FEBS Letters 583 (2009) 1736–1743

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Venoms, venomics, antivenomics

Juan J. Calvete a,*, Libia Sanz a, Yamileth Angulo b, Bruno Lomonte b, José María Gutiérrez b

a Instituto de Biomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spainb Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

a r t i c l e i n f o

Article history:Received 17 February 2009Revised 12 March 2009Accepted 13 March 2009Available online 20 March 2009

Edited by Miguel De la Rosa

Keywords:Proteomics of snake venomSnake venomicsSnake venomAntivenomAntivenomics

0014-5793/$36.00 � 2009 Federation of European Biodoi:10.1016/j.febslet.2009.03.029

* Corresponding author. Fax: +34 96 369 0800.E-mail address: jcalvete@ibv.csic.es (J.J. Calvete).

a b s t r a c t

Venoms comprise mixtures of peptides and proteins tailored by Natural Selection to act on vital sys-tems of the prey or victim. Here we review our proteomic protocols for uncoiling the composition,immunological profile, and evolution of snake venoms. Our long-term goal is to gain a deep insightof all viperid venom proteomes. Knowledge of the inter- and intraspecies ontogenetic, individual,and geographic venom variability has applied importance for the design of immunization protocolsaimed at producing more effective polyspecific antivenoms. A practical consequence of assessing thecross-reactivity of heterologous antivenoms is the possibility of circumventing the restricted avail-ability of species-specific antivenoms in some regions. Further, the high degree of target specificitymakes toxins valuable scaffolds for drug development.� 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. The Ying and Yang of animal venoms

Venomous organisms are widely spread throughout the animalkingdom, comprising more than 100,000 species distributedamong all major phyla, such as chordates (reptiles, fishes, amphib-ians, mammals), echinoderms (starfishes, sea urchins), molluscs(cone snails, octopi), annelids (leeches), nemertines, arthropods(arachnids, insects, myriapods) and cnidarians (sea anemones, jel-lyfish, corals). In any habitat there is a competition for resources,and every ecosystem on Earth supporting life contains poisonousor venomous organisms. One of the most fascinating techniquesof capturing prey or defending oneself is the use of poisons or ven-oms. Venom represents an adaptive trait and an example of con-vergent evolution [1–3]. Venoms are deadly cocktails, eachcomprising unique mixtures of peptides and proteins naturally tai-lored by Natural Selection to act on vital systems of the prey or vic-tim. Venom toxins disturb the activity of critical enzymes,receptors, or ion channels, thus disarranging the central andperipheral nervous systems, the cardiovascular and the neuromus-cular systems, blood coagulation and homeostasis. On the otherhand, due to their high degree of target specificity, venom toxinshave been increasingly used as pharmacological tools and as proto-types for drug development. The medicinal value of venoms hasbeen known from ancient times. The snake is a symbol of medicinedue to its association with Asclepius, the Greek god of medicine.

chemical Societies. Published by E

The medical uses of scorpion and snake venoms are well docu-mented in folk remedies, and in Western and Chinese traditionalmedicine [4,5]. However, extensive investigations on venom com-pounds as natural leads for the generation of pharmaceutical prod-ucts have only been performed in the last decades, after abradykinin-potentiating peptide isolated from the venom of theBrazilian viper Bothrops jararaca was developed in the 1950s intothe first commercial angiotensin I-converting enzyme (ACE)-inhib-iting drug, captopril�, for the treatment of renovascular hyperten-sion [6,7]. The latest example of development of a toxin into anapproved drug by the US Food and Drug Administration (FDA)(December 2004) is ziconotide (Prialt�), a synthetic non-opioid,non-NSAID, non-local anesthetic drug originating from the conesnail Conus magus peptide x-conotoxin M-VII-A, an N-type calciumchannel blocker. Discovered in the early 1980s [8], zicotonide is arare example of a molecule used unaltered from a creature’s chem-istry. Prialt� is used for the amelioration of chronic untreatablepain, and due to the profound side effects or lack of efficacy whendelivered through more common routes, such as orally or intrave-nously, Prialt� must be administered directly into the spine.

Venoms represent a huge and essentially unexplored reservoirof bioactive components that may cure disease conditions whichdo not respond to currently available therapies. The great pharma-cological cornucopia accumulated by Nature over evolution has re-sulted in true combinatorial libraries of hundreds of thousands ofpotentially active and useful molecules synthesised in the venomsof the 700 or so known species of Conus, the roughly 725 species of

lsevier B.V. All rights reserved.

