Expression of Liver/Bone/Kidnet-type alkaline phosphatase and metabolism of bone

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A chimeric protein (alphaGL-PLAP) consisting of alpha_<2u>-globulin (alphaGL)fused in frame to a COOH-terminal propeptide derived from placental alkaline phosphatase (PLAP) was constructed and expressed transiently in the COS-1 cell. Immunofluorescence study and digestion with phosphatidylinositol-specific phospholipase Cdemonstrated that the chimeric protein is anchored on thecell surface via a glycosylphosphatidylinositol (GPI). These results indicate that the COOH-terminal propeptide of PLAP serves as a GPI-anchor signal and render an otherwise soluble protein a GPI-linked membrane-bound protein. In contrast, a mutant chimeric protein (alphaGL-PLAP) in which Asp159 was replaced with Trp using a site-directed mutagenesis failed to be cleaved and modified by GPI.Immunoelectron microscopic observation revealed that the mutant chimeric protein never appeared on the cell surface and was retained in the endoplasmic reticulum (ER) and nuclear envelope . Immunoprecipitation with antibody ag … More ainst an ER-retrieval signal KDEL showed that prochimeric protein with either a cleavable or an uncleavable propeptide, but not a GPI-linked mature form, was associated with Bip/GRP78, an ER resident molecular chaperone. Pulse-chase experiment showed that the mutant chimeric protein remained associated with Bip/GRP78 throughout the experiment and eventually was degraded in the ER (or its subcompartment). Thus retention by Bip/GRP78 and degradation of the mutant chimeric protein in the pre-Golgi compartment ensure that only the wild type chimericprotein leave the ER.A chimeric protein consisting of alphaGL and COOH-terminal 30 amino acids derived from liver/bone/kidney type alkaline phosphatase (BALP) was also found to be anchored onthe cell surface when the chimeric protein was expressed in the COS-1 cell, indicating that the chimeric protein contains a cleavage/attachment site of GPI of BALP in a COOH-terminal 30 amino acids length. We are currently identifying the cleavage/attachment site of GPI of BALP by means of a series of site-directed mutagenesis experiments. Less