Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization.
Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.
Ikezawa
H
H
Faculty of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan.
Taguchi
R
R
Tamura
H
H
Kobayashi
T
T
D015720:5'-Nucleotidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010727:Phosphoric Diester Hydrolases / Q000502:physiology
D017261:Glycosylphosphatidylinositols; D015720:5'-Nucleotidase; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase