Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito

Abstract

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na⁺ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (β-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide β-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Full text

Citation: Restano-Cassulini, R.;
Olamendi-Portugal, T.;
Riaño-Umbarila, L.; Zamudio, F.Z.;
Delgado-Prudencio, G.; Becerril, B.;
Possani, L.D. Characterization of
Sodium Channel Peptides Obtained
from the Venom of the Scorpion
Centruroides bonito.Toxins 2024,16,
125. https://doi.org/10.3390/
toxins16030125
Received: 7 December 2023
Revised: 20 February 2024
Accepted: 27 February 2024
Published: 1 March 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
toxins
Article
Characterization of Sodium Channel Peptides Obtained from
the Venom of the Scorpion Centruroides bonito
Rita Restano-Cassulini 1, † , Timoteo Olamendi-Portugal 1,† , Lidia Riaño-Umbarila 2, Fernando Z. Zamudio 1,
Gustavo Delgado-Prudencio 1, Baltazar Becerril 1and Lourival D. Possani 1, *
1Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional
Autónoma de México, Cuernavaca 62210, Mexico; rita.restano@ibt.unam.mx (R.R.-C.);
timoteo.olamendi@ibt.unam.mx (T.O.-P.); fernando.zamudio@ibt.unam.mx (F.Z.Z.);
gustavo.delgado@ibt.unam.mx (G.D.-P.); baltazar.becerril@ibt.unam.mx (B.B.)
2Investigadora por México CONAHCYT, Instituto de Biotecnología, Universidad Nacional Autónoma de
México, Cuernavaca 62250, Mexico; lidia.riano@ibt.unam.mx
*Correspondence: possani@ibt.unam.mx
†These authors contributed equally to this work.
Abstract:
Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito
by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and
were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one
contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from
about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na
+
channels
expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological
effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the
channels and belong to the beta type scorpion toxin (
β
-ScTx), affecting mainly the Nav1.6 channels.
A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities
with other known bona fide
β
-ScTx. The envenomation caused by this venom in mice is treated by
using commercially horse antivenom available in Mexico. The potential neutralization of the toxic
components was evaluated by means of surface plasmon resonance using four antibody fragments
(10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody
fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4
toxins to various degrees.
Keywords:
antivenoms; Cbo1–Cbo5; Centruroides bonito; scorpion toxin; single-chain antibodies;
voltage-gated sodium channels
Key Contribution:
Five new primary sequences of scorpion toxins are described. Their effects on
Na+-channels were evaluated.
1. Introduction
Scorpions are arthropods distributed worldwide, except on the poles and some ocean
islands, and are represented by 2798 different species, classified into 23 different families
(https://www.ntnu.no/ub/scorpion-files) (accessed on 27 November 2023) [1].
In some regions of the world, scorpion stings can cause lethal effects on humans,
as revised by Chippaux and Goyffon [
2
,
3
]. The most dangerous species belong to the
family Buthidae, among which are the genera Androctonus,Leiurus,Buthus,Buthotus, and
Heterometrus, which are geographically distributed in the Old World, mainly North Africa,
the Middle East, and India [
4
]. The genus Parabutus is found in South Africa [
5
], whereas
the genus Centruroides is located in the Southern part of the USA, Mexico, and Central
America [
6
–
8
]. Brazil, among other countries of South America, like Argentina, Colombia,
and Venezuela, have the genus Tityus as a dangerous species [
7
,
8
]. The venomous gland
Toxins 2024,16, 125. https://doi.org/10.3390/toxins16030125 https://www.mdpi.com/journal/toxins

Toxins 2024,16, 125 2 of 14
of scorpions contains many active components: enzymes, peptides, nucleotides, lipids,
mucoproteins, biogenic amines, heterocyclic components, and a variety of unknown sub-
stances [
9
,
10
]. Thus far, the scorpion venom components best studied are the polypeptides
that recognize ion channel receptors in excitable membranes and are harmful to different
organisms, including humans [
11
–
16
]. Different families of toxins have been described,
which specifically interact with ion channels of the following types: Na
+
channels, K
+
chan-
nels, Cl
–
channels, and Ca
2+
channels. It is generally accepted that the toxins of medical
importance are those that affect voltage-gated Na
+
channels since they are neurotoxins
which cause nerve impulse impairment that could lead to death [17].
Mexico is home to 281 different species, from which at least 21 are potentially danger-
ous to humans [
18
,
19
]. In the State of Guerrero, México, 12 different species of scorpions
of the genus Centruroides are known, among which C. bonito is one of the most recently
described [
20
]. This species is known to cause human envenoming [
21
], but thus far, the
venom of this scorpion has not been fully characterized. Here we describe the isolation
of unknown peptides from this venom. The amino acid sequence of the peptides was
determined, and their effect was evaluated using electrophysiological experiments on
cells recombinantly expressing human Na
+
channels. Four peptides (Cbo1 to Cbo4) were
shown to affect the function of Na
+
channels to different extents. The protecting effect
of antitoxins of the single-chain antibody fragment (scFv) type generated in our group
by phage-display and directed evolution is discussed. A phylogenetic analysis of these
peptides is also reported.
2. Results
2.1. Venom LD50 Determination
The soluble venom from C. bonito is toxic to mice since abdominal contraction, hyper-
ventilation, respiratory distress, cyanosis, and death have been observed. The lethal dose
that kills 50% of animals (LD
50
) was determined and found to be 16.7
µ
g/20 g mouse body
weight. For LD
50
determination, 16 mice were injected by intraperitoneal via following the
up and down method. Results are reported in the supplementary data (Table S1).
2.2. Purification and Sequencing
In total, 80 mg of soluble venom was separated through a Sephadex G-50 column,
obtaining three different well-resolved fractions (I to III, see Figure 1A). All the absorbing
material from the Sephadex G-50 column was fully recovered and based on our previ-
ous experience with Centruroides venoms; fraction II was the one we chose to be further
separated by means of an ion-exchange column containing carboxyl–methyl–cellulose
resins (CMC). The CMC separation resolved 14 different fractions (fraction II–1 to II–14,
Figure 1B). Fractions II–11 to II–14 were further submitted to High-Performance Liquid
Chromatography (HPLC), and the corresponding profiles are shown in Figure 1C–F. The
HPLC separation of fraction II–11 resolved at least 13 components (Figure 1C), from which
the peptide eluting at 38.8 min was homogeneous, showed a molecular mass of 7578
±
1 Da,
and was named Cbo1. The abundance of this component estimated from the initial soluble
venom corresponded to 1.7%. The HPLC of fraction II.12 allowed for the separation of nine
components (Figure 1D), from which the peptides eluting at 39.30 min corresponded to 12%
of the total venom. The determination of the experimental molecular mass (MW
exp
) of this
component gave two ionic series corresponding to masses of 7666
±
1 Da and
7638 ±1 Da
(Cbo2 and Cbo3). We proceeded to their sequence determination even though it was not
possible to obtain the two components in separated form. The HPLC separation of fraction
II–13 permitted obtaining circa 20 components (Figure 1E), from which the most abundant
was eluted at around 46 min, obtained in pure form, and was denoted Cbo4. It showed a
molecular mass of 7846
±
1 Da., and it corresponded to 1.1% of the total soluble venom.
Finally, HPLC separation of fraction II–14 allowed for obtaining approximately 15 compo-
nents (Figure 1F), from which the component eluting at 33.1 min was homogeneous and
showed a molecular mass of 7483 ±1 Da, and this was denoted as Cbo5.

