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

构建了α-球蛋白(AlphaGL)与胎盘碱性磷酸酶(PLAP)COOH末端前肽的融合蛋白(AlphaGL-PLAP)，并在COS-1细胞中瞬时表达。免疫荧光研究和磷脂酰肌醇特异性磷脂酶C的酶切表明，嵌合蛋白通过糖基磷脂酰肌醇(GPI)锚定在细胞表面。这些结果表明，PLAP的COOH末端前肽作为GPI锚定信号，使原本可溶的蛋白质成为GPI连接的膜结合蛋白。相反，用Trp定点突变Asp159的突变型嵌合蛋白(AlphaGL-PLAP)未能被GPI切割和修饰。免疫电子显微镜观察发现，突变型嵌合蛋白从未出现在细胞表面，而是保留在内质网和核膜中。抗Ag-…抗体的免疫沉淀此外，ER恢复信号KDEL表明，具有可切割或不可切割前肽的前嵌合蛋白，而不是GPI连接的成熟形式，与内质网驻留的分子伴侣Bip/GRP78有关。脉冲追逐实验表明，突变的嵌合蛋白在整个实验过程中一直与Bip/GRP78结合，并最终在内质网(或其亚室)中被降解。因此，Bip/GRP78保留和突变的嵌合蛋白在高尔基体前隔室的降解确保了只有野生型嵌合蛋白离开内质网。当嵌合蛋白在COS-1细胞中表达时，还发现由来自肝/骨/肾型碱性磷酸酶(BALP)的αGL和COOH末端30个氨基酸组成的嵌合蛋白锚定在细胞表面，表明该嵌合蛋白含有BALP的GPI的切割/附着部位，长度为COOH-末端30个氨基酸。我们目前正在通过一系列的定点突变实验来确定BALP的GPI的切割/附着位点。较少

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