Three structurally related, highly potent, peptides from the venom
of Parabuthus transvaalicus possess divergent biological activity
Bora Inceoglua, Jozsef Langob, Isaac N. Pessahc, Bruce D. Hammocka,*
aDepartment of Entomology and Cancer Research Center, University of California at Davis, CA 95616, USA
bDepartment of Chemistry and Superfund Analytical Laboratory, University of California Davis, CA 95616, USA
cDepartment of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis CA 95616, USA
Received 26 October 2004; revised 19 January 2005; accepted 21 January 2005
The venom of South African scorpion Parabuthus transvaalicus contains a novel group of peptide toxins. These peptides
resemble the longchain neurotoxins (LCN) of 60–70residueswith fourdisulfide bridges;however they are 58residues longand
have only three disulfide bridges constituting a new family of peptide toxins. Here we report the isolation and characterization
of three new members of this mammal specific group of toxins. Dortoxin is a lethal peptide, bestoxin causes writhing in mice
and altitoxin is a highly depressant peptide. Binding abilityof these peptidesto rat brainsynaptosomes is tested. While the crude
venom of P. transvaalicus enhances the binding of [3H] BTX to rat brain synaptosomes none of these individual toxins had a
positive effect on binding. Although the primary structures of these toxins are very similar to birtoxin, their 3D models indicate
significant differences. Dortoxin, bestoxin and altitoxin cumulatively constitute at least 20% of the peptide contained in the
venom of P. transvaalicus and contribute very significantly to the toxicity of the venom of this medically important scorpion
species. Therefore the aminoacid sequences presented here can beused to make more specific and effective antivenins.Possible
approaches to a systematic nomenclature of toxins are suggested.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Scorpion; Toxin; NaCchannel; Birtoxin; Ikitoxin; Nomenclature
Mining the rich pool of scorpion venoms has proved
valuable in isolation of specific modulators of ion channel
function and the development of targeted antivenins.
Possani et al. (2000) predicted that each scorpion species
may possess at least 100 peptide toxins. A conservative
calculation than indicates that there may at least be 125,000
peptides from 1250 scorpion species around the world. This
estimate does not include the intra-species variation, which
may raise the above estimate considerably. Considering
the 250 or so individual toxins currently identified, it is clear
that only a small fraction of the existing structural diversity
of scorpion peptide toxins have been elucidated so far.
Classification of scorpion toxins is evolving rapidly as
new members are being isolated and characterized. One way
to classify peptide toxins is based on their site of action.
Indeed, conserved primary sequences among scorpion
toxins are known to target particular types of ion channels.
For example, long chain neurotoxins (LCNs) act on sodium
channels, short chain neurotoxins (SCNs) act on potassium
and chloride channels with the exception of Kb toxins,
which are ‘long-chain’ toxins of 60–64 amino acid residues
with three disulfide bridges acting on potassium channels,
maurocalcine-like peptides act on ryanodine sensitive
calcium channels (Possani et al., 2000; Legros et al.,
Toxicon 45 (2005) 727–733
0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: C1 530 752 7519; fax: C1 530 752
E-mail address: email@example.com (B.D. Hammock).
1998). Among the LCNs the peptides are classified as alpha
and beta toxins. Alpha toxins bind to site 3 of the voltage
gated sodium channel and prolong the inactivation phase,
inhibiting sodium current inactivation. Beta toxins on the
other hand bind to site 4 of the voltage gated sodium channel
and induce both a shift in the voltage dependence of channel
activation in the hyperpolarizing direction and a reduction
of the peak sodium current amplitude (Ceste `le and Catterall,
2000). Additionally, insect specific excitatory and depress-
ant toxins also constitute different structural classes that
target unique binding sites and modes of action.
