Oxyopinins, Large Amphipathic Peptides Isolated from the Venom of the Wolf Spider Oxyopes kitabensis...

Oxyopinins, Large Amphipathic Peptides Isolated from the Venomof the Wolf Spider Oxyopes kitabensis with Cytolytic Properties andPositive Insecticidal Cooperativity with Spider Neurotoxins*

Received for publication, January 17, 2002, and in revised form, April 25, 2002Published, JBC Papers in Press, April 25, 2002, DOI 10.1074/jbc.M200511200

Gerardo Corzo‡§, Elba Villegas‡, Froylan Gomez-Lagunas¶, Lourival D. Possani�,Olga S. Belokoneva‡, and Terumi Nakajima‡

From the ‡Suntory Institute for Bioorganic Research, Mishima-Gun, Shimamoto-Cho, Wakayamadai 1-1-1,Osaka 618-8503, Japan, the ¶Department of Physiology, School of Medicine, National Autonomous University of Mexico(UNAM), Cd. Universitaria, Mexico City 04510, Mexico, and the �Department of Molecular Recognition and StructuralBiology, Institute of Biotechnology, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico

Five amphipathic peptides with antimicrobial, hemo-lytic, and insecticidal activity were isolated from thecrude venom of the wolf spider Oxyopes kitabensis. Thepeptides, named oxyopinins, are the largest linear cati-onic amphipathic peptides from the venom of a spiderthat have been chemically characterized at present. Ac-cording to their primary structure Oxyopinin 1 is com-posed of 48 amino acid residues showing extended se-quence similarity to the ant insecticidal peptideponericinL2 and to the frog antimicrobial peptide der-maseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly sim-ilar sequences. At least 27 out of 37 amino acid residuesare conserved. They also show a segment of sequencesimilar to ponericinL2. Circular dichroism analysesshowed that the secondary structure of the five peptidesis essentially �-helical. Oxyopinins showed disruptingactivities toward both biological membranes and artifi-cial vesicles, particularly to those rich in phosphatidyl-choline. Electrophysiological recordings performed oninsect cells (Sf9) showed that the oxyopinins produce adrastic reduction of cell membrane resistance by open-ing non-selective ion channels. Additionally, a new par-alytic neurotoxin named Oxytoxin 1 was purified fromthe same spider venom. It contains 69 amino acid resi-due cross-linked by five disulfide bridges. Application ofmixtures containing oxyopinins and Oxytoxin 1 to in-sect larvae showed a potentiation phenomenon, bywhich an increase lethality effect is observed. Theseresults suggest that the linear amphipathic peptides inspider venoms and neuropeptides cooperate to captureinsects efficiently.

Amphipathic and cationic �-helical peptides are broadlyfound in animals as a part of their biological defense system (1).

Since the discovery of melittins (2) and cecropins (3) a largenumber of amphiphilic and cationic peptides have been char-acterized in invertebrates, especially from the phyla Arthrop-oda and in vertebrates from the class Amphibia. Linear cationicpeptides with �-helical conformation share some common char-acteristics such as antimicrobial activities at low micromolarconcentrations and �-helix formation in hydrophobic environ-ments. However, other features distinguish them such as theirdisruptive activity toward eukaryotic cells, particularly redblood cells, and their hydrophilic/hydrophobic amino acid dis-tribution along the structure. In arthropods, based on thesource of the antimicrobial peptides, these molecules could beroughly classified in two types. The first type is cecropin-likepeptides, which are antimicrobial with low hemolytic activityacting as a part of the insect immune defense system againstthe invasion of pathogenic microorganisms. This type of mole-cule is mainly found in the hemolymph of various arthropodssuch as sarcotoxin A in house fly (4), cecropins in lepidoptera(3), and spinigerin in termites (5). The second type is melittin-like peptides, which are antimicrobial with hemolytic activityhigher than that of the first type acting mainly as weapons forcapturing prey or as self-defense against other animals. Thistype of peptide is mostly found in the venom glands of bees (6),wasps (7), spiders (8), ants (9), and scorpions (10, 11). Am-phipathic �-helical peptides cause mainly the disruption of cellmembranes by molecular mechanisms depending principallyon the charge distribution of the peptide and on the chemicalcomposition of the cell membranes. In the order Arachnida, thebiological function of the linear amphipathic peptides may playan important role in capturing insects by dissipating ion andvoltage gradients across the membrane of excitable cells (8)and as a defense mechanism against external pathogens (10) orpossible microbial infection after digestion caused by decompo-sition of the prey (8). In this work, we report the isolation andchemical characterization of five amphiphilic peptides (oxyopi-nins) and one neurotoxin (Oxytoxin 1) from the venom of thespider Oxyopes kitabensis. Oxyopinins are the largest linearcationic amphipathic peptides from the venom of spiders thathave been chemically characterized thus far. We have analyzedtheir membrane lytic activities in biological and in artificialphospholipid vesicles and their mode of action in insect cells.

For comparative purposes we have included here a discus-sion of the effects of the pandinins, extracted from the venom ofthe scorpion Pandinus imperator. Additionally, we have dem-onstrated a positive cooperativity effect of oxyopinins with thatof neurotoxic peptides found in spider venom.

* This work was supported by a grant from the Research for theFuture Program from the Japanese Society for the Promotion of Scienceand by Grant z-005 from the National Council of Science and Technol-ogy (to F. G. L. and L. D. P.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The amino acid sequences reported in this paper has been submitted tothe Swiss Protein Database under Swiss-Prot accession no. P83247 forOxyopinin 1 (Oxki1), P83248 for Oxyopinin 2a (Oxki2a), P83249for Oxyopinin 2b (Oxki2b), P83250 for Oxyopinin 2c (Oxki2c), P83251 forOxyopinin 2d (Oxki2d), and P83288 for Oxytoxin 1 (OxyTx1).

§ To whom correspondence should be addressed. Tel.: 81-75-962-8792; Fax: 81-75-962-2115; E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 26, Issue of June 28, pp. 23627–23637, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 23627

EXPERIMENTAL PROCEDURES

Biological Materials—O. kitabensis crude venom was purchasedfrom SpiderPharm (Yarnell, AZ). Endoproteinase Glu-C (EC 3.4.21.19)was from Sigma Chemical Co. (St. Louis, MO). L-�-Phosphatidylcholine(PC)1 and L-�-phosphatidic acid (PA) from egg yolk were purchasedfrom Nacalai Tesque (Osaka, Japan). Phosphatidylethanolamine (PE)from Escherichia coli was from Doosan Serdary Research Laboratories(Toronto, Canada). All phospholipids were of reagent grade with purityhigher than 98%. The microbial strains E. coli (ATCC 11775) andBacillus subtilis (ATCC 1175) were obtained from the American TypeCulture Collection. Staphylococcus aureus (IAM 1098, from the Insti-tute of Applied Microbiology Culture Collection, University of Tokyo)was obtained from the Suntory Institute for Fundamental Research.Pandinins and �-paluIT1 were obtained according to Corzo et al. (11, 12).

