New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay.
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.
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.
Xue-Wen
XW
Li
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.
Johanna S
JS
Rees
From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Peng
P
Xue
From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China.
Hong
H
Zhang
the Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom.
Samir W
SW
Hamaia
the Department of Biological Sciences, DePaul University, Chicago, Illinois 60604, and.
Bailey
B
Sanderson
the Department of Biological Sciences, DePaul University, Chicago, Illinois 60604, and.
Phillip E
PE
Funk
the Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom.
Richard W
RW
Farndale
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.
Kathryn S
KS
Lilley
From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China, sarah.perrett@cantab.net.
Sarah
S
Perrett
From the National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China, the Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom, apj10@cam.ac.uk.
Antony P
AP
Jackson
D000818:Animals; D030161:Avian Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D001402:B-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D045744:Cell Line, Tumor; D002645:Chickens; D016023:Integrins / Q000276:immunology / Q000378:metabolism; D007519:Isoantigens / Q000276:immunology / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D021962:Membrane Microdomains / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding / Q000276:immunology; D020543:Proteome / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D014439:Tyramine / Q000737:chemistry
D030161:Avian Proteins; D016023:Integrins; D007519:Isoantigens; D020543:Proteome; D011947:Receptors, Antigen, B-Cell; D001710:Biotin; D014439:Tyramine
B Cell Receptor; Evi2a; Immunology; Integrin; Lymphocyte; Mass Spectrometry (MS); Proteomics; Raftlin; SILAC; chB6