Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans

Journal Pre-proof Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans I.A. García-Guerrero, E. Cárcamo-Noriega, F. Gómez-Lagunas, E. GonzálezSantillán, F.Z. Zamudio, G.B. Gurrola, L.D. Possani PII:

S0041-0101(19)30737-8

DOI:

https://doi.org/10.1016/j.toxicon.2019.11.004

Reference:

TOXCON 6237

To appear in:

Toxicon

Received Date: 9 May 2019 Revised Date:

8 November 2019

Accepted Date: 11 November 2019

Please cite this article as: García-Guerrero, I.A., Cárcamo-Noriega, E., Gómez-Lagunas, F., GonzálezSantillán, E., Zamudio, F.Z., Gurrola, G.B., Possani, L.D., Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans, Toxicon (2019), doi: https://doi.org/10.1016/j.toxicon.2019.11.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Biochemical characterization of the venom from the Mexican scorpion Centruroides

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ornatus, a dangerous species to humans.

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García-Guerrero, I.A1+, Cárcamo-Noriega, E1+, Gómez-Lagunas, F.2, González-Santillán,

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E.1, Zamudio, F.Z1, Gurrola, G.B.1, Possani, L.D.1,*

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+

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1

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Universidad Nacional Autonoma de Mexico, Avenida Universidad, 2001, Cuernavaca,

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Morelos, 62210, Mexico.

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Contributed equally in this work.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia,

Departamento de Fisiología, Facultad de Medicina, Universidad Nacional Autonoma de

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Mexico, Ciudad de Mexico 04510, Mexico

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*Corresponding author: Lourival Domingos Possani, Departmento de Medicina Molecular y

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Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico,

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Avenida Universidad, 2001, Colonia Chamilpa, Cuernavaca, Morelos, 62210, Mexico.Tel.:

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+52 (777) 3171209 E-mail: [email protected]

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ABSTRACT

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Every year in Mexico, around 300,000 people suffer from accidents related to scorpion

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stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose

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venom characterization is described here. From this venom, a total of 114 components

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were found using chromatographic separation and mass spectrometry analysis. The most

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abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da

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respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using

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intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three

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new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond

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to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these

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peptides using heterologously expressed human Na+-channels revealed a typical β-toxin

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effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in

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vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3

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among other β-toxins from Centruroides scorpions. Peptide Co52 segregates among

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peptides of unknown defined functions.

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Running title: Mammalian sodium-channel toxins from Centruroides ornatus

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Keywords: Centruroides ornatus; mass-spectrometry analysis; primary structure, scorpion

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toxin; sodium-channel

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1. Introduction

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The official legislation of the Health Service of Mexico (Norma Oficial Mexicana NOM-033-

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SSA2-2011) uses the Spanish term “alacranismo” (in English scorpionism) to define the

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public health problem caused by scorpion stings in communities of certain areas of

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Mexico. This is an important issue because close to 300,000 people in Mexico suffer

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accidents related to scorpion stings every year (Jiménez-Vargas et al., 2017). Mexico

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harbors the highest biodiversity of scorpion species in the world, with 289 species

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described until now (Santibanez-Lopez et al., 2015). Although, and fortunately, only about

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10% of them are considered dangerous to humans. The species with medical relevance all

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belong to the genus Centruroides of the Buthidae family (Ponce-Saavedra et al., 2016 ).

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The LD50 was determined for 13 different species of scorpions of the genus Centruroides

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of Mexico (Riaño-Umbarila et al., 2017). However, a more recent publication shows that at

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least 21 different species of Mexican scorpions are dangerous to humans (González-

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Santillán and Possani, 2018).

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Scorpion venoms contain a complex mixture of compounds, including enzymes such as

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hyaluronidase, protease and phospholipase. They might also contain mucoproteins, free

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amino acids, nucleotides, lipids, biogenic amines, heterocyclic compounds, inorganic salts,

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and mainly peptides that recognize ion-channels (Na+, K+, Ca2+ and Cl-) (Quintero-

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Hernández et al., 2013). The peptides that modify the gating mechanism of Na+- channels,

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or block K+-channels, have been widely studied in Mexican scorpion venoms because they

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are known to produce the symptoms of poisoning (Possani et al., 1999; Santibanez-Lopez

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et al., 2015). From a medical point of view, the sodium-channel scorpion toxins (NaScTxs)

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that affect mammalian channels represent the most relevant components for human

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intoxication (Ortiz et al., 2015). These peptides are single-chain polypeptides of 58-76

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amino acids cross-linked by four disulfide bonds. Depending on their mechanism in which

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affects the channels they are classified into two main categories: i) α-scorpion toxins (α-

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NaScTxs), which delay the fast inactivation process; and ii) β-scorpion toxins (β-NaScTxs)

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that trigger the channel opening at more negative potentials (Couraud et al., 1982;

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Quintero-Hernández et al., 2013; Rodriguez de la Vega and Possani, 2005). Both classes

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of toxins produce an anomalous depolarization, and interfere with cell communication, thus

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leading to severely impaired biological functions (mainly in muscles and nerves), and may

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cause death (Possani et al., 1999).

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Among the well-studied venom of scorpions from Mexico that represent medical relevance

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are those from the species: Centruroides noxius, Centruroides suffusus, Centruroides

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tecomanus, Centruroides limpidus, Centruroides sculpturatus, Centruroides infamatus and

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Centruroides elegans (Possani et al., 1999). Nonetheless, there are still other species from

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which the venom composition has not been studied. One of these species is Centruroides

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ornatus that was considered a subspecies of Centruroides infamatus for a long time.

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Recent studies on morphological and genetic aspects (Towler et al., 2001) lead to

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reestablishing its status as an independent species (De Armas and Martin-Frias, 2008). C.

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ornatus is mainly found in the states of Michoacán, Jalisco and Guanajuato. The lethal

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dose of the venom of C. ornatus is among those recently described (Riaño-Umbarila et al.,

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2017). It has a LD50 of 13 µg of venom per 20 g of mouse body weight. Its venom

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composition however, is not well-defined (subject of this communication).

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This report is aimed at identifying the components from the soluble venom of C. ornatus,

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with emphasis on peptides that affect the function of mammalian sodium-channels. Using

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chromatographic and mass-spectrometry (MS) analysis, we found 114 compounds from

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which we isolated three peptides lethal to mice (Co1, Co2 and Co3). Electrophysiology

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analysis of these peptides in human sodium-channels classified them among the other β-

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NaScTxs from scorpions from Centruroides genus. A phylogenetic analysis of the purified

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peptides is included. A non-human toxic peptide (Co52) was also characterized.

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2. Materials and Methods

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2.1 Source of venom

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Under permission of the Mexican authority SEMARNAT (reference number FAUT-0305),

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we collected C. ornatus specimens in Morelia, Michoacán. The scorpions were brought to

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our lab and milked by electrical stimulation of the telson as previously recommended

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(Rabia et al., 2016). Venom collected was dissolved in water and centrifuged at 10,000 g

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for 10 min at 4 ºC. The supernatant (soluble fraction) was lyophilized and kept at -20 ºC

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until use. Insoluble components and cellular debris were discarded. For a protein

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estimation, we assumed that one unit of absorbance at a wavelength of 280 nm

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corresponds to a concentration of 1 mg of protein per mL.

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2.2 Chromatographic procedures

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In order to evaluate the peptide composition of the venom, 1.2 mg of lyophilized venom

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was applied in to a HPLC system, using an analytical C18 reverse-phase column (4.6 ×

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250 nm) from Vydac (Hisperia, CA, USA), and eluted with a gradient from solution A

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(0.12% TFA in water) to 60% solution B (0.10% TFA in acetonitrile) at 1 mL/min for 60 min.

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Principal RP-HPLC peaks were analyzed by mass spectrometry (MS).

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The isolation of the peptides was performed in a 3 steps chromatographic scheme

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previously reported with modification to scale to minimal venom requirement (Ramirez-

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Dominguez et al., 2002). First, the venom of 128 adult scorpions (89 females and 39

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males, approximately 28 mg) was fractioned by gel filtration using Sephadex G50-fine

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(Sigma Aldrich) into a column (0.9 x 50 cm) equilibrated and run with 20 mM ammonium

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acetate buffer, pH 4.7. The flow rate was 15 mL/h and fractions of 1 mL per tube were

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collected and pooled into three fractions according to the absorbance at 280 nm. Fraction

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II was further separated by cation-exchange chromatography using a column of carboxy-

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methyl cellulose (CMC) (3 cm x 8 cm) equilibrated with 20 mM ammonium acetate buffer.

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Fractions were eluted by a linear gradient from 20 mM to 500 mM ammonium acetate, pH

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4.7 at 2 mL/min during 240 minutes. Pooled fractions were lyophilized and stored at -20°C

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until used. Finally, material from these fractions was solubilized in solution A and peptides

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isolated by RP-HPLC under condition above described.

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2.3 Amino acid sequence by Edman degradation

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Isolated peptides were sequenced by Edman degradation. For this, approximately 0.5

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nmol of the peptide was analyzed using a Shimadzu Protein Sequencer PPSQ-31A/33A

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(Columbia, Maryland, USA). The N-terminus sequence of the peptides was obtained in

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native state and reduced and alkylated form in order to identify cysteine residues,

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according to the protocol described previously (Olamendi-Portugal et al., 2016). In order to

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complete sequencing, reduced and alkylated peptides were digested using specific

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endoproteases. The proteolytic enzymes were from the company Roche Diagnostics

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GmbH (Mannheim, Germany).

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(peptide/enzyme): Glu-C endoprotease incubated at 37 ºC for 4.5 hours in a 25 mM

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ammonium bicarbonate buffer pH 8.4; Asp-N endoprotease incubated over-night at 37ºC

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in a 50 mM sodium phosphate buffer pH 8.2; and the enzyme Lys-C endoprotease was

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added into a 25 mM Tris-HCl buffer pH 8.0 containing 1 mM EDTA and incubated at 37 ºC

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for 18 hours. The peptide fragments obtained after enzymatic digestion were separated by

Three enzymes were used in a ratio of 32:1

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RP-HPLC under conditions previously mentioned and sequenced.

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2.4 Mass spectrometry

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Fractions obtained from HPLC were dissolved in 60% acetonitrile, 0.1% acetic acid and

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analyzed in a LCQFleet apparatus from Thermo Fisher Scientific Inc. (San Jose, CA,

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USA). The precision of the MS analysis with this equipment for peptides on the range of

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7000 Da is circa 1 Da.

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2.5 Toxic activity

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Fraction and isolated peptides were tested in animal model following bioethical standards,

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using a minimal number of animal and with the approval of the Animal Welfare Committee

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of our Institute. For toxicity assays we used mice (Mus musculus), chicks (Gallus gallus),

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house crickets (Acheta domesticus) and woodlice (terrestrial crustacean from the

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Armadillidiidae family). Sample administration for mice was performed by intraperitoneal

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(i.p) injection using a volume of 100 µL. For chick the sample was subcutaneously injected

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under the left wing using a sample of 100 µL volume. For crickets the sample was

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intraabdominally injected between the 4th and 5th spiracles using a sample with 10 µL

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volume. For woodlice was an injection into the pleon using a sample volume of 5 µL.

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Administered doses for i.p. injection in mice corresponded to 15 µg of whole soluble

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venom and 5 µg of fractions from CM-cellulose and/or isolated peptides. Doses for the

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assay in chicks, crickets and woodlice were 10 µg, 10 µg and 0.5 µg of isolated peptides

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respectively. Injected animals were supplied with water and food ad libitum and observed

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concerning possible envenomation symptoms. After 24 h of observation the surviving

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animals were disposed.

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2.6 Electrophysiology procedures

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For the electrophysiology evaluation, HEK293 cells stably expressing human voltage-

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gated sodium channels (VGSCs) of subtypes hNav1.1, hNav1.2, hNav1.3, hNav1.4,

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hNav1.5 and hNav1.6 channels and CHO cells stably expressing hNav1.7 were used

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(kindly donated by Prof. Enzo Wanke, Milano University, Italy, described in (Oliveira et al.,

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2004).

