title: Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol.
abstract: Alkaline phosphatase is anchored to the membrane via glycosylphosphatidylinositol (GPI). Mannose residues of the GPI glycan are suggested to be derived from dolichol-P-mannose. In the present study we examined the effect of 2-fluoro-2-deoxy-D-glucose (F-Glc), an inhibitor of dolichol-P-mannose synthesis, on the biosynthesis and processing of alkaline phosphatase in JEG-3 cells. In control cells, a proform precursor (64.5 kDa) with a hydrophobic peptide domain at the COOH terminus was immediately processed into an intermediate form (63 kDa) by proteolytic removal of the COOH-terminal extension and replacement with the GPI anchor, and then to a mature form (66 kDa) by terminal glycosylation of its N-linked oligosaccharides. In contrast, when cells were treated with F-Glc (1 mM), the protein was synthesized as a proform of 61 kDa. The reduction in its molecular mass was mostly due to the inhibition in maturation of N-linked oligosaccharides by F-Glc. The 61-kDa proform identified by antibodies to the COOH-terminal peptide was detectable even at 3 h after the synthesis, and was gradually processed to doublet forms of 58-59 kDa which were finally secreted into the medium. None of these forms were labeled with [3H]ethanolamine and [3H]stearic acid, components of the GPI anchor, and expressed on the cell surface as a membrane-bound form. Taken together, these results suggest that the inhibition of the GPI synthesis causes a prolonged accumulation of the proform, which is then gradually processed into secretory forms by proteolytic removal of the COOH-terminal hydrophobic peptide.
authors.affiliation: Department of Biochemistry, Fukuoka University School of Medicine, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Takami
authors.forename: K
authors.initials: K
authors.lastname: Oda
authors.forename: Y
authors.initials: Y
authors.lastname: Ikehara
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D003847:Deoxyglucose / Q000031:analogs & derivatives; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D019788:Fluorodeoxyglucose F18; D006017:Glycolipids / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols; D006801:Humans; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D019788:Fluorodeoxyglucose F18; D003847:Deoxyglucose; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases