Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol.
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.
Takami
N
N
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Oda
K
K
Ikehara
Y
Y
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
D004792:Enzyme Precursors; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D010716:Phosphatidylinositols; D019788:Fluorodeoxyglucose F18; D003847:Deoxyglucose; D000469:Alkaline Phosphatase; D010738:Type C Phospholipases