FEBS 27793 FEBS Letters 554 (2003) 211^218 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Isolation of N-missulenatoxin-Mb1a, the major vertebrate-activeprovided by spiderElsevier - Publisher Connector N-toxin from the venom of Missulena bradleyi (Actinopodidae)1 Simon J. Gunninga, Youmie Chonga, Ali A. Khalifea, Peter G. Hainsb, Kevin W. Broadyc, Graham M. Nicholsona;Ã aDepartment of Health Sciences, University of Technology, Sydney, NSW 2007, Australia bDepartment of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australia cDepartment of Cell and Molecular Biology, University of Technology, Sydney, NSW 2007, Australia Received 1 September 2003; revised 23 September 2003; accepted 3 October 2003 First published online 17 October 2003 Edited by Maurice Montal N-MSTX-Mb1a caused signi¢cant insect toxicity at doses up to Abstract The present study describes the isolation and phar- 2000 pmol/g. N-MSTX-Mb1a therefore provides evidence of a macological characterisation of the neurotoxin N-missulenatox- N N highly conserved spider -toxin from a phylogenetically distinct in-Mb1a ( -MSTX-Mb1a) from the venom of the male Austra- spider family that has not undergone signi¢cant modi¢cation. lian eastern mouse spider, Missulena bradleyi.This toxin was ß 2003 Published by Elsevier B.V. on behalf of the Federation isolated using reverse-phase high-performance liquid chromatog- of European Biochemical Societies. raphy and was subsequently shown to cause an increase in rest- ing tension, muscle fasciculation and a decrease in indirect Key words: Spider; Peptide; N-Missulenatoxin; twitch tension in a chick biventer cervicis nerve-muscle bioassay. Sodium channel; Scorpion toxin; N-Atracotoxin Interestingly, these e¡ects were neutralised by antivenom raised against the venom of the Sydney funnel-web spider Atrax ro- bustus.Subsequent whole-cell patch-clamp electrophysiology on rat dorsal root ganglion neurones revealed that N-MSTX-Mb1a caused a reduction in peak tetrodotoxin (TTX)-sensitive sodium 1.Introduction current, a slowing of sodium current inactivation and a hyper- polarising shift in the voltage at half-maximal activation.In Australian mouse spiders (Araneae, Mygalomorphae, Acti- addition, N-MSTX-Mb1a failed to a¡ect TTX-resistant sodium nopodidae) are one of the oldest groups of spiders in the currents.Subsequent Edman degradation revealed a 42-residue Australo-Papuan region. Importantly, the male eastern mouse peptide with unusual N- and C-terminal cysteines and a cysteine spider (Missulena bradleyi), restricted to the east coast of triplet (Cys14À16).This toxin was highly homologous to a family mainland Australia, can cause systemic envenomation in hu- N N of -atracotoxins ( -ACTX) from Australian funnel-web spiders mans. The symptoms of envenomation, including muscle fas- including conservation of all eight cysteine residues.In addition ciculation, tachycardia, dyspnoea, and profuse sweating, are to actions on sodium channel gating and kinetics to N-ACTX, remarkably similar to those reported for Australian funnel- web spiders (Aranaea, Mygalomorphae, Hexathelidae, Atra- cinae). The only reported case of severe envenomation in- *Corresponding author. Fax: (61)-2-9514 2228. volved a 19-month-old girl who lost consciousness within 30 E-mail address: [email protected] (G.M. Nicholson). min of being bitten on the ¢nger by a male M. bradleyi. The 1 The protein sequence of N-missulenatoxin-Mb1a reported in this administration of Sydney funnel-web spider antivenom, raised paper has been submitted to the Swiss Protein Database under the against Atrax robustus venom, surprisingly produced a dra- SwissProt accession code P83608. matic improvement in her condition and a full recovery was made following a second dose of antivenom [1]. This N N Abbreviations: -MSTX-Mb1a, -missulenatoxin-Mb1a from the prompted a recent study to characterise the pharmacological eastern mouse spider Missulena bradleyi; TTX, tetrodotoxin; N-ACTX, N-atracotoxins from Australian funnel-web spiders; actions of mouse spider venom. Whole male M. bradleyi ven- N-ACTX-Ar1a, N-atracotoxin-Ar1a (formerly robustoxin) from the om caused rapid muscle fasciculation, a large contracture and Sydney funnel-web spider Atrax robustus; N-ACTX-Hv1a, N-atraco- a rapid decrease in indirectly evoked twitches in the chick toxin-Hv1a (formerly versutoxin) from the Blue Mountains funnel- biventer cervicis muscle, with activity blocked by A. robustus web spider Hadronyche versuta; N-ACTX-Hv1b, N-atracotoxin-Hv1b from Hadronyche versuta; N-ACTX-Hs20.1a, N-atracotoxin-Hs20.1a antivenom [2]. These actions were found to be due to a re- from Hadronyche sp. 20; Lqh II, anti-mammal K-toxin from the scor- duction in the rate of tetrodotoxin (TTX)-sensitive sodium pion Leiurus quinquestriatus hebraeus; Aah-II, anti-mammal K-toxin current inactivation and a hyperpolarising shift in the thresh- from the scorpion Androctonus australis hector; LqhKIT, K-insect tox- old of activation of TTX-sensitive sodium currents [2]. The in from the scorpion Leiurus quinquestriatus hebraeus; SDS^PAGE, modulation of sodium channel gating and kinetics, and rever- sodium dodecyl sulphate^polyacrylamide gel electrophoresis; PVDF, polyvinylidene £uoride; TBS, Tris-bu¡ered saline; FWS-AV, Sydney sal by A. robustus antivenom, indicate that the venom from funnel-web spider antivenom; RP-HPLC, reverse-phase high-perfor- male M. bradleyi may contain vertebrate-active neurotoxins mance liquid chromatography; TFA, tri£uoroacetic acid; DRG, dor- that have a similar action to those found in Australian fun- sal root ganglion; HEPES, N-2-hydroxyethylpiperazine-N-2-ethane- nel-web spider venoms, despite the fact that mouse spiders are sulfonic acid; TEA, tetraethylammonium; (+)-TC, (+)-tubocurarine; taxonomically distinct from funnel-web spiders. LD50, median lethal dose; PD50, median paralytic dose; ApB, antho- pleurin-B from the sea anemone Anthopleura xanthogrammica; Magi Previous studies on the venom of Australian funnel-web 4, sodium channel toxin from the spider Macrothele gigas spiders have revealed a number of toxins, called atracotoxins 0014-5793 / 03 / $22.00 ß 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/S0014-5793(03)01175-X FEBS 27793 23-10-03 212 S.J. Gunning et al./FEBS Letters 554 (2003) 211^218 (ACTX), that are responsible for the neurotoxic action seen M. bradleyi venom was performed under reducing conditions accord- with whole venom (for a review see [3]). However, the lethal ing to the method of Laemmli [21] using 10^20% Tris^tricine gradient gels (Novex, San Diego, CA, USA). The molecular weight marker toxins responsible for the major primate-speci¢c symptoms of lane consisted of markers of 3.4^26.6 kDa (Life Science Research envenomation are the N-ACTX shown to target the voltage- Products, Regents Park, NSW, Australia). Following electrophoresis, gated sodium channel (for a review see [4]). To date, four N- protein bands were stained using silver nitrate. ACTX have been identi¢ed, these include N-ACTX-Ar1a from Western blotting was performed following initial SDS^PAGE elec- N trophoresis as described above. Proteins were transblotted onto poly- the Sydney funnel-web spider (A. robustus) [5], -ACTX-Hv1a vinylidene £uoride (PVDF) membrane (Bio-Rad Laboratories, Her- [6] and -Hv1b [7] from the Blue Mountains funnel-web spider cules, CA, USA) in Tris^tricine^methanol transfer bu¡er using 200 (Hadronyche versuta) and N-ACTX-Hs20.1a from Hadronyche mA constant current for 1 h. The PVDF membrane was incubated sp. 20 [4]. All N-ACTX are highly homologous 42-residue pep- with 3% w/v bovine serum albumin (BSA) in phosphate-bu¡ered sa- tides with a high proportion of basic residues and are cross- line for 1 h at room temperature. It was then washed in Tris-bu¡ered saline (pH 8.0)^Tween 0.05% v/v (TBS^Tween), followed by incuba- linked by four conserved intramolecular disul¢de bonds. tion with funnel-web spider antivenom raised against male Sydney These peptide, like many spider neurotoxins, also contain an funnel-web spider (A. robustus) venom (FWS-AV) (Commonwealth ‘inhibitor cystine-knot’ motif [8^10]. N-ACTX produce a slow- Serum Laboratories, Melbourne) at a dilution of 1:500 for 1 h. The ing of TTX-sensitive sodium current inactivation, a modest washing step was repeated and the blot was then incubated with a 1:5000 dilution of the secondary antibody, alkaline phosphatase con- hyperpolarising shift in the voltage dependence of activation jugated goat-anti-rabbit IgG(Sigma Laboratories, St Louis, MO, and a reduction in peak sodium current [4,7,11,12]. This re- USA), for 1 h. It was then washed again with TBS^Tween and devel- sults in spontaneous repetitive ¢ring accompanied by plateau oped with 5-bromo-4-chloro-indoyl phosphate p-toluidine plus nitro- potentials [13,14]. Of great interest has been the ¢nding that blue tetrazolium. N -ACTX, in addition to being mammalian toxic, are also in- 2.2. Toxin puri¢cation and sequencing secticidal. Both N-ACTX-Ar1a and -Hv1a are toxic by later- Pooled venom was initially fractionated using reverse-phase high- oventral injection into crickets [15] and target the insect vol- performance liquid chromatography (RP-HPLC) employing a Vydac W tage-gated sodium channel to cause similar actions on analytical column (C18, 4.6U250 mm, 5 m) on a Shimadzu HPLC neuronal excitability as seen in vertebrate preparations [13]. system. Peptide peaks were monitored at an absorbance of 215 nm. Elution of venom peptide components was achieved using a linear Notably, these actions on insect and mammalian voltage- gradient of 5^60% v/v acetonitrile/0.1% v/v tri£uoroacetic acid gated sodium channels are similar, but not identical, to the (TFA) over 55 min at a £ow rate of 1 ml/min. Selected fractions mechanism of action of scorpion K-toxins and sea anemone were subjected to vertebrate toxicity bioassays using a chick biventer toxins [16,17].