2009_REVIEW_PLA2_ProtPeptLett_Lomonte

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

0929-8665/09 $55.00+.00 2009 Bentham Science Publishers Ltd.

The Phospholipase A2 Homologues of Snake Venoms: Biological Activitiesand Their Possible Adaptive Roles

Bruno Lomonte1,*

, Yamileth Angulo1,2

, Mahmood Sasa1

and Jos Mara Gutirrez1

1Instituto Clodomiro Picado, School of Microbiology, and2Department of Biochemistry, School of Medicine, Universi-dad 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 PLA 2 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 PLA 2 homologues is presented, em-

phasizing 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 hypo-

thetical 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 mostcommon and abundant components in the venom of ad-vanced snakes of the superfamily Colubroidea, including thehighly poisonous Viperidae and Elapidae families, whichevolved nearly 60-80 million years ago [1,2]. The growingnumber of snake venom proteomes, or venomes, being stud-ied reveal that PLA2s may constitute in some species asmuch as 60% of the proteins in these complex secretions [3],where they play critical roles in toxicity [4]. In the venomsof some species of snakes, PLA2 can be the main toxic factorresponsible for prey immobilization and death [5,6]. Thus,toxic PLA2s have had a major adaptive value for the evolu-tion of venomous snakes, and it has been demonstrated that

these venom enzymes evolved by means of an acceleratedprocess [7,8] to acquire diverse and potent toxic functions.Noteworthy among these functions are the targeting of skele-tal muscle, the neuromuscular junction, and the haemostaticsystem [9].

According to phylogenetic evidence, snake venom PLA2sprobably arose by two independent recruitment events,originating from the non-toxic pancreatic (group I) digestiveenzymes in the family Elapidae, and from inflammatory(group II) enzymes in the family Viperidae [1]. The overallthree-dimensional structure, as well as the catalytic machin-ery of all these interfacial enzymes have been highly con-served [10-12]. However, subtle changes in surface-exposed

amino acid residues during evolution provided to snakevenom PLA2s the ability to exert a wide range of toxic ac-tivities [13], by gradually adapting to the recognition of par-ticular molecular targets, hence interfering with physiologi-cal processes [14].

*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: [email protected]

Since early studies, the toxic effects of snake venomPLA2s 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 activityand toxicity of PLA2s gradually disclosed that enzymaticactivity alone, as determined in vitro using artificial substrates, could not always predict toxic potency, i.e. somehighly catalytic enzymes have lower toxicity than othershowing lower catalytic activity, and vice versa [15]. Thipuzzling finding has been subsequently clarified, since theinterfacial binding properties and the target specificity othese PLA2s vary considerably, and are also known to playimportant roles in their overall mechanisms of toxicity, o

which phospholipid hydrolysis is only one of the steps involved [4,9]. Moreover, although most of the toxic effects othe catalytically-active PLA2s are dependent on phospholipidhydrolysis, effects that are unrelated to catalysis in this groupof enzymes have also been demonstrated [16-18].

Snake PLA2 toxins possess a conserved catalytic networkformed by four amino acid residues, His48, Asp49, Tyr52and Asp99, present in both group I and group II enzymes[10]. Together with residues of the "calcium-binding loop"Asp49 coordinates the essential Ca

2+ion cofactor for phos

pholipid hydrolysis [11]. For this reason, the presence oAsp49 and the ability to bind Ca

2+are considered as absolute

requirements for the expression of catalytic activity in alsecreted PLA2s. Strikingly, the occurrence of two proteins in

which the highly conserved Asp49 was substituted by Lyswas first reported in the venoms ofAgkistrodon p. piscivoruand Bothrops atrox[19], soon to be followed by reports onthe presence of similar proteins in numerous other Viperidaesnake species, mostly of the Crotalinae subfamily (reviewedin [20]). Initially, these "Lys49" venom proteins were considered to possess a low, but detectable and intrinsic PLAactivity [19]. However, this notion was subsequently questioned by structural and biochemical evidence that stronglyargued against such interpretation [21-23]. Studies using


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Activities of Phospholipase A2 Homologues Protein & Peptide Letters, 2009, Vol. 16, No. 8 861

recombinant Lys49 [24] and Ser49 [25] toxins have unambi-guously resolved this controversy, demonstrating that theseproteins lack PLA2 activity. Thus, the earlier observations onlow enzymatic activity of Lys49 proteins isolated from natu-ral sources can now be re-interpreted to be the consequenceof minor contamination with catalytically-active Asp49PLA2 isoforms present in the same venoms. Due to their es-sential difference with true PLA2 enzymes, the Lys49-

substituted proteins, as well as variants with other substitu-tions 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 rapidlygrown to currently include not less than 60 proteins, as listedin Table 1. Among these, the most frequent type of toxinspresents the Lys49 substitution, but other amino acids suchas Ser, Arg, Gln, and Asn have also been found in this posi-tion. Several of these toxins have been thoroughly character-ized as to their structural properties and functional abilities,and although significant progress has been made in under-standing the molecular basis of their toxic effects, there arestill fundamental details of their mode of action that remainunknown. This review will briefly mention few aspects onthe structure-function relationships of PLA2 homologues, asthese have been extensively reviewed [10,20,26-28] and willbe covered in other contributions of this special issue. Themain focus of the present work will be to summarize thediverse activities displayed by these toxins, and to discussthe possible biological significance and adaptive roles oftheir emergence in snake venoms.

3. STRUCTURAL AND FUNCTIONAL ASPECTS OF

PLA2 HOMOLOGUES

PLA2 homologues possess the general structural featuresof group II secreted PLA

2s, with subunits of 120-122 amino

acids that are stabilized by seven conserved disulfide bridges[10]. Most commonly the PLA2 homologues exist as ho-modimers in solution, with subunits held together by non-covalent forces which do not dissociate in the presence ofstrong anionic detergents such as sodium dodecylsulphate. Amonomeric state has been observed for some of these pro-teins when crystallized, although the possibility should beconsidered that acidic conditions utilized during chroma-tographic procedures or crystallization protocols could havean influence on these findings, since it has been shown, atleast for some of these toxins, that their dimers dissociate atpH values 5 [29,30].

All of the PLA2 homologues share the property of having

slightly to markedly basic isoelectric points, being rich inLys and Arg residues. This cationic character is proposed tohave an important role in target recognition, as well as in theeffector activities of these toxins [26,31-34]. The key struc-tural determinants for the toxic actions of the Lys49 PLA2homologues lie at their C-terminal region, within residues115-129 (numbering system of Renetseder [35]), as initiallyrevealed by studies using synthetic peptides [36-39] and neu-tralizing agents [40-43], and clearly supported by more re-cent site-directed mutagenesis analyses [24,27,28,44]. In athoroughly characterized Lys49 protein, bothropstoxin I,detailed mapping analyses of the C-terminal residues in-

volved in toxicity have revealed subtle differences for thediverse activities, with a partial overlapping of the delineatedbioactive sites confined within this region [28,44].

The current view on the mode of action of the Lys49PLA2 homologues involves their binding to anionic membrane targets, still unidentified, followed by the interactionof the C-terminal region with phospholipid bilayers, eithebiological or artificial, leading to drastic permeability distur

bances [20]. The effector role of the C-terminal region uponmembrane integrity might additionally be enhanced by atransition in the quaternary structure of the toxin dimers, asthere is structural evidence that their dimerization interfacemight function as a "molecular hinge" that would facilitatepenetration into the bilayer [45,46]. This potential enhancingmechanism, however, does not appear to be an absolute re-quirement for exerting membrane damage by Lys49 PLAhomologues, since it has been shown that the dissociatedmonomeric proteins still retain significant toxicity [30]. Inaddition to this quaternary structural change, crystallographievidence for a conformational change in the C-terminal re-gion, possibly associated with the binding of a phospholipidor a fatty acid into the nominal (in)active "catalytic" site of aLys49 toxin, has been documented [47]. In ACL myotoxinsuch ligand-induced conformational change exposes twohydrophobic residues, Phe121 and Phe124, towards themembrane bilayer, a phenomenon that is likely to be relevanfor the toxic actions of this protein [47].

4. BIOLOGICAL ACTIVITIES OF THE PLA2 HOMO

LOGUES

Myotoxicity in vivo was the first biological activity reported for a Lys49 PLA2 homologue [48], an effect now welestablished to be common to all proteins within the group oPLA2 homologues tested so far. The myotoxic effect oLys49 proteins is exerted only locally, at the site of injection

in contrast to the systemic action of other types of myotoxinsuch as PLA2s or PLA2 complexes from elapids and someviperids [34,49]. A comprehensive list of the biological activities described for PLA2 homologues is presented in Table2. As indicated, some of these actions are observed in vivoand therefore bear relevance to the pathophysiological alterations induced by snakebites, while others are restricted to exvivo or in vitro phenomena which are important from a basicpoint of view, but are not necessarily linked to the effectscaused by these toxins in human and animal envenomingsAn effect that belongs to this category, still lacking evidencefor its occurrence in experimental or clinical envenomings, ithe neuromuscular blockade activity. Such an effect has beendemonstrated in neuromuscular preparations [50-57], bu

neurotoxicity is not observed when these toxins are injectedin vivo, as indicated by their relatively low lethal potencie(see below) and by the lack of neurotoxic manifestationsSimilarly, renal damage [58] and alterations in bladder watetransport [59,60] have been studied only in ex vivo models operfusion or bathing. Despite not having been demonstratedin vivo, the study of these phenomena is relevant, since imay reveal molecular and cellular clues to understand themodes of action of PLA2 homologues, and at the same timemay exploit these toxins as experimental tools to gain deepeinsights into the diverse physiological processes affected bythem.


