New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay

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.

Keywords: B Cell Receptor; Evi2a; Immunology; Integrin; Lymphocyte; Mass Spectrometry (MS); Proteomics; Raftlin; SILAC; chB6.

FIGURE 1.

Outline of the SPPLAT protocol. A, structure of the biotin-tyramide proximity labeling reagent. B, principle of the method. The antibody-directed targeting of HRP to a surface protein of interest, followed by brief labeling with biotin-tyramide enables proteins in the immediate vicinity of the target to be biotinylated. These are isolated by incubation of the cell lysate with streptavidin-agarose (SA), and elution with reducing agent.

FIGURE 2.

SPPLAT analysis of the BCR-induced clusters in DT40 cells. A, co-localization of BCR and deposited biotin. Cells were preincubated with HRP-conjugated anti-chicken IgM and tyramide biotinylated. The distribution of BCR and deposited biotin was detected using FITC-conjugated anti-goat IgG and Alexa 568-labeled avidin as described under “Experimental Procedures.” Three representative images are shown with corresponding images rendered as pseudo-three-dimensional. Bar = 5 μm. B, tyramide-biotinylated cells were stripped of biotin by treatment with impermeant reducing agent as described under “Experimental Procedures.” Cells were stained with FITC-conjugated anti-goat IgG to detect BCR, and with Texas Red-conjugated avidin to detect remaining biotin. Bar = 5 μm.

FIGURE 3.

Summary of quantitative SILAC data. A, scatter plot showing the isotope ratios for each protein quantitatively identified in both independent SILAC experiments. The organelle locations of the proteins are indicated. Broken lines represent the median value plus 1 S.D. for each data set. B, proteins chB6 and CDC42 were immunoprecipitated from specifically labeled (S) and nonspecifically labeled cells (NS), then separately probed with antibodies against chB6 or CDC42 and with HRP-streptavidin to detect biotin labeling as indicated. These samples were run under non-reducing conditions. C, immunofluorescence image showing accumulation of mitochondria underneath the BCR surface cluster. Mitochondria were detected using MitoTracker staining, and BCR was detected by immunofluorescence as described under “Experimental Procedures.” Bar = 5 μm.

FIGURE 4.

BCR cross-linking drives the close association of BCR with raftlin. A, PLA signal between raftlin and BCR in cells cross-linked with anti-BCR to induce BCR clustering. Bar = 10 μm. B, control cells fixed before anti-BCR addition showing no PLA stain in the absence of BCR clustering. Bar = 10 μm. C, quantification of data shown in A and B.

FIGURE 5.

The chicken allotypic marker chB6 partitions into lipid rafts and becomes associated with BCR following BCR cross-linking. A, lipid raft and non-raft fractions were isolated from the DT40 plasma membrane (∼10⁶ cells), separated by SDS-PAGE under reducing conditions, and blotted for chB6 expression as described under “Experimental Procedures.” B, DT40 cells were incubated with anti-chB6 for 20 min on ice, and double stained with anti-mouse FITC and Alexa 594 cholera toxin B. Cells were visualized by epifluorescence microscopy. C, PLA signal between chB6 and BCR in cells cross-linked with anti-BCR to induce BCR clustering. Bar = 10 μm. D, control cells fixed before anti-BCR addition showing no PLA stain in the absence of BCR clustering. Bar = 10 μm. E, quantification of data shown in C and D.

FIGURE 6.

Putative functional regions and regions of Ig domain sequence similarity identified in chB6. A, relationship between amino acid sequence and putative functional regions. Red sequence: ER targeting signal. Blue sequence: transmembrane domain identified using SMART. Orange underline: putative palmitoylation site. Black underline: putative SH3-domain binding site. Purple line: region of sequence similarity to CD2 detected by PSI-BLAST. Dark green line: regions of sequence similarity to Ig domains detected by SMART. Light green lines: regions of sequence similarity to Ig domains detected with FUGUE. Specific proteins identified by FUGUE are: (i) growth arrest specific protein (Z score 16.23); (ii) junction adhesion molecule (Z score 15.31); (iii) high affinity Fc receptor (Z score 14.55); (iv) CD43 (Z score 14.34); (v) SLAMf6 (Z score 11.76); (vi) T lymphocyte activation antigen CD80. For further details of analysis methods see “Results.” B, schematic summary of putative functional regions in chB6.

FIGURE 7.

Cross-linking the BCR and the chB6 alloantigen in DT40 cells stimulates integrin-mediated cell adhesion. A, co-localization of cross-linked BCR with β1 integrin. Bar = 10 μm. B, RT-PCR for α3 integrin, α4 integrin, and actin. C and D, cell binding to the integrin substrates. Cells were preincubated with goat anti-chicken IgM or non-immune goat antibody (C), or with mouse anti-chB6 or non-immune mouse antibody (D), as described under “Experimental Procedures.” Cells were incubated with substrate-coated wells (laminin, gelatin, or albumin), and binding was assayed as the shift in peak wavelength value (PWV) after a 2-h incubation, using the SRU Biosystems BIND Explorer system. Values are mean ± S.D. of 3 replicates. The p values for experiments with and without antibody cross-linking are indicated.

FIGURE 8.

Summary of the key proteins identified by SPPLAT. Proteins with specific/nonspecific SILAC isotope ratios consistently greater than 1 S.D. above the median for both SILAC replicates are indicated as schematics, and labeled in blue. Additional key cytosolic proteins identified by SPPLAT and discussed in the text are indicated in green. Black arrows indicate known interactions between proteins.