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Bothrops jararaca accessory venom gland is an ancillary source of toxins to the

snake

Article  in  Journal of Proteomics · January 2018

DOI: 10.1016/j.jprot.2017.12.009

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 Journal of Proteomics 177 (2018) 137–147

Contents lists available at ScienceDirect

Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot

Bothrops jararaca accessory venom gland is an ancillary source of toxins to T

the snake
Richard Hemmi Valenteb,f, Milene Schmidt Lunaa, Ursula Castro de Oliveirac,
Milton Yutaka Nishiyama-Juniorc, Inácio de Loiola Junqueira-de-Azevedoc,
José Antonio Portes-Juniord, Patricia Bianca Clissad, Luciana Godoy Vianaa, Leonardo Sanchese,
⁎
Ana Maria Moura-da-Silvad,e, Jonas Peralesb,f, Norma Yamanouyea,f,
a
Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil
b
Laboratório de Toxinologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil, 4365, 21040-900 Rio de Janeiro, Brazil
c
Laboratório Especial de Toxinologia Aplicada, CeTICS, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil
d
Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil
e
Laboratório de Ecologia e Evolução, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil
f
Instituto Nacional de Ciência e Tecnologia em Toxinas (INCTTox/CNPq), Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: In Viperidae snakes, it has been attributed to the main venom gland, a component of the venom gland apparatus,
Accessory gland the function of synthesizing all venom toxins and storing them inside a basal-central lumen. However, the role of
Proteomics the accessory gland is still unknown. Here, we analyzed the proteome and the transcriptome of the accessory
Transcriptomics gland during venom production and secretion cycle. We showed that the accessory gland expresses and syn-
Toxins
thesizes toxins that are similar to those produced by the main venom gland such as C-type lectin/C-type lectin-
Bothrops jararaca
Snake
like proteins, metalloproteinase, phospholipase A2, cysteine rich secretory protein, nerve growth factor, vascular
endothelial growth factor, serine proteinase, and L-amino acid oxidase. Our data have shown that toxin synthesis
in the accessory gland is asynchronous when compared to the same process in the venom gland. Moreover, this
gland also expresses inhibitors of venom phospholipases A2 and metalloproteinases. Transcriptome analysis
showed that the transcripts that correspond to toxins in the accessory gland have a good correlation to the main
venom gland transcripts. Therefore, it is proposed that the accessory gland is an ancillary source of toxins to the
snake, and provides inhibitors that could control venom toxicity (and integrity) during storage.
Significance: In this study, we propose that the accessory venom gland acts as an important ancillary source of
toxins to the snake, in lieu of a depleted main venom gland, and provides inhibiting agents that control venom
toxicity (and integrity) during its storage.

1. Introduction
Bothrops jararaca (Serpentes, Viperidae) is a Brazilian solenogly-
phous venomous snake. The venom glandular apparatus from this snake
is composed of the main venom gland, the primary duct, the accessory
gland, and the secondary duct that connects to the fang [1]. The main
venom gland (venom gland, for short) of Viperidae contains at least
four distinct cell types: secretory, mitochondria-rich, horizontal, and
dark [2]. All venom toxins are synthesized in this gland and stored
inside its basal-central lumen. Following a bite or manual venom ex-
traction, the amount of venom inside the lumen decreases, which sti-
mulates the noradrenergic innervation that triggers the venom
production cycle [3–7]. During this new cycle of venom synthesis, the
secretory epithelium undergoes morphological and biochemical
changes [2, 8–13].
On the other hand, the role of the accessory gland is still unclear.
Bothrops jararaca accessory gland consists of two parts. The anterior
region has a simple secretory epithelium with at least six cell types:
secretory (two kinds), mitochondria-rich without secretion, horizontal,
dark, and basal. The posterior region has a simple epithelium with two
cell types: flattened secretory (with an elongated nucleus) and hor-
izontal [14]. According to morphological studies, the accessory gland
displays a different cycle of synthesis and secretion which is not syn-
chronized with the secretory cycle of the venom gland. The accessory

⁎
Corresponding author at: Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil, 1500, 05503-900 São Paulo, Brazil.
E-mail address: norma.yamanouye@butantan.gov.br (N. Yamanouye).

