Residues in Torpedo californica acetylcholinesterase necessary for processing to a glycosyl phosphatidylinositol-anchored form.
Acetylcholinesterase from Torpedo californica (TcAChE) can be found as a glycosyl phosphatidylinositol (GPI)-anchored, membrane associated form. The C-terminal amino-acid sequence of the precursor protein resembles the signal peptide sequence found in proteins and enzymes destined for GPI-modification. Characteristics of such a signal peptide are a relatively polar stretch of amino acids, separating a cleavage- and modification-site (omega-site) residue from a hydrophobic C-terminus. We have introduced mutations, both at putative omega-sites and in the hydrophobic region, and analysed their effects on GPI-anchoring of TcAChE. Our results show that substitution of all three Ser residues in the region Ser542-Ser544 prevents GPI-modification and membrane anchoring. Individual substitution of each of these residues resulted in no or only a minor effect on the modification. We therefore conclude that more than one residue within this sequence can be utilised as the omega-site. Our analyses of double substitutions indicated that Ser543 and Ser544 are the preferred residues for GPI-modification. Moreover, the hydrophobic region is shown to be essential for GPI-anchoring of TcAChE.
Bucht
G
G
Department of NBC Defence, National Defence Research Establishment, Umeå, Sweden.
Hjalmarsson
K
K
D000110:Acetylcholinesterase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014101:Torpedo; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000110:Acetylcholinesterase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D003545:Cysteine