Toxins 2024,16, 125 3 of 14
Toxins 2024, 16, x FOR PEER REVIEW 3 of 15
and was denoted Cbo4. It showed a molecular mass of 7846 ± 1 Da., and it corresponded
to 1.1% of the total soluble venom. Finally, HPLC separation of fraction II–14 allowed for
obtaining approximately 15 components (Figure 1F), from which the component eluting
at 33.1 min was homogeneous and showed a molecular mass of 7483 ± 1 Da, and this was
denoted as Cbo5.
Figure 1. Purification of C. bonito peptides. (A) Separation of 80 mg of soluble venom by means of
Sephadex G-50 column. (B) CMC separation of fraction II from Sephadex G-50 column. A column
of 5 mL capacity was equilibrated and run with 20 mM ammonium acetate buffer pH 4.7. The elu-
tion was performed with a linear gradient of 0 to 0.5 M sodium chloride, during 1000 min at 0.5
mL/min. Fractions II–11 to II–14 were further separated by HPLC. The peptides of interest are in-
dicated with an asterisk (*) and correspond to the following factions: (C) II–11–38.8 (Cbo1), (D)
II–12–39.30, and 39.31 (Cbo2 and Cbo3) that correspond to the ascendant and descendant section of
the indicated peak, both containing Cbo2 and Cbo3. Panels (E,F) correspond to fractions II–13–46.8
(Cbo4) and II–14–33.1 (Cbo5), respectively.
The sequences of Cbo1–Cbo5 peptides were determined by means of direct se-
quencing with the native and alkylated peptides, which allowed for the identification of
the first 55 amino acid residues for Cbo1, the first 54 for Cbo2 and Cbo3, the first 50 for
Cbo4, and the first 25 for Cbo5 (Figure 2, Edman). The C–terminal sequence was obtained
after endopeptidase digestion of the carboxymethylated peptide by means of the en-
zymes listed in Figure 2 (AspN for Cbo1–Cbo5 plus GluC for Cbo4). The sequence of the
peptide fragments resulting from digestion is indicated in Figure 2 after the enzyme
name and the corresponding elution time of each obtained fragment were determined.
The final sequences of Cbo1–Cbo5 were obtained by overlapping the sequence of the
fragments, and they are listed in bold in Figure 2, preceded by the sequence ID and the
formal name. The determination of the amino acid sequence confirmed that Cbo2 and
Cbo3 sequences differ for one amino acid in position 30, where in Cbo2 there is an R30
and in Cbo3 a K30 which are both positively charged amino acids, and this is probably
the reason why it was not possible to separate these two peptides.
Figure 1.
Purification of C. bonito peptides. (
A
) Separation of 80 mg of soluble venom by means of
Sephadex G-50 column. (
B
) CMC separation of fraction II from Sephadex G-50 column. A column of
5 mL capacity was equilibrated and run with 20 mM ammonium acetate buffer pH 4.7. The elution
was performed with a linear gradient of 0 to 0.5 M sodium chloride, during 1000 min at 0.5 mL/min.
Fractions II–11 to II–14 were further separated by HPLC. The peptides of interest are indicated with
an asterisk (*) and correspond to the following factions: (
C
) II–11–38.8 (Cbo1), (
D
) II–12–39.30, and
39.31 (Cbo2 and Cbo3) that correspond to the ascendant and descendant section of the indicated
peak, both containing Cbo2 and Cbo3. Panels (
E
,
F
) correspond to fractions II–13–46.8 (Cbo4) and
II–14–33.1 (Cbo5), respectively.
The sequences of Cbo1–Cbo5 peptides were determined by means of direct sequencing
with the native and alkylated peptides, which allowed for the identification of the first
55 amino acid residues for Cbo1, the first 54 for Cbo2 and Cbo3, the first 50 for Cbo4,
and the first 25 for Cbo5 (Figure 2, Edman). The C–terminal sequence was obtained after
endopeptidase digestion of the carboxymethylated peptide by means of the enzymes listed
in Figure 2(AspN for Cbo1–Cbo5 plus GluC for Cbo4). The sequence of the peptide
fragments resulting from digestion is indicated in Figure 2after the enzyme name and
the corresponding elution time of each obtained fragment were determined. The final
sequences of Cbo1–Cbo5 were obtained by overlapping the sequence of the fragments, and
they are listed in bold in Figure 2, preceded by the sequence ID and the formal name. The
determination of the amino acid sequence confirmed that Cbo2 and Cbo3 sequences differ
for one amino acid in position 30, where in Cbo2 there is an R30 and in Cbo3 a K30 which
are both positively charged amino acids, and this is probably the reason why it was not
possible to separate these two peptides.

Toxins 2024,16, 125 4 of 14
Toxins 2024, 16, x FOR PEER REVIEW 4 of 15
Figure 2. Amino acid sequence obtained from the Edman degradation of the fractions highlighted
with an asterisk (*) in Figure 1. The first line of each one of the 5 peptides/toxins contains the full
sequence obtained (listed in bold). The second line shows the sequence of the same sample reduced
and alkylated (indicated as Edman). The subsequent lines in each toxin are the sequences obtained
from the peptides after enzymatic digestion, necessary to obtain the entire sequence by means of
overlapping the corresponding fragments. The time of elution from the HPLC of each digested
peptide is indicated on the left after the name of the enzyme. Numbers at the left and the right of
the sequences indicate the first and the last position according to the length of the complete se-
quence. On the right side of the figure, the theoretical molecular weight (MWth) calculated from the
given sequences and the experimental molecular weight (MWexp) obtained in the ESI–MS deter-
mination are reported.
The theoretical molecular weight (MWth), based on the sequence, was obtained for
each peptide from the Expasy protein param tool (https://web.expasy.org/protparam/
accessed 14 March 2023) and is reported on the right side of Figure 2 (MWth) along with
the experimental molecular weight (MWexp). The difference between MWth and MWexp of
each peptide is around 8 Da that corresponds to the difference between eight cystine
reduced or forming four disulfides bonds.
The toxicity of Cbo1–Cbo5 was evaluated in in vivo assays using mice. Cbo1 to Cbo4
were toxic to mice (Mus musculus CD1 strain), while Cbo5 peptide showed no toxic effects
on mice and was further assayed in crickets (Sphenarium purpurascens) and sweet water
shrimps (Procambarus acanthophorus) using doses of 20 and 50 µg per animal, where no
appreciable toxic effects were observed either.
2.3. Phylogenetic Analysis
The amino acid sequence of the peptides from C. bonito was compared to similar
toxins from the venom of other scorpions (Figure 3). The sequences recovered by
BLASTP, and phylogenetic analysis, showed that these toxins belong to the subfamily
beta of scorpion Na+ toxins (β–NaScTx). The amino acid sequence of Cbo1 toxin is iden-
tical to antimammalian amidated toxins Cll2b (UniProt accession P59899) from Centru-
roides limpidus [22], Cii1 (UniProt accession P59897) from Centruroides infamatus [23], and
CpoNaTBet09 from Centruroides possanii [24]. The toxins closest to Cbo1 in the phyloge-
netic tree and with the highest percentage identity are the antimammalian toxins Co1
(UniProt accession C0HLF2) from Centruroides ornatus [25] and Cb1 (UniProt accession
C0HLR3) from Centruroides baergi [26], with which Cbo1 shares 98% and 97% amino acid
Figure 2.
Amino acid sequence obtained from the Edman degradation of the fractions highlighted
with an asterisk (*) in Figure 1. The first line of each one of the 5 peptides/toxins contains the full
sequence obtained (listed in bold). The second line shows the sequence of the same sample reduced
and alkylated (indicated as Edman). The subsequent lines in each toxin are the sequences obtained
from the peptides after enzymatic digestion, necessary to obtain the entire sequence by means of
overlapping the corresponding fragments. The time of elution from the HPLC of each digested
peptide is indicated on the left after the name of the enzyme. Numbers at the left and the right of the
sequences indicate the first and the last position according to the length of the complete sequence.
On the right side of the figure, the theoretical molecular weight (MW
th
) calculated from the given
sequences and the experimental molecular weight (MW
exp
) obtained in the ESI–MS determination
are reported.
The theoretical molecular weight (MW
th
), based on the sequence, was obtained for
each peptide from the Expasy protein param tool (https://web.expasy.org/protparam/
accessed 14 March 2023) and is reported on the right side of Figure 2(MW
th
) along with
the experimental molecular weight (MW
exp
). The difference between MW
th
and MW
exp
of each peptide is around 8 Da that corresponds to the difference between eight cystine
reduced or forming four disulfides bonds.
The toxicity of Cbo1–Cbo5 was evaluated in
in vivo
assays using mice. Cbo1 to Cbo4
were toxic to mice (Mus musculus CD1 strain), while Cbo5 peptide showed no toxic effects
on mice and was further assayed in crickets (Sphenarium purpurascens) and sweet water
shrimps (Procambarus acanthophorus) using doses of 20 and 50
µ
g per animal, where no
appreciable toxic effects were observed either.
2.3. Phylogenetic Analysis
The amino acid sequence of the peptides from C. bonito was compared to similar toxins
from the venom of other scorpions (Figure 3). The sequences recovered by BLASTP, and
phylogenetic analysis, showed that these toxins belong to the subfamily beta of scorpion
Na
+
toxins (
β
–NaScTx). The amino acid sequence of Cbo1 toxin is identical to antimam-
malian amidated toxins Cll2b (UniProt accession P59899) from Centruroides limpidus [
22
],
Cii1 (UniProt accession P59897) from Centruroides infamatus [
23
], and CpoNaTBet09 from
Centruroides possanii [
24
]. The toxins closest to Cbo1 in the phylogenetic tree and with
the highest percentage identity are the antimammalian toxins Co1 (UniProt accession
C0HLF2) from Centruroides ornatus [
25
] and Cb1 (UniProt accession C0HLR3) from Cen-
truroides baergi [
26
], with which Cbo1 shares 98% and 97% amino acid identity, respectively

