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Protein & Peptide Letters, 2009, 16, 860-876

The Phospholipase A2 Homologues of Snake Venoms: Biological Activities and Their Possible Adaptive Roles
Bruno Lomonte1,*, Yamileth Angulo1,2, Mahmood Sasa1 and Jos Mara Gutirrez1
1

Instituto Clodomiro Picado, School of Microbiology, and 2Department of Biochemistry, School of Medicine, Universidad de Costa Rica, San Jos, Costa Rica
Abstract: A particular subgroup of toxins with phospholipase A2 (PLA2) structure, but devoid of this enzymatic activity, is commonly found in the venoms of snakes of the family Viperidae, and known as the PLA2 homologues. Among these, the most frequent type presents a lysine residue at position 49 (Lys49), in substitution of the otherwise conserved aspartate (Asp49) of catalytically-active PLA2s. A brief and updated overview of these toxic PLA2 homologues is presented, emphasizing their various biological activities, both in vivo and in vitro. The relevance of these bioactivities in relation to their possible adaptive roles for the snakes is discussed. Finally, experiments designed to assess the validity of such hypothetical roles are suggested, to stimulate future studies in this field.

Keywords: Snake, venom, myotoxin, phospholipase A2, Lys49 homologues. 1. PHOSPHOLIPASE A2 TOXINS IN SNAKE VENOMS Phospholipase A2 (PLA2) enzymes are among the most common and abundant components in the venom of advanced snakes of the superfamily Colubroidea, including the highly poisonous Viperidae and Elapidae families, which evolved nearly 60-80 million years ago [1,2]. The growing number of snake venom proteomes, or venomes, being studied reveal that PLA2s may constitute in some species as much as 60% of the proteins in these complex secretions [3], where they play critical roles in toxicity [4]. In the venoms of some species of snakes, PLA2 can be the main toxic factor responsible for prey immobilization and death [5,6]. Thus, toxic PLA2s have had a major adaptive value for the evolution of venomous snakes, and it has been demonstrated that these venom enzymes evolved by means of an accelerated process [7,8] to acquire diverse and potent toxic functions. Noteworthy among these functions are the targeting of skeletal muscle, the neuromuscular junction, and the haemostatic system [9]. According to phylogenetic evidence, snake venom PLA2s probably arose by two independent recruitment events, originating from the non-toxic pancreatic (group I) digestive enzymes in the family Elapidae, and from inflammatory (group II) enzymes in the family Viperidae [1]. The overall three-dimensional structure, as well as the catalytic machinery of all these interfacial enzymes have been highly conserved [10-12]. However, subtle changes in surface-exposed amino acid residues during evolution provided to snake venom PLA2s the ability to exert a wide range of toxic activities [13], by gradually adapting to the recognition of particular molecular targets, hence interfering with physiological processes [14]. Since early studies, the toxic effects of snake venom PLA2s were attributed to their enzymatic activity, i.e. hydrolysis of the sn-2 ester bond of 1,2-diacyl-3-sn-phosphoglycerids to produce lysophospholipids and fatty acids [12]. Later analyses on the correlation between catalytic activity and toxicity of PLA2s gradually disclosed that enzymatic activity alone, as determined in vitro using artificial substrates, could not always predict toxic potency, i.e. some highly catalytic enzymes have lower toxicity than others showing lower catalytic activity, and vice versa [15]. This puzzling finding has been subsequently clarified, since the interfacial binding properties and the target specificity of these PLA2s vary considerably, and are also known to play important roles in their overall mechanisms of toxicity, of which phospholipid hydrolysis is only one of the steps involved [4,9]. Moreover, although most of the toxic effects of the catalytically-active PLA2s are dependent on phospholipid hydrolysis, effects that are unrelated to catalysis in this group of enzymes have also been demonstrated [16-18]. Snake PLA2 toxins possess a conserved catalytic network formed by four amino acid residues, His48, Asp49, Tyr52, and Asp99, present in both group I and group II enzymes [10]. Together with residues of the "calcium-binding loop", Asp49 coordinates the essential Ca2+ ion cofactor for phospholipid hydrolysis [11]. For this reason, the presence of Asp49 and the ability to bind Ca2+ are considered as absolute requirements for the expression of catalytic activity in all secreted PLA2s. Strikingly, the occurrence of two proteins in which the highly conserved Asp49 was substituted by Lys was first reported in the venoms of Agkistrodon p. piscivorus and Bothrops atrox [19], soon to be followed by reports on the presence of similar proteins in numerous other Viperidae snake species, mostly of the Crotalinae subfamily (reviewed in [20]). Initially, these "Lys49" venom proteins were considered to possess a low, but detectable and intrinsic PLA 2 activity [19]. However, this notion was subsequently questioned by structural and biochemical evidence that strongly argued against such interpretation [21-23]. Studies using
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*Address correspondence to this author at the Instituto Clodomiro Picado, Facultad de Microbiologa, Universidad de Costa Rica, San Jos, Costa Rica; Fax (+506) 2292-0485; E-mail: bruno.lomonte@ucr.ac.cr

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recombinant Lys49 [24] and Ser49 [25] toxins have unambiguously resolved this controversy, demonstrating that these proteins lack PLA2 activity. Thus, the earlier observations on low enzymatic activity of Lys49 proteins isolated from natural sources can now be re-interpreted to be the consequence of minor contamination with catalytically-active Asp49 PLA2 isoforms present in the same venoms. Due to their essential difference with true PLA2 enzymes, the Lys49substituted proteins, as well as variants with other substitutions at position 49, have been commonly referred to as '"PLA2-like", or "PLA2 homologues". 2. PHOSPHOLIPASE A2 HOMOLOGUES The subgroup of PLA2 homologue toxins has rapidly grown to currently include not less than 60 proteins, as listed in Table 1. Among these, the most frequent type of toxins presents the Lys49 substitution, but other amino acids such as Ser, Arg, Gln, and Asn have also been found in this position. Several of these toxins have been thoroughly characterized as to their structural properties and functional abilities, and although significant progress has been made in understanding the molecular basis of their toxic effects, there are still fundamental details of their mode of action that remain unknown. This review will briefly mention few aspects on the structure-function relationships of PLA2 homologues, as these have been extensively reviewed [10,20,26-28] and will be covered in other contributions of this special issue. The main focus of the present work will be to summarize the diverse activities displayed by these toxins, and to discuss the possible biological significance and adaptive roles of their emergence in snake venoms. 3. STRUCTURAL AND FUNCTIONAL ASPECTS OF PLA2 HOMOLOGUES PLA2 homologues possess the general structural features of group II secreted PLA2s, with subunits of 120-122 amino acids that are stabilized by seven conserved disulfide bridges [10]. Most commonly the PLA2 homologues exist as homodimers in solution, with subunits held together by noncovalent forces which do not dissociate in the presence of strong anionic detergents such as sodium dodecylsulphate. A monomeric state has been observed for some of these proteins when crystallized, although the possibility should be considered that acidic conditions utilized during chromatographic procedures or crystallization protocols could have an influence on these findings, since it has been shown, at least for some of these toxins, that their dimers dissociate at pH values 5 [29,30]. All of the PLA2 homologues share the property of having slightly to markedly basic isoelectric points, being rich in Lys and Arg residues. This cationic character is proposed to have an important role in target recognition, as well as in the effector activities of these toxins [26,31-34]. The key structural determinants for the toxic actions of the Lys49 PLA2 homologues lie at their C-terminal region, within residues 115-129 (numbering system of Renetseder [35]), as initially revealed by studies using synthetic peptides [36-39] and neutralizing agents [40-43], and clearly supported by more recent site-directed mutagenesis analyses [24,27,28,44]. In a thoroughly characterized Lys49 protein, bothropstoxin I, detailed mapping analyses of the C-terminal residues in-

volved in toxicity have revealed subtle differences for the diverse activities, with a partial overlapping of the delineated bioactive sites confined within this region [28,44]. The current view on the mode of action of the Lys49 PLA2 homologues involves their binding to anionic membrane targets, still unidentified, followed by the interaction of the C-terminal region with phospholipid bilayers, either biological or artificial, leading to drastic permeability disturbances [20]. The effector role of the C-terminal region upon membrane integrity might additionally be enhanced by a transition in the quaternary structure of the toxin dimers, as there is structural evidence that their dimerization interface might function as a "molecular hinge" that would facilitate penetration into the bilayer [45,46]. This potential enhancing mechanism, however, does not appear to be an absolute requirement for exerting membrane damage by Lys49 PLA 2 homologues, since it has been shown that the dissociated monomeric proteins still retain significant toxicity [30]. In addition to this quaternary structural change, crystallographic evidence for a conformational change in the C-terminal region, possibly associated with the binding of a phospholipid or a fatty acid into the nominal (in)active "catalytic" site of a Lys49 toxin, has been documented [47]. In ACL myotoxin, such ligand-induced conformational change exposes two hydrophobic residues, Phe121 and Phe124, towards the membrane bilayer, a phenomenon that is likely to be relevant for the toxic actions of this protein [47]. 4. BIOLOGICAL ACTIVITIES OF THE PLA2 HOMOLOGUES Myotoxicity in vivo was the first biological activity reported for a Lys49 PLA2 homologue [48], an effect now well established to be common to all proteins within the group of PLA2 homologues tested so far. The myotoxic effect of Lys49 proteins is exerted only locally, at the site of injection, in contrast to the systemic action of other types of myotoxins such as PLA2s or PLA2 complexes from elapids and some viperids [34,49]. A comprehensive list of the biological activities described for PLA2 homologues is presented in Table 2. As indicated, some of these actions are observed in vivo, and therefore bear relevance to the pathophysiological alterations induced by snakebites, while others are restricted to ex vivo or in vitro phenomena which are important from a basic point of view, but are not necessarily linked to the effects caused by these toxins in human and animal envenomings. An effect that belongs to this category, still lacking evidence for its occurrence in experimental or clinical envenomings, is the neuromuscular blockade activity. Such an effect has been demonstrated in neuromuscular preparations [50-57], but neurotoxicity is not observed when these toxins are injected in vivo, as indicated by their relatively low lethal potencies (see below) and by the lack of neurotoxic manifestations. Similarly, renal damage [58] and alterations in bladder water transport [59,60] have been studied only in ex vivo models of perfusion or bathing. Despite not having been demonstrated in vivo, the study of these phenomena is relevant, since it may reveal molecular and cellular clues to understand the modes of action of PLA2 homologues, and at the same time, may exploit these toxins as experimental tools to gain deeper insights into the diverse physiological processes affected by them.

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Table 1.

Phospholipase A2 Homologues Isolated from Snake Venoms

Snake Species Agkistrodon piscivorus piscivorus Agkistrodon bilineatus Agkistrodon contortrix laticinctus Atropoides (Bothrops) mexicanus (nummifer) Atropoides (Bothrops) mexicanus (nummifer) Atropoides (Bothrops) mexicanus (nummifer) Bothrops alternatus Bothrops asper Bothrops asper Bothrops asper Bothrops asper Bothrops atrox Bothrops atrox Bothrops atrox Bothrops brazili Bothrops jararacussu Bothrops leucurus Bothrops moojeni Bothrops moojeni Bothrops neuwiedi Bothrops neuwiedi pauloensis Bothrops pirajai Bothrops pirajai Bothriechis (Bothrops) schlegelii Bothriechis (Bothrops) schlegelii Calloselasma (Agkistrodon) rhodostoma Calloselasma (Agkistrodon) rhodostoma Cerrophidion (Bothrops) godmani Cerrophidion (Bothrops) godmani Crotalus atrox Crotalus molossus molossus Cryptelytrops (Trimeresurus) albolabris Cryptelytrops (Trimeresurus) albolabris Cryptelytrops (Trimeresurus) albolabris Deinagkistrodon (Agkistrodon) acutus Deinagkistrodon (Agkistrodon) acutus Toxin AppK49 PLA2-II ACL myotoxin myotoxin I myotoxin I-h myotoxin II BaTX myotoxin II myotoxin IV myotoxin IVa M1-3-3 Ba-K49 BaPLA2-I myotoxin I myotoxin bothropstoxin I bl/K-PLA 2 myotoxin I myotoxin II myotoxin I BnSP-7 piratoxin I piratoxin II myotoxin I Bsc-K49 CRV-K49 Cr5 myotoxin II PgoK49 Cax-K49 Cmm-K49 Tal-K49 GPV-K49 GPV-N49 Dac-K49 Dac-K49b 49 K * K * * K K K * K K K K K * K K K K * K K K * K K K K K K * * K N K K Sequence P04361 Q9PSF9 P49121 u p P82950 ** P24605 Q9PR7, p P0C616 Q9PVE3 ** **, p Q6JK69 u Q90249 **, p P82114 Q9I834 **, p Q9IAT9 P58399 P82287 **, p P80963 Q9PVF3 ** P81165 Q8UVU7 Q8UVZ7 **, p **, p ** ** x77650 O57385 PDB 1PPA u 1S8G ** u 2AOZ u 1CLP u u u u u u u 2H8I u u 1XXS u 1PC9 u 1QLL u u u u 1GOD u u u u u u u u References [19, 139, 140] [141] [47, 142, 143] [48, 144] [145] [80, 146, 147] [57] [135, 148, 149] [150] [151] unpublished [19] [152] [153] [71] [33, 154, 155] [156] [157, 158] [157, 159, 160] [161] [162-164] [165, 166] [165, 167, 168] [169] [170] [170] [171] [86, 172, 173] [79] [79] [79] [79] [174] [174] [72] [79, 175]

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(Table 1) contd.

