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Activation of Bothrops jararaca snake venom gland and venom production: A proteomic approach Milene Schmidt Lunaa , Richard Hemmi Valenteb,d , Jonas Peralesb,d , Mônica Larucci Vieirac , Norma Yamanouyea,d,⁎ a

Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil Laboratório de Toxinologia, Instituto Osvaldo Cruz, FIOCRUZ, Av. Brasil, 4365, 21040-900 Rio de Janeiro, Brazil c Centro de Biotecnologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil d Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq), Brazil b

AR TIC LE I N FO

ABS TR ACT

Article history:

Viperidae venom glands have a basal–central lumen where the venom produced by

Received 18 July 2013

secretory cells is stored. We have shown that the protein composition of venom gland

Accepted 18 October 2013

changes during the venom production cycle. Here, we analyzed the venom gland proteins during the venom production cycle by proteomic approach. We identified specific proteins in each stage of the cycle. Protein species from endoplasmic reticulum (PDI and GPR78) and

Keywords:

cytoplasm (actin, vimentin, tropomyosin, proteasome subunit alpha type-1, thioredoxin,

Venom gland

and 40S ribosomal protein) are more abundant in the activated stage, probably increasing

Protein synthesis

the synthesis and secretion of toxins. We also showed for the first time that many toxins

Venom production cycle

are present in the secretory cells during the quiescent stage. C-type lectin-like and serine

Proteomic analysis

proteinases were more abundant in the quiescent stage, and GPIb-BP and coagulation factor

Snake

IX/X were present only in this stage. Metalloproteinases, L-amino acid oxidases, PLA2 and snake venom metalloproteinase and PLA2 inhibitors, and disintegrins were more abundant in the activated stage. Regarding metalloproteinases, the presence of peptides corresponding to the pro-domain was observed. These results allow us to better understand the mechanism of venom gland activation and venom production, contributing to studies about snake toxins and their diversity. Biological significance In this study we identified, for the first time, the presence of different toxins in the snake venom gland in its quiescent stage. Furthermore, we showed that not all toxins are synthesized during the activated stage of the gland, suggesting an asynchronous synthesis for different toxins. Besides, the synthesis of some protein species from endoplasmic reticulum and cytoplasm, which are related to the synthesis and secretion processes, are more abundant in the activated stage of this gland. The knowledge of the proteomic composition of the venom gland in different stages of the venom production cycle will give us new insights into the mechanism of venom gland activation and venom production, contributing to studies about snake toxins and their diversity. © 2013 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil. Tel.: +55 11 2627 9763; fax: +55 11 2627 9752. E-mail address: [email protected] (N. Yamanouye). 1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jprot.2013.10.026

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1.

Introduction

Bothrops jararaca is a Brazilian venomous snake (Serpentes, Viperidae, Crotalinae). The venom gland of Viperidae snakes is an oral exocrine gland that is related to salivary glands and has the capacity to secrete toxic proteins [1]. This venom gland has two important characteristics: (1) the presence of a basal–central lumen where most of the venom produced is stored, and (2) a long venom production cycle with two distinct steps, a quiescent stage when the lumen is full of venom, and an activated stage after the lumen is emptied of venom; this cycle lasts around 30–50 days [2–4]. Many studies have shown that the production of new venom by epithelial cells starts with morphological and biochemical changes in the secretory epithelium. The epithelial cells change from a cuboidal to a columnar shape, and the total volume and membrane surface of the rough endoplasmic reticulum, Golgi apparatus, subcomponents, secretory vesicles and mitochondria increase up to the eighth day after venom removal [5–10]. The maximal synthetic activity of the secretory cells and the highest mRNA concentrations are observed after 4–8 days [3,6–8]. Using labeled amino acids, it has been shown that the amount of labeled proteins synthesized by the glands increases during the venom production cycle [9,11]. The incorporation of labeled amino acids peaks at 8 days after venom removal. Afterwards, the synthetic activity decreases, and venom gradually accumulates inside the gland lumen, with the gland returning to the quiescent stage [5–10]. Thus, a complete venom production cycle is longer than the protein production cycle in mammalian salivary and pancreatic glands [12–14]. Although the highest mRNA concentrations are observed after 4 to 8 days, the synthesis of new venom toxins is asynchronous and is responsible for a non-parallel secretion in the venom gland of viperid snake during the first few days after venom removal [11]. We have shown that the cytosolic protein composition of the venom gland (venom-free) changes during the venom production cycle and striking changes occur 4 and 7 days after venom removal in female and male snakes, respectively. Protein composition of the activated venom gland is similar as in the quiescent stage only 15 days after venom removal [15]. Here, we investigated further the proteomic composition of the venom gland at different stages of the venom production cycle. Our goal was to identify proteins that were differently expressed in these different stages to better understand the physiology of the venom gland and, consequently, venom production. Comparative and statistical analyses of protein patterns from venom gland at different stages of venom production cycle allowed the identification of specific protein species in each stage. Among these, endoplasmic reticulum and cytoplasm proteins were synthesized more in the activated stage, probably improving the synthesis and secretion of the toxic proteins. Regarding the proteins present in the secretory glands, we demonstrated for the first time that many toxins are present in the so-called “quiescent” stage and that the synthesis of new toxins is asynchronous. Besides, we also detected the presence of metalloproteinase pro-domain in the venom gland.

2.

Experimental procedures

2.1.

Animal and venom gland

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B. jararaca female adults (n = 9), weighing 150–400 g, were classified by the Laboratory of Herpetology at the Instituto Butantan, and kept in a room under controlled conditions [16]. These snakes had no access to food for 40 days to prevent loss of venom and to make sure that all cells were in the quiescent state. Fasting periods of 1–2 months are common in snakes living in the wild, but fasts can exceed 1 year [17]. Animal care and procedures used were in accordance with guidelines of the Animal Ethics Committee of the Instituto Butantan (555/2008), the Biomedical Science Institute of the University of São Paulo (138/2009) and the Brazilian Institute for Environment and Renewable Natural Resources, a Brazilian Ministry of the Environment enforcement agency (IBAMA, License 01/2009). Snakes were anesthetized with sodium pentobarbital (30 mg/kg, s.c.), and decapitated. The venom glands were removed from snakes from which no venom was previously removed (quiescent stage, n = 3) and snakes that had their venom removed manually 4 and 7 days (activated stages, n = 3 for each group) before they were killed by decapitation. After venom gland removal, the residual venom was expelled from the lumen (to avoid any interference during proteomic analysis), washed with saline solution, freed from connective tissue [18], frozen in liquid nitrogen, and kept at − 80 °C until sample processing. The venom glands were then processed for two-dimensional gel electrophoresis (2D-PAGE) as described below. To remove the venom to activate the venom gland, snakes were anesthetized with sodium pentobarbital (20 mg/kg, s.c.) and the venom was removed manually [19].

2.2.

Protein extraction

Frozen venom glands (venom free) were pulverized in liquid nitrogen and homogenized in lysis buffer containing 2 M thiourea, 7 M urea, 4% CHAPS, 30 mM Tris–HCl, pH 8.5, and 1% protease inhibitor cocktail (P8340 Sigma-Aldrich, St Louis, MO, USA). The homogenates were incubated on ice for 1 h and then centrifuged at 12,000 ×g for 10 min at 4 °C. Protein concentration of the supernatants was determined by the Bradford assay [20] using bovine albumin as standard and the supernatants were kept at −80 °C.

2.3.

2D-PAGE

Protein samples (750 μg protein) were first precipitated with acetone [21]. The pellet was suspended in 340 μL of DeStreak rehydration solution and 0.5% immobilized pH gradient (IPG) buffer pH 3–10 (GE Healthcare, Uppsala, Sweden). IPG strips of 18 cm, pH range 3–10, were passively rehydrated overnight at room temperature. Isoelectric focusing (IEF) was performed on an Ettan IPGphor II (GE Healthcare) using a program for a total of 25,500 Vh according to the manufacturer's suggestion, including an initial step at low voltage of 100 V for 15 h to allow salt to migrate out of the strip. After IEF, the strips were

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initially incubated with equilibration buffer (75 mM Tris–HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 1% dithiothreitol for 15 min. Next, solutions were discarded and the strips were incubated in equilibration buffer containing 2.5% iodoacetamide for 15 min at room temperature. After equilibration, the strips were applied to the second dimension to separate proteins on a 12.5% SDS-PAGE and run on an Ettan DALTsix vertical system (GE Healthcare) according to manufacturer's instructions. After running the gels, they were fixed (5% acetic acid, 20% methanol) for at least 30 min and stained with Coomassie Brilliant Blue G (CBB-G) for 5 days. The stained gels were scanned on ImageScanner III (GE Healthcare) and the digitized images (8-bit grayscale, 300 dpi) were analyzed using the ImageMaster 2D Platinum v7.0 software (GE Healthcare). Technical triplicates of three different extracts per group were analyzed. Spots and matches were automatically detected and manually edited when necessary. Differences between the spots were analyzed by ANOVA and comparisons with p-values smaller than 0.05 were considered statistically significant.

2.4.

In-Gel trypsin digestion

Only the most abundant specific spots from each group were analyzed. Unless otherwise specified, all steps were carried out at room temperature. All tubes used were washed with methanol, water and methanol prior to use. Protein spots were excised from the gel, transferred to 0.5 mL tubes and cut into smaller pieces. Four hundred microliters of 50% acetonitrile, 25 mM ammonium bicarbonate, pH 8.0, aqueous solution was added to each tube, shaken for 15 min and discarded. This washing procedure was repeated twice and the last solution left overnight for complete destaining and detergent removal. Washing solution was then removed and the gel was dehydrated by the addition of 200 μL acetonitrile for 5 min. This procedure was repeated until gel pieces became opaque white. Solvent was removed and samples were completely dried using a SpeedVac (Thermo Fisher, Bremen, Germany) vacuum centrifuge concentrator for approximately 20 min. Gels were rehydrated with 15 μL of ice-cold porcine trypsin solution (20 ng/μL in 40 mM ammonium bicarbonate, pH 8.0 — cat. V511, Promega, WI, USA) and left on ice for 45 min. Excess trypsin was removed and 20 μL of 40 mM ammonium bicarbonate, pH 8.0, was added followed by incubation for 16 to 24 h at 37 °C. After digestion, each sample was submitted to sonication for 10 min, followed by a 20-s vortex cycle, and transferred to 0.5 mL tubes. Further peptide extraction was performed by 2 cycles of the following procedure: addition of 30 μL of a 50% acetonitrile, 5% (v/v) formic acid aqueous solution to the gel pieces, followed by vigorous vortexing for 20 s, 15 min rest at room temperature, sonication for 2 min, vigorous vortexing for 20 s, and transfer to the previously described 0.5 mL tubes. The final 80 μL tryptic peptide extracts were completely dried with the SpeedVac concentrator.

