Acetylcholinesterase from snake venom as a model for its nerve and muscle counterpart.

Abstract

Acetylcholinesterase (AChE) plays a key role in cholinergic transmission. At the neuromuscular junction of vertebrates, for example, 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 is abundant in the venoms of Elapid snakes, except mambas. AChE purified from snake venom consists of soluble, hydrophilic monomers. Cloning of cDNA of the AChE from Bungarus fasciatus venom showed that its C-terminal peptide is very different from those of other AChEs. The partial sequence of the Bungarus fasciatus AChE gene shows that 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. AChEs from snake venoms vary in their sensitivity to peripheral site inhibitors, notably to fasciculins from mamba venoms. 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 intermediate IC50 of 10(-8) M. Analysis of its sequence reveals two major differences in the peripheral site region, 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). Modification of these residues by site-directed mutagenesis, and enzymatic analysis of modified recombinant enzymes, confirmed that these two residues are implicated in the properties of the Bungarus AChE peripheral site. The presence of an alternative exon, which generates 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 pre-exist, for expression in other contexts? Snake venom AChEs offer an exceptional system for analyzing the mechanism of peripheral site inhibition, because of their wide range of activities.