Isolation of Oxyopinins and Oxytoxin—O. kitabensis crude venom (70�l) was resuspended in 0.1% aqueous trifluoroacetic acid containing10% acetonitrile (CH3CN), and the insoluble material was removed bycentrifugation at 14,000 � g for 5 min. The supernatant was useddirectly for HPLC separation. The diluted venom was fractionatedusing a reverse-phase semipreparative C18 column (5C18MS, 10 � 250mm, Nacalai Tesque, Japan) equilibrated in 0.1% trifluoroacetic acidand eluted with a linear gradient of acetonitrile from 0 to 60% in 0.1%trifluoroacetic acid, run for 60 min at a flow rate of 2 ml/min. Effluentabsorbance was monitored at 215 nm. Fractions with antimicrobial(oxyopinins) or insecticidal (oxytoxin) activity were further fractionatedby cation exchange HPLC on a TSK-gel sulfopropyl column (TosohSP-5PW, 7.5 � 75 mm, Japan). The column was equilibrated with 5 mM

sodium phosphate buffer, pH 6.5, containing 30% CH3CN. A lineargradient from 10 mM to 1.0 M sodium chloride was applied using thesame buffer and run at 1 ml/min. The peptides were finally purified bya C4 reverse-phase column (4.6 � 250 mm, Nacalai Tesque, Japan)using the same gradient as that for HPLC described above but withHFBA as an ion pairing instead of trifluoroacetic acid, run at a flow rateof 1 ml/min.

Sequence Analysis—Oxyopinins (Oxki1–5) and oxytoxin (OxyTx1)were reduced with tributylphosphine (Nacalai Tesque, Japan) and werealkylated with 4-vinyl-pyridine (Wako, Japan), in 0.5 M NaHCO3 buffer(pH 8.3) for 2 h at 37 °C in the dark prior to sequencing. The molecularmasses of oxyopinins did not change after reduction and alkylation.However, the mass of the pyridylethylated peptide OxyTx1, 9,109.4 Da,exceeded that of the native peptide by 1,050 Da, indicating the presenceof five disulfide bonds. All peptides were directly sequenced on a Shi-madzu PPSQ-10 automated gas-phase sequencer. The spider peptideswere dissolved in 30 �l of a 37% CH3CN solution and applied totrifluoroacetic acid-treated glass fiber membranes, precycled with Po-lybrene (Aldrich, Milwaukee, WI). Data were recorded on a ShimadzuCR-7A integrator.

Enzymatic Digestions—The peptides were subjected to enzymatichydrolysis. Hydrolysis with type XVII-B endoproteinase Glu-C fromStaphylococcus aureus V8 was carried out in 0.1 M sodium bicarbonatebuffer (pH 7.6), at 37 °C for 3 h, using a 1:20 (w/w) enzyme to substrateratio. The endoproteinase digest was fractionated by reverse-phaseHPLC using a C4 column (250 � 4.6 mm, Nacalai Tesque, Japan) anda linear gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid.The endoproteinase fractions were analyzed by MALDI-TOF MS, andchemically sequenced.

Mass Spectrometry—MALDI mass spectra were obtained on a Per-Septive Voyager Elite time-of-flight (TOF) spectrometer (PerSeptiveBiosystems Inc., League City, TX) equipped with a model VSL-337NDnitrogen laser (Laser Science, Cambridge, MA). The accelerating volt-age in the ion source was set to 20 kV, and data were acquired in the

positive linear mode of operation. Time-to-mass conversion wasachieved by external and/or internal calibration using standards ofbradykinin (m/z 1061.2), bovine pancreatic beta insulin (m/z 3496.6),bovine pancreatic insulin (m/z 5734.5), and ubiquitin (m/z 8565.8) (Sig-ma). All experiments were performed using �-cyano-4-hydroxycinnamicacid (Aldrich) as the matrix.

Peptide Synthesis of Oxyopinins—Oxki1 was chemically synthesizedby a solid-phase method using the Fmoc methodology on an AppliedBiosystems 433 A peptide synthesizer. Fmoc-Gln(tBu)-PEG resin(Watanabe Ltd., Hiroshima, Japan) was used to provide a free carboxylat the C terminus of synthetics Oxki1-OH and Oxki2b-OH. Chemicalsynthesis, cleavage, and deprotection of peptide resins were performedaccording to the instructions from the 1999 Novabiochem peptide syn-thesis handbook. The crude synthetic peptide was dissolved in a 30%aqueous acetonitrile solution and separated by reverse-phase HPLC ona semipreparative C18 column (10 � 250 mm, Nacalai Tesque, Japan).Cation exchange chromatography and C4 reverse-phase HPLC, as de-scribed above, were further used to purify the synthetic peptides. Thestructural identity of the synthetic and natural peptides was verified byco-elution experiments using capillary zone electrophoresis and cationexchange HPLC. The mass identity between synthetic and naturalpeptides was verified by MALDI-TOF mass spectrometry.

Circular Dichroism (CD) Measurements—CD spectra were obtainedon a Jasco J-725 spectropolarimeter (Jasco, Japan). The spectra weremeasured from 260 to 180 nm in 10 mM potassium phosphate buffer, pH7.2, and in 60% TFE, pH 7.1, at room temperature, with a 1-mmpath-length cell. Data were collected at 0.1 nm with a scan rate of 100nm/min and a time constant of 0.5 s. The concentration of the peptideswas 40 �g/ml. Data were the average of 10 separate recordings andwere analyzed by the method of Bohm et al. (13).

Antimicrobial and Hemolytic Assays—Detection and isolation ofoxyopinins was done following plate growth inhibition of E. coli and B.subtilis by vacuum-dried HPLC fractions that were resuspended in 20�l of distilled water according to Corzo et al. (11). Growth inhibitioncurves and minimal inhibitory concentrations (MIC) were obtainedusing pure peptides at concentrations of 1.6, 3.1, 6.2, 12.5, 25, and 50�M. Briefly, the inoculum was prepared from fresh bacteria cultures.Serial dilutions of peptides were arranged, and an aliquot of cell sus-pension was added to each vial. The final volume in each vial was 100�l, and the cell count was 1.2 � 106 CFU/ml for S. aureus and 1.6 � 106

colony-forming units/ml for E. coli. The final peptide concentrationranged from 1.6 to 50 �M. After 16–18 h of incubation at 37 °C, theoptical density (OD) of each vial was measured at 630 nm in an enzyme-linked immunosorbent assay reader (Bio-Rad, model 450, Hercules,CA). The positive control contained only the bacterial suspension, andthe negative control contained only sterile culture medium. The MICvalues were defined as the lowest concentration of peptide at which100% growth inhibition was observed. Hemolytic activity was deter-mined by incubating suspensions of sheep, pig, or guinea pig red bloodcells with serial dilutions of each selected peptide. Red blood cells (10%v/v) were rinsed several times in PBS by centrifugation for 3 min at3000 � g until the OD of the supernatant reached the OD of the control(PBS only). Red blood cells were counted by an hematocytometer andadjusted to approximately 7.7 � 106 � 0.3 � 106 cells/ml. Red blood cellswere then incubated at room temperature for 1 h in 10% Triton X-100(positive control), in PBS (blank), or with amphipathic peptides atconcentrations of 1.6, 3.1, 6.2, 12.5, 25, and 50 �M. The samples werethen centrifuged at 10,000 � g for 5 min; the supernatant was sepa-rated from the pellet, and its absorbance was measured at 570 nm. Therelative optical density compared with that of the suspension treatedwith 10% Triton X-100 defined the percentage of hemolysis.

Artificial Vesicles—Small unilamellar vesicles (SUVs) containing cal-cein were prepared according to Wieprecht et al. (14). Calcein leakagefrom vesicles was monitored fluorometrically using a Hitachi fluores-cence spectrophotometer (F-4500, Tokyo, Japan) by measuring the time-dependent increase in fluorescence of calcein release. As calcein leaksfrom the vesicles by the disrupting activity of oxyopinins it becomesdiluted and therefore dequenched, and an increase in fluorescence isrecorded (excitation � 490 nm, emission � 520 nm). A final volume of0.95 ml of SUVs was placed in a stirred cuvette at room temperature.An aliquot of peptide solution (50 �l) was added to the cuvette. Thepercentage of calcein released by the addition of peptides was evaluatedby the equation: 100 � (F � Fo)/(Ft � Fo), where F is the fluorescenceintensity achieved by the peptides. Fo is the fluorescence intensityobserved without the peptides, and Ft is the fluorescence intensitycorresponding to 100% calcein release determined by the addition of 50�l of 10% Triton X-100. The phosphorus content in phospholipid vesi-cles was estimated by spectrophotometric analysis (15).

1 The abbreviations used are: PC, phosphatidylcholine; PE, phos-phatidylethanolamine; PA, phosphatidic acid; CD, circular dichroism;CZE, capillary zone electrophoresis; MALDI-TOF MS, matrix-assistedlaser desorption-ionization time-of-flight mass spectrometry; OD, opti-cal density; Oxki1, oxyopinin 1; Oxki2a–d, oxyopinins 2a, 2b, 2c, and2d; Pin1, pandinin 1; Pin 2, pandinin 2; PBS, phosphate-buffered saline;TFE, trifluoroethanol; HFBA, hepta-fluorobutyric acid; MIC, minimuminhibitory concentration; SUV, small unilamellar vesicles; Fmoc, N-(9-fluorenyl)methoxycarbonyl; LD50, lethal dose that kills 50% of the an-imals; �-paluIT1, insecticidal toxin from the spider Paracoelotes luctuo-sus; OxyTx1, insecticidal toxin from the spider O. kitabensis; HPLC,high performance liquid chromatography; CD, circular dichroism; CE,cation-exchange chromatography; HP, holding potential; AaIT, insectspecific neurotoxin from the venom of the scorpion Androctonusaustralis.

Large Amphipathic Peptides from Spider Venom23628

Hemocytes—The hemolymph of Spodoptera litura larvae was col-lected by an aseptic puncture in the prolegs in 1.5-ml polypropylenevials containing 0.5 ml of an anticoagulant solution (16). The dilutedhemolymph was centrifuged at 720 � g for 5 min. The supernatant wasdiscarded, and the pellet containing the hemocytes was washed twicewith the anticoagulant solution. Hemocytes were resuspended inGrace’s insect media (Invitrogen). Eighty microliters of Grace’s mediumcontaining hemocytes was added to each vial containing 20 �l of thecultured medium with serial dilutions of peptides. The final suspension(100 �l) contained �2 � 106 hemocytes/ml (total hemocytes count).Viability of hemocytes was detected after 60 min of incubation at roomtemperature by a lactic dehydrogenase-based assay kit (Sigma). Thepositive control (100% hemolysis) was determined by applying a lyticsolution (supplied with the kit) to disrupt hemocyte cells. The hemo-cytes in Grace’s medium were used as a negative control.

Sf9 Insect Cell Culture and Electrophysiological Recordings—InsectSf9 cells (Spodoptera frugiperda cells) were kept in culture in Grace’smedia at 27 °C as previously reported (17). The membrane resistancewas monitored by recording the ionic current elicited by test pulsesapplied every second from a holding potential of 0 mV under whole-cellpatch clamp, using an Axopatch-1D amplifier (Axon Instruments,Burlingame, CA). Currents were filtered at 5 KHz and sampled at 100�s per point, with a TL1 interface (Axon Instruments). Electrodes werepulled from borosilicate glass (KIMAX 51) to a final resistance of 2 M�.The bath solution contained (millimolar): 115 NaCl, 10 CaCl2, 10HEPES-NaOH buffer, pH 7.2. The internal solution contained (milli-molar): 90 KF, 30 KCl, 10 EGTA, 10 HEPES-KOH buffer, pH 7.2.Oxyopinins were diluted in the bath solution before being added to therecording chamber.

Microinjection Assay—Insect paralytic activity was evaluated with apreviously described microinjection assay using S. litura (tobacco cut-worm) early third-instar larvae (2–3 mg) (18). Larvae were injected inthe pronotum with glass capillary pipettes and up to 300 nl of neuro-toxic and/or cytolytic peptide resuspended in water and placed in 55

mm-diameter plastic Petri dishes with an artificial diet. Paralytic andlethal effects were observed at different time intervals up to 24 h. Thelethal dose corresponding to 50% of mortality (LD50) was calculated byprobit analysis (19).

Statistical Analysis—The last significant difference method was usedto determine whether statistically significance differences occurredamong the mean values obtained.

RESULTS

Purification and Sequence Analysis of Oxyopinins and Oxy-toxin—The crude venom of O. kitabensis was fractionated usingreverse-phase HPLC (Fig. 1). Sixty fractions were manuallycollected and vacuum-dried. Dried HPLC fractions were resus-pended in distilled water and assayed for antimicrobial activitytoward E. coli and B. subtilis. Fractions 40, 41, and 45 showedantimicrobial activity toward both E. coli and B. subtilis, andthey were further fractionated by cation-exchange chromatog-raphy (CE). Fraction 45 yielded a pure peptide under CE, andit was named oxyopinin1 (Oxki1). Fractions 40 and 41 pro-duced two antimicrobial peptides each by CE, and they werenamed oxyopinins 2, 3, 4, and 5 (Oxki2–5). All five antimicro-bial peptides (Oxki1–5) were further resolved under reverse-phase HPLC conditions using HFBA for ion pairing. Oxyopin-ins were obtained in homogenous form as analyzed by capillaryzone electrophoresis (data not shown). Direct Edman degrada-tion sequencing of Oxki1 allowed unequivocal identification ofthe first 42 amino acid residues. The complete amino acidsequence of Oxki1 was obtained after its enzymatic digestionwith endoproteinase Glu-C. The endoproteinase digestion ofOxki1 yielded two fragments that permitted the full identifica-

FIG. 1. RP-HPLC chromatogram of crude O. kitabensis venom. The numbers 40, 41, and 45 represent HPLC fractions that gave positiveresults in antimicrobial bioassays. The asterisk represents the major insecticidal neurotoxin OxyTx1 from the crude venom.

Large Amphipathic Peptides from Spider Venom 23629

tion of all amino acid residues in Oxki1 (Fig. 2A). Oxyopinin1 iscomposed of 48 amino acid residues, and its molecular massdetermined by mass spectrometry was 5221.2 Da, in goodagreement with the theoretically expected molecular weight of5221.3. Direct sequencing of Oxki2 to Oxki5 permitted theidentification of their 37 amino acid residues and their theo-retical molecular weights were 4126.9, 4146.9, 4064.8, and4156.9, which also agree with their masses found by massspectrometry of 4127.1, 4146.9, 4064.7, and 4156.8 Da. Becausethe oxyopinins 2, 3, 4, and 5 had similar number of residuesand they were identical in at least 27 of 37 amino acid residues(see Fig. 2A), they were named oxyopinins 2a, 2b, 2c, and 2d(Oxki 2a–2d). All four Oxki2 analogs were digested with Glu-C,and, as expected from their primary structure, only Oxki2b andOxki2c produced peptide fragments, which is a confirmatoryresult of the primary structure obtained. Analysis of knownantimicrobial peptide sequences using the data base(bbcm1.univ.trieste.it/�tossi/pag1.htm) showed that at least240 �-linear peptides from animals and plants have been re-ported thus far. From the entire repertoire of peptides, we havechosen some antimicrobial and insecticidal �-helical peptidesfrom arthropods and frogs to compare their structural similar-ities. The primary structure of Oxyopinin 1 is 29% identical tothat of the frog antimicrobial peptide dermaseptin and to that

of the ant insecticidal peptide ponericinL2. Similarly, the Oxyo-pinin 2 analogs are about 20% identical to that of the am-phipathic peptide ponericinL2 (Fig. 2A).

