Drosophila acetylcholinesterase: effect of post-translational [correction of post-traductional] modifications on the production in the baculovirus system and substrate metabolization.
Acetylcholinesterase cDNAs from Drosophila melanogaster modified on its primary sequence were cloned into baculovirus and were expressed in Sf9 cells with the aim to identify a mutant form that produces the enzyme at a high level. Directed mutagenesis was used in order to independently knockout different sites of post-translational modifications: exchange of the C-terminal hydrophobic peptide for a glycolipid molecule, dimerization by disulfide bridge, N-linked glycosylation at the five accessible sites, and subunit formation by proteolytic cleavage of a hydrophilic peptide found in the precursor. Another mutation involved the elimination of a free cysteine in the mature protein. All mutations involving post-translational modifications resulted in lower recoveries, suggesting that they are useful for maintaining high amounts of protein in the synapse. By contrast, elimination of a free cysteine in the mature protein permitted an increase in the level of production of the enzyme. These mutations did not affect specific activity of the enzyme at substrate concentrations ranging from 3 microM to 200 mM, suggesting that activation and inhibition of the enzyme activity does not originate from a polymorphism in post-translational modifications.
Estrada-Mondaca
S
S
Laboratoire d'Entomologie Appliquée, Université Paul Sabatier, Cedex, Toulouse, France.
Lougarre
A
A
Fournier
D
D
D000109:Acetylcholine / Q000032:analysis; D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009154:Mutation / Q000502:physiology*; D011499:Protein Processing, Post-Translational
D018076:DNA, Complementary; D000110:Acetylcholinesterase; D003545:Cysteine; D000109:Acetylcholine