Studies on the venom proteome of Bothrops asper: Perspectivesand applications

Toxicon 54 (2009) 938–948

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Studies on the venom proteome of Bothrops asper: Perspectives and applications Alberto Alape-Giro´n a, b, c, *, Marietta Flores-Dı´az a, Libia Sanz d, Marvin Madrigal a, b, Jose´ Escolano d, Mahmood Sasa a, Juan J. Calvete d a

Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, 2060 San Jose´, Costa Rica Departamento de Bioquı´mica, Escuela de Medicina, Universidad de Costa Rica, San Jose´, Costa Rica c ´ picas, Universidad de Costa Rica, San Jose´, Costa Rica Centro de Investigaciones en Estructuras Microsco d Instituto de Biomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spain b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 April 2009 Received in revised form 7 June 2009 Accepted 9 June 2009 Available online 17 June 2009

Bothrops asper is responsible for the vast majority of snakebite accidents in Central America and several studies have demonstrated that specific toxic and enzymatic activities of its venom vary with the geographic origin and age of the specimens. Variability in venom proteins and enzymes between specimens from the Caribbean and the Pacific versants of Costa Rica has been reported since 1964. Recently, we performed a comparative proteomic characterization of the venoms from one population of each versant. Proteins belonging to several families, including disintegrin, phospholipases A2, serine proteinases, C-type lectins, CRISP, L-amino acid oxidase, and Zn2þ-dependent metalloproteinases show a variable degree of relative occurrence in the venoms of both populations. The occurrence of prominent differences in the protein profile between venoms from adults and newborns, and among venom samples from individual specimens of the same region or developmental stage, further demonstrated the existence of geographic, ontogenetic and individual variability in the venom proteome of this species. These findings provide new insights towards understanding the biology of B. asper, contribute to a deeper understanding of the pathology induced by its venom and underscore the importance of the use of venoms pooled from specimens from both regions for producing antivenom exhibiting the broadest cross-reactivity. Furthermore, knowledge of the protein composition of B. asper venom paves the way for detailed future structure–function studies of individual toxins as well as for the development of new protocols to study the reactivity of therapeutic antivenoms. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Snake venom protein families Proteomics Viperid toxins Snake venomics Antivenoms

1. Introduction Snake venoms are secretions produced by specialized exocrine glands, containing a complex mixture of toxic proteins which contribute to subdue, kill and/or digest the prey (Fry et al., 2006; Vonk et al., 2008). Venom toxins * Corresponding author at: Instituto Clodomiro Picado, Facultad de Microbiologı´a, Universidad de Costa Rica, 2060 San Jose´, Costa Rica. Tel.: þ506 2511 3135; fax: þ506 2292 0485.

E-mail address: alberto.alape@ucr.ac.cr (A. Alape-Giro´n).

0041-0101/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2009.06.011

likely evolved from a reduced set of proteins with normal physiological functions which were recruited into the venom proteome before the diversification of the advanced snakes (Fry and Wu¨ster, 2004; Fry, 2005). Toxins from Viperidae can be grouped into just a few protein families, including enzymes (serine proteinases, Zn2þ-metalloproteinases, L-amino acid oxidase, group II PLA2) and proteins without enzymatic activity (disintegrins, C-type lectin-like molecules, bradykinin-potentiating peptides, ohanin, myotoxins, cysteine-rich secretory proteins, nerve and vascular endothelium growth factors, cystatin and

´n et al. / Toxicon 54 (2009) 938–948 A. Alape-Giro

Kunitz-type proteinase inhibitors) (Calvete et al., 2007).

Each toxin family is usually represented in the same venom by multiple proteins, which often differ in their pharmacological activities, but might share remarkable structural and antigenic similarities (Fry and Wu¨ster, 2004; Fry, 2005). These proteins are encoded by paralogous genes, probably originated by gene duplications and accelerated Darwinian evolution processes (Nakashima et al., 1995; Deshimaru et al., 1996; Ogawa et al., 2005). The specific proteins found in the venoms of different species vary in amino acid sequence and abundance and thereby contribute to the differences in the overall biological activity of individual venoms. Variability in protein composition among snake venoms from individuals of different geographic origins, sex, or age is a well documented phenomenon (Chippaux et al., 1991) and this variability has very important implications for the production and use of therapeutic antivenoms in the treatment of snakebite envenomings.

A deep analysis of the variability in the toxin composition of venom proteomes is now possible using high-throughput approaches. The combination of 2D electrophoresis or RP-HPLC with Edman sequencing, MALDI-TOF/MS and ESI/ MS/MS has opened the opportunity to study snake venom proteomes, making possible to identify the complete set of proteins from several viperid venoms and to perform quantitative comparisons of related venoms (Gutie´rrez et al., 2009; Calvete et al., 2007, 2009a). The understanding of the protein/peptide content of snake venoms could help in unravelling the evolution of the venom protein families, for the development of novel drugs and also has several potential applications in antivenom preparation (Gutie´rrez et al., 2009). In this work we review the studies performed to characterize the venom proteome of B. asper and discuss their potential applications in the development of more effective therapeutic antivenoms.

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leucurus, Bothrops marajoensis, Bothrops moojeni, Bothrops colombiensis and B. asper (da Saloma˜o et al., 1997) (Fig. 1).

However, the status and phylogenetic alliances of many of the conventionally recognized species within the asper– atrox group are still under debate (Wu¨ster et al., 1999, 2002; Castoe and Parkinson, 2006).

B. asper is widely distributed in humid lowlands. It is the only Bothrops species that occurs in Trinidad Island; in Mexico and Central America, it occurs up to 1500 m altitude, whereas in South America it occurs up to 2500 m (Campbell and Lamar, 2004). The Caribbean distribution of B. asper extends continuously along the coast of the Gulf of Mexico, the Yucatan Peninsula, Central America, Panama, the Caribbean coast and through the inter-Andean valleys of Colombia, thence eastward across northern and Central Venezuela. The Pacific distribution of B. asper includes the region from the Central and South part of Costa Rica, through Panama, continuing along western Colombia, northern Ecuador and the northeastern extreme of Peru.

Specimens assigned to B. asper have a long history of taxonomical change. Cope (1887) recognized the species in the synonymy of B. atrox, Smith and Taylor (1945) recognized it as a subspecies of B. atrox from South America, and therefore the trinomial B. atrox–asper was adopted by earlier researchers of its toxins (Jime´nez-Porras, 1964a).

The binomial name B. asper was adopted after Hoge (1966) and is currently the accepted nomenclature. Some of the common names applied to this species are: terciopelo and barba amarilla (Central America), nauyaca (Mexico and Guatemala), mapepire (Trinidad) and equis (South America).

Many people are bitten each year in the New World by B. asper because it has a widespread distribution and is capable to adapt to altered environments such as agricultural fields and peridomestic areas (Gutie´rrez, 1995; Warrell, 2004). Most cases occur in rural areas and affect

2. Bothrops asper: the medically most important pitviper in Central America and Northern South America The genus Bothrops (Subfamily Crotalinae of Viperidae) comprises at least five lineages of pitvipers commonly referred as lanceheads, which inhabit from northeastern Mexico, through Central and South America to Argentina and are also present in some Caribbean islands (Campbell and Lamar, 2004). Bothrops snakes are terrestrial or semiarboreal, nocturnal pitvipers, and in tropical regions activity peaks usually correspond to rainy periods, during which most reproductive activities occur (Campbell and Lamar, 2004). Most species of Bothrops feed largely on exothermic prey as juveniles but shift to endothermic prey when they reach a size sufficient to swallow rodents, marsupials, birds, and other bulky prey items (Campbell and Lamar, 2004). Bothrops species are included into various groups according to their phylogenetic relationships (da Saloma˜o et al., 1997). The asper–atrox group represents a monophyletic clade of medium to large-sized pitvipers widely spread throughout the tropical parts of Central and South America northward of the Amazon Basin, and includes Bothrops atrox, Bothrops isabelae, Bothrops

Fig. 1. Distribution of some snake species of the Bothrops asper–atrox complex in Central and South America. Physical map of Central and northern South America highlighting the geographic distribution of B. asper (closed circles), B. atrox (gray), and B. colombiensis (white and yellow). B. asper is found in the Atlantic lowlands of eastern Mexico and Central America, including Guatemala, Belize, Honduras, Nicaragua, Costa Rica and Panama.

In northern South America it is found in the Pacific versant of the Colombian and Ecuadorian Andes and the adjacent coastal lowlands, and in northern Venezuela. The picture shown at the lower left corner corresponds to an adult specimen of Costa Rican B. asper.

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young agricultural workers and children, many of whom survive but are left with permanent disability (Jorge et al., 1999; Gutie´rrez et al., 2006). In Mexico, B. asper together with Crotalus simus is the leading cause of snakebite in Yucatan. In Costa Rica, B. asper is responsible for almost half of all snakebites (Arroyo et al., 1999; Sasa and Va´zquez, 2003). In the Colombian states of Antioquia and Choco´ it causes 50–70% of all snakebites (Otero et al., 1992) whereas in the state of Lara, Venezuela, it is responsible for 78% of all envenomations and snakebite fatalities (Dao-L., 1971).

Envenomations by B. asper are characterized by conspicuous local alterations at the site where the venom is injected, including edema, local hemorrhage, blistering, dermonecrosis, and myonecrosis. In severe cases, systemic signs and symptoms of envenoming include defibrin(ogen)ation, thrombocytopenia, platelet hypoaggregation, systemic bleeding, disseminated intravascular coagulation, cardiovascular shock and acute renal failure (Saborı´o et al., 1998; Otero et al., 2002). Death might result from hypotension secondary to hypovolemia, renal failure, or intracranial hemorrhage (Saborı´o et al., 1998; Otero et al., 2002).

Adequate treatment of B. asper envenoming is critically dependent on the intravenous administration of an efficient antivenom (Gutie´rrez, 1995; Warrell, 2004).

Therapeutic antivenoms currently used in the region for the treatment of B. asper envenomed patients are polyvalent and consist of either native IgG molecules or F(ab0 )2 fragment preparations obtained from animals hyperimmunized either with venom mixtures of different Bothrops species (Suero Antiofidico produced by Instituto Nacional de Higiene y Medicina Tropical ‘‘Leopoldo Izquieta Perez’’, Guayaquil, Ecuador, and Suero antibotro´pico produced by Centro Nacional de Produccio´n de Biolo´gicos, Instituto Nacional de Salud, Lima, Peru´), mixtures of Bothrops and Crotalus venoms (Suero ABC produced by Centro de Biotecnologı´a, Universidad Central de Venezuela, Suero polivalente antibotro´pico–anticrota´lico produced by Instituto Nacional de Salud, Bogota´, Colombia, and Antivipmyn produced by Instituto Bioclon, Mexico) or mixtures of Bothrops, Crotalus and Lachesis venoms (Suero antiofidico polivalente produced by Instituto Clodomiro Picado, San Jose´, Costa Rica, and Suero polivalente antibotro´pico, anticrota´lico, antilaque´sico produced by Probiol Laboratories, Bogota´, Colombia) (Espino-Solı´s et al., 2009).

Fig. 2. Distribution of B. asper in Costa Rica. Physical map of Costa Rica showing the geographic distribution of B. asper (scratched area) and the regions where the venoms were collected at the Caribbean Bas(C) and the Pacific Bas(P) versants. Reproduced from J. Proteome Res. 7, 3556–3571 with permission from ACS.

specimens, a major band corresponding to protein(s) with relative molecular mass of about 23 kDa and a minor band corresponding to protein(s) with relative mol. mass of about 17 kDa were exclusively found in the venom from the Pacific versant, whereas a band corresponding to protein(s) with relative mol. mass of about 25 kDa was exclusively present in the venom form the Caribbean versant. When the protein patterns from venoms of adults and newborns were compared, those from adults showed mainly protein(s) with relative mol. mass in the range between 14

3. Geographic and ontogenetic variability in the venom proteome of B. asper The Caribbean and Pacific populations of B. asper from Costa Rica and Panama are separated since the late Miocene or early Pliocene (8–5 Mya) by the mountain ridge constituted by the Cordillera Volca´nica Central, Cordillera de Talamanca, Cordillera de Chiriquı´ and Serranı´a de Tabasara´, which is a barrier to the dispersal of this species (Campbell and Lamar, 2004). The electrophoretic protein patterns of venom pools from adult and newborn B. asper specimens collected at the Caribbean versant (Distrito Quesada, San Carlos) and at the Pacific versant (Distrito de Sabanillas, Acosta) of Costa Rica (Fig. 2) and separated by SDS-PAGE revealed the presence of conspicuous qualitative and quantitative differences (Fig. 3). In the venoms from adult

Fig. 3. SDS-PAGE of the proteins from pooled B. asper venoms obtained from adults (CA) or newborns (CN) from the Caribbean, adults (PA) or neonates (PN) from the Pacific versants of Costa Rica. The proteins contained in 30 mg of each venom were separated on a 15% acrylamide gel under reducing conditions and stained with Coomassie Brilliant Blue. Molecular markers (MM) are shown in lines 1 and 6.

´ n et al. / Toxicon 54 (2009) 938–948 A. Alape-Giro

and 45 kDa, whereas the venoms from newborn specimens displayed predominantly proteins with relative mol. mass between 20 and 67 kDa. Intraspecific variability in the enzymatic and toxic activities of B. asper venoms from Costa Rica, according to geographic origin, has been previously documented in several studies (Jime´nez-Porras, 1964a; Gutie´rrez et al., 1980; Arago´n and Gubensek, 1981; Moreno et al., 1988). In the first report, which dates from 1964, starch gel electrophoresis was used to fractionate the venom of specimens from 15 different localities in Costa Rica and the enzymatic activities and the toxicity of the fractions were evaluated (Jime´nez-Porras, 1964a). The most relevant conclusions of this work were that the most toxic fractions corresponded to those containing chymotryptic and thrombotic activities and that the venom from specimens of the Caribbean and Pacific versants was distinguishable based on the occurrence and the relative amounts of some enzymes. Enzymatic activities of adenosine-triphosphatase, deoxyribonuclease and ribonuclease were about twice as high in the venom of specimens from the Pacific versant than in the venoms from those of the Caribbean zones. On the other hand, the enzymatic activities of adenosine 50 -monophosphatase, nicotinamide-adenine-dinucleotidase and phosphatidase A were lowest in venoms from the Pacific zone (Jime´nezPorras, 1964a).

In 1980, Gutie´rrez et al. reported a comparative study of the toxic activities of venoms from the Caribbean and from the Pacific versants. They found that the venom from snakes of the Caribbean regions had greater hemorrhagic and myonecrotic effects than that from the Pacific versant, which had a higher proteolytic activity against casein.

Afterwards, Arago´n and Gubensek (1981), using PAGE and isoelectric focusing, reported differences in the electrophoretic pattern of B. asper venom from the two versants of Costa Rica. Furthermore, they found that the venom from specimens of the Pacific versant had a higher proteolytic activity against casein and fibrinogen, but that the venom from specimens from the Caribbean versant had a higher pro-coagulant activity. It has been also reported that, depending on geographical origin, the venom from B. asper shows qualitative and quantitative variations in the PLA2 isoforms (Moreno et al., 1988). Six acidic and tree basic PLA2 isoforms were detected in the venom from specimens of the Caribbean versant, whereas four acidic, one neutral, and at least one basic isoform were detected in the venom from specimens of the Pacific versant (Moreno et al., 1988).

The detailed comparative proteomic analysis of venom pools from B. asper specimens collected in the Caribbean region (Distrito Quesada, San Carlos) and in the Pacific versant (Distrito de Sabanillas, Acosta) of Costa Rica (Fig. 2) revealed similarities and differences between the proteins secreted by snakes from these two geographically separated locations (Alape-Giro´n et al., 2008). Distinct relative abundance of the protein families occurs between the two populations isolated by mountain barriers, probably as a consequence of allopatric variability. The venom from snakes of the Caribbean versant has a higher content of serine proteinases (410%), L-amino acid oxidases (LAO) (200%), and disintegrin (160%) than venom from specimens of the Pacific versant, whereas the latter is enriched in

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PLA2s (160%) compared to the venom from the Caribbean specimens (Table 1). These results confirmed and extended the results of Moreno et al. (1988), showing that venoms of B. asper specimens from the Caribbean and Pacific regions of Costa Rica contain both identical and different PLA2 isoforms.

Venoms pooled from adult specimens collected at the Caribbean and Pacific versants of Costa Rica were subjected to 2D electrophoresis (Fig. 4) (Alape-Giro´n et al., 2008). At least 80 separated spots can be detected in the venom from the Caribbean versant and 86 spots in the venom from the Pacific versant. In both cases, the proteins are spread through the gel displaying a wide range of isoelectric points and molecular masses. The isoelectric point, relative mol. mass, and proportion in the venom of each of these proteins are indicated in Tables 2 and 3. Only twelve and ten spots represent more than 2% of the venom proteins of specimens from the Caribbean and the Pacific versants, respectively. In both cases, proteins having isoelectric point between 9 and 10.5, and relative mol. mass of about 14–14.5 kDa represent more than 20% of venom proteins, and likely correspond to the myotoxic PLA2s and PLA2s homologues. A visual comparison revealed that twelve spots were exclusively present in the venom from the Caribbean versant and nine spots exclusively present in the venom from the Pacific versant. The geographic distinctlysecreted spots were subjected to automated reduction, carbamidomethylation and in-gel tryptic digestion and the resulting tryptic peptides were analyzed by MALDI-TOF mass fingerprinting followed by amino acid sequence determination of selected doubly- and triply-charged peptide ions by collision-induced dissociation tandem mass spectrometry (Alape-Giro´n et al., 2008). The sequences were submitted to BLAST similarity searches, which allowed their unambiguous assignment to known protein families (Tables 2 and 3). Two of the SVMPs exclusively found in the venom from specimens of the Pacific versant correspond to cDNAs which have been already sequenced. The protein in spot number 64 is the hemorrhagic PI-SVMP BaP1 (Watanabe et al., 2003), which represents almost 10% of the venom proteins and has the accession number Q072L4 at Swiss-Prot. The cDNA

Table 1 Relative occurrence of protein families (in percentage of the total HPLC separated proteins) in pools of venoms from adult specimens of B. asper from populations of the Caribbean and the Pacific versants of Costa Rica.

Protein family

Medium size disintegrin DC-fragments Total PLA2 K49 PLA2 D49 PLA2 CRISP Serine proteinase L-Amino acid oxidase C-type lectin-like Total SMVPs PI-SMVPs PII-SMVPs PIII-SMVPs

% of total venom proteins Caribbean

Pacific

2.1 <0.1 28.8 18.8 10.0 0.1 18.2 9.2 0.5 41.0 32.2 – 8.8

1.4 <0.1 45.1 36.0 9.1 0.1 4.4 4.6 0.5 44.0 30.0 0.7 13.3

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Fig. 4. Two dimensional electrophoresis of the proteins from pooled B. asper venoms from adults of the Caribbean (A) and the Pacific (B) versants of Costa Rica. The proteins contained in 200 mg of each venom were separated on an IPG strip with a pH gradient from 3 to 11 and the second dimension was done on a 12% polyacrylamide gel. The separated proteins were stained with Comassie Brillant blue and densitometric analysis was performed using the program ImageQuant TL from GE healthcare. For each protein the relative molecular mass, isoelectric point (IP) and relative proportion (in percentage of the total proteins in the gel) are indicated in Tables 2 and 3.

corresponding to the PII-SVMP in spot 54 (accession number Q072L5 at Swiss-Prot) has been used to generate antibodies which neutralize the hemorrhagic activity of the whole B. asper venom (Arce-Estrada et al., 2009).

Prominent ontogenetic changes occur in the enzymatic and toxic activities of B. asper venoms from Costa Rica (Gutie´rrez et al., 1980; Chaves et al., 1992). Venoms from newborn specimens of each of the two versants are more proteolytic, hemorrhagic, edema-forming and lethal than venoms from adult specimens from the same versant, whereas adult venoms display higher myotoxic and phospholipase A2 activities (Gutie´rrez et al., 1980). Accordingly, in venoms from the newborns of both versants the major PLA2s are acidic proteins (Moreno et al., 1988). Furthermore, the basic PLA2 homologues are completely absent in the venom from newborn specimens of the Caribbean, but reach a high proportion of the venom from all adults analyzed using a cathodic electrophoretic system for basic

proteins under native conditions (Lomonte and Carmona, 1992). In experimental envenomations, the venom from newborns of the Caribbean versant was also reported to have a higher defibrinating activity than that from adult specimens from the same versant (Chaves et al., 1992).

Similar ontogenetic differences in the toxic profile of venoms from newborns and adults have been reported to occur in B. asper specimens from Antioquia, Colombia, and furthermore, the venom from newborns was found to have a higher coagulant activity than the venom from adults (Saldarriaga et al., 2003).

In accordance with the above-cited biochemical and toxinological data, the detailed comparative proteomic analysis of venom proteins of newborns and adults from both versants in Costa Rica revealed prominent ontogenetic changes in these geographic populations (Alape-Giro´n et al., 2008). The variation is particularly striking, regarding the balance between two of the most common venom protein families, i.e. metalloproteinases and PLA2 (Fig. 5).

Among Caribbean snakes, neonate specimens express in their venom neither the major K49 PLA2 molecules nor the abundant PI-metalloproteinases characterized in adult venoms, but instead contained a larger proportion of D49 PLA2s and of PIII-metalloproteinases, including at least three molecules not observed in adults. These neonate specific PIII-metalloproteinases account for about 14% of the total venom proteins. A similar ontogenetic trend occurs in specimens from the Pacific versant: neonates express larger amounts of D49 PLA2 than K49 PLA2s, the content of PI-metalloproteinases is lower in neonate than in adult venoms, and the secreted PIII-metalloproteinases also show age-dependent qualitative and quantitative variations. The ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to subdue prey may change with the age of the snake. The higher content of PIII-metalloproteinases in venoms of neonates than in those of adults is in agreement with their reported more potent hemorrhagic activity (Gutie´rrez et al., 1980; Chaves et al., 1992) and the lower content of basic PLA2 homologues with their lower myotoxic activity (Gutie´rrez et al., 1980).

4. Individual variability in the venom proteome of B. asper Individual variations were also observed in the pattern of proteins of 116 specimens analyzed using starch gel electrophoresis (Jime´nez-Porras, 1964a). These electrophoretic variations did not correlate with either gender, size of the animal, duration of its captivity or differences in climate, season or diet. Individual variations in the pattern of PLA2 homologues was also described for 17 individuals of the Caribbean versant and 41 individuals of the Pacific versant analyzed using a cathodic electrophoretic system for basic proteins under native conditions (Lomonte and Carmona, 1992). In both cases, some individuals from different versants are reported to have the same pattern than individuals of the other versant.

To investigate venom variability among B. asper specimens from Costa Rica, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11

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Table 2 Proteins separated by 2D electrophoresis from B. asper venom from Costa Rica (Caribbean versant).

Spot number

Rel. mol. mass

I.P.

% in gel

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

112 104 65 65–60 56 56 56 55 55 51 51 47 42 42 42 42 38 38 38 38 38 36 35 35 34 34 34 34 42 36-28 34 34 34 33 33 33 28 33 32 32

8.0–8.5 7.2 8.0 5.5–6.5 7.2 8.0 7.5 6.8 6.0 7.0 9.2 9.7 4.5 6.0 6.2 6.3 6.3 6.7 8.3 0.9 7.3 7.7 9.1 6.0 5.1 8.2 5.4 9.2 6.2 5.7 6.5 8.8 10.7 6.5 9.5 8.5 8.5 10.0 6.8 10.7

1.2 2.3 0.8 8.4 2.1 0.4 0.6 2.1 2.1 0.6 0.2 0.2 0.2 0.5 0.3 0.2 0.1 0.9 0.1 0.1 0.8 0.7 0.4 0.1 0.3 0.6 0.7 0.6 0.5 1.2 0.5 0.4 0.6 0.6 0.7 0.5 0.1 1.2 2.6 0.6

Prot. family

PIII-SVMP

Serine Prot.

PIII-SVMP

Serine Prot.

Serine Prot.

