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

高鸟氨酸血症引起的脉膜和视网膜旋转性萎缩（GACR）是由先天性鸟氨酸转氨酶（OAT）缺乏引起的，OAT是一种核编码的线粒体基质酶。我们研究了一名伴有GACR和高鸟氨酸血症的OAT缺乏患者OAT缺乏的分子基础，我们得到了以下结果。用ebv感染淋巴细胞测定OAT活性。患者细胞显示的OAT活性相当于健康人对照细胞的3.5%。对骨骼肌活检标本进行OAT蛋白免疫组化染色，患者呈阴性。利用患者细胞的总mRNA， RT和PCR扩增OATcDNA。对扩增的OAT cDNA进行核苷酸序列分析，发现成熟蛋白编码序列中有一个碱基从C变为G，导致Gln90 （CAA）被Glu (GAA) （Q90E）取代。2 .患者患有GACR的兄弟也携带Q90E突变。我们利用CHO细胞检测了Q90EOAT蛋白的瞬时表达。western blot分析发现，表达Q90EOAT的细胞中45 kda成熟OAT （mOAT）对应的单个免疫反应带强度明显降低。利用杆状病毒-昆虫细胞表达系统高水平表达OAT蛋白，western blot和免疫细胞化学分析表明，Q90EOAT定位在49-kDa前体（pOAT）形式的细胞质游离核糖体范围内，没有任何线粒体进入。另一方面，western blot分析显示，在表达正常OAT的细胞中，除了pOAT外，还存在与46-kDa中间体（iOAT）和mOAT相对应的两条强免疫反应带。为了解释iOAT与pOAT或mOAT之间的分子量差异，我们对iOAT的氨基端进行了测序。氨基末端序列分析表明，iOAT在Gly17和Val18之间被切割，表明pOAT通过iOAT（信号肽的两步处理）转化为mOAT。在OATcDNA的定点诱变分析中，用极性残基（带正电或负电的残基）取代Asn89、Gln90或Gly91会导致细胞质中前体的积累。这些发现表明，成熟OAT蛋白编码序列中的Q90E突变导致线粒体前体靶向功能受损，导致酶活性不足，Q90EOAT被合成并由于在细胞质中积累而迅速降解。近年来，包括Hsp70在内的细胞质线粒体输入因子在线粒体前体标记中发挥着重要作用。可能包含Gln90的编码序列对于这些因子的特定残基的相互作用是至关重要的。少

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。

Matsuzawa T., Kobayashi T., Tashiro K., and Kasahara M.: "Changes in ornithine metabolic enzymes induced by dietary protein in small intesine and liver: intestine liver relationship in ornithine supply to liver" Journal of Biochemistry. 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：鸟氨酸向肝脏供应的肠肝关系”《生物化学杂志》。