Biosynthesis of Torpedo acetylcholinesterase in mammalian cells. Functional expression and mutagenesis of the glycophospholipid-anchored form.
The catalytic subunits of asymmetric and hydrophobic forms of acetylcholinesterase arise from a single gene by alternative mRNA splicing. Each protein is encoded in three exons, with exons 1 and 2 encoding sequence common to both forms and exons 3A and 3H specifying unique carboxyl-terminal domains. We examined the expression of cDNAs for the two forms by transient transfection in COS-1 cells. The catalytic subunit of the asymmetric form expressed by transfected cells exhibits low activity and is retained within the cell. The cDNA encoding hydrophobic acetylcholinesterase directs the synthesis of enzyme with much greater activity, which is expressed on the outer surface of the cell membrane and can be released by phosphatidylinositol-specific phospholipase C. A mutant truncated acetylcholinesterase which lacks either carboxyl-terminal sequence encoded by the alternative exons is secreted into the medium. An exon 1-3H fusion mutant, created by deletion of coding exon 2 from the hydrophobic form cDNA, is glycophospholipid-linked. The 30-amino acid carboxyl-terminal domain specified by exon 3H appears necessary and sufficient to direct glycophospholipid attachment. Thus, heterologous expression of wild-type and mutant acetylcholinesterase proteins indicates that the carboxyl-terminal domains specified by alternative coding exons determine the cellular dispositions of acetylcholinesterase.
Gibney
G
G
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Taylor
P
P
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D005455:Fluorescent Antibody Technique; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D012995:Solubility; D013696:Temperature; D014101:Torpedo; D014162:Transfection
D009841:Oligonucleotides; D012333:RNA, Messenger; D004247:DNA; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase