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

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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