Toxins 2024,16, 125 5 of 14
(Figure 3A,B)
. Toxins Cbo2 and Cbo3 share 98% amino acid identity, with Cbo3 differing by
a single amino acid at position 30 relative to Cbo2 (K30R) (Figure 3B). The toxin phylogenet-
ically closest to Cbo2 and Cbo3 is toxin Cll4 (UniProt accession Q7Z1K8) from C. limpidus,
the antimammalian toxin Co2 (UniProt accession C0HLF3) from C. ornatus [
25
], and Chui5
(UniProt accession C0HM18) from Centruroides huichol [
27
], with which Cbo2 shares 95,
92, and 95% amino acid identity, respectively (Figure 3A,B). The Cbo4 toxin shares 98%
identity with the antimammalian Cl13 toxin of C. limpidus [
28
] and 91% identity with Cb2
of Centruroides baergi [
26
] (Figure 3B). The Cbo5 toxin is in a major clade distinct from the
other Cbo toxins. In the Cbo5 clade is the CsEl toxin (UniProt accession P01491) from C.
sculpturatus, with 83% amino acid identity; this toxin preferentially affects nonmammalian
vertebrates (affects channels from chicken and frog) [
11
,
29
]. Another toxin with the same
percentage of similarity is Co52 (UniProt accession C0HLF8) from C. ornatus, a toxin with
no activity against mice, chickens, crickets, or woodlice [25] (Figure 3B).
2.4. Physiological Characterization
The five peptides were evaluated for their activity on human voltage-gated sodium
channels stably expressed in HEK (hNav 1.1 to 1.6) and CHO (hNav 1.7) cells. Currents
were elicited in control condition (black traces) and in the presence of a toxin applied at a
final concentration of 200 nM for 30–60 s (olive and green traces). Of the five peptides, Cbo5
was the only one that showed no activity in the sodium channel subtypes evaluated (results
are shown in Supplementary Figure S2). The other four peptides act as beta scorpion toxins;
thus, they shift the activation threshold to more negative potential and reduce the total
current in a different magnitude depending on the toxin and the channel (Figure 4). Cbo1
was shown to be selective for hNav 1.6 channels. As previously explained, toxins Cbo2
and Cbo3 have just one conservative amino acid change in position 30 (arginine in Cbo2;
lysine in Cbo3). For this reason, it was almost impossible to obtain pure peptides and they
were applied as a mix of both toxins at a final concentration of 200 nM. Cbo2–Cbo3 mix
was weakly active on hNav1.6, where they shifted the activation threshold but did not
significantly reduce the peak current. In the hNav 1.4 channels, the current reduction was
also not significant, as well as the activation shift. All the other channels were insensitive
to the Cbo2–Cbo3 toxin mix. On the contrary, Cbo4 is promiscuous, affecting several
sodium channel subtypes. It significantly modifies the gaiting properties of hNav 1.4 and
hNav 1.6, shifting the activation process and reducing the peak currents. Like other beta
scorpion toxins [
30
], Cbo4 reduces the peak current of the hNav 1.5 without affecting the
activation threshold. Moreover, Cbo4 slows down the inactivation process in hNav 1.5 and
hNav 1.6. Cbo4 also slightly acts on hNav 1.1 and hNav 1.2, where modifications on the
activation threshold and peak current were present, but at a 200 nM toxin concentration,
these modifications were not statistically significant (at p= 0.05 level), and no apparent
activity was observed in hNav 1.3 and hNav 1.7.
2.5. Neutralization with Single-Chains
In our group, following the interest of generating a protective antidote, we have
obtained a series of neutralizing antibody fragments against toxins and venoms of Mexican
scorpions [
27
,
31
–
33
]. In preliminary assays of protection against 1 LD
50
of C. bonito venom
(without knowing the characteristics of the toxins and their proportion in this venom),
we observed a delay in the appearance of envenoming signs, with respect to the control
group (1 LD
50
of venom), where the initial signs of envenoming are sensitivity to noise and
uncontrolled movements. At 30 min after the venom injection, oscillatory movement of
the tail and cyanosis was observed. Subsequently, respiratory distress was evident. Finally,
death occurred in a timelapse of 2 or 4 h. The animals treated with 1 LD
50
of venom and
the mixture of single-chain variable fragments (scFvs) LR and 10FG2 were able to survive.