2.5.

MALDI-TOF/TOF MS

Samples were initially resuspended in 10 μL of 0.1% (v/v) trifluoroacetic acid aqueous solution followed by sonication

for 10 min and desalting with ZipTip C18 (Millipore, Billerica, MA, USA), according to the manufacturer's instructions, with peptides being eluted in a 1.5-μL total volume. All MS spectra were acquired in positive ion reflector mode on an AB Sciex MALDI-TOF/TOF 5800 mass spectrometer using Data Explorer software, version 4.0.0. An aliquot (0.3 μL) of the desalted tryptic digest was deposited onto the target plate immediately before the addition of an equal volume of a saturated matrix solution [10 mg/mL α-cyano-4-hydroxycinnamic acid (Aldrich, Milwaukee, WI) in 50% acetonitrile, 0.1% trifluoroacetic acid aqueous solution]. After sample drying at room temperature, both MS and MS/MS data were acquired with laser acquisition rates up to 400 Hz in MS and MS/MS mode. Typically, 2000 shots were accumulated for spectra in both MS and MS/MS modes. Up to 10 of the most intense ion signals with a signal-to-noise ratio above 30 were selected as precursors for MS/MS acquisition, excluding common trypsin autolysis peaks and matrix ion signals. External calibration in MS mode was performed using a mixture of four peptides: des-Arg1-bradykinin (m/z) 904.468; angiotensin I (m/z) 1296.685; Glu1-fibrinopeptide B (m/z) 1570.677 and ACTH (18–39) (m/z) 2465.199. MS/MS spectra were externally calibrated using known fragment ion masses observed in the spectrum of angiotensin I.

2.6.

NanoESI-LTQ/Orbitrap XL

One microliter of ZipTip desalted tryptic digest (see above) was added to 9 μL of 1% (v/v) formic acid. From the resulting solution, 4 μL was initially applied to a 2-cm long (100 μm internal diameter) trap column packed with 5 μm, 200 A Magic C18 AQ matrix (Michrom Bioresources, USA) followed by separation on a 10.5-cm long (75 μm internal diameter) column that was packed with the same matrix, directly on an empty 15 μm PicoTip (New Objective, USA). Chromatography was carried out on an EASY-nLC II instrument (Thermo Fisher Scientific). Samples were loaded onto the trap column at 2000 nL/min, while chromatographic separation occurred at 200 nL/min. Mobile phase A consisted of 0.1% (v/v) formic acid in water while mobile phase B consisted of 0.1% (v/v) formic acid in acetonitrile, and gradient conditions were as follows: 2 to 40% B in 32 min; up to 80% B in 4 min, maintaining this concentration for 2 min longer. Eluted peptides were directly introduced into an LTQ/Orbitrap XL mass spectrometer (Thermo Fisher Scientific) for analysis. Source voltage was set to 1.9 kV, capillary temperature to 200 °C and tube lens voltage to 100 V. Ion trap full and MSn AGC target values were 30,000 and 10,000, respectively, while FTMS full AGC target was set to 500,000. MS1 spectra were acquired on the Orbitrap analyzer (300 to 1700 m/z) at 60,000 resolution (for m/z 445.1200). For each spectrum, the 10 most intense ions were submitted to CID fragmentation (minimum signal required of 10,000; isolation width of 2.5; normalized collision energy of 35.0; activation Q of 0.25 and activation time of 30 s) followed by MS2 acquisition on the linear trap analyzer. Dynamic exclusion option was enabled and set with the following values for each parameter: repeat count = 1; repeat duration = 30 s; exclusion list size = 500; exclusion duration = 45 s and exclusion mass width = 10 ppm.

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2.7.

Data analysis

All MS/MS data were analyzed using PEAKS Studio 5.3 build 20110719 (Bioinformatics Solutions, Canada). Data were refined with the precursor correction option and de novo analysis was run assuming trypsin digestion with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 100 ppm for MALDI-TOF/TOF MS and fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 20 ppm for nanoESI-LTQ/Orbitrap XL MS. Oxidation of methionine as well as iodoacetamide and acrylamide derivatives of cysteine was specified as variable modifications. PEAKS Database analysis was performed using these same parameters plus the possibility of up to two missed enzyme cleavages. Searches were made against two subsets of NCBI non-redundant database from August 19, 2011: Metazoa and Serpentes. False discovery rate (FDR) was estimated through the use of decoy sequences. Finally, data from all searches were consolidated and only those results with calculated FDR values ≤1% were considered as reliable identifications. Peptide sequence inferred from MS/MS data from spots identified as snake venom metalloproteinase or snake venom serine proteinase was localized in the primary structure of these toxins and the graphic representation was generated by CAITITU software version 2.0.1.4 [22].

2.8.

Western blotting analysis

The protein extracts (40 μg) derived from each stage of the venom production cycle (n = 3) were submitted to 1D-PAGE and electroblotted on nitrocellulose membranes. Nonspecific binding was blocked with 5% nonfat milk in PBS-T (1.5 mM NaH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 0.5% Tween-20, pH 7.4) overnight at 4 °C. Blocked membranes were incubated for 2 h at room temperature with antibodies against actin (1:500, Sigma-Aldrich, St. Louis, MO, USA), and serine proteinases (1:500, kindly supplied by Dr. SMT Serrano [23]). The membranes were then probed with HRP-conjugated secondary antibodies (1:2000) for 30 min at room temperature. Immunoreactive protein spots were visualized using an enhanced chemiluminescence detection system (SuperSignal West Pico Substrate, Thermo Fisher Scientific, Bremen, Germany). Protein bands were detected with a ChemiDoc XRS photodocumentation system (Bio-Rad, Hercules, CA). The density of the bands was quantified with Quantity One software (Bio-Rad). Statistical comparisons were made by one-way analysis of variance (ANOVA) followed by the Mann–Whitney test for multiple comparisons with Excel software. Data were presented as mean ± S.E.M and p-values less than 0.05 were considered statistically significant.