J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743 1737

venomous snakes, the �1500 species of scorpions, or the ca. 37,000known species of spiders, just to cite only a few examples of ven-omous animals. The accelerated Darwinian evolution acting to pro-mote high levels of variation in venom proteins may be part of apredator–prey arms race that allows the predator to adapt to avariety of different prey, each of which are most efficiently sub-dued with a different venom formulation. In addition to under-standing how venoms evolve, a major aim of venomic projects isto gain a deeper insight into the spectrum of medically importanttoxins in venoms to uncover clues in order to solve the riddle of themedical effectiveness of venoms and to learn how to convertdeadly toxins into lifesaving drugs [9,10]. On the other hand,envenomation constitutes a highly relevant public health issueon a global basis, as there are venomous organisms in every conti-nent and almost every country. However, venomous animals areparticularly abundant in tropical regions, which represent thekitchen of evolution. Arthropod stings inflicted by bees, wasps,ants, spiders, scorpions, and – to a lesser extent – millipedes andcentipedes, constitute the most common cause of envenoming byanimals, although around 80% of deaths by envenomation world-wide are caused by snakebite, followed by scorpion stings, whichcause 15%. Envenoming constitutes a highly relevant public healthissue on a global basis, although it has been systematically ne-glected by health authorities in many parts of the world [11–15].Being a pathology mainly affecting young agricultural workers liv-ing in villages far from health care centers in low-income countriesof Africa, Asia and Latin America, it must be regarded as a ‘ne-glected tropical pathology’ [11]. Adequate treatment of envenom-ing is critically dependent on the ability of antivenoms toneutralize the lethal toxins reversing thereby the signs of enven-oming. A long-term research goal of venomics of applied impor-tance for improving current antivenom therapy is to understandthe molecular mechanisms and evolutionary forces that underlievenom variation. Thus, a robust knowledge of venom compositionand of the onset of ontogenetic, individual, and geographic venomvariability may have an impact in the treatment of bite victims andin the selection of specimens for the generation of improved anti-dotes [16] (see below).

2. The evolution of the advanced snakes and their venoms

The suborder of snakes (Serpentes) of the reptilian order Squa-mata, named for their scaly skin, includes about 3000 extant spe-cies placed in approximately 400 genera and 18 families (http://www.reptile-database.org). The timing of major events in snakeevolution is not well understood, however, owing in part to a rel-atively patchy and incomplete fossil record [17,18]. Nevertheless,after more than 100 years of research, the most generalized phylo-genetic view is that the group evolved from a family of terrestriallizards during the time of the dinosaurs in the Jurassic period,about 200 million years (Myr) ago [19]. After the end of the non-avian dinosaurs reign, around the Cretaceous-Tertiary boundary65 Myr ago [20], the boids (the ancestors of boas, pythons and ana-condas) were the dominant snake family on Earth. Within theCenozoic era that followed, advanced snakes (colubrids) arose aslong ago as in the Oligocene epoch (35–25 Myr). Colubrids, thefamily which we regard today as typical snakes, remained a smalltaxon until the tectonic plates drifted apart from the equator andthe cool climate pushed boids to disappear from many ecologicalniches. Colubrids quickly colonized these empty habitats and thisfamily today comprises over two-thirds of all the living snake spe-cies [21]. Colubroidea encompasses Viperidae (30 genera, 230 spe-cies of vipers and pitvipers), Elapidae (63 genera, 272 species ofcorals, mambas, cobras and their relatives), Atractaspididae (14genera, 65 species of Stiletto snakes and mole vipers), and Colubri-

dae (290 genera, almost 1700 species of rear-fanged and ‘‘harm-less” colubrids) (http://www.reptile-database.org). Noteworthy,the front-fanged venom-delivery system appeared three timesindependently in Viperidae, Elapidae, and Atractaspididae [22].

The presence of a venom-secreting oral gland is a shared de-rived character of the advanced (Caenophidia) snakes. All venom-ous squamates, snakes and venomous lizards such as gilamonster, beaded lizard, komodo dragon, etc., share a common ven-omous ancestor [1]. Given the central role that diet has played inthe adaptive radiation of snakes [23], venom thus represents akey adaptation that has played an important role in the diversifica-tion of these animals. Venoms represent the critical innovation inophidian evolution that allowed advanced snakes to transitionfrom a mechanical (constriction) to a chemical (venom) means ofsubduing and digesting prey larger than themselves, and as such,venom proteins have multiple functions including immobilizing,paralyzing, killing and digesting prey. Venoms of snakes in thefamilies Viperidae and Elapidae are produced in paired specializedvenom glands located in the upper jaw, ventral and posterior to theeyes [24], and introduced deeply into prey tissues via elongate,rotatable fangs. Viperids and elapids possess the most widely stud-ied types of animal toxins [9,25,26]. These snake venoms containcomplex mixtures of hundreds of important pharmacologically ac-tive molecules, including low molecular mass organic and inor-ganic components (histamine and other allergens, polyamines,alkaloids), small peptides and proteins [9,27,28]. The biological ef-fects of venoms are complex because different components havedistinct actions and may, in addition, act in concert with other ve-nom molecules. The synergistic action of venom proteins may en-hance their activities or contribute to the spreading of toxins.According to their major toxic effect in an envenomed animal,snake venoms may be conveniently classified as neurotoxic andhaemotoxic. Among the first group are the Elapidae snakes (mam-bas, cobras, and particularly the Australian snakes, which are wellknown to be the most toxic in the world). On the other hand,snakes of the family Viperidae (vipers and pitvipers) containnumerous proteins that typically disrupt the function of the coag-ulation cascade, the haemostatic system and tissue integrity,which are manifested as bleeding and incoagulable blood and localtissue necrosis in human victims of envenoming [9,26,27].