Despite the abundance of knowledge about the primary
structure and modes of action of scorpion toxins, few studies
have addressed the structure–activity relationships of these
peptides. Identification of the bioactive surfaces of toxins
responsible for modulating ion channels can lead to the
synthesis of non-peptidic effectors of these channels that
may have beneficial uses in therapeutics or pest manage-
ment. Birtoxin, from the venom of Parabuthus transvaali-
cus for example resembles LCNs, with the exception of
being slightly smaller and more importantly having three
disulfide bridges instead of the four disulfide bridges of
other members of this group (Inceoglu et al., 2001). The
primary structure of birtoxin indicated that its site of action
could be the sodium channel. Indeed electrophysiological
characterization of the effects of birtoxin revealed that it is a
beta group peptide (Inceoglu et al., 2002).
Here we are reporting the discovery of three new
members of the birtoxin family, each with its unique
biology. These peptides are named dortoxin (lethal),
bestoxin (writher) and altitoxin (depressant-lethal). Despite
their close similarity to birtoxin, the 3D structural models
for each of these toxins indicate sufficiently significant
differences to sub-categorize these peptides into a separate
2. Materials and methods
2.1. Peptide purification and characterization
Birtoxin, dortoxin, bestoxin and altitoxin are purified
through three steps of RP-HPLC guided by murine
bioassays as described previously (Inceoglu et al., 2001).
Fractions P5 for dortoxin and P6 for bestoxin and P6B for
altitoxin from this column are collected, then several runs
are pooled and freeze dried. These fractions are then run on
a Michrom C18 microbore column on a Magic 2002
Microbore HPLC system equipped with a peptide trap. The
main peaks from the C18 column purifications are collected
and polished by running them through a microbore phenyl
column on the same system. Purity is confirmed by ESI-
TOF. Mass spectra of crude venom, separated fractions and
isolated peptides are analyzed off-line as described
previously (Inceoglu et al., 2001). Protein sequencing
and peptide quantification are accomplished in the same
manner as for birtoxin (Inceoglu et al., 2001).
Biological activity of peptides is monitored by injecting
mice and insects as described previously (Inceoglu et al.,
2001). Briefly, fractions are concentrated to dryness using a
Heto Speed Vac (ATR, Inc., Emeryville, CA). Dried
samples are resuspended in 10 ml 20 mM ammonium acetate
buffer with 1 mg/ml BSA and incubated overnight at 4 8C to
insure proper refolding before injection to the test animals.
Mice are anesthetized using ethyl ether and intracerebro-
ventricular injections of peptide solutions are executed
immediately. Control animals injected with BSA in buffer
do not show any symptoms when recovering from
anesthesia. All symptoms are observed and recorded up to
24 h post-injection. Blowfly (Sarcophaga spp.), and crickets
(Acheta domesticus) were purchased from Carolina Biolo-
gicals (Burlington, NC), Cotton bollworms (Heliothis
virescens) were obtained from USDA/ARS (Stoneville,
MI) and reared on artificial diet.
2.3. Binding assays
Tritiated BTX-A-20-a-benzoate ([3H] BTX-B) is pur-
chased from Dupont-NEN (specific activity, 37.2 Ci/mmol).
Binding of toxins to rat brain synaptosomes is measured by
utilizing the ability of site 3 toxins to enhance the binding of
batrachotoxin (Catterall et al., 1981; Little et al., 1998). Rat
brain synaptosomes are prepared from two Springer-
Dowley male rats as described previously (Catterall et al.,
1981). A crude synaptosomal fraction is prepared by
centrifuging the brain homogenate at 10,000!g for
10 min and taking the supernatant and centrifuging it for
1 h at 100,000!g. For binding assays, rat brain synapto-
somes (300 mg/mL) are suspended in 250 mL of binding
buffer (choline chloride 130 mM, CaCl2 1.8 mM, KCl
5.4 mM, MgSO4 0.8 mM, b D-glucose 5.5 mM, HEPES
50 mM, pH 7.4, BSA 1 mg/ml) in the presence of 1 mM
tetrodotoxin together with 25 nM [3H] BTX-A (NEN) and
appropriateconcentrations oftoxins.Non-specific binding is
determined in the presence of 300 mM veratridine (Calbio-
chem) and is subtracted from the total binding to determine
specific binding. The reaction is incubated for 50 min at
37 8C and terminated by filtering through GF-C glass fiber
filters that are equilibrated in wash buffer (choline
chloride 163 mM, CaCl21.8 mM, KCl 5.4 mM, MgSO4
0.8 mM, HEPES 5 mM, pH 7.4, BSA 1 mg/ml). The
filtrate is then rinsed three times with cold wash buffer
(Catterall et al., 1981).
