Dynamic Rearrangement in Snake Venom Gland Proteome: Insights

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Dynamic rearrangement in snake venom gland proteome: insights into Bothrops jararaca intraspecific venom variation César Augusto de-Oliveira, Daniel R. Stuginski, Eduardo S. Kitano, Débora AndradeSilva, Tarcísio Liberato, Isabella Fukushima, Solange M.T. Serrano, and André Zelanis J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00561 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on September 1, 2016

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Journal of Proteome Research

Dynamic rearrangement in snake venom gland proteome: insights into Bothrops jararaca intraspecific venom variation

César Augusto-de-Oliveira1, Daniel R. Stuginski2, Eduardo S. Kitano3, Débora Andrade-Silva3, Tarcísio Liberato1, Isabella Fukushima1, Solange. M. T. Serrano3, André Zelanis1*

1

Laboratório de Proteômica Funcional, Departamento de Ciência e Tecnologia,

Universidade Federal de São Paulo (ICT-UNIFESP), São José dos Campos, SP, Brazil. 2

Laboratório de Herpetologia, Instituto Butantan, São Paulo, Brazil.

3

Laboratório Especial de Toxinologia Aplicada, Center of Toxins, Immune-

Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo, Brazil.

*

Corresponding author: André Zelanis Department of Science and Technology, Federal University of São Paulo (ICT-UNIFESP), São José dos Campos, SP, Brazil, Rua Talim, 330, 12231-280, Tel.: +55 11 3385-4135 ext. 9727. E-mail: [email protected] Running title: Bothrops jararaca venom gland proteome 1 ACS Paragon Plus Environment


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Abstract We carried out an analysis of the venom gland proteome of Bothrops jararaca taking into account two distinct phases of its ontogenetic development (i.e. newborn and adult) and the marked sexual dimorphism recently reported on its venom proteome. Proteomic data analysis showed a dynamic rearrangement in the proteome landscape of B. jararaca venom gland upon development and gender-related changes. Differentially expressed proteins covered a number of biological pathways related to protein synthesis, including proteins associated to transcription and translation, which were found to be significantly higher expressed in the newborn venom gland. Our results suggest that the variation in the expression levels of cellular proteins might give rise to an even higher variation in the levels of the expressed toxins. Upon ageing the venom gland proteome repertoire related to the protein synthesis together with ecological traits would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan. Proteomic data are available via ProteomeXchange with identifier PXD004186.

Keywords: snake venom proteome, venom gland proteome, venom variability, Bothrops jararaca

2 ACS Paragon Plus Environment

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Abbreviations svVEGF: snake venom Vascular Endothelium Growth Factor; 5NUCL: Nucleotidase; AP: Aminopeptidase; BPP: Bradykynin Potentiating Peptides; CRISP: Cysteine-Rich Secretory Protein; CTL: C-Type lectin; CVF:Cobra Venom Factor; DIEST: Diesterase; DPP: Dipeptidyl Peptidase; NGF: Nerve Growth Factor; PLAI: phospholipase inhibitor gamma-like; PLB: phospholipase B; SVMP: Snake Venom Metalloproteinase; SVSP: Snake Venom Serine Proteinase; LAAO: L-Amino Acid Oxidase.



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Introduction Investigators have long appreciated the composition and diversity of snake venoms and a number of venom proteomes have been reported so far.1-4 As a biological product derived from an intricate process of protein synthesis and secretion, the composition of snake venoms may vary according to several factors, such as habitat, gender, ontogenetic development and others.5-11 Owing to their highly specialized secretory function, snake venom glands represent exceptional models for the study of protein synthesis, secretion and long-term storage of potentially harmful components, such as proteases and myotoxins.12-18 Moreover, venom gland secretory cells encode rich information regarding toxin diversity as well as comprise the molecular machinery responsible for the venom production. As such, they are the main biological source of starting material for the study of the gene expression profiles. Nevertheless, with some exceptions, little attention has been given to the cellular transcripts/proteins composing snake venom glands or the biological processes related to snake venom synthesis. Even though a number of venom gland transcriptomes have been characterized so far, little is known regarding the composition of the venom gland proteome of any snake species. Bothrops jararaca is among the most studied South American snake species both in terms of ecological and toxinological points of view.10,11,19-26 According to recent epidemiological data from Brazilian Ministry of Health, this species is responsible to the majority of the accidents with humans in Southeastern Brazil, a highly populated region.27 Although this species is primarily generalist, it displays a significant change in feeding habits during lifespan with newborn specimens feeding on small ectothermic prey such as small lizards, amphibians 4 ACS Paragon Plus Environment

