A system for reconstructing B cell antigen receptor signaling in the mouse myeloma J558L cell line.
A system for reconstructing B cell antigen receptor signaling in the mouse myeloma J558L cell line.
复制标题
用于在小鼠骨髓瘤 J558L 细胞系中重建 B 细胞抗原受体信号传导的系统。
DOI：
10.1016/j.abb.2013.02.008
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发表时间：
2013
影响因子：
3.9
通讯作者：
Adachi T.
中科院分区：
文献类型：
--
作者：
Harumiya S;Yoshino A;Hayashizaki K;Mizuno K;Yakura H;Adachi T.
B细胞抗原受体（BCR）信号传导受各种细胞表面受体如CD 19和CD 45的正和负调节。利用基因打靶技术对这些受体进行了功能分析，这是阐明其功能的有效方法。然而，当同时评估多个分子时，这种类型的分析受到限制。从不同的角度来看，合成生物学为分析各种分子提供了高度的自由度。在这里，我们开发了一个系统，使用J558L骨髓瘤细胞系与基于蛋白质的Ca2+指示剂YC 3.60组合来重建BCR信号传导。携带YC 3.60的BCR重建J558L细胞（J558Lμv11细胞）允许监测Ca2+动员。在J558Lμv11细胞中重建CD19导致可检测的BCR诱导的Ca2+动员，但动力学与表达CD45的细胞不同。此外，我们评估的有效性的J558L系统的酪氨酸磷酸化蛋白质组学分析抗原刺激后。在J558 L μv11细胞中鉴定了100多个BCR诱导的酪氨酸磷酸化蛋白，发现与B细胞中的蛋白相似，并发现了一个新的非受体蛋白酪氨酸激酶Fer。
B cell antigen receptor (BCR) signaling is positively and negatively regulated by various cell surface receptors such as CD19 and CD45. Functional analysis of these receptors has been performed using gene targeting technology, which is a valid approach to elucidate their functions. However, this type of analysis is restricted when multiple molecules are evaluated simultaneously. From a different perspective, synthetic biology provides a high degree of freedom for analyzing various molecules. Here we developed a system to reconstruct BCR signaling using the J558L myeloma cell line in combination with the protein-based Ca2+indicator YC3.60. BCR-reconstituted J558L cells harboring YC3.60 (J558Lμv11 cells) permitted monitoring of Ca2+mobilization. Reconstituting CD19 in J558Lμv11 cells resulted in detectable BCR-induced Ca2+mobilization but with kinetics different from that of CD45-expressing cells. Furthermore, we evaluated the validity of the J558L system by proteomic analysis of tyrosine-phosphorylated proteins after antigen stimulation. Identification of more than 100 BCR-induced tyrosine-phosphorylated proteins in J558Lμv11 cells revealed a similarity to that observed in B cells, and a novel member, non-receptor protein tyrosine kinase Fer, was found. Thus, this reconstruction system using J558L cells appeared to be valid for comprehensively investigating BCR signaling.