Optimising the signal peptide for glycosyl phosphatidylinositol modification of human acetylcholinesterase using mutational analysis and peptide-quantitative structure-activity relationships.
Glycosyl phosphatidylinositol (GPI)-modified proteins have a C-terminal signal peptide (GPIsp) that mediates the addition of a GPI-anchor to an amino acid residue at the cleavage and modification site (omega-site). Within the GPIsp, a stretch of hydrophilic amino acid residues are found which constitutes the spacer region that separates the omega-site residue from a hydrophobic C-terminus. Deletions and insertions into the spacer region of human acetylcholinesterase (AChE) show that the length of this spacer region is very important for efficient GPI-modification. Surprisingly, the natural length of the spacer region in human AChE was not optimal for the highest degree of GPI modification. The importance of the two adjacent residues downstream of the omega-site, the omega+1 and omega+2 residues, was investigated by peptide-quantitative structure-activity relationships (Peptide-QSAR). A model was made that predicts the efficiency of the GPI modification when these residues are substituted with others, and suggests important features for these residues. The most preferred omega+1 and omega+2 residues, predicted by the model, in combination with an ideal spacer length resulted in an optimised GPIsp. This mutant protein is more efficiently GPI-modified than any mutant AChE tested thus far.
Bucht
G
G
Department of Microbiology, National Defence Research Establishment, Cementvägen 20, S-901 82, Umeå, Sweden. bucht@ume.foa.se
Wikström
P
P
Hjalmarsson
K
K
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006801:Humans; D009154:Mutation; D010449:Peptide Mapping; D021382:Protein Sorting Signals / Q000235:genetics*; D013329:Structure-Activity Relationship; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D021382:Protein Sorting Signals; D000110:Acetylcholinesterase