A new neurotoxic peptide named Oxytoxin 1 (OxyTx1) wasalso isolated from the same spider venom. OxyTx1 is the majorparalytic neurotoxin of O. kitabensis, which elutes from HPLCat the position indicated with an asterisk in Fig. 1. It contains69 amino acid residues with a molecular mass of 8059.2 Daclosely packed by five disulfide bridges (Fig. 2B). OxyTx1 is abasic peptide (calculated pI 9.28) with a carboxylated free C-terminal residue as suggested by mass spectrometry data.OxyTx1 has some sequence similarity to insecticidal neuropep-tides isolated from the crude venom of the Brazilian spiderPhoneutria nigriventer (20) (Fig. 2B). Tx4(6–1), from P. ni-griventer, is an insecticidal toxin that is non-toxic to mice (21)and slows down the inactivation of the sodium channel in insectCNS via binding to receptor site 3 (22). Similarly to Tx4(6–1),OxyTx1 is an insecticidal toxin that is non-toxic to mice up to 1�g/20-g mouse, but it is surmised to be specific for sodiumchannels of insects (complete characterization to be describedelsewhere). The use of mixtures of OxyTx1 with oxyopininsincreased the lethality effect of individual components, as willbe briefly described below.

Circular Dichroism and Peptide Synthesis of Oxyopinins—

FIG. 2. Amino acid sequences of oxyopinins and oxytoxin (OxyTx1). A, alignment of oxyopinins with known amphipathic antimicrobialpeptides. The sequences compared are: esculentin 1 from Rana esculenta (24); cecropin A from Hyalophora cecropia (3); sarcotoxin IA fromSarcohaga peregrina (4); dermaseptin from Phyllomedusa sauvagii (23), pandinins from P. imperator (11); lycotoxins from Lycosa carolinensis (8);hadrurin from Hadrurus aztecus (10); ponericins from Pachycondyla goeldii (9). Percentage of identity is shown based in either oxyopinin 1 oroxyopinin 2a. B, alignment of oxytoxin with known insecticidal neurotoxins. The sequences compared are; Tx2–1, Tx2–5, Tx2–6, and Tx4(6–1) fromP. nigriventer (20, 21). The percent identity is shown based on OxyTx1. The sequence alignment was obtained from www.ch.embnet.org/software/ClustalW.html. Gaps (hyphens) have been introduced to enhance similarities.


The secondary structures of Oxki1 and Oxki2 analogs wereanalyzed by circular dichroism spectrometry in phosphatebuffer and in 60% solution of TFE. Aqueous solutions of TFEpromote hydrogen bonding, and it is considered to mimic a cellmembrane environment (25). Oxyopinins adopted unorderedstructures in potassium phosphate buffer with negative ellip-ticities around 195–200 nm (data not shown). Oxyopininsadopted �-helix structures with negative ellipticities at 208 and222 nm in 60% TFE (Fig. 3). Similar CD spectra of oxyopininsin hydrophobic environments have been previously observedwith other amphiphilic peptides containing �-helix structures(11, 23, 26, 27).

To conduct biological experiments and to confirm the pri-mary and secondary structures of oxyopinins, Oxki1 andOxki2b were chemically synthesized. Despite the fact thatOxki1 and Oxki2b are peptides of 48 and 37 amino acid resi-dues long, the final yield of their chemical synthesis was 44 and56%. In our experience, these good yields are probably a con-sequence of their linear nature, which would favor amino acidassembly throughout the automatic synthesis. Shortly aftertheir chemical synthesis and purification, co-elution analysis ofthe synthetic peptides with their respective native forms wasverified. Mass spectrometry determinations confirmed theidentity of the synthetic and native oxyopinins (data notshown). Further biological experiments were carried out usingthe synthetic oxyopinins with the prevailing names of Oxki1and Oxki2.

Antimicrobial Assays—The antibacterial and hemolytic ac-tivities of oxyopinins were compared with those of pandinins(Pin1 and Pin2), which are �-linear antimicrobial peptidesisolated from the venom of the scorpion P. imperator. Pandinin1 is a low hemolytic and antimicrobial peptide. Pandinin 2 is ahighly hemolytic and antimicrobial peptide with biological andstructural characteristics similar to melittin (11). The minimalinhibitory concentrations against S. aureus were 6.2, 6.2, 25,and 1.6 �M for Oxki1, Oxki2, Pin1, and Pin2, respectively (Fig.4A). The minimal inhibitory concentrations against E. coli were1.6, 12.5, 50, and 6.2 �M for Oxki1, Oxki2, Pin1, and Pin2,respectively (Fig. 4B). Oxki1 showed strong antimicrobial ac-tivity toward both E. coli and S. aureus. Oxki2 and the com-parative controls Pin1 and Pin2 were more lytic to S. aureus(Gram-positive) than to E. coli. The high antimicrobial activityof Oxki1 toward E. coli could be of a great significance as apotential antibiotic, because most of the antimicrobial peptidesalready discovered usually had low activity toward Gram-neg-ative bacteria.

Hemolytic Assays and Calcein Leakage from PhospholipidVesicles—The content of PC in mammalian red blood cellsvaries depending on the animal source. The percentages of PCof the total membrane phospholipids in guinea pig, pig, andsheep red blood cells are 41, 23, and 1%, respectively (28, 29).The hemolytic activities of Oxki1, Oxki2, and Pin1 in guineapig, pig, and sheep erythrocytes were lower compared with thatof the hemolytic activities of Pin2 (Fig. 5, A–C). With pig andsheep blood the hemolytic activity of Oxki1 was slightly higherthan that of Oxki2, whereas with all blood cells the hemolyticactivity of Oxki2 was higher than that of the peptide Pin1. Pigand guinea pig erythrocytes whose cell membranes are rich inPC were more susceptible to the hemolysis induced by all fourarachnic peptides. Sheep erythrocytes were less susceptible tothe hemolysis induced by all four amphipathic peptides. More-over, at higher concentrations of sheep erythrocytes (approxi-mately 6.6 � 107 cells/ml) non-significant hemolytic activity ofOxki1, Oxki2, and Pin1 was observed using peptide concentra-tions up to 50 �M, except for Pin2.

A comparison of pandinins and oxyopinins in their ability torelease calcein from SUVs with different lipid composition isshown in Fig. 5 (D–F). Oxki1 was more effective in releasingcalcein from zwitterionic and anionic vesicles when comparedwith Oxki2 and pandinins. All four amphipathic peptides weremore lytic toward PC vesicles than toward PE and PA vesicles.Although pandinin 2 is shorter than Oxki2, it was more effec-tive than Oxki2 in releasing calcein from artificial vesicles. The

FIG. 3. Circular dichroism spectra of native oxyopinins in 60%TFE. The secondary structures of Oxki1 and Oxki2 analogs were ana-lyzed by circular dichroism spectrometry in phosphate buffer and in60% solution of TFE. The concentration of the peptides was 40 �g/ml.Data are the average of 10 separate recordings.

