Cousin_1997_C.R.Seances.Soc.Biol.Fil_191_381

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. For example, located at the neuro-muscular junction of vertebrates, it allows a fine temporal control of muscle contraction. The presence of AChE in tissues devoid of cholinergic function is also well known and raises the question of its role. In particular, AChE occurs at high level in the venoms of Elapid snakes, except Mambas. In contrast, the venom of snakes belonging to Viperid or Colubrid families does not contain any AChE. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning the cDNA of the venom AChE from Bungarus fasciatus showed that its C-terminal peptide is very different from those of other AChEs. This peptide is encoded by a new alternative exon, called S for Soluble and Snake. It is a short very basic peptide of 15 residues. Analysis of the venom enzyme and in vitro expression experiments showed that the last eight residues are removed in the mature protein. This cleavage is not necessary for enzymatic activity and occurs before secretion of the enzyme. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to Mambas toxins, fasciculins. While Ophiophagus AChE is as sensitive as Torpedo enzyme (IC50 around 10(-10) M), Naja and Heamacatus AChEs are insensitive to the toxin up to a concentration of 10(-6) M. Bungarus AChE has an intermediary IC50 of 10(-8) M. The analysis of its sequence shows two major differences, in the peripheral site region, when compared to Torpedo or mammalian AChEs: at position 70 it contains a methionine instead of a tyrosine and at position 285, it contains a lysine instead of an acidic residue (glutamic or aspartic acid). The modification of these residues by site-directed mutagenesis and the enzymatic analysis of modified recombinant enzymes confirmed the implication of these two residues in the properties of Bungarus AChE peripheral site. The presence of an alternative exon, used for generating a soluble form of AChE in venoms, raises interesting evolutionary questions: does it exist in snakes whose venom does not contain AChE, e.g. Mambas? did this exon preexist, for expression in other contexts? In addition snake venoms offer an exceptional system for analysing the mechanism of peripheral site inhibition, because of its wide range of sensitivity.