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DMEM/F12 media supplemented with 10% fetal serum bovine (FSB), and the selected

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antibiotic G418 bisulphate salt (100 µg/1mL). On the day of the experiments the cells were

Cells were kept in culture at 37 ºC in a humidified 5% CO2 atmosphere in

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plated on glass coverslips for further experimentation, as previously reported for HEK293

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cells stably expressing ion channels (Gómez-Lagunas et al., 2017).

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Peptides were tested at concentration up to 200 nM according to their extinction coefficient

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determined from their amino acid sequence based on absorbance at 280 nm using

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ExPASy-ProtParam tool. . The values obtained were for Co1= 2.89 (mg/ml)-1cm-1, Co2=

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3.07 (mg/ml)-1cm-1, Co3= 3.39 (mg/ml)-1cm-1 and Co52= 2.89 (mg/ml)-1cm-1. Sodium

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currents were recorded with the patch-clamp technique during step depolarization from -

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120 to 30 mV in 100 ms steps of 10 mV from the resting potential (-120 mV).

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For evaluating the voltage-dependence of inactivation, each depolarization step was

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followed by 50 ms step at full-activation potential (−10 mV or −20 mV in the case of hNav

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1.4 and hNav 1.5 channels).

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In order to enhance any shift to more negative potential caused by the peptides, a short

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pre-pulse (5 ms at 50 mV) was applied 50 ms before the depolarization steps. Sodium

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current (INa+) reads were performed using a MultiClamp 700 B amplifier coupled to an

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analog-digital converter Digidata 1440A and software pCalmp10 (Molecular Devices,

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Sunnyvale CA, USA). The extracellular solution used was: NaCl 130 mM, KCl 5 mM,

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CaCl2 2 mM, MgCl2 2 mM, HEPES 10 mM, glucose 5 mM, pH adjusted to 7.3 with NaOH.

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Intracellular solution contained: CsF 105 mM, CsCl 27 mM, NaCl 5 mM, MgCl2 2 mM,

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EGTA 10 mM, HEPES 10 mM, pH adjusted to 7.3 with CsOH.

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The data represent the mean of at least three independent experiments for each channel

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sub-type. The channel conductance (G) was calculated for each depolarization step using

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the relation: G = I/(Vm-ENa) where “I” is the peak maximal current at “Vm” potential; “ENa”

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represent the experimental Nernst equilibrium potential for Na+ obtained for each cell.

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Conductance was normalized to its maximal value (G/Gmax) and data was fitted to the

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Boltzmann equation:

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Y = G/Gmax = 1/(1+exp[-z(F/RT)(Vm-V1/2)]),

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where z is the apparent gating valence; and V1/2 is the voltage at which G/Gmax=0.5. R,

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T, F have their usual meaning. Conductance curves in the presence of toxin were fitted to

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a sum of two Boltzmann equations:

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Y= gc/(1+exp[-z,c(F/RT)(Vm-V1/2,c)]) + gh/(1+exp[-z,h(F/RT)(Vm-V1/2,h)]),

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where gc + gh=1, and where the subscript c stands for the parameters of control and h

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stands for the parameters of the hyperpolarized (toxin modified). The shift in the activation

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potential was represented by ∆V1/2, which represent the difference between the V1/2

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(calculated using the software Clampfit10) from control minus toxin.

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The apparent affinity Kd of the toxin to the Na channel was estimated using the saturation

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Michaelis-Menten equation:

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gh/(gh+gc)=[Toxin]/([Toxin]+Kd)

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2.7 Phylogenetic analysis

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To retrieve putative homologous amino acid sequences and to construct the hypothesis of

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the relation among the newly recognized toxins, the web BLAST suite blastp version

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2.10.0+ (Altschul et al., 1997) search engine was used. The search was limited to

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Scorpiones (taxid:6855) using the Non-redundant UniProtKB/SwissProt sequence

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database. Since we were interested in sequences exclusively belonging to the genus

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Centruroides, rather than making a thorough phylogenetic analysis of the species, we

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used a cutoff E-value of 2e-14 for Co1, 4e-16 for Co2, 9e-19 for Co3 and 6e-16 for Co52.

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The retrieved sequences were compiled to remove redundancy and once filtered, were

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aligned in the online server MAAFT using default settings (Katoh et al., 2017). To obtain

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the appropriated substitution model and conduct the phylogenetic analyses we used the

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pipeline of IQ-Tree server 1.6.11. The best-fit model found for the Bayesian information

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criterion was WAG+I+G4 implemented in ModelFinder module of the pipeline. The tree

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reconstruction using maximum likelihood as optimality criterion included a basic

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perturbation strength of 0.5 and an IQ-Tree stopping rule of 100 (Trifinopoulos et al.,

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2016). The support of the topology was obtained through the ultrafast bootstrap algorithm

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(Hoang et al., 2017) and the SH-aLRT branch test (Guindon et al., 2010) with 1000

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replicates each.

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3. Results

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3.1 Peptides Purification

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The components from 1.2 mg of whole soluble venom were separated by RP-HPLC as

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shown in Figure 1. From this, we collected 85 peaks-fractions, represented as a single

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peak in the chromatogram, and evaluated by MS. A total of 114 compounds were

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identified within a range of molecular weights from 458 to 39246 Da (Table 1).

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Components with molecular mass around 4000 Da were eluted from the column at 20 to

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29 minutes, components of 6000 to 8000 Da eluted from at 30 to 39 minutes and

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components of greater than 10,000 Da eluted after 40 minutes. This chromatographic

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behavior is similar to other scorpion venoms analyzed in similar conditions (Batista et al.,

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2004; Schwartz et al., 2008; Valdez-Velázquez et al., 2013). From all components found,

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the 23% have molecular masses between 3500 and 4500 Da and the 32% masses

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between 6000 and 7500 Da, corresponding to typical molecular weights of potassium-

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channel scorpion toxins (KScTxs) and sodium-channel scorpion toxins (NaScTxs)

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respectively (Possani et al., 1999; Valdez-Velázquez et al., 2013).

