University of Southern Denmark
GiTx1(β/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae) Isolation, structural assessments and activity on voltage-gated ion channels Montandon, Gabriela Gontijo; Cassoli, Juliana Silva; Peigneur, Steve; Braga, Thiago Verano; Moreira Dos Santos, Daniel; Bittencourt Paiva, Ana Luiza; Ricardo de Moraes, Éder; Kushmerick, Christopher; Borges, Márcia Helena; Richardson, Michael; Monteiro de Castro Pimenta, Adriano; Kjeldsen, Frank; Vasconcelos Diniz, Marcelo Ribeiro; Tytgat, Jan; Elena de Lima, Maria Published in: Biochimie
DOI: 10.1016/j.biochi.2020.07.008
Publication date: 2020
Document version: Accepted manuscript
Document license: CC BY-NC-ND
Citation for pulished version (APA): Montandon, G. G., Cassoli, J. S., Peigneur, S., Braga, T. V., Moreira Dos Santos, D., Bittencourt Paiva, A. L., Ricardo de Moraes, É., Kushmerick, C., Borges, M. H., Richardson, M., Monteiro de Castro Pimenta, A., Kjeldsen, F., Vasconcelos Diniz, M. R., Tytgat, J., & Elena de Lima, M. (2020). GiTx1(β/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae): Isolation, structural assessments and activity on voltage-gated ion channels. Biochimie, 176, 138-149. https://doi.org/10.1016/j.biochi.2020.07.008
Go to publication entry in University of Southern Denmark's Research Portal
Terms of use This work is brought to you by the University of Southern Denmark. Unless otherwise specified it has been shared according to the terms for self-archiving. If no other license is stated, these terms apply:
• You may download this work for personal use only. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying this open access version If you believe that this document breaches copyright please contact us providing details and we will investigate your claim. Please direct all enquiries to [email protected]
Download date: 05. Oct. 2021 1 GiTx1(β/κ-theraphotoxin-Gi1a), a novel toxin from the venom of Brazilian 2 tarantula Grammostola iheringi (Mygalomorphae, Theraphosidae): 3 isolation, structural assessments and activity on voltage-gated ion channels 4 5 Gabriela Gontijo Montandona; Juliana Silva Cassolia,b; Steve Peigneurb; Thiago Verano 6 Bragac,d; Daniel Moreira dos Santosa; Ana Luiza Bittencourt Paivaf; Éder Ricardo de 7 Moraesc; Christopher Kuschmerickc; Márcia Helena Borgesf; Michael Richardsonf; Adriano 8 Monteiro de Castro Pimentaa; Frank Kjeldsend; Marcelo Ribeiro Vasconcelos Dinizf; Jan 9 Tytgatb; Maria Elena de Limaa,h* 10 11 Affiliations 12 13 aDepartamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais - UFMG, 14 Av. Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil. 15 16 bPharmacology and Toxicology, University of Leuven (KULeuven) - Campus Gasthuisberg, 17 PO Box 922, Herestraat 49, 3000 Leuven, Belgium. 18 19 cDepartamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais - UFMG, Av. 20 Antônio Carlos 6627, 31270-901 Belo Horizonte, MG, Brazil. 21 22 dProtein Research Group, Department of Biochemistry and Molecular Biology, University of 23 Southern Denmark, Campusvej 55, 5230 Odense M, Denmark. 24 25 fDiretoria de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Rua Conde Pereira 26 Carneiro 80, 30510-010 Belo Horizonte, MG, Brazil. 27 28 hSanta Casa de Belo Horizonte: Ensino e Pesquisa, Rua Domingos Vieira, 590, 30.150-240 29 Belo Horizonte, MG, Brazil. 30 31 32 * Corresponding author: Maria Elena de Lima - Santa Casa de Belo Horizonte: Ensino e 33 Pesquisa, Rua Domingos Vieira, 590, 30.150-240 Belo Horizonte, MG, Brazil. 34 E-mail: [email protected]; [email protected] (M.E. de Lima). 35
36 Keywords 37 Grammostola, tarantula venom, GiTx1, β/κ-TRTX-Gi1a, spider toxin 38
39
40
41
42
43
44
1
45 Abstract
46 Spider venoms, despite of their toxicity, represent rich sources of active compounds with
47 biotechnological potential. However, in view of the diversity of the spider species, the full
48 potential of their venom molecules is still far from being known. In this work, we report the
49 purification, the structural and functional characterization of GiTx1 (β/κ-TRTX-Gi1a), the first
50 toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. This
51 toxin was isolated by chromatographic procedures, completely sequenced through automated
52 Edman’s degradation and tandem mass spectrometry and its spatial structure was predicted by
53 molecular modeling. It has a MW of 3.585 Da and the following sequence of amino acids:
54 SCQKWMWTCDQRPCCEDMVCKLWCKIIK. The pharmacological activities of GiTx1
55 were characterized by electrophysiology using two different approaches: (i) whole-cell patch
56 clamp on dorsal root ganglia neurons (DRG) and (ii) two-electrode voltage-clamp on voltage-
57 gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1,
58 at 2 μM, caused a partial block of inward ( 40 %) and outward ( 20 %) currents in DRG
59 cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a noticeable effect on VdNav, an arachnid
60 sodium channel isoform, among others. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and
61 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and
62 hERG isoforms. However, it was not effective on Shaker IR and rKv2.1 potassium channel
63 subtypes. These multiple effects on ion channels characterize GiTx1 as a promiscuous toxin.
64
65
66
67 68
69
70
71
2
72 1. Introduction
73 Spider venoms are complex mixtures of hundreds of compounds that act on different
74 targets when introduced into other organisms due to predatory or defense senses [1]. The
75 chemical nature of these molecules is very diverse, including salts, nucleotides, free amino
76 acids, glucose, neurotransmitters, acylpolyamines, and mainly peptide toxins and proteins
77 [2,3]. Because of the diverse mixture of components, spider venoms venom represent an
78 important source of novel biologically active compounds potentially useful as tools for
79 pharmacological and agricultural researches – e.g. development of drugs and insecticides (for
80 review see: [4,5]).
81 Spiders from the Theraphosidae family (infraorder Mygalomorphae), also called
82 tarantulas and “caranguejeiras”, are a sizable group of large, hairy and diverse spiders species,
83 distribute nearly all over globe. Example are species belonging the genus Theraphosa,
84 Lasiodora and Grammostola, the last mainly found in the Southern region of Brazil, Argentina,
85 Chile, Paraguay Bolivia and Uruguay.
86 Tarantula toxins comprise a group of peptides with 31 - 41 amino acids residues and 3
87 - 4 disulfide bridges, displaying DDH or ICK structural motifs [3,6,7]. Several studies have
88 demonstrated the wide molecular repertoire of toxins from tarantula venoms [3,8–12]. To date,
89 information on 450 toxins from the Theraphosidae family have been deposited in the
90 Arachnoserver database [13], however, few of these toxins have been functionally
91 characterized.
