Individual Variability in the Venom Proteome of Juvenile Bothrops

Subscriber access provided by Library, Special Collections and Museums, University of Aberdeen

Article

Individual variability in the venom proteome of juvenile Bothrops jararaca specimens. Gabriela Silva Dias, Eduardo Shigueo Kitano, Ana Helena Pagotto, Sávio Stefanini Sant’anna, Marisa Maria Teixeira Rocha, André Zelanis, and Solange M.T. Serrano J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr4007393 • Publication Date (Web): 02 Sep 2013 Downloaded from http://pubs.acs.org on September 8, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Individual variability in the venom proteome of juvenile Bothrops jararaca specimens.

Gabriela S. Dias1, Eduardo S. Kitano1,2, Ana H. Pagotto1, Sávio S. Sant'anna3, Marisa M. T. Rocha3, André Zelanis1, Solange M. T. Serrano1*

1

Laboratório Especial de Toxinologia Aplicada - CeTICS, Instituto Butantan,

Brazil 2

Instituto de Química, Departamento de Bioquímica, Universidade de São

Paulo, Brazil 3

Laboratório de Herpetologia, Instituto Butantan, Brazil

*Corresponding author: Solange M. T. Serrano Laboratório Especial de Toxinologia Aplicada Instituto Butantan Av. Vital Brasil 1500, 05503-000, São Paulo, Brazil Tel.: +55 11 3726-1024 E-mail: [email protected]

Keywords: individual variability; peptidome; proteinase; proteome; snake venom.

Abbreviations: AP, aminopeptidase; CRISP, cysteine-rich secretory protein; CTL, C-type lectin; LAAO, L-amino acid oxidase; M.C.D., minimum coagulant dose; SVMP, snake venom metalloproteinase; SVSP, snake venom serine proteinase; VEGF, vascular endothelial growth factor.

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 55

Abstract Snake venom proteomes/peptidomes are highly complex and subject to ontogenetic changes. Individual variation in the venom proteome of juvenile snakes is poorly known. We report the proteomic analysis of venoms from 21 juvenile specimens of Bothrops jararaca of different geographical origins and correlate it with the evaluation of important venom features. Individual venoms showed similar caseinolytic activities, however their amidolytic activities were significantly different. Rather intriguingly, plasma coagulant activity showed remarkable variability among the venoms but not the prothrombin-activating activity. LC-MS analysis showed significant differences between venoms, however, an interesting finding was the ubiquitous presence of the tripeptide ZKW, an endogenous inhibitor of metalloproteinases. Electrophoretic profiles of proteins submitted to reduction showed significant variability in total proteins, glycoproteins, and in the subproteomes of proteinases. Moreover, identification of differential bands revealed variation in most B. jararaca toxin classes. Profiles of venoms analyzed under non-reducing conditions showed less individual variability and identification of proteins in a conserved band revealed the presence of metalloproteinases and L-amino acid oxidase as common components of these venoms. Taken together, our findings suggest that individual venom proteome variability in B. jararaca exist since very early animal age and is not a result of ontogenetic and diet changes.

2


Page 3 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction

The complexity of snake venoms has long been investigated to reveal a myriad of biologically active proteins and peptides that are responsible for the pathological effects observed upon envenomation.

1-3

At the same time, snake

venom components have proved to be useful tools in biomedical investigation and in drug design.

4-7

Viperid venoms are highly complex mixtures of

enzymatic and non-enzymatic components, most of which are involved in the rapid immobilization, killing and digestion of envenomed prey. In recent years, a growing number of studies have illustrated the complexity of venoms from viperid snakes using a variety of proteomic approaches.

2,8

Notably, the

complexity of viperid venom proteomes is contributed to by various molecular mechanisms including: i) the high degree of amino acid sequence variation in non-conserved regions of toxins; ii) the variable glycosylation levels within toxin families; iii) the modular structure of metalloproteinase precursors which may undergo different processing events resulting in variable mature protein structures; iv) the variable sites of processing of the precursor of bradykinin potentiating peptides (BPPs) that results in a variety of BPPs of different amino acid sequences and length. 9-14 The proteome determined by a given genome can be variable depending on various endogenous and/or exogenous factors. In the case of venomous snakes, variation in the venom proteome has been illustrated by different electrophoretic profiles and pharmacological activities between venoms. It is a ubiquitous phenomenon that is detected at all taxonomical levels including 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 55

species occurring in different geographical regions and habitats15,16, and specimens belonging to the same litter.

12,17-19

Moreover, intraspecific venom

variation occurs between individual specimens, and also in individual specimens, due to seasonal variation, diet, age, and sexual dimorphism.

13,19-24

Studies of captive-bred snakes indicated that intraspecific variation in venom is genetically inherited rather than environmentally induced25,26, however, microevolutionary forces other than selection for local prey might influence the high variation of venom components among populations.

27

Moreover, there is

also indirect evidence that venom variation is under genetic control likely due to the presence or absence of alleles that encode specific venom proteins, substitutions of amino acids in the regions of genes that encode mature proteins including sequons, and modulation of venom composition by miRNAs. 28-32 The knowledge on venom variation is relevant to the selection of snake donors that are used in venom production for research and antivenom preparation, to evolution analyses, and to the management of snake envenomation. Hence, there have been several studies focusing on individual variation in snake venoms. More recently, proteomic and peptidomic approaches have been applied to underscore in some cases significant differences and in others subtle aspects of individual venom variation. Bothrops jararaca is one of the most abundant venomous snake species in Brazil and shows a broad geographical distribution. It is responsible for a high number of accidents in Brazil and the envenomation cases are characterized by local and systemic effects, such as local edema, hemorrhage, myonecrosis, and severe coagulatopathy.

33

Interestingly, the comparative analysis of envenomation cases caused by newborn and adult B. jararaca snakes has shown distinct patterns, mainly 4


Page 5 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


concerning the coagulopathy, which is more prominent in accidents with newborn specimens.

34

Indeed, the pharmacological activities displayed by B.

jararaca venom undergo a significant ontogenetic shift and, in parallel, the diet of this species changes from ectothermic prey in early life to endothermic prey in adulthood. Recently, we have reported the variability in the proteome and peptidome of newborn and adult B. jararaca venoms which showed differences in their different protein profiles, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and content of concanavalin A-binding proteins.

13

We also showed that upon snake development, the subproteome of

metalloproteinases undergoes a shift from a P-III-rich to a P-I-rich profile while the serine proteinase profile does not vary significantly.

20,21

Quantitative

proteomic changes were also detected by iTRAQ analysis in various toxin classes, especially the proteinases, confirming the ontogenetic variation of B. jararaca venom. Similarly, using a transcriptomic approach we demonstrated a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which were correlated with changes in the venom proteome complexity and pharmacological activities. 21

However, the N-glycome profiling of newborn and adult venoms did not reveal

significant differences in the N-glycan composition. 35 The first study that used two-dimensional electrophoresis for the analysis of individual variation among B. jararaca adult venoms showed distinct images for the venoms from six individual adult specimens.

36

Furthermore, sex-based

venom variation was demonstrated by the analysis of the venom proteomes of eighteen B. jararaca adult specimens of a single litter by one-dimensional and two-dimensional electrophoresis along with the determination of various 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

proteolytic activities.

18

Page 6 of 55

Likewise, the individual analysis of the peptidomes of B.

jararaca venoms from sibling and non-sibling snakes showed for the first time sex-based differences among the BPPs. 12 Although the venom of B. jararaca has been the focus of extensive studies, the variation of newborn venom composition is completely unknown given that most studies on snake venoms are usually carried out using venom from adult specimens. Moreover, the individual variability demonstrated for venoms from adult specimens raised the question as to how often the venom of a newborn specimen exhibits the phenotype of the adult venom. Therefore the aim of this investigation was to analyze individual venom samples from B. jararaca juvenile specimens in order to evaluate sex-based and geographic origin differences in venom composition. Using electrophoretic techniques coupled to mass spectrometric analysis and various protocols for measuring the proteolytic activities of the individual venoms, we have highlighted proteomic similarities and differences among sibling and non-sibling juvenile snakes. The findings of this work suggest that individual venom variability in B. jararaca exist since very early animal age and is not a result of ontogenetic changes.

Experimental section

B. jararaca venom samples Individual venom samples were extracted from 21 juvenile specimens of B. jararaca (not older than 9 months) for analysis. Snakes were born in the Laboratory of Herpetology at Instituto Butantan by pregnant specimens that originated from São Paulo (SP), Minas Gerais (MG), and Santa Catarina (SC) 6


Page 7 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


states (Supplemental table 1; supplemental figure 1) and maintained in individual containers under identical climatic conditions. The specimens originated from 10 mothers (mother #1, from São Bento do Sul-SC, specimens #1-8; mother #2, from Pilar do Sul-SP, specimens #9-10; mother #3, from Ibiúna-SP, specimens #11-12; mother #4, from São Lourenço da Serra-SP, specimens #13-15; mother #5, from Extrema-MG, specimen #16; mother #6, from Pilar do Sul-SP, specimen #17; mother #7, from Araçariguama-SP, specimen #18; mother #8 Santana do Parnaíba-SP, specimen #19; mother #9, from Ribeirão Pires-SP, specimen #20; mother #10, from Tapiraí-SP, specimen #21). They were fed the same diet, and the technique and frequency of venom extraction were identical for all animals. Venom from each specimen was manually milked during nine months using glass capillaries and individually pooled. Then each pool was centrifuged for 30 min at 2000 x g, 4°C, to remove any scales or mucus and stored at -20°C until analysis. Venom protein concentrations were determined using the Bradford reagent (Sigma, St. Louis, MO, USA) and bovine serum albumin (Sigma, St. Louis, USA) as a standard.

SDS-polyacrylamide gel electrophoresis and Western blot analysis SDS-PAGE was carried out according to Laemmli

37

and proteins were stained

with colloidal Coomassie G-250 (Sigma, St. Louis, MO, USA), or with the fluorescent dye Pro-Q-Emerald (Molecular Probes, Eugene, OR) according to the manufacturers’ instructions. Western blot analysis was carried out as described elsewhere

38

using (i) anti-Bothropasin rabbit polyclonal antibody

raised against the P-III class metalloproteinase bothropasin from B. jararaca 39,

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 55

and (ii) anti-MSP1/2 rabbit polyclonal antibody, raised against a mixture of the basic serine proteinases MSP1 and MSP2 from B. moojeni. 40

In-gel protein digestion and MS identification Protein bands were excised and in-gel trypsin digestion was performed according to Hanna et al.

41

An aliquot (4.5 µL) of the resulting peptide mixture

was injected into a trap column packed with C18 (180 µm id x 20 mm) (Waters, Milford, MA) for desalting with 100% solvent A (0.1% formic acid) at 15 µL/min for 3 min. Peptides were then eluted onto an analytical C18 column (100 µm id x 100 mm) (Waters, Milford, MA) using a 20 min gradient at a flow rate of 600 nL/min where solvent A was 0.1% formic acid and solvent B was 0.1% formic acid in acetonitrile. The gradient was 0–3% of solvent B in 1 min, 3–60% B in 14 min, 60–80%B in 2.5 min, 80%B for 1 min, then back to 3%B in 1.5 min. A QTOF Ultima mass spectrometer (Waters, Milford, MA) was used to acquire spectra. Spray voltage and temperature were set at 3.4 kV and 100 °C, respectively, and the instrument was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 200–2000 followed by MS/MS acquisition using collision induced dissociation of the three most intense ions from the MS scan. Dynamic peak exclusion was applied to avoid the same m/z to be selected for the next 90 s. Raw data files were processed by ProteinLynx 2.2 (Waters, Milford, MA) and converted to *pkl format and searched against the NCBI NR database restricted to the taxa Serpentes (25,211 entries; downloaded in June 20th, 2011) with a parent and fragment ion tolerance of 0.6 Da. Iodoacetamide derivative of cysteine and oxidation of methionine were specified in MASCOT 2.2.04 (Matrix Science, London, UK) as 8


Page 9 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fixed and variable modifications, respectively, for database search. Scaffold 3 Q+ (version Scaffold_3_00_01, Proteome Software, Portland, OR, USA) was used to validate MS/MS based peptide and protein identifications. MASCOT identifications required ion scores greater than both the associated identity scores and 10, 20, 20, and 40 for singly, doubly, triply, and quadruply charged peptides, respectively.

LC-MS analysis Venom samples (1 µg in 4.5 µL 0.1% formic acid) were injected into a nanoAcquity UPLC system (Waters, Milford, MA) using a trap column packed with C18 (180 µm id x 20 mm) and an analytical C18 column (100 µm id x 100 mm) coupled to a Q-TOF Ultima mass spectrometer (Waters, Milford, MA). The gradient was 0–3% of solvent B in 1 min, 45% B for 57.5 min, 80%B for 1 min, then back to 3%B in 1 min. Base peak intensity chromatograms were analyzed using MassLynx 4.1 (Waters, Milford, MA).

Caseinolytic activity N,N-dimethylated casein (Sigma, St. Louis, MO, USA) was used as a substrate in a system containing 0.4 mL buffer solution (0.1 M Tris-HCl buffer, pH 8.8, 0.01 M CaCl2), 0.1 mL venom solution (containing 10 µg or 20 µg protein) and 0.5 mL 2% casein solution (solubilized in the same buffer), for 30 min, at 37°C. The reaction was stopped by adding 1 mL 5% trichloroacetic acid, the mixture was centrifuged at 14,000 rpm for 15 min and absorbance at 280 nm was measured. One unit of activity was defined as the amount of venom yielding an

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 55

increase in O.D. of 1.0 per min at 280 nm. Specific activity was expressed as units/mg protein.

Amidolytic activity Amidolytic activity was determined on Bz-Arg-pNA (Merck, Germany) at 37°C for 30 mim) in a system containing 0.40 mL of 0.1 M Tris–HCl buffer, pH 8.0 (buffer A), 0.45 mL of substrate solution (1 mM) and 0.05 mL of venom (10 µg or 20 µg protein) diluted in buffer A. Reactions were stopped by adding 0.1 mL of 30% acetic acid and release of p-nitroaniline was monitored at 405 nm. Amidolytic activity was calculated using a molar absorbance of 10200 for pnitroaniline. Specific amidolytic activity was expressed as micromole of substrate hydrolysed per minute per mg protein.

Coagulant activity Minimum Coagulant Dose (M.C.D.) was used to measure coagulant activity of venom samples. M.C.D. 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). ,

Prothrombin activation Prothrombin (500 nM, final concentration) (Sigma, St. Louis, MO, USA) was incubated with 50 ng of venom samples in a final volume of 100 µL of 20 mM Tris-HCl, pH 8.3 containing 5 mM CaCl2, and H-D-Phe-Pip-Arg-pNA (400 µM, 10


Page 11 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


final concentration) (Chromogenix, Italy), a chromogenic peptide substrate for thrombin, and the hydrolysis was monitored at 405 nm for 60 min at 37°C. Amidolytic activity was calculated using a molar absorbance of 10200 for pnitroaniline and expressed as picomole of peptide substrate hydrolyzed per minute.

Multivariate analysis The cluster analysis was performed using the unsupervised hierarchical clustering method based on Euclidean distance and complete linkage. The variables used in the analysis were protein concentration and enzymatic activities

(caseinolytic,

amidolytic,

plasma

coagulant

and

prothrombin-

activating) of the venoms. Since it is an unsupervised approach, information about gender and geographic origin of the specimens was omitted from the analysis, so that any clustering that might separate the samples according to their gender or origin would be only based on venom features. The statistical analysis

was

carried

out

using

the

ActionTM

(Estatcamp)

package

(http://www.portalaction.com.br) installed as a complement to MicrosoftTM Excel.

Results and discussion

Total protein and glycoprotein profiles For this study, venom from 21 B. jararaca juvenile specimens (9 male and 12 female), originated from 10 litters, was monthly collected over 9 months. Snakes were born at Instituto Butantan by pregnant specimens that originated from 9 localities of 3 Brazilian states (São Paulo, Minas Gerais and Santa 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 55

Catarina) (Supplemental figure 1; supplemental table 1) and kept under controlled laboratory conditions. All experiments were carried out with nonlyophilized venoms whose protein concentration ranged between 110 mg/mL and 120 mg/mL, except for two samples that showed values of 87 mg/mL and 135 mg/mL (Table 1). To determine the extent to which gender and geographical origin may be reflected in individual venom variation, we first examined the venom protein profiles by SDS-PAGE (12% gel) under reducing conditions (Figure 1A). We observed a remarkable variation in SDS-PAGE protein profile between venom samples of individual snakes irrespective of gender or origin. Profile variation concerning presence/absence of bands and their intensities was visualized at molecular masses between 10 kDa and 70 kDa. Even the venoms of the largest litter (venoms #1-8) showed significant variable profiles particularly at the molecular mass range of 10-40 kDa. Nevertheless, a large protein band of ~50 kDa showed similar intensity in all venoms. These findings are in agreement with our previous analysis of venoms from 18 B. jararaca adult siblings which also showed a high degree of variability of protein bands. Interestingly, the analysis of venoms from 17 newborn siblings of Deinagkistrodon acutus showed very similar electrophoretic protein profiles under reducing conditions.19 Therefore, it seems that the individual variability of venom polypeptide chains in B. jararaca species is a feature present in the proteome since early life stage. Protein glycosylation is a key post-translational modification important to a range of biological phenomena including the toxins of B. jararaca venom.

13,43

We used a fluorescent glycoprotein specific stain, Pro-Q-Emerald, to assess the glycoproteome of the individual venoms of juvenile B. jararaca. Figure 1B 12


Page 13 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shows variable profiles of protein bands stained with this fluorescent dye between all venoms, and, interestingly, the region of molecular mass of ~50 kDa, which showed a rather similar profile with Coomassie staining, was revealed to contain a variable content of glycoproteins. Also notable was the variable staining of proteins of low molecular mass (below 14 kDa) with Pro-QEmerald. The glycoproteomic analysis of B. jararaca venom showed that Nglycosylation seems to be the most prominent post-translational modification in toxins and the N-glycosylation profiles differed for newborn and adult venom toxins.

13

Moreover, the subproteomes of glycoproteins from B. jararaca

newborn and adult venoms with affinity for Concanavalin A lectin were shown to be composed mainly by SVSPs and SVMPs.

13

Hence, the variable profiles of

proteins stained with Pro-Q-Emerald shown in this study suggest that the degree of glycosylation in SVSPs and SVMPs likely differ between individual specimens of B. jararaca.

Proteinase subproteomes We next analyzed the subproteome of metalloproteinases of B. jararaca juvenile venoms by Western blot using an antibody against the P-III metalloproteinase bothropasin, from B. jararaca, which theoretically, based on its structure, could have some cross-reactivity with all classes of SVMPs as well as disintegrinlike/cysteine-rich proteins and disintegrins in the venoms.

11,13,39

As would be

expected for the B. jararaca venom proteome, the profiles of most juvenile venoms showed a significant presence of SVMPs in the molecular range of 2050 kDa (Figure 2A). Interestingly, proteins of venoms #11 and #12 were poorly recognized, while venoms #8 and #14 showed a wider range of SVMPs which 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 55

cross-reacted with anti-bothropasin antibody. Overall, immunostaining by antibothropasin indicated that individual variability of SVMPs occurs in juvenile venoms, as observed by the different molecular masses and intensities of the stained bands, and is independent of the animal gender and geographical origin. The subproteome of serine proteinases of B. jararaca juvenile venoms was analyzed using an antibody generated against two basic serine proteinases from B. moojeni venom, MSP1 and MSP2. 40 Most SVSPs are composed of 232 amino residues but show molecular masses higher than 25 kDa due to glycosylation. Accordingly, in the majority of juvenile venoms nearly all recognized bands were of molecular mass between 25 kDa and 35 kDa which is consistent with those reported for SVSPs analyzed by SDS-PAGE10, however, the anti-MSP1/2 antibody showed clear differences in cross-reactivity among the SVSPs present in the juvenile venoms (Figure 2B). While some bands were more intensely stained by the antibody, for example, in the venoms #8 and #12, in others there was no significant recognition (venoms #9-11 and #15-17), and in venom #13 no band was recognized. On the other hand, venoms #1-8, which belong to the same litter, showed a rather similar profile of bands recognized by the antibody. The transcriptomic analysis of venom glands from newborn and adult specimens showed that transcripts of SVSPs account for only 3.3% total toxin transcripts.

21

Nevertheless, our current results suggest

that despite the relatively low amount of SVSPs predicted to be present in the venom of B. jararaca at early life stage, there is clear variability concerning presence/absence and molecular masses of these enzymes between individuals. 14


Page 15 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Proteolytic activities Snake venom proteinases are important players of the pathological aspects of viperid envenomation including hemorrhage and inflammation.

11,44-47

In venom

glands of newborn B. jararaca snakes over 56% of toxin transcripts were shown to encode proteolytic enzymes.

13

. We had previously shown that venom from

18 adult B. jararaca snakes of a single litter showed considerable individual variation of fibrinogenolytic, fibrinolytic, gelatinolytic, and amidolytic activities.

18

In this study, to assess the variability of proteolytic activities in individual juvenile venoms, we initially assayed their caseinolytic and amidolytic activities. Casein may be degraded by both SVMPs and SVSPs, and, although some of the individual venoms have shown significantly low or high values of specific activity (0.6 U/mg and 1.2 U/mg), most of them showed values around 0.75 U/mg, a fact that indicates that there was no marked variation in their ability to degrade casein (Table 1). On the other hand, snake venom amidolytic activity on benzoyl-arginil-p-nitroanilide is only exerted by serine proteinases, and the individual juvenile venoms clearly displayed variable amidolytic activities on this substrate (Table 1) while, notably, five venoms showed very low or no activity (#14 and #16-19). The coagulopathy caused 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.

10

On the other hand, many

SVMPs display unspecific fibrinogenolytic activity and yet other enzymes possess pro-coagulant activity since they specifically activate factor II 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(prothrombin) or factor X of the coagulation cascade.

Page 16 of 55

11,48,49

We had previously

shown that the coagulant activity of newborn B. jararaca venom upon human plasma is ten times higher than that of adult venom by SVMPs that activate factors II and X.

13

13

and is contributed mainly

Here, the determination of the

M.C.D. of the individual juvenile venoms showed that most of them displayed as high coagulant activity as the pool of newborn venoms used in our previous study and their specific activities were significantly variable. Interestingly, three venoms (#16, #18 and #20) showed very low plasma coagulant activity while venoms #19 and #21 did not clot human plasma with doses up to 50 µg (Table 1). In contrast, the analysis of the ability of the venoms to activate prothrombin to generate thrombin showed less variation between the individual venoms indicating that the subproteome of P-III class SVMPs active on prothrombin is rather conserved in juvenile B. jararaca venoms, however, venoms #19 and #21 showed very low activity on prothrombin (Table 1). Interestingly, specimens #19 and #21 had each a sibling which was not included in this study due to the very low amount of venom obtained from them during the period of venom collection; nevertheless we tested their coagulant activity and surprisingly they were able to clot human plasma with doses similar to those of the other juvenile specimens (not shown). Hence, the lack of enzymes involved in plasma clotting in venoms #19 and #21 was restricted to these individuals and was not a feature of the litter to which they belonged. Thus, one interesting observation derived from these results is that in spite of the importance of the coagulant activity in the effects of newborn and adult B. jararaca venom, some individuals may simply be devoid of enzymes active upon the coagulation cascade. Venoms #19 and #21 did show protein bands that cross-reacted with anti16


Page 17 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SVMP and anti-SVSP antibodies (Figure 2) and yet these enzymes apparently are unable to convert fibrinogen into fibrin or activate prothrombin. And these facts lead one to wonder what makes a B. jararaca venom “a B. jararaca venom”? If the venoms of specimens #19 and #21 were analyzed without species identification based on their physical phenotype, the determination of their coagulant activity would not even indicate that they belong to Bothrops genus due to their inability to clot human plasma. Variation in the coagulant activity was also observed among the venoms of 18 adult B. jararaca siblings

18

and similarly, in the present study the juvenile

specimens that belonged to the same litter (#1-8) displayed variable M.C.D. Hence, it seems clear that even among the enzymes that contribute to one the hallmarks of B. jararaca envenomation, i.e. the coagulopathy, there is no pressure or genetic control for their conserved expression in all individual specimens.

Protein identification of variable protein bands As an attempt to characterize the individual variability of juvenile B. jararaca venoms we submitted 41 differential bands (indicated in Figure 1) to in gel trypsin digestion coupled to mass spectrometric analysis resulting in the identification of proteins present in 39 bands. By LC-MS/MS analysis, proteins of the main viperid toxin families including SVMP, SVSP, PLA2, CTL and LAAO as well as the less abundant components CRISP and VEGF were identified in the bands of variable intensity at the molecular mass range of 10-40 kDa (Table 2; supplemental table 2) confirming the significant variability observed on the venom SDS-PAGE profiles (Figure 1). Moreover, two bands of molecular mass 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 55

around 70 kDa (bands #24 and #31) that were variable in intensity among the venoms contained SVMPs. Notably, some bands of molecular mass ~14 kDa and lower contained PLA2 and CTL along with SVMPs indicating that the latter might be derived from protein autolysis (bands # 3 and #4). Bands of variable intensity at the molecular mass range of 25-40 kDa contained SVSPs as well as SVMPs. Overall, the majority of the identified bands contained more than one type of protein indicating the co-migration of polypeptide chains of different toxins yet with similar molecular masses. Considering that protein identification showed individual variability in most toxin classes of B. jararaca venom, it is possible to suggest that starting from early life individual variation is a stochastic phenomenon concerning geographical origin, gender and litter.

Protein identification of conserved protein bands In view of the broad variability of protein bands and toxins detected in the individual juvenile venoms, we decided to characterize the components that could be conserved in these venoms. For this purpose the venoms were submitted to SDS-PAGE (12% gel) under non-reducing conditions in order to visualize their real protein complexity since multimeric toxins would migrate according to their whole molecular masses. In Figure 3 is seen the electrophoretic profiles of the 21 juvenile venoms and one very interesting observation is that the non-reducing profiles of the venoms are somewhat less variable to each other than the reducing profiles shown in Figure 1. Nevertheless, various bands of different staining intensity between the venoms are visualized in the gel under non-reducing conditions, especially in the region of molecular mass between 16 kDa and 40 kDa. The profile of the region 18


Page 19 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


located at 45-50 kDa is however fairly similar between the venoms and was selected for in gel trypsin digestion and mass spectrometric analysis (Figure 3). Interestingly, protein identification indicated the presence of SVMPs and LAAO in this region in all venoms and notably, in 10 venoms, aminopeptidase A was detected for the first time in B. jararaca venom (Table 3; supplemental table 3). The identification of LAAO at the molecular mass of 45-50 kDa is rather unusual and suggests that this protein band may correspond to a monomeric and less glycosylated form of LAAO or to a fragment of LAAO that resulted from proteolysis within the venom, as recently reported for the adult venoms of B. jararaca, B. cotiara and B. fonsecai.14 The presence of conserved SVMPs of ~45-50 kDa in all venoms is notable, however, it is not in agreement with the variable potencies to clot human plasma and to activate prothrombin displayed by the venoms. It is therefore possible that these conserved SVMPs are not involved in the juvenile venom coagulant activity and rather play a role in other B. jararaca venom effects.

