| Project/Area Number |
14571759
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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, Graduate School of Medical and Dental Sciences, Professor, 大学院・医歯学総合研究科, 教授 (10122681)
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| Co-Investigator(Kenkyū-buntansha) |
ITO Masahiro NIIGATA UNIVERSITY, Graduate School of Medical and Dental Sciences, Assistant, 大学院・医歯学総合研究科, 助手 (40313522)
AMAYA Yoshihiro NIIGATA UNIVERSITY, Graduate School of Medical and Dental Sciences, Associate Professor, 大学院・医歯学総合研究科, 助教授 (50193032)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2003: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2002: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Keywords | Hypophosphatasia / Alkaline phosphatase / Defect of Calcification / Calcium / polyubiquitination / proteasome / inborn error of bone metabolism / 遺伝子疾患 / 石灰化 / 先天性代謝異常 / ユビキチン / 分解 |
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| Research Abstract |
Two missense mutations(N153D,D289V) of tissue-nonspecific alkaline phosphatase(TNSALP) gene associated with perinatal hypophosphatasia were analyzed. First off, specific mutations were introduced into the wild-type TNSALP cDNA using site-directed mutation and resultant plasmids were transfected into COS-1 cells. The cells expressing each TNSALP mutant was examined biochemically and morphologically.
1.Contrast to dimeric structure of the wild type, each TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate, indicative of defective folding and incorrect assembly. Resultantly, each TNSALP mutant exhibited no measurable alkaline phosphatase acitivity.
2.Both TNSALP mutants failed to reach the cell surface. Instead, TNSALP(D289V) was found to be localized mainly to the endoplasmic reticulum, while TNSALP(N153D) was in the cis-Golgi.
3.Both TNSALP mutants were degraded in the proteasome at different rates, presumably reflecting difference in their intracellular localization.
4.TNSALP(D289V) was found to be polyubiqutinated prior to degradation in the proteasome.
5.Aspartic acid at position 289 of TNSALP is assumed to be involved in the binding of calcium ion. So we analyzed another TNSALP mutant(E218G), which is also involved in coordination of calcium ion TNSALP mutant(E218G) quite resembled TNSALP(D289V) regarding to their molecular properties. These results raise the possibility that calcium binding is critical for acquisition of three dimensional structure of TNSALP.
Taken together, sever forms of hypophosphatasia caused by two missense mutations(N153D,D289V) of TNSALP can be classified as folding disease.
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