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      New Insights into the DT40 B Cell Receptor Cluster Using a Proteomic Proximity Labeling Assay*

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          Abstract

          Background: B cell receptor (BCR) clusters modulate BCR signaling in B-lymphocytes.

          Results: We used a quantitative proteomic proximity assay to analyze the BCR cluster in DT40 cells.

          Conclusion: Our proximity labeling assay identified novel components of the BCR cluster linked to integrin signaling.

          Significance: We provide new insights into BCR assembly and identify new and unexpected targets for further functional analysis.

          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.

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          Most cited references 62

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          FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties.

          FUGUE, a program for recognizing distant homologues by sequence-structure comparison (http://www-cryst.bioc.cam.ac.uk/fugue/), has three key features. (1) Improved environment-specific substitution tables. Substitutions of an amino acid in a protein structure are constrained by its local structural environment, which can be defined in terms of secondary structure, solvent accessibility, and hydrogen bonding status. The environment-specific substitution tables have been derived from structural alignments in the HOMSTRAD database (http://www-cryst.bioc. cam.ac.uk/homstrad/). (2) Automatic selection of alignment algorithm with detailed structure-dependent gap penalties. FUGUE uses the global-local algorithm to align a sequence-structure pair when they greatly differ in length and uses the global algorithm in other cases. The gap penalty at each position of the structure is determined according to its solvent accessibility, its position relative to the secondary structure elements (SSEs) and the conservation of the SSEs. (3) Combined information from both multiple sequences and multiple structures. FUGUE is designed to align multiple sequences against multiple structures to enrich the conservation/variation information. We demonstrate that the combination of these three key features implemented in FUGUE improves both homology recognition performance and alignment accuracy. Copyright 2001 Academic Press.
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            Iterated profile searches with PSI-BLAST--a tool for discovery in protein databases.

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              Increased ratio of targeted to random integration after transfection of chicken B cell lines.

              Constructs of four different genetic loci were transfected into the avian leukosis virus-induced chicken B cell line DT40, which continues diversification of its rearranged light chain immunoglobulin gene by gene conversion. Analysis of stable transfectants revealed an unexpectedly high frequency of targeted integration into the homologous gene loci of DT40. Transcriptional activity of the target gene locus is not required, since a construct of the untranscribed ovalbumin gene also integrated predominantly by homologous recombination. A construct derived from the beta-actin locus was transfected into other chicken cell lines to determine the cell type specificity of the phenomenon. Targeted integration still occurred at high frequency in two other B cell lines that do not have the gene conversion activity. However, the ratios of targeted to random integration were reduced by at least one order of magnitude in three non-B cell lines.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                23 May 2014
                4 April 2014
                4 April 2014
                : 289
                : 21
                : 14434-14447
                Affiliations
                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,
                the []Cambridge Centre for Proteomics, Tennis Court Road, University of Cambridge, Cambridge CB2 1QR, United Kingdom,
                the [** ]Department of Biological Sciences, DePaul University, Chicago, Illinois 60604, and
                the [§ ]University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China
                Author notes
                [4 ] To whom correspondence may be addressed. Tel.: 86-10-64889870; E-mail: sarah.perrett@ 123456cantab.net .
                [5 ] Supported by a Chinese Academy of Sciences Visiting Professorship for Senior International Scientists Grant 2010T1S11. To whom correspondence may be addressed: Dept. of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QW, United Kingdom. Tel.: 44-1223-765951; E-mail: apj10@ 123456cam.ac.uk .
                [1]

                Supported by Grants BB/J021091 and H024085/1 from the Biotechnology and Biological Sciences Research Council (UK).

                [2]

                Supported by Grant G0500707 from the Medical Research Council (UK) and Grant 094470/Z/10/Z from the Wellcome Trust.

                [3]

                Supported by the DePaul University Research Council.

                Article
                M113.529578
                10.1074/jbc.M113.529578
                4031500
                24706754
                © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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                Creative Commons Attribution Unported License applies to Author Choice Articles

                Product
                Categories
                Immunology

                Biochemistry

                chb6, silac, raftlin, evi2a, b cell receptor, proteomics, mass spectrometry (ms), lymphocyte, integrin, immunology

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