Corso di Perfezionamento Tecnologie per l'autonomia e l'integrazione sociale delle persone disabili Anno Accademico 1999/2000 Storia di ordinaria sclerosi multipla CANDIDATO: Bianca Tovo Abstract. Il caso di studio riguarda Roberta, 61 anni, che ha presentato i primi disturbi motori e sensitivi a 31anni e che dall'età di 43 anni si sposta esclusivamente in carrozzina. Ha conservato un discreto utilizzofunzionale dell'arto superiore sinistro.Ha uno spirito vivace e sensibile e ha affrontato la sua disabilità con coraggio e buon senso.Negli ultimi anni, anche in seguito ad una malattia del marito oltre che al proprio aggravamento motorio, haperso entusiasmo e ha rinunciato poco per volta alle attività che comportano fatica per lei e per chi l'assiste.Il progetto si propone di:• migliorare la stazione seduta in carrozzina
CSTX-13, a highly synergistically acting two-chain
neurotoxic enhancer in the venom of the spider
Cupiennius salei (Ctenidae)
Benno Wullschleger*, Lucia Kuhn-Nentwig*†, Jan Tromp‡, Urs Ka¨mpfer‡, Johann Schaller‡, Stefan Schu¨rch‡,
and Wolfgang Nentwig*
*Zoological Institute, University of Bern, Baltzerstrasse 6, CH-3012 Bern, Switzerland; and ‡Department of Chemistry and Biochemistry, University of Bern,Freiestrasse 3, CH-3012 Bern, Switzerland Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved June 22, 2004 (received for review April 1, 2004) The survival of the spider Cupiennius salei depends on its hunting
cysteine in their composition. Some peptides have already been success, which largely relies on its immediately paralyzing multi-
characterized and named the cupiennin 1 family. These peptides component venom. Here, we report on the isolation and charac-
exhibit strong bactericidal activities in the submicromolar terization of CSTX-13, a neurotoxic enhancer in the spider venom.
range, but also cytolytic and insecticidal activities. In addition, De novo elucidation of the disulfide bridge pattern of CSTX-13 and
cupiennin 1a shows a high synergistic effect with the main the neurotoxin CSTX-1 by tandem MS revealed an identical ar-
neurotoxin CSTX-1, facilitating a rapid paralysis (12, 13). Pos- rangement. However, in contrast to CSTX-1, CSTX-13 is a two-chain
itive insecticidal cooperativity between the cytolytically active peptide with two interchain and two intrachain disulfide bridges.
oxyopinins and neurotoxins is also reported for the spider Furthermore, the insecticidal activity of CSTX-13 is synergistically
Oxyopes kitabensis (14).
increased in the presence of Kⴙ ions as well as of the cytolytic
The second group involves neurotoxically active peptides with peptide cupiennin 1a. We demonstrated that the weakly neuro-
molecular masses of ⬇8 kDa, which we named Cupiennius salei toxic CSTX-13 enhances the paralytic activity of the neurotoxin
toxins (CSTX-1 to -13) (11). There is evidence that CSTX-1 CSTX-1 by 65% when it is administered with the latter at its entirely
inhibits L-type Ca2⫹ channels of GH3 cells (J. S. Cruz, personal nontoxic physiological concentration, which is 440 times below its
communication). To date, sequence data for the neurotoxins CSTX-1 and CSTX-9 are available. The four disulfide bridges of CSTX-9 form linkages between C1–C4; C2–C5; C3–C8; and Spidersandscorpionsusetheirvenomtoparalyzepreyand兾or C6–C7 (15–17). This arrangement is also found in other spider
to defend against predators. These venoms are complex neurotoxins belonging to the inhibitor cystine knot (ICK) struc- mixtures of different components, and the knowledge about tural motif (18).
their interactions and role in the envenomation process is still So far, a roughly comparable two-chain peptide structure has limited. During their evolution, these arthropods have developed only been reported for the spider Agelenopsis aperta: the -aga- a large number of neurotoxins that act simultaneously on various toxins IA and G block presynaptic calcium channels in insect invertebrate and兾or vertebrate membrane-bound sodium, po- neuromuscular junction (19, 20). A two-chain structure has also tassium, and calcium channels. Also, interactions with acid- been proposed for the Hololena toxin, a presynaptic antagonist sensing ion channels, glutamate receptors, and as yet unidenti- of insect neuromuscular transmission (21).