92 The spider Grammostola iheringi is one of best-known spider species from the
93 Theraphosidae family in Brazil. With a broad characterization approach based on
94 Multidimensional Protein Identification Technology (MudPIT) and bioinformatic analysis
95 using PepExplorer tool, our group has identified more than three hundred proteins in the venom
96 of Grammostola iheringi [14]. Through alignments searches in databases, we were able to
97 identify about 30% of these sequences, which include proteins (metalloproteinases, cysteine
3
98 proteinases, aspartic proteinases, carboxypeptidases and cysteine-rich secretory enzymes –
99 CRISP) and toxins. We have found in the G. iheringi venomous extract 63 toxins with 20-86
100 amino acid residues (MW 2-10 kDa) and 6-12 cysteines. Through sequence homology, it was
101 possible to infer functional activity for some of these toxins, including analgesic,
102 antiarrhythmic and insecticidal activity, likely through modulation of ion channels since
103 tarantula’s toxins have been described mainly as ion channel modulators. However, several
104 neurotoxins are yet to be characterized, such as the 24 neurotoxins in the venom of G. iheringi
105 identified by Borges et al, 2016, with remaining unknown targets.
106 In this work, a new short ICK toxin from G. iheringi venom, GiTx1 (β/κ-TRTX-Gi1a),
107 was purified and structurally characterized. Furthermore, its actions on voltage-gated sodium
108 and potassium channels were characterized using whole-cell patch clamp and two electrode
109 voltage-clamp techniques. GiTx1 is the first toxin purified from the venom of G. iheringi and
110 its structural and pharmacological characterization provide a relevant contribution on the
111 venom study of this spider.
112 2. Material and Methods
113 2.1. Animal collection and Venom Extraction
114 Specimens of Grammostola iheringi (Sisgen #A9BCC40) were collected at the border
115 of Santa Catarina and Rio Grande do Sul states (Coordinates: 27°31'31"S, 51°47'7" W), in the
116 south region of Brazil. The species were identified by Dr. Sylvia Lucas from the Arthropod
117 Laboratory of Butantan Institute (Brazil). The venom was obtained from eighteen adult
118 individuals by electrical stimulation (5V) of the chelicerae. Soluble venom was pooled,
119 lyophilized and stored at -20 oC until use.
120 2.2. Purification procedures
121 G. iheringi crude venom was fractionated by reverse phase chromatography on HPLC
122 system, according Richardson et al., 2006 [15]. A preparative column Vydac C4 (22 x 250 mm)
123 (Grace – Deerfield, USA) previously equilibrated with 0.1 % aqueous trifluoroacetic acid
4
124 (TFA) (Eluent A), was used in this procedure. For each run, 30 mg of the lyophilized venom
125 were dissolved in 2 mL of Eluent A. Samples were centrifuged and supernatants loaded into
126 column. Components were eluted with 0.1% trifluoroacetic acid in acetonitrile (Eluent B) using
127 the following gradient system: 0 to 20 min, 100% A; 20 to 30 min, 0-20% B; 30 to 110 min;
128 20-40% B; 110 to 130 min, 40-50% B; 130 to 150 min, 50-70% B, being Eluent B a solution
129 of 0.1% trifluoroacetic acid in acetonitrile (ACN). Flow rate was 5 mL/min and elution was
130 monitored by absorbance at 214 nm. Eluted fractions were manually collected and lyophilized.
131 The fraction of interest was purified by another reverse phase chromatography on HPLC, using
132 an analytical column Sephasil Peptide C18 5µ ST (4.6 x 250 mm) (GE Healthcare - Uppsala,
133 Sweden) equilibrated with Eluent A. The peptide was eluted by a linear gradient of 20 – 50 %
134 of eluent B during 40 min. Flow rate was 1 mL/min and chromatographic runs monitored by
135 absorbance at 214 nm. Samples were collected and lyophilized for structural and functional
136 experiments.
137 2.3. Peptide sequencing by Edman’s degradation
138 The purified peptide was previously S-reduced and alkylated with vinyl pyridine as
139 described in Yuen et al, 1988 [16]. The reduced and alkylated sample was desalted by RPC-
140 HPLC using a Vydac C4 column (22 x 250 mm) (Grace - Deerfield, USA) and one half was
141 digested in solution with trypsin. Digested peptides were purified by RPC-HPLC using a Vydac
142 C18 column (4.6 x 250 mm, Small Pore). Both reduced/alkylated and digested peptides were
143 sequenced by Edman degradation using the automated PPSQ-21A protein sequencer
144 (Shimadzu - Kyoto, Japan) coupled to reverse phase separation of PTH-amino acids on a
145 WAKOSIL-PTH (4.6 x 250 mm) column (Wako, Osaka, Japan), according to the
146 manufacturer’s instructions.
147 2.4. Mass Spectrometry (MS) Analyses
148 2.4.1. Sample preparation and sample desalting.
5
149 Eighty micrograms of the purified peptide were resuspended in 40 µL of 20 mM
150 triethylammonium bicarbonate (Sigma). Disulfide-bridged cysteine residues were reduced
151 with 10 mM TCEP (Pierce) for 20 min at 57 oC, followed by 40 min at room temperature. Thiol
152 groups were blocked with 30 mM iodoacetamide (Sigma) for 45 min at room temperature and
153 protected from light. Acetylation of the primary amines was achieved by incubating at room
154 temperature the peptide with acetic anhydride/dichloromethane (1:1) for 1 h.
155 The native sample and its chemically derived forms (i.e., reduced, alkylated and
156 acetylated) were desalted prior mass spectrometry analyses using self-made microcolumns
157 packed with Poros R2 resin (PerSeptive Biosystems) with a small plug of C18 extraction disk
158 (3M Bioanalytical Technologies) to close the tip. Samples were acidified with TFA (0.1% final
159 concentration) and loaded onto microcolumns previously equilibrated with 0.1% TFA and
160 washed 3 times with this solution. Peptides were eluted from the column with 70 % ACN/ 0.1
161 % TFA, lyophilized and stored at -20 ºC until required.
162 2.4.2. MALDI-TOF MS
163 MALDI-TOF mass spectrometry analyses were performed using an UltrafleXtreme
164 mass spectrometer (Bruker Daltonics), in positive ion mode and controlled by the software
165 FlexControl 3.4 (Bruker Daltonics). Samples were mixed with α-ciano-4-hydroxycynamic acid
166 (1:1, v/v) directly on the MALDI target and dried at room temperature. Analyses were
167 performed using reflected mode with external calibration using a peptide calibration standard
168 (Bruker Daltonics). Results were visualized using the software FlexAnalysis 3.4 (Bruker
169 Daltonics).
170 2.4.3. Reverse-phase nanoLC-MS/MS
171 Samples were resuspended in 0.1% formic acid (FA) and loaded onto a reverse-phase
172 in-house packed Reprosil-Pur C18-AQ column (3 μm; Dr. Maisch GmbH) with 15 cm length
173 and 75 μm inner diameter using an Easy-LC nanoHPLC system (Thermo Fisher Scientific).
174 Peptides were eluted from the column with a chromatography gradient from 0−34% solvent B
6
175 (90% ACN, 0.1% FA) for 23 at a flow rate of 250 nL/min directly into the mass spectrometer.
176 The Orbitrap XL (Thermo Fisher Scientific) was operated in a data-dependent mode (DDA).
177 After a survey scan (3 µscan; mass range m/z 300 – 1800; max injection time = 500 ms / AGC
178 target = 1x106; 30,000 FWHM at 400 m/z), the 2 most intense ion species. Were selected for
179 ETD fragmentation (1 µscan; max injection time = 100 ms / AGC target = 2x105; 30,000
180 FWHM at 400 m/z).
181 2.5. Similarity searches
182 Similarity searches were performed using BLAST tool against the UniProtKB/Swiss-
183 Prot database [17] and Clustal Omega [18] was used to align similar sequences.
184 2.6. Molecular modeling
185 Modeller v9.22 software [19] was used to determine the structure by molecular
186 modeling. The three-dimensional homology models of toxin targets were constructed using the
187 NMR structures of the templates (GsmTx2 or κ-TRTX-Gr2a, PDB: 1LUP, PaTx1 or κ-TRTX-
188 Ps1a, PDB: 1V7F and ProTx-II or β/ω-TRTX-Tp2a, PDB: 2N9T) assuming alignment between
189 targets and template. The Python script was written, and 50 best models were generated. The
190 best predicted model with least DOPE (discrete optimized protein energy) score was selected
191 for further investigation. Models were evaluated by the lowest DOPE by PROCHECK [20]
192 statistics and subsequently checked by VERIFY- 3D [21], ProSA [22] and Ramachandran plot
193 [23]. Tertiary structure models were visualized using Discovery Studio Visualizer 17.2.
194 2.7. Toxicity assays
195 Toxicity assays on mammals were performed on male Swiss mice (Mus musculus) of
196 about 20 g. Samples with different concentrations of crude venom or toxin, diluted in saline
197 with 0.1% BSA were injected intraperitoneal (i.p) or intracerebral (i.c). Toxicity to insects
198 were evaluated by intrathoracic injections into adult house flies (Musca domestica), as
199 described (Figueiredo et al., 1995). Previously to injections, flies were cold anesthetized at 4
200 oC during 2-3 min. Injections (1 L/fly) were carried out with a syringe (Hamilton, Reno, USA)
7
201 coupled to an automatic volume dispenser and a capillary glass needle. Control groups (mouse
202 and fly) received saline solution containing 0.1% of bovine serum albumin (BSA). Observation
203 of the intoxication symptoms were carried out immediately after injection and 2 and 24 h later.
204 The median lethal dose 50 % (LD50) value was estimated by the probit method (Finney, 1964).
205 The experimental procedures involving mice were approved by the Animal Experimentation
206 Ethics Committee (CETEA) of the Federal University of Minas Gerais (protocol 43/2005).
207 2.8. Culture of rat dorsal root ganglia (DRG) neurons cells
208 Culture of DRG neurons was done as previously described in Moraes et al., 2011 [24].
209 Briefly, male adults Wistar rats were killed by decapitation. Ganglia from all spinal segments
210 were removed and placed in cold Ca2+ -free Ringer solution. Ganglia were sectioned with
211 iridectomy scissors and incubated at 37°C for 20 minutes in Ca2 + -free Ringer plus papain (1
212 mg/ml) activated by cystein (0.03 mg/ml), followed by 20 minutes in Ca2 + -free Ringer plus
213 collagenase (2.5 mg/ml), also at 37°C. The enzyme incubation was stopped with F-12 media
214 supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. The
215 cells were released by gentle trituration through Pasteur pipettes with fire-polished tips. Cells
216 were platted on glass discs pre-coated with poly-D-lysine and laminin inside Petri dishes. After
217 2 hours in an incubator at 37°C to allow the cells to settle, the media was exchanged with
218 Leibovitz’s L-15 medium supplemented with 10% FBS, 5 mM glucose, 5 mM NaHEPES and
219 100 U/ml penicillin/ streptomycin. Cultures were stored in a normal atmosphere at room
220 temperature and used within 72 h.
221 2.9. Heterologous expression of voltage-gated ion channels
222 For the expression of the Voltage Gated Potassium Channels (Shaker IR, rKv2.1, rKv4.3
223 and hERG) and the Voltage Gated Sodium Channels (rNav1.2, rNav1.3, rNav1.4, hNav1.5,
224 mNav1.6, rNav1.8, DmNav1, BgNav1, VdNav1) in Xenopus laevis oocytes, the linearized
225 plasmids were transcribed using the T7 or SP6 mMESSAGEmMACHINE transcription kit
226 (Ambion, Life TechnologiesTM - Carlsbad, USA). The harvesting of stage V-VI oocytes from
8
227 anaesthetized female X. laevis frog was performed according to Liman et al. (1992). Oocytes
228 were injected with 50 nL of cRNA at a concentration of 1 ng/nL using a micro-injector
229 (Drummond Scientific, USA). The oocytes were incubated in a solution containing (in mM):
230 NaCl, 96; KCl, 2; CaCl2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4), supplemented with 50 mg/L
231 gentamycin sulfate. Whole-cell currents from oocytes were recorded 2–5 days after injection.
232 2.10. Electrophysiological recordings
233 2.10.1. DRG neurons
234 Currents from DRG neurons were accessed using whole cell patch-clamp technique and
235 were recorded at room temperature using a patch clamp amplifier (Axopatch 200B - Axon
236 Instruments, USA), controlled by pClamp7 software (Axon Instruments, USA) (Hamill et al.,
237 1981; Moraes et al., 2011). Pipettes open-tip resistance were < 1.5 MΩ. Pipettes were covered
238 with dental wax to approximately 0.1 mm from tips to reduce their electric capacitance and
239 thus facilitate cancellation of capacitive currents. Pipette solution was constituted by (in mM):
240 KCl, 30, K-Gluconate, 110; NaCl, 4; HEPES, 10; EGTA, 10; MgCl2, 5; MgATP, 2; NaGTP,
241 0.3; pH 7.0. The composition of bath solution was (in mM): NaCl, 140; KCl, 2.5; HEPES,
242 10;MES, 10; CaCl2, 2; MgCl2, 1; pH 7.4. Cell capacitance and series resistance were properly
243 compensated. Leak subtraction was performed using a -P/4 protocol. Elicited currents were
244 filtered at 1 kHz and sampled at 0.5 kHz (for outward currents) or at 20 kHz (for inward
245 currents) using a four-pole low-pass Bessel filter. Cells were held at -80 mV. Inward currents
246 were elicited by 100 ms depolarization pulses to 0 mV preceded by a 50-ms pre-pulse to -120
247 mV. Outward currents were evoked by a 200 ms depolarization pulses to 0 mV also from a
248 holding potential of -80 mV.
