An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action

In 1984, the first venom phospholipase A₂ (PLA₂) with a lysine substituting for the highly conserved aspartate 49 was discovered, in the North American crotalid snake Agkistrodon p. piscivorus [J. Biol. Chem. 259 (1984) 13839]. Ten years later, the first mapping of a ‘toxic region’ on a Lys49 PLA₂ was reported, in Bothrops asper myotoxin II [J. Biol. Chem. 269 (1994) 29867]. After a further decade of research on the Lys49 PLA₂s, a better understanding of their structural determinants of toxicity and mode of action is rapidly emerging, with myotoxic effector sites identified at the C-terminal region in at least four proteins: B. asper myotoxin II, A. p. piscivorus K49 PLA₂, A. c. laticinctus ACL myotoxin, and B. jararacussu bothropstoxin I. Although important features still remain to be established, their toxic mode of action has now been understood in its more general concepts, and a consistent working hypothesis can be experimentally supported. It is proposed that all the toxic activities of Lys49 PLA₂s are related to their ability to destabilize natural (eukaryotic and prokaryotic) and artificial membranes, using a cationic/hydrophobic effector site located at their C-terminal loop. This review summarizes the general properties of the Lys49 PLA₂ myotoxins, emphasizing the development of current concepts and hypotheses concerning the molecular basis of their toxic activities.     

Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water

The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA₂ from Agkistrodon contortrix laticinctus snake venom and Lys49PLA₂-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA₂-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.     

The C-terminal region of a Lys49 myotoxin mediates Ca2+ influx in C2C12 myotubes

Myotoxins are abundant components of snake venoms, being a significant public health problem worldwide. Among them, Lys49 phospholipase A₂ homologue myotoxins cause extensive necrosis in skeletal muscle tissue. Their mechanisms of action are still poorly understood, but there is evidence that the C-terminal region is involved in membrane damage leading to myotoxicity. To investigate the effect of the C-terminal peptide 115–129 of Agkistrodon contortrix laticinctus myotoxin on the plasma membrane of myoblasts and myotubes, the entry of Ca²⁺ was monitored by fluorescence imaging, and the ensuing cytotoxicity was determined. The myotoxin synthetic peptide was found to act selectively on myotubes, which were rapidly overloaded with Ca²⁺ with ensuing necrosis. The profile of intracellular Ca²⁺ increase induced by the C-terminal peptide, but not by its scrambled version control, reproduces the second, prominent wave of the biphasic response documented in previous studies using whole Lys49 myotoxins. These observations provide relevant insights into the mechanism of action of this family of toxins, with implications for the understanding of their structure–function relationships.     

Understanding the in vitro neuromuscular activity of snake venom Lys49 phospholipase A2 homologues

Phospholipases A₂ (PLA₂s) with a lysine substituting for the highly conserved aspartate 49, Lys49 PLA₂ homologues, are important myotoxic components in venoms from snakes of Viperidae family. These proteins induce conspicuous myonecrosis by a catalytically-independent mechanism. Traditionally, the Lys49 PLA₂ homologues are classified as non-neurotoxic myotoxins given their inability to cause lethality or paralytic effects when injected in vivo, even at relatively high doses. However, a series of in vitro studies has shown that several Lys49 PLA₂ homologues from Bothrops snake venoms induce neuromuscular blocking activity on nerve-muscle preparations in vitro. The interpretation of these findings has created some confusion in the literature, raising the question whether the Lys49 PLA₂ homologues present some neurotoxic activity. The present article reviews the in vitro neuromuscular effects of Lys49 PLA₂ homologues and discusses their possible mechanisms of action. It was concluded that the neuromuscular blockade induced by Lys49 PLA₂ homologues in isolated preparations is mainly a consequence of the general membrane-destabilizing effect of these toxins.     

Snake venom Lys49 myotoxins: From phospholipases A2 to non-enzymatic membrane disruptors

Snake venoms often contain toxins that cause a rapid necrosis of skeletal muscle fibers, referred to as myotoxins. The most common among them are phospholipases A₂ (PLA₂s), enzymes that have two independent evolutionary origins in snake venoms. Within the group II PLA₂s found in viperid venoms, a particular subgroup emerged, in which the otherwise conserved Asp49 of their catalytic center is replaced by Lys49. These intriguing proteins, referred to as Lys49 myotoxins, lost the ability to catalyze phospholipid hydrolysis, but still induce myonecrosis by a non-enzymatic mechanism based on membrane permeabilization as the critical event. Such mechanism is only partially understood. This review briefly describes the general structural and functional characteristics of the Lys49 myotoxins, and summarizes four proposed models of their functional “toxic site”. Finally, it discusses some novel insights into their mode of action, in particular examining arguments and experimental observations that could shed light on the possible nature of their membrane target on skeletal muscle cells, which remains elusive.     

