Aberrant processing of alkaline phosphatase precursor caused by blocking the synthesis of glycosylphosphatidylinositol. |
| |
Authors: | N Takami K Oda Y Ikehara |
| |
Institution: | Department of Biochemistry, Fukuoka University School of Medicine, Japan. |
| |
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. |
| |
Keywords: | |
|
|