Analysis of individual venoms by LC-MS We have previously described the individual, variable LC-MS profiles of venoms from 18 B. jararaca adult siblings.

12

Our aim here was to further investigate the

individual variation in juvenile snake venoms and indeed significantly variable profiles of total nanoLC–ESI-MS ion chromatograms were observed regardless of the gender or geographical origin of the specimen (Figure 4). The different profiles observed among components eluted between 22 min and 40 min from the column indicated a considerable variability of molecular masses and hydrophobicity levels among venom components. The only peak that showed a 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 55

similar profile in all venoms was detected at ~20 min and was selected for MS/MS analysis. The fragmentation of the component present in this peak resulted in the identification of the ion of m/z 444.241+[M+H]+ whose amino acid sequence corresponded to the tripeptide ZKW (pyroglutamyl-lysyl-tryptophan) (Supplemental figure 2). This tripeptide is known for its ability to inhibit SVMPs and is present in snake venoms along with other homologs (ZNW and ZQW) as a strategy to avoid uncontrolled proteolysis within the venom gland.

50-52

Venom

proteome homeostasis may undergo significant changes depending on sampling procedures

14

and, in this context, the proteome landscape of a given

venom may vary as a result of early proteolytic events triggered by active venom proteinases. Since all venom samples used in this study were not submitted to lyophilisation, the presence of ZKW in all juvenile venoms reinforces its importance in the maintenance of venom gland and venom components integrity in all stages of snake life.

Multivariate analysis In view of the considerable individual variability detected in most venom parameters evaluated in this study, we decided to conduct a multivariate analysis of venom features using a clustering method based on Euclidean distance and complete linkage to estimate variation of protein concentration, and enzymatic activities (caseinolytic, amidolytic, coagulant and prothrombinactivating). Since the simple inspection of results of venom electrophoretic profiles (Figures 1 and 3) and enzymatic activities (Table 1) showed no clear correlation to specimen gender and geographical origin, we omitted these parameters in this analysis to avoid their influence in the clustering. The 20


Page 21 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dendrogram shown in Figure 5 indicates the separation of the 21 venoms in two groups: group 1 is composed of all specimens from Santa Catarina state and one specimen from the South region of São Paulo state (Pilar do Sul) (Supplemental figure 1; Supplemental table 1), and group 2 is composed of all remaining specimens. Although the group of Santa Catarina is composed mainly of male specimens, it is interesting to note that the parameter ‘gender’ did not clearly influence the venom grouping since none of the groups is homogeneous, i.e. no group is composed of only specimens of the same gender. Moreover, in group 2 the geographic origin of the specimens was not decisive for the formation of sub-groups (Figure 5). Therefore, considering that the number of specimens that composed this study is relatively low and that 8 of them had the same geographic origin (Santa Catarina), it is not possible to establish: i) if the clustering analysis grouped the specimens according to their origins, and ii) whether group 1 (composed mainly of specimens from Santa Catarina) was defined as such because most specimens belonged to the same litter. Interestingly, Grazziotin and colleagues

53

carried out a large-scale survey

of the genetic variation at the mitochondrial cytochrome b gene of B. jararaca and showed by phylogenetic and network analyses the existence of two wellsupported clades, which showed a Southern and a Northern distribution in the Brazilian Atlantic Forest. Santa Catarina state is located in the region of the southern-distributed clade while in the region of São Paulo state is found the Northern clade. However, it is not known whether the clade separation of B. jararaca specimens is also present in their venoms (juvenile or adult). Thus, future studies using a higher number of venom samples from both clades are required to elucidate this hypothesis. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 55

Conclusions

Diversity in toxin content and changes in venom proteomes are considered a consequence of gene duplication and accelerated molecular evolution to endow the venom with new proteins and increase the diversity of molecules to deal with prey targets. Thus, the proteome variability among 21 juvenile B. jararaca venoms showed here by the determination of various enzymatic activities and proteomics approaches cannot be attributed to variation of environmental conditions, age or diet of the specimens analyzed since these were born and raised under strictly homogeneous laboratory conditions. On the contrary, the proteome diversity observed in the venom of these juvenile specimens seems to be genetically inherited and under control of evolutionary forces. One important aspect of the intraspecific variability of snake venoms is the variability of specific venom components, which may influence the whole venom phenotype as illustrated by the significant ontogenetic change documented for B. jararaca which moves from a newborn venom very active upon the coagulation cascade components to an adult venom rather less coagulant and yet very potent to cause local damage. In this respect, the identification in all juvenile venoms of a protein band of ~50 kDa containing SVMPs and LAAO, and of the tripeptide ZKW, suggests that a minimum core of conserved components is important to define the juvenile venom and that variations in other toxins result in the different venom profiles. Furthermore, variation can be so widespread among toxin families that some juvenile individuals may display a venom phenotype that differ from others of similar age and adult specimens. 22


Page 23 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Acknowledgements This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (grants 98/14307-9, 07/54626-7, 11/08514-8 and 11/11308-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (grant 1214/2011).

Supporting Information Available: This material is available free of charge via the Internet at http://pubs.acs.org.

References 1. Calvete, J. J.; Juárez, P.; Sanz, L. Snake venomics. Strategy and applications. J. Mass. Spectrom. 2007, 42 (11), 1405–1414.

2 Fox, J. W.; Serrano, S. M. Exploring snake venom proteomes: multifaceted analyses for complex toxin mixtures. Proteomics 2008, 8 (4), 909–920.

3 Doley, R.; Kini, R. M. Protein complexes in snake venom. Cell. Mol. Life. Sci. 2009, 66 (17), 2851–2871

4 Fox, J. W.; Serrano, S. M. Approaching the golden age of natural product pharmaceuticals from venom libraries: an overview of toxins and toxinderivatives currently involved in therapeutic or diagnostic applications. Curr. Pharm. Des. 2007, 13 (28), 2927–2934

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 55

5. Kini, R. M. Toxins in thrombosis and haemostasis: potential beyond imagination. J. Thromb. Haemost. 2011, 9 (1), 195–208.

6 Camargo, A. C.; Ianzer, D.; Guerreiro, J. R.; Serrano, S. M. Bradykininpotentiating peptides: beyond captopril. Toxicon 2012, 59 (4), 516–523.

7 McCleary, R. J.; Kini, R. M. Non-enzymatic proteins from snake venoms: a gold mine of pharmacological tools and drug leads. Toxicon 2013, 62, 56–74.

8 Calvete, J. J. Snake venomics: From the inventory of toxins to biology. Toxicon 2013, doi: 10.1016/j.toxicon.2013.03.020.

9 Nakashima, K.; Nobuhisa, I.; Deshimaru, M.; Nakai, M.; Ogawa, T.; Shimohigashi, Y.; Fukumakit, Y.; Hatfrorit, M.; Sakakit,Y.; Hattori, S.; Ohno, M. Accelerated evolution in the protein-coding regions is universal in crotalinae snake venom gland phospholipase A2 isozyme genes. Proc. Natl. Acad. Sci. U. S. A. 1995, 92 (12), 5605–5609.

10 Serrano, S. M.; Maroun, R. C. Snake venom serine proteinases: sequence homology vs. substrate specificity, a paradox to be solved. Toxicon 2005, 45 (8), 1115–1132.

11 Fox, J. W.; Serrano, S. M. Insights into and speculations about snake venom metalloproteinase (SVMP) synthesis, folding and disulfide bond formation and their contribution to venom complexity. FEBS J. 2008, 275 (12), 3016–3030. 24


Page 25 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


12 Pimenta, D. C.; Prezoto, B. C.; Konno, K.; Melo, R. L.; Furtado, M. F.; Camargo A. C.; Serrano, S. M. Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence for sex-based variation among the bradykinin-potentiating peptides. Rapid Commun. Mass Spectrom. 2007, 21(6), 1034–1042.

13 Zelanis, A.; Tashima, A. K.; Rocha, M. M.; Furtado, M. F.; Camargo, A. C.; Ho, P. L.; Serrano, S. M. Analysis of the ontogenetic variation in the venom proteome/peptidome of Bothrops jararaca reveals different strategies to deal with prey. J. Proteome Res. 2010, 9 (5), 2278–2291.