3.

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stage, 4- and 7-days activated stages were prepared and analyzed by 2D-PAGE. Comparison between groups pointed out that different protein species were detected. Extracts of venom gland in quiescence and in the 4- and 7-days activated stages displayed 93, 148 and 243 specific spots, respectively (Fig. 1). Comparison between these three groups revealed just 3 common spots. When the comparison was made between quiescent and 4-days activated stage, quiescent and 7-days activated stage, and 4- and 7-days activated stages, only 8, 10 and 10 spots were detected in common, respectively (Fig. 1).

3.2.

Protein identification

The most abundant specific spots from each group were selected to be identified to detect which proteins are present in each stage of the venom production cycle. Representative gels with selected spots present in the venom gland in quiescent, 4- and 7-days activated stages are demonstrated in Fig. 2A, B, C, respectively. To identify the specific proteins from each group, the spots with density higher than 0.02 were excised from the gels, submitted to in-gel digestion with trypsin, and identified by MALDI-TOF/TOF MS. Some spots that were not identified by MALDI-TOF/TOF MS were analyzed by nanoESI-LTQ/Orbitrap MS. Information on the proteins identified is summarized in Supplementary Tables 1 to 3 according to information collected on UniProt (www.uniprot.org). The proteins identified in each group were distributed according to subcellular localization (Fig. 3). Comparisons of the quiescent stage relative to the 4- and 7-days activated stages showed that the percentage of protein species identified as cytoplasmic proteins increased in activated venom glands whereas membrane proteins increased in 4-days but were not detected in 7-days activated stages. Moreover, mitochondrial proteins decreased in both activated stages. Finally, the percentage of the protein species identified as secreted protein was smaller in the activated stages of the

Results

3.1. Differential protein expression in venom glands during venom production cycle To evaluate changes in protein expression profile in the venom gland of B. jararaca snake during the venom production cycle, whole extracts from venom gland in the quiescent

Fig. 1 – 2D-PAGE spot distribution for the different stages in venom production cycle of Bothrops jararaca snake venom gland. Venn diagram depicting overlap in spots detected in quiescent stage, 4- and 7-days after venom extraction (activated) stages. Data for each condition/group are a result of nine gels (triplicate biological samples ran in technical triplicate).

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Fig. 2 – Representative proteome maps of different spots selected during venom production cycle. 2D-PAGE of the extracts of venom glands in (A) quiescent, (B) 4-days and (C) 7-days after venom extraction (activated) stages. Proteins were separated on 18 cm IPG strips pH 3–10 followed by 12.5% SDS-PAGE. The gels were stained with Coomassie Brilliant Blue G. Specific spots indicated in each gel were identified by MALDI-TOF/TOF MS or nanoESI-LTQ/Orbitrap XL, as reported in Supplementary Tables 1, 2 and 3.

Fig. 3 – Distribution according to subcellular localization of protein species identified in Bothrops jararaca snake venom gland during venom production cycle. (A) Quiescent stage, (B) 4-days and (C) 7-days after venom extraction (activated) stages. Classification of protein species was obtained by UniProt database. A detailed list of protein species identifications is given in Supplementary Tables 1, 2 and 3.

venom production cycle, when compared to the quiescent stage. A more detailed analysis of the occurrence of cytoplasmic proteins during the venom production cycle displayed an increase in the number of protein species related to cytoskeleton such as actin, vimentin and tropomyosin, although the last protein increased in the 4-days activated stage and it was not identified at the 7-days activated stage (Fig. 4A). Other cytoplasmic proteins such as proteasome subunit alpha type-1, thioredoxin and 40S ribosomal protein also showed increased values when compared to the quiescent stage, where they were not detected (Fig. 4B). Finally, although the overall analysis did not indicate a change in endoplasmic reticulum (ER) proteins (Fig. 3), individual analysis of two ER-related proteins displayed a higher number of protein species identified in the 4-days activated stage (protein disulfide isomerase — PDI) or 7-days activated stage (78 kDa glucose regulated protein — GRP78) when compared to quiescent stage (Fig. 4C). Regarding secreted protein distribution, we detected the presence of most classes of venom toxins in the quiescent stage of the venom gland: C-type lectin proteins (CTL), serine proteinase (SVSP), cysteine-rich secretory protein (CRISP), metalloproteinase (SVMP), phospholipase A2 (PLA2), L-amino acid oxidase (LAAO) and disintegrin (Fig. 5). It is important to point out that during venom gland dissection, all precautions were taken for complete venom removal before gland proteomic processing. Moreover, if some residual venom was still present in the lumen, one would expect the number of protein species identified in the quiescent stage to be similar as in the two activated stages (4-days and 7-days activated) of the venom production cycle. The synthesis of SVMP, PLA2, LAAO, PLA2 inhibitor (PLI), snake venom metalloproteinase inhibitor (SVMPI) and disintegrin seemed to increase during the venom production cycle, since the number of specific protein species identified increased in the activated stage (4-days and/or 7-days) of the

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Fig. 5 – Distribution of secreted proteins identified in Bothrops jararaca snake venom gland during venom production cycle. C-type lectin-like protein (CTL), serine proteinase (SVSP), cysteine rich secretory protein (CRISP), metalloproteinase (SVMP), phospholipase A2 (PLA2), L-amino acid oxidase (LAAO), phospholipase A2 inhibitor (PLI), metalloproteinase inhibitor (SVMPI). Numbers above the bars indicate the number of protein species identified.