https://doi.org/10.1016/j.jprot.2017.12.009
Received 30 October 2017; Received in revised form 27 November 2017; Accepted 17 December 2017
Available online 08 January 2018
1874-3919/ © 2018 Elsevier B.V. All rights reserved.
R.H. Valente et al.
gland synthesis and secretion cycle lasts around 15 days with the se-
cretion occurring after venom release or extraction. The exocytosis in
the accessory gland starts about 1 h after venom extraction; a massive
exocytosis occurs four days after venom extraction [14].
However, the relevance of the accessory gland secretion to the
toxicity of the venom is still controversial in the literature. Current data
suggest that this secretion could be not only a supplementary source of
toxins but also a site for activation of venom toxins, increasing their
biological activities [15, 16]. On the other hand, no differences were
found in the protein profiles from venoms collected from the intact
venom gland apparatus and from the main gland, when analyzed by
electrophoresis and reversed-phase HPLC [17]. The transcriptome of
the accessory gland of Ophiophagus hannah (Elapidae) identified mRNAs
coding for most of the toxin groups produced by the venom gland;
however, the expression of these toxins is low. Interestingly, the lectin
toxin family has a higher expression in the accessory gland than in the
venom gland, suggesting an evolutionary model in which transcription
of venom-like lectin paralogs has been repeatedly activated in the ac-
cessory gland and deactivated in the venom gland [18].
The aim of this study was to further characterize the proteome and
the transcriptome of the accessory venom gland from Bothrops jararaca
(Viperidae) at different stages of the secretion production cycle. We
showed that the accessory gland expressed and synthesized toxins that
are also found in the venom gland. Our results indicate that the toxins
from the accessory gland can be an ancillary source of toxins to the
snake, given that the release of the secretion from the accessory gland
starts 1 h after venom extraction [14], when the production of toxins in
the venom gland is still incipient. Furthermore, the accessory gland
secretes toxin inhibitors variants that could protect the venom gland
apparatus during venom storage, as well as maintain venom integrity.
2. Experimental procedures
2.1. Animals and venom glands
Bothrops jararaca female adults (n = 18) from São Paulo state
(Southeastern, Brazil), weighing 150–400 g, were classified by the
Special Laboratory of Zoological Collections at the Instituto Butantan,
and kept in a room under controlled conditions [19]. 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 same stage. Fasting periods of one to two
months are common in snakes living in the wild, and can exceed one
year [20]. Animal care and procedures used were in accordance with
guidelines of the Animal Ethics Committee of the Instituto Butantan
(374/07 and 1643020615), 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 accessory glands were removed from snakes
that had no venom previously removed (0-day group, n = 9) and snakes
that had their venom removed manually 4 and 7 days (4-days, n = 6
and 7-days groups, n = 3) before they were killed by decapitation.
Venom glands and liver were also collected from 0-day and 4-days
groups (n = 3 for each group). After accessory gland, venom gland and
liver removals, the tissues were washed with saline solution and freed
from connective tissue [21]. All tissues were frozen in liquid nitrogen
and kept at −80 °C until sample processing. To remove the venom from
the main venom gland lumen, snakes were anesthetized with sodium
pentobarbital (20 mg/kg, s.c.) and the venom was removed manually
[22].
2.2. Protein extraction
Frozen accessory glands (0-day, 4-days and 7-days groups, n = 3 for
each group) were pulverized in liquid nitrogen and homogenized in
138
Journal of Proteomics 177 (2018) 137–147
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 12,000g for 10 min at 4 °C. Protein concentration of
the supernatants was determined by the Bradford assay [23] using
bovine albumin as standard and the supernatants were kept at −80 °C.
2.3. 1D-PAGE
The extracts of the accessory glands (30 μg protein/sample) were
denatured in sample buffer containing 10% β-mercaptoethanol [24] for
5 min at 100 °C. Proteins were separated on 4–20% gradient SDS-PAGE
with the buffer system described by Laemmli [24]. After running, the
gels were fixed (5% acetic acid, 20% methanol) for at least 30 min and
stained with Coomassie Brilliant Blue G. The gels were scanned and the
bands were detected by using Quantity One software (Bio-Rad, Her-
cules, CA, USA). Technical triplicates of three different extracts per
group were analyzed. After that, the bands detected were cut, resulting
in 15 slices per lane (group).
2.4. In-gel trypsin digestion
We used the method described in [25], with modifications. After the
destaining, washing and drying steps, the gel pieces were incubated
with 100 μL of reducing solution (65 mM DTT, 100 mM NH4HCO3) for
30 min at 56 °C. The solution was then removed and gel pieces were
incubated with 200 μL of alkylating solution (200 mM iodoacetamide,
100 mM NH4HCO3) for 30 min. The alkylating solution was removed
and the gel pieces were dehydrated by the addition of 200 μL 100%
acetonitrile for 5 min. Solvent was removed and samples were com-
pletely dried using a SpeedVac (Thermo Fisher) vacuum centrifuge
concentrator for approximately 20 min. After that, gel rehydration in
trypsin solution and further processing followed the original protocol
[25].
2.5. Mass spectrometry analysis
In order to identify the generated peptides, samples were submitted
to reversed-phase nanochromatography separation (EASY-nLC II,
Thermo Fisher Scientific). Samples were initially loaded onto a trap
column [100 μm i.d., 2 cm, packed with Magic C18 AQ matrix
(Michrom Bioresources, USA)] at 2000 nL/min. Analytical chromato-
graphic separation was performed at 200 nL/min on a 10.5-cm long
(75 μm i.d.) column, packed with the same matrix described above.