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Snake Species Deinagkistrodon (Agkistrodon) acutus Echis carinatus sochureki Gloydius (Agkistrodon) halys Gloydius (Agkistrodon) blomhoffi ussurensis Ovophis (Trimeresurus) okinavensis Protobothrops (Trimeresurus flavoviridis Protobothrops (Trimeresurus) flavoviridis Protobothrops (Trimeresurus) mucrosquamatus Protobothrops (Trimeresurus) mucrosquamatus Protobothrops (Trimeresurus) mucrosquamatus Protobothrops (Trimeresurus) mucrosquamatus Trimeresurus borneensis Trimeresurus gramineus Trimeresurus gramineus Trimeresurus puniceus Trimeresurus puniceus Viridovipera (Trimeresurus) stejnegeri Viridovipera (Trimeresurus) stejnegeri Viridovipera (Trimeresurus) stejnegeri Viridovipera (Trimeresurus) stejnegeri Viridovipera (Trimeresurus) stejnegeri Viridovipera (Trimeresurus) stejnegeri Vipera ammodytes Zhaoermia (Trimeresurus) mangshanensis

Toxin acutohemolysin ecarpholin S Gh-N49 Gln49-PLA2 To-3 BP-I BP-II TMV-K49 Tm-K49 TM-N49 promutoxin Tbo-K49 PLA2-V PLA2-VII Tpu-K49a Tpu-K49b Ts-K49a Ts-K49b Ts-K49b' Ts-K49c Ts-R6-N49 CTs-R6-N49 ammodytin L zhaoermiatoxin

49 K S N Q K K K K K N R K K K K K K K K K N N S R

Sequence ** P48650 O42188 ** Q92152 P20381 E48188 P22640 x77647 DQ212913 DQ299948 AY355177 P70090 P70089 AY211935 AAR14166 AAP48893 AAP48896 AAP48895 AAP48894 AAP48891 AAP48890 P17935 P84776

PDB 1MC2 2QHE u u u u u u u u u u u u u u u u u u u u u 2PH4

References [176] [177] [178] [179] [180] [181] [182] [183] [72] [184] [184] [73] [185] [7] [73, 79] [79] [89] [89] [89] [89] [89] [89] [186] [81, 187]

Species nomenclature follows Castoe and Parkinson [188]. Former names for genera or species are indicated in parentheses, to facilitate tracking of previous literature on the toxins. * Not determined; considered as PLA2 homologue on the basis of sequence homology and/or lack of PLA2 activity. ** Not submitted to databases; p: partial sequence; u: undetermined.

The activities of PLA2 homologues (Table 2) can be grouped into several categories. Lethality should be ruled out as a main biological purpose of these toxins. Although death can be induced by the injection of PLA2 homologues via intraperitoneal or intravenous routes in mice, very high doses are needed, in the range of mg/kg of body weight. For example, the lethal dose 50% for the Lys49 protein of A. p. piscivorus is 25 mg/kg (intravenously) [61], and for B. moojeni myotoxin II this value corresponds to 7.6 mg/kg (intraperitoneally) [62]. These potencies are three orders of magnitude lower than those recorded for the catalytically-active PLA2s or PLA2 complexes that have evolved as paralyzing and lethal neurotoxins [15], strongly suggesting that PLA2 homologues have not evolved for the main purpose of killing prey, at least in the case of rodents.

By analyzing the activities listed in Table 2, it is evident that one group of toxic effects of the PLA2 homologues is directly related to membrane bilayer disorganization, which can be exerted on either artificial (liposome disruption), prokaryotic (bactericidal action), or eukaryotic (antifungal, antiparasite, cytotoxic, and myotoxic activities) membranes. Several of these activities have been reproduced by synthetic peptides corresponding to their C-terminal 115-129 region (Table 2), highlighting the key role of this molecular site for toxicity. On the other hand, a number of other activities can be grouped as being related to the inflammatory reactions triggered by these toxins (edema, leukocyte chemotaxis and activation, cytokine release, mast cell degranulation, and hyperalgesia). Some of these effects might arise as an indirect consequence of necrotic damage to skeletal muscle

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Table 2.
Type

Activities Described for Phospholipase A2 Homologues

Activity Original Report Activity Reproduced by C-Terminal Peptides * [38]

in vivo

myotoxicity (local) lethality edema cytokine release leukocyte recruitment hyperalgesia and mechanical allodynia analgesic action tumor growth inhibition

[48] [48] [135] [63] [189] [110] [190]

[38]

[110]

[191] ** [48] [61] [50] [59] [85] [192] [193] [113] [113] [66] [194] [195] [58] [196] [197] [87] [198] [67] [62] [62] [120] [117] ** [118] ** [36] [36] [113] [113]

ex vivo

myotoxicity neuromuscular blockade presynaptic neuromuscular blockade increased water transport in bladder

in vitro

liposome disruption cytotoxicity (on many cell types) heparin (and other polyanions) binding bactericidal action LPS-binding mast cell degranulation inhibition of HIV replication neutrophil chemotaxis kidney damage cell proliferation apoptosis KDR (VEGF receptor)-binding factor Xa-binding macrophage phagocytosis activation fungicidal action antiparasite action blockade of proinflammatory effects of LPS

For simplicity, only the reference of the first report where the activity was described is indicated, although some of these activities have been extensively confirmed for several of the toxins classified as PLA2 homologues. * Synthetic peptides corresponding to the C-terminal region 115-129 of the parent toxins, or ** modified peptides derived from such sequences.

tissue, which induces a complex inflammatory host response [63-65], but several direct in vitro actions of the PLA2 homologue toxins have also been demonstrated, such as mast cell degranulation [66], macrophage activation [67] (Table 2), or endothelial cell damage [68]. Finally, other activities such as the recently reported high affinity binding to the KDR receptor for vascular endothelial growth factor (VEGF), or the

induction of apoptosis (Table 2) still require further studies to assess their pathophysiological consequences in vivo, and thus rationalize their biological purpose. For example, recent data evidenced that Lys49 PLA2 homologues may act synergistically with VEGF to promote vascular permeability in vivo, through the binding to the KDR receptor [69].

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5. PLA2 HOMOLOGUES ARE NUMEROUS AND ABUNDANT IN VIPERID VENOMS Several observations are noteworthy when analyzing the PLA2 homologues so far described in the venoms of viperids. First, they occur in a large number of viperid taxa, although not ubiquitously in all, intriguingly being absent in the venoms of some species that are phylogenetically very close to others that contain them (Table 1 and Fig. 1). The character reconstructions presented in (Fig. 1) show that PLA2 homologues were recruited in Viperidae before the separation of Viperinae and Crotalinae, as evidenced by their presence in both lineages. Nevertheless, the vast majority of these toxins have been found in crotalines, where their widespread occurence is also in support of an early presence of these genes in the evolution of this subfamily. Ambiguous ancestral states in (Fig. 1) derive from the absence of toxin expression in Bothrops jararaca and B. erythromelas, which in the pruned tree pooled together within the same group. Similarly, our analysis reveals that several terminal taxa appear to have independently lost the capacity to express this type of toxin. A second observation is that, in species where they occur, PLA2 homologue toxins may be produced in considerably high amounts by the venom glands. For example, a single PLA2 homologue isoform may comprise as much as 10-25% of the whole venom protein content, as noted in Bothrops asper, B. brazilii, Trimeresurus mucrosquamatus and T. borneensis [70-73]. In Protobothrops (formerly Trimeresurus) flavoviridis, the sum of basic Asp49 PLA2 and PLA 2 homologues constitutes 30% of the venom proteins [74], and in Bothrops jararacussu, 58% of the expressed sequence tags generated from venom glands corresponded to PLA2s, out of which 83% corresponded to its major Lys49 PLA2 homologue, bothropstoxin I [75]. Third, a number of toxin isoforms exist, both at the population level and in single specimens. Genomic analyses evidenced six venom PLA2 isozyme genes in Protobothrops flavoviridis, two of which correspond to Lys49 homologues [76]. At the protein level, several isoforms of PLA2 homologues have been identified and isolated from single snake species (see, for example, Viridovipera (Trimeresurus) stejnegeri in Table 1), and it is common to find at least two isoforms of these toxins in a single species (Table 1). Individual snakes express various isoforms, as analyzed in the venom of Bothrops asper [77]. Thus, the widespread and abundant presence of PLA2 homologues in the venom of many viperids, together with the existence of a number of isoforms, suggest that these toxins play an important biological role for these snakes. The multiplicity of protein isoforms at the single specimen level, originating from gene duplication and divergence mechanisms, might have provided a redundancy that allowed the PLA2 homologues to escape the pressure of negative selection and mutate under the positive accelerated evolution scheme evidenced by Ohno and colleagues [8]. 6. RATIONALE FOR THE EMERGENCE OF PLA 2 HOMOLOGUES: DO BIOLOGICAL ACTIVITIES AND DIET PROVIDE CLUES? Many aspects concerning the PLA2 homologue toxins are still intriguing, which makes their study challenging. Phylo-

genetic studies indicate that PLA2 homologues diverged from ancestral group II Asp49 PLA2s [78-82]. Many basic Asp49 PLA2s in viperid venoms possess myotoxic activity, inducing a pattern of muscle damage that develops with morphological and temporal features that are indistinguishable from those induced by PLA2 homologues [49,83]. Interestingly, the myotoxic potency of Lys49 toxins was found to be higher than that of Asp49 PLA2s of the same venom, in the case of Protobothrops flavoviridis [84], therefore suggesting a rationale for the emergence of the PLA2 homologues during evolution. However, in other species such as Bothrops asper, Lys49 proteins show a weaker myotoxic potency than basic Asp49 PLA2s [85], and in Cerrophidion (Bothrops) godmani, no difference in myotoxic potency between a Lys49 homologue and an Asp49 PLA2 was observed [86]. Therefore, a gain in myotoxic potency over Asp49 PLA2 ancestors does not appear to be a general principle to explain the emergence and widespread presence of PLA2 homologues in viperids. In addition to myotoxicity, most biological activities of PLA2 homologues are shared also by their basic Asp49 PLA2 counterparts in viperid venoms. For example, both types of toxins have in common the ability to induce edema, hyperalgesia and other inflammatory responses, to disrupt artificial membranes such as liposomes, to cause cytolysis of a variety of cell lines in culture, and to kill diverse types of bacteria. These similarities raise the question as to what advantage did the emergence of PLA2 homologues provide. One clear difference in biological activities between Lys49 PLA2 homologues and their Asp49 counterparts is the strong binding of the former, but not of the latter, to the KDR receptor for VEGF [87,88]. This finding might provide novel clues to understand the biological role of PLA2 homologues, but at this moment the relationship of this activity to the mechanisms of toxicity of Lys49 proteins is unknown. Another puzzling observation is the fact that PLA2 homologues may coexist with basic Asp49 PLA2 myotoxins in some venoms, may be the only basic myotoxins in others, and may be absent in still other venoms. Moreover, a striking ontogenetic regulation exists in the expression of basic myotoxins, both Asp49 PLA2s and PLA2 homologues in some species. The venoms of newborn and juvenile specimens lack these proteins, whereas the venom of adults contains high amounts of them [77,89,90]. Additional results presented in (Fig. 2) for Atropoides mexicanus (formerly A. nummifer) and Cerrophidion (Bothrops) godmani support the notion that such ontogenetic regulation for the expression of basic PLA2 myotoxins may be a general rule among crotalines. This phenomenon may be related to diet, involving prey capture and/or digestion, but such analyses are limited by the scarcity of data on feeding habits of newborn and juvenile snakes in nature. In a study of Viridovipera (Trimeresurus) stejnegeri, the bamboo viper from Taiwan and China, an association between the presence of a Lys49 PLA2 homologue in venom and a rodent-rich diet was noted [89]. In contrast, the lack of Lys49 toxins in the venom of juvenile snakes was associated with a diet in which frogs predominate [89]. A similar observation was reported in a study of Protobothrops flavoviridis from Okinawa island, in which the lack of Lys49 PLA2 homologues appeared to be linked to a frogrich diet [91,92]. However, the association between a rodent-

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Figure 1. Evolutionary transformations of phospholipase A2 homologues and diet in Viperidae. Ancestral character states for 46 species were reconstructed using the Farris optimization algorithm [199,200] implemented in MacClade [201]. Absence of the toxin was assumed only in those taxa in which immunochemical and chromatographic analyses to detect expression yielded negative results [83,202]. All states were treated as unordered, and transitions between them as equally probable. Character reconstruction is mapped over Castoe and Parkinson [188] pruned viper phylogeny, following Wster et al. [203] resolution for the Bothrops clade (including snakes formerly assigned to the genus Bothriopsis). As the position of the bushmaster genus Lachesis remains controversial within the clade of Neotropical crotalines [204,205, and references therein], we examined the effect of several alternative topologies regarding its location in the phylogenetic tree. No differences were noticed in the overall ancestral patterns reported here. Black lines: PLA2 homologue present; white lines: PLA2 homologue absent; cross-hatched lines: equivocal data. Records for diet follow Daltry et al. [206], Gloyd and Conant [207], Martins et al. [96], Solrzano [94], and our unpublished data. Generalist: consumes at least four different prey types; variable diet: consumes two or three prey types; mammal specialist: known to feed only on mammals.