14 Tashima, A. K.; Zelanis, A.; Kitano, E. S.; Ianzer, D, Melo, R. L.; Rioli, V, Sant'anna, S. S.; Schenberg, A. C.; Camargo, A. C.; Serrano, S. M. Peptidomics of three Bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes. Mol Cell Proteomics 2012, 11 (11), 245–262.

15 Alape-Girón, A.; Sanz, L.; Escolano, J.; Flores-Díaz, M.; Madrigal, M.; Sasa, M.; Calvete, J. J. Snake venomics of the lancehead Bothrops asper. Geographical, individual and ontogenetic variations. J. Proteome Res. 2008, 7 (8), 3556– 3571.

16 Calvete, J. J.; Sanz, L.; Pérez, A.; Borges, A.; Vargas, A. M.; Lomonte, B.; Angulo, Y.; Gutiérrez, J. M.; Chalkidis, H. M.; Mourão, R. H.; Furtado, M. F.; 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 55

Moura-Da-Silva, A. M. Snake population venomics and antivenomics of Bothrops atrox: Paedomorphism along its transamazonian dispersal and implications of geographic venom variability on snakebite management. J. Proteomics 2011, 74 (4), 510–527.

17 Chippaux, J. P.; Williams, V.; White, J. Snake venom variability: methods of study, results and interpretation. Toxicon 1991, 29 (11), 1279–1303.

18 Menezes, M. C.; Furtado, M. F.; Travaglia-Cardoso, S. R.; Camargo, A. C.; Serrano, S. M. Sex-based individual variation of snake venom proteome among eighteen Bothrops jararaca siblings. Toxicon 2006, 47 (3), 304–312.

19 Gao, J. F.; Qu, Y. F.; Zhang, X. Q.; Ji, X. Within-clutch variation in venoms from hatchlings of Deinagkistrodon acutus (Viperidae). Toxicon 2011, 57 (7–8), 970–977.

20 Zelanis, A.; Tashima, A. K.; Pinto, A. F.; Leme, A. F.; Stuginski, D. R.; Furtado, M. F.; Sherman, N. E.; Ho, P. L.; Fox J. W.; Serrano S. M. Bothrops jararaca venom proteome rearrangement upon neonate to adult transition. Proteomics 2011, 11 (21), 4218–4228

21 Zelanis, A.; Andrade-Silva, D.; Rocha, M. M.; Furtado, M. F.; Serrano, S. M.; Junqueira-de-Azevedo, I. L.; Ho, P. L. A transcriptomic view of the proteome variability of newborn and adult Bothrops jararaca snake venoms. PLoS Negl. Trop. Dis. 2012, 6 (3), e1554. 26


Page 27 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


22 Massey, D. J.; Calvete. J. J.; Sánchez. E. E.; Sanz, L.; Richards, K.; Curtis, R.; Boesen, K. Venom variability and envenoming severity outcomes of the Crotalus scutulatus scutulatus (Mojave rattlesnake) from Southern Arizona. J Proteomics. 2012, 75 (9), 2576–2587.

23 Modahl, C. M.; Doley, R.; Kini, R. M. Venom analysis of long-term captive Pakistan cobra (Naja naja) populations. Toxicon 2010, 55 (2–3), 612–618.

24 Gibbs, H. L.; Sanz, L.; Chiucchi, J. E.; Farrell, T. M.; Calvete, J. J. Proteomic analysis of ontogenetic and diet-related changes in venom composition of juvenile and adult Dusky Pigmy rattlesnakes (Sistrurus miliarius barbouri). J. Proteomics 2011, 74 (10), 2469–2179.

25 Daltry, J. C.; Wüster, W.; Thorpe, R. S. Diet and snake venom evolution. Nature 1996, 379 (6565), 537–540.

26 Daltry, J.C., Ponnudurai, G., Shin, C.K., Tan, N.H., Thorpe, R.S., Wuster, W.,. Electrophoretic profiles and biological activities: intraspecific variation in the venom of the Malayan pit viper Calloselasma rhodostoma. Toxicon 1996, 34 (1), 67–79.

27 Sasa, M., Diet and snake venom evolution: can local selection alone explain intraspecific venom variation? Toxicon 1999, 37 (2), 249– 252.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 55

28 Deshimaru, M.; Ogawa, T,.; Nakashima, K.; Nobuhisa, I.; Chijiwa, T.; Shimohigashi, Y.; Fukumaki, Y.; Niwa, M.; Yamashina, I.; Hattori, S.; Ohno, M. Accelerated evolution of crotalinae snake venom gland serine proteases. FEBS Lett. 1996, 397 (1), 83–88.

29 Moura-da-Silva, A. M.; Theakston, R. D.; Crampton, J. M. Evolution of disintegrin cysteine-rich and mammalian matrix-degrading metalloproteinases: gene duplication and divergence of a common ancestor rather than convergent evolution. J. Mol. Evol. 1996, 43 (3), 263–269.

30 Chijiwa, T.; Deshimaru, M.; Nobuhisa, I.; Nakai, M.; Ogawa, T.; Oda, N.; Nakashima, K.; Fukumaki, Y.; Shimohigashi, Y.; Hattori, S.; Ohno, M. Regional evolution of venom-gland phospholipase A2 isoenzymes of Trimeresurus flavoviridis snakes in the southwestern islands of Japan. Biochem. J. 2000, 347 (2), 491–499.

31 Wooldridge, B. J.; Pineda, G.; Banuelas-Ornelas, J. J; Dagda, R. K.; Gasanov, S. E.; Rael, E. D.; Lieb, C. S. Mojave rattlesnakes (Crotalus scutulatus scutulatus) lacking the acidic subunit DNA sequence lack Mojave toxin in their venom. Comp. Biochem. Physiol. 2011, 130 (2),169–179.

32 Durban, J.; Pérez, A.; Sanz, L.; Gómez, A.; Bonilla, F.; Rodríguez, S.; Chacón, D.; Sasa, M.; Angulo, Y.; Gutiérrez, J. M.; Calvete, J. J. Integrated "omics" profiling indicates that miRNAs are modulators of the ontogenetic

28


Page 29 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


venom composition shift in the Central American rattlesnake, Crotalus simus simus. BMC Genomics 2013, doi: 10.1186/1471-2164-14-234.

33 Rosenfeld, G. Symptomatology, pathology and treatment of snake bites in South America. In: Bücherl, W.; Buckley, E. E.: Venomous Animals and their Venoms, Vol. 2. Academic Press, New York, 1971, 345–384.

34 Rosenfeld, G.; Hampe, O. G.; Kelen, E. M. A. Coagulant and fibrinolytic activity of animal venoms; determination of coagulant and fibrinolytic index of different species. Mem. Inst. Butantan 1959, 29, 143–163.

35 Zelanis, A.; Serrano, S. M.; Reinhold, V. N. N-glycome profiling of Bothrops jararaca newborn and adult venoms. J. Proteomics 2012, 75(3), 774–782.

36. Fox, J. W.; Shannon, J. D.; Stefansson, B.; Kamiguti, A. S.; Theakston, R. D. G.; Serrano, S. M. T.; Camargo, A. C. M.; Sherman, N. Role of discovery science in toxinology: examples in venom proteomics, In: Perspectives in Molecular Toxinology (Ménez, A., ed). John Wiley & Sons, Ltd.: Chichester, UK, 2002, 97–108.

37 Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (5259), 680–685.

38 Paes-Leme, A. F.; Kitano, E. S.; Furtado, M. F.; Valente, R. H.; Camargo, A. C.; Fox, J. W.; Serrano, S. M. T. Analysis of the subproteomes of proteinases 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 55

and heparin-binding toxins of eight Bothrops venoms. Proteomics 2009, 9 (3), 733–745.

39 Assakura, M. T.; Silva C. A; Mentele, R, Camargo, A. C.; Serrano, S. M. Molecular cloning and expression of structural domains of bothropasin, a P-III metalloproteinase from the venom of Bothrops jararaca. Toxicon 2003, 41 (2), 217–227.

40 Serrano, S. M. T.; Matos, M. F. C.; Mandelbaum, F. R.; Sampaio, C. A. M. Basic proteinases from Bothrops moojeni (caissaca) venom - I. Isolation and activity of two serine proteinases, MSP 1 and MSP 2, on synthetic substrates and on platelet aggregation. Toxicon 1993, 31 (4), 471–483.

41 Hanna, S. L.; Sherman, N. E.; Kinter, M. T.; Goldberg, J. B. Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry. Microbiology 2000 (pt 10), 146, 2495–2508.

42 Theakston, R. D. G.; Reid, H. A. Development of simple standard assay procedures for the characterization of snake venoms. Bull. World Health Org. 1983, 61 (6), 949–956.

43 Oliveira, A. K.; Paes Leme, A. F.; Asega, A. F.; Camargo, A. C.; Fox, J. W; Serrano, S. M. New insights into the structural elements involved in the skin 30


Page 31 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


haemorrhage induced by snake venom metalloproteinases. Thromb Haemost. 2010,104 (3), 485–497.

