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首页> 外文期刊>The FEBS journal >Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406
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Molecular basis of perinatal hypophosphatasia with tissue-nonspecific alkaline phosphatase bearing a conservative replacement of valine by alanine at position 406

机译：围产期低磷性伴组织非特异性碱性磷酸酶的分子基础，在406位的位置由丙氨酸保守替代缬氨酸

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摘要

Hypophosphatasia, a congenital metabolic disease related to the tissue-nonspecific alkaline phosphatase gene (TNSALP), is characterized by reduced serum alkaline phosphatase levels and defective mineralization of hard tissues. A replacement of valine with alanine at position 406, located in the crown domain of TNSALP, was reported in a perinatal form of hypophosphatasia. To understand the molecular defect of the TNSALP (V406A) molecule, we examined this missense mutant protein in transiently transfected COS-1 cells and in stable CHO-K1 Tet-On cells. Compared with the wild-type enzyme, the mutant protein showed a markedly reduced alkaline phosphatase activity. This was not the result of defective transport and resultant degradation of TNSALP (V406A) in the endoplasmic reticulum, as the majority of newly synthesized TNSALP (V406A) was conveyed to the Golgi apparatus and incorporated into a cold detergent insoluble fraction (raft) at a rate similar to that of the wild-type TNSALP. TNSALP (V406A) consisted of a dimer, as judged by sucrose gradient centrifugation, suggestive of its proper folding and correct assembly, although this mutant showed increased susceptibility to digestion by trypsin or proteinase K. When purified as a glycosylphosphatidylinositol-anchorless soluble form, the mutant protein exhibited a remarkably lower K-cat/K-m value compared with that of the wild-type TNSALP. Interestingly, leucine and isoleucine, but not phenylalanine, were able to substitute for valine, pointing to the indispensable role of residues with a longer aliphatic side chain at position 406 of TNSALP. Taken together, this particular mutation highlights the structural importance of the crown domain with respect to the catalytic function of TNSALP.
机译：低磷血症是与组织非特异性碱性磷酸酶基因（TNSALP）相关的先天性代谢疾病，其特征在于血清碱性磷酸酶水平降低和硬组织矿化不良。据报道，围产期发育不全的低磷血症是在TNSALP冠状结构域的406位上的丙氨酸替代缬氨酸。为了了解TNSALP（V406A）分子的分子缺陷，我们在瞬时转染的COS-1细胞和稳定的CHO-K1 Tet-On细胞中检查了这种错义突变蛋白。与野生型酶相比，该突变蛋白显示出明显降低的碱性磷酸酶活性。这不是运输缺陷和内质网中TNSALP（V406A）降解的结果，因为大多数新合成的TNSALP（V406A）被输送到高尔基体中，并在50℃下被引入冷洗涤剂不溶物级分中。速率与野生型TNSALP相似。 TNSALP（V406A）由蔗糖梯度离心法判断为二聚体，提示其适当折叠和正确组装，尽管该突变体显示出对胰蛋白酶或蛋白酶K消化的敏感性增加。与野生型TNSALP相比，突变蛋白的K-cat / Km值低得多。有趣的是，亮氨酸和异亮氨酸而不是苯丙氨酸能够代替缬氨酸，这表明在TNSALP 406位具有较长脂肪族侧链的残基起着不可或缺的作用。总体而言，这种特殊的突变突出了冠状结构域在TNSALP催化功能方面的结构重要性。

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