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首页> 外文期刊>The FEBS journal >Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells
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Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

机译：抗Wiskott-Aldrich综合征蛋白EVH1结构域的体内抗体抑制转基因小鼠T细胞中的T细胞受体信号传导

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摘要

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.
机译：细胞内表达的抗体（体内抗体）已被用来抑制细胞内各种蛋白质的功能。然而，胞浆中胞内抗体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，在N端以重链可变（V（H））前导信号序列构建的单链可变片段（scFv）抗体从内质网转移到T淋巴细胞的胞质中并被抑制靶分子Wiskott-Aldrich综合征蛋白（WASP）的功能。 WASP以多功能衔接子分子的形式存在于细胞质中，并在T细胞受体（TCR）信号传导途径中介导肌动蛋白聚合和白介素（IL）-2合成。已经提出，WASP的N-末端区域中的EVH1结构域可能参与IL-2合成。在表达衍生自产生WASP-EVH1 mAb的杂交瘤细胞的抗EVH1 scFv的转基因小鼠中，通过免疫沉淀分析检测到细胞质中大量的scFv和胞浆中抗EVH1 scFv与天然WASP之间的结合。此外，在scFv转基因T细胞中证实了由TCR刺激诱导的增殖反应和IL-2产生的损害，其不影响TCR的上限。我们先前描述了过表达EVH1结构域的WASP转基因小鼠中相同的T细胞缺陷。这些结果表明，EVH1抗体仅抑制调节IL-2合成信号的EVH1结构域功能，而不会影响WASP的整体结构域。此处介绍的新方法是用于细胞溶质蛋白体内功能分析的有价值的工具。

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