44 Gutiérrez, J. M.; Rucavado, A. Snake venom metalloproteinases: their role in the pathogenesis of local tissue damage. Biochimie 2000, 82 (9–10), 841–850.

45 Gallagher, P. G.; Bao, Y.; Serrano, S. M.; Kamiguti, A. S.; Theakston, R. D.; Fox, J. W. Use of microarrays for investigating the subtoxic effects of snake venoms: insights into venom-induced apoptosis in human umbilical vein endothelial cells. Toxicon 2003, 41 (4), 429–440.

46 Escalante, T.; Rucavado, A.; Fox, J. W.; Gutiérrez, J. M. Key events in microvascular

damage

induced

by

snake

venom

hemorrhagic

metalloproteinases. J. Proteomics 2011, 74 (9), 1781–1794.

47 Takeda, S.; Takeya, H.; Iwanaga, S. Snake venom metalloproteinases: structure, function and relevance to the mammalian ADAM/ADAMTS family proteins. Biochim. Biophys. Acta. 2012, 1824 (.1), 164–176.

48 Tans, G.; Rosing, J. Snake venom activators of factor X: an overview. Haemostasis 2001, 31 (3–6), 225–233

49 Silva, M. B.; Schattner, M.; Ramos, C. R.; Junqueira-de-Azevedo, I. L.; Guarnieri, M. C.; Lazzari, M. A.; Sampaio, C. A.; Pozner, R. G.; Ventura, J. S.; Ho, P. L.; Chudzinski-Tavassi, A. M. A. Prothrombin activator from Bothrops 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 55

erythromelas (jararaca-da-seca) snake venom: characterization and molecular cloning. Biochem. J. 2003, 369 (1), 129–139.

50 Kato, H.; Iwanaga, S.; Suzuki, T. The isolation and amino acid sequence of new pyroglutamylpeptides from snake venoms. Experientia 1966, 22, 49–50.

51 Zhang, D.; Botos, I.; Gomis-Ruth, F. X.; Doll, R.; Blood, C., Njoroges, F. G.; Fox, J. W.; Bode, W.; Meyer, E. F. Structural interaction of natural and synthetic inhibitors with the venom metalloproteinase, atrolysin C (form d). Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (18), 8447–8451.

52 Wagstaff, S. C.; Favreau, P.; Cheneval, O.; Laing, G. D.; Wilkinson, M. C.; Miller, R. L.; Stöcklin, R.; Harrison, R. A. Molecular characterization of endogenous snake venom metalloproteinase inhibitors. Biochem. Biophys. Res. Commun. 2008, 365 (4), 650–656.

53 Grazziotin, F. G., Monzel, M., Echeverrigaray, S., Bonatto, S. L. Phylogeography of the Bothrops jararaca complex (serpentes: Viperidae): past fragmentation and island colonization in the Brazilian Atlantic Forest. Mol. Ecol. 2006, 15 (13), 3969–3982.

Legends for figures

Figure 1. Electrophoretic profiles of venom proteins from juvenile B. jararaca specimens under reducing conditions. SDS-PAGE (12% gel) of venom proteins 32


Page 33 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


stained with Coomassie blue (A) or of venom glycoproteins stained with Pro-QEmerald (B). Molecular mass markers (lane M). White rectangles indicate bands that were submitted to in gel trypsin digestion and mass spectrometric analysis.

Figure 2. Western blot analysis of venoms from juvenile B. jararaca specimens. After SDS-PAGE separation (12% gel) proteins were transferred to a nitrocellulose membrane and immunostained with polyclonal antibodies. (A) anti-bothropasin antibody. (B) anti-MSP1/2 antibody. Numbers on the left indicate molecular mass marker mobility.

Figure 3. Electrophoretic profiles of venom proteins from juvenile B. jararaca specimens under non-reducing conditions. SDS-PAGE (12% gel) of venom proteins stained with Coomassie blue. Molecular mass markers (lane M). White rectangles indicate bands that were submitted to in gel trypsin digestion and mass spectrometric analysis.

Figure 4. LC-MS profiles of juvenile B. jararaca venoms. Venom solutions (1 µg in 4.5 µL 0.1% formic acid) were analyzed in the nanoAcquity UPLC/Q-ToF system as described in the Experimental Section. The arrow indicates a peak present in all individual venoms.

Figure 5. Cluster dendrogram of protein concentration and caseinolytic, amidolytic, plasma coagulant and prothrombin-activating activities of 21 B. jararaca juvenile venoms using unsupervised hierarchical clustering and 33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 55

complete linkage method. Red and green outlines indicate two groups resulted from clustering. Pink and blue circles indicate venom from female and male specimens, respectively; the dashed line indicates specimens that belong to a single litter.

34


Page 35 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Table 1. Protein concentration and enzymatic activities of venoms from juvenile B. jararaca specimens. Venom #

Protein concentration

Amidolytic activity

Caseinolytic activity

(µmole/min/mg)

(U/mg)a

(mg/mL)

Plasma coagulant activity

Prothrombin activation

M.C.D.

Specific activity

(pmole/min)

(µg)

(U/mg)

1♂

86.64 ± 3.9

0.041 ± 0.004

0.955 ± 0.110

0.20 ± 0.010

4965

211.44 ± 35.84

2♀

108.28 ± 4.4

0.108 ± 0.004

0.792 ± 0.041

0.16 ± 0.007

6277

212.25 ± 16.43

3♂

113.15 ± 3.3

0.089 ± 0.005

1.047 ± 0.115

0.17 ± 0.012

6753

224.75 ± 39.01

4♂

90.9 ± 10.7

0.086 ± 0.008

1.135 ± 0.142

0.09 ± 0.001

11231

225,49 ± 36,14

5♀

110.86 ±14.2

0.087 ± 0.002

0.892 ± 0.052

0.10 ± 0.011

10033

224.18 ± 38.78

6♂

128.16 ± 3.9

0.042 ± 0.002

0.952 ± 0.120

0.13 ± 0.004

7942

224.84 ± 32.99

7♂

116.67 ± 16.2

0.075 ± 0.003

0.888 ± 0.119

0.07 ± 0.021

15026

216.34 ± 32.64

8♂

131.24 ± 12.0

0.103 ± 0.005

0.993 ± 0.164

0.11 ± 0.012

8846

210.13 ± 29.42

9♂

118.97 ± 10.0

0.137 ± 0.005

0.887 ± 0.107

0.14 ± 0.023

7130

208.58 ± 16.43

10♀

122.38 ± 11.5

0.023 ± 0.002

0.888 ± 0.092

0.52 ± 0.085

1937

159.15 ± 18.76

11♀

110.09 ±5.7

0.011 ± 0.001

0.752 ± 0.073

0.67 ± 0.022

1486

164.05 ± 9.32

12♀

129.71 ± 8.7

0.056 ± 0.006

0.852 ± 0.061

0.59 ± 0.437

1709

138.56 ± 10.48

13♂

123.35 ± 10.1

0.046 ± 0.005

0.745 ± 0.071

0.57 ± 0.038

1756

156.21 ± 32.64

14♀

110.50 ± 11.4

0.002 ± 0

0.993 ± 0.134

0.56 ± 0.039

1773

145.42 ± 15.51

15♀

118.91 ± 16.6

0.016 ± 0.001

0.818 ± 0.031

0.59 ± 0.344

1682

157.52 ± 8.90 35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 36 of 55

16♀

127.76 ± 17.4

0.001 ± 0

0.767 ± 0.039

2.45 ± 0.017

409

139.46 ± 4.41

17♂

127.24 ± 18.0

0.007 ± 0

0.802 ± 0.067

0.20 ± 0.096

5020

197.55 ± 5.46

18♀

106.15 ± 10.3

0.008 ± 0

0.557 ± 0.028

6.03 ± 0.349

166

169.36 ± 6.27

19♀

134.66 ± 13.1

0.004 ± 0

0.757 ± 0.065

NDb

ND

20.26 ± 3.96

20♀

119.13 ± 15.9

0.058 ± 0.025

0.637 ± 0.012

3.55 ± 0.356

282

150.74 ± 9.07

21♀

108.92 ± 15.7

0.048 ± 0.003

0.823 ± 0.072

NDb

ND

43.14 ± 2.59

Data are means ±SE (n=3). a One unit of casein-hydrolyzing activity is defined as the amount of enzyme yielding an increase in O.D. of 1.0 min-1 at 280 nm. b ND, not detected using doses up to 50 µg of venom.

