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Molecular pathological analysis of inborn error of metabolism with mineralization defects

矿化缺陷先天性代谢缺陷的分子病理学分析

基本信息

批准号：
16591854
负责人：
ODA Kimimitsu
金额：
$ 2.11万
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：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

Hypophosphatase is a genetic disease characterized by defective mineralization of bone and tooth and reduction in serum alkaline phosphatase activity. Various mutations in the tissue-nonspecific alkaline phosphatase gene (TNSALP) are responsible for a wide variety of clinical symptoms of the disease. So far 178 mutations have been reported, though little is known about the molecular basis of this disease. We have been studying effects of several missense mutations on the biosynthesis and catalytic function of TNSALP molecule in mammalian cells expressing each TNSALP mutant. The aim of this research was conducted to elucidate the effect of two mutations : a frame shift caused by a thymidine deletion at 1559 of cDNA (1559delT) and replacement of alanine with threonine at position 99 of TNSALP polypeptide (A99T).1. 1559delT : In combination with an in vitro translational system, we confirmed that 1559delT is synthesized as a larger polypeptide with 80 amino acid residues at C-terminus. Due to this extra amino acids, 1559delT is no longer a membrane bound enzyme via GPI (glycosylphosphatidyl-inositol), but a soluble enzyme possessing alkaline phosphatase activity. However, since majority of newly synthesized 1559delT forms a disulfide-bonded high-molecular-mass aggregate in the ER, undergo polyubiquitination and is degraded in the proteasome, only a small portion of the 1559delT was secreted into the medium.2. A99T : Like wild-type TNSALP, A99T was found to expressed as a 80 kDa mature form on the cell surface via GPI, though in contrast to the wild type, though A99T exhibited no enzyme activity. This indicates that alanine at position 99 is involved in catalytic function of TNSALP. Importantly, A99T was found to form a heterodimer with the wild-type polypeptide when coexpressed with the wild type, presumably accounting for its dominant mode of transmission.

下磷酸酶是一种遗传性疾病，其特征是骨和牙齿矿化缺陷和血清碱性磷酸酶活性降低。组织非特异性碱性磷酸酶基因(TNSALP)的各种突变导致了该病的各种临床症状。到目前为止，已经报告了178个突变，尽管人们对这种疾病的分子基础知之甚少。我们一直在研究几个错义突变对表达每个TNSALP突变体的哺乳动物细胞中TNSALP分子生物合成和催化功能的影响。本研究的目的是阐明两种突变的影响：cDNA1559位胸苷缺失(1559delT)引起的帧移位和TNSALP多肽(A99T)第99位的丙氨酸被苏氨酸取代。1559delT：结合体外翻译系统，我们证实了1559delT是一个C末端有80个氨基酸残基的大分子多肽。由于这些额外的氨基酸，1559delT不再是通过GPI(糖基磷脂酰肌醇)与膜结合的酶，而是具有碱性磷酸酶活性的可溶性酶。然而，由于大多数新合成的1559delT在内质网中形成二硫键结合的高分子聚集体，经历多泛素化，并在蛋白酶体中被降解，所以只有一小部分1559delT分泌到介质中。A99T：与野生型TNSALP一样，A99T通过GPI在细胞表面以80 kDa的成熟形式表达，但与野生型相反，尽管A99T没有显示酶活性。这表明99位丙氨酸参与了TNSALP的催化功能。重要的是，当A99T与野生型多肽共表达时，发现A99T与野生型多肽形成异源二聚体，这可能是其主要传播方式。