cDNA Cloning, In Vitro Expression, and Biochemical Characterization of Cholinesterase 1 and Cholinesterase 2 from Amphioxus

Abstract

We have isolated cDNA’s coding for the complete amino acid sequences of Cholinesterase 1 (ChEl) and Cholinesterase 2 (ChE2) from amphioxus. Both ChE transcripts appear to code for H catalytic subunits, which may become inserted in the membrane via an ethanolamine-glycan-phosphatidylinositol anchor. For ChE2, the acyl pocket, a region of the enzyme important for substrate binding, resembles those of invertebrate ChE’s. A novel acyl-binding pocket characterizes ChEl. In vitro expression in COS-7 cells demonstrates that ChE2 is eserine-sensitive and hydrolyzes acetylthiocholine. In contrast, ChEl is eserine-resistant and hydrolyzes both acetylthiocholine and butyrylthiocholine. Velocity sedimentation and nondenaturing gel electrophoresis in conjunction with digestion by phosphatidylinositol-specific phospholipase C suggest that ChEl and ChE2 are present as ethanolamine-glycan-phosphatidylinositol-anchored G₂ forms in vivo and in vitro. Supported by NSF-RUI grant IBN-9319354 to L.P.