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From the separation by RP-HPLC we pooled the principal peaks into 12 fractions (FI-FXII,

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Figure 1) and then assayed each one injecting mice. From this, only the fraction FIX

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resulted toxic corresponding to components with retention time between 33.5 and 37.5

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minutes. Experiments aimed at purifying the toxins directly from FIX by RP-HPLC resulted

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in more than one peptide each time. Thus, in order to isolate the peptides, the venom was

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fractionated by a three-step chromatographic scheme. First, the venom was separated into

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3 fractions by gel filtration (Figure 2A), of which the fraction FII resulted lethal to mice. It is

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known that under the condition applied, the scorpion venoms can be separated into at

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least 3 fractions by gel filtration from where the fraction FII contains the toxic peptides

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(Olamendi-Portugal et al., 2017; Vandendriessche et al., 2010). Fraction FI and FIII did not

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show any toxicity against mice. A further separation of fraction FII by cation-exchange

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chromatography resulted in 11 sub-fractions (Figure 2B) where only the fractions FII.7 and

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FII.9 exhibited toxicity in mice. Finally, the purification of these sub-fractions by RP-HPLC

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resulted in three homogeneous peptides: Co1 (FII.7-34.3 min, MW= 7561.2 Da), Co2

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(FII.9-35.8 min, MW= 7614.3 Da) and Co3 (FII.7-37.1 min, MW= 7774.9 Da). We estimate

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that Co1, Co2, and Co3 toxins represent 5.0%, 0.69% and 0.80% of the protein content of

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the venom, respectively.

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In addition, the most abundant component in the venom (named Co52), which represents

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approximately 10% of the soluble venom, was also isolated. This peptide was recovered

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from the fraction 52 (Table 1) of the initial separation of the whole venom (Figure 1) and

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re-purified by RP-HPLC in order to obtain the isolated form with a molecular mass of

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7343.6 Da (Figure 3C).

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3.2 Peptides Sequence

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The full-length amino acids sequences of Co1, Co2, Co3 and Co52 were determined by

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Edman degradation as described in the Material and Methods section. Their full-length

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sequences were obtained by digesting the reduced and alkylated peptides using

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endoproteinases Glu-C from Staphylococcus aureus, Endoproteinase Lys-C from

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Lysobacter enzymogenes and Asp-N from Pseudomonas fragi separation of the fragments

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by HPLC and sequencing the resulted peptides. In the case of Co3 the digestion was

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performed in native condition since the reduced and alkylated form of the peptide

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precipitates. After enzymatic digestion with Glu-C and Lys-C endopeptidases, the peptide

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was alkylated and separated by RP-HPLC. The corresponding fragments were used for

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sequence determination. Two independent experiments were performed, one for each

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enzyme. The sequence assembly of the N-terminus and the digested peptides was

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confirmed by comparing theoretical and experimental molecular-mass values (Figure 3)

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with an error of ± 1 Dalton. A total of 66 amino acids residues (65 amino acids in the case

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of Co52) were determined for the peptides, including 8 cysteine residues forming four

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disulfide bonds with the typical pattern of NaScTx. These sequences have been deposited

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in UniProt (COHLF2 for Co1, C0HLF3 for Co2, C0HLF4 for Co3 and C0HLF8 for Co52).

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3.3 Toxic Activity

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Toxicity of peptides Co1, Co2, Co3 and Co52 was tested in mice at a unique dose of 5 µg

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of isolated peptide. The peptides Co1, Co2 and Co3 injected in mice produced an

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envenomation during the first 10 minutes and death after 40 minutes. Some of the

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symptoms exhibited were: tail and mouth with cyanosis, eyes wide-opened, excessive

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salivation, bristling, tail wagging, strong spasms in the abdomen, paralysis of hind legs,

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abnormal respiratory frequency and death before the hour. The Co52 peptide did not show

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activity in mice. Additionally, the Co52 was tested in chicks, crickets and woodlice showing

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negative toxicity. Similarly, peptides Co1, Co2 and Co3 were injected in crickets and

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woodlice at amount up to 5 µg showing no apparent effects.

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3.4 Electrophysiological effects

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All peptides described in this communication were evaluated at a concentration of 200 nM

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by electrophysiological recordings in human VGSCs sub-types hNav1.1–hNav1.7 as

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described previously (Olamendi-Portugal et al., 2017). According to the results, Co1, Co2

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and Co3 affect the activation process of the human sodium channels. Co1 is targeted

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specific to the hNav1.6 producing a shift in the activation process to more negative

288

potentials with a ∆V1/2= 29 mV (Figure 4). Considering that of the three toxic components

289

of the venom, Co1 is the most abundant; we thereafter used it for further investigation of

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its affinity to VGSCs. In order to assess the affinity of Co1 toxin, a dose-response assay

291

was performed in order to fit the fraction of toxin modified channels, assessed as its

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fractional contribution to the total Na+ conductance (gh/(gh+gc) (where gc stands for

293

control, unmodified channels conductance, and gh stands for hyperpolarization-shifted)

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against Co1 toxin concentration. The data was fitted with a Michaelis-Menten equation

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estimating an apparent Kd of 45 nM (Figure 5).

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The toxin Co2 affect multiples VGSCs altering the activation process and showing a

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classic effect of β-NaScTx against hNav1.1 (∆V1/2= 7.7 mV), hNav1.2 (∆V1/2= 16.7 mV),

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hNav1.4 (∆V1/2= 13.7 mV) and hNav1.6 (∆V1/2= 21.2 mV) (Figure 6). Additionally, Co2

299

affect the hNav1.5 by reducing the sodium current (INa+) at all potentials. Similarly, the toxin

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Co3 affects the activation process of multiples human sodium-channels: hNav1.2 (∆V1/2=

301

20.5 mV), hNav1.4 (∆V1/2= 8.5 mV) and hNav1.6 (∆V1/2= 28 mV) and reducing the INa+ of

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the hNav1.5 at all potential (Figure 7).

303

4. Discussion

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The scorpion Centruroides ornatus has as wide distribution in geographical areas between

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the states of Michoacán, Jalisco and Guanajuato in the Southwest region of Mexico, where

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it causes a great number of human accidents. It was important to know the composition of

307

its venom, to identify the possibly toxic peptides and to determine their possible functions.

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In this communication we describe for the first time the isolation and characterization of the

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toxic components of this venom. We found three peptides (Co1, Co2 and Co3) that affect

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human sodium channels modulating the voltage-dependent activation process. This

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malfunction of the channels is thought to be responsible for the envenomation symptoms

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shown by people stung by this species. In addition another peptide was identified (Co52)

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whose function is still not clear.

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These four newly described peptides are closely related to previously known NaScTxs

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from scorpion of the Centruroides genus as shown in a multi-sequence alignment (Figure

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8). Particularly, the Co1 toxin has very high identity to Cll2 from Centruroides limpidus with

317

only one amino acid change (Thr49 for a Asn) showing virtually the same behavior on the

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activation process of the hNav1.6 (Schiavon et al., 2012). In the same way, Co1 exhibit

319

high identity to Cell9, a β-NaScTx of Centruroides elegans, with two amino acid changes

320

(Lys30 for Arg and in the Gln32 for Gly). Interestingly, Cll9 cause a negative-shift in the

321

activation process in the hNav1.4 (Vandendriessche et al., 2010). This effect was not

322

observed in Co1 suggesting a key role of the Arg30 and Gly32 in the affinity of the

323

hNav1.4. The effect of these amino acid changes in the affinity to hNav1.6 cannot be

324

addressed since the hNav1.6 has not been evaluated for Cell9.

325

Differently to Co1, the Co2 and Co3 toxins are more promiscuous showing a reduction of

326

the INa+ and a negative-shift the activation process of several VGSCs (hNav1.1, hNav1.2,

327

hNav1.4, and hNav1.6) (Figure 6 and 7). In this matter, multi-sequence alignment of the

328

Co2 and Co3 shows that these toxins are more related to Cn8 from Centruroides noxius

329

and to Cl13 from Centruroides limpidus, toxins with higher target promiscuity (Olamendi-

330

Portugal et al., 2017; Schiavon et al., 2012). These toxin including Co2, Co3, Cn8 and

331

Cl13 differ to others β-NaScTxs from Centruroides scorpions like Cn2, Css2, Css4, Clt1,

332

Cell9 and Cll1 and Cll2 (Figure 8) particularly in the region comprising the pharmacophore

333

involved in the interaction of β-NaScTxs with receptor site-4 on the VGSCs (Cohen et al.,

334

2004; Schiavon et al., 2012). It is worth observing that amino acid residue Gln32, in the

335

case of Co2 and Co3 is Arg32. According to Cohen et al., the residue Gln32 in Css4 (from

336

C. suffusus) is involved in the function of the toxin and its side chain is projected to the

337

solvent and flanks the Glu28 (amino acid crucial for the binding interaction and highly

338

conserved among Centruroides β-NaScTxs) (Cohen et al., 2005). It has been described

339

that the Glu28 in Css4 interacts with site-4 of the receptor, constituted by residues of

340

channel domains II and III (Glu592 at DII/S1–S2, Glu650 at DII/S3-S4 and Glu1251 at

341

DIII/SS2–S6) (Gurevitz, 2012). Since Arg32 in Co2 and Co3 (Gln32 in the case of Css4)

342

flanks the Glu28 and its side chain is exposed to the solvent, it could well be interacting by

343

electrostatic forces with the glutamic residues on the site-4 improving the affinity to the

344

site. This could explain the promiscuity of β-effect observed in these toxins with several

345

VGSCs. Nevertheless to prove this further investigation is needed.

346

In addition, Co2 and Co3 toxins strongly decreased INa+ (nearly 70%) at all voltages in the

347

hNav1.5, without a negative-shifting the activation process, on the contrary a slightly

348

positive-shift is notice in voltage-dependent activation curve (Figure 6 and 7; better seen in

349

Supplementary Figure 1). This effect could be attributed to a true channel blocker,

350

nevertheless this effect has been observed previously for other β-NaScTxs of Centruroides

12

351

scorpions. In the case of Cl13 of C. limpidus, a positive-shift is observed in the activation

352

process with a great reduction of the INa+ (about 80%) in the hNav1.5 (Olamendi-Portugal

353

et al., 2017) similar to the case of Co2 and Co3. This behavior has been further detailed in

354

the interaction of Css4 with the rH1 sodium channel (rat Nav1.5). Here it was determined

355

that the prepulse-dependent negative-shift in the voltage dependence of gating by a

356

voltage sensor-trapping mechanism is absent in the Nav1.5 due the mutation Gly845Asn

357

in the DIIS3-S4 (Cestèle et al., 1998).

358

A phylogenetic analysis of the peptides described in this work comprised only scorpion β-

359

NaScTxs reveal two main clades weakly supported with 85.3/66 of SH-aLRT and ultrafast

360

bootstrap respectively (Figure 9). Roughly, these two clades correspond to the anti-

361

mammal and to the anti-insect functional grouping proposed by Pedraza and Possani

362

(2013). The basal monophyletic group comprises β-NaScTxs binding to voltage-

363

independent at site-4 of the sodium-channel, producing paralysis to insects and

364

crustaceans but not to vertebrates (Cook et al., 2002; Corona et al., 2001; Pedraza

365

Escalona and Possani, 2013). For instance Cn10(Q94435) from C. noxius is considered

366

specific to insects and Cn5(P45663) from the same scorpion is considered specific to

367

crustaceans (Pedraza Escalona and Possani, 2013; Vazquez et al., 1995). Additional

368

sequences were found in the clade that although have never been tested for their

369

pharmacological function, are similarly in their sequence and have been retrieved in

370

genomic and proteomic studies. None of these peptides were directly verified in

371

experimental assays. The second clade includes peptides affecting both invertebrates and

372

vertebrates such as CsEl(P01491) from C. sculpturatus that affects preferentially

373

vertebrates and Cn2(P01495) from C. noxius the most abundant and toxic peptide found in

374

the most dangerous species of Centruroides in Mexico. Included in this clade was

375

Cn1(P15223) from C. noxius that is specific to crustaceans (Pedraza Escalona and

376

Possani, 2013; Vazquez et al., 1995).

