New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay
Funk, Phillip E
The Journal of Biological Chemistry
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Li, X., Rees, J., Xue, P., Zhang, H., Hamaia, S., Sanderson, B., Funk, P. E., et al. (2014). New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay. The Journal of Biological Chemistry, 289 14434-14447. https://doi.org/10.1074/jbc.M113.529578
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.
Immunology, Integrin, Lymphocyte, Mass Spectrometry (MS), Proteomics, B Cell Receptor, Evi2a, Raftlin, SILAC, chB6
JSR supported by Grants BB/J021091 and H024085/1 from the Biotechnology and Biological Sciences Research Council (UK). SWH & RWF supported by Grant G0500707 from the Medical Research Council (UK) and Grant 094470/Z/10/Z from the Wellcome Trust. BS & PEF supported by the DePaul University Research Council. This work was supported in part by grants from the Chinese Ministry of Science and Technology 973 Program (2012CB911000 and 2013CB910700) and the National Natural Science Foundation of China (31110103914, 31070656, 31000342, and 31270794).
Wellcome Trust (094470/Z/10/Z)
British Heart Foundation (RG/09/003/27122)
External DOI: https://doi.org/10.1074/jbc.M113.529578
This record's URL: https://www.repository.cam.ac.uk/handle/1810/253318
Attribution 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/
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