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Table 1. Phospholipase A2 Homologues Isolated from Snake Venoms

Snake Species Toxin 49 Sequence PDB References

Agkistrodon piscivorus piscivorus AppK49 K P04361 1PPA [19, 139, 140]

Agkistrodon bilineatus PLA2-II * Q9PSF9 u [141]

Agkistrodon contortrix laticinctusACL myotoxin K P49121 1S8G [47, 142, 143]

Atropoides (Bothrops) mexicanus (nummifer) myotoxin I * u ** [48, 144]

Atropoides (Bothrops) mexicanus (nummifer) myotoxin I-h * p u [145]

Atropoides (Bothrops) mexicanus (nummifer) myotoxin II K P82950 2AOZ [80, 146, 147]

Bothrops alternatus BaTX K ** u [57]

Bothrops asper myotoxin II K P24605 1CLP [135, 148, 149]

Bothrops asper myotoxin IV * Q9PR7, p u [150]

Bothrops asper myotoxin IVa K P0C616 u [151]

Bothrops asper M1-3-3 K Q9PVE3 u unpublished

Bothrops atrox Ba-K49 K ** u [19]

Bothrops atrox BaPLA2-I K **, p u [152]

Bothrops atrox myotoxin I K Q6JK69 u [153]

Bothrops brazili myotoxin * u u [71]

Bothrops jararacussu bothropstoxin I K Q90249 2H8I [33, 154, 155]

Bothrops leucurus bl/K-PLA2 K **, p u [156]

Bothrops moojeni myotoxin I K P82114 u [157, 158]

Bothrops moojeni myotoxin II K Q9I834 1XXS [157, 159, 160]

Bothrops neuwiedi myotoxin I * **, p u [161]

Bothrops neuwiedi pauloensis BnSP-7 K Q9IAT9 1PC9 [162-164]

Bothrops pirajai piratoxin I K P58399 u [165, 166]

Bothrops pirajai piratoxin II K P82287 1QLL [165, 167, 168]

Bothriechis (Bothrops) schlegelii myotoxin I * **, p u [169]

Bothriechis (Bothrops) schlegelii Bsc-K49 K P80963 u [170]

Calloselasma (Agkistrodon) rhodostoma CRV-K49 K Q9PVF3 u [170]

Calloselasma (Agkistrodon) rhodostoma Cr5 K ** u [171]

Cerrophidion (Bothrops) godmani myotoxin II K P81165 1GOD [86, 172, 173]

Cerrophidion (Bothrops) godmani PgoK49 K Q8UVU7 u [79]

Crotalus atrox Cax-K49 K Q8UVZ7 u [79]

Crotalus molossus molossus Cmm-K49 * **, p u [79]

Cryptelytrops (Trimeresurus) albolabris Tal-K49 * **, p u [79]

Cryptelytrops (Trimeresurus) albolabris GPV-K49 K ** u [174]

Cryptelytrops (Trimeresurus) albolabris GPV-N49 N ** u [174]

Deinagkistrodon (Agkistrodon) acutus Dac-K49 K x77650 u [72]

Deinagkistrodon (Agkistrodon) acutus Dac-K49b K O57385 u [79, 175]


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(Table 1) contd.

Snake Species Toxin 49 Sequence PDB References

Deinagkistrodon (Agkistrodon) acutus acutohemolysin K ** 1MC2 [176]

Echis carinatus sochureki ecarpholin S S P48650 2QHE [177]

Gloydius (Agkistrodon) halys Gh-N49 N O42188 u [178]

Gloydius (Agkistrodon) blomhoffi ussurensis Gln49-PLA2 Q ** u [179]

Ovophis (Trimeresurus) okinavensis To-3 K Q92152 u [180]

Protobothrops (Trimeresurus flavoviridis BP-I K P20381 u [181]

Protobothrops (Trimeresurus) flavoviridis BP-II K E48188 u [182]

Protobothrops (Trimeresurus) mucrosquamatus TMV-K49 K P22640 u [183]

Protobothrops (Trimeresurus) mucrosquamatus Tm-K49 K x77647 u [72]

Protobothrops (Trimeresurus) mucrosquamatus TM-N49 N DQ212913 u [184]

Protobothrops (Trimeresurus) mucrosquamatus promutoxin R DQ299948 u [184]

Trimeresurus borneensis Tbo-K49 K AY355177 u [73]

Trimeresurus gramineus PLA2-V K P70090 u [185]

Trimeresurus gramineus PLA2-VII K P70089 u [7]

Trimeresurus puniceus Tpu-K49a K AY211935 u [73, 79]

Trimeresurus puniceus Tpu-K49b K AAR14166 u [79]

Viridovipera (Trimeresurus) stejnegeri Ts-K49a K AAP48893 u [89]

Viridovipera (Trimeresurus) stejnegeri Ts-K49b K AAP48896 u [89]

Viridovipera (Trimeresurus) stejnegeri Ts-K49b' K AAP48895 u [89]

Viridovipera (Trimeresurus) stejnegeri Ts-K49c K AAP48894 u [89]

Viridovipera (Trimeresurus) stejnegeri Ts-R6-N49 N AAP48891 u [89]

Viridovipera (Trimeresurus) stejnegeri CTs-R6-N49 N AAP48890 u [89]

Vipera ammodytes ammodytin L S P17935 u [186]

Zhaoermia (Trimeresurus) mangshanensis zhaoermiatoxin R P84776 2PH4 [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 PLA 2 activity.

** Not submitted to databases; p: partial sequence; u: undetermined.

The activities of PLA2 homologues (Table 2) can begrouped into several categories. Lethality should be ruled outas a main biological purpose of these toxins. Although deathcan be induced by the injection of PLA2 homologues via

intraperitoneal or intravenous routes in mice, very high dosesare needed, in the range of mg/kg of body weight. For exam-ple, the lethal dose 50% for the Lys49 protein of A. p.piscivorus is 25 mg/kg (intravenously) [61], and forB. moo-jeni myotoxin II this value corresponds to 7.6 mg/kg (intrap-eritoneally) [62]. These potencies are three orders of magni-tude lower than those recorded for the catalytically-activePLA2s or PLA2 complexes that have evolved as paralyzingand lethal neurotoxins [15], strongly suggesting that PLA2homologues have not evolved for the main purpose of killingprey, at least in the case of rodents.

By analyzing the activities listed in Table 2, it is evidenthat one group of toxic effects of the PLA2 homologues idirectly related to membrane bilayer disorganization, whichcan be exerted on either artificial (liposome disruption), pro

karyotic (bactericidal action), or eukaryotic (antifungal, antiparasite, cytotoxic, and myotoxic activities) membranesSeveral of these activities have been reproduced by syntheticpeptides corresponding to their C-terminal 115-129 region(Table 2), highlighting the key role of this molecular site fotoxicity. On the other hand, a number of other activities canbe grouped as being related to the inflammatory reactionstriggered by these toxins (edema, leukocyte chemotaxis andactivation, cytokine release, mast cell degranulation, andhyperalgesia). Some of these effects might arise as an indirect consequence of necrotic damage to skeletal muscle


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Table 2. Activities Described for Phospholipase A2 Homologues

Type Activity Original Report Activity Reproduced by

C-Terminal Peptides*

in vivo myotoxicity (local) [48] [38]

lethality [48]

edema [135] [38]

cytokine release [63]

leukocyte recruitment [189]

hyperalgesia and mechanical allodynia [110] [110]

analgesic action [190]

tumor growth inhibition [191] **

ex vivo myotoxicity [48]

neuromuscular blockade [61]

presynaptic neuromuscular blockade [50]

increased water transport in bladder [59]

in vitro liposome disruption [85]

cytotoxicity (on many cell types) [192] [36]

heparin (and other polyanions) binding [193] [36]

bactericidal action [113] [113]

LPS-binding [113] [113]

mast cell degranulation [66]

inhibition of HIV replication [194]

neutrophil chemotaxis [195]

kidney damage [58]

cell proliferation [196]

apoptosis [197]

KDR (VEGF receptor)-binding [87]

factor Xa-binding [198]

macrophage phagocytosis activation [67]

fungicidal action [62] [118] **

antiparasite action [62]

blockade of proinflammatory effects of LPS [120] [117] **

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 th

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 homo-logue toxins have also been demonstrated, such as mast celldegranulation [66], macrophage activation [67] (Table 2), orendothelial cell damage [68]. Finally, other activities such asthe recently reported high affinity binding to the KDR recep-tor for vascular endothelial growth factor (VEGF), or the

induction of apoptosis (Table 2) still require further studieto assess their pathophysiological consequences in vivo, andthus rationalize their biological purpose. For example, recendata evidenced that Lys49 PLA2 homologues may act synergistically with VEGF to promote vascular permeability invivo, 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 thePLA2 homologues so far described in the venoms of vi-perids. First, they occur in a large number of viperid taxa,although not ubiquitously in all, intriguingly being absent inthe venoms of some species that are phylogenetically veryclose to others that contain them (Table 1 and Fig. 1). The

character reconstructions presented in (Fig. 1) show thatPLA2 homologues were recruited in Viperidae before theseparation of Viperinae and Crotalinae, as evidenced by theirpresence in both lineages. Nevertheless, the vast majority ofthese toxins have been found in crotalines, where their wide-spread occurence is also in support of an early presence ofthese genes in the evolution of this subfamily. Ambiguousancestral states in (Fig. 1) derive from the absence of toxinexpression in Bothrops jararaca and B. erythromelas, whichin the pruned tree pooled together within the same group.Similarly, our analysis reveals that several terminal taxa ap-pear to have independently lost the capacity to express thistype of toxin.

A second observation is that, in species where they occur,PLA2 homologue toxins may be produced in considerablyhigh amounts by the venom glands. For example, a singlePLA2 homologue isoform may comprise as much as 10-25%of the whole venom protein content, as noted in Bothropsasper, B. brazilii, Trimeresurus mucrosquamatus and T.borneensis [70-73]. In Protobothrops (formerly Trimeresu-rus) flavoviridis, the sum of basic Asp49 PLA2 and PLA2homologues constitutes 30% of the venom proteins [74], andinBothrops jararacussu , 58% of the expressed sequence tagsgenerated from venom glands corresponded to PLA2s, out ofwhich 83% corresponded to its major Lys49 PLA2 homo-logue, bothropstoxin I [75].

