Structure-function relationships of sea anemone toxin II from Anemonia sulcata

Abstracte STRDCTDR$-FUNCTION RELATIONSHIPS OF SEA ANEMONE TOXIN II FROM ANSMONIA SDLCATA Jacques BARHANIN , Michel HDGDES, Hugues SCHHEITZ Jean-Pierre VINCENT, Michel LAZDDNSRI Centre de Biochimie du Centre tlational de la Recherche Scientifique, Dniversité de Nice, Parc Valrose, 06034 NICE CEDEX, France . Several neurotoxins are ab~e to selectively alter the functioning of the action potential Na ionophore in excitable membranes . A series of neurotoxins has recently been isolated in pure form from sea anemones of different origins . The toxins are small polypeptides comprising less than 50 aminoacids . Chemical modifications of sea anemone toxin II from Anemonia sulcata have been used to study the residues involved in is tox c ac~onon crabs and mice and in its binding properties to the Na channel of rat brain synaptoeomes . Guanidination of the a -aminogroups of lysines 35, 36 and 46 with o-methylisourea hydrogen sulfate did not change the net charge of the toxin molecule and had no effect upon its toxic and binding properties . Sither acetylation or fluorescamine treatment of the toxin that destroyed the positive charges of the three e-amino-groups and of the a-amino function of Gly produced an almost complete loss of toxicity and a considerable decease in the binding activity . Iodination of the toxin on His induced practically no lôss of toxic or binding properties . Carb~~ thoxylation of both histidines 32 and 37 with diethylpyrocarbonate provoked an important decrease of both the toxicity and the binding activity . Modification of the guanidine side-chain of Arg with 1,2-cyclohexanedion~ fully destroyed both toxicity and bulging of the toxin to the Na channel . Modification of the carboxylate func tions of Asp7 , Asp and of the C-terminal Gln with glycine ethyl ester in the presence of a soluble carbodümig~ completely abolished the toxicity but left the affinity for the sea anemone toxin receptor unchanged . The antagonist character of this carboxylate-m~if~ed derivative was further confirmed by electrophysiological and Na flux experiments . Ionizable functions of AS can be placed in three different groups according to their func~~onal role, (i) The guanidine function of Arg 4 is the only single ionizable group the integrity of which is es~ential for both the neurotoxic and the binding properties of AS . (ii) Carboxylic functions of Asp , Asp and Gln play an importa~~ role for the expression of the to~ic properties g~ AS II but are not involved in the binding of the toxin to its specific receptor . (iii) The amino groups of Gly , Lys , Lys and Lys or imidazole functions of His and His ~re pr2~~ably i~~t importg~t individually for the toxic3~nd bindü~~ properties of AS . A striking result from these studies is that modification of ~~ie three carboxylic functions of AS does not impair its ability to associate to the specific sea ane~f~ne toxin receptor but induces a complete loss of its toxic properties . This result indicates that there must exist at least two different steps in the mode of action of AS II . I II ` AS II + R ~- (ASII - R) l ,r-..._ (AS IZ - R) 2 Step I represents the bimolecular association of AS to the specific sea anemone toxin receptor, R, which is an inte~~al part of the action potential Na channel . Step II is a rearrangement of the first (AS - R) complex that follows the binding step and that leads to ~l~e expression of neurotoxicity . [x] Poster

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