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Analysis of mutated alkaline phosphateses involved in calcification defect of hard tissyes

参与硬蒂西斯钙化缺陷的突变碱性磷酸酶分析

基本信息

批准号：
18592027
负责人：
ODA Kimimitsu
金额：
$ 2.43万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

1. Molecular defect of two tissue-nonspecific alkaline phosphatase (TNSALP) mutant proteins associated with severe hypophosphatasia was examined by using transiently expressed COS-1 cells and conditional stable cell line (Tet-On CHO-K1 cells) harboring each plasmid encoding the cDNA of a TNSALP mutant. In contrast to the wild-type enzyme, a TNSALP mutant with an Arg433-Cys substitution was found to be cross-linked via disulfide bond. The formation of disulfide bond occurred in the endoplasmic reticulum, however, its intracellular transport rate was comparable to the wild type, in which two subunits associated with each other noncovalently. TNSALP (R433C) was localized to the cell surface, the site of function, though it showed a much reduced activity probably due to distortion of the structure of TNSALP resulting from the inter-subunit disulfide formation. Another missense mutation of TNSALP, replacement of valine with alanine at position of 406 of TNSALP, did not affect the migration of TNSALP (V406A) to the cell surface. However, this mutant protein showed a markedly decreased enzyme activity compared to the wild type. This was further confirmed by the kinetic study of a purified GPI-anchor-less enzyme. The catalytic efficiency of TNSALP (V406A) was only one tenth of the wild-enzyme. Mutagenesis experiments showed that leucine and isoleuicine, but not phenylalanine well substituted for the valine at 406, suggesting that not only hydrophobicity, but also the length of the side chain is crucial for the catalytic function of TNSALP.2. A new monoclonal antibody was raised against human TNSALP. This monoclonal antibody is more specific to liver-derived enzyme rather than bone-derived one, suggestive of its clinical application for liver diseases. In addition, new enzyme immunoassay (ELISA) system was developed using this antibody for the detection of IgG-TNSALP complex in serum.

1. 通过使用含有编码 TNSALP 突变体 cDNA 的每种质粒的瞬时表达 COS-1 细胞和条件稳定细胞系（Tet-On CHO-K1 细胞），检查了与严重低磷酸酯酶症相关的两种组织非特异性碱性磷酸酶 (TNSALP) 突变蛋白的分子缺陷。与野生型酶相反，发现具有 Arg433-Cys 取代的 TNSALP 突变体通过二硫键交联。二硫键的形成发生在内质网中，然而，其细胞内转运速率与野生型相当，其中两个亚基彼此非共价结合。 TNSALP (R433C) 定位于细胞表面，即功能位点，但其活性大大降低，这可能是由于亚基间二硫键形成导致 TNSALP 结构的扭曲。 TNSALP 的另一个错义突变，即在 TNSALP 的 406 位上用丙氨酸替换缬氨酸，并不影响 TNSALP (V406A) 向细胞表面的迁移。然而，与野生型相比，这种突变蛋白的酶活性显着降低。纯化的 GPI 无锚定酶的动力学研究进一步证实了这一点。 TNSALP(V406A)的催化效率仅为野生酶的十分之一。诱变实验表明亮氨酸和异亮氨酸，但苯丙氨酸不能很好地取代406位的缬氨酸，表明疏水性和侧链长度对于TNSALP的催化功能都至关重要。2。针对人 TNSALP 产生了一种新的单克隆抗体。这种单克隆抗体对肝源性酶比骨源性酶更具特异性，这表明其在肝脏疾病方面的临床应用。此外，使用该抗体开发了新的酶联免疫分析（ELISA）系统，用于检测血清中的IgG-TNSALP复合物。