Third, a number of toxin isoforms exist, both at the popu-

lation level and in single specimens. Genomic analyses evi-denced six venom PLA2 isozyme genes in Protobothropsflavoviridis, two of which correspond to Lys49 homologues[76]. At the protein level, several isoforms of PLA2 homo-logues have been identified and isolated from single snakespecies (see, for example, Viridovipera (Trimeresurus) ste-jnegeri in Table 1), and it is common to find at least twoisoforms of these toxins in a single species (Table 1). Indi-vidual snakes express various isoforms, as analyzed in thevenom of Bothrops asper[77]. Thus, the widespread andabundant presence of PLA2 homologues in the venom ofmany viperids, together with the existence of a number ofisoforms, suggest that these toxins play an important bio-logical role for these snakes. The multiplicity of protein iso-

forms at the single specimen level, originating from geneduplication and divergence mechanisms, might have pro-vided a redundancy that allowed the PLA2 homologues toescape the pressure of negative selection and mutate underthe positive accelerated evolution scheme evidenced byOhno and colleagues [8].

6. RATIONALE FOR THE EMERGENCE OF PLA2

HOMOLOGUES: DO BIOLOGICAL ACTIVITIES

AND DIET PROVIDE CLUES?

Many aspects concerning the PLA2 homologue toxins arestill intriguing, which makes their study challenging. Phylo-

genetic studies indicate that PLA2 homologues divergedfrom ancestral group II Asp49 PLA2s [78-82]. Many basicAsp49 PLA2s in viperid venoms possess myotoxic activityinducing a pattern of muscle damage that develops withmorphological and temporal features that are indistinguishable from those induced by PLA2 homologues [49,83]. Interestingly, the myotoxic potency of Lys49 toxins was found tobe 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 a Bothrops asper, Lys49 proteins show a weaker myotoxicpotency 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 Asp49PLA2 ancestors does not appear to be a general principle toexplain the emergence and widespread presence of PLAhomologues in viperids.

In addition to myotoxicity, most biological activities ofPLA2 homologues are shared also by their basic Asp49 PLAcounterparts in viperid venoms. For example, both types otoxins have in common the ability to induce edema, hyperalgesia and other inflammatory responses, to disrupt artificiamembranes such as liposomes, to cause cytolysis of a varietyof cell lines in culture, and to kill diverse types of bacteriaThese similarities raise the question as to what advantage didthe emergence of PLA2 homologues provide. One clear difference in biological activities between Lys49 PLA2 homologues and their Asp49 counterparts is the strong binding ofthe former, but not of the latter, to the KDR receptor forVEGF [87,88]. This finding might provide novel clues tounderstand the biological role of PLA2 homologues, but athis 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 insome venoms, may be the only basic myotoxins in othersand may be absent in still other venoms. Moreover, a strikingontogenetic regulation exists in the expression of basic myotoxins, both Asp49 PLA2s and PLA2 homologues in somespecies. The venoms of newborn and juvenile specimenlack these proteins, whereas the venom of adults containhigh amounts of them [77,89,90]. Additional results presented in (Fig. 2) for Atropoides mexicanus (formerly Anummifer) and Cerrophidion (Bothrops) godmani supporthe notion that such ontogenetic regulation for the expressionof 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 limitedby the scarcity of data on feeding habits of newborn and juvenile snakes in nature. In a study ofViridovipera (Trimeresurus) stejnegeri, the bamboo viper from Taiwan and Chinaan association between the presence of a Lys49 PLA2 homologue in venom and a rodent-rich diet was noted [89]. Incontrast, the lack of Lys49 toxins in the venom of juvenilesnakes was associated with a diet in which frogs predominate[89]. A similar observation was reported in a study ofProtobothropsflavoviridis from Okinawa island, in which the lackof 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 specie

were reconstructed using the Farris optimization algorithm [199,200] implemented in MacClade [201]. Absence of the toxin was assumedonly in those taxa in which immunochemical and chromatographic analyses to detect expression yielded negative results [83,202]. All state

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 th

genus Bothriopsis). As the position of the bushmaster genus Lachesis remains controversial within the clade of Neotropical crotaline

[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 homologuesmay not be a general trend. For example, a study on thevenom proteome of Bothriechis lateralis, an arboreal viperid

from Costa Rica, noted the lack of Lys49 PLA 2 homologues[93], despite this species having a diet mainly based on ro-dents [94]. Similarly, the venom of Bothrops jararaca fromBrazil is practically devoid of basic PLA2s [95], despite theobservation that rodents constitute 50-75% of its diet [96].These apparent discrepancies prompted us to analyze therelationships between diet and expression of PLA2 homo-logues (Fig. 1).

Diet reconstruction supports the previous notion that pitvipers evolved from diet generalists, some of which alsoinclude invertebrates in their menu [96,97]. Mammal spe-

cialization (as well as other monotypic diets) is an apomor-phic 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 mammalianspecialists included in our analysis do not express PLAhomologue toxins. In fact, there is an inverse correlationbetween the absence of the toxins and the number of preytypes known to be consumed by viper species (Spearmanr=0.6, p=0.012), even after restricting the analysis to the ap-propriate degrees of freedom using Felsensteins [99method of evolutionary independent contrasts. Thus, diverging with other studies [91], loss of expression of PLAhomologue toxins is associated with mammal-restricted dietin this data set. Nevertheless, there are four generalist osemi-generalist species that also do not express these toxins


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Figure 2. Ontogenetic regulation in the expression of venom myo-

toxic 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 myo-

toxic 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. nummiferis a Lys49 variant

[80,146]. Note the absence of bands in venom samples from juve-

nile (less than one month of age) specimens of both species. Gel

polarity is indicated.

Bothrops jararaca, B. taeniata, Porthidum nasutum, and P.ophryomegas, adding difficulties to the interpretation of thepattern. Other examples of mammal specialists that lackPLA2 homologues in venom are Atropoides picadoi, Crota-lus simus, Crotalus durissus, Bothrops cotiara, Lachesismuta, andL. stenophrys (Fig. 1).

In spite of the difficulties to understand the biologicalmeaning for the emergence of the PLA2 homologues in vi-perid venoms, as outlined above, their abundance and fre-quent occurrence in many species strongly suggest that theyhave provided an important adaptive value in this family ofsnakes. In the following sections, we propose and discusssome hypotheses on what may be the biological significanceand adaptive value of the Lys49- and related PLA2 homo-logues. Where possible, experiments to address such hy-potheses will be suggested, attempting to stimulate furthermultidisciplinary research in this challenging subject.

7. ADAPTIVE BIOLOGICAL ROLES OF LYS49 PLA2HOMOLOGUES

7.1. Contribution to the Digestion of Muscle Mass

Adult viperid snakes mostly prey on mammals [98].Mammalian species present a relatively high body vol-ume:surface ratio [98], which makes digestion of large prey

by gastric and pancreatic digestive secretions a difficult taskThe venom, therefore, is likely to play a relevant role in thedigestion of such bulky prey, and the abundance and activityof proteinases in viperid snake venoms, especially of metalloproteinases [100], constitutes an adaptation to accomplishthis role. Muscle tissue represents a large percentage of thebody 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 oprey. It is proposed that necrotic muscle tissue, i.e. tissue thahas been affected by myotoxic components of snake venomsis more readily digested by proteinases present in the venomitself and in the gastric and pancreatic secretions. MyotoxicPLA2s and PLA2 homologues directly affect muscle cells bydamaging the plasma membrane, allowing a prominent influx of Ca

2+from the extracellular milieu, which activates a

complex series of intracellular degenerative events leading toirreversible cell damage [49]. One of the consequences osuch Ca

2+influx is the activation of calpains, Ca

2+-dependen

cysteine proteinases that cleave a number of cytoskeletaproteins [103]. A rapid degradation of structurally-relevan

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 actinand myosin, and is likely to facilitate the widespread hydrolysis of these predominant muscle proteins which in vivois accomplished by proteinases from inflammatory cells[104,107] and, in the case of natural envenomations, wouldbe performed by venom proteinases and by digestive gastricand pancreatic proteinases. Therefore, by inducing rapidmyonecrosis, the myotoxic action of PLA2s and PLA2 homologues is likely to facilitate the further digestion of muscleproteins. Such a digestion-promoting role has been proposedfor the myotoxic PLA2s present in the venom of the Australian elapid Oxyuranus scutellatus [108].

PLA2 homologues exert local myotoxicity, i.e. inducemyonecrosis in the anatomical region where venom is in jected, and do not induce systemic myotoxicity [34]. However, in the natural setting, the usually large volume ovenom injected by viperid snakes in natural prey wouldguarantee a relatively widespread distribution of venomwithin the prey, thus reaching many muscle compartments aa rapid rate. This would ensure a widespread myonecrosithat facilitates muscle mass digestion, as discussed aboveNevertheless, this hypothetic role of myotoxins could beaccomplished by both PLA2 homologues and by myotoxicAsp49 PLA2 enzymes, thus not providing a selective advantage of the former in performing this digestive task. Moreo

ver, the fact that PLA2 homologues are absent in the venomof many species that only prey on mammals argues againsthis digestive role when handling prey having a high volumeto surface body ratio. Consequently, additional biologicaroles might be at work behind the conspicuous presence ofPLA2 homologues in these venoms.

