Department for Molecular Immunology, Max Planck-Institute for Immunobiology, Freiburg, Germany.
Immunol Lett. 2010 May 4;130(1-2):43-50. doi: 10.1016/j.imlet.2009.12.011. Epub 2010 Jan 6.
The T cell antigen receptor (TCR-CD3) complex contains 12 different cytoplasmic tyrosines, each of which is part of an immunoreceptor tyrosine-based activation motif and thus occurs in similar sequence context. Since phosphorylation of individual tyrosines can be correlated with the quality of the T cell response, monitoring their phosphorylation is important. We thus generated novel antibodies against phospho-tyrosines of the TCR-CD3 complex and tested the specificity in a synthetic biology approach. We utilized the Drosophila S2 reconstitution system testing several kinases and stimulation conditions that lead to optimal phosphorylation of the TCR-CD3 subunit zeta. Expressing TCR-CD3 subunits and tyrosine mutants thereof we tested the specificity of the novel antibodies in Western blot and immunopurification experiments. In particular, we generated and characterized the monoclonal antibody EM-26 that specifically recognizes phosphorylation of the membrane proximal tyrosine of zeta (phospho-zetaY1) and antisera raised against the first and the second phospho-tyrosine of CD3epsilon (phospho-epsilonY1 and phospho-epsilonY2).
T 细胞抗原受体 (TCR-CD3) 复合物包含 12 个不同的胞质酪氨酸，每个酪氨酸都是免疫受体酪氨酸基激活基序的一部分，因此它们出现在类似的序列环境中。由于单个酪氨酸的磷酸化可以与 T 细胞反应的质量相关，因此监测其磷酸化很重要。因此，我们针对 TCR-CD3 复合物的磷酸酪氨酸生成了新型抗体，并在合成生物学方法中测试了其特异性。我们利用果蝇 S2 重组系统测试了几种激酶和刺激条件，这些激酶和刺激条件可导致 TCR-CD3 亚基 ζ 的最佳磷酸化。表达 TCR-CD3 亚基及其酪氨酸突变体，我们在 Western blot 和免疫沉淀实验中测试了新型抗体的特异性。特别是，我们生成并表征了单克隆抗体 EM-26，该抗体特异性识别 ζ 膜近端酪氨酸的磷酸化（磷酸化-zetaY1），并针对 CD3epsilon 的第一和第二个磷酸酪氨酸（磷酸化-epsilonY1 和磷酸化-epsilonY2）产生了抗血清。
