Snake Venomics of the Lancehead Pitviper Bothrops asper: Geographic, Individual, and Ontogenetic Variations Alberto Alape-Giro ´ n,†,‡ Libia Sanz,§ Jose´ Escolano,§ Marietta Flores-Dı´az,† Marvin Madrigal,† Mahmood Sasa,† and Juan J. Calvete*,§ Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose´, Costa Rica, Departamento de Bioquı´mica, Escuela de Medicina, Universidad de Costa Rica, San Jose´, Costa Rica, and Instituto de Biomedicina de Valencia, C.S.I.C., Jaume Roig 11, 46010 Valencia, Spain Received April 30, 2008
We report the comparative proteomic characterization of the venoms of adult and newborn specimens of the lancehead pitviper Bothrops asper from two geographically isolated populations from the Caribbean and the Pacific versants of Costa Rica. The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The two B. asper populations, separated since the late Miocene or early Pliocene (8-5 mya) by the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range, contain both identical and different (iso)enzymes from the PLA2, serine proteinase, and SVMP families. Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. To investigate venom variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions were compared. Within each B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The occurrence of intraspecific individual allopatric variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations. Analysis of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat revealed prominent ontogenetic changes in both geographical populations. Major ontogenetic changes appear to be a shift from a PIII-SVMP-rich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA2 molecules than in the neonates. In addition, the ontogenetic venom composition shift results in increasing venom complexity, indicating that the requirement for the venom to immobilize prey and initiate digestion may change with the size (age) of the snake. Besides ecological and taxonomical implications, the geographical venom variability reported here may have an impact in the treatment of bite victims and in the selection of specimens for antivenom production. The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different gegraphical location and age has long been apreciated by herpetologist and toxinologists, though detailed comparative proteomic analysis are scarce. Our study represents the first detailed characterization of individual and ontogenetic venom protein profile variations in two geographical isolated B. asper populations, and highlights the necessity of using pooled venoms as a statistically representative venom for antivenom production. Keywords: Snake venomics • Bothrops asper • snake venom protein families • proteomics • viperid toxins • N-terminal sequencing • mass spectrometry • geographical venom variation • individual venom variation • ontogenetic shift
Introduction The genus Bothrops (subfamily Crotalinae of Viperidae) comprises 32 (http://www.reptile-database.org) or 37 species1 of pitvipers, commonly referred as lanceheads, which are widely distributed in tropical Latin America, from northeastern Mexico * Address correspondence to: Juan J. Calvete, Instituto de Biomedicina de Valencia, C.S.I.C., Jaime Roig 11, 46010 Valencia, Spain. Phone, +34 96 339 1778; fax, +34 96 369 0800; e-mail,
[email protected]. † Instituto Clodomiro Picado, Universidad de Costa Rica. ‡ Departamento de Bioquı´mica, Escuela de Medicina, Universidad de Costa Rica. § Instituto de Biomedicina de Valencia.
3556 Journal of Proteome Research 2008, 7, 3556–3571 Published on Web 06/17/2008
to Argentina, and the southern parts of the lower Caribbean islands.1 The species of this genus are responsible for the vast majority of snakebites in Central and South America.2 In this regard, the most important species are Bothrops asper (Central America and northern South America), Bothrops atrox (tropical lowlands of northern South America east of the Andes) and Bothrops jararaca (southern Brazil, Paraguay and northern Argentina). Without treatment, the fatality rate is estimated to be about 7%, but with an appropriate antivenom therapy, it can be reduced to 0.5-3%.1 Nevertheless, many victims of B. asper snakebite suffer from life threatening sequelae due to the tissuedamaging effects of the venom.2,3 10.1021/pr800332p CCC: $40.75
2008 American Chemical Society
research articles
Proteomics of Bothrops asper During the period 1990-2000, a total of 5550 snakebite accidents were reported in health centers in Costa Rica.4 Most of the snakebites occurred in the Caribbean, South and Central Pacific, and North regions of the country, where B. asper, the only Bothrops species present in Central America, is very common.4 In Costa Rica, B. asper (commonly named ”terciopelo”) is responsible for almost half of all snakebite accidents.5 The victims of B. asper bites show conspicuous local tissue damage characterized by dermonecrosis, blistering, edema, local hemorrhage and myonecrosis,6,7 and, in severe cases, defibrin(ogen)ation, thrombocytopenia, platelet hypoaggregation, bleeding distant from the bite site, disseminated intravascular coagulation, cardiovascular shock and acute renal failure.6–8 Venoms represent the critical innovation in ophidian evolution that allowed advanced snakes to transition from a mechanical (constriction) to a chemical (venom) means of subduing and digesting prey and represents a key adaptation that has played an important function in the diversification of these animals. Venom toxins 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.9–12 Venoms produced by snakes of the family Viperidae (vipers and pit vipers) contain proteins that interfere with the coagulation cascade, the normal hemostatic system and tissue repair.13,14 Snake venom proteins belong to only a few major protein families, including enzymes (serine proteinases, Zn2+-metalloproteinases, L-amino acid oxidase, group II PLA2) and proteins without enzymatic activity (disintegrins, C-type lectins, natriuretic peptides, myotoxins, CRISP toxins, nerve and vascular endothelium growth factors, cystatin and Kunitz-type proteinase inhibitors). However, viperid venoms depart from each other in the composition and the relative abundance of toxins.15 Snake venom composition may retain information on its evolutionary history, and may thus have a potential taxonomical value.16 In addition to understanding how venoms evolve, characterization of the protein/peptide content of snake venoms also has a number of potential benefits for basic research, clinical diagnosis, development of new research tools and drugs of potential clinical use, and for antivenom production strategies.17 To explore the putative venom components, several laboratories have carried out transcriptomic analyses of the venom glands of viperid (Bitis gabonica,18 Bothrops insularis,19 Bothrops jararacussu,20 B. jararaca,21 Agkistrodon acutus,22,23 Echis ocellatus,24 Lachesis muta,25 and Sistrurus catenatus edwardsii26), elapid (Oxyuramus scutellatus27), and colubrid (Philodryas olfersii28) snake species. Transcriptomic investigations provide catalogues of partial and full-length transcripts that are synthesized by the venom gland. However, transcriptomes include translated and nontranslated mRNAs, transcripts encoding nonsecreted proteins, housekeeping and cellular, in addition to toxin precursor genes. Moreover, the transcriptome does not reflect within-species ontogenetic,29,39 individual and geographic31 heterogeneity of venoms, which may account for differences in the clinical symptoms observed in accidental envenomations. Geographic variability concerning the toxic and enzymatic activites of B. asper venoms from the Caribbean versus the Pacific regions of Costa Rica has been documented.32–34 Venom of specimens from the Caribbean region exhibited enhanced procoagulant hemorrhagic and myonecrotic effects, whereas the venom of specimens from the Pacific versant displayed a
higher proteolytic activity. Variations in the pattern of PLA2 isoforms in relation to geographic origin have been also reported.35,36 Prominent ontogenetic changes in the toxic and enzymatic activites of B. asper venoms from Costa Rica34,37 and Colombia38 have also been noticed. This phenomenon appears to be linked to a shift in the feeding habits of juvenile versus adult snakes, from cold-blooded (frogs and lizards) to warm-blooded (mammals) prey.38 Clinical reports indicated that envenomation by juvenile B. asper specimens is associated to prominent blood coagulation alterations and hemorrhagic symptoms,39 despite the low amount of venom that a small size specimens may inject in a bite. In line with the clinical observations, the venom from newborns appeared to be more procoagulant in vitro and, in experimental envenomations, showed a higher defibrinating and hemorrhagic activities and a lower myotoxicity than the venom from adult specimens.34,37 On the other hand, the latter displayed higher phospholipase A2 activity and had a higher number of PLA2 isoforms.35 Besides its intrinsic biological relevance, the characterization of the ontogenetic variability of B. asper venom also has implications to understand the characteristics of envenomations in humans. To address the need for detailed proteomic studies of snake venoms, we have initiated a snake venomics project whose long-term goal is the in-depth analysis of viperid venom proteomes. To date, we have reported the protein composition of the venoms from the North American rattlesnakes Sistrurus miliarius barbouri,40,41 Sistrurus catenatus (subspecies catenatus, tergeminus and edwardsii),41 the Tunisian vipers Cerastes cerastes, Cerastes vipera and Macrovipera lebetina,42 the Afrotropical species Bitis arietans (Ghana),43 B. gabonica gabonica,44 Bitis gabonica rhinoceros, Bitis nasicornis, and Bitis caudalis,16 and the Central and South American pitvipers Atropoides nummifer,45 Atropoides picadoi,45 L. muta,31 Lachesis stenophrys,31 Bothriechis lateralis,46 and Bothriechis schlegeli.46 Here, we describe the proteomes of the venoms of B. asper adult and neonate speciments from the Caribbean and the Pacific versants of Costa Rica. Geographic, individual, and ontogenetic venom composition variations are reported.
Experimental Section Venom Samples. Venom samples were obtained from B. asper specimens collected in the Caribbean (Distrito Quesada, San Carlos, Alajuela province) and the Pacific (Distrito de Sabanillas, Acosta, San Jose´ province) regions of Costa Rica (Figure 1, Table 1) and kept in captivity at the Serpentarium of Instituto Clodomiro Picado (Universidad de Costa Rica, San Jose´). In both cases, the collection areas comprised 145-150 Km2. Venoms from adult specimens (15 from the Caribbean and 11 from the Pacific regions) and from 6-7 weeks old snakes (at least 20 from each versant) were collected by snake biting on a parafilm-wrapped jar. Crude venoms were centrifuged at low speed to remove cells and debris, lyophilized, weighed on a microbalance, and stored at -20 °C until used. Venom pools were prepared by mixing equal amounts of samples from at least 11 specimens from both sexes from the Caribbean or from the Pacific regions. Isolation and Proteomic Characterization of Venom Proteins. Proteins from 2-5 mg of crude, lyophilized venoms were separated by reverse-phase HPLC using an ETTAN LC HPLC system (Amersham Biosciences) and a Lichrosphere RP100 C18 column (250 × 4 mm, 5 µm particle size) as described.15,16,31,41,44–46 The relative abundances (% of the total Journal of Proteome Research • Vol. 7, No. 8, 2008 3557
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Figure 1. Geographic origin of the venoms. Physical map of Costa Rica showing the geographical origin of the B. asper venoms from the Pacific, Bas(P), and the Caribbean, Bas(C), versants of Costa Rica (Table 1), whose proteomic characterization is reported in this work. Collection areas comprised 145-150 Km2. The Guanacaste, Tilara´n, Central Volcanic and Talamanca, mountainous chains extending the entire length of the country from the Northwest to the Southeast and dividing the Caribbean and the Pacific regions, are labeled.
Table 1. Phenotypic Characteristics of the B. asper Specimens from the Caribbean and Pacific Versants of Costa Rica (Figure 1) Whose Venoms Are Described in This Work Caribbean versant
Pacific versant
(Distrito Quesada, Canto´n de San Carlos, Provincia de Alajuela)
(Distrito de Sabanillas, Canto´n de Acosta, Provincia de San Jose´)
specimen no.
sex
size
specimen no.
sex
size
472 545 355 384 733 734 544 446 449 447 406 560 473 489 404
Female Female Female Female Female Female Female Female Male Male Male Male Male Male Male
76 cm 139cm 139cm 146cm 147cm 152cm 161cm 164cm 82 cm 110cm 113cm 124cm 136cm 140cm 146cm
389 497 341 496 499 429 735 580 607 611 736
Female Female Female Female Female Female Female Male Male Male Male
104cm 120cm 123cm 132cm 144cm 152cm 154cm 146cm 129cm 117cm 134cm
venom proteins) of the different protein families in the venoms were estimated from the relation of the sum of the areas of the reverse-phase chromatographic peaks containing proteins from the same family to the total area of venom protein peaks. Isolated protein fractions were subjected to N-terminal sequence analysis using a Procise instrument (Applied Biosystems, Foster City, CA). Amino acid sequence similarity searches were performed against the available databanks using the BLAST program47 at http://www.bork.embl-heidelberg.de. The molecular masses of the purified proteins were determined by SDS-PAGE (on 12-15% polyacrylamide gels) and by electrospray ionization (ESI) mass spectrometry using an Applied Biosystems QTrap 2000 mass spectrometer48 operated in Enhanced Multiple Charge mode in the range m/z 600-1700. Protein bands of interest were excised from a Coomassie Brilliant Blue-stained SDS-PAGE and subjected to automated 3558
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in-gel digestion, mass fingerprinting, and CID-MS/MS as described.15,16,31,41,44–46 CID spectra were interpreted manually or using a licensed version of the MASCOT program (http:// www.matrixscience.com) against a private database containing 927 viperid protein sequences deposited in the Swiss-Prot/ TrEMBL database (Knowledgebase Release 12 of July 2007; http://us.expasy.org/sprot/; 212 in Swiss-Prot, 715 in TrEMBL) plus the previously assigned peptide ion sequences from snake venomics projects carried out in our laboratory.15,16,31,40–46 Variation in venom protein composition between taxa was estimated using a Protein Similarity Coefficient [PSCab ) [2 × (no. of proteins shared between a and b)/(total number of distinct proteins in a + total number of distinct proteins in b)] × 100] based on bandsharing coefficient used to compare individual genetic profiles based on multilocus DNA fingerprints,49 and previously described criteria.15,16,31,40–46
Proteomics of Bothrops asper
Figure 2. Reverse-phase HPLC separation of the proteins from a pool of venoms of B. asper adult specimens from the Caribbean region of Costa Rica. Two milligrams of total venom proteins was applied to a Lichrosphere RP100 C18 column, which was then developed with the following chromatographic conditions: isocratically (5% B) for 10 min, followed by 5-15% B for 20 min, 15-45% B for 120 min, and 45-70% B for 20 min. Fractions were collected manually and characterized by N-terminal sequencing, ESI mass spectrometry, tryptic peptide mass fingerprinting, and CID-MS/MS of selected doubly or triply charged peptide ions. The results are shown in Table 2.
2-D SDS-PAGE. The proteins of the venom pools were separated by 2-D SDS-PAGE using an IPGphor (Amersham Bioscience, Uppsala, Sweden) instrument. For isolectric focusing, 200 µg of total venom proteins (in 250 µL of 8 M urea, 4% CHAPS and 0.5% IPG buffer) was loaded on a 13 cm IPG strip (pH range 3-11) and the following focusing conditions were used: 30 V for 6 h, 60 V for 6 h, 500 V for 1 h, 1000 V for 1 h, and 8000 V for 2 h. SDS-PAGE was done in a 16 cm 12% polyacrylamide gel. Coomassie blue was employed for protein staining.
Results and Discusion Geographical Variation between the Venom Proteomes of B. asper Specimens from the Caribbean and Pacific Regions of Costa Rica. Previous studies have shown that venoms from adult B. asper specimens from the Caribbean versant of Costa Rica are more hemorrhagic and myonecrotic, whereas those from Pacific regions are more proteolytic, having similar lethality, edema-forming activity, and hemolytic effect.34 For the characterization of their overall protein composition and gaining a deeper insight into geographic and individual variations, the venoms of 15 (from Caribbean regions) and 11 (from Pacific regions) (Figure 1, Table 1) adult B. asper specimens were analyzed, both as geographic pools and individually. Pooled crude venoms (herein called Bas(C) and Bas(P) according to their Caribbean or Pacific origin) were fractionated by reverse-phase HPLC (Figures 2 and 4), followed by analysis of each chromatographic fraction by SDS-PAGE (Figures 3 and 5), N-terminal sequencing, and MALDI-TOF mass spectrometry (Table 2). Protein fractions showing single electrophoretic band, molecular mass, and N-terminal sequence were straightforwardly assigned by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST) to a known protein family. Protein fractions showing heterogeneous or blocked Ntermini were analyzed by SDS-PAGE and the bands of interest were subjected to automated reduction, carbamidomethylation,
research articles and in-gel tryptic digestion. The resulting tryptic peptides were then 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. Despite its medical relevance, the Swiss-Prot/TrEMBL UniProt Knowledgebase (release of 15 January 2008) contains only 6 full-length B. asper venom toxin sequences: four PLA2 myotoxins (P20474, P24605, Q9PVE3, and P0C616), a serine proteinase (Q072L6), and one PI (P83512 and Q072L4) and one PII (Q072L5) metalloproteinases. Thus, except for these proteins, all of which were found in the pooled venoms (Table 2), the peptide mass fingerprinting approach alone was unable to identify any protein in the databases. In addition, as expected from the rapid amino acid sequence divergence of venom proteins evolving under accelerated evolution,50–56 with a few exceptions, the product ion spectra did not match any known protein. Hence, mass spectra were manually interpreted for de novo sequencing and the CID-MS/ MS-deduced peptide ion sequences (Table 2) were submitted to BLAST similarity searches. High-quality MS/MS peptide ion fragmentation spectra yielded sufficient amino acid sequence information derived from almost complete series of sequencespecific b- and/or y-ions to unambiguously identify a homologue protein in the current databases. All the identified proteins displayed strong similarity with entries from Bothrops species, highlighting the close phylogenetic relationship of B. asper with the New World South American bothopoid genera.57 Closest homologues to B. asper proteins were venom proteins from B. atrox (medium-sized disintegrin P18618), B. jararacussu (serine proteinases AAB30013 and P81824; PIII-SVMPs Q7T1T5; PLA2 Q8AXY1; LAO Q6TGQ9), and B. jararaca (C-type lectinlike proteins P22028, P22029, and AAB47092; PIII-SVMP AAG48931), supporting the allocation of B. asper within the clade that includes B. atrox, B. jararaca and B. jararacussu.57 The 30-31 fractions isolated by reverse-phase HPLC from the Bas(A) (Figures 2 and 3) and Bas(P) (Figures 4 and 5) venoms comprised, respectively, about 30 and 27 different proteins (Table 2), which in both cases belong to 8 different groups of toxins, distributed into 4 major (SVMP, PLA2, serine protease, and L-amino acid oxidase (LAO)) and 4 minor (disintegrin, DC-fragment, C-type lectin-like, and cysteine-rich secretory protein (CRISP)) protein families (Figure 6). However, the venoms from the two B. asper populations exhibited distinct relative protein family abundances, which are listed in Table 3. Specifically, venom pooled from Caribbean specimens contained higher content of serine proteinases (410%), LAO (200%), and disintegrin (160%) than venom pooled from Pacific snakes, whereas the latter was enriched in PLA2s (160%) compared to the Caribbean specimens (Figure 7). The two B. asper populations also depart in the relative proportion of PI and PIII SVMPs. In addition to their different overall venom compositions, the venoms from each geographic population also showed distinct protein expression profiles, as judged by both reverse-phase HPLC (Figures 2 and 4) and 2D-SDS-PAGE (Figure 6). Knowledge of the within geographic locality venom variability is essential for isolating pharmacologically active fractions from crude venoms. Thus, except for the LAO molecule(s), which appears to be highly conserved in venoms from both B. asper populations, each protein family comprised different complements of molecules (Tables 2 and 3). For instance, the disintegrin found in the venom of B. asper (Caribbean) appears to be identical to medium-sized RGDdisintegrins from other Bothrops species, as B. atrox [P18618], Journal of Proteome Research • Vol. 7, No. 8, 2008 3559
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Figure 3. SDS-PAGE of reverse-phase separated fractions from the venom of adult B. asper from the Caribbean region of Costa Rica. SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom protein fractions displayed in Figure 2 and run under nonreduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 2.
B. insularis [AY736107], and B. jararacussu [DQ408681], but departs in residues 10GT11 from bothrasperin (10DA11) [Q072L5] secreted into the venom of B. asper from the Pacific versant. Moreover, B. asper (Caribbean) DC-fragment shows similarity to the DC-domains of B. jararacussu metalloprotease BOJUMET II [AY255004], whereas the N-terminal sequence of the DCfragment from B. asper (Pacific) is conserved in the DCdomains from a variety of PIII-metalloproteases from species of the genera Bothrops, Agkistrodon, Trimeresurus, Gloydius, Crotalus, Echis. The two B. asper populations contain both identical and different (iso)enzymes from the PLA2, serine proteinase, and SVMP families (Table 2). Our results confirm and extend previous studies by Moreno and co-workers35 and Lomonte and Carmona36 showing clear differences in the PLA2 isoform electrophoregrams from venoms of B. asper specimens from the Caribbean and Pacific regions of Costa Rica. Similarly, among the major proteins found in Sistrurus venoms, PLA2 proteins appear to be exceptionally divergent at both the intraand the interspecific level,41 suggesting that they have been the subject of strong balancing selection58 within, and diversifying selection between, taxa. Other studies have also shown that PLA2 genes show high levels of divergence between species and high levels of variation in the composition of myotoxic PLA2 molecules in different geographical populations of several pitvipers, including L. muta,31 Trimeresurus flavoviridis,59,60 Trimeresurus stejneri,61 and Bothrops neuwedi.62 Using a similarity coefficient, we estimate that the similarity of venom proteins between the two B. asper populations may be around 52%. Compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The uplift of the mountains of lower Central America, including the Guanacaste Mountain Range, Central Mountain Range, and Talamanca Mountain Range which presently separates the Caribbean and Pacific regions of Costa Rica (Figure 1), occurred in the late Miocene or early Pliocene (8-5 Mya) and culminated in the Pliocene with the closure of the Panamanian Portal.63 This uplift may have fragmented the original homogeneous lowland Costa Rican herpetofauna into allopatric Caribbean 3560
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and Pacific populations.64,65 In Costa Rica, B. asper occurs in lowlands up to an elevation of about 1500 m above sea level in both the Pacific and the Caribbean versants. These two snake populations, separated by the high mountain ridge ranging from approximately 1000 to 2000 m, and extending diagonally across the center the entire length of the country, represent reproductive isolated communities. The formation of such a physical barrier may have promoted (sub)speciation66 of the two separated Costa Rican B. asper populations. A subspecies is a taxonomic subdivision that ranks just below a species and comprises a geographically separated group of genetically distinct individuals whose members can interbreed. Subspecies usually arise as a consequence of geographical isolation within a species. In this sense, although our comparative proteomic analysis would support the classification of B. asper Caribbean and Pacific populations as subspecies, additional venomic analyses from areas of sympatry (i.e., Central Panama, where both forms might converge) are needed. In addition, detailed genomic analyses are also required to address this point and to assess whether the distinct venom protein profiles of Caribbean and Pacific B. asper populations arise from expression of population-specific genes or from distinct translation patterns of the same genome. Individual Variations among the Venoms of B. asper Specimens from Caribbean and Pacific Regions of Costa Rica. The use of pooled venoms stems from a requirement for a statistically representative venom, that is, for antivenom production, but identification of individual variations is removed from the researcher’s control. To investigate the degree of variability among specimens from the two B. asper populations, the reverse-phase HPLC protein profiles of 15 venoms from Caribbean specimens and 11 venoms from snakes from Pacific regions (Figure 1) were compared. Individual venoms from each population consistenly exhibited the characteristic Caribbean or Pacific protein profile, clearly showing that reverse-phase HPLC can be employed for unambiguously tracing the geographic origin of Costa Rican B. asper snakes. Nevertheless, the concentration of specific components varied between specimens from each of the two B. asper populations. Major differences between individuals from the Caribbean and