predominant diet and presence of venom PLA2 homologues may not be a general trend. For example, a study on the venom proteome of Bothriechis lateralis, an arboreal viperid from Costa Rica, noted the lack of Lys49 PLA2 homologues [93], despite this species having a diet mainly based on rodents [94]. Similarly, the venom of Bothrops jararaca from Brazil is practically devoid of basic PLA2s [95], despite the observation that rodents constitute 50-75% of its diet [96]. These apparent discrepancies prompted us to analyze the relationships between diet and expression of PLA2 homologues (Fig. 1). Diet reconstruction supports the previous notion that pit vipers evolved from diet generalists, some of which also include invertebrates in their menu [96,97]. Mammal spe-

cialization (as well as other monotypic diets) is an apomorphic character in the crotalines, probably resulting from ecological forces related to prey availability in the areas they inhabit [96,98]. Eight out of the nine species of mammalian specialists included in our analysis do not express PLA 2 homologue toxins. In fact, there is an inverse correlation between the absence of the toxins and the number of prey types known to be consumed by viper species (Spearman r=0.6, p=0.012), even after restricting the analysis to the appropriate degrees of freedom using Felsensteins [99] method of evolutionary independent contrasts. Thus, diverging with other studies [91], loss of expression of PLA2 homologue toxins is associated with mammal-restricted diets in this data set. Nevertheless, there are four generalist or semi-generalist species that also do not express these toxins:

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Figure 2. Ontogenetic regulation in the expression of venom myotoxic phospholipases A2 in Cerrophidion godmani (left panel) and Atropoides mexicanus (right panel) from Costa Rica. Basic proteins were electrophoretically separated in 12% polyacrylamide gels at pH 4.3 under native conditions, as previously described [77], and stained with Coomassie blue R-250. Bands corresponding to myotoxic phospholipases in venom samples from adults are indicated: myotoxins I and II in C. godmani are Asp49 and Lys49 variants, respectively [86], myotoxin II in A. nummifer is a Lys49 variant [80,146]. Note the absence of bands in venom samples from juvenile (less than one month of age) specimens of both species. Gel polarity is indicated.

by gastric and pancreatic digestive secretions a difficult task. The venom, therefore, is likely to play a relevant role in the digestion of such bulky prey, and the abundance and activity of proteinases in viperid snake venoms, especially of metalloproteinases [100], constitutes an adaptation to accomplish this role. Muscle tissue represents a large percentage of the body mass in mammals, in the range of 40-50 % [101,102]. Thus, digestion of the complex array of muscle proteins, especially actin and myosin constituting the contractile machinery of muscle, is fundamental for a proper digestion of prey. It is proposed that necrotic muscle tissue, i.e. tissue that has been affected by myotoxic components of snake venoms, is more readily digested by proteinases present in the venom itself and in the gastric and pancreatic secretions. Myotoxic PLA2s and PLA2 homologues directly affect muscle cells by damaging the plasma membrane, allowing a prominent influx of Ca2+ from the extracellular milieu, which activates a complex series of intracellular degenerative events leading to irreversible cell damage [49]. One of the consequences of such Ca2+ influx is the activation of calpains, Ca2+-dependent cysteine proteinases that cleave a number of cytoskeletal proteins [103]. A rapid degradation of structurally-relevant proteins, such as desmin, -actinin and titin, has been described in muscle injected with myotoxic PLA2s [104-106]. Such degradation affects the mechanical integration of actin and myosin, and is likely to facilitate the widespread hydrolysis of these predominant muscle proteins which in vivo is accomplished by proteinases from inflammatory cells [104,107] and, in the case of natural envenomations, would be performed by venom proteinases and by digestive gastric and pancreatic proteinases. Therefore, by inducing rapid myonecrosis, the myotoxic action of PLA2s and PLA2 homologues is likely to facilitate the further digestion of muscle proteins. Such a digestion-promoting role has been proposed for the myotoxic PLA2s present in the venom of the Australian elapid Oxyuranus scutellatus [108]. PLA2 homologues exert local myotoxicity, i.e. induce myonecrosis in the anatomical region where venom is injected, and do not induce systemic myotoxicity [34]. However, in the natural setting, the usually large volume of venom injected by viperid snakes in natural prey would guarantee a relatively widespread distribution of venom within the prey, thus reaching many muscle compartments at a rapid rate. This would ensure a widespread myonecrosis that facilitates muscle mass digestion, as discussed above. Nevertheless, this hypothetic role of myotoxins could be accomplished by both PLA2 homologues and by myotoxic Asp49 PLA2 enzymes, thus not providing a selective advantage of the former in performing this digestive task. Moreover, the fact that PLA2 homologues are absent in the venom of many species that only prey on mammals argues against this digestive role when handling prey having a high volume to surface body ratio. Consequently, additional biological roles might be at work behind the conspicuous presence of PLA2 homologues in these venoms. 7.2. On the Toxic Role of PLA2 Homologues in Particular Prey As discussed before, PLA2 homologues are not highly lethal for rodents when injected by several routes. However, this does not necessarily imply that such proteins are devoid

Bothrops jararaca, B. taeniata, Porthidum nasutum, and P. ophryomegas, adding difficulties to the interpretation of the pattern. Other examples of mammal specialists that lack PLA2 homologues in venom are Atropoides picadoi, Crotalus simus, Crotalus durissus, Bothrops cotiara, Lachesis muta, and L. stenophrys (Fig. 1). In spite of the difficulties to understand the biological meaning for the emergence of the PLA2 homologues in viperid venoms, as outlined above, their abundance and frequent occurrence in many species strongly suggest that they have provided an important adaptive value in this family of snakes. In the following sections, we propose and discuss some hypotheses on what may be the biological significance and adaptive value of the Lys49- and related PLA2 homologues. Where possible, experiments to address such hypotheses will be suggested, attempting to stimulate further multidisciplinary research in this challenging subject. 7. ADAPTIVE BIOLOGICAL ROLES OF LYS49 PLA 2 HOMOLOGUES 7.1. Contribution to the Digestion of Muscle Mass Adult viperid snakes mostly prey on mammals [98]. Mammalian species present a relatively high body volume:surface ratio [98], which makes digestion of large prey

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of toxicity for other types of prey. The expression of PLA2 homologues in the venoms of viperids evidences an early appearance of their genes in the diversification of pit vipers and an early recruitment of these toxins in the venom proteomes (Fig. 1). Many species of vipers present a highly generalistic prey repertoire, which includes groups as diverse as arthropods, amphibians, reptiles, birds and mammals. It might be that PLA2 homologues are more toxic for some of these prey groups than for rodents. In agreement with this concept, species having a generalistic diet repertoire are those that express PLA2 homologues in their venoms. The recently described strong affinity of a Lys49 PLA2 homologue for the KDR VEGF receptor [87,88] may have implications in the species of prey in which possible orthologues of this receptor could have still unknown physiological roles. The toxicity of PLA2 homologues needs to be investigated in a wide variety of potential prey species. 7.3. The Induction of Pain as an Effective Immobilizing Mechanism Viperid snakebite envenomings are associated with excruciating pain [109]. Pharmacological investigations have demonstrated that myotoxic Asp49 PLA2s and Lys49 PLA 2 homologues are potent pain inducers in rat models of hyperalgesia and allodynia [110-112]. Such hyperalgesic effect is of very rapid onset and results from the action of endogenous mediators that interact with afferent nerve fibers associated with pain [110]. We suggest that this pain effect of rapid onset is a highly effective mechanism for prey immobilization, since mammalian prey injected with crude viperid venoms or isolated Lys49 PLA2 homologues remain stationary at the site of injection licking the injected limb (our unpublished observations). Such an effect would preclude a rapid escape of the prey, and would facilitate their location and ingestion by snakes that present a strike and release pattern of biting, such as the majority of viperid snakes [98]. Thus, the acquisition of pain-inducing effect in the evolution of PLA2s and PLA2 homologues may have represented an adaptive advantage for prey immobilization. Again, as in the case of the proposed digestive role of myotoxic PLA2s, there does not seem to be a special advantage of PLA2 homologues over Asp49 PLA2s in the fulfilment of this biological role. This hypothesis implies that the mechanisms of prey immobilization by snake venoms go beyond the well-known paralytic, hemorrhagic and hypotensive effects, and might include the ability of venoms to provoke immediate pain. 7.4. Is Microbicidal Activity the Clue? Myotoxic Asp49 PLA2s and Lys49 PLA2 homologues are potent microbicidal components in snake venoms [113-117]. The structural determinant of this effect in Lys49 PLA 2 homologues is a stretch of cationic and hydrophobic residues located at the C-terminal region of these proteins [28,37,113,118,119]. Lys49 PLA2 homologues bind with high affinity to bacterial lipopolysaccharide (LPS) [113,120], being able to block the effects of LPS on macrophages and other targets [117,120]. It is therefore proposed that a possible adaptive role of the high concentration of PLA2 homologues in many viperid venoms has to do with their microbicidal activity, with two important functional implications: (a) to maintain the venom stored in the venom gland free of mi-

crobial contamination. Owing to the high protein concentration of venom, and to the fact that venom is stored in the venom gland lumen before injection [121], the risk of contamination in the venom gland is high. The presence of these microbicidal components in relatively high concentration in the venom would preclude this contamination; and (b) to avoid bacterial-induced putrefaction of the bulky prey that viperid snakes ingest, as suggested by Tsai et al. [120], thus contributing to an appropriate digestive process. In both cases, the high concentrations of PLA2 homologues described in many venoms would favor the accomplishment of these roles. PLA2 homologues are likely to have an advantage over catalytically-active Asp49 PLA2 in performing this bactericidal role, due to the fact that venoms contains a high concentration of citrate as a mechanism to keep some enzymes inactive during storage [122,123]. Citrate concentrations in viperid venoms are high enough to inhibit the enzymatic activity of a myotoxic Asp49 PLA2 [122]. In addition, the characteristic acidic pH in the venom represents another inhibitory factor for several enzymatic components, including proteinases and PLA2s [124]. Therefore, in the venom gland milieu, Asp49 PLA2s are unlikely to be effective microbicidal compounds owing to the inhibition of catalytic activity in these conditions. In contrast, Lys49 PLA2 homologues exert microbicidal action by a catalytically-independent mechanism [113] and, therefore, would not be inhibited by citrate or acidic pH in the venom gland. The ability of PLA2 homologues to kill bacteria and fungi in the venom gland, as well as in the body of the prey during digestion may therefore accomplish a powerful adaptive role. In the light of these hypotheses, the presence of a highly cationic face in PLA2 homologues, and the typically high pIs of these proteins, represent an adaptive advantage. The presence of this cationic face [31,32,34] enables these proteins to bind to negatively-charged membranes, such as those of bacteria, via anionic moieties such as lipopolysaccharide, lipid A, and teichoic acids [113,120]. This property, which reduces its specificity to physiological tissue targets in eukaryotic cells [34], enables them to effectively bind to bacterial membranes, thus contributing to the widespread bactericidal activity of these proteins [113,116]. 7.5. Synergism between Myotoxic Asp49 PLA2s and PLA 2 Homologues The possibility that Asp49 PLA2s and PLA2 homologues, by acting in combination, may enhance their actions has not been analyzed thoroughly. A single study using these two types of toxins purified from Agkistrodon p. piscivorus venom, alone or in combination, provided evidence for a synergistic effect on phospholipid bilayer permeabilization in vitro [125]. It was proposed that the catalytically-active Asp49 PLA2 would create anionic patches of reaction products on the bilayer surface, which in turn may facilitate electrostatic interactions with the Lys49 toxin, thus increasing the bilayer permeabilization effect [125]. Also related to the concept of a synergistic action between Asp49 PLA2s and PLA2 homologues, it would be reasonable to envisage that the former may provide a readily available source of free fatty acids, in the vicinity of the lat-

