Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells
Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells
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DOI：
10.1111/j.1742-4658.2005.05011.x
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发表时间：
2005-12-01
期刊：
影响因子：
5.4
通讯作者：
Sekikawa, K
中科院分区：
文献类型：
--
作者：
Sato, M;Iwaya, R;Sekikawa, K
细胞内表达的抗体（胞内抗体）已被用于抑制细胞内各种蛋白质的功能。然而，胞内抗体在胞质溶胶中的稳定性和功能性表达的问题仍未解决。在这项研究中，我们表明，单链可变片段（scFv）的胞内抗体与重链可变区（V-H）的前导信号序列在N-末端构建从内质网易位到T淋巴细胞的胞质溶胶和抑制的目标分子，Wiskott-Aldrich综合征蛋白（WASP）的功能。WASP作为多功能衔接分子存在于细胞溶质中，并介导T细胞受体（TCR）信号传导途径中的肌动蛋白聚合和白细胞介素（IL）-2合成。已经表明，在WASP的N-末端区域中的EVH 1结构域可能参与IL-2的合成。在表达来自产生WASP-EVH 1 mAb的杂交瘤细胞的抗EVH 1 scFv的转基因小鼠中，通过免疫沉淀分析检测到胞质溶胶中的大量scFv以及抗EVH 1 scFv与T细胞中的天然WASP之间的结合。此外，在scFv转基因T细胞中证明了由TCR刺激诱导的增殖反应和IL-2产生的损伤，其不影响TCR加帽。我们先前描述了相同的T细胞缺陷WASP转基因小鼠过表达EVH 1域。这些结果表明EVH 1胞内抗体仅抑制调节IL-2合成信号传导的EVH 1结构域功能，而不影响WASP的整体结构域结构。这里提出的新方法是一个有价值的工具，在体内功能分析的胞质蛋白。
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.