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Purification And Characterization Of A New Toxin From Lycosa
erythrognatha With Antimicrobial And Insecticidal Activities.
1,2Santos,
1 Lab.Venenos
5Lab.
D.M., 1,2Pimenta, A.M.C., 2,3Richardson, M., 2Bemquerer, M.P; 4Galuppo, C.D., 4Farias, L.M; 4Carvalho, M.A.R.; 5 De Maria, M.; 1,2De Lima, M.E.
e Toxinas Animais; 2Núcleo de Biomoléculas - Depto. Bioquímica e Imunologia, 4Lab. de Microbiologia Oral e Anaeróbios – Depto. De Microbiologia;
Aracnologia, Depto. Zoologia – ICB – Universidade Federal de Minas Gerais. 3Centro de Pesquisa e Desenvolvimento – Fundação Ezequiel Dias. Belo Horizonte, MG. Brasil.
•Introduction
ESI-Q-TOF mass espectrometry analyses
Wolf spiders from Lycosa genus, are very common in urban areas in southeastern region of Brazil. Their
venoms are poorly studied and knowing their components can be a step forward to prospect new molecules with
biological activity. In this work, we initiate the biochemical characterization of the L. erythrognatha venom by using
ion-exchange high performance liquid chromatography (RP-HPLC). We verify interspecifc variations in the
composition of the venom related to the sex. Using ion-exchange and reverse phase high performance liquid
chromatography we purify new peptide (LyeTx I, II, III and IV) with molecular mass of and 2830,90 and 2847.3 Da
determined for ESI-Q-TOF mass spectrometry. LyeTx was sequenced by automatic Edman degradation and ESIQ-TOF and its predicted secondary structure display amphipathic a-helix, character typical of antimicrobial poreforming peptides. Antimicrobial assays showed this toxin potently inhibits the growth of pathogenic bacteria at
micromolar concentrations. Yet, biological assays with the fraction 12C.4 have demonstrated a high lethality in
house fly (Musca domestica) at nanomolar concentrations. Chemical synthesis of this molecule is in progress to
obtain sufficient material to improve biological and structural studies.
•Objectives
2831.10±0.02
2847.10±0.03
709.2862
A4
708.7852
B4
713.0342
713.2850
%
945.0499
945.3839
713.5367
A3
944.7153 950.3821
A5
567.4257
567.6243
950.7171
713.7865
A5
B5
567.2230 570.6227
951.0465
570.8254
571.0231
450
500
714.0401
A4
708.5255
571.2308
473.0190
544.8091
0
The main objective of this work was to initiate the characterization of Lycosa erythrognatha venom and the
purification of bioatives compounds.
A:
B:
A4
709.0356
100
952.3806
717.0351
721.0239
1417.0796
958.3829
m/z
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
Figure 3. Mass spectrometry (ESI-Q-TOF) of fraction 12C.4 (fig. 2). It is observed presence of two main
masses. :A:2830,90 e B: 2847,30. Electrospray ionization source operated in positive ion mode. Samples
were diluted in 50% acetonitrile/0.1% TFA in Milli-Q® water and applied with flow rates of 5-10 mL/min.
•Material and methods:
Simulation of the secondary structures of toxins obtained from the
venom of L. erythrognatha.
Animals and venom obtention.
Spider were collected from both Belo Horizonte, MG, and Santa Bárbara, MG, regions, maintened in the
aracnidarium at Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia (ICB–
UFMG),. Spiders were classified as Lycosa erythrognatha and the venom was obtained by electrical stimulation of
spider´s fangs.
Cation exchange chromatography in HPLC system.
Liophylized venoms were solubilized in Milli-Q water and loaded onto HPLC system using a Tosoh (TSK gel CMSW, 25cm x 4,6 mm) column.
Reverse phase chromatography in HPLC system.
Liophylized frations of venom were solubilized in 0.1% TFA and loaded onto HPLC system using a supelco C18
(25cm x 4.6mm) column. Gradient was carried out with 0,1% TFA in acetonitrile.
ESI-Q-TOF mass espectrometry analyses.
ESI-Q-TOF mass spectrometry analyses were carried out using a Q-TOF MicroTM (Micromass, UK) equipped with
an electrospray ionization source operated in positive ion mode. Capillary voltage was 3-3.5 kV and sample cone
voltages were 40-60V. Mass spectrometer calibrations were made by using sodium iodide with ceasium iodide in
2000 Da range. Samples were diluted in 50% acetonitrile/0.1% TFA in Milli-Q® water and introduced by using a
syringe pump with flow rates of 5-10 mL/min in electrospray source. Collision induced dissociation (CID) was
carried out using argon and collision energies in the range 30-45V. Data were analyzed by MassLynx® 3.5
software.
LyeTx I and II
LyeTx III
LyeTx IV
Figure 4. a-helical wheel plot of LyeTx I, II, III and IV showing amphipathic character. The toxins had been
sequenced from the fraction 12C.4 for automatic degradation of Edman and spectrometry of mass (ESI-Q-TOF and
MALDI-TOF-TOF), The obtained sequences presented great similarity with the Lycotoxin I purified from venom of Lycosa
carolinensis (YAN & ADAMS, 1998). The peptides in the a-helical wheel plot present periodic variation in the
hydrophobicity value of the residues along the peptide backbone with a 3.6 residues/cycle period characterizes an ahelix. The amino acid sequence of LyeTx begins with isoleucine (blue arrow) and proceeds in a clockwise direction.
Hydrophobic aminoacids;
Polars aminoacids;
Charged aminoacids (+);
Charged
aminoácids (-).
Tests of antimicrobian activity
Antimicrobial tests.
The bacterias were cultered on BHI (brain Heart Infusion) plus levedure extract (5%). Tha agar diffusion method
was performade for screening the antibacterial activity. The fractions of venom were applied on the discs (0,5mm
diameter) and added to petri plates previously inoculated with the bacterial strain. After 24 h at 37o C, the
inhibition zone was evaluated.
A
B
C
•Results
Cation exchange chromatography in HPLC system.
A
200
100
80
Figure 5. Tests of inhibition of the growth of bacteria: (A) Staphylococcus epidermides; (B) Staphylococcus aureus;
(C) Escherichia coli.
Paper of filter containig 5 mg of the fraction 12C.4.
Bacteria.
Halo of inhibition of the growth.
150
60
Biological assay in house fly.
100
40
20
50
0
The effect of the intratoraxic injections of the fraction 12C.4 in flies had started immediately. The flies
had presented a fast contraction of the members. The minimum dose for activity was of 0,75 ng/fly and
the calculated DL50 was of 14 ng/fly
0
0
20
40
60
80
100
Time (min)
B
200
100
80
•Conclusions
150
60
100
40
*
The venom of L. erythrognatha presents intraspecific variations.
20
50
*
*
0
Five peptides were sequenced from the venom of L. erythrognatha.
0
0
20
40
60
80
100
Time (min)
The obtained sequences presented great similarity with the Lycotoxin I purified from venom
of Lycosa carolinensis.
Figure 1: Gender variations in Lycosa erythrognatha venoms. HPLC cation exchange profiles of venoms
from adult male (A) and female (B) specimens. ( ) red marks show fractions present only in female specimens.
*
Green arrow show the fraction 12C. Tosoh (TSK gel CM-SW, 25cm x 4,6 mm) column.Gradient
was carried out
with NaCl (1M) in sodium acetate (10mM), pH 5,0. Flow rate was 1,0 mL/min
The fraction 12C.4 presented antibacterial action in pathogenic bacterias.
Reverse phase chromatography in HPLC system
500
100
400
80
300
60
200
40
100
20
Biological assays with the fraction 12C.4 have demonstrated a high lethality in house fly.
•References
YAM, L., ADAMS, M. E. Lycotoxins, antimicrobial peptides from venom of the wolf spider Lycosa
carolinensis. J. Biol. Chem., 273, 2059-2066, 1998.
0
0
20
Time (min)
40
60
Figure 2. HPLC reverse phase profile of fraction 12 C. Red arrow show the fraction 12C.4. Supelco (C18
Supecosil) column, equilibrated with 0,1 % TFA and eluted by linear gradient of 0,1 % TFA in acetonitrile.
Supported by CAPES, CAPES-PRODOC, FAPEMIG, MCT-INFRA and CNPq.