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著作: Naganawa, Yasunori/[伊藤 孝司]/Shimmoto, Michie/Kamei, Sachiko/Takiguchi, Kyoko/Doi, Hirofumi/Sakuraba, Hitoshi/Stable expression of protective protein/cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient./[The Biochemical Journal]

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EID
142465
EOID
680515
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LastModified
2013年2月28日(木) 10:49:37
Operator
三木 ちひろ
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Owner
伊藤 孝司
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種別 必須 学術論文(審査論文)
言語 必須 英語
招待 推奨
審査 推奨
カテゴリ 推奨
共著種別 推奨
学究種別 推奨
組織 推奨
著者 必須
  1. (英) Naganawa, Yasunori
    役割 任意
    貢献度 任意
    学籍番号 推奨
  2. 伊藤 孝司([徳島大学.大学院医歯薬学研究部.薬学域.薬科学部門.統合医薬創製科学系.創薬生命工学])
    役割 任意
    貢献度 任意
    学籍番号 推奨
  3. (英) Shimmoto, Michie
    役割 任意
    貢献度 任意
    学籍番号 推奨
  4. (英) Kamei, Sachiko
    役割 任意
    貢献度 任意
    学籍番号 推奨
  5. (英) Takiguchi, Kyoko
    役割 任意
    貢献度 任意
    学籍番号 推奨
  6. (英) Doi, Hirofumi
    役割 任意
    貢献度 任意
    学籍番号 推奨
  7. (英) Sakuraba, Hitoshi
    役割 任意
    貢献度 任意
    学籍番号 推奨
題名 必須

(英) Stable expression of protective protein/cathepsin A-green fluorescent protein fusion genes in a fibroblastic cell line from a galactosialidosis patient.

副題 任意
要約 任意

(英) Fibroblastic cell lines derived from a galactosialidosis patient, stably expressing the chimaeric green fluorescent protein variant (EGFP) gene fused to the wild-type and mutant human lysosomal protective protein/cathepsin A (PPCA) cDNA, were first established as a model system for revealing the sorting and processing of lysosomal enzymes and for investigating the molecular bases of their deficiencies. In the cell line expressing the wild-type PPCA-EGFP chimaera gene (EGFP-PPwild), an 81 kDa form (27 kDa EGFP fused to the C-terminus of the 54 kDa PPCA precursor) was produced, then processed into the mature 32/20 kDa two-chain form free of the EGFP domain. The intracellular cathepsin A, alpha-N-acetylneuraminidase and beta-galactosidase activities, which are deficient in the parent fibroblastic cells, could also be significantly restored in the cells. In contrast with the uniform and strong fluorescence throughout the cytoplasm and nucleus in the mock-cell line expressing only EGFP cDNA, weak reticular and punctate fluorescence was distributed throughout the EGFP-PPwild cell line. Bafilomycin A1, a potent inhibitor of vacuolar ATPase and intracellular acidification, induced the distribution of Golgi-like perinuclear fluorescence throughout the living and fixed cells, in which only the 81 kDa product was detected. After removal of the agent, time-dependent transport of the chimaeric protein from the Golgi apparatus to the prelysosomal structure in living cells was monitored with a confocal laser scanning microscope system. Leupeptin caused the distribution of lysosome-like granular fluorescence throughout the cytoplasm in the fixed cells, although it was hardly observed in living cells. The latter agent also dose-dependently induced an increase in the intracellular amount of the 81 kDa product containing the EGFP domain and inhibited the restoration of cathepsin A activity in the EGFP-PPwild cells after the removal of bafilomycin A1. In parallel, both the mature two-chain form and PPCA function disappeared. These results suggested that the chimaera gene product was transported to acidic compartments (endosomes/lysosomes), where proteolytic processing of the PPCA precursor/zymogen, quenching of the fluorescence, and random degradation of the EGFP portion occurred. A cell line stably expressing a chimaeric gene with a mutant PPCA cDNA containing an A1184-->G (Y395C) mutation, commonly detected in Japanese severe early-infantile type of galactosialidosis patients, showed an endoplasmic reticulum (ER)-like reticular fluorescence pattern. The PPCA-immunoreactive gene product was hardly detected in this cell line. The mutant chimaeric product was suggested to be degraded rapidly in the ER before transport to post-ER compartments. A cell line expressing the chimaeric gene with a T746-->A (Y249N) PPCA mutation exhibited both ER-like reticular and granular fluorescence on the reticular structure that was stronger than that in the EGFP-PPwild cells. Some of them contained large fluorescent inclusion-body-like structures. The ineffectiveness of transport inhibitors in the distribution changes in the two mutant chimaeric proteins suggested that they were not delivered to acidic compartments. Therefore this expression system can possibly be applied to the direct analysis of the sorting defects of mutant gene products in living cells and will be useful for the molecular investigation of lysosomal diseases, including galactosialidosis.

キーワード 推奨
  1. (英) Amino Acid Sequence
  2. (英) Anti-Bacterial Agents
  3. (英) Base Sequence
  4. (英) Biological Transport
  5. (英) Carboxypeptidases
  6. (英) Cathepsin A
  7. (英) Cell Line
  8. (英) DNA, Complementary
  9. (英) Fibroblasts
  10. (英) Green Fluorescent Proteins
  11. (英) Humans
  12. (英) Leupeptins
  13. (英) Luminescent Proteins
  14. (英) Lysosomal Storage Diseases
  15. (英) Lysosomes
  16. (英) Macrolides
  17. (英) Microscopy, Fluorescence
  18. (英) Models, Biological
  19. (英) Molecular Sequence Data
  20. (英) Mutation
  21. (英) Protein Processing, Post-Translational
  22. (英) Recombinant Fusion Proteins
発行所 推奨
誌名 必須 The Biochemical Journal([The Biochemical Society])
(pISSN: 0264-6021, eISSN: 1470-8728)
ISSN 任意 0264-6021
ISSN: 0264-6021 (pISSN: 0264-6021, eISSN: 1470-8728)
Title: The Biochemical journal
Title(ISO): Biochem J
Publisher: Portland Press, Ltd.
 (NLM Catalog  (Scopus  (CrossRef (Scopus information is found. [need login])
必須 340
必須 ---
必須 467 474
都市 任意
年月日 必須 1999年 6月 1日
URL 任意
DOI 任意
PMID 任意 10333491    (→Scopusで検索)
NAID 任意
WOS 任意
Scopus 任意
評価値 任意
被引用数 任意
指導教員 推奨
備考 任意
  1. (英) Article.Affiliation: Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113-8613, Japan.

  2. (英) Article.PublicationTypeList.PublicationType: Journal Article

  3. (英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't

  4. (英) OtherID: PMC1220273