MUTATIONAL ANALYSIS OF THE C-TERMINAL SIGNAL PEPTIDE OF BOVINE LIVER 5'-NUCLEOTIDASE FOR GPI ANCHORING - A STUDY ON THE SIGNIFICANCE OF THEHYDROPHILIC SPACER REGION

Bovine liver 5'-nucleotidase is a GPI-anchored protein whose Ser(523) attaches to GPI as the omega-site. For GPI-modification, pro-protein o f the enzyme possesses a signal peptide at the C-terminus, comprising a hydrophilic spacer sequence of 8 amino acid residues and the followi ng hydrophobic region of 17 amino acid residues. The C-terminal signal peptide is replaced by GPI on a luminal leaflet of endoplasmic reticu lum. To characterize the C-terminal signal peptide for GPI modificatio n, we constructed a series of deletion and elongation mutant genes, al tering length of the hydrophilic spacer sequence by site-directed muta genesis. Systematic deletion and Ala insertion of the sequence showed that the sequence of 6-14 residues were compatible for GPI modificatio n. For GPI transfer to the pro-protein, the optimum length of spacer s equence would be 8, being consistent with natural selection. The space r sequence may play a role for leading the omega-residue correctly to the active site of putative GPI transamidase. The elongation of the sp acer is more permissible than deletion. Nevertheless, the length of th e spacer sequence may influence efficiency of GPI modification by its positive or negative control. (C) 1997 Elsevier Science B.V.