7.2. On the Toxic Role of PLA2 Homologues in ParticularPrey

As discussed before, PLA2 homologues are not highlylethal for rodents when injected by several routes. Howeverthis does not necessarily imply that such proteins are devoid


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of toxicity for other types of prey. The expression of PLA2homologues in the venoms of viperids evidences an earlyappearance of their genes in the diversification of pit vipersand an early recruitment of these toxins in the venom pro-teomes (Fig. 1). Many species of vipers present a highlygeneralistic prey repertoire, which includes groups as diverseas arthropods, amphibians, reptiles, birds and mammals. Itmight be that PLA2 homologues are more toxic for some of

these prey groups than for rodents. In agreement with thisconcept, species having a generalistic diet repertoire arethose that express PLA2 homologues in their venoms. Therecently described strong affinity of a Lys49 PLA2 homo-logue for the KDR VEGF receptor [87,88]

may have impli-

cations in the species of prey in which possible orthologuesof this receptor could have still unknown physiological roles.The toxicity of PLA2 homologues needs to be investigated ina wide variety of potential prey species.

7.3. The Induction of Pain as an Effective ImmobilizingMechanism

Viperid snakebite envenomings are associated with ex-cruciating pain [109]. Pharmacological investigations havedemonstrated that myotoxic Asp49 PLA2s and Lys49 PLA2homologues are potent pain inducers in rat models of hyper-algesia and allodynia [110-112]. Such hyperalgesic effect isof very rapid onset and results from the action of endogenousmediators that interact with afferent nerve fibers associatedwith pain [110]. We suggest that this pain effect of rapidonset is a highly effective mechanism for prey immobiliza-tion, since mammalian prey injected with crude viperid ven-oms or isolated Lys49 PLA2 homologues remain stationaryat the site of injection licking the injected limb (our unpub-lished observations). Such an effect would preclude a rapidescape of the prey, and would facilitate their location andingestion by snakes that present a strike and release patternof biting, such as the majority of viperid snakes [98]. Thus,the acquisition of pain-inducing effect in the evolution ofPLA2s and PLA2 homologues may have represented an adap-tive advantage for prey immobilization. Again, as in the caseof the proposed digestive role of myotoxic PLA2s, there doesnot seem to be a special advantage of PLA2 homologues overAsp49 PLA2s in the fulfilment of this biological role. Thishypothesis implies that the mechanisms of prey immobiliza-tion by snake venoms go beyond the well-known paralytic,hemorrhagic and hypotensive effects, and might include theability 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 2homologues is a stretch of cationic and hydrophobic residueslocated at the C-terminal region of these proteins[28,37,113,118,119]. Lys49 PLA2 homologues bind withhigh affinity to bacterial lipopolysaccharide (LPS) [113,120],being able to block the effects of LPS on macrophages andother targets [117,120]. It is therefore proposed that a possi-ble adaptive role of the high concentration of PLA2 homo-logues in many viperid venoms has to do with their microbi-cidal 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 thevenom gland lumen before injection [121], the risk of contamination in the venom gland is high. The presence of thesemicrobicidal components in relatively high concentration inthe venom would preclude this contamination; and (b) toavoid bacterial-induced putrefaction of the bulky prey thaviperid snakes ingest, as suggested by Tsai et al. [120], thu

contributing to an appropriate digestive process. In bothcases, the high concentrations of PLA2 homologues described in many venoms would favor the accomplishment othese roles.

PLA2 homologues are likely to have an advantage ovecatalytically-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 enzymeinactive during storage [122,123]. Citrate concentrations inviperid venoms are high enough to inhibit the enzymaticactivity of a myotoxic Asp49 PLA2 [122]. In addition, thecharacteristic acidic pH in the venom represents another inhibitory factor for several enzymatic components, includingproteinases and PLA2s [124]. Therefore, in the venom glandmilieu, Asp49 PLA2s are unlikely to be effective microbicidal compounds owing to the inhibition of catalytic activityin these conditions. In contrast, Lys49 PLA2 homologueexert microbicidal action by a catalytically-independenmechanism [113] and, therefore, would not be inhibited bycitrate or acidic pH in the venom gland. The ability of PLAhomologues to kill bacteria and fungi in the venom gland, aswell 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 highlycationic face in PLA2 homologues, and the typically high pIof 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, lipidA, 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 bacteriamembranes, thus contributing to the widespread bactericidaactivity of these proteins [113,116].

7.5. Synergism between Myotoxic Asp49 PLA2s and PLAHomologues

The possibility that Asp49 PLA2s and PLA2 homologuesby acting in combination, may enhance their actions has nobeen analyzed thoroughly. A single study using these twotypes of toxins purified from Agkistrodon p. piscivoru

venom, alone or in combination, provided evidence for asynergistic effect on phospholipid bilayer permeabilizationin vitro [125]. It was proposed that the catalytically-activeAsp49 PLA2 would create anionic patches of reaction products on the bilayer surface, which in turn may facilitate electrostatic interactions with the Lys49 toxin, thus increasingthe bilayer permeabilization effect [125].

Also related to the concept of a synergistic action be-tween Asp49 PLA2s and PLA2 homologues, it would be reasonable to envisage that the former may provide a readilyavailable 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-inducedconformational shift in Lys49 myotoxins proposed by Am-brosio et al. [47], as a relevant step in their mechanism oftoxicity. If future studies demonstrate synergistic(Lys49/Asp49) effects to be at play using biologically-relevant models of toxicity, this would help to understand theevolutive forces that might have driven the emergence andconsolidation of PLA2 homologues in viperids.

7.6. PLA2 Homologues May Mediate the Strike-InducedChemosensory 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 envenomedprey, then follows its trail [126]. This second strategy is gen-erally conceived as a mechanism to protect the snake frombites and injuries inflicted by the prey, thus it is observedchiefly when striking at rodents or other dangerous prey[127-129]. There is, however, a cost associated to this strat-egy: the snakes success as a predator relies on its ability tofind, recognize, and follow the trail of the envenomed prey.In several vipers (some of which are included in Table 1), ithas been well documented that there is a strong discrimina-tion toward envenomed preys, usually manifested throughvarious behavioral responses, from tongue flicking to ac-tively selecting the trails left by their prey [126,130]. Che-mosensory detection of envenomed prey could result fromthe ability to detect the enzymatic effects of major venomcomponents. Conversely, it is possible that other non-enzymatic components of the venom evolved to serve aschemosensitive signals that allow to discern the trail of en-venomed prey [129]. Either way, the identification of venomcomponents acting as enhancers of chemosensory function isstill pending, and PLA2 homologues may be studied for theirpossible involvement in this biologically-relevant role, asaddressed in the following sections.

8. TESTING THE HYPOTHESES ON POSSIBLEADAPTIVE ROLES

8.1. On the Digestive Role

Several methods to estimate rates of digestion are avail-able, each having advantages and caveats. The rates of diges-tion have been measured through estimation of the meal pas-sage rate (the retention time of Stevenson et al. [131]), ratesof O2 consumption following feeding (measured in an auto-mated respirometer [132]), as well as small intestinal brush-border 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 homologuesin favoring the digestion of muscle mass could be tested inexperimental settings similar to that described by Nicholsonet al. [108]. An anatomically-defined muscle obtained from arodent can be injected with various amounts of toxin, or withsaline solution as a control. Injected muscle would then beplaced in an experimental setting similar to the chamber de-scribed by Nicholson et al. [108], to resemble the conditionsof the stomach or small intestine of the snake. In the case ofthe stomach, pH should be between 1.2 and 1.7 and tempera-ture should be 25 C, under microanaerobic conditions. Pep-sin should be added, at a concentration resembling that of the

gastric content of snakes, and the muscle tissue would beregularly sampled to assess the extent of protein digestionThis can be done by quantifying the proteins released to thesupernatant or by assessing protein digestion in homogenateprepared from the muscle mass. Additionally, electrophoreticprocedures can be used to analyze hydrolysis of specificmuscle proteins, particularly actin and myosin, the mosabundant 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. Thepredicted outcome of this type of experiment is that muscleinjected with PLA2 homologues, and then exposed to gastricor intestinal model environments, should be digested to agreater extent than muscle injected with saline solutionalone. An alternative experimental strategy to assess thihypothesis would be to determine the time of digestion in thesnake of mice injected with PLA2 homologue myotoxinprior 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 bio-logically more meaningful setting would be to use venomwith or without Lys49 PLA2s, instead of just injecting Lys49PLA2s. In these conditions, the digestive role of these PLAhomologues would be assessed in the context of the totavenom. If the proposed digestive role is indeed relevant, thenmuscle injected with venom depleted of Lys49 PLA2 wouldbe digested to a lower extent than muscle injected with totavenom. The depletion of Lys49 PLA2s could be achieved bychromatographic techniques, for example using cationexchangers that exploit the high pI of these toxins. Immunoaffinity chromatography, i.e. using a column containingimmobilized anti-Lys49 PLA2 polyclonal antibodies, mighbe difficult as there is a strong antigenic cross-reactivity between basic Asp49 PLA2s and Lys49 PLA2 homologue[86,135,136]. Monoclonal antibodies have been obtained thaare able to discriminate between different isoforms [137]and thus could be useful for immunoaffinity procedures todeplete PLA2 homologues in crude venoms.

8.2. On the Toxic Role of PLA2s Homologues in DifferenTypes of Prey

The possibility that PLA2 homologues are toxic for certain species of prey can be tested by performing a screeningof toxicity in a wide spectrum of species that constitute preyitems for viperid snakes. These include a number of arthro-pods, amphibians, reptiles, birds and mammals. Initially, thescreening of toxicity may be based on the classical analysisof 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 beimportant to test in parallel the toxicity of Asp49 PLA2s, inorder to highlight toxic activities acquired by Lys49 variantsor related homologues, that are not present in their catalytically-active counterparts.

8.3. On the Role of PLA2 Homologues for Pain-RelatedPrey 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 ofLys49 PLA2, of total venom, of venom depleted of Lys49PLA2, or of saline solution would be injected intramuscularlyin mice or rats. Once the treatments are applied, the behaviorof tested animals is recorded during a time interval (usually15-20 min). Videos are then scored by a single researcherunder a double-blind protocol. This researcher should scorethe frequency and duration of anticipated behaviors. Differ-

ences among treatments are examined by mean or mediancomparisons. The prediction is that rodents injected withLys49 PLA2s, or with total venom, would show a more re-stricted movement range than animals injected with salinesolution or with venom depleted of Lys49 PLA2s. This gen-eral protocol has been extensively used in assessing the ef-fect 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 ofthe venom gland are reproduced, including venom concen-tration, pH (around 5.4) [124], and temperature (22-25 C)[108]. A solution of venom is then prepared from lyophilizedstored venom, and immediately sterilized by filtrationthrough a 0.22 m membrane. Then, sterile venom, at theconcentration observed in the venom gland, has to bespiked with inocula of several bacterial and fungal species.At various time intervals, aliquots of the venom would becollected and microbial counts determined. In this case, acomparison should be made between total venom and venomdepleted of Lys49 PLA2s, in order to test whether thesePLA2 homologues play a key microbicidal role in the contextof total venom. The hypothesis predicts that the ability ofvenom to exert microbicidal effect largely depends on theaction of Lys49 PLA2s. Therefore, venom depleted of thesePLA2 variants would be less effective in terms of reducingthe bacterial and fungal counts after spiking.

8.5. On the Synergistic Effects of Asp49 PLA2s and Lys49PLA2 Homologues

Experiments using purified Asp49 and Lys49 isoforms,ideally isolated from the venom of a single species wherethey coexist, could be performed to quantify various biologi-cal activities of the toxins, either alone or in combination.Activities such as invivo myotoxicity in mice, induction offootpad edema, in vitro cytolysis of differentiated myotubes,microbicidal action, liposome permeabilization, etc. (Table2) could be quantitatively determined. The hypothesis pre-dicts that the combination of both types of toxins should re-sult in an effect higher than the effect corresponding to thesum 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 toeuthanized mice, and presented to snakes. Tongue flicks di-rected toward envenomed and control mice can be recordedfor a given length of time, and compared. This design can bemodified to compare behavioral responses among differentvenom components. A second set of experiments can beplanned by comparing responses on a Y-shape testing arena.Snakes are allowed to follow trails made by euthanized ro-dents 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 variableto be measured.

CONCLUDING REMARKS

To understand the biological role and adaptive value fothe emergence of PLA2 homologue toxins in viperid snakevenoms is a difficult and ambitious task. However, as basic

knowledge on the diverse activities exerted by these proteinsand on their mechanisms of action and structure-functionrelationships grows, such goal may not seem unrealistic toapproach. The wide distribution and high concentration othese proteins in the venoms of many viperid speciestrongly suggests that selective pressures have been at workfor their expression. It is hoped that this review will fostethe 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 Ne

Tropica network is gratefully acknowledged. Thanks are dueto 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 pub-lished, demonstrating the synergism between a Lys49 PLA2homologue and an Asp49 PLA2, in exerting toxicity uponcultured 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 analyseof toxin sequences.Mol. Biol. Evol., 2004,21(5), 870-883.

[2] Fry, B.G. From genome to venome: molecular origin and evolution of the snake venom proteome inferred from phylogeneti

analysis of toxin sequences and related body proteins. GenomRes., 2005, 15(3), 403-420.

[3] Calvete, J.J.; Jurez, P.; Sanz, L. Snake venomics. Strategy anapplications.J. Mass Spectrometry, 2007, 42(11), 1405-1414.

[4] Kini, R.M. Excitement ahead: structure, function and mechanism osnake venom phospholipase A2 enzymes. Toxicon, 2003, 42(8)827-840.

[5] dos Santos, M.C.; Diniz, C.R.; Pacheco, M.A. ; Dias da Silva, W

Phospholipase A2 injection in mice induces immunity against thlethal effects ofCrotalus durissus terrificus venom. Toxicon, 1988

26(2), 207-213.[6] Freitas, T.V.; Fortes-Dias, C.L.; Diniz, C.R. Protection against th

lethal effects of Crotalus durissus terrificus (South American rattlesnake) venom in animals immunized with crotoxin. Toxicon

1990, 28(12), 1491-1496.[7] 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 re

gions is universal in crotalinae snake venom gland phospholipaseA2 isozyme genes. Proc. Natl. Acad. Sci. USA, 1995, 92(12), 5605

5609.[8] Ohno, M.; Chijiwa, T.; Oda-Ueda, N.; Ogawa, T.; Hattori, S. Mo

lecular evolution of myotoxic phospholipases A2 from snakvenom. Toxicon, 2003, 42(8), 841-854.


12/17


[9] Montecucco, C.; Gutirrez, J.M.; Lomonte, B. Cellular pathologyinduced by snake venom phospholipase A2 myotoxins and neuro-toxins: common aspects of their mechanisms of action. Cell. Mol.

Life Sci., 2008, 65(18), 2897-2912.[10] Arni, R.K.; Ward, R.J. Phospholipase A2 - a structural review.

Toxicon, 1996, 34(8), 827-841.[11] Scott, D.L.; White, S.P.; Otwinowski, Z.; Yuan, W.; Gelb, M.H.;

Sigler, P.B. Interfacial catalysis: the mechanism of phospholipaseA2. Science, 1990, 250(4987), 1541-1546.

[12] Scott, D.L. Phospholipase A2: structure and catalytic properties. In:

Venom phospholipase A2 enzymes: structure, function, and mecha-nism, Kini, R.M., Ed.; John Wiley & Sons: England, 1997,pp. 97-128.

[13] Kini, R.M.; Chan, Y.M. Accelerated evolution and molecular sur-face of venom phospholipase A2 enzymes.J. Mol. Evol., 1999, 48,125-132.

[14] Kini, R.M.; Evans, H.J. A model to explain the pharmacologicaleffects of snake venom phospholipases A2. Toxicon, 1989, 27(6),613-635.

[15] Rosenberg, P. Phospholipases. In:Handbook of Toxinology, Shier,W.T.; Mebs, D., Eds.; Marcel Dekker: New York, 1990, pp. 67-277.

[16] Chwetzoff, S.; Couderc, J.; Frachon, P.; Menez, A. Evidence thatthe anti-coagulant and lethal properties of a basic phospholipase A 2from snake venom are unrelated. FEBS Lett., 1989, 248(1-2), 1-4.

[17] Stefansson, S.; Kini, R.M.; Evans, H.J. The basic phospholipase A2from Naja nigricollis venom inhibits the prothrombinase complex

by a novel nonenzymatic mechanism. Biochemistry, 1990, 29(33),1742-7746.[18] Kini, R.M.; Evans, H.J. The role of enzymatic activity in inhibition

of the extrinsic tenase complex by phospholipase A2 isoenzymesfromNaja nigricollis venom. Toxicon, 1995, 33(12), 1585-1590.

[19] Maraganore, J.M.; Merutka, G.; Cho, W.; Welches, W.; Kzdy,F.J.; Heinrikson, R.L. A new class of phospholipases A2 with lysinein place of aspartate 49. J. Biol. Chem., 1984, 259(22), 13839-13843.

[20] Lomonte, B.; Angulo, Y.; Caldern, L. An overview of Lysine-49phospholipase A2 myotoxins from crotalid snake venoms and theirstructural determinants of myotoxic action. Toxicon, 2003, 42(8),885-901.

[21] 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 phospholi-pase 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.[22] Scott, D.L.; Achari, A.; Vidal, J.C.; Sigler, P.B. Crystallographic

and biochemical studies of the (inactive) Lys-49 phospholipase A2from the venom of Agkistrodon piscivorus piscivorus. J. Biol.Chem., 1992, 267(31), 22645-22657.

[23] Li, Y.; Yu, B.; Zhu, H.; Jain, M.; Tsai, M. Phospholipase A2 engi-neering. Structural and functional roles of the highly conserved ac-tive site residue aspartate-49. Biochemistry, 1994, 33(49), 14714-14722.

[24] Ward, R.J.; Chioato, L.; de Oliveira, A.H.C.; Ruller, R.; S, J.M.Active-site mutagenesis of a Lys49-phospholipase A2: biologicaland membrane-disrupting activities in the absence of catalysis.

Biochem. J., 2002, 362(1), 89-96.[25] Petan, T.; Kriaj, I.; Pungerar, J. Restoration of enzymatic activity

in a Ser-49 phospholipase A2 homologue decreases its Ca2+-

independent membrane-damaging activity and increases its toxic-

ity.Biochemistry, 2007, 46(44), 12795-12809.[26] Ownby, C.L.; Selistre de Araujo, H.S.; White, S.P.; Fletcher, J.E.Lysine 49 phospholipase A2 proteins. Toxicon, 1999, 37(3), 411-445.

[27] Chioato, L.; Ward, R.J. Mapping structural determinants of bio-logical activities in snake venom phospholipases A2 by sequenceanalysis and site directed mutagenesis. Toxicon, 2003, 42(8), 869-883.

[28] Chioato, L.; Arago, E.A.; Ferreira, T.L.; de Medeiros, A.I.; Fac-cioli, L.H.; Ward, R.J. Mapping of the structural determinants ofartificial and biological membrane damaging activities of a Lys49phospholipase A2 by scanning alanine mutagenesis. Biochim. Bio-

phys. Acta, 2007, 1768(5), 1247-1257.[29] 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-

sine 49-phospholipase A2 abolishes Ca2+- independent membran

damaging activity.Biochemistry, 2001, 40(23), 6912-6920.[30] Angulo, Y.; Gutirrez, J.M.; Soares, A.M.; Cho, W.; Lomonte, B

Myotoxic and cytolytic activities of dimeric Lys49 phospholipasA2 homologues are reduced, but not abolished, by a pH-induceddissociation. Toxicon, 2005, 46(3), 291-296.

[31] Falconi, M.; Desideri, A.; Rufini, S. Membrane-perturbing activitof Viperidae myotoxins: an electrostatic surface potential approachto a puzzling problem.J. Mol. Recogn., 2000, 13(1), 14-19.

