Isolation and Pharmacological Characterization of Cannitoxin, a Presynaptic Neurotoxin from the Venom of the Papuan Taipan (Oxyuranus scutellatus canni)

Abstract

The Papuan taipan (Oxyuranus scutellatus canni) is widely distributed throughout much of Papua New Guinea. Although neurotoxicity is a major symptom of envenomation, no neurotoxins have been isolated from this venom. Using a series of size exclusion chromatography steps, we report the isolation of cannitoxin, a presynaptic neurotoxin (44,848 Da) that represents approximately 16% of the whole venom. The toxin displayed high phospholipase A 2 (PLA 2 ) activity (330 Ϯ 5 mol/ min/mg) and caused concentration-dependent (11-66 nM) inhibition of indirect (0.2 ms; 0.1 Hz; supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation without effecting nicotinic receptor agonists. Prior addition of CSL Taipan antivenom (5 U/ml) or inhibition of phospholipase A 2 activity by incubation with 4-bromophenacyl bromide prevented the inhibition of twitches. Cannitoxin is composed of three different subunits, ␣, ␤, and ␥, with the possibility of two ␤ isomers. However, only the ␣ subunit displayed in vitro neurotoxic activity of its own. Thus, cannitoxin is similar in structure and pharmacology to taipoxin, which has been isolated from the closely related Australian species O. scutellatus scutellatus (coastal taipan).Three species of taipans (genus Oxyuranus) have been identified and are considered to be the world's most venomous snakes (Sutherland and Tibballs, 2001 (Broad et al., 1979). However, t 90 (i.e., time to produce 90% inhibition of indirect twitches) data obtained in the chick biventer cervicis nerve-muscle (CBCNM) preparation indicated that O. scutellatus scutellatus venom is more neurotoxic than that of O. scutellatus canni venom (Crachi et al., 1999b). They also showed that CSL Taipan antivenom (CTPV) was markedly more effective in neutralizing the neurotoxic effects of the Papuan or coastal taipan venoms than that of the inland taipan (Crachi et al., 1999b).Presynaptic neurotoxins act at the motor nerve terminal to either facilitate (e.g., dendrotoxin) or inhibit (e.g., ␤-bungarotoxin, taipoxin, and paradoxin) the release of neurotransmitter. All of the presynaptic inhibitors have phospholipase A 2 (PLA 2 ) activity (Harris, 1991). Taipoxin and paradoxin were isolated from the venoms of O. scutellatus scutellatus (Fohlman et al., 1976) and O. microlepidotus, respectively. Taipoxin is a ternary complex of three subunits, ␣-, ␤-, and ␥-taipoxin, that exists in a stoichiometry of 1:1:1 held together by noncovalent interactions. The ␣ subunit is the only subunit with toxic activity on its own. The ␤ subunit is neutral and exists in two isoforms (i.e., ␤ 1 and ␤ 2 ), which are interchangeable in the complex. The ␥ subunit is the largest of the three subunits, and it seems to be glycosylated (FohlArticle, publication date, and citation information can be found at

Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: Role of neurotoxic and procoagulant effects in venom toxicity

“…The percentage of taipoxin in the venome of Australian O. scutellatus (20%) roughly corresponds to the previously described yield of this toxin [12]. In contrast, the percentage of taipoxin (56%) in PNG O. scutellatus deduced by proteomic analysis is much higher than the 16% yield of "cannitoxin" obtained from Papuan O. scutellatus [13]. This discrepancy might suggest additional variation between the venoms of the eastern PNG and western New Guinea (Western Province of PNG and Indonesian West Papua) populations which are physically disjoint from one another, an issue that deserves further investigation.…”

“…More recently, an analogous toxin, named cannitoxin, was characterized from the venom of Papuan O. scutellatus [13], although this can be regarded as taipoxin given that the PNG and Australian populations belong to the same species, and indeed, we identified the N-terminal sequence for taipoxin (NLLQFGFMIR), rather than cannitoxin (NLLQFGYMIR), in venom of our PNG O. scutellatus suggesting that both isoforms are likely to exist in this venom. In both populations, the trimers are comprised of subunit α, which is an active neurotoxic PLA 2 , and by subunits β and γ, which are PLA 2 homologues without enzymatic activity [12,13,[32][33][34]. Despite the lack of catalytic and toxic activities of subunits β and γ, they significantly potentiate the toxicity of subunit α [27,35].…”

“…In addition, coagulopathy associated with spontaneous bleeding has been described in these patients [10], together with myotoxicity and cardiac disturbances in some cases [11]. The toxins responsible for these effects have been isolated and characterized, such as the potent neurotoxic and myotoxic heterotrimeric phospholipase A 2 (PLA 2 ) complex taipoxin (named cannitoxin for the West Papuan population) [12,13], two monomeric PLA 2 s, named OS1 and OS2 [14], Oscutarin-C, a prothrombin activator responsible for the characteristic coagulopathy [15], post-synaptically acting α-neurotoxins [16], and taicatoxin, a blocker of voltage-dependent calcium channels [17]. In addition, cDNA analysis derived from venom gland transcripts detected various putative toxin genes, including PLA 2 s, neurotoxins, cysteine-rich secretory proteins (CRISPs), a venom natriuretic peptide and a nerve growth factor [18].…”

Antivenomic Characterization of Two Antivenoms Against the Venom of the Taipan, Oxyuranus scutellatus, from Papua New Guinea and Australia

“…14 The ability of antivenoms to neutralize the lethal effect of O. scutellatus venom depends, to a large extent, on their capacity to neutralize taipoxin, the most potent neurotoxin in the venoms of both populations of taipan. 15,20 In this regard, the two antivenoms bound taipoxin α-, β-, and γ-subunits to varying degrees in venoms from both PNG and Australian O. scutellatus in the affinity column. In addition, SPR observations corroborated the ability of both antivenoms to bind taipoxin, with a higher affinity in the ICP antivenom.…”

Protein complexes in snake venom

“…Paradoxin isolated from the venom of Parademansia microlepidotus (currently known as Oxyuranus microlepidotus) [70,71] and Cannitoxin isolated from the venom of Oxyuranus scutellatus canni [72] are homologs of taipoxin. Structurally, they have three subunits, a, b and c, and functionally they act as presynaptic neurotoxins similar to taipoxin.…”