The existence in the same venom of a diversity of proteins of thesame family but differing from each other in their pharmacologicaleffects reflects an accelerated positive Darwinian evolution. Venomtoxins likely evolved from proteins with a normal physiologicalfunction and appear to have been recruited into the venom prote-ome before the diversification of the advanced snakes, at the baseof the Colubroid radiation [1,29,30]. Gene duplication followed byfunctional divergence is the main source of molecular novelty.Gene duplication creates redundancy and allows a gene copy tobe selectively expressed in the venom gland, escaping the pressureof negative selection and evolving a new function through positiveselection and adaptative molecular evolution [31–33]. The occur-rence of multiple isoforms within each major toxin family evi-dences the emergence of paralogous groups of multigene familiesacross taxonomic lineages where gene duplication events occurredprior to their divergence, and suggests an important role for bal-ancing selection in maintaining high levels of functional variationin venom proteins within populations. The mechanism leading tothis mode of selection is unclear but it has been speculated thatit may be related to unpredictability with which a sit-and-waitpredator like a rattlesnake encounters different types of prey, eachof which are most efficiently subdued with different venom pro-teins [34,35]. Thus, to deal with this uncertainty, snakes are re-quired to have a variety of proteins ‘‘available” in their venom atall times to deal with different prey. The selection pressure leadingto high levels of variation in venom genes may parallel the selec-

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tion pressures acting by the birth-and-death model of protein evo-lution [36]. Understanding how toxin genes are regulated and howtoxins mutate in an accelerated fashion may reveal not only themolecular basis for adaptive variations in snake phenotypes [35],but also to learn how to use deadly toxins as therapeutic agents[37].

3. Snakebite envenoming and the challenge of generatingeffective antivenoms

Snakebite envenoming constitutes a highly relevant publichealth issue on a global basis, although it has been systematicallyneglected by health authorities in many parts of the world [11–14].The actual incidence of snakebite envenoming world wide and itsassociated mortality are difficult to estimate, since there are manycountries where this pathology is not appropriately reported, andsince epidemiological data are often fragmentary. Nevertheless, arecently published study estimated at least 421,000 cases ofenvenoming and 20,000 deaths yearly, though these largely hospi-tal-based figures may be as high as 1,841,000 envenomings result-ing in 94,000 deaths [14], and a previous report had estimated atotal of 2.5 million envenomings and over 125,000 deaths [38].Even realizing that the actual impact of this pathology is likely tobe underestimated, because many of snakebite victims seek tradi-tional treatment and may die at home unrecorded, it is evident thatsnakebite envenoming occupies a prominent position as a publichealth issue in many regions of the world. Moreover, an unknownpercentage of snakebite victims end up with permanent physicaldisability, due to local necrosis, and with psychological sequelae,both of which greatly jeopardize the quality of their lives. There-fore, if this pathology is analyzed in terms of DALYs (‘disability-ad-justed life years’) lost, its impact is even greater [39].

Adequate treatment of snakebite envenoming is criticallydependent on the ability of antivenoms to reverse venom-inducedcoagulopathy, hemorrhage, hypotensive shock and other signs ofsystemic envenoming. First generation antivenoms, described over100 years ago [40,41], comprised unpurified serum from animalshyperimmunised with venom. Current antivenoms consist of puri-fied immunoglobulins which have reduced the incidence andseverity of treatment-induced serum-sickness, anaphylactic shock,and other adverse reactions. However, the venom-immunizationprotocols have not changed in over a century and make no attemptto direct the immune response to the most pathogenic venom pro-teins (many venom proteins are not toxic and many low molecularweight venom proteins are highly toxic but weakly immunogenic).Consequently, the dose-efficacy of antivenoms is thought to sufferfrom the presence of redundant antibodies to non-toxic moleculesand a lack of potent neutralizing antibodies to small molecularweight toxins. This in turn results in the need for high volumesof antivenom to effect treatment and a consequent increase inthe risk of serum-sickness and anaphylactic adverse effects. Onthe other hand, the immunization mixtures used for antivenomproduction are specific for every country or region, due to theintraspecific venom variability and to the fact that different snakespecies are responsible for the majority of envenomings in differ-ent countries. The inter- and intraspecies heterogeneity in venomcomposition may account for differences in the clinical symptomsobserved in human victims of envenoming by the same snake spe-cies in different geographical regions [42,43]. Understanding thevariation in antigenic constituents of venoms from snakes of dis-tinct geographic origin represents thus a key challenge towardsthe design of novel, toxin-specific approaches for the immunother-apy of snake bite envenoming. Further, the high levels of intra- andinterspecific variation [42], which reflects local adaptations confer-ring fitness advantages to the snake population, and age-related

(ontogenetic) changes in venom composition (possibly related todiet differences between juvenile and adults of the same species)[35], may also have an impact in the treatment of bite victimsand highlights the need of using pooled venoms as a substratefor antivenom production. Developing toxin-specific antivenomsis critically dependent upon a detailed knowledge of the venomtoxin composition and immunological profiles.

Qualitative individual differences in the composition of the ve-nom of the same ophidian species are of fundamental importancein snakebite pathology and therapeutics, since, as a rule, ophio-toxicosis results from the venom of a single snake. Thus, knowl-edge of inter- and intraspecies variability is necessary for the selec-tion of the regions from which snake specimens have to becollected for the preparation of venom pools. The reference venompool has to be obtained from a relatively large number of speci-mens collected from different geographic regions within the distri-bution range of the species. Intraspecific, geographic, andontogenetic variability in venom composition can be convenientlyanalyzed using a combination of proteomic tools and toxicologicaland biochemical functional assays. Below we describe the proto-cols we have developed for analyzing in detail the compositionof venom proteomes (‘‘snake venomics”) and for investigatingwhich venom proteins bear epitopes recognized by an antivenomand which ones escaped the immunological response of the hyper-immunized animal (‘‘snake antivenomics”).

4. Snake venomics: proteomic tools for studying the proteincomposition of venoms

Venom proteomics has been under investigation since the veryearliest biochemical studies, though the field has began to flourishowing to the recent application of mass spectrometry, coupledwith improvements of databases of venom protein sequences pro-vided by transcriptomic analyses of venom glands, for the charac-terization of toxin proteomes [44–46]. The significance ofproteomic methodologies for biological research has been grantedby the pace of genome sequencing projects. However, organismswith unsequenced genomes, including venomous animals andsnakes in particular, still represent the overwhelming majority ofspecies in the biosphere. We have designed a snake venomics ap-proach (Fig. 1) which starts with the fractionation of the crude ve-nom by reverse-phase HPLC (Fig. 1A), followed by the initialcharacterization of each protein fraction by combination of N-ter-minal sequencing, SDS–PAGE (or 2DE), and mass spectrometricdetermination of the molecular masses and the cysteine (SH andS–S) content [44].

Detection of proteins separated by reverse-phase HPLC is per-formed at 215 nm. Given that the wavelength of absorbance for apeptide bond is 190–230 nm, protein detection at 215 nm allowsan estimation of the relative abundances (expressed as percentageof the total venom proteins) of the different protein families asdetermined from the relationship between the sum of the areasof the reverse-phase chromatographic peaks containing proteinsfrom the same family, and the total area of venom protein peaksin the reverse-phase chromatogram. In a strict sense, and accord-ing to the Lambert–Beer law, the calculated relative amounts cor-respond to the ‘‘% of total peptide bonds in the sample”, which is agood estimate of the % by weight (g/100 g) of a particular venomcomponent. Understanding how venoms work requires quantita-tive data on the occurrence of individual toxins in a given venom.Abundant venom proteins may perform generic killing and diges-tive functions that are not prey specific whereas low abundanceproteins may be more plastic either in evolutionary or ecologicaltimescales. Mutations provide the ground on which natural selec-tion operates to create functional innovations. A subset of low

Fig. 1. Snake venomics. Schematic representation of the steps typically followed in a snake venomics project. (A) Reverse-phase chromatographic separation of the venomproteins; (B and C) SDS–PAGE of the RP-HPLC isolated proteins and determination of the molecular masses of the proteins isolated in panel A; (C) in-gel tryptic digestion ofprotein bands; (D) amino acid sequence determination by nanospray-ionization CID–MS/MS of selected tryptic peptide ions; and (E) integration of results into toxin familycomposition pies. For more details consult [44].

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abundance proteins may serve to ‘‘customize” an individual venomto feeding on particular prey or may represent orphan moleculesevolving under neutral selection ‘‘in search for a function”. Hence,whereas abundant proteins are the primary targets for immuno-therapy, minor components may represent scaffolds for biotechno-logical developments.

Protein fractions showing single electrophoretic band, molec-ular mass, and N-terminal sequence (Fig. 1B) can be straightfor-wardly assigned by BLAST analysis to a known protein family.Thus, although few toxins from any given species are annotatedin the public-accessible databases, representative members ofmost snake venom toxin families are present among the�1100 viperid toxin protein sequences belonging to 157 speciesdeposited to date in the SwissProt/TrEMBL database (Knowl-edgebase Release 56.5 of November 2008; http://us.expasy.org/sprot/). On the other hand, protein fractions showing heteroge-neous or blocked N-termini are analyzed by SDS–PAGE and thebands of interest subjected to automated reduction, carbami-domethylation, and in-gel tryptic digestion (Fig. 1C). The result-ing tryptic peptides are then analyzed by MALDI-TOF massfingerprinting followed by amino acid sequence determinationof selected doubly- and triply-charged peptide ions by colli-sion-induced dissociation tandem mass spectrometry. Exceptfor a few proteins, the peptide mass fingerprinting approachalone is unable to identify any protein in the databases. In addi-

tion, as expected from the rapid amino acid sequence divergenceof venom proteins evolving under accelerated evolution [9,47],with a few exceptions, the product ion spectra do not matchany known protein using the ProteinProspector (http://prospec-tor.ucsf.edu) or the MASCOT (http://www.matrixscience.com)search programs against the UniProtKB/Swiss-Prot entries fromtaxon Serpentes (http://ca.expasy.org/cgi-bin/get-entries?view=full&KW=Toxin&OC=Serpentes). Furthermore, it is not too unu-sual that a product ion spectrum matched with high MASCOTscore to a particular peptide sequence corresponds actually toa tryptic peptide of a homolog snake toxin containing one ormore nearly isobaric amino acid substitutions. Hence, it is neces-sary to revise manually all the CID–MS/MS spectra (to confirmthe assigned peptide sequence or for performing de novosequencing), and submit the deduced peptide ion sequences toBLAST similarity searches. Although the lack of any completesnake genome sequence is a serious drawback for the identifica-tion of venom proteins, high-quality MS/MS peptide ion frag-mentation spectra usually yield sufficient amino acid sequenceinformation derived from almost complete series of sequence-specific b- and/or y-ions (Fig. 1D) to unambiguously identify ahomolog protein in the current databases. The combined venom-ics strategy allows us to assign unambiguously all the isolatedvenom toxins representing over 0.05% of the total venom pro-teins to known protein families (Fig. 1E, Table 1).

Table 1Overview of the relative occurrence of proteins (in percentage of the total HPLC-separated proteins) of the toxin families in the venoms of Sistrurus catenatus catenatus (SCC),Sistrurus catenatus tergeminus (SCT), and Sistrurus catenatus edwardsii (SCE) from USA [34]; Sistrurus miliarius barbouri (SMB) from USA [52]; the Tunisian snakes Cerastes cerastescerastes (CCC), Cerastes vipera (CV) and Macrovipera lebetina transmediterranea (MLT) [48]; African Bitis arietans (BA) [50]; Bitis gabonica gabonica (BGG) [49]; Bitis gabonicarhinoceros (BGR), Bitis nasicornis (BN), and Bitis caudalis (BC) [56]; Echis ocellatus (EO) [51]; Lachesis muta (LM) [53]; Crotalus atrox (CA), and Agkistrodon contortrix contortrix (ACC)from USA (Calvete et al., unpublished); Armenian vipers Macrovipera lebetina obtusa (Mlo), and Vipera raddei (Vr) [54]; Atropoides picadoi (Api), and Atropoides mexicanus (Amex)[57] from Costa Rica; Bothrops asper (Bas) from the Caribbean (C) and the Pacific versants of Costa Rica [60]; Lesser Antillean pitvipers Bothrops caribbaeus (Bcar) (Santa Lucía), andBothrops lanceolatus (Blan) (Martinique) [58]; Brazilian Bothrops fonsecai (Bfon), and Bothrops cotiara (Bco) [59]; Bothriechis lateralis (Bolat), and Bothriechis schlegelii (Bosch) [55]from Costa Rica; and Lachesis stenophrys (Lste) [53] from Costa Rica. Major toxin families in each venom are highlighted in boldface.

Venom

SCC SCT SCE SMB CCC CV MET BA BGG BGR BN BC EO LM CA ACC

Protein family % of total venom proteinsDisintegrins

– Long – – – – – – – 17.8 – – – – – – – –– Medium 2.5 4.2 0.9 7.7 – – – – – – – – – – 6.5 –– Dimeric – – – – 8.1 <1 6.0 – 3.4 8.5 3.5 – 4.2 – – 1.5– Short – – – – – – <1 – – – – – 2.6 – – –Myotoxin 0.4 <0.1 – – – – – – – – – – – – – –C-type BPP/NP – – <0.1 <0.1 – – <1 – 2.8 0.3 – – – 14.7 2.1 <0.1Kunitz-type inhibitor – – <0.1 <0.1 – – – 4.2 3.0 7.5 – 3.2 – – – –Cystatin – – – – – – – 1.7 9.8 5.3 4.2 – – – – –DC-fragment <0.1 <0.1 <0.1 1.3 – – 1.0 – 0.5 0.6 <0.1 – 1.7 – – <0.1NGF/svVEGF <0.1 <0.1 <0.1 <0.1 – – 2.1 – 1.0 – – – – – – –Ohanin-like – – – – – – – – – – – – – – – <0.1CRISP 0.8 1.3 10.7 2.9 – – – – 2.0 1.2 1.3 1.2 1.5 1.8 4.2 –PLA2 29.9 31.6 13.7 32.5 20.0 21.1 4.0 4.3 11.4 4.8 20.1 59.8 12.6 8.7 16.3 18.5Serine proteinase 18.2 20.4 24.4 17.1 9.1 20.0 9.2 19.5 26.4 23.9 21.9 15.1 2.0 31.2 10.1 13.8C-type lectin-like <0.1 <0.1 <0.1 <0.1 24.0 0.9 10.1 13.2 14.3 14.1 4.2 4.9 7.0 8.1 1.6 –L-amino acid oxidase 4.2 1.6 2.5 2.1 12.0 9.0 – – 1.3 2.2 3.2 1.7 1.4 2.7 8.0 2.2Zn2+-metalloproteinase 43.8 40.6 48.6 36.1 37.0 48.1 67.1 38.5 22.9 30.8 40.9 11.5 67.0 31.9 51.1 63.6

