Drosophila acetylcholinesterase. Expression of a functional precursor in Xenopus oocytes.
In insects, acetylcholinesterase is mainly found in the central nervous system. It is expressed in the synapse where it hydrolyzes the neurotransmitter acetylcholine. Maturation of this protein involves several post-translational modifications. The precursor polypeptide is cut at three sites; the N-terminal signal peptide is removed, the C-terminal hydrophobic polypeptide is clipped off and replaced by a glycolipid anchor and the resulting peptide is cut into two polypeptides, corresponding to active subunits. Two of these active subunits are associated to form the final active glycosylated protein. We have expressed the protein via microinjection of an expression vector into Xenopus oocyte nuclei. When the complete cDNA is injected, the acetylcholinesterase formed is biochemically similar to the Drosophila-head acetylcholinesterase. However, the hydrophobic C-terminal peptide is not replaced by a glycolipid anchor. As a consequence, the enzyme is no longer externalized, the proteolytic cutting of the main peptide does not occur and a new polymerization form occurs. Although incompletely processed, this protein is enzymatically active. When a cDNA lacking the coding region of the C-terminal hydrophobic peptide is injected, the resulting acetylcholinesterase is hydrophilic, cleaved into two subunits and secreted into the incubation medium free of contaminants.
Fournier
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Institut National de la Recherche Agronomique, Antibes, France.
Mutero
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Rungger
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D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004330:Drosophila / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011233:Precipitin Tests; D014981:Xenopus
D004792:Enzyme Precursors; D004247:DNA; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase