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

肝/骨/肾型碱性磷酸酶的表达及骨代谢

• 批准号: 06404065
• 负责人: ODA Kimimitsu
• 金额: $ 11.14万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (A)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06404065/
• 关键词: alkaline phosphatase; glycosylphosphatidylinositol; bone; placenta; chaperone; Bip; GRP78; membrane protein; ER-retrieval signal; 骨アルカリホスファターゼ; GPIアンカー; 膜結合型酵素; キメラ蛋白質; トランスフェクション

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

构建了由α _<2u>-球蛋白（alphaGL）与胎盘碱性磷酸酶（PLAP）衍生的cooh末端前肽在框架内融合而成的嵌合蛋白（alphaGL-PLAP），并在COS-1细胞中短暂表达。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，使其他可溶性蛋白成为gpi连接的膜结合蛋白。相比之下，使用位点定向诱变将Asp159替换为Trp的突变体嵌合蛋白（alphaGL-PLAP）无法被GPI切割和修饰。免疫电镜观察显示，突变嵌合蛋白未出现在细胞表面，保留在内质网和核膜中。针对内质网检索信号KDEL的免疫沉淀表明，具有可切割或不可切割前肽的原嵌合蛋白与内质网分子伴侣Bip/GRP78相关，而不是gpi连接的成熟形式。脉冲追踪实验表明，突变嵌合蛋白在整个实验过程中仍与Bip/GRP78相关，并最终在内质网（或其亚室）中降解。因此，Bip/GRP78的保留和突变嵌合蛋白在前高尔基区室的降解确保只有野生型嵌合蛋白离开内质网。来源于肝/骨/肾型碱性磷酸酶（BALP）的由α gl和cooh末端30个氨基酸组成的嵌合蛋白在COS-1细胞中表达时也被发现锚定在细胞表面，表明该嵌合蛋白在cooh末端30个氨基酸的长度上含有BALP GPI的切割/附着位点。我们目前正在通过一系列定点诱变实验确定BALP中GPI的切割/附着位点。少

项目成果

Nishimura,Y.et al.: "Intracellular sorting of lysosomal β-glucuronidase is altered due to administration of dibutylphosphate" J.Biochem.118. 46-55 (1995)

Nishimura, Y. 等人：“溶酶体 β-葡萄糖醛酸酶的细胞内分选因磷酸二丁酯的施用而改变”J.Biochem.118 (1995)。

Oda, K., Wada, I., Takami, N., et al.: "Bip/GRP78 but not calnexin associates with a precursor of glycosylphosphatidylinositol-anchored protein" Biochem.J.(in press). (1996)

Oda, K.、Wada, I.、Takami, N. 等人：“Bip/GRP78 但不是钙联蛋白与糖基磷脂酰肌醇锚定蛋白的前体结合”Biochem.J.（出版中）。

K.Oda: "Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor of alkaline phophatase" Biochem.J.301. 577-583 (1994)

K.Oda：“通过与碱性磷酸酶的糖基磷脂酰肌醇锚融合，将分泌蛋白转化为膜蛋白”Biochem.J.301。

Oda,K.et al.: "Bip/GRP78 but not calnexin associates with a precursor of glycosyl-phosphatidylinositol anchored protein" Biochem.J.(in press). (1996)

Oda,K.等人：“Bip/GRP78 但不是钙联蛋白与糖基磷脂酰肌醇锚定蛋白的前体相关”Biochem.J.（正在出版）。

Oda, K., Cheng J., Saku, T., et al.: "Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase" Biochem.J.301. 577-583 (1994)

Oda, K.、Cheng J.、Saku, T. 等人：“通过与碱性磷酸酶的糖基磷脂酰肌醇锚信号融合将分泌蛋白转化为膜蛋白”Biochem.J.301。