Fig. 4 – Number of protein species from cytoplasm (A, B) and endoplasmic reticulum (C) identified in Bothrops jararaca snake venom gland during venom production cycle. Actin, vimentin and tropomyosin, are cytoskeleton proteins; proteasome subunit alpha, thioredoxin and 40S ribosomal protein are cytoplasmic proteins involved in proteolysis, disulfide reductase activity and translation process, respectively. Protein disulfide isomerase (PDI) and glucose regulated protein 78 kDa (GRP78) are endoplasmic reticulum proteins involved in protein folding and post-translational modifications, respectively. Venom glands in quiescent stage, 4- and 7-days after venom extraction (activated) stages. venom gland (Fig. 5). On the other hand, the number of specific protein species identified as CTL and SVSP decreased during venom gland activation. Finally, there was no shift in CRISP abundance for quiescent and activated venom glands (Fig. 5). True CTLs and CTL-like toxins such as glycoprotein IB-binding proteins (GPIb-BP), coagulation factor IX/X and bothroinsularin were detected in the quiescent stage of the venom gland (Fig. 6A). The number of specific protein species

Fig. 6 – Protein family distribution across different classes of toxins in snake venom gland of Bothrops jararaca during venom production cycle. (A) Lectin family, (B) metalloproteinase family. GPIb-BP, glycoprotein IB-binding protein; SVMP, snake venom metalloproteinase.

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identified as true CTLs increased in the activated stages (Fig. 6A). Regarding the CTL-like toxin class, we detected the presence of GPIb-BP and coagulation factor IX/X only in the quiescent stage, and galactose-specific lectin was detected only in the activated stages. The number of specific protein species identified as bothroinsularin decreased in the 4-days activated venom gland but returned to high levels (33% more than in quiescent stage) in the 7-days activated venom gland (Fig. 6A). Snake venom metalloproteinases are classified as SVMP-PI, SVMP-PII and SVMP-PIII [24]; during the venom production cycle, the number of specific protein species of SVMP-I and SVMP-II increased. The increase in SVMP-PII was only detected in the 7-days activated venom gland (Fig. 6B). Noteworthy, the number of specific protein species identified as SVMP-III decreased in the 4-days activated venom gland but returned to high levels (25% more than in quiescent stage) in the 7-days activated venom gland (Fig. 6B). To corroborate the presence of specific proteins identified by proteomic approach in the venom gland during the venom production cycle, two proteins (actin and serine proteinase) were quantified by Western blotting. As shown in Fig. 7, the density of actin was significantly (p < 0.05) higher in the activated stages of venom gland than in the quiescent stage. On the other hand, the density of serine proteinase was significantly (p < 0.05) higher in the quiescent stage than in the activated stages of the venom gland (Fig. 8). These data were consistent with the findings from MS analysis.

3.3. In-depth analysis of spots identified as SVMPs and SVSPs The data presented in Fig. 2 (panels A–C) and Supplementary Tables 1–3 indicate a wide range of molecular masses and pI values for spots identified as SVMP (masses from 22 to 102 kDa/pI from 4.38 to 9.42) and SVSP (masses from 9 to 57 kDa/pI from 4.10 to 9.54). Using the software CAITITU version 2.0.1.4 [22], we have undertaken a detailed analysis of peptide sequence coverage (Fig. 9 and Supplementary

Figs. 1 to 10) as well as compared experimentally determined molecular masses and pI with calculated ones (Supplementary Tables 4A/B to 6A/B). Regarding SVMPs, one of the most striking features that could be observed was the overwhelming presence of peptides corresponding to the pro-domain region, for all classes (P-I, P-II and P-III) in almost all conditions (quiescent, 4- and 7-days activated gland). The presence of SVMP pro-domain peptides has already been described in the literature during Bothrops insularis snake venomics analysis [25] but this is the first time that it is described in the gland. As for SVSPs, only one spot presented a molecular mass smaller than calculated (Spot 72, Supplementary Tables 2 and 4B) being most likely a degradation product (Supplementary Fig. 7). A more detailed discussion on the processing of precursor molecules and/or the existence of post-translational modifications will be presented ahead.

4.

Discussion

In this study, we showed for the first time that most toxin classes are present in the secretory cells of venom gland during its quiescent stage and that GPIb-BP and coagulation factor IX/X were only detected in this stage. After venom extraction and protein synthesis activation, the detection of LAAO, PLI, SVMPI and disintegrin was higher in the 4- and 7-days activated glands when compared to the quiescent stage; the detection of SVMP was lower at 4-days but higher at 7-days whereas PLA2 was higher at 4-days but undetectable at 7-days stages. CRISP displayed no changes in abundance, while SVSP and CTLs were higher in the quiescent stage. Furthermore, some proteins from endoplasmic reticulum and cytoplasm showed increased synthesis during venom production cycle. The venom produced by the specialized gland of Viperidae snakes is stored in a basal–central lumen and when this venom is expelled, after biting or venom extraction, the epithelial cells increase in size to produce more venom [4–10]. The morphological changes are followed by an increase in total volume and membrane surface of many organelles

Fig. 7 – Corroboration of the upregulation of actin synthesis, in Bothrops jararaca snake venom gland, during venom production cycle by immunoblotting. Extracts of venom gland in quiescent stage, 4- and 7-days, after venom extraction, activated stages. (A) Immunostained western blotting showing differential synthesis of actin species in the different stages of the venom production cycle. (B) Densitometry of the band revealed by chemiluminescence. The values are expressed as mean ± S.E.M. of three independent experiments. (*) Indicates significant difference when compared to quiescent stage (ANOVA, p < 0.05).

