Review
Proteomics in Venom Research: a Focus on PLA2 Molecules
Juan J. Calvete*
Instituto de Biomedicina de Valencia, Consejo Superior de Investigaciones Científicas, Jaime Roig 11, 46010 Valencia, Spain
* Corresponding author: E-mail: jcalvete@ibv.csic.es
Received: 13-06-2011
Dedicated to the memory of Professor Franc Guben{ek
Abstract
This paper focuses on the application of proteomic tools to study the composition and natural history of snake venoms, and their crossreactivity with current homologous and heterologous antivenoms. Proteomic analyses on Bothrops indicated the suitability of using PLA2 molecules as taxonomic and population-specific markers. The lack of phylogenetic clustering among Neotropical and Neartic rattlesnakes with neurotoxic PLA2 molecules in their venoms suggests that phylogeny may not be an important consideration in venom evolution. Proteomic-guided identification of evolutionary and immunological trends among venoms may aid replacing the traditional geographic- and phylogenetic-driven hypotheses for antivenom production strategies by a more rationale approach based on venom proteome phenotyping and im-munological profile similarities. Recent proteomic and antivenomic surveys on Bothrops, Crotalus, and Bothriechis illustrate the feasibility of this view.
Keywords: Snake venomics, mass spectrometry, venom toxins, PLA2 molecule, taxonomic marker, population marker
1. Introduction
Venom represents an adaptive trophic trait that allowed snakes to transition from a mechanical (constriction) to a chemical means of subduing and digesting prey. Venoms comprise unique mixtures of deadly toxins tailored by Natural Selection in a prey-predator co-evolutionary arms race.1 Venoms also represent a sophisticated natural source of chemical and pharmacological novelty. The parallelism between the biological systems impaired by en-venomation and dysregulated in certain pathological conditions, such as hypertension, thrombosis, ischemia, chronic pain, type-II diabetes, tumor angiogenesis, etc., has opened up new exciting opportunities for the modern pharmaceutical industry for the development of new bi-oactive compounds for innovative therapeutic intervention based on venom toxin activities.2-4 While research on venoms ("venomics") has emerged as a discovery science, snake envenoming constitutes a highly relevant public health issue in many tropical and subtropical countries,5'6 and has been recognised by the World Health Organiza-
tion as a "neglected tropical disease" (http://www.who. int/neglected_diseases/diseases_en). Adequate treatment of envenoming is critically dependent on the ability of antivenoms to neutralize the symptoms of systemic envenoming.7 A robust knowledge of the toxin composition and pathophysiological activities of venom proteomes is instrumental for the treatment of envenomed victims and for the selection of specimens for the generation of improved antidotes.6'8
Research on venoms has been continuously enhanced by advances in technology. Technological developments in classical protein chemistry and molecular biology protocols' such as high-performance protein separation techniques (i.e., reverse-phase HPLC and two-dimensional electrophoresis using isoelectric focusing in immobilized pH-gradients) and high-sensitivity protein and DNA automated sequencers, catalyzed an explosion of knowledge on structure-function correlations of individual toxins during the last quarter of the 20th century. The emergence of "omic" technologies (genomics, transcrip-tomics, and proteomics) in the field of toxinology at the
turn of the 21st century offered the unprecedented possibility to explore global biological trends and expand our understanding of the clinical correlation of the global toxin composition of venoms.9 In particular, the development of hyphenated mass spectrometric techniques has been crucial for unraveling the complexity of venoms.10,11 Our approaches using proteomic tools ("venomics", "anti-venomics", and "venom phenotyping") to study the composition and natural history of snake venoms, and the crossreactivity of antivenoms with homologous and heterologous venoms, has been extensively reviewed8,9,12-14 and will not be repeated here. The focus of this paper is the application of mass spectrometry-based phospholipa-se A2 (PLA2) profiling. PLA2 molecules are among the most common and abundant toxins in viperid and elapid snake venoms.15 The adaptive role PLA2s play in the radiation of venomous snakes is highlighted by the demonstration of this multigene toxin family being subjected to neofunctionalization through accelerated evolution.16-21 Gene gain and loss and protein sequence evolution via positive selection are important evolutionary forces driving adaptive divergence in venom proteins in closely related species of venomous snakes.21 A goal of snake venomics of applied importance in human therapy is to understand
the molecular mechanisms and evolutionary forces that underlie venom variation.22-24 A comprehensive understanding of the evolutionary diversification of venoms may aid in taxonomy.25-27 In addition, a robust knowledge of the onset of ontogenetic, individual, and geographic venom variability may have an impact in the treatment of envenomed victims and in the selection of specimens for the generation of improved antidotes.6,8,28 The following examples will illustrate these views.
