BOVINE ACETYLCHOLINESTERASE - CLONING, EXPRESSION AND CHARACTERIZATION

The bovine acetylcholinesterase (BoAChE) gene was cloned from genomic DNA and its structure was determined. Five exons coding for the AChE T -subunit and the alternative H-subunit were identified and their organ ization suggests high conservation of structure in mammalian AChE gene s. The deduced amino acid sequence of the bovine T-subunit is highly s imilar to the human sequence, showing differences at 34 positions only . However, the cloned BoAChE sequence differs from the published amino acid sequence of AChE isolated from fetal bovine serum (FBS) by: (1) 13 amino acids, 12 of which are conserved between BoAChE and human ACh E, and (2) the presence of four rather than five potential N-glycosyla tion sites. The full coding sequence of the mature BoAChE T-subunit wa s expressed in human embryonal kidney 293 cells (HEK-293). The catalyt ic properties of recombinant BoAChE and its reactivity towards various inhibitors were similar to those of the native bovine enzyme. Soluble recombinant BoAChE is composed of monomers, dimers and tetramers, yet in contrast to FBS-AChE, tetramer formation is not efficient. Compara tive SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that re combinant BoAChE comigrates with FBS-AChE, A major difference between the recombinant enzyme and the native enzyme was observed when clearan ce from circulation was examined. The HEK-293-derived enzyme was clear ed from the circulation at a much faster rate than FBS-AChE. This diff erence in behaviour, together with previous studies on the effect of p ost-translation modification on human AChE clearance [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959 -967] suggests that cell-dependent glycosylation plays a key role in A ChE circulatory residence.