FIG. 4. Antimicrobial activity of oxyopinins and pandinins. A,S. aureus; B, E. coli. Data are the average of three independent exper-iments. Error bars represent the standard deviation.


disrupting activities of Oxki1 and Oxki2 to the three differentphospholipid vesicles demonstrated that the mode of action ofthese peptides might be phospholipid-mediated. Moreover,oxyopinins are able to bind to the phospholipid bilayers with

different specificities and eventually can produce leakage oftheir content. Overall, oxyopinin 1 was more effective in dis-rupting both biological and artificial vesicles than oxyopinin 2.

Hemocytes and Whole-cell Patch Clamp of Sf9 Insect Cells—

FIG. 5. Hemolytic activity and percent release of calcein from artificial vesicles following the addition of oxyopinins andpandinins. A, guinea pig; B, pig; C, sheep; D, phosphatidic acid; E, phosphatidylethanolamine; and F, phosphatidylcholine. Data are the averageof three independent experiments for the pig and sheep red blood cells and the average of two independent experiments for guinea pig red bloodcells and the artificial vesicles. Error bars represent the standard deviation.


Because oxyopinins were obtained from the venom gland of apredator specialized in insect prey capture; we looked for apossible significant biological effect in insect cells. Therefore,the membrane integrity of insect hemocytes from S. lituralarvae in the presence of oxyopinins and pandinins was meas-ured. However, no cytolytic or membrane disruption effect us-ing up to 200 �M amphipathic peptides was observed whencompared with both positive and negative controls under theassay conditions. The positive control gave a clear reactionproduced by the release of cytoplasmic lactate dehydrogenasefrom insect hemocytes. Although the cell surface of insect he-mocytes could be a target of amphipathic peptides, becausethey are negatively charged (30), hemocytes respond to foreignmolecules by adhesion mechanisms that could subdue the ac-tivity of pore-forming peptides. Moreover we specifically testedthe effect of oxyopinins on the membrane resistance of S. fru-giperda pupal ovary cells (Sf9). After the whole-cell configura-tion of the patch clamp was established, the stability of themembrane was monitored with the delivery of �10 mV/3.5-mstest voltage pulses applied every second from the holding po-tential (HP) of 0 mV. Fig. 6A shows the last three superimposedcurrents elicited by the test pulses, after compensation of thestray capacitance. The traces show the typical resistor-capaci-tor passive electrical behavior of biological membranes (31).

The large capacitive current transients mark the beginningand the end of the test pulses. Once the stability of the mem-brane was verified, Oxki1 was added to the bath solution at afinal concentration of 1 �M. Fig. 6B shows that the addition ofOxki1 produced a sudden fall of the membrane resistance,which is seen by the increase in both the holding current(labeled I1) and the current during the pulse (labeled I2). In-terestingly, it was observed that (a) the holding current tran-siently changes its sign (further more detailed studies areneeded to determine the reason of this change) and (b) after itsinitial increase, both the holding and the pulse currents re-laxed to a smaller value (I1 dropped from 0.6 to 0.01 nA) wherethey remained stable for some minutes (variable from cell tocell) until the cell was finally lost (not shown). The latter is bestseen in Fig. 6C, which shows the membrane resistance duringeach pulse. The arrow points to the addition of Oxki1.

Finally, to explore the properties of the conductance pathwayopen by Oxki1, a current versus voltage (I-V) relationship wasconstructed (Fig. 6D) by stepping up the membrane potentialfrom �50 to �30 mV in 10-mV increments after a former�10-mV test pulse (applied to verify the constancy of the mem-brane resistance during the I-V). Fig. 6E shows the I-V rela-tionship of both the traces in D (circles) and the traces (trian-gles) recorded 3 min later. Fig. 6E illustrates that Oxki1 dropsthe membrane resistance by opening a non-selective (reversalpotential � 0 mV) ohmic (approximately linear) pathway in themembrane.

It is important to mention that qualitatively similar resultswere also observed with all other four native oxyopinins 2a–dtested, including the initial drop in membrane resistance fol-lowed by the subsequent partial relaxation shown in Fig. 6C. Itis also important to mention that appropriate controls wereconducted to show that the effects registered were due neitherto buffer components nor to mechanical artifacts.

Insecticidal Cooperativity between Oxyopinins and Neuro-toxic Peptides—It is well known that the primary function ofspider venom is to paralyze their prey (32). Therefore, a possi-ble toxic effect of oxyopinins in insects was tested. Oxki1,Oxki2, and Pin2 were injected into S. litura larvae. Uponpeptide injection the larvae presented necrotic spots after60–90 min post-injection at the site of needle insertion. Fur-thermore, depending on the concentration of peptide injected,the size of such a necrotic spot would increase with time. TheLD50 values of Oxki1, Oxki2, and Pin2 were 166.3 � 13.4 (n �3), 500.4 � 26.3 (n � 3), and 553.3 � 90.4 (n � 3) nmol/g oflarvae, respectively (Fig. 7). The insecticidal activities of oxyo-pinins were compared with those of the spider neurotoxinsOxyTx1 and �-paluIT1 (12). OxyTx1 is insecticidal to the lepi-dopteran larvae S. litura with an LD50 of 5.1 � 0.5 (n � 2)nmol/g of larvae (Fig. 7). Also, the neurotoxin �-paluIT1 islethal to the same insect with a LD50 of 1.8 � 0.2 (n � 2) nmol/gof larvae (Fig. 7). Comparative studies of the insecticidal activ-ity of oxyopinins to those of the spider neurotoxins showed thatthe latter were from 30 to 90 times more potent than theinsecticidal activity of the oxyopinin 1. Because the paralysisinflicted by the venom on insects is considered to be a syner-gistic effect caused by several factors, such as neurotransmit-ters, neuropeptides, and enzymes, the insecticidal activity ofthe neurotoxins OxyTx1 and �-paluIT1 were tested in the pres-ence of the individual oxyopinins. The relative concentrationsof the pure peptides Oxki1, Oxki2, and OxyTx1 in the solublevenom, as estimated by the chromatogram areas obtained fromFig. 1, were 7.2, 12.0, and 4.1%, respectively. Based on thesedata, the area ratio of amphipathic peptides to OxyTx1 isroughly 4.6. Therefore, a molar mixture 1:5 (neurotoxin:Oxki1)was prepared for use for injection into S. litura larvae. Al-

FIG. 6. Effect of Oxki1 on Sf9 cells. A, control currents elicited bythree test pulses of �10 mV/3.5 ms. B, currents after the addition of 1�M Oxki1 to the external solution, rate of pulsing was 1 Hz. C, mem-brane resistance versus time of the whole traces in B. D, current elicitedby stepping up the membrane potential from �50 to �30 mV (labeledI3) after the initial �10-mV test pulse. The horizontal scale bar indi-cates 2.3 ms in panels A, B, and D. The vertical scale bar indicates 1.1nA in A, 0.93 nA in B, and 1.7 nA in D. E, current versus voltagerelationship of the traces in D (circles) and the traces recorded 3 minlater (not shown in D, triangles). HP � 0 mV, Cm � 8 picofarads. Thedotted line in the traces indicates the zero current level.