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.
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 pep-
tides were directly introduced into a high resolution mass spectrometer
(LTQ/Orbitrap XL, Thermo Fisher Scientific) for analysis, using the
same instrument parameters previously described [25].
2.6. Data analysis
2.6.1. Protein database construction
We used a database resulting from a merge of UniProt (taxonomy
Serpentes) entries as well as transcriptomic data from venom gland and
other snake tissues, and from accessory gland (this work), as previously
described [26]. Additionally, this non-redundant protein database was
composed by the most complete annotation, performed by multiple
annotation methods. The proteins were aligned by BLASTp [27] against
the protein databases of UniProt/Swissprot and NCBI non-redundant
(NR) to assess the protein description with cut-off e-value of 1 × 10−5,
and according to the criterion of longer protein similarity. The analysis
of PFAM domains retained for the protein annotation was performed
with hmmsearch tool [28], against PFAM domains database (version
R.H. Valente et al.
29) [29]. The priority order of UniProt/Swissprot, PFAM database, and
NR-NCBI was used for annotation. The database is available upon re-
quest.
2.6.2. Search engine parameters
All MS/MS data were analyzed using PEAKS Studio 8.0 build
20160908 (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 0.60 Da and a
parent ion tolerance of 10 ppm. Carbamidomethylation of cysteine was
set as fixed modifications. Variable modifications included: (1) oxida-
tion of methionine; (2) carbamidomethylation of aspartic acid, histi-
dine, lysine, glutamic acid, and N-terminus; (3) carbamylation; (4)
deamidation of asparagine and glutamine; (5) pyroglutamic acid from
glutamine or glutamic acid. PEAKS Database analysis was performed
using these same parameters plus the possibility of up to two missed
enzyme cleavages. False discovery rate (FDR) was estimated through
the use of decoy sequences. Finally, data from all searches were con-
solidated and only those results with calculated FDR values ≤ 1%, and
at least two unique peptides, were considered as reliable identifications.
Quantitative values for identified proteins were expressed as unique
peptide spectral count, corresponding to the number of unique spectra
attributed to a single protein. Peptide sequences inferred from MS/MS
data from bands identified as snake venom metalloproteinases were
localized in the primary structure of these toxins and the graphic re-
presentation was generated by CAITITU software version 2.0.1.4 [30].
2.7. Total RNA isolation and cDNA preparation
Total RNA from the venom gland, accessory gland, and liver of three
animals from 0-day and 4-days groups (n = 3 each group) were ob-
tained with RNeasy® Mini Kit (Qiagen). The quantification was done
with NanoDrop 2000c (Thermo scientific, Wilmington, USA) and total
RNA concentration was presented as mean ± S.E.M. One microgram of
total RNA was reverse transcribed using SuperScript™ III reverse tran-
scriptase according to the manufacturer's protocol (Invitrogen,
Carlsbad, USA).
2.8. Transcriptomic analysis (RNAseq)
Total RNA from the venom glands and from the accessory glands of
three animals from 0-day group and of three animals from 4-days group
were quantified by Quant-iT™ RiboGreen® RNA reagent and Kit (Life
Technologies). cDNA library were generated following the standard
TruSeq RNA Sample Prep Kit protocol (Illumina, San Diego, CA).
Briefly, the cDNA was synthesized from fragmented mRNA using
random hexamers primers, followed by a ligation with appropriate se-
quencing adaptors. The size distribution of the cDNA libraries was
measured by 2100 Bioanalyzer with DNA1000 assay (Agilent
Technologies) and an ABI StepOnePlus Real-Time PCR System were
used in quantification of the sample library before sequencing. The
cDNA library was sequenced on the Illumina HiSeq 1500 System, into a
Rapid paired end flowcell in a 300 cycles of 2 ∗ 151 bp paired end
technique, according to the standard manufacturer's protocol
(Illumina). All sequences were processed for removing adaptors and
low quality ends. The paired-end reads from the accessory glands of the
six specimens were first merged to generate longer continuous reads
that were further assembled with seqMan NGen software (DNAstar),
using De Novo assembly module for transcriptomic data, with standard
parameter except for “Match Window Length: 40; Min Match Percent:
98; Mismatch Penalty: 25; Match Repeat Percent: 400”. Sequencing
reads from accessory glands and from venom glands samples in the 0-
day and 4-days groups (n = 3, per each group) were mapped to as-
sembled accessory gland reference transcriptome using CLC Genomics
suite (Qiagen), RNAseq module, considering mapping up to 0.8% of the
read with 97% identity. The RPKM (Reads Per Kilobase of exon model
139
Journal of Proteomics 177 (2018) 137–147
per million mapped reads [31] was calculated in order to allow a re-
lative comparison of the level of expression of accessory gland tran-
scripts in each sample and among samples. The reads were also mapped
under the same conditions to a venom gland referential transcriptome
of B. jararaca [32] in order to evaluate the level of expression of venom
gland transcripts. The raw sequence data is available on GenBank SRA
(Sequence Read Archive) database under accession numbers
(SRR5328950 to SRR532896) and the assembled accessory venom
gland transcriptome is deposited in GenBank TSA (Transcriptome
Shotgun Assembly) under accession number GFJM00000000, linked to
Bioproject PRJNA262899.
2.9. Immunohistochemistry analysis
To detect the presence of toxins in the accessory glands we used
snakes from 0-day group (n = 3), since we verified previously that
under this condition the cytoplasm of secretory cells are completely
filled with secretory vesicles and the tubular lumina are narrow and
empty [14]. The accessory gland was removed as described above and
the tissue included in optimal cutting temperature. Sections of 7 μm