[32] Murakami, M.; Arni, R.K. A structure based model for liposom

disruption and the role of catalytic activity in myotoxic phospholipase A2s. Toxicon, 2003, 42(8), 903-913.[33] 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.KInterfacial surface charge and free accessibility to the PLA2-activsite-like region are essential requirements for the activity of Lys4PLA2 homologues. Toxicon, 2007, 49(3), 378-387.

[34] Gutirrez, J.M.; Ponce-Soto, L.A.; Marangoni, S.; Lomonte, BSystemic and local myotoxicity induced by snake venom group Iphospholipases A2: comparison between crotoxin and a Lys4PLA2 homologue. Toxicon, 2008, 51(1), 80-92.

[35] Renetseder, R.; Brunie, S.; Dijkstra, B.W.; Drenth, J.; Sigler, P.BA comparison of the crystal structures of phospholipase A2 frombovine pancreas and Crotalus atrox venom.J. Biol. Chem., 1985260(21), 11627-11634.

[36] Lomonte, B.; Moreno, E.; Tarkowski, A.; Hanson, L..; Maccarana, M. Neutralizing interaction between heparins and myotoxi

II, a Lys-49 phospholipase A2 from Bothrops aspersnake venomIdentification of a heparin-binding and cytolytic toxin region by thuse of synthetic peptides and molecular modeling. J. Biol. Chem1994, 269(47), 29867-29873.

[37] Lomonte, B.; Pizarro-Cerd, J.; Angulo, Y.; Gorvel, J.P.; MorenoE. Tyr/Trp-substituted peptide 115-129 of a Lys49 phospholipasA2 expresses enhanced membrane-damaging activities and reproduces its in vivo myotoxic effect. Biochim. Biophys. Acta, 19991461(1), 19-26.

[38] Nez, C.E.; Angulo, Y.; Lomonte, B. Identification of the myotoxic site of the Lys49 phospholipase A2 from Agkistrodo

piscivorus piscivorus snake venom: synthetic C-terminal peptidefrom Lys49, but not from Asp49 myotoxins, exert membranedamaging activities. Toxicon, 2001, 39(10), 1587-1594.

[39] Lomonte, B.; Angulo, Y.; Santamara, C. Comparative study osynthetic peptides corresponding to region 115-129 in Lys49 myotoxic phospholipases A2 from snake venoms. Toxicon, 2003, 42(3)

307-312.[40] Lomonte, B.; Tarkowski, A.; Bagge, U.; Hanson, L.. Neutralization of the cytolytic and myotoxic activities of phospholipases AfromBothrops aspersnake venom by glycosaminoglycans of thheparin/heparan sulfate family.Biochem. Pharmacol., 1994, 47(9)1509-1518.

[41] Caldern, L.; Lomonte, B. Immunochemical characterization anrole in toxic activities of region 115-129 of myotoxin II, a Lys49phospholipase A2 from Bothrops aspersnake venom. Archs. Biochem. Biophys.,1998, 358(2), 343-350.

[42] Caldern, L.; Lomonte, B. Inhibition of the myotoxic action oBothrops aspermyotoxin II in mice by immunization with its synthetic peptide 115-129. Toxicon, 1999, 37(4), 683-687.

[43] 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.

[44] Chioato, L.; de Oliveira, A.H.C.; Ruller, R.; S, J.M.; Ward, R.J

Distinct sites for myotoxic and membrane-damaging activities inthe C-terminal region of a Lys49-phospholipase A2. Biochem. J2002, 366(3), 971-976.

[45] da Silva Giotto, M.T.; Garrat, R.C.; Oliva, G.; Mascarenhas, Y.PGiglio, J.R.; Cintra, A.C.O.; de Azevedo, Jr.; W.F., Arni; R.KWard, R.J. Crystallographic and spectroscopic characterization of molecular hinge: conformational changes in bothropstoxin I, a dimeric Lys49 phospholipase A2 homologue. Prot. Struct. FunctGenet., 1998, 30(4), 442-454.

[46] Magro, A.J.; Soares, A.M.; Giglio, J.R.; Fontes, M.R.M. Crystastructures of BnSP-7 and BnSP-6, two Lys49-phospholipases A2quaternary structure and inhibition mechanism insights. Biochem

Biophys. Res. Comm., 2003, 311(3), 713-720.[47] 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


13/17


molecular mechanism for Lys49-phospholipase A2 activity basedon ligand-induced conformational change. J. Biol. Chem., 2005,280(8), 7326-7335.

[48] Gutirrez, J.M.; Lomonte, B.; Cerdas, L. Isolation and partial char-acterization of a myotoxin from the venom of the snake Bothropsnummifer. Toxicon, 1986, 24(9), 885-894.

[49] Gutirrez, J.M.; Ownby, C.L. Skeletal muscle degeneration in-duced by venom phospholipases A2: insights into the mechanismsof local and systemic myotoxicity. Toxicon, 2003, 42(8), 915-931.

[50] Heluany, N.F.; Homsi-Brandeburgo, M.I.; Giglio, J.R.; Prado-

Franceschi, J.; Rodrigues-Simioni, L. Effects induced by bothrop-stoxin, a component from Bothrops jararacussu snake venom, onmouse and chick muscle preparations. Toxicon, 1992, 30(10),1203-1210.

[51] Rodrigues-Simioni, L.; Prado-Franceschi, J.; Cintra, A.C.O.;Giglio, J.R.; Jiang, M.S.; Fletcher, J.E. No role for enzymatic activ-ity or dantrolene-sensitive Ca2+ stores in the muscular effects ofbothropstoxin, a Lys49 phospholipase A2 myotoxin. Toxicon, 1995,33(11), 1479-1489.

[52] 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 charac-terization of BnSP-7, a lysine-49 myotoxic phospholipase A2homologue fromBothrops neuwiedi pauloensis venom.Archs. Bio-chem. Biophys., 2000, 378(2), 201-209.

[53] Oshima-Franco, Y.; Hyslop, S.; Cintra, A.C.O.; Giglio, J.R.; daCruz-Hofling, M.A.; Rodrigues-Simioni, L. Neutralizing capacity

of commercial bothropic antivenom against Bothrops jararacussuvenom and bothropstoxin-I. Muscle Nerve, 2000, 23(12), 1832-1839.

[54] Oshima-Franco, Y.; Leite, G.B.; Silva, G.H.; Cardoso, D.F.;Hyslop, S.; Giglio, J.R.; da Cruz-Hofling, M.A.; Rodrigues-Simioni, L. Neutralization of the pharmacological effects ofbothropstoxin-I fromBothrops jararacussu (jararacuu) venom bycrotoxin antiserum and heparin. Toxicon, 2001, 39(10), 1477-1485.

[55] Tsai, I.H.; Chen, Y.H.; Wang, Y.M. Comparative proteomics andsubtyping of venom phospholipases A2 and disintegrins ofProto-bothrops pit vipers. Biochim. Biophys. Acta, 2004, 1702(1), 111-119.

[56] Gallacci, M.; Oliveira, M.; Pai-Silva, M.; Cavalcante, W.L.;Spencer, P.J. Paralyzing and myotoxic effects of a recombinantbothropstoxin-I (BthTX-I) on mouse neuromuscular preparations.

Exp. Toxicol. Pathol., 2006, 57(3), 239-245.[57] Ponce-Soto, L.A.; Lomonte, B.; Gutirrez, J.M.; Rodrigues-

Simioni, L.; Novello, J.C.; Marangoni, S. Structural and functionalproperties of BaTX, a new Lys49 phospholipase A2 homologue iso-lated from the venom of the snake Bothrops alternatus. Biochim.

Biophys. Acta, 2007, 1770(4), 585-593.[58] 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.; NovelloJ.C.; Monteiro H.S.A. Renal toxicity of Bothrops moojeni snakevenom and its main myotoxins. Toxicon, 2002, 40(10), 1427-1435.

[59] Leite, R.S.; Franco, W.; Ownby, C.L.; Selistre-de-Araujo, H.S.Effects of ACL myotoxin, a Lys49 phospholipase A2 from Ag-kistrodon contortrix contortrix, on water transport in the isolatedtoad urinary bladder. Toxicon, 2004, 43(1), 77-83.

[60] Leite, R.S.; Giuliani, C.D.; Lomonte, B.; Franco, W.; Selistre-de-Araujo, H.S. Effect of a recombinant Lys49 PLA2 myotoxin andLys49 PLA2-derived synthetic peptides from Agkistrodon specieson membrane permeability to water. Toxicon, 2004, 44(2), 157-159.

[61] Dhillon, D.S.; Condrea, E.; Maraganore, J.M.; Heinrikson, R.L.;Benjamin, S.; Rosenberg, P. Comparison of enzymatic and phar-macological activities of lysine-49 and aspartate-49 phospholipasesA2 fromAgkistrodon piscivorus piscivorus snake venom.Biochem.Pharmacol., 1987, 36(10), 1723-1730.

[62] 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 myo-toxin-II, a Lys49-phospholipase A2 homologue: an example offunction versatility of snake venom proteins. Comp. Biochem.Physiol. C, 2006, 142(3-4), 371-381.

[63] Lomonte, B.; Tarkowski, A.; Hanson, L.. Host response to Bothrops aspersnake venom: analysis of edema formation, in-flammatory cells, and cytokine release in a mouse model. Inflam-mation, 1993, 17(2), 93-105.

[64] Rucavado, A.; Escalante, T.; Texeira, C.F.; Fernandes, C.M.; DazC.; Gutirrez, J.M. Increments in cytokines and matrix metalloproteinases in skeletal muscle after injection of tissue-damaging toxinfrom the venom of the snakeBothrops asper.Med. Inflamm., 200211(2), 121-128.

[65] Chaves, F.; Teixeira, C.F.P.; Gutirrez, J.M. Role of TNF-alphaIL-1beta and IL-6 in the local tissue damage induced by Bothropaspersnake venom: an experimental assessment in mice. Toxicon2005, 45(2), 171-178.

[66] Landucci, E.C.T.; Castro, R.C.; Pereira, M.F.; Cintra, A.C.O

Giglio, J.R.; Marangoni, S.; Oliveira, B.; Cirino, G.; Antunes, EDe 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.