Toxins 2024,16, 125 6 of 14
Toxins 2024, 16, x FOR PEER REVIEW 5 of 15
identity, respectively (Figures 3A,B). Toxins Cbo2 and Cbo3 share 98% amino acid iden-
tity, with Cbo3 differing by a single amino acid at position 30 relative to Cbo2 (K30R)
(Figure 3B). The toxin phylogenetically closest to Cbo2 and Cbo3 is toxin Cll4 (UniProt
accession Q7Z1K8) from C. limpidus, the antimammalian toxin Co2 (UniProt accession
C0HLF3) from C. ornatus [25], and Chui5 (UniProt accession C0HM18) from Centruroides
huichol [27], with which Cbo2 shares 95, 92, and 95% amino acid identity, respectively
(Figure 3A and 3B). The Cbo4 toxin shares 98% identity with the antimammalian Cl13
toxin of C. limpidus [28] and 91% identity with Cb2 of Centruroides baergi [26] (Figure 3B).
The Cbo5 toxin is in a major clade distinct from the other Cbo toxins. In the Cbo5 clade is
the CsEl toxin (UniProt accession P01491) from C. sculpturatus, with 83% amino acid
identity; this toxin preferentially affects nonmammalian vertebrates (affects channels
from chicken and frog) [11,29]. Another toxin with the same percentage of similarity is
Co52 (UniProt accession C0HLF8) from C. ornatus, a toxin with no activity against mice,
chickens, crickets, or woodlice [25] (Figure 3B).
Figure 3.
Phylogenetic analysis and multiple alignment of C. bonito toxins and other related
β
–NaScTx.
(
A
) Tree topology obtained from Bayesian analysis of Cbo1–5 toxins and other 35 related
β
–NaScTx
from scorpions of Centruroides genus. ID indicates identical sequences. The numbers below the
nodes indicate percentage values of posterior probability greater than 50. The scale bar represents the
number of amino acid substitutions per site. Analysis was performed using mature chains. Sequence
names are composed of the accession code UniProt, followed by the toxin name and the species
name. Sequences recovered from the NCBI are indicated with their accession numbers, followed by
toxin name and the species name. CpoNaTBet09 was retrieved from the original publication [
24
].
Three
α
–NaScTx (BTN, Aah3, and Os3) were used as outgroups and to root the tree. (
B
) Multiple
alignment of C. bonito toxins and other
β
–NaScTxs from scorpions of Centruroides genus. UniProt
sequences closely related to Cbo1, Cbo2, Cbo3, Cbo4, and Cbo5 are shown. Len indicates mature
chain length; %ID indicates percent amino acid identity. %SI indicates the percentage of amino acid
similarity. Conserved cysteine residues are highlighted in yellow. Identical positions to C. bonito
toxins are indicated by dots. (*) indicates toxins where the C–terminal amidation has been reported.
The UniProt access code is shown on the left, followed by the toxin name and species name.

Toxins 2024,16, 125 7 of 14
Toxins 2024, 16, x FOR PEER REVIEW 7 of 15
Figure 4.
Activity of Cbo1, Cbo2 plus Cbo3, and Cbo4 toxins on human voltage-gated sodium
channels hNav 1.1–hNav 1.7. The figure shows the curves of activation (circles) and inactivation
(squares) processes of hNav 1.1 to hNav1.7. Data were obtained in control conditions and during
application of toxin at 200 nM. Lines are the best fit obtained by a Boltzmann equation or the sum of
two Boltzmann equations when toxin modifies the voltage dependence of activation process. This was
the case of hNav 1.6 with Cbo1–Cbo4 and hNav 1.2 and 1.4 with Cbo4. Cbo4 is also capable to slowing
down the inactivation process in channels hNav 1.5 and hNav 1.6 as shown here. Representative traces
of currents affected by the toxins are shown in the small panels, where black traces are currents in
control recorded at sub-threshold and full-activation potentials; in olive and green are sub-threshold
and full-activated currents with toxin. Dashed traces in green are currents recorded at –30 mV for
hNav1.4 or –10 mV for hNav 1.5 and scaled to the control traces to better appreciate the inactivation
delay. Symbols and line colors correspond to the legend. The effect of toxins on the peak currents is
graphed in the bottom row. Data are the mean of
3–7 experiments ±standard
error. The asterisk (*)
indicates significance at 0.05 level of the difference between control and toxin conditions by means of
a “Paired Sample t Test”. The activation and inactivation fitting parameters, along with the fractional
residual current values, are reported in Supplementary Table S2.

Toxins 2024,16, 125 8 of 14
The appearance of the described signs was not observed. However, there was a delay
in the onset of severe respiratory distress, suggesting that there was a partial neutralization
of the venom. To explain these results, we performed real-time analyses of the interactions
of scFvs with the identified toxins of C. bonito (see sensograms, Figure 5). We found that
Cbo toxins 1–3 were not recognized by scFv LR, whereas they were well recognized by
scFv 10FG2. Considering these results, it is likely that 10FG2 neutralizes them. The Cbo4
toxin was not well recognized by either LR or 10FG2, which explains the appearance of the
envenoming signs. Recently, new antibody fragments have been developed, such as scFv
HV [
27
], which shows good interaction with Cb2 and Cbo3 toxins. Its low dissociation from
the toxins is remarkable. In the case of the Cbo4 toxin, there is no good interaction with
the scFvs evaluated, and the only one that does interact is scFv 11F, although it showed
fast dissociation.
Toxins 2024, 16, x FOR PEER REVIEW 9 of 15
Figure 5. Sensorgrams of interaction between scFvs (at 100 nM) and toxins (A) with Cbo1 toxin, (B)
with Cbo2–Cbo3 mix, and (C) with Cbo4 toxin. Biosensor measurements were performed at 25 °C
with a flow rate of 50 µL/min. RU stands for resonance units, a standard parameter for surface
plasmon resonance analysis. 11F, HV, 10FG2, and LR are the arbitrary names for the single-chain
variable fragment (scFvs).
3. Discussion
Since C. bonito is a scorpion species which has recently been described and its venom
contains toxic components to humans, we decided to obtain venom from these animals,
aiming to conduct formal studies of its components. The venom components separated
by chromatographic procedures were assayed in vivo using mice. Several components
Figure 5.
Sensorgrams of interaction between scFvs (at 100 nM) and toxins (
A
) with Cbo1 toxin,
(
B
) with Cbo2–Cbo3 mix, and (
C
) with Cbo4 toxin. Biosensor measurements were performed at
25
◦
C with a flow rate of 50
µ
L/min. RU stands for resonance units, a standard parameter for surface
plasmon resonance analysis. 11F, HV, 10FG2, and LR are the arbitrary names for the single-chain
variable fragment (scFvs).

