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

将α-<2u>球蛋白（alphaGL）与胎盘碱性磷酸酶（PLAP）羧基端前肽（COOH-terminal propeptide）按读码框融合，构建了一种嵌合蛋白（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的消化证明嵌合蛋白通过糖基化磷脂酰肌醇（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，并使原本可溶的蛋白质成为GPI连接的膜结合蛋白。与此相反，突变嵌合蛋白（alphaGL-PLAP），其中Asp 159被替换为色氨酸使用定点突变不能被GPI切割和修饰。免疫电镜观察显示，突变嵌合蛋白从来没有出现在细胞表面，并保留在内质网（ER）和核膜。抗体ag免疫沉淀 ...更多信息 ER检索信号KDEL显示，具有可裂解或不可裂解前肽但不具有GPI连接的成熟形式的前嵌合蛋白与Bip/GRP 78（ER驻留分子伴侣）相关。脉冲追踪实验表明，突变嵌合蛋白在整个实验过程中保持与Bip/GRP 78结合，并最终在ER（或其亚区室）中降解。因此，Bip/GRP 78的保留和突变嵌合蛋白在前高尔基体区室中的降解确保了只有野生型嵌合蛋白离开ER。当嵌合蛋白在COS-1细胞中表达时，还发现由源自肝/骨/肾型碱性磷酸酶（BALP）的α GL和COOH末端30个氨基酸组成的嵌合蛋白锚定在细胞表面上，表明嵌合蛋白在COOH末端30个氨基酸长度中含有BALP的GPI的切割/附着位点。我们目前正在确定的切割/连接位点的GPI的BALP通过一系列的定点突变实验。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