ARTICLE IN PRESS
Toxicon 50 (2007) 861–867
www.elsevier.com/locate/toxicon
Purification and characterization of a novel short-chain
insecticidal toxin with two disulfide bridges from the
venom of the scorpion Liocheles australasiae$
Nobuto Matsushita, Masahiro Miyashita, Atsushi Sakai,
Yoshiaki Nakagawa, Hisashi Miyagawa
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received 23 May 2007; received in revised form 20 June 2007; accepted 21 June 2007
Available online 26 June 2007
Abstract
Scorpion venoms contain a variety of peptides toxic to mammals, insects and crustaceans. Most of the scorpion toxins
have been isolated from the venoms of scorpions in the family Buthidae, but little interest has been paid to non-Buthidae
scorpions. In this study, we isolated a short-chain insecticidal toxin (LaIT1) from the venom of the scorpion Liocheles
australasiae belonging to the Hemiscorpiidae family. This toxin showed insect toxicity against crickets at a dose of 1.0 mg/
insect, but no toxicity was observed against mice even after injection of 1.0 mg of LaIT1 via the intracerebroventricular
route, suggesting that the effect of the toxin is insect-selective. Edman sequencing and mass spectrometric analysis revealed
that the toxin is composed of 36 amino acid residues and cross-linked by only two disulfide bridges. The pattern of the
disulfide bridges was assigned by LC/MS analysis after enzymatic digestion. LaIT1 shows no sequence homology to any
other known toxins, suggesting that this toxin represents a novel structural motif class.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Scorpion venom; Insecticidal peptide; Disulfide bridge; Neurotoxin; Short-chain toxin
1. Introduction
$
Ethical statement: On behalf of, and having obtained
permission from all the authors, Masahiro Miyashita declares
that: (a) the material has not been published in whole or in part
elsewhere; (b) the paper is not currently being considered for
publication elsewhere; (c) all authors have been personally and
actively involved in substantive work leading to the report, and
will hold themselves jointly and individually responsible for its
content; (d) all relevant ethical safeguards have been met in
relation to animal experimentation. Masahiro Miyashita testifies
to the accuracy of the above on behalf of all the authors.
Corresponding author. Tel.: +81 75 753 6116;
Fax: +81 75 753 6123.
E-mail address: [email protected]
(M. Miyashita).
0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2007.06.014
Scorpion venoms contain a variety of peptides
toxic to mammals, insects and crustaceans (Loret
and Hammock, 2001; Zlotkin, 2005). These toxic
peptides can interact with ion channels with high
affinity and selectivity. These biological features
make scorpion toxins useful tools for probing the
structures of different ion channels and evaluating
their physiological contribution to cell and organ
behavior (Massensini et al., 2002; Wanke and
Restano-Cassulini, 2007). In addition, analyzing
the relationships between the biological activity and
three-dimensional structure of scorpion toxins may
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N. Matsushita et al. / Toxicon 50 (2007) 861–867
provide useful information for the design of novel
insecticides and therapeutics (Menez, 1998; Zlotkin
et al., 2000).
Scorpion toxins can be divided into two groups
according to their molecular size. One is the longchain peptide group with 60–80 residues crosslinked by three or four disulfide bridges (Rodriguez
de la Vega and Possani, 2005), and the other is the
short-chain peptide group with 20–40 residues
cross-linked by two to four disulfide bridges
(Rodriguez de la Vega and Possani, 2004). The
majority of the long- and short-chain peptides are
specific for voltage-dependent Na+ and K+ channels, respectively. However, several long-chain
peptides act on K+ (Legros et al., 1998; Srairi-Abid
et al., 2005; Yao et al., 2005) and Ca2+ (Chuang
et al., 1998) channels and several short-chain
peptides act on Cl (Debin et al., 1993) and Ca2+
(Fajloun et al., 2000) channels. Although a number
of insect-specific toxins have been characterized in
scorpions, they mostly belong to the long-chain
peptide group (Zlotkin, 2005).
To date, about 400 toxic peptides have been
identified in scorpion venoms, mainly from those of
the Buthidae family (Tan et al., 2006). The main
focus has been placed upon the Buthidae family
because some species within this family possess
particularly potent venoms that can be harmful to
humans. However, it is worth exploring the nonButhidae scorpion venoms, because it has been
demonstrated that several peptides isolated from the
non-Buthidae scorpion venoms possess unique
primary and secondary structures (Chagot et al.,
2005; Dai et al., 2001; Torres-Larios et al., 2000).
The scorpion Liocheles australasiae belonging to the
Hemiscorpiidae family is widely found in the
western Pacific region including Japan and Australia, but few studies have been carried out to date
on its venoms (Miyashita et al., 2007). Here, we
report the purification and characterization of an
insecticidal toxin from the venom of the scorpion
L. australasiae.
2. Materials and methods
2.1. Collection of venom
The scorpions L. australasiae were collected in
Ishigaki Island located at the southern end of the
Ryukyu island chain in Japan. They were maintained in the laboratory in a humid environment at
28 1C and fed crickets (Acheta domestica) purchased
from Cyber Cricket (Shiga, Japan). The venom was
collected by allowing a scorpion telson to sting
Parafilm membrane set on a microfuge tube. The
venom was dissolved in aqueous 2% acetic acid and
centrifuged at 1200g for 10 min and the supernatant
was lyophilized and stored at 80 1C.
2.2. Bioassay
Insect toxicity was examined against crickets
(A. domestica, 5075 mg body weight) by injection
of 1 ml of the sample solution in distilled water into
their abdominal region. Distilled water was injected
as a negative control. Paralysis and death were
monitored after 1 and 48 h, respectively. Six animals
were used for each measurement. Toxicity to
mammals was evaluated by injecting the sample
solution in PBS buffer (10 ml) intracerebroventricularly (i.c.v.) into mice (male Slc:ICR strain, 20 g
body weight) after anesthesia using diethyl ether.
PBS buffer was injected as a negative control. Two
animals were used for each measurement. The toxic
symptoms were monitored up to 24 h.
2.3. Mass spectrometric analysis
MALDI-TOF MS measurements were carried
out on a Voyager DE Pro mass spectrometer with a
nitrogen-pulsed laser (337 nm) (Applied Biosystems,
Foster City, CA). Samples were dissolved in a
matrix solution containing 10 mg/ml 2,5-dihydroxybenzoic acid, 50% acetonitrile and 0.1% TFA, and
1 ml of the solution was spotted on the MALDI
sample target, then allowed to dry at room
temperature. External calibration of the mass scale
was carried out using peptides of known molecular
masses. LC/MS measurements were carried out in
positive ion mode on an LCMS-IT-TOF mass
spectrometer (Shimadzu, Kyoto, Japan) equipped
with an electrospray ion source. Reversed-phase
high-performance liquid chromatography (RPHPLC) separation was performed on a C18 microbore column (1 250 mm, Grace Vydac, Hesperia,
CA). The column was eluted with 0.1% formic acid
in water (solvent A) and 0.1% formic acid in
acetonitrile (solvent B) at a flow rate of 0.05 ml/min
using a linear gradient of 5–65% solvent B over
15 min. Mass spectra were obtained in the positive
mode. For MS/MS analysis, precursor ions were
selected manually, and collision-induced dissociation spectrum was obtained using a collisional
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energy of 50%. The mass scale was calibrated
externally using a TFA-Na solution.
2.4. Purification of peptides
The crude venom (7.3 mg) was dissolved in
distilled water and applied to a C4 semipreparative
RP-HPLC column (10 250 mm, Grace Vydac).
The column was eluted with 0.1% trifluoroacetic
acid (TFA) in water (solvent C) and 0.08% TFA in
acetonitrile (solvent D) at a flow rate of 2 ml/min
using a linear gradient of 5–60% solvent D over
55 min. The elution was monitored by UV absorbance at 215 nm. The fractions were collected every
5 min and lyophilized. Each fraction was submitted
to the insect toxicity test, and fractions showing
toxicity were applied to a C18 analytical RP-HPLC
column (4.6 250 mm, Grace Vydac). The column
was eluted at a flow rate of 0.8 ml/min using a linear
gradient of 5–35% solvent B over 60 min. Each
HPLC peak was collected individually and lyophilized. A toxic fraction was further purified by RPHPLC on a C18 microbore column (1.0 250 mm,
Grace Vydac). The column was eluted with 0.1%
heptafluorobutyric acid (HFBA) in water (solvent
E) and 0.1% HFBA in acetonitrile (solvent F) at a
flow rate of 0.05 ml/min using a linear gradient of
10–50% solvent F over 80 min. The insecticidal
peptide (25 mg) was obtained and the purity was
checked by LC/MS analysis as described above.