Structural and functional characterization of brazilitoxins II and III (BbTX-II and -III), two myotoxins from the venom of Bothrops brazili snake

We report the purification and biochemical/pharmacological characterization of two myotoxic PLA₂ (BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂) from Bothrops brazili venom. Both were purified by a single chromatographic step on reverse phase HPLC, showing M_r 14 kDa for both myotoxins, showing high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂, had a sequence of 121 amino acid residues. BbTX-II: SLFELGKMILQETGKNPAKSYGAYGCYCGVLGRGKPKDATDRCCYVHKCCYKLTGCDNKKKDRYSYSWKDKTIVCGENNPCLKELCECDKAVAICLRENLNTYNKKYRYHLKPLCKKADAC with pI value 8.73. BbTX-III: SLWEWGQMILKETGKNPFPYYGAYGCYCGWGGRRKPKDATDRCCFVHDCCRYKKLTGCPKTNDRYSYSRLDYTIVCGEDDPCKEICECDKAAAVCFRENLRTYNKKYMAHLRVLCKKDKPC with a pI value of 8.46. BbTX-III presented PLA₂ activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35–45 °C. Maximum PLA₂ activity required Ca²⁺. In vitro, BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂ caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other Bothrops species. In mice, BbTX-II K49 PLA₂ homologue and BbTX-III PLA₂ induces myonecrosis and edema-forming activity. All these biological effects induced by the BbTX-II K49 PLA₂ homologue, occur in the absence of a measurable PLA₂ activity in vitro, further supporting the concept of catalytic independent mechanisms exerted by Lys49 proteins.     

Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis

In addition to their catalytic activity, snake venom phospholipases A₂ (vPLA₂) present remarkable diversity in their biological effects. Sequence alignment analyses of functionally related PLA₂ are frequently used to predict the structural determinants of these effects, and the predictions are subsequently evaluated by site directed mutagenesis experiments and functional assays. In order to improve the predictive potential of computer-based analysis, a simple method for scanning amino acid variation analysis (SAVANA) has been developed and included in the analysis of the lysine 49 PLA₂ myotoxins (Lys49-PLA₂). The SAVANA analysis identified positions in the C-terminal loop region of the protein, which were not identified using previously available sequence analysis tools. Site directed mutagenesis experiments of bothropstoxin-I, a Lys49-PLA₂ isolated from the venom of Bothrops jararacussu, reveals that these residues are exactly those involved in the determination of myotoxic and membrane damaging activities. The SAVANA method has been used to analyse presynaptic neurotoxic and anti-coagulant vPLA₂s, and the predicted structural determinants of these activities are in excellent agreement with the available results of site directed mutagenesis experiments. The positions of residues involved in the myotoxic and neurotoxic determinants demonstrate significant overlap, suggesting that the multiple biological effects observed in many snake vPLA₂s are a consequence of superposed structural determinants on the protein surface.     

Characterization of a basic phospholipase A2-homologue myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A₂ activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A₂ myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A₂-homologues with myotoxic, cytolytic, and edema-inducing activities.     

Characterization of Two Myotoxic Proteins from Venom of Crotalus viridis concolor

Abstract

The fractionation of the previously unstudied venom for Crotalus viridis concolor (midget faded rattlesnake) provided a protein fraction (FD-1) that generated myotoxic responses from envenomated mice. Highly basic homologous myotoxic proteins have been purified from the protein fraction, FD-1. The two major components, designated myotoxin I and myotoxin II, have considerable sequence homology with crotamine from C. d. terrificus, myotoxin a from C. v. viridis and peptide c from C. v. helleri. The amino acid sequences of the toxins from C. v. concolor contain at least two more residues of amino acid than previously described homologous toxins and myotoxin II contains a sequence region with microheterogeneity. The myotoxins cause extensive damage to the sarcoplasmic reticulum as evidenced by electron microscopic studies of envenomated muscle. The destruction is comparable to that observed with the homologous toxins. Studies with isolated sarcoplasmic reticulum vesicles failed to demonstrate any significant changes in Ca²⁺-Mg²⁺-ATPase or in uptake or release of Ca²⁺. Current evidence indicates that a closely related group of homologous toxins are present in rattlesnake venoms but the biochemical mode of action of these proteins remains obscure.     