though the concentration of Oxki1 was lower than the total ofOxki2a–d peptides in the crude venom, individually, Oxki1was the most potent among all five amphipathic peptides. TheLD50 values of the OxyTx1/Oxki1 and �-paluIT1/Oxki1 mix-tures containing molar ratios 1:5 were 0.4 � 0.1 (n � 2) and0.1 � 0.01 (n � 2), respectively. They were significant lower(p � 0.05, n � 2) than the LD50 values of the individualneurotoxins, suggesting the existence of a cooperative lethalityeffect when mixtures of both neurotoxins and Oxki1 were usedfor bioassay. Additionally, mixtures at molar ratio 1:1 (neuro-toxin:Oxki1) were also tested (see the legend of Fig. 7). In theseconditions, the LD50 values for the pair OxyTx1/Oxki1 and�-paluIT1/Oxki1 were 5.8 � 1.6 (n � 2) and 1.6 � 0.1 (n � 2),respectively. They were not significant different (p 0.05, n �2) from those of the insecticidal activities of pure neurotoxinsOxyTx1 and �-paluIT1. Furthermore, the time required for thelarvae to become paralyzed or eventually to die, under theeffect of mixtures (ratio 1:5) of neurotoxin/amphipathic pep-tides, were greatly reduced (Fig. 8). After the injection of themixtures the S. litura larvae showed a faster paralytic effect.Both neurotoxins OxyTx1 and �-paluIT1 act gradually overseveral hours depending on the concentration injected into theinsects (Fig. 8, A and B). Therefore, co-injection of neurotoxinsand amphipathic peptides enhanced the paralytic and lethalactivity of neurotoxins in a time- and dose-dependent manner.

DISCUSSION

Analysis of the primary structure of all five oxyopinins showsthe absence of the amino acid residues tryptophan and tyro-sine. The only aromatic residue present in these amphipathic

peptides was phenylalanine, which has the lowest molar ex-tinction of all aromatic amino acids. Consequently, oxyopininswere detected at low UV wavelengths. Oxyopinin 1 and theanalogs of oxyopinin 2 were eluted at a high percentage ofacetonitrile from the reverse-phase HPLC column (Fig. 1) in-dicating their hydrophobic character. Oxyopinin 1 is more hy-drophobic than the oxyopinin 2 analogs, and this characteristiccould reflect possible differences in their biological activity. Thedegree of hydrophobicity and the distribution of cationiccharges in oxyopinins certainly play a crucial role in determin-ing the cytotoxic and the antimicrobial effects. These charac-teristics also should define their ability to interact with mem-brane phospholipids of different compositions and chemicalstructures. Oxyopinins belong to a large group of �-helicalantimicrobial peptides that may contain two different am-phipathic �-helices separated by flexible hinge regions such asVal22-Leu23-Pro24 and Gly19-Val20-Gly21, respectively. Sepa-rated segments of �-helices have been determined in the anti-microbial peptides cecropin A (33) and sarcotoxin A (34). Forthe first peptide, Pro23 was described as the kink-forming pointof the structure, whereas for the second a segment of fourresidues in positions Gly24-Ile27 were the ones disrupting thecontinuity of the of �-helices. The length occupied by these 37-to 48-amino acid peptides was estimated to be 55.5–72 Å (1.5 Åper residue), which is therefore enough to span at least oncethrough the 30-Å-thick phospholipid bilayer of the biologicalmembranes, facilitating the formation of a pore-like structure.Although an extended shape of oxyopinins would be largeenough to span twice through the lipid bilayer, the apparent

FIG. 7. Insecticidal activity of oxyopinins and neurotoxins toward insect paralysis. Dose response of the individual and the mixturesof amphipathic and neurotoxic peptides. The concentration on the abscissa scale corresponds to nanomoles of individual peptide per gram of larvaefor the peptides Pin2, Oxki1, Oxki2, �-paluIT1, and OxyTx1. In the case of the peptide mixtures OxyTx1/Oxki1 and �-paluIT1/Oxki1, the scalecorresponds to the values based on the concentration of the neurotoxins. The lethal dose corresponding to 50% of mortality (LD50) was calculatedby probit analysis. The LD50 values are Oxki1, 166.3 � 13.4 (n � 3); Oxki2, 500.4 � 26.3 (n � 3); Pin2, 553.3 � 90.4 (n � 3); �-PaluIT1, 1.8 � 0.2(n � 2); OxyTx1, 5.1 � 0.5 (n � 2); �-PaluIT1/Oxki1(1:5), 0.1 � 0.01 (n � 2); OxyTx1/Oxki1(1:5), 0.4 � 0.1 (n � 2); �-PaluIT1/Oxki1(1:1), 1.6 � 0.1(n � 2); and OxyTx1/Oxki1(1:1), 5.8 � 1.6 (n � 2). Each point represents the average of two to three independent experiments using at least tenS. litura larvae. Error bars represent the standard deviation.


absence of a secondary structure, such as disulfide bridges,�-turns, or �-sheets would preclude such physical occurrence.The possibility that oxyopinins form helicoidal structures in alipidic environment seems to be plausible, as shown by theresults of Fig. 3, obtained in the presence of TFE. Natural(35–37) and artificial linear (38, 39) amphipathic peptideslarger than 24 amino acid residues can form well-defined mem-brane ion channels. Thus, oxyopinins are more likely pore-forming peptides. This view is partially supported by the ex-periments performed with the Sf9 ovarian cell membranes(Fig. 6), but it was not directly proven here. Moreover, it hasbeen recently observed that the pore-forming peptide ma-gainin2 and PGLa form a heterodimer, and this heterodimer

exhibited membrane permeabilization activity an order of mag-nitude higher compared with the monomeric species (40).Therefore, there is an additional possibility of synergistic in-teraction between Oxki1 and Oxki2, either for increasing thecytolytic activity or for enhancement of the neurotoxic effects ofthe venom.

For amphipathic peptides several mechanisms of membranepermeabilization have been proposed. Cecropin and dermasep-tin are suggested to disrupt membranes by the so-called “car-pet-like” mechanism (41). In the case of alamethicin, the “bar-rel-stave” model of pore formation was proposed (42), and in thecase of magainin, a peptide-lipid toroidal pore (43, 44). Theoxyopinins 1 and 2 act on bacterial cells and also lyse red bloodcell membranes (Figs. 4 and 5). The results in vivo correspondwell with in vitro studies, which demonstrated that peptidesbind and permeate both negatively charged and zwitterionicvesicles. However, the mode of action could differ betweenacidic and zwitterionic phospholipids. The dose dependencecurves obtained with these peptides assayed on PC vesicles aresigmoidal, and peptides permeabilize vesicles at a low peptide-to-lipid ratio, which is consistent with the pore-forming model.On the other hand, the curves for PA and PE vesicles requireda 5 to 10 times higher peptide-to-lipid ratio for vesicle perme-abilization. The latter is consistent with a non-pore-formingprocess whereby cationic peptides first adsorbed onto the neg-atively charged membrane surface to form a carpet-like layerand then (when a threshold concentration of peptides bound tomembrane has been reached) form transient holes, capable ofproducing complete lysis. It is thought that the density or sizeof the lipid headgroup present in the bilayers plays an impor-tant function in this behavior. The headgroups of PE or PA aresubstantially smaller than that of PC (45, 46). Therefore, PC-rich membranes would be less dense or less compact permittingthe insertion of oxyopinins into the bilayers. Interestingly, theaddition of PE to PC artificial vesicles has resulted in a decre-ment of the disruptive activity of some natural antimicrobialpeptides such as alamethicin (46) and PGLa (47). Moreover,protegrins have a deleterious effect toward human erythro-cytes that are rich in PC lipids but non-toxic to those of pigsthat are less rich in PC lipids (29, 48). Oxyopinins have ahigher propensity to lyse PC vesicles rather than PE and PAvesicles. This concept correlates well with the increasing he-molytic activity observed in red blood cells containing higheramounts of PC (Fig. 5, A–C). The phospholipid composition ofcell membranes may influence parameters of the pore struc-ture, for example, the increase of membrane fluidity shortensthe pore lifetime (44). Therefore, the different lytic potency ofoxyopinins against erythrocytes of different lipid compositionalso suggest the pore-forming mechanism.

