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FEBS Letters 583 (2009) 1736–1743
journal homepage: www.FEBSLetters.org
Minireview 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, Spain b Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica
article info abstract
Article history: Venoms comprise mixtures of peptides and proteins tailored by Natural Selection to act on vital sys- Received 17 February 2009 tems of the prey or victim. Here we review our proteomic protocols for uncoiling the composition, Revised 12 March 2009 immunological profile, and evolution of snake venoms. Our long-term goal is to gain a deep insight Accepted 13 March 2009 of all viperid venom proteomes. Knowledge of the inter- and intraspecies ontogenetic, individual, Available online 20 March 2009 and geographic venom variability has applied importance for the design of immunization protocols Edited by Miguel De la Rosa aimed at producing more effective polyspecific antivenoms. A practical consequence of assessing the cross-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 specificity Keywords: Proteomics of snake venom makes toxins valuable scaffolds for drug development. Snake venomics Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Snake venom Antivenom Antivenomics
1. The Ying and Yang of animal venoms The medical uses of scorpion and snake venoms are well docu- mented in folk remedies, and in Western and Chinese traditional Venomous organisms are widely spread throughout the animal medicine [4,5]. However, extensive investigations on venom com- kingdom, comprising more than 100,000 species distributed pounds as natural leads for the generation of pharmaceutical prod- among all major phyla, such as chordates (reptiles, fishes, amphib- ucts have only been performed in the last decades, after a ians, mammals), echinoderms (starfishes, sea urchins), molluscs bradykinin-potentiating peptide isolated from the venom of the (cone snails, octopi), annelids (leeches), nemertines, arthropods Brazilian viper Bothrops jararaca was developed in the 1950s into (arachnids, insects, myriapods) and cnidarians (sea anemones, jel- the first commercial angiotensin I-converting enzyme (ACE)-inhib- lyfish, corals). In any habitat there is a competition for resources, iting drug, captoprilÒ, for the treatment of renovascular hyperten- and every ecosystem on Earth supporting life contains poisonous sion [6,7]. The latest example of development of a toxin into an or venomous organisms. One of the most fascinating techniques approved drug by the US Food and Drug Administration (FDA) of capturing prey or defending oneself is the use of poisons or ven- (December 2004) is ziconotide (PrialtÒ), a synthetic non-opioid, oms. Venom represents an adaptive trait and an example of con- non-NSAID, non-local anesthetic drug originating from the cone vergent evolution [1–3]. Venoms are deadly cocktails, each snail Conus magus peptide x-conotoxin M-VII-A, an N-type calcium comprising unique mixtures of peptides and proteins naturally tai- channel blocker. Discovered in the early 1980s [8], zicotonide is a lored by Natural Selection to act on vital systems of the prey or vic- rare example of a molecule used unaltered from a creature’s chem- tim. Venom toxins disturb the activity of critical enzymes, istry. PrialtÒ is used for the amelioration of chronic untreatable receptors, or ion channels, thus disarranging the central and pain, and due to the profound side effects or lack of efficacy when peripheral nervous systems, the cardiovascular and the neuromus- delivered through more common routes, such as orally or intrave- cular systems, blood coagulation and homeostasis. On the other nously, PrialtÒ must be administered directly into the spine. hand, due to their high degree of target specificity, venom toxins Venoms represent a huge and essentially unexplored reservoir have been increasingly used as pharmacological tools and as proto- of bioactive components that may cure disease conditions which types for drug development. The medicinal value of venoms has do not respond to currently available therapies. The great pharma- been known from ancient times. The snake is a symbol of medicine cological cornucopia accumulated by Nature over evolution has re- due to its association with Asclepius, the Greek god of medicine. sulted in true combinatorial libraries of hundreds of thousands of potentially active and useful molecules synthesised in the venoms * Corresponding author. Fax: +34 96 369 0800. of the 700 or so known species of Conus, the roughly 725 species of E-mail address: [email protected] (J.J. Calvete).
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.03.029 J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743 1737 venomous snakes, the 1500 species of scorpions, or the ca. 37,000 dae (290 genera, almost 1700 species of rear-fanged and ‘‘harm- known species of spiders, just to cite only a few examples of ven- less” colubrids) (http://www.reptile-database.org). Noteworthy, omous animals. The accelerated Darwinian evolution acting to pro- the front-fanged venom-delivery system appeared three times mote high levels of variation in venom proteins may be part of a independently in Viperidae, Elapidae, and Atractaspididae [22]. predator–prey arms race that allows the predator to adapt to a The presence of a venom-secreting oral gland is a shared de- variety of different prey, each of which are most efficiently sub- rived character of the advanced (Caenophidia) snakes. All venom- dued with a different venom formulation. In addition to under- ous squamates, snakes and venomous lizards such as gila standing how venoms evolve, a major aim of venomic projects is monster, beaded lizard, komodo dragon, etc., share a common ven- to gain a deeper insight into the spectrum of medically important omous ancestor [1]. Given the central role that diet has played in toxins in venoms to uncover clues in order to solve the riddle of the the adaptive radiation of snakes [23], venom thus represents a medical effectiveness of venoms and to learn how to convert key adaptation that has played an important role in the diversifica- deadly toxins into lifesaving drugs [9,10]. On the other hand, tion of these animals. Venoms represent the critical innovation in envenomation constitutes a highly relevant public health issue ophidian evolution that allowed advanced snakes to transition on a global basis, as there are venomous organisms in every conti- from a mechanical (constriction) to a chemical (venom) means of nent and almost every country. However, venomous animals are subduing and digesting prey larger than themselves, and as such, particularly abundant in tropical regions, which represent the venom proteins have multiple functions including immobilizing, kitchen of evolution. Arthropod stings inflicted by bees, wasps, paralyzing, killing and digesting prey. Venoms of snakes in the ants, spiders, scorpions, and – to a lesser extent – millipedes and families Viperidae and Elapidae are produced in paired specialized centipedes, constitute the most common cause of envenoming by venom glands located in the upper jaw, ventral and posterior to the animals, although around 80% of deaths by envenomation world- eyes [24], and introduced deeply into prey tissues via elongate, wide are caused by snakebite, followed by scorpion stings, which rotatable fangs. Viperids and elapids possess the most widely stud- cause 15%. Envenoming constitutes a highly relevant public health ied types of animal toxins [9,25,26]. These snake venoms contain issue on a global basis, although it has been systematically ne- complex mixtures of hundreds of important pharmacologically ac- glected by health authorities in many parts of the world [11–15]. tive molecules, including low molecular mass organic and inor- Being a pathology mainly affecting young agricultural workers liv- ganic components (histamine and other allergens, polyamines, ing in villages far from health care centers in low-income countries alkaloids), small peptides and proteins [9,27,28]. The biological ef- of Africa, Asia and Latin America, it must be regarded as a ‘ne- fects of venoms are complex because different components have glected tropical pathology’ [11]. Adequate treatment of envenom- distinct actions and may, in addition, act in concert with other ve- ing is critically dependent on the ability of antivenoms to nom molecules. The synergistic action of venom proteins may en- neutralize the lethal toxins reversing thereby the signs of enven- hance their activities or contribute to the spreading of toxins. oming. A long-term research goal of venomics of applied impor- According to their major toxic effect in an envenomed animal, tance for improving current antivenom therapy is to understand snake venoms may be conveniently classified as neurotoxic and the molecular mechanisms and evolutionary forces that underlie haemotoxic. Among the first group are the Elapidae snakes (mam- venom variation. Thus, a robust knowledge of venom composition bas, cobras, and particularly the Australian snakes, which are well and of the onset of ontogenetic, individual, and geographic venom known to be the most toxic in the world). On the other hand, variability may have an impact in the treatment of bite victims and snakes of the family Viperidae (vipers and pitvipers) contain in the selection of specimens for the generation of improved anti- numerous proteins that typically disrupt the function of the coag- dotes [16] (see below). ulation cascade, the haemostatic system and tissue integrity, which are manifested as bleeding and incoagulable blood and local tissue necrosis in human victims of envenoming [9,26,27]. 2. The evolution of the advanced snakes and their venoms The existence in the same venom of a diversity of proteins of the same family but differing from each other in their pharmacological The suborder of snakes (Serpentes) of the reptilian order Squa- effects reflects an accelerated positive Darwinian evolution. Venom mata, named for their scaly skin, includes about 3000 extant spe- toxins likely evolved from proteins with a normal physiological cies placed in approximately 400 genera and 18 families (http:// function and appear to have been recruited into the venom prote- www.reptile-database.org). The timing of major events in snake ome before the diversification of the advanced snakes, at the base evolution is not well understood, however, owing in part to a rel- of the Colubroid radiation [1,29,30]. Gene duplication followed by atively patchy and incomplete fossil record [17,18]. Nevertheless, functional divergence is the main source of molecular novelty. after more than 100 years of research, the most generalized phylo- Gene duplication creates redundancy and allows a gene copy to genetic view is that the group evolved from a family of terrestrial be selectively expressed in the venom gland, escaping the pressure lizards during the time of the dinosaurs in the Jurassic period, of negative selection and evolving a new function through positive about 200 million years (Myr) ago [19]. After the end of the non- selection and adaptative molecular evolution [31–33]. The occur- avian dinosaurs reign, around the Cretaceous-Tertiary boundary rence of multiple isoforms within each major toxin family evi- 65 Myr ago [20], the boids (the ancestors of boas, pythons and ana- dences the emergence of paralogous groups of multigene families condas) were the dominant snake family on Earth. Within the across taxonomic lineages where gene duplication events occurred Cenozoic era that followed, advanced snakes (colubrids) arose as prior to their divergence, and suggests an important role for bal- long ago as in the Oligocene epoch (35–25 Myr). Colubrids, the ancing selection in maintaining high levels of functional variation family which we regard today as typical snakes, remained a small in venom proteins within populations. The mechanism leading to taxon until the tectonic plates drifted apart from the equator and this mode of selection is unclear but it has been speculated that the cool climate pushed boids to disappear from many ecological it may be related to unpredictability with which a sit-and-wait niches. Colubrids quickly colonized these empty habitats and this predator like a rattlesnake encounters different types of prey, each family today comprises over two-thirds of all the living snake spe- of which are most efficiently subdued with different venom pro- cies [21]. Colubroidea encompasses Viperidae (30 genera, 230 spe- teins [34,35]. Thus, to deal with this uncertainty, snakes are re- cies of vipers and pitvipers), Elapidae (63 genera, 272 species of quired to have a variety of proteins ‘‘available” in their venom at corals, mambas, cobras and their relatives), Atractaspididae (14 all times to deal with different prey. The selection pressure leading genera, 65 species of Stiletto snakes and mole vipers), and Colubri- to high levels of variation in venom genes may parallel the selec- 1738 J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743 tion pressures acting by the birth-and-death model of protein evo- (ontogenetic) changes in venom composition (possibly related to lution [36]. Understanding how toxin genes are regulated and how diet differences between juvenile and adults of the same species) toxins mutate in an accelerated fashion may reveal not only the [35], may also have an impact in the treatment of bite victims molecular basis for adaptive variations in snake phenotypes [35], and highlights the need of using pooled venoms as a substrate but also to learn how to use deadly toxins as therapeutic agents for antivenom production. Developing toxin-specific antivenoms [37]. is critically dependent upon a detailed knowledge of the venom toxin composition and immunological profiles. Qualitative individual differences in the composition of the ve- 3. Snakebite envenoming and the challenge of generating nom of the same ophidian species are of fundamental importance effective antivenoms in snakebite pathology and therapeutics, since, as a rule, ophio- toxicosis results from the venom of a single snake. Thus, knowl- Snakebite envenoming constitutes a highly relevant public edge of inter- and intraspecies variability is necessary for the selec- health issue on a global basis, although it has been systematically tion of the regions from which snake specimens have to be neglected by health authorities in many parts of the world [11–14]. collected for the preparation of venom pools. The reference venom The actual incidence of snakebite envenoming world wide and its pool has to be obtained from a relatively large number of speci- associated mortality are difficult to estimate, since there are many mens collected from different geographic regions within the distri- countries where this pathology is not appropriately reported, and bution range of the species. Intraspecific, geographic, and since epidemiological data are often fragmentary. Nevertheless, a ontogenetic variability in venom composition can be conveniently recently published study estimated at least 421,000 cases of analyzed using a combination of proteomic tools and toxicological envenoming and 20,000 deaths yearly, though these largely hospi- and biochemical functional assays. Below we describe the proto- tal-based figures may be as high as 1,841,000 envenomings result- cols we have developed for analyzing in detail the composition ing in 94,000 deaths [14], and a previous report had estimated a of venom proteomes (‘‘snake venomics”) and for investigating total of 2.5 million envenomings and over 125,000 deaths [38]. which venom proteins bear epitopes recognized by an antivenom Even realizing that the actual impact of this pathology is likely to and which ones escaped the immunological response of the hyper- be underestimated, because many of snakebite victims seek tradi- immunized animal (‘‘snake antivenomics”). tional treatment and may die at home unrecorded, it is evident that snakebite envenoming occupies a prominent position as a public health issue in many regions of the world. Moreover, an unknown 4. Snake venomics: proteomic tools for studying the protein percentage of snakebite victims end up with permanent physical composition of venoms disability, due to local necrosis, and with psychological sequelae, both of which greatly jeopardize the quality of their lives. There- Venom proteomics has been under investigation since the very fore, if this pathology is analyzed in terms of DALYs (‘disability-ad- earliest biochemical studies, though the field has began to flourish justed life years’) lost, its impact is even greater [39]. owing to the recent application of mass spectrometry, coupled Adequate treatment of snakebite envenoming is critically with improvements of databases of venom protein sequences pro- dependent on the ability of antivenoms to reverse venom-induced vided by transcriptomic analyses of venom glands, for the charac- coagulopathy, hemorrhage, hypotensive shock and other signs of terization of toxin proteomes [44–46]. The significance of systemic envenoming. First generation antivenoms, described over proteomic methodologies for biological research has been granted 100 years ago [40,41], comprised unpurified serum from animals by the pace of genome sequencing projects. However, organisms hyperimmunised with venom. Current antivenoms consist of puri- with unsequenced genomes, including venomous animals and fied immunoglobulins which have reduced the incidence and snakes in particular, still represent the overwhelming majority of severity of treatment-induced serum-sickness, anaphylactic shock, species in the biosphere. We have designed a snake venomics ap- and other adverse reactions. However, the venom-immunization proach (Fig. 1) which starts with the fractionation of the crude ve- protocols have not changed in over a century and make no attempt nom by reverse-phase HPLC (Fig. 1A), followed by the initial to direct the immune response to the most pathogenic venom pro- characterization of each protein fraction by combination of N-ter- teins (many venom proteins are not toxic and many low molecular minal sequencing, SDS–PAGE (or 2DE), and mass spectrometric weight venom proteins are highly toxic but weakly immunogenic). determination of the molecular masses and the cysteine (SH and Consequently, the dose-efficacy of antivenoms is thought to suffer S–S) content [44]. from the presence of redundant antibodies to non-toxic molecules Detection of proteins separated by reverse-phase HPLC is per- and a lack of potent neutralizing antibodies to small molecular formed at 215 nm. Given that the wavelength of absorbance for a weight toxins. This in turn results in the need for high volumes peptide bond is 190–230 nm, protein detection at 215 nm allows of antivenom to effect treatment and a consequent increase in an estimation of the relative abundances (expressed as percentage the risk of serum-sickness and anaphylactic adverse effects. On of the total venom proteins) of the different protein families as the other hand, the immunization mixtures used for antivenom determined from the relationship between the sum of the areas production are specific for every country or region, due to the of the reverse-phase chromatographic peaks containing proteins intraspecific venom variability and to the fact that different snake from the same family, and the total area of venom protein peaks species are responsible for the majority of envenomings in differ- in the reverse-phase chromatogram. In a strict sense, and accord- ent countries. The inter- and intraspecies heterogeneity in venom ing to the Lambert–Beer law, the calculated relative amounts cor- composition may account for differences in the clinical symptoms respond to the ‘‘% of total peptide bonds in the sample”, which is a observed in human victims of envenoming by the same snake spe- good estimate of the % by weight (g/100 g) of a particular venom cies in different geographical regions [42,43]. Understanding the component. Understanding how venoms work requires quantita- variation in antigenic constituents of venoms from snakes of dis- tive data on the occurrence of individual toxins in a given venom. tinct geographic origin represents thus a key challenge towards Abundant venom proteins may perform generic killing and diges- the design of novel, toxin-specific approaches for the immunother- tive functions that are not prey specific whereas low abundance apy of snake bite envenoming. Further, the high levels of intra- and proteins may be more plastic either in evolutionary or ecological interspecific variation [42], which reflects local adaptations confer- timescales. Mutations provide the ground on which natural selec- ring fitness advantages to the snake population, and age-related tion operates to create functional innovations. A subset of low J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743 1739
Fig. 1. Snake venomics. Schematic representation of the steps typically followed in a snake venomics project. (A) Reverse-phase chromatographic separation of the venom proteins; (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 of protein bands; (D) amino acid sequence determination by nanospray-ionization CID–MS/MS of selected tryptic peptide ions; and (E) integration of results into toxin family composition pies. For more details consult [44]. abundance proteins may serve to ‘‘customize” an individual venom tion, as expected from the rapid amino acid sequence divergence to feeding on particular prey or may represent orphan molecules of venom proteins evolving under accelerated evolution [9,47], evolving under neutral selection ‘‘in search for a function”. Hence, with a few exceptions, the product ion spectra do not match whereas abundant proteins are the primary targets for immuno- any known protein using the ProteinProspector (http://prospec- therapy, minor components may represent scaffolds for biotechno- tor.ucsf.edu) or the MASCOT (http://www.matrixscience.com) logical developments. search programs against the UniProtKB/Swiss-Prot entries from Protein fractions showing single electrophoretic band, molec- taxon Serpentes (http://ca.expasy.org/cgi-bin/get-entries?view= ular mass, and N-terminal sequence (Fig. 1B) can be straightfor- full&KW=Toxin&OC=Serpentes). Furthermore, it is not too unu- wardly assigned