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

1.用瞬时表达的COS-1细胞和条件稳定细胞系（Tet-On CHO-K1细胞）检测了与严重低磷酸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。与野生型酶相比，发现具有Arg 433-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，但其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（R433 C）定位于细胞表面，即功能位点，尽管其显示出大大降低的活性，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V406 A不影响TNSALP向细胞表面的迁移。然而，该突变蛋白质显示出与野生型相比显著降低的酶活性。这进一步证实了纯化的GPI锚无酶的动力学研究。TNSALP（V406 A）的催化效率仅为野生型酶的十分之一。突变实验表明，亮氨酸和异亮氨酸，而不是苯丙氨酸很好地取代了406位的缬氨酸，这表明不仅疏水性，而且侧链的长度对TNSALP的催化功能至关重要.本文报道了一株新的抗人TNSALP单克隆抗体。该单克隆抗体对肝源性酶的特异性高于骨源性酶，提示其在肝脏疾病的临床应用。此外，利用该抗体建立了新的酶免疫分析（ELISA）系统，用于检测血清中IgG-TNSALP复合物。

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.