Toxins 2024,16, 125 9 of 14
3. Discussion
Since C. bonito is a scorpion species which has recently been described and its venom
contains toxic components to humans, we decided to obtain venom from these animals,
aiming to conduct formal studies of its components. The venom components separated
by chromatographic procedures were assayed
in vivo
using mice. Several components
(Cbo1, Cbo4, and Cbo5) were obtained in pure form, while for two components (Cbo2 and
Cbo3), it was impossible to separate them due to the high similarity in their sequences;
in fact, they differ by one conservative change (R30K). The full amino acid sequences
were obtained by using Edman degradation and enzymatic digestions. The molecular
weights found by mass spectroscopy correspond to those expected from the full sequences
obtained with a difference of about 8 Daltons (8 D), which explains the oxidized state of the
cysteines. Phylogenetic analysis performed with this information and with known scorpion
toxins from venoms of other Centruroides species suggested that these C. bonito venom
components belong to another known beta type of sodium scorpion toxins (
β
–NaScTxs).
This analysis showed two strongly supported clades (branch support of 90) in which C.
bonito and related
β
–NaScTxs are clustered (Figure 3). The clade in which the Cbo5 toxin
is grouped comprises other toxins with diverse activities, including those affecting insect–
crustaceans, such as Cn1 from Centruroides noxius [
34
,
35
], vertebrates, such as CsE1 [
11
,
29
],
and toxins with functions not yet elucidated (e.g., Co52, Ct17, and others) [
25
,
36
]. Like
the latter, Cbo5 has no detectable effect on sodium channels from human origin, as well
as in
in vivo
experiments on mice, crickets, and sweet water shrimps. These negative
results suggest that these components may act on a different type of receptor or on a
different type of organism [
22
,
37
,
38
]. In a similar way, the poor branch support observed
in Cbo5 (<50) could be explained by a limited sampling of the sequences available in the
databases; this effect can be observed in the percentage identity shared by Cbo5 with
other nearby toxins, which do not exceed 83% amino acid identity (Figure 3B). The second
clade includes phylogenetically more recent toxins, including toxins Cbo1 to Cbo4 and
other antimammalian toxins. Toxins such as Cbo1, whose sequence has been reported in
other species of the genus Centruroides (see Figure 3), may have been present in common
ancestors and preserved during the speciation process of the Centruroides genus, which
appeared at the end of the tertiary period of the cenozoic period when mammals, potential
predators of these animals, diversified [
18
]. Cbo1 and Cbo2–Cbo3 have an effect on Nav1.6
channels. This confirms the
in vivo
assays performed with mice. Cbo4 is the most active
toxin, modifying the function of several channels, mainly hNav.1.4, hNav1.5, and hNav1.6.
This toxin is highly similar to Cl13 (they differ in position 5, where Cbo4 has the amino acid
I and Cl13 has L) and, as expected, they have shown very similar effects in voltage-gated
sodium channels [
39
]. Moreover, it is important to mention that due to the sensibility of the
equipment utilized for mass determination, it was not possible to show if the Cbo toxins
here described have been amidated in the C–terminal like many toxins of the same group
(see multiple alignment in Figure 3B).
From the characterization of the toxic components of any given venom, it is possible,
through scFv affinity maturation, to implement rational strategies to achieve a good neu-
tralization of each of the toxic components [
40
–
43
]. The scFvs 10FG2 and HV evaluated in
this work show a good level of interaction with the evaluated toxins similar to those that
have already been neutralized [
27
,
33
]. These scFvs could contribute to the neutralization
of toxins Cbo1, Cbo2, and Cbo3, which are important toxic components of the venom
(together they correspond to 13.7%). In the case of Cbo4 toxin, which is also an important
component (1.1% of the venom), we do not yet have an scFv that shows a good interaction
which could contribute to the neutralization of the venom. The scFv 11F is a good candidate
to be matured
in vitro
, and then neutralization of this toxin could be achieved. These results
are promising since three of the four toxins are well recognized, and there is a good chance
that neutralization of the entire venom will be achieved relatively soon.

Toxins 2024,16, 125 10 of 14
4. Materials and Methods
4.1. Venom Extraction, LD50 Determination, and Purification
Venom was obtained from approximately 500 animals collected in the State of Guerrero
(region of Costa Chica) by means of electric stimulation (SEMARNAT, official permission
SCPA/DGVS/00367/22). The venom was dissolved in water, centrifuged at 15,000
×
gfor
15 min, and the supernatant was lyophilized and kept at
−
20
◦
C until use. The lethal dose
50% (LD
50
) of whole venom was estimated using 16 mice (Mus musculus strain CD1) by
intraperitoneal via following the method of up and down described in [
44
]. The use of
animals in this work complies with the guidelines established by Mexican legislation and
internationally recommended procedures and was approved by the Bioethics Committee of
the Instituto de Biotecnología of Universidad Nacional Autónoma de México, number 413.
Initial purification was performed using 80 mg of C. bonito soluble venom separated
in a Sephadex G-50 column (2 m high, 0.9 cm diameter) run in the presence of 20 mM
ammonium acetate buffer, pH 4.7. From our previous experiences with Centruroides venoms
of different species, we have learned that fraction II of Sephadex is the one that contains
most of the venom toxic component. With this idea, Fraction II was further separated
by ion-exchange chromatography on a carboxy–methyl–cellulose (CMC) column (5 mL
volume) run in the same buffer and eluted with a sodium chloride gradient from 0 to 0.5 M
salt, during 1000 min at 0.5 mL/min, in a New Generation Chromatography (NGC
TM
) sys-
tem connected to a fraction collector (both from BioRAD, Mexico City, Mexico). Fractions
from the CMC column were further separated through high-performance liquid chro-
matography (HPLC) using a Waters model 1525 binary HPLC pump, detector 2489 UV/vis
chromatographer (Mexico City, Mexico) in the conditions earlier described [
45
]. Basically,
an analytical C18 reverse-phase column from Vydac (Hisperia, CA, USA) was used (C18
250
×
4.6 mm, wakopac/wakosil pTH–II 4.6
×
250 mm). The components were eluted with
an acetonitrile gradient from 0 to 60%, and the mobile phase was prepared with solution A,
containing 0.12% trifluoroacetic acid (TFA) in water, and solution B contained 0.10% TFA
in acetonitrile. For each component, the columns were run for 60 min at a 1mL/min flow
rate. Our main interest is to find the venom components principally responsible for the
venom intoxication and, in Centruroides venom, these are mainly the sodium channel toxins.
Our first approach was to select the most abundant peaks obtained from HPLC and those
eluting after 30 min because we empirically know that in this interval, the sodium channel
toxins should be eluted. The relative concentration of the fractions was estimated from the
chromatogram, dividing the area of each peak by the sum of the area of all the peaks. The
chosen components were analyzed for purity by mass spectrometry analysis and sequenc-
ing determination by Edman degradation, as mentioned below. Toxicity to mammals of
the sequenced peptides (Cbo1–Cbo5) was evaluated using two mice (Mus musculus CD1
strain) injected with 10
µ
g/20 g mouse weight. Toxin Cbo5 was also assayed in crickets
(Sphenarium purpurascens) and in sweet water shrimps (Procambarus acanthophorus) using
the technique earlier described [
26
]. For these experiments, 2 crickets were injected with
20
µ
g and 2 were injected with 50
µ
g of Cbo5, while 2 sweet water shrimps were assayed
with 20 µg of toxin.
4.2. Mass Spectrometry and Sequence Determination
The purified fractions were reconstituted to a final concentration of 500 pmol/5
µ
L
of 50% acetonitrile with 0.1% acetic acid and directly applied into a LCQFleet ion trapp
mass spectrometer (Thermo Fisher Scientific Inc. San Jose, CA, USA) using a Surveyor MS
syringe pump delivery system. The eluate at 10
µ
L/min was split to allow for only 5% of
the sample to enter the nano spray source (0.5
µ
L/min). The spray voltage was set from
1.0 to 2.0 kV, and the capillary temperature was set from 100 to 200
◦
C. All spectra were
obtained in positive-ion mode. The equipment used for this analysis has a precision of
±
1 Da when using peptides of about 7 to 8 kDa. The purified peptides were subjected to
Edman degradation in a Shimadzu Protein Sequencer PPSQ–31A/33A (Columbia, MD,
USA). Homogeneous peptides were analyzed in two formats: native peptide and reduced

