Synthesis and characterization of delta-atracotoxin-Ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus).
Delta-atracotoxin-Ar1a (delta-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 alpha-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 delta-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. (1)H 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 delta-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 delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.
Institute for Molecular Bioscience, University of Queensland, Queensland 4072 Australia. P.Alewood@imb.uq.edu.au
Dianne
D
Alewood
Liesl C
LC
Birinyi-Strachan
Paul K
PK
Pallaghy
Raymond S
RS
Norton
Graham M
GM
Nicholson
Paul F
PF
Alewood
D000200:Action Potentials / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D004351:Drug Resistance; D005727:Ganglia, Spinal / Q000166:cytology; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009475:Neurons, Afferent / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D009498:Neurotoxins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D019906:Nuclear Magnetic Resonance, Biomolecular; D018408:Patch-Clamp Techniques; D010452:Peptide Biosynthesis; D051381:Rats; D017208:Rats, Wistar; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism; D013111:Spider Venoms / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D013112:Spiders; D013779:Tetrodotoxin / Q000494:pharmacology
D009498:Neurotoxins; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013111:Spider Venoms; D013779:Tetrodotoxin; C044536:robustoxin