2.5. Determination of amino acid sequence and
disulfide bridge pattern
The purified peptide (1 nmol) was subjected to
automated Edman sequencing (Procise 491-HT,
Applied Biosystems) without reduction and alkylation of disulfide bridges. Cysteine residues in the
sequence were identified by the appearance of the
dithiothreitol adduct of phenylthiohydantoin
(PTH)–dehydroalanine, which is a known byproduct of Edman degradation of cysteine residues
(Marti et al., 1987). To obtain the C-terminal
fragment and analyze the disulfide bridge pattern,
the peptide (1 nmol) without reduction and alkylation of disulfide bridges was digested with endoproteinase Lys-C (Roche diagnostics K.K., Tokyo,
Japan) at a peptide/enzyme ratio of 42:1 (w/w).
Digestion was performed in 25 mM Tris-HCl buffer
(pH 7.1) for 18 h at 37 1C. Digested peptide
fragments were subjected to LC/MS analysis. The
unknown C-terminal sequence was determined by
863
comparison of its retention time and product ion
spectrum with those of synthesized peptides possessing all possible sequences, which were prepared by
the standard Fmoc solid-phase method. The pattern
of disulfide bridges in the peptide was determined by
comparison of the observed molecular masses of
fragment peptides with those calculated from all
peptides with possible disulfide bridge patterns.
3. Results
3.1. Purification of the insecticidal toxin
The crude venom was fractionated every 5 min
using a C4 RP-HPLC column to obtain 11 fractions
(I–XI) (Fig. 1A). These fractions were tested for
insecticidal activity against A. domestica. Fraction
VI, eluting at 30–35 min, and fraction VII, eluting at
40–45 min, showed activity. Fraction VI was the
most toxic and was therefore further separated into
nine fractions (A–I) using a C18 RP-HPLC column
(Fig. 1B), and these individual sub-fractions were
tested for insecticidal activity against A. domestica.
The toxic fraction F eluting at 48 min was further
purified by RP-HPLC with a C18 microbore column
(Fig. 1C) to give a single pure peptide, which was
named LaIT1. Approximately 25 mg (0.34%) of
LaIT1 was obtained from 7.3 mg of the crude
venom. When 1.0 mg of LaIT1 was injected into
crickets, limb spasms were observed initially,
followed by whole-body paralysis within 30 min
and death within 48 h. Even though we could not
isolate a sufficient quantity of LaIT1 to estimate
LD50 values for crickets, LaIT1 was toxic as shown
above. Since no toxic effect was observed against
mice even after injection of 1.0 mg of LaIT1 via the
i.c.v. route, this toxin is thought to be insectselective.
3.2. Structural characterization
The mass spectrometric analysis of LaIT1 demonstrated that this toxin has a monoisotopic
molecular mass of 4200.02 Da. To examine the
number of disulfide bridges in LaIT1, cysteine residues
were reduced with DTT, alkylated with iodoacetamide
and measured by MALDI-TOF MS (data not
shown). Based on the mass shift (D ¼ 232), which is
attributed to carboxyamidomethylation of four thiol
groups, LaIT1 was found to contain two disulfide
bridges. The sequence of 35 N-terminal residues,
DFPLSKEYET CVRPRKCQPP LKCNKAQICV
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864
II
III
IV
V
VI* VII VIII* IX
X
XI
Absorbance (215nm)
I
0
20
40
60
Time (min)
Absorbance (215nm)
E
F*
C
D
B
G
A
35
H
45
I
55
Time (min)
Absorbance (215nm)
*
seven different possible amino-acid sequences for the
unknown C-terminal region (GE, EG, AD, DA, VS,
SV and W). To obtain the fragment containing the
C-terminal region, intact LaIT1 was digested with
endoproteinase Lys-C, and the resulting peptides were
characterized by LC/MS/MS analysis. The C-terminal
fragment peptide (m/z ¼ 262.1) was detected at an
elution time of 9 min (Fig. 2A). Since no amino acid
sequence of this peptide could be determined from its
product ion spectrum (Fig. 2B), all possible peptides
(GGE, GEG, GAD, GDA, GVS, GSV and GW) for
this fragment were chemically synthesized and measured by LC/MS/MS (data not shown except for
GW). Comparison of retention times and product
ion spectra between native and synthetic samples
revealed that the sequence of this fragment was GW
(Fig. 2A and C), resulting in the total sequence
DFPLSKEYET CVRPRKCQPP LKCNKAQICV
DPKKGW (Fig. 2D). LaIT1 has no sequence
homology to other known natural peptides reported
in the public databases.
