toxins

Article Venomic, Transcriptomic, and Bioactivity Analyses of Pamphobeteus verdolaga Venom Reveal Complex Disulfide-Rich Peptides That Modulate Calcium Channels

Sebastian Estrada-Gomez 1,2,* , Fernanda Caldas Cardoso 3 , Leidy Johana Vargas-Muñoz 4 , 4 5, Juan Carlos Quintana-Castillo , Claudia Marcela Arenas Gómez † , Sandy Steffany Pineda 6,7,8 and Monica Maria Saldarriaga-Cordoba 9

1 Programa de Ofidismo/Escorpionismo—Serpentario, Universidad de Antioquia UdeA, Carrera 53 No 61–30, Medellín, Antioquia, CO 050010, Colombia 2 Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia UdeA, Calle 70 No 52–21, Medellín, Antioquia, CO 050010, Colombia 3 Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St Lucia, QLD 4072, Australia 4 Facultad de Medicina, Universidad Cooperativa de Colombia, Calle 50 A No 41–20 Medellín, Antioquia, CO 050012, Colombia 5 Grupo de Génetica, Regeneración y Cáncer, Universidad de Antioquia UdeA, Carrera 53 No 61–30, Medellín, Antioquia, CO 050010, Colombia 6 Brain and Mind Centre, University of Sydney, Camperdown, NSW 2052, Australia 7 Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW 2010, Australia 8 St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW 2010, Australia 9 Centro de Investigación en Recursos Naturales y Sustentabilidad, Universidad Bernardo O’Higgins, Avenida Viel 1497, Santiago 7750000, Chile * Correspondence: [email protected]; Tel.: +57-(4)-219-2315; Fax: +57-(4)-263-1914 Current address: Marine Biological Laboratory, Eugene Bell Center for Regenerative Biology and Tissue † Engineering, Woods Hole, MA 02543, USA.



Received: 23 June 2019; Accepted: 14 August 2019; Published: 27 August 2019


Abstract: Pamphobeteus verdolaga is a recently described Theraphosidae spider from the Andean region of Colombia. Previous reports partially characterized its venom profile. In this study, we conducted a detailed analysis that includes reversed-phase high-performance liquid chromatography (rp-HPLC), calcium influx assays, tandem mass spectrometry analysis (tMS/MS), and venom-gland transcriptome. rp-HPLC fractions of P. verdolaga venom showed activity on CaV2.2, CaV3.2, and NaV1.7 ion channels. Active fractions contained several peptides with molecular masses ranging from 3399.4 to 3839.6 Da. The tMS/MS analysis of active fraction displaying the strongest activity to inhibit calcium channels showed sequence fragments similar to one of the translated transcripts detected in the venom-gland transcriptome. The putative peptide of this translated transcript corresponded to a toxin, here named ω-theraphositoxin-Pv3a, a potential ion channel modulator toxin that is, in addition, very similar to other theraphositoxins affecting calcium channels (i.e., ω-theraphotoxin-Asp1a). Additionally, using this holistic approach, we found that P. verdolaga venom is an important source of disulfide-rich proteins expressing at least eight superfamilies.

Keywords: theraphosidae; Pamphobeteus; peptides; disulfide-rich peptide (DRP); inhibitory cysteine knot (ICK); venomics; transcriptome; ion channels

Key Contribution: Pamphobeteus verdolaga is an important source of active disulfide rich peptides modulators of ion channels, which are commonly found in the Theraphosids. Our bioactivity analysis

Toxins 2019, 11, 496; doi:10.3390/toxins11090496 www.mdpi.com/journal/toxins Toxins 2019, 11, 496 2 of 21

revealed that P. verdolaga’s venom has a number of peptides that potentially modulate voltage-gated calcium channels.

1. Introduction Spider venoms are a complex mixture of neurotoxins, enzymes, proteins, antimicrobial, neurotoxic and cytolytic peptides, nucleotides, salts, amino acids, and neurotransmitters [1–6]. The production of this arsenal “manufactured” in the venom glands can be divided into three major groups: low (<1 kDa), medium (<10 kDa), and high (>10 kDa) molecular weight compounds. Medium molecular weight compounds correspond mainly to peptides that enhance neurotoxic activity. Their molecular masses range from 3 to 10 kDa, and their main targets are voltage-dependent sodium (NaV), potassium (KV), and calcium (CaV) channels as well as calcium and potassium ligand-dependent channels (pre- and post-synaptic) and cholinergic receptors [1–7]. Although spider venom seems to be an important source of toxins modulating CaV channels [8], there are no reports describing these types of toxins in P. verdolaga venom. Disulfide-rich peptides (DRPs) adopting the inhibitory cysteine knot fold comprise the most abundant components in spider venoms and are responsible for their toxic activities [9,10]. This inhibitory cysteine knot (ICK) scaffold has a key role in the structure/function and neurotoxicity, and it is defined as antiparallel β sheets stabilized by a cysteine knot formed by three disulfide bridges [9]. Additionally, the ICK motif can confer structural rigidity to maintain the active conformation for receptor binding and improved stability against thermal and enzymatic degradation [3]. Most ICK peptides described to date have been isolated mainly from the mygalomorphae suborder [9], specially from the Theraphosidae family, and in several genera including Acanthoscuria, Chilobrachys, Grammosotola, Cyriopagopus (Haplopelma), Davus, and Thrixopelma, with the majority of them having different pharmacological activities and molecular masses ranging from 3 to 5 kDa [11–16]. The main targets of these peptides include calcium-activated potassium (KCa) channels, voltage-gated calcium (CaV) channels, voltage-gated sodium (NaV) channels and voltage-gated potassium (KV) channels [3,4,8,15,16]. A previous report from P. verdolaga showed that this venom contains low and high molecular mass proteins (i.e., phospholipase A2, sphingomyelinase D, hyaluronidase, Kunitz-type serine protease inhibitors, and compounds similar to lycotoxins) [17]. Several ICK fragments that matched theraphotoxins and affected voltage-gated calcium channels (U2-theraphotoxin-Asp1a and ω-theraphotoxin-Hh1a) were also described in this venom [18]. However, their full-length sequences and bioactivities remain unknown. In this study, we further characterized P. verdolaga venom and showed that, by using a combined approach, we were able describe a number of disulfide-rich peptide superfamilies with strong activity to inhibit CaV channels.

2. Results

2.1. rp-HPLC Profile rp-HPLC fractionation of crude P. verdolaga venom yielded a total of 35 fractions divided into two main regions, eluting between 15%–20% and 35%–40% of acetonitrile (ACN), as shown in Figure1. To test the bioactivity of P. verdolaga venom, fractions from five different rp-HPLC runs were collected and analyzed using calcium and sodium channel assays. All runs showed the same chromatographic profile as observed in Figure1. In some cases, fractions with large elution volumes were separated into different RNA-free plastic vials. To perform the mass spectrometry (MS/MS) analysis of active fractions, an additional run was carried out to collect the fraction of interest. Toxins 2019, 11, 496 3 of 21 Toxins 2019, 11, x FOR PEER REVIEW 3 of 20

Figure 1. Pamphobeteus verdolaga venom reversed-phase high-performance liquid chromatography Figure 1. Pamphobeteus verdolaga venom reversed-phase high-performance liquid chromatography (rp-HPLC) chromatographic profile of 1 mg of crude venom using a C18 column (250 4.6 mm). (rp-HPLC) chromatographic profile of 1 mg of crude venom using a C18 column (250 × 4.6× mm). The The run was monitored at 215 nm using a flow rate of 1 mL/min and a gradient of 5% B for 5 min, run was monitored at 215 nm using a flow rate of 1 mL/min and a gradient of 5% B for 5 min, followed followed by a linear gradient of 5%–15% B for 10 min, 15%–45% B for 60 min, and 45%–70% B for by a linear gradient of 5%–15% B for 10 min, 15%–45% B for 60 min, and 45%–70% B for 12 min. 12 min. Fractions collected are numbered/labeled on the top of the peaks. Fractions collected are numbered/labeled on the top of the peaks. 2.2. Bioactivity of Pamphobeteus verdolaga Venom 2.2. Bioactivity of Pamphobeteus verdolaga Venom Spider venoms are known for their exquisite ability to modulate voltage-gated ion channels [10]. TheSpider bioactivities venoms of areP. known verdolaga for theirvenom exquisite fractions ability were to modulate evaluated voltage-gated in endogenously ion channels expressed [10]. voltage-gatedThe bioactivities sodium of P. andverdolaga calcium venom channels fractions in neuroblastoma were evaluated cell in lines endogenously or recombinantly expressed expressed voltage- in gated sodium and calcium channels in neuroblastoma cell lines or recombinantly expressed in HEK293T cells. Calcium influx assays revealed that the venom was able to inhibit hCaV2.2, hCaV3.2, HEK293T cells. Calcium influx assays revealed that the venom was able to inhibit hCaV2.2, hCaV3.2, and hNaV1.7 responses (Figure2). Venom fractions (according to Figure1) from five independent fractionationsand hNaV1.7 responses by rp-HPLC (Figure (venom 2). Venom fractionations fractions A(according to E) were to assayedFigure 1) for from bioactivity. five independent Venom fractionations by rp-HPLC (venom fractionations A to E) were assayed for bioactivity. Venom fractionations A and B were evaluated for their activity on hCaV2.2 (in duplicate), showing that fractionsfractionations 21b and A 22,and corresponding B were evaluated to elution for their times activity of 54 and on 55 hCa minV2.2 and (in 33% duplicate), and 34% B,showing respectively, that hadfractions thestrongest 21b and inhibitory22, corresponding activities (Figureto elution2A). times Venom of fractionations54 and 55 min C andand D 33% were and evaluated 34% B, respectively, had the strongest inhibitory activities (Figure 2A). Venom fractionations C and D were for their activity on hCaV3.2 (in duplicate), showing that fractions 21a and 22, corresponding to elutionevaluated times for of their 53 and activity 55 min andon 32%hCaV and3.2 34%(in B,duplicate), respectively, showing had the that strongest fractions inhibitory 21a activitiesand 22, corresponding to elution times of 53 and 55 min and 32% and 34% B, respectively, had the strongest (Figure2B). Venom fractionation E was evaluated for its activity on hNa V1.7, showing that fractions 3inhibitory and 22,corresponding activities (Figure to elution2B). Venom times fractionation of 14 and 55 minE was and evaluated 14% to 34% for B,its respectively,activity on hNa hadV1.7, the strongestshowing that inhibitory fractions activities 3 and (Figure22, corresponding2C). Fluorescent to elution traces times from theof 14 calcium and 55 influx min and assays 14% showed to 34% no B, increaserespectively, in intracellular had the strongest Ca2+ upon inhibitory the addition activities of the (Figure venom 2C). fractions, Fluorescent which traces suggests from the the observed calcium 2+ activitiesinflux assays were showed only inhibitory no increase (Figure in intracellular2D). Ca upon the addition of the venom fractions, which suggests the observed activities were only inhibitory (Figure 2D). Mass spectrometry (MS) analyses of fractions 21a-b, 22, and 25, using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), showed strong inhibition of hCaV2.2 and hCaV3.2 and approximately 50% inhibition of hNaV1.7. The MALDI trace also showed multiple masses ranging from 3399.4 to 3839.61 Da (Figure 2E,F). The peptide masses obtained from the MS analysis revealed a similar mass of 3810.6 Da in fraction number 25 (data not shown), which was approximately 1 Da different from the mass 3809.61 Da found in the CaV bioactive fractions. This suggests the native peptide displaying 3809.61 Da is the C-terminal amide form of the 3810.6 Da peptide. C-terminal amidation is a common post-translational modification found in spider ICK

Toxins 2019, 11, x FOR PEER REVIEW 4 of 20 peptides and usually confers stronger bioactivity compared to the respective C-terminal carboxy version [15,19]. Further, venom E fraction 3 did not show any masses above 1000 Da, which suggests the inhibition of Na2+ influx responses observed was induced by polyamines [20] or other small molecules present in larger quantities in venom E compared to venoms A to D. Also in venom E, the masses 3809.61 and 3839.61 Da were found at high intensities in fractions 21a and 21b, and with less Toxinsintensity2019 , in11, 496fractions 25 and 27, suggesting these newly found peptides had a weak effect in4 ofthe 21 hNaV1.7 activity tested in our bio-assays.

Figure 2. BioactivityBioactivity of of the venom of Pamphobeteus verdolaga. Venoms Venoms fractions fractions were were tested tested for modulation of of voltage-gated voltage-gated calcium calcium and and sodium sodium channels. channels. (A ()A Venoms) Venoms A and A and B were B were evaluated evaluated for

bioactivityfor bioactivity on hCa on hCaV2.2,V revealing2.2, revealing that thatthe fractions the fractions 21b 21band and22 had 22 hadthe strongest the strongest inhibitory inhibitory effect. eff ect.(B)

(VenomsB) Venoms B and B and C were C were evaluated evaluated for for bioactivity bioactivity on on hCa hCaV3.2,V3.2, revealing revealing that that the the fractions fractions 21a 21a and and 22 had the strongest inhibitory eeffect.ffect. ( C) Venom EE waswas evaluatedevaluated forfor bioactivitybioactivity onon hNahNaVV1.7, revealing the fractions 3 and 22 had the st strongestrongest inhibitory effects. effects. ( D) Fluorescent traces of the calcium influx influx assay showed responses over baseline for the entire ti timeme course of the bio-assay. Positive and negative controls areare representedrepresented in in blue blue and an redd red lines, lines, respectively, respectively, and specificand specific inhibition inhibition controls controls are in orange.are in orange.Calcium Calcium response response traces aretraces represented are represented in green in green for venom for venom A fraction A fraction 21b (21bA)21b (21bA) and and purple purple for

venomfor venom B fraction B fraction 22 (22B)22 (22B) in the in the hCa hCaV2.2V2.2 assay, assay, green green for for venom venom C fraction C fraction 22 (22C)22 (22C) and and purple purple for venomfor venom D fraction D fraction 21a (21aD)21a (21aD) in the in hCa theV 3.2hCa assay,V3.2 assay, and in and green in forgreen venom for Evenom fraction E fraction 22 in the 22 hNa inV the1.7 assay. (E,F) Mass spectrometry analysis using matrix-assisted laser desorption/ionization time-of-flight hNaV1.7 assay. (E,F) Mass spectrometry analysis using matrix-assisted laser desorption/ionization (MALDI-TOF) of the fractions 21a-b (E) and 22 (F) with strong inhibition of hCa 2.2 and hCa 3.2, and time-of-flight (MALDI-TOF) of the fractions 21a-b (E) and 22 (F) with strong inhibitionV of hCaVV2.2 and approximately 50% inhibition of hNa 1.7, respectively. hCaV3.2, and approximately 50% inhibitionV of hNaV1.7, respectively.

Mass spectrometry (MS) analyses of fractions 21a-b, 22, and 25, using matrix-assisted laser 2.3. Tandem Mass Spectrometry (MS)/MS Analysis of the Active Fraction desorption/ionization time-of-flight (MALDI-TOF), showed strong inhibition of hCaV2.2 and hCaV3.2 and approximatelyActive fractions 50% 21 inhibitionand 22 (according of hNaV 1.7.to Figure The MALDI 1) were trace collected also showedin a single multiple RNA-free masses plastic ranging vial andfrom subjected 3399.4 to to 3839.61 tandem Da mass (Figure spectrometry2E,F). The peptide (tMS/MS). masses The obtainedseparation from of venom the MS components analysis revealed using Tris-Tricinea similar mass gels of 3810.6showed Da that in fractionthe likely number active 25 fractions (data not of shown), P. verdolaga which venom was approximately corresponded 1 Dato componentsdifferent from with the mass a molecular 3809.61 Damass found ranging in the from CaV bioactive 6 to 21 kDa fractions. (Figure This 3B, suggests each red the box native highlights peptide bandsdisplaying a, b, 3809.61c, and d). Da Mass is the spectrometric C-terminal amide analysis form of of the the in-gel 3810.6 digestion Da peptide. of C-terminalprotein bands amidation revealed is thea common presence post-translational of 3 fragments that modification matched peptides found in with spider molecular ICK peptides masses and below usually 6 kDa. confers No fragments stronger withbioactivity molecular compared masses to si themilar respective to high C-terminalmolecular carboxymass compou versionnds [15 (HMMCs),,19]. Further, above venom 10 kDa, E fraction were 3 did not show any masses above 1000 Da, which suggests the inhibition of Na2+ influx responses observed was induced by polyamines [20] or other small molecules present in larger quantities in venom E compared to venoms A to D. Also in venom E, the masses 3809.61 and 3839.61 Da were found at high intensities in fractions 21a and 21b, and with less intensity in fractions 25 and 27, suggesting these newly found peptides had a weak effect in the hNaV1.7 activity tested in our bio-assays. Toxins 2019, 11, 496 5 of 21

2.3. Tandem Mass Spectrometry (MS)/MS Analysis of the Active Fraction Active fractions 21 and 22 (according to Figure1) were collected in a single RNA-free plastic vial and subjected to tandem mass spectrometry (tMS/MS). The separation of venom components using Tris-Tricine gels showed that the likely active fractions of P. verdolaga venom corresponded to components with a molecular mass ranging from 6 to 21 kDa (Figure3B, each red box highlights bands a, b, c, and d). Mass spectrometric analysis of the in-gel digestion of protein bands revealed the presenceToxins 2019 of, 11 3, x fragments FOR PEER REVIEW that matched peptides with molecular masses below 6 kDa. No fragments5 of 20 with molecular masses similar to high molecular mass compounds (HMMCs), above 10 kDa, were availably detected, although the Tris-Tricine gel allowed the detection of different bands availably detected, although the Tris-Tricine gel allowed the detection of different bands corresponding corresponding to these HMMCs. to these HMMCs.

FigureFigure 3. 3.( A(A)) The The single single peak peak from fromP. verdolagaP. verdolagavenom venom highlighted highlighted in red in boxes,red boxes, along along with with sodium sodium and calciumand calcium active active fractions. fractions. (B) Active (B) Active fractions fractions Tris-Tricine Tris-Tricine gels showing gels showing the overall the sizes overall of di sizesfferent of peptidesdifferent that peptides were subjected that were to automated subjected reduction to automated/alkylation withreduction/alkylation DTT and iodoacetamide. with DTT Peptides and wereiodoacetamide. digested with Peptides porcine were trypsin digested using with a ProGest porcine™ digestor.trypsin using a ProGest™ digestor.

tMStMS/MS/MS analysisanalysis ofof in-gelin-gel digestiondigestion ofof proteinprotein bandsbands separatedseparated byby Tris-TricineTris-Tricine allowedallowed thethe detectiondetection of of four four diff differenterent fragment fragment sequences sequences (see Table (see1 ).Table After 1). a local After protein a local Basic protein Local AlignmentBasic Local SearchAlignment Tool (BLASTP)Search Tool analysis (BLASTP) using analysis the translated using transcriptomicthe translated informationtranscriptomic of P. information verdolaga venom of P. glandverdolaga as the venom local database,gland as tMSthe /localMS sequences database, showedtMS/MS hits sequences exhibiting, showed in some hits cases, exhibiting, a resemblance in some identitycases, a ofresemblance 100% with identity translated of transcripts100% with (seetransl Tableated1 ).transcripts The transcriptomic (see Table analysis1). The transcriptomic allowed the detectionanalysis allowed of a sequence the detection that matched of a sequence 66% of the that MS matched/MS results 66% (see of the below). MS/MS results (see below).

