Cloning and Expression of a Rat Acetylcholinesterase Subunit: Generation of Multiple Molecular Forms and Complementarity with a Torpedo Collagenic Subunit

Abstract: We obtained a cDNA clone encoding one type of catalytic subunit of acetylcholinesterase (AChE) from rat brain (T subunit). The coding sequence shows a high frequency of (G + C) at the third position of the codons (66%), as already noted for several AChEs, in contrast with mammalian butyrylcholinesterase. The predicted primary sequence of rat AChE presents only 11 amino acid differences, including one in the signal peptide, from that of the mouse T subunit. In particular, four alanines in the mouse sequence are replaced by serine or threonine. In northern blots, a rat AChE probe indicates the presence of major 3.2-and 2.4-kb mRNAs, expressed in the CNS as well as in some peripheral tissues, including muscle and spleen. In vivo, we found that the proportions of G₁, G₂, and G₄ forms are highly variable in different brain areas. We did not observe any glycolipid-anchored G₂ form, which would be derived from an H subunit. We expressed the cloned rat AChE in COS cells: The transfected cells produce principally an amphiphilic G₁^a form, together with amphiphilic G₂^a and G₄^a forms, and a nonamphiphilic G₄^na form. The amphiphilic G₁^a and G₂^a forms correspond to type II forms, which are predominant in muscle and brain of higher vertebrates. The cells also release G₄^na, G₂^a, and G₁^a in the culture medium. These experiments show that all the forms observed in the CNS in vivo may be obtained from the T subunit. By cotransfecting COS cells with the rat T subunit and the Torpedo collagenic subunit, we obtained chimeric collagentailed forms. This cross-species complementarity demonstrates that the interaction domains of the catalytic and structural subunits are highly conserved during evolution.