249 2.10.3. Xenopus laevis oocytes
250 For electrophysiological measurements in oocytes, two-electrode voltage clamp
251 recordings were performed at room temperature (18 - 22 oC) using a Geneclamp 500 amplifier
252 (Axon Instruments - Sunnyvale, California, USA) controlled by a pClamp data acquisition
9
253 system (Axon Instruments - Sunnyvale, California, USA). Bath solution composition was
254 ND96 (in mM): NaCl, 96; KCl, 2; CaCl2, 1.8; MgCl2, 2 and HEPES, 5 (pH 7.4). Voltage and
255 current electrodes were filled with 3M KCl. Resistances of both electrodes were kept between
256 0.7 and 1.5 MΩ. Elicited currents were sampled at 1 kHz and filtered at 0.5 kHz (for potassium
257 currents) or sampled at 20 kHz and filtered at 2 kHz (for sodium currents) using a four-pole
258 low-pass Bessel filter. Leak subtraction was performed using a-P/4 protocol. Shaker currents
259 were evoked by 500 ms depolarizations to 0 mV followed by a 500 ms pulse to -50 mV, from
260 a holding potential of -90 mV. Current traces of hERG channels were elicited by applying a
261 +40 mV pre-pulse for 2 s followed by a step to -120 mV for 2 s. Kv2.1 and Kv4.3 currents were
262 elicited by 500 ms pulses to +20 mV from a holding potential of -90 mV. Sodium current traces
263 were evoked by 100 ms depolarization to 0 mV from a holding potential of -90 mV. To assess
264 the concentration dependency of the peptide induced inhibitory effects, a concentration-
265 response curve was constructed, in which the percentage of current inhibition was plotted as a
266 function of toxin concentration. Data were fitted with the Hill equation: y = 100/[1 +
h 267 (IC50/[toxin]) ], were y is the amplitude of the toxin-induced effect, IC50 is the toxin
268 concentration at half-maximal efficacy, [toxin] is the toxin concentration, and h is the Hill
269 coefficient.
270 3. Results and Discussion
271 3.1. Toxicity, fractionation and mass fingerprinting of G. iheringi venom and isolation of
272 GiTx1
273 Toxicity of the total venom was checked in mouse by i.p. (n=2), i.c. (n=2) routes and in flies
274 (n=10) by intrathoracic injections. The first effects caused in mice by the i.p. injection of the
275 venom (800, 1,600, 3,200, 6,400 and 12,800 ng/mouse), occurred on average 30 minutes after
276 its application. These effects included paralysis and limited mobility at the lowest doses (800
277 ng and 1600 ng/animal), complete paralysis and death occurred in the highest doses (3,200 ng;
278 6,400 ng and 12,800 ng/animal). For all doses, when non-lethal, the effects were reversible and
10
279 after 24 hours, the mice had no visible symptoms. Higher toxicity in mice was observed when
280 the venom was injected by i.c via (100, 200, 400, 800 and 1,600 ng/mouse) being lethal at
281 1,600 ng/mouse. In insects that receive 15, 31, 62, 125, 250, 500 and 1000 ng/fly, toxicity was
282 visible from doses of 31 ng/fly, being lethal from 500 ng/fly. The LD50 calculated was 205.45
283 ng/fly (or 10.3 μg/g of fly). As most of the values reported from the literature were obtained
284 in tests using crickets the comparison of our results with these data is not adequate. The LD50
285 was not calculated to mouse, due to limitation on animal number and also because our first
286 objective was the characterization of novel insecticidal toxins. However, neurotoxic effects on
287 mouse were observed in all i.c tested doses (100, 200, 400, 800, 1400 ng), such as rotating
288 movements, tail erection, disorientation, piloerection, difficulty in locomotion followed by
289 paralysis. Grunts, screw movements and cyanosis were also noted in higher doses (800 and
290 1,600 ng/mouse). Few studies show comparative toxicity data from the venoms of tarantulas.
291 Lethal doses described vary between 210-950 μg of dry venom per mouse (20g). Despite our
292 values are far below that, Bücherl et al. 1971 [25] demonstrated that the toxicity and effects of
293 the venom vary widely with the injected route. Thus, the strict comparison of these data remains
294 complicated by the divergence of doses and routes used in the scarce studies with these venoms.
295 Crude venom fractionation and toxin purification were performed in two
296 chromatographic steps. Initially, 30 mg from G. iheringi venom were applied to a reversed-
297 phase C4 column, resulting in 33 fractions (Fig 1A). Each obtained fraction was submitted to
298 mass spectrometry analyses. The mass spectra results were summarized in Fig. 1B, which
299 represents the correspondence between molecular masses vs % ACN elution of venom
300 components.
11
301
302 Figure 1: G. iheringi venom fractionation and venom molecular mass profile obtained by
303 MALDI-TOF MS analysis. (A) Preparative RPC profile of G. iheringi venom on HPLC
304 system. The column (Vydac C4 250/22 mm) was equilibrated with 0.1% aqueous
305 trifluoroacetic acid, and 33 fractions eluted by a gradient of 0.1% trifluoroacetic acid in
306 acetonitrile (5 mL/min flow rate) as described in item 2.2. The elution was monitored at 214
307 nm. (B) Molecular mass vs % ACN elution plots of G. iheringi venom.
12
308
309 The results showed that the peptide fractions of G. iheringi venom comprised molecular
310 masses ranging from m/z 3,585 Da to 43,202 Da. Most of the eluted components seem to depict
311 a linear correlation between molecular masses and hydrophobicity. As other tarantula venoms
312 [3], G. iheringi components also displayed such bimodal behavior.
313 Following MS analysis, insect toxicity assays were performed in all eluted fractions
314 from the first chromatographic step, in exception of the fraction 20 that seemed to be pure and
315 was selected for further characterization. Table 1 shows a summary of the results with the
316 fractions obtained. Fractions 12 to 21 displayed high toxicity to adult flies including prolonged
317 paralyses and inability to fly. LD50 ranged from 3 to 171 ng/fly, hereby confirming the presence
318 of insecticidal toxins in G. iheringi venomous extract, as reported by Borges et al., 2016 [14].
319 Table 1. Insecticidal fractions from Grammostola iheringi venom and their respective 320 molecular masses. 321 Fractions Molecular weight of [M+H]+ in Da 12 nd* 13 6683; 6859; 6194; 6033; 7063 14 3867; 5182 15 5822; 5198; 3867; 3899 16 3978; 3884; 3633 17 6855; 3980; 3897; 4044 18 3999; 4165; 7998; 7999; 8909 19 4165; 4000; 6855 20 3588 21 3581; 3681 nd (not determinated) 322 323 The assessment of molecular masses was performed using reverse phase chromatography (RPC) on 324 HPLC followed by matrix assisted laser desorption/ionization mass spectrometry in offline mode. 325 Attempts to reach a reasonable amount of insecticidal pure peptide were carried out,
326 and, due to the highest protein concentration among the insecticidal fractions and its high state
327 of purity, the fraction 20 was further investigated. This sample was submitted to analytical RPC
328 using a linear gradient of ACN and a pure peptide - toxin GiTx1- was eluted with 36% of ACN
329 (Fig 2A).
13
330 Its toxicity was evaluated in flies and in mouse. Toxicity in flies ranged from 100 to
331 400 ng (causing paralyses) and death when 900 ng or above. Comparing the toxicities of GiTx1
332 (LD50 = 20.9 µg/g and LD100 = 45 µg/g ) to that of HwTx-II (LD50 = 127 + 54 µg/g and LD100
333 = 200 µg/g) from the Chinese spider Selenocosmia huwena [26], it shows that GiTx1 is six
334 times more toxic and also more lethal to the insects, than the HwTx-II. The toxicity trials in
335 mouse showed different effects: within the testing doses (i.p = 5 µg; i.c = 0.5 µg and 1
336 µg/animal) the toxin showed very low toxicity – causing reversible paralyses at the first hour
337 when injected i.c, at dose of 1µg/animal.
338 3.2. Peptide sequencing and sequence analyses
339 The molecular mass of GiTx1 was determined by MALDI-TOF/MS as 3,585.2 Da
340 (upper panel of Figure 2B). Because of a mass increase of 6 Da following the reduction of the
341 disulfide bonds, it is possible to assume that the native toxin contains 3 disulfide-bridges
342 (Figure 2B middle panel), and 6 cysteine residues, confirmed by the mass increase of 342 Da
343 (6 x 57 Da) after alkylation of thiol groups (Figure 2B, lower panel).
14
344
345
15
346 Figure 2: Purification of GiTx1 and MALDI-TOF MS analysis. (A) Analytical RPC of
347 subfraction 20 obtained from the preparative RPC step. The column (Sephasil Peptide C18 5µ
348 ST - 4.6 x 250 mm) was equilibrated with 0.1% aqueous trifluoroacetic acid, and GiTx1 was
349 eluted by a linear gradient of 0.1 % trifluoroacetic acid in acetonitrile according to item 2.2.
350 The elution was monitored at 214 nm. (B) Spectra of the native toxin and its chemically derived
351 forms. Each spectrum contains an insert representing the isotope distribution of GiTx1. In the
352 upper panel the monoisotopic mass of the native toxin is indicated. Mass increase of 6 Da
353 following the reduction of the disulfide bonds is depicted in middle panel. The 3 dissulfide-
354 bridged cysteines (6 cysteine residues) were confirmed by the mass increase of 342 Da (6x57
355 Da) following alkylation of the thiol groups in the lower panel.
356
357 In addition, GiTx1 was submitted to Edman’s sequencing in order to assess its primary
358 amino acids sequence. The sequence obtained of the reduced, alkylated and digested sample
359 and of the native sample comprises 30 amino acid residues, including 6 cysteine residues, that
360 were previously detected by MALDI-MS. Sequencing by Edman's degradation had
361 confidentially identified 29 amino acid residues out of 30 cycles, exception to the last cycle.
362 Comparing the theoretical mass of the obtained sequence and the experimental mass resulted
363 in nominal 128 Da, suggesting that a residue of lysine, glutamine or even an amidated glutamic
364 acid, could be the candidates for last residue of GiTx1 sequence. In order to solve the toxin
365 entire amino acid sequence, the reduced and alkylated toxin was subjected to ETD LTQ-
366 Orbitrap MS/MS analysis. Its primary structure was de novo sequenced, assisted by Edman
367 degradation’s data (Fig. 3A). Acetylation of primary amines was also carried out to confirm
368 the number of lysine residues that could be present in toxin sequence. The observed mass
369 increase of 252 Da is consistent with six primary amines, five in lysine residues and the free
370 amine at the N-terminus (Fig. 3B). The amino acids sequence data reported for GiTx1 toxin
371 was deposited in UniProt database under the accession number C0HJJ7.
16
372
373 Figure 3: High-resolution ETD Orbitrap MS/MS analysis of GiTx1 and acetylation of its
374 primary amines. (A) The reduced and alkylated GiTx1was subjected to ETD fragmentation
375 and its primary structure was de novo sequenced manually and assisted with the Edman
376 degradation result. The figure represents a deconvoluted MS/MS spectrum of the ion 656.80
377 m/z (z = 6). (B) The reduced and alkylated toxin was acetylated in order to confirm the number
378 of lysine residues present in the toxin. The observed mass increase is consistent with 6 primary
379 amines, which mean that the toxin indeed contains 5 lysine residues and a free N-terminal.
380 Acetylation = 42 Da.
381
17
382 3.3. Sequence alignments and molecular modeling
383 Searches on protein databases revealed a high similarity index of GiTx1 to other
384 tarantula peptides. The multiple alignment with such peptides is shown in Fig. 4.
385
386 Figure 4: Sequence and alignment of GiTx1 with other tarantula’s toxins. Full sequence
387 and alignment of GiTx1 with other similar proteins from spiders found at UniProtKB database.
388 Cysteine residues are highlighted in red. Identical and similar residues are boxed in black and
389 grey, respectively. The percentages of similarity (Sim.) of each sequence with GiTx1 are
390 indicated next to each sequence. The peptides κ-TRTX-Ps1b and κ-TRTX-Ps1a are from
391 Paraphysa scrofa venom. β-TRTX-Gr1a, β-TRTX-Gr1b, κ-TRTX-Gr2a and κ-TRTX-Gr2b
392 are from Grammostola rosea venom. Finally, β/ω-TRTX-Tp2a is from Thrixopelma pruriens
393 venom. Information about these sequences can be accessed in ArachnoServer [13].
394
395 GiTx1 displays cysteine residues framework similar to the aligned toxins, which
396 disulfide bridge pattern is CysI-CysIV, CysII-CysV and CysIII-CysIV. Following the remarkable
397 similarity concerning the cysteine positions, it is proposed that GiTx1 has a disulfide bridge
398 pattern similar to this group of spider toxins, adopting the ICK (Inhibitory Cystine Knot)
399 conformation [6], as a short loop ICK type toxin [3]. Apart from cysteine residues, it is possible
400 to notice also in the Fig.4, similarities and differences among amino acid residues that may
401 interfere in their pharmacological activities. The first residue of the GiTx1 sequence is differing
402 from the other peptides. It distinguishes itself from the others for being the only N-terminal
403 residue that is a serine and not a tyrosine. At position 11th, a glutamine is a distinct residue
18
404 facing the glutamic acid and serine residues. The same situation can be seen for the 14th
405 residue, the presence of a proline residue in GiTx1 contrasts the conserved lysine residues in
406 other known sequences. Other non-conservative amino acid residues from GiTx1 in
407 comparison to aligned sequences are also present at positions 27th and 29th. Despite the
408 observed differences, it is also valid to emphasize some similarities among the aligned
409 sequences. Overall, the toxin sequences from figure 5 displays a positive net charge (+1 to +4).
410 Other resemblance is in terms of positioning of hydrophobic residues. They are mainly located
411 between the first and the second cysteines residues, just before the fifth and the sixth cysteine
412 residues.
413 Among the aligned toxin sequences shown in fig.4, three of them had their solution
414 structures determined by two-dimensional NMR spectroscopy [27,28]. Solution structures of
415 κTrTx-Gr2a (GsmTx2), β/ω-TRTX-Tp2a (ProTx-II) and κTrTx-Ps1a (PaTx1), are available at
416 Protein Data Bank (PDB) under accession numbers 1LUP, 2N9T and 1V7F, respectively.
417 Using these three structures, it was achievable to predict the solution structure of GiTx1 by
418 molecular modeling tools (see item 2.6). The GiTx1 predicted structure is presented in Fig. 5.
19
419
420 Figure 5: Molecular model of GiTx1. (A) Stereo view of the peptide and (B) its rotated view
421 180 degrees to the left of the orientation in panel A (counterclockwise). The β-strands are in
422 light blue, loop regions in green and the disulfide bridges in yellow. Blue colors represent
423 positive charges, as red colors represent negative charges. (C) Electrostatic potential of the
424 molecular surface of GiTx1. (D) Electrostatic potential of GiTx1, rotated 180 degrees to the
425 left of the orientation in panel C (counterclockwise).
