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 (V(H)) 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 region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.
Sato
Mitsuru
M
Department of Molecular Biology and Immunology, National Institute of Agrobiological Sciences, Ibaraki, Japan.
Iwaya
Ryo
R
Ogihara
Kazumasa
K
Sawahata
Ryoko
R
Kitani
Hiroshi
H
Chiba
Joe
J
Kurosawa
Yoshikazu
Y
Sekikawa
Kenji
K
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D049109:Cell Proliferation; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2 / Q000276:immunology; D042541:Intracellular Space / Q000276:immunology*; D008221:Lymphoid Tissue / Q000276:immunology / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D051316:Wiskott-Aldrich Syndrome Protein