CRISP PI-SVMP

K49 PLA2

venoms from snakes from Pacific regions were compared (Alape-Giro´n et al., 2008). Among Caribbean B. asper venoms, PLA2 molecules, serine proteinases, Znþ2-dependent metalloproteinases and, to a minor extent, LAO, displayed the largest variability. A similar trend was observed in venoms from the Pacific B. asper population, with PLA2s, serine proteinases, LAO and the Znþ2-dependent metalloproteinases exhibiting the most noticeable expression variation. Hence, within both B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations.

The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different geographical locations and of different ages has long been appreciated by herpetologists and toxinologists, and appears to be a general feature of venoms (Chippaux et al., 1991). The disparity of symptoms in victims of the same species of snake has long alerted clinicians to the requirement for more specific antivenoms (Jime´nez-Porras, 1964b; Chippaux et al., 1991).

Thus, besides ecological and taxonomical implications, knowledge of the venom variability may have an impact in

Spot number

Rel. mol. mass

I.P.

% in gel

41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

32 32 30 30 32-31 29 28 28 28 27 28 28 26 24 24 24 24 22 22 22 21 14.5-16 14.5–16 16 15 15 14.5 14.5 14.5 14.5 14–14.5 14.5 12 13 12.6 13 13 13 13 13

10.0 7.0 8.2 7.7 5.1 5.7 9.0 7.3 8.8 9.3 5.7 6.0 9.5-10 4.7 5.2 8.3 6.2 8.5 5.4 10.7 9.2 4.7 5.0 5.7 9.0 8.8 5.2 4.5 6.1 5.2

2.3 2.4 0.9 0.2 0.4 0.2 0.8 1.6 0.4 5.7 1.0 1.5 3.8 0.1 0.1 0.1 0.2 0.3 0.2 0.2 0.1 0.3 2.9 0.1 0.4 0.9 0.2 0.7 2.0 0.8 27.4 3.1 0.5 0.2 0.3 0.1 0.2 0.2 0.5 0.2

6.2 5.0 7.2 6.1 8.5 10.5 6.7 10 8.2

Prot. family

PI-SVMP

PI-SVMP PI-SVMP

D49 PLA2

Disintegrin

the treatment of snakebite victims and in the selection of specimens for antivenom production (Gutie´rrez et al., 2009).

5. Potential applications of proteomics in the production of therapeutic antivenoms Adequate treatment of snakebite envenoming is critically dependent on the intravenous administration of heterologous antivenom exhibiting the ability to reverse venom-induced coagulopathy, hemorrhage, hypotensive shock and other signs of systemic envenoming (Gold et al., 2002; Gutie´rrez et al., 2006). Conventional antivenoms are produced by fractionation of plasma from horses or sheep hyperimmunized with a single whole venom or a mixture of venoms from different species from a defined geographical area (Lalloo and Theakston, 2003; Gutie´rrez et al., 2006). Snake venoms are a complex mixture of proteins and many venom proteins are not toxic and many low molecular mass venom proteins are highly toxic but weakly immunogenic. Consequently the dose-efficacy of antivenoms is thought to suffer from the presence of

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Table 3 Proteins separated by 2D electrophoresis from B. asper venom from Costa Rica (Pacific versant).

Spot number

Rel. mol. mass

I.P.

% in gel

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

116 97 97 95 93 93 90 90 66 66 66 64 64 64 63 57 57 57 57 55 49 49 49 48 48 47 46 46 43 37 36 36 36 35 35 35 35 35 35 34.5 34 34 32

7.0 7.0 8.5 7.0 8.0 8.3 8.1 8.3 5.5–7.0 8.0–8.7 8.2 8.7–8.2 5.8 5.8 5.8 6.2–7.2 8.0 8.7 8.2 7.2 8.2 8.3 8.5 5.2 7.5 7.7 10.3 9.2 5.1 8.3 6.3 8.3 8.6 6.7 5.8 6.2 6.5 6.7 9.0 5.7 6.7 8.3 8.0

0.1 0.2 0.9 0.2 0.1 0.1 0.1 0.1 9.6 1.2 0.1 1.6 0.1 0.1 0.1 2.2 0.1 0.1 0.05 0.1 0.1 0.1 0.1 0.1 0.05 0.1 1.9 0.5 0.1 0.2 0.3 0.1 0.4 0.1 0.7 1.2 0.8 0.2 0.3 0.05 0.1 0.5 0.1

Prot. family

LAO

PIII-SVMP

CRISP

redundant antibodies to non-toxic molecules and a lack of potent neutralizing antibodies to small molecular weight toxins. This in turn results in the need for high volumes to effect treatment and a consequent increase in the risk of serum sickness and anaphylactic adverse effects. Further, current antivenoms consist of purified immunoglobulins or immunoglobulin fragments which have reduced the incidence and severity of treatment-induced serum sickness and anaphylactic shock. However, the venom-immunization protocols have not significantly changed in over a century and make no attempt to direct the immune response to the most toxic venom proteins. The administration of non-toxin-specific immunoglobulins to the snake-bitten patients increases the risk of adverse effects, and thus, the cost of medical care (Gold et al., 2002; Gutie´rrez et al., 2006). Therefore, the therapeutic value of the antivenoms could be enhanced by restricting antibody specificity to the venom proteins playing a role in the pathophysiology of envenomation. In B. asper venom from adult specimens Zn2þ-dependent metalloproteinases

Spot number

Rel. mol. mass

I.P.

% in gel

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86

31 31 31 31 31 31 30 30 31 29 31 29 29 29 28 28 28 28 28 28 26 26 24 21.5 18 17 16 15 15 15 15 15 14.5 14.5 14.5 14 14 14 13.5 13.5 13.5 13.5 13

10.0 7.2 6.2 8.4 9.4 8.6 6.2 6.4 9.5 6.7 7.4 10.0 9.5 7.5 8.5 9.8 5.5 10.5 10.0 5.6 9.2 6.5 6.0 5.8 9.8 8.6 5.6 8.3 9.0 5.8 7.0 6.0 5.2 8.8 7.2 6.0 9.2–11.0 6.5 5.8 6.3 6.0 6.5 8.0

5.4 5.7 0.3 0.2 0.5 2.0 0.8 0.2 3.8 0.3 0.7 0.4 0.4 0.3 0.3 0.5 0.1 0.8 0.7 0.1 9.85 0.2 0.2 0.2 0.2 0.3 1.8 0.6 0.7 0.3 0.8 4.1 1.6 0.8 0.3 1.7 24.1 3.5 0.1 0.2 0.2 0.1 0.3

Prot. family

PII-SVMP

PI-SVMP

PI-SVMP

D49 PLA2 C-type lectin

D49 PLA2

Disintegrin Disintegrin

account for 41–43.8% of the venom proteins (Alape-Giro´n et al., 2008). Specific antibodies against a PIII or a PII metalloproteinase, which are minor venom components, have been shown to neutralize the hemorrhagic activity of the whole venom (Rucavado et al., 1995; Arce-Estrada et al., 2009). Therefore, it seems likely that despite of the complexity in venom protein composition, a defined selection of few toxins having wide cross-reactivity with toxins playing key roles in the pathophysiology of envenoming, will be suitable to prepare novel immunogenic mixtures for the production of more effective therapeutic antivenoms. Clearly, the design of such immunogenic mixtures depends on a detailed knowledge of the venom proteomes, and therefore the proteomic characterization of B. asper venom could help to generate better antivenoms to treat patients bitten by this species.

Several antivenoms are produced in Latin America using different venoms in the immunization schemes (EspinoSolı´s et al., 2009). Each of these antivenoms is effective in variable degree against envenomations by snake venoms

´ n et al. / Toxicon 54 (2009) 938–948 A. Alape-Giro

Fig. 5. Ontogenetic comparison of protein families B. asper venom from adults and neonates of the Caribbean (A) and the Pacific (B) versants from Costa Rica. DC, disintegrin/cysteine-rich fragment from PIII SVMPs; C-lectin, C-type lectin-like protein; PLA2, phospholipase A2; CRISP, cysteine-rich secretory protein; LAO, L-amino acid oxidase; SP, serine proteinase.

not included in the immunization protocol, demonstrating the high degree of immunological cross-reactivity between Central and South American crotaline snake venoms. A practical consequence of this fortunate circumstance is the possibility of using these heterologous antivenoms to circumvent the restricted availability of species-specific antivenoms in some regions. However, before testing in clinical trials, antivenoms need to be evaluated experimentally by assessing their neutralizing ability against the most relevant toxic and enzymatic activities of snake venoms. To this end, we have developed a simple proteomic protocol, which we call ‘‘antivenomics’’, for investigating the immunoreactivity, and thus the potential therapeutic usefulness, of antivenoms towards homologous and heterologous venoms (Lomonte et al., 2008).

Antivenomics is based on the immunodepletion of toxins upon incubation of whole venom with antivenom followed by the addition of a secondary antibody. Antigen–antibody complexes immunodepleted from the reaction mixture contain the toxins against which antibodies in the antivenom are directed. By contrast, venom components that

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remain in the supernatant are those which failed to raise antibodies in the immunized animals, or which triggered the production of low-affinity antibodies. These components can be easily identified by comparison of reversephase HPLC separation of the non-precipitated fraction with the HPLC pattern of the whole venom previously characterized by venomics. According to their immunoreactivity towards antivenoms, toxins may be conveniently classified as: C-toxins, completely immunodepleted toxins; P-toxins, partly immunodepleted toxins; and N-toxins, non-inmunodepleted proteins. Assuming a link between the in vitro toxin immunodepletion capability of an antivenom and its in vivo neutralizing activity towards the same toxin molecules, improved immunization protocols should make use of mixtures of immunogens to generate high-affinity antibodies against class P- and class N-toxins.