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ter, which in turn may bind to and induce the ligand-induced conformational shift in Lys49 myotoxins proposed by Ambrosio et al. [47], as a relevant step in their mechanism of toxicity. If future studies demonstrate synergistic (Lys49/Asp49) effects to be at play using biologicallyrelevant models of toxicity, this would help to understand the evolutive forces that might have driven the emergence and consolidation of PLA2 homologues in viperids. 7.6. PLA2 Homologues May Mediate the Strike-Induced Chemosensory Search (SICS) Response Vipers are known to follow two striking strategies: (a) the snake strikes and holds the prey while it struggles in its jaws, or (b) the snake strikes and releases the envenomed prey, then follows its trail [126]. This second strategy is generally conceived as a mechanism to protect the snake from bites and injuries inflicted by the prey, thus it is observed chiefly when striking at rodents or other dangerous prey [127-129]. There is, however, a cost associated to this strategy: the snakes success as a predator relies on its ability to find, recognize, and follow the trail of the envenomed prey. In several vipers (some of which are included in Table 1), it has been well documented that there is a strong discrimination toward envenomed preys, usually manifested through various behavioral responses, from tongue flicking to actively selecting the trails left by their prey [126,130]. Chemosensory detection of envenomed prey could result from the ability to detect the enzymatic effects of major venom components. Conversely, it is possible that other nonenzymatic components of the venom evolved to serve as chemosensitive signals that allow to discern the trail of envenomed prey [129]. Either way, the identification of venom components acting as enhancers of chemosensory function is still pending, and PLA2 homologues may be studied for their possible involvement in this biologically-relevant role, as addressed in the following sections. 8. TESTING THE HYPOTHESES ON POSSIBLE ADAPTIVE ROLES 8.1. On the Digestive Role Several methods to estimate rates of digestion are available, each having advantages and caveats. The rates of digestion have been measured through estimation of the meal passage rate (the retention time of Stevenson et al. [131]), rates of O2 consumption following feeding (measured in an automated respirometer [132]), as well as small intestinal brushborder uptake rates of sugars (L-D-glucose) and amino acids, change in wet mass of intestine and other organs, and blood chemistry [133,134]. The possible role of PLA2 homologues in favoring the digestion of muscle mass could be tested in experimental settings similar to that described by Nicholson et al. [108]. An anatomically-defined muscle obtained from a rodent can be injected with various amounts of toxin, or with saline solution as a control. Injected muscle would then be placed in an experimental setting similar to the chamber described by Nicholson et al. [108], to resemble the conditions of the stomach or small intestine of the snake. In the case of the stomach, pH should be between 1.2 and 1.7 and temperature should be 25 C, under microanaerobic conditions. Pepsin should be added, at a concentration resembling that of the

gastric content of snakes, and the muscle tissue would be regularly sampled to assess the extent of protein digestion. This can be done by quantifying the proteins released to the supernatant or by assessing protein digestion in homogenates prepared from the muscle mass. Additionally, electrophoretic procedures can be used to analyze hydrolysis of specific muscle proteins, particularly actin and myosin, the most abundant myofibrillar proteins. A similar experiment can be designed for assessing the digestion under conditions resembling the small intestine. In this case the pH should be increased and trypsin, instead of pepsin, should be added. The predicted outcome of this type of experiment is that muscle injected with PLA2 homologues, and then exposed to gastric or intestinal model environments, should be digested to a greater extent than muscle injected with saline solution alone. An alternative experimental strategy to assess this hypothesis would be to determine the time of digestion in the snake of mice injected with PLA2 homologue myotoxins prior to ingestion by the snake, as compared with the digestion of mice previously treated only with saline solution. An approach to further explore this hypothesis in a biologically more meaningful setting would be to use venom with or without Lys49 PLA2s, instead of just injecting Lys49 PLA2s. In these conditions, the digestive role of these PLA 2 homologues would be assessed in the context of the total venom. If the proposed digestive role is indeed relevant, then muscle injected with venom depleted of Lys49 PLA2 would be digested to a lower extent than muscle injected with total venom. The depletion of Lys49 PLA2s could be achieved by chromatographic techniques, for example using cationexchangers that exploit the high pI of these toxins. Immunoaffinity chromatography, i.e. using a column containing immobilized anti-Lys49 PLA2 polyclonal antibodies, might be difficult as there is a strong antigenic cross-reactivity between basic Asp49 PLA2s and Lys49 PLA2 homologues [86,135,136]. Monoclonal antibodies have been obtained that are able to discriminate between different isoforms [137], and thus could be useful for immunoaffinity procedures to deplete PLA2 homologues in crude venoms. 8.2. On the Toxic Role of PLA2s Homologues in Different Types of Prey The possibility that PLA2 homologues are toxic for certain species of prey can be tested by performing a screening of toxicity in a wide spectrum of species that constitute prey items for viperid snakes. These include a number of arthropods, amphibians, reptiles, birds and mammals. Initially, the screening of toxicity may be based on the classical analysis of lethal effect in these species. On the basis of the results, more in-depth studies on the mechanisms of toxicity (neurotoxicity, cardiotoxicity, etc.) can be performed. It would be important to test in parallel the toxicity of Asp49 PLA2s, in order to highlight toxic activities acquired by Lys49 variants, or related homologues, that are not present in their catalytically-active counterparts. 8.3. On the Role of PLA2 Homologues for Pain-Related Prey Immobilization In general, the methodology is to construct a testing arena (a box of dimensions that allow free movement). Animals

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are acclimated for a period of time. Various amounts of Lys49 PLA2, of total venom, of venom depleted of Lys49 PLA2, or of saline solution would be injected intramuscularly in mice or rats. Once the treatments are applied, the behavior of tested animals is recorded during a time interval (usually 15-20 min). Videos are then scored by a single researcher under a double-blind protocol. This researcher should score the frequency and duration of anticipated behaviors. Differences among treatments are examined by mean or median comparisons. The prediction is that rodents injected with Lys49 PLA2s, or with total venom, would show a more restricted movement range than animals injected with saline solution or with venom depleted of Lys49 PLA2s. This general protocol has been extensively used in assessing the effect of presence of predators in the behavior of rodents [138]. 8.4. On the Role of Microbicidal Effect The conditions in which venom is stored in the lumen of the venom gland are reproduced, including venom concentration, pH (around 5.4) [124], and temperature (22-25 C) [108]. A solution of venom is then prepared from lyophilized stored venom, and immediately sterilized by filtration through a 0.22 m membrane. Then, sterile venom, at the concentration observed in the venom gland, has to be spiked with inocula of several bacterial and fungal species. At various time intervals, aliquots of the venom would be collected and microbial counts determined. In this case, a comparison should be made between total venom and venom depleted of Lys49 PLA2s, in order to test whether these PLA2 homologues play a key microbicidal role in the context of total venom. The hypothesis predicts that the ability of venom to exert microbicidal effect largely depends on the action of Lys49 PLA2s. Therefore, venom depleted of these PLA2 variants would be less effective in terms of reducing the bacterial and fungal counts after spiking. 8.5. On the Synergistic Effects of Asp49 PLA2s and Lys49 PLA2 Homologues Experiments using purified Asp49 and Lys49 isoforms, ideally isolated from the venom of a single species where they coexist, could be performed to quantify various biological activities of the toxins, either alone or in combination. Activities such as in vivo myotoxicity in mice, induction of footpad edema, in vitro cytolysis of differentiated myotubes, microbicidal action, liposome permeabilization, etc. (Table 2) could be quantitatively determined. The hypothesis predicts that the combination of both types of toxins should result in an effect higher than the effect corresponding to the sum of each individual toxin's effect. 8.6. On the Role as Chemosensitive Signals To evaluate their potential as chemosensitive signals, different venom components can be isolated, injected to euthanized mice, and presented to snakes. Tongue flicks directed toward envenomed and control mice can be recorded for a given length of time, and compared. This design can be modified to compare behavioral responses among different venom components. A second set of experiments can be planned by comparing responses on a Y-shape testing arena. Snakes are allowed to follow trails made by euthanized rodents envenomed with different venom components, in a

pair-wise design at each arm of the Y-maze. The trail selected, and the time expended in it are the response variables to be measured. CONCLUDING REMARKS To understand the biological role and adaptive value for the emergence of PLA2 homologue toxins in viperid snake venoms is a difficult and ambitious task. However, as basic knowledge on the diverse activities exerted by these proteins, and on their mechanisms of action and structure-function relationships grows, such goal may not seem unrealistic to approach. The wide distribution and high concentration of these proteins in the venoms of many viperid species strongly suggests that selective pressures have been at work for their expression. It is hoped that this review will foster the discussion of new hypotheses on this subject and stimulate future experimental work to assess their validity. ACKNOWLEDGEMENTS The support from Vicerrectora de Investigacin, University of Costa Rica, and the Sweden-Central America NeTropica network is gratefully acknowledged. Thanks are due to colleagues and students with whom the authors have collaborated. We specially thank Julin Fernndez for electrophoretic analyses of juvenile snake venoms. NOTE ADDED IN PROOF During editorial processing of this review, a paper relevant to the discussion presented in Section 7.5 was published, demonstrating the synergism between a Lys49 PLA2 homologue and an Asp49 PLA2, in exerting toxicity upon cultured skeletal muscle cells (Cintra-Francischinelli et al., Cell. Mol. Life Sci., 2009, Apr. 17, E-pub ahead of print). REFERENCES
[1] Fry, B.G.; Wster, W. Assembling an arsenal:origin and evolution of the snake venom proteome inferred from phylogenetic analyses of toxin sequences. Mol. Biol. Evol., 2004, 21(5), 870-883. Fry, B.G. From genome to venome: molecular origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences and related body proteins. Genome Res., 2005, 15(3), 403-420. Calvete, J.J.; Jurez, P.; Sanz, L. Snake venomics. Strategy and applications. J. Mass Spectrometry, 2007, 42(11), 1405-1414. Kini, R.M. Excitement ahead: structure, function and mechanism of snake venom phospholipase A2 enzymes. Toxicon, 2003, 42(8), 827-840. dos Santos, M.C.; Diniz, C.R.; Pacheco, M.A. ; Dias da Silva, W. Phospholipase A2 injection in mice induces immunity against the lethal effects of Crotalus durissus terrificus venom. Toxicon, 1988, 26(2), 207-213. Freitas, T.V.; Fortes-Dias, C.L.; Diniz, C.R. Protection against the lethal effects of Crotalus durissus terrificus (South American rattlesnake) venom in animals immunized with crotoxin. Toxicon, 1990, 28(12), 1491-1496. Nakashima, K.-I.; Nobuhisa, I.; Deshimaru, M.; Nakai, M.; Ogawa, T.; Shimohigashi, Y.; Fukumaki, Y.; Hattori, M.; Sakaki, Y.; Hattori, S.; Ohno, M. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc. Natl. Acad. Sci. USA, 1995, 92(12), 56055609. Ohno, M.; Chijiwa, T.; Oda-Ueda, N.; Ogawa, T.; Hattori, S. Molecular evolution of myotoxic phospholipases A2 from snake venom. Toxicon, 2003, 42(8), 841-854.

[2]

[3] [4] [5]

[6]

[7]

[8]

Activities of Phospholipase A2 Homologues [9] Montecucco, C.; Gutirrez, J.M.; Lomonte, B. Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action. Cell. Mol. Life Sci., 2008, 65(18), 2897-2912. Arni, R.K.; Ward, R.J. Phospholipase A2 - a structural review. Toxicon, 1996, 34(8), 827-841. Scott, D.L.; White, S.P.; Otwinowski, Z.; Yuan, W.; Gelb, M.H.; Sigler, P.B. Interfacial catalysis: the mechanism of phospholipase A2. Science, 1990, 250(4987), 1541-1546. Scott, D.L. Phospholipase A2: structure and catalytic properties. In: Venom phospholipase A2 enzymes: structure, function, and mechanism, Kini, R.M., Ed.; John Wiley & Sons: England, 1997, pp. 97128. Kini, R.M.; Chan, Y.M. Accelerated evolution and molecular surface of venom phospholipase A2 enzymes. J. Mol. Evol., 1999, 48, 125-132. Kini, R.M.; Evans, H.J. A model to explain the pharmacological effects of snake venom phospholipases A2. Toxicon, 1989, 27(6), 613-635. Rosenberg, P. Phospholipases. In: Handbook of Toxinology, Shier, W.T.; Mebs, D., Eds.; Marcel Dekker: New York, 1990, pp. 67277. Chwetzoff, S.; Couderc, J.; Frachon, P.; Menez, A. Evidence that the anti-coagulant and lethal properties of a basic phospholipase A2 from snake venom are unrelated. FEBS Lett., 1989, 248(1-2), 1-4. Stefansson, S.; Kini, R.M.; Evans, H.J. The basic phospholipase A2 from Naja nigricollis venom inhibits the prothrombinase complex by a novel nonenzymatic mechanism. Biochemistry, 1990, 29(33), 1742-7746. Kini, R.M.; Evans, H.J. The role of enzymatic activity in inhibition of the extrinsic tenase complex by phospholipase A2 isoenzymes from Naja nigricollis venom. Toxicon, 1995, 33(12), 1585-1590. Maraganore, J.M.; Merutka, G.; Cho, W.; Welches, W.; Kzdy, F.J.; Heinrikson, R.L. A new class of phospholipases A2 with lysine in place of aspartate 49. J. Biol. Chem., 1984, 259(22), 1383913843. Lomonte, B.; Angulo, Y.; Caldern, L. An overview of Lysine-49 phospholipase A2 myotoxins from crotalid snake venoms and their structural determinants of myotoxic action. Toxicon, 2003, 42(8), 885-901. van den Bergh, C.J.; Slotboom, A.J.; Verheij, H.M.; de Haas, G.H. The role of aspartic acid-49 in the active site of phospholipase A2. A site-specific mutagenesis study of porcine pancreatic phospholipase A2 and the rationale of the enzymatic activity of [lysine49] phospholipase A2 from Agkistrodon piscivorus piscivorus venom. Eur. J. Biochem., 1988, 176(2), 353-357. Scott, D.L.; Achari, A.; Vidal, J.C.; Sigler, P.B. Crystallographic and biochemical studies of the (inactive) Lys-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. J. Biol. Chem., 1992, 267(31), 22645-22657. Li, Y.; Yu, B.; Zhu, H.; Jain, M.; Tsai, M. Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-49. Biochemistry, 1994, 33(49), 1471414722. Ward, R.J.; Chioato, L.; de Oliveira, A.H.C.; Ruller, R.; S, J.M. Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis. Biochem. J., 2002, 362(1), 89-96. Petan, T.; Kri aj, I.; Punger ar, J. Restoration of enzymatic activity in a Ser-49 phospholipase A2 homologue decreases its Ca2+independent membrane-damaging activity and increases its toxicity. Biochemistry, 2007, 46(44), 12795-12809. Ownby, C.L.; Selistre de Araujo, H.S.; White, S.P.; Fletcher, J.E. Lysine 49 phospholipase A2 proteins. Toxicon, 1999, 37(3), 411445. Chioato, L.; Ward, R.J. Mapping structural determinants of biological activities in snake venom phospholipases A2 by sequence analysis and site directed mutagenesis. Toxicon, 2003, 42(8), 869883. Chioato, L.; Arago, E.A.; Ferreira, T.L.; de Medeiros, A.I.; Faccioli, L.H.; Ward, R.J. Mapping of the structural determinants of artificial and biological membrane damaging activities of a Lys49 phospholipase A2 by scanning alanine mutagenesis. Biochim. Biophys. Acta, 2007, 1768(5), 1247-1257. de Oliveira, A.H.C.; Giglio, J.R.; Andrio-Escarso, S.H.; Ito, A.S.; Ward, R.J. A pH-induced dissociation of the dimeric form of a ly-