were obtained using cryostat, mounted onto slides, and processed for
immunohistochemistry. The slices were fixed in 3.7% formaldehyde for
15 min at room temperature, then incubated with methanol for 5 min at
room temperature, washed with PBS containing 0.1% Tween-20, and
incubated with blocking solution (1% Triton X-100, 5% normal goat
serum, 0.5% BSA, 0.5%, glycine, 0.5% fish skin gelatin) for 1 h at room
temperature. The slices were then incubated with mouse anti-B. jar-
araca venom serum (1:150) for 2 h at room temperature. The slices
were submitted to a new wash in PBS containing 0.1% Tween-20 fol-
lowed by incubation with anti-mouse IgG conjugated with Alexa 488
(succinimidyl-ester – Molecular Probes, USA) (1:500) and nucleus la-
belling with DAPI (4′,6-amino-2phenylindole, dihydrochloride – Mo-
lecular Probes, 1:500). Histochemical sections were analyzed by con-
focal microscope (LSM-780-NLO, Zeiss, Germany). For negative control,
the slices were incubated with normal mouse serum and anti-mouse IgG
conjugate with Alexa 488 at the same dilutions.
2.10. Quantitative real time polymerase chain reaction (qPCR)
cDNA from the venom gland, accessory gland, and liver from 0-day
group (n = 3) and from 4-days group (n = 3) were used to assess re-
lative gene expression of the metalloproteinase inhibitor BJ46a and the
α, γ, and venom gland phospholipase inhibitors (α-PLI, γ-PLI, and VG-
PLI). The primers (Invitrogen) were designed with IDT integrated DNA
technology program (www.idtdna.com/primerquest/home/index) and
spanned exon–exon boundaries after checking the gene sequence of
BJ46a, α-PLI, and γ-PLI from Bothrops jararaca DNA genome. The oli-
gonucleotide properties were identified with IDT oligo analysis tools
(www.idtdna.com/calc/analyzer). 60S ribosomal L32 was used as en-
dogenous control for normalization. The sequences and product size for
each gene are listed in Supplementary Table 5. For quantitative PCR we
used qPCR – Sybr Green Plus (LCG Biotechnology). All primers designed
have approximately the same amplification efficiency (around 1).
Controls without cDNA were included in each assay. An aliquot of 1 μL
of the cDNA was used in the PCR, in a final volume of 25 μL. Samples
were run in triplicate in Real Time PCR system BioEr – LineGene K
(Hangzhou Bo Science and Technology, China). The thermal cycling
conditions for RT-qPCR were: activation of enzyme for 3 min at 95 °C,
40 cycles at 95 °C for 5 s, 58 °C for 20 s, and 72 °C for 20 s. Cycle
threshold (CT) was determined by LineGene K software, version 4.2.20.
After RT-qPCR amplification the relative gene expression levels were
calculated by comparative ΔΔCT method. Means of CT from 0-day group
of each tissue analyzed independently was used as a calibrator of ex-
periment for the respective tissue at 4-days group. Data were expressed
as mean ± S.E.M. of 2 ΔΔCT from 3 snakes.
R.H. Valente et al.
3. Results
3.1. Protein identification in accessory gland during secretion production
cycle
The production and secretion cycle of the accessory gland lasts
around 15 days [14]. Based on the morphology study, we decided to use
accessory glands from snakes that had no venom previously removed
(0-day - secretory cells full of secretory vesicles) and from snakes that
had their venom removed manually 4 and 7 days before collections (4-
days and 7-days, respectively). Four days after venom extraction, there
is a massive apocrine type secretion into the tubular lumina and 7 days
after venom extraction the secretory cells present some secretory ve-
sicles. Fifteen days after venom extraction, the morphology of the cells
is quite similar to 0-day [14]. Electrophoretic protein profile of the
accessory gland of Bothrops jararaca snake during the secretion pro-
duction cycle (0-day, 4-days, and 7-days) was analyzed. For each ex-
perimental group, fifteen gel slices were excised from the gels, sub-
mitted to in-gel digestion with trypsin, and analyzed by nanoESI-LTQ/
Orbitrap MS (Supplementary Fig. 1). Information on the proteins
identified is summarized in Supplementary Tables 1A/B to 3A/B ac-
cording to information collected on UniProt (www.uniprot.org).
The proteins identified in each experimental group (0-day, 4-days,
and 7-days) were distributed according to subcellular localization into
proteins from cytoplasm, endoplasmic reticulum, exosome, Golgi ap-
paratus, hemoglobin complex, lysosome, membrane, mitochondria,
mitochondrial membrane, nucleus, peroxisome, and secreted proteins
(Fig. 1, Supplementary Tables 1A to 3A). The distribution of proteins
from each subcellular localization was based on the percentage of un-
ique peptide spectral count for proteins from each subcellular locali-
zation relatively to the total unique peptide spectral count for all pro-
teins identified in each group (0-day, 4-days, and 7-days). Analyzing the
proteins identified for each subcellular localization, the majority of
proteins detected was either cytoplasmatic or secreted (Fig. 1). During
the production and secretion cycle, the percentage of cytoplasmatic
proteins is higher in 0-day group and endoplasmic reticulum proteins
increase during the cycle. Secreted proteins concentration is higher in
4-days group (Fig. 1).
A more detailed analysis of the occurrence of secreted proteins
showed us the presence of venom toxins which are also found in the
venom gland (Fig. 2 and Supplementary Tables 1B to 3B); secreted
proteins usually found in salivary glands were also identified (Supple-
mentary Tables 1A to 3A).
Overall, the proteome analysis of accessory gland extracts revealed
31 protein families previously associated to B. jararaca venom com-
position [26], that is: α-2-antiplasmin, α-2-macroglobulin, collagen I,
collagen VI, cysteine-rich protein (CRISP), cobra venom factor-like
140
Journal of Proteomics 177 (2018) 137–147
protein (CVF-like), cystatin, deleted in malignant brain tumors 1 pro-
tein, dipeptidyl peptidase 4, fibronectin, glutaminyl-peptide cyclo-
transferase, IgM chain C region, L-amino acid oxidase (LAAO), me-