36


Page 37 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Identification of protein bands indicated in Figure 1, by LC-MS/MS. Band Protein accession Protein NUPb Toxin classc a no. number gi|20069135|

Metalloproteinase precursor

2

MP

gi|123883733|

Venom serine proteinaselike

2

SP

gi|31742525|

Hemorrhagic metalloproteinase HF3

1

MP

gi|231997|

Metalloproteinasedisintegrin jararhagin

1

MP

gi|20069135|


3

MP

gi|1839441|

Platelet glycoprotein Ibbinding protein alpha subunit, GPIb-BP alpha subunit

5

CTL

gi|3914258|

Phospholipase A2

1

PLA2

gi|292630844|

Phospholipase A2

1

PLA2

gi|20069135|


2

MP

gi|20069135|


1

MP

gi|1839441|


1

CTL

gi|32450776|

Echicetin B-chain

1

CTL

gi|1839442|

Platelet glycoprotein Ibbinding protein beta subunit, GPIb-BP beta subunit

3

CTL

gi|31742525|


1

MP

gi|229621685|

Bothroinsularin

2

CTL

gi|33341212|

Stejaggregin-A beta chain-1

1

CTL

gi|13399948|

Chain A, Crystal Structure Of Botrocetin

1

CTL

gi|11967287|

Agkicetin beta subunit precursor

1

CTL

gi|292630844|

Phospholipase A2 2

1

PLA2

gi|190195339|

Cysteine-rich secretory protein Da-CRPb

1

CRISP

1

2

3

4

6

37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7

gi|205278803|

Batroxstatin-1

1

MP

gi|24266796|

Stejnefibrase 1

1

SP

gi|110610035|

Scutiarin

1

MP

gi|308212496|

MP_III1 SVMP precursor

2

MP

gi|20069139|

Serine proteinase precursor

1

SP

gi|13194760|

Bothrostatin precursor

1

MP

gi|52426579|

Insularinase and insularin precursor

4

MP

gi|172044592|

Zinc metalloproteinase BnP2

4

MP

gi|308212516|

MP_IIa SVMP precursor

4

MP

gi|1127129|

Metalloproteinase, Atrolysin C (Ht-D)

1

MP

gi|109254970|

Metalloproteinase isoform 6

1

MP

gi|205278803|

Batroxstatin-1

1

MP

gi|54650276|

Hypothetical protein

4

SP

gi|123883733|


1

SP

gi|32396014|

Serine protease

1

SP

gi|308212514|

MP_IIx3 SVMP precursor

2

MP

gi|52426579|


3

MP

gi|172044592|


3

MP

gi|1839441|


7

CTL

gi|86450426|

Phospholipase A2 precursor

1

PLA2

gi|300394|

Phospholipase A2 monomers

1

PLA2

gi|1839441|


1

CTL

gi|190195339|


3

CRISP

8

9

10

11

12

13

14

15

Page 38 of 55

38


Page 39 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


gi|6093643|

Platelet-aggregating proteinase PA-BJ

1

SP

gi|52426579|


5

MP

gi|172044592|


3

MP

gi|308212516|


5

MP

gi|32469800|

Thrombin-like enzyme contortrixobin

1

SP

gi|292630844|

Phospholipase A2 2

1

PLA2

gi|3122187|

Halystase

2

SP

gi|54650276|


2

SP

gi|123883733|


3

SP

gi|32396014|

Serine protease

3

SP

gi|297593770|

Serine protease

3

SP

gi|6093643|

Platelet-aggregating proteinase PA-BJ

1

SP

gi|1127129|

Metalloproteinase, Atrolysin C (Ht-D)

1

MP

gi|123912829|

Metalloprotease

3

MP

gi|308212516|


2

MP

gi|52426579|


4

MP

gi|52426579|


1

MP

gi|320579375|

Group III snake venom metalloproteinase

1

MP

gi|13194760|


4

MP

22

gi|3914258|

Phospholipase A2

1

PLA2

23

gi|158518414|

Phospholipase A2

4

PLA2

gi|308212514|


1

MP

gi|20069135|


1

MP

gi|123908731|

Zinc metalloproteinasedisintegrin bothrojarin-1

1

MP

gi|52426579|


1

MP

16

17

18

20

21

24

25

39



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 55

gi|172044592|


1

MP

gi|13194760|


1

MP

26

gi|292630844|

Phospholipase A2

1

PLA2

27

gi|3914258|

Phospholipase A2

2

PLA2

28

gi|109254978|

C-type lectin isoform 3

1

CTL

gi|229621684

Bothroinsularin subunit alpha

4

CTL

gi|33341212|

Stejaggregin-A beta chain-1

1

CTL

gi|3023232|

Alboaggregin-A subunit 4

1

CTL

gi|229621685|

Bothroinsularin subunit beta

2

CTL

gi|15072462|

Vascular endothelial growth factor precursor

1

VEGF

gi|1127129|

Atrolysin C (Ht-D)

1

MP

gi|308212516|


3

MP

gi|52426579|


3

MP

gi|308212514|


3

MP

gi|123908731|


1

MP

gi|126035656|

Putative serine protease

1

SP

gi|123883733|


1

SP

gi|54650276|


4

SP

gi|52426579|


2

MP

gi|123905786|


1

MP

gi|190195339|


2

CRISP

gi|1127129|

Atrolysin C (Ht-D)

1

MP

gi|1839441|


5

CTL

gi|3914258|

Phospholipase A2

1

PLA2

gi|320579375|

group III snake venom

1

MP

29

30

31

32

33

34

35

40


Page 41 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


metalloproteinase gi|1839441|

Platelet glycoprotein Ibbinding protein alpha subunit, GPIb-BP alpha

1

CTL

gi|13194760|


7

MP

gi|20069135|


1

MP

gi|205278803|

Batroxstatin-1

2

MP

gi|6760464|

Non-hemorrhagic fibrin(ogen)olytic metalloprotease

1

MP

gi|258618066|

Metalloproteinase VMP-II precursor

1

MP

gi|319759240|

Metalloproteinase PMMP-1

1

MP

gi|31742525|


1

MP

gi|297593862|

Metalloproteinase

1

MP

gi|109254996|

L-amino acid oxidase

1

LAAO

gi|225354041|

Leberagin-C

1

MP

gi|20069135|


1

MP

gi|308212512|


4

MP

gi|190358877|

Zinc metalloproteinasedisintegrin brevilysin-H6;

1

MP

gi|308212514|


4

MP

gi|54650276|


5

SP

gi|123883733|


3

SP

gi|190195339|


4

SP

gi|86450426|


1

PLA2

gi|300394|


1

PLA2

gi|292630844|

Phospholipase A2 2

1

PLA2

gi|3023230|

Alboaggregin-A subunit 2

1

CTL

gi|86450426|


3

PLA2

gi|300394|


2

PLA2

gi|229621684|

Bothroinsularin subunit

1

CTL

36

37

38

39

40

41

41



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 55

alpha gi|292630844|

Phospholipase A2 2

3

PLA2

gi|158518414|

Phospholipase A2 2; A

3

PLA2

gi|38146948|

Acidic phospholipase A2

4

PLA2

gi|3914258| Phospholipase A2 2 PLA2 According to National Center for Biotechnology Information (NCBI). b NUP: Number of unique peptides. c Toxin classes: CRISP, cysteine-rich secretory protein; CTL, C-type lectin; LAAO, L-amino acid oxidase; MP, metalloproteinase; PLA2, phospholipase A2; SP, serine proteinase; VEGF, vascular endothelial growth factor. a

42


Page 43 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Identification of protein bands indicated in Figure 3, by LC-MS/MS. Protein NUPb Toxin classc Venom# Protein accession a number 1

