Abstract
The box jellyfish Chironex fleckeri produces extremely potent and rapid-acting venom that is harmful to humans and lethal to prey. Here, we describe the characterization of two C. fleckeri venom proteins, CfTX-A (∼40 kDa) and CfTX-B (∼42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography. Full-length cDNA sequences encoding CfTX-A and -B and a third putative toxin, CfTX-Bt, were subsequently retrieved from a C. fleckeri tentacle cDNA library. Bioinformatic analyses revealed that the new toxins belong to a small family of potent cnidarian pore-forming toxins that includes two other C. fleckeri toxins, CfTX-1 and CfTX-2. Phylogenetic inferences from amino acid sequences of the toxin family grouped CfTX-A, -B, and -Bt in a separate clade from CfTX-1 and -2, suggesting that the C. fleckeri toxins have diversified structurally and functionally during evolution. Comparative bioactivity assays revealed that CfTX-1/2 (25 μg kg(-1)) caused profound effects on the cardiovascular system of anesthetized rats, whereas CfTX-A/B elicited only minor effects at the same dose. Conversely, the hemolytic activity of CfTX-A/B (HU50 = 5 ng ml(-1)) was at least 30 times greater than that of CfTX-1/2. Structural homology between the cubozoan toxins and insecticidal three-domain Cry toxins (δ-endotoxins) suggests that the toxins have a similar pore-forming mechanism of action involving α-helices of the N-terminal domain, whereas structural diversification among toxin members may modulate target specificity. Expansion of the cnidarian toxin family therefore provides new insights into the evolutionary diversification of box jellyfish toxins from a structural and functional perspective.
Highlights
Box jellyfish produce a unique family of toxic venom proteins
We describe the characterization of two C. fleckeri venom proteins, CfTX-A (ϳ40 kDa) and CfTX-B (ϳ42 kDa), which were isolated from C. fleckeri venom using size exclusion chromatography and cation exchange chromatography
Size Exclusion Chromatography (SEC) and CEX—Crude C. fleckeri venom proteins eluted from the Superdex 200pg SEC column in six major peaks with native molecular masses ranging from 30 to Ͼ600 kDa (Fig. 1a)
Summary
Box jellyfish produce a unique family of toxic venom proteins. Results: The toxins are structurally similar, yet two subgroups confer different cytolytic activities in red blood cells and cardiovascular effects in rats. The first toxins in C. fleckeri venom to be sequenced were CfTX-1 and -2 [4] These highly abundant venom proteins belong to a family of taxonomically restricted cnidarian toxins (42– 46 kDa) that includes CqTX-A, CrTX-A, and CaTX-A from box jellyfish species Chironex yamaguchii [5] (as Chiropsalmus quadrigatus [6]), Carybdea rastonii [7], and Alatina moseri [8] (as Carybdea alata [9]), respectively, as well as other representatives from Cubozoa, Scyphozoa, and Hydrozoa. Molecular and Functional Studies of Box Jellyfish Toxins cation and molecular characterization of two CfTX-like toxins from C. fleckeri venom that are closely related in sequence to CaTX-A as well as a third, putative toxin that is homologous to CaTX-A. Through computational analyses and bioactivity assays, we examine the structural and functional characteristics of the new toxins, explore the molecular diversity of the expanded toxin family, and discuss the implications for the biological role of these toxins in box jellyfish stings
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