Proteomics of the Neurotoxic Fraction from the Sea Anemone Bunodosoma cangicum.Novel Peptides Belonging to a New Class of Neurotoxins.

^{1,2Zaharenko, A.J.; 1,2Oliveira, J.S; 2Pimenta, D.C.; 2Konno, K.; 3Hirata, I.Y. and 1,2Freitas, J.C.}

1 Departamento de Fisiologia, IB, USP, São Paulo, Brazil; 2 Center for Applied Toxinology, CAT/CEPID- FAPESP, Instituto Butantan, São Paulo, Brazil; 3 Departamento de Biofísica, INFAR- UNIFESP, São Paulo, Brazil.

Since sea anemones employ several different polypeptides in order either to capture preys or to defense against predators, it is assumed that very diverse physiological targets may exist. Up to now, sodium channel and potassium channel toxins were mainly isolated and characterized over channels subtypes. Cytolisins were also extensively investigated. As most sea anemones have a similar profile of venom fractionation on gel filtration chromatography, their neurotoxic fractions (peptides ranging from 2000-5000amu) are eluted in the same peak of the chromatogram. Even phylogenetically distant species present this characteristics. Neurotoxins and hemolysins were reported previously from the sea anemone Bunodosoma cangicum, and here we present the molecular masses, primary sequence and neurotoxicity of individually collected peptides from its venom neurotoxic fraction. Different peaks were purified and pooled to homogeneity by a linear gradient of RP-HPLC (C-18, 5mm, 4.6X150mm), being collected “offline” in order to yield great amounts of material. The peaks were subjected to MALDI-TOF and ESI-QTof mass spectrometry analyses. From this neurotoxic fraction we obtained at least 35 different molecules, with molecular masses of near 2000 till 5000. In the crab leg nerve bioassay we reported a ~3500amu toxin probably acting on potassium channels. A complete novel type-I sodium channel peptide was purified and partially sequenced by Edman degradation. The most interesting result was the obtainment of two novel neurotoxins (4157 and 4370amu). They are active on crab leg nerve and belong to the group of the recently reported APETx1 and APETx2 from Anthopleura elegantissima (Diochot et al., 2003; Diochot et al., 2004; respectively). These two novel peptides were also partially sequenced by Edman degradation and are the first ones in the genus Bunodosoma. From the same neurotoxic fraction we have not observed any kind of effect on the crab nerve from the molecules with molecular mass near 2000amu. As a major conclusion we can assume that two distinct groups of active peptides are eluted in the RP-HPLC chromatography, providing us with completely novel toxins and some others to come. Probably different ion channels subtypes or other targets may be reported in the future.

Support: FAPESP, CAT/CEPID-FAPESP and CNPq.

Correspondence to: a.j.zaharenko@ig.com.br