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

脉络膜和视网膜的回旋状萎缩（GACR）伴高鸟氨酸血症是由先天性鸟氨酸氨基转移酶（OAT）缺乏引起的，OAT是一种核编码的线粒体基质酶。我们对一例OAT缺乏伴GACR和高鸟氨酸血症患者的OAT缺乏的分子基础进行了研究，并获得了以下结果.用EBV感染的淋巴细胞测定OAT活性。患者细胞显示的OAT活性相当于来自健康个体的对照细胞的3.5%。通过对骨骼肌活检标本进行免疫组织化学染色，观察到患者骨骼肌活检标本中OAT蛋白的表达为阴性.用RT和PCR方法扩增出OAT cDNA，经核苷酸序列分析发现，OAT成熟蛋白编码序列中有一个碱基由C变为G，导致一个氨基酸由Gln 90（CAA）变为Glu（GAA）（Q90 E）。患者的兄弟患有GACR a ...更多信息 也携带Q90 E突变。我们用CHO细胞检测了Q90 EOAT蛋白的瞬时表达。Western blot分析显示，表达Q90 EOAT的细胞中，对应于45-kDa成熟OAT（mOAT）的单一免疫反应条带强度明显降低.在使用杆状病毒-昆虫细胞表达系统高水平表达OAT蛋白时，蛋白质印迹和免疫细胞化学分析表明Q90 EOAT以49-kDa前体（pOAT）形式定位于细胞质游离核糖体的范围内，而没有任何线粒体进入。另一方面，western blot分析显示，除了pOAT形式外，细胞表达正常OAT，还显示两条强免疫反应条带，对应于46-kDa中间体（iOAT）和mOAT。为了解释iOAT与pOAT或mOAT之间分子量的差异，我们对iOAT的氨基端进行了测序。氨基端序列分析显示，iOAT在Gly 17和Val 18之间被切割，表明pOAT通过iOAT（信号肽的两步加工）转化为mOAT。在OATcDNA的定点突变分析中，Asn 89，Gln 90，或具有极性残基的Gly 91（带正电荷或负电荷的残基）这些发现表明，成熟OAT蛋白编码序列内的Q90 E突变导致酶活性的缺乏，这是由于前体的线粒体靶向受损，并且Q90 EOAT被缩小，由于在细胞质中积累，降解迅速。近年来，包括热休克蛋白70在内的线粒体输入因子在前体蛋白的线粒体标记中发挥重要作用。可能包括Gln 90的编码序列对于与这些因子的特定残基的相互作用是关键的。少

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