On the other hand, our antivenomics approach is simple and easy to implement in any protein chemistry laboratory, and may thus represent another useful protocol for investigating the immunoreactivity, and thus the potential therapeutic usefulness, of antivenoms towards homologous and heterologous venoms (Gutie´rrez et al., 2009). At this respect, Calvete et al. from Venezuela and Costa Rica have compared the venom proteomes of B. colombiensis, B. asper, and B. atrox using venomics and antivenomics approaches (Calvete et al., 2009b). This study showed that the closest homologues 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 approximately 65– 70% of their venom proteomes (Fig. 6A and B). Further, the virtually indistinguishable immunological cross-reactivity of a Venezuelan ABC antiserum (raised against a mixture of B. colombiensis and Crotalus durissus cumanensis venoms) and the Costa Rican ICP polyvalent antivenom (generated against a mixture of B. asper, C. simus, and Lachesis stenophrys venoms) towards the venoms of B. colombiensis and B. asper (Fig. 6C and D), supports this view and suggests the possibility of indistinctly using these antivenoms for the management of snakebites by any of these Bothrops species.

6. Concluding remarks Costa Rica is bisected northwest-to southeast by a series of mountain ranges, which define two versants, one which faces the Caribbean Sea and another towards the Pacific Ocean. B. asper is distributed in both versants and the studies on the venom proteomes from specimens collected in the Caribbean and in the Pacific regions evidenced marked geographical, ontogenetic, and individual variations. The most abundant toxins in the B. asper venom proteome in populations from the Caribbean and Pacific versants have been identified and the relative amounts of different toxin families determined, thus providing a survey of the gene expression within the venom gland of this species and opening novel perspectives in the study of this venom.

The studies on the B. asper venom proteome raised very interesting questions in our search for understanding the biological role of this venom. Are the differences between Caribbean and Pacific populations from Costa Rica the

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A. Alape-Giro´n et al. / Toxicon 54 (2009) 938–948

Fig. 6. Comparison of the composition and crossed immunoreactivity of B. asper and B. colombiensis venoms. Panels A and B display the reverse-phase HPLC separations of the venom proteins of B. asper from the Pacific versant of Costa Rica, and B. colombiensis, respectively. Proteins exhibiting identical chromatographic elution time and containing common tryptic peptide sequences are labeled with asterisks. PLA2 molecules Bas(P)-12 and Bcol-17, labeled with closed circles, share peptide ion sequences but elute at different times. For detailed information consult Calvete et al. (2009b). Panels C and D show, respectively, reversephase separations of B. colombiensis venom proteins recovered after incubation of the crude venom with the anti-bothropic and anti-crotalic Venezuelan antivenom and with the polyvalent (Crotalinae) Costa Rican antivenom, followed by rabbit anti-horse IgG antiserum and immunoprecipitation. The inserts show an SDS-PAGE analysis of b-mercaptoethanol-reduced fractions labeled in the chromatograms. Peaks 13–17 were identified as PLA2 molecules; peaks 21 and 28 correspond to PI-SVMPs. Peak labeled ‘‘a’’ in panel D correspond to IgG fragments. CID-MS/MS of tryptic peptide ions from the 50 kDa and the 25 kDa protein bands in fractions h–l (panel C), g–j (panel D) were identified by MS/MS as IgG fragments of the primary and secondary antibodies used for immunoprecipitation.

result of adaptive processes to different diets and ecological scenarios? Or are these differences the result of more ‘neutral’ evolutionary mechanisms operating in the variations of venom composition? Which is the composition of the venom proteome in other B. asper populations from different geographic locations? Are the variations in diet composition between juvenile and adult specimens of B. asper directly related to the strikingly different venom proteomes?

Snake composition is under genetic control and therefore proteome studies can provide molecular markers for taxonomical purposes. The characterization of B. asper venom proteome and its comparison with the venom proteomes of B. atrox and B. colombiensis from Venezuela, has contributed to clarify the taxonomic relationships among those species (Calvete et al., 2009b), and has expanded the therapeutic range of existing antivenoms against B. asper and B. colombiensis.

Besides their biological relevance, the characterization of the variability of B. asper venom has also implications for understanding the characteristics of envenomations in humans. The results explain previous findings that venoms from newborn B. asper (whether from the Caribbean or the

Pacific versant of Costa Rica) are more lethal and tend to induce bleeding and proteolysis, whereas those of adult specimens have a higher myotoxic activity. Clinical reports also indicate that envenomations by juvenile B. asper specimens are associated with prominent blood coagulation alterations and haemorrhagic symptoms (Otero et al., 1996), despite the low amount of venom that small size specimens may inject in a bite.

Finally, the geographic, ontogenetic and individual intraspecific variability in B. asper venom composition revealed by the proteomic studies highlights the necessity of using pooled venoms in antivenom production and provides information towards a more rational selection of immunogenic mixtures in the future search of more effective therapeutic antivenoms.

Acknowledgments This work has been financed by grants from the Ministerio de Educacio´n y Ciencia, Madrid, Spain (BFU2007-61563), CRUSA-CSIC (2007CR0004), and Vicerrectorı´a de Investigacio´n (741-A7-611) from the University of Costa Rica.

´ n et al. / Toxicon 54 (2009) 938–948 A. Alape-Giro

Conflicts of interest All authors declare that there are no conflicts of interest.

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