377

The peptide Co52 described in this communication segregates within the second clade in

378

a subclade well supported (95.9/97) which links two clades, one containing CsEl(P01491)

379

and the other Cn1(P15223). The former affecting vertebrates and the latter specific for

380

crustaceans, nevertheless our results showed no toxicity against any group of animal

381

tested. This peptide was also tested in our

382

unusual position of Co52 in the topology may be an artifact created by incomplete

383

sampling. It is important to remember that our knowledge of scorpion β-NaScTxs is

13

human VGSCs, showing no effect. This

384

preliminary and there should be in the order of thousands the number of peptides to be

385

discovered. On the other hand, the toxins Co1-Co3 are intermixed with the main bulk of

386

anti-mammal paralog toxins, which was expected since they are similar in tertiary structure

387

and activity on VGSCs. This phylogenetic analysis reinforces our characterization and

388

classification of the new toxins and further suggests that this family gene evolved from

389

duplication and neo-functionalization.

390

5. Conclusions

391

The venom of Centruroides ornatus, a widely distributed scorpion in Mexico, is highly toxic

392

to human and its toxicity resides in three peptides Co1, Co2 and Co3. These peptides are

393

closely related to NaScTxs from others scorpion of the Centruroides genus. Their activity

394

against several human sodium-channels classifies these peptides as β-toxins affecting the

395

current and the activation process of the channels. In addition, the most abundant peptide

396

in the venom (Co52) was characterized. Despite the fact that the amino acid sequence of

397

Co52 is similar to those proved to display NaScTxs activity, the toxicity analysis of this

398

peptide was not conclusive and its function remains unknown.

399

6. Acknowledgements

400

Partially supported by grant IN202619 from Dirección General de Asuntos del Personal

401

Academico of UNAM given to LDP. The authors greatly acknowledged the support

402

received by Dr. Rita Restano Cassulini from our Department for helping with the analyses

403

of the newly obtained electrophysiological experiments. IAGG received a scholarship

404

(number 740685) from CONACyT.

405

7. Conflicts of interest:

406

The authors declare no conflicts of interest

407

14

408

8. References

409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455

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genus Centruroides for production of antivenoms. Toxicon : official journal of the International Society on Toxinology 128, 5-14. Katoh, K., Rozewicki, J., Yamada, K.D., 2017. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics. Olamendi-Portugal, T., Bartok, A., Zamudio-Zuñiga, F., Balajthy, A., Becerril, B., Panyi, G., Possani, L.D., 2016. Isolation, chemical and functional characterization of several new K+channel blocking peptides from the venom of the scorpion Centruroides tecomanus. Toxicon : official journal of the International Society on Toxinology 115, 1-12. Olamendi-Portugal, T., Restano-Cassulini, R., Riaño-Umbarila, L., Becerril, B., Possani, L.D., 2017. Functional and immuno-reactive characterization of a previously undescribed peptide from the venom of the scorpion Centruroides limpidus. Peptides 87, 34-40. Oliveira, J.S., Redaelli, E., Zaharenko, A.J., Cassulini, R.R., Konno, K., Pimenta, D.C., Freitas, J.C., Clare, J.J., Wanke, E., 2004. Binding Specificity of Sea Anemone Toxins to Nav 1.1-1.6 Sodium Channels: UNEXPECTED CONTRIBUTIONS FROM DIFFERENCES IN THE IV/S3-S4 OUTER LOOP. Journal of Biological Chemistry 279, 33323-33335. Ortiz, E., Gurrola, G.B., Schwartz, E.F., Possani, L.D., 2015. Scorpion venom components as potential candidates for drug development. Toxicon : official journal of the International Society on Toxinology 93, 125-135. Pedraza Escalona, M., Possani, L.D., 2013. Scorpion beta-toxins and voltage-gated sodium channels: interactions and effects. Frontiers in bioscience 18, 572-587. Ponce-Saavedra, J., Francke-B, O.F., Quijano-Ravell, A.F., Cortes-Santillan, R., 2016 Alacranes (Arachnida: Scorpiones) de importancia para la salud pública en México. Folia Entomológica Mexicana 2. Possani, L.D., Becerril, B., Delepierre, M., Tytgat, J., 1999. Scorpion toxins specific for Na+-channels. European journal of biochemistry 264, 287-300. Quintero-Hernández, V., Jiménez-Vargas, J.M., Gurrola, G.B., Valdivia, H.H., Possani, L.D., 2013. Scorpion venom components that affect ion-channels function. Toxicon : official journal of the International Society on Toxinology 76, 328-342. Rabia, Y., Hafiz, M.T., Muhammad, A., Sajida, N., Muhammad, M.A., 2016. Optimization of the Conditions for Maximum Recovery of Venom from Scorpions by Electrical Stimulation. Pakistan Journal of Zoology 48, 265-269. Ramirez-Dominguez, M.E., Olamendi-Portugal, T., Garcia, U., Garcia, C., Arechiga, H., Possani, L.D., 2002. Cn11, the first example of a scorpion toxin that is a true blocker of Na+ currents in crayfish neurons. Journal of Experimental Biology 205, 869-876. Riaño-Umbarila, L., Rodríguez-Rodríguez, E.R., Santibañez-López, C.E., Güereca, L., Uribe-Romero, S.J., Gómez-Ramírez, I.V., Cárcamo-Noriega, E.N., Possani, L.D., Becerril, B., 2017. Updating knowledge on new medically important scorpion species in Mexico. Toxicon : official journal of the International Society on Toxinology 138, 130-137. Rodriguez de la Vega, R.C., Possani, L.D., 2005. Overview of scorpion toxins specific for Na+ channels and related peptides: biodiversity, structure-function relationships and evolution. Toxicon : official journal of the International Society on Toxinology 46, 831-844. Santibanez-Lopez, C.E., Francke, O.F., Ureta, C., Possani, L.D., 2015. Scorpions from Mexico: From Species Diversity to Venom Complexity. Toxins 8. Schiavon, E., Pedraza-Escalona, M., Gurrola, G.B., Olamendi-Portugal, T., Corzo, G., Wanke, E., Possani, L.D., 2012. Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels. Toxicon : official journal of the International Society on Toxinology 59, 283-293. Schwartz, E.F., Camargos, T.S., Zamudio, F.Z., Silva, L.P., Bloch, C., Caixeta, F., Schwartz, C.A., Possani, L.D., 2008. Mass spectrometry analysis, amino acid sequence and biological activity of venom components from the Brazilian scorpion Opisthacanthus cayaporum. Toxicon : official journal of the International Society on Toxinology 51, 1499-