2

3

4

gi|39841346


4

LAAO

gi|48425312

L-Amino Acid Oxidase from Agkistrodon halys pallas

3

LAAO

gi|710354

catrocollastatin precursor

3

MP

gi|20069135

metalloproteinase precursor

14

MP

gi|195927838

L-amino acid oxidase precursor

11

LAAO

gi|39841344


10

LAAO

gi|3426324


6

LAAO

gi|258618060

metalloproteinase VMP-III precursor

9

MP

gi|4106001

metalloprotease

8

MP

gi|15887054

M-LAO

6

LAAO

gi|308387832

L-Amino Acid Oxidase from Vipera ammodytes ammodytes

4

LAAO

gi|33355627


5

LAAO

gi|4689408

acutolysin e precursor

3

MP

gi|213030

preprometalloproteinase

1

MP

gi|308212496


1

MP

gi|58003506

calbindin D28K

1

gi|195927838


8

LAAO

gi|39841344


7

LAAO

gi|4895110

bothropasin precursor

7

MP

gi|205278803

batroxstatin-1

2

MP

gi|3426324


3

LAAO

gi|48425312

Chain A, L-Amino Acid Oxidase From Agkistrodon Halys Pallas

4

LAAO

gi|4106001

metalloprotease

6

MP

gi|195927838


10

LAAO 43



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

5

6

7

8

Page 44 of 55

gi|20069135

metalloproteinase precursor

11

MP

gi|39841344


10

LAAO

gi|3426324


4

LAAO

gi|258618060


7

MP

gi|48425312


6

LAAO

gi|297593790

metalloproteinase

2

MP

gi|320579375

group III snake venom metalloproteinase

2

MP

gi|297593806

metalloproteinase

2

MP

gi|4689408


2

MP

gi|126035653


3

LAAO

gi|39841346


4

LAAO

gi|126035653


2

LAAO

gi|62468

jararhagin

1

MP

gi|64408

trigramin

1

MP

gi|148367284

aminopeptidase A

1

MP

gi|320579347


6

MP

gi|39841344


2

LAAO

gi|62468

jararhagin

1

MP

gi|205278803

batroxstatin-1

2

MP

gi|148367284

aminopeptidase A Gloydius brevicaudus

1

AP

gi|320579347


1

MP

gi|32306927

metalloprotease BOJUMET II

2

MP

gi|39841344


2

LAAO

gi|39841344


5

LAAO

gi|62468

jararhagin

7

MP

gi|70797645


3

LAAO

gi|4106001

metalloprotease

4

MP 44


Page 45 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9

10

11

12

gi|195927838


8

LAAO

gi|205278803

batroxstatin-1

4

MP

gi|3426324


5

LAAO

gi|308212498


2

MP

gi|32306927


2

MP

gi|308387832

L-Amino Acid Oxidase from Vipera ammodytes ammodytes

4

LAAO

gi|126035653


3

LAAO

gi|319759240

metalloproteinase PMMP-1

1

MP

gi|148367284


2

AP

gi|320579347


2

MP

gi|205278803

batroxstatin-1

4

MP

gi|39841344


3

LAAO

gi|320579347


1

MP

gi|148367284


1

AP

gi|297593814

metalloproteinase

1

MP

gi|205278803

batroxstatin-1

6

MP

gi|39841344


3

LAAO

gi|308212498


3

MP

gi|126035653


2

LAAO

gi|148367284


1

AP

gi|320579347


1

MP

gi|205278803

batroxstatin-1

4

MP

gi|3426324


4

LAAO

gi|195927838


6

LAAO

gi|48425312

L-Amino Acid Oxidase from

6

LAAO 45



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 55

Agkistrodon halys pallas

13

14

gi|33355627


LAAO

gi|126035653


3

LAAO

gi|290454890

aminopeptidase A Bitis gabonica rhinoceros

2

AP

gi|320579347


1

MP

gi|195927838


13

LAAO

gi|205278803

batroxstatin-1

3

MP

gi|3426324


7

LAAO

gi|48425312


8

LAAO

gi|126035653


6

LAAO

gi|62468

jararhagin

2

MP

gi|308212498


2

MP

gi|308212500


2

MP

gi|5305427

alpha enolase

2

gi|109254964

metalloproteinase isoform 3

2

MP

gi|19701420

unnamed protein product

1

MP

gi|13194760

bothrostatin precursor

1

MP

gi|144905068

vascular apoptosis-inducing protein 2A

2

MP

gi|4106005

metalloprotease

2

MP

gi|320579347


3

MP

gi|148367284


1

AP

gi|63146080

elongation factor 2

1

gi|20530121

hemorrhagic metalloproteinase HR1b

1

MP

gi|223635807

Zinc metalloproteinase leucurolysin-B; Short=LeucB

1

MP

gi|4895110


8

MP

gi|195927838


6

LAAO 46


Page 47 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


precursor

15

16

gi|205278803

batroxstatin-1

3

MP

gi|258618060


8

MP

gi|48425312

L-Amino Acid Oxidase From Agkistrodon halys pallas

gi|4106001

metalloprotease

5

MP

gi|40311128


2

MP

gi|7630286

contortrostatin precursor

2

MP

gi|320579347


3

MP

gi|4689408


2

MP

gi|148367284


1

AP

gi|290454890

aminopeptidase A Bitis gabonica rhinoceros

2

AP

gi|195927838


11

LAAO

gi|3426324


6

LAAO

gi|205278803

batroxstatin-1

3

MP

gi|62468

jararhagin

6

MP

gi|48425312


7

LAAO

gi|126035653


4

LAAO

gi|40311128


2

MP

gi|258618060


3

MP

gi|4106001

metalloprotease

3

MP

gi|320579347


2

MP

gi|402262

prepro-hemorrhagic toxin c

1

MP

gi|19701420


1

MP

gi|13194760


1

MP

gi|195927838


9

LAAO

gi|205278803

batroxstatin-1

3

MP

MP

47



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

17

18 19

Page 48 of 55

gi|4895110


6

MP

gi|48425312


6

LAAO

gi|258618060


4

MP

gi|40311128


2

MP

gi|308212498


2

MP

gi|320579347


2

MP

gi|148367284


1

AP

gi|195927838


12

LAAO

gi|205278803

batroxstatin-1

4

MP

gi|3426324


6

LAAO

gi|48425312


7

LAAO

gi|70797645


8

LAAO

gi|402262

prepro-hemorrhagic toxin c

1

MP

gi|19701420


1

MP

gi|13194760


1

MP

gi|308212498


2

MP

gi|109254964

metalloproteinase isoform 3

2

MP

gi|148367284


1

AP

gi|320579347


1

MP

gi|39841344


1

LAAO

gi|39841344


1

LAAO

gi|195927838


gi|4895110


11

MP

gi|39841344


10

LAAO

gi|32306927


1

MP

gi|4106001

metalloprotease

6

MP

LAAO

48


Page 49 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20 21

gi|48425312


6

LAAO

gi|258618060


7

MP

gi|4689408


3

MP

gi|297593790

metalloproteinase

2

MP

gi|39841344


2

LAAO

gi|126035653


2

LAAO

gi|205278803

batroxstatin-1

2

MP

gi|195927838


6

LAAO

gi|3426324


4

LAAO

gi|4895110 bothropasin precursor 2 MP According to National Center for Biotechnology Information (NCBI). b NUP: Number of unique peptides. c Toxin classes: AP, aminopeptidase; LAAO, L-amino acid oxidase; MP, metalloproteinase. a

49



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 50 of 55

A kDa

♂ ♀ ♂ ♂ 1 2 3 4

M

♀ 5

♂ ♂ 6 7

♂ 8

M

♂ 9

♀ ♀ ♀ ♂ ♀ 10 11 12 13 14

♀ ♀ ♂ ♀ ♀ ♀ ♀ 15 16 17 18 19 20 21

M

kDa

kDa

6850-

685043-

24

685043-

7

43-

23.5-

11 10 9 8

23.5-

1 2

143 4

B

14-

6

68-

23.5-

17

M

♂ 8

22

14-

♂ ♀ ♀ ♀ ♂ ♀ 9 10 11 12 13 14

3 4

5

6

7

38 39

M

25 26 27 28 29

30 33 35 34

40 41

♀ ♀ ♂ ♀ ♀ ♀ ♀ 15 16 17 18 19 20 21

kDa

68-

6850-

50-

2

20 21

19

kDa

1

23.5-

5

kDa

43- P

14

37

23

♂ ♀ ♂ ♂ ♀ ♂ ♂ 1 2 3 4 5 6 7

M

13

36

32

18 15 16

12

31

50-

kDa 43-

43-

23.523.5-

14-

14-

14-

50

Figure 1 ACS Paragon Plus Environment

Page 51 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


♂ ♀ 1 2

A

♂ 8

♂ ♂ ♀ ♂ ♂ 3 4 5 6 7

♂ ♀ ♀ ♀ ♂ ♀ 9 10 11 12 13 14 kDa

kDa

kDa

♀ ♀ ♂ ♀ ♀ ♀ ♀ 15 16 17 18 19 20 21

755035-

7550-

7550-

30-

35-

35-

25-

30-

30-

25-

25-

15-

15-

10-

10-

1510-

B kDa 685043-

♂ 1

♀ 2

♂ ♂ ♀ ♂ ♂ 3 4 5 6 7 kDa

♂ 8

♂ ♀ 9 10

♀ ♀ ♀ ♀ ♂ ♀ 11 12 13 14 kDa 15 16

6850-

6850-

43-

43-

23.5-

23.5-

♂ ♀ ♀ ♀ ♀ 17 18 19 20 21

23.5-

14-

14-

14-

51

Figure 2



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

M

♂ ♀ ♂ ♂ ♀ ♂ ♂ 1 2 3 4 5 6 7

M

Page 52 of 55

♂ ♂ ♀ ♀ ♀ ♂ ♀ 8 9 10 11 12 13 14

kDa

kDa

kDa

685043-

685043-

685043-

23.5-

23.5-

23.5-

14-

14-

14-

M

♀ ♀ ♂ ♀ ♀ ♀ ♀ 15 16 17 18 19 20 21

Figure 3

52


Page 53 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Venom # 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

♀ ♀ ♀ ♀ ♂ ♀ ♀ ♀ ♂ ♀ ♀ ♀ ♂ ♂ ♂ ♂ ♀ ♂ ♂ ♀ ♂

F Retention time (min)

Figure 4 ACS Paragon Plus Environment

53


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 54 of 55

Figure 5 54


Page 55 of 55

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Proteomic analysis of 21 juvenile Bothrops jararaca venoms. 254x190mm (96 x 96 DPI)


Individual Variability in the Venom Proteome of Juvenile Bothrops

Recommend Documents