fied targets lead to rapid paralysis or death of the envenomed Here, we present the amino acid sequence of CSTX-13, an animals (1, 2).
enhancer peptide from the venom of C. salei, and the de novo Cupiennius salei (Keyserling, 1877) is a nocturnal hunting determination of the disulfide bridge pattern of CSTX-1 and spider living in the Central American rain forest (3). The spider CSTX-13. Despite its sequence similarity to the neurotoxins relies on an immediately paralyzing venom activity because, in its CSTX-1 and CSTX-9, CSTX-13 acts as a neurotoxic enhancer.
arboreal environment, a prey item that escapes is lost. The spider Moreover, its low neurotoxic activity is also augmented by other also loses its venom investment and reduces its chance of venom compounds.
successfully subduing a subsequent prey item, because its venom storage is limited, regeneration takes ⬇16 days (4), and its Materials and Methods
production involves high metabolic costs. Behavioral, ecological, Chemicals. Chemicals were of analytical grade and purchased
and biochemical investigations of the venom economy of C. salei from Merck unless otherwise specified.
indicate that it alters the amount of venom injected according to the size, mobility, and defense behavior of its prey (5–8).
Isolation of CSTX-13. Spider maintenance, venom collection, and
This economical venom use is paralleled on the physiological separation of 425 l of venom by FPLC and HPLC methods were and biochemical levels by the interactions of different venom performed as described (ref. 11; see also Supporting Text, which components (9). In the venom, low molecular mass compounds is published as supporting information on the PNAS web site).
such as histamine (5.7 mM) and free amino acids, basically Final purification of CSTX-13 was achieved by RP-HPLC on a taurine (70 M) and glycine (43.3 M), are present. K⫹, Na⫹, nucleosil 100–5 C8 column (4 ⫻ 250 mm, Macherey & Nagel) and Ca2⫹ ions have also been identified. Remarkably, K⫹ ions using 22% solvent B (0.1% trifluoroacetic acid in acetonitrile) in are abundant in the venom and rare in the hemolymph (10, 11).
Furthermore, C. salei possesses a complex multicomponent system consisting of a few proteins with molecular masses ⬎10 This paper was submitted directly (Track II) to the PNAS office.
kDa, among them a highly active hyaluronidase. About 100 Abbreviation: ESI, electrospray ionization different peptides with molecular masses between 2 and 8 kDa Data deposition: The sequences reported in this paper have been deposited in the Swiss- have been detected by electrospray ionization (ESI)-MS. The Prot and TrEMBL databases [accession nos. P83919 (CSTX-13 chain A) and P83920 (CSTX-13 peptides can be roughly divided into two groups. The first group contains the smaller peptides with molecular masses of ⬇3–4 †To whom correspondence should be addressed. E-mail: [email protected]
kDa, which are mainly highly cationic ␣-helical peptides without 2004 by The National Academy of Sciences of the USA PNAS 兩 August 3, 2004 兩 vol. 101 兩 no. 31 兩 11251–11256
Isolation of CSTX-13 from the venom of C. salei. (A) Crude venom was first separated by gel filtration on a Superdex 75 column. (B) Further separation of the pooled fraction was achieved by cationic exchange chromatography on a Mono S HR column. (C) When RP-HPLC was used, CSTX-13 was finally isolatedon a nucleosil 100 –5 C8 column, and the purity was controlled by SDS兾PAGE. (D) Lanes: 1, native CSTX-13; 2, reduced CSTX-13; 3, native CSTX-1; 4, molecular massmarkers (14.4 –97.4 kDa; Bio-Rad); and 5, molecular mass markers (2.5–16.9 kDa; Amersham Pharmacia). (E) RP-HPLC of reduced and alkylated CSTX-13 on anucleosil 100 –5 C8 column resulted in the separation of chain A (first arrow) and chain B (second arrow).
solvent A (0.1% trifluoroacetic acid in water) with a flow rate of supernatant was recovered for separation by RP-HPLC. The 0.5 ml兾min. Directly after injection of the sample, the gradient digested and purified peptides were subjected to collision- (22–28% solvent B) was started for 20 min (Fig. 1C). This step induced dissociation using nitrogen as the collision gas. Collision was repeated several times to obtain CSTX-13 (variability be- energies were in the range of 20–80 eV.
tween different preparations: 0.5–1.3 mg).