Toxins 2024,16, 125 11 of 14
and alkylated cysteines with iodoacetic acid for the purpose of determining their position
in the sequence. In our conditions, most pure peptides in their native format allow for
the identification of approximately the first 50 amino acid residues, starting from the
N–terminal amino acid. In order to obtain the full sequence, the reduced and alkylated
format of the peptides was submitted to enzymatic hydrolysis [
45
]. The corresponding
peptides after treatment with the endopeptidases (GluC and AspN) were subjected to
HPLC separation, and their sub-peptides were placed into the sequencer. Both enzymes,
from the company Roche Diagnostics GmbH (Mannheim, Germany), were used according
to the protocol suggested by the provider. The overlapping of the sequences and the
verification of the completeness of the sequence were confirmed by mass spectrometry
determination of the pure native peptides and compared to the expected molecular weight
based on the amino acid structure experimentally determined. The amino acid sequence of
the five peptides was registered at UNIPROT and was given the following identification
numbers: Cbo1 is C0HMA3; Cbo2 is C0HMA4; Cbo3 is C0HMA5; Cbo4 is C0HMA6; and
Cbo5 is C0HMA7.
4.3. Phylogenetic Analysis
The search for potential homologs of C. bonito toxins was performed using BLASTP
v2.13.0+ [
46
] in a command line. All 569793 proteins listed in UniProtKB/Swiss–Prot
Release 2024_01 (UniProt, 2023) (accessed on 26 September 2023) were used as a database.
Each of the C. bonito sequences was used as a query using an evalue = 1
×
10
–20
as the
significance limit. Redundant UniProt IDs from BLASTP analysis were removed using
unix commands. An additional BLASTP search was performed in the NCBI nr database.
The sequences of the mature chains of 56 scorpion Na
+
toxins from the beta subfamily
(β–NaScTx)
recovered from BLASTP and 3 Na
+
toxins from the alpha subfamily (
α
–NaScTx)
(outgroup) were aligned with MAFFT v7.490 [
47
] using the progressive FFT–NS–2 method.
Construction of an initial phylogenetic tree was performed by Bayesian inference using
MrBayes v3.2.7 [
48
] with the options lset rates =gamma with prset aamodelpr =fixed. The
WAG model [
49
] was chosen as the amino acid substitution model. The analysis was run for
1
×
10
7
generations in eight chains with a sampling frequency of every 1000 trees. During
the construction of the consensus tree, the first 1
×
10
6
trees were discarded (Burn-in).
The initial tree was pruned by eliminating clades poorly related to the C. bonito sequences.
A new phylogenetic analysis by Bayesian inference was performed using 41 remaining
β
–NaScTx; the 3
α
–NaScTx retained as an outgroup. Percentage identity and amino
acid similarity between sequences were determined with “The sequence manipulation
suite” [50].
4.4. Cell Culture and Electrophysiology Characterization
Cell culture and the electrophysiology experiment were performed according to the
protocols described in [
26
]. Briefly, HEK cells expressing human voltage-gated sodium
channels of the subtypes 1.1 to 1.6 (hNav 1.1–hNav 1.6) and CHO cells expressing hNav
1.7 were cultivated in Dulbeco’s Modified Eagle Medium supplemented with 10% fetal
bovine serum and 500
µ
g/mL of antibiotic G418 and were maintained at 37
◦
C and 5%
CO
2
in a humidified atmosphere. Cells were kindly gifted from Prof. Enzo Wanke lab
(University of Milan-Bicocca, Italy). Currents were elicited by depolarizing steps from
−
120 to 40 mV with 10 mV increment, preceded by a strong and short depolarization (5 ms
at +50 mV), and then they were tested at their full activation potential (
−
30 or 10 mV). For
the activation and inactivation curves, current and conductance were plotted against the
corresponding membrane potential. Data were fitted with single or double Boltzmann
equation [51] and are presented as a mean of 3–7 experiment ±standard deviations.
4.5. Evaluation of the Interaction of scFvs and C. bonito Toxins by Surface Plasmon Resonance
The scFv variants were tested against C. bonito toxins in a biosensor that detects
molecular interactions in real time (Biacore X100, Uppsala, Sweden). Each toxin was

Toxins 2024,16, 125 12 of 14
dissolved in 10 mM 2–(N–morpholino) ethanesulfonic acid (pH 6) and immobilized on cell
2 of a CM5 sensor chip using the amino coupling kit, reaching binding levels of 200 RUs.
Cell 1 in the sensor chip without antigen was used as a control. The scFvs LR, 10FG2, HV,
and 11F [
27
,
31
–
33
] were used to evaluate binding to each toxin. The samples were diluted
in HBS–EP buffer (Biacore) at 100 nM concentration, and 100
µ
L of the scFvs was injected
over the chip at a flow rate of 50
µ
L min
−1
at 25
◦
C with a delay time of 500 s. The chip
surfaces were regenerated with 10 mM glycine–HCl pH 2. The sensorgrams were corrected
by subtracting the values from the reference flow cell and comparing the results using
BIA-evaluation software version 3.1.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/toxins16030125/s1, Figure S1: Mass spectrometry (MS) data.
Figure S2: Voltage-gated sodium channels hNav 1.1–hNav 1.7 are insensible to Cbo5. Table S1:
Determination of LD
50
of C. bonito venom using the “up and down” method. Table S2: Fitting
parameters of data from activation, inactivation, and total current of hNav 1.1–1.7 channels. Table S3:
Plain text and FASTA format for toxins Cbo1 to Cbo5.
Author Contributions:
Conceptualization, L.D.P.; methodology, F.Z.Z., G.D.-P., L.R.-U., R.R.-C. and
T.O.-P.; formal analysis, F.Z.Z., G.D.-P., L.R.-U., R.R.-C. and T.O.-P.; investigation, F.Z.Z., G.D.-P.,
L.R.-U., R.R.-C. and T.O.-P.; data curation, G.D.-P.; writing—original draft preparation, L.D.P., G.D.-P.,
L.R.-U. and R.R.-C.; writing—review and editing, B.B., F.Z.Z., G.D.-P., L.D.P., L.R.-U., R.R.-C. and
T.O.-P.; supervision, L.D.P.; funding acquisition, L.D.P. and B.B. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was funded by CONAHCYT (Consejo Nacional de Humanidades, Ciencias y
Tecnologías), grant number 303045, and by Dirección General de Asuntos de Personal Académico–
UNAM, project IN200323.
Institutional Review Board Statement:
The animal study protocol was approved by the Institutional
Ethics Committee of the Institute of Biotecnology of UNAM (protocol code 413 of 24 September 2021).
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
The authors acknowledged Pedro Saucedo Ramirez for graphics design and
Cipriano Balderas Altamirano for injecting the arthropods. We also thank Ilse V. Gómez Ramírez for
helping on the LD50 determination.
Conflicts of Interest: The authors declare that there is no conflict of interest.
References
1. Rein, J.O. The Scorpion Files; NTNU: Trondheim, Norway, 2023.
2. Chippaux, J.P.; Goyffon, M. Epidemiology of scorpionism: A global appraisal. Acta Trop. 2008,107, 71–79. [CrossRef]
3.
Abroug, F.; Ouanes-Besbes, L.; Tilouche, N.; Elatrous, S. Scorpion envenomation: State of the art. Intensive Care Med.
2020
,46,
401–410. [CrossRef]
4.
Balozet, L. Scorpionism in the old world. In Venomous Animal and Their Venoms; Bücherl, W., Buckley, E.E., Deulofeu, V., Eds.;
Academic Press: New York, NY, USA, 1971; Volume 3, pp. 349–371.
5.
Debont, T.; Swerts, A.; Van der Walt, J.J.; Muller, G.J.; Verdonck, F.; Daenens, P.; Tytgat, J. Comparison and characterization of
the venoms of three Parabuthus scorpion species occurring in southern Africa. Toxicon Off. J. Int. Soc. Toxinol.
1998
,36, 341–352.
[CrossRef]
6.
Mazzotti, L.; Bravo-Becherelle, M.A. Scorpionism in the Mexican Republic. In Venomous and Poisonous Animals and Noxious Plants
of the Pacific Region: A Collection of Papers Based on a Symposium in the Public Health and Medical Science Division at the Tenth Pacific
Science Congress; Keegan, H.L., Macfarlane, W.V., Eds.; Macmillan: London, UK, 1963; pp. 119–131.
7.
Bücherl, W.; Diniz, C.R. Venoms of Tityinae. In Arthropod Venoms. Handbuch der Experimentellen Pharmakologie, 1st ed.; Bettini, S.,
Ed.; Springer: Berlin/Heidelberg, Germany, 1978; Volume 48.
8.
Watt, D.D.; Simard, J.M.; Babin, D.R.; Mlejnek, R.V. Physiological characterization of toxins isolated from scorpion venom. In
Toxins: Animal, Plant and Microbial; Rosenber, P., Ed.; Pergamon Press: Oxford, UK, 1978; pp. 647–660.
9.
Al-Asmari, A.K.; Kunnathodi, F.; Al Saadon, K.; Idris, M.M. Elemental analysis of scorpion venoms. J. Venom Res.
2016
,7, 16–20.
10.
Zlotkin, E.; Miranda, F.; Rochat, H. Chemistry and pharmacology of Buthinae scorpion venoms. In Handbook of Experimental
Physiology; Bettini, S., Ed.; Springer: Berlin/Heidelberg, Germany, 1978; Volume 48.

