Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia.
Defective intracellular transport of tissue-nonspecific alkaline phosphatase with an Ala162-->Thr mutation associated with lethal hypophosphatasia.
复制标题
组织非特异性碱性磷酸酶的细胞内运输缺陷，具有与致死性低磷酸酯酶相关的 Ala162-->Thr 突变。
DOI：
--
复制
发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Kimimitsu Oda
中科院分区：
文献类型：
--
作者：
Hisanobu Shibata;Mariko Fukushi;Atsuko Igarashi;Yoshio Misumi;Yukio Ikehara;Yasushi Ohashi;Kimimitsu Oda
我们研究了瞬时表达的组织非特异性碱性磷酸酶（TNSALP）在COS-1细胞中的生物合成和胞内转运。将突变引入TNSALP中以检查单个氨基酸取代对TNSALP的活性和生物合成的影响。表达野生型TNSALP的细胞表现出比未转染的细胞高200倍以上的碱性磷酸酶活性。脉冲追踪实验表明，TNSALP合成为66 kDa的内切氨基葡萄糖苷酶H（Endo H）敏感的形式，并转化为具有异质性分子量（约80 kDa）的EndoH抗性形式，最终出现在细胞表面上，通过用磷脂酰肌醇特异性磷脂酶C（PI-PLC）消化判断。相比之下，具有Glu 218->Gly突变的TNSALP根本不表现出磷酸酶活性，并且66-kDa Endo H-敏感形式是转染细胞中整个追踪过程中唯一的分子种类。根据这一发现，用PI-PLC消化和免疫荧光观察证实该突变体从未在细胞表面上表达。另一种突变体具有Ala 162-->Thr取代，其天然地与致死性低磷酸酶症相关，表现出低活性，并且只有一小部分66-kDa形式获得Endo-H抗性并到达细胞表面。由于野生型和突变体TNSALPs标记的[3 H]乙醇胺，糖基磷脂酰肌醇（GPI）的一个组成部分，这是不太可能的，受损的细胞内转运的两个突变体是由于失败的修饰GPI。有趣的是，发现TNSALP突变体的66-kDa Endo H-敏感形式而不是野生型的形式在细胞内形成链间二硫键合的高分子量聚集体。这些结果表明，TNSALP（Ala 162->Thr）分子聚集引起的细胞内转运受损是携带该突变的致死性低磷酸酶症的分子基础。
We have studied the biosynthesis and intracellular transport of tissue-nonspecific alkaline phosphatase (TNSALP) transiently expressed in COS-1 cells. Mutations were introduced into TNSALP to examine the effects of a single amino acid substitution on the activity and biosynthesis of TNSALP. The cells expressing wild-type TNSALP exhibited more than 200-fold higher alkaline phosphatase activity than untransfected ones. Pulse-chase experiments showed that TNSALP was synthesized as a 66-kDa endoglucosaminidase H (Endo H)-sensitive form and converted to EndoH-resistant forms with heterogenous molecular masses ( approximately 80 kDa), which finally appeared on the cell surface as judged by digestion with phosphatidylinositol-specific phospholipase C (PI-PLC). In contrast, a TNSALP with a Glu218-->Gly mutation exhibited no phosphatase activity at all and the 66-kDa Endo H-sensitive form was the only molecular species throughout the chase in the transfected cells. In accordance with this finding, digestion with PI-PLC and immunofluorescence observation confirmed that this mutant was never expressed on the cell surface. Another mutant with a Ala162-->Thr substitution, which naturally occurs in association with a lethal hypophosphatasia, exhibited a low activity and only a small fraction of the 66-kDa form acquired Endo-H resistance and reached the cell surface. Since the wild-type and the mutant TNSALPs were labeled with [3H]ethanolamine, a component of glycosylphosphatidylinositol (GPI), it is unlikely that the impaired intracellular transport of the two mutants is due to a failure in their modification by GPI. Interestingly, the 66-kDa Endo H-sensitive form of the TNSALP mutants but not that of the wild-type, was found to form an interchain disulfide-bonded high-molecular-mass aggregate within the cells. These results suggest that impaired intracellular transport of the TNSALP (Ala162-->Thr) molecule caused by its aggregation is the molecular basis for the lethal hypophosphatasia carrying this mutation.