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 sig nal 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 consti tutes the spacer region that separates the omega-site residue from a hydrop hobic C-terminus. Deletions and insertions into the spacer region of human acetylcholinesterase (AChE) show that the length of this spacer region is v ery important for efficient GPI-modification. Surprisingly, the natural len gth of the spacer region in human AChE was not optimal for the highest degr ee of GPI modification. The importance of the two adjacent residues downstr eam of the w-site, the omega+1 and omega+2 residues, was investigated by pe ptide-quantitative structure-activity relationships (Peptide-QSAR). A model was made that predicts the efficiency of the GPI modification when these r esidues are substituted with others, and suggests important features for th ese residues. The most preferred omega+1 and omega+2 residues, predicted by the model, in combination with an ideal spacer length resulted in an optim ised GPIsp. This mutant protein is more efficiently GPI-modified than any m utant AChE tested thus far. (C) 1999 Elsevier Science B.V. All rights reser ved.