Tx4(5-2), a new toxin from Phoneutria nigriventer venom elicites the glutamate uptake inhibition displayed by PhTx4 toxic fraction

Oliveira, L.C.(1,2), Figueiredo, S.G.(5), Pimenta, A.M.C.(2), Mansuelle, P.(3), Cordeiro, M.N.(4), Richardson, M.(4), Diniz, C.R.(4), Rochat, H.(3), De Lima, M.E.(2)

(1)Departamento de  Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, (2)Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, (3)Laboratoire de Biochimie - Ingénierie des Protéines, Marseille, France, (4)Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, (5)Departamento de Ciências Fisiológicas, Centro Biomédico, Universidade Federal do Espírito Santo.

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic toward insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a peptide class that is able to inhibit glutamate uptake in preparations of mammalian central nervous systems. In this work a new toxin Tx4(5-2) was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Some biological features and the complete amino acid sequence were established. This toxin leads to immediate excitatory effects when injected in house flies and cockroaches) by intrathoracical injections. Intracerebroventricular injections of 0.03 mg of Tx4(5-2) in mice resulted in no apparent signs of intoxication. Pharmacological characterization carried out in rat brain synaptosomes by using [³H]-L-glutamate, showed that the whole PhTx4 fraction as well as Tx4(5-2),  Tx4(6-1) and Tx4(5-5) pure toxins were able inhibit the glutamate uptake in the micromolar concentration range. Tx4(5-2) inhibits the glutamate uptake in a dose dependent manner, with an IC₅₀ of approximately 1mM. Edman sequencing of Tx4(5-2) revealed a 48 residues long polypeptide, containing 10 cysteines and cross-linked by 5 disulfide bridges. Molecular mass measured by MALDI-TOF mass spectrometry was 5203.99 Da, which is very close from calculated MM (5199.99). Tx4(5-2) is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.

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LeidaCalegário de Oliveira, Rua Carlos do Carmo, nº 22, Belo Horizonte, MG, CEP: 30642-420, Brasil, Email: leida@mono.icb.ufmg.br