tailieunhanh - Báo cáo khoa học: Intrabodies against the EVH1 domain of Wiskott–Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (VH) leader signal sequence at the N-terminus were translocated from the endoplasmic reti-culum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott–Aldrich syndrome protein (WASP) | ềFEBS Journal Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells Mitsuru Sato1 Ryo Iwaya1 2 Kazumasa Ogihara1 3 Ryoko Sawahata1 3 Hiroshi Kitani1 Joe Chiba2 Yoshikazu Kurosawa4 and Kenji Sekikawa1 5 1 Department of Molecular Biology and Immunology National institute of AgrobiologicalSciences Ibaraki Japan 2 Department of BiologicalScience and Technology Tokyo University of Science Chiba Japan 3 Institute for Antibodies Co. Ltd National institute of AgrobiologicalSciences Ibaraki Japan 4 Institute for Comprehensive MedicalScience Fujita Health University Toyoake Aichi Japan 5 Kitasato University Schoolof Veterinary Medicine and AnimalSciences Aomori Japan Keywords cytosolic protein functional knockdown intrabody T-cellreceptor signaling Wiskott-Aldrich syndrome protein WASP Correspondence K. Sekikawa Department of Molecular Biology and Immunology NationalInstitute of AgrobiologicalSciences 3-1-5 Kannondai Tsukuba Ibaraki 305-0856 Japan Tel Fax 81 29 8386039 E-mail sekiken@ Received 2 August 2005 revised 4 October 2005 accepted 10 October 2005 doi Intracellularly expressed antibodies intrabodies have been used to inhibit the function of various kinds of protein inside cells. However problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study we show that single-chain variable fragment scFv intrabodies constructed with a heavy chain variable VH leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule Wiskott-Aldrich syndrome protein WASP . WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin IL -2 synthesis in the T-cell receptor TCR signaling pathway. It has been suggested that an EVH1 domain in the N-terminal .