1. 1. A PLA2 Molecule as a Taxonomy Signature of B. Fonsecai
The genus Bothrops (subfamily Crotalinae of Vipe-ridae) comprises 32 (http://www.reptile-database.org) or 37 species29 of primarily South and Central American pi-tvipers, commonly referred as lanceheads. Except for southwestern South America, the extreme highlands of the Andes, and southernmost Patagonia, this genus is widely distributed in tropical Latin America, from northeastern Mexico to Argentina, and the southern parts of the lower Caribbean islands.29 Bothrops are diverse in their morphology and natural history, and represent a particularly interesting group because of the wide array of bioto-
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Figure 1. The venom proteomes of B. cotiara (a) and B. fonsecai (b). Both species are moderately heavy-bodied snake (adult length usually 0. 7-1. 0 m) that inhabit very specialized biotopes (Araucaria angustifolia pine forests on highlands). Although B. cotiara and B. fonsecai are not sympa-tric, they are morphologically extremely similar (lower left panel). A comparative proteomic analysis26 showed that compositional differences between their venoms, particularly the unique presence in B. fonsecai venom of a G6-D49-PLA molecule of isotope-averaged molecular mass 13980 Da (lower right panel) and N-terminal sequence NLWQFGMMIQHTTRENPLFKYFSYGCYCGWGGGGPLDATDRCCFVHDCCYG can be employed as a taxonomic signature for unambiguous species identification independently of geographic origin and morphological characteristics.
pes they inhabit, such as lowland evergreen forests, montane semideciduous forests, savannas, and montane open formations. B. cotiara and B. fonsecai inhabit similar, highly specialized habitats (Araucaria angustifolia pine forests), in different geographical regions of Brazil. Bot-hrops cotiara's habitat include the Araucaria forests of southern Brazil in the states of Sao Paulo, Paraná, Santa Catarina and Rio Grande do Sul. It is also found sporadically in northeastern Argentina in the province of Misiones, with a vertical distribution from sea level to at least 1,800 m. B. fonsecai is endemic to Southeastern Brazil (northeastern Sao Paulo, southern Rio de Janeiro and extreme southern Minas Gerais). Its elevational distribution ranges from 1000 to 1600 m. Both species are mammal specialists and are morphologically very difficult to distinguish. A comparative proteomic analysis shows the overall composition of B. cotiara and B. fonsecai venoms highlighting compositional differences. In particular, B. fonsecai expresses a high abundance PLA2 molecule (13890 Da) whereas B. cotiara's venom is devoid of PLA2 proteins. The absence of PLA2 proteins is a unique feature among all viperid venoms characterized to date and defines a taxonomy signature that can be employed for the unambiguous differentiation of B. cotiara and B.
fonsecai independently of geographical and morphological factors.