426
427 The molecular model for GiTx1 presents two distinct loops, and two short stranded
428 antiparallel β sheets. The model shows an overall charge distribution that reveals an
429 electrostatic anisotropy represented by the dipole moment shown in Fig. 5C and 5D, in which
430 electrostatic potential of the molecular surface of GiTx1 is shown. Charged residues are visibly
431 located in one side of the structure, while the opposite side is composed by hydrophobic amino
432 acids (TRP5, MET6, TRP7, THR8, LEU23 and TRP24). These characteristics are similar to
433 β/ω-TRTX-Tp2a [29,30]. Henriques et al., 2016 demonstrated the membrane binding of the
20
434 hydrophobic face of β/ω-TRTX-Tp2a and its importance for the interaction with the sodium
435 channel voltage sensor domain, proposing a correlation between membrane binding affinity
436 and its inhibitory potential. β/ω-TRTX-Tp2a insertion in the membrane favors its correct
437 orientation with the channel, facilitating the electrostatic interactions between the polar amino
438 acids of the toxin with the channel [30].
439 3.4. Electrophysiological recordings
440 Considering the structural similarity with other tarantula toxins and their biological
441 activities, electrophysiological experiments were conducted to determine if GiTx1 modulates
442 inward or outward ionic currents in DRG neurons. These experiments showed that the toxin,
443 at a concentration of 2M, GiTx1 reversibly inhibited 40% of inward current and 20% of
444 outward currents, suggesting its action upon voltage-gated sodium and potassium channels
445 (Fig. 6).
446
447
448 Figure 6: Normalized current plots from whole-cell patch clamp recordings. Effects of
449 GiTx1 (2M) upon sodium and potassium (inward and outward) currents from DRG cells
450 (n=8).
451 In addition, the action of GiTx1 was investigated upon a broad variety of sodium and
452 potassium channels subtypes expressed in X. laevis oocytes. The mammalian (rNav1.2,
453 rNav1.3, rNav1.4, hNav1.5, mNav1.6, hNav1.8), insect (DmNav1, BgNav1) and arachnid
21
454 (VdNav1) voltage gated sodium channel (VGSC) subtypes, as well as the voltage gated
455 potassium (VGPC) insect channel Shaker (Shaker IR) and mammalian channels Shab (rKv2.1),
456 Shal (rKv4.3) and erg1 (hERG) - were tested by two electrode voltage-clamp technique using
457 2µM of GiTx1. The electrophysiological recordings of the action of GiTx1 on these channels
458 are shown in Fig. 7A. The results show that at 2 µM, GiTx1 was able to inhibit the ionic currents
459 through all tested VGSCs isoforms (Fig. 7A). Among the VGPCs, the toxin showed complete
460 and partial inhibition on Kv4.3 and hERG channels respectively, but did not have any effect
461 on Shaker IR and Kv2.1 (Fig.7A). Fig. 7B summarizes GiTx1 effects in terms of percentage of
462 inhibition on the tested channels. The toxin effectiveness upon the inhibition of the ionic
463 currents through the sodium channels Nav1.6 and VdNav and the potassium channel hERG
464 was determined by dose-response curves, as shown in Fig. 7C. Measured IC50 values were
465 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, indicating that GiTx1a has
466 a similar affinity for these channels. In contrast, the IC50 value obtained for hERG channel
467 was 3695.17 127.22 nM. This concentration is 25 times higher than for the sodium channel
468 subtypes, suggesting that GiTx1 has a much higher affinity for sodium channels.
469
22
470 23
471 Figure 7. Effects of GiTx1 upon voltage-gated ion channels subtypes, expressed in 472 Xenopus laevis oocytes. (A) Screening of GiTx1 at 2µM. The tested mammalian isoforms were 473 raised from rat (r), mouse (m) and human (h). The invertrebrate subtypes were originated from 474 fruit-fly (Dm, Drosophyla melanogaster), cockroach (Bg, Blatella germanica) and mite (Vd, 475 Varroa destructor). Representative whole-cell current traces in the absence and in the presence 476 of 2 µM GiTx1 are shown for each channel. Dotted line indicates the zero-current level. The * 477 marks steady state current traces after application of GiTx1 (2µM) (n 3). (B) GiTx1 (2µM) 478 blocking effect on tested sodium and potassium channels isoforms (n 3). (C) Dose-response 479 curves of GiTx1 on mNav1.6, VdNav and hERG channels. Curves were obtained by plotting 480 the percentage of blocked current as a function of increasing toxin concentrations. All data are 481 presented as mean ± standard error (n ≥ 3). 482 483 Despite the differences in affinities presented for sodium and potassium voltage-gated
484 sodium channels, GiTx1 toxin can be considered a promiscuous toxin. The action of
485 promiscuous toxins is not specific for an ion channel family and they can act on ion channels
486 with different selectivity [3,31]. This promiscuous activity upon ion channels is largely found
487 in ICK toxins from tarantula venoms. For instance, Hainan toxins showed high affinity for Kv
488 channels and low affinity for Cav channels, whereas the opposite was observed for ω-TRTX-
489 Gr1a (GsTxSIA) [32]. Likewise, several works have been reporting the promiscuous activity
490 of tarantula’s toxins [33–36]. As consequence of the promiscuity displayed by these toxins, its
491 functional prediction becomes a difficult task. For example, κ-TRTX-Ps1a and κ-TRTX-Gr2a,
492 that were taken as templates for the prediction of the GiTx1 structure, are toxins that share 90%
493 of similarity in terms of sequence, however, their pharmacological actions have been reported
494 as modulators of Kv4.2 and Kv4.3 channels [37] or as blockers of mechano-sensitive channel
495 [27]. Unlike, μ-TRTX-Hh2a (HwTx4) and β-TRTX-Ps1a (PaurTx3) toxins share only 40% of
496 structural similarity; nevertheless they modulate neuronal sodium channels with equivalent
497 affinities [38]. Besides that, it was reported that the activity of some toxins upon specific ion
498 channels can be dose-dependent, which suggests that spider toxins have a number of putative
499 binding sites able to alter the function of a voltage-gated ion channel in different modes, at the
24
500 same time [34]. These findings emphasize the importance of the functional characterization of
501 spider toxins activity upon different ion channels and at different toxin concentrations. Thus,
502 additional studies may further illustrate other possible pharmacological actions of GiTx1.
503 Nevertheless, considering the ion channel activities reported for GiTx1 in this study and its
504 high homology to tarantula toxins with β action on sodium channels (Fig. 4), this toxin should
505 be renamed as β/κ-theraphotoxin-Gi1a according to the rational nomenclature proposed by
506 King et al, 2008 [39].
507 4. Final remarks
508 In this report, we have described the isolation, sequencing and the structural and
509 pharmacological characterization of GiTx1, the first peptide purified from the venom of the
510 spider G. iheringi. Additionally, the peptide mass diversity of G. iheringi venom was obtained
511 by mass spectrometry analysis.
512 A current database investigation revealed a similarity of GiTx1 with theraphosid toxins
513 bearing the short loop ICK toxin group. This was confirmed by its predicted 3D structure,
514 which showed that GiTx1 has two loops, two short stranded antiparallel β and an electrostatic
515 anisotropy, that are some features of short loop ICK toxin group. The electrophysiological
516 experiments performed on DRG neurons and X. laevis oocytes show that GiTx1 was effective
517 upon voltage-gated sodium and potassium channels. The action on invertebrate channels was
518 one of the identified targets of the toxin underlying the toxic effects observed in flies injected
519 with the venom as well as with this toxin. In summary, the present work provided a relevant
520 contribution for the description of the toxicity and the molecular diversity of the spider G.
