Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: A new member of the cholinesterase family resistant to diisopropyl fluorophosphate

Acetylcholinesterase cDNA was cloned by screening a library from Loligo opa lescens optic robes; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20-41% amino acid ident ity with the acetylcholinesterases studied so far. The characteristic struc ture of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfde bonds) was conserved in t he protein. The heterologous expression of acetylcholinesterase in COS cell s gave a recovery of acetylcholinesterase activity 20-fold higher than in c ontrols. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholin esterase expressed in vivo, which displays a high catalytic efficiency. Bot h enzymes are true acetylcholinesterase corresponding to phosphatidylinosit ol-anchored G(2)(a) dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some par ticular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F (288) in Torpedo] in the acyl pocket of the active site could justify the h igh substrate specificity of the enzyme, the absence of hydrolysis with but yrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.