TableTable 1. 1. AssignmentAssignment ofof thethe rp-HPLCrp-HPLC fractionsfractions fromfrom ofof P.P. verdolaga verdolagavenom, venom,with withprotein proteinfamilies families matchingmatching the the nESI-MS nESI-MS/MS/MS collision-induced collision-induced dissociation dissociation of peptide of pept ionside generated ions generated by in-gel digestionby in-gel and the respective homologous venom gland transcript. Transcript abundance estimation is supported digestion and the respective homologous venom gland transcript. Transcript abundance estimation by transcripts per million (TPM) values. Cysteine residues are carbamidomethylated. Apparent is supported by transcripts per million (TPM) values. Cysteine residues are carbamidomethylated. molecular masses (Mapp, in kDa) were estimated by Tris-Tricine gel of β-mercaptoethanol-reduced Apparent molecular masses (Mapp, in kDa) were estimated by Tris-Tricine gel of β-mercaptoethanol- (H) samples. reduced (▼) samples. rp-HPLC Fraction Best Transcriptomic rp-HPLC Fraction Mapp (kDa) m/z z Peptide Sequence Score Best Transcriptomic TPM Number/Tris-Tricine Band Mapp (kDa) m/z z Peptide Sequence Score Match TPM Number/Tris-Tricine Band Match 21-2221-22/a-b_1/a-b_1 6–106–10H▼ 444.36444.362 2 K.IKLCLKI.- K.IKLCLKI.- 40% 40% c7142_g1_i1c7142_g1_i1 27806.1927806.19 21-2221-22/a-b_2/a-b_2 773.98773.982 2 -.IFECVFSCDIEK.E-.IFECVFSCDIEK.E 100%100% c7142_g1_i1c7142_g1_i1 27806.1927806.19 21-22/a-b_3 618.64 4 -.IFECVFSCDIEKEGKPCKPK.G 100% c7142_g1_i1 27806.19 21-22/a-b_3 618.64 4 -.IFECVFSCDIEKEGKPCKPK.G 100% c7142_g1_i1 27806.19 21-22/c_1 10H 579.10 2 CVFSCDXEK 81.9% c7142_g1_i1 27806.19 21-22/c_1 10▼ 579.10 2 CVFSCDXEK 81.9% c7142_g1_i1 27806.19

2.4. Transcriptomic Results

2.4.1. General Transcriptomic Assembly Analysis A de novo reference transcriptome of P. verdolaga was generated from the RNAs isolated from the venom gland. A total 46,598,494 high-quality pair-end reads were assembled using Trinity v.2. A total of 78,088 contigs were obtained with a GC content of 39.19%, with an average and maximum contig length of 596 and 13,249 base pairs (bp), respectively. The length distribution ranged from 201 to 13,249 bp, 68% < 500 bp, 18% 500–1000 bp, and 14% > 1000 bp (Supplementary File S1). Based on read coverage, the E90N50 statics showed ~1300 bp (Supplementary File S1), and the reference

Toxins 2019, 11, 496 6 of 21

2.4. Transcriptomic Results

2.4.1. General Transcriptomic Assembly Analysis A de novo reference transcriptome of P. verdolaga was generated from the RNAs isolated from the venom gland. A total 46,598,494 high-quality pair-end reads were assembled using Trinity v.2. A total of 78,088 contigs were obtained with a GC content of 39.19%, with an average and maximum contig length of 596 and 13,249 base pairs (bp), respectively. The length distribution ranged from 201 Toxinsto 13,249 2019, bp,11, x 68%FOR PEER< 500 REVIEW bp, 18% 500–1000 bp, and 14% > 1000 bp (Supplementary File S1). Based6 of 20 on read coverage, the E90N50 statics showed ~1300 bp (Supplementary File S1), and the reference transcriptome contained 79.8% of the highly conserved sequences among arthropods (850 out of 1066) transcriptome contained 79.8% of the highly conserved sequences among arthropods (850 out of 1066) and 84.2% (255 out of 303) among eukaryotes. and 84.2% (255 out of 303) among eukaryotes.

2.4.2.2.4.2. Transcriptomic Transcriptomic Annotation Annotation TheThe reference reference transcriptome transcriptome of of the the venom venom gland gland from from P.P. verdolaga was annotated with with the the TrinotateTrinotate pipeline (https://github.com/Trinotate/Trinot (https://github.com/Trinotate/Trinotate).ate). Utilizing Utilizing this this pipeline, pipeline, a a total total of of 16,030 transcriptstranscripts were were annotated annotated (Supplementary (Supplementary file file 2), 2), and, and, subsequently, subsequently, the the list list of of nonredundant nonredundant genes genes ((n == 71377137 genes)genes) was was clustered clustered in genein gene ontology ontology (GO) (GO) categories categories (Figure (Figure4). The 4). main The biological main biological process processwas cellular was componentcellular component organization, organization, or biogenesis or bi (GO:0071840),ogenesis (GO:0071840), the most representativethe most representative molecular molecularprocess was process translation was regulatortranslation activity regulator (GO:0045182), activity (GO:0045182), and the principal and cellular the principal component cellular was componentsynapse (GO:0045202). was synapse (GO:0045202).

Figure 4. Gene ontology (GO) categories for annotated Pamphobeteus verdolaga transcripts.

The graph shows the Level 2 categories detected for biological processes, molecular function, and Figurecellular 4. components. Gene ontology (GO) categories for annotated Pamphobeteus verdolaga transcripts. The graph shows the Level 2 categories detected for biological processes, molecular function, and cellular components.After acquiring GO annotation for all transcripts, we used a three-step approach to extract the maximum number of sequences containing hits to toxin/toxin-like transcripts and venom protein transcripts.After acquiring Briefly, we GO used, annotation as the first for step,all transcripts, a search that we utilizedused a three-step Basic Local approach Alignment to Searchextract Toolthe maximumsearching translatednumber of nucleotide sequences databases containing using hits a to translated toxin/toxin-like nucleotide transcripts and protein and queryvenom (tBLASTX protein transcripts.and tBLASTN). Briefly, Using we used, this strategy,as the first a step, total a of se 128arch sequences that utilized were Basic identified. Local Alignment Similarly, Search using Tool the searchingTox|Note pipeline,translated a totalnucleotide of 119 databases peptides were using identified. a translated Furthermore, nucleotide Toxand| Noteprotein provided query (tBLASTX important andinformation tBLASTN). about Using the processingthis strategy, predictions a total of for 128 peptides. sequences Finally were (and identified. additionally), Similarly, using using a manual the Tox|Notedirected search, pipeline, a total a oftotal 39 sequencesof 119 peptides were identified. were identified. The majority Furthermore, of these sequences Tox|Note corresponded provided importantmainly to highinformation molecular about mass the compounds processing (HMMCs) predictions and included:for peptides. phospholipases, Finally (and hyaluronidases, additionally), usingKunitz-type a manual peptides, directed EF-hand search, proteins. a total and of cysteine-rich39 sequences secretory were identified. proteins (CRISP; The majority data not of shown). these sequences corresponded mainly to high molecular mass compounds (HMMCs) and included: phospholipases, hyaluronidases, Kunitz-type peptides, EF-hand proteins. and cysteine-rich secretory proteins (CRISP; data not shown). The Venn diagram shows the coincidence detected in each analysis with a final count of 265 different hits (see Figure 5). From the 265 contigs identified using tBLASTX, tBLASTN, and SpiderPro|HMM, 21% corresponded to hits with known neurotoxins affecting different ion channels (NTAIC); 11% corresponded to cysteine-rich proteins (CRPs) like theraphotoxins, hainantoxins, huwetoxins (different to CRISP), or colipase-like proteins; 22% showed different biological activities (i.e., hyaluronidases, calcium-binding (EF-hand), and serine protease inhibitor); 40% were toxins with unknown activity; and 5% did not show a match (Figure 3). Eighty-three percent of these contigs are similar to spider proteins, and 78% of transcripts showed similitude with spider proteins corresponding to proteins reported in the Theraphosidae family (Figure 5).

Toxins 2019, 11, 496 7 of 21

The Venn diagram shows the coincidence detected in each analysis with a final count of 265 different

Toxinshits (see2019, Figure 11, x FOR5). PEER REVIEW 7 of 20

FigureFigure 5. 5. StrategiesStrategies used used for for toxin toxin identifica identificationtion based based on on transcriptomic transcriptomic data. data. (A (A) )Figure Figure shows shows the the relativerelative proportion proportion of of the the transcripts transcripts identified identified in in the the venom venom gland gland of of P.P. verdolaga verdolaga followingfollowing a a BLAST BLAST searchsearch using using tBLASTN, tBLASTN, tBLASTX, tBLASTX, and and Tox|Note. Tox|Note. Dash Dasheded boxes boxes show show the the relative relative proportion proportion of of the the transcriptstranscripts identified identified by manual analysis.analysis. ((BB)) VennVenn diagram diagram showing showing the the degree degree of of overlap overlap between between all allstrategies strategies used. used.

TheFrom translated the 265 contigs and identified identified transcripts using tBLASTX, also show tBLASTN,ed high and similarity SpiderPro with|HMM, toxins 21% that corresponded have been previouslyto hits with reported known in neurotoxins the Theraphosidae affecting family. different Most ion ofchannels the transcripts (NTAIC); displayed 11% corresponded similarity with to toxinscysteine-rich isolated proteins from Chilobrachys (CRPs) like theraphotoxins,guangxiensis, Cyriopagopus hainantoxins, schmidti huwetoxins (H. schmidti (different), Cyriopagopus to CRISP), or hainanuscolipase-like (H. hainanum proteins;), 22% G. rosea, showed and diPelinobiusfferent biological muticus (Figure activities 6A). (i.e., Seventy-one hyaluronidases, translated calcium-binding transcripts showed(EF-hand), homology and serine with protease different inhibitor); theraphotoxins 40% were toxinsknown with to unknownhave insecticidal activity; and activity 5% did and not block/modulateshow a match (Figurepotassium3). Eighty-threechannels. Seventy-three percent of these percent contigs of the are theraphotoxins similar to spider had proteins,an unknown and target78% of or transcripts function (Figure showed 6B). similitude with spider proteins corresponding to proteins reported in the Theraphosidae family (Figure5). The translated and identified transcripts also showed high similarity with toxins that have been previously reported in the Theraphosidae family. Most of the transcripts displayed similarity with toxins isolated from Chilobrachys guangxiensis, Cyriopagopus schmidti (H. schmidti), Cyriopagopus hainanus

Toxins 2019, 11, 496 8 of 21

(H. hainanum), G. rosea, and Pelinobius muticus (Figure6A). Seventy-one translated transcripts showed homology with different theraphotoxins known to have insecticidal activity and block/modulate potassium channels. Seventy-three percent of the theraphotoxins had an unknown target or function

Toxins(Figure 20196B)., 11, x FOR PEER REVIEW 8 of 20

FigureFigure 6. 6. SimilaritySimilarity of of P.P. verdolaga verdolaga transcriptstranscripts identified identified using using tBLAST tBLAST or or Tox|Note Tox|Note with: with: ( (AA)) other other spidersspiders venom venom contents contents and and ( (BB)) theraphotoxins. theraphotoxins.

