Two classes of paralytic toxins were isolated from the venom of Agelenopsis aperta and their chemical and larvicidal properties characterized. Five acyl-.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 264, No. 4, Issue of February 5, pp. 2150-2155.1989 Printed in U.S. A.
D 1989 by The American Society for Biochemistry and Molecular Biology, Inc
Purification and Characterizationof Two Classes of Neurotoxins from the FunnelWeb Spider, Agelenopsis aperta” (Received for publication, September 8,1988)
Wayne S. Skinner$§,Michael E. Adamsll, GaryB. Quistadt, Hiroshi KataokaS,Blake J. CesarinS, Frances E. EnderlinS, and David A. SchooleyS From SZoecon Research Institute, Sandoz Crop Protection Corporation, Palo Alto, California 94304 and the VDivisionof Toxicology and Physiology, University of California, Riuerside, California92521
Two classes of paralytic toxins were isolated from the venom of Agelenopsis aperta and their chemical and larvicidal properties characterized. Fiveacylpolyamine toxins(a-agatoxins) ofmolecular masses 452, 488, 489, 504, and 505 Da produce immediate but reversible paralysis in Manduca sextu following injection. Six insecticidal peptides (p-agatoxins) produce agradual but irreversible paralysis. The complete amino acid sequences (36-38 residues) of the p-agatoxins are presented. These peptides contain eight halfcystines and are quite similar in sequence. At least four of these toxins areamidated at the carboxyl terminus. The secondary structure of one of these toxins (F-Aga V) was investigated.
addition, we investigated the secondary structure of one of these pagatoxins. EXPERIMENTAL PROCEDURES’ RESULTS
Purification of A. aperta Toxins-Fig. 1 depicts the profile of toxins in crude venom observed during initial LC2 purification. Two groups of toxins are resolved, an early eluting group of five a-agatoxins, and asecond group of six p-agatoxins. The very hydrophilic a-agatoxins were collected asa group and rechromatographed as shown in Fig. 2. The relative abundance of a- and p-agatoxins in A. aperta venom is given in Table I. ‘Typically,3 or 4 LC steps were necessary to purify each toxin by using various combinations of organic modifier (acetonitrile or 1-propanol) and ion-pairing agents (trifluoSpider venoms are known to contain several distinct classes roacetic acid or heptafluorobutyric acid). The degree of purity of synaptic toxins. Toxins which act on glutamatergic neuro- was determined by peak shape under different LC conditions. muscular junctions at postsynaptic sites recently have been The purity of a-agatoxins was analyzed also by comparing identified from the venoms of orb weaver spiders (Argiope ratios of UV signals at various wavelengths throughout LC and Nephila species). Each of these toxins contains an aryl peaks using a diode array detector. p-Agatoxins were hydromoiety (either 2,4-dihydroxyphenylaceticacid or 4-hydrox- lyzed for amino acid analyses (Table 11). yindoleacetic acid) and a strongly basic region with the folBioassay-Lethal doses (LD50) of N-agatoxins and paralytic lowing generalized structure: arylacetyl-asparaginyl-polya- doses (EDs0) of wagatoxins upon injection in M . sexta are mine-(arginine) (1-3). Neurotoxic peptides which cause pre- given in Table I. The LDsoand EDsovalues for whole venom synaptic blockage of neurotransmitter release in Drosophila in M. sexta are 2.8 and 3.1 pl/g, respectively. p-Agatoxins I, larvae were recently isolated from Hololena and Plectreurys 111, IV, V, and VI have LD50 values ranging from 28 to 48 pg/ spiders (4, 5). Another class of high molecular weight toxins g while for pagatoxin 11, the LD, is 2-fold higher. The first symptoms of intoxication resulting from LD50 doses of pwhichform ion-permeable channels (e.g. a-latrotoxin)has agatoxins (sluggishness, paralysis) occur 2-3 h post-injection. been isolated from Theridiid spiders (6, 7). We have investigated two classes of toxins from the venom The a-agatoxins have ED50 values in M . sexta of 39-124 pg/g of the funnel web spider Agelenopsis aperta. One class Consists (excluding AGso4).These doses are similar to thelethal doses of acylpolyamine toxins which cause immediate, but reversible of p-agatoxins (on a mass basis). However, the p-agatoxins paralysis in insects which is associated with postsynaptic have about 10-fold higher activity on a molar basis (nanomole of toxin/g insect) since the molecular weights of p-agatoxins blockade of glutamate-sensitive receptor channels (8). We are 8-fold higher. Paralysis from a-agatoxins is instantaneous have named these transitory paralytic agents a-agatoxins. upon injection, but is often reversible. The onset of toxicity The second class of toxins includes six highly disulfidefrom p-agatoxins is slower, but irreversible. bridged peptides which cause irreversible paralysis in lepidopCharacterization of a-Agatoxins-Aliquots of a-agatoxins terous insects (Manduca sexta, Heliothis virescem) and flies were hydrolyzedand subjected to amino acid anaiysis. Unlike (Musca domestica; Ref. 8). These toxins, like a number of the p-agatoxins or the argiotoxins (3), no amino acids were scorpion toxins (9), cause repetitive firing and massive trans- detected in thehydrolysates. Analysis of the pure a-agatoxins mitter release from presynaptic stores at neuromuscular junc- by fast atombombardment-mass spectrometry showed molections (8).The amino acid sequences of these toxic peptides, I Portions of this paper (including “Experimental Procedures,” henceforth referred to as pagatoxins, are presented here. In
* The costs of publication
of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 To whom correspondence should be addressed: Sandoz Crop Protection, 975 California Ave., Palo Alto, CA 94304.
Tables II-VI, and Figs. 4 and 5 ) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. The abbreviations used are: LC, reversed-phase liquid chromatography; RCM, reduced and carboxymethylated; TFA, trifluoroacetic acid.
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TABLE I Relative amounts of toxins in venomof A . aperta and their toxicity to lepidopteran larvae Individual toxins were injected into early third stadium larvae of M. sexta. ED50 values for wagatoxins, A h s s , and spermine are effective dose to paralyze 50% of treated insects at 1 h post-injection. LD50 values for the p-agatoxins are lethal dose for 50% of treated insects at 24 h post-injection. ED, and LD5o values were obtained by probit analysis of data from three or more groups of at least 15 animals. The whole venom LD60 = 2.8 pl/g, and the whole venom ED50 = 3.1 pl/g. All masses are for free bases (not salts). Toxin Time ( m i d
FIG.1. Profile of agatoxins during LC purification of A. uperta venom. Shown here is an LC chromatogram of 2 pl of venom using a Vydac C1 300-A column (15 X 0.46 cm) and a gradient of acetonitrile in a constant 0.1% trifluoroacetic acid. The flow rate was 1.0 ml/min.
20
40
Time (min)
FIG.2. Fractionation of a-agatoxins by LC. LC employed a Vydac CIS300-A column (15 X 0.46 cm) and a gradient of acetonitrile in a constant 0.1% heptafluorobutyric acid. The flow rate was 1.5 ml/ min.
a-Agatoxin AGw, AG45z AG~B AG505 AGm ARm' Spermine
M,
Concentration in whole venom
EDm
nmoljpl (pgjpl)
P&?k
3.2 (1.6) 3.9 (2.0) 6.3 (3.1) 1.4 (0.71) 11.5 (5.6)
274" 39 f 25' 124 & 56 80 f 21 54 f 61 62 f 20 >280
LDw p-Agatoxin 4264 0.38 (1.6) 28 f 7 p-Aga I 4137 0.74 (3.1) 75 f 27 p-Aga I1 4188 0.58 (2.4) 28 .+ 12 p-Aga I11 4199 2.7 (11) 40 f 9 p-Aga IV 48 & 11 p-Aga V 4199 1.4 (5.9) 38 -+ 12 p-Aga VI 4159 1.6 (6.7) No effect a t this dose. *Variability in values given as plus or minus half of the 95% fiducial limit. e Argiotoxin A h 9 from A. aurantia (3).