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Fig. 8 – Corroboration of the downregulation of serine proteinase synthesis, in Bothrops jararaca snake venom gland, during venom production cycle by immunoblotting. Extracts of venom gland in quiescent stage, 4- and 7-days, after venom extraction, activated stages. (A) Immunostained western blotting showing differential synthesis of serine proteinase species in the different stages of the venom production cycle. (B) Densitometry of the band revealed by chemiluminescence. The values are expressed as mean ± S.E.M. of three independent experiments. (*) Indicates significant difference when compared to quiescent stage (ANOVA, p < 0.05).

inside the secretory cells [6]. Protein synthesis, degradation and secretion are important to ensure the maintenance of protein regulation. Therefore, the increase in the number of protein species such as actin, vimentin, tropomyosin, PDI,

GRP78, proteasome subunit alpha type-1, thioredoxin and 40S ribosomal protein in the activated stage of the venom gland suggests that these proteins could improve the synthesis and secretion processes in the secretory cells.

Fig. 9 – Graphical representation of the sequence coverage for class P-II snake venom metalloproteinases identified in the 7-days activated Bothrops jararaca venom gland. (A) gi 82219563; (B) gi 82197476. The different domains are represented: pre-domain or signal peptide (PRE), pro-domain (PRO), metalloproteinase and disintegrin domains. MS/MS sequencing data were combined to generate the figures using the software CAITITU version 2.0.1.4 [22].

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Among the cytoplasmic proteins, we identified proteins from the cytoskeleton such as actin, vimentin and tropomyosin. Actin is a major structural element of the cytoskeleton and is involved in changes in the cytoskeleton organization and regulation of saliva secretion [26–28]. The cytoskeletal tropomyosins are a family of actin-associated proteins that form head-to-tail polymers arranged in the major groove of polymerized actin filaments [29]. In resting parotid acinar cells, the cytoskeletal protein fodrin and actin appear as a continuous subplasmalemmal ring in most cells, which disappears almost completely upon stimulation with secretagogues. These cytoskeletal changes occur in parallel with amylase secretion [26]. Studies that investigated the role of cytoskeleton and motor proteins in the secretory process of salivary glands showed that amylase secretion depends on the integrity of the microfilaments, since treatment with the actin-stabilizing agent jasplakinolide and the actin-disrupting agent cytochalasin D decreased the amylase release induced by both β-adrenergic and muscarinic–cholinergic agonists [27–30]. Vimentin, a major intermediate filament protein, has a role as an organizer of proteins, both structural and signaling-associated (for review see [31]). In relation to endoplasmic reticulum proteins, PDI constitutes a family of structurally related enzymes that catalyze disulfide bond formation or reduction or isomerization of newly synthesized proteins in the lumen of the endoplasmic reticulum. PDI is rapidly induced under different endoplasmic reticulum stresses, showing its importance in protein folding quality control besides disulfide bonding [32,33]. GRP78 is found in a range of secretory cell types and may have a function in the post-translational modification, assembly or packaging of proteins prior to secretion, or some other part of the cellular secretory mechanism [34,35]. The detection of PDI was higher in the 4-days activated stage of the venom gland and these data are also in good agreement with recent data from the literature showing that venom transcriptome (identification and quantification of mRNA present in the venom secretion) is a representative venom gland transcriptome and that the expression of PDI increases in activated stages of the venom gland [36]. Ribosomes are among the most essential molecular machines in cells, where they are required for protein synthesis. In eukaryotes, small (40S) and large (60S) ribosomal subunits assemble on mRNA with the start codon and a methionyl initiator tRNA at the peptidyl (P) site during translation initiation [37]. Moreover, thioredoxin (TRX), a 12-kDa protein, is found mainly in the cytoplasm and occasionally in the nucleus. TRX is an ubiquitous antioxidant protein that is well known for its disulfide reductase activity, which is important for protein secondary structure. This protein also modulates additional redox-dependent post-translational modifications, including the transnitrosylation and denitrosylation of specific proteins. TRX is involved in many different aspects of cellular function, regulating transcription, translation, protein–protein interactions, and post-translational modifications of proteins (for review see [38]). Taking together the data in the literature about these proteins along with our results, we suggest that the increase