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.     

Cloning and characterization of Trimeresurus gracilis venom phospholipases A2: Comparison with Ovophis okinavensis venom and the systematic implications

This study focuses on the structural and functional characterizations of novel venom phospholipases A₂ (PLA₂s) from Trimeresurus gracilis, an endemic Taiwanese pitviper. The PLA₂ cDNAs were cloned from venom glands and sequenced. The majority of the clones encoded a Glu6-containing PLA₂ (designated as Tgc-E6) whose deduced amino acid sequence resembled those of other Crotalinae acidic PLA₂s. Tgc-E6 was also purified and constituted about 6% (w/w) of the total venom proteins. For human platelet rich plasma, Tgc-E6 inhibited the ADP- and collagen-induced aggregation with an IC₅₀ of 272 nM and 518 nM, respectively. Like Ovophis okinavensis venom, T. gracilis venom did not contain any Lys49-PLA₂s, although a cDNA encoding Lys49-PLA₂ has been cloned from each of the species. Their predicted protein sequences are 94% identical, and their pI values 8.3 are lower than those of other Lys49-PLA₂s, mainly due to the acidic substitutions within positions 78-111, which are apparently more similar to those in Tgc-E6 than to those in other Lys49-PLA₂s. This unique structural feature of the venom PLA₂s thus render evidence for close phylogenetic relationship between both species. The structural variations in the venom acidic PLA₂s of the two species possibly have resulted from adaptation to different prey ecology.     

Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation

Lys49 phospholipase A₂ (PLA₂) homologues are myotoxic proteins devoid of catalytic activity. Their toxic determinants map to the C-terminal region 115-129, which plays an effector role in membrane damage. The dimeric state was reported to be essential for a Lys49 PLA₂ which lost its liposome-disrupting activity after dissociating into monomers at pH 5.0. This study, evaluated the effects of a pH-induced dissociation on the toxicity of four Lys49 PLA₂s, using biological targets instead. Both their cytolytic and myotoxic activities were lower at pH 5.0 than at pH 7.2. However, in contrast with experiments using artificial bilayers, toxic effects upon biological targets were not abolished at pH 5.0. Importantly, C-terminal synthetic peptides of two Lys49 PLA₂s also showed lower cytolytic action at pH 5.0 than at pH 7.2, indicating that factors other than the dimeric/monomeric state of the proteins may also be involved in these differences of toxicity. Results support the view that the dimeric state of Lys49 PLA₂s could play an enhancing, although not essential role, in their C-terminal region-mediated mechanism of myotoxicity.     

Amino acid sequence of a myotoxin from venom of the eastern diamondback rattlesnake (Crotalus adamanteus)

The complete amino acid sequence of a myotoxin isolated from Crotalus adamanteus venom (CAM-toxin) was determined. The total number of amino acid residues was 45, giving a mol. wt of 5202. The amino acid sequence was compared with those of previously determined Crotalus myotoxins. The CAM-toxin shows a surprisingly close relationship to those of peptide c (C. v. helleri), myotoxin a (C. v. viridis), crotamine (C. d. terrificus), myotoxin I and II (C. v. concolor) with homologies of approximately 98, 83, 90, 95 and 91%, respectively. The hydropathy profile of the CAM-toxin is almost identical to peptide c and myotoxin I, as can be assumed from the sequence homology. Despite the absence of any close sequence homology with the myotoxic phospholipase A2 enzymes, CAM-toxin contains a similar cationic myotoxic region at residues 2-10 as found in the other five myotoxic peptides.     

Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 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 pI 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(2)s, 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.     

An overview of lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action.