Protein & Peptide Letters, 2009, Vol. 16, No. 8

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[30]

[10] [11]

[31]

[12]

[32] [33]

[13] [14]

[34]

[15] [16]

[35]

[17]

[36]

[18] [19]

[37]

[20]

[38]

[21]

[39]

[40]

[22]

[23]

[41]

[24]

[42]

[43] [44]

[25]

[26]

[45]

[27]

[28]

[46]

[29]

[47]

sine 49-phospholipase A2 abolishes Ca2+- independent membrane damaging activity. Biochemistry, 2001, 40(23), 6912-6920. Angulo, Y.; Gutirrez, J.M.; Soares, A.M.; Cho, W.; Lomonte, B. Myotoxic and cytolytic activities of dimeric Lys49 phospholipase A2 homologues are reduced, but not abolished, by a pH-induced dissociation. Toxicon, 2005, 46(3), 291-296. Falconi, M.; Desideri, A.; Rufini, S. Membrane-perturbing activity of Viperidae myotoxins: an electrostatic surface potential approach to a puzzling problem. J. Mol. Recogn., 2000, 13(1), 14-19. Murakami, M.; Arni, R.K. A structure based model for liposome disruption and the role of catalytic activity in myotoxic phospholipase A2s. Toxicon, 2003, 42(8), 903-913. Murakami, M.T.; Vicoti, M.M.; Abrego, J.R.B.; Lourenzoni, M.R.; Cintra, A.C.O.; Arruda, E.Z.; Tomaz, M.A.; Melo, P.A.; Arni, R.K. Interfacial surface charge and free accessibility to the PLA2-active site-like region are essential requirements for the activity of Lys49 PLA2 homologues. Toxicon, 2007, 49(3), 378-387. Gutirrez, J.M.; Ponce-Soto, L.A.; Marangoni, S.; Lomonte, B. Systemic and local myotoxicity induced by snake venom group II phospholipases A2: comparison between crotoxin and a Lys49 PLA2 homologue. Toxicon, 2008, 51(1), 80-92. Renetseder, R.; Brunie, S.; Dijkstra, B.W.; Drenth, J.; Sigler, P.B. A comparison of the crystal structures of phospholipase A2 from bovine pancreas and Crotalus atrox venom. J. Biol. Chem., 1985, 260(21), 11627-11634. Lomonte, B.; Moreno, E.; Tarkowski, A.; Hanson, L..; Maccarana, M. Neutralizing interaction between heparins and myotoxin II, a Lys-49 phospholipase A2 from Bothrops asper snake venom. Identification of a heparin-binding and cytolytic toxin region by the use of synthetic peptides and molecular modeling. J. Biol. Chem., 1994, 269(47), 29867-29873. Lomonte, B.; Pizarro-Cerd, J.; Angulo, Y.; Gorvel, J.P.; Moreno, E. Tyr/Trp-substituted peptide 115-129 of a Lys49 phospholipase A2 expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect. Biochim. Biophys. Acta, 1999, 1461(1), 19-26. Nez, C.E.; Angulo, Y.; Lomonte, B. Identification of the myotoxic site of the Lys49 phospholipase A2 from Agkistrodon piscivorus piscivorus snake venom: synthetic C-terminal peptides from Lys49, but not from Asp49 myotoxins, exert membranedamaging activities. Toxicon, 2001, 39(10), 1587-1594. Lomonte, B.; Angulo, Y.; Santamara, C. Comparative study of synthetic peptides corresponding to region 115-129 in Lys49 myotoxic phospholipases A2 from snake venoms. Toxicon, 2003, 42(3), 307-312. Lomonte, B.; Tarkowski, A.; Bagge, U.; Hanson, L.. Neutralization of the cytolytic and myotoxic activities of phospholipases A2 from Bothrops asper snake venom by glycosaminoglycans of the heparin/heparan sulfate family. Biochem. Pharmacol., 1994, 47(9), 1509-1518. Caldern, L.; Lomonte, B. Immunochemical characterization and role in toxic activities of region 115-129 of myotoxin II, a Lys49 phospholipase A2 from Bothrops asper snake venom. Archs. Biochem. Biophys., 1998, 358(2), 343-350. Caldern, L.; Lomonte, B. Inhibition of the myotoxic action of Bothrops asper myotoxin II in mice by immunization with its synthetic peptide 115-129. Toxicon, 1999, 37(4), 683-687. Angulo, Y.; Lomonte, B. Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A2. Biochem. Pharmacol., 2003, 66(10), 1993-2000. Chioato, L.; de Oliveira, A.H.C.; Ruller, R.; S, J.M.; Ward, R.J. Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2. Biochem. J., 2002, 366(3), 971-976. da Silva Giotto, M.T.; Garrat, R.C.; Oliva, G.; Mascarenhas, Y.P.; Giglio, J.R.; Cintra, A.C.O.; de Azevedo, Jr.; W.F., Arni; R.K.; Ward, R.J. Crystallographic and spectroscopic characterization of a molecular hinge: conformational changes in bothropstoxin I, a dimeric Lys49 phospholipase A2 homologue. Prot. Struct. Funct. Genet., 1998, 30(4), 442-454. Magro, A.J.; Soares, A.M.; Giglio, J.R.; Fontes, M.R.M. Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A2: quaternary structure and inhibition mechanism insights. Biochem. Biophys. Res. Comm., 2003, 311(3), 713-720. Ambrosio, A.L.B.; Nonato, M.C.; Selistre de Araujo, H.S.; Arni, R.K.; Ward, R.J.; Ownby, C.L.; de Souza, D.H.F.; Garrat, R.C. A

872 Protein & Peptide Letters, 2009, Vol. 16, No. 8 molecular mechanism for Lys49-phospholipase A2 activity based on ligand-induced conformational change. J. Biol. Chem., 2005, 280(8), 7326-7335. Gutirrez, J.M.; Lomonte, B.; Cerdas, L. Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer. Toxicon, 1986, 24(9), 885-894. Gutirrez, J.M.; Ownby, C.L. Skeletal muscle degeneration induced by venom phospholipases A2: insights into the mechanisms of local and systemic myotoxicity. Toxicon, 2003, 42(8), 915-931. Heluany, N.F.; Homsi-Brandeburgo, M.I.; Giglio, J.R.; PradoFranceschi, J.; Rodrigues-Simioni, L. Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations. Toxicon, 1992, 30(10), 1203-1210. Rodrigues-Simioni, L.; Prado-Franceschi, J.; Cintra, A.C.O.; Giglio, J.R.; Jiang, M.S.; Fletcher, J.E. No role for enzymatic activity or dantrolene-sensitive Ca2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A2 myotoxin. Toxicon, 1995, 33(11), 1479-1489. Soares, A.M.; Guerra-S, R.; Borja-Oliveira, C.; Rodrigues, V.M.; Rodrigues-Simioni, L.; Rodrigues, V.; Fontes, M.R.M.; Lomonte, B.; Gutirrez, J.M.; Giglio, J.R. Structural and functional characterization of BnSP-7, a lysine-49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Archs. Biochem. Biophys., 2000, 378(2), 201-209. Oshima-Franco, Y.; Hyslop, S.; Cintra, A.C.O.; Giglio, J.R.; da Cruz-Hofling, M.A.; Rodrigues-Simioni, L. Neutralizing capacity of commercial bothropic antivenom against Bothrops jararacussu venom and bothropstoxin-I. Muscle Nerve, 2000, 23(12), 18321839. Oshima-Franco, Y.; Leite, G.B.; Silva, G.H.; Cardoso, D.F.; Hyslop, S.; Giglio, J.R.; da Cruz-Hofling, M.A.; RodriguesSimioni, L. Neutralization of the pharmacological effects of bothropstoxin-I from Bothrops jararacussu (jararacuu) venom by crotoxin antiserum and heparin. Toxicon, 2001, 39(10), 1477-1485. Tsai, I.H.; Chen, Y.H.; Wang, Y.M. Comparative proteomics and subtyping of venom phospholipases A2 and disintegrins of Protobothrops pit vipers. Biochim. Biophys. Acta, 2004, 1702(1), 111119. Gallacci, M.; Oliveira, M.; Pai-Silva, M.; Cavalcante, W.L.; Spencer, P.J. Paralyzing and myotoxic effects of a recombinant bothropstoxin-I (BthTX-I) on mouse neuromuscular preparations. Exp. Toxicol. Pathol., 2006, 57(3), 239-245. Ponce-Soto, L.A.; Lomonte, B.; Gutirrez, J.M.; RodriguesSimioni, L.; Novello, J.C.; Marangoni, S. Structural and functional properties of BaTX, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus. Biochim. Biophys. Acta, 2007, 1770(4), 585-593. Barbosa, P.S.F.; Havta, A.; Faco, P.E.G.; Sousa, T.M.; Bezerra, I.S.A.M.; Fonteles, M.C.; Toyama, M.H.; Marangoni, S.; Novello J.C.; Monteiro H.S.A. Renal toxicity of Bothrops moojeni snake venom and its main myotoxins. Toxicon, 2002, 40(10), 1427-1435. Leite, R.S.; Franco, W.; Ownby, C.L.; Selistre-de-Araujo, H.S. Effects of ACL myotoxin, a Lys49 phospholipase A2 from Agkistrodon contortrix contortrix, on water transport in the isolated toad urinary bladder. Toxicon, 2004, 43(1), 77-83. Leite, R.S.; Giuliani, C.D.; Lomonte, B.; Franco, W.; Selistre-deAraujo, H.S. Effect of a recombinant Lys49 PLA2 myotoxin and Lys49 PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water. Toxicon, 2004, 44(2), 157159. Dhillon, D.S.; Condrea, E.; Maraganore, J.M.; Heinrikson, R.L.; Benjamin, S.; Rosenberg, P. Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom. Biochem. Pharmacol., 1987, 36(10), 1723-1730. Stbeli, R.G.; Amui, S.F.; Sant'Ana, C.D.; Pires, M.G.; Nomizo, A.; Monteiro, M.C.; Romo, P.R.T.; Guerra-S, R.; Vieira, C.A.; Giglio, J.R.; Fontes, M.R.M.; Soares, A.M. Bothrops moojeni myotoxin-II, a Lys49-phospholipase A2 homologue: an example of function versatility of snake venom proteins. Comp. Biochem. Physiol. C, 2006, 142(3-4), 371-381. Lomonte, B.; Tarkowski, A.; Hanson, L.. Host response to Bothrops asper snake venom: analysis of edema formation, inflammatory cells, and cytokine release in a mouse model. Inflammation, 1993, 17(2), 93-105. [64]

