Analysis of molecular mechanism of hypophosphatasia

低磷酸酯酶症的分子机制分析

• 批准号: 14571759
• 负责人: ODA Kimimitsu
• 金额: $ 2.5万
• 依托单位: NIIGATA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571759/
• 关键词: 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

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.

分析与围产期低磷血症相关的组织非特异性碱性磷酸酶(TNSALP)基因2个错义突变(N153D、D289V)。首先，用定点突变的方法将野生型TNSALP基因导入野生型TnSALP基因中，并将其导入COS-1细胞。1.与野生型的二聚体结构相比，每个TNSALP突变体都形成了一个二硫键结合的高分子聚集体，这表明存在折叠缺陷和组装错误。结果，每一株TNSALP突变体均未表现出明显的碱性磷酸酶活性。2.两株突变体均未能到达细胞表面。相反，TNSALP(D289V)主要定位于内质网，而TNSALP(N153D)主要定位于顺式结构。3.两种突变体在蛋白酶体中的降解速度不同，可能反映了它们在细胞内定位的差异。4.TNSALP(D289V)在蛋白酶体降解之前被发现是多泛化的。5.推测TNSALP第289位天冬氨酸参与了钙离子的结合。因此，我们分析了另一个与钙离子配位有关的TNSALP突变体(E218G)，该突变体与TNSALP(D289V)的分子性质非常相似。这些结果提出了钙结合对获得TNSALP三维结构至关重要的可能性。因此，由TNSALP的两个错义突变(N153D和D289V)引起的多种形式的低磷血症可归类为折叠病。

项目成果

Kawai M., et al.: "Ectopic bone formation by human bone morphologenic protein-2 gene transfer to skeletal muscle using trancutaneous electroporation"Human Gene Therapy. 14-16. 1547-1556 (2003)

Kawai M.等人：“使用经皮电穿孔将人骨形态发生蛋白2基因转移到骨骼肌来形成异位骨”人类基因疗法。

Ishida Y., et al.: "Tissue-nonspecific alkaline phosphatase with an Asp289-Val mutation fails to reach the cell membrane and undergoes proteasome-mediated degradation"Journal of Biochmistry. 134-1. 65-70 (2003)

Ishida Y. 等人：“具有 Asp289-Val 突变的组织非特异性碱性磷酸酶无法到达细胞膜并经历蛋白酶体介导的降解”《生物化学杂志》。

Ito, M.et al.: "Jaw bone remodeling at the invention front of gingival squamous cell carcinomas"J. Oral Path. Med.. 32. 10-17 (2003)

Ito, M.等人：“牙龈鳞状细胞癌发明前沿的颌骨重塑”J.

Ito M., et al.: "Retention at the cis-Golgi and delayed degradation of tissue-non-specific alkaline phosphatase with an Asn153-Asp substitution, a cause of perinatal hypophosphatasia"Biochemical Journal. 361-3. 473-480 (2002)

Ito M. 等人：“Asn153-Asp 取代保留在顺式高尔基体并延迟组织非特异性碱性磷酸酶的降解，这是围产期低磷酸酯酶症的原因”《生化杂志》。

Ito, M., et al.: "Jaw bone remodeling at the invasion front of gingival squamous cell carcinoma"Journal of Oral Pathology and Medicine. 32・1. 10-17 (2003)

Ito, M., et al.：“牙龈鳞状细胞癌侵袭前部的颌骨重塑”《口腔病理学与医学杂志》32·1（2003）。