dase fragment Ly-1 (residues30-36), allowed the assignment of the complete sequence of p-agatoxin I. The applied Biosystems Model 477A Sequencerandon-line analyzer(Model ular weights of 452, 488, 489, 504, and 505 with relatively 120A) gave initial yields of 40-70% with average repetitive little fragmentation. The toxins are hereafter designated by yields of 95% or better. The complete sequencesof H-agatoxins their molecular weight. The UV spectra were determined with are given in Fig. 3; regions of sequence identity are enclosed a diode array detector during the final purification. AG504 and in a box. T h e enzyme fragments used to determine the primary AGSo5 haveidenticalspectra, extremely similartothe 4- structures are shown in Fig. 4 (Miniprint Section). Amino hydroxyindoleacetyl chromophore of argiotoxin 659 (3). AG4, acid compositions of p-agatoxins, their RCMderivatives, and and AG489 also have identical spectra resembling a n indole fragment peptides thereof aregiven in Tables 11,111, and IV. chromophore while AG452has a third and rather different type Characterization of Carboxyl Terminus-Synthetic samples of UV spectrum. Fragmentation patternsof native a-agatox- of the COOH-terminal heptapeptides from lysyl endopeptiins uponchemicalionization (NHJ mass spectral analysis dase digestion of RCM pagatoxins I, IV, and V, and chymoconfirm the presence of polyamine moieties in these toxins. tryptic digestion of RCM p-agatoxin 111 were prepared in the Full structural detailsfor these acylpolyamines (a-agatoxins) acid andamideform. LCcomparison of syntheticRCMwill be publishedelsewhere. CICRNNN acid and amide to the COOH-terminal heptapepSequence Determination of p-Agatoxins-Sequence and tides from 3H-labeled RCM p-agatoxins I, IV, and V demonamino acid composition analyses of 100-500 pmol of RCM p- strated that these toxins are amidated at the COOH terminus. agatoxins 11-VI resulted in the identificationof nearly com- Likewise, LC comparison of CRCRSDS acid and amide to the plete primary structures. Due to carryover during sequence COOH-terminal heptapeptide from 3H-labeled RCM p-agaanalysis, the carboxyl-terminal residue of these toxins was toxin I11 showed this toxin to be amidated at the COOH not obvious, particularly when repetitive residues are present terminus.When %-labeled RCMfragmentpeptides were at the COOH terminus (i.e. p-agatoxins 11,IV,V, and VI). coinjected (LC) with nonlabeled RCM-CICRNNN or RCMEnzymic cleavage of p-agatoxins 11-VI followed by sequence CRCRSDS acid and amide standards, the radioactivity coeand amino acid composition analyses of the COOH-terminal luted with the amide and not the acid. peptides clarified the sequence at thecarboxyl terminus. The Position of Disulfide Bonds of p-Agatoxin V-Fig. 5 shows sequence analysis of p-agatoxin I was complicated by a high the secondary structure of pagatoxin V and indicates the degree of carryover imparted by the Pro-Glu and Gly-His thermolysin fragments used in locating the disulfide bonds. linkages which are particularlyslow to cleave (11). Extending Purified thermolysinfragments weresequenced. As these the time of cleavage for these residues (AB1 PRO-1 program) fragments contain two or three chains linked by disulfide decreased the amount of carryover, but gave very poor yields bonds, we observed simultaneous sequencing of the separate for subsequent residues.Digestion of p-agatoxin I with a - peptide chains, the results of which are shown in Table V. chymotrypsin gave two major fragments (residues 1-21 and During sequence analysis, the disulfide bonds arereduced due 22-36). Sequence analysis of these, and the lysyl endopepti- to the presence of dithiothreitol in certain solvents (i.e. n-
Toxins f r o m the S p i d e r A. aperta
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"
30
20
C C E G F Y C S C R Q P P K C I C R N N N N H 2
p-Aga
I
Y D E
p-Aga
I1
A G P W C C D G L Y C S C R S Y P G C M C R P S S
p-Aga
I11
p-Aga
IV
p-Aga
V
Q C A D W A G P H C C D G Y Y C T C R Y F P K C I C R N N N - . N H 2
p-Aga
VI
Q C A D W A G P H C C D G Y Y C T C R Y F P K C I C
-3-
20
-
20
A D
C
30
A G P Y C C S G Y Y C S C R S M P Y S R C R S D S - N H 2
c
20
30
A C V G E N Q Q C A D W A G P H C C D G Y Y C T C R Y F P K C I C R N N N . - N H z 10 10
20
20
30 30
FIG. 3. Primary structures of p-agatoxins. Residues within boxes are identical between peptides, allowing €or one more amino acid a t t.he NH2 terminus of p-agatoxin 111 and one deletion between residues 1 2 and 16 for pagatoxin I.
