Analysis of mutated alkaline phosphateses involved in calcification defect of hard tissyes
| Project/Area Number |
18592027
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional basic dentistry
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| Research Institution | Niigata University |
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Principal Investigator |
ODA Kimimitsu Niigata University, Institute of Medicine and Dentistry, Professor (10122681)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| 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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| Keywords | Calcification / Alkaline phosphatase / Hypophosphatasia / Inborn-error of bone metabolism / Molecular basis of diseases / genetic diseases / 病気発症メカニズム |
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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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Report
(3 results)
Research Products
(49 results)
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[Journal Article] Involvement of the Klotho protein in dentin formation and mineralization2008
Author(s)
Suzuki, H, Amizuka, N, Oda, K, Noda, M, Ohshima, H, Maeda, T
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Journal Title
Anat. Rec 291
Pages: 183-190
Description
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[Journal Article] Histological evaluation on bone regeneration of dental implant placement sites grafted with a self-setting alpha-tricalcium phosphate cement2008
Author(s)
Nakadate, M, Amizuka, N, Li, M, Freitas, PH, Oda, K, et. al.
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Journal Title
Micros. Res. Tech 71
Pages: 93-104
Description
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[Journal Article] Histological and elemental analyses of impaired bone mineralization in klotho-deficient mice2008
Author(s)
Suzuki, H, Amizuka, N, Oda, K, Noda, M, Ohshima, H, Maeda, T
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Journal Title
J. Anat 212
Pages: 275-285
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[Journal Article] Development of an ELISA method for detecting immune complexes between tissue-nonspecific alkaline phosphatase and immunoglobulinG2007
Author(s)
Hocchi, K, Ohashi, T, Miura, T, Sasagawa, K, Oda, K, et. al.
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Journal Title
J. Clin. LAB Anal 21
Pages: 322-329
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[Journal Article] Histological assessments on the abnormalitiesof mouse epiphyseal chondrocytes with short term centrifugal loading2007
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de, Freitas, PH, Kojima, T, Ubaidus, S, Li, M, Oda, K, et. al.
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[Journal Article] A histological assessment on the distribution of the osteocytoc lacnar canalicular systems using silver staining2007
Author(s)
Hirose, S, Li, M, Kojima, T, de, Freitas, PH, Oda, K, et. al.
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Journal Title
J. Bone Miner. Metab 25
Pages: 374-382
Description
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[Journal Article] Generation of a Monoclonal Antibody Specific for Tissue-Nons-pecific Alkaline Phosphatase and Its Use in a Clinical Diagnostic Study2007
Author(s)
Ohashi, T, Sunaga, M, Miura, T, Sasagawa, K, Sato, Y, Oda, K., et. al.
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Journal Title
Hybridoma 26
Pages: 401-406
Description
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[Journal Article] Aberrant interchain disulfide bridge of tissue-nonspecific alkaline phosphatas with an Arg433->Cys substitution associated with severe hypophosphatasia2006
Author(s)
Nasu M, Ito, M, Ishida, Y, Numa, N, Komaru, K, Oda, K
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Journal Title
FEBS J 273
Pages: 5612-5624
Description
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[Journal Article] High dose glucocorticoid hampers bone formation and resorption after bone marrow ablation in rat2006
Author(s)
Kondo, N, Tokunaga, K, Arai, K, Amizuka, N, Oda, K, et. al.
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Journal Title
Microsc. Res.Tech 69
Pages: 839-846
Description
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[Presentation] Intermittent PTH stimulates cells of the osteopoietic lineage even in the absence of osteoclasts2007
Author(s)
Freitas, PHL, Li, M, Ninomiya, T , Nakamura, M, Oda, K, et. al.
Organizer
ASBMR 29th Annual Meeting
Place of Presentation
in Hawaii, USA
Year and Date
2007-09-19
Description
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[Presentation] Effects of functional single nucleotide polymorphism in the tissue-nonspecific alkaline phosphatase gene(787T>C)associated with BMD2007
Author(s)
Goseki-Sone, M, Sogabe, N, Oda, K, Nakamura, H, Orimo, H, et. al.
Organizer
ASBMR 29th Annual Meeting
Place of Presentation
in Hawaii, USA
Year and Date
2007-09-18
Description
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