2001 Fiscal Year Final Research Report Summary
Cloning ofagene encoding 70 kDa heat shock protein expressed on the cell surface of mammalian cells
| Project/Area Number |
11680703
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | Sapporo Medical University |
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Principal Investigator |
TORIGOE Toshihiko Sapporo Medical University School of Medicine, Associate Professor, 医学部, 助教授 (20301400)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Noriyuki Sapporo Medical University School of Medicine, Professor, 医学部, 教授 (50158937)
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| Project Period (FY) |
1999 – 2001
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| Keywords | Chaperone / heat shock protein / HSP70 / HSP40 / HEDJ / T-cell receptor / Antigen peptide / TAP |
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| Research Abstract |
(1) Monoclonal antibodies #067 and NT22, which detected HSP70-like molecules on the cell surface, were established and characterized. The epitope of NT22 was mapped on HSP70 by using recombinant deletion mutant HSP70. PCR cloning of a gene encoding NT22 antigen was performed using degenerated primers from the amino acid sequence of the NT22 epitope. A novel gene was amplified from cDNA library of Daudi cells. The sequence and expression of the gene are on the way to elucidation.
(2) Expression cloning of a gene encoding #067 antigen or NT22 antigen was performed by a signal sequence trap method. No positive clone has been obtained so far. Retroviral expression cloning is still on the way.
(3) Characterization of #067 antigen suggested that it could present peptide antigens to double negative T-cells. G/d T-cell hybridoma clones that were responsive to #067 antigen were established. Analysis of TCR gene usage of the clones revealed that a unique set of g/d TCR gene was utilized in the clo
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nes. It was indicated that #067 antigen might present peptides to a specific subset of T-cells.
(4) Molecular interaction between TAP and HSP70 was examined. It was found that HSP70 was associated with TAP. In addition, synthetic polyamine compound that was capable of binding to HSP70 inhibited the ssociation, thereby decreasing TAP-mediated translocation of cytosolic peptides. Analysis of HSP70 affinity of various antigenic peptides revealed that peptides with high HSP70 affinity could be preferentially transported by TAP. Our study showed for the first time that MHC class I-presented peptides might be selected by HSP70 in the cytosol.
(5) A gene encoding HEDJ-1, HSP40 family protein in the endoplasmic reticulum, was cloned. Specific antibody recognizing HEDJ-1 was developed by immunizing a recombinant protein. Expression and fuction of HEDJ-1 have been analyzed. It was indicated that HEDJ-1 might be involved in the quality control of secretary proteins in epitheial cells and plasma cells. Less
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[Publications] Akazawa, T., Hirai, I., Hirohashi, Y., Kamiguchi, K., Sahara, H., Torigoe, T., Nagasawa, S., Tamura, Y. and Sato, N.: "A novel negative regulator Cho-1 molecule is involved in the cytotoxicity by human natural killer cells but not in cytotoxic T lymphocytes"Microbiol. Immunol.. 43. 285-291 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Suzuki, K., Sahara, H., Okada, Y., Yasoshima, T., Hirohashi, Y., Nabeta, Y., Hirai, I., Torigoe, T., Takahashi, S, Matsuura, A., Takahashi, N., Sasaki, A., Suzuki, M., Hamuro, J., Ikeda, H., Wada, Y., Hirata, K., Kikuchi, K. and Sato, N.: "Identification of natural antigenic peptides of a human gastricbn signet ring cell carcinoma recognized by HLA-A31-restricted cytotoxic T lymphocytes"J. Immunol.. 163. 2783-2791 (1999)
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「研究成果報告書概要(欧文)」より
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[Publications] Kanki, K., Torigoe, T., Hirai, I., Sahara, H., Kamiguchi, K., Tamura, Y., Yagihashi, A., Sato, N.: "Molejbular cloning of rat NK target structure-the possibility of CD44 involvement in NK cell-mediated lysis"Microbiol. Immunol.. 44. 1051-1061 (2000)
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「研究成果報告書概要(欧文)」より
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[Publications] Maeda, A., Ohguro, H., Nabeta, Y., Hirohashi, Y., Sahara, H., Maeda, T., Wada, Y., Sato, T., Chuns, Y., Nishimura, Y., Kuroki, Y. and Sato, N.: "Identification of human antitumor cytotoxic T lymphocytes epitopes of recoverin, cancer associated retinopathy antige, to achieve a clinical better prognosis in a paraneoplastic syndrome"Eur. J. Immunol.. 31. 563-572 (2001)
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[Publications] Kishi, A., Ichinohe, T., Kinebuchi, M., Hirai, I., Kamiguchi, K., Tamura, Y., Matsuura, A., Torigoe, T., and Sato, N.: "The cell surface-expressed HSC70-like molecule preferentially reacts with rat T cell recptor δ6 family"Immunogenetics. 53. 401-409 (2001)
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「研究成果報告書概要(欧文)」より
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