heptane, ethyl acetate, and n-butyl chloride). Phenylthiohy- to occur. By contrast, paralysis resultingfrom injection of adantoin-cysteine is notidentified by the sequenator under the agatoxins (at lower doses) is immediate and begins to disapconditions employed. Thermolysin fragment peptideT h 1was pear at about the time the p-agatoxins begin t o intoxicate M. identified by combinations of sequence and amino acid anal- sexta. yses (Tables V and VI). The results fromsequencing this Physiological studies (8) show that the p-agatoxins, like fragmentdemonstratethat Cys2 and Cys33 formdisulfide certain scorpion toxins (9), cause repetitive firing in presynbonds withCys17and Cys18, respectively. The exact placement aptic motoraxons. It thus appears that venom the of A. aperta of these disulfide bonds could not be determined as cleavage acts both pre- and postsynaptically,possibly t o achieve synof t h e C y ~ ' ~ - C y speptide '~ bond was not accomplished. Se- ergism in neuromuscular block. These different biochemical quence analyses of T h 4 (native and RCM fragment peptides)activities may also extend thevenom's biological spectrum of identified the sequence TCRYFPLC. This information cou- activity toa wider range of insect prey. pled with the fact thatonly 1 cystine residue was found upon The sequences of the p-agatoxins are extremely similar amino acid analyses of native and oxidized material confirms (Fig. 3). p-Agatoxin IV and p-agatoxin V differ only at the a disulfide link between CysZ5and Cys31. The disulfide bond seventh residue, while p-agatoxin VI differs from p-agatoxin between Cysg and CysZ3is inferred since all other cystine IV at three residues. The sequences of p-agatoxin I1 and 111 residues are accounted for. The remote possibility that Cysg show more variance from p-agatoxins IV-VI; the presence of and CYS*~ are free sulfhydryls was discounted since nativep- distinctly different tripeptide sequences at the carboxyl teragatoxin V does not alkylatein the presence of ['H]iodoacetic minus is interesting. p-Agatoxin 111 is extended at the amino acid (in 6 M guanidine hydrochloride, pH 8.5). with the other toxins, terminus by 1residue (Ala) as compared whereas p-agatoxin I has a 1-residue deletion at position 16 DISCUSSION as comparedwith p-agatoxin I1 and p-agatoxin IV-VI. pWe havedescribedtwoclasses of toxins from A. aperta Agatoxins show no obvious homology to other identified arvenom which show distinct chemical properties and paralytic thropod toxins although the presenceof four disulfide bonds actions on insects. The a-agatoxins induce immediate but is shared by most scorpion toxins (14, 15) andtwo previously pidentifiedneurotoxic peptides from Australian funnel web only temporaryincapacitation.Ontheotherhand,the agatoxins produce gradual but lethal paralysis. The combined spiders of the genus Atrax (16, 17). With 8 half-cystines, native p-agatoxins are highly folded actions of these toxins appear provide to for rapid, irreversible and are quite resistant to proteolytic digestion using a-chyparalysis. Initially, we utilized adult cockroaches (Periplaneta ameri- motrypsin and trypsin. This prevented us from conducting cleavages while investigating the secondary cana) and house 5ies (Musca domestica) for injection bioassay more specific (12). The sensitivity of the housefly bioassay allowed selection structure. However, thermolysin has advantages in that it of venom components for further analysis of chemical struc- hydrolyzes peptides at many sites and at pHlevels which are tures. In this study,we report the sequences of six insecticidal not likely to result in thescrambling of disulfide bonds. Also, peptides (p-agatoxins)as well as themolecular weights of five the thermostability of the enzyme allows the use of elevated acylpolyamines (a-agatoxins). The paralytic and insecticidal temperatures (e.g. 55 "C) which, at least theoretically, allows activit.y of pure, isolated toxins was also studied in lepidop- the enzyme better access to attack partially denatured peptide. The secondary structure of p-agatoxin V bridges the ends of terans ( M . sexta, H . uirescens, and Spodoptera exigua). We purified six peptide toxins from the venom of A. aperta the peptide to the center, forming a very compact structure by reversed-phase LC. These p-agatoxins are moderately toxic (Fig. 6), which is also the case with the scorpion toxin, AaH to M . sexta upon injection, with LDS0values of 7-11 nmol/g IT (18). Presumably this compactnessis essential for biolog(28-48 pg/g), excluding p-agatoxin I1 (LD,, = 18 nmol/g, 75 ical activity. For example, when disulfide bonds are reduced p g / g ) . Two p-agatoxins, IV, and V, are 2 to 3 times less active and the sulf%ydryls carboxymethylated, the resultant peptide when injected into another lepidopterin, fourth stadium H. is not toxic to M.sexta, even at twice the LDSodose. We showed conclusively that p-agatoxins I, 111, IV, and V virescens (LDbo = 100 t 32 and 96 -+ 58 pg/g, respectively), while in fifth stadium S.exigua the LD50 of p-agatoxin IV is are amidated at the COOH terminus. Lysyl endopeptidase 16 4 7 gg/g or about half the LD50 in M.sexta. For comparison, and a-chymotrypsinwere very specific and active in cleaving producing thedesiredCOOH-terminal the insect-specific toxin AaH I T from the scorpion Androc- thesepeptidesand tonus australis hasan LD50of 13 pg/g in the armyworm fragments. The use of synthetic acid and amide formsof the Spodopteralittoralis (13). Theg-agatoxins havea delayed COOH-terminal peptides made defining the COOH terminus effect on M.sexta with paralysis typically taking several hours relatively simple by comparative LC behavior. Presumably,
Spider the
fromToxins
A. aperta
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Acknowledgments-We thank C. Kristensen for supplying spider venom, Dr. R. Cone for separating samples of p-agatoxins 11, 111, and VI for initial sequence analyses, Dr. E. T. Fu, G. C. Jamieson, and C. C. Reuter for mass spectral analysis, and Dr. J. P. Lifor synthesis of two heptapeptides. REFERENCES 1. Grishin, E. V., Volkova, T. M., Arseniev, A. S., Reshetova, 0. S., Onoprienko, V.V., Magazanic, L.G., Antonov, S. M., and Fedorova, I. M. (1986) Bioorg. Khim. 12, 1121-1124 2. Aramaki, Y., Yasuhara, T., Higashijima, T., Miwa, A., Kawai, N., and Nakajima, T. (1987) Biomedical Res. 8 , 167-173 3. Adams, M. E., Carney, R. L., Enderlin, F. E., Fu, E. T., Jarema, M. A., Li, J . P., Miller, C. A., Schooley, D. A. Shapiro, M. J., and Venema, V. J . (1987) Biochem.Biophys.Res.Commun. 