Watanabe Hisashi, Goseki-Sone Masae, Orimo Hideo, Hamatani Ryoko, Takinami Hiroyuki, Ishikawa Isao
Department of Hard Tissue Engineering, Graduate School, Tokyo Medical and Dental University, Japan.
J Bone Miner Res. 2002 Nov;17(11):1945-8. doi: 10.1359/jbmr.2002.17.11.1945.
Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue-nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In our previous study, we found the novel point mutations (G1144A and T979C) from the genomic TNSALP gene of a patient with childhood HOPS. In this study, we have characterized the protein translated from the mutant G1144A gene. Wild-type and G1144A mutant-type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS-1 cells by lipofectin technique. After 48-h or 72-h transfection, cells were collected and homogenized using polytron homogenizer. After centrifugation at 10,000 g for 10 minutes, the supernatant was assayed. ALP activity was determined with 10 mM of p-nitrophenylphosphate as a substrate in 100 mM of 2-amino-2-methyl-1,3-propanediol-HCl buffer containing 5 mM of MgCl2. ALP activity of cells transfected with the mutant cDNA (G1144A) plasmid after 48-h or 72-h transfection exhibited 0.063 +/- 0.012 U/mg and 0.054 +/- 0.012 U/mg, respectively. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 2.7% and 1.7%, respectively. These values were not significantly different from those found with mock-transfected cells, that is, 2.5% and 1.5%, respectively. This study indicated that the mutation (G1144A) produced the inactive ALP enzyme and would be a disease-causing mutation of the childhood-type HOPS.
低磷酸酯酶症（HOPS）是一种临床异质性遗传性疾病，其特征为骨骼矿化缺陷、组织非特异性碱性磷酸酶（TNSALP）活性缺乏以及乳牙过早脱落。TNSALP基因位于1号染色体p34 - 36.1，由12个外显子和11个内含子组成。在我们之前的研究中，我们从一名儿童HOPS患者的基因组TNSALP基因中发现了新的点突变（G1144A和T979C）。在本研究中，我们对由突变G1144A基因翻译的蛋白质进行了表征。构建了野生型和G1144A突变型TNSALP cDNA表达载体pcDNA3，并通过脂质体转染技术将其转染至COS - 1细胞。转染48小时或72小时后，收集细胞并用聚能器匀浆器匀浆。以10000g离心10分钟后，测定上清液。以10 mM对硝基苯磷酸酯为底物，在含有5 mM MgCl2的100 mM 2 - 氨基 - 2 - 甲基 - 1,3 - 丙二醇 - HCl缓冲液中测定碱性磷酸酶（ALP）活性。转染突变cDNA（G1144A）质粒48小时或72小时后的细胞ALP活性分别为0.063±0.012 U/mg和0.054±0.012 U/mg。以野生型的酶活性为100%，突变型的值分别估计为2.7%和1.7%。这些值与空载体转染细胞的值（分别为2.5%和1.5%）无显著差异。本研究表明，突变（G1144A）产生了无活性的ALP酶，可能是儿童型HOPS的致病突变。
