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

1.利用瞬时表达的COS-1细胞和编码组织非特异性碱性磷酸酶(TNSALP)突变蛋白的条件稳定细胞系(Tet-on CHO-K1细胞)，检测了与严重低磷相关的两个组织非特异性碱性磷酸酶(TNSALP)突变蛋白的分子缺陷。与野生型酶不同，带有Arg433-Cys取代的TNSALP突变体是通过二硫键进行交联的。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，其中两个亚基相互非共价连接。TNSALP(R433C)定位于细胞表面的功能部位，但其活性明显降低，这可能是由于亚基间二硫键的形成导致了TNSALP结构的扭曲。TNSALP的另一个错义突变，即在TNSALP的406位用丙氨酸取代了Valine，并没有影响TNSALP(V406A)向细胞表面的迁移。然而，与野生型相比，该突变蛋白的酶活性明显降低。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，亮氨酸和异亮氨酸很好地替代了406位的缬氨酸，而苯丙氨酸则不能很好地替代，这表明不仅疏水性，而且侧链的长度对TNSALP2的催化功能也是至关重要的。研制了一种新的抗人TNSALP的单抗。该单抗对肝源性酶比骨源性酶更具特异性，提示其在肝病中的临床应用。此外，利用该抗体建立了一种新的检测血清中免疫球蛋白-TNSALP复合体的酶免疫分析体系。

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.