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

Research Project

Project/Area Number 06671780
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field Ophthalmology
Research InstitutionSchool of Medicine, Fujita Health University

Principal Investigator

KOBAYASHI Tatsuhiko  School of Med., Fujita Health Univ., Biochem., Assistant Prof., 医学部, 講師 (90186750)

Co-Investigator(Kenkyū-buntansha) MATSUZAWA Takeo  School of Med., Fujita Health Univ., Biochem., Prof., 医学部, 教授 (30084502)
KASAHARA Masao  School of Med., Fujita Health Univ., Pathol., Prof., 医学部, 教授 (70084506)
OGAWA Hisamitsu  School of Med., Fujita Health Univ., Biol., Associate Prof., 医学部, 助教授 (80101658)
Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1994: ¥1,600,000 (Direct Cost: ¥1,600,000)
Keywordsgyrate 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
Research Abstract

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

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] 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)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 小林 龍彦: "アラニンアミノ基転移酵素(GPT)アイソザイム及び脳回転状網脈絡膜萎縮症の発症機構に関する研究" 石田財団研究助成報告. 22. 131-135 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 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. 284-291 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ogawa H., Hayashi N., Hori I., Kobayashi T., and Hosono R.: "Expression, purification, and characterization of recombinant C. elegans UNC-18" Neurochemistry International. (in press).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 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)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kobayashi T.: "Studies on alanine aminotransferase (GPT) isozyme and gyrate atrophy of the choroid and retina (Japanese)" Ishida Foundation Report. 22. 131-135 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kobayashi T., Ogawa H., Kasahara M., Shiozawa Z., and Matsuzawa T.: "A single amino acid substitution within the mature sequence of ornithine aminotransferase obstructs mitochondrial entry of the precursor" Am.J.Hum.Genet.57. 284-291 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 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).

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] 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. 284-291 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Ogawa H., Hayashi N., Hori I., Kobayashi T., and Hosono R.: "Expression, purification, and characterization of recombinant C. elegans UNC-18" Neurochemistry International. (in press).

    • Related Report
      1995 Annual Research Report
  • [Publications] 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)

    • Related Report
      1994 Annual Research Report
  • [Publications] 小林龍彦: "アラニンアミノ基転移酵素(GPT)アイソザイム及び脳回転状網脈絡膜萎縮症の発症機構に関する研究" 石田財団研究助成報告. 22. 131-135 (1995)

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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