Lomonte et al. Rucavado, A.; Escalante, T.; Texeira, C.F.; Fernandes, C.M.; Daz, C.; Gutirrez, J.M. Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxins from the venom of the snake Bothrops asper. Med. Inflamm., 2002, 11(2), 121-128. Chaves, F.; Teixeira, C.F.P.; Gutirrez, J.M. Role of TNF-alpha, IL-1beta and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice. Toxicon, 2005, 45(2), 171-178. Landucci, E.C.T.; Castro, R.C.; Pereira, M.F.; Cintra, A.C.O.; Giglio, J.R.; Marangoni, S.; Oliveira, B.; Cirino, G.; Antunes, E.; De Nucci, G. Mast cell degranulation induced by two phospholipase A2 homologues: dissociation between enzymatic and biological activities. Eur. J. Pharmacol., 1998, 343(2-3), 257-263. Zuliani, J.P.; Gutirrez, J.M.; Casais, L.L.; Sampaio, S.C.; Lomonte, B.; Teixeira, C.F.P. Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2. Toxicon, 2005, 46(5), 523-532. Lomonte, B.; Gutirrez, J.M.; Borkow, G.; Ovadia, M.; Tarkowski, A.; Hanson, L.. Activity of hemorrhagic metalloproteinase BaH-1 and myotoxin II from Bothrops asper snake venom on capillary endothelial cells in vitro. Toxicon, 1994, 32(4), 505-510. Yamazaki, Y.; Nakano, Y.; Imamura, T.; Morita, T. Augmentation of vascular permeability of VEGF is enhanced by KDR-binding proteins. Biochem. Biophys. Res. Comm., 2007, 355(3), 693-699. Gutirrez, J.M.; Lomonte, B. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon, 1995, 33(11), 1405-1424. Pantigoso, C.; Escobar, E.; Yarlequ, A. Aislamiento y caracterizacin de una miotoxina del veneno de la serpiente Bothrops brazili Hoge, 1953 (Ophidia: Viperidae). Rev. Per. Biol., 2001, 8(2), 110. Wang, Y.M.; Wang, J.H.; Pan, F.M.; Tsai, I.H. Lys-49 phospholipase A2 homologs from venoms of Deinagkistrodon acutus and Trimeresurus mucrosquamatus have identical protein sequence. Toxicon, 1996, 34(4), 485-489. Wang, Y.M.; Peng, H.F.; Tsai, I.H. Unusual venom phospholipases A2 of two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis. FEBS J., 2005, 272(12), 3015-3025. Ogawa, T.; Onoue, H.; Nakagawa, K.; Nomura, S.; Sueishi, K.; Hattori, S.; Kihara, H.; Ohno, M. Localization and expression of phospholipases A2 in Trimeresurus flavoviridis (Habu snake) venom gland. Toxicon, 1995, 33(12), 1645-1652. Kashima, S.; Roberto, P.G.; Soares, A.M.; Astolfi-Filho, S.; Pereira, J.O.; Giuliati, S.; Faria, M.; Xavier, M.A.S.; Fontes, M.R.M.; Giglio, J.R.; Franca, S.C. Analysis of Bothrops jararacussu venomous gland transcriptome focusing on structural and functional aspects: gene expression profile of highly expressed phospholipases A2. Biochimie, 2004, 86(3), 211-219. Nakashima, K.; Ogawa, T.; Oda, N.; Hattori, M.; Sakaki, Y.; Kihara, H.; Ohno, M. Accelerated evolution of Trimeresurus flavoviridis venom gland phospholipase A2 isozymes. Proc. Natl. Acad. Sci., 1993, 90(13), 5964-5968. Lomonte, B.; Carmona, E. Individual expression patterns of myotoxin isoforms in the venom of the snake Bothrops asper. Comp. Biochem. Physiol. B, 1992, 102(2), 325-329. Moura-da-Silva, A.M.; Paine, M.J.I.; Diniz, M.R.V.; Theakston, R.D.G.; Crampton, J.M. The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: evolution of venom group II phospholipase A2s's may imply gene duplications. J. Mol. Evol., 1995, 41(2), 174-179. Tsai, I.H.; Chen, Y.H.; Wang, Y.M.; Tu, M.C.; Tu, A.T. Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A2 from the venoms of rattlesnakes and other pit vipers. Archs. Biochem. Biophys., 2001, 394(2), 236-244. Angulo, Y.; Olamendi-Portugal, T.; Alape-Giron, A.; Possani, L.D.; Lomonte, B. Structural characterization and phylogenetic relationships of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a Lys49 phospholipase A2 homologue. Int. J. Biochem. Cell Biol., 2002, 34(10), 1268-1278. Mebs, D.; Kuch, U.; Coronas, F.I.V.; Batista, C.V.F.; Gumprecht, A.; Possani, L.D. Biochemical and biological activities of the venom of the Chinese pitviper Zhaoermia mangshanensis, wit the complete amino acid sequence and phylogenetic analysis of a novel Arg49 phospholipase A2 myotoxin. Toxicon, 2006, 47(7), 797-811.

[48] [49]

[65]

[50]

[66]

[51]

[67]

[68]

[52]

[69]

[53]

[70] [71]

[54]

[72]

[55]

[73] [74]

[56]

[75]

[57]

[58]

[76]

[59]

[77]

[78]

[60]

[79]

[61]

[80]

[62]

[81]

[63]

Activities of Phospholipase A2 Homologues [82] Lynch, V.J. Inventing an arsenal: adaptive evolution and neofunctionalization of snake venom phospholipase A2 genes. BMC Evolut. Biol., 2007, 7, 2, doi:10.1186/1471-2148-7-2. Gutirrez, J.M.; Lomonte, B. Phospholipase A2 myotoxins from Bothrops snake venoms. In: Venom phospholipase A2 enzymes: structure, function, and mechanism, Kini, R.M., Ed.; John Wiley & Sons: England, 1997, pp. 321-352. Kihara, H.; Uchikawa, R.; Hattori, S.; Ohno, M. Myotoxicity and physiological effects of three Trimeresurus flavoviridis phospholipases A2. Biochem. Int., 1992, 28(5), 895-903. Daz, C.; Gutirrez, J.M.; Lomonte, B.; Gen, J.A. The effect of myotoxins isolated from Bothrops snake venoms on multilamellar liposomes: relationship to phospholipase A2, anticoagulant and myotoxic activities. Biochim. Biophys. Acta, 1991, 1070(2), 455460. Daz, C.; Gutirrez, J.M.; Lomonte, B. Isolation and characterization of basic myotoxic phospholipases A2 from Bothrops godmani (Godman's pit viper) snake venom. Archs. Biochem. Biophys., 1992, 298(1), 135-142. Yamazaki, Y.; Matsunaga, Y.; Nakano, Y.; Morita, T. Identification of vascular endothelial growth factor receptor-binding protein in the venom of Eastern cottonmouth: a new role of snake venom myotoxic Lys49-phospholipase A2. J. Biol. Chem., 2005, 280(34), 29989-29992. Fujisawa, D.; Yamazaki, Y.; Lomonte, B.; Morita, T. Catalytically inactive phospholipase A2 homologue binds to vascular endothelial growth factor receptor-2 via C-terminal loop region. Biochem. J., 2008, 411(3), 515-522. Tsai, I.H.; Wang, Y.M.; Chen, Y.H.; Tsai, T.S.; Tu, M.C. Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries. Biochem. J., 2004, 377(1), 215-223. Alape-Girn, A.; Sanz, L.; Escolano, J.; Flores-Daz, M.; Madrigal, M.; Sasa, M.; Calvete, J.J. Snake venomics of the lancehead pitviper Bothrops asper. Geographic, individual, and ontogenetic variations. J. Proteome Res., 2008, 7(8), 3556-3571. Chijiwa, T.; Deshimaru, M.; Nobuhisa, I.; Nakai, M.; Ogawa, T.; Oda, N.; Nakashima, K.I.; Fukumaki, Y.; Shimohigashi, Y.; Hattori, S.; Ohno, M. Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem. J., 2000, 347(2), 491-499. Chijiwa, T.; Yamaguchi, Y.; Ogawa, T.; Deshimaru, M.; Nobuhisa I.; Nakashima, K.I.; Oda-Ueda, N.; Fukumaki, Y.; Hattori, S.; Ohno, M. Interisland evolution of Trimeresurus flavoviridis venom phospholipase A2 isozymes. J. Mol. Evol., 2003, 56(3), 286-293. Lomonte, B.; Escolano, J.; Fernndez, J.; Sanz, L.; Angulo, Y.; Gutirrez, J.M.; Calvete, J.J. Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii. J. Proteome Res., 2008, 7(6), 2445-2457. Solrzano, A. Serpientes de Costa Rica, Editorial InBio: San Jos, 2004. Cidade, D.A.P.; Simo, T.A.; Dvila, A.M.R.; Wagner, G.; Junqueira-de-Azevedo, I.; Ho, P.L.; Bon, C.; Zingali, R.B.; Albano, R.M. Bothrops jararaca venom gland transcriptome: analysis of the gene expression pattern. Toxicon, 2006, 48(4), 437-461. Martins, M.; Marques, O.A.V.; Sazima, I. Ecological and phylogenetic correlates of feeding, habits in neotropical pitvipers of the genus Bothrops. In: Biology of the Vipers, Shuett, G. W.; Hggren, M.D.; Douglas, M.; Greene, H.W., Eds.; Eagle Mountain Publishing: Utah, 2002, pp.307-328. Campbell, J.A.; Solrzano, A. The distribution, variation, and natural history of the Middle American montane pitviper Porthidium godmani. In: Biology of the Pitvipers, Campbell, J.A.; Brodie, E.D. Jr., Eds.; Selva Tyler: Texas, 1992, pp.223-250. Greene, H. Snakes. The Evolution of Mystery in Nature. University of California Press: Berkeley, 1997. Felsenstein, J. Phylogenies and the comparative method. Amer. Nat., 1985, 125(1), 1-15. Bjarnason, J.B.; Fox, J.W. Hemorrhagic metalloproteinases from snake venoms. Pharmac. Ther., 1994, 62(3), 325-372. Brobeck, J.R. Best & Taylors Physiological Basis of Medical Practice. Baltimore: Williams & Wilkins, 1979. Harris, J.B.; Cullen, M.J. Muscle necrosis caused by snake venoms and toxins. Electron Microsc. Rev., 1990, 3(2), 183-211. Bertipaglia, I.; Carafoli, E. Calpains and human disease. Subcell. Biochem., 2007, 45(0), 29-53. [104]

Protein & Peptide Letters, 2009, Vol. 16, No. 8

873

[83]

[105]

[84] [85]

[106] [107]

[108]

[86]

[109]

[87]

[110]

[88]

[111]

[89]

[90]

[112]

[91]

[113]

[92]

[114]

[93]

[115]

[94] [95]

[116]

[96]

[117]

[97]

[118]

[98] [99] [100] [101] [102] [103]

[119]

[120] [121]

Gutirrez, J.M.; Arce, V.; Brenes, F.; Chaves, F. Changes in myofibrillar components after skeletal muscle necrosis induced by a myotoxin isolated from the venom of the snake Bothrops asper. Exp. Mol. Pathol., 1990, 52(1), 25-36. Vater, R.; Cullen, M.J.; Harris, J.B. The fate of desmin and titin during the degeneration and regeneration of the soleus muscle of the rat. Acta Neuropathol., 1992, 84(3), 140-148 Harris, J.B.; Vater, R.; Wilson, M.; Cullen, M.J. Muscle fibre breakdown in venom-induced muscle degeneration. J. Anat., 2003, 202(4), 363-372. Gutirrez, J.M.; Chaves, F.; Cerdas, L. Inflammatory infiltrate in skeletal muscle injected with Bothrops asper venom. Rev. Biol. Trop., 1986, 34(2), 209-219. Nicholson, J.; Mirtschin, P.; Madaras, F.; Venning, M.; Kokkim, M. Digestive properties of the venom of the Australian Coastal Taipan, Oxyuranus scutellatus (Peteres, 1867). Toxicon, 2006, 48(4), 422-428. Warrell, D.A. Snakebites in Central and South America: Epidemiology, clinical features and clinical management. In The Venomous Reptiles of the Western Hemisphere; Campbell, J.A.; Lamar, W.W., Eds. Ithaca: Comstock Publishing Associates, 2004, pp. 709-761. Chacur, M.; Longo, I.; Picolo, G.; Gutirrez, J.M.; Lomonte, B.; Guerra, J.L.; Teixeira, C.F.P.; Cury, Y. Hyperalgesia induced by Asp49 and Lys49 phospholipases A2 from Bothrops asper snake venom: pharmacological mediation and molecular determinants. Toxicon, 2003, 41(6), 667-678. Chacur, M.; Milligan, E.D.; Sloan, E.M.; Wieseler-Frank, J.; Barrientos, R.M.; Martin, D.; Poole, S.; Lomonte, B.; Gutirrez, J.M.; Maier, S.F.; Cury, Y.; Watkins, L.R. Snake venom phospholipase A2s (Asp49 and Lys49) induce mechanical allodynia upon perisciatic administration: involvement of spinal cord glia, proinflammatory cytokines and nitric oxide. Pain, 2004, 108(1-2), 180-191. Chacur, M.; Gutirrez, J.M.; Milligan, E.D.; Wieseler-Frank, J.; Britto, L.R.G.; Maier, S.F.; Watkins, L.R.; Cury, Y. Snake venom components enhance pain upon subcutaneous injection: an initial examination of spinal cord mediators. Pain, 2004, 111(1-2), 65-76. Pramo, L.; Lomonte, B.; Pizarro-Cerd, J.; Bengoechea, J.A.; Gorvel, J.P.; Moreno, E. Bactericidal activity of Lys49 and Asp49 myotoxic phospholipases A2 from Bothrops asper snake venom: synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region. Eur. J. Biochem., 1998, 253(2), 452-461. Soares, A.M.; Guerra-S, R.; Borja-Oliveira, C.; Rodrigues, V.M.; Rodrigues-Simioni, L.; Rodrigues, V.; Fontes, M.R.M.; Lomonte, B.; Gutirrez, J.M.; Giglio, J.R. Structural and functional characterization of BnSP-7, a lysine-49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Archs. Biochem. Biophys., 2000, 378(2), 201-209. Soares, A.M.; Andrio-Escarso, S.H.; Bortoleto, R.K.; RodriguesSimioni, L.; Arni, R.K.; Ward, R.J.; Gutirrez, J.M.; Giglio, J.R. Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom. Archs. Biochem. Biophys., 2001, 387(2), 188196. Santamara, C.; Larios, S.; Angulo, Y.; Pizarro, J.; Gorvel, J.P.; Moreno, E.; Lomonte, B. Antimicrobial activity of myotoxic phospholipases A2 from crotalid snake venoms and synthetic peptide variants derived from their C-terminal region. Toxicon, 2005, 45(7), 807-815. Santamara, C.; Larios, S.; Quirs, S.; Pizarro, J.; Gorvel, J.P.; Lomonte, B.; Moreno, E. Bactericidal and anti-endotoxic properties of short cationic peptides derived from a snake venom Lys49 phospholipase A2. Antimicrob. Agents Chemother., 2005, 49(4), 13401345. Murillo, L.A.; Lan, C.Y.; Agabian, N.M.; Larios, S.; Lomonte, B. Fungicidal activity of a phospholipase A2-derived synthetic peptide variant upon Candida albicans. Rev. Esp. Quimioter., 2007, 20(3), 330-333. Arago, E.A.; Chioato, L.; Ward, R.J. Permeabilization of E. coli K12 inner and outer membranes by bothropstoxin-I, a Lys49 phospholipase A2 from Bothrops jararacussu. Toxicon, 2008, 51(4), 538-546. Tsai, S.H.; Chen, Y.C.; Chen, L.; Wang, Y.M.; Tsai, I.H. Binding of a venom Lys-49 phospholipase A2 to LPS and suppression of its effects on mouse macrophages. Toxicon, 2007, 50(7), 914-922. Kochva, E. The origin of snakes and evolution of the venom apparatus. Toxicon, 1987, 25(1), 65-106.

