Molecular basis of neonatal-onset multiple carboxylase deficiency

新生儿发病的多种羧化酶缺乏症的分子基础

• 批准号: 05454282
• 负责人: NARISAWA Kuniaki
• 金额: $ 4.03万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454282/
• 关键词: multiple carboxylase deficiency; biotine; holocarboxylase synthetase; HAS; holocarboxylase synthetase deficiency; HCScDNA; mutation; genetic diagnosis; ビオチン依存症; PCR-SSCP; HCScDNAの発現; 新生児マルチプルカルボキシラーゼ欠損症; cDNA; クローニング; ノーザンブロット

Neonatal-onset multiple carboxylase deficiency is known to be caused by deficiency of holocarboxylase synthetase (HCS). HCS plays an essential role in biotin utilization in cells. We purified HCS from bovine liver cytosol and found that it consists of a single subunit with a molecular mass of 64,000 Da. Identification of this polypeptide from bovine liver enabled us to determine the partial amino acid sequence of eukaryotic HCS.We have cloned the human HCS cDNA,which maps to chromosome 21q22.1. Two mutations in the HCS genes of Japanese patients with HCS deficiency have been identified : a transition from T to C which causes an amino acid substitution of proline for leucine at position 237 (L237P) and A single guanine base deletion (DELTAG1067) followed by premature termination. Transient expression analysis in cultured fibroblasts from a patient after site-directed mutagenesis demonstrated that the L237P mutant construct produce markedly decreased HCS activity, despite the presence of normal amounts ofimmunoreactive protein by Western blotting. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the prevalance of the mutations-L237P and DELTAG1067-was 50% and 30%, respectively, among Japanese patients with HCS deficiency.

已知新生儿发病的多种羧化酶缺乏症是由全羧化酶合成酶（HCS）缺乏引起的。 HCS 在细胞生物素利用中发挥重要作用。我们从牛肝细胞质中纯化了 HCS，发现它由分子量为 64,000 Da 的单个亚基组成。从牛肝脏中鉴定出该多肽使我们能够确定真核HCS的部分氨基酸序列。我们克隆了人HCS cDNA，定位于染色体21q22.1。已经鉴定出日本 HCS 缺乏症患者的 HCS 基因中的两个突变：从 T 到 C 的转变，导致第 237 位脯氨酸被亮氨酸取代 (L237P)，以及单个鸟嘌呤碱基缺失 (DELTAG1067)，然后提前终止。定点诱变后对患者培养的成纤维细胞进行瞬时表达分析表明，尽管通过蛋白质印迹法存在正常量的免疫反应蛋白，但 L237P 突变体构建体产生显着降低的 HCS 活性。使用等位基因特异性寡核苷酸探针的杂交分析表明，在日本 HCS 缺陷患者中，L237P 和 DELTAG1067 突变的患病率分别为 50% 和 30%。

项目成果

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