2.4. Alignment analysis and homology modeling
Multiple alignment analysis is done by using the
T-coffee software and visualized with ESPript software
B. Inceoglu et al. / Toxicon 45 (2005) 727–733728
(Notredame et al., 2000; Gouet et al., 1999). Peptide amino
acid sequences are submitted to Swiss-Model automated
modeling server (http://www.expasy.org/swissmod/SWISS-
MODEL.html) and modeled according to previously
determined NMR structures (Peitsch, 1995; Pintar et al.,
1999; Cook et al., 2002; Zhao et al., 1992; Lee et al., 1994;
Jablonsky et al., 1999). Results are retrieved by email.
3.1. Bioassay driven peptide purification
Three HPLC peaks separated from the venom of
P. transvaalicus having potent biological activity toward
mammals are identified (Fig. 1). Fraction P5 is very
potent against mice but shows minor insecticidal activity
at O10 mg/insect using 200 mg Trichoplusia ni larvae
indicating its mammal specificity. This fraction is well
resolved on the C4 column from the preceding peak that
contains birtoxin (Inceoglu et al., 2001). Further purifi-
cation on a C18 microbore column results in a single
major peak with shoulder peaks on both sides. These
shoulders are eliminated by re-running the major peak on
a phenyl microbore column and collecting the middle
part of the peak. Molecular mass and purity of dortoxin
is then confirmed by ESI-TOF (Fig. 2(A)). Biological
activity of dortoxin from fraction P5 is assessed by
injecting 4-week-old mice. This fraction shows very high
toxicity to mice. Immediately after pure dortoxin is
injected, the mice display hyperactivity that persists until
Other symptoms include tremors, convulsions, profuse
salivation, lacrymation, continuous urination and vocali-
zation. In contrast to birtoxin whose effect increases over
a 10-min period following injection, dortoxin has a more
rapid onset of symptoms that progress to generalized
tremors and seizures with intermittent hyperactivity
lasting several seconds. Death occurs after the last of
the runs, which is significantly more intense and contains
uncontrolled movements (e.g. jumps, whole body
twitches) and urination. After death, limb muscles
continue to twitch at least for another 30 s. The LD99
is 200 ng of peptide per 20 g mouse. The amino acid
sequence of this peptide is in agreement with the MS
determined molecular mass of 6644.3 Da.
Fraction P6 and purified bestoxin both cause intense
writhing in injected animals. Immediately after injection
these animals start displaying a unique posture with their
neck twisted and the body following the neck thus
completing a full turn around the body axis. With time,
the severity of this behavior increases and the mice start to
spin around. When manually restrained the mice do not
respond to the handler. However, they involuntarily
continue to writhe; first the neck twists then the forelegs
and the body following to complete a turn. After 24 h post-
injection the test animals still continue to writhe albeit
exhausted, the rate of writhing decreases gradually to half to
one turn per second. No lethality is observed at doses
studied. The ED99is 100 ng of peptide per 20 g mouse.
Fraction P6 is further purified as for fraction P5 and
Fig. 1. Reverse phase HPLC profile of the crude venom of Parabuthus transvaalicus. Crude venom was injected to a Vydac C18 RP columnand
a linear gradient of 5–60% mobile phase was formed. Peaks were collected separately and amino acid sequences were determined as described
in Section 2. Fully identified and characterized peaks are shown with assigned names and determined molecular masses.
B. Inceoglu et al. / Toxicon 45 (2005) 727–733729
submitted for amino acid sequencing after the purity is
confirmed by MS. The calculated amino acid sequence is in
agreement with the determined molecular mass of 6603 Da
(Fig. 2(B)). The sequence of bestoxin has only two amino
acid differences from the sequence of dortoxin (Fig. 3).