Twenty-sixTwenty-six translated translated transcripts transcripts were were classi classifiedfied as as CRP CRP molecules molecules and and were were divided divided into into 8 8 superfamiliessuperfamilies according according to to their their cysteine cysteine pattern, pattern, as as summarized summarized in in Table Table 22.. Length and number of cysteines differ between Superfamilies, and only two showed the common inhibitory cystine knot (ICK) motif. A detailed description of each superfamily is described in the following section.

Toxins 2019, 11, 496 9 of 21 cysteines differ between Superfamilies, and only two showed the common inhibitory cystine knot (ICK) motif. A detailed description of each superfamily is described in the following section.

Table 2. P. verdolaga cysteine-rich proteins grouped by cysteine pattern.

Superfamily Cysteine Pattern Number of Cysteines Mature Peptide Length SF1 C-C-CC-C 5 54 SF2 C-C-C-CC-C 6 48 SF3 C-C-CC-C-C-C 7 43–84 SF4 C-C-CC-C-C-C-C 8 44–74 SF5 C-C-C-CC-C-C-C 8 61–65 SF6 C-C-C-C-CC-C-C 8 43–71 SF7 C-C-C-C-C-CC-C 8 76–80 SF8 C-C-CC-C-C-C-C-C-C 10 67–88

Superfamily 1 This group included only one sequence characterized by the presence of 5 cysteine residues and a length of 54 amino acids. The predicted disulfide pattern suggested that cysteine bond connectivities were between CysII–CysIII and CysIV–CysV, with cysteine I not forming any bridge. This protein is similar to the U35-theraphotoxin-Cg1a (B1P1J4) from C. guangxiensis with an e-value of 7 10 24. × − The activity of this peptide is still unknown.

Superfamily 2 Group 2 included one sequence with an amino acid length of 48 residues and 6 cysteines. The disulfide bond pattern was predicted between amino acids CysI–CysIV, CysII–CysV, and CysIII–CysVI. This peptide is similar to a hypothetical protein reported on the Theraphosidae spider Parasteatoda tepidariorum with an e-value of 1 10 13 and with an unknown biological activity. × − Superfamily 3 Two sequences were included in this group characterized by 7 cysteine residues and three disulfide bridges formed between CysII–CysV, CysIII–CysVI, and CysIV-CysVII, with cysteine I not forming a bridge. SF3 members had between 43 and 84 residues. Both peptides from this group did not show similarity with any known protein.

Superfamily 4 Four sequences were identified in this group displaying a length between 44 and 74 residues. This family was characterized by the presence of 4 disulfide bridges between either cysteines CysI–CysVIII, CysII–CysIII, CysIV–CysV, and CysVI-CysVII or CysI–CysVII, CysII–CysVIII, CysIII–CysVI, and CysIV–CysV. Members of this knottin group, for example, U54-theraphositoxin-Pv1a_1, a putative toxin precursor that has a similar cysteine pattern (“X20CX6CX5CCX11CX14CX2CX6CX2”; X is any amino acid), showed similarity with the cystine knot toxin and domains of G. rosea GTx6-1 (M5AXR5) and to cysteine knot toxins of the tarantula C. jinzhao (“guangxiensis”), assigned by Chen et al. (2008) in orphan family 2 (JZTX-72; B1P1J5) (see Figure7)[12]. This cluster of toxins was named group 4a. In addition, putative toxin precursors U27-theraphositoxin-Pv1a_1 and U82-theraphositoxin-Pv1a_1 had similar cysteine patterns (“X1CX6CX6CCX4CX1CX6CX1CXn”; X is any amino acid, and n is an uncertain number) to the spider toxin U8-agatoxin-Ao1a from Stegodyphus mimosarum (A0A087TBW8) and Parasteatoda tepidariorum (A0A2L2YIL4, house spider). Furthermore, they showed similarity with the U8-agatoxin-Ao1a from Daphnia magna (A0A0P6I6W0) and putative U8-agatoxin-ao1a-like isoform x2 from Ixodes ricinus (A0A147BFN0, common tick) (see Figure8). This cluster of toxins was named group 4b. ToxinsToxins2019 2019, ,11 11,, 496 x FOR PEER REVIEW 1010 ofof 2120 Toxins 2019, 11, x FOR PEER REVIEW 10 of 20

FigureFigure 7.7. MultipleMultiple alignmentalignment ofof cystinecystine knotknot toxinstoxins (CKTs)(CKTs) presentpresent ininPamphobeteus Pamphobeteus verdolagaverdolagavenom venom Figure 7. Multiple alignment of cystine knot toxins (CKTs) present in Pamphobeteus verdolaga venom withwith CKTCKT previously previously characterized characterized (A )( andA) and sequence sequence logos logos (B). Group (B). Group 4a U54-theraphositoxin-Pv1a_1 4a U54-theraphositoxin- with CKT previously characterized (A) and sequence logos (B). Group 4a U54-theraphositoxin- putativePv1a_1 putative toxin precursor toxin precursor present similitude present withsimilitudeGrammostola with Grammostola rosea GTx6-1, rosea M5AXR5, GTx6-1, and M5AXR5, Cystine knot and Pv1a_1 putative toxin precursor present similitude with Grammostola rosea GTx6-1, M5AXR5, and toxinsCystine of knot the tarantula toxins of Chilobrachysthe tarantula jinzhao Chilobrachysassigned jinzhao by Chenassigned et al. by 2008 Chen in et orphan al. 2008 family in orphan 2 (JZTX-72; family Cystine knot toxins of the tarantula Chilobrachys jinzhao assigned by Chen et al. 2008 in orphan family B1P1J5).2 (JZTX-72; Polymorphic B1P1J5). Polymorphi sites are indicatedc sites are in indicated gray. in gray. 2 (JZTX-72; B1P1J5). Polymorphic sites are indicated in gray.

Figure 8. Multiple alignment of spider toxins present in Pamphobeteus verdolaga venom with agatoxin Figure 8. Multiple alignment of spider toxins present in Pamphobeteus verdolaga venom with agatoxin previously characterized (A) and sequence logos (B). Group 4b U27-theraphositoxin-Pv1a_1 and Figurepreviously 8. Multiple characterized alignment (A) ofand sp sequenceider toxins logos present (B). Groupin Pamphobeteus 4b U27-theraphositoxin-Pv1a_1 verdolaga venom with agatoxinand U82- U82-theraphositoxin-Pv1a_1 putative toxin precursors present similitude with U8-agatoxin-Ao1a from previouslytheraphositoxin-Pv1a_1 characterized (putativeA) and sequence toxin precursors logos (B). presentGroup 4b similitude U27-theraphositoxin-Pv1a_1 with U8-agatoxin-Ao1a and U82-from Stegodyphus mimosarum (A0A087TBW8), Parasteatoda tepidariorum (A0A2L2YIL4, house spider), Daphnia theraphositoxin-Pv1a_1Stegodyphus mimosarum putative (A0A087TBW8), toxin precursors Parasteatoda present tepidariorum similitude (A0A2L2YIL4,with U8-agatoxin-Ao1a house spider), from magna (A0A0P6I6W0), and putative U8-agatoxin-ao1a-like isoform x2 from Ixodes ricinus (A0A147BFN0, StegodyphusDaphnia magna mimosarum (A0A0P6I6W0), (A0A087TBW8), and putative Parasteatoda U8-agatoxin-ao1a-like tepidariorum (A0A2L2YIL4,isoform x2 from house Ixodes spider), ricinus common tick). Daphnia(A0A147BFN0, magna (A0A0P6I6W0),common tick). and putative U8-agatoxin-ao1a-like isoform x2 from Ixodes ricinus Superfamily(A0A147BFN0, 5 common tick). Superfamily 5 SuperfamilyThis family 5 included two sequences characterized by the presence of 8 cysteine residues and an aminoThis acid family length included between two 61 and sequences 65 residues. characterized The predicted by the disulfide presence pattern of 8 cysteine suggested residues that cysteine and an This family included two sequences characterized by the presence of 8 cysteine residues and an bondsamino formedacid length between between CysI–CysVI, 61 and CysII–CysVII,65 residues. The CysIII–CysIV, predicted anddisulfide CysV-CysVIII. pattern suggested Both peptides that amino acid length between 61 and 65 residues. The predicted disulfide pattern suggested that fromcysteine this bonds family formed did not between show any CysI–CysVI, similarity with CysI anyI–CysVII, known CysIII–CysIV, peptides. and CysV-CysVIII. Both cysteinepeptides bonds from thisformed family between did not CysI–CysVI, show any similarity CysII–CysVII, with any CysIII–CysIV, known peptides. and CysV-CysVIII. Both peptides from this family did not show any similarity with any known peptides. Superfamily 6 Superfamily 6

Toxins 2019, 11, 496 11 of 21

Toxins 2019, 11, x FOR PEER REVIEW 11 of 20 Superfamily 6 Six peptides were identified in this group, with amino acid lengths between 43 and 71 residues, and Sixwere peptides predicted were to form identified four disulfide in this group, bridges. with Cysteine amino acidbond lengths formations between were 43 predicted and 71 residues, between andCysI–CysVI, were predicted CysII–CysVII, to form CysIII– fourCysV, disulfide and bridges. CysIV–CysVIII. Cysteine All bond memb formationsers of this group were predicteddisplayed betweenthe whey CysI–CysVI, acid protein CysII–CysVII, (WAP)-type CysIII–CysV, four-disu andlfide CysIV–CysVIII.core domain Allwith members a cysteine of this pattern group displayed(“XnCPX6/8 theCX6 wheyCX5CX acid5CCX protein3/4CX3CX (WAP)-typen”; X is any four-disulfideamino acid and core n is domain an uncertain with anumber) cysteine similar pattern to (“XWAPnCPX domain6/8CX6 identifyCX5CX5 CCXin Eriocheir3/4CX3CX sinensisn”; X is(A0A0N6XEM9) any amino acid and and Nephila n is an uncertainpilipes (A0A076L0S4) number) similar (Figure to WAP9). domain identify in Eriocheir sinensis (A0A0N6XEM9) and Nephila pilipes (A0A076L0S4) (Figure9).