1. 2. Population-specific PLA2 Molecules Provide Clues to Trace the Dispersal Pattern of B. atrox in Northern South America
Bothrops atrox (Viperidae: Crotalinae), the Common Lancehead, is a terrestrial, generally nocturnal, and highly adaptable pitviper found in tropical lowlands and rainforest up to 1200 m of northern South America east of the Andes, including southern and eastern Venezuela, southeastern Colombia, eastern Ecuador, eastern Perú, northern Bolivia, the northern half of Brazil, and throughout Guyana, Suriname, and French Guiana.29 Despite its wide range of ecological and geographical habitats, no subspecies are currently recognized.28 B. atrox is a very dangerous species being notorious as the leading cause of more human fatalities than any other South American reptile.30 Comparative proteomic investigations of B. atrox venoms from 19 localities from Colombia, Ecuador, Perú, Venezuela, and Brazil (highlighted in Figure 2)31,32 showed that PLA2 molecules exhibit large interpopulational
Figure 2. Physical map of northern South America highlighting the Amazon River basin and the sampling localities for the B. atrox venoms investigated by Núñez et al.31 and Calvete and co-workers,32 including specimens from Colombia: Meta (M), Magdalena Medio (Antioquia Department) (1); Ecuador (E), Perú (P), Venezuela: El Paují (2) (Orinoquia) and Puerto Ayacucho (3) (Amazonia); and Brazil: Sao Gabriel de Cachoeira (4) and Presidente Figueiredo (5) (Amazonas), Sao Bento (7) (Maranhao), and and Monte Alegre (6), Ananindeua (8), Santa Isabel (9), Tucuruí (10), Icoaraci (11), Barcarena (12), Acara (13), Belém (14), Ilha de Mosqueiro (15), and Santarém (16) (Pará). The isotope-averaged molecular masses of PLAj molecules (13-15 kDa), a conserved CRISP protein (24854 ± 2 Da), and PI-SVMPs (22-25 kDa) found in the different venoms are indicated. Neighbour populations sharing identical toxins are grouped by ovals.
variation, with the venoms from neighboring locations expressing common and variable molecules (Figure 2). This pattern of geographic intraspecific variability of PLA2 loci has been reported in other species, i.e. Vipera palesti-nae,33 B. asper,34 Trimeresurus (Protobothrops) flavoviri-dis,35-37 and Lachesis muta.38 This phenomenon is often linked to differences in diet among populations.39 Snake venom PLA2 genes are members of a large, rapidly-evolving multigene family with many diverse functions.15,20,21 Positive Darwinian selection is common in group-II vipe-rid snake venom PLA2 genes and is associated with the evolution of new toxin functions and speciation events,20 suggesting adaptation of the PLA2 arsenal to novel prey species after niche shifts. Mapping the molecular diversity between conspecific populations onto a physical map (Figure 2) provides clues for tracing dispersal routes that account for the current biogeographic distribution of the species. The emerging phylogeographic hypothesis summarized in Figure 2 is consistent with an intricate model of southeast and southwest dispersal and allopatric fragmentation northern of the Amazon Basin, and trans-Amazonian expansion through the Andean Corridor and across the Amazon river. On the other hand, venoms from Sao Bento (Maranhao State), and Ananindeua, Santa Isabel, Tucurui, Icoaraci, Barcarena, Acara, Belem, and Ilha de Mosqueiro (Para State), located south of the mouth of the Amazon river (Figure 2), share two PLA2 molecules (13930 and 13875 Da), but have no common molecules with the venoms from specimens inhabiting regions north of the Amazon river. These populations may have been established in Para and Maranhao by ancient vicariance of a B. atrox population which managed to cross the Amazon river or from a dispersal event involving B. atrox populations not sampled in the proteomic survey.
Venoms from snakes collected northern of the Amazon Basin (Magdalena Medio Valley and Orinoquia) exhibited the ontogenetic phenotype reported in adult specimens of Venezuelan B. colombiensis,40 and Costa Rican B. asper,41 (characterized by %PI-SVMP (snake venom metalloproteinase) > %PIII-SVMP and %K49-PLA2 > %D49-PLA2), whereas Amazonian B. atrox venoms showed a paedomorphic phenotype comprised predominantly of PIII-SVMPs and %D49-PLA2 > %K49-PLA2. Antivenoms raised in Costa Rica and Brazil using different Bothrops venoms in the immunization mixtures im-munodepleted very efficiently the major toxins (PIII-SVMPs, serine proteinases, CRISP, LAO) of paedomorp-hic venoms, but had impaired reactivity towards PLA2 and P-I SVMP molecules abundantly present in the onto-genetic venoms. These results clearly illustrate that knowledge of evolutionary and immunological trends among conspecific populations may aid replacing the traditional geographic- and phylogenetic-driven hypotheses for antivenom production strategies by a more rationale approach based on proteome phenotype and immunologi-cal profile similarities.
