Background
Scorpion venom is a rich source of various polypeptides
with diverse physiological and pharmacological activities
which generally exert their action via target specific
modulation of ion channel function [ 1 2 3 ] . Among the
well characterized peptide toxins are those from the venom
of scorpions belonging to the family Buthidae. Buthoid
venom has been reported to strongly affect a wide variety
of vertebrate and invertebrate organisms [ 4 ] and its
toxicity is attributed to the presence of a large variety
of basic polypeptides cross-linked by 3-4 disulfide bridges
[ 1 4 ] . Based on their molecular size and pharmacological
activity, these toxins are classified into two main groups.
The first group contains short toxins (30-40 amino acid
residues) with 3-4 disulfide bridges which mainly affect
the voltage dependent K +channels and the large conductance
calcium activated K +channels [ 5 6 ] . The second group
includes long-chain (60-70 amino acids) peptides cross
linked by 4 disulfide bridges which mainly have an effect
on voltage dependent sodium channels of excitable cells [ 1
2 ] . According to their species selectivity sodium channel
toxins have been divided into mammalian and insect toxins.
Furthermore, depending on their binding affinities and
electrophysiological properties, the mammalian toxins are
sub-classified into α- and β- toxins [ 7 8 ] , and the
insect-specific toxins are subdivided into excitatory,
depressant and α- insect toxins [ 9 ] . Several toxins
which specifically affect sodium and potassium channels
have been extensively studied with respect to their
structure, mode of action and pharmacological properties [
4 6 10 ] . In addition to the two main groups of toxins,
several other toxins targeted toward Ca 2+ [ 5 ] and Cl - [
11 ] channels have also been isolated during the past
decade.
Owing to their species selectivity, increasing attention
has been paid in recent years to identify insect-selective
toxins that can be used to develop recombinant
biopesticides as a safer alternative to broad spectrum
chemical insecticides [ 12 13 14 ] . Several
insect-selective toxins have been identified and purified
from scorpions collected in different geographical
locations [ 2 11 15 16 17 18 ] . However, the Indian red
scorpion,
Mesobuthus tamulus, known for its
severe toxicity [ 19 20 ] received little attention in this
regard. The few studies documented on
Buthus tamulus are restricted to the
isolation and purification of neurotoxins, protease
inhibitors, histamine releasers [ 19 ] iberiotoxin, an
inhibitor of high conductance Ca 2+-activated K +channel [
21 ] and neurotoxin Bt-II [ 20 ] . In the quest for natural
insect-selective toxins, we have identified a novel short
lepidopteran-selective toxin from the venom of
Mesobuthus tamulus having 37 amino
acids residues and 8 half-cystines. We named it as ButaIT (
B. tamulus insect toxin) which has
high sequence homology with a few short toxins [ 11 22 23
24 25 26 27 ] and shows high selectivity to a lepidopteran
insect,
Heliothis virescens. This paper
describes isolation, purification, and sequence
determination of this toxin.
Results and Discussion
Bioassay driven purification of ButaIT
The CM-52 cation exchange column yielded 7 peaks at
280 nm (Fig- 1). When CM fractions were tested for
lethality on mice, only CM-fraction III and crude venom
were found to be toxic at a dose of 3 μg/g body weight.
Among all the CM fractions, CM fraction IV accounted for
almost all of the lethality to tobacco budworm. Hence
further studies were conducted using only CM fraction IV.