Mlo Vr Api Amex Bas(C) Bas(P) Bear Blan Bco Bfon Bolat Bosch Lste

– Long – – – – – – 1.5 – – – – – –– Medium – – <0.1 2.5 2.1 1.4 – – 1.2 4.4 – – –– Dimeric 8.5 9.7 – – – – – – – – – – –– Short 2.8 – – – – – – – – – – – –Myotoxin – – – – – – – – – – – – –C-type BPP/NP 5.3 6.0 1.8 8.6 – – – – – – 11.1 13.4 14.7Kunitz-type inhibitor – 0.1 – – – – – – – – – – –Kazal-type inhibitor – – – – – – – – – – – 8.3 –Cystatin – – – – – – – – – – – – –DC-fragment 1.7 0.1 <0.1 <0.1 <0.1 <0.1 <0.2 – 0.5 0.7 – – –NGF/svVEGF – 2.4 <0.1 <0.1 – – – – 3.3 3.9 0.5 – 0.4Ohanin-like – – – – – – – – – – – – –3-Finger toxin – – – <0.1 – – – – – – – – –CRISP 2.6 7.4 4.8 1.9 0.1 0.1 2.6 – 3.6 2.4 6.5 2.1 –PLA2 14.6 23.8 9.5 36.5 28.8 45.1 12.8 8.6 – 30.1 8.7 43.8 12.3Serine proteinase 14.9 8.4 13.5 22.0 18.2 4.4 4.7 14.4 14.4 4.1 11.3 5.8 25.6C-type lectin-like 14.8 9.6 1.8 1.3 0.5 0.5 – <0.1 <0.1 9.8 0.9 – 3.6L-amino acid oxidase 1.7 0.2 2.2 9.1 9.2 4.6 8.4 2.8 3.8 1.9 6.1 8.9 5.3Zn2+-metalloproteinase 32.1 31.6 66.4 18.2 41.0 44.0 68.6 74.3 73.1 42.5 55.1 17.7 38.2

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The long-term goal of our Snake Venomics project is a detailedanalysis of all viperid venomes. To date, this methodology has beenapplied to explore the venom proteomes (Table 1) of the medi-cally-relevant Tunisian vipers Cerastes cerastes, Cerastes vipera,Macrovipera lebetina [48]; African Bitis gabonica [49], Bitis arietans[50], and Echis ocellatus [51]; North American Sistrurus miliariusbarbouri [52] and Sistrurus catenatus subspecies [34]; the Southand Central American Lachesis sp. [53]; the Armenian mountain vi-pers Vipera raddei and Macrovipera lebetina obtusa [54]; the arbo-real Neotropical pitvipers Bothriechis lateralis and Bothriechisschlegelii [55]; to infer phylogenetic alliances within genus Bitis[56] and Sistrurus [34]; to rationalize the envenomation profilesof Atropoides [57] and Bothrops [58] species; to define venom-associated taxonomic markers [59]; to establish the molecular ba-sis of geographic, individual, and ontogenetic venom variations inBothrops asper [60]; and to contribute to reconstruct the naturalhistory and cladogenesis of Bothrops colombiensis [61], a medicallyimportant pitviper of the Bothrops atrox-asper complex endemic toVenezuela. A few other laboratories have reported qualitative pro-teomic studies on several venoms, including those from the Euro-

pean vipers, Vipera ammodytes ammodytes and Vipera ammodytesmeridionalis [62], Asian Daboia russelli siamensis [63], AmazonianBothrops atrox [64], and Brazilian Bothrops insularis [65].