Amino Acid Analysis and Amino Acid Sequence Analysis. Samples
PAGE. SDS兾PAGE and silver staining of native and reduced
were hydrolyzed in the gas phase with 6 M hydrochloric acid (2-mercaptoethanol) CSTX-13 were performed with the Phast- containing 0.1% (by volume) phenol for 24 h at 115°C under N2 System using high density PhastGel (Amersham Pharmacia).
vacuum according to Chang and Knecht (22). N-terminal se- quence analysis was carried out either in a Procise cLC 492 Reduction and Alkylation. Fifty micrograms of CSTX-13 was
protein sequencer or in a pulsed liquid-phase sequencer 477A, reduced and alkylated according to the published procedure (ref.
both from Applied Biosystems.
15, see Supporting Text). Chains A and B were further desalted and separated by RP-HPLC on a nucleosil 100-5 C8 column (4 ⫻ Experiments with Spider Digestive Liquid. Digestive liquid from C.
250 mm; Macherey & Nagel) using 100% solvent A with a flow salei was obtained by electrical stimulation and collected in glass rate of 0.5 ml兾min for 0–5 min followed by a 55-min gradient of capillary tubes, and 8 l of diluted digestive liquid (1:100 with 0.73% solvent B in solvent A per min (Fig. 1E).
water) was mixed with 17 g of CSTX-13 in 8 l of water. The mixture was kept at 24°C and, after 0.5, 1, and 24 h, aliquots of MS. Mass spectrometric analyses were performed on a QSTAR
2 l were analyzed by ESI-MS.
Pulsar hybrid quadrupole time-of-flight mass spectrometer (Ap- For further experiments, 50 g of CSTX-13 was dissolved in plied Biosystems) equipped with a nanoelectrospray ion source.
31.8 l of water, mixed with 31.8 l of diluted digestive liquid The instrument was tuned for a mass resolving power of 12,000 (1:100 with water), and incubated for 24 h at 24°C, and the (m兾⌬m, full width at half maximum) and calibrated with caesium fragment was isolated by RP-HPLC on a nucleosil 120–5 C18 iodide and reserpine (Sigma). Samples were dissolved in meth- column (2 ⫻ 125 mm; Macherey & Nagel) using a gradient of anol兾water (1:1 vol兾vol) containing 1% formic acid. The final 0.2% B in A兾min for 200 min and a flow rate of 0.5 ml兾min. For peptide concentration was 5 pmol兾l. All analyses were per- ESI-MS analysis, the CSTX-13 fragment was again dissolved in formed in the positive ion mode. Numbers represent monoiso- 100 l of buffer [100 mM Tris䡠HCl, pH 8.0, containing 135 M topic masses.
TLCK (N␣-p-tosyl-L-lysine chloromethyl ketone, Sigma) and 220 For elucidation of the disulfide bridge pattern, 50 g of native M TPCK (N-tosyl-L-phenylalanine chloromethyl ketone, CSTX-13 was cleaved with immobilized trypsin (23 l wet gel Sigma)], reduced, alkylated, and separated as described above.
containing 0.5 units of trypsin (Sigma) in 50 l of 0.1 M Tris䡠HCl buffer, pH 8.1, and 1.0 mM iodoacetamide, Fluka) under gentle Bioassays and Calculations. Bioassays were performed according
shaking for 17 h at 24°C. The suspension was centrifuged, and the to Escoubas et al. (23) using 1- to 3-day-old Drosophila melano- Wullschleger et al.
Sequence comparison and disulfide bridge arrangement of CSTX-13, CSTX-1, CSTX-9 (C. salei), and -agatoxin IA (A. aperta). Identical amino acids are shaded gray, the disulfide bridges are represented by lines, and the corresponding cysteine residues are shaded black. The disulfide bridge patterns of CSTX-13and CSTX-1 were determined by nanoelectrospray tandem MS of the corresponding disulfide-linked tryptic fragment.
gaster female flies. The injected volume was 0.05 l of 0.1 M successive RP-HPLC (Fig. 1C). The retention profile of ammonium acetate, pH 6.1 (control), and all further injections CSTX-13 revealed no impurities. CSTX-13 was characterized by with different components were carried out in this solution, ESI-MS and amino acid composition. The yield of CSTX-13 except nifedipine (Calbiochem), which was injected in ⱖ0.06 M obtained by purification of crude venom was 1.2–3.0 g兾l ammonium acetate, pH 6.1, and ⱕ5.6 M dimethyl sulfoxide. To depending on the separation protocol.
estimate the LD50 (24 h after injection), 20 flies were used as N-terminal sequence analysis of native CSTX-13 provided control, and 20 flies were used for each concentration.
evidence for a two-chain molecule: Ser兾Ala–Asp兾Lys–Xaa兾Lys– To investigate synergistic effects between CSTX-13 and fur- ther venom components such as cupiennin 1a (9.6 M) (in Xaa兾Gln. Therefore, CSTX-13 was reduced and alkylated, and nontoxic concentration), histamine (5.7 mM, Sigma), taurine chains A and B were separated by RP-HPLC. Both chains were (0.07 mM, Sigma), and KCl (215 mM) (all in physiological venom sequenced by Edman degradation from the N to the C termini concentrations), bioassays were performed with 12.6 pmol of without any ambiguity. Chain A is composed of 34 residues CSTX-13 per mg of fly. We tested CSTX-13 alone and in (measured, 4,342.73 Da; calculated, 4,342.76 Da), and chain B is combination with each of the above mentioned venom compo- composed of 29 residues. ESI-MS of chain B gave a monoiso- nents [n ⫽ 2 ⫻ (15 ⫻ 5) for each assay]. Venom components in topic mass of 3,475.80 Da, which is one mass unit less than the above-mentioned concentrations were injected alone as control expected theoretical mass of 3,476.83 Da, thus indicating C- (n ⫽ 20). The paralytic activity of physiological KCl concentra- terminal amidation of chain B. The determined amino acid tion was measured by comparing the awake time of a control sequences of both chains agree well with the amino acid com- group (n ⫽ 20) and a treated group (n ⫽ 20) (Mann–Whitney U position of native CSTX-13 as well as with the individual chains.
test, SPSS 10 software).
Taking into account the four disulfide bridges of both peptide To highlight synergistic effects between the neurotoxins chains and the amidation, the calculated monoisotopic mass of CSTX-1 and CSTX-13, corresponding to their molar ratio in the CSTX-13 is in agreement with the measured mass of native crude venom (9:1), bioassays were performed with 0.315 pmol of CSTX-13 (measured, 7,354.51 Da; calculated, 7,354.37 Da) (Fig.
CSTX-1 per mg of fly alone and in combination with 0.035 pmol 2 and Fig. 5, which is published as supporting information on the of CSTX-13 per mg of fly, and three further concentrations down PNAS web site).
to 0.63 fmol of CSTX-13 per mg of fly [n ⫽ 2 ⫻ (12 ⫻ 5) for each SDS兾PAGE analysis of purified native CSTX-13 revealed a assay]. CSTX-13 alone was used in the above mentioned con- single band at 12 kDa, whereas reduced CSTX-13 revealed a centrations as a control (n ⫽ 2 ⫻ 20).
single band at ⬇3 kDa, obviously containing the peptide chains The influence of CSTX-13 on two different Ca2⫹ channel A and B. This supports the two-chain structure of the native blockers was further investigated. NiCl2 was administered in a CSTX-13 (Fig. 1 C and D).
concentration of 5.26 nmol兾mg of fly alone and in combination To exclude the possibility that the two-chain structure of with 0.035 pmol of CSTX-13 per mg of fly [n ⫽ 2 ⫻ (12 ⫻ 5) for CSTX-13 is a proteolytic artifact because of contamination of the each assay]. Nifedipine was tested in a concentration of 0.105 venom with digestive liquid (24), CSTX-13 was incubated with nmol兾mg of fly alone and in combination with 0.035 pmol of fresh digestive liquid. After 0.5, 1, and 24 h of incubation, the CSTX-13 per mg of fly [2 ⫻ (6 ⫻ 5) for each assay], and three obtained mass indicates a proteolytic degradation of the 14 further concentrations up to 5.5 pmol of CSTX-13 per mg of fly C-terminal amino acid residues of chain B (measured, 5,746.51 [n ⫽ 6 ⫻ 5 for each assay].
Da; calculated, 5,746.51 Da). The CSTX-13 fragment was puri- The relative mortality of D. melanogaster was arcsin square fied by RP-HPLC, and reduced and alkylated in the presence of root-transformed and treated as the dependent variable, two protease inhibitors. ESI-MS analysis of the purified com- whereas the venom components or CSTX-13 were treated as pounds revealed an intact chain A (measured, 4,342.80 Da; nominal independent variables. The experiment was analyzed by calculated, 4,342.76 Da) and a truncated chain B (measured, generalized linear models. The means of the nominal indepen- 1,868.03 Da; calculated, 1,867.98 Da). These findings are in dent variables venom components or CSTX-13, respectively, accordance with the result described above and support the were compared pairwise by the Bonferroni method. Fulfillment assumption of a native two-chain structure of CSTX-13 in the of the model assumptions was checked by visual inspection of the venom. CSTX-13 seems to be present in the venom as a residuals distribution for every statistical test conducted. Statis- two-chain molecule and to the best of our knowledge does not tics were performed with S-PLUS 6.0 PROFESSIONAL software.
represent a purification artifact.
Because of the unique amino acid sequences of CSTX-1 and CSTX-13 with cysteine residues arranged in close proximity, Purification and Sequence Analysis of CSTX-13. The crude venom
classical approaches to determine the disulfide bridge pattern, (425 l) was separated in a four-step protocol using gel filtration based on specific enzymatic or chemical cleavages, failed. Con- (Fig. 1 A), cationic exchange chromatography (Fig. 1B), and sequently, the disulfide bridge patterns of CSTX-1 and CSTX-13 Wullschleger et al. PNAS 兩 August 3, 2004 兩 vol. 101 兩 no. 31 兩 11253
Sequence of the cystine-containing fragment obtained from tryptic digest of native CSTX-13 (4,473.73 Da). The asterisk (*) indicates the [M ⫹ 8H]8⫹ ion with m兾z 560.23, which was selected as the precursor for CID. Typical fragmentation pathways include loss of terminal amino acids in conjunction with loss ofwater (ions I, I, and III), disulfide cleavage (ion ), and cleavage of peptide bonds between cystines (disulfide bridge-defining ions A–Z). The enlargement showsthe isotopic pattern of fragment ion C with m兾z 500.22, which defines, in combination with fragment ion A, the Cys 1–Cys 4 and Cys 2–Cys 5 bridges.
were identified de novo by nanoelectrospray tandem MS. Diges- calculated masses. Fig. 3 also shows a section of the product ion tion of the native toxin with immobilized trypsin yielded main spectrum obtained by CID of the [M ⫹ 8H]8⫹ precursor ion of fragments consisting of five short peptide chains cross-linked by the tryptic CSTX-13 fragment. The high mass accuracy and four disulfide bridges.
resolving power of the tandem mass spectrometer allow unam- Multiply charged [M ⫹ nH]n⫹ ions (n ⫽ 3–9) of the cystine biguous peak assignment, as demonstrated for the quadruply containing tryptic fragment (measured, 4,473.75 Da; calculated, charged fragment ion C. Additional information was obtained by 4,473.76 Da) of CSTX-13 were selected as precursor ions for assigning peaks generated by disulfide bridge cleavage. The same subsequent collision-induced dissociation (CID). The resulting strategy was applied for the elucidation of the disulfide bridge product ion spectra are characterized by abundant peaks of pattern of CSTX-1, which exhibits identical disulfide bridges fragment ions generated by cleavage of the disulfide bridges.
These ions define the order of peptide chains. Further abundant peaks indicate repetitive loss of amino acids from the termini of Synergistic Insecticidal Effects. To evaluate the biological impor-
the peptide chains, often occurring in conjunction with the loss tance of CSTX-13, comparative bioassays with D. melanogaster of water. Detailed information on the disulfide bridge pattern were performed. The LD50 of 16.3 pmol兾mg of fly (14.5–27.1; was obtained by detection of the less abundant fragment ions of 95% confidence limits) indicates a lower toxicity than other mass 968.43 Da (A), 1,895.80 Da (B), 1,995.85 Da (C), 1,289.45 neurotoxins of C. salei. CSTX-13 is ⬇49 times less toxic than the Da (Y), and 1,390.49 Da (Z), generated by cleavage of the neurotoxin CSTX-1, and 2.8 times less toxic than the cytolytically peptide backbone between adjacent cystines. The corresponding active peptide cupiennin 1a, both key components identified in cleavage sites are indicated in Fig. 3 (see Fig. 6, which is the venom of C. salei (24, 12) (Table 1).
published as supporting information on the PNAS web site).
Synergistic interactions of different venom components (taurine, Measurements exhibit a maximum deviation of 0.02 Da from the histamine, KCl) with the paralytic activity of CSTX-13 were ana- Table 1. Insecticidal activity of spider venom components
Physiological venom Synergistically tested concentration, mM Estimation of the lethal doses (LD50) in a Drosophila bioassay, where 50% of the test flies died of intoxication 24 h after injection. Different amounts of peptides, histamine, taurine, and KCl were dissolved in 0.1 M ammoniumacetate, at a pH of 6.1, and 0.05 l was injected into the flies. The physiological concentrations of CSTX-1 (13),taurine (13), histamine (13), KCl (13), CSTX-9 (15), and cupiennin 1a (12) in the venom were reported.
Wullschleger et al.
only in high concentrations (LD50 309 M) when applied alone, but synergistically enhances the paralytic activity of the main neurotoxin CSTX-1 at low concentrations (0.7 M).
In the venom, CSTX-13 is constitutively present at a 7–8 times lower concentration than the main neurotoxin CSTX-1 and in an up to 2.8 times lower concentration than a further neurotoxin CSTX-9. Similarly, its insecticidal activity, expressed as a LD50 value, is 49 times lower than the activity of CSTX-1 and 1.5 times lower than that of CSTX-9 (Table 1) (24). Protein database search using BLASTP 2.2.8 (29) resulted in a high sequence identity of 56% (70% similarity) between CSTX-1 and CSTX-9, but in lower sequence identities of 35% between CSTX-13 and CSTX-1 (51% similarity), and of 31% between CSTX-9 and CSTX-13 Synergistic effects between CSTX-13 and venom components. (A) (49% similarity). Nevertheless, all three peptides exhibit iden- Synergistic effects between CSTX-13 and low molecular venom components.
tical disulfide bridge patterns (15–17) (Fig. 2). Sequence com- In a Drosophila bioassay, the lethal effect of CSTX-13 injected alone (239.4 M) parison implies that, upon processing, a short peptide is excised was compared with the lethal effect of coinjected CSTX-13 (239.4 M) with in the loop forming the disulfide bridge C6–C7, thus leading to taurine (0.07 mM; not significant), histamine (5.7 mM; not significant), KCl the two-chain structure of CSTX-13.
(215 mM; *, P ⬍ 0.05), or cupiennin 1a (9.6 M; ***, P ⬍ 0.001). As controls,
No sequence similarities were detected with further neuro- taurine, histamine, KCl, and cupiennin 1a showed no toxic effect when toxins and the two other sequenced two-chain calcium channel administered alone. (B) Synergistic effects of CSTX-13 on the toxicity ofCSTX-1. The lethal effect of CSTX-1 (5.99 M) was compared with the lethal -agatoxin IA (66 and 3 residue chains) and -agatoxin effect of coinjected CSTX-1 (5.99 M) with CSTX-13 (0.67 M兾mg of fly; G (62 and 3 residue chains) (20) from the spider Agelenopsis P ⬍ 0.001) (molar ratio of 9:1) corresponding to their concentrations in the aperta. In contrast to CSTX-13, which contains two interchain venom. CSTX-13 was also injected alone as control and showed no toxic effect and two intrachain disulfide linkages, these two neurotoxins on the flies. Statistical analysis was done by using the Bonferroni method.
possess four intrachain and one interchain disulfide linkage. - Standard error bars are shown for every treatment.
agatoxin IA is formed from its precursor by excision of an internal heptapeptide leading to a major peptide chain that is lyzed. Taurine itself was not toxic to D. melanogaster up to 8.9 connected to the minor peptide chain (three residues) by one nmol兾mg of fly. The LD disulfide bridge (19) (Fig. 2).
50 of histamine was 51.0 nmol兾mg of fly (44.2–61.2; 95% confidence limits) and KCl showed a LD Unlike the above mentioned -agatoxins, CSTX-13 is neuro- nmol兾mg of fly (91.7–118.4; 95% confidence limits) (Table 1).
toxic by itself only at a high micromolar concentration. This When tested alone at physiological concentrations, taurine circumstance raised the question of whether the two-chain (3.88 pmol兾mg of fly) and histamine (316.67 pmol兾mg of fly) structure of CSTX-13 might be the result of a purification artifact showed no effects in a Drosophila bioassay. Injection of KCl caused by contamination with proteases. The purification (11.94 nmol兾mg of fly) showed a short significant paralytic effect protocols of CSTX-13 over the last 5 years have always resulted (564.7 s ⫾ SD 288.9 s; P ⬍ 0.001) when compared with the in a pure peptide with identical molecular masses. Experiments control group (234.5 s ⫾ SD 116.5 s). An injection of 12.6 pmol with spider digestive liquid, which could be the major source of of CSTX-13兾mg of fly resulted in a mortality of 39%. No protease contaminations, resulted only in a C-terminal trunca- statistically significant differences were observed by coinjection tion of 14 residues of chain B. As shown previously, C-terminal of CSTX-13 with taurine (mortality of 34%) or with histamine proteolytic degradation of CSTX-1 by spider digestive liquid (mortality of 42%). In contrast, coinjection of CSTX-13 with stopped at position 49 (Gly) (24). Therefore, we conclude that KCl significantly increased the mortality to 59% (P ⬍ 0.05) (Fig.
the two-chain structure of CSTX-13 is not a purification artifact 4A). When injected alone, the cytolytic cupiennin 1a is not toxic or protease degradation product, but a valid constitutive to D. melanogaster in a concentration of 0.53 pmol兾mg of fly, but component of the C. salei venom. Whether the two-chain it increases the mortality of CSTX-13 from 39% to 97% (P ⬍ structure is posttranslationally generated, however, remains 0.001) (Fig. 4A).
to be investigated.
In addition, we investigated the synergistic effect of CSTX-13 on the toxicity of CSTX-1. At physiological concentrations in the Biological Function of CSTX-13 in the Venom. To analyze the bio-
venom, the molar ratio of CSTX-1 and CSTX-13 is 9:1. With logical function of CSTX-13, we investigated possible interac- administration of one peptide alone, 0.315 pmol of CSTX-1 per tions between CSTX-13 and different venom components in a mg of fly caused a mortality of 31%, and injection of 0.035 pmol Drosophila bioassay. Previously, we have shown that the neuro- of CSTX-13 per mg of fly had no effect. Surprisingly, coinjection toxicity of the main neurotoxin CSTX-1 to blow flies (Proto- of CSTX-1 and CSTX-13 in the above mentioned molar ratio of phormia sp.) could be increased when coinjected with taurine 9:1 significantly increased the mortality to 96% (P ⬍ 0.001) (Fig.
and histamine (9). However, coinjection of CSTX-13 with 4B), and, even in a molar ratio of 500:1, the enhancing effect of taurine or histamine in its physiological venom concentration did CSTX-13 was observed (45%, not significant).
not increase its insecticidal activity. Nevertheless, histamine as a In view of the fact that CSTX-1 inhibits L-type Ca2⫹ channels neurotransmitter and taurine as a neuromodulator play an and that, in Drosophila muscle, a 1,4-dihydropyridine-sensitive important role in the insect nerve system (30–33).
(25) homolog of the mammalian L-type兾␣1D (Dmca1D) subunit Remarkably, the venom of C. salei exhibits a very high K⫹ ion gene is expressed (26), the influence of CSTX-13 on the activity concentration that is 32-fold higher than in the hemolymph, and of the L-type calcium channel blocker nifedipine (27) as well as even 2.7-fold higher than in the prevenom of the scorpion NiCl2, a general inhibitor of calcium channels (28), was inves- Parabuthus transvaalicus (11, 34). Hammock and coworkers (34) tigated. No synergistic effects between NiCl2 or nifedipine and suggest an economically motivated strategy in venom utilization CSTX-13 were detected.
for this scorpion. P. transvaalicus first secretes a prevenom containing a high K⫹ ion concentration at a low protein content, whereas the subsequently secreted venom is characterized by a The Structure of CSTX-13. In CSTX-13, we have characterized a
high protein content and a 15-fold lower K⫹ ion concentration.
two-chain peptide from the venom of C. salei. It paralyzes flies The synergistic activity in the prevenom between the ‘‘inexpen- Wullschleger et al. PNAS 兩 August 3, 2004 兩 vol. 101 兩 no. 31 兩 11255
sive'' K⫹ ion and the assumed inhibitors of rectifier K⫹ channels neurotoxic enhancer CSTX-13 show that it enhances the efficacy is proposed as a means of conserving metabolically expensive of the neurotoxin CSTX-1 at a concentration of 440 times below neuropeptides in the venom (34). In part, C. salei also uses this its LD50. Tests with different concentrations of CSTX-13 re- strategy to enhance its venom efficacy. Coinjection of CSTX-13 vealed a positive correlation between the amount of CSTX-13 with K⫹ ions increases the mortality of the flies by 20%. The and the efficacy of CSTX-1. The cooperation between CSTX-1 synergistic cooperation of K⫹ ions is also detectable when and CSTX-13 seems to be highly specific, because no synergistic applied together with CSTX-1, a suggested L-type Ca2⫹ channel interactions between CSTX-13 and other Ca2⫹ channel blockers, blocker (B.W. and L.K.-N., unpublished data). The high K⫹ such as nifedipine and NiCl2, were found.
concentration in the venom alone caused an immediate short paralysis, and there seems to be a general cooperation between K⫹ ions and various ion channel blockers described here for a In summary, the structural and biological characterization of labidognath spider.
CSTX-13 provide further insight into the complexity of C. salei Enhancement of insecticidal efficacy through the cooperative venom as more multiple interactions between different venom interaction of different venom peptide neurotoxins in spiders components become apparent. After venom injection into a (35) and scorpions (36, 37) has been well investigated. Addi- prey animal, the hyaluronidase seems to act as a spreading tionally, synergistic interactions between acylpolyamines and factor, followed by the dual cytolytic activity of the cupiennins.
cysteine-rich peptide neurotoxins (38) as well as between cyto- They facilitate the activity of the neurotoxins and at the same lytic peptides and neurotoxins have been described (12–14).
time protect the venom duct and glands against bacterial These positive interactions were principally demonstrated by invasion by membrane disturbance and pore building. Addi- applying both components in toxic concentrations.
tionally, antimicrobial peptides may also modulate intracellu- In contrast, a nontoxic concentration of the cytolytically active lar signaling by increasing intracellular Ca2⫹, as reported for cupiennin 1a (20 times lower than its LD parabutoporin and opistoporin from scorpion venoms (39).
50) dramatically en- Simultaneously, the inhibition of ion channels by the neuro- hances the efficacy of CSTX-1 (12, 13). The same effect was toxins is further enhanced by the high K⫹ ion concentration in observed when testing CSTX-13 and cupiennin 1a. It is assumed the venom, shifting the K⫹ equilibrium potential (34). Finally, that, in both cases, mainly through the nonspecific cytolytic the neurotoxins act on different ion channels with a concom- activity of cupiennin 1a, CSTX-1 and CSTX-13 have better itant enhancement by CSTX-13.
access to their targets.
Surprisingly, when CSTX-1 and CSTX-13 were administered We thank Dr. Patrik Kehrli and Dr. Sven Bacher for statistical advice, together at their venom concentrations, a strong positive coop- Dr. Heather Murray for critical comments on the manuscript, and the eration was found. The data presented here on the two-chain Swiss National Science Foundation for funding.
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clinical therapeutics Metformin for the Treatment of the Polycystic Ovary Syndrome John E. Nestler, M.D. This Journal feature begins with a case vignette that includes a therapeutic recommendation. A discussion of the clinical problem and the mechanism of benefit of this form of therapy follows. Major clinical studies, the clinical use of this therapy, and potential adverse effects are reviewed. Relevant formal guidelines,