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 organization suggests high conservation of structure in mammalian AChE genes. The deduced amino acid sequence of the bovine T-subunit is highly similar 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 AChE, and (2) the presence of four rather than five potential N-glycosylation sites. The full coding sequence of the mature BoAChE T-subunit was expressed in human embryonal kidney 293 cells (HEK-293). The catalytic 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. Comparative SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that recombinant BoAChE comigrates with FBS-AChE. A major difference between the recombinant enzyme and the native enzyme was observed when clearance from circulation was examined. The HEK-293-derived enzyme was cleared from the circulation at a much faster rate than FBS-AChE. This difference in behaviour, together with previous studies on the effect of post-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 AChE circulatory residence.
Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel.
I
I
Mendelson
C
C
Kronman
N
N
Ariel
A
A
Shafferman
B
B
Velan
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D019281:Dimerization; D019143:Evolution, Molecular; D005312:Fetal Blood / Q000201:enzymology; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000110:Acetylcholinesterase