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Articles by Wolfgang W. A. Schamel in JoVE
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Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates
Gina J. Fiala1,2,3, Wolfgang W. A. Schamel*2,3, Britta Blumenthal*3
1Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, 2Centre for Biological Signalling Studies (bioss) and Biology III, Faculty of Biology, University of Freiburg, 3Department of Molecular Immunology, Max-Planck-Institute of Immunology and Epigenetics
In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.
Other articles by Wolfgang W. A. Schamel on PubMed
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A High-molecular-weight Complex of Membrane Proteins BAP29/BAP31 is Involved in the Retention of Membrane-bound IgD in the Endoplasmic Reticulum
Proceedings of the National Academy of Sciences of the United States of America.
Aug, 2003 |
Pubmed ID: 12886015 B cell antigen receptors (BCRs) are multimeric transmembrane protein complexes comprising membrane-bound immunoglobulins (mIgs) and Ig-alpha/Ig-beta heterodimers. In most cases, transport of mIgs from the endoplasmic reticulum (ER) to the cell surface requires assembly with the Ig-alpha/Ig-beta subunits. In addition to Ig-alpha/Ig-beta, mIg molecules also bind two ER-resident membrane proteins, BAP29 and BAP31, and the chaperone heavy chain binding protein (BiP). In this article, we show that neither Ig-alpha/Ig-beta nor BAP29/BAP31 nor BiP bind simultaneously to the same mIgD molecule. Blue native PAGE revealed that only a minor fraction of intracellular mIgD is associated with high-molecular-weight BAP29/BAP31 complexes. BAP-binding to mIgs was found to correlate with ER retention of chimeric mIgD molecules. On high-level expression in Drosophila melanogaster S2 cells, mIgD molecules were detected on the cell surface in the absence of Ig-alpha/Ig-beta. This aberrant transport was prevented by coexpression of BAP29 and BAP31. Thus, BAP complexes contribute to ER retention of mIg complexes that are not bound to Ig-alpha/Ig-beta. Furthermore, the mechanism of ER retention of both BAP31 and mIgD is not through retrieval from a post-ER compartment, but true ER retention. In conclusion, BAP29 and BAP31 might be the long sought after retention proteins and/or chaperones that act on transmembrane regions of various proteins.
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Biochemical Differences in the Alphabeta T Cell Receptor.CD3 Surface Complex Between CD8+ and CD4+ Human Mature T Lymphocytes
The Journal of Biological Chemistry.
Jun, 2004 |
Pubmed ID: 15060077 We have reported the existence of biochemical and conformational differences in the alphabeta T cell receptor (TCR) complex between CD4(+) and CD8(+) CD3gamma-deficient (gamma(-)) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (gamma(+)) individuals. Surface TCR.CD3 components from CD4(+) gamma(-) T cells, other than CD3gamma, were detectable and similar in size to CD4(+) gamma(+) controls. Their native TCR.CD3 complex was also similar to CD4(+) gamma(+) controls, except for an alphabeta(deltaepsilon)(2)zeta(2) instead of an alphabetagammaepsilondeltaepsilonzeta(2) stoichiometry. In contrast, the surface TCRalpha, TCRbeta, and CD3delta chains of CD8(+) gamma(-) T cells did not possess their usual sizes. Using confocal immunofluorescence, TCRalpha was hardly detectable in CD8(+) gamma(-) T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR.CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (gamma(+)) donors, we performed a broad epitopic scan. In contrast to all other TCR.CD3-specific monoclonal antibodies, RW2-8C8 stained CD8(+) better than it did CD4(+) T cells, and the difference was dependent on glycosylation of the TCR.CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8(+) T cells was shown to be more dependent on lipid raft integrity than that of CD4(+) T cells. Finally, immunoprecipitation studies on purified primary CD4(+) and CD8(+) T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR.CD3 from CD4(+) and CD8(+) wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.
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Subproteomic Analysis of Metal-interacting Proteins in Human B Cells
Proteomics.
Sep, 2005 |
Pubmed ID: 16097032 Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, T cell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific T cell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.
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Two Dimensional Blue Native-/SDS-PAGE Analysis of SLP Family Adaptor Protein Complexes
Immunology Letters.
Apr, 2006 |
Pubmed ID: 16356554 SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.
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Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for the Identification and Analysis of Multiprotein Complexes
Science's STKE : Signal Transduction Knowledge Environment.