Fractionation of CM-IV yielded 14 peaks using ion-pair
reversed phase HPLC (Fig. 2). Bioassay of the individual
HPLC fractions of CM-IV fraction on tobacco budworm,
blowfly larvae and mice led to the localization of
lepidopteran selective toxicity in CM-IV fraction 6
(Table 1). Administration of CM-IV HPLC fractions to
tobacco budworm at a dose of 1 μg/100 mg larva showed
slow and progressive flaccid paralysis only with fraction
6. Other fractions were ineffective at this dose. Hence,
fraction 6 (CM-IV-6) was collected, lyophilized and
resolved further using HPLC gradient system-II which
yielded two peaks: CM-IV-6A and CM-IV-6B. Among the two,
fraction CM-IV-6A was found to induce progressive,
irreversible flaccid paralysis at the dose of 1 μg/100 mg
per larva of
Heliothis virescens. Neither of
these fractions induced any significant toxic symptoms in
blowfly larvae or mice. This fraction was further
purified to apparent homogeneity by microbore HPLC and
the purity was confirmed by capillary electrophoresis
(Fig. 3). This fraction is estimated to account for
0.026% of the total protein content of the dry venom.
Structural analysis
The molecular mass of the peptide toxin determined by
ESI-MS was 3856.7Da (Fig. 4) which is in agreement with
the molecular mass calculated from the amino acid
sequence when the toxin is in its native form with four
disulfide bridges as observed in other similar toxins. An
examination of multiple sequence alignment with known
insect toxins using BLAST search [ 28 ] revealed that
ButaIT toxin shows considerable homology (Fig. 5) with
short insect toxins, and shows highest percentage
identity with peptide I (68%) and LQH-8/6 (68%) followed
by neurotoxin P2 (66%), chlorotoxin (64%), insectotoxin
I5A (60%), insect toxin I5 (60%) and insectotoxin II
(52%) [ 11 22 23 24 25 26 27 ] .
Toxins of buthid venoms constitute a family of closely
related peptides which could be classified into several
structurally related peptides depending on their amino
acid [ 1 ] . Although the information available on the
mode of action of these toxins is limited, it appears
that different short toxins exhibit considerable
diversity in their physiological mechanisms in spite of
their structural homology. For example Fazal
et al. , [ 25 ] reported that
peptide I is related to neurophysin, agglutinins [ 29 30
] and other peptides of the four-disulfide core family
with toxin/agglutinin fold. The neurotoxin P2 (35 ammo
acids), another short toxin and a structural homologue of
short insectotoxin II and 15, shows toxicity towards
Sarcophaga falculata and
crustaceans [ 26 ] . Chlorotoxin, isolated from
Leiurus quinquestriatus
haebraeus, shows Cl -channel-blocking activity and
causes paralysis due to the inhibition of structurally
related anion channels such as the extrajunctional
channels of arthropod muscles [ 11 ] . It has also been
reported that the short insectotoxin I5A may act on a
"glutamate receptor of the postsynaptic membrane" [ 22 ]
. Although no specific experimental evidence is available
on the mode of action of ButaIT, it is assumed that
ButaIT exerts similar ion channel blocking activity as
that of other short insect toxins. However a detailed
physiological and pharmacological analysis is necessary
to define the exact mechanism of ButaIT in spite of its
high sequence and structural homology with other insect
toxins.
Considering the high degree of sequence similarity, it
is presumed that ButaIT will have a similar 3-D structure
to that of other short toxins. The 3-D structures of
several short toxins such as charybdotoxin (ChTX) [ 17 ]
, iberiotoxin (IbTx) [ 31 ] , kaliotoxin (KxTx) [ 32 33 ]
margatoxin (MgTx) [ 34 ] , maurotoxin [ 35 ] , agitoxin
(AgTx) [ 36 ] , noxiustoxin (NTX) [ 37 ] , PiL [ 10 ] ,
insectotoxin I5A [ 22 ] , LQH-8/6 [ 27 ] and chlorotoxin
[ 38 ] have been determined by NMR spectroscopy and X-ray
crystallography. A structure model of ButaIT, generated
from the Swiss-model protein modeling server based on the
structural coordinates of chlorotoxin [ 38 ] and
insectotoxin I5A [ 22 ] showed that ButaIT adopts a
typical core structure of scorpion toxins primarily
composed of one α-helix and three β-strands with four
disulfide bridges, a feature similar to other toxins of
similar size that act on K +and Cl -channels [ 17 31 34
38 ] . It is also evident from the data that disulfide
bridge formation in ButaIT likely follows a common motif,
with covalent links between cysl6-cys32, cys5-cys27,
cys20-cys34 and an additional disulfide bridge between
cys2 and cys 19 which cross-links the N-terminal strand
of the β-sheet to the α-helix. Experiments are underway
to purify larger quantities of ButaIT using baculovirus
expression system in order to establish a refined
structure/function relationship.