148,678-683 4. Bowers, C. W., Phillips, H. S., Lee, P., Jan, Y. N., and Jan,L. Y. FIG. 6. Secondary structure of pagatoxin V. Cys2 and Cys33 (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3506-3510 form disulfide bonds with Cys" and Cys" (exact placement unknown, 5. Branton, W . D., Kolton, L., Jan, Y. N., and Jan, L. Y. (1987) J . i.e. either C y ~ ~ - C y s ' ~ , C y ~ 'or ~ -Cys2-Cys", Cys~~ Cy~'~-Cys~~). Neurosci. 7,4195-4200 6. Tu, A. T., and Keeler, R. F. (1986) J. Toxicol. Tox. Reu. 5, 161170 p-agatoxin VI exists asa carboxyl-terminal amide as well, but 7. Cavalieri, M., D'Urso, D., Lassa, A., Pierdominici, E., Robello, we did not have synthetic standards for comparison to its M., and Grasso, A. (1987) Toxicon 25, 965-974 slightly different carboxyl-terminal fragment from lysyl en8. Adams, M. E., Herold, E. E., and Venema, V. J . (1989) J . Comp. dopeptidase. p-Agatoxin 11 and p-agatoxin I11 have substanPhysiol. A , in press 9. Zlotkin, E., Kadouri, D., Gordon, D., Pelhate, M., Martin, M. F., tially less sequence similarity in the carboxyl termini comand Rochat, H. (1985) Arch. Biochem. Biophys. 240,877-887 pared to the other p-agatoxins. However, p-agatoxin I11 is amidated at the COOH terminus. We assume that p-agatoxin 10. Bohlen, P., and Schroeder, R. (1982) Anal. Biochern. 126, 144152 I1 is probably amidated at the carboxyl terminus. 11. Tarr, G. E. (1977) Methods Enzymol. 47, 335-357 Five a-agatoxins havebeenidentifiedwithmolecular 12. Adams, M. E., Enderlin, F. E., Cone, R. I., and Schooley, D. A. masses of 452,488,489,504, and 505. These paralytic agents (1986) in Insect Neurochemistry and Neurophysiology (Borkovec, A. B., and Gelman, D. B., eds) pp. 397-400, Humana Press, are acylpolyamines, whose structures will be reported elseClifton, N J where. The a-agatoxins appear toformed be from twounusual polyamines which are acylated by three different acids. The 13. DeDianous, S., Hoarau, F., and Rochat, H. (1987) Tonicon 25, 411-417 M. sexta similar to an argiotoxin 14. Martin, M. F., Garcia Y. Perez, L. G., Ayeb, M. E., Kopeyan, C., a-agatoxins have potency in Argiope aurantia (Table I; Ref. 3). (ARBBY) from Bechis, G., Jover, E., and Rochat, H. (1987) J. Biol. Chem. Insummary, A. aperta producestwo distinct classes of 262,4452-4459 neurotoxins. We characterized five acylpolyamines (cu-agatox- 15. Lazarovici, P., Yanai, P., Pelhate, M., and Zlotkin, E. (1982) J. Biol. Chem. 257,8397-8404 ins) which produce immediateparalysisin M. sextu upon injection. Six neurotoxic peptides which produce delayed pa- 16. Sheumack, D. D., Claassens, R., Whiteley, N. M., and Howden, M. E. H. (1985) FEBS Lett. 181, 154-156 ralysis and death in injected M. sexta were identified ( p - 17. Brown, M. R., Sheumack, D. D., Tyler, M. I., and Howden, M. agatoxins). We believe these toxins will be of use as pharmaE. H. (1988) Biochem. J . 250,401-405 cological tools in studiesof ion channelsinvolved in synaptic 18. Darbon, H., Zlotkln, E., Kopeyan, J., Van Rietschoten, J., and transmission. Rochat, H. (1982) Znt. J . Peptide Protein Res. 20, 320-330
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