New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay.
New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay.
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DOI：
10.1074/jbc.m113.529578
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发表时间：
2014-05-23
期刊：
影响因子：
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通讯作者：
Jackson AP
中科院分区：
文献类型：
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作者：
Li XW;Rees JS;Xue P;Zhang H;Hamaia SW;Sanderson B;Funk PE;Farndale RW;Lilley KS;Perrett S;Jackson AP
背景：B细胞受体（BCR）簇调节B淋巴细胞中的BCR信号。结果：我们使用定量蛋白质组学邻近分析来分析DT 40细胞中的BCR簇。结论：我们的邻近标记法鉴定了与整合素信号传导相关的BCR簇的新组分。意义：我们为BCR组装提供了新的见解，并为进一步的功能分析确定了新的和意想不到的目标。在脊椎动物免疫系统中，每个B淋巴细胞表达表面IgM类B细胞受体（BCR）。当与抗原或抗IgM抗体交联时，BCR与其他蛋白质聚集成不同的表面簇，从而激活细胞信号传导、分裂或细胞凋亡。然而，这些簇的分子组成并没有很好地定义。在这里，我们描述了一种定量分析，我们称之为选择性蛋白质组邻近标记使用酪胺（SPPLAT）。它允许靶附近的蛋白质被选择性地生物素化，从而分离用于质谱分析。使用鸡B细胞系DT 40作为模型，我们使用SPPLAT提供第一个蛋白质组学分析的任何BCR集群使用邻近标记。我们检测BCR簇的已知组分，包括整合素，以及以前认为与BCR无关的蛋白质。特别是，我们确定了鸡B淋巴细胞同种异型标记chB 6。我们表明，chB 6移动到约30-40 nm的BCR后BCR交联，我们表明，交联chB 6激活细胞结合整合素基板层粘连蛋白和明胶。我们的工作为BCR簇的性质和组成提供了新的见解，并证实SPPLAT是分子和细胞蛋白质组学中有用的研究工具。
Background: B cell receptor (BCR) clusters modulate BCR signaling in B-lymphocytes. Results: We used a quantitative proteomic proximity assay to analyze the BCR cluster in DT40 cells. Conclusion: Our proximity labeling assay identified novel components of the BCR cluster linked to integrin signaling. Significance: We provide new insights into BCR assembly and identify new and unexpected targets for further functional analysis. In the vertebrate immune system, each B-lymphocyte expresses a surface IgM-class B cell receptor (BCR). When cross-linked by antigen or anti-IgM antibody, the BCR accumulates with other proteins into distinct surface clusters that activate cell signaling, division, or apoptosis. However, the molecular composition of these clusters is not well defined. Here we describe a quantitative assay we call selective proteomic proximity labeling using tyramide (SPPLAT). It allows proteins in the immediate vicinity of a target to be selectively biotinylated, and hence isolated for mass spectrometry analysis. Using the chicken B cell line DT40 as a model, we use SPPLAT to provide the first proteomic analysis of any BCR cluster using proximity labeling. We detect known components of the BCR cluster, including integrins, together with proteins not previously thought to be BCR-associated. In particular, we identify the chicken B-lymphocyte allotypic marker chB6. We show that chB6 moves to within about 30–40 nm of the BCR following BCR cross-linking, and we show that cross-linking chB6 activates cell binding to integrin substrates laminin and gelatin. Our work provides new insights into the nature and composition of the BCR cluster, and confirms SPPLAT as a useful research tool in molecular and cellular proteomics.