Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2kDa, which represent the smallest peptide toxins found in sea anemones.
Crotamine, a 5-kDa peptide, possesses a unique biological versatility. Not only has its cell-penetrating activity become of clinical interest but, moreover, its potential selective antitumor activity is of great pharmacological importance. In the past, several studies have attempted to elucidate the exact molecular target responsible for the crotamine-induced skeletal muscle spasm. The aim of this study was to investigate whether crotamine affects voltagegated potassium (K V ) channels in an effort to explain its in vivo effects. Crotamine was studied on ion channel function using the two-electrode voltage clamp technique on 16 cloned ion channels (12 K V channels and 4 Na V channels), expressed in Xenopus laevis oocytes. Crotamine selectively inhibits K V 1.1, K V 1.2, and K V 1.3 channels with an IC 50 of ϳ300 nM, and the key amino acids responsible for this molecular interaction are suggested. Our results demonstrate for the first time that the symptoms, which are observed in the typical crotamine syndrome, may result from the inhibition of K V channels. The ability of crotamine to inhibit the potassium current through K V channels unravels it as the first snake peptide with the unique multifunctionality of cell-penetrating and antitumoral activity combined with K V channel-inhibiting properties. This new property of crotamine might explain some experimental observations and opens new perspectives on pharmacological uses.
Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Na v 1.1 to Na v 1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Na v 1.3 and Na v 1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na ؉ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools.
Sea anemone venoms have become a rich source of peptide toxins which are invaluable tools for studying the structure and functions of ion channels. In this work, BcsTx3, a toxin found in the venom of a Bunodosoma caissarum (population captured at the Saint Peter and Saint Paul Archipelago, Brazil) was purified and biochemically and pharmacologically characterized. The pharmacological effects were studied on 12 different subtypes of voltage-gated potassium channels (K V 1.1-K V 1.6; K V 2.1; K V 3.1; K V 4.2; K V 4.3; hERG and Shaker IR) and three cloned voltagegated sodium channel isoforms (Na V 1.2, Na V 1.4 and BgNa V 1.1) expressed in Xenopus laevis oocytes. BcsTx3 shows a high affinity for Drosophila Shaker IR channels over rKv1.2, hKv1.3 and rKv1.6, and is not active on Na V channels. Biochemical characterization reveals that BcsTx3 is a 50 amino acid peptide crosslinked by four disulfide bridges, and sequence comparison allowed BcsTx3 to be classified as a novel type of sea anemone toxin acting on K V channels. Moreover, putative toxins homologous to BcsTx3 from two additional actiniarian species suggest an ancient origin of this newly discovered toxin family.
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