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CSTX-13 and the Multi-Component Venom of the Spider Cupiennius Salei

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CSTX-13 and the Multi-Component Venom of the Spider Cupiennius Salei CCSSTTXX--1133 aanndd tthhee mmuullttii--ccoommppoonneenntt vveennoomm ooff tthhee ssppiiddeerr CCuuppiieennnniiuuss ssaalleeii Inauguraldissertation der Philosophisch-naturwissenschaftlichen Fakultät der Universität Bern vorgelegt von Benno Wullschleger von Zofingen Leiter der Arbeit: Prof. Dr. Wolfgang Nentwig Zoologisches Institut der Universität Bern CSTX-13 and the multi-component venom of the spider Cupiennius salei Inauguraldissertation der Philosophisch-naturwissenschaftlichen Fakultät der Universität Bern vorgelegt von Benno Wullschleger von Zofingen Leiter der Arbeit: Prof. Dr. Wolfgang Nentwig Zoologisches Institut der Universität Bern Von der Philosophisch-naturwissenschaftlichen Fakultät angenommen. Bern, den 16.12.2004 Der Dekan: Prof. Dr. P. Messerli für Monika INHALTSVERZEICHNIS INHALTSVERZEICHNIS Einleitung 001 Publikationen 003 MANUSKRIPT I 004 Benno Wullschleger, Lucia Kuhn-Nentwig, Jan Tromp, Urs Kämpfer, Johann Schaller, Stefan Schürch & Wolfgang Nentwig (2004). CSTX-13, a highly synergistically acting two-chain neurotoxic enhancer in the venom of the spider Cupiennius salei (Ctenidae). Proc. Natl. Acad. Sci. USA 101, 11251–11256. MANUSKRIPT II 015 Benno Wullschleger, Wolfgang Nentwig & Lucia Kuhn-Nentwig. Spider venom: Enhancement of venom efficacy mediated by different synergistic strategies in Cupiennius salei. Submitted. MANUSKRIPT III 032 Benno Wullschleger, Wolfgang Nentwig & Lucia Kuhn-Nentwig. Different effects of neurotoxic and cytolytically acting peptides of Cupiennius salei venom to insects. Zusammenfassung 046 Literatur 048 Danksagung 051 Curriculum vitae 052 EINLEITUNG EINLEITUNG Spinnen gehören mit 38'663 rezenten Arten (PLATNICK, 2004) nach den Insekten zu der erfolgreichsten Tiergruppe. Die ebenfalls zu den Arachniden zählenden Skorpione gelten als die ältesten Landtiere, welche sich wahrscheinlich schon vor 400 Millionen Jahren entwickelt haben. Die ältesten fossilen Spinnen stammen aus dem Devon und dürften über 350 Millionen Jahre alt sein (WUNDERLICH, 1986). Trotz unterschiedlicher Habitattypen und Verhaltensmustern ist allen Spinnen gemeinsam, dass sie Prädatoren sind und sich in erster Linie von Arthropoden (v. a. Insekten) ernähren. Wohl nur ausnahmsweise werden Wirbeltiere wie Kaulquappen, kleine Fische, Reptilien, Nagetiere oder Vögel erbeutet (FOELIX, 1996). Ihrer Jagdtechnik entsprechend lassen sich Spinnen in Netzspinnen und Laufspinnen einteilen, wobei sich beide Gruppen durch eine grosse Variabilität auszeichnen. Allen gemeinsam ist aber, dass Beutetiere im Endeffekt mit Gift, welches mit Hilfe von Cheliceren inijziert wird, immobilisiert werden. Dabei nimmt die letale Wirkung des Giftes, im Gegensatz zur Lähmung, eher eine sekundäre Rolle ein (FRIEDEL & NENTWIG, 1989). Als giftlose Spinnen gelten lediglich die sehr ursprünglichen Liphistiidae, welche noch keine Giftdrüsen entwickelt haben (HAUPT, 2003) und die Uloboridae respektive Holarchaea (MARETIC, 1987), welche vermutlich ihre Giftdrüsen sekundär reduziert haben. Nur gerade von vier Gattungen sind Arten bekannt, die auch für den Menschen als potentiell gefährlich einzustufen sind. Hierzu gehören Latrodectus (Theridiidae), Atrax (Hexathelidae), Loxosceles (Sicariidae) und Phoneutria (Ctenidae) (HABERMEHL, 1976). Dagegen sind einheimische Spinnen nicht von medizinischer Bedeutung. Spinnengifte sind Multi-Komponenten-Systeme mit einer noch nicht gut verstandenen Komplexizität. Die Giftkomponenten können grob in drei chemische Klassen eingeteilt werden: Organische Moleküle und Ionen mit einem geringen Molekulargewicht (< 1 kDa), Polypeptide (3–10 kDa) und Proteine (> 10 kDa) (ESCOUBAS et al., 2000). Einige dieser Komponenten haben sich als ideale Werkzeuge erwiesen, um Strukturen und Funktionen von Ionenkanälen zu erforschen (SACCOMANO & AHLIJANIAN, 1994; SWARTZ & MACKINNON, 1997; SUCHYNA et al., 2004; LEE & MACKINNON, 2004). Andere haben das Potential, als Insektizide eingesetzt zu werden (KING et al., 2002; TEDFORD et al., 2004), sie werden verwendet, um Antiseren zu entwickeln (FISHER et al., 1981; SUTHERLAND, 1983) oder könnten Basis für zukünftige Medikamente sein (BODE et al., 2001). KING et al. (2002) bezeichnen Spinnengifte als pharmakologische Goldminen und die Autoren gehen bei der momentan bekannten Anzahl Spinnenarten von mehr als einer Million verschiedener, 1 EINLEITUNG pharmakologisch aktiver Peptide aus. Um so erstaunlicher ist es, dass Spinnengifte im Gegensatz zu anderen Tiergiften vergleichsweise wenig intensiv untersucht wurden. Cupiennius salei (Ctenidae; KEYSERLING, 1877), eine polyphage Spinne aus den tropischen Regionen Zentralamerikas, ist als Jäger in höheren Vegetationsschichten besonders auf ein effektives Gift angewiesen, um ihre Beute nicht zu verlieren (BARTH & SEYFARTH, 1979; NENTWIG, 1986; BARTH, 2001; KUHN-NENTWIG et al., 2004). Da Gift eine wertvolle Ressource ist und die Regeneration bis zu 16 Tage in Anspruch nimmt (BOEVE et al., 1995), geht die Spinne haushälterisch mit ihrem Gift um (MALLI et al., 1999; WIGGER et al., 2002; WULLSCHLEGER & NENTWIG, 2002). Das Gift von C. salei beinhaltet im Wesentlichen Cupiennius salei Toxine (CSTX-1 bis 13), zytolytisch aktive Cupiennine, niedermolekulare Substanzen wie Ionen und einzelne Aminosäuren und hochmolekulare Substanzen (Bsp. Hyaluronidase). Insgesamt setzt sich das Rohgift wohl aus über 100 verschiedenen Substanzen zusammen (KUHN-NENTWIG et al., 1994; KUHN-NENTWIG et al., 2004). Bei diesem ausgesprochen komponentenreichen Gift stellt sich natürlich die Frage nach dessen Zweck. Da C. salei ein breites Beutespektrum hat, wären beispielsweise beutetierspezifische Komponenten denkbar. Die hier vorliegende Dissertation zeigt indes, dass die Funktion der einzelnen Komponenten wohl eher darin liegt, mit anderen Komponenten synergistisch zu interagieren. Allerdings lassen die einzelnen Sensitivitätsexperimente mit Rohgift an verschiedenen Beutetierarten bereits den Schluss zu (KUHN-NENTWIG et al., 1998), dass Komponenten je nach Testorganismus unterschiedlich gut funktionieren. Die drei vorliegenden Manuskripte geben einerseits Auskunft über Struktur und Wirkung von CSTX-13 und stellen andererseits einen Ansatz dar, die Komplexität des Giftes von Cupiennius salei besser verstehen zu können. 2 PUBLIKATIONEN PUBLIKATIONEN 3 MANUSKRIPT I MANUSKRIPT I Wullschleger, B., Kuhn-Nentwig, L., Tromp, J., Kämpfer, U., Schaller, J., Schürch, S. & Nentwig, W. (2004). CSTX-13, a highly synergistically acting two-chain neurotoxic enhancer in the venom of the spider Cupiennius salei (Ctenidae). Proc. Natl. Acad. Sci. USA 101, 11251–11256. 4 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 K 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 Ϸ8 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 ϩ 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 LD50 concentration. CSTX-1 and CSTX-9 are available. The four disulfide bridges of CSTX-9 form linkages between C1–C4; C2–C5; C3–C8; and piders and scorpions use their venom to paralyze prey and͞or C6–C7 (15–17). This arrangement is also found in other spider Sto 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
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