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Volume 271, Number 25, Issue of June 21, 1996 pp. 15099-15108
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Cloning and Expression of Acetylcholinesterase from Bungarus fasciatus Venom
A NEW TYPE OF COOH-TERMINAL DOMAIN; INVOLVEMENT OF A POSITIVELY CHARGED RESIDUE IN THE PERIPHERAL SITE

(Received for publication, December 18, 1995, and in revised form, March 29, 1996)

Xavier Cousin ^§ , Suzanne Bon ^§ , Nathalie Duval , Jean Massoulié ^§ and Cassian Bon

From the  Unité des Venins, Institut Pasteur, 28 rue du Dr Roux, 75015 Paris, France and ^§ Laboratoire de Neurobiologie, CNRS URA 1857, 46 rue d'Ulm, 75005 Paris, France

As deduced from cDNA clones, the catalytic domain of Bungarus fasciatus venom acetylcholinesterase (AChE) is highly homologous to those of other AChEs. It is, however, associated with a short hydrophilic carboxyl-terminal region, containing no cysteine, that bears no resemblance to the alternative COOH-terminal peptides of the GPI-anchored molecules (H) or of other homomeric or heteromeric tailed molecules (T). Expression of complete and truncated AChE in COS cells showed that active hydrophilic monomers are produced and secreted in all cases, and that cleavage of a very basic 8-residue carboxyl-terminal fragment occurs upon secretion. The COS cells produced Bungarus AChE about 30 times more efficiently than an equivalent secreted monomeric rat AChE. The recombinant Bungarus AChE, like the natural venom enzyme, showed a distinctive ladder pattern in nondenaturing electrophoresis, probably reflecting a variation in the number of sialic acids.

By mutagenesis, we showed that two differences (methionine instead of tyrosine at position 70; lysine instead of aspartate or glutamate at position 285) explain the low sensitivity of Bungarus AChE to peripheral site inhibitors, compared to the Torpedo or mammalian AChEs. These results illustrate the importance of both the aromatic and the charged residues, and the fact that peripheral site ligands (propidium, gallamine, D-tubocurarine, and fasciculin 2) interact with diverse subsets of residues.

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