Jul, 2006 |
Pubmed ID: 16868305 Multiprotein complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCs--for example, multisubunit receptors or transcription factors; and (ii) signal-induced, transient, low copy number MPCs--for example, complexes that form upon binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a separation method with a higher resolution than gel filtration or sucrose density ultracentrifugation that can be used to analyze abundant, stable MPCs from 10 kD to 10 MD. In contrast to immunoprecipitation and two-hybrid approaches, it allows the determination of the size, the relative abundance, and the subunit composition of an MPC. In addition, it shows how many different complexes exist that share a common subunit, whether free monomeric forms of individual subunits exist, and whether these parameters change upon cell stimulation. Here, we give a detailed protocol for the separation of MPCs from total cellular lysates or of prepurified MPCs by one-dimensional BN-PAGE or by two-dimensional BN-PAGE and SDS-PAGE.
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Overlapping Functions of Human CD3delta and Mouse CD3gamma in Alphabeta T-cell Development Revealed in a Humanized CD3gamma-mouse
Blood.
Nov, 2006 |
Pubmed ID: 16888097 Humans lacking the CD3gamma subunit of the pre-TCR and TCR complexes exhibit a mild alphabeta T lymphopenia, but have normal T cells. By contrast, CD3gamma-deficient mice are almost devoid of mature alphabeta T cells due to an early block of intrathymic development at the CD4(-)CD8(-) double-negative (DN) stage. This suggests that in humans but not in mice, the highly related CD3delta chain replaces CD3gamma during alphabeta T-cell development. To determine whether human CD3delta (hCD3delta) functions in a similar manner in the mouse in the absence of CD3gamma, we introduced an hCD3delta transgene in mice that were deficient for both CD3delta and CD3gamma, in which thymocyte development is completely arrested at the DN stage. Expression of hCD3delta efficiently supported pre-TCR-mediated progression from the DN to the CD4(+)CD8(+) double-positive (DP) stage. However, alphabetaTCR-mediated positive and negative thymocyte selection was less efficient than in wild-type mice, which correlated with a marked attenuation of TCR-mediated signaling. Of note, murine CD3gamma-deficient TCR complexes that had incorporated hCD3delta displayed abnormalities in structural stability resembling those of T cells from CD3gamma-deficient humans. Taken together, these data demonstrate that CD3delta and CD3gamma play a different role in humans and mice in pre-TCR and TCR function during alphabeta T-cell development.
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A Native Antibody-based Mobility-shift Technique (NAMOS-assay) to Determine the Stoichiometry of Multiprotein Complexes
Journal of Immunological Methods.
Jul, 2007 |
Pubmed ID: 17568608 Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view of protein interaction networks and prerequisite for a molecular understanding of how a certain MPC functions. Here, we present a technique utilizing monoclonal subunit-specific antibodies for an electrophoretic immunoshift assay in Blue Native-gels (NAMOS-assay), which allows the determination of the stoichiometry of MPCs. First, we use the B cell antigen receptor as a model MPC whose stoichiometry is known, confirming the HC(2)LC(2)Igalpha/beta(1) stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of alphabetaepsilon(2)gammadeltazeta(2). We then show that the NAMOS-assay does not require purified MPCs, since it can determine the stoichiometry of an MPC in cell lysates. The NAMOS-assay is also compatible with use of epitope tags appended to the protein of interest, as e.g. the widely used HA-tag, and anti-epitope antibodies for the assay. Given its general applicability, this method has a wide potential for MPC research.
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Different Composition of the Human and the Mouse Gammadelta T Cell Receptor Explains Different Phenotypes of CD3gamma and CD3delta Immunodeficiencies
The Journal of Experimental Medicine.
Oct, 2007 |
Pubmed ID: 17923503 The gammadelta T cell receptor for antigen (TCR) comprises the clonotypic TCRgammadelta, the CD3 (CD3gammaepsilon and/or CD3deltaepsilon), and the zetazeta dimers. gammadelta T cells do not develop in CD3gamma-deficient mice, whereas human patients lacking CD3gamma have abundant peripheral blood gammadelta T cells expressing high gammadelta TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human gammadelta TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR-specific antibodies. The gammadelta TCR isolated in digitonin from primary and cultured human gammadelta T cells includes CD3delta, with a TCRgammadeltaCD3epsilon(2)deltagammazeta(2) stoichiometry. In CD3gamma-deficient patients, this may allow substitution of CD3gamma by the CD3delta chain and thereby support gammadelta T cell development. In contrast, the mouse gammadelta TCR does not incorporate CD3delta and has a TCRgammadeltaCD3epsilon(2)gamma(2)zeta(2) stoichiometry. CD3gamma-deficient mice exhibit a block in gammadelta T cell development. A human, but not a mouse, CD3delta transgene rescues gammadelta T cell development in mice lacking both mouse CD3delta and CD3gamma chains. This suggests important structural and/or functional differences between human and mouse CD3delta chains during gammadelta T cell development. Collectively, our results indicate that the different gammadelta T cell phenotypes between CD3gamma-deficient humans and mice can be explained by differences in their gammadelta TCR composition.