[67] Zuliani, J.P.; Gutirrez, J.M.; Casais, L.L.; Sampaio, S.C.; Lomonte, B.; Teixeira, C.F.P. Activation of cellular functions inmacrophages by venom secretory Asp-49 and Lys-49 phospholipases A2. Toxicon, 2005, 46(5), 523-532.

[68] Lomonte, B.; Gutirrez, J.M.; Borkow, G.; Ovadia, M.; TarkowskiA.; Hanson, L.. Activity of hemorrhagic metalloproteinase BaH-and myotoxin II from Bothrops aspersnake venom on capillarendothelial cells in vitro. Toxicon, 1994, 32(4), 505-510.

[69] Yamazaki, Y.; Nakano, Y.; Imamura, T.; Morita, T. Augmentationof vascular permeability of VEGF is enhanced by KDR-bindingproteins.Biochem. Biophys. Res. Comm., 2007, 355(3), 693-699.

[70] Gutirrez, J.M.; Lomonte, B. Phospholipase A2 myotoxins fromBothrops snake venoms. Toxicon, 1995, 33(11), 1405-1424.

[71] 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.

[72] Wang, Y.M.; Wang, J.H.; Pan, F.M.; Tsai, I.H. Lys-49 phospholipase A2 homologs from venoms of Deinagkistrodon acutus anTrimeresurus mucrosquamatus have identical protein sequenceToxicon,1996, 34(4), 485-489.

[73] Wang, Y.M.; Peng, H.F.; Tsai, I.H. Unusual venom phospholipaseA2 of two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis. FEBS J., 2005, 272(12), 3015-3025.

[74] Ogawa, T.; Onoue, H.; Nakagawa, K.; Nomura, S.; Sueishi, KHattori, S.; Kihara, H.; Ohno, M. Localization and expression ophospholipases A2 in Trimeresurus flavoviridis (Habu snakevenom gland. Toxicon, 1995, 33(12), 1645-1652.

[75] Kashima, S.; Roberto, P.G.; Soares, A.M.; Astolfi-Filho, SPereira, 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 andfunctional aspects: gene expression profile of highly expressephospholipases A2.Biochimie, 2004, 86(3), 211-219.

[76] Nakashima, K.; Ogawa, T.; Oda, N.; Hattori, M.; Sakaki, Y.; Kihara, H.; Ohno, M. Accelerated evolution ofTrimeresurus flavoviridis venom gland phospholipase A2 isozymes. Proc. Natl. AcadSci., 1993, 90(13), 5964-5968.

[77] 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.[78] 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 phospholipasA2 from Bothrops jararacussu snake venom: evolution of venomgroup II phospholipase A2s's may imply gene duplications. J. Mo

Evol., 1995, 41(2), 174-179.[79] Tsai, I.H.; Chen, Y.H.; Wang, Y.M.; Tu, M.C.; Tu, A.T. Purifica

tion, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A2 from the venoms of rattlesnakes and other pit vipersArchs. Biochem. Biophys., 2001, 394(2), 236-244.

[80] Angulo, Y.; Olamendi-Portugal, T.; Alape-Giron, A.; PossaniL.D.; Lomonte, B. Structural characterization and phylogenetic relationships of myotoxin II from Atropoides (Bothrops) nummifesnake venom, a Lys49 phospholipase A2 homologue. Int. J. Biochem. Cell Biol., 2002, 34(10), 1268-1278.

[81] Mebs, D.; Kuch, U.; Coronas, F.I.V.; Batista, C.V.F.; GumprechtA.; Possani, L.D. Biochemical and biological activities of thevenom of the Chinese pitviper Zhaoermia mangshanensis, wit thcomplete amino acid sequence and phylogenetic analysis of a noveArg49 phospholipase A2 myotoxin. Toxicon, 2006, 47(7), 797-811


14/17


[82] Lynch, V.J. Inventing an arsenal: adaptive evolution and neofunc-

tionalization of snake venom phospholipase A2 genes.BMC Evolut.Biol., 2007, 7, 2, doi:10.1186/1471-2148-7-2.

[83] Gutirrez, J.M.; Lomonte, B. Phospholipase A2 myotoxins fromBothrops snake venoms. In: Venom phospholipase A2 enzymes:

structure, function, and mechanism, Kini, R.M., Ed.; John Wiley &Sons: England, 1997,pp. 321-352.

[84] Kihara, H.; Uchikawa, R.; Hattori, S.; Ohno, M. Myotoxicity andphysiological effects of three Trimeresurus flavoviridis phospholi-

pases A2.Biochem. Int., 1992, 28(5), 895-903.

[85] Daz, C.; Gutirrez, J.M.; Lomonte, B.; Gen, J.A. The effect ofmyotoxins isolated from Bothrops snake venoms on multilamellarliposomes: relationship to phospholipase A2, anticoagulant and

myotoxic activities. Biochim. Biophys. Acta, 1991, 1070(2), 455-460.

[86] Daz, C.; Gutirrez, J.M.; Lomonte, B. Isolation and characteriza-tion of basic myotoxic phospholipases A2 from Bothrops godmani

(Godman's pit viper) snake venom. Archs. Biochem. Biophys.,1992, 298(1), 135-142.

[87] Yamazaki, Y.; Matsunaga, Y.; Nakano, Y.; Morita, T. Identifica-tion of vascular endothelial growth factor receptor-binding protein

in the venom of Eastern cottonmouth: a new role of snake venommyotoxic Lys49-phospholipase A2. J. Biol. Chem., 2005, 280(34),

29989-29992.[88] Fujisawa, D.; Yamazaki, Y.; Lomonte, B.; Morita, T. Catalytically

inactive phospholipase A2 homologue binds to vascular endothelialgrowth factor receptor-2 via C-terminal loop region. Biochem. J.,

2008, 411(3), 515-522.[89] Tsai, I.H.; Wang, Y.M.; Chen, Y.H.; Tsai, T.S.; Tu, M.C. Venom

phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): mo-lecular characterization, geographic variations and evidence of

multiple ancestries.Biochem. J., 2004, 377(1), 215-223.[90] Alape-Girn, A.; Sanz, L.; Escolano, J.; Flores-Daz, M.; Madrigal,

M.; Sasa, M.; Calvete, J.J. Snake venomics of the lancehead pitvi-perBothrops asper. Geographic, individual, and ontogenetic varia-

tions.J. Proteome Res., 2008, 7(8), 3556-3571.[91] Chijiwa, T.; Deshimaru, M.; Nobuhisa, I.; Nakai, M.; Ogawa, T.;

Oda, N.; Nakashima, K.I.; Fukumaki, Y.; Shimohigashi, Y.; Hat-tori, S.; Ohno, M. Regional evolution of venom-gland phospholi-

pase A2 isoenzymes of Trimeresurus flavoviridis snakes in thesouthwestern islands of Japan.Biochem. J., 2000, 347(2), 491-499.

[92] Chijiwa, T.; Yamaguchi, Y.; Ogawa, T.; Deshimaru, M.; NobuhisaI.; Nakashima, K.I.; Oda-Ueda, N.; Fukumaki, Y.; Hattori, S.;

Ohno, M. Interisland evolution ofTrimeresurus flavoviridis venom

phospholipase A2 isozymes.J.Mol.Evol., 2003, 56(3), 286-293.[93] 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 andBothriechis schlegelii.J. Proteome Res., 2008, 7(6), 2445-2457.

[94] Solrzano, A. Serpientes de Costa Rica, Editorial InBio: San Jos,2004.

[95] Cidade, D.A.P.; Simo, T.A.; Dvila, A.M.R.; Wagner, G.; Jun-queira-de-Azevedo, I.; Ho, P.L.; Bon, C.; Zingali, R.B.; Albano,

R.M. Bothrops jararaca venom gland transcriptome: analysis ofthe gene expression pattern. Toxicon, 2006, 48(4), 437-461.

[96] Martins, M.; Marques, O.A.V.; Sazima, I. Ecological and phyloge-netic correlates of feeding, habits in neotropical pitvipers of the ge-

nus Bothrops. In: Biology of the Vipers, Shuett, G. W.; Hggren,M.D.; Douglas, M.; Greene, H.W., Eds.; Eagle Mountain Publish-

ing: Utah, 2002, pp.307-328.[97] Campbell, J.A.; Solrzano, A. The distribution, variation, and

natural history of the Middle American montane pitviper Porthid-ium godmani. In:Biology of the Pitvipers, Campbell, J.A.; Brodie,

E.D. Jr., Eds.; Selva Tyler: Texas, 1992, pp.223-250.[98] Greene, H. Snakes. The Evolution of Mystery in Nature . University

of California Press: Berkeley, 1997.[99] Felsenstein, J. Phylogenies and the comparative method. Amer.

Nat., 1985, 125(1), 1-15.[100] Bjarnason, J.B.; Fox, J.W. Hemorrhagic metalloprote inases from

snake venoms. Pharmac. Ther., 1994, 62(3), 325-372.[101] Brobeck, J.R. Best & Taylors Physiological Basis of Medical

Practice. Baltimore: Williams & Wilkins, 1979.[102] Harris, J.B.; Cullen, M.J. Muscle necrosis caused by snake venoms

and toxins.Electron Microsc. Rev., 1990, 3(2), 183-211.[103] Bertipaglia, I.; Carafoli, E. Calpains and human disease. Subcell.

Biochem., 2007, 45(0), 29-53.

[104] Gutirrez, J.M.; Arce, V.; Brenes, F.; Chaves, F. Changes in myo

fibrillar components after skeletal muscle necrosis induced by myotoxin isolated from the venom of the snake Bothrops aspe

Exp. Mol. Pathol., 1990, 52(1), 25-36.[105] Vater, R.; Cullen, M.J.; Harris, J.B. The fate of desmin and titi

during the degeneration and regeneration of the soleus muscle othe rat. Acta Neuropathol.,1992, 84(3), 140-148

[106] Harris, J.B.; Vater, R.; Wilson, M.; Cullen, M.J. Muscle fibrbreakdown in venom-induced muscle degeneration.J. Anat., 2003

202(4), 363-372.