874 Protein & Peptide Letters, 2009, Vol. 16, No. 8 [122] Francis. B.; Seebart, C.; Kaiser, I.I. Citrate is an endogenous inhibitor of snake venom enzymes by metal-ion chelation. Toxicon, 1992, 30(10), 1239-1246. Odell, G.V.; Ferry, P.C.; Vick, L.M.; Fenton, A.W.; Decker, L.S.; Cowell, R.L.; Ownby, C.L.; Gutirrez, J.M. Citrate inhibition of snake venom proteases. Toxicon, 1998, 36(12), 1801-1806. Mackessy, S.P.; Baxter, L.M. Bioweapons synthesis and storage: The venom gland of front-fanged snakes. Zoolog. Anzeiger, 2006, 245(3-4), 147-159. Shen, Z.; Cho, W. Membrane leakage induced by synergetic action of Lys-49 and Asp-49 Agkistrodon piscivorus piscivorus phospholipases A2: implications in their pharmacological activities. Int. J. Biochem. Cell Biol., 1995, 27(10), 1009-1013. Stiles, K.; Stara, P.; Chiszar, D.; Smith, H.M. Strike-induced chemosensory searching (SICS) and trail-following behavior in copperheads (Agkistrodon contortrix). In: Biology of the Vipers, Shuett, G. W.; Hggren, M.D.; Douglas, M.; Greene, H.W., Eds.; Eagle Mountain Publishing: Utah, 2002, pp.413-418. Kardong, K.V. The strike behavior of the rattlesnake Crotalus viridis oreganus. J. Comp. Psychol., 1986, 100(3), 314-324. Chiszar, D.; Lee, R.K.K.; Smith, H.M.; Radcliffe, C.W. Searching behaviors by rattlesnakes following predatory strikes. In: Biology of the Pitvipers Campbell, J.A.; Brodie, E.D., Eds.; Selva Tyler: Texas, 1992, pp.369-382. Chiszar, D.; Walters, A.; Urbaniak, J.; Smith, H.M.; Mackessy, S.P. Discrimination between envenomated and nonevenomated prey by western diamondback rattlesnakes (Crotalus atrox): chemosensory consequences of venom. Copeia, 1999, 3(0), 640-648. Lavn-Murcio, P.A.; Kardong, K.V. Scent related to venom and prey as cues in the post-strike training behavior of rattlesnakes Crotalus viridis oreganus. Herpetologica, 1995, 51(1), 39-44. Stevenson, R.D.; Peterson, C.R.; Tsuji, J.S. The thermal dependence of locomotion, tongue flicking, digestion and oxygen consumption in the wandering garter snake. Physiol. Zool., 1985, 58(1), 46-57. Andrade, D.V.; Cruz-Neto, A.P.; Abe, A.S. Meal size and specific dynamic action in the rattlesnake Crotalus durissus (Serpentes: Viperidae). Herpetologica, 1997, 53(4), 485-493. Secor, S.; Diamond, J.M. Evolution of the adaptative response to feeding among snakes. Amer. Zoologist, 1994, 34(5), 48A. Secor, S.; Diamond, J.M. Adaptative responses to feeding in Burmese Pythons: pay before pumping. J. Exp.Zool., 1995, 198(6), 1313-1325. Lomonte, B.; Gutirrez, J.M. A new muscle damaging toxin, myotoxin II, from the venom of the snake Bothrops asper (terciopelo). Toxicon, 1989, 27(7), 725-733. Lomonte, B.; Gutirrez, J.M.; Carmona, E.; Rovira, M.E. Equine antibodies to Bothrops asper myotoxin II: isolation from polyvalent antivenom and neutralizing ability. Toxicon, 1990, 28(4), 379-384. Lomonte, B.; Kahan, L. Production and partial characterization of monoclonal antibodies to Bothrops asper (terciopelo) myotoxin. Toxicon, 1988, 26(7), 675-689. Pillay, N.; Alexander, G.J.; Lazenvy, S.L. Responses of striped mice Rhabdomys pumilio, to faeces of a predatory snake. Behaviour, 2003, 140(1), 125-135. Maraganore, J.M.; Heinrikson, R.L. The lysine-49 phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. Relation of structure and function to other phospholipases A2. J. Biol. Chem., 1986, 261(11), 4797-4804. Holland, D.R.; Clancy, L.L.; Muchmoreg, S.W.; Rydell, T.J.; Einspahr, H.M.; Finzel, B.C.; Heinrikson, R.L.; Watenpaugh, K.D. The crystal structure of a lysine 49 phospholipase A2 from the venom of the cottonmouth snake at 2.0- resolution. J. Biol. Chem., 1990, 265(29), 17649-17658. Nikai, T.; Komori, Y.; Ohara, A.; Yagihashi, S.; Ohizumi, Y.; Sugihara, H. Characterization and amino-terminal sequence of phospholipase A2-II from the venom of Agkistrodon bilineatus (common cantil). Int. J. Biochem., 1994, 26(1), 43-48. Johnson, E.K.; Ownby, C.L. Isolation of a myotoxin from the venom of Agkistrodon contortrix laticinctus (broad-banded copperhead) and pathogenesis of myonecrosis induced by it in mice. Toxicon, 1993, 31(3), 243-245. Selistre de Araujo, H.S.; White, S.P.; Ownby, C.L. Sequence analysis of Lys49 phospholipase A2 myotoxins: a highly conserved class of proteins. Toxicon, 1996, 34(11-12), 1237-1242. [144]

Lomonte et al. de Azevedo, W.F.; Ward, R.J.; Gutirrez, J.M.; Arni, R.K. Structure of a Lys49-phospholipase A2 homologue isolated from the venom of Bothrops nummifer (jumping viper). Toxicon, 1999, 37(2), 371-384. Rojas, E.; Saravia, P.; Angulo, Y.; Arce, V.; Lomonte, B.; Chavez, J.J.; Velsquez, R.; Thelestam, M.; Gutirrez, J.M. 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. Comp. Biochem. Physiol. C, 2001, 129(2), 151-162. Angulo, Y.; Olamendi-Portugal, T.; Possani, L.D.; Lomonte, B. Isolation and characterization of myotoxin II from Atropoides (Bothrops) nummifer snake venom, a new Lys49 phospholipase A2 homologue. Int. J. Biochem. Cell Biol., 2000, 32(1), 63-71. Murakami, M.T.; Melo, C.C.; Angulo, Y.; Lomonte, B.; Arni, R.K. Structure of myotoxin-II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom. Acta Cryst. F , 2006, 62(5), 423-426. Francis, B.; Gutirrez, J.M.; Lomonte, B.; Kaiser, I.I. Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2. Archs. Biochem. Biophys., 1991, 284(2), 352-359. Arni, R.K.; Ward, R.J.; Gutirrez, J.M.; Tulinsky, A. Structure of a calcium-independent phospholipase-like myotoxic protein from Bothrops asper venom. Acta Cryst. D, 1995, 51(3), 311-317. Daz, C.; Lomonte, B.; Zamudio, F.; Gutirrez, J.M. Purification and characterization of myotoxin IV, a phospholipase A2 variant, from Bothrops asper snake venom. Natural Toxins, 1995, 3(1), 2631. Lizano, S.; Lambeau, G.; Lazdunski, M. Cloning and cDNA sequence analysis of Lys49 and Asp49 basic phospholipase A2 myotoxin isoforms from Bothrops asper. Int. J. Biochem. Cell Biol., 2001, 33(2), 127-132. Kanashiro, M.M.; Escocard, R.C.M.; Petretski, J.H.; Prates, M.V.; Alves, E.W.; Machado, O.L.T.; Dias da Silva, W.; Kipnis, T.L. Biochemical and biological properties of phospholipases A2 isolated from Bothrops atrox snake venom. Biochem. Pharmacol., 2002, 64(7), 1179-1186. Nez, V.; Arce, V.; Gutirrez, J.M.; Lomonte, B. Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops atrox. Toxicon, 2004, 44(1), 91-101. Homsi-Brandeburgo, M.I.; Queiroz, L.S.; Santo-Neto, H.; Rodrigues-Simioni, L.; Giglio, J.R. Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin. Toxicon, 1988, 26(7), 615-627. Cintra, A.C.O.; Marangoni, S.; Oliveira, B.; Giglio, J.R. Bothropstoxin-I: amino acid sequence and function. J. Prot. Chem., 1993, 12(1), 57-64. Higuchi, D.A.; Barbosa, C.M.V.; Bincoletto, C.; Chagas, J.R.; Magalhaes, A.; Richardson, M.; Sanchez, E.F.; Pesquero, J.B.; Araujo, R.C.; Pesquero, J.L. Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (whitetailed-jararaca) snake venom. Biochimie, 2007, 89(3), 319-328. Lomonte, B.; Gutirrez, J.M.; Furtado, M.F.; Otero, R.; Rosso, J.P.; Vargas, O.; Carmona, E.; Rovira, M.E. Isolation of basic myotoxins from Bothrops moojeni and Bothrops atrox snake venoms. Toxicon, 1990, 28(10), 1137-1146. Soares, A.M.; Andrio-Escarso, S.H.; Angulo, Y.; Lomonte, B.; Gutirrez, J.M.; Toyama, M.H.; Marangoni, S.; Arni, R.K.; Giglio, J.R. Structural and functional characterization of myotoxin I, a Lys49 phospholipase A2 homologue from Bothrops moojeni (caissaca) snake venom. Archs. Biochem. Biophys., 2000, 373(1), 7-15. Soares, A.M.; Rodrigues, V.M.; Homsi-Brandeburgo, M.I.; Toyama, M.H.; Lombardi, F.R.; Arni, R.K.; Giglio, J.R. A rapid procedure for the isolation of the Lys-49 myotoxin II from Bothrops moojeni (caissaca) venom: biochemical characterization, crystallization, myotoxic and edematogenic activity. Toxicon, 1998, 36(3), 503-514. Watanabe, L.; Soares, A.M.; Ward, R.J.; Fontes, M.R.M.; Arni, R.K. Structural insights for fatty acid binding in a Lys49phospholipase A2: crystal structure of myotoxin II from Bothrops moojeni complexed with stearic acid. Biochimie, 2005, 87(2), 161167. Geoghegan, P.; Angulo, Y.; Cangelosi, A.; Daz, M.; Lomonte, B. Characterization of a basic phospholipase A2-homologue myotoxin

[123] [124]

[145]

[125]

[146]

[126]

[147]

[127] [128]

[148] [149]

[129]

[150]

[130]

[151]

[131]

[152]

[132] [133] [134]

[153]

[154]

[135] [136]

[155]

[137] [138]

[156]

[157]

[139]

[158]

[140]

[159]

[141]

[142]

[160]

[143]

[161]