Wolf spiders are vagabonds that lie in ambush or freely hunttheir prey (49). Although the antimicrobial activity of oxyopi-nins from O. kitabensis could be just fortuitous, it is unlikelythat bacteria were the exclusive targets of these amphipathicmolecules from a venom gland specialized in prey capture.Despite the fact that several antimicrobial peptides are pro-duced by organisms throughout the phylogenetic tree (50), thebiological function of antimicrobial �-linear peptides makessense when they are produced and excreted on the skin ofamphibians or produced inside cells specialized in host immu-nity. In the case of oxyopinins, because of their origin, it seemsthat the main targets are insect cells rather than microorgan-isms. The insecticidal activity of amphipathic molecules hasbeen previously shown by Orivel et al. (9). They found thatsome amphipathic molecules from the ant Pachycondyla goeldiiare insecticidal to house crickets. Yan and Adams (8) proposedthat the biological function of linear amphipathic peptides from

FIG. 8. Insecticidal cooperativity of neurotoxins and oxyopin-ins. A, insecticidal effect over time of the individual neurotoxin OxyTx1and the peptide mixtures OxyTx1/Oxki1, OxyTx1/Oxki2, and OxyTx1/Pin2. (Concentration used: 0.5 nmol of OxyTx1 plus 2.5 nmol of am-phipathic peptide per gram of larvae.) B, insecticidal effect over time ofindividual neurotoxin �-paluIT1 and the peptide mixtures �-paluIT1/Oxki1, �-paluIT1/Oxki2, and �-paluIT1/Pin2. (Concentration used: 0.5nmol of �-paluIT1 plus 2.5 nmol of amphipathic peptide per gram oflarvae.) A control sample of 2.5 nmol of amphipathic peptide/g of larvaedid not produce paralysis of insects (data not shown). Each point rep-resents the average of two independent experiments using at least tenS. litura larvae. Error bars represent the standard deviation.


spiders might play an important role in capturing insect preyby dissipating ion and voltage gradients across the membranesof excitable cells. The whole-cell patch clamp experiments inSf9 cells showed that oxyopinins can effectively disturb themembrane potential of insect cells leading to its complete lysisat higher concentrations (Fig. 6). This effect was also observedwith the enlargement of necrotic spots in larvae after post-injection of oxyopinins. However, it seems that not all theinsect cell lines are susceptible to oxyopinins as demonstratedhere with the hemocytes, which were not susceptible to lysis inthe presence of high concentrations of these peptides. There-fore, the detrimental effect of oxyopinins could be more pro-nounced in insect cell lines containing a substantial amount ofPC lipids in their membranes. This hypothesis is supported bythe enhanced hemolytic activity of red blood cells with a highercontent of PC.

The insecticidal cooperativity between neurotoxins and oxyo-pinins is not a particular characteristic of these amphipathicmolecules from spider venoms. Insecticidal cooperativity wasobserved in the mixtures OxyTx1/Pin2 and �-paluIT1/Pin2(Fig. 8), where Pin2 is an amphipathic molecule from the scor-pion P. imperator venom. Additionally, positive cooperativity invenoms of spiders has been observed between polyamines andneuropeptides (51) as well as between adenosine triphosphateand venom toxins (52). Therefore, other arachnid venoms maycontain amphipathic molecules used for potentiation of theirneurotoxic activities, as demonstrated for the case of scorpions(53). Recently Elazar et al. (54) have shown that lower levels ofneurotoxin AaIT (35-fold lower) are needed to kill or paralyzelepidoptera larvae when the neurotoxin AaIT is expressed in-ternally by baculovirus infection than that when AaIT is in-jected directly to the hemolymph of the larvae. This phenome-non is called toxin potentiation. Usually upon prey injection,the venom components have to overcome accessibility barriersand internal processes of degradation before reaching theirtarget (55). The larger components and the hydrophilic mole-cules from spider venom would find it more difficult to diffusetoward their receptor sites. Therefore, neurotoxin diffusiontoward their target molecules, inside the prey, might be facil-itated by the presence of amphipathic peptides acting as co-factors of insect toxicity. Oxyopinins as well as other linearamphipathic peptides could function as spreading factors, byclearing the way and helping the larger neurotoxins to reachtheir receptor sites. Spreading factors have been often observedin the venom of spiders or poisonous animals such as snakesand lizards. However, the best known spreading factors areenzymes like hyaluronidase and other proteases that degradethe extracellular matrix molecules of tissues and disrupt cellmembranes spreading the deleterious effect over the injuredtissue (56). The hypothesis of oxyopinins as spreading factorsor co-factors could explain the positive insecticidal cooperativ-ity of oxyopinins and the neurotoxic peptides toward lepidop-tera larvae. Oxyopinins, therefore, are an additional ingredientin the lethal mixture of gland venom components that are usedby the spiders to achieve a quick and effective mode of captur-ing their prey.

The antimicrobial properties of oxyopinins are certainly amodel for the design and development of alternative drugs to beuse in the therapeutic treatment of infection by pathogenicbacteria. Unfortunately, their cytolytic effect should still beaddressed in this context. In addition, the oxyopinins could beused to enhance the insecticidal activity of genetically modifiedinsect-specific baculoviruses in the field of biopesticides.

Acknowledgments—We are grateful to T. Maeda for insect rearingand to Dr. Pierre Escoubas for helping with mass spectrometryanalysis.

Addendum—During the original submission of this work, Kuhn-Nentwig et al. (57) reported the antimicrobial peptide structures of fourcupiennins from the spider Cupiennius salei (Ctenidae). Although thesespiders are of a different genus and from different geographic zones, itseems that they are evolutionarily related.

REFERENCES

1. Andreu, D., and Rivas, L. (1998) Biopolymers 47, 415–4332. Habermann, E., Jentsch, J., and Hoppe-Seyler’s, Z. (1967) Physiol. Chem. 348,

37–503. Steiner, H., Hultmark, D., Engstrom, A., Bennich, H., and Boman, H. G. (1981)

Nature 292, 246–2484. Okada, M., and Natori, S. (1985) J. Biol. Chem. 260, 7174–71775. Lamberty, M., Zachary, D., Lanot, R., Bordereau, C., Robert, A., Hoffmann,

J. A., and Bulet, P. (2001) J. Biol. Chem. 276, 4085–40926. Fennell, J. F., Shipman, W. H., and Cole, L. J. (1968) Proc. Soc. Exp. Biol. Med.

127, 707–7107. Nakajima, T., Yasuhara, T., Horikawa, R., Pisano, J. J., and Erspamer, V.

(1990). Adv. Exp. Med. Biol. 247A, 215–2208. Yan, L., and Adams, M. E. (1998) J. Biol. Chem. 273, 2059–20669. Orivel, J., Redeker, V., Le Caer, J. P., Krier, F., Revol-Junelles, A. M.,

Longeon, A., Chaffotte, A., Dejean, A., and Rossier, J. (2001) J. Biol. Chem.276, 17823–17829

10. Torres-Larios, A., Gurrola, G. B., Zamudio, F. Z., and Possani, L. D. (2000) Eur.J. Biochem. 267, 5023–5031

11. Corzo, G., Escoubas, P., Villegas, E., Barnham, K. J., He, W., Norton, R. S., andNakajima, T. (2001) Biochem. J. 359, 35–45

12. Corzo, G., Escoubas, P., Stankiewicz, M., Pelhate, M., Kristensen, C. P., andNakajima, T. (2000) Eur. J. Biochem. 267, 5783–5795

13. Bohm, G., Muhr, R., and Jaenicke, R. (1992) Protein Eng. 5, 191–19514. Wieprecht, T., Apostolov, O., and Seelig, J. (2000) Biophys. Chem. 85, 187–19815. Bottcher, C. J. F., Van Gent, C. M., and Pries, C. (1961) Anal. Chim. Acta 24,

203–20416. Soderhall, K., and Smith, V. J. (1983) Dev. Comp. Immunol. 7, 229–23917. Gomez-Lagunas, F., Olamendi-Portugal, T., Zamudio, F., and Possani, L. D.

(1996) J. Membr. Biol. 152, 49–5618. Escoubas, P., Palma, M. F., and Nakajima, T. (1995) Toxicon 33, 1549–155519. Finney, D. J. (1962) Probit Analysis: A Statistical Treatment of the Sigmoid

Response Curve, pp. 318–323, Cambridge University Press, Cambridge20. Cordeiro, M. d. N., Diniz, C. R., Valentim, A. do C., von Eickstedt, V. R., Gilroy,

J., and, Richardson, M. (1992) FEBS Lett. 310, 153–15621. Figueiredo, S. G., Garcia, M. E., Valentim, A. C., Cordeiro, M. N., Diniz, C. R.,

and Richardson, M. (1995) Toxicon 33, 83–9322. de Lima, M. E., Stankiewicz, M., Hamon, A., de Figueiredo, S. G., Cordeiro,

M. N., Diniz, C. R., Martin-Eauclaire, M. F., and M. Pelhate. (2002) J. InsectPhysiol. 48, 53–61

23. Mor, A., Nguyen, V. H., Delfour, A., Migliore-Samour, D., and Nicolas, P.(1991) Biochemistry 30, 8824–8830

24. Simmaco, M., Mignogna, G., Barra, D., and Bossa, F. (1993) FEBS Lett. 324,159–161

25. Sonnichsen, F. D., Van Eyk, J. E., Hodges, R. S., and Sykes, B. D. (1992)Biochemistry 31, 8790–8798

26. Batista, C. V. F., Rosendo da Silva, L., Sebben, A., Scaloni, A., Ferrara, L.,Paiva, G. R., Olamendi-Portugal, T., Possani, L. D., and Bloch, C., Jr. (1999)Peptides 20, 679–686

27. Wong, H., Bowie, J. H., and Carver, J. A. (1997) Eur. J. Biochem. 247, 545–55728. Nelson, G. J. (1967) Biochim. Biophys. Acta 144, 221–23229. White, D. A. (1973) in Form and Function of Phospholipids (Ansell, B.,

Dawson, R. M. C., and Hawthorne, J. N., eds) p. 454, Elsevier, Amsterdam30. Takle, G., and Lackie, A. M. (1985) J. Cell Sci. 75, 207–21431. Hille, B. (1992) Ionic Channels of Excitable Membranes, 2nd Ed., Sinauer

Associates, Inc., Sunderland, MA32. Friedel, T., and Nentwig, W. (1989) Toxicon 27, 305–31633. Steiner, H., Andreu, D., and Merrifield, R. B. (1988) Biochim. Biophys. Acta

939, 260–26634. Iwai, H., Nakajima, Y., Natori, S., Arata, Y., and Shimada, I. (1993) Eur.

J. Biochem. 217, 639–64435. Silvestro, L., Gupta, K., Weiser, J. N., and Axelsen, P. H. (1997) Biochemistry

36, 11452–1146036. Christensen, B., Fink, J., Merrifield, R. B., and Mauzerall, D. (1988) Proc.

Natl. Acad. Sci. U. S. A. 85, 5072–507637. Duclohier, H., Molle, G., and Spach, G. (1989) Biophys. J. 56, 1017–102138. Agawa, Y., Lee, S., Ono, S., Aoyagi, H., Ohno, M., Taniguchi, T., Anzai, K., and

Kirino, Y. (1991) J. Biol. Chem. 266, 20218–2022239. Higashimoto, Y., Kodama, H., Jelokhani-Niaraki, M., Kato, F., and Kondo, M.

(1999) J. Biochem. (Tokyo) 125, 705–71240. Hara, T., Mitani, Y., Tanaka, K., Uematsu, N., Takakura, A., Tachi, T.,

Kodama, H., Kondo, M., Mori, H., Otaka, A., Nobutaka, F., and Matsuzaki,K. (2001) Biochemistry 40, 12395–12399

41. Sansom, M. S. (1991) Prog. Biophys. Mol. Biol. 55, 139–23542. Mak, D. O., and Webb, W. W. (1995) Biophys J. 69, 2323–233643. Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35,

11361–1136844. Matsuzaki, K., Nakamura, A., Murase, O., Sugishita, K., Fujii, N., and Miyajima,

K. (1997) Biochemistry 36, 2104–211145. Heller, W. T., He, K., Ludtke, S. J., Harroun, T. A., and Huang, H. W. (1997)

Biophys. J. 73, 239–24446. Chen, F. Y., Lee, M. T., and Huang, H. W. (2002) Biophys. J. 82, 908–91447. Blazyk, J., Wiegand, R., Klein, J., Hammer, J., Epand, R. M., Epand, R. F.,

Maloy, W. L., and Kari, U. P. (2001) J. Biol. Chem. 276, 27899–2790648. Huang, H. W. (2000) Biochemistry 39, 8347–835249. Foelix, R. F. (1996) Biology of Spiders, 2nd Ed., pp. 7–9, Oxford University


Press, New York50. Silva, P. I., Jr., Daffre, S., and Bulet, P. (2000) J. Biol. Chem. 275,

33464–3347051. Adams, M. E., Herold, E. E., and Venema, V. J. (1989) J. Comp. Physiol. [A]

164, 333–34252. Chan, T. K., Geren, C. R., Howell, D. E., and Odell, G. V. (1975) Toxicon 13,

61–6653. Herrmann, R., Moskowitz, H., Zlotkin, E., and Hammock, B. D. (1995) Toxicon

33, 1099–110254. Elazar, M., Levi, R., and Zlotkin, E. (2001) J. Exp. Biol. 204, 2637–264555. Herrmann, R., Fishman, L., and Zlotkin, E. (1990) Insect Biochem. 20,

625–63756. Veiga, S. S., Zanetti, V. C., Braz, A., Mangili, O. C., and Gremski, W. (2001)

Braz. J. Med. Biol. Res. 34, 843–85057. Kuhn-Nentwig, L., Muller, J., Schaller, J., Walz, A., Dathe, M., and Nentwig,

W. (2002) J. Biol. Chem. 277, 11208–11216


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