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and arthropods and adults feeding mainly on mammals.19,20,28 Such changes correlate with the venom proteome rearrangement upon neonate to adult transition and may reflect different strategies to deal with distinct prey types.10,23 It has been shown that in B. jararaca noradrenergic innervation is a key activator of venom gland and that the stimulation of α1-adrenoreceptors triggers snake

venom

production

phosphatidylinositol

cycle

in

4,5-bisphosphate

secretory hydrolysis

cells, and

by

activating

ERK

signaling

pathway.16,29 Interestingly, Luna and coworkers29 reported sexual dimorphism in the activation of NFκB and AP-1 transcription factors in adult male and female B. jararaca venom gland. Furthermore, the authors also reported gender-related differences in the venom production cycle after 4 and 7 days after venom extraction in adult female and male snakes, respectively. Indeed, sexual dimorphism in adult B. jararaca venom was recently reported in which quantitative label-free proteomic analysis revealed the higher expression of Nerve Growth Factor in male venom whereas C-type lectins displayed more than 3 fold increase in expression in female venom.26 Moreover, peptidomic analysis confirmed the ubiquitous presence of the four Bradykinin Potentiating Peptides (BPPs) that lack the C-terminal Q-I-P-P sequence only in the female venom as gender molecular markers.26,30 Despite the number of reports on qualitative/quantitative variation in B. jararaca venom proteome as well as its venom gland transcriptomic profiles22,25 the molecular mechanisms underlying venom variability in this species remains poorly understood. Therefore, the aim of this work was to analyze the venom gland proteome of B. jararaca taking into account two distinct phases of its ontogenetic development (i.e. newborn and adult) and the sexual dimorphism 5 ACS Paragon Plus Environment


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recently reported on its venom proteome26. Our results point to a striking rearrangement of B. jararaca venom gland proteome in two distinct phases of the ontogenetic development as well as gender-related differences and shed light into the remarkable intraspecific venom variability found in this species.

Material and Methods Bothrops jararaca venom and venom gland 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 São Paulo State (Brazil) was used in this study. The venom was extracted and centrifuged for 30 min at 2,000 x g, at 4 °C, to remove any scales or mucus, lyophilized and stored at -20°C until use. Venom protein concentrations were determined using the Bradford reagent (Sigma, USA) and bovine serum albumin (Sigma, USA) as a standard. B. jararaca specimens were obtained from the Herpetology Laboratory, Instituto Butantan, São Paulo, Brazil. Twelve animals were used to obtain venom glands, including 6 newborns (up to 45 days old; 3 male and 3 female specimens) and 6 wild caught adults (3 male and 3 female specimens), from different geographic regions of São Paulo State, Brazil (Supplementary table 1). The venom was extracted and 3 days later the animals were sacrificed by CO2 inhalation. The venom glands were carefully dissected, frozen in liquid nitrogen and kept at -80°C until use. All animal work has been conducted in agreement with the Ethical Principles in Animal Research, adopted by the Brazilian College of Animal Experimentation and was approved by the Ethical Committee for Animal Research of Butantan Institute (protocol # 1371/15). 6 ACS Paragon Plus Environment

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Protein extraction and quantitation Three milliliters of lysis buffer (50 mM HEPES, 200 mM NaCl, 2% CHAPS, pH 7.5) containing the HALTTM protease Inhibitor cocktail (Thermo Fisher, USA) and 5 mM EDTA (final concentration) were added to each venom gland pool and the samples were subjected to homogenization using a tissue homogenizer Polytron PT3100 (Kinematica AG, Switzerland) using 18,000 rpm cycles for 30 s in ice bath. Homogenates were clarified by centrifugation at 14,000 x g for 10 min, at 4°C, to remove debris and the clear supernatant was carefully removed. Due to the low weight of the individual newborn venom glands (data not shown), we chose to pool the 6 pairs of newborn venom glands (3 male + 3 female), whereas a total of 6 venom glands was used for the adult comparison: a male sample (a pool of 3 venom glands derived from 3 male individuals), a female sample (a pool of 3 venom glands derived from 3 female individuals) and an adult pool (a mixture where equal amounts of proteins were added from each adult pool to make up an 1 mg/mL protein solution) Protein concentrations were determined using the Bradford reagent (Sigma, USA) and bovine serum albumin (Sigma, USA) as a standard.

SDS-polyacrylamide gel electrophoresis and Western blot analysis SDS-PAGE was carried out according to Laemmli31 and proteins were stained with Colloidal Coomassie Blue G-250. Western blot analysis was carried out as described elsewhere32 using anti-bothropic antivenom from Instituto Butantan (batch # 0610188), generated by the immunization of horses with a mixture of adult venoms of B. jararaca (50%), B. alternatus (12.5%), B. jararacussu



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(12.5%), B. moojeni (12.5%), B. neuwiedi (synonyms B. n. goyazensis, B. n. meridionalis, B. n. paranaensis and B. n. urutu)33 (6.25%) and B. pauloensis (6.25%). The secondary antibody used was anti-Horse IgG- peroxidase (Sigma, USA) with PierceTM ECL substrate (Thermo Scientific, USA). As a negative control, the membrane was incubated with non-immune horse serum as the primary antibody, followed by the incubation with the same secondary antibody.

In-solution trypsin digestion and reductive isotopic dimethylation labeling The in-solution trypsin digestion was performed according to the protocol described by Kleifeld et al.34 with slight modifications. Briefly, a solution of 6 M guanidine hydrochloride (GuHCl) was added to a sample of 100 µg of protein from each venom gland sample to a final concentration of 3 M GuHCl, followed by the addition of 5 mM dithiothreitol (DTT) (final concentration). The mixture was incubated at 65 °C for 60 min. Iodoacetamide (IAA) was then added to a final concentration of 15 mM and the samples were incubated for 60 min at room temperature, in the dark. To quench the excess of IAA, DTT was added to a final concentration of 15 mM. Clean-up of samples was performed by the addition of ice cold acetone (8 volumes) and methanol (1 volume), followed by the incubation of samples for 3 h at -80° C. After centrifugation at 14,000 x g for 10 min, protein pellets were washed twice with one volume of ice cold methanol and then resolubilized with NaOH solution (final concentration of 2.5 mM), followed by the addition of 50 mM HEPES buffer, pH 7.5, to a final volume of 100 µL. Trypsin (Proteomics grade; Sigma, USA) was added at 1:100 ratio (enzyme/substrate) and protein samples were incubated at 37 °C for 18 h.


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Tryptic peptides were differentially labeled via stable-isotope dimethyl labeling, as previously described35. In brief, tryptic peptides were submitted to reductive dimethylation with either light or heavy formaldehyde/cyanoborohydride solutions, as follows: newborn venom gland peptides (light) vs. adult venom gland peptides (heavy); adult female venom gland peptides (light) vs. adult male venom gland peptides (heavy). Tryptic peptides (pH 7.5) from each sample were incubated overnight at 37 °C with either light or heavy sodium cyanoborohydride

(NaBH3CN,

light,

or

NaBD3CN,

heavy)

concentration of 20 mM followed by the addition of formaldehyde or

13

to 12

a

final

CH2O (light)

CD2O (heavy) to a final concentration of 40 mM, resulting in mass

differences of +28.031300 Da and +36.075670 Da for the light and heavylabeled samples, respectively. The reaction was terminated by adding 1 M Tris (pH 6.8; to a final concentration of 200 mM) to each sample and the mixture was incubated for 2 h at 37 °C. Samples were then combined at 1:1 ratio into two pools: (i) newborn + adult and (ii) adult female + adult male. After desalting using C-18 cartridges (3M EmporeTM SPE Extraction disks, USA) peptide samples were dried in a SpeedVac and redissolved in 50 µL of 0.1% formic acid prior

to

nanoflow

liquid

chromatography/tandem

mass

spectrometry

(LC−MS/MS) analysis.