by BLAST analysis to a known protein family. sual that a product ion spectrum matched with high MASCOT Thus, although few toxins from any given species are annotated score to a particular peptide sequence corresponds actually to in the public-accessible databases, representative members of a tryptic peptide of a homolog snake toxin containing one or most snake venom toxin families are present among the more nearly isobaric amino acid substitutions. Hence, it is neces- 1100 viperid toxin protein sequences belonging to 157 species sary to revise manually all the CID–MS/MS spectra (to confirm deposited to date in the SwissProt/TrEMBL database (Knowl- the assigned peptide sequence or for performing de novo edgebase Release 56.5 of November 2008; http://us.expasy.org/ sequencing), and submit the deduced peptide ion sequences to sprot/). On the other hand, protein fractions showing heteroge- BLAST similarity searches. Although the lack of any complete neous or blocked N-termini are analyzed by SDS–PAGE and the snake genome sequence is a serious drawback for the identifica- bands of interest subjected to automated reduction, carbami- tion of venom proteins, high-quality MS/MS peptide ion frag- domethylation, and in-gel tryptic digestion (Fig. 1C). The result- mentation spectra usually yield sufficient amino acid sequence ing tryptic peptides are then analyzed by MALDI-TOF mass information derived from almost complete series of sequence- fingerprinting followed by amino acid sequence determination specific b- and/or y-ions (Fig. 1D) to unambiguously identify a of selected doubly- and triply-charged peptide ions by colli- homolog protein in the current databases. The combined venom- sion-induced dissociation tandem mass spectrometry. Except ics strategy allows us to assign unambiguously all the isolated for a few proteins, the peptide mass fingerprinting approach venom toxins representing over 0.05% of the total venom pro- alone is unable to identify any protein in the databases. In addi- teins to known protein families (Fig. 1E, Table 1). 1740 J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743
Table 1 Overview 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 cerastes cerastes (CCC), Cerastes vipera (CV) and Macrovipera lebetina transmediterranea (MLT) [48]; African Bitis arietans (BA) [50]; Bitis gabonica gabonica (BGG) [49]; Bitis gabonica rhinoceros (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), and Bothrops 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 proteins Disintegrins – 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 <0.1 Kunitz-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 – – <0.1 NGF/svVEGF <0.1 <0.1 <0.1 <0.1 – – 2.1 – 1.0 ––––––– Ohanin-like ––––––––– ––––––<0.1 CRISP 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 18.5 Serine 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 13.8 C-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 2.2 Zn2+-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 14.7 Kunitz-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 – 0.4 Ohanin-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 12.3 Serine 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 25.6 C-type lectin-like 14.8 9.6 1.8 1.3 0.5 0.5 – <0.1 <0.1 9.8 0.9 – 3.6 L-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 5.3 Zn2+-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
The long-term goal of our Snake Venomics project is a detailed pean vipers, Vipera ammodytes ammodytes and Vipera ammodytes analysis of all viperid venomes. To date, this methodology has been meridionalis [62], Asian Daboia russelli siamensis [63], Amazonian applied to explore the venom proteomes (Table 1) of the medi- Bothrops atrox [64], and Brazilian Bothrops insularis [65]. cally-relevant Tunisian vipers Cerastes cerastes, Cerastes vipera, Qualitative discrepancies in the compositional agreement of ve- Macrovipera lebetina [48]; African Bitis gabonica [49], Bitis arietans nom acquired using proteomic and transcriptomic approaches [50], and Echis ocellatus [51]; North American Sistrurus miliarius have been reported, i.e. for Lachesis muta [53], Bitis gabonica gabo- barbouri [52] and Sistrurus catenatus subspecies [34]; the South nica [49], and Echis ocellatus [51]. Venom composition may be and Central American Lachesis sp. [53]; the Armenian mountain vi- influenced by a number of different factors, most notably the po- pers Vipera raddei and Macrovipera lebetina obtusa [54]; the arbo- tential for genetic drift on account of (i) gender based variation real Neotropical pitvipers Bothriechis lateralis and Bothriechis and (ii) ontogenetic variations. Conversely, the occurrence of schlegelii [55]; to infer phylogenetic alliances within genus Bitis mRNAs that are abundant and readily identifiable in the transcrip- [56] and Sistrurus [34]; to rationalize the envenomation profiles tome, yet apparently absent from the venom proteome may also be of Atropoides [57] and Bothrops [58] species; to define venom- explained by a number of factors. Firstly, these messengers may associated taxonomic markers [59]; to establish the molecular ba- exhibit transient, individual or a temporal expression patterns over sis of geographic, individual, and ontogenetic venom variations in the life time of the snake. Alternatively, mRNAs whose predicted Bothrops asper [60]; and to contribute to reconstruct the natural polypeptides were not found to be secreted in venom may repre- history and cladogenesis of Bothrops colombiensis [61], a medically sent very low abundance toxins that play a hitherto unrecognised important pitviper of the Bothrops atrox-asper complex endemic to physiological function in the venom gland, or may simply repre- Venezuela. A few other laboratories have reported qualitative pro- sent a hidden repertoire of non-translated orphan molecules which teomic studies on several venoms, including those from the Euro- may 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 that with antivenom followed by the addition of a secondary antibody. the final composition of venom is influenced by transcriptional and Antigen-antibody complexes immunodepleted from the reaction post-translational mechanisms that may be more complex than mixture contain the toxins against which antibodies in the anti- previously appreciated. Nevertheless, a major conclusion drawn venom are directed. By contrast, venom components that remain from snake venomics and transcriptomic analyses of viperid ve- in the supernatant are those which failed to raise antibodies in nom glands is that despite venoms being complex mixture of pro- the antivenom, or which triggered the production of low-affinity teins, venom proteins belong to only a few major families, antibodies. These components can be easily identified by compar- including enzymes (serine proteinases, Zn2+-metalloproteases, ison of reverse-phase HPLC separation of the non-precipitated frac-
L-amino acid oxidase, PLA2) and proteins without enzymatic activ- tion with the HPLC pattern of the whole venom previously ity (disintegrins, C-type lectins, natriuretic peptides, ohanin, myo- characterized by venomics. According to their immunoreactivity toxins, CRISP toxins, nerve and vascular endothelium growth towards antivenoms, toxins may be conveniently classified as: C- factors, cystatin and Kunitz-type protease inhibitors) [16,44] toxins, completely immunodepletable toxins; P-toxins, partly (Table 1), though different venoms exhibit a distinct toxin family immunodepleted toxins; and N-toxins, non-inmunodepleted pro- distributional profile (Table 1). This situation may reflect the fact teins (Fig. 2). Assuming a link between the in vitro toxin immun- that toxins likely evolved from a restricted set of protein families, odepletion capability of an antivenom and its in vivo neutralizing and opens the door for a rationale design of intra-, but also inter-, activity towards the same toxin molecules, improved immuniza- species family-specific neutralizing antibodies. tion protocols should make use of mixtures of immunogens to gen- Several antivenoms are produced in Latin America using differ- erate high-affinity antibodies against class P and class N toxins. On ent venoms in the immunization schemes [66]. Each of these anti- the other hand, our antivenomics approach is simple and easy to venoms is effective in variable degree against envenomations by implement in any protein chemistry laboratory, and may thus rep- snake venoms not included in the immunization protocol, demon- resent another useful protocol for investigating the immunoreac- strating the high degree of immunological cross-reactivity be- tivity, and thus the potential therapeutic usefulness, of tween Central and South American crotaline snake venoms. A antivenoms towards homologous and heterologous venoms [16]. practical consequence of this fortunate circumstance is the possi- The deficit (‘crisis’) of antivenom supply in some regions of the bility of using these heterologous antivenoms to circumvent the world can be addressed to a certain extent by optimizing the use of restricted availability of species-specific antivenoms in some re- existing antivenoms and through the design of novel immuniza- gions. However, before testing in clinical trials, antivenoms need tion mixtures for producing broad-range polyspecific antivenoms. to be evaluated experimentally by assessing their neutralizing abil- Proteomics-based immunochemical analysis (antivenomics) pro- ity against the most relevant toxic and enzymatic activities of vides relevant information for outlining which venom mixtures snake venoms. To this end, we have developed a simple proteomic cross-react with the most important components in medically- protocol for investigating the immunoreactivity, and thus the po- relevant venoms from a particular region. This type of approach tential therapeutic usefulness, of antivenoms towards homologous may set the basis for the development of antivenoms on an and heterologous venoms. immunologically sound basis. The potential value of antivenomics, together with preclinical neutralization tests, in assessing antiven- 5. Antivenomics: proteomic tools for studying the om cross-reactivity is clearly illustrated by the following examples. Ò immunological profile of venoms A highly effective antivenom (Sanofi-Pasteur ‘Bothrofav ’) has been developed for the treatment of envenomings by Bothrops We have coined the term ‘‘antivenomics” for the identification lanceolatus, endemic to the Lesser Antillean island of Martinique. of venom proteins bearing epitopes recognized by an antivenom It exhibits an excellent preclinical profile of neutralization [67] using proteomic techniques [55,58,61]. Antivenomics is based on and its timely administration prevents the development of the the immunodepletion of toxins upon incubation of whole venom most serious effects of envenoming, including thrombosis
Fig. 2. Antivenomics. Panel A displays the reverse-phase HPLC separation of the venom proteins from Crotalus simus (from Costa Rica). Red arrows indicate venom proteins not immunodepleted by the Costa Rican polyvalent (Crotalinae) ICP antivenom. These N-toxins were identified as disintegrins (4–8), crotoxin subunits (9–12), and a PLA2 molecule (15). The orange arrow points to the major snake venom metalloproteinase (SVMP), which was partly immunodepleted by the ICP antivenom. The antivenomics results fully explain the incapacity of the ICP antivenom to neutralize the neurotoxicity of the venom of South American Crotalus durissus subspecies, which is associated to a large content of the heterodimeric PLA2 Crotoxin (Panel B). Similarly, antivenoms produced in South America against C.d. terrificus venom neutralize the lethality of Central American Crotalus simus but are ineffective at neutralizing the hemorrhagic activity of venom from genus Crotalus. Such neutralizing profile is also fully explained by the almost absence of hemorrhagic SVMPs (fractions 17–18 in reverse-phase chromatogram shown in B) in the C.d. terrificus venom used in the immunization protocol. The antivenomic findings suggest that an antivenom raised against a mixture of C. simus and C.d. terrificus may effectively neutralize both the hemorrhagic and the neurotoxic effects of Middle and South American Crotalus species. 1742 J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743
[68,69]. However, the restricted availability of the antivenom in Summerhayes, E. and Kochva, E. (2006) Early evolution of the venom system the neighboring island of Saint Lucia and in zoos and herpetariums in lizards and snakes. Nature 439, 584–588. [2] Fry, B.G., Scheib, H., van der Weerd, L., Young, B., McNaughtan, J., Ramjan, S.F., is a matter of concern. Gutiérrez and colleagues have performed Vidal, N., Poelmann, R.E. and Norman, J.A. (2008) Evolution of an arsenal: detailed proteomic studies of the venoms of Bothrops caribbaeus structural and functional diversification of the venom system in the advanced and B. lanceolatus and have evaluated the immunoreactivity of a snakes (Caenophidia). Mol. Cell Proteomics 7, 215–246. [3] Fry, B.G., Vidal, N., van der Weerd, L., Kochva, E. and Renjifo, C. (2009) Crotalinae polyvalent antivenom produced in Costa Rica (by immu- Evolution and diversification of the Toxicofera reptile venom system. J. nization of horses with a mixture of equal amounts of the venoms Proteomics 72, 127–136. of B. asper, Crotalus simus, and Lachesis stenophrys) towards the ven- [4] Koh, D.C.I., Armugam, A. and Jeyaseelan, K. (2006) Snake venom components and their applications in biomedicine. Cell Mol. Life Sci. 63, 3030–3041. oms of B. caribbaeus and B. lanceolatus [58]. This study showed that [5] Harvey, A.L., Bradley, K.N., Cochran, S.A., Rowan, E.G., Pratt, J.A., Quillfeldt, J.A. the antivenom immunodepleted 80% of the proteins from both B. and Jerusalinsky, D.A. (1998) What can toxins tell us for drug discovery? caribbaeus and B. lanceolatus venoms, and was effective in neutral- Toxicon 36, 1635–1640. [6] Rocha e Silva, M., Beraldo, W.T. and Rosenfeld, G. (1949) Bradykinin, a izing the lethal, hemorrhagic, PLA2 and proteolytic activities of the hypotensive and a smooth muscle stimulating factor released from plasma two venoms. globulin by snake venom and by trypsin. Am. J. Physiol. 156, 261–270. Calvete and co-workers from Venezuela and Costa Rica have [7] Fernandez, J.H., Neshich, G. and Camargo, A.C.M. (2004) Using bradykinin- compared the venom proteomes of B. colombiensis, B. asper, and potentiating peptide structures to develop new antihypertensive drugs. Genet. B. atrox using venomics and antivenomics approaches [61]. This Mol. Res. 3, 554–563. [8] McIntosh, M., Cruz, L.J., Hunkapiller, M.W., Gray, W.R. and Olivera, B.M. (1982) study did not support the suggested synonymy between B. colom- Isolation and structure of a peptide toxin from the marine snail Conus magus. biensis and B. atrox. On the other hand, the closest homologs to B. Arch. Biochem. Biophys. 218, 329–334. colombiensis venom proteins appeared to be toxins from B. asper. [9] Ménez, A. (2002) Perspectives in Molecular Toxinology, John Wiley & Sons Ltd., Chichester, UK. A rough estimation of the similarity between the venoms of B. [10] Ménez, A., Stöcklin, R. and Mebs, D. (2006) ‘‘Venomics” or: the venomous colombiensis and B. asper indicated that these species share approx- systems genome project. Toxicon 47, 255–259. imately 65–70% of their venom proteomes. The close kinship of B. [11] Gutiérrez, J.M., Theakston, R.D.G. and Warrell, D.A. (2006) Confronting the neglected problem of snake bite envenoming: the need for a global colombiensis and B. asper points at the ancestor of B. colombiensis as partnership. PLoS Med. 3, e150. the founding Central American B. asper ancestor. This finding may [12] Gutiérrez, J.M., Higashi, H.G., Wen, F.H. and Burnouf, T. (2007) Strengthening be relevant for reconstructing the natural history and cladogenesis antivenom production in Central and South American public laboratories: report of a workshop. Toxicon 49, 30–35. of Bothrops. Further, the virtually indistinguishable immunological [13] Stock, R.P., Massougbodji, A., Alagón, A. and Chippaux, J.-P. (2007) Bringing cross-reactivity of a Venezuelan ABC antiserum (raised against a antivenoms to Sub-Saharan Africa. Nat. Biotechnol. 2, 173–177. mixture of B. colombiensis and Crotalus durissus cumanensis ven- [14] Kasturiratne, A., Wickremasinghe, A.R., de Silva, N., Gunawardena, N.K., oms) and the Costa Rican ICP polyvalent antivenom (generated Pathmeswaran, A., Premaratna, R., Savioli, L., Lalloo, D.G. and de Silva, H.J. (2008) The global burden of snakebite: a literature analysis and modelling against a mixture of B. asper, C. simus, and L. stenophrys venoms) to- based on regional estimates of envenoming and deaths. PLoS Med. 5, e218. wards the venoms of B. colombiensis and B. asper, supports this [15] Chippaux, J.-P. and Goyffon, M. (2008) Epidemiology of scorpionism: a global view and suggests the possibility of indistinctly using these anti- appraisal. Acta Tropica 107, 71–79. [16] Gutiérrez, J.M., Lomonte, B., León, G., Alape-Girón, A., Flores-Díaz, M., Sanz, L., venoms for the management of snakebites by any of these Bothr- Angulo, Y. and Calvete, J.J. (2009) Snake venomics and antivenomics: ops species. proteomic tools in the design and control of antivenoms for the treatment An antivenom manufactured in Costa Rica using venom of the of snakebite envenoming. J. Proteomics 72, 165–182. [17] Scanlon, J.D. and Lee, M.S.Y. (2000) The pleistocene serpent Wonambi and the Central American rattlesnake (Crotalus simus simus) population, is early evolution of snakes. Nature 403, 416–420. ineffective for neutralizing the neurotoxic activity of South Amer- [18] Apesteguía, S. and Zaher, H. (2006) A cretaceous terrestrial snake with robust ican Crotalus durissus terrificus. Similarly, antivenoms produced in hindlimb and a sacrum. Nature 440, 1037–1040. [19] Vidal, N. and Hedges, S.B. (2005) The phylogeny of squamate reptiles (lizards, South America against C.d. terrificus venom neutralize the neuro- snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes. toxicity of Central American venoms but are ineffective at neutral- C.R. Biol. 328, 1000–1008. izing the hemorrhagic activity of venoms from genus Crotalus [70]. [20] Alvarez, L.W., Alvarez, W., Asaro, F. and Michel, H.V. (1980) Extraterrestrial cause for the Cretaceous-Tertiary extinction. Science 208, 1095–1108. Such neutralizing profile is fully explained by the proteomic and [21] Greene, H.W. (1997) Snakes: The Evolution of Mystery in Nature, University of antivenomic characterization of Crotalus (simus and durissus) ven- California Press, Berkeley. oms described in Fig. 2. The case of Crotalus illustrates how the [22] Vidal, N. and Hedges, S.B. (2002) Higher-level relationships of snakes inferred knowledge of venom variations and their geographical distribution from four nuclear and mitochondrial genes. C.R. Biol. 325, 977–985. [23] Greene, H.W. (1983) Dietary correlates of the origin and radiation of snakes. can lead to securing venoms with a more defined composition for Am. Zool. 23, 431–441. preparing venom mixtures for the generation of antivenoms effec- [24] Mackessy, S.P. (1991) Morphology and ultrastructure of the venom glands of tive against the venoms of rattlesnakes from Central and South the northern Pacific rattlesnake Crotalus viridis oreganus. J. Morphol. 208, 109– 128. America. [25] Meier, J. and White, J. (1995) Handbook of Clinical Toxicology of Animal Venoms and Poisons, CRC Press, Boca Raton. [26] Fox, J.W. and Serrano, S.M.T. (2005) Snake toxins and hemostasis. Toxicon 45, Acknowledgments 951–1181. [27] Markland, F.S. (1998) Snake venoms and the hemostatic system. Toxicon 36, Research in Costa Rica has been supported by Vicerrectoría de 1749–1800. [28] Fry, B.G. (1999) Structure-function properties of venom components from Investigación, Universidad de Costa Rica (Project 741-A8-521), Australian elapids. Toxicon 37, 11–32. CRUSA-CSIC (Project 2007CR0004), CYTED (Project 206AC0281) [29] Fry, B.G. and Wüster, W. (2004) Assembling an arsenal: origin and evolution of and CONARE (Fondos del Sistema). Snake venomics and antive- the snake venom proteome inferred from phylogenetic analysis of toxin sequences. Mol. Biol. Evol. 21, 870–883. nomic studies at the Spanish IBV have been financed by grants [30] Fry, B.G. (2005) From genome to ‘‘venome”: molecular origin and evolution of BFU2004-01432 and BFU2007-61563 from the Ministerios de Edu- the snake venom proteome inferred from phylogenetic analysis of toxin cación y Ciencia and Ciencia e Innovación, Madrid. Travelling be- sequences and related body proteins. Genome Res. 15, 403–420. tween Spain and Costa Rica was financed by Acciones Integradas [31] Lynch, V.J. (2007) Inventing an arsenal: adaptative evolution and neofunctionalization of snake venom phospholipase A2 genes. BCM Evol. 2006CR0010 between CSIC and the University of Costa Rica (UCR). Biol. 7, 2. [32] Gibbs, H.L. and Rossiter, W. (2008) Rapid evolution by positive selection and References gene gain and loss: PLA(2) venom genes in closely related Sistrurus rattlesnakes with divergent diets. J. Mol. Evol. 66, 151–166. [33] Juárez, P., Comas, I., González-Candelas, F. and Calvete, J.J. (2008) Evolution of [1] Fry, B.G., Vidal, N., Norman, J.A., Vonk, F.J., Scheib, H., Ramjan, S.F., Kuruppu, S., snake venom disintegrins by positive Darwinian selection. Mol. Biol. Evol. 25, Fung, K., Hedges, S.B., Richardson, M.K., Hodgson, W.C., Ignjatovic, V., 2391–2407. J.J. Calvete et al. / FEBS Letters 583 (2009) 1736–1743 1743
[34] Sanz, L., Gibbs, H.L., Mackessy, S.P. and Calvete, J.J. (2006) Venom proteomes of [55] Lomonte, B., Escolano, J., Fernández, J., Sanz, L., Angulo, Y., Gutiérrez, J.M. and closely related Sistrurus rattlesnakes with divergent diets. J. Proteome Res. 5, Calvete, J.J. (2008) Snake venomics and antivenomics of the arboreal 2098–2112. neotopical pitvipers Bothriechis lateralis and Bothriehis schlegelii. J. Proteome [35] Gibbs, H.L., Sanz, L. and Calvete, J.J. (2009) Snake population venomics: Res. 7, 2445–2457. proteomics-based analyses of individual variation reveals significant gene [56] Calvete, J.J., Escolano, J. and Sanz, L. (2007) Snake venomics of Bitis species regulation effects on venom protein expression in Sistrurus rattlesnakes. J. reveals large intragenus venom toxin composition variation: application to Mol. Evol. 68, 113–125. taxonomy of congeneric taxa. J. Proteome Res. 6, 2732–2745. [36] Nei, M., Gu, X. and Sitnikova, T. (1997) Evolution by the birth-and-death [57] Angulo, Y., Escolano, J., Lomonte, B., Gutiérrez, J.M., Sanz, L. and Calvete, J.J. process in multigene families of the vertebrate immune system. Proc. Natl. (2008) Snake venomics of Central American pitvipers: clues for rationalizing Acad. Sci. USA 94, 7799–7806. the distinct envenomation profiles of Atropoides nummifer and Atropoides [37] Calvete, J.J. (2009) Digging into the evolution of venomous systems and picadoi. J. Proteome Res. 7, 708–719. learning to twist nature to fight pathology. J. Proteomics 72, 121–126. [58] Gutiérrez, J.M., Sanz, L., Escolano, J., Fernández, J., Lomonte, B., Angulo, Y., [38] Chippaux, J.-P. (1998) Snake-bites: appraisal of the global situation. Bull. Rucavado, A., Warrell, D.A. and Calvete, J.J. (2008) Snake venomic of the Lesser World Health Org. 76, 515–524. Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation [39] World Health Organization (2007). Rabies and Envenomings. A Neglected with toxicological activities and immunoreactivity of a heterologous Public Health Issue: Report of a Consultative Meeting. Geneva: WHO.