| Budget Amount *help |
¥3,790,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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.
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