Mass spectrometric analysis An aliquot (5 µL) of the resulting peptide mixture was injected into a trap column packed with C18 (100 µm i.d. × 2 cm) for desalting with 100% solvent A (0.1% formic acid). Peptides were then eluted onto an analytical column (75 µm i.d. × 100 mm) packed in house with Aqua® C-18 5 µm beads (Phenomenex, 9 ACS Paragon Plus Environment


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USA). Nanoflow liquid chromatography was performed on an Easy nanoLC system (Thermo Fisher Scientific, USA) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, USA). Peptides were loaded onto the column with solvent A (0.1% formic acid) and eluted with a 120 min linear gradient from 3 to 30% of solvent B (acetonitrile in 0.1% formic acid) at a flow rate of 200 nL/min. Spray voltage was set at 2.1kV, at 200 °C and the mass spectrometer was operated in data dependent mode, in which one full MS scan was acquired in the m/z range of 300-1650 followed by MS/MS acquisition using Collisional Induced Dissociation (CID) of the fifteen most intense ions from the MS scan. MS spectra were acquired in the Orbitrap analyzer at 60,000 resolution (at 400 m/z). Dynamic exclusion was defined by a list size of 500 features and exclusion duration of 60 s. For the survey (MS) scan AGC target value of 1,000,000 was set whereas the AGC target value for the fragment ion (MS/MS) spectra was set to 10,000 ions. The lower threshold for targeting precursor ions in the MS scans was 3,000 counts. Samples were analyzed as three technical replicates.

Proteomics data processing Mass spectrometric (RAW) data were analyzed with MaxQuant software36 (version 1.5.3.17). A False Discovery Rate (FDR) of 1% was required for both protein and peptide-to-spectrum match identifications. LTQ-Orbitrap Velos raw data were searched against a target database restricted to the taxonomy ‘Serpentes’ (UniProt release 09_2015; 58,895 sequences appended with 15,276 sequences derived from the translation of cDNAs encoding tissue proteins and toxins from B. jararaca venom gland.18 This database was also 10 ACS Paragon Plus Environment

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combined with the sequences of 245 common contaminants and concatenated with the reversed versions of all sequences. Enzyme specificity was set to trypsin

and

at

least

two

missed

cleavages

were

allowed;

cysteine

carbamidomethylation was selected as fixed modification whereas methionine oxidation, glutamine/asparagine deamidation and protein N-terminal acetylation were selected as variable modifications. Multiplicity was set to 2 to account for the two labeling states that were used (light and heavy dimethyl labeling at peptide N-terminus and lysine side chains). Peptide identification was based on a search with an initial mass deviation of the precursor ion of 7 ppm and the fragment mass tolerance was set to 20 ppm. For protein quantification, a minimum of two ratio counts was set and the ‘Re-quantify’ and ‘match between runs’ functions of MaxQuant software were enabled. As is observed from complex proteomes such as vertebrates, peptides can be shared between homologous proteins or splice variants, leading to “protein groups”. For each protein group in the MaxQuant’s ‘proteinGroups.txt’ file, the first protein entry was selected as representative. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE37 partner repository with the dataset identifier PXD004186.

Bioinformatic analysis All the light and heavy peptide intensities were log2-transformed and quantile-normalized using the ‘preprocessCore’ library in R scripting and statistical environment38,39 to correct for intra-experimental variation. Protein identifications in which light or heavy singletons were found were not considered for further analysis. Statistical analyses were performed using the 11 ACS Paragon Plus Environment


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‘limma’ package in R/Bioconductor.40,41 After fitting a linear model to the data, an empirical Bayes moderated t-test was used for the comparisons (Female x Male and Newborn x Adult), and P values were adjusted for multiple testing with the Benjamini-Hochberg method. Proteins with an adjusted P-value < 0.05 and log2(fold change) > 1 and 170 kDa to


