Author/Editor		Prijatelj, Petra
Title		Mutacijska analiza amoditoksina A, presinaptično nevrotoksične fosfolipaze A2, iz strupa modrasa (Vipera ammodytes ammodytes)
Translated title		Mutational analysis of ammoditoksin A, a presynaptically neurotoxic phospholipase A2, from the venom of the long-nosed viper (Vipera ammodytes ammodytes)
Type		monografija
Place		Ljubljana
Publisher		Medicinska fakulteta
Publication year		2003
Volume		str. 92
Language		slo
Abstract		Ammodytoxins (Atx) are presynaptically neurotoxic phospholipases A2 (PLA2) from the venom of the long-nosed viper (Vipera ammodytes ammodytes), whose molecular mechanism of neurotoxicity is not completely understood. To elucidate which parts on the molecule are important for neurotoxicity and for interaction with specific binding proteins, we prepared an array of different mutants and chimeric proteins. First, we transformed basic ammodytoxin A (AtxA) into an acidic protein by substituting certain basic residues in the Cterminal region, which is involved in presynaptic neurotoxicity. The six-site mutant of AtxA (K108N/K111N/K127T/K128E/E129T/K132E), in which five out of seven C-terminal basic amino acid residues were substituted with neutral or acidic ones, was approximately 30times less lethal than AtxA, but still neurotoxic. In accordance with this, the binding affinities for neuronal binding proteins were lowered only by a factor of five. Additionally, a single mutant of AtxA with the substitution at only one site of the six-site mutant (K127T) has been prepared. Its toxicity shows that most, if not all, of the six substituted sites partially contribute to lower lethality of the multiple mutant. We also prepared two chimeric PLA2s with substitution of the C-terminal end of a nontoxic PLA2 from the venom, ammodytin I2 (AtnI2), with the corresponding part of neurotoxic AtxA(K108N). The chimeric mutants were not toxic, but were able to bind, in contrast to AtnI2, strongly, to neuronal binding protein R25, specific for Atx. The binding affinity on neuronal M-type PLA2-receptor was largely unchanged. The results showed that the C-terminal region of AtxA is critical for interaction with R25, but also other parts of the molecule are important for neurotoxicity. (Abstract truncated at 2000 characters).
Descriptors		VIPER VENOMS PHOSPHOLIPASES A PROTEIN BINDING CHIMERIC PROTEINS RECOMBINATION, GENETIC NEUROTOXINS CALMODULIN VIPERIDAE