Post-translational modifications of Drosophila acetylcholinesterase. In vitro mutagenesis and expression in Xenopus oocytes.
Drosophila acetylcholinesterase (EC 3.1.1.7) is a 150-kDa glycoprotein anchored in plasmic membranes via a glycolipid. It is composed of two active subunits which are themselves made of two noncovalently linked polypeptides of 18 and 55 kDa resulting from the proteolysis of a single precursor of 75 kDa. Active Drosophila acetylcholinesterase can be expressed in Xenopus oocytes as an excreted protein. We have identified some of the amino acids essential in post-translational modifications of the protein by site-directed mutagenesis and expression of mutants in this system. The intersubunit disulfide bond involves cysteine at position 615. Cleavage of the 75-kDa precursor, as observed in Drosophila, originates from a hydrophilic peptide (in position 148 to 180) which does not exist in cholinesterase sequences from vertebrates. This cleavage is associated with excretion out of the cell. Drosophila acetylcholinesterase exhibits four effective sites of asparagine-linked glycosylation in positions 126, 174, 331, and 531. We show that glycosylations and dimerization protect the protein against proteolytic digestion. In contrast, none of these post-translational modifications significantly affects the activity of acetylcholinesterase or affinity for its substrate.
Mutero
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Institut National de la Recherche Agronomique, Antibes, France.
Fournier
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D000110:Acetylcholinesterase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000502:physiology*; D010449:Peptide Mapping; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014981:Xenopus
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000110:Acetylcholinesterase; D010450:Endopeptidases