Mutational analysis of the C-terminal signal peptide of bovine liver 5'-nucleotidase for GPI anchoring: a study on the significance of the hydrophilic spacer region.
Bovine liver 5'-nucleotidase is a GPI-anchored protein whose Ser523 attaches to GPI as the omega-site. For GPI-modification, pro-protein of the enzyme possesses a signal peptide at the C-terminus, comprising a hydrophilic spacer sequence of 8 amino acid residues and the following hydrophobic region of 17 amino acid residues. The C-terminal signal peptide is replaced by GPI on a luminal leaflet of endoplasmic reticulum. To characterize the C-terminal signal peptide for GPI modification, we constructed a series of deletion and elongation mutant genes, altering length of the hydrophilic spacer sequence by site-directed mutagenesis. Systematic deletion and Ala insertion of the sequence showed that the sequence of 6-14 residues were compatible for GPI modification. For GPI transfer to the pro-protein, the optimum length of spacer sequence would be 8, being consistent with natural selection. The spacer sequence may play a role for leading the omega-residue correctly to the active site of putative GPI transamidase. The elongation of the spacer is more permissible than deletion. Nevertheless, the length of the spacer sequence may influence efficiency of GPI modification by its positive or negative control.
Furukawa
Y
Y
Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Tsukamoto
K
K
Ikezawa
H
H
D015720:5'-Nucleotidase / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004252:DNA Mutational Analysis; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*
D017261:Glycosylphosphatidylinositols; D011498:Protein Precursors; D021382:Protein Sorting Signals; D015720:5'-Nucleotidase