521 iheringi venom and for the structural and functional characterization of a novel toxin belonging
522 to the short loop ICK family.
523
524 Acknowledgments
25
525 The authors are thankful to Dr. Sylvia Lucas (Laboratory of Arthropods from Butantan Institute
526 – São Paulo, SP, Brazil) by careful identification of specimens Grammostola iheringi.
527 We are grateful D. J. Snyders for sharing rKv2.1 and rKv4.3. The Shaker IR clone was kindly
528 provided by G. Yellen. We thank M. Keating for sharing hERG. We are grateful to G. Mandel
529 (Stony Brook University, Stony Brook, NY) for providing the rNav1.4 clone, R.G. Kallen
530 (University of Pennsylvania, PA) for providing hNav1.5, A.L. Goldin (University of
531 California, Irvine, CA) for providing mNav1.6, J.N. Wood (University College, London, UK)
532 for providing rNav1.8, S.H. Heinemann (Friedrich-Schiller-Universita, Jena, Germany) for
533 sharing rβ1, and M.S. Williamson (Institute of Arable Crops Research Rothamsted, Harpenden,
534 UK) for providing DmNav1 and tipE clone. J.T. was funded by GOC2319N and GOA4919N
535 (FWO-Vlaanderen) and CELSA/17/047 (BOF - KU Leuven). KU Leuven funding
536 (PDM/19/164) supports S.P. We thank the following Brazilian Agencies for the financial
537 support: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES, Fundação
538 de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG and Conselho Nacional de
539 Desenvolvimento Científico e Tecnológico – CNPq.
540
541 References
542 [1] L. Kuhn-Nentwig, R. Stöcklin, W. Nentwig, Venom composition and strategies in
543 spiders. is everything possible?, 2011. https://doi.org/10.1016/B978-0-12-387668-
544 3.00001-5.
545 [2] E. Grishin, Polypeptide neurotoxins from spider venoms, Eur. J. Biochem. (1999).
546 https://doi.org/10.1046/j.1432-1327.1999.00622.x.
547 [3] P. Escoubas, L. Rash, Tarantulas: Eight-legged pharmacists and combinatorial
548 chemists, Toxicon. (2004). https://doi.org/10.1016/j.toxicon.2004.02.007.
549 [4] G.F. King, M.C. Hardy, Spider-Venom Peptides: Structure, Pharmacology, and
550 Potential for Control of Insect Pests, Annu. Rev. Entomol. (2013).
26
551 https://doi.org/10.1146/annurev-ento-120811-153650.
552 [5] N.J. Saez, V. Herzig, Versatile spider venom peptides and their medical and
553 agricultural applications, Toxicon. (2019).
554 https://doi.org/10.1016/j.toxicon.2018.11.298.
555 [6] R.S. Norton, P.K. Pallaghy, The cystine knot structure of ion channel toxins and
556 related polypeptides, in: Toxicon, 1998. https://doi.org/10.1016/S0041-
557 0101(98)00149-4.
558 [7] X.H. Wang, M. Connor, R. Smith, M.W. Maciejewski, M.E.H. Howden, G.M.
559 Nicholson, M.J. Christie, G.F. King, Discovery and characterization of a family of
560 insecticidal neurotoxins with a rare vicinal disulfide bridge, Nat. Struct. Biol. (2000).
561 https://doi.org/10.1038/75921.
562 [8] Z. Liao, J. Cao, S. Li, X. Yan, W. Hu, Q. He, J. Chen, J. Tang, J. Xie, S. Liang,
563 Proteomic and peptidomic analysis of the venom from Chinese tarantula Chilobrachys
564 jingzhao, Proteomics. (2007). https://doi.org/10.1002/pmic.200600785.
565 [9] J. Chen, L. Zhao, L. Jiang, E. Meng, Y. Zhang, X. Xiong, S. Liang, Transcriptome
566 analysis revealed novel possible venom components and cellular processes of the
567 tarantula Chilobrachys jingzhao venom gland, Toxicon. (2008).
568 https://doi.org/10.1016/j.toxicon.2008.08.003.
569 [10] E. Diego-García, S. Peigneur, E. Waelkens, S. Debaveye, J. Tytgat, Venom
570 components from Citharischius crawshayi spider (Family Theraphosidae): Exploring
571 transcriptome, venomics, and function, Cell. Mol. Life Sci. 67 (2010) 2799–2813.
572 https://doi.org/10.1007/s00018-010-0359-x.
573 [11] X. Tang, Y. Zhang, W. Hu, D. Xu, H. Tao, X. Yang, Y. Li, L. Jiang, S. Liang,
574 Molecular diversification of peptide toxins from the tarantula haplopelma hainanum
575 (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic
576 analyses, J. Proteome Res. (2010). https://doi.org/10.1021/pr1000016.
27
577 [12] C.B.F. Mourão, F.N. Oliveira, A.C. E Carvalho, C.J. Arenas, H.M. Duque, J.C.
578 Gonçalves, J.K.A. Macêdo, P. Galante, C.A. Schwartz, M.R. Mortari, M. de F.M.
579 Almeida Santos, E.F. Schwartz, Venomic and pharmacological activity of
580 Acanthoscurria paulensis (Theraphosidae) spider venom, Toxicon. (2013).
581 https://doi.org/10.1016/j.toxicon.2012.11.008.
582 [13] V. Herzig, D.L.A. Wood, F. Newell, P.A. Chaumeil, Q. Kaas, G.J. Binford, G.M.
583 Nicholson, D. Gorse, G.F. King, ArachnoServer 2.0, an updated online resource for
584 spider toxin sequences and structures, Nucleic Acids Res. (2011).
585 https://doi.org/10.1093/nar/gkq1058.
586 [14] M.H. Borges, S.G. Figueiredo, F. V. Leprevost, M.E. De Lima, M. do N. Cordeiro,
587 M.R.V. Diniz, J. Moresco, P.C. Carvalho, J.R. Yates, Venomous extract protein profile
588 of Brazilian tarantula Grammostola iheringi: Searching for potential biotechnological
589 applications, J. Proteomics. (2016). https://doi.org/10.1016/j.jprot.2016.01.013.
590 [15] M. Richardson, A.M.C. Pimenta, M.P. Bemquerer, M.M. Santoro, P.S.L. Beirao, M.E.
591 Lima, S.G. Figueiredo, C. Bloch, E.A.R. Vasconcelos, F.A.P. Campos, P.C. Gomes,
592 M.N. Cordeiro, Comparison of the partial proteomes of the venoms of Brazilian
593 spiders of the genus Phoneutria, Comp. Biochem. Physiol. - C Toxicol. Pharmacol.
594 142 (2006) 173–187. https://doi.org/10.1016/j.cbpc.2005.09.010.
595 [16] S. Yuen, M.W. Hunkapiller, J. Wilson, P.A.U.M. Yuan, Sylvia yuen, michael w.
596 hunkapiller, 15 (1988) 5–15.
597 [17] A. Bateman et al. UniProt: The universal protein knowledgebase, Nucleic Acids Res.
598 (2017). https://doi.org/10.1093/nar/gkw1099.
599 [18] F. Sievers, A. Wilm, D. Dineen, T.J. Gibson, K. Karplus, W. Li, R. Lopez, H.
600 McWilliam, M. Remmert, J. Söding, J.D. Thompson, D.G. Higgins, Fast, scalable
601 generation of high-quality protein multiple sequence alignments using Clustal Omega,
602 Mol. Syst. Biol. (2011). https://doi.org/10.1038/msb.2011.75.
28
603 [19] A. Šali, T.L. Blundell, Comparative protein modelling by satisfaction of spatial
604 restraints, J. Mol. Biol. (1993). https://doi.org/10.1006/jmbi.1993.1626.
605 [20] R.A. Laskowski, M.W. MacArthur, D.S. Moss, J.M. Thornton, PROCHECK: a
606 program to check the stereochemical quality of protein structures, J. Appl. Crystallogr.
607 (1993). https://doi.org/10.1107/s0021889892009944.
608 [21] D. Eisenberg, R. Lüthy, J.U. Bowie, VERIFY3D: Assessment of protein models with
609 three-dimensional profiles, Methods Enzymol. (1997). https://doi.org/10.1016/S0076-
610 6879(97)77022-8.
611 [22] M. Wiederstein, M.J. Sippl, ProSA-web: Interactive web service for the recognition of
612 errors in three-dimensional structures of proteins, Nucleic Acids Res. (2007).
613 https://doi.org/10.1093/nar/gkm290.
614 [23] S.C. Lovell, I.W. Davis, W.B.A. Iii, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S.
615 Richardson, D.C. Richardson, Structure validation by Calpha geometry: Phi,psi and
616 Cbeta deviation, Proteins. (2003).
617 [24] E.R. Moraes, E. Kalapothakis, L.A. Naves, C. Kushmerick, Differential effects of
618 tityus bahiensis scorpion venom on tetrodotoxin-sensitive and tetrodotoxin-resistant
619 sodium currents, Neurotox. Res. (2011). https://doi.org/10.1007/s12640-009-9144-8.
620 [25] W. BÜCHERL, Classification, Biology, and Venom Extraction of Scorpions, in:
621 Venom. Anim. Their Venoms, 1971. https://doi.org/10.1016/b978-0-12-138903-
622 1.50019-3.
623 [26] Q. Shu, S.P. Liang, Purification and characterization of huwentoxin-II, a neurotoxic
624 peptide from the venom of the Chinese bird spider Selenocosmia huwena, J. Pept. Res.
625 (1999). https://doi.org/10.1034/j.1399-3011.1999.00039.x.
626 [27] R.E. Oswald, T.M. Suchyna, R. McFeeters, P. Gottlieb, F. Sachs, Solution structure of
627 peptide toxins that block mechanosensitive ion channels, J. Biol. Chem. (2002).
628 https://doi.org/10.1074/jbc.M202715200.
29
629 [28] B. Chagot, P. Escoubas, E. Villegas, C. Bernard, G. Ferrat, G. Corzo, M. Lazdunski,
630 H. Darbon, Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4
631 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus ,
632 Protein Sci. (2004). https://doi.org/10.1110/ps.03584304.
633 [29] S.T. Henriques, E. Deplazes, N. Lawrence, O. Cheneval, S. Chaousis, M. Inserra, P.
634 Thongyoo, G.F. King, A.E. Mark, I. Vetter, D.J. Craik, C.I. Schroeder, Interaction of
635 tarantula venom peptide ProTx-II with lipid membranes is a prerequisite for its
636 inhibition of human voltage-gated sodium channel NaV 1.7, J. Biol. Chem. (2016).
637 https://doi.org/10.1074/jbc.M116.729095.
638 [30] H. Xu, T. Li, A. Rohou, C.P. Arthur, F. Tzakoniati, E. Wong, A. Estevez, C. Kugel, Y.
639 Franke, J. Chen, C. Ciferri, D.H. Hackos, C.M. Koth, J. Payandeh, Structural Basis of
640 Nav1.7 Inhibition by a Gating-Modifier Spider Toxin, Cell. (2019).
641 https://doi.org/10.1016/j.cell.2018.12.018.
642 [31] A.J. Agwa, S. Peigneur, C.Y. Chow, N. Lawrence, D.J. Craik, J. Tytgat, G.F. King,
643 S.T. Henriques, C.I. Schroeder, Gating modifier toxins isolated from spider venom:
644 Modulation of voltage-gated sodium channels and the role of lipid membranes, J. Biol.
645 Chem. (2018). https://doi.org/10.1074/jbc.RA118.002553.
646 [32] Y. Li-Smerin, K.J. Swartz, Gating modifier toxins reveal a conserved structural motif
647 in voltage-gated Ca2+ and K+ channels, Proc. Natl. Acad. Sci. U. S. A. (1998).
648 https://doi.org/10.1073/pnas.95.15.8585.
649 [33] S. Sokolov, R.L. Kraus, T. Scheuer, W. a Catterall, Inhibition of sodium channel
650 gating by trapping the domain II voltage sensor with protoxin II., Mol. Pharmacol. 73
651 (2008) 1020–1028. https://doi.org/mol.107.041046 [pii]\r10.1124/mol.107.041046.
652 [34] E. Redaelli, R.R. Cassulini, D.F. Silva, H. Clement, E. Schiavon, F.Z. Zamudio, G.
653 Odell, A. Arcangeli, J.J. Clare, A. Alago, R.C. Rodríguez De La Vega, L.D. Possani,
654 E. Wanke, Target promiscuity and heterogeneous effects of tarantula venom peptides
30
655 affecting Na+ and K+ ion channels, J. Biol. Chem. (2010).
656 https://doi.org/10.1074/jbc.M109.054718.
657 [35] J.K. Klint, S. Senff, D.B. Rupasinghe, S.Y. Er, V. Herzig, G.M. Nicholson, G.F. King,
658 Spider-venom peptides that target voltage-gated sodium channels: Pharmacological
659 tools and potential therapeutic leads, Toxicon. 60 (2012) 478–491.
660 https://doi.org/10.1016/j.toxicon.2012.04.337.
661 [36] J.J. Smith, V. Herzig, M.P. Ikonomopoulou, S. Dziemborowicz, F. Bosmans, G.M.
662 Nicholson, G.F. King, Insect-active toxins with promiscuous pharmacology from the
663 African theraphosid spider Monocentropus balfouri, Toxins (Basel). (2017).
664 https://doi.org/10.3390/toxins9050155.
665 [37] S. Diochot, M.D. Drici, D. Moinier, M. Fink, M. Lazdunski, Effects of phrixotoxins on
666 the Kv4 family of potassium channels and implications for the role of Itoj in cardiac
667 electrogenesis, Br. J. Pharmacol. (1999). https://doi.org/10.1038/sj.bjp.0702283.
668 [38] F. Bosmans, L. Rash, S. Zhu, S. Diochot, M. Lazdunski, P. Escoubas, J. Tytgat, Four
669 novel tarantula toxins as selective modulators of voltage-gated sodium channel
670 subtypes., Mol. Pharmacol. 69 (2006) 419–429.
671 https://doi.org/10.1124/mol.105.015941.
672 [39] G.F. King, M.C. Gentz, P. Escoubas, G.M. Nicholson, A rational nomenclature for
673 naming peptide toxins from spiders and other venomous animals, Toxicon. 52 (2008)
674 264–276. https://doi.org/10.1016/j.toxicon.2008.05.020.
675
31