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The Extracellular Part of Zeta is Buried in the T Cell Antigen Receptor Complex
Immunology Letters.
Mar, 2008 |
Pubmed ID: 18215426 The zeta chain is a key component of the T cell antigen receptor (TCR-CD3) complex, required for the expression of the receptor on the cell surface. It contains an extremely small extracellular (EC) part of nine amino acids. Interestingly, the length, but not the sequence, of the zeta EC has been highly conserved through evolution. Here, we examined the effect of increasing the length of human zeta EC on TCR-CD3 assembly and surface expression. Appending a 30 kDa polypeptide to the N-terminus of zeta completely abolished assembly and transport of the TCR-CD3 to the cell surface. Addition of only 17 amino acids, including the HA-tag (HAzeta), strongly reduced the efficiency of TCR-CD3 assembly and led to reduced expression on the surface, suggesting that the short zeta EC region is located within the receptor complex. In Blue Native gels (BN-PAGE) these receptors had a normal size, indicating that they have a stoichiometry of alphabetagammaepsilondeltaepsilonzetazeta. In resting TCR-CD3s the HA-tag, and thus the zeta EC region, was not accessible for anti-HA antibody binding, demonstrating that it was indeed buried in a cavity within the receptor complex. However, prolonged stimulation with antigen permitted the access of the anti-HA antibody, thus suggesting that stimulation led to architectural changes in the TCR-CD3.
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Segregation Models
Advances in Experimental Medicine and Biology.
2008 |
Pubmed ID: 19065785 Many antigen receptors of the immune system belong to the family of multichain immune recognition receptors (MIRRs). Binding of ligand (antigen) to MIRR results in receptor phosphorylation, triggering downstream signaling pathways and cellular activation. How ligand binding induces this phosphorylation is not yet understood. In this Chapter, we discuss two models exploring the possibility that kinases and phosphatases are intermingled on the cell surface. Thus, in resting state, MIRR phosphorylation is counteracted by dephosphorylation. Upon ligand binding, phosphatases are removed from the vicinity of the MIRR and kinases, such that phosphorylated MIRRs can accumulate (segregation models). In the first model, clustering of MIRRs by multivalent ligand leads to their concentration in lipid rafts where kinases, but not phosphatases, are localized. The second model takes into account that the MIRR-ligandpair needs dose apposition of the two cell membranes, in cases where ligand is presented by an antigen-presenting cell. The intermembrane distance is too small to accommodate transmembrane phosphatases, which possess large ectodomains. Thus, phosphatases become spatially separated from the MIRRs and kinases (kinetic-segregation model).
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Target-dependent T-cell Activation by Coligation with a PSMA X CD3 Diabody Induces Lysis of Prostate Cancer Cells
Journal of Immunotherapy (Hagerstown, Md. : 1997).
Jul-Aug, 2009 |
Pubmed ID: 19483653 Recently, we have described a bispecific PSMA x CD3 diabody with one binding site for the T-cell antigen receptor (TCR-CD3) and another for the Prostate Specific Membrane Antigen (PSMA). It effectively eliminates human prostate cancer cells by redirecting T-lymphocytes in vitro and in vivo. Here, we show that activation of the T-cells and killing of the tumor cells, only occurred when the T-cells were coincubated with PSMA-positive tumor cells and the PSMA x CD3 diabody. Both CD4+ and CD8+ human T-lymphocytes were activated. Surprisingly, they were equally potent in their cytotoxic activity, proliferation, and up-regulation of activation markers. Both, CD4+, and CD8+ T-cells mainly used the perforin-granzyme- based pathway and to a somewhat lesser extent the FasL pathway to lyse tumor cells. When Jurkat T-cells were stimulated with the diabody alone, the TCR-CD3 was not triggered. In contrast, when the diabody was clustered with a secondary antibody the TCR-CD3 was stimulated as detected by Ca(2+)-influx and Erk, IkappaB, and linker of activated T-cell phosphorylation. Clustering of the diabody could also be achieved by the dimeric PSMA antigