Analysis of the Ontogenetic Variation in the Venom Proteome/ Peptidome of Bothrops jararaca Reveals Different Strategies to Deal with Prey Andre´ Zelanis,†,‡ Alexandre K. Tashima,†,§ Marisa M. T. Rocha,| Maria F. Furtado,| Antonio C. M. Camargo,† Paulo L. Ho,‡,⊥ and Solange M. T. Serrano*,† Laborato´rio Especial de Toxinologia Aplicada-CAT/cepid, Instituto Butantan, Brazil, Instituto de Quı´mica, Departamento de Bioquı´mica, Universidade de Sa˜o Paulo, Brazil, Departamento de Cieˆncias Exatas e da Terra, Universidade Federal de Sa˜o Paulo, Brazil, Laborato´rio de Herpetologia, Instituto Butantan, Brazil, and Centro de Biotecnologia, Instituto Butantan, Brazil Received November 11, 2009
Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species. Keywords: glycosylation • ontogenetic venom variation • peptidome • proteome • proteinase • snake venom
Introduction Snake venom proteomes are complex mixtures of a large number of distinct proteins and peptides with biological activity. Each individual venomous snake family is comprised of genera with venoms that contain common protein families. However, the specific toxin families found in the different snake families are composed of related toxins that vary in amino acid sequence and abundance and thereby contribute to the differences in the overall biological activity of individual venoms.1,2 * To whom correspondence should be addressed. S. M. T. Serrano, Laborato´rio Especial de Toxinologia Aplicada-CAT/cepid, Instituto Butantan, Av. Vital Brasil 1500, 05503-900, Sa˜o Paulo, Brazil. Tel.: +55 11 3726-1024. E-mail:
[email protected]. † Laborato´rio Especial de Toxinologia Aplicada-CAT/cepid, Instituto Butantan. ‡ Universidade de Sa˜o Paulo. § Universidade Federal de Sa˜o Paulo. | Laborato´rio de Herpetologia, Instituto Butantan. ⊥ Centro de Biotecnologia, Instituto Butantan.
2278 Journal of Proteome Research 2010, 9, 2278–2291 Published on Web 02/10/2010
An important aspect of snake venom proteomes is the venom variability, a well-documented phenomenon that has long been appreciated by investigators. The composition of snake venoms is a result of multiple factors, and its inherent variability is often related to environmental and ecological traits, which can change from species to species. In this context, diet is believed to exert a key role on venom composition, acting as a selective factor3-10 and providing means of immobilizing, killing, and digesting the prey and of self-defense.11,12 The existence of a relationship between venom and diet was elegantly shown by Daltry and colleagues,4 and these findings are in agreement with observed differences in prey type susceptibility to snake toxins as well as with simultaneous ontogenetic changes in diet and venom composition reported for some species.5,6 On the other hand, it was recently shown that the venom composition of Crotalus simus changes dramatically during development, from a neurotoxic to a hemorrhagic phenotype, while the species feeds from its birth primarily on small rodents and, less 10.1021/pr901027r
2010 American Chemical Society
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Venom Proteome/Peptidome of Bothrops jararaca frequently, on lizards, indicating that the ontogenetic mechanism of venom change does not apply to this species.13In contrast, changes in venom composition may also represent a neutral feature and would become permanent within a snake population only if the resulted venom change conferred reproductive suitability. The complexity of viperid venom proteomes is contributed to by various molecular mechanisms including the high degree of amino acid sequence variation in nonconserved regions of some toxin families such as the proteinases and phospholipase A2s, variable glycosylation levels within toxin families, and the modular structure of metalloproteinase precursors, which allow for variable processing patterns and hence mature protein structures.14-16 These features result in proteins with variable biophysical and biochemical properties within the same toxin family, which often display different biological activities upon the prey, as illustrated, for instance, by the family of venom serine proteinases that shows enzymes with similar primary structures, variable glycosylation levels, molecular masses, and isoelectric points and different specificities upon macromolecular substrates.15 Snake venom variability has been verified at several levels, such as intraspecies, interspecies, sex-based, geographic, and ontogenetic variation, and in most of the cases, snake diet is considered the variability driving factor.9,17-21 Snakes belonging to Bothrops genus are among the most studied neotropical viperids, both in terms of ecological features and toxin families. Bothrops jararaca is one of the most abundant species in Brazil, inhabiting rain forests as well as more open vegetation areas.22 This species is primarily nocturnal and generalist, however, it exhibits a notable ontogenetic shift in diet, feeding on ectothermic prey (mainly arthropods, lizards, and amphibians) through juvenile phase and on endothermic animals (mainly small mammals) during adult life.23,24 This pattern of ontogenetic shift in B. jararaca diet seems to be related to venom lethality upon different prey types.25 Due to its broad geographical distribution, this species is responsible for the majority of the accidents by Bothrops genus in Brazil and envenomation cases are complex, with local and systemic effects, such as hemorrhage, myonecrosis, bleeding and severe coagulatopathy.26,27 Studies on envenomation cases with newborn and adult B. jararaca snakes have shown distinct patterns, mainly related to coagulantion disorders, which seems to be prominent in accidents with newborn specimens.26 The first report on the variability of coagulant activity of B. jararaca venom was made by Gasta˜o Rosenfeld and co-workers,28 in which a prominent coagulant activity was reported for venom from young specimens. Recently, two-dimensional electrophoresis coupled with LC-MS/MS has been used for examining B. jararaca venom complexity along with other interesting approaches to selectively delineate subpopulations of venom toxins based on particular characteristics of the proteins such as antibody crossreactivity or enzymatic activities.29 In another work, the venom of B. jararaca was subjected to in-solution digestion with trypsin and ion-currents of tryptic peptides with FT-ICR LC-MS/MS were used to investigate the venom proteomic content showing 42 individual proteins representing 12 venom protein classes.30 Furthermore, the analysis of the venom peptidome of B. jararaca venom from sibling and nonsibling snakes showed for the first time sex-based differences among the bradykinin potentiating peptides (BPPs).20 In another recent study, we extended the analysis of Bothrops venoms with focus on the comparison of subproteomes of
snake venom metalloproteinases (SVMPs) and serine proteinases (SVSPs) of eight species including B. jararaca, using specific antibodies in immunostaining analyses of 2-DE and 2D-gelatin zymography and demonstrated the diversity of their proteinase profiles.31 In addition, we used heparin chromatography and protein identification by mass spectrometry to characterize the subproteome of toxins with high-affinity for heparin in these Bothrops venoms as composed of SVSPs and C-type lectins. Furthermore, we assessed the subset of B. jararaca venom toxins with affinity for heparin under physiological conditions, and interestingly, we observed a clear separation between the subproteome of more toxic components, composed of mainly SVMPs, that displayed lethal and hemorrhagic activities and were found in the nonbound fraction and the subproteome of less toxic components associated with coagulant and fibrinogenolytic activities, composed of mainly SVSPs, found in the bound fraction.31 Although B. jararaca venom has been the focus of extensive studies, the structure of the toxins present in the newborn venom is completely unknown since most studies on snake venoms are usually carried out using venom from adult specimens. Moreover, the degree to which the snake venom proteome and peptidome vary between newborn and adult specimens is poorly understood. The aim of this investigation was to explore some aspects of the variability of B. jararaca venom, mainly concerning the differences in the biological activities of newborn and adult venoms that could be related to the differential expression of certain toxin families. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. Moreover, the coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of: (i) SDS-PAGE; (ii) gelatin zimography; (iii) immunostaining using antibothropic antivenom and antiproteinase antibodies; (iv) glycoprotein staining and deglycosylation pattern; and (v) glycoproteins, as illustrated by the mass spectrometric identification of Con A-binding proteins. Furthermore, we also report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides. However, the profile of identified bradykinin potentiating peptides was similar in both venoms, and these peptides were detected showing their canonical sequences32,33 and also novel sequences corresponding to forms of BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.
Experimental Section B. jararaca Venom Samples. Venom from 694 two-week old newborns (359 male and 335 female specimens) and 110 adults (49 male and 61 female specimens older than 3 years) from Sa˜o Paulo State (Brazil) was used in this study. As for the newborn venom, considering that the mean offspring number for the species B. jararaca is 10.7 animals,23 the 694 specimens that composed this study derived from ∼65 different mothers. The venom was milked, centrifuged for 30 min at 2000× g, 4 °C, to remove any scales or mucus, lyophilized and stored at -20 Journal of Proteome Research • Vol. 9, No. 5, 2010 2279
research articles °C until use. Venom protein concentrations were determined using the Bradford reagent34 (Sigma, St. Louis, MO) and bovine serum albumin (Sigma, St. Louis, MO) as a standard. SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis. SDS-PAGE was carried out according to Laemmli35 and proteins were stained with silver.36 Western blot analysis was carried out as described elsewhere31 using (i) antiBothropasin rabbit polyclonal antibody raised against the P-III class metalloproteinase bothropasin from B. jararaca,37 (ii) antiMSP1/2 rabbit polyclonal antibody, raised against a mixture of the basic serine proteinases MSP1 and MSP2 from B. moojeni,38 and (iii) antibothropic antivenom (batch # 0610188), generated by the immunization of horses with a mixture of adult venoms of B. jararaca (50%), B. alternatus (12.5%), B. jararacussu (12.5%), B. moojeni (12.5%), B. neuwiedi (synonyms B. n. goyazensis, B. n. meridionalis, B. n. paranaensis and B. n. urutu)39 (6.25%) and B. pauloensis (6.25%) at Instituto Butantan. Gelatin Zymography. For gelatin zymography, venoms (25 µg) were submitted to SDS-PAGE on 12% SDS-polyacrylamide gels containing 1 mg/mL gelatin (Sigma, St. Louis, MO) under nonreducing conditions. After electrophoresis, the gel was incubated for 30 min at room temperature on a rotary shaker in 0.05 M Tris-HCl, pH 7.4, containing 2.5% Triton X-100 to remove traces of SDS. The gel was washed with deionized water to remove excess of Triton X-100 and then incubated in zymography incubation buffer (0.05 M Tris-HCl, pH 8.0, 0.15 M NaCl, 0.01 M CaCl2, 0.02% CHAPS) at 37 °C for 12 h. The gel was stained with Coomassie blue and destained. Gelatin digestion was identified as clear zones of lysis against a blue background. Staining of Glycoproteins and Deglycosylation Analysis. Venom glycoproteins were detected after SDS-PAGE (50 µg venom samples) using 8%-18% SDS-polyacrylamide gels and staining with the fluorescent dye Pro-Q-Emerald (Molecular Probes, Eugene, OR) according to the manufacturers’ instructions. Gel images were acquired after exposure to an UVtransilluminator at 300 nm. For protein deglycosylation under denaturing conditions, venom samples (15 µg) were incubated in 10% SDS for 1 min at 95 °C. After adding 0.02 M sodium phosphate buffer, 0.08% sodium azide, 0.01 M EDTA, 2% Triton X-100, pH 7.0, incubation was prolonged for 2 min at 95 °C. After cooling, 1 U of N-glycosidase F or 2.5 mU of O-glycosidase (Roche, Mannheim, Germany) was added, and the mixture was incubated for 18 h at 37 °C. The deglycosylation profiles were evaluated by SDS-PAGE as described above. In-Gel Protein Digestion and MS Identification. Protein bands were excised and in-gel trypsin digestion was performed according to Hanna et al.40 An aliquot (5 µL) of the resulting peptide mixture was separated by C18 (75 µm i.d. × 100 mm) (Waters, Milford, MA) RP-HPLC coupled with nanoelectrospray MS/MS on a LTQ XL mass spectrometer (Thermo, San Jose, CA) at a flow rate of 400 nL/min. The gradient was 0-80% acetonitrile in 0.5% formic acid over 45 min. The instrument was operated in the “top ten” mode, in which one MS spectrum is acquired followed by MS/MS of the top 10 most intense peaks detected. Full dynamic exclusion was used to enhance dynamic rangesone spectrum before exclusion for 120 s. The resulting fragment spectra were searched using MASCOT search engine (Matrix Science, UK) against the NCBI NR database restricted to the taxa Serpentes (9500 entries, including 4037 entries corresponding to snake venom components; downloaded on September 1, 2009) with a parent tolerance of 1.50 Da and fragment tolerance of 0.8 Da. Iodoacetamide derivative 2280
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Zelanis et al. of cysteine and oxidation of methionine were specified in MASCOT as fixed and variable modifications, respectively. Con A-Chromatography. Venom samples (5 mg in 1 mL binding buffers20 mM Tris-HCl, 500 mM NaCl, 5 mM CaCl2, pH 7.4) were submitted to chromatography on a Con ASepharose (Sigma, Saint Louis, MO) column (0.8 cm × 2.0 cm) at room temperature. Venom proteins were loaded on the column, which was kept closed for 20 min to allow binding to concanavalin A. After elution of the nonbound proteins with 5 mL binding buffer, bound proteins were eluted with 5 mL of 0.5 M D-glucose in binding buffer. Eluted fractions were pooled and concentrated to 400 µL using CentriconYM-3 (Millipore, Bradford, MA) and then submitted to in solution trypsin digestion. Mass Spectrometric Protein Identification by LC-MS/ MS. For protein identification a sample of 100 µg protein was incubated at 95 °C for 30 min with 20 µL of 10 mM DTT in 100 mM ammonium bicarbonate, followed by addition of 20 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate and additional incubation at room temperature for 30 min in the dark. Ten microliters of a trypsin solution containing 0.2 µg/ µL (Proteomics grade; Sigma, St. Louis, MO) was added to each sample and incubation was prolonged for 16 h at 37 °C. An aliquot (10 µL) of the resulting peptide mixture was injected into a trap column packed with C18 (180 µm i.d. × 20 mm) (Waters, Milford, MA) for desalting with 100% solvent A (0.5% formic acid) at 15 µL/min for 12.5 min. Peptides were then eluted onto an analytical C18 column (75 µm i.d. × 100 mm) (Waters, Milford, MA) using a 105 min gradient at a flow rate of 400 nL/min where solvent A was 0.5% formic acid and solvent B was 0.5% formic acid in acetonitrile. The gradient was 15-50% of solvent B in 50 min, hold at 50% B for 20 min, 50-65% B in 22.5 min, then back to 100% A in 12.5 min. A LTQ XL mass spectrometer (Thermo, San Jose, CA) was used to acquire spectra. Spray voltage was set at 2.5 kV and the instrument was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 300-1600 followed by MS/MS acquisition using collision induced dissociation of the 10 most intense ions from the MS scan. A dynamic peak exclusion was applied to avoid the same m/z of being selected for the next 120 s. Tandem mass spectra were extracted by Xcalibur software (version 2.0; Thermo scientific). The resulting MS/MS spectra were searched using MASCOT search engine (Matrix Science, UK) against the NCBI NR database restricted to the taxa Serpentes (9500 entries, including 4037 entries corresponding to snake venom components; downloaded on September 1, 2009) with a parent tolerance of 1.50 Da and fragment tolerance of 0.8 Da. Iodoacetamide derivative of cysteine and oxidation of methionine were specified in MASCOT as fixed and variable modifications, respectively. Scaffold (version Scaffold_2_04_00, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they exceeded specific database search engine thresholds. MASCOT identifications required ion scores greater than both the associated identity scores and 20, 30, 40, and 40 for singly, doubly, triply, and quadruply charged peptides. X! Tandem identifications required at least -Log(Expect Scores) scores of greater than 2.0. Protein identifications were accepted if they contained at least 2 identified peptides. Any peptides/
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Venom Proteome/Peptidome of Bothrops jararaca proteins corresponding to toxins were confirmed by manual examination of the spectra. Venom Peptidome Analysis by MALDI-TOF Mass Spectrometry. Prior to mass spectrometry analysis, venoms (5 µL of a solution containing 0.2 mg/mL in 0.1% trifluoroacetic acid (TFA) were subjected to desalinization using ZipTip C-18 (Millipore Co., Billerica, MA) according to the manufacturer’s instructions. The eluted peptide mixture was spotted onto the sample plate of a Ettan MALDI-TOF/Pro instrument (GE Healthcare). The sample (0.35 µL, in triplicate) was mixed on the plate with the same volume of a saturated solution of alphacyano-4-hydroxycinnamic acid (Sigma, St. Louis, MO) in 50% acetonitrile containing 0.1% TFA, dried, and analyzed in delayed extraction method and reflectron mode using P14R [(M+H)+ 1533.8582] and Angiotensin II [(M+H)+ 1046.5423] (Sigma, St. Louis, MO) as external calibrants. Alternatively, prior to mass spectrometric analysis, the venom samples were incubated with a mixture of proteinase inhibitors (2 mM PMSF, 10 mM EDTA, final concentrations) for 30 min, at room temperature. Venom Peptidome Analysis by LC-MS/MS. Venoms (10 mg) were dissolved in 1 mL 0.1% TFA and submitted to solid phase extraction (SPE) using Sep-Pak C18 cartridges (Waters, Milford, MA), according to Menin et al.41 Venom samples were eluted with 30% acetonitrile containing 0.1% TFA. The eluates were dried in a SpeedVac concentrator and the dried venom eluates (DVEs) were stored at -20 °C for further analysis. For LC-MS/ MS analysis the DVEs were dissolved in 200 µL 0.1% formic acid and aliquots of 2.5 µL were automatically injected into a LTQ XL (Thermo, San Jose, CA) or into a nanoUPLC (nanoAcquity, Waters, Milford, MA) coupled with a Q-TOF Ultima mass spectrometer (Waters, Milford, MA) using the parameters described for protein identification. Database searching was carried out using MASCOT algorithm, as described above. The search parameters were: no enzyme restriction, and as variable modification, pyroglutamic acid at N-terminal Gln or Glu, peptide mass tolerance of ( 1.5 Da, and MS/MS mass tolerance of 0.8 Da (for LTQ XL analysis) or of ( 1.2 Da, and MS/MS mass tolerance of 0.6 Da (for Q-TOF analysis). Coagulant Activity. Minimum Coagulant Dose (M.C.D.) was used to measure coagulant activity of venom samples. MCD is defined as the minimum amount of venom resulting in clot formation within 60 s at 37 °C.42 Coagulant activity was determined on human citrated plasma (200 µL) using serial dilutions of venom samples in a final volume of 100 µL (dissolved in 20 mM Tris-HCl, 0.5 mM CaCl2, pH 8.0). Venom samples were also incubated for 1 h, at room temperature, with metalloproteinase or serine proteinase inhibitors (5 mM EDTA or 2 mM PMSF, respectively) prior to determining the M.C.D. Factor II and Factor X Activation. Factor II (HYPHEN Biomed, France) or factor X (HYPHEN Biomed, France) (90 nM) was incubated with 0.2 µg or 0.1 µg of venom samples, respectively, in a final volume of 100 µL of 20 mM Tris-HCl, pH 8.3, containing 5 mM CaCl2, at 37 °C. After 10 min of incubation, S-2238 (H-D-Phe-Pip-Arg-pNA · 2HCl) or S-2765 (NR-Z-D-Arg-Gly-Arg-pNA · 2HCl) (Chromogenix, Italy), chromogenic peptide substrates for factors IIa and Xa, respectively, were added at the final concentration of 400 µM, and the hydrolysis was monitored at 405 nm for 30 min at 37 °C. No amidolytic activity of newborn and adult venoms upon both peptide substrates was detected under the same conditions but in the absence of factor II or factor X.
Hemorrhagic Activity. For determination of Minimum Hemorrhagic Dose (M.H.D.) the method described by Kondo et al.43 and modified by Gutie´rrez et al.44 was used. Groups of male Swiss mice (n ) 4 for each dose) (18-22 g), were injected intradermically in the abdomen with five doses of venom (0.25-2.0 µg) dissolved in 0.15 M NaCl. MHD was the venom dose that caused a hemorrhagic area of 10 mm diameter. Caseinolytic Activity. Caseinolytic activity was determined as described elsewhere19 using N,N-dimethylated casein (Sigma, St. Louis, MO) as a substrate. One unit of activity was defined as the amount of venom yielding an increase in O.D. of 1.0 per min at 280 nm. Specific activity was expressed as units/ mg protein. Fibrinogenolytic Activity. Fibrinogenolytic activity was determined as described elsewhere45 using human fibrinogen (Sigma, St. Louis, MO) as a substrate. One unit of activity is defined the amount of enzyme yielding an increase in O.D. of 1.0 per min at 280 nm. Specific activity was expressed as units/ mg protein. Median Lethal Dose (LD50). Groups of male Swiss mice (n ) 4 for each dose) (18-22 g), were injected intraperitoneally with five doses of venom (25-80 µg) dissolved in 0.5 mL 0.15 M NaCl. The dose that killed 50% of animals was calculated by probit analysis,46 with a computer program, taking into consideration deaths occurring within 48 h after venom injection. For the determination of LD50 in chicks (Gallus domesticus), groups of one-day old male chicks (Bovans White; provided by Aviary Farm Kunitomo (Mogi das Cruzes, Brazil), of 40 g-45 g, n ) 4 for each dose, were subjected to an intramuscular injection on the left pectoralis muscle with five doses of venom solubilized in 100 µL 0,15 M NaCl (newborn venom: 0.5-6 µg; adult venom: 30-150 µg). The LD50 values were calculated as mentioned above, taking into consideration deaths occurring 24 h after venom injection. Animals had free access to food and water ad libitum during the assays. All assays involving animals had the approval of the Ethical Committee of Animal Experimentation of Instituto Butantan.
Results and Discussion Electrophoretic Profile. To compare and contrast the newborn and adult venom electrophoretic profiles, we first analyzed them by SDS-PAGE (8-18% gradient gel) under reducing and nonreducing conditions (Figure 1). One very interesting observation is that the venom profiles under reducing conditions are not identical but show various common bands; however, under nonreducing conditions, the profiles are rather different in appearance with the adult venom showing various protein bands that are absent in the newborn venom. Of especial note is a group of protein bands higher than 100 kDa observed in the adult venom under nonreducing conditions which is absent in the newborn venom. Moreover, as a common feature of both venoms we observed the presence of 9 kDa-12 kDa bands under reducing conditions which are not visible when disulfide bonds are not reduced indicating that these proteins are present in the venom in oligomeric state as is the case of some dimeric disintegrins, C-type lectins, and SVMPs of the P-IId, P-IIe and P-IIId classes.16,47,48 The identification of proteins differentially expressed in the venoms by in gel trypsin digestion followed by LC-MS/MS analysis showed five bands present only in the newborn venom which contained SVMPs (Figure 1; Table 1). Three bands observed in the adult venom Journal of Proteome Research • Vol. 9, No. 5, 2010 2281
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Figure 1. SDS-PAGE (8-18% SDS-polyacrylamide gel) profile of B. jararaca newborn and adult venoms (50 µg). Mobility of molecular mass markers is indicated on the left. The arrows indicate bands of proteins differentially expressed among the venoms and the toxin class identified by mass spectrometry. Proteins were stained with Coomassie blue.
contained SVMPs, including two bands of high molecular mass under nonreducing conditions, which may correspond to P-IIIc or to P-IIId SVMPs,16 and one band of ∼20 kDa under reducing conditions (Figure 1; Table 1), which is likely a P-I SVMP.16 Moreover, one band corresponding to a LAAO and one band containing a SVSP were observed as present only in the adult venom. A general observation derived from these experiments is that the adult venom shows a higher degree of complexity compared to the newborn venom, as illustrated by the higher number of protein bands visualized under nonreducing conditions (Figure 1). Furthermore, most of proteins identified as differentially expressed in newborn and adult venoms are SVMPs which is in close agreement with previous analyses of both the venom proteome and the venom gland transcriptome showing that SVMPs comprise ∼50% of toxins of B. jararaca, however, the strong protein band containing a SVSP present only in the adult venom suggests a role for this toxin in the biological activities displayed by the adult venom. Taken together, these data suggest that likely as a result of ontogenetic shift in animal size and diet the composition of B. jararaca venom changes allowing the animal to deal with different types of prey during adult life. Gelatin Zymography and Immunostaining with Antiproteinase Antibodies. Gelatin zymography of newborn and adult B. jararaca venoms was performed to visualize their proteinase proteomes since gelatin is degraded by both SVMPs and SVSPs (Figure 2). Adult venom showed clearly higher gelatinolytic activity than newborn venom as illustrated by intense clear zones on the gel at the molecular mass range of around 40-100 kDa. The newborn venom showed only two regions of discrete gelatinolytic acivity at ∼35 kDa and at ∼100 2282
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Zelanis et al. kDa (Figure 2). Whether this reflects a difference in the general substrate specificities of the proteinases from the two venoms is unknown. This was nonetheless surprising since both venoms are very rich in SVMPs and SVSPs, as observed by the analysis of Western blots of the venoms immunostained with antimetalloproteinase and antiserine proteinase antibodies which showed similar profiles for both venoms (Figure 3). SVMPs and SVSPs are the most abundant components of B. jararaca venom2 and theoretically, based on the structures of these toxins as isolated from adult venoms, those antibodies should have some cross-reactivity with all classes of SVMPs and with SVSPs. The fact that both antibodies recognized similar protein bands in newborn and adult venoms indicate that metalloproteinases and serine proteinases are structurally related in these venoms and there is no detectable ontogenetic shift in their contents. A different observation was made by Alape-Giro´n and colleagues10 in their analysis of the ontogenetic variation concerning metalloproteinases in B. asper venom. These authors reported a shift from newborn venom rich in P-III SVMPs to adult venom rich P-I SVMPs. Similar results were reported by Guercio and colleagues18 by the proteomic comparison of juvenile and adult B. atrox venoms. In the case of B. jararaca venom composition, despite the fact that no clear ontogenetic shift from P-I SVMP to P-III SVMP was observed, there is a striking change in the ability to hydrolyze gelatin from newborn to adult venom that is in agreement with the necessity of the snake to initiate digestion of larger preys. On the other hand, the immunostaining of B. jararaca venoms with the antibothropic antivenom, which is generated in horses by the immunization with adult venom from five Bothrops genera including B. jararaca revealed a rather different profile for newborn and adult venoms suggesting that other classes of toxins may vary in the venom along with the ontogenetic variation (Figure 3). Proteolytic Activity upon Casein and Fibrinogen. We also determined the proteolytic activity of the newborn and adult venoms on casein and fibrinogen, which are broadly used substrates that can be degraded by both SVSPs and SVMPs. The specific activity on casein of the adult venom is ∼1.7 times higher than that of the newborn venom (newborn venom ) 0.73 ( 0.05 U/mg; adult venom ) 1.25 ( 0.16 U/mg). Likewise, when fibrinogen was used as a substrate the adult venom showed a specific activity ∼1.9 times higher than that of the newborn venom (newborn venom ) 5.6 ( 0.72 U/mg; adult venom ) 10.5 ( 1.62 U/mg). These data are in agreement with the higher gelatinolytic activity displayed by the adult venom (Figure 2). Coagulant Activity. The coagulopathy observed upon envenomation with viperid venoms is contributed by both SVSPs and SVMPs. Among the SVSPs there are enzymes capable of converting fibrinogen into fibrin by the specific cleavage of fibrinogen alpha chain with the release of fibrinopeptide A.15 Some SVMPs display unspecific fibrinogenolytic activity and yet other enzymes, especially those of the P-III class, are capable of specifically activating factor II or factor X of the coagulation cascade.49-51 Considering the difference observed in the general proteolytic activity illustrated by the higher gelatinolytic activity showed by the adult venom, we next examined the coagulant activity of newborn and adult venoms. As shown in Table 2, despite its low proteolytic activity upon gelatin, the newborn venom possesses a coagulant activity upon human plasma 10 times higher than that of the adult venom. Interestingly, the nature
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Venom Proteome/Peptidome of Bothrops jararaca
Table 1. Identification of Differentially Expressed Proteins in Newborn and Adult Venoms, Indicated in Figure 1, by LC-MS/MS band no.
1
Jerdonitin Trimeresurus jerdonii HF3 Bothrops jararaca Insularinase-A
2 3
Bothrops insularis Jararafibrase-2 Bothrops jararaca Insularinase-A
4 5
6 7 8 9 10
a
protein name/organism
Bothrops insularis Metalloproteinase VMP-III Agkistrodon contortrix laticinctus Metalloprotease BOJUMET II Bothrops jararacussu Metalloprotease BOJUMET II Bothrops jararacussu L-amino-acid oxidase Bothrops moojeni Serine proteinase HS114 Bothrops jararaca
NCBI accession number.
b
protein accession numbera
Mowse score
peptide sequenceb
gi|34329647|
59
BXSBXMXPPEBBR, ESMCDPBR, XTPGSBCADGXCCDBCR
gi|82219706|
56
LHSWVECESGECCDQCR
gi|82197476|
39
SCXMASTXSB, ABCAEGXCCDBCR, GDNPDDRCTGBSADCPR, CTGBSADCPR
gi|82219563|
124
ARGDDMDDYCNGISAGCPR
gi|82197476|
584
YXEXAVVADHGMFTB, YNSNXNTXR, TRVHEMVNTXNGFFR, ASXANXEVWSB, TXTSFGEWR
c
gi|258618056|
TPEBBAYXDAB
gi|32306927|
30
YXXDNRPPCXXNXPXR, VCNSNRECVDVSTAY
gi|32306927|
26
YLIDNRPPCILNIPLR
gi|82127389|
51
SAGBXYEESXBB
gi|82233395|
1175
NDDALDKDLMLVR, LDSPVSDSEHIAPLSLPSSPPSVGSVCR, IMGWGSITPIQK, TNPDVPHCANINLLDDAVCR, AAYPELPAEYR
X, Ile or Leu. B, Lys or Gln; C-terminal peptides are shown in italics. c De novo sequencing (BlastP e-value ) 2 × 10-5).
Figure 2. Gelatinolytic activity of B. jararaca newborn and adult venoms (25 µg) evaluated by zymography. Numbers on the left indicate molecular mass marker mobility. The gel was stained with Coomassie blue.
of the proteinases contributing to the coagulant activity displayed by the venoms is rather different. The incubation of the venoms with a specific SVSP inhibitor (PMSF) and a metal chelating agent (EDTA) showed that the coagulant activity of the venom from newborns is mainly related to metalloproteinases while in the case of adult venom both proteinase classes contribute to the observed coagulant activity (Table 2). These data clearly point to an ontogenetic variation in venom composition that leads to a significant change in strategy by the snake to deal with prey larger in size. Therefore, the potent coagulant activity of the newborn venom upon human plasma is likely the result of the
Figure 3. Western blot analysis of newborn and adult B. jararaca venoms (25 µg) using antibothropic, antimetalloproteinase, and antiserine proteinase antibodies. After SDS-PAGE separation on 12% SDS-polyacrylamide gels, proteins were transferred to a nitrocellulose membrane and immunostained with polyclonal antibodies. (A) Antibothropasin antibody; (B) anti-MSP1/2 antibody; (C) antibothropic antivenom. The arrows indicate protein band differences among the venoms.
activation of factor II or factor X while the rather far less potent adult venom clots plasma by a combination of effects upon these factors and upon fibrinogen. To test this hypothesis, we tested the ability of newborn and adult venoms to directly activate factor II (prothrombin) and factor X and detected a significantly higher and faster generation of thrombin and factor Xa in vitro by the newborn venom (Figure 4). These results confirmed the role played by metalloproteinases of the P-III class in the potent coagulant activity of the newborn venom and underscored a clear shift Journal of Proteome Research • Vol. 9, No. 5, 2010 2283
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Table 2. Human Plasma Coagulant Activity of B. jararaca Newborn and Adult Venoms newborn venom +10 mM EDTA
Coagulant activity (µg)† Specific activity (U/mg) Residual specific activity (U/mg)§
0.15 6514 100%
† Minimum Coagulant Dose (see Experimental Section). after previous incubation of venom with inhibitors.
6.85 146 2.24% §
adult venom +10 mM EDTA
+2 mM PMSF
0.16 6018 92.4%
1.44 696 100%
5.90 169 24.3%
+2 mM PMSF
2.98 335 48.1%
Residual activity is expressed as percentage of specific activity (U/mg of venom) remaining
Figure 4. Activation of Factor II and Factor X by newborn and adult B. jararaca venoms, as described in the Experimental Section. Table 3. LD50 and Minimum Hemorrhagic Dose (M.H.D.) of B. jararaca Newborn and Adult Venoms
Newborn venom Adult venom
LD50 (mouse) (mg/kg)a
LD50 (chick) (mg/kg)a
3.33 (3.04-3.71) 2.26 (1.86-2.89)
0.05 (0.02-0.09) 1.36 (0.86-2.07)
MHD (µg)b
0.44 ( 0.09 0.34 ( 0.02
a
Values between parentheses mean 95% upper and lower confidence intervals. b MHD determined in mice (µg) (see Experimental Section). Results are expressed as mean of four determinations ( standard deviation.
in metalloproteinase substrate specificity between newborn and adult venoms Hemorrhagic and Lethal Activities. Local hemorrhage is an important feature of B. jararaca envenomation related to the activity of SVMPs in which both the metalloproteinase and the noncatalytic domains of these enzymes play a role.16 Despite the significantly higher gelatinolytic activity displayed by the adult venom, the determination of the skin hemorrhagic activity of newborn and adult venoms showed similar MHD for both venoms (Table 3), corroborating the similar profiles obtained by immunostaining of newborn and adult venoms with antimetalloproteinase antibodies (Figure 3). We next evaluated the lethal potency of B. jararaca venoms in mice which showed LD50 values of 3.33 mg/kg for the 2284
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Figure 5. Glycoproteome analysis of newborn and adult B. jararaca venoms. (A) SDS-PAGE (8-18% SDS-polyacrylamide gel) profile of venom proteins (50 µg) stained with Pro-Q-Emerald. (B) SDS-PAGE (8-18% SDS-polyacrylamide gel) profiles of newborn and adult venoms (15 µg) incubated in the presence and in the absence of N-glycosidase F and O-glycosidase. Arrows indicate shifts in molecular mass after deglycosylation. (C) SDSPAGE (12% SDS-polyacrylamide gel) profile of B. jararaca venoms submitted to Con A-Sepharose chromatography. (NB) nonbound and (B) bound venom fractions. Proteins were stained with silver.
newborn venom and 2.26 mg/kg for the adult venom (Table 3). These results are in agreement with those previously reported by Andrade and Abe25 which found that venom from adult specimens of B. jararaca was more potent to kill rodents in comparison to newborn ones. This was not surprising since adult snakes feed mainly on bulky (endothermic) prey in comparison to newborn specimens. On the other hand, the determination of LD50 in chicks revealed a significant difference
research articles
Venom Proteome/Peptidome of Bothrops jararaca
Table 4. Identification of B. jararaca Newborn and Adult Venom Proteins with Affinity for Con A-Sepharose, Indicated in Figure as Bound (B) Fraction, by LC-MS/MS NUPc acc. number
Venom serine proteinase HS114 Venom serine proteinase-like Venom serine protease homologue Platelet-aggregating proteinase PA-BJ Kinin-releasing and fibrinogen-clotting serine proteinase 2 Thrombin-like enzyme batroxobin Putative serine protease Zinc metalloproteinase-disintegrin BITM06A Batroxstatin-1 Zinc metalloproteinase-disintegrin HF3 Zinc metalloproteinase-disintegrin bothrojarin-2 Zinc metalloproteinase-disintegrin atrolysin-A Zinc metalloproteinase-disintegrin bothrojarin-3 Zinc metalloproteinase/disintegrin Zinc metalloproteinase-disintegrin berythractivase Batroxstatin-3 L-amino acid oxidase precursor Coagulation factor IX/factor X-binding protein subunit B Glycoprotein IB-binding protein subunit beta
a
gi|82233395| gi|123883733| gi|82240434| gi|6093643| gi|13959622| gi|114837| gi|126035656| gi|82214993| gi|205278803| gi|82219706| gi|123905787| gi|75570463| gi|123905786| gi|52000724| gi|82216043| gi|205278807| gi|195927838| gi|82116580| gi|82116883|
toxin class
SP SP SP SP SP SP SP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP SVMP LAAO CTL CTL
b
N
d
7 7 7 4 2 2 4 4 2 6 2 2 4 3 2 15 2 2
A
5
SC(%)e d
5 7 3 2 2 2 6 2 6 -
d
N
Ad
glycosylation sitesf
41.1 36.8 42.7 17.5 13.3 12.4 14.3 20.6 12 39 8.83 25.5 12.2 10.3 9.66 35.8 93.3 24.4
36.8 39.4 24.2 19.8 12.8
N20 N20 N59 N20; S23 N78;N145;N226 N225 N23;N27;N117;N131;N135 N183 N29;N74 N69;N123;N183;N329;N394 N72;N326 N107; N159 N73;N161;N187 N7;T27;N70 N172; T332 -
12.4 17.2 6.93
24.1
a NCBI accession number. b SP: serine proteinase; SVMP: snake venom metalloproteinase; LAAO: L-amino acid oxidase; CTL:C-type lectin. c NUP: Number of unique peptides (proteins identified with only one peptide were not considered for further analysis). d N: Newborn venom proteins; A: Adult venom proteins. e SC(%): Percentage of sequence coverage. f N and O-glycosylation sites were predicted from mature protein sequences using NetNGlyc Server 1.0 and OGPET v 1.0, respectively (available at http://www.cbs.dtu.dk/services/NetNGlyc/ and http://ogpet.utep.edu/OGPET/index.php, respectively).
in lethal potency between the venoms. Not only both venoms were more lethal to chicks compared to mice but also the newborn venom showed a value of LD50 (0.05 mg/kg) which is 27 times lower than that of adult venom (1.36 mg/kg) (Table 3). Zelanis and collagues9 showed ontogenetic changes in proteinase expression in the venom of an insular pitviper species, B. insularis, which shows a striking diet shift during ontogeny as it feeds on ectothermic prey (amphibians, arthropods and lizards) when juvenile and on endothermic prey (migratory birds) in adulthood. Regulation of venom proteinase expression must be a key step in species whose diet changes ontogenetically. Being smaller and having a lower size, as well as having a small amount of venom in their venom glands, newborns have to face serious challenges in order to survive and become sometimes prey of other animals. Therefore, ontogenetic changes in expression of venom proteins and peptides could ensure not only suitable toxins for specific types of prey, but also protection against potential predators. The significance of the higher lethal activity of B. jararaca newborn venom upon chicks is unknown, however, considering that neonate specimens are potential prey to birds, it is possible to hypothesize that the newborn venom may contain toxins that target specific vital survival systems to immobilize and kill this type of predator as illustrated by the high coagulant activity of the newborn venom that could potentially cause an acute imbalance in the bird blood coagulation system leading to death. Glycoproteome. Protein glycosylation is a key post-translational modification important to a range of biological phenomena. We used the fluorescent glycoprotein specific stain, Pro-Q-Emerald, to assess the glycoproteome of the newborn and adult venoms of B. jararaca. Figure 5A shows a similar profile of protein bands stained with this fluorescent dye in both venoms, however, the adult venom showed a stained band of ∼20 kDa absent in the newborn venom. Moreover, the
glycoprotein complexity of B. jararaca venom, as illustrated by the staining with this reagent, appears somewhat less than that observed after the deglycosylation with N-glycosidase F. Indeed, Figure 5B shows a significant shift in the molecular masses of proteins of both newborn and adult venoms after removal of N-linked carbohydrate chains indicating a rather high level of N-glycosylation in the B. jararaca proteome which seems to be even more prominent in the adult venom. It is notable that even after N-deglycosylation the newborn and adult venoms showed a distinct electrophoretic profile indicating that their proteomes differ not only at the N-glycosylation level but also at the protein core itself. On the other hand, O-glycosylation does not seem to occur in most newborn and adult venom proteins as no significant change in the pattern of protein migration was observed after incubation of venoms with O-glycosidase (Figure 5B). We next explored the subproteome of glycoproteins with affinity for the lectin concanavalin A, which binds mannosecontaining carbohydrate ligands,52 using Con A-Sepharose chromatography. This simple process to enrich post-translationally modified proteins allowed for the rapid fractionation of glycoproteins from the complex mixtures of newborn and adult venoms and confirmed the dramatic change in molecular mass observed after N-deglycosylation of both venoms. As shown in Figure 5C many venom proteins were found in the fraction eluted with D-glucose from the Con A-Sepharose column. Most of the glycoproteins with affinity for Con A showed molecular masses between 24 kDa and 80 kDa, however, a few protein bands of ∼14 kDa and lower were also observed. Interestingly, the electrophoretic profiles of newborn and adult venom proteins with affinity for Con A are rather similar indicating the presence of common carbohydrate chains in both venoms despite the fact that their glycoproteins contain different levels of glycosylation, as observed in Figure 5B. On the basis of the identification of Con A-binding proteins by in solution trypsin digestion and LC/MS/MS analysis some Journal of Proteome Research • Vol. 9, No. 5, 2010 2285
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Figure 6. (Left) MALDI-TOF profiles of peptides of B. jararaca newborn and adult venoms. (Right) Profiles of venoms incubated with a mixture of proteinase inhibitors prior to mass spectrometric analysis.
generalizations can be made as to what classes of proteins undergo glycosylation in the venom gland. As shown in Table 4, SVMPs, SVSPs, L-amino acid oxidase and C-type lectins were detected among the proteins with affinity for Con A-Sepharose. Using as a criterion for protein identification the number of unique peptides and restricting the identification to those proteins for which at least two peptides matched a protein in the database we identified 18 proteins in the newborn venom and 9 proteins in the adult venom with affinity for Con A (Supplemental Table 1, Supporting Information). This was surprising since the electrophoretic profiles of proteins of newborn and adult venoms that bound to Con A showed similar complexity by SDS-PAGE (Figure 5C). Moreover, considering that most snake venom protein information deposited in databases is derived from investigation on adult venom or adult venom gland, it was unexpected to identify a lower number of glycoproteins from adult venom than from newborn venom. Among the venom components with affinity for Con A, there are four proteins detected only in the newborn venom which do not contain putative glycosylation sites: two SVMPs (zinc metalloproteinase-disintegrin bothrojarin-2 and zinc metalloproteinase-disintegrin bothrojarin-3) and two C-type lectins (coagulation factor IX/factor X-binding protein subunit B and glycoprotein IB-binding protein subunit beta). We hypothesize 2286