sencephalic astrocyte-derived neurotrophic factor, phosphodiesterase
(PDE) 1, phospholipase A2 (PLA2), phospholipase B, α-phospholipase
inhibitor (PLI), β-PLI, γ-PLI, pulmonary surfactant-associated protein A,
serum albumin, snake C-type lectin/C-type lectin-like (CTL/CTL-like),
class P-I snake venom metalloproteinase (SVMP), class P-II SVMP, class
P-III SVMP, snake venom metalloproteinase inhibitor (SVMPI), snake
venom nerve growth factor (svNGF), snake venom serine proteinase
(SVSP), snake venom vascular endothelial growth factor (svVEGF), and
transferrin (Fig. 2).
Regarding known venom toxins, the most abundant proteins were
represented by CTLs/CTL-like proteins and classes P-II and P-III SVMPs,
while those of intermediate abundance were PLA2 and CRISP. The other
identified toxin classes (SVSP, class P-I SVMP, and LAAO) were present
at lower amounts. To the exception of CRISPs, toxins´ abundance varied
during the production and secretion cycle (Fig. 2). CTLs/CTL-like were
detected at all periods analyzed, but the intragroup percentage of un-
ique peptide spectral count was higher for 0-day and 7-days groups
(6.9% and 12.3%, respectively) than in 4-days group (1.6%). Classes P-
II and P-III SVMPs were detected in all groups analyzed. P-II were more
abundant in 7-days group (11.2%) than in 4-days and 0-day group
(3.1% and 4.1%, respectively). P-III were more abundant in 0-day
group (18.6%) than in 4-days and 7-days (12.0% and 13.5%, respec-
tively). Class P-I SVMP was identified only in 0-day group (0.4%). PLA2
was mainly detected in 7-days group, while SVSP was detected only in
4-days group. LAAO was marginally detected in 0-day and 4-days
groups.
Since SVMP was one of the most prominent toxin family detected in
the accessory venom gland, we decided to undertake a detailed analysis
of the peptide sequence coverage of toxins identified as Bothrops jar-
araca SVMPs, using the software CAITITU version 2.0.1.4 [30]. The
analysis showed the presence of peptides corresponding to the pro-
domain regions for P-II and P-III classes for all SVMP representatives
(Fig. 3), except for the P-II SVMP represented by accession number
BJARALL23953|m.13239.
In addition, α, β, and γ phospholipase A2 inhibitors, as well as a
metalloproteinase inhibitor, were also detected (Fig. 2). The α-PLI was
most abundant (3.1%) in 7-days group and marginally detected (0.4%)
in 4-days; β-PLI was only detected (0.4%) in 7-days; γ-PLI cycled along
0-day to 4-days to 7-days groups (5.8%, 2.4%, and 4.6%, respectively).
The SVMPI BJ46a also displayed an abundance cycling behaviour from
0-day to 7-days (2.1%, 5.0%, and 1.7%).
Other identified secreted proteins, previously associated to B. jar-
araca venom composition [26], such collagens VI and I, serum albumin,
and transferrin were among the top 10 most abundant proteins.
Fig. 1. Distribution of secreted or endogenous proteins (according to
subcellular localization) identified in the accessory gland of Bothrops
jararaca snake, during the secretory cycle. Reported data are for
accessory glands from snakes that had no venom previously removed
(0-day) and from snakes that had their venom removed manually 4
and 7 days before (4- and 7-days, respectively). Classification of
protein species were obtained from UniProt database. The distribu-
tion of proteins was based on the percentage of unique peptide
spectral count for proteins from each subcellular localization rela-
tively to the total unique peptide spectral count for all proteins
identified in each group (0-day, 4-days, and 7-days). A detailed list
of protein species identification is given in Supplementary Tables 1A
to 3A.
R.H. Valente et al.
3.2. Transcriptomic profiles of venom and accessory glands
In order to check (i) if the venom present in the accessory gland of
B. jararaca resulted from a transcription in this tissue and (ii) if there
was a biased predominance of specific toxin classes, we carried out an
RNAseq analysis of mRNAs from both accessory and venom glands of
the same individuals within the 0-day and 4-days groups.
Initially, the extracted total RNA from each sample was quantified
revealing similar quantities of RNA per mg of tissue, in both tissues at
both times, with a discrete augment in accumulated RNA on the 4-days
group (Supplementary Table 4, Fig. 4A). This result indicated that both
tissues were transcriptionally active and that, at the resting state (0-
day), the RNA was actually present in the cells.
Each sample was then sequenced with deep coverage with Illumina
reads (2 million good quality reads per triplicate of each sample) and
the reads from each sample were initially mapped to a master-set of B.
jararaca venom gland transcripts [32] to identify expressed toxin
classes and to comparatively calculate the relative expression of refer-
ential toxin transcripts in both tissues. This analysis showed that toxin
coding transcripts were highly represented in the venom gland (~50%
of the transcriptome), not only in the 4-days group, as previously ob-
served in this and other related species [32–34], but also in the 0-day
group (Fig. 4B), meaning that the produced (or accumulated) RNA in
the resting state is equally composed of toxin transcripts when com-
pared to the active gland in the 4-days group. In the accessory glands,
transcripts corresponding to toxins were also well transcribed and re-
presented the most expressed kind of functional product in that tissue,
but they occupied a lower proportion of the transcriptome when com-
pared to the venom gland (Fig. 4B). In this case, the proportion of toxin-
coding transcripts in the 4-days group was higher (18.3%) than that of
resting state (9.5%), though there was a big variation among replicates.
141
Journal of Proteomics 177 (2018) 137–147
Fig. 2. Distribution of accessory venom secreted protein
families, previously associated to Bothrops jararaca venom
composition [26], during its secretory cycle. Reported data
are for accessory glands from snakes that had no venom
previously removed (0-day) and from snakes that had their
venom removed manually 4 and 7 days before (4- and 7-
days, respectively). The distribution of proteins was based
on the percentage of unique peptide spectral count for
proteins from each family relatively to the total unique
peptide spectral count for all proteins identified in each
group (0-day, 4-days, and 7-days). A detailed list of protein
species identification is given in Supplementary Tables 1B
to 3B. CRISP, cysteine-rich protein; CVF-like, cobra venom
factor-like protein; LAAO, L-amino acid oxidase; PDE,
phosphodiesterase; PLA2, phospholipase A2; PLI, phospho-
lipase inhibitor; CTL/CTL-like; C-type lectin/C-type lectin-
like; SVMP, snake venom metalloproteinase; SVMPI, snake
venom metalloproteinase inhibitor; svNGF, snake venom
nerve growth factor; SVSP, snake venom serine proteinase;
svVEGF, snake venom vascular endothelial growth factor.
The specific differential expression of each gene in both tissue types can
be observed in scattered plots (Fig. 4C and D), where it was possible to
observe a good general correlation on the expression levels for the
majority of the transcripts in both tissues in a time independent
manner. However, venom genes (red dots), despite being all expressed
in both tissues, showed a slightly higher expression in the venom glands
at both times (0-day and 4-days).
For a more detailed view of the accessory gland transcriptome, all
reads from this tissue were assembled together generating 45,000
contigs that were annotated by comparison to known toxin-related
proteins. Reads from accessory and venom gland tissues were then
mapped back to these accessory gland contigs and the count was nor-
malized according to the size of the contig and to the total reads
mapped in each case, proving a comparative quantification of contig
expression in the samples. It is possible to observe that toxin classes
were expressed at the same proportions in both tissues, being SVMPs
and CTLs (including CTL-like proteins) by far the most expressed classes
of toxins (Fig. 4E), as we verified in the proteome (Fig. 2). Considering
the expression of toxin transcripts in the accessory gland in the 0-day
and 4-days groups, a very similar profile could be observed, meaning
that, on average, no particular class of toxins was transcribed earlier or
later in this tissue (Fig. 4F). Although some individual transcripts
showed a differential expression at each time (data not show), it is
difficult to predict if this was a consequence of a differential regulation,
individual variability or due to the conserved sequence strings among
assembled contigs. Actually, we observed that the different individuals
analyzed in each condition showed the presence or absence of several
toxin transcripts, suggesting a high genetic variability on the population
studied.
R.H. Valente et al.
Fig. 3. Graphical representation of the sequence coverage for classes P-II and P-III SVMPs from Bothrops jararaca identified in the accessory gland in the 0-day, 4-days and 7-days groups.
MS/MS sequencing data were combined to generate the figures using the software CAITITU version 2.0.1.4 [30].
3.3. Detection of venom in the accessory gland
Proteome and transcriptome analyses detected many toxin families
in the accessory gland. This gland has two different regions with dif-
ferent morphologies [14]. To verify in which region the venom was
produced, we carried out an immunohistochemistry assay using mouse
anti-B. jararaca venom sera. In this assay we used an accessory gland
from a snake that had no venom recently extracted (0-day group) to
detect the toxins; these particular sample and conditions were chosen
since the amount of secretion inside the vesicles in the cytoplasm was
expected to be higher than in other conditions [14]. Mouse anti-B.
jararaca venom serum strongly stained the apical region of the epi-
thelium of the accessory gland at both the anterior (Fig. 5A) and pos-
terior (Fig. 5B) regions. Normal mouse serum stained the tissues very
weakly; this was considered the basal level of fluorescence (Fig. 5C and
D).
3.4. Expression of inhibitors in accessory and venom glands
In this work, we detected the presence of metalloproteinase and
phospholipase inhibitors in the accessory gland proteome; such protein
families were also previously detected in venom gland proteome [25].
However, in the transcriptome of these glands (accessory and main)
only the transcript coding for a protein related to γ-PLI was detected. In
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Journal of Proteomics 177 (2018) 137–147
order to verify whether the inhibitors identified in proteomic studies
were bona fide proteins expressed by the accessory gland (or eventual
residual blood contaminants), we performed an RT-qPCR aiming the
detection of the mRNA of one SVMPI (accession AF294836), one α-PLI
(accession EU421915), one γ-PLI (accession EU155170), and one γ-PLI
(accession GEXA01000036), reported to be found only in venom gland
[32], named PLI-VG. The mRNA for all inhibitors were detected in the
main venom gland and in the accessory gland. In the liver, only PLI-VG
was not detected, but the CT values for other inhibitors were lower than
for venom or accessory glands (Supplementary Table 6), showing that
the expression of these inhibitors is much higher in the liver than in the
glands. Comparing the expression of these inhibitors between 0-day and
4-days groups, across three snake tissues (liver, venom gland, and ac-
cessory gland), the fold-change value was higher than 2.0 for γ-PLI,
mainly in venom gland (30.9 ± 21.1), followed by accessory gland
(6.3 ± 4.0), and liver (3.3 ± 0.5); α-PLI had an increase in the ac-
cessory gland only (2.6 ± 2.4) (Table 1).
4. Discussion
This paper contributes to a better functional characterization of a
Viperidae snake accessory gland, a subject which is quite lacking in the
literature. We showed that both the anterior and the posterior regions
of B. jararaca accessory gland express and produce toxins, stored in the
R.H. Valente et al. Journal of Proteomics 177 (2018) 137–147

Fig. 4. Transcriptomic analysis of accessory and venom glands. (A) Total RNA obtained from each sample (n = 3); (B) relative proportion of venom gland transcripts annotated as “toxin”
among total transcriptome in each sample (n = 3); (C) scattered plot showing the average level of expression of all venom gland transcripts (blue spots) in each tissue at time 0-day,
stressing the transcripts coding for toxins (red spots); D) the same as (C) but for 4-days group; (E) relative expression level of accessory gland transcripts according to the toxin class in
each tissue and time; (F) proportion of each toxin class among toxin annotated transcripts from the accessory gland in groups 0-day and 4-days, with indication of summed RPKM and

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R.H. Valente et al. Journal of Proteomics 177 (2018) 137–147

Fig. 5. Detection of venom in the secretory cells of both anterior (A,

C) and posterior (B, D) regions of the accessory gland. Sectioned
(7 μm thickness) samples were stained with mouse anti-Bothrops
jararaca (Bj) venom serum (A, B) or with normal mouse serum (C, D)
as a negative control. The nuclear staining was performed with DAPI
(blue). Note that both anterior and posterior regions were strongly
stained with mouse anti-Bj venom serum at the apical region of the
epithelium (green). CT: connective tissue; L: lumen; S: secretory
epithelium.

Table 1 gland of Viperidae snakes. It has been attributed to this gland the
Relative expression of the SVMPI BJ46a, the γ-PLI from venom gland (PLI-VG), the γ-PLI, function of (i) being a venom reservoir; (ii) acting as a valve to keep the
and the α-PLI in liver, venom gland, and accessory gland of Bothrops jararaca snake, four venom from draining continuously into the mouth; (iii) being a source
days after venom extraction.
of toxic principles or (iv) being a source of diluents that facilitate
Tissue SVMPI PLI-VG γ-PLI α-PLI venom flow through the fang (Fontana, 1787, Mitchell, 1861; Wolter,
1924, Soubeiran, 1861, apud [35]). Gans and Kochva [35] observed
Liver 1.5 ± 0.3 – 3.3 ± 0.5 1.5 ± 1.2 different electrophoretic patterns between the venom from the venom
Venom gland 0.7 ± 0.5 1.5 ± 0.6 30.9 ± 21.1 1.3 ± 1.0
gland and the whole venom. However, using reversed-phase high-per-
Accessory gland 0.5 ± 0.2 1.9 ± 1.0 6.3 ± 4.0 2.6 ± 2.4
formance liquid chromatography, Mackessy and Baxter [17] did not
mRNA levels were measured by RT-qPCR, and changes in mRNA expression in 4-days report any differences in the protein profile between the venoms col-
group were expressed as fold change. cDNA from 0-day group obtained from the re- lected from the intact venom gland apparatus and from the main gland
spective tissues were used as a calibrator. 60S ribosomal L32 was used as endogenous only, of the diamondback Crotalus atrox. There is a hypothesis that the
control for normalization. Results were expressed as mean ± SEM of technical triplicate
secretion of the accessory gland from Agkistrodon piscivorus piscivorus
of 4 independent experiments.
can be a source of specific toxins different from the main venom gland
or even a possible site for venom toxins activation, increasing their
secretory vesicles inside the cytoplasm, that can be used as an ancillary
toxicity [15, 16].
source to compose to the animal's venom. Moreover, this gland can
As previously shown, the accessory gland has a long cycle of se-
produce variants of PLA2 and SVMP inhibitors, which might help pro-
cretion production that lasts up to 15 days [14]. In the present study,
tecting the secretory epithelium from the venom toxicity during its
we verified that the accessory gland could express and synthesize toxins
storage.
that are present in the venom gland during the full cycle. These data
We have previously shown that the epithelium of the anterior region
were confirmed by immunohistochemistry assay using mouse anti-B.
of the accessory gland has two different types of secretory cells with
jararaca venom serum (Fig. 5). Moreover, at least for B. jararaca, it has
microvilli in the apical region. One type is a goblet-like cell, with its
been recently shown that activation of SVMPs (involving prodomain
cytoplasm filled with low electron-density vesicles, that secretes mucus;
removal) occurs in the lumen of the venom gland [36, 37], although
the other type is a cell with smaller apical vesicles with moderate
unprocessed forms of SVMPs were still detected in pooled samples of
electron-density. The epithelium of the posterior region has flattened
manually extracted venom [26]. Our data indicate that there are un-
secretory cells with apical vesicles with moderate electron-density [14].
processed forms of classes P-II and P-III SVMPs in the accessory gland
Immunohistochemistry data showed that the secretory cells with ve-
during the whole cycle time studied (Fig. 3); however, it is impossible
sicles with moderate electron-density in both regions are responsible for
to state if most SVMP molecules are activated or in their zymogen
venom production; however, we cannot exclude the participation of
forms.
goblet-like cells in toxins production in the anterior region. The mucins
When comparing the proteomes of the accessory and the main
identified in this gland (Supplementary Tables 1A to 3A) probably are
venom glands, during the venom production cycle, a striking observa-
produced by goblet-like cells.
tion was that, in the accessory gland, one of the most abundant toxin
There are many controversies about the function of the accessory