Activities of Phospholipase A2 Homologues isolated from the venom of the snake Bothrops neuwiedii (yarar chica) from Argentina. Toxicon, 1999, 37(12), 1735-1746. Rodrigues,V.M.; Soares, A.M.; Mancin, A.C.; Fontes, M.R.M.; Homsi-Brandeburgo, M.I.; Giglio, J.R. Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activity. Comp. Biochem. Physiol. A, 1998, 121(3), 215-222. Soares, A.M.; Guerra-S, R.; Borja-Oliveira, C.; Rodrigues, V.M.; Rodrigues-Simioni, L.; Rodrigues, V.; Fontes, M.R.M.; Lomonte, B.; Gutirrez, J.M.; Giglio, J.R. Structural and functional characterization of BnSP-7, a lysine-49 myotoxic phospholipase A2 homologue from Bothrops neuwiedi pauloensis venom. Archs. Biochem. Biophys., 2000, 378(2), 201-209. Magro, A.J.; Soares, A.M.; Giglio, J.R.; Fontes, M.R.M. Crystal structures of BnSP-7 and BnSP-6, two Lys49-phospholipases A2: quaternary structure and inhibition mechanism insights. Biochem. Biophys. Res. Comm., 2003, 311(3), 713-720. Toyama, M.H.; Mancuso, L.C.; Giglio, J.R.; Novello, J.C.; Oliveira, B.; Marangoni, S. A quick procedure for the isolation of dimeric piratoxins-I and II, two myotoxins from Bothrops pirajai snake venom: N-terminal sequencing. Biochem. Mol. Biol. Int., 1995, 37(6), 1047-1055. Toyama, M.H.; Soares, A.M.; Vieira, C.A.; Novello, J.C.; Oliveira, B.; Giglio, J.R.; Marangoni, S. Amino acid sequence of piratoxin-I, a myotoxin from Bohtrops pirajai snake venom and its biological activity after alkylation with p-bromophenacyl bromide. J. Prot. Chem., 1998, 17(7), 713-728. Toyama, M.H.; Soares, A.M.; Wen-Hwa, L.; Polikarpov, I.; Giglio, J.R.; Marangoni, S. Amino acid sequence of piratoxin-II, a myotoxic lys49 phospholipase A2 homologue from Bothrops pirajai venom. Biochimie, 2000, 82(3), 245-250. Lee, W.H.; da Silva Giotto, M.T.; Marangoni, S.; Toyama, M.H.; Polikarpov, I.; Garrat, R.C. Structural basis for low catalytic activity in Lys49 phospholipases A2 - a hypothesis: the crystal structure of piratoxin II complexed to fatty acid. Biochemistry, 2001, 40(1), 28-36. Angulo, Y.; Chaves, E.; Alape, A.; Rucavado, A.; Gutirrez, J.M.; Lomonte, B. Isolation and characterization of a myotoxic phospholipase A2 from the venom of the arboreal snake Bothriechis (Bothrops) schlegelii from Costa Rica. Archs. Biochem. Biophys., 1997, 339(2), 260-267. Tsai, I.H.; Wang, Y.M.; Au, L.C.; Ko, T.P.; Chen, Y.H.; Chu, Y.F. Phospholipases A2 from Callosellasma rhodostoma venom gland. Cloning and sequencing of 10 of the cDNAs, three-dimensional modelling and chemical modification of the major isozyme. Eur. J. Biochem., 2000, 267(22), 6684-6691. Bonfim, V.L.; Ponce-Soto, L.A.; Novello, J.C.; Marangoni, S. Structural and functional properties of Cr5, a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Calloselasma rhodostoma. Prot. J., 2006, 25(7-8), 492-502. de Sousa, M.V.; Morhy, L.; Arni, R.K.; Ward, R.J.; Daz, C.; Gutirrez, J.M. Amino acid sequence of a myotoxic Lys49phospholipase A2 homologue from the venom of Cerrophidion (Bothrops) godmani. Biochim. Biophys. Acta, 1998, 1384(2), 204208. Arni, R.K.; Fontes, M.R.M.; Barberato, C.; Gutirrez, J.M.; Daz, C.; Ward, R.J. Crystal structure of myotoxin II, a monomeric Lys49-phospholipase A2 homologue isolated from the venom of Cerrophidion (Bothrops) godmani. Archs. Biochem. Biophys., 1999, 366(2), 177-182. Rojnuckaring, P.; Muanpasitporn, C.; Chanhome, L.; Arpijuntarangkoon, J.; Itragumtornchai, T. Molecular cloning of novel serine proteases and phospholipases A2 from green pit viper (Trimeresurus albolabris) venom gland cDNA library. Toxicon, 2006, 47(3), 279-287. Fan, C.Y.; Qian, Y.C.; Yang, S.L.; Gong,Y. cDNA cloning and sequence analysis of Lys-49 phospholipase A2 from Agkistrodon acutus. Genet. Anal. Tech. Appl., 1999, 15(1), 15-18. Liu, Q.; Qingqiu, H.; Teng, M.; Weeks, C.M.; Jelsch, C.; Zhang, R.; Niu, L. The crystal structure of a novel, inactive, Lysine 49 PLA2 from Agkistrodon acutus venom: an ultrahigh resolution, ab initio structure determination. J. Biol. Chem., 2003, 278(42), 41400-41408. Polgr, J.; Magnenat, E.M.; Peitsch, M.C.; Wells, T.N.C.; Clemetson, K.J. Asp-49 is not an absolute prerequisite for the enzymic activity of low-Mr phospholipases A2: purification, characterization

Protein & Peptide Letters, 2009, Vol. 16, No. 8

875

[162]

[178]

[163]

[179]

[180]

[164]

[181]

[165]

[182]

[166]

[183]

[167]

[168]

[184]

[169]

[185]

[170]

[186]

[187]

[171]

[172]

[188]

[189]

[173]

[190] [191]

[174]

[192] [193]

[175] [176]

[194]

[177]

[195]

and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper). Biochem. J., 1996, 319(3), 961-968. Pan, H.; Liu, X.L.; Ou-Yang, L.L.; Yang, G.Z.; Zhou, Y.C.; Li, Z.P.; Wu, X.F. Diversity of cDNAs encoding phospholipase A2 from Agkistrodon halys pallas venom, and its expression in E. coli. Toxicon, 1998, 36(8), 1155-1163. Bao, Y.; Bu, P.; Jin, L.; Hongxia, W.; Yang, Q.; An, L. Purification, characterization and gene cloning of a novel pospholipase A2 from the venom of Agkistrodon blomhoffii ussurensis. Int. J. Biochem. Cell Biol., 2005, 37(3), 558-565. Nobuhisa, I.; Nakashima, K.; Deshimaru, M.; Ogawa, T.; Shimohigashi, Y.; Fukumaki, Y.; Sakaki, Y.; Hattori, S.; Kihara, H.; Ohno, M. Accelerated evolution of Trimeresurus okinavensis venom gland phospholipase A2 isozyme-encoding genes. Gene, 1996, 172(2), 267-272. Yoshizumi, K.; Liu, S.Y.; Miyata, T.; Saita, S.; Ohno, M.; Iwanaga, S.; Kihara, H. Purification and amino acid sequence of basic protein I, a lysine-49 phospholipase A2 with low activity, from the venom of Trimeresurus flavoviridis (Habu snake). Toxicon, 1990, 28(1), 43-54. Liu, S.Y.; Yoshizumi, K.; Oda, N.; Ohno, M.; Tokunaga, F.; Iwanaga, S.; Kihara, H. Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trimeresurus flavoviridis venom. J. Biochem., 1990, 107(3), 400408. Liu, C.S.; Chen, J.M.; Chang, C.H.; Chen, S.W.; Teng, C.M.; Tsai, I.H. The amino acid sequence and properties of an edema-inducing Lys-49 phospholipase A2 homolog from the venom of Trimeresurus mucrosquamatus. Biochim. Biophys. Acta, 1991, 1077(3), 362370. Wei, J.F.; Li, T.; Wei, X.L.; Sun, Q.Y.; Yang, F.M.; Chen, Q.Y.; Wang, W.Y.; Xiong, Y.L.; He, S.H. Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A2 from the venom of Protobothrops mucrosquamatus. Biochimie, 2006, 88(10), 1331-1342. Nakai, M.; Nakashima, K.I.; Ogawa, T.; Shimohigashi, Y.; Hattori, S.; Chang, C.C.; Ohno, M. Purification and primary structure of a myotoxic lysine-49 phospholipase A2 with low lipolytic activity from Trimeresurus gramineus venom. Toxicon, 1995, 33(11), 1469-1478. Krizaj, I.; Bieber, A.L.; Ritonja, A.; Guben ek, F. The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom. Eur. J. Biochem., 1991, 202(3), 1165-1168. Murakami, M.T.; Kuch, U.; Betzel, C.; Mebs, D.; Arni, R.K. Crystal structure of a novel myotoxic Arg49 phospholipase A2 homolog (zhaoermiatoxin) from Zhaoermia mangshanensis snake venom: insights into Arg49 coordination and the role of Lys122 in the polarization of the C-terminus. Toxicon, 2008, 51(5), 723-735. Castoe, T.A.; Parkinson, C.L. Bayesian mixed models and the phylogeny of pitvipers (Viperidae: Serpentes). Mol. Phyl. Evol., 2006, 39(1), 91-110. de Castro, R.C.; Landucci, E.T.C.; Toyama, M.H.; Giglio, J.R.; Marangoni, S.; De Nucci, G.; Antunes, E. Leucocyte recruitment induced by type II phospholipases A2 into the rat pleural cavity. Toxicon, 2000, 38(12), 1773-1785. Chang, Y.; Li, Y.; Bao, Y.; An, L. Neurotoxic activity of Gln49 phospholipase A2 from Gloydius ussuriensis snake venom. J. Appl. Toxicol., 2007, 27(5), 447-452. Araya, C.; Lomonte, B. Antitumor effects of cationic synthetic peptides derived from Lys49 phospholipase A2 homologues of snake venoms. Cell Biol. Int., 2007, 31(3), 263-268. Bruss, J.L.; Capaso, J.; Katz, E.; Pilar, G. Specific in vitro biological activity of snake venom myotoxins. J. Neurochem., 1993, 60(3), 1030-1042. Melo, P.A.; Homsi-Brandeburgo, M.I.; Giglio, J.R.; Suarez-Kurtz, G. Antagonism of the myotoxic effect of Bothrops jararacussu venom and bothropstoxin by polyanions. Toxicon, 1993, 31(3), 285-291. Fenard, D.; Lambeau, G.; Valentin, E.; Lefebvre, J.C.; Lazdunski, M.; Doglio, A. Secreted phospholipases A2, a new class of HIV inhibitors that block virus entry into host cells. J. Clin. Invest., 1999, 104(5), 611-618. Gambero, A.; Landucci, E.C.T.; Toyama, M.H.; Marangoni, S.; Giglio, J.R.; Nader, H.B.; Dietrich, C.P.; De Nucci, G.; Antunes, E.

876 Protein & Peptide Letters, 2009, Vol. 16, No. 8 Human neutrophil migration in vitro induced by secretory phospholipases A2: a role for cell surface glycosaminoglycans. Biochem. Pharmacol., 2002, 63(1), 65-72. Rufini, S.; Cesaroni, M.P.; Luly, P. Proliferative effect of ammodytin L from the venom of Vipera ammodytes on 208F rat fibroblasts in culture. Biochem. J., 1996, 320(2), 467-472. Mora, R.; Valverde, B.; Daz, C.; Lomonte, B.; Gutirrez, J.M. A Lys49 phospholipase A2 homologue from Bothrops asper snake venom induces proliferation, apoptosis and necrosis in a lymphoblastoid cell line. Toxicon, 2005, 45(5), 651-660. Mounier, C.M.; Luchetta, P.; Lecut, C.; Koduri, R.S.; Faure, G.; Lambeau, G.; Valentin, E.; Singer, A.; Ghomashchi, F.; Bguin, S.; Gelb, M.; Bon, C. Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition. Eur. J. Biochem., 2000, 267(16), 4960-4969. Farris, J.S. Methods for computing Wagner trees. Syst. Zool., 1970, 19(1), 83-92. Brooks, D.R.; McLennan, D.A. Phylogeny, Ecology, and Behaviour: A Research Program in Comparative Biology. University of Chicago Press: Chicago, 1991. Maddison, W.P.; Maddison, D.R. MacClade: analysis of phylogeny and character evolution. Sinauer Associates: Massachusetts, 1992. Lomonte, B.; Furtado, M.F.; Rovira, M.E.; Carmona, E.; Rojas, G.; Aymerich, R.; Gutirrez, J.M. South American snake venom pro-

Lomonte et al. teins antigenically-related to Bothrops asper myotoxins. Braz. J. Med. Biol. Res., 1990, 23(5), 427-435. Wster, W.; da Graca Slomao, M.; Quijada-Mascareas, J.A.; Thorpe, R.S. Butantan-British Bothrops Systematic Project 2002. Origin and evolution of the South American pitviper fauna: evidence from mitochondrial DNA sequence data. In: Biology of the Vipers, Shuett, G. W.; Hggren, M.D.; Douglas, M.; Greene, H.W., Eds.; Eagle Mountain Publishing: Utah, 2002, pp.111-128. Parkinson, C.L.; Campbell, J.A.; Chippindale, P.T. () Multigene phylogenetic analyses of pitvipers: with comments on the biogeographical history of the group. In: Biology of the Vipers, Shuett, G. W.; Hggren, M.D.; Douglas, M.; Greene, H.W., Eds.; Eagle Mountain Publishing: Utah, 2002, pp.93-110. Gutberlet Jr., R.L.; Harvey, M.B. The evolution of New World venomous snakes. In: The Venomous Reptiles of the Western Hemisphere, Campbell, J.A.; Lamar, W.W., Eds.; Cornell University Press: New York, 2004, pp.634-682. Daltry, J.C.; Wster, W.; Thorpe, R.S. Intraspecific variation in the feeding ecology of the crotaline snake Calloselasma rhodostoma in Southeast Asia. J. Herpetol., 1998, 32(2), 198-205. Gloyd, H.K.; Conant, R. Snakes of the Agkistrodon complex: a monographic review. SSAR Contributions to Herpetology 6: New York, 1990.

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[196] [197]

[204]

[198]

[205]

[199] [200] [201] [202]

[206] [207]

Received: August 18, 2008

Revised: September 08, 2008

Accepted: September 09, 2008