To assign the disulfide bridge pattern of LaIT1,
LC/MS analysis was performed for the same
digested peptides used above, in which the disulfide
bridges remained intact. Fragment peptides with
monoisotopic molecular masses of 3305.6, 1682.9
and 1554.8 were detected at elution times of 17.1,
17.7 and 18.0 min, respectively (Fig. 2A and 3A).
These fragments can be generated only from the
peptide with the disulfide bridge pattern shown in
Fig. 3B. Thus LaIT1 is cross-linked by two disulfide
bridges, Cys (11)–Cys (23) and Cys (17)–Cys (29).
4. Discussion
40
50
Time (min)
60
Fig. 1. RP-HPLC purification of LaIT1 from the L. australasiae
venom. The fractions labeled with asterisks showed insecticidal
activity. (A) First separation of the crude venom using a C4
semipreparative column. (B) Second separation of the fraction VI
using a C18 analytical column. (C) Final separation of the
fraction F using a C18 microbore column. See Section 2 for
experimental details.
DPKKG, was determined by Edman sequencing
analysis. From the difference between the calculated
molecular mass of the 35 N-terminal residues and
the observed molecular mass of LaIT1, there were
We isolated the novel insecticidal toxin LaIT1
from the Hemiscorpiidae scorpion L. australasiae.
LaIT1 is categorized into a short-chain toxin group
by its molecular size. Over 400 scorpion toxins have
been reported to date, but most of the insecticidal
toxins isolated from scorpion venoms are longchain toxins with 6–8 kDa molecular masses and act
on Na+ channels (Gurevitz et al., 2007; Zlotkin,
2005). Several short-chain peptides have been
reported to show insecticidal properties (Dhawan
et al., 2002; Tytgat et al., 1998; Wudayagiri et al.,
2001). In general, the potency of the insecticidal
short-chain toxins is relatively low (mg/insect level)
as compared with that of long-chain toxins (ng/
insect level). Although the biological targets of these
insecticidal short-chain toxins are not characterized,
most of the short-chain toxins are known to act on
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865
244.11
Intensity
TIC
216.11
205.10
132.08
Intensity
100
159.09 188.07
150
200
250
m/z
m/z = 262.1
Intensity
244.10
216.11
205.09
Gly-Trp
132.08
0
10
20
30
100
159.09
150
200
250
m/z
Time (min)
1
188.07
10
20
30
36
DFPLSKEYET CVRPRKCQPP LKCNKAQICV DPKKGW
Edman degradation
MS/MS
Fig. 2. LC/MS/MS analysis of digested LaIT1 and synthetic glycyltryptophan. (A) Chromatograms for total ion (upper) and extracted ion
with m/z ¼ 262.1 from digested LaIT1 (middle) and total ion from synthetic glycyltryptophan (bottom). Product ion spectra of the
C-terminal fragment (B) and synthetic glycyltryptophan (C). (D) Primary sequence of LaIT1.
K+, Cl and Ca2+ channels in mammals. Several
insecticidal spider toxins with molecular masses of
4 kDa have been shown to act on insect K+ and
Ca2+ channels (Escoubas, 2006; Tedford et al.,
2004). LaIT1 showed insecticidal toxicity comparable to that of other short-chain insecticidal toxins.
These facts suggest that LaIT1 might also target
K+, Cl and Ca2+ channels rather than Na+
channels, although further biochemical and neurophysiological experiments are required.
One of the characteristic structural features of
LaIT1 is the presence of a Pro–Pro sequence in the
central region. Pro–Pro sequences have also been
observed in several short-chain scorpion toxins such
as margatoxin and BmKK2 (Fig. 3B) (Rodriguez de
la Vega and Possani, 2004), which are blockers of
mammalian K+ channels, but their contributions
to the secondary structure formation are different
(Johnson et al., 1994; Zhang et al., 2004). In the case
of margatoxin, the Pro–Pro sequence, which is
located in the a-helix region, is thought to help in
folding the peptide chain as an aid to helix
nucleation. On the other hand, the Pro–Pro sequence
that is located in the loop region of BmKK2 provides
a smaller and condensed core structure that is
different from those of other short-chain toxins.