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1508. Towler, W.L., Ponce-Saavedra, J., Gantenbein, B., Fet, V., 2001. Mitocondrial DNA reveals a divergent phylogeny in tropical Centruroides (Scorpiones: Buthidae) from Mexico. Biogeographica, 157-172. Trifinopoulos, J., Nguyen, L.-T., von Haeseler, A., Minh, B.Q., 2016. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res 44, W232W235. Valdez-Velázquez, L.L., Quintero-Hernández, V., Romero-Gutiérrez, M.T., Coronas, F.I.V., Possani, L.D., 2013. Mass Fingerprinting of the Venom and Transcriptome of Venom Gland of Scorpion Centruroides tecomanus. PLOS ONE 8, e66486. Vandendriessche, T., Olamendi-Portugal, T., Zamudio, F.Z., Possani, L.D., Tytgat, J., 2010. Isolation and characterization of two novel scorpion toxins: The α-toxin-like CeII8, specific for Nav1.7 channels and the classical anti-mammalian CeII9, specific for Nav1.4 channels. Toxicon : official journal of the International Society on Toxinology 56, 613-623. Vazquez, A., Tapia, J.V., Eliason, W.K., Martin, B.M., Lebreton, F., Delepierre, M., Possani, L.D., Becerril, B., 1995. Cloning and characterization of the cDNAs encoding Na+ channel-specific toxins 1 and 2 of the scorpion Centruroides noxius Hoffmann. Toxicon : official journal of the International Society on Toxinology 33, 1161-1170.

526 527 528 529 530 531 532 533 534 535 536 537 538

17

539 540 541

Figure Legends

542 543 544 545

Figure 1. Separation of the venom from C. ornatus by RP-HPLC. An amount of 1.2 mg protein was separated by RP-HPLC. Each peak observed represent a fraction collected and analyzed by mass spectrometry analysis. Pooled peaks into the fractions FI-FXII represent those used for the determination of lethality in mice.

546 547 548 549 550 551 552

Figure 2. Peptides isolation. In order to isolate the active peptides a chromatographic scheme was performed. (A) First a gel filtration on Sephadex G-50 fine of whole soluble venom. Three fractions (FI, FII and FIII) were collected as shown in the figure. Only FII was lethal to mice. FI corresponds to 3.8%, fraction II to 70.1% and fraction III to 16.5% of protein recovered. (B) Second, a cation-exchange separation of Fraction II using carboxymethyl cellulose. Eleven subfractions were collected; subfractions FII.7 and FII.9 were lethal to mice as indicated by the signal (+ and ++).

553 554 555 556 557 558 559 560 561 562 563 564 565

Figure 3. Peptide isolation and amino acid sequence determination of Co1, Co2, Co3 and Co52. (A) Separation of sub-fraction FII.9 by RP-HPLC. Stars (***) indicate peak of Co2 which elutes at retention time (RT) of 35.8 min with a molecular mass of 7614.3 Da. (B) Separation of sub-fraction FII.7 by RP-HPLC showing the isolation of Co1, indicated with a star (*) with a RT of 34.3 min and molecular mass of 7561.2 Da. Double stars (**) show peak of Co3 toxin, eluting at RT of 37.1 min with a molecular mass of 7774.9 Da. (C) The fraction VIII from the separation of the whole venom of C. ornatus (Figure 1) was applied into an analytical C18 column and eluted with a linear gradient from 10 to 40% of solution B run for 60 min. Under these conditions the Co52 toxin (‡) eluted at 38.24 min with a molecular mass of 7343.7 Da. Table show the sequence assembly from the fragments digested and the N-terminus determined by Edman degradation. The molecular mass of the sequence assembled was calculated and compared with the experimental mass obtained by MS.

566 567 568 569 570 571 572 573

Figure 4. Co1 toxin effect on human sodium-channels. Panels (hNav1.1-hNav1.7) show the voltage-dependence of the activation process and the inactivation process in control (in black) and in presence of 200 nM of Co1 (in gray) fitted to Boltzmann equation. Centerbottom panel show the shift in the activation potential expressed as the difference between the V1/2 (membrane potential at which the conductance is half-maximal) from control minus toxin. Right-bottom panel show the reduction of the sodium current by the action of the toxin. The Co1 toxins have effect against only hNav1.6 by left-shifting the activation process and reducing the sodium current.

574 575 576

Figure 5. The affinity of Co1 toxin on Nav1.6 channels. (A) Nav1.6 channels were activated with a 15 ms pulse of -70 mV applied from the HP of - 110 mV, before (left panel) and upon addition of 100 nM Co1 to the extracellular solution (right panel, as indicated).