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~10 kDa. Since a small portion of MS/MS spectra matched to venom peptides, we decided to perform a Western blot analysis of venom gland extracts using a polyspecific antivenom (anti-bothropic antivenom, produced by Instituto Butantan, Brazil) in order to get a better picture of the residual (or newly synthesized) venom proteome. For this purpose, the best profile was acquired after analyzing venom gland extracts in 5-fold protein excess compared to venom proteins (i.e. 25 µg of venom gland protein extract and 5 µg of venom proteins). Immunostaining of B. jararaca venom gland extracts with antibothropic antivenom revelaed several protein bands ranging from 110 kDa to 10 kDa, with a similar pattern of recognition among all samples (Figure 2B). For several protein bands, the Western blot profile of venom gland extracts somehow mirrored the venom protein profiles, with some exceptions found for high molecular mass proteins. It is interesting to note that the protein profiles at the range of 20-15 kDa were considerably similar between venom gland extracts and venom proteins. On the other hand, a number of high molecular mass proteins (> 100 kDa) were detected in all the samples derived from venom gland extracts, which might correspond to still non-processed precursor protein forms.

Differentially expressed cellular proteins Reductive isotopic dimethylation allowed for the identification and quantification of proteins derived from distinct biological scenarios: (i) two stages of B. jararaca ontogenetic development (newborn and adult) and (ii) gender-related differences. All technical replicates presented an overall good



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correlation as observed by their Pearson correlation coefficient ranging from 0.89 to 0.94 (Supplementary figures 1 and 2; Supplementary table 3). Almost 8% of the quantified proteins were found to be differentially expressed (51 proteins in total; 21 in newborn and 30 in adult; Supplementary table 4). A protein disulfide-isomerase (U3FBW7) was quantified with the highest expression among all of the identified proteins (Figure 3A).Protein disulfide-isomerases (PDIs) are Endoplasmic Reticulum (ER)-resident proteins that catalyze the formation, breakage and rearrangement of disulfide bonds and therefore are key enzymes responsible for protein folding and the maintenance of protein native structures.43 The identification of PDIs and molecular chaperones among differentially expressed proteins is a recurrent feature in quantitative proteomic studies of eukaryotic samples.44 However, this observation is often related to experimental/biological stress conditions (tumor tissues, tumor derived cell lines or experimentally induced toxic states). In fact, protein synthesis requires continuous activity of PDIs, which might explain the high levels of this protein in tissues where biosynthetic process are taking place, a feature already observed in the proteome of B. jararaca venom gland.17 In relation to gender-related differences the male x female comparison yielded almost 5% of differentially expressed proteins (31 proteins in total; 19 in male and 12 in female; Supplementary table 4). Structural proteins such as collagen alpha-2(I)chain (V8NPI7) and the calcium binding protein, (sorcin-like protein, P30626) were highly expressed in female’s venom gland whereas ribosomal and histone proteins displayed significantly higher expression values in male’s venom gland (Figure 3B). In addition, we also found higher expression of PDIs in male’s venom gland, a feature that might correlate with the higher abundance


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of high molecular mass disulfide-linked oligomeric toxins recently reported in the electrophoretic profiles of adult male B. jararaca venom.26 Clustering of newborn and adult differentially expressed proteins revealed specific patterns of expression (Figures 4A) in which proteins belonging to ‘General metabolism’ category were prominent and, in general, displayed a similar pattern of high expression between both samples. Further analysis revealed a striking higher expression of proteins related to biosynthetic process (transcription/translation category) among newborn’s differentially expressed

proteins,

followed

by

proteins

related

to

post-translational

modifications (Figure 4B). Inhibitors such as the antihemorrhagic factor BJ46a (Q9DGI0), small serum protein-1 (D9N567), murinoglobulin-2 (V8NK36) and Complement C3a protein (A0A081DUA7) were highly expressed in adult venom gland, followed by structural proteins such as tropomyosin and collagen. It is possible that these proteins might be present due to the contamination of venom gland extracts with muscle or blood proteins (in the case of structural and inhibitor proteins, respectively), a feature that was previously reported.17 Clustering of adult male and female differentially expressed proteins and the subsequent functional analysis revealed a number of proteins related to the process of transcription/translation as well as PTM/processing in male venom gland whereas ‘general metabolism’ and ‘inhibitor’ categories were prominent in female venom gland (Figure 4C and D).

