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Analysis of mutated alkaline phosphatases associated with hypophosphatasia

与低磷酸酯酶症相关的突变碱性磷酸酶分析

基本信息

批准号：
09671890
负责人：
ODA Kimimitsu
金额：
$ 1.98万
依托单位：
Niigata University School of Dentistry
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

Hypophosphatasia is a congenital genetic disorder caused by mutations of tissue-nonspecific alkaline phosphatase (TNSALP) gene. In order to define the molecular defect of mutated TNSALP molecules, COS-1 cells were transfected with either wild-type or mutated TNSALP cDNA and transiently expressed TNSALP molecules in the cells were studied. We focused two mutated TNSALPs associated with severe form of hypophosphatasia in the present study.1. The TNSALP with an Ala162-Thr substitutionWhen synthesized in GOS-1 cells, only a fraction of newly synthesized TNSALP molecules underwent oligosaccharide processing and reached the cell surface as an active enzyme, while the remaining molecules were found to form a disulfide-bonded high molecular mass aggregate and to be arrested along the secretory pathway before it reached the Golgi apparatus.2.The TNSALP with a Gly3 17-Asp substitutionWhen expressed in COS-I cells, the cell surface appearance of this mutated molecule was totally blocked and the mutant protein formed a disulfide-bonded high molecular mass aggregate within the cell, presumably in the endoplasmic reticulum or a pre-Golgi compartment. Eventually the mutant protein was found to be degraded. The degradation of mutant protein was inhibited by lactacystin, a specific inhibitor of proteasome, Since both the TNSALP (Ala162-Thr) and the TNSALP(Gly3 17-Asp) were labeled with [3H]ethanolamine, the mutant proteins were modified with a glycosylphosphatidylinositol, through which TNSALP molecule is anchored to the cell membrane.Taken together, it is highly likely that missense mutataions found in patients with severe form of hypophosphatasia bring about a three-dimensional structural change of the protein, leading to the formation of an aggregate within the cells. As a consequense functional TNSALP molecules with alkaline phosphatase activity are greately decreased in number or totally absent from the cell surface.

低磷酸酯酶症是一种由组织非特异性碱性磷酸酶（TNSALP）基因突变引起的先天性遗传性疾病。为了确定突变的TNSALP分子的分子缺陷，用野生型或突变的TNSALP cDNA转染COS-1细胞，并研究细胞中瞬时表达的TNSALP分子。在本研究中，我们重点关注与严重低磷酸酯酶症相关的两种突变 TNSALP。1．具有 Ala162-Thr 取代的 TNSALP 在 GOS-1 细胞中合成时，只有一小部分新合成的 TNSALP 分子经过寡糖加工并作为活性酶到达细胞表面，而发现其余分子形成二硫键高分子量聚集体，并在到达细胞之前沿着分泌途径被捕获。高尔基体。2.Gly3 17-Asp 取代的 TNSALP 当在 COS-I 细胞中表达时，该突变分子的细胞表面外观被完全封闭，突变蛋白在细胞内形成二硫键键合的高分子质量聚集体，可能在内质网或前高尔基体区室中。最终发现突变蛋白被降解。突变蛋白的降解受到蛋白酶体特异性抑制剂乳胞素的抑制，由于TNSALP（Ala162-Thr）和TNSALP（Gly3 17-Asp）均被[3H]乙醇胺标记，因此突变蛋白被糖基磷脂酰肌醇修饰，通过糖基磷脂酰肌醇将TNSALP分子锚定在总而言之，在患有严重低磷酸酯酶症的患者中发现的错义突变很可能会导致蛋白质的三维结构变化，从而导致细胞内形成聚集体。结果，具有碱性磷酸酶活性的功能性 TNSALP 分子的数量大大减少或在细胞表面完全消失。