1. 3. Neurotoxic PLA2 Molecules in Rattlesnake Type II Venoms. Clues for the Management of Rattlesnake Envenomings
The monophyletic clade of the rattlesnakes (genera Crotalus and Sistrurus) had its origin ~20 Mya in the Sierra Madre Occidental in the north-central Mexican Plate-au.42 Rattlesnakes dispersed northward into North America and southward into South America, and today genus Crotalus groups approximately 70 species and subspecies (http://www.reptile-database.org) of venomous pitvipers widely and discontinuosly distributed from southern Canada to northern Argentina.29,43 Rattlesnake venoms belong to one of two distinct phenotypes, which broadly correspond to type I (high levels of SVMPs and low toxicity, LD50 >1 mg/g mouse body weight) and type II (low metal-loproteinase activity and high toxicity, LD50 <1 mg/g mouse body weight) defined by Mackessy.44 The high to-xicity of type II venoms and the characteristic systemic neuro- and myotoxic effects observed in envenomations appear to be directly related to the expression of the presy-naptic P-neurotoxic heterodimeric PLA2 molecules, cro-toxin (in Central and South American rattlesnake venoms),45-47 Mojave toxin (in Neartic rattlesnakes),48 and sistruxin (in Sistrurus catenatus catenaus and S.c. terge-minus venoms).49,50 Crotoxin, Mojave toxin, and sistruxin are composed of two subunits, a non-toxic acidic subunit (CA) (named crotapotin in crotoxin), which lacks PLA2 activity, and a weakly toxic basic subunit (CB), which exhibits PLA2 activity. The acidic subunit undergoes pro-teolytic processing to form three polypeptides held together by disulphide bridges. Although the CB subunit exhibits neurotoxic activity, the native crotoxin complex is at least one order of magnitude more potent than CB alone. The increase of toxicity in mice, rats and rabbits, due to crotapotin acting as a chaperone blocking the binding to non-specific sites and guiding the CB subunit to its specific target site, is the crucial feature of crotoxin.51
The New World pitvipers represent a monophyletic radiation of a single unidirectional invasion by an ancestral crotaline form of Old World origin across the Bering Land Bridge in the late Cretaceous to early Tertiary.52 Although PLA2 enzymes mostly exist as monomers, both covalent and noncovalent oligomeric complexes with other PLA2 (or PLA2-like molecules) or with other proteins have been described in a number of Old World and New World Elapi-dae and Viperidae taxa.53 Venoms of Vipera include postsynaptic (e.g., vipoxin from Vipera ammodytes meridionals)54,55 and presynaptic acting toxins (e.g., ammody-toxin from V. a. ammodytes,56 and vipertoxin F from Da-boia russellii formosensis51), where the neurotoxic PLA2 either works alone, or is complexed with an inhibitor or a chaperone.53 Within the Old World crotaline taxa, monomeric or dimeric neurotoxic PLA2s have been described in the genera Gloydius and Protobothrops. However, the fact
that few Old World species possess neurotoxic components, and none to date are found with P-neurotoxic hete-rodimeric crotoxin-like PLA2s, indicates that the emergence of these presynaptic-acting complexes must be dated after the derivation of rattlesnakes in the New World.
The increased concentration of crotoxin in South American rattlesnake venoms represents a paedomorphic trend.58,59 Gain of neurotoxicity and lethal venom activities to mammals (rodents) have represented the key axis along which overall venom toxicity has evolved during Crotalus radiation in South America.58,59 Mojave toxin positive Neartic type II venoms, such as C. tigris (Tiger rattlesnake), C. horridus (Timber rattlesnake), C. scutula-tus scutulatus type A (Mohave rattlesnake), the midget-faded rattlesnake (C. oreganus concolor), and C. oreganus helleri (Southern Pacific rattlesnake), exhibit toxin venom phenotypes closely resembling those of neurotoxic South American Crotalus durissus subspecies (terrificus, casca-vella, collilineatus) (Figure 3), and the subunits of the Mojave toxin (SwissProt accession codes P18998 and P62023) share respectively 95% and 100% amino acid sequence identity with the acidic (A, P08878) and the basic
(CB1, P62022) subunits of crotoxin. Powell and Lieb60 have predicted that the extremely high neurotoxicity exhibited by North American rattlesnakes represents a transitory populational phenomenon associated with novel prey bases. The phylogeny and evolution of P-neurotoxic PLA2s present in the venoms of rattlesnakes has been investigated by Werman.61 Maximum parsimony phyloge-netic reconstructions support the view of gene duplication and subsequent independent evolution for the origin of the two subunits of the heterodimeric sistruxin, crotoxin, and Mojave toxin from pre-existing PLA2 sequences already present in non-neurotoxic species.61 However, the distribution of PLA2 P-neurotoxins among rattlesnakes shows very little phylogenetic structure, as there are no clades that have neurotoxic PLA2 complexes in all members. Only terminal clades of recently divergent taxa (e.g., subs-pecific taxa within Sistrurus catenatus, Crotalus durissus, C. simus, and C. scutulatus) appear to express neurotoxin in adult venoms.61
The lack of phylogenetic clustering among rattlesnakes with neurotoxin indicates that phylogeny may not be an important consideration in venom evolution. Wer-
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Figure 3. Comparison of the reverse-phase HPLC separations of the venom proteins of (a) C. scutullatus scutulatus (type A), (b) C. oreganus helleri, (c) C. horridus, and (d) C. durissus collilineatus. Crotoxin/Mojave toxin acidic and basic subunits are labelled "a" and "b", respectively. Inserts in panels A-C, ESI-MS spectra of the basic subunits of Mojave toxin from C. scutullatus scutullatus (type A) (N-terminal sequence, HLLQFNKMIKFETRK, isotope-averaged mass, 14186 Da), C. oreganus helleri (HLLQFNKMIKFETRK, 14177 Da), and C. horridus (HLLQFNKMIKFETRK, 14156 Da), respectively. The homologous crotoxin B-subunit from C. durissus collilineatus has N-terminal sequence HLLQFNKMIKFETRK and an ESI-MS of 14187 Da. 59
man61 has speculated that the presence of neurotoxins among rattlesnakes may have resulted from the influence of a combination of factors, including paedomorphosis in terminally-derived clades and gene transfer through ancestral hybridization events. According to this author, S. ca-tenatus venom containing both heterodimeric neurotoxin (sistruxin) and other PLA2 molecules could represent the archetype of rattlesnake type I venoms. The archetype for neurotoxic type II venoms may be represented by C. si-mus, where juveniles are highly neurotoxic, but adults have hemorrhagic type I venoms.61
An anti-crotalic antivenom produced at Instituto Bu-tantan against C. d. terrificus venom showed a very high effectiveness in the neutralization of the lethal, myotoxic, and neurotoxic effects of venoms of C. durissus subspecies and newborn C. simus.62 However, this antivenom failed to neutralize the hemorrhagic activity of adult C. si-mus and C. d. cumanensis and C. d. ruruima venoms. In contrast, a polyvalent antivenom produced by Instituto Clodomiro Picado (San José, Costa Rica) effectively neutralized the hemorrhagic activity of Crotalus venoms but not against C. durissus venoms, and showed a very low neutralizing activity against newborn C. simus enveno-ming.63 Such neutralizing profile is fully explained by the proteomic finding that adult C. simus and C.d. cumanensis
synthesize type I venoms while the venoms of juvenile C. simus and C.d. durissus, C.d. ruruima, C.d. collilineatus, C.d. cascavella, and C.d. terrificus belong to the type II class. The evolutionary trend observed in Crotalus suggests that an effective pan-American anti- Crotalus antivenom should primarily neutralize the toxic actions of four major toxin groups, PIII-SVMPs, crotoxin, crotamine, and thrombin-like serine proteinases. Such antivenom might be achievable by hyperimmunizing with a mixture of type I and type II venoms comprising conserved antigenic determinants for each of the major toxin families of the genus.
1. 4. Extreme Venom Variabilty Among Palm Viper Venoms. A Vrotoxin-like PLA2 Molecule in B. Nigroviridis Venom
The genus Bothriechis comprises 9 species (B. auri-fer, B. bicolor, B. lateralis, B. marchi, B. nigroviridis, B. rowleyi, B. schlegelii, B. supraciliaris, B. thalassinus) of relatively slender to medially robust, arboreal, prehensile-tailed, New World pitvipers29 (Figure 4). Documentation of human accidents by Bothriechis snakebites is scarce, and although Bothriechis venoms investigated seem to be of moderate toxicity, bites may have dire consequences due to the arboreal nature of these snakes which results in
Figure 4. Mapping the venom toxin profiles of B. lateralis, B. schlegelii, and B. nigroviridis onto the phylogenetic tree of Bothriechis, reveals the extreme venom composition divergence among congeneric taxa. CRISP, cysteine-rich secretory protein; C-lectin, C-type lectin-like molecule; BPP, bradykinin-potentiating peptide; svVEGF, snake venom vascular endothelial growth factor; LAO, L-amino acid oxidase; SVMP, snake venom me-talloproteinase; PLA2, phospholipase A2.
many of the bites being inflicted in the head, neck, and shoulder regions. Venomic studies have revealed a high divergence in the venom compositions of B. lateralis, B. schlegelii, and B. nigroviridis64,65 (Figure 4), in spite of the fact that these species have evolved to adapt to arboreal habits and seem to have similar generalist-type diets.
The major toxin families of B. lateralis and B. schlegelii venoms are SVMP (55% of the total proteins) and PLA2 (44%), respectively (Figure 4). Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation in experimental animals caused by B. schlegelii and B. lateralis.64 The venom from B. nigroviridis is devoid of hemorrhagic activity, has low edematogenic and coagulant effects, presents modest myotoxic and phospholipase A2 activities, but has higher lethality than the venoms of other Bothriechis species. Strikingly, the venom proteome of B. nigroviridis does not possess detectable Zn2+-dependent SVMPs, and is uniquely characterized by a high content of crotoxin-like PLA2 subunits and vasoactive peptides, each of these groups of toxins representing as much as 38% of total venom proteins65 (Figure 4). These data support the view that different evolutionary solutions have evolved within the arboreal genus Bothriechis for the same trophic purpose, and underscore the versatility of venoms as adaptive traits in these viperid snakes. On the other hand, the presence of crotoxin-like PLA2 subunits in the venom of B. nigroviridis could not have been guessed through a phylogenetic hypothesis. However, neutralization of the lethal activity of B. ni-groviridis venom by an anti-crotalic antivenom,65 manufactured by Instituto Butantan using venom of C. d. terrificus as immunogen, points to a major role of crotoxin-like PLA2 in B. nigroviridis venom-induced lethality, and highlights the relevance of in vivo neutralization assays and antiveno-mic profiling for expanding the clinical use of heterologous antivenoms on an immunologically sound basis.
2. Acknowledgements
The author gratefully acknowledge the many colleagues who have provided venoms and antivenoms within the framework of collaborative projects. Funding for conducting the research described in this paper in the author's laboratory was provided by grants BFU2007-61563 and BFU2010-17373 from the Ministerios de Educación y Ciencia and Ciencia é Innovación, Madrid; CRUSA-CSIC (2007CR0004 and 2009CR0021); and PROME-TE0/2010/005 (Generalitat Valenciana).
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Povzetek
Članek je osredotočen na uporabo proteomskih orodij pri raziskavah sestave in razvoja kačjih strupov ter njihove nav-skrižne reaktivnosti s trenutno razpoložljivimi homolognimi in heterolognimi protistrupi. Proteomske analize strupov kač iz družine Bothrops so izpostavile primernost uporabe PLA2 molekul kot taksonomskih in populacijsko-specifičnih označevalcev. Izostanek filogenetskega združevanja neotropskh in nearktičnih klopotač, ki v strupih vsebujejo nevrotok-sične PLA2, nakazuje, da filogenija živali ni pomembna pri obravnavi evolucije njihovih strupov. Na proteomski analizi osnovana identifikacija evolucijskih in imunoloških smeri razvoja kačjih strupov bi utegnila nadomestiti tradicionalne geografsko- in filogenetsko-pogojene hipoteze za oblikovanje strategij za pripravo protistrupov z bolj racionalnim pristopom, ki bi temeljil na primerjavi proteomov in imunoloških profilov strupov. Najnovejše analize protistrupov in proteom-ske analize strupov kač iz družin Bothrops, Crotalus in Bothriechis kažejo na obetaven potencial takega pristopa.