in the synthesis of cytoskeleton proteins, PDI, GRP78, ribosomal protein and TRX in activated stage ensures the synthesis of the new proteins or toxins, the correct folding, the reorganization of cytoskeleton and the correct structure and post-translational modifications of the newly synthesized proteins. In other words, these proteins may play an essential role in building the synthetic apparatus, consequently regulating venom production and secretion. On the other hand, the ubiquitin–proteasome system is a main non-lysosomal protein degradation pathway. This system is required for the specific and irreversible removal of critical regulatory proteins, as well as the general control of protein content and quality in the cell. The 26S proteasome is responsible for the degradation of ubiquitinated protein targets in all eukaryotic cells. The proteasome core particle comprises seven different alpha subunits and seven different beta subunits that are related to restrict the access to the internal cavity and proteolysis activity, respectively (for review see [39]). In activated venom gland we detected an increase in the alpha subunit species, which could be related to the control of internal cavity opening. However, it is important to stress that no evidence of ubiquitination could be found in our samples. Many studies have shown that after venom is expelled from the lumen of the gland, morphological and biochemical changes occur in the secretory epithelium, and maximal synthetic activity of the secretory cells and the highest mRNA concentrations are observed after 4 to 8 days [4–10]. The pattern of accumulation of toxins is different and is related to the different rate of synthesis [40]. Concerning the toxins identified, our data indicate that most venom toxins are present in the quiescent stage gland and that some of them are present only in this stage, such as GPIb-BP and coagulation factor IX/X. The presence of metalloproteinase in the quiescent stage was also verified in B. jararaca venom gland using immunoelectron microscopy [41]. Besides the differences between venom of Bitis arietans specimens from different origins, Currier at al [36] correlated the mRNA found in the venom with synthesis of toxin by venom gland. In this study they analyzed mature venom that corresponds with venom from quiescent gland, 0–1 d, 0–3 d, 0–7 d and verified the presence of mRNA in those venoms. They observed that the venom from individual specimens varied in terms of expression of toxins at different times after venom extraction corroborating with our data, showing an asynchronous synthesis of different toxins. Moreover, the 0–1 d venom could correspond to toxins already present in the quiescent gland that, after venom extraction, are released to the lumen. This hypothesis can be supported by morphological studies showing that in the quiescent stage, the Golgi apparatus is less developed than in the activated stage [42], thus the venom extraction can activate the secretion process by increasing the synthesis of proteins involved in this process and promote the release of the toxins already synthesized. GPIb-BPs and coagulation factor IX/X are only observed in the venom gland in quiescent stage. One day after venom extraction these proteins are not detected anymore (data not shown). The different spots identified as snake venom metalloproteinase (SVMP) or snake venom serine proteinase (SVSP) toxin species covered a wide range of molecular mass and pI values.

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This leads us to investigate the possibility of proteolytic processing and/or occurrence of post-translational modifications. For this reason, we have made a global analysis based on data presented in Figs. 2(panels A–C) and 9, Supplementary Figs. 1–10 and Supplementary Tables 4A/B to 6A/B. Part of the class P-I SVMPs identified in the present work (gi 82214994, 82214994 and 187608847) had full transcript sequences (pro-domain included) determined. To the exception of one spot, all experimental molecular masses ranged from 22 to 24 kDa, which could be perceived as compatible with mature P-I SVMPs. However, when we take into account peptide sequence distribution (Supplementary Figs. 1 and 8) and calculated molecular masses for pro-domain and metalloproteinase domains (Supplementary Tables 4A and 6A), it is clear that they are truncated/processed forms of P-I SVMPs, containing mostly the pro-domain region. The biological implication of this is still unclear. Moreover, spot 9 (Fig. 2C) has also been identified as gi 82214994 and could represent a dimeric P-I, due to its 70 kDa molecular mass. The other identified P-I SVMPs matched to gi 172044591, 172044592 and 187655925. The published molecular masses for these proteins are 24, 24 and 22.83 kDa, respectively [43,44]. Unfortunately, the deposited sequences for these gi are only partial and, although most of the masses (27–29 kDa) from 2DE are compatible with these values, we cannot rule out the presence of these molecules in their processed form (with pro-domain), as well. It is important to notice, that spot 43 (38 kDa — Fig. 2C) could be a full pro-domain + metalloproteinase representative of gi 172044591 while spot 5 (73 kDa — Fig. 2B) is compatible with a dimeric form of gi 172044592. Regarding class P-II SVMPs, they were only detected and identified in the gland's quiescent and 7-days activated stages. For the quiescent stage, one representative (Fig. 2A, spot 14) with an apparent molecular mass of 57 kDa was identified as gi 82197476, with coverage for the pro-, metallo- and disintegrin domains (Supplementary Fig. 2). The experimental mass was 8.6 kDa bigger than the calculated mass for these three domains, which raises the possibility for the presence of glycosylation or, at least, for an anomalous electrophoretic migration. For the 7-days activated gland, several spots (Fig. 2C, spots 12 to 21) matched P-II SVMPs. Spots 12 to 15 matched gi 82219563, also known as bothrostatin, while spots 16 to 21 matched gi 82197476, named insularinase A. For these spots, no peptide matching the disintegrin domains could be detected (Fig. 9), which is in accordance with the literature [45,46], that reports that the disintegrin domains are processed post-translationally. However, the experimental masses (60 kDa for gi 82219563 and 53–57 kDa for gi 82197476) are quite higher than the theoretical calculated masses of 43.1 kDa and 42.7 kDa, respectively, for the pro- and metalloproteinase domains together (Supplementary Table 6A). Again, the possibility of post-translational modification arises and could be investigated in the future. However, it should be stressed that, for insularinase A, the authors specifically reported the absence of glycosylation [46]. Finally, the pI variation (6.43 to 6.78) for spots assigned to gi 82219563 and gi 82197476 (6.07 to 6.49), can be explained by amino acid substitutions, which would not be detected by our methodology, since we could not achieve 100% sequence coverage. The interpretation of the results for class P-III SVMPs is less clear. Although the presence of peptides related to the pro-domain