SLVELGKMILQETGK
11
13
SLFELGKMILQETGK
SLFELGKMILQETGK
SPPVCGNELLEVGEE SLVELGKMILQETGK
13957.7 28-31 kDa9
13781.6 26 kDa9
13757.6 26 kDa9
13739.6 26 kDa9
25 kDa9/1 13792.8 26 kDa9
7828 29 kDa9/1
GEECDCDAPENPCC SPPVCGNYFVEVGEE
SLFELGKMILQETGK
10
8 9
7602 7401 7215 7836
EAGEECDCGTPENP GEECDCGTPENPCC ECDCCGTPENPCCD EAGEECDCDAPENP
molecular mass
7810
N-terminal sequence
n.p EAGEECDCGTPENP
12 C14
11
10
9
8
5 P3
Bas(P)
1-4, 6,7
Bas(C)
1-4, 6,7 5 C1
HPLC fraction
972.9 697.8 538.2 1183.6 1736.9 1347.2 1475.3 1424.6 2861.4 766.9 697.8 538.2 2002.3 2845.4 559.1 538.3 566.6 1329.3 1437.8 1533.7 1736.9 1944.6 559.1 538.3 1533.7 1736.9 1944.6 538.3 566.6 1124.3 1347.2
502.1 898.6
575.8 683.7
576.1 683.7
m/z
peptide ion
2 2 2 1 1 1 1 1 1 2 2 2 1 1 2 2 2 1 1 1 1 1 2 2 1 1 1 2 2 1 1
2 3
2 3
2 3
z
NPVTSYGAYGCNCGVLGR TIVCGENNSCLK YSYSWKDK NNYLKPFCK ELCECDKAVAICLR DRYSYSWKDK YKNNYLKPFCK DATDRCCYVHK M(ox)ILGETGKNPVTSYGAYGCNCGVLGR SYGAYGCNCGVLGR TIVCGENNSCLK YSYSWKDK LTGCNPKKDRYSYSWK MILGETGKNPVTSYGAYGCNCGVLGR YYLKPFCK YSYSWKDK LTGCNPKKDR MILQETGKNPAK YRYYLKPFCK SYGAYGCNCGVLGR ELCECDKAVAICLR NPAKSYGAYGCNCGVLGR YYLKPFCK YSYSWKDK SYGAYGCNCGVLGR ELCECDKAVAICLR NPAKSYGAYGCNCGVLGR YSYSWKDK LTGCNPKKDR ENLNTYNKK DRYSYSWKDK
GQGTYYCR SECDIAESCTGQSPECPTDDFHR
CTGQSADCPR LRPGAQCAEGLCCDQCR
CTGQSADCPR LRPGAQCAEGLCCDQCR
MS/MS-derived sequence
[∼P18618] 1-71 [∼P18618] 3-71 [∼P18618] 5-71 [Q072L5] 1-73
K49-PLA2 [∼P0C616] Myotoxin IVa
K49-PLA2 [∼P24605 F114]
K49-PLA2 [P24605 F114]
K49-PLA2 [∼P24605 L114] Myotoxin II
DC-fragment K49-PLA2 [Q9PVE3 M8-ox] Myotoxin 1-3-3
Disintegrin [Q072L5] 3-73 DC-fragment [∼Q7T1T5]
Disintegrin Disintegrin Disintegrin Disintegrin
Disintegrin [∼P18618] 1-73
protein family
Table 2. Assignment of the Reverse-Phase Chromatographic Fractions of B. asper Venom from the Caribbean (BasC-) and the Pacific (BasP-) Versants of Costa Rica, Isolated as in Figures 1 and 3, Respectively, To Protein Families by N-Terminal Edman Sequencing, MALDI-TOF Mass Fingerprinting, and Collision-Induced Fragmentation by nESI-MS/MS of Selected Peptide Ions from in-Gel Digested Protein Bands (Separated by SDS-PAGE as in Figures 3 and 5, Respectively)a
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N.D.
13 P9
13
14
16 C13
16
17 C12
N.D.
23 kDa9
25 kDa9
VIGGDECNINEHRSL
C3
27 kDa9
VVGGRPCKINIHRSL
19
36 kDa9/9
VIGGDECNINEHRFL
13 kDa
SLIEFAKMILEETKR
18 C6,7
29 kDa9
13998.3
23/28 kDa1
32 kDa9
14211.6
32 kDa9/9
14220.2
molecular mass
VIGGDECNINEHRSL
SLIEFAKMILEETKR
Blocked
16
NLWQFGQMMSDVMR
15
12 P2,4
VIGGDECNINEHRSL
15 C8
N-terminal sequence
NLWQFGQMMSDVMR
Bas(P)
14 C2
Bas(C)
HPLC fraction
Table 2. Continued
z
2 2 2 2 2 2 2 2 3 2 2 1 2 2 2 2 2 3 2 2 2 1 1 3 2 2 2 3 3 2 2 1 3 2 2 2 3 2 3 2 2 3
731.6 868.9 753.1 467.3 570.6 756.8 644.8 595.8 826.4 753.1 538.3 980.0 467.3 563.9 614.6 569.6 768.9 635.6 752.9 753.6 864.6 1983.6 2064.8 919.6 570.6 595.8 756.8 768.9 826.4 552.9 864.6 1983.6 919.6 558.6 715.3 604.9 784.9 604.9 826.4 716.3 847.1 777.9
peptide ion m/z
TXVCDENNSCXK ELCECDKAVAICLR CCFVHDCCYGK YWFYGAK VSDYTEWIK VIGGDECNINEHR NFQMQLGVHSK IMGWGTISPTK VSNSEHIAPLSLPSSPPSVGSVCR CCFVHDCCYGK YSYSWKDK QICECDR YWFYGAK XYGDFNAATR FGQFASSXER SVDFDSESPR MEWYPEAAANAER KPNEIQNEIVDLHNSLR CCFVHDCCYGK SGVIICGEGTPCEK QICECDKAAAVCFR TYKKRYMAYPDFLCK DATDRCCFVHDCCYGK RLPFPYYTTYGCYCGWGGQGQPK VSDYTEWIK IMGWGTISPTK VIGGDECNINEHR AAYPWQPVSSTTLCAGILQGGK VSNSEHIAPLSLPSSPPSVGSVCR CCFVHDCCYGK QICECDKAAAVCFR TYKKRYMAYPDFLCK RLPFPYYTTYGCYCGWGGQGQPK SVANDDEVIR NVITDKDIMLIR INILDHAVCR AAYTWWPATSTTLCAGILQGGK INILDHAVCR VSNSEHIAPLSLPSSPPSVGSVCR VSXTNXEXWTTR YIELAVVADHGMFTK (661.2)QXXTAVVFNENVXR
MS/MS-derived sequence
PI-metalloproteinase [∼P83512]
Serine proteinase [∼AAB30013] Serine proteinase [∼P81824] Serine proteinase
D49-PLA2 [∼P20474]
Serine proteinase [Q072L6]
D49-PLA2 [P20474 F113] Myotoxin III/I
CRISP
unknown
D49-PLA2 [∼Q8AXY1]
Serine proteinase [Q072L6]
D49-PLA2 [∼Q8AXY1]
protein family
research articles Alape-Giro ´ n et al.
N.D. N.D.
23 23-26
25
ADDRNPLEECFRETD
Blocked
20,21 P8
22 P6
23-25
N.D.
Blocked
18 P11-13
22,25
Blocked
18,19
110 kDa9/9 56,58 kDa9
26 kDa9/9
56 kDa9/9
65 kDa9/9
28, 23 kDa9/9
27 kDa9
26 kDa9/13 kDa1
DCPS(G/D)WSSYEGHCYR
26 kDa9/9 13 kDa
30 kDa9/9 13 kDa
28 kDa9/9
21 C15-17
molecular mass
48 kDa9
VIGGDECNINEHRSL
17
VIGGDECNINEHRSL SLFE(L/F)AKMILQETGK
16
N-terminal sequence
VVGGDECNINEHRSL SLIEFAKMILEETGKNPAKSYGAYGCNCKV
N.D.
15
Bas(P)
20
C4,5
Bas(C)
HPLC fraction
Table 2. Continued
552.9 919.6 604.9 826.4 507.1 964.8 790.6 548.3 1126.3 1220.3 1661.6 1765.5 547.3 790.3 563.9 838.1 1765.4 2264.5 688.3 859.8 725.3 807.6 532.6 857.1 630.3 647.3 676.6 756.8 1486.2 1914.4 790.6 548.3 838.1 868.8 1732.3 1837.3 776.8 688.3
552.9 864.6 1983.6 919.6
716.3 847.1 777.9
m/z
peptide ion z
3 3 2 3 2 2 2 2 1 1 1 1 2 2 2 2 1 1 2 2 2 3 2 2 2 2 2 3 1 1 2 2 2 3 1 1 2 2
3 2 1 3
2 2 3
MS/MS-derived sequence
CCFVHDCCYGK RLPFPYYTTYGCYCGWGGQGQPK INILDHAVCR VSNSEHIAPLSLPSSPPSVGSVCR NFVCEFQA DCPPDWSSYEGHCYR VHEMLNTVNGFYR TLTSFGEWR AYTGGMCDPR HSVGVVRDHSK SFGEWRERDLLPR SVDVHAPLANLEVWSK YNSNLNTIR VHEMLNTVNGFYR AYTGGMCDPR YIELAVVADHGIFTK SVDVHAPLANLEVWSK SHDHAQLLTAVVFDGNTIGR YVEFVVVXDHR (231.2)PVXSXPGXTSXSFR (199.2)SXNVXCTECR (1199.6)HXPVXYNNVR NPLEECFR DPGVLEYPVKPSEVGK FWEDDGIHGGK EGWYANLPGMR SAGQLYEESLQK IYFAGEYTAQAHGWIDSTIK ETDYEEFLEIAK ETLSVTADYVIVCTTSR VHEMLNTVNGFYR TLTSFGEWR YIELAVVADHGIFTK EVLSYEFSDCSQNQYETYLTK SVNVTASLASLEVWSKK TRVHEMLNTVNGFYR VCSNGHCVDVATAY YVEFVVVXDHR
CCFVHDCCYGK QICECDKAAAVCFR TYKKRYMAYPDFLCK RLPFPYYTTYGCYCGWGGQGQPK
VSXTNXEXWTTR YIELAVVADHGMFTK (661.2)QXXTAVVFNENVXR
protein family
PIII-metalloproteinase PIII-metalloproteinase
PII-metalloproteinase [Q072L5]
L-amino acid oxidase [∼Q6TGQ9]
PIII-metalloproteinase
P1-metalloproteinase BaP1 [P83512/Q072L4]
(Rβ) C-type lectin-like [∼P22029/P22030] PI-metalloproteinase [∼ABB76282]
Serine proteinase
Serine proteinase D49-PLA2 [∼P20474]
Serine proteinase D49-PLA2 [∼P20474]
PIII-metalloproteinase [∼P83512]
Proteomics of Bothrops asper
research articles
Journal of Proteome Research • Vol. 7, No. 8, 2008 3563
3564
Journal of Proteome Research • Vol. 7, No. 8, 2008
n.d. n.d
37 35-38
58 kDa9 23 kDa9
52 kDa9
48-72 kDa9 72 kDa9 58 kDa9
72 kDa 9
48 kDa9
51 kDa9 2 2 2 3 2 2 2 2 2 2 2 2 2 3 2 2
2 2 2 2 2 2 2 2 2 2
898.2 670.3 534.9 867.6 559.1 766.6 548.3 548.3 810.9 822.9
526.8 514.8 915.3 656.3 670.3 999.6 474.6 773.9 591.3 656.9 773.9 716.3 847.1 777.9 611.1 790.6
2
2 2 2 2 2 1 1 2 2 2 1 1 3
902.3 902.3 548.3 933.3 790.6 1676.3 1765.5 547.3 611.1 790.6 1676.3 1765.5 718.6 591.3
z
peptide ion m/z
GNYYGYCR IPCAPEDVK YFVEVGEECDCGXPR YXXDNRPPCXXNXPXR (263.2)EXVXVADYR XTPGSQCADGXCCDQCR FCTQQHK VFNEPQNWADAEK ZXVVTPEQQR VYNDNXYPCR VFNEPQNWADAEK VSXTNXEXWTTR YIELAVVADHGMFTK (661.2)QXXTAVVFNENVXR HSVGVVRDHSK VHEMLNTVNGFYR
VTXSADDTXDAFGEWR (263.2)EXVXVADYR YYVWIGLR DTPFECPSDWSTHR YYLKPFCK SYGAYGCNCGVLGR TLTSFGEWR TLTSFGEWR XYEXVNXXNVXYR (292.8)YXEXVXVWYR
ZXVVTPEQQR
YFVEVGEECDCGSPR YFVEVGEECDCGSPR TLTSFGEWR IENDADSTASISACNGLK VHEMLNTVNGFYR YIELAVVADHGIFTK SVDVHAPLANLEVWSK YNSNLNTIR HSVGVVRDHSK VHEMLNTVNGFYR YIELAVVADHGIFTK SVDVHAPLANLEVWSK LHSWVECESGECCGQCR
MS/MS-derived sequence
C-type lectin-type PI-metalloproteinase [∼P83512]
PIII-metalloproteinase
PIII-metalloprotease [∼Q7T1T5] PIII-metalloproteinase PIII-metalloproteinase C-type lectin-type
D49-PLA2 [∼Q8AXY1] Serine proteinase [∼Q8QG86] PIII-metalloprotease [∼P30431] PIII-metalloprotease
(PI)-metalloproteinase (PI)-metalloproteinase PIII-metalloprotease
PIII-metalloproteinase [∼AAG48931] PIII-metalloproteinase [∼AAG48931] PIII-metalloproteinase PIII-metalloproteinase C-type lectin-like [∼AAB47092] K49-PLA2 [P24605 F114]
PI-metalloproteinase [∼P83512]
PIII-metalloproteinase PIII-metalloproteinase PI-metalloproteinase [∼ABB76282]
protein family
a The table includes also the proteomic characterization of 2D-electrophoresic-separated venom proteins from adult specimens from Caribbean (BasC) and Pacific (BasP) specimens (Figure 6), as well as the identification of proteins distinctly secreted into the venoms of neonate snakes from Caribbean and Pacific origin (see Figures 9 and 10). X, Ile or Leu; Z, pyrrolidone carboxylic acid; M(ox), methionine sulphoxide. Unless other stated, for MS/MS analyses, cysteine residues were carbamidomethylated. Molecular masses of native proteins were determined by electrospray-ionization ((0.02%) or MALDI-TOF ((0.2%) mass spectrometry. Apparent molecular mass determined by SDS-PAGE of non-reduced (9) and reduced (1) samples; n.p., non peptidic material found. M and m, denote mayor and minor products within the same HPLC fraction. Previously reported venom protein from B. asper (or other Bothrops species) are identified by their databank accession codes; the symbol ”∼” denotes ”highest similarity to”.
Blocked Blocked n.d.
Blocked
Blocked
AFTAEQRRYLNTRKY
35-38 35 36
34
33
34
33
13 kDa9 38 kDa9
NLWQFGQMMSDVMR VVGGDECDINEHPFLAFLYSHGYFC
31 32
13 kDa9
42 kDa / 56 kDa9/9 26 kDa9 9 9
52, 46 kDa9/9
P7
N.D.
N.D. N.D.
Blocked
48 kDa9/9
23 kDa9/9
91,96 kDa / 48 kDa9 26 kDa9/9 9 9
molecular mass
33 kDa9 36 kDa9 48 kDa9
30
30 30 P10
32
C9 C11
27-31
29 30,31
29
28
Blocked
Blocked
27 P5
28
N.D. N.D. TPEQQRFSPRYIELA
N-terminal sequence
26
Bas(P)
27
26 C10
26
Bas(C)
HPLC fraction
Table 2. Continued
research articles Alape-Giro ´ n et al.
Proteomics of Bothrops asper
Figure 4. Reverse-phase HPLC separation of the proteins from a pool of venoms of B. asper adult specimens from the Pacific region of Costa Rica. Two milligrams of total venom proteins was applied to a Lichrosphere RP100 C18 column, which was then developed as in Figure 2. Fractions were collected manually and characterized by N-terminal sequencing, ESI mass spectrometry, tryptic peptide mass fingerprinting, and CID-MS/MS of selected doubly or triply charged peptide ions. The results are shown in Table 2.
