Molecular mechanism of hereditary ornithine aminotransferase deficiency causing gyrate atrophy of the choroid and retina

遗传性鸟氨酸转氨酶缺乏导致脉络膜和视网膜回旋萎缩的分子机制

• 批准号: 06671780
• 负责人: KOBAYASHI Tatsuhiko
• 金额: $ 1.34万
• 依托单位: School of Medicine, Fujita Health University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06671780/
• 关键词: gyrate atrophy of the choroid and retina; ornithine aminotransferase; import of mitochondrial protein; cytosolic mitochondrial import factor; two-step processing of the siganal peptides; 多段階プロセシング; シグナル配列; 成熟配列; ミトコンドリアタンパク質前駆体輸送

Gyrate atrophy of the choroid and retina (GACR) with hyperornithinemia is caused by a congenital deficiency of ornithine aminotransferase (OAT), a nuclear-encoded, mitochondrial matrix enzyme. We studied the molecular basis of OAT deficiency in an OAT-deficient patient with GACR and hyperornithinemia, and we obtained following results.1. OAT activity was determined with EBV-infected lymphocytes. The patient's cells showed OAT activity equivalent to 3.5% that of control cells from a healthy individual. By immunohistichemical staining of skeletal muscle biopsy specimens for the presence of OAT protein, negative staining was observed in the patient.2. Using total mRNA from the patient's cells, OATcDNA was amplified by RT and PCR.Nucleotide-sequence analysis of the amplified OAT cDNA revealed a single base change from C to G within the coding sequence of the mature protein, resulting in a single amino acid substitution of Gln90 (CAA) with Glu (GAA) (Q90E). The patient's brother with GACR a … More lso carried Q90E mutation.3. We examined the transient expression of Q90EOAT protein by using CHO cells. A marked reduciton in intensity of a single immunoreactive band corresponding with 45-kDa mature OAT (mOAT) from the cells expressed Q90EOAT was observed by western blot analysis.4. In high level expression of OAT protein using the baculovirus-insect cell expression system, western blot and immunocytochemical analyzes demonstrated that Q90EOAT was localized within the limits of cytoplasmic free ribosomes in 49-kDa precursor (pOAT) form without any mitochondrial entry. On the other hand, western blot analysis showed two strong immunoreactive bands corresponding with 46-kDa intermediate (iOAT) and mOAT in addition to pOAT form the cells expressed normal OAT.To explain that the difference in molecular weight between iOAT and either pOAT or mOAT,we sequenced amino-terminus of iOAT.Amino-terminal sequence analysis showed that iOAT was cleaved between Gly17 and Val18, suggesting that pOAT was converted into mOAT via an iOAT (two-step processing of the signal peptides).5. In site-directed mutagenesis analysis of OATcDNA,substitution of Asn89, Gln90, or Gly91 with polar residue (positively or negatively charged residue) caused accumulation of the precursor in the cytosol.These findings suggest that Q90E mutation within the coding sequence of the mature OAT protein results in a deficiency of enzymatic activity due to impairment of mitochondrial targeting of the precursor and Q90EOAT is synthsized and rapidly degraged because of accumulaiton in the cytosol. Recently, cytosolic mitochondrial import factors including Hsp70 play essential role in mitochondrial tageting of precursor. It is possible that the coding sequence including Gln90 is critical for interaction with specific residues of these factors. Less

脉络膜和视网膜（GACR）的回旋萎缩伴有高义血症是由鸟氨酸氨基转移酶（OAT）的先天性缺乏引起的，鸟氨酸氨基转移酶（OAT）是一种核编码的线粒体基质酶。我们研究了燕麦缺乏症患者的GACR和高义氨酸血症患者的燕麦缺乏症的分子基础，我们获得了以下结果。1。用EBV感染的淋巴细胞确定燕麦活性。患者的细胞显示燕麦活性相当于健康个体的对照细胞的3.5％。通过对燕麦蛋白存在的骨骼肌活检标本的免疫组织化染色，在患者中观察到阴性染色。2。使用来自患者细胞的总mRNA，通过RT和PCR进行了燕麦片。对放大燕麦cDNA的核苷酸 - 序列分析显示，在成熟蛋白的编码中，单个碱基从C到G的单个碱基变化，从而导致单个氨基酸替代GLN90（CAA）用GLU（GAA（GAA））（Q90E）（Q90E）。患者的兄弟与GACR A…更多的LSO携带了Q90E突变3。我们通过使用CHO细胞检查了Q90EOAT蛋白的瞬时表达。通过Western blot分析观察到单个免疫反应性带的显着的降量强度，与来自表达的Q90EOAT的细胞相对应的45 kDa成熟燕麦（护城河）。4。在使用杆状病毒 - 诱导细胞表达系统的高水平表达中，蛋白质印迹和免疫细胞化学分析表明，Q90EOAT位于49 kDa Promosursor（POAT）的细胞质游离核糖体范围内，而无需任何线粒体的进入。 On the other hand, western blot analysis showed two strong immunoreactive bands corresponding with 46-kDa intermediate (iOAT) and mOAT in addition to pOAT form The cells expressed normal OAT.To explain that the difference in molecular weight between iOAT and either pOAT or mOAT,we sequenced amino-terminus of iOAT.Amino-terminal sequence analysis shown that iOAT was cleaved between Gly17 and Val18, suggesting that pOAT通过IOAT（信号肽的两步处理）被转换为护城河。5。在燕麦片的定位定向诱变分析中，具有极性保留（正面或负电荷的居住）的替代ASN89，GLN90或GLY91导致细胞质中的前体加速。这些发现表明，成熟燕麦蛋白的编码序列内的Q90E突变导致酶促活性的缺乏，这是由于细胞溶胶中累积的累积，前体和Q90EOAT的线粒体靶向受损而被合成并迅速降解。最近，包括HSP70在内的胞质线粒体进口因子在前体的线粒体标记中起重要作用。包括GLN90在内的编码序列对于与这些因素的特定残差相互作用至关重要。较少的

项目成果

松澤健夫: "Changes in Ornithine Metabolic Enzymes Induced by Dietary Protein in Small Intestine and Liver:Intestine-Liver Relationship in Ornithine Supply to Liver" J.Biochem.116. 721-727 (1994)

Takeo Matsuzawa：“小肠和肝脏中膳食蛋白质引起的鸟氨酸代谢酶的变化：鸟氨酸供应到肝脏中的肠-肝关系”J.Biochem.116（1994）。

Ogawa H., Hayashi N., Hori I., Kobayashi T., and Hosono R.: "Expression, purification, and characterization of recombinant C.elegans UNC-18" Neurochem.Intern.(in press).

Okawa H.、Hayashi N.、Hori I.、Kobayashi T. 和 Hosono R.：“重组线虫 UNC-18 的表达、纯化和表征” Neurochem.Intern.（正在印刷中）。

Kobayashi T., Ogawa H., Kasahara M., Shiozawa Z., and Matsuzawa T.: "A single amino acid substitution withn the mature sequence of ornithine aminotransferase obstructs mitochondrial entry of the precursor" The American Journal of Human Genetics. 57. 284-2

Kobayashi T.、Okawa H.、Kasahara M.、Shiozawa Z. 和 Matsuzawa T.：“鸟氨酸转氨酶成熟序列中的单个氨基酸取代会阻碍前体进入线粒体”《美国人类遗传学杂志》。

Matsuzawa T., Kobayashi T., Tashiro K., and Kasahara M.: "Changes in ornithine metabolic enzymes induced by dietary protein in small intestine and liver : instestine-liver relationship in ornithine supply to liver" J.Biochem.116. 721-727 (1994)

Matsuzawa T.、Kobayashi T.、Tashiro K. 和 Kasahara M.：“小肠和肝脏中膳食蛋白质诱导的鸟氨酸代谢酶的变化：向肝脏供应鸟氨酸的肠-肝脏关系”J.Biochem.116。

Kobayashi T, Ogawa H., Kasahara M., Shiozawa Z., and Matsuzawa T.: "A single amino acid substitution within the mature sequence of ornithine aminotransferase obstructs mitoc hondrial entry of the precursor" The American Journal of Human Genetics. 57. 284-

Kobayashi T、Okawa H.、Kasahara M.、Shiozawa Z. 和 Matsuzawa T.：“鸟氨酸转氨酶成熟序列中的单个氨基酸取代会阻碍前体进入线粒体”美国人类遗传学杂志。