Conclusions
Here we report the isolation of a short insecticidal
toxin from the venom of
Mesobuthus tamulus. The most
important characteristic of this novel toxin is its
specificity towards
Heliothis virescens, the tobacco
budworm, a worldwide pest of numerous valuable crops such
as cotton. When expressed in baculovirus system as a
biopesticide scorpion toxins offer the advantage of faster
kill without chemical insecticides [ 13 ] . Although the
safety of recombinant baculoviruses is less of a concern
ButaIT offers an extra level of safety as a recombinant
biopesticide due to its specificity to tobacco budworm ion
channels. Moreover this toxin can be utilized as a specific
probe of tobacco budworm ion channels. Chemical libraries
can be screened for the ability to displace ButaIT leading
to the discovery of better pesticides.
Materials and Methods
Toxin purification
The crude venom of
Mesobuthus tamulus was purchased
from Haffkine Institute (Bombay, India). Lyophilized
venom (50 mg) was homogenized in 2 ml of 10 mM ammonium
acetate (pH 6.4) using Potter-Elvehjam homogenizer and
the insoluble material was removed by centrifugation at
15,000 rpm for 20 min. An additional 1 ml of ammonium
acetate was added to the pellet, the homogenization was
repeated, the contents were centrifuged and the
supernatant was collected. The above procedure was
repeated 4 to 5 times to maximize the yield of peptide
toxins from the crude venom. The supernatant fractions
were pooled, passed through a pre-cycled CM-52 column and
eluted with a gradient of 0.01 M to 0.5 M ammonium
acetate (pH 6.4) at a flow rate of 3 ml/h using a
Pharmacia peristaltic pump. Fractions (3 ml), were
collected using a BioRad (Model 2110) fraction collector.
All the chromatographic separations were carried out at
4°C. Individual fractions were monitored at 280 nm using
a Shimadzu spectrophotometer (UV 2101 PC). The active
fractions under the same absorbency peak were pooled and
freeze dried. Chromatographic analysis of CM-fractions
was done by HPLC (Perkin-Elmer Series 410 pump) which was
equipped with a LC-235 diode array detector. The UV
measurements were performed simultaneously at 215 and 280
nm. A 10 μm particle size Protein C4 column (25 cm × 4.5
mm ID; Vydac, Hesperia, CA, USA) was used in conjunction
with a C4 guard column cartridge. The column was
maintained at room temperature (25°C). Chromatographic
peaks were integrated on an Everex 386/20 PC using Omega
software and Perkin-Elmer GP-100 graphics integrator. The
freeze dried CM-fractions were resuspended in water and
aliquots of 1 mg each were loaded onto the C4 reversed
phase column equilibrated in Solvent A (A= 5%
acetonitrile, 0.1% trifluroacetic acid, TFA). The
individual proteins were eluted from the column with a
linear gradient reaching 60% Solvent B (B= 95%
acetonitrile; 0.1% TFA) in 70 min at a flow rate of 0.6
ml/min. (Gradient system-I). All the fractions were
collected, freeze dried and assayed for biological
activity. The active fractions were further purified
using a Protein C4 Column (Vydac 25 cm × 4.6 mm ID)
equilibrated with Solvent A (A= 5% acetonitrile; 0.1%
heptafluorobutyric acid (HFBA) and resolution of peaks
was accomplished using gradient elution of solvent B (B=
95% acetonitrile; 0.1% HFBA) reaching 60% in 70 min at a
flow rate of 0.6 ml/min. (Gradient system-II). Individual
peaks were collected, lyophilized and used for bioassays.
The protein concentrations of the samples were determined
using the BCA protein assay (Pierce) with bovine serum
albumin as the standard. Following two HPLC steps, the
toxins were further purified using a Reliasil C18
reversed-phase column (Michrom 10 cm × 1 mm ID)on a
Microbore HPLC system (Michrom Bioresources Inc., USA).
The insect-selective toxin was loaded onto the C18 column
and eluted in a linear gradient of 10-60 % Solvent B (95%
acetonitrile; 0.1% TFA) over 60 min at a flow rate of 100
μl/min.
Purity determination
The purity of the toxins was evaluated by free
solution capillary electrophoresis on an Applied
Biosystems capillary electrophoresis Model 270A. The 75
cm capillary was equilibrated for 4 minutes with 20 mM
sodium citrate buffer (pH 2.9) and the toxin (0.2 mg/ml)
was loaded using vacuum for 2 seconds. Resolution of
peaks were performed using 20 mM sodium citrate buffer
(pH 2.9) at 20 kV electric potential and the absorbance
was monitored at 215 nm.
Mass spectrometry
The molecular mass of ButaIT was determined using
electrospray ionization mass spectrometry on a VG/Fisons
Quattro-BD mass spectrometer (VG Biotech, Altrincham,
UK). The delivery of mobile phase (50% acetonitrile/0.05%
formic acid; v/v) was performed with ISCO μLC-500 syringe
pump at a flow rate of 5 μl/min. The purified toxin
collected from microbore HPLC was analyzed by direct flow
injection of 10 μl. The spectral analysis was done in a
positive ion mode at a capillary voltage of +3.5 kV, a
cone voltage of 50 V and at a source temperature of 70°C.
Spectra were scanned over the range of 200-2000 m/z at a
rate of 20 sec/scan ; 20 scans were combined using the
VGMCA acquisition mode. The molecular weight of the toxin
was determined using the maximum entropy deconvolution
algorithm (MaxEnt) to transform the range of 650/1500 m/z
to give a true mass scale spectrum. The mass calibration
was performed using heart myoglobin (Sigma)
Bioassay of toxins
The individual lyophilized fractions were dissolved in
deionized water, and an appropriate concentration of each
fraction was injected into fourth instar
Heliothis virescens (tobacco
budworm) and last instar larvae of
Sarcophaga falculata (blowfly). The
neurotoxic symptoms in the larvae were observed
continuously for 30 minutes and also after 24 hr.
post-injection. Complete immobility or paralysis was
taken as an indication for the neurotoxic activity in
insect larvae. Each fraction was also injected
subcutaneously into mice in order to check for any
mammalian toxicity.
Sequence determination
The purified ButaIT was reduced and carboxymethylated
by incubating in 6 M guanidine hydrochloride, 0.1 M
Tris-HCl (pH 8.3), 1 mM EDTA and 20 mM dithiothreitol for
1 hr at 37°C. Iodoacetic acid was added to a final
concentration of 50 mM and incubated for an additional
hour at 37°C in the dark. The N-terminal sequence of the
toxin was determined using HP GS1000 sequence analyzer by
automated Edman degradation at the Molecular Structure
Facility of UC Davis.
Homology modeling
The amino acid sequence of ButaIT was submitted to
Swiss-Model Protein modeling server (Guex and Peitsch,
1997; Peitsch, 1995; Peitsch, 1996; Preitsch and Guex,
1997) and the three dimensional model structure was
extracted as PDB file based on the NMR coordinates of
apparently homologous scorpion toxins such as chlorotoxin
(PDB entry : 1Chl.pdb) and insectotoxin I5A (PDB entry:
1SIS.pdb). Visualization of the 3D structure was
performed using RasMol Molecular PDB visualization
software.
Note: The amino acid sequence of novel
lepidopteran-selective toxin reported in this paper will
appear in the SWISS-PROT protein data bank under the
accession number P81761.