The shoulder peak of fraction P5 was subsequently
resolved into a distinct peak with the continuous use of the
C4 column. These two peaks were separated and their
masses were determined by ESI-TOF. Peak A has a
molecular mass of 6603 Da which is bestoxin and peak B,
named altitoxin, has a molecular mass of 6599 Da.
Injection of altitoxin to mice results in a very profound
state of akinesia, depression and death. The ED99is 100 ng
of peptide per 20 g mouse. The peptide is then submitted for
amino acid sequencing. Complete sequencing shows that
altitoxin has a mutation, a deletion and an insertion at the C
terminus compared to bestoxin. Calculated molecular mass
is in agreement with determined molecular mass of
3.2. Binding assays
The crude venom of P. transvaalicus increases the
binding of [3H] BTX by 6-fold compared to 12-fold increase
seen with equal concentration of venom of Leiurus
quinquestriatus (Fig. 4(A)). In contrast none of the toxins
identified from this venom (birtoxin, ikitoxin, dortoxin,
bestoxin and altitoxin) cause any enhancement in the
binding of [3H] BTX to rat brain synaptosomes (Fig. 4(B)).
3.3. Structural analysis
Homology modeling gave us an opportunity to compare
the structures of all five toxins isolated from this venom for
the first time. Birtoxin and ikitoxin form a structural group
whereas dortoxin, bestoxin and altitoxin have significantly
different structure from the first group despite the sequence
homology between all of the toxins. This seems to be due to
the difference in the surface electrostatic potential of these
Fig.2. ESI-MSdeterminedmolecularmasses foreach toxinis presented.2Ashows dortoxin andbestoxintogetherwhereas2Bpresentsaltitoxin
B. Inceoglu et al. / Toxicon 45 (2005) 727–733730
two groups of peptides. More specifically birtoxin and
ikitoxin have a-helices that do not contain the typical basic
residues; instead they begin with two acidic residues. In
contrast to this dortoxin, bestoxin and altitoxin have
a-helices that are lined up with basic residues, typical of
known scorpion toxins, all protruding from the helix. In
addition, all five toxins uniquely have localized positively
and negatively charged surfaces. Moreover, the amino acid
differences between the toxins are concentrated to these
charged regions, indicating their functional importance.
Novel peptides with unique biology are isolated and
characterized from the venom of South African scorpion
P. transvaalicus. These peptides can be included in the
group of LCNs. However they possess three disulfide
bridges instead of the usual four disulfide bridges of other
members of the group and they are uniquely shorter than
other LCNs. Therefore, these peptides should be included in
a new sub-structural group of scorpion toxins. We predict
that the majority of the toxicity of P. transvaalicus venom to
mammals is due to these peptides. Dortoxin and altitoxin are
lethal at about 200 ng each, whereas bestoxin and ikitoxin
although potent are non-lethal and birtoxin is lethal at low
microgram quantities. Possible synergism among these
peptides may increase their cumulative effect significantly.
Therefore an antibody directed towards these peptides is
expected to reduce most of the toxicity of this venom.
Among these peptides birtoxin and ikitoxin are of the beta
group toxins. These two toxins do not enhance the binding
of [3H] BTX to rat brain synaptosomes, consistent with their
action on voltage gated NaCcurrent (Inceoglu et al., 2002).
Dortoxin, bestoxin and altitoxin also do not enhance the
binding of [3H] BTX to rat brain synaptosomes. This could
be explained in several different ways. One possibility is
that these toxins could be acting on KCchannels rather than
voltage gated NaCchannels. Another possibility is that they
could belong to the beta group of toxins that act on NaC
Control 100101 0.1
Fig. 4. The ability of the venom and purified peptides of P. transvaalicus to enhance the binding of [3H] BTX was tested and compared to that of
L. quinquestriatus (4A). Although the crude venom enhanced the binding of [3H] BTX none of the purified peptides had any effect (4B).
Fig. 3. Sequences of presented peptides are compared with known toxins. Multiple alignment is done using the T-coffee software and visualized
using ESPript software both located on the Expasy proteomics server (www.expasy.ch). A consensus of all analyzed sequences is given at the
bottom. The aligned toxins are cen3; Centruroides exilicauda neurotoxin 3, cngtii; Centruroides noxius toxin II, cngtiii; Centruroides noxius
toxin III cngtiv; Centruroides noxius toxin IV, cen1; Centruroides exilicauda neurotoxin 1, aahvi; Androctonus australis neurotoxin VI.
B. Inceoglu et al. / Toxicon 45 (2005) 727–733 731
channels. Alternatively, they may have different binding
sites on the voltage gated NaCchannel or specificity
towards a sub-type of ion channel that may not be expressed
in the rat brain. Multiple sequence alignment implies that
dortoxin bestoxin and altitoxin are more related to beta
scorpion toxins in primary structure. However, caution
should be taken when making this assumption. Ultimately,
electrophysiological measurements have to be taken into
account to classify these peptides.
As suggested by Olivera (1997), rapid mutation of
venom peptides would be an optimum evolutionary strategy
when prey, predators, and competitors change very rapidly
due to a sudden climate change or a geological catastrophe.
Special mechanisms then may have evolved that accelerate
the generation of new venom peptides such as frequent point
mutations of the toxin encoding sequence, for example as
seen at the C-termini of scorpion toxins. It should be noted
however that the disulfide bridge framework is highly
conserved in all scorpion peptides. In spite of the lack of the
wrapper disulfide in birtoxin-like peptides the other three
disulfide bridges are still well conserved (Inceoglu et al.,
The fact that birtoxin-like peptides are highly conserved
but produce distinct in vivo effects strongly reiterates that
scorpions-like other venomous animals employ a combina-
torial library strategy to evolve new peptides in their
venoms. Although in the venom mixture multiple peptides
that bind to the same target site seem to coexist, it is
hypothesized that, in vivo, no functional interference occurs
between these potentially antagonistic venom components.
Therefore it is possible to speculate that each one of these
three peptides may be binding to a different channel sub-
type, thus displaying a range of activities.
Homology model of the birtoxin-like peptides illuminate
an interesting aspect of these peptides. Their surfaces have
distinct negatively and positively charged regions. This is
quite striking because both charged regions are concentrated
into separate domains, which are highly correlated with the
amino acid differences between toxins. The differences
observed in toxicosis among these peptides may stem from
targeting different channel sub-types and/or differentially
modulating their channel targets. In any case further
characterization, including solution structures and electro-
physiological effects, will improve our understanding of
how exactly these peptides interact with their targets and the
basis of their selectivity.
With the ever-increasing number of new toxins identified
there is a need in the field to classify and unify the
nomenclature of these peptides (Possani et al., 1999). As
with other peptides and proteins one could name scorpion
toxins based on their mechanism of toxicity, their
specificity, their physical properties or other systems.
Some toxins are named for the species they were isolated
from (Lqh), their specificity (IT for insect toxin) and a
number such as LqhIT2. The system is systematic, and has
high information content, but raises ambiguities such as how
to deal with a peptide toxic to highly divergent organisms or
with similar peptides from different scorpion species.
We have been less systematic in naming toxins from P.
transvaalicus. As the numbers of known toxins increases
none of the above systems are sustainable. A systematic
procedure for naming toxins must be able to handle the
rapidly expanding numbers of identical toxins as well as
show phylogenic relationships. The system also should be
based on a consensus of scientists in the field. For example,
a system emphasizing the gene or peptide sequence to form
sub-families, families and clans rather than function
or organism as achieved with cytochrome P450 area
may serve as an illustrative model for this purpose
(Nelson et al., 1996).
This study was supported by grants from NIEHS
Superfund Basic Research Program P42 ES04699, NIEHS
Center for Environmental Health Sciences P42 ES05707,
NIEHS Center for Children’s Environmental Health &
Diseases Prevention, 1 P01ES11269 and USDA Competi-
tive Research Grants Program, 2003-35302-13499.
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