FEBS 23521
FEBS Letters 471 (2000) 165^168
Scorpine, an anti-malaria and anti-bacterial agent puri¢ed from scorpion
venom
Renaud Condea , Fernando Z. Zamudioa , Mario H. Rodr|¨guezb , Lourival D. Possania; *
a
Department of Molecular Recognition and Structural Biology, Institute of Biotechnology, National Autonomous University of Mexico,
Avenida Universidad, 2001, P.O. Box 510-3, Cuernavaca 62210, Mexico
b
Centro de Investigaciön sobre Enfermedades Infecciosas, National Institute of Public Health, Avenida Universidad, 655, Cuernavaca 62508, Mexico
Received 7 March 2000
Edited by Maurice Montal
Abstract A novel peptide, scorpine, was isolated from the
venom of the scorpion Pandinus imperator, with anti-bacterial
activity and a potent inhibitory effect on the ookinete (ED50 0.7
WM) and gamete (ED50 10 WM) stages of Plasmodium berghei
development. It has 75 amino acids, three disulfide bridges with a
molecular mass of 8350 Da. Scorpine has a unique amino acid
sequence, similar only to some cecropins in its N-terminal
segment and to some defensins in its C-terminal region. Its gene
was cloned from a cDNA library.
z 2000 Federation of European Biochemical Societies.
Key words: Antibiotic; Malaria; Scorpine;
Pandinus imperator ; Plasmodium
1. Introduction
Scorpion venoms are rich sources of peptides with a variety
of pharmacological functions, specially those that a¡ect membrane permeability for Na , K , Ca2 and Cl3 of excitable
and non-excitable cells (review [1]). They have been excellent
tools to study ion-channel structure and function [2]. However, these venoms contain other interesting peptides, such as
phospholipin [3] and imperatoxin-I [4], both with phospholipase activity, and the anti-microbial and anti-malaria peptide
described here. Other peptides that could be e¡ective in the
control of malaria, a parasitic disease caused by Plasmodium
spp and transmitted to vertebrates by mosquitoes, were also
investigated lately [5,6]. The long range aim of the work is to
incorporate into mosquitoes the genes encoding the bioactive
peptides to produce transgenic vectors resistant to malaria [7].
Among the peptides isolated from the venom of the African
scorpion Pandinus imperator, we identi¢ed a molecule, named
scorpine. Due to the hybrid similarity of this peptide with the
amino acid sequence of known cecropins and defensins [8], the
idea came of assaying its e¡ect on microbial growth and Plasmodium berghei stages that develop in mosquitoes. The description of the structure and biological function of scorpine
is the object of this communication.
2. Materials and methods
2.1. Chemicals and puri¢cation procedure
All chemicals were analytical grade reagents, obtained from sources
already described [3,4]. The venom of the scorpion was obtained in
*Corresponding author. Fax: (52)-73-172388.
E-mail: possani@ibt.unam.mx
the laboratory by electrical stimulation, and prepared for chromatographic separations as previously reported [4]. The soluble venom
applied to a Sephadex G-50 column (0.9U190 cm) in 20 mM ammonium acetate bu¡er, pH 4.7, resolved ¢ve well-de¢ned fractions, from
which fraction number II was subsequently applied to a carboxymethyl-cellulose (CM-cellulose) column, as described earlier for the isolation of imperatoxin-I [3] and phospholipin [4]. Scorpine was ¢nally
obtained in homogeneous form by high performance liquid chromatography (HPLC) separation, using a C18 reverse-phase column (Vydac, Hisperia, CA, USA).
2.2. Amino acid sequence
Scorpine was sequenced either as a native peptide or after reduction
and alkylation, using the method described [3,4]. An aliquot (100 Wg)
of alkylated peptide was cleaved with endopeptidase AspN and separated by HPLC (data not shown), following the conditions described
elsewhere [4]. Each independent sub-peptide was placed into the sequencer for obtaining overlapping amino acid sequences. Microsequence determination was performed on a 6400/6600 Milligen/Biosearch prosequencer, using the peptide adsorbed protocol on CD
Immobilon membranes [3,4].
2.3. Mass spectrometry determination
The molecular weight of pure scorpine was determined by mass
spectrometry, using a Kratos Kompact MALDI 3 V 3.0.2, apparatus.
2.4. Cloning and sequencing
Isolation of RNA and preparation of a cDNA library was performed as described [9]. The screening of the library was performed
using a degenerated oligonucleotide (ATG GCN AAY ATG GAY
ATG CT, where N means any nucleotide and Y means T or C), which
encodes for the amino acid sequence of residues 40 to 46 (Met-AlaAsn-Met-Asp-Met-Leu) of scorpine, synthesized as previously described [6]. The clone detected with this oligonucleotide was ampli¢ed
by polymerase chain reaction (PCR) using (lambda) gt11 forward and
reverse primers. PCR products were subcloned into pBluescript (pKS)
phagemid and sequenced using the Sequenase0 kit V. 2.0. (US Biochemical corp.). Oligo M13-20 and M13 reverse were used for sequencing [9].
2.5. Anti-bacterial assays
The classical method of Fleming was used for testing the anti-bacterial activity of scorpine. Escherichia coli ATCC 25922, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis and Klebsiella pneumoniae, were initially assayed. Inhibition zones were recorded around
wells in thin agar plates with bacteria, as described by Hultmark et al.
[15].
2.6. Parasites
The gametocyte-producing clone of P. berghei Anka strain 2.34
(kindly provided by R.S. Sinden, Imperial College of Science Technology and Medicine, UK) was used. Parasites were maintained in
BALB/c mice by mechanical passage. Mice, with parasitemias ranging
between 50 and 60% and gametocytemia around 10% were bled by
heart puncture using heparin (100 IU/ml blood). This usually occurred
after 8^10 days post-infection.
2.7. Susceptibility of P. berghei sexual stages to scorpine
Ookinete cultures were carried out as described [5]. Leucocyte-de-
0014-5793 / 00 / $20.00 ß 2000 Federation of European Biochemical Societies. All rights reserved.
PII: S 0 0 1 4 - 5 7 9 3 ( 0 0 ) 0 1 3 8 4 - 3
166
pleted infected-mouse blood was suspended 1:5 in culture medium
and tested in 100 Wl aliquots in £at-bottom 96-well plates. Scorpine
at concentrations of 0.049 to 100 WM was added to triplicate wells and
the number of rosettes, indicative of active fecundation, was assessed
by bright ¢eld microscopy between 10 and 30 min. later. The number
of ookinetes were assessed 24 h later in Giemsa-stained blood smears.
3. Results and discussion
Fig. 1 shows the pro¢le of CM-Cellulose separation of fraction 5, from the soluble venom of P. imperator after gel ¢ltration on Sephadex G-50 [4]. This chromatogram shows a better
resolution of that earlier reported [4], because it was applied
in a much greater amount of protein and separated with a
slightly di¡erent gradient. The component eluting at about
0.35 M NaCl (labeled with an asterisk) was further applied
to a C18 reverse phase column providing homogeneous scorpine (marked with an asterisk in the inset-¢gure). Small contaminants with weak phospholipase activity were eliminated
by this HPLC step. Scorpine puri¢ed by this procedure corresponds to approximately 1.4% of the total venom.
Direct automatic Edman degradation of native scorpine
permitted the identi¢cation of the ¢rst 38 amino acid residues
of the N-terminal segment, whereas a sample of scorpine reduced and carboxymethylated, permitted the unequivocal assignment of the ¢rst 47 amino acid residues of the primary
structure, as shown in Fig. 2A. Two additional sub-peptides
obtained by HPLC, after digestion with AspN endopeptidase
(data not shown), allowed the identi¢cation of the C-terminal
part of the molecule (residues in position 44^75), as well as an
extended overlapping region of scorpine (residues from posi-
Fig. 1. Puri¢cation of scorpine. Fraction II (63 mg) of the venom
from P. imperator separated by Sephadex G-50 gel ¢ltration (see ¢gure 1 in [4]) was loaded into a CM-cellulose chromatographic column (0.9U30 cm) dissolved in 20 mM ammonium acetate bu¡er,
pH 4.7. The column was eluted with a linear gradient of sodium
chloride, from 0.0 to 0.6 M salt. The fraction labeled with an asterisk contained anti-bacterial activity, and some residual phospholipase activity and was further puri¢ed by HPLC in a C18 reversephase column, as shown in the inset. Scorpine corresponds to the
major component, labeled with an asterisk. A linear gradient was
used from solvent A (0.12% tri£uoroacetic acid in water) to 60% B
(0.10% tri£uoroacetic acid in acetonitrile).
R. Conde et al./FEBS Letters 471 (2000) 165^168
tions 14 to 43). Additional segments with excellent overlapping positions were also obtained, but not included in Fig.
2A, for simpli¢cation of the picture. The completion of the
sequence was con¢rmed by the results of molecular weight
determination using mass spectrometry analysis. The molecular mass of scorpine, 8350 is compatible both with the direct
amino acid sequence obtained from the sequencer, and that
deduced from the cloned gene. Fig. 2B shows the nucleotide
sequence of a gene cloned using the internal oligonucleotide
for hybridization and the (lambda) gt11 forward and reverse
primers for ampli¢cation. A clone containing 487 nucleotides
was sequenced. The internal segment contains the information
(indicated by capital letters) that encodes the amino acids in
position 1^75 of mature scorpine, ending in the stop codon at
position 76. Two additional stretches of nucleotides were also
found, one situated at the 5P region, where a putative signal
peptide containing the information for 19 amino acids is
shown, and another at the 3P untranslated region, where a
putative adenylation site was identi¢ed (see lower case letters
in bold, in Fig. 2B).
When the amino acid sequence of scorpine was compared
with other known peptides, using data banks (SwissProt and
FASTA program), none of them showed substantial similarities to that of scorpine, except for a small stretch of the Cterminal segment, that contains three-disul¢de bridges like
those of defensins [10,11]. Also, the N-terminal region was
similar to cecropins [12^14]. Fig. 3C shows the comparison
of the primary structure of scorpine with these peptides (only
representative examples were chosen for this ¢gure). It is clear
that scorpine is a unique peptide, much longer than other
anti-bacterial peptides (practically double size). It seems to
be a hybrid of cecropin and defensin, showing some identical
amino acids (labeled with an asterisk in Fig. 2C) in certain
stretches of the sequence, when appropriate gaps are manually
introduced in order to increase similarities.
Due to the fact that the N-terminal segment of scorpine
showed similarity to cecropins, the ¢rst idea was to test its
e¡ect against bacteria.
The method described by Hultmark et al. [15] was used to
access the e¡ect of scorpine on the inhibition of bacterial
growth. Clear inhibition was obtained for B. subtilis (Minimum inhibitory concentration (MIC) around 1.0 WM) and for
K. pneumoniae (MIC of approximately 10 WM), respectively.
The biological function of scorpine in P. imperatur awaits
investigation. It is possible that it may function as microbicide
within the venom gland which is open to the exterior and in
contact with the hemocele of the scorpion prey during the
sting.
Transmission of malaria parasites by mosquito vectors is
dependent on the successful development of Plasmodium infective forms following ingestion of a blood-meal infected
with gametocytes. This process is complex and includes a
series of morphological and physiological transformations
within the mosquito midgut (gametogenesis, fecundation,
and ookinete formation) [16]. We previously observed in in
vitro and in vivo tests that 100 WM Shiva-3, a synthetic cecropin, inhibited P. berghei ookinetes development [5^7]. The
similarity of scorpine to cecropin prompted us to investigate
its e¡ect on the same P. berghei sexual stages. Our results
showed that scorpine completely inhibited both fecundation
and ookinete formation at 50 and 3 WM, respectively (Fig. 3),
and that it had a toxic e¡ect on gametes and on ookinetes at
167
R. Conde et al./FEBS Letters 471 (2000) 165^168
Fig. 2. Amino acid and nucleotide sequence of scorpine. A: Primary structure of scorpine as determined by direct sequencing the native peptides (underlined DS) and fragments produced by enzymatic hydrolysis with endopeptidase AspN (labeled ASP-N). B: Nucleotide sequence
written from the 5P to the 3P end of the clone that encodes scorpine. The sequence corresponding to the putative signal is underlined. The mature peptide is indicated from residues 1^75, ending with the stop codon. The lower case letter in bold at the 3P region indicates the possible
poly-adenylation site, whereas lower case letters at the extreme 5P and 3P ends the non-coding region of the gene. Numbers on the right side of
the page correspond to the positions of nucleotides from the signal peptide to the stop codon (upper lines) and to their corresponding amino
acids (lower lines). C: Amino acid sequence comparison of scorpine with that of cecropin and defensin antibiotic peptides. The sequences were
aligned with extended blank spaces, because of the di¡erence in size of the peptides and lack of extended similarities. The segments with some
consensus sequences are shown pairwise with arti¢cial blank spaces (small dots, to enhance similarity) and whenever an identical residue is situated in equivalent position, an asterisk is placed either down or up to the sequence of scorpine. The sequences compared are: cecropin B from
Antheraea pernyi [12]; sarcotoxin Ic from Sarcophaga peregrina [13]; cecropin P1 from Sus scrofa [14]; scorpine from this work; defensin from
Aeschna cyanea [10] and a defensin from the scorpion Leiurus quinquestriatus hebraeus [11].
lower concentration than that of Shiva-3. The calculated ED50
were 10 WM for the gamete and 0.7 WM for the ookinete
stages of development. These results indicate that scorpine
could be a better candidate than Shiva-3, to be incorporated
into the genome of genetically engineered malaria-resistant
anophelines.
Acknowledgements: This work was supported in part by a Grant from
Howard Hughes Medical Institute (No. 75197-527107) to LDP,
WHO/TDR (ID 930812), and CONACyT-Mexico (G021-M9607) to
MHR and LDP. R. Conde was a fellow of the N.G.O. (Service Civil
Internacional, branche Belge) of the Belgium Government. The technical assistance of Mr. Fredy Coronas and Jorge A. Torres is acknowledged.
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Fig. 3. Dose response curve of the e¡ect of scorpine on the ookinete
and fecundation (rosette) phases of the development of P. berghei.
Inhibition of the ookinete development (closed triangles) and inhibition of rosette formation (open rhomboids) of P. berghei in vitro by
the scorpine peptide. For the ookinete phase, at least 10 000 red
blood cells were counted, whereas for the rosette formation a minimum of 10 microscopic ¢elds were directly examined, for each scorpine concentration.
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