catalytic activity on synthetic substrates than BaPLA 2I. Comparison of the N-terminal residues of BaPLA 2I with other PLA 2 proteins from snake venoms showed that it has the highest homology (94%) with B. asper myotoxin II and homology with a PLA 2 Lys 49 from B. atrox (89%). In contrast, BaPLA 2III demonstrated 75, 72, and 71% homology with PLA 2 from Vipera ammodytes meridionalis, B. jararacussu, and B. jararaca, respectively. BaPLA 2I and BaPLA 2III were capable, in vitro, of inducing mast cell degranulation and, in vivo, of causing creatine kinase release, edema, and myonecrosis typical of PLA 2s from snake venoms, characterized by rapid disruption of the plasma membrane as indicated by clumping of myofilaments and necrosis of affected skeletal muscle cells. BaPLA 2I- and BaPLA 2III-specific monoclonal and polyclonal antibodies, although incapable of neutralizing PLA 2 edematogenic activity, blocked myonecrosis efficiently in an in vivo neutralization assay. The results presented herein suggest that the biological active site responsible for edema induction by these two PLA 2 enzymes is distinct from the myonecrosis active site and is not dependent upon the catalytic activity of the PLA 2 enzyme.

Identification of the myotoxic site of the Lys49 phospholipase A 2 from Agkistrodon piscivorus piscivorus snake venom: synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membrane-damaging activities

Group II phospholipase A 2 (PLA 2) myotoxins found in the venoms of Crotalidae snakes can be divided into ‘Asp49’ and ‘Lys49’ isoforms, the latter being considered catalytically-inactive variants. Previous studies on one Lys49 isoform, myotoxin II from Bothrops asper, indicated that its myotoxic activity is due to the presence of a short cationic/hydrophobic sequence (115–129) near its C-terminus, which displays membrane-damaging properties. Since the C-terminal region of different group II PLA 2 myotoxins presents considerable sequence variability, synthetic peptides homologous to region 115–129 of myotoxin II, but corresponding to B. asper myotoxin III (Asp49), Agkistrodon piscivorus piscivorus Asp49 PLA 2 and Lys49 PLA 2, were studied to determine the possible functional relevance of such region for the toxic activities of these proteins. Results showed that both Lys49-derived peptides (p-BaK49 and p-AppK49) were able to lyse skeletal muscle C2C12 cells in culture, and to induce edema in the mouse footpad assay. Moreover, p-AppK49, which showed a markedly stronger cytotoxic potency than p-BaK49, additionally induced skeletal muscle necrosis when injected into mice. These observations unequivocally identify the sequence 115–129 (KKYKAYFKLKCKK) of the Lys49 PLA 2 of A. p. piscivorus as containing the key structural determinants needed for myotoxicity, and represent the first report of an unmodified, PLA 2-derived short synthetic peptide with the ability to reproduce this effect of a parent toxin in vivo. On the other hand, the two Asp49-derived peptides did not show any toxic effects in vitro or in vivo, even at high concentrations. These findings suggests that Lys49 and Asp49 group II PLA 2s might exert their myotoxic actions through different molecular mechanisms, by implying that the latter may not utilize their C-terminal regions as main membrane-destabilizing elements.

Venom of the crotaline snake Atropoides nummifer (jumping viper) from Guatemala and Honduras: comparative toxicological characterization, isolation of a myotoxic phospholipase A 2 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 A 2 activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A 2 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 A 2 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 A 2 activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A 2 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.

Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A 2 from Bothrops asper snake venom

Myotoxic class II phospholipases A 2 from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115–129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1–15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A 2, showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins ( Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A 2, regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.

Structural and Functional Characterization of BnSP-7, a Lys49 Myotoxic Phospholipase A2 Homologue from Bothrops neuwiedi pauloensis Venom

BnSP-7, a Lys49 myotoxic phospholipase A2 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 A2 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 Ca2+-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 and characterization of myotoxin II from Atropoides ( Bothrops) nummifer snake venom, a new Lys49 phospholipase A 2 homologue

Myotoxic phospholipases A 2 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 A 2 homologue, isolated from the venom of Atropoides ( Bothrops) nummifer from Costa Rica. A. nummifer myotoxin II 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 μg) has no detectable phospholipase A 2 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 A 2. Sequence comparison with other phospholipases A 2 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 a myotoxic Lys49-phospholipase A 2 homologue from the venom of Cerrophidion ( Bothrops) godmani

The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A 2 (PLA 2) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA 2 showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca 2+, since the residues involved in forming the catalytic network of PLA 2s (His-48, Tyr-52 and Asp-99) are conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA 2s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA 2. The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca 2+-independent membrane damaging activity.

Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper.

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23-25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23-25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I-IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.