Qualitative discrepancies in the compositional agreement of ve-nom acquired using proteomic and transcriptomic approacheshave been reported, i.e. for Lachesis muta [53], Bitis gabonica gabo-nica [49], and Echis ocellatus [51]. Venom composition may beinfluenced by a number of different factors, most notably the po-tential for genetic drift on account of (i) gender based variationand (ii) ontogenetic variations. Conversely, the occurrence ofmRNAs that are abundant and readily identifiable in the transcrip-tome, yet apparently absent from the venom proteome may also beexplained by a number of factors. Firstly, these messengers mayexhibit transient, individual or a temporal expression patterns overthe life time of the snake. Alternatively, mRNAs whose predictedpolypeptides were not found to be secreted in venom may repre-sent very low abundance toxins that play a hitherto unrecognisedphysiological function in the venom gland, or may simply repre-sent a hidden repertoire of non-translated orphan molecules whichmay eventually become functional for the adaptation of snakes to

J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743 1741

changing ecological niches and prey habits. These data suggest thatthe final composition of venom is influenced by transcriptional andpost-translational mechanisms that may be more complex thanpreviously appreciated. Nevertheless, a major conclusion drawnfrom snake venomics and transcriptomic analyses of viperid ve-nom glands is that despite venoms being complex mixture of pro-teins, venom proteins belong to only a few major families,including enzymes (serine proteinases, Zn2+-metalloproteases,L-amino acid oxidase, PLA2) and proteins without enzymatic activ-ity (disintegrins, C-type lectins, natriuretic peptides, ohanin, myo-toxins, CRISP toxins, nerve and vascular endothelium growthfactors, cystatin and Kunitz-type protease inhibitors) [16,44](Table 1), though different venoms exhibit a distinct toxin familydistributional profile (Table 1). This situation may reflect the factthat toxins likely evolved from a restricted set of protein families,and opens the door for a rationale design of intra-, but also inter-,species family-specific neutralizing antibodies.

Several antivenoms are produced in Latin America using differ-ent venoms in the immunization schemes [66]. Each of these anti-venoms is effective in variable degree against envenomations bysnake venoms not included in the immunization protocol, demon-strating the high degree of immunological cross-reactivity be-tween Central and South American crotaline snake venoms. Apractical consequence of this fortunate circumstance is the possi-bility of using these heterologous antivenoms to circumvent therestricted availability of species-specific antivenoms in some re-gions. However, before testing in clinical trials, antivenoms needto be evaluated experimentally by assessing their neutralizing abil-ity against the most relevant toxic and enzymatic activities ofsnake venoms. To this end, we have developed a simple proteomicprotocol for investigating the immunoreactivity, and thus the po-tential therapeutic usefulness, of antivenoms towards homologousand heterologous venoms.

5. Antivenomics: proteomic tools for studying theimmunological profile of venoms

We have coined the term ‘‘antivenomics” for the identificationof venom proteins bearing epitopes recognized by an antivenomusing proteomic techniques [55,58,61]. Antivenomics is based onthe immunodepletion of toxins upon incubation of whole venom

Fig. 2. Antivenomics. Panel A displays the reverse-phase HPLC separation of the venomnot immunodepleted by the Costa Rican polyvalent (Crotalinae) ICP antivenom. These Nmolecule (15). The orange arrow points to the major snake venom metalloproteinase (Sresults fully explain the incapacity of the ICP antivenom to neutralize the neurotoxicity olarge content of the heterodimeric PLA2 Crotoxin (Panel B). Similarly, antivenoms produAmerican Crotalus simus but are ineffective at neutralizing the hemorrhagic activity of valmost absence of hemorrhagic SVMPs (fractions 17–18 in reverse-phase chromatogramantivenomic findings suggest that an antivenom raised against a mixture of C. simus aneffects of Middle and South American Crotalus species.

with antivenom followed by the addition of a secondary antibody.Antigen-antibody complexes immunodepleted from the reactionmixture contain the toxins against which antibodies in the anti-venom are directed. By contrast, venom components that remainin the supernatant are those which failed to raise antibodies inthe antivenom, or which triggered the production of low-affinityantibodies. These components can be easily identified by compar-ison of reverse-phase HPLC separation of the non-precipitated frac-tion with the HPLC pattern of the whole venom previouslycharacterized by venomics. According to their immunoreactivitytowards antivenoms, toxins may be conveniently classified as: C-toxins, completely immunodepletable toxins; P-toxins, partlyimmunodepleted toxins; and N-toxins, non-inmunodepleted pro-teins (Fig. 2). Assuming a link between the in vitro toxin immun-odepletion capability of an antivenom and its in vivo neutralizingactivity towards the same toxin molecules, improved immuniza-tion protocols should make use of mixtures of immunogens to gen-erate high-affinity antibodies against class P and class N toxins. Onthe other hand, our antivenomics approach is simple and easy toimplement in any protein chemistry laboratory, and may thus rep-resent another useful protocol for investigating the immunoreac-tivity, and thus the potential therapeutic usefulness, ofantivenoms towards homologous and heterologous venoms [16].

The deficit (‘crisis’) of antivenom supply in some regions of theworld can be addressed to a certain extent by optimizing the use ofexisting antivenoms and through the design of novel immuniza-tion mixtures for producing broad-range polyspecific antivenoms.Proteomics-based immunochemical analysis (antivenomics) pro-vides relevant information for outlining which venom mixturescross-react with the most important components in medically-relevant venoms from a particular region. This type of approachmay set the basis for the development of antivenoms on animmunologically sound basis. The potential value of antivenomics,together with preclinical neutralization tests, in assessing antiven-om cross-reactivity is clearly illustrated by the following examples.A highly effective antivenom (Sanofi-Pasteur ‘Bothrofav�’) hasbeen developed for the treatment of envenomings by Bothropslanceolatus, endemic to the Lesser Antillean island of Martinique.It exhibits an excellent preclinical profile of neutralization [67]and its timely administration prevents the development of themost serious effects of envenoming, including thrombosis

proteins from Crotalus simus (from Costa Rica). Red arrows indicate venom proteins-toxins were identified as disintegrins (4–8), crotoxin subunits (9–12), and a PLA2

VMP), which was partly immunodepleted by the ICP antivenom. The antivenomicsf the venom of South American Crotalus durissus subspecies, which is associated to aced in South America against C.d. terrificus venom neutralize the lethality of Centralenom from genus Crotalus. Such neutralizing profile is also fully explained by the

shown in B) in the C.d. terrificus venom used in the immunization protocol. Thed C.d. terrificus may effectively neutralize both the hemorrhagic and the neurotoxic

1742 J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743

[68,69]. However, the restricted availability of the antivenom inthe neighboring island of Saint Lucia and in zoos and herpetariumsis a matter of concern. Gutiérrez and colleagues have performeddetailed proteomic studies of the venoms of Bothrops caribbaeusand B. lanceolatus and have evaluated the immunoreactivity of aCrotalinae polyvalent antivenom produced in Costa Rica (by immu-nization of horses with a mixture of equal amounts of the venomsof B. asper, Crotalus simus, and Lachesis stenophrys) towards the ven-oms of B. caribbaeus and B. lanceolatus [58]. This study showed thatthe antivenom immunodepleted �80% of the proteins from both B.caribbaeus and B. lanceolatus venoms, and was effective in neutral-izing the lethal, hemorrhagic, PLA2 and proteolytic activities of thetwo venoms.

Calvete and co-workers from Venezuela and Costa Rica havecompared the venom proteomes of B. colombiensis, B. asper, andB. atrox using venomics and antivenomics approaches [61]. Thisstudy did not support the suggested synonymy between B. colom-biensis and B. atrox. On the other hand, the closest homologs to B.colombiensis venom proteins appeared to be toxins from B. asper.A rough estimation of the similarity between the venoms of B.colombiensis and B. asper indicated that these species share approx-imately 65–70% of their venom proteomes. The close kinship of B.colombiensis and B. asper points at the ancestor of B. colombiensis asthe founding Central American B. asper ancestor. This finding maybe relevant for reconstructing the natural history and cladogenesisof Bothrops. Further, the virtually indistinguishable immunologicalcross-reactivity of a Venezuelan ABC antiserum (raised against amixture of B. colombiensis and Crotalus durissus cumanensis ven-oms) and the Costa Rican ICP polyvalent antivenom (generatedagainst a mixture of B. asper, C. simus, and L. stenophrys venoms) to-wards the venoms of B. colombiensis and B. asper, supports thisview and suggests the possibility of indistinctly using these anti-venoms for the management of snakebites by any of these Bothr-ops species.

An antivenom manufactured in Costa Rica using venom of theCentral American rattlesnake (Crotalus simus simus) population, isineffective for neutralizing the neurotoxic activity of South Amer-ican Crotalus durissus terrificus. Similarly, antivenoms produced inSouth America against C.d. terrificus venom neutralize the neuro-toxicity of Central American venoms but are ineffective at neutral-izing the hemorrhagic activity of venoms from genus Crotalus [70].Such neutralizing profile is fully explained by the proteomic andantivenomic characterization of Crotalus (simus and durissus) ven-oms described in Fig. 2. The case of Crotalus illustrates how theknowledge of venom variations and their geographical distributioncan lead to securing venoms with a more defined composition forpreparing venom mixtures for the generation of antivenoms effec-tive against the venoms of rattlesnakes from Central and SouthAmerica.

Acknowledgments

Research in Costa Rica has been supported by Vicerrectoría deInvestigación, Universidad de Costa Rica (Project 741-A8-521),CRUSA-CSIC (Project 2007CR0004), CYTED (Project 206AC0281)and CONARE (Fondos del Sistema). Snake venomics and antive-nomic studies at the Spanish IBV have been financed by grantsBFU2004-01432 and BFU2007-61563 from the Ministerios de Edu-cación y Ciencia and Ciencia e Innovación, Madrid. Travelling be-tween Spain and Costa Rica was financed by Acciones Integradas2006CR0010 between CSIC and the University of Costa Rica (UCR).

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