Analysis of mutated alkaline phosphatases associated with hypophosphatasia

低磷酸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病。为了确定突变TNSALP分子的分子缺陷，我们分别用野生型和突变TNSALP cDNA转染COS-1细胞，研究细胞中TNSALP分子的瞬时表达。在本研究中，我们重点研究了两种与严重形式的低磷血症相关的突变tnsalp。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分子进行寡糖加工并作为活性酶到达细胞表面，而其余分子形成二硫键结合的高分子质量聚集体，在到达高尔基体之前沿着分泌途径被阻滞。Gly3 - 17-Asp取代的TNSALP在COS-I细胞中表达时，该突变分子的细胞表面外观被完全阻断，突变蛋白在细胞内形成二硫键结合的高分子量聚集体，可能是在内质网或前高尔基区室中。最终发现突变蛋白被降解了。由于TNSALP（Ala162-Thr）和TNSALP（Gly3 17-Asp）都被[3H]乙醇胺标记，因此突变蛋白被糖基磷脂酰肌醇修饰，TNSALP分子通过糖基磷脂酰肌醇锚定在细胞膜上。综上所述，在严重形式的低磷酸症患者中发现的错义突变极有可能导致蛋白质的三维结构变化，导致细胞内形成聚集体。因此，具有碱性磷酸酶活性的功能性TNSALP分子数量大大减少或完全不存在于细胞表面。

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.