Alewood Dianne, Birinyi-Strachan Liesl C, Pallaghy Paul K, Norton Raymond S, Nicholson Graham M, Alewood Paul F
Institute for Molecular Bioscience, University of Queensland, Queensland 4072 Australia.
Biochemistry. 2003 Nov 11;42(44):12933-40. doi: 10.1021/bi030091n.
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.
δ-蛛毒环肽-Ar1a（δ-ACTX-Ar1a）是从雄性悉尼漏斗网蜘蛛（Atrax robustus）毒液中分离出的主要多肽神经毒素。这种神经毒素作用于昆虫和哺乳动物的电压门控钠通道，在该通道上它与蝎α-毒素竞争神经毒素受体位点3，从而减缓钠通道失活。由于天然来源的纯毒素供应有限，对这种毒素的结构和作用机制的研究进展受到了阻碍。在本文中，我们描述了含四对二硫键的δ-ACTX-Ar1a首次成功的化学合成及氧化重折叠。该合成过程采用双偶联的固相Boc化学法，随后使用2 M盐酸胍和还原型谷胱甘肽/氧化型谷胱甘肽以1:1混合于2-丙醇（pH 8.5）的缓冲液对纯化后的肽进行氧化折叠。通过化学和药理学表征证实了成功的氧化和重折叠。采用离子喷雾质谱法确认分子量。氢核磁共振（1H NMR）分析显示天然毒素和合成毒素具有相同的化学位移，表明合成毒素具有天然构象。使用大鼠背根神经节神经元的全细胞膜片钳记录进行的药理学研究证实，合成的δ-ACTX-Ar1a使钠电流失活减慢，并且激活和失活的电压依赖性发生超极化偏移，类似于天然毒素。在电流钳制条件下，我们首次表明δ-ACTX-Ar1a产生了中毒时所见临床症状背后的自发重复平台电位。合成的δ-ACTX-Ar1a成功的氧化重折叠为未来确定毒素药效基团的构效关系研究铺平了道路。