In 1984, the first venom phospholipase A₂ (PLA₂) with a lysine substituting for the highly conserved aspartate 49 was discovered, in the North American crotalid snake Agkistrodon p. piscivorus [J. Biol. Chem. 259 (1984) 13839]. Ten years later, the first mapping of a ‘toxic region’ on a Lys49 PLA₂ was reported, in Bothrops asper myotoxin II [J. Biol. Chem. 269 (1994) 29867]. After a further decade of research on the Lys49 PLA₂s, a better understanding of their structural determinants of toxicity and mode of action is rapidly emerging, with myotoxic effector sites identified at the C-terminal region in at least four proteins: B. asper myotoxin II, A. p. piscivorus K49 PLA₂, A. c. laticinctus ACL myotoxin, and B. jararacussu bothropstoxin I. Although important features still remain to be established, their toxic mode of action has now been understood in its more general concepts, and a consistent working hypothesis can be experimentally supported. It is proposed that all the toxic activities of Lys49 PLA₂s are related to their ability to destabilize natural (eukaryotic and prokaryotic) and artificial membranes, using a cationic/hydrophobic effector site located at their C-terminal loop. This review summarizes the general properties of the Lys49 PLA₂ myotoxins, emphasizing the development of current concepts and hypotheses concerning the molecular basis of their toxic activities.     

Characterization of a basic phospholipase A2-homologeu myotoxin isolated from the venom of the snake Bothrops neuwiedii (yarará chica) from Argentina.

A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A₂ activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A₂ myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A₂-homologues with myotoxic, cytolytic, and edema-inducing activities.     

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(2)s 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.     

Pharmacological modulation of edema induced by Lys-49 and Asp-49 myotoxic phospholipases A2 isolated from the venom of the snake Bothrops asper (terciopelo)

The pharmacological modulation of edema-forming activity of Bothrops asper myotoxins II and III, Lys-49 and Asp-49 phospholipases A₂, respectively, was studied plethysmographically in the mouse foot pad model. Myotoxin III had phospholipase A₂ activity, whereas myotoxin II was devoid of enzymatic activity when tested on egg yolk phosphatidylcholine. Both toxins induced a dose-dependent edema of rapid onset. Chemical modification of myotoxin III with p-bromophenacyl bromide abrogated enzymatic activity and significantly reduced edema-forming activity, although a residual effect remained. Pretreatment of animals with diphenhydramine, dexamethasone, indomethacin and prazosin significantly reduced the effect of both myotoxins. It is concluded that (a) these myotoxins are important edema-forming components of B. asper venom, (b) enzymatic activity is not a strict requirement to exert this effect, although in the case of myotoxin III it contributes to its development, and (c) several inflammatory mediators participate in mouse foot pad edema induced by these myotoxins.     

Phospholipases A2 of Asian Snake Venoms

Abstract

This review up-dated the structural and functional information of various phospholipase A₂ (PLA₂) isoforms purified from Asian snake venoms. A phylogenic tree of group I PLA₂s was constructed herein based on many recently resolved amino acid sequences of the venom enzymes. It was found that PLA₂s of Asian elapid venoms are structurally different from those of sea-snake/Australian elapid venoms, and are usually associated with cardiovascular effect, although exceptions such as β-bungarotoxins do exist. Two types of venom PLA₂s appear to be present in the venom of Asiatic viperinae such as Daboia and Echis, one has a N-terminal residue Asn and the other has the residue Ser. In the venom of Asiatic crotalinae, up to four subgroups of PLA₂ isoforms are present and each of them is characterized by a distinct substitution at residue 6 (Glu, Asn or Arg) or residue 49 (Asp or Lys) in their sequences. The venom PLA₂s in each of the subgroup show more or less functional similarity specific for the subgroup: the Glu6-PLA₂s are usually antiplatelet, the Asn6-PLA₂s are neurotoxic and/or myotoxic and many Arg6-PLA₂s are anticoagulating, while the Lys49-PLA₂s are myotoxic and edema-inducing. Mechanisms for the pharmacological actions of venom PLA₂s have been discussed, including neurotoxicity, myotoxicity, antiplatelet activity, anticoagulating activity, heparin-binding, protein-acylation and deacylation. Conclusions derived from many recent studies on pancreatic PLA₂ by method of protein engineering render valuable information about the structure-activity relationship of the secretory PLA₂ superfamily. Site-directed-mutagenesis methods coupled with relevant and dissecting functional assays are essential for understanding the structure-activity relationship of snake venom PLA₂s with special function or toxicity.     

Identification of the Molecular Determinants of the Antibacterial Activity of LmutTX,a Lys49 Phospholipase A2 Homologue Isolated from Lachesis muta muta Snake Venom (Linnaeus, 1766)

Snake venom phospholipases A₂ (PLA₂s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA₂ homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA₂s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 μg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram‐positive and Gram‐negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA₂ from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.