However, a common motif (CXXPXXCXPP) exists
on the N-terminal side of the Pro–Pro sequence in
both LaIT1 and margatoxin, suggesting that LaIT1
contains an a-helix structure in the central region
similar to that of margatoxin.
Another uniqueness is that LaIT1 possesses only
two disulfide bridges. To date, no insecticidal
scorpion toxin with only two disulfide bridges has
been identified, although several short-chain toxins
possess two disulfide bridges. k-Hefutoxin, a weak
K+ channel blocker isolated from Scorpionidae
scorpion Heterometrus fulvipes, adopts a unique
three-dimensional structure comprising two parallel
a-helices cross-linked by two disulfide bridges
(cysteine-stabilized a/a) (Srinivasan et al., 2002).
In the structure of k-hefutoxin, two disulfide
bridges are formed between the first and fourth
cysteines and between the second and third cysteines
(Fig. 3B). However, LaIT1 has a different disulfide
bridge pattern, in which the first and third cysteines,
and the second and fourth cysteines are linked,
indicating that the three-dimensional structure of
LaIT1 is very different from that of k-hefutoxin.
Most of the short-chain scorpion toxins form a
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866
Monoisotopic
MM
Fragment peptide
3305.6
EYETCVRPRKCQPPLK
1682.9
CQPPLK
AQICVDPKK
1554.8
CQPPLK
AQICVDPK
LaITI
Margatoxin
BmKK2
κ-Hefutoxin
CNK
AQICVDPKK
DFPLSKEYETCVRPRKCQPPLKCNKAQICVDPKKGW
TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
TPFAIKCATDADCSRKCPGNPPCRNGFCACT
GHACYRNCWREGNDEETCKERC
Fig. 3. Determination of the disulfide bridge pattern of LaIT1. (A) Fragment peptides identified by LC/MS analysis. (B) Pattern of
disulfide bridges of LaIT1 and its comparison with other short-chain scorpion toxins. The regions of a-helix are denoted with underlines,
and the residues in the common motif (CXXPXXCXPP) between LaIT1 and margatoxin are shaded in gray.
cysteine-stabilized a/b (CS-a/b) scaffold, in which
an a-helix is connected to a double- or triplestranded b-sheet by three or four disulfide bridges
(Rodriguez de la Vega and Possani, 2004). If we
assume that LaIT1 forms a structure similar to the
CS-a/b scaffold, the question arises whether two
disulfide bridges are sufficient for the stabilization
of the structure. In the study of leiurotoxin I, a
short-chain K+ channel blocker with the CS-a/b
scaffold, two disulfide bridges have been shown to
be particularly important to form an active conformation (Zhu et al., 2002). In addition, there are
two proline residues in the N- and C-terminal
sequence of LaIT1, which may stabilize the structure to compensate for lack of one or two additional
disulfide bridges found in typical short-chain toxins
with the CS-a/b scaffold. These facts suggest that
LaIT1 can form a CS-a/b scaffold with just two
disulfide bridges. However, it is also possible that
LaIT1 adopts a totally new class of structural motif.
Since LaIT1 was isolated from a Hemiscorpiidae
scorpion, it could have evolved independently of
other K+ channel blockers of Buthidae family
scorpion origin, as in the case of k-hefutoxin from
the Scorpionidae scorpion.
Future studies on the three-dimensional structure
of LaIT1 and its relationship with the insecticidal
activity will provide invaluable information for a
better understanding of the evolution of scorpion
toxins as well as the development of novel
insecticides.
Acknowledgements
We are grateful to Drs. Tadafumi Nakata and
Ken Nakamura of the Japan International Research
Center for Agricultural Sciences for the scorpion
collection. We also thank Dr. Yoshinao Wada and
Michiko Tajiri of the Osaka Medical Center and
Research Institute for Maternal and Child Health
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for MALDI-TOF-MS measurements. This study
was supported, in part, by the 21st century COE
program for Innovative Food and Environmental
Studies Pioneered by Entomomimetic Sciences,
from the Ministry of Education, Culture, Sports,
Science and Technology of Japan.
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