Toxins Regardless of age and gender-related differences (i.e. when protein identification data were merged together) the venom proteome correlated well 17 ACS Paragon Plus Environment


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with B. jararaca venom proteomes reported so far, both quantitatively (as observed from the number of proteins derived from each toxin family) and qualitatively (as illustrated by the diversity of the identified toxins).10,2225

Proteases comprised the majority of venom toxins (almost 60% of the overall

toxin composition) with SVMPs being the most abundant class (Figure 5A). Actually, among all peptide identifications, only a small portion matched to venom toxins (16%). Whether these toxins resulted from newly synthesized proteins or are remnants from an incomplete emptying of venom gland is difficult to address. We were able to identify peptides belonging to the precursor regions of SVMPs and SVSPs (Supplementary table 5). Sequence logos of the set of the identified peptides resulted in the recurrence of the canonical sequences ‘MCGVT’, and ‘SEL’, part of the cysteine-switch and pro-peptide regions in SVMPs and SVSPs, respectively (Figure 5B). These results suggest that newly synthesized (or remnant) proteinases are inactive either within the secreting cells or within the lumen of the venom gland, or both. In the case of SVMPs, our results are in line with the hypothesis that their processing starts within secretory vesicles and continues into the lumen of the venom gland just after enzyme secretion.45 Moreover, we also found peptides derived from the predicted mature Ntermini form of some C-type lectins and a peptide derived from the signal peptide from a phospholipase A2 enzyme (Supplementary figure 3 and Supplementary table 5). The latter observation illustrates a case where the protein had still not left the endoplasmic reticulum and, therefore, was inside the venom gland cells, a likely newly synthesized protein. These results corroborate with the previous report on B. jararaca venom gland activation in which C-type 18 ACS Paragon Plus Environment

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lectins were found to be present in the quiescent stage of secreting cells whereas PLA2s where more abundant in the activated stage17. Statistical

analysis

of

differentially

expressed

proteins

strongly

corroborated previous findings related to the venom proteome and venom gland transcriptomes of newborn and adult B. jararaca as well as adult male and female venoms/venom gland transcriptomes/proteomes (Figure 6 A and B) 8,10,22-26,30

. SVMPs comprised more than 60% of the differentially expressed

toxins within newborn venom glands, whereas SVSPs and CTLs accounted for more than 70% of the differentially expressed proteins in adult venom gland (Figure 6A-D). A previous work on the ontogenetic changes in B. jararaca venom showed that the main role of SVMPs in the venom of newborns is acting upon coagulation cascade components, including the activation of Factor X and prothrombin, a feature that likely enables newborn specimens to deal with distinct prey types at an early stage of their ontogeny.10,46 As the snake grows, there might be a diversification in the biological targets of venom toxins as illustrated by the qualitative and quantitative differences found in B. jararaca venom proteome and venom gland transcriptome.23,25 Changes in the dynamics of protein expression and proteome diversity may impact the composition of male and female venoms. In fact, we have recently identified gender molecular markers in adult B. jararaca venoms.26 The quantitative results reported herein also corroborate the higher expression of NGF and CTLs in male and female venoms, respectively (Figures 6E and F). In addition, our results suggest a higher expression of LAAO enzymes in male venom, a feature also observed in the two-dimensional venom protein profile of adult male venoms26. Indeed, changes in the protein composition of B. jararaca 19 ACS Paragon Plus Environment


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venom gland during the venom production cycle were reported, with striking changes occurring 4 and 7 days after venom removal in adult female and male snakes, respectively.29 Therefore, the results reported herein regarding genderrelated differences in adult B. jararaca venom gland might also be related to our (venom) extraction protocol. As sexual maturation and ontogeny display a paramount role in numerous biological features, one might expect that differences in the venom phenotype would mirror the main developmental changes that occur in newborn and adult and male and female specimens. It was shown that the regulation of protein synthesis in B. jararaca venom gland is triggered by noradrenergic stimulation, after the activation of α1-and βadrenoreceptors16,29 and that there is a marked sexual dimorphism in the activation of the transcription factors NFκB and AP-1, whose expression peaks earlier in female B. jararaca venom glands.

Insights into the intraspecific variability in B. jararaca venom proteome We separately evaluated the quantification data from toxins and cellular proteins and, as a general observation, regardless of age or gender, the expression range of cellular proteins showed less variation than the toxin expression range (Figure 7). While cellular transcripts encode rich information that provide key features to understand the molecular basis of snake venom variation, their presence/ abundance does not necessary imply/correlate in the translation of a functional (protein) product. Therefore, a proteomic approach is desirable as a complementary approach. Additionally, several reports have shown a low to


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moderate correlation of mRNA and protein levels in eukaryotes including vertebrates47,48. Based on our results it is possible to hypothesize that at an early stage of B. jararaca development the protein biosynthetic process might be prominent in comparison to the adult phase. A number of reports showed that the expression of genes related to protein synthesis is diminished upon aging in the salivary glands of other vertebrates, such as mouse and humans.49-51 The biological meaning behind this hypothesis might be related to the snake metabolism and growing. Growing involves, besides metabolic adjustments, an increase in protein synthesis, which could explain the higher expression of proteins related to transcription and translation when compared to adults. In the same way, due to their higher metabolic rates and the necessity to grow, it is expected that juveniles need to eat more frequently than adults, in order to fuel this process, therefore an increase in the protein synthesis could also play an important role in food processing during the juvenile phase of these animals. The limiting step in protein synthesis is the translation initiation, when the ribosome subunits are assembled to begin the polypeptide formation52. Actually, in addition to a number of ribosomal proteins we found higher expression of several translation initiation factors among newborn venom gland proteins (Supplementary table 4). In any living organism, the maintenance of protein homeostasis

is

acquired

through

a

fine-tuned

balance

of

molecular

mechanisms, namely transcriptional and translational rates coupled with the stability of individual proteins which, in turn, results from the balance between the rate of protein synthesis and the rate of degradation.48,53-55 Therefore, an increase in the rate of protein synthesis should usually be followed by an 21 ACS Paragon Plus Environment


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increase in the degradation rate, which ultimately results in a faster protein turnover and allows a rapid adaptation to a variation of nutritional and/or pathophysiological conditions55. Upon ageing, the cellular proteome repertoire related to the protein synthesis together with ecological traits, would have an impact on the toxin repertoire, which, in the case of B. jararaca species, would enable the species to deal with different prey types during its lifespan.

Conclusions The knowledge of the proteome of B. jararaca venom gland revealed specific profiles related to aging and sexual dimorphism and, in light of the understanding of the diversity of B. jararaca venom, it is possible to infer a dynamic proteome rearrangement of cellular proteins that likely results in the striking intraspecific variability of the venom in this species. It turns out that the variation in the expression levels of cellular proteins gives rise to an even higher variation in the dynamic range of the expressed toxins. Underlying mechanisms that modulate the expression of toxins may likely involve hormonal regulation, which, in turn, might account for the ontogenetic and gender-related differences found in the expression profiles of B. jararaca venom gland. Further analysis (i.e. metabolomics) would also assist in the integration of data related to venom production in this species and bridge the gaps towards the comprehensive knowledge of snake venom proteomes.

SUPPORTING INFORMATION


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The

following

files

are

available

free

of

charge

at

ACS

website http://pubs.acs.org:

Figures (S1-S3) and table S1.docx. Supplementary information on proteomic analysis and biological samples Supplementary tables(S2-S6).xls. Detailed analysis on proteomic data.

Acknowledgements We thank to Dr. Inácio Junqueira-de-Azevedo for providing the B. jararaca transcrpitome dataset used in this work and Dr. Sávio S. Sant’Anna for his assistance in providing access to B. jararaca specimens. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (grant 441804/2014-4), Fundação de Amparo à Pesquisa do Estado de São Paulo (grants 2015/18096-0, 2014/06579-3 and 2013/074671) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Figure legends

Scheme 1. Schematic overview of the analysis of B. jararaca venom gland proteome.

Figure 1. Overall composition of B. jararaca venom gland proteome (A) and biological annotation of cellular proteins into functional categories (B). The diversity of Post Translational Modification (PTM)/processing category (C).

Figure 2. B. jararaca venom and venom gland protein profiles. (A) SDS-PAGE (12% SDS-polyacrylamide gel) profile of B. jararaca venoms and venom gland extracts (25 µg of proteins) under reducing conditions. Mobility of molecular mass markers is indicated on the left. (B) Western blot analysis of B. jararaca venoms (5 µg) and venom gland extracts (25 µg). After SDS-PAGE separation on 12% SDS-polyacrylamide gels proteins were transferred to a nitrocellulose membrane and immunostained with anti-bothropic antivenom.