144
R.H. Valente et al.
group was comprised by the SVMPs (classes P-II + P-III) during the
whole cycle period studied (0 to 7 days), but was higher in the 0-day
group. However, in the venom gland, SVMPs were more abundant in
the 7-days group [25]. If the comparison is made with venom collected
from B. jararaca from the Southeast region of Brazil, which is supposed
to have all toxins synthesized during the full cycle, the most abundant
toxin is also SVMP [26, 38–40]. Another important toxin, SVSP, was a
highly abundant toxin in 0-day and 4-days groups in the venom gland
[25]; in the accessory gland, however, SVSP was only detected (at low
levels) in 4-days group. CTL/CTL-like proteins were detected in high
abundance in 0-day and 7-days group. Nevertheless, the number of
specific protein species identified as CTL/CTL-like in the venom gland
was higher in 0-days group and decreased during venom gland acti-
vation [25]. Therefore, our data support that toxin synthesis in the
accessory gland occurs in an asynchronous manner when compared to
toxin synthesis in the venom gland. It is important to stress that a good
correlation was observed for the transcription patterns of toxins in 0-
day and 4-days groups, when the venom gland and the accessory gland
were compared (Fig. 4). Hence, regulatory factors related to the
translation process should be responsible for the different expression
patterns observed in the proteomic studies of the venom gland [25] and
the accessory gland (this work). This remains to be investigated.
The expression of toxins in the accessory gland of king cobra
Ophiophagus hannah, an Elapidae snake, seems to be different from
Viperidae snake. In that species, the pattern of toxin transcription for
the accessory gland was very different than that for the venom gland;
toxins were expressed at low levels in the accessory gland, except for
the lectin toxin family. However, no lectins were detected in the king
cobra venom [18]. The structure and morphology of the venom gland
apparatuses of Elapidae and Viperidae snakes are different [12];
therefore, it would not be surprising if the functions of the accessory
glands from members of these two snake families were different.
Knowing that in Viperidae the synthesis of different toxins does not
occur at the same time in the venom gland [25], but that the tran-
scription pattern was similar during the whole venom production cycle
(Fig. 4), one could explain the origin of the known discrepancy between
several snake venom transcriptomic and proteomic profiles [38].
Another interesting finding in the accessory venom gland proteome
was the presence of metalloproteinase and phospholipase A2 inhibitors.
The SVMPI BJ46a is a plasmatic 46 kDa glycoprotein purified from B.
jararaca, that interacts with classes P-I and P-III SVMPs through a tight-
binding non-covalent complex formation [41], with a preliminary KD
determination in the low nanomolar range (unpublished results). BJ46a
was also detected in B. jararaca's main venom gland proteome [25, 42]
as well as in the transcriptomes of the main venom and accessory glands
of Ophiophagus hannah (see Supplementary Table S2 from [18]). How-
ever, it seems unlikely that such a tight-binding inhibitor would act as a
regulator of stored venom's metalloproteinase activity, since this in-
hibition would hardly be reversed, even after dilution due to venom
inoculation into the prey. One possible explanation for this apparent
paradox is the occurrence of BJ46a-like proteoforms, presenting dif-
ferent affinities for SVMPs: low-affinity (reversible inhibition) in the
venom glands, aiming to venom integrity maintenance and high-affinity
(irreversible-like inhibition) in the plasma, involved with the snake's
innate immunity system [43, 44]. Regarding the PLA2 inhibitors, α-, β-,
and γ-types were detected in the accessory glands, similarly to the ac-
tivated stage of the main venom gland of Bothrops jararaca, where α-
and γ-PLIs were found [25, 42]. Transcriptomic studies showed that
these inhibitors are expressed in the venom gland of different species of
the Bothrops genus [34, 45, 46]. It is important to remember that
SVMPIs and PLIs are plasmatic proteins, considered to be members of
the snake's innate immune system [47, 48] and their presence in the
accessory venom gland is paradoxical, as discussed above. The possi-
bility that plasmatic inhibitors were detected in accessory glands by
contamination with snake blood during dissection procedure was ruled
out by the detection of transcripts of such inhibitors in the accessory
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Journal of Proteomics 177 (2018) 137–147
glands (Supplementary Table 6). RT-qPCR results showed that these
inhibitors are expressed in the venom and accessory glands (Table 1),
although at lower expression levels than in the liver, where these pro-
teins are mostly produced [49–52]. Therefore, the accessory gland se-
cretes toxin inhibitors variants which could participate in the protection
of the epithelium of the venom gland apparatus during venom storage,
in addition to an already hypothesized mechanism, regarding the po-
tential reduced enzymatic activity due to the low pH inside the lumen
[17].
Another interesting point was the identification of albumin and
mucin in the accessory gland. Albumin was not detected in the main
venom gland proteome [25], but the expression of albumin was shown
in the transcriptome of the venom gland and the accessory gland of
Ophiophagus hannah (Supplementary Table S2 from [18]). Albumin and
mucin are currently expressed and secreted by the salivary glands
[53–56]. The morphology of the accessory gland is very similar to that
of the salivary gland [57]. Furthermore, both secrete proteins and
mucus; the secretion is stored inside secretory vesicles in the cytoplasm.
Assuming that the snake venom gland developed from a salivary gland
[58], it is feasible that the accessory gland would share some char-
acteristics with a salivary gland, such as morphology and secretion of
some proteins. However, since it also secretes toxins, it is possible that
the accessory gland is an anterior phase of the evolutionary process that
led to the main venom gland. Analyzing the evolution of snake venom
toxins, Hargreaves et al. [59] concluded that venom could be simply
considered a modified form of saliva, and that snake venom toxins are
likely derived from pre-existing salivary proteins.
Other non-toxic secreted proteins identified were collagens I and VI,
and annexin A2. Collagen is also expressed in parotid gland [60].
Nevertheless, another possible explanation for the high abundance of
the collagen identified would be the presence of a dense connective
tissue that covers the whole accessory gland of venom gland apparatus
[14], which could have been a source of contamination during the
accessory gland processing. Annexin A2 is a 36-kDa protein produced
by endothelial cells, monocytes, macrophages, dendritic cells, tropho-
blast cells, epithelial cells, tumor cells, among others [61] and is known
to regulate hemostasis in vivo [62]. It has been detected in venom gland
proteomic studies [25, 42].
In a previous study, we showed that the accessory gland has a long
cycle of production and secretion, which is asynchronous to the main
venom gland secretory cycle. This accessory gland also showed a de-
layed exocytosis that starts 1 h after venom extraction, peaking at four
days, when the toxins are still being synthesized by the main venom
gland [14]. Now, we have shown that the accessory gland is an ancil-
lary source of toxins to the snake, in lieu of a depleted main venom
gland, and provides inhibiting agent(s) that could control venom toxi-
city (and integrity) during its storage.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jprot.2017.12.009.
Transparency document
The Transparency document associated with this article can be
found, in the online version.
Acknowledgements
This study was supported by the following Brazilian funding agen-
cies: (i) grants 2007/50083-9 (NY), 2013/07467-1 (ILMJA), 2014/
26058-8 (AMMS), and 2016/50127-5 (AMMS and ILMJA) from São
Paulo Research Foundation (FAPESP); (ii) grants AuxPE 1209/2011
and 1519/2011 (AMMS), AuxPE 1214/2011 (JP), AuxPE 1224/2011
(RHV) from Coordenação de Aperfeiçoamento de Pessoal de Nível
Superior (CAPES); (iii) grant 131820/2014-1 (AMMS) from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq). MSL
was a CNPq fellow as a graduate student in the Department of Cell and
R.H. Valente et al. Journal of Proteomics 177 (2018) 137–147

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