Molecular pathological analysis of inborn error of metabolism with mineralization defects

低磷酸酶症是一种遗传性疾病，其特征是骨和牙齿的矿化缺陷和血清碱性磷酸酶活性降低。组织非特异性碱性磷酸酶基因（TNSALP）的各种突变是导致该疾病各种临床症状的原因。到目前为止，已经报道了178个突变，尽管对这种疾病的分子基础知之甚少。我们一直在研究几种错义突变对表达每种TNSALP突变体的哺乳动物细胞中TNSALP分子的生物合成和催化功能的影响。本研究的目的是阐明两种突变的影响：由cDNA 1559处的胸苷缺失（1559 delT）引起的移码以及TNSALP多肽（A99 T）第99位的丙氨酸被苏氨酸取代。1. 1559 delT：结合体外翻译系统，我们证实了1559 delT是作为一个较大的多肽合成的80个氨基酸残基在C-末端。由于这些额外的氨基酸，1559 delT不再是通过GPI（糖基磷脂酰肌醇）的膜结合酶，而是具有碱性磷酸酶活性的可溶性酶。然而，由于大多数新合成的1559 delT在ER中形成二硫键结合的高分子量聚集体，经历多泛素化并在蛋白酶体中降解，因此只有一小部分1559 delT被分泌到培养基中. A99 T：与野生型TNSALP一样，发现A99 T通过GPI在细胞表面上表达为80 kDa的成熟形式，尽管与野生型相反，但A99 T没有表现出酶活性。这表明99位的丙氨酸参与TNSALP的催化功能。重要的是，发现A99 T与野生型共表达时与野生型多肽形成异源二聚体，这可能是其主要传播模式的原因。

Hypophosphatase is a genetic disease characterized by defective mineralization of bone and tooth and reduction in serum alkaline phosphatase activity. Various mutations in the tissue-nonspecific alkaline phosphatase gene (TNSALP) are responsible for a wide variety of clinical symptoms of the disease. So far 178 mutations have been reported, though little is known about the molecular basis of this disease. We have been studying effects of several missense mutations on the biosynthesis and catalytic function of TNSALP molecule in mammalian cells expressing each TNSALP mutant. The aim of this research was conducted to elucidate the effect of two mutations : a frame shift caused by a thymidine deletion at 1559 of cDNA (1559delT) and replacement of alanine with threonine at position 99 of TNSALP polypeptide (A99T).1. 1559delT : In combination with an in vitro translational system, we confirmed that 1559delT is synthesized as a larger polypeptide with 80 amino acid residues at C-terminus. Due to this extra amino acids, 1559delT is no longer a membrane bound enzyme via GPI (glycosylphosphatidyl-inositol), but a soluble enzyme possessing alkaline phosphatase activity. However, since majority of newly synthesized 1559delT forms a disulfide-bonded high-molecular-mass aggregate in the ER, undergo polyubiquitination and is degraded in the proteasome, only a small portion of the 1559delT was secreted into the medium.2. A99T : Like wild-type TNSALP, A99T was found to expressed as a 80 kDa mature form on the cell surface via GPI, though in contrast to the wild type, though A99T exhibited no enzyme activity. This indicates that alanine at position 99 is involved in catalytic function of TNSALP. Importantly, A99T was found to form a heterodimer with the wild-type polypeptide when coexpressed with the wild type, presumably accounting for its dominant mode of transmission.