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Figure 3. Volcano plots showing the statistically differentially expressed cellular proteins (t-test; adjusted p-value ≤ 0.05 and log2 fold change 1) in adult and newborn venom glands (A) and adult male and female venom glands (B). Colored dots refer to statistically differentially expressed proteins in adult (blue) or newborn (red) and adult male (blue) and female (red) cellular proteins.

Figure 4. The heatmaps show the normalized (z-score) expression values for differentially expressed proteins (left) together with their functional annotation presented as the percentage of identifications matching specific functional categories (right). Newborn vs. adult venom gland proteins (A and B) and adult male vs. female cellular proteins (C and D). The expression values were measured across three technical replicates. Median expression values were clustered as described in Materials and Methods.

Figure 5. The overall composition of B. jararaca venom proteome as inferred from the analysis of the venom gland proteome (A). (B) Sequence logos of the set of the identified peptides showing the canonical sequences ‘MCGVT’, and ‘SEL’, part of the cysteine-switch and propeptide regions in SVMPs and SVSPs, respectively. Sequence logos were generated with IceLogo57 .

Figure 6. Volcano plot showing the statistically differentially expressed toxins (ttest; adjusted p-value ≤ 0.05 and log2 fold change 1) in adult and newborn venom glands (A) and adult male and female venom glands (B). Differentially expressed proteins in newborn (C) and adult (D) and male (E) and female (F) are presented as the percentages matching to specific toxin classes.


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Colored dots refer to statistically differentially expressed proteins in adult (red) or newborn (green) and adult male (blue) and female (pink) venom proteins.

Figure 7. Variation in the log2 median of newborn/adult ratio (A) and male/female ratio found for cellular proteins and toxins (C and D), respectively. The dashed red lines correspond to the standard deviation related to the measures.



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Dynamic proteome rearrangement in Bothrops jararaca venom gland 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Venom gland 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Bothrops jararaca 41 (Photo courtesy of André Zelanis, Copyright 2016) 42

Ontogenetic changes

Biological annotation

Sexual dimorphism Proteome diversity

Quantitative analysis

Intraspecific venom variation Venom gland proteome

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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

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Functional categories 3%

4%

4% Degradation

General metabolism

16%

Inhibitor

18% PTM/processing

47%

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13%

84%

Transcription/translation Transport/sorting

1% 9% Toxins

Secretion

Cellular proteins ACS Paragon Plus Figure 1 Environment

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PTM/processing

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2% 1% 2%

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6%

Glycation 17%

Glycosylation

2%

70%

N-terminal modification N-terminal processing (secretion) Protein processing

Figure 1



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B

kDa

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170 130 100 70

102 76 52

55

38 31

40 35

24

25

17 15

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Venoms

Venom gland extracts

Venoms


Figure 2

Venom gland extracts

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A

B

V8NPI7

T1E4R4

U3FBW7

V8NPI7

3 2 V8NSS7

-log10(adj.Pvalue)

V8PA09

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Up Female

Up Newborn Journal of Proteome Research Up Male

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PTM/processing

Structural

General metabolism

0


Figure 4

20

40 60 Percentage

80

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PTM/processing

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Figure 4

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40 60 Percentage

80

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B

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4.0

svVEGF SVSP Bits

SVMP PLB

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SVSP 3%

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Male

E CTL 9% DPP 3%

DIEST 3%

SVSP 18%

PLA2 9%

SVMP 64%

NGF 9%

CTL 14%

LAAO 9% PLA2 9%

SVMP 41%

PLB 3%

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D SVSP 36%

Female

F

VF 8%

SVSP 20%

CTL 36%

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SVMP 32% LAAO 5% PLAI 5%

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log2(ratio adult/newborn)

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-8

8 4 0 0

-8

5

0 0

50

100

150

-5

Quantified proteins

10

D

-4

10

-10

100

200

300

Quantified proteins

400

500

600

log2 (ratio male/female)

B

C

Cellular proteins

log2 ratio (male/female)

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

5 0 0

40

60

-5

Quantified proteins -10

Figure 7

20


80

100

120

Dynamic Rearrangement in Snake Venom Gland Proteome: Insights

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