FigureFigure 9.9. Multiple alignment of whey acidacid proteinprotein (WAP)-type(WAP)-type toxinstoxins presentpresent inin PamphobeteusPamphobeteus verdolagaverdolaga venomvenom with with WAP WAP protein protein previously previously characterized characterized (A) and (sequenceA) and logos sequence (B). Group logos 6 (U9,B). GroupU16-theraphositoxin-Pv1a_1, 6 U9, U16-theraphositoxin-Pv1a_1, U22-theraphosito U22-theraphositoxin-Pv1a_1,xin-Pv1a_1, U24-theraphositoxin-Pv1a_1, U24-theraphositoxin-Pv1a_1, U26- U26-theraphositoxin-Pv1a_1,theraphositoxin-Pv1a_1, and and U26-ther U26-theraphositoxin-Pv1b_1aphositoxin-Pv1b_1 putative putative toxin toxin precursors precursors are similar toto EriocheirEriocheir sinensis sinensis(A0A0N6XEM9) (A0A0N6XEM9) and andNephila Nephila pilipes pilipes(A0A076L0S4). (A0A076L0S4). The The WAP-type WAP-type ‘four-disulfide ‘four-disulfide core’ domaincore’ domain profile profile is indicated is indicated in the box.in the box. Superfamily 7 Superfamily 7 This group was characterized by 2 proteins with amino acid lengths between 76 and 80 residues This group was characterized by 2 proteins with amino acid lengths between 76 and 80 residues and the presence of 4 disulfide bridges between cysteines CysI–CysV, CysII–CysVI, CysIII–CysVII, and the presence of 4 disulfide bridges between cysteines CysI–CysV, CysII–CysVI, CysIII–CysVII, and CysIV-CysVIII. All members of this group displayed the single domain von Willebrand factor and CysIV-CysVIII. All members of this group displayed the single domain von Willebrand factor type C domain with a cysteine pattern (“X13CX18/19CX4CX9CX8/11/12CX11CCX4C”; X is any amino acid) type C domain with a cysteine pattern (“X13CX18/19CX4CX9CX8/11/12CX11CCX4C”; X is any amino acid) similar to SVWC domain identify in Nephila pilipes (A0A076KZ59) (Figure 10). similar to SVWC domain identify in Nephila pilipes (A0A076KZ59) (Figure 10). Superfamily 8 This group included eight sequences characterized by the formation of 5 disulfide bridges and amino acid lengths between 67 and 88 residues. Predicted disulfide bounds were either between CysI–CysVI, CysII–CysIV, CysIII–CysV, CysVII–CysX and CysVIII–CysIX or CysI–CysIII, CysII–CysIV, CysV–CysVI, CysVII–CysVIII, and CysIX–CysX. Six members from this group did not show similarity with any known domain. Only two putative toxin precursors had similar cysteine patterns (“XnCX5/6CX4CCX11CX9CX15/28/30CX1CX5CX4/6CXn”; X is any amino acid and n is an uncertain number) with other identified invertebrates and spider toxin cytokines as astakines (Figure 11). U29-theraphositoxin-Pv1c_1 is similar to the previously isolated Pl astakine 1 from Pacifastacus leniusculus (Q56R11), while U29-theraphositoxin-Pv1b_1 is similar to the previously isolated Pl astakine 2a isolated from Pacifastacus leniusculus (A5HTU2), Stegodyphus mimosarum (A0A087TV92), Hyalomma excavatum (A0A131XC84), and Rhipicephalus zambeziensis (A0A224YLN4, IxodegrinFigure B). 10. The Multiple U29C-like alignment Pl astakineof single-domain 1 has 15 von amino Willebrand acid residuesfactor type between C (SVWC) C#7 domain and C#8.toxins The U29-theraphositoxin-Pv1b_1-likepresent in Pamphobeteus verdolaga Pl astakine venom with 2a has SVWC an insert protein of 13previously amino acid characterized residues between (A) and C#7 sequence logos (B). Group 7 U14-theraphositoxin-Pv1a_1, U25-theraphositoxin-Pv1a_1, and U36- theraphositoxin-Pv1a_1 putative toxin precursors are similar to Nephila pilipes (A0A076KZ59).

Toxins 2019, 11, x FOR PEER REVIEW 11 of 20

Six peptides were identified in this group, with amino acid lengths between 43 and 71 residues, and were predicted to form four disulfide bridges. Cysteine bond formations were predicted between CysI–CysVI, CysII–CysVII, CysIII–CysV, and CysIV–CysVIII. All members of this group displayed the whey acid protein (WAP)-type four-disulfide core domain with a cysteine pattern (“XnCPX6/8CX6CX5CX5CCX3/4CX3CXn”; X is any amino acid and n is an uncertain number) similar to WAP domain identify in Eriocheir sinensis (A0A0N6XEM9) and Nephila pilipes (A0A076L0S4) (Figure 9).

Figure 9. Multiple alignment of whey acid protein (WAP)-type toxins present in Pamphobeteus verdolaga venom with WAP protein previously characterized (A) and sequence logos (B). Group 6 U9, U16-theraphositoxin-Pv1a_1, U22-theraphositoxin-Pv1a_1, U24-theraphositoxin-Pv1a_1, U26- theraphositoxin-Pv1a_1, and U26-theraphositoxin-Pv1b_1 putative toxin precursors are similar to Eriocheir sinensis (A0A0N6XEM9) and Nephila pilipes (A0A076L0S4). The WAP-type ‘four-disulfide core’ domain profile is indicated in the box.

Superfamily 7 Toxins 2019, 11, 496 12 of 21 This group was characterized by 2 proteins with amino acid lengths between 76 and 80 residues and the presence of 4 disulfide bridges between cysteines CysI–CysV, CysII–CysVI, CysIII–CysVII, andToxins C#8CysIV-CysVIII. 2019 (Boxed, 11, x FOR in PEER Figure All REVIEW members 11). The of motif this group GX2RYSX(P displayed/R/A) the XC single was founddomain in von both Willebrand toxin precursors 12factor of 20 isolated in this study and all invertebrate sequences shown in Figure 10. The LXYP motif present in 2 type C domain with a cysteine pattern (“X13CX18/19CX4CX9CX8/11/12CX11CCX4C”; X is any amino acid) Superfamily 8 wassimilar not to present SVWC in domain U29-theraphositoxin-Pv1b_1. identify in Nephila pilipes (A0A076KZ59) (Figure 10). This group included eight sequences characterized by the formation of 5 disulfide bridges and amino acid lengths between 67 and 88 residues. Predicted disulfide bounds were either between CysI–CysVI, CysII–CysIV, CysIII–CysV, CysVII–CysX and CysVIII–CysIX or CysI–CysIII, CysII– CysIV, CysV–CysVI, CysVII–CysVIII, and CysIX–CysX. Six members from this group did not show similarity with any known domain. Only two putative toxin precursors had similar cysteine patterns (“XnCX5/6CX4CCX11CX9CX15/28/30CX1CX5CX4/6CXn”; X is any amino acid and n is an uncertain number) with other identified invertebrates and spider toxin cytokines as astakines (Figure 11). U29- theraphositoxin-Pv1c_1 is similar to the previously isolated Pl astakine 1 from Pacifastacus leniusculus (Q56R11), while U29-theraphositoxin-Pv1b_1 is similar to the previously isolated Pl astakine 2a isolated from Pacifastacus leniusculus (A5HTU2), Stegodyphus mimosarum (A0A087TV92), Hyalomma excavatum (A0A131XC84), and Rhipicephalus zambeziensis (A0A224YLN4, Ixodegrin B). The U29C-like Pl astakine 1 has 15 amino acid residues between C#7 and C#8. The U29-theraphositoxin-Pv1b_1-like Pl astakine 2a has an insert of 13 amino acid residues between C#7 and C#8 (Boxed in Figure 11). The Figure 10. Multiple alignment of single-domain von Willebrand factor type C (SVWC) domain motifFigure GX2RYSX(P/R/A) 10. Multiple alignment XC was of single-domainfound in both von toxi Willen brandprecursors factor typeisolated C (SVWC) in this domain study toxins and all toxins present in Pamphobeteus verdolaga venom with SVWC protein previously characterized (A) invertebratepresent insequences Pamphobeteus shown verdolaga in Figure venom 10. with The SVWCLXYP proteinmotif present previously in 2characterized was not present (A) and in U29- and sequence logos (B). Group 7 U14-theraphositoxin-Pv1a_1, U25-theraphositoxin-Pv1a_1, and sequence logos (B). Group 7 U14-theraphositoxin-Pv1a_1, U25-theraphositoxin-Pv1a_1, and U36- theraphositoxin-Pv1b_1.U36-theraphositoxin-Pv1a_1 putative toxin precursors are similar to Nephila pilipes (A0A076KZ59). theraphositoxin-Pv1a_1 putative toxin precursors are similar to Nephila pilipes (A0A076KZ59).

FigureFigure 11.11. MultipleMultiple alignmentalignment ofof astakineastakine isoformsisoforms presentpresent inin PamphobeteusPamphobeteus verdolagaverdolaga venomvenom withwith astakineastakine previouslypreviously characterizedcharacterized ((AA)) andand sequencesequence logoslogos ((BB).). GroupGroup 88 U29-theraphositoxin-Pv1c_1U29-theraphositoxin-Pv1c_1 isis similar to to the the previously previously isolated isolated Pl astakine Pl astakine 1 from 1 fromPacifastacusPacifastacus leniusculus leniusculus (Q56R11)(Q56R11).. U29B- U29B-theraphositoxin-Pv1b_1theraphositoxin-Pv1b_1 is similar is similar to the to previous the previouslyly isolated isolated compound. compound. Pl astakine Pl astakine 2a from 2a fromPacifastacus Pacifastacus leniusculus leniusculus (A5HTU2), (A5HTU2), Steg Stegodyphusodyphus mimosarum mimosarum (A0A087TV92), HyalommaHyalomma excavatumexcavatum (A0A131XC84) (A0A131XC84) and and Rhipicephalus Rhipicephalus zambeziensis zambeziensis (A0A224YLN4, (A0A224YLN4, Ixodegrin Ixodegrin B). U_29C- B). U_29C-theraphositoxin-Pv1c_1theraphositoxin-Pv1c_1 like Pl astakine like Pl astakine1 have 15 1 amino have 15 acid amino residues acid between residues C#6 between and C#7. C#6 U29B and C#7.like Pl U29B astakine like Pl 2a astakine have an 2a haveinsert an ofinsert 13 amin of 13o aminoacid residues acid residues between between C#6 and C#6 C#7 and (Boxed). C#7 (Boxed). The Theconserved conserved motifs motifs GX2RYSX(P/R/A)XC GX2RYSX(P/R/A)XC and and CPC CPC are are underlined. underlined. Conserved Conserved LXYP LXYP motif motif in in astakine. astakine. 2.4.3. Transcriptomics Reveal Sequences of Potential Voltage-Gated Calcium Channel Modulators 2.4.3. Transcriptomics Reveal Sequences of Potential Voltage-Gated Calcium Channel Modulators After a local BLAST using the venom-gland transcriptomic results as the database and the tMS/MS After a local BLAST using the venom-gland transcriptomic results as the database and the fragments as the query, all hits were similar to transcript c7142 (c7142_g1_i1). This transcript, with a tMS/MS fragments as the query, all hits were similar to transcript c7142 (c7142_g1_i1). This transcript, with a transcripts per million (TPM) value of 27806.19, corresponds to a sequence that is 82 residues long with a theoretical molecular weight of 8896.18 Da. After processing the signal and propetide cleaving sites, the mature peptide of c7142 is a 32 residue peptide with a theoretical monoisotopic

Toxins 2019, 11, 496 13 of 21 Toxins 2019, 11, x FOR PEER REVIEW 13 of 20 Toxins 2019, 11, x FOR PEER REVIEW 13 of 20 transcriptsmolecular perweight million of (TPM)3495.69 value Da of(considering 27806.19, correspondsall cysteines to aare sequence oxidized) that and is 82 is residues named long ω- molecularwiththeraphositoxin-Pv3a a theoretical weight molecular of (Figure3495.69 weight 12A).Da of(considering MALDI-TOF 8896.18 Da. Afterall MS cysteines processinganalyses are of the voltage-gatedoxidized) signal and and propetide activeis named fractions cleaving ω- theraphositoxin-Pv3asites,showed the a mature molecular peptide mass(Figure of range c7142 12A). between is aMALDI-TOF 32 residue 3399.41 peptide andMS analyses3839.61 with a Da theoretical of (Figurevoltage-gated monoisotopic2E, F). Inactive an attempt molecularfractions to showedweightdetermine ofa 3495.69molecular the complete Da mass (considering sequence range betweenof all ω cysteines-theraphositoxin-Pv3a, 3399.41 are and oxidized) 3839.61 andwe Da combined is (Figure named theω2E,-theraphositoxin-Pv3a tMS/MSF). In an analysis attempt and to determine(Figurevenom-gland 12 A).the MALDI-TOFcompletetranscriptomics. sequence MS analysesFragments of ω-theraphositoxin-Pv3a, of voltage-gatedcorresponding active to we 21-22/a-b_1 fractionscombined showed thematched tMS/MS a molecularthe analysis propeptide mass and venom-glandrangeregion between (data transcriptomics. 3399.41not shown), and 3839.61 whileFragments Da (Figure21-22/a-b_2, corresponding2E, F). In21-22/a-b_3 an attempt to 21-22/a-b_1 toand determine 21-22/c_1 matched the completematched the propeptidesequence the ω- regionoftheraphositoxin-Pv3a_1,ω-theraphositoxin-Pv3a, (data not shown), covering wewhile combined 66% 21-22/a-b_2, of thethe tMStoxin. /21-22/a-b_3MS Fragments analysis and and 21-22/a-b_2 venom-gland21-22/c_1 and matched transcriptomics.21-22/c_1 the were ω- theraphositoxin-Pv3a_1,Fragmentscontained in corresponding fragments 21-22/a-b_3covering to 21-22 66%/a-b_1 and of werematched the 100%toxin. the identicalFragments propeptide (see 21-22/a-b_2 regionTable 1). (data Figure and not 21-22/c_1 12B shown), shows whilewere the contained21-22pairwise/a-b_2, sequencein 21-22fragments/a-b_3 alignment 21-22/a-b_3 and 21-22 of ω/c_1 -theraphositoxin-Pv3a_1and matched were 100% the ω identical-theraphositoxin-Pv3a_1, sequence (see Table with 1). theFigure covering tMS/MS 12B shows 66%results of the the of pairwisetoxin.fraction Fragments 21/22.sequence tMS/MS 21-22 alignment /fractionsa-b_2 andof didω 21-22-theraphositoxin-Pv3a_1 not/c_1 show were any contained similarities insequence fragmentswith any with other 21-22 the transcribed/a-b_3 tMS/MS and weretranscript.results 100% of fractionidenticalThis theraphositoxin 21/22. (see Table tMS/MS1). Figurefrom fractions P.12 Bverdolaga did shows not the showwas pairwise similarany si sequencemilarities (e-value alignment with >2 × any 10− of4other) toω -theraphositoxin-Pv3a_1 othertranscribed theraphositoxins transcript. Thissequencereported theraphositoxin within the the Theraphosidae tMS /fromMS results P. verdolaga family, of fraction inwas the 21 similar/22.species tMS (e-value/(MSAphonopela fractions >2 ×sp.did 10 and− not4) to showcalifornicum other any theraphositoxins similarities), Brachypelma with any other transcribed transcript. This theraphositoxin from P. verdolaga was similar (e-value > 2 10 4) reported(Brachypelma in the albicans Theraphosidae and Brachypelma family, insmithi the species), and Acanthoscuria(Aphonopela sp. paulensis and californicum, all distributed), Brachypelma ×in the− (toAmericanBrachypelma other theraphositoxins continent albicans and(United reportedBrachypelma States, in the Mexico,smithi Theraphosidae), andand AcanthoscuriaBrazil, family, respectively, in paulensis the species ,according all ( Aphonopeladistributed to [21]).sp. in andtheω- Americancalifornicumtheraphositoxin-Pv3a continent), Brachypelma (Unitedshows(Brachypelma high Stat sequencees, albicansMexico, similarityand andBrachypelma Brazil,to ω-theraphotoxin-Asp1a respectively, smithi), and Acanthoscuriaaccording (e-value to paulensis of[21]). 2 × 10,ω all−4-) theraphositoxin-Pv3adistributedfrom the related in the spider American shows Aphonopelma continent high sequence sp, (United which similarity States, is known Mexico, to andω-theraphotoxin-Asp1a and proved Brazil, to respectively,inhibit voltage-gated (e-value according of 2calcium to× 10 [21−4]).) ω-theraphositoxin-Pv3a shows high sequence similarity to ω-theraphotoxin-Asp1a (e-value of 2 10 4) fromchannels the related (see Figure spider 12C) Aphonopelma [22–24]. Itsp, was which similar is known as well and (62.5% proved identity) to inhibit with voltage-gated the calcium calcium× active− channelsfrompeptide, the identified related(see Figure spider in 12C)theAphonopelma theraphosid [22–24]. It sp, wasCoremiocnemiswhich similar is known as tropixwell and .(62.5% Nevertheless, proved identity) to inhibit the with full voltage-gated the sequence calcium and calcium active bio- peptide,channelsactivity ofidentified (see Pv3a Figure still in 12has theC) yet [theraphosid22 to–24 be]. confirmed It was Coremiocnemis similar in further as well tropix venomic, (62.5%. Nevertheless, identity) transcriptomic, with the the full calcium and sequence pharmacological active and peptide, bio- activityidentifiedstudies. of Pv3a in the still theraphosid has yet toCoremiocnemis be confirmed in tropix further. Nevertheless, venomic, transcriptomic, the full sequence and and pharmacological bio-activity of studies.Pv3a still has yet to be confirmed in further venomic, transcriptomic, and pharmacological studies.

Figure 12. (A) ω-theraphositoxin-Pv3a mature sequence derived from the combination of the tandem FigureFigureMS/MS 12. 12. analysis ((AA)) ωω-theraphositoxin-Pv3a and-theraphositoxin-Pv3a venom-gland transcriptomics. mature mature sequence sequence (B) Pairwise derived derived alignmentfrom from the the combination combinationof ω-theraphositoxin-Pv3a of of the the tandem tandem MS/MSMSand/MS the analysis analysistandem and andMS/MS venom- venom-gland fragments.gland transcriptomics. transcriptomics. Asterisks (*) indicate ( (BB)) Pairwise Pairwise an identical alignment alignment match. of of ωω (-theraphositoxin-Pv3aC-theraphositoxin-Pv3a) Pairwise alignment andandof ωthe the-theraphositoxin-Pv3a tandem tandem MS/MS MS/MS fragments. and ω Asterisk Asterisks-theraphositoxin-Pv3a-Asp1as (*) (*) indicate indicate an an identical from match. Aphonopelma ( (CC)) Pairwise Pairwise sp. alignment alignment (proved ofoftheraphotoxin ω-theraphositoxin-Pv3a-theraphositoxin-Pv3a to inhibit volt ageand gated ωω-theraphositoxin-Pv3a-Asp1a calcium channels). from AphonopelmaAphonopelma sp.sp. (proved (proved theraphotoxintheraphotoxin to to inhibit inhibit volt voltageage gated gated calcium calcium channels) channels).. Besides Pv3a, other theraphositoxins detected in the transcriptome showed high identity to calciumBesidesBesides channel Pv3a, Pv3a, inhibitors other other theraphositoxins theraphositoxins such as ω-theraphotoxin-Bs2a detected detected in in the the (e-value: transcriptome transcriptome 3 × 10−18) showedisolated showed fromhigh high Brachypelmaidentity identity to to 18 calcium channel inhibitors such as ω-theraphotoxin-Bs2a (e-value: 3 10−18 ) isolated from Brachypelma calciumsmithi [25] channel (see inhibitorsFigure 13). such This as novelω-theraphotoxin-Bs2a peptide has a theoretical (e-value: 3 ×monoisotopic× 10− ) isolated mass from ofBrachypelma 4900 kDa smithismithi(considering [25][25] (see (see all Figure cysteines 13).13 ).are This This oxidized) novel novel peptide peptideand corresponds has has a a theoretical theoretical to a DRP monoisotopic monoisotopic named here massω mass-theraphositoxin- of of 4900 4900 kDa kDa (considering(consideringPv2a. allall cysteinescysteines are are oxidized) oxidized) and and corresponds corresponds to ato DRP a DRP named named here hereω-theraphositoxin-Pv2a. ω-theraphositoxin- Pv2a.

Figure 13. ω-theraphotoxin-Pv2a-theraphotoxin-Pv2a mature mature peptide peptide sequence detected in the transcriptome analysis and Figurepairwise 13. alignment ω-theraphotoxin-Pv2a withwith ωω-theraphotoxin-Bs2a mature peptide from sequence Brachypelma detected smithismithi in the.. transcriptome analysis and pairwise alignment with ω-theraphotoxin-Bs2a from Brachypelma smithi. 3. Discussion 3. Discussion

Toxins 2019, 11, 496 14 of 21

3. Discussion The complexity of Theraphosidae venoms is commonly underestimated because of the lack of high-throughput methods for toxin identification [14,26]. Venomics allows the study of this venom by integrating proteomic, genomic, and transcriptomic analyses [27]. To date, only a handful of Theraphosidae venoms have been extensively characterized, including the venoms of the Chinese black earth tiger tarantula C. hainanus (H. hainanum), C. schmidti (H. schmidti), and C. guangxiensis, among others [28]. P. verdolaga is a recently described Theraphosidae spider distributed mainly in the region of Antioquia (Colombia) [29]. MS/MS and transcriptomics analyses of the venom-gland content showed that P. verdolaga venom is not only composed of theraphotoxins, but also by larger proteins, in agreement with what has been previously reported for tarantulas [17]. Tarantula venoms are a rich source of bio-active molecules specialized in modulating ion channels [10,19]. Such peptides are believed to have an important role in spider behavior during prey capture and defense from predators. Indeed, the venom of P. verdolaga contained peptides that were able to modulate the ion channels hCaV2.2, hCaV3.2, and hNaV1.7. Interestingly, our bioactivity assay results suggest that the venom of P. verdolaga has the ability to preferentially modulate CaV channels over NaV channels, although more detailed experiments are required to explore these observations further at the single peptide level using ion channel electrophysiology assays. These findings are in agreement with previous reports of P. verdolaga venom that described the presence of peptides with high identity with CaV channel modulators from other Theraphosids [17,18]. Disulfide-bonded neurotoxins are the most common toxins in spider venoms as well as in scorpion and cone snail venoms [1,3,11–14,28,30]. Interestingly, non-Theraphosidae spider peptides that potently inhibit CaV2 channels often display disulfide bridge patterns other than a typical ICK scaffold, as reported for toxins in the venom of the genus Phoneutria, for example—ω-ctenitoxins Pn2a (Tx3-3) [31], Pn3a (Tx3-4) [32], Pn4A (Tx3-6) [33] as well as Pr1a (PRTx3-7) [34]—and for ω-segestritoxin of the genus Segestria [35]. Most of the groups of toxins found in the venom of P. verdolaga displayed disulfide patterns that resembled these CaV2 inhibitory toxins. Bioactive results were consistent with the MS/MS findings, where the active fractions displaying the inhibitory activity on CaV channels possessed peptides showing homology with peptides assumed to inhibit voltage-gated calcium channels [22]. These findings encourage more detailed studies of the components of P. verdolaga venom to further characterize the peptides responsible for the interesting pharmacological effects observed. Only one fraction of P. verdolaga venom showed strong inhibitory activity on hNaV1.7. Interestingly, another species belonging to the genera Pamphobeteus, P. nigricolor, was previously described to contain bio-active peptides but with a strong and selective inhibition of NaV1.7 channels [18]. The tMS/MS analysis of the active fraction showed the presence of fragments similar to the mature peptide of transcribed transcript c7142. This mature peptide was named ω-theraphositoxin-Pv3a, which may be responsible for the activity on Cav channels. Additionally, the similarity of this theraphotoxin with ω-theraphotoxin-Asp1a, which is also known to affect voltage-gated calcium channel in vertebrates (based on sequence homologies), supports this hypothesis [8,22]. ω-theraphositoxin-Pv3a may correspond to a fragment of the mature peptide that is very similar to other theraphotoxins, like ω-theraphotoxin-Asp1a from Aphonopelma sp and Ct1a from Coremiocnemis tropix (both calcium active peptides), but missing at least two cysteines to allow the three disulfide bridges common in ICK peptides [36]. The full sequence and bio-activity of Pv3a is still to be confirmed in further venomic, transcriptomic, and pharmacological studies. P. verdolaga transcriptomic results are in agreement with the Chen et al. report for the spider C. hainanus (H. hainanum)[14]. Both analyses showed the presence of CRISP, the Kunitz-type toxin, insecticidal toxins, and a significant number of proteins with unknown functions [14]. The last group of unknown proteins is of special interest since it could indicate several new variants of potential novel toxins as described in other Theraphosidae venoms [14]. Cystein-rich peptides forming cysteine knots, obtained from the transcriptome, are similarly clustered as described in the Theraphosidae spiders G. rosea, C. schmidti (H. schmidti), C. guangxiensis, and C. hainanus (H. hainanum)[12,14,28,37]. Toxins 2019, 11, 496 15 of 21

We found two different cytokines in P. verdolaga venom, astakine 1 and astakine 2, which have been found in several invertebrate species: astakine 1 in Pacifastacus leniusculus (crayfish), Acanthoscurria gomesiana (tarantula spider), and Ornithoctonus huwena (the Chinese bird spider) and astakine 2 in crustaceans, ticks, and spiders as well as in blattodean, hymenopteran, and hemipteran insects [38]. Surprisingly, both astakine sequences have been described as having different roles in hemocyte lineage differentiation and show conserved motifs among invertebrate groups. An interesting finding is that the astakine-2 transcript found in this study may not exhibit the same function in granular cell differentiation as previously described by Lin et al. 2010 [38], as the new sequence does not contain the conserved motif LXYP (L > Y; Y > N; P > S) required for that function. It is also known that when leucine (Leu72) and tyrosine (Tyr74) in this conserved motif are mutated into glycines in r-astakine 2Mut, its activity in promoting the maturation of granular cells is abolished, further offering support for the hypothesis that astrakine-2 has a different function.

4. Materials and Methods

4.1. Spider Collection and Venom Extraction Five female Pamphobeteus verdolaga specimens were collected in the locality of La Estrella-Pueblo Viejo, Antioquia Province, Colombia. Venom from five specimens was obtained as previously described [18]. Venom extraction was carried out using electro-stimulation. Metal electrodes, impregnated with a saline solution, were carefully positioned on the chelicerae, and a block signal with an amplitude of 18 V at 40–60 Hz was applied twice with an interval of 5 s using a custom-made electro-stimulator (model 01). Collected venom was transferred to dry, low-protein binding vials, lyophilized, and stored at 20 C until use. All tissue and venom collections were conducted in − ◦ accordance with: (a) the ethical principles in animal research adopted by the World Health Organization for the characterization of venoms [39,40] and (b) the “Comité Institucional para el Cuidado y Uso de Animales” (CICUA). After each extraction, all animals were kept alive in captivity.

4.2. Venom Fractionation The venom profile of P. verdolaga was obtained using reversed-phase high-pressure liquid chromatography (rp-HPLC). One milligram of crude venom was dissolved in 200 µL of solution A (0.1% TFA in water) and centrifuged at 3500 g for 5 min at room temperature. The supernatant was × fractionated using a C rp-HPLC analytical column (250 4.6 mm), equilibrated and eluted at a flow 18 × rate of 1.0 mL/min, first isocratically (5% B for 5 min), followed by a linear gradient of 5%–15% B for 10 min, 15%–45% B for 60 min, and 45%–70% B for 12 min [41]. The chromatographic separation was monitored at 215 nm, and fractions were collected manually, lyophilized, and stored 20 C until used. − ◦ Chromatographic fractions were analyzed by gel electrophoresis using 10% Tris-Tricine gels or 12% sodium dodecyl sulfate polyacrylamide gels [42] as explained below. Genetic accession was carried out under contract 155 signed by the University of Antioquia with the Environmental Ministry of the Republic of Colombia.

4.3. Sample Preparation for Proteomic Analysis Bands/spots selected for proteomic analysis were excised from Tris-Tricine gels and subjected to automated reduction/alkylation with DTT and iodoacetamide, respectively. Peptides were digested with porcine trypsin (Promega, Madison, WI, USA) using a ProGest™ digestor (Genomic solutions, model PRO100001, v2.00.07, Ann Arbor, MI, USA) following the manufacturer’s instructions. The dried digests were re-suspended in 8 µL of 0.1% formic acid and subjected to LC-MS/MS analysis using an aSynapt G2 ESI-QTOF instrument (Waters, Manchester, UK) (see below). Toxins 2019, 11, 496 16 of 21

4.4. Data Analysis MS/MS spectra were interpreted manually or using a licensed version of ProteinLynx Global (Server v2.5.2 software from Waters, Waters, Manchester, UK) or a free version of MASCOT (http: //www.matrixscience.com). ProteinLynx searches were made using a tryptic digestion with two missed cleavages. The peptide tolerance was set to 10 ppm, while fragment tolerance and estimated calibration error were set to 0.05 and 0.005 Da, respectively. Carbamidomethyl cysteine and oxidation of methionine were fixed as well as variable modifications [43].

4.5. LC-MS/MS Six microliter aliquots were subjected to nano-LC separation (nanoACQUITY-UPLC, Waters, Manchester, UK) equipped with a C18 (100 µm 100 mm, 1.7 µm particle size) BEH130 column × operated at 0.6 µL/min and coupled to ESI-Q-TOF (Synapt G2, Waters, Manchester, UK) MS system. The nLC column was eluted with a gradient of 0.1% formic acid in water (solution A) and acetonitrile (solution B). Samples were separated using the following gradient: 1%–12% B for 1 min; 12%–40% B for 16 min; 40%–85% B for 2 min; 85%–100% B for 2 min; and 1.0% B for 10 min. The autosampler was maintained at 10 ◦C. All analyses were performed in a data-dependent analysis (DDA).

4.6. Venom Gland RNA Extraction and Library Construction Two Female P. verdolaga specimens (from Antioquia Province in the locality of La Estrella-Pueblo Viejo, Colombia) were anesthetized under CO2. Venom glands were excised and placed in plastic vials containing TRIzol® reagent (ThermoFisher Scientific, Waltham, MA USA). Total RNA was extracted following the manufacturer’s protocol. RNAquality and concentration were measured using the bioanalyzer capillary system (Bioanalyzer Agilent 2100, Agilent, Santa Clara, CA, USA). mRNA was purified using the kit, and library preparation was carried out using the Illumina mRNA TruSeq kit v2. A 100 bp pair-end library was sequenced in a Hiseq 2500 instrument. Reads were cleaned using PRINSEQ-LITE (v0.2.) using default settings [44]. Genetic accession was carried out under contract 155 signed by the University of Antioquia with the Environmental Ministry of the Republic of Colombia.

4.7. De Novo Transcriptome Assembly and Gene Annotation De novo assembly of transcripts was done using the TRINITY (v2.) assembler package under default settings [45]. Transcriptome assembly quality was assessed based on the calculated E90N50 contig length and Benchmarking Universal Single-Copy Orthologs (BUSCO) annotation [46]. Sequence reads were aligned back to the reference transcriptome using Bowtie2 [47], and RNA-seq by expectation maximization (RSEM) [48] was used to estimate transcript abundance for all transcripts as transcripts per million (TPM). After assembly, all contigs/singlets were translated in six frames (16), and the Trinotate pipeline [45] was used to annotate the transcriptome. Briefly, homology searches were performed against the protein sequences contained in Genbank and UniProt databases using BLASTX with an e-value cutoff of 1 10 5. HMMER and Pfam databases were used to predict protein domains contained within × − each transcript. Presence of a signal peptide was determined using SignalP version 4.0. The resulting annotation information was then combined and pooled into an SQLite database. Also, to identify transcript homologs to spider toxins, BLASTX and tBLASTN programs (Sweet Version 2.28) were used. Cleaving signals for each transcript were predicted using the standalone tool and Tox|Note [22], which uses a combination of SignalP v4.1 [49] and an HMM to predict signal and propeptide sites, respectively. After the prediction of putative cleavage sites, mature peptides were aligned using the Clustal omega program [50]. Fasta36 (20) was used perform multiple high-scoring local alignments between the MS/MS sequences and the translated transcripts. Toxins 2019, 11, 496 17 of 21

4.8. Nomenclature Peptides and proteins were named following the nomenclature proposed by King et al. [51], with some modifications for proteins (masses above 20 kDa) i.e., protein group, followed by the isoform number and the species name. Names for all peptides and proteins annotated from the transcriptome were generated automatically using the Tox|Name module in the Tox|Note pipeline [22]. Spider taxonomic names were assigned according to the World Spider Catalog v20.0 (available at: https://wsc.nmbe.ch/)[21].

4.9. Protein Domain Searching and Sequence Logos A total of 26 mature peptides were screened against online software (http://web.expasy.org/blast/) 5 using the Blast program with an e-value cutoff set to <10− to identify similar sequences and putative functions of the new peptides. In order to identify the domain location within the query sequence, a multiple sequence alignment was performed among query and homology sequences, according to their cysteine pattern using the Geneious software package (v8.1.9) [52]. Resulting alignments were manually curated using GeneDoc [53]. Logo analysis of consensus sequences derived from alignments were generated using WEBLOGO (v2.8.2 https://weblogo.berkeley.edu/).

4.10. Disulfide Bond Prediction Disulfide bonds were predicted using the Cysteines Disulfide Bonding State and Connectivity Predictor (DISULFIND http://disulfind.dsi.unifi.it/), a web-based tool for disulfide engineering in proteins [54].

4.11. Calcium Influx Assays Five milligrams of P. verdolaga venom was fractionated by rp-HPLC (1 mg per run, venoms A–E) as described above, and fractions were evaluated for bioactivities using calcium influx imaging Tetra assays and the FLIPR instrument (Molecular Devices, CA, USA) [15,55]. Briefly, hCaV2.2 and hNaV1.7 responses were evaluated in neuroblastoma SH-SY5Y maintained in Roswell Park Memorial Institute (RPMI) media supplemented with 15% FBS and 2 mM L-glutamine. The hCaV3.2 responses were evaluated using recombinant hCaV3.2 expressed in HEK293T cells maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 750 µg/mL geneticin. Cells were seeded at 40,000 and 10,000 per well for SH-SY5Y and HEK293T cells, respectively, and cultured in a humidified atmosphere for 48 h before the assay. Media was removed, and 20 µL of Calcium 4 dye (Molecular Devices) prepared in an assay buffer—(in mM) 140 NaCl, 11.5 glucose, 5.9 KCl, 1.4 MgCl2, 1.2 NaH2PO4, 5 NaHCO3, 1.8 CaCl2, and 10 HEPES (pH 7.4)—was added to the wells. 2+ Cells were incubated for 30 min at 37 ◦C 5% CO2. Ca fluorescence responses were recorded at an excitation of 470–495 nm and emission of 515–575 nm for 10 s to set the baseline, at 600 s after addition of 20% of each venom fraction reconstituted in assay buffer, and for a further 300 s after addition of 90 mM KCl CaCl2 for CaV2.2 activation, 40 mM KCl CaCl2 for hCaV3.2 activation, and 3 µM veratridine and 30 nM of the scorpion toxin OD1 for hNaV1.7 activation. For the hCaV2.2 assay, nifedipine at 10 µM was added to the Calcium 4 dye to inhibit endogenous hCaV1 responses. 2+ The controls for the inhibition of Ca fluorescence responses were CVID at 1 µM for hCaV2.2, TTA-A2 at 50 µM for hCaV3.2, and tetrodotoxin (TTX) at 1 µM for hNaV1.7 responses. Data were normalized against the baseline (F/F0) and fluorescence responses plotted using GraphPad Prism 7. Venom fractions showing bioactivity were submitted to mass spectrometry analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a 4700 Proteomics Bioanalyser Model (Applied Biosystems, CA, USA). Peptides dissolved in water were mixed 1:1 (v/v) with matrix (7 mg/mL α-cyano-4-hydroxy-cinnamic acid in 50% ACN) and mass spectra acquired in positive reflector mode. Reported masses are for monoisotopic M + H+ ions. Toxins 2019, 11, 496 18 of 21

4.12. Data Deposition Metadata and annotated nucleotide sequences were deposited to the European Nucleotide Archive (ENA) under accessions: PRJEB21288/ERS1788422/ERX2067777-ERR2008012.

5. Conclusions P. verdolaga venom is a rich source of cysteine-rich peptides that form cystine knots, which can affect vertebrate CaV and NaV channels. Transcriptomic analysis showed the presence of at least 265 novel compounds. The venom content is similar to other Theraphosidae spiders like G. rosea, C. schmidti (H. schmidti), C. guangxiensis, and C. hainanus (H. hainanum).

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6651/11/9/496/s1. Supplementary File S1. Trinity assembly summary statistics of de novo reference transcriptome for venom gland of Pamphobeteus verdolaga. Base pair length distribution of transcripts from Pamphobeteus verdolaga venom gland. E90N50 statistic for de novo reference transcriptome of Pamphobeteus verdolaga. Supplementary File S2. Annotation of 16,030 transcripts of the venom gland of Pamphobeteus verdolaga. Author Contributions: S.E.-G. and F.C.C. conceived and designed the experiments; L.J.V.-M. and F.C.C. performed the experiments; J.C.Q.-C., S.S.P., F.C.C., C.M.A.G., and M.M.S.-C. analyzed the data; F.C.C. assisted writing and reviewing the manuscript; S.E.-G. wrote the manuscript. Funding: This research received no external funding. Acknowledgments: Authors are grateful to: Sostenibilidad program of the Universidad de Antioquia (UdeA), and Comité para el Desarrollo de la Investigación CONADI, Universidad Cooperativa de Colombia. This research was funded by the Comité para el Desarrollo de la Investigación (CODI-UdeA), Project CIQF-284. Authors thank Emmanuel Bourinet (Institute of Functional Genomics, Montpellier University, France) and Edward Perez-Reyes (School of Medicine, University of Virginia, USA) for kindly donating the hCaV3.2 cell line, and Glenn F King for the guidance and suggestions (Institute for Molecular Bioscience, University of Queensland, AU). Special thanks to the researchers in the Laboratorio de Venómica Evolutiva y Traslacional, Consejo Superior de Investigaciones Científicas, Valencia, Spain. Conflicts of Interest: The authors declare no conflict of interest.

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