Toxins 2024,16, 125 13 of 14
11.
Meves, H.; Rubly, N.; Watt, D.D. Effect of toxins isolated from the venom of the scorpion Centruroides sculpturatus on the Na
currents of the node of Ranvier. Pflug. Arch. Eur. J. Physiol. 1982,393, 56–62. [CrossRef] [PubMed]
12.
Catterall, W.A. Neurotoxins that act on voltage-sensitive sodium channels in excitable membranes. Annu. Rev. Pharmacol. Toxicol.
1980,20, 15–43. [CrossRef] [PubMed]
13.
Catterall, W.A. Purification of a toxic protein from scorpion venom which activates the action potential Na
+
ionophore. J. Biol.
Chem. 1976,251, 5528–5536. [CrossRef] [PubMed]
14.
Mozhayeva, G.N.; Naumov, A.P.; Nosyreva, E.D.; Grishin, E.V. Potential-dependent interaction of toxin from venom of the
scorpion Buthus eupeus with sodium channels in myelinated fibre: Voltage clamp experiments. Biochim. Biophys. Acta
1980
,597,
587–602. [CrossRef] [PubMed]
15.
Gordon, D.; Martin-Eauclaire, M.F.; Cestele, S.; Kopeyan, C.; Carlier, E.; Khalifa, R.B.; Pelhate, M.; Rochat, H. Scorpion toxins
affecting sodium current inactivation bind to distinct homologous receptor sites on rat brain and insect sodium channels. J. Biol.
Chem. 1996,271, 8034–8045. [CrossRef] [PubMed]
16.
Barhanin, J.; Giglio, J.R.; Leopold, P.; Schmid, A.; Sampaio, S.V.; Lazdunski, M. Tityus serrulatus venom contains two classes
of toxins. Tityus gamma toxin is a new tool with a very high affinity for studying the Na+ channel. J. Biol. Chem.
1982
,257,
12553–12558. [CrossRef] [PubMed]
17.
Mendes, L.C.; Viana, G.M.M.; Nencioni, A.L.A.; Pimenta, D.C.; Beraldo-Neto, E. Scorpion Peptides and Ion Channels: An
Insightful Review of Mechanisms and Drug Development. Toxins 2023,15, 238. [CrossRef] [PubMed]
18.
Santibanez-Lopez, C.E.; Aharon, S.; Ballesteros, J.A.; Gainett, G.; Baker, C.M.; Gonzalez-Santillan, E.; Harvey, M.S.; Hassan, M.K.;
Abu Almaaty, A.H.; Aldeyarbi, S.M.; et al. Phylogenomics of Scorpions Reveal Contemporaneous Diversification of Scorpion
Mammalian Predators and Mammal-Active Sodium Channel Toxins. Syst. Biol. 2022,71, 1281–1289. [CrossRef]
19.
Santibanez-Lopez, C.E.; Francke, O.F.; Ureta, C.; Possani, L.D. Scorpions from Mexico: From Species Diversity to Venom
Complexity. Toxins 2015,8, 2. [CrossRef]
20.
Quijano-Ravell, A.F.; Teruel, R.; Ponce-Saavedra, J. A new Centruroides Marx, 1890 (Scorpiones, Buthidae) from Southern Guerrero
State, Mexico. Rev. Ibérica Aracnol. 2016,28, 25–34.
21.
Gonzalez-Santillan, E.; Possani, L.D. North American scorpion species of public health importance with a reappraisal of historical
epidemiology. Acta Trop. 2018,187, 264–274. [CrossRef] [PubMed]
22.
Alagon, A.C.; Guzman, H.S.; Martin, B.M.; Ramirez, A.N.; Carbone, E.; Possani, L.D. Isolation and characterization of two toxins
from the Mexican scorpion Centruroides limpidus limpidus Karsch. Comp. Biochem. Physiol. B Comp. Biochem.
1988
,89, 153–161.
[CrossRef] [PubMed]
23.
Dehesa-Davila, M.; Ramirez, A.N.; Zamudio, F.Z.; Gurrola-Briones, G.; Lievano, A.; Darszon, A.; Possani, L.D. Structural and
functional comparison of toxins from the venom of the scorpions Centruroides infamatus infamatus,Centruroides limpidus limpidus
and Centruroides noxius.Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 1996,113, 331–339. [CrossRef]
24.
Garcia-Villalvazo, P.E.; Jimenez-Vargas, J.M.; Lino-Lopez, G.J.; Meneses, E.P.; Bermudez-Guzman, M.J.; Barajas-Saucedo, C.E.;
Delgado Enciso, I.; Possani, L.D.; Valdez-Velazquez, L.L. Unveiling the Protein Components of the Secretory-Venom Gland and
Venom of the Scorpion Centruroides possanii (Buthidae) through Omic Technologies. Toxins 2023,15, 498. [CrossRef]
25.
Garcia-Guerrero, I.A.; Carcamo-Noriega, E.; Gomez-Lagunas, F.; Gonzalez-Santillan, 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 2020,173, 27–38. [CrossRef]
26.
Gomez-Ramirez, I.V.; Riano-Umbarila, L.; Olamendi-Portugal, T.; Restano-Cassulini, R.; Possani, L.D.; Becerril, B. Biochemical,
electrophysiological and immunological characterization of the venom from Centruroides baergi, a new scorpion species of medical
importance in Mexico. Toxicon Off. J. Int. Soc. Toxinol. 2020,184, 10–18. [CrossRef]
27.
Valencia-Martinez, H.; Olamendi-Portugal, T.; Restano-Cassulini, R.; Serrano-Posada, H.; Zamudio, F.; Possani, L.D.; Riano-
Umbarila, L.; Becerril, B. Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its
Neutralization by a Single Recombinant Antibody Fragment. Toxins 2022,14, 369. [CrossRef]
28.
Lopez-Giraldo, A.E.; Olamendi-Portugal, T.; Riano-Umbarila, L.; Becerril, B.; Possani, L.D.; Delepierre, M.; Del Rio-Portilla, F. The
three-dimensional structure of the toxic peptide Cl13 from the scorpion Centruroides limpidus.Toxicon Off. J. Int. Soc. Toxinol.
2020
,
184, 158–166. [CrossRef] [PubMed]
29.
Babin, D.R.; Watt, D.D.; Goos, S.M.; Mlejnek, R.V. Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing.
Arch. Biochem. Biophys. 1975,166, 125–134. [CrossRef] [PubMed]
30.
Cestele, S.; Qu, Y.; Rogers, J.C.; Rochat, H.; Scheuer, T.; Catterall, W.A. Voltage sensor-trapping: Enhanced activation of sodium
channels by beta-scorpion toxin bound to the S3-S4 loop in domain II. Neuron 1998,21, 919–931. [CrossRef]
31.
Fernandez-Taboada, G.; Riano-Umbarila, L.; Olvera-Rodriguez, A.; Gomez-Ramirez, I.V.; Losoya-Uribe, L.F.; Becerril, B. The
venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two
recombinant antibody fragments. Mol. Immunol. 2021,137, 247–255. [CrossRef] [PubMed]
32.
Riano-Umbarila, L.; Contreras-Ferrat, G.; Olamendi-Portugal, T.; Morelos-Juarez, C.; Corzo, G.; Possani, L.D.; Becerril, B.
Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment. J. Biol. Chem.
2011,286, 6143–6151. [CrossRef]