[107] Gutirrez, J.M.; Chaves, F.; Cerdas, L. Inflammatory infiltrate iskeletal muscle injected with Bothrops aspervenom. Rev. BioTrop., 1986, 34(2), 209-219.

[108] Nicholson, J.; Mirtschin, P.; Madaras, F.; Venning, M.; KokkimM. Digestive properties of the venom of the Australian Coasta

Taipan, Oxyuranus scutellatus (Peteres, 1867). Toxicon, 200648(4), 422-428.

[109] Warrell, D.A. Snakebites in Central and South America: Epidemiology, clinical features and clinical management. In The Venomou

Reptiles of the Western Hemisphere; Campbell, J.A.; Lamar, W.WEds. Ithaca: Comstock Publishing Associates, 2004, pp. 709-761.

[110] Chacur, M.; Longo, I.; Picolo, G.; Gutirrez, J.M.; Lomonte, BGuerra, J.L.; Teixeira, C.F.P.; Cury, Y. Hyperalgesia induced by

Asp49 and Lys49 phospholipases A2 from Bothrops aspersnakvenom: pharmacological mediation and molecular determinants

Toxicon, 2003, 41(6), 667-678.[111] Chacur, M.; Milligan, E.D.; Sloan, E.M.; Wieseler-Frank, J.; Barri

entos, R.M.; Martin, D.; Poole, S.; Lomonte, B.; Gutirrez, J.M.Maier, S.F.; Cury, Y.; Watkins, L.R. Snake venom phospholipas

A2s (Asp49 and Lys49) induce mechanical allodynia upon perisciatic administration: involvement of spinal cord glia, proinflam

matory cytokines and nitric oxide. Pain, 2004, 108(1-2), 180-191.[112] Chacur, M.; Gutirrez, J.M.; Milligan, E.D.; Wieseler-Frank, J

Britto, L.R.G.; Maier, S.F.; Watkins, L.R.; Cury, Y. Snake venomcomponents enhance pain upon subcutaneous injection: an initia

examination of spinal cord mediators.Pain, 2004,111(1-2), 65-76[113] Pramo, L.; Lomonte, B.; Pizarro-Cerd, J.; Bengoechea, J.A

Gorvel, J.P.; Moreno, E. Bactericidal activity of Lys49 and Asp4myotoxic phospholipases A2

from Bothrops aspersnake venom

synthetic Lys49 myotoxin II-(115-129)-peptide identifies its bactericidal region.Eur. J. Biochem., 1998, 253(2), 452-461.

[114] Soares, A.M.; Guerra-S, R.; Borja-Oliveira , C.; Rodrigues, V.MRodrigues-Simioni, L.; Rodrigues, V.; Fontes, M.R.M.; Lomonte

B.; Gutirrez, J.M.; Giglio, J.R. Structural and functional charac

terization of BnSP-7, a lysine-49 myotoxic phospholipase Ahomologue fromBothrops neuwiedi pauloensis venom.Archs. Biochem. Biophys., 2000, 378(2), 201-209.

[115] 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 Bothrop

pirajai snake venom.Archs. Biochem. Biophys., 2001, 387(2), 188196.

[116] Santamara, C.; Larios, S.; Angulo, Y.; Pizarro, J.; Gorvel, J.PMoreno, E.; Lomonte, B. Antimicrobial activity of myotoxic phos

pholipases A2 from crotalid snake venoms and synthetic peptidvariants derived from their C-terminal region. Toxicon, 2005

45(7), 807-815.[117] Santamara, C.; Larios, S.; Quirs, S.; Pizarro, J.; Gorvel, J.P

Lomonte, B.; Moreno, E. Bactericidal and anti-endotoxic propertieof short cationic peptides derived from a snake venom Lys49 phos

pholipase A2. Antimicrob. Agents Chemother., 2005, 49(4), 13401345.

[118] Murillo, L.A.; Lan, C.Y.; Agabian, N.M.; Larios, S.; Lomonte, BFungicidal activity of a phospholipase A2-derived synthetic peptid

variant upon Candida albicans.Rev. Esp. Quimioter., 2007, 20(3)330-333.

[119] Arago, E.A.; Chioato, L.; Ward, R.J. Permeabilization ofE. coK12 inner and outer membranes by bothropstoxin-I, a Lys49 phos

pholipase A2 from Bothrops jararacussu. Toxicon, 2008, 51(4)538-546.

[120] Tsai, S.H.; Chen, Y.C.; Chen, L.; Wang, Y.M.; Tsai, I.H. Bindingof a venom Lys-49 phospholipase A2 to LPS and suppression of it

effects on mouse macrophages. Toxicon, 2007, 50(7), 914-922.[121] Kochva, E. The origin of snakes and evolution of the venom appa

ratus. Toxicon, 1987, 25(1), 65-106.


15/17


[122] Francis. B.; Seebart, C.; Kaiser, I.I. Citrate is an endogenous inhibi-tor of snake venom enzymes by metal-ion chelation. Toxicon, 1992,30(10), 1239-1246.

[123] 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 ofsnake venom proteases. Toxicon, 1998, 36(12), 1801-1806.

[124] 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.

[125] Shen, Z.; Cho, W. Membrane leakage induced by synergetic action

of Lys-49 and Asp-49Agkistrodon piscivorus piscivorus phosphol-ipases A2: implications in their pharmacological activities. Int. J.Biochem. Cell Biol., 1995, 27(10), 1009-1013.

[126] Stiles, K.; Stara, P.; Chiszar, D.; Smith, H.M. Strike-induced che-mosensory searching (SICS) and trail-following behavior in cop-perheads (Agkistrodon contortrix). In: Biology of the Vipers, Shu-ett, G. W.; Hggren, M.D.; Douglas, M.; Greene, H.W., Eds.; Ea-gle Mountain Publishing: Utah, 2002, pp.413-418.

[127] Kardong, K.V. The strike behavior of the rattlesnake Crotalusviridis oreganus. J. Comp. Psychol., 1986, 100(3), 314-324.

[128] Chiszar, D.; Lee, R.K.K.; Smith, H.M.; Radcliffe, C.W. Searchingbehaviors by rattlesnakes following predatory strikes. In: Biologyof the Pitvipers Campbell, J.A.; Brodie, E.D., Eds.; Selva Tyler:Texas, 1992, pp.369-382.

[129] Chiszar, D.; Walters, A.; Urbaniak, J.; Smith, H.M.; Mackessy,S.P. Discrimination between envenomated and nonevenomatedprey by western diamondback rattlesnakes (Crotalus atrox): che-

mosensory consequences of venom. Copeia, 1999, 3(0), 640-648.[130] Lavn-Murcio, P.A.; Kardong, K.V. Scent related to venom andprey as cues in the post-strike training behavior of rattlesnakes Cro-talus viridis oreganus.Herpetologica, 1995, 51(1), 39-44.

[131] Stevenson, R.D.; Peterson, C.R.; Tsuji, J.S. The thermal depend-ence of locomotion, tongue flicking, digestion and oxygen con-sumption in the wandering garter snake. Physiol. Zool., 1985,58(1), 46-57.

[132] Andrade, D.V.; Cruz-Neto, A.P.; Abe, A.S. Meal size and specificdynamic action in the rattlesnake Crotalus durissus (Serpentes: Vi-peridae).Herpetologica, 1997, 53(4), 485-493.

[133] Secor, S.; Diamond, J.M. Evolution of the adaptative response tofeeding among snakes.Amer. Zoologist, 1994, 34(5), 48A.

[134] Secor, S.; Diamond, J.M. Adaptative responses to feeding in Bur-mese Pythons: pay before pumping. J. Exp.Zool., 1995, 198(6),1313-1325.

[135] Lomonte, B.; Gutirrez, J.M. A new muscle damaging toxin, myo-

toxin II, from the venom of the snake Bothrops asper(terciopelo).Toxicon, 1989, 27(7), 725-733.[136] Lomonte, B.; Gutirrez, J.M.; Carmona, E.; Rovira, M.E. Equine

antibodies toBothrops aspermyotoxin II: isolation from polyvalentantivenom and neutralizing ability. Toxicon, 1990, 28(4), 379-384.

[137] Lomonte, B.; Kahan, L. Production and partial characterization ofmonoclonal antibodies to Bothrops asper(terciopelo) myotoxin.Toxicon, 1988, 26(7), 675-689.

[138] Pillay, N.; Alexander, G.J.; Lazenvy, S.L. Responses of stripedmice Rhabdomys pumilio, to faeces of a predatory snake. Behav-iour, 2003, 140(1), 125-135.

[139] Maraganore, J.M.; Heinrikson, R.L. The lysine-49 phospholipaseA2 from the venom ofAgkistrodon piscivorus piscivorus. Relationof structure and function to other phospholipases A2. J. Biol.Chem., 1986, 261(11), 4797-4804.

[140] 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 thevenom of the cottonmouth snake at 2.0- resolution. J. Biol.Chem., 1990, 265(29), 17649-17658.

[141] Nikai, T.; Komori, Y.; Ohara, A.; Yagihashi, S.; Ohizumi, Y.;Sugihara, H. Characterization and amino-terminal sequence ofphospholipase A2-II from the venom of Agkistrodon bilineatus(common cantil).Int. J. Biochem., 1994, 26(1), 43-48.

[142] Johnson, E.K.; Ownby, C.L. Isolation of a myotoxin from thevenom ofAgkistrodon contortrix laticinctus (broad-banded copper-head) and pathogenesis of myonecrosis induced by it in mice. Toxi-con, 1993, 31(3), 243-245.

[143] Selistre de Araujo, H.S.; White, S.P.; Ownby, C.L. Sequenceanalysis of Lys49 phosph

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