Chemical modification of histidine and lysine residues of myotoxic phospholipases A 2 isolated from Bothrops asper and Bothrops godmani snake venoms: Effects on enzymatic and pharmacological properties

Lysine and histidine residues of two myotoxic phospholipases A 2, Bothrops asper myotoxin III and Bothrops godmani myotoxin I, were chemically modified in order to study the effects of these treatments on enzymatic and pharmacological properties. After lysine acetylation the overall basicity of these toxins was lost and their enzymatic activity was significantly reduced, although a residual effect remained, which corresponded to 25% of the activity of native toxins. This treatment abolished both myotoxic and anticoagulant effects, and partially reduced liposome-disrupting activity. Histidine alkylation with P-bromophenacyl bromide affected phospholipase A 2, myotoxic and anticoagulant effects in a parallel way. After 24 hr of incubation with the alkylating reagent, these three activities were totally inhibited, in contrast to the liposome-disrupting effect which was only partially affected by this treatment. It is suggested that: (1) catalytic activity plays a role in the pharmacological effects of these myotoxins; (2) lysine residues are relevant for the toxic effects induced by these phospholipases A 2; and (3) despite the apparent relevance of enzymatic activity to the pharmacological properties of these toxins, the dissociation observed in lysine acetylation experiments suggests that these myotoxins have a molecular region, different from the catalytic site, which might be also involved in the toxic effects observed.

Skeletal Muscle Necrosis and Regeneration after Injection of BaH1, A Hemorrhagic Metalloproteinase Isolated from the Venom of the Snake Bothrops asper (Terciopelo)

The effects of BaH1, a hemorrhagic metalloproteinase isolated from Bothrops asper venom, on mouse gastrocnemius muscle was investigated. The toxin induced severe hemorrhage within minutes after injection. Groups of necrotic muscle fibers were observed after the sixth hour, with evident disorganization of myofibrillar material. At the ultrastructural level myofibrils in these cells lost their regular arrangement, Z lines were absent, and sarcomeres were disoriented, although there was no evidence of myofilament clumping or hypercontraction. Plasma membrane was interrupted in many portions. Mitochondrial alterations included swelling and the formation of flocculent densities and dense intracristal plates. At 12, 24, and 48 hr necrotic cells had amorphous masses of myofilaments and abundant phagocytic cells were observed both within necrotic fibers and in the interstitial space. Fraction D-1, which contains the three hemorrhagic metalloproteinases BaH1, BH2, and BH3, did not cause direct muscle damage when incubated with gastrocnemius muscle in vitro. Upon intramuscular injection in mice this fraction induced a small but significant increment in muscle lactic acid levels. Observations carried out 7 and 14 days after BaH1 injection revealed some regenerating muscle fibers with centrally located nuclei. However, other areas had few regenerating fibers of reduced diameter, surrounded by abundant fibroblasts, fibrosis, calcification, and remnants of necrotic muscle cells. When BaH1 was injected together with B. asper myotoxin III, a myotoxic phospholipase A2 that induces myonecrosis but does not affect blood vessels, a poor muscle regeneration was observed. In contrast, if B. asper myotoxin III was injected alone, regeneration proceeded normally and successfully.

Purification and characterization of myotoxin IV, a phospholipase A2 variant, from Bothrops asper snake venom.

A basic myotoxic protein was purified from Bothrops asper venom. Like other basic Bothrops myotoxins, myotoxin IV induces acute muscle damage after intramuscular injection in mice and disrupts negatively charged liposomes but not positively charged ones. Furthermore, this protein exerts a weak anticoagulant effect only at concentrations of 40 micrograms/ml or higher, and is devoid of phospholipase A2 activity. Rabbit polyclonal antibodies to B. asper myotoxin II, a related lysine-49 isoform, cross-react strongly with myotoxin IV by enzyme immunoassay, indicating a high degree of antigenic similarity between these two proteins. The fact that both toxins have similar amino acid compositions and amino-terminal sequences, including leucine-5 and glutamine-11, 2 amino acid residues conserved in all lysine-49 phospholipase A2 variants purified, strongly suggests that B. asper myotoxin IV is a lysine-49 phospholipase A2.

Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A 2 of Bothrops asper snake venom

B. Lomonte, A. Tarkowski and L. Å. Hanson. Broad cytolytic specificity of myotoxin II, a lysine-49 phospholipase A 2 of Bothrops asper snake venom. Toxicon 32, 1359–1369, 1994.—The cytotoxic activity of Bothrops asper myotoxin II, a lysine-49 phospholipase A 2 isoform, on different cell types in culture, was investigated. Myotoxin II caused a dose-dependent cytolytic effect on all cell types tested, characterized by rapid release of cytoplasmic lactic dehydrogenase and drastic morphological cell alterations. Quantitative differences in the susceptibility to myotoxin II among cell types fell within a relatively narrow range, and in general, the toxin was cytolytic at concentrations of 50–100 μg/ml (3–7 μM), when assays were performed using culture medium as a diluent. Toxin activity was markedly enhanced if phosphate-buffered saline was utilized instead of medium. The cytotoxic activity of myotoxin III, an aspartate-49 isoform from the same venom, on both endothelial cells and skeletal muscle myoblasts was higher than that of myotoxin II, suggesting that, although phospholipase A 2 activity is clearly not required for the induction of cell damage, it may have an enhancing role. In contrast to B. asper myotoxins, other basic phospholipases A 2 with myotoxic activity in vivo (notexin from Notechis scutatus, and two enzymes isolated from Vipera russelli venom) did not affect endothelial cells and myoblasts. Pretreatment of cells with neuraminidase, tunicamycin, or protamine, did not alter their susceptibility to myotoxin II. At low temperatures (2–4°C) myotoxin II was devoid of cytolytic effect. Washing and neutralization experiments using heparin with low affinity for antithrombin or mouse monoclonal antibody MAb-3 suggest that at low temperatures myotoxin II binds very weakly to the cells, and that its normal interaction with the putative target is probably not only based on charge, but that a membrane penetration event may be required.