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New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay
Xue-Wen Li1, Johanna S Rees2, Peng Xue3
1From 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.
Insights
Researchers developed a new method, selective proteomic proximity labeling using tyramide (SPPLAT), to analyze B cell receptor (BCR) clusters. This technique identified novel proteins associated with BCR clusters, revealing new insights into immune cell signaling.
Area of Science:
- Immunology
- Proteomics
- Cell Biology
Background:
- B-lymphocytes utilize B cell receptors (BCRs) for immune surveillance.
- BCR cross-linking triggers signaling cascades, but cluster composition remains unclear.
Purpose of the Study:
- To define the molecular composition of BCR clusters.
- To introduce and validate a novel proximity labeling technique for proteomic analysis.
Main Methods:
- Development and application of selective proteomic proximity labeling using tyramide (SPPLAT).
- Quantitative proteomic analysis of BCR clusters in chicken B cells (DT40 model).
- Mass spectrometry to identify biotinylated proteins in proximity to BCRs.
Main Results:
- SPPLAT successfully identified known BCR cluster components, including integrins.
- Novel proteins, such as the chicken B-lymphocyte allotypic marker chB6, were identified.
- chB6 proximity to BCRs and its role in integrin-mediated cell adhesion were demonstrated.
Conclusions:
- SPPLAT is a valuable tool for studying protein complexes like BCR clusters.
- New insights into BCR cluster composition and function were gained, highlighting chB6's role.
Abstract:
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.
