Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C201-Y or C489-S substitution associated with severe hypophosphatasia
Disulfide bonds are critical for tissue-nonspecific alkaline phosphatase function revealed by analysis of mutant proteins bearing a C201-Y or C489-S substitution associated with severe hypophosphatasia
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DOI：
10.1016/j.bbadis.2012.01.007
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发表时间：
2012-04-01
影响因子：
6.2
通讯作者：
Oda, Kimimitsu
中科院分区：
文献类型：
--
作者：
Satou, Yasuhito;Al-Shawafi, Hiba A.;Oda, Kimimitsu
低磷症(HPP)是一种罕见的遗传性疾病，以血清碱性磷酸酶(ALP)活性降低和骨和牙齿矿化失败为特征，由组织非特异性ALP(TNSALP)基因突变引起。在被诊断为围产期HPP的患者中，报告了两个TNSALP错义突变(C201Y和C489S，标准化命名法)，涉及链内二硫键(Taillandier A.等人)。哼。穆塔特。13(1999)171-172，嗯。穆塔特。15(2000)293)。为了研究二硫键在TNSALP中的作用，我们在COS-1细胞中瞬时表达了TNSALP(C201Y)和TNSALP(C489S)。与野生型酶[TNSALP(W)]相比，两个突变体的细胞内ALP活性均降低，其中以66 kDa的未成熟形式为主，边际含量为80 kDa的成熟形式。对Tet-on CHO建立的表达TNSALP(W)或TNSALP(C201Y)的细胞系的详细研究表明，66 kDa形式的TNSALP(C201Y)以单体形式存在，而不是TNSALP(W)的二聚体。只有一小部分TNSALP(C201Y)以80 kDa成熟形式到达细胞表面，尽管66 kDa形式中的大部分被发现是内切-β-N-乙酰氨基葡萄糖苷酶H敏感的，并在多泛素化后在蛋白酶体中迅速降解。综上所述，这些结果不仅表明亚基内的二硫键对于TNSALP正确折叠和组装成二聚体酶是至关重要的，而且与TNSALP(C201Y)或TNSALP(C489S)相关的HPP的发展归因于功能酶细胞表面出现的减少。(C)2012爱思唯尔B.V.保留所有权利。
Hypophosphatasia (HPP), a rare genetic disease characterized by reduced serum alkaline phosphatase (ALP) activity and failure in bone and tooth mineralization, is caused by mutations in tissue-nonspecific ALP (TNSALP) gene. Two missense mutations (C201Y and C489S, standardized nomenclature) of TNSALP, involved in intra-chain disulfide bonds, were reported in patients diagnosed with perinatal HPP (Taillandier A. et al. Hum. Mutat. 13 (1999) 171-172, Hum. Mutat. 15 (2000) 293). To investigate the role of the disulfide bond in TNSALP, we expressed TNSALP (C201Y) and TNSALP (C489S) in COS-1 cells transiently. Compared with the wild-type enzyme [TNSALP (W)], both the TNSALP mutants exhibited a diminished ALP activity in the cells, where a 66 kDa immature form was predominant with a marginal amount of a 80 kDa mature form of TNSALP. Detailed studies on Tet-On CHO established cell line expressing TNSALP (W) or TNSALP (C201Y) showed that the 66 kDa form of TNSALP (C201Y) exists as a monomer in contrast to a dimer of TNSALP (W). Only a small fraction of the TNSALP (C201Y) reached cell surface as the 80 kDa mature form, though most of the 66 kDa form was found to be endo-beta-N-acetylglucosaminidase H sensitive and rapidly degraded in proteasome following polyubiquitination. Collectively, these results indicate not only that the intra-subunit disulfide bonds are crucial for TNSALP to properly fold and assemble into the dimeric enzyme, but also that the development of HPP associated with TNSALP (C201Y) or TNSALP (C489S) is attributed to decreased cell surface appearance of the functional enzyme. (C) 2012 Elsevier B.V. All rights reserved.