Synthesis and Characterization of δ-Atracotoxin-Ar1a, the Lethal Neurotoxin from Venom of the Sydney Funnel-Web Spider (Atrax robustus)

Alewood, D; Birinyi-Strachan, LC; Pallaghy, PK; Norton, RS; Nicholson, GM; Alewood, PF

Synthesis and Characterization of δ-Atracotoxin-Ar1a, the Lethal Neurotoxin from Venom of the Sydney Funnel-Web Spider (Atrax robustus)

Alewood, D Birinyi-Strachan, LC Pallaghy, PK Norton, RS Nicholson, GM

Alewood, PF

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Publication Type:: Journal Article
Citation:: Biochemistry, 2003, 42 (44), pp. 12933 - 12940
Issue Date:: 2003-11-11

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Field	Value	Language
dc.contributor.author	Alewood, D	en_US
dc.contributor.author	Birinyi-Strachan, LC	en_US
dc.contributor.author	Pallaghy, PK	en_US
dc.contributor.author	Norton, RS	en_US
dc.contributor.author	Nicholson, GM https://orcid.org/0000-0002-4277-4296	en_US
dc.contributor.author	Alewood, PF	en_US
dc.date.issued	2003-11-11	en_US
dc.identifier.citation	Biochemistry, 2003, 42 (44), pp. 12933 - 12940	en_US
dc.identifier.issn	0006-2960	en_US
dc.identifier.uri	http://hdl.handle.net/10453/3713
dc.description.abstract	δ-Atracotoxin-Ar1a (δ-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion α-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing α-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. 1H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic δ-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that δ-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic δ-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.	en_US
dc.relation	http://purl.org/au-research/grants/arc/DP0346325
dc.relation.ispartof	Biochemistry	en_US
dc.relation.isbasedon	10.1021/bi030091n	en_US
dc.subject.classification	Biochemistry & Molecular Biology	en_US
dc.subject.mesh	Ganglia, Spinal	en_US
dc.subject.mesh	Neurons, Afferent	en_US
dc.subject.mesh	Cells, Cultured	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Rats	en_US
dc.subject.mesh	Rats, Wistar	en_US
dc.subject.mesh	Spiders	en_US
dc.subject.mesh	Tetrodotoxin	en_US
dc.subject.mesh	Sodium Channels	en_US
dc.subject.mesh	Sodium Channel Blockers	en_US
dc.subject.mesh	Spider Venoms	en_US
dc.subject.mesh	Neurotoxins	en_US
dc.subject.mesh	Nuclear Magnetic Resonance, Biomolecular	en_US
dc.subject.mesh	Patch-Clamp Techniques	en_US
dc.subject.mesh	Amino Acid Sequence	en_US
dc.subject.mesh	Membrane Potentials	en_US
dc.subject.mesh	Peptide Biosynthesis	en_US
dc.subject.mesh	Action Potentials	en_US
dc.subject.mesh	Drug Resistance	en_US
dc.subject.mesh	Molecular Sequence Data	en_US
dc.subject.mesh	Male	en_US
dc.title	Synthesis and Characterization of δ-Atracotoxin-Ar1a, the Lethal Neurotoxin from Venom of the Sydney Funnel-Web Spider (Atrax robustus)	en_US
dc.type	Journal Article
utslib.citation.volume	44	en_US
utslib.citation.volume	42	en_US
utslib.for	0601 Biochemistry and Cell Biology	en_US
utslib.for	0304 Medicinal and Biomolecular Chemistry	en_US
utslib.for	1101 Medical Biochemistry and Metabolomics	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Strength - CHT - Health Technologies
utslib.copyright.status	closed_access
pubs.issue	44	en_US
pubs.publication-status	Published	en_US
pubs.volume	42	en_US

Abstract:

δ-Atracotoxin-Ar1a (δ-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion α-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing α-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. 1H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic δ-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that δ-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic δ-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

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http://hdl.handle.net/10453/3713