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is ubiquitous (Supplementary Figs. 3, 6 and 9), it is better supported (higher sequence coverage) for the data related to the 7-days activated gland. This is also true for the metalloproteinase and cysteine-rich domains. One possible explanation is the higher intensity of the spots (higher protein abundance) for these protein species in that condition (Fig. 2C). For P-III SVMPs, the experimental molecular masses higher than expected can be attributed to glycosylations, as reported in the literature [47,48], although one 102 kDa spot (Fig. 2B, spot 2) could represent a dimeric P-III. Spot 53 (Fig. 2B and Supplementary Table 5A) and spots 47 and 53 (Fig. 2C and Supplementary Table 6) represent degraded products containing mainly pro-domain peptides (Supplementary Figs. 6 and 9). SVSPs were identified in all conditions analyzed, although they were more prominent in the gland's quiescent stage. When analyzing the peptide sequence coverage obtained (Supplementary Figs. 4, 7 and 10) along with the calculated and experimental molecular masses (Supplementary Tables 4B, 5B and 6B), one spot displayed a low molecular mass of 9 kDa (Fig. 2B, spot 72) and can represent a degraded form of the toxin (Supplementary Fig. 7). The other spots had masses ranging from 26 to 57 kDa and pI values from 4.10 to 9.54. Regarding the molecular mass, it is known from the literature that SVSPs can be found as several proteoforms [49] due to differential post-translational modification, i.e., glycosylation. On the other hand, the different pI values observed can be due to amino acid substitutions and/or glycan composition (ex. different number of sialic acids) [47,48,50]. Therefore, there are probably several post-translational modifications during the cycle and such modifications could have a functional significance that should be taken into consideration in toxin studies and requires an additional investigation. Since most molecular biology and transcriptomic studies used the 4-days activated venom glands, there is a strong possibility that considerable important information about toxin diversities was missed. Probably the transcription of toxins, present in the quiescent stage, is regulated to occur later in the venom production cycle. Considering that only the most abundant specific spots in each stage were identified, the data presented in this study clearly indicate that different toxin species are secreted independently from each other, suggesting that the synthesis and secretion of toxins are not synchronized, which corroborates previous data from the literature [11]. The asynchronous synthesis of toxins may be due to different intracellular signaling to induce gene expression of different toxins, modulation of mRNA translation by miRNA [51] or even a way to avoid the stress of the cells in producing efficiently all toxins at the same time. Another interesting finding of this study was the detection of snake venom metalloproteinase and phospholipase A2 inhibitors in activated venom glands. Several SVMPIs have been described in snake plasma, with one of them (BJ46a) shown to be synthesized in the liver [52]. These inhibitors are present in the plasma of animals, such as B. jararaca, naturally resistant to envenomation and are considered to be part of the natural immunity system of these organisms (for a review see [53,54]). One possible explanation for the presence of these

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inhibitors in the 4- and 7-days activated venom glands (Fig. 5) would be a cross-contamination by snake blood during venom gland dissection procedure. However, if that were the case, one would expect the detection of those inhibitors in the quiescent stage gland samples too, which did not happen. Another possibility would be the cross-contamination with blood during the venom extraction procedures that preceded the 4- and 7-days stage protein synthesis; we tried to prevent that possibility by using only glands that did not display any visible damage (swelling/hematoma). On the other hand, the venom accumulated inside the lumen is a protease mixture, and many factors that help protect the venom gland from the toxicity of the venom have been described, such as low pH, ionic strength and concentrations of small molecules such as citrate [55,56], the secretion of inactive enzyme zymogen precursors and the expression of endogenous peptides as specific inhibitors of venom enzymes [57,58]. One could speculate that the presence of these inhibitors in the B. jararaca venom gland contributes to maintaining metalloproteinase and myotoxic PLA2 in an inactive form inside the lumen, providing new mechanisms that control venom toxicity during its synthesis and storage. However, recent data (unpublished) from our research group have determined that the interaction between BJ46a (SVMPI) and jararhagin (P-III SVMP) has a dissociation constant (Kd) in the low nanomolar range. This kind of interaction is a very strong non-covalent one, that cannot be easily broken and venom efficacy would be jeopardized. From an evolutionary point of view, this situation should not be favored by natural selection. Our working hypothesis is that due to their natural immunity function, these inhibitors are proteins that are ubiquitously produced by all of the organism cells, including the specialized venom gland, although their levels of expression differ between different cell types. In conclusion, the data generated by this work extend our knowledge of the proteomic composition of the venom gland in different stages of the venom production cycle, leading to new insights into the mechanism of venom gland activation and venom production, thereby contributing to the studies of snake toxins and their diversity. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jprot.2013.10.026.

Acknowledgments This study was supported by grants 07/50083-9, São Paulo Research Foundation (FAPESP) to N.Y. and Rio de Janeiro Research Foundation (FAPERJ) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) to J.P. M.S.L. is supported by a CNPq graduate fellowship and is a graduate student in the Department of Cell and Developmental Biology at the University of São Paulo, Brazil. The authors would like to thank the Programa de Desenvolvimento Tecnológico em Insumos para a Saúde — PDTIS/FIOCRUZ for the use of its mass spectrometry platform and Dr. André Teixeira da Silva Ferreira, B.Sc. Monique Nunes da Costa and Ms. Andreía de Souza for their technical assistance. We are also grateful to Dr. A. Leyva for his help with English editing of the manuscript.

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