Li Xue-Wen, Rees Johanna S, Xue Peng, Zhang Hong, Hamaia Samir W, Sanderson Bailey, Funk Phillip E, Farndale Richard W, Lilley Kathryn S, Perrett Sarah, Jackson Antony P
From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China, the University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China.
the Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom, the Cambridge Centre for Proteomics, Tennis Court Road, University of Cambridge, Cambridge CB2 1QR, United Kingdom.
J Biol Chem. 2014 May 23;289(21):14434-47. doi: 10.1074/jbc.M113.529578. Epub 2014 Apr 4.
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.
在脊椎动物免疫系统中，每个B淋巴细胞都表达一种表面IgM类B细胞受体（BCR）。当被抗原或抗IgM抗体交联时，BCR会与其他蛋白质聚集形成不同的表面簇，从而激活细胞信号传导、分裂或凋亡。然而，这些簇的分子组成尚未明确界定。在这里，我们描述了一种定量分析方法，我们称之为使用酪胺的选择性蛋白质组邻近标记法（SPPLAT）。它能使靶标附近的蛋白质被选择性地生物素化，进而分离出来用于质谱分析。以鸡B细胞系DT40为模型，我们使用SPPLAT通过邻近标记对任何BCR簇进行了首次蛋白质组分析。我们检测到了BCR簇的已知成分，包括整合素，以及以前认为与BCR无关的蛋白质。特别是，我们鉴定出了鸡B淋巴细胞同种异型标记物chB6。我们发现，BCR交联后，chB6会移动到距BCR约30 - 40纳米的范围内，并且我们还发现，交联chB6会激活细胞与整合素底物层粘连蛋白和明胶的结合。我们的工作为BCR簇的性质和组成提供了新的见解，并证实了SPPLAT是分子和细胞蛋白质组学中一种有用的研究工具。
