Molecular characterization of tissue-nonspecific alkaline phosphatase with an Ala to Thr substitution at position 116 associated with dominantly inherited hypophosphatasia
Molecular characterization of tissue-nonspecific alkaline phosphatase with an Ala to Thr substitution at position 116 associated with dominantly inherited hypophosphatasia
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DOI：
10.1016/j.bbadis.2010.12.002
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发表时间：
2011-03-01
影响因子：
6.2
通讯作者：
Oda, Kimimitsu
中科院分区：
文献类型：
--
作者：
Ishida, Yoko;Komaru, Keiichi;Oda, Kimimitsu
组织非特异性碱性磷酸酶(TNSALP)基因突变是导致低磷症的原因，低磷症是一种与血清碱性磷酸酶活性降低相关的先天性骨和牙齿代谢错误。根据标准化命名法，在显性遗传性低磷酸盐血症患者中报道了TNSALP基因的错义突变(C.346G和GT；A)，该基因在第116位将丙氨酸转化为苏氨酸(A.S.lia-Baldini等人)。哼，吉内特。109(2001)99-108)。为了研究TNSALP(A116T)的分子表型，我们在COS-1细胞和Tet-on CHO K1细胞中进行了表达。TnSALP(A116T)与野生型酶共表达时，其碱性磷酸酶活性几乎可以忽略不计，但显性负效应较弱。与TNSALP(W，野生型)不同的是，TNSALP(A116T)主要以非共价组装的同源二聚体形式存在，而TNSALP(A116T)则以单体和通过二硫键连接的异质缔合聚集体的形式存在。有趣的是，TnSALP(A116T)的单体和聚集态都能进入细胞表面，并通过糖基磷脂酰肌醇(GPI)固定在细胞膜上。分泌形式的TNSALP(W)和TNSALP(A116T)的共表达导致由TNSALP(W)-His和TNSALP(A116T)-FLAG组成的异构体复合体的释放。综上所述，这些发现有力地表明，TNSALP(A116T)未能正确折叠并形成二硫键结合的聚集体，尽管它确实能够与野生型相互作用并到达细胞表面，从而解释了其优势传递。(C)2010爱思唯尔B.V保留所有权利。
Mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene are responsible for hypophosphatasia, an inborn error of bone and teeth metabolism associated with reduced levels of serum alkaline phosphatase activity. A missense mutation (c.346G>A) of TNSALP gene, which converts Ala to Thr at position 116 (according to standardized nomenclature), was reported in dominantly transmitted hypophosphatasia patients (A.S. Lia-Baldini et al. Hum Genet. 109 (2001) 99-108). To investigate molecular phenotype of TNSALP (A116T), we expressed it in the COS-1 cells or Tet-On CHO K1 cells. TNSALP (A116T) displayed not only negligible alkaline phosphatase activity, but also a weak dominant negative effect when co-expressed with the wild-type enzyme. In contrast to TNSALP (W, wild-type), which was present mostly as a non-covalently assembled homodimeric form, TNSALP (A116T) was found to exist as a monomer and heterogeneously associated aggregates covalently linked via disulfide bonds. Interestingly, both the monomer and aggregate forms of TNSALP (A116T) gained access to the cell surface and were anchored to the cell membrane via glycosylphosphatidylinositol (GPI). Co-expression of secretory forms of TNSALP (W) and TNSALP (A116T), which are engineered to replace the C-terminal GPI anchor with a tag sequence (his-tag or flag-tag), resulted in the release of heteromeric complexes consisting of TNSALP (W)-his and TNSALP (A116T)-flag. Taken together, these findings strongly suggest that TNSALP (A116T) fails to fold properly and forms disulfide-bonded aggregates, though it is indeed capable of interacting with the wild-type and reaching the cell surface, therefore explaining its dominant transmission. (C) 2010 Elsevier B.V All rights reserved.