Toxins 2024,16, 125 14 of 14
33.
Riano-Umbarila, L.; Gomez-Ramirez, I.V.; Ledezma-Candanoza, L.M.; Olamendi-Portugal, T.; Rodriguez-Rodriguez, E.R.;
Fernandez-Taboada, G.; Possani, L.D.; Becerril, B. Generation of a Broadly Cross-Neutralizing Antibody Fragment against Several
Mexican Scorpion Venoms. Toxins 2019,11, 32. [CrossRef]
34.
Rendon-Anaya, M.; Delaye, L.; Possani, L.D.; Herrera-Estrella, A. Global transcriptome analysis of the scorpion Centruroides
noxius: New toxin families and evolutionary insights from an ancestral scorpion species. PLoS ONE 2012,7, e43331. [CrossRef]
35.
Vazquez, A.; Tapia, J.V.; Eliason, W.K.; Martin, B.M.; Lebreton, F.; Delepierre, M.; Possani, L.D.; Becerril, B. Cloning and
characterization of the cDNAs encoding Na+ channel-specific toxins 1 and 2 of the scorpion Centruroides noxius Hoffmann. Toxicon
Off. J. Int. Soc. Toxinol. 1995,33, 1161–1170. [CrossRef]
36.
Valdez-Velazquez, L.L.; Romero-Gutierrez, M.T.; Delgado-Enciso, I.; Dobrovinskaya, O.; Melnikov, V.; Quintero-Hernandez, V.;
Ceballos-Magana, S.G.; Gaitan-Hinojosa, M.A.; Coronas, F.I.; Puebla-Perez, A.M.; et al. Comprehensive analysis of venom from
the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities. Toxicon
2016
,118,
95–103. [CrossRef]
37.
Herzig, V.; Ikonomopoulou, M.; Smith, J.J.; Dziemborowicz, S.; Gilchrist, J.; Kuhn-Nentwig, L.; Rezende, F.O.; Moreira, L.A.;
Nicholson, G.M.; Bosmans, F.; et al. Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin
from the African spider Augacephalus ezendami.Sci. Rep. 2016,6, 29538. [CrossRef]
38.
Kasheverov, I.E.; Oparin, P.B.; Zhmak, M.N.; Egorova, N.S.; Ivanov, I.A.; Gigolaev, A.M.; Nekrasova, O.V.; Serebryakova, M.V.;
Kudryavtsev, D.S.; Prokopev, N.A.; et al. Scorpion toxins interact with nicotinic acetylcholine receptors. FEBS Lett.
2019
,593,
2779–2789. [CrossRef]
39.
Olamendi-Portugal, T.; Restano-Cassulini, R.; Riano-Umbarila, L.; Becerril, B.; Possani, L.D. Functional and immuno-reactive
characterization of a previously undescribed peptide from the venom of the scorpion Centruroides limpidus.Peptides
2017
,87,
34–40. [CrossRef] [PubMed]
40.
Pucca, M.B.; Cerni, F.A.; Janke, R.; Bermudez-Mendez, E.; Ledsgaard, L.; Barbosa, J.E.; Laustsen, A.H. History of Envenoming
Therapy and Current Perspectives. Front. Immunol. 2019,10, 1598. [CrossRef] [PubMed]
41.
Roncolato, E.C.; Campos, L.B.; Pessenda, G.; Costa e Silva, L.; Furtado, G.P.; Barbosa, J.E. Phage display as a novel promising
antivenom therapy: A review. Toxicon Off. J. Int. Soc. Toxinol. 2015,93, 79–84. [CrossRef] [PubMed]
42.
Alonso Villela, S.M.; Kraiem-Ghezal, H.; Bouhaouala-Zahar, B.; Bideaux, C.; Aceves Lara, C.A.; Fillaudeau, L. Production of
recombinant scorpion antivenoms in E. coli: Current state and perspectives. Appl. Microbiol. Biotechnol.
2023
,107, 4133–4152.
[CrossRef]
43.
von Reumont, B.M.; Anderluh, G.; Antunes, A.; Ayvazyan, N.; Beis, D.; Caliskan, F.; Crnkovic, A.; Damm, M.; Dutertre, S.;
Ellgaard, L.; et al. Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal
venom research. Gigascience 2022,11, giac048. [PubMed]
44.
Dixon, W.J.; Mood, A.M. A Method for Obtaining and Analyzing Sensitivity Data. J. Am. Stat. Assoc.
1948
,43, 109–126. [CrossRef]
45.
Shakeel, K.; Olamendi-Portugal, T.; Naseem, M.U.; Becerril, B.; Zamudio, F.Z.; Delgado-Prudencio, G.; Possani, L.D.; Panyi, G. Of
Seven New K(+) Channel Inhibitor Peptides of Centruroides bonito, alpha-KTx 2.24 Has a Picomolar Affinity for Kv1.2. Toxins
2023
,
15, 506. [CrossRef] [PubMed]
46.
Camacho, C.; Coulouris, G.; Avagyan, V.; Ma, N.; Papadopoulos, J.; Bealer, K.; Madden, T.L. BLAST+: Architecture and
applications. BMC Bioinform. 2009,10, 421. [CrossRef]
47.
Katoh, K.; Standley, D.M. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability.
Mol. Biol. Evol. 2013,30, 772–780. [CrossRef] [PubMed]
48. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D.L.; Darling, A.; Hohna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck,
J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol.
2012
,61,
539–542. [CrossRef] [PubMed]
49.
Whelan, S.; Goldman, N. A general empirical model of protein evolution derived from multiple protein families using a
maximum-likelihood approach. Mol. Biol. Evol. 2001,18, 691–699. [CrossRef] [PubMed]
50.
Stothard, P. The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences.
Biotechniques 2000,28, 1102–1104. [CrossRef]
51.
Schiavon, E.; Sacco, T.; Cassulini, R.R.; Gurrola, G.; Tempia, F.; Possani, L.D.; Wanke, E. Resurgent current and voltage sensor
trapping enhanced activation by a beta-scorpion toxin solely in Nav1.6 channel. Significance in mice Purkinje neurons. J. Biol.
Chem. 2006,281, 20326–20337. [CrossRef]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.