the Pacific regions are highlighted in panels A and B of Figure 8, respectively. The variability at this level provides supportive evidence to venom composition being under genetic control. Among Caribbean B. asper venoms, PLA2 molecules (BasC9-15), serine proteinases (BasC18-20), SVMPs (BasC26 and 27), and to a minor extent LAO (BasC23 and 24), displayed the largest variability (Figure 8A). A similar trend was observed in venoms from the Pacific B. asper population, with PLA2s (BasP9-13), serine proteinases (BasP14 and 17), LAO (BasP20,21) and the SVMPs (BasP18, 25-30) exhibiting the most noticeable expression variation (Figure 8B). Hence, within both B. asper geographic populations, all major venom protein families appeared to be subjected to individual variations. The sources of such variability are not obvious. On one hand, no obvious genderspecific trend could be established, and venom components appear to vary independently. On the other hand, individual venom variation seems to be a stochastic phenomenon, at least concerning ecological niches, because venoms from specimens from close geographic locations do not follow related venom composition variability. The occurrence of intraspecific individual variability highlights the concept that a species, B. asper in our case, should be considered as a group of metapopulations.67 The fact that venoms exhibit larger compositional variation between the two isolated geographic populations (Caribbean vs Pacific) than among specimens from the same geographic range may be the consequence of allopatric speciation of the two B. asper subpopulations isolated by mountain barriers. Evidence from mitochondrial gene sequences indicates that the two Costa Rican B. asper populations split during Pleistocene, 3-3.5 mya ago (Saldariaga and Sasa, unpublished results). The Modern Synthesis of Evolution emphasizes the importance of populations as the units of evolution. Local adaptation within a restricted population can occur if the strength of selection exceeds the rate of gene flow.68 Hence, Natural Selection acting on individual venom variations may endow specimens within a reproductive community the necessary versatility to adapt to the changing environments of a geographical range. Individual venom variation is well-documented in the literature and appears to be a general feature of venoms.69 In
research articles particular, Taborska and Kornalik70 reported considerable individual variability in both pathophysiological and enzymatic activities between parents and siblings of a family of B. asper snakes. The disparity of symptoms in victims of the same species of snake has alerted clinicians to the requirement for more specific antivenoms.69,71 Thus, knowledge of the geographical and the individual variability in venoms, as reported here, could be relevant for antivenom production. Defining Ontogenetic Changes in the Venom Proteomes among B. asper Specimens from Caribbean and Pacific Regions of Costa Rica. In line with a previous paper by Gutie´rrez and colleagues50 who reported marked differences in electrophoretic and immunoelectrophoretic patterns between newborn and adult venoms from two Costa Rican populations (San Carlos in the Caribbean versant and Puriscal in the Pacific), comparison of the HPLC separation profiles of pooled venoms of neonate specimens from Caribbean and Pacific regions with those of adult snakes from the same geographical habitat (Figure 9 vs 2 and Figure 10 vs 4) revealed prominent protein expression changes. Thus, among Caribbean snakes, neonate venoms (Figure 9) express neither the major K49-PLA2 molecules Bas(C)-9-13 nor the abundant PI-SVMPs Bas(C)-21 and -26 characterized in adult venoms (Figure 2). On the other hand, neonate venoms contained a larger proportion of D49-PLA2s 15 and 16, and of PIII-SVMPs, including at least three molecules (numbered 32-34 in Figure 9 and labeled with asterisks) not observed in adults. The N-terminal sequence of Bas(C)_neo-32, AFTAEQRRYLNTRKY, shows extensive similarity with the region 147-160 of PIII-metalloprotease HF2 precursor from B. jararaca [P30431]. Bas(C)_neo-33 and 34 contained blocked N-termini and were identified by in-gel tryptic digestion and ESI-MS/MS as PIII-SVMPs. These neonatespecific PIII-SVMPs account for about 14% of the total venom proteins (32, 3%; 33, 8.3%; 34, 2.7%). Comparison of the protein profiles of pooled venoms from Pacific neonates and adult specimens (Figure 10 vs Figure 4) indicated the occurrence of a similar ontogenetic trend than in Caribbean snakes. Thus, Pacific neonates express larger amounts of D49-(PLA2 Bas(P)-12 and 31) than K49-PLA2s (Bas(P)-10 and 11), though the total amount of PLA2 molecules is only 40% than that of adults (Table 3). The concentration of PI-SVMPs 18 and 19 is also lower in neonate versus adult venoms, and the secreted complement of PIII-SVMPs also show age-dependent qualitative and quantitative variations. Protein peaks labeled with asterisks in Figure 10 represent neonatespecific toxins not found in adult venoms. Bas(P)_ neo-31-34 were characterized by N-terminal sequencing, respectively, as a D49-PLA2 molecule, a 38 kDa serine proteinase, and PIIISVMPs apparently identical to Bas(C)_neo-32 and -34 (Table 2). Bas(P)_neo-35-38 contain PIII-SVMPs (48-72 kDa), which share at least ion 670.3(2+), (263.2)EXVXVADYR, (Rβ)2 C-type lectin-like proteins, and a PI-SVMP (23 kDa) highly similar or identical to Bas(P)-27 (Table 2). The overall protein composition of pooled venoms from neonate B. asper snakes from Caribbean and Pacific versants are displayed in Figure 6C,D and listed in Table 3. Major ontogenetic changes appear to be a shift from a PIII-SVMPrich to a PI-SVMP-rich venom and the secretion in adults of a distinct set of PLA2 molecules than in the neonates. The agedependent P-III to PI SVMP ontogenetic variation has also been reported in B. atrox.29 However, whether it represents a general or a genus-specific phenomenon requires detailed analysis in a higher number of species. Journal of Proteome Research • Vol. 7, No. 8, 2008 3565
research articles
Alape-Giro ´ n et al.
Figure 5. SDS-PAGE of reverse-phase separated fractions from the venom of adult B. asper from the Pacific region of Costa Rica. SDS-PAGE showing the protein composition of the reverse-phase HPLC separated venom fractions (see Figure 4) run under nonreduced (upper panels) and reduced (lower panels) conditions. Molecular mass markers (in kDa) are indicated at the left of each gel. Protein bands were excised and characterized by mass fingerprinting and CID-MS/MS. The results are shown in Table 2. Table 3. Overview of the Relative Occurrence of Proteins (in Percentage of the Total HPLC-Separated Proteins) of the Different Families in the Venoms of B. asper Populations from the Caribbean and the Pacific Versants of Costa Rica % of total venom proteins Caribbean
Figure 6. Overall protein compositions of B. asper venoms. Comparison of the protein composition of the pooled venoms of adult B. asper from the Caribbean region (A) and from the Pacific region of Costa Rica (B), and of the pooled venoms from neonate specimens from the Caribbean region (C) and from the Pacific region (D). DC, disintegrin/cysteine-rich fragment from PIII snake venom metalloproteinase (SVMPs); C-lectin, C-type lectinlike protein; PLA2, phospholipase A2; CRISP, cysteine-rich secretory protein; LAO, L-amino acid oxidase; SerProt, serine proteinase. Details of the individual proteins characterized in adult venoms are shown in Table 2 (but see also Table 3).
Interestingly, in neonate B. asper venoms, the ratio K49/D49 PLA2s is about 80%/20%, adult Caribbean and Pacific venoms contain, respectively, about 65% of K49- and 35% of D49-PLA2s 3566
Journal of Proteome Research • Vol. 7, No. 8, 2008
Pacific
protein family
adult
neonate
adult
neonate
Medium-sized disintegrin DC-fragments PLA2 (- K49 (- D49 CRISP Serine proteinase L-amino acid oxidase C-type lectin-like Zn2+-metalloproteinase (- PI-SVMPs (- PIII-SVMPs
2.1