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that these proteins are actually unknown glycoproteins present in the newborn venom which show homology with other nonglycosylated proteins of adult venom whose sequences are deposited in the databanks (Table 4; Supplemental Table 1, Supporting Information). Glycosylation, particularly N-linked glycosylation, affects significantly protein folding, oligomerization and stability,53 and might be important for protein function. The exact contribution of carbohydrate moieties to the biological activities of venom toxins is poorly understood, however, there are some reports on the role of glycosylation in the stability and specificity of SVMPs, SVSPs, and LAAO.54-56 Moreover, the varying glycosylation levels occurring in proteins within the same toxin family considerably contributes to the complexity of the snake venom proteome.2,57,58 Our results indicate that the proteomes of both newborn and adult B. jararaca venoms are comprised mostly of glycoproteins with affinity for Con A and that there does not seem to be a shift in the glycosylation level of these toxins upon ontogenetic development. Peptidome. In the past few years, efforts to study snake venoms by proteomic approaches have built up, unveiling the complexity of venoms from several species. Although snake venom proteomes and transcriptomes are rapidly advancing research fields, the study of venom peptidomes do not progress
research articles
Venom Proteome/Peptidome of Bothrops jararaca
Table 5. Monoisotopic Masses of Peptides Identified in B. jararaca Newborn and Adult Venoms by MALDI-TOF Analysis [M + H]+a
venom
Newborn
Newborn (+2 mM PMSF and 10 mM EDTA) Adult
Adult (+2 mM PMSF and 10 mM EDTA) a
825.1; 844.4; 1044.1; 1095.7; 1101.7; 1117.7; 1123.7; 1189.7; 1196.7; 1213.7; 1215.7; 1237.7; 1240.6; 1259.7; 1279.8; 1296.8; 1297.8; 1299.8; 1331.8;1321.7; 1343.9; 1353.8; 1357.9; 1368.8; 1370.8; 1386.8; 1392.8; 1402.8;1408.8; 1414.8; 1486.9; 1512.4; 1557.9; 1564.0; 1685.0; 1813.1 607.2; 1095.5; 1101.5; 1189.6; 1196.5; 1215.6; 1275.6; 1279.6; 1296.6; 1357.6;1370.6; 1392.6; 1414.6; 1486.7 761.4; 780.4; 811.2; 825.2; 841.4; 844.6; 864.5; 1015.2; 1044.3; 1095.8; 1101.8; 1117.7; 1189.8; 1196.8; 1215.9; 1240.9; 1280. 0; 1296.4; 1297.2; 1298.1; 1300.0; 1358.0; 1370.3; 1386.9; 1393.1; 1402.5; 1408.8; 1415.0; 1487.1; 1524.2; 1685.1 607.2; 761.2;780.3; 844.4;1095.5; 1101.5; 1185.5; 1189.5; 1196.5; 1215.5; 1279.6; 1297.5 1357.6; 1370.6; 1392.5
Masses shown in bold face correspond to those of known BPPs.59
at the same rate due to the need of database searching of MS/ MS spectra without specific enzyme cleavage and, for unknown sequences, the need of large scale de novo sequencing of new peptides. Hence, snake venom peptidome variation is largely unknown. In order to assess the ontogenetic variation of B. jararaca venom peptide fraction, the MALDI-TOF mass spectra of venoms from newborn and adult specimens were compared (Figure 6). Table 5 shows that a total of 36 and 31 peptide monoisotopic masses up to 2 kDa were identified in the newborn and adult venom, respectively. A comparison of the peptide masses revealed a high variability between the venoms. The number of identified masses of known BPPs (10 and 9, in newborn and adult venom, respectively) was similar. Most of the measured peptide masses do not correspond to known BPP sequences and likely represent unknown peptides. These peptides varied considerably between the venoms but the significance of this phenomenon is unknown. However, when the venoms were treated with proteinase inhibitors (PMSF and EDTA) prior to desalinization using ZipTip C-18 and MALDITOF analysis, the profile of monoisotopic masses changed clearly showing a significantly lower number of peaks (Table 5; Figure 6) indicating that many peptides detected in the venom without treatment with inhibitors were generated by the cleavage of proteins by venom proteinases and the different content observed for those peptides suggest that different proteinases in the newborn and adult venoms played a role in the proteolysis. Nevertheless, the number of BPPs detected in the treated venoms is similar to that observed in the untreated venoms indicating that these peptides are in general resistant to proteolysis both in the adult and newborn venom (Table 6). The venom peptide fraction was also analyzed by LC-MS/ MS. After submitting the venoms to solid phase extraction using Sep-Pak C18 cartridges, peptides were submitted to chromatography on an analytical C18 column and MS/MS using two mass spectrometers, a linear LTQ ion trap and a Q-TOF. As was the case with the MALDI-TOF analysis, the LC-MS/MS approach also revealed that the general profiles of the venom peptidomes are different. The total ion count (TIC) chromatograms of LC-MS/MS runs of peptides and the plots of elution times from LC versus ion intensity showed different profiles for newborn and adult venoms (Figure 7), as was observed with the analysis by MALDI-TOF. On the other hand, the profile of bradykinin potentiating peptides was not so diverse between the venoms. These peptides were detected in the venoms showing their canonical sequences32,33 and also novel sequences corresponding to forms of BPPs processed from their
Table 6. BPPs Identified in B. jararaca Newborn and Adult Venoms by MALDI-TOF Analysis theoretical mass
BPPa
newborn
adult
Untreated venom 1094.6 1100.5 1188.6 1195.6 1214,6 1296.7 1298.6 1369.6 1485.8 1683.8
11d 9a 11e 10c 10b 13b 11a 13a 12c 14a
Y Y Y Y Y Y Y Y Y Y
Y Y Y Y Y Y Y Y
Venom treated with 2 mM PMSF and 10 mM EDTA 1094.6 11d Y 1100.5 9a Y 1188.6 11e Y 1195.6 10c Y 1214,6 10b Y 1296.7 13b 1369.6 13a Y 1485.8 12c Y a
Y Y Y Y Y Y Y Y
BPP nomenclature is according to Ianzer et al.59
precursor protein at sites so far not described (Table 7). A total of 11 and 10 BPPs were identified in the newborn and adult venoms, respectively (Table 7). Other known BPPs were also detected in the database search of MS/MS spectra, however, their scores were below the 95% confidence level (p > 0.05) according to MASCOT criteria and were not listed in Table 7 as identified peptides. Nevertheless, these MS/MS spectra showed signals, such as the intense y2 peak at m/z 213.1, corresponding to Pro-Pro and the complementary bn-2 in some spectra, typically attributed to BPPs. In addition, BPPs are proline-rich peptides, and y-ions with prolines at the Nterminal are the most abundant in the spectra.60-62 As a consequence, other fragments are detected in low abundance or are absent, a fact that we also observed in these BPP spectra (not shown). Three BPPs (12c, 13a, 14a) were detected showing their characteristic structure containing a pyroglutamic acid residue (
