Molecular Cloning and Characterization of a cDNA Encoding AChE from Optic Lobe of Loligo Opalescences

Abstract

The optic lobe of Cephalopods shows the highest acetylcholine content at any neural tissue. Therefore, we intended to characterize acetylcholinesterase in such at organ from Loligo opalescens and then to perform cloning and sequencing of the relative cDNA from an expression library of the same species. The purified enzyme shows a high catalytic efficiency like that of vertebrate AChEs. It is present with two distinct forms, a G2 amphiphilic one (90% of total activity), likely anchored to the cell membrane through a phosphatidylinositol tail, and a G2 hydrophilic enzyme. The cDNA regarding the prevailing amphiphilic form has been cloned, sequenced and expressed in COS cells. It shows that the aminoacid residues of catalytic triad (Ser, His, Glu) are in conserved position, as well as the aromatic residues at the border of catalytic gorge. The aminoacid sequence deduced from cDNA shows a hydrophobic C-terminus. The enzyme obtained from expression in COS cells displays pharmacological and kinetic features quite similar to those of the enzyme extracted and purified from the fresh tissue (sensitivity to AChE-specific inhibitors, excess- substrate inhibition, membrane anchoring via phosphatidylinositol).