18

577 578 579 580 581 582 583 584 585 586 587 588 589 590

Notice the significant inward Na+ current in the presence of toxin. (B) As in A, but the activation pulse was -40 mV. (C) As in A, but the activation voltage was 0 mV. Notice that, in contrast to what is seen at more negative activation potentials, at this fully depolarized voltage Na+ current in the presence Co1 is smaller than that in control conditions. (D) Normalized chord conductance (G/Gmax) vs. membrane voltage (Vm), obtained under the following conditions: Control (Filled circles); 5 nM Co1 in the external solution (Open circles); 20 nM Co1 (open squares); 100 nM Co1 (open rhombs). The lines are the leastsquares fit of points with either a single (control) or two Boltzmann functions (at the indicated [Co1]), with parameters (see Methods): Control: zC=+3.2, V1/2,C= -17.2 mV; 5 nM Co1: zh=+3.7; V1/2,h= -47.4 mV, gh=0.06; 20 nM Tco1: zh=+3.8; V1/2,h=-51 mV; gh=0.38; 100 nM Tco1: zh=+3.2; V1/2,h= -54.5 mV; gh=0.63. Relative second Boltzmann component (gh/(gh+gc)) vs. [Co1]. With the available data, and considering the known 1:1 stoichiometry of beta toxins binding (see Text), a Kd of 45 nM was estimated (slashed line).

591 592 593 594 595 596 597 598 599

Figure 6. Co2 toxin effect on human sodium-channels. Panels (hNav1.1-hNav1.7) show the voltage-dependence of the activation process and the inactivation process in control (in black) and in presence of 200 nM of Co2 (in gray) fitted to Boltzmann equation. Centerbottom panel show the shift in the activation potential expressed as the difference between the V1/2 (membrane potential at which the conductance is half-maximal) from control minus toxin. Right-bottom panel show the reduction of the sodium current by the action of the toxin. The Co2 toxins exhibit promiscuous effect against several sodium-channels affecting the activation process and reducing the sodium current of hNav1.1, hNav1.2, hNav1.4 and hNav1.6. In addition, Co2 significantly reduce the current of the hNav1.5

600 601 602 603 604 605 606 607 608

Figure 7. Co3 toxin effect on human sodium-channels. Panels (hNav1.1-hNav1.7) show the voltage-dependence of the activation process and the inactivation process in control (in black) and in presence of 200 nM of Co3 (in gray) fitted to Boltzmann equation. Centerbottom panel show the shift in the activation potential expressed as the difference between the V1/2 (membrane potential at which the conductance is half-maximal) from control minus toxin. Right-bottom panel show the reduction of the sodium current by the action of the toxin. The Co3 toxins exhibit promiscuous effect against several sodium-channels affecting the activation process and reducing the sodium current of hNav1.2, hNav1.4 and hNav1.6. In addition, Co3 significantly reduce the current of the hNav1.5.

609 610 611 612

Figure 8. Multi-sequence alignment of the peptides described in this communication with NaScTxs from scorpions of Centruroides genus: C. limpidus (Cll1, Cll2 and Cl13), C. elegans (Cell9), C. tecomanus (Clt1), C. suffusus (Css2, Css4 and Css6) and C. noxius (Cn2 and Cn8). Cysteine residues involved in disulfide bonds are in bold.

613 614 615 616

Figure 9. Phylogenetic analysis of the peptides described in this communication. The final matrix comprised 57 OTUs and 67 aligned amino acids with 16 constant sites, 41 parsimony informative sites, and 61 distinct site patters. The phylogenetic analysis produced a tree with an ML of -1895.2109.

19

617 618 619 620

20

621 622 623

Table 1. Mass spectrometry analysis of Centruroides ornatus venom. Determination of molecular masses by mass spectrometry from the RP-HPLC separated fractions of the soluble venom.

Fraction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Retention time (min) 2.85 3.38 4.13 4.64 5.69 8.96 9.74 12.13 12.43 13.06 13.28 14.97 15.42 17.04 17.30 18.86 19.61 19.92 20.44 20.89 21.14 21.89 22.10 22.60 22.84 23.44 23.73 24.29 24.68 24.88 25.25 25.82 26.24 26.81 27.44 28.04 28.42 28.86 29.49 29.77 30.11 30.71 31.11 31.24 31.75

Molecular mass (Da)

Fraction

1

ND ND

485.0

51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85

693.0 693.0 510.0 ND ND 1180.7 ND ND ND ND

863.0

495.0 561.0 881.0 458.0

545.0

738.0 473.0 2511.0

658.0

486.0 477.0

876.0 2610.9 2567.0 678.0

2517.6 2568.0 2610.5 642.0 1801.6 1673.5 3664.9 3731.7

3596.0 4337.4 4338.1 4098.1 4270.3 4123.0 4269.8 4008.0

4121.4 4760.7 7799.1 2982.0 4156.1

690.0 4434.0 3419.7 4338.2 4121.9 4269.7 4034.1 4283.2 4008.0 4206.7 3987.1 4083.3

4270.4 4083.2 2827.0 7799.0 3480.2

6977.5 7276.7 7180.0 6584.8 7179.5 7277.4 7179.9 7375.2 6439.2 7179.0

624 625

1ND: no data.

21

Retention time (min) 32.19 32.46 33.38 33.76 34.11 34.34 34.87 35.37 36.00 36.50 37.41 38.90 39.39 40.16 41.31 42.13 42.40 42.77 43.65 47.55 53.38

Molecular mass (Da) 7246.8

7375.2 7343.6

5162.3 6989.6 7560.3 7560.5 7560-0 7560.8 7173.2 7339.3 7297.5 7168.6 ND 7167.4 7150.6 6046.2 7467.6 4956.0 3795.8 5394.3 16082.0 3250.7 3579.6 7399.7 6134.4 ND ND ND 16054.9 1811.0 ND 35327.4 31196.7 ND 17566.0 11665.0 39246.0 ND

733 5592.7 7612.4 7377.4 7365.3 7313.8

6968.7 5929.2 2270.0 3579.5

7401.0

7551.6

20287.0

HIGHLIGHTS 1.

Venom from Centruroides ornatus scorpion contains at least 114 different components.

2.

Four peptides from the soluble venom were characterized: Co1, Co2, Co3 and Co52.

3.

Co1, Co2 and Co3 are toxins that produce a β-effect in human sodium-channels.

4.

The function of peptide Co52 was not identified, using the assays described here.

Ethical Statment The authors declare that this work was performed according to the ethical procedures approved by our Universities *

Declaration of interests X The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

All authors have read and accepted what is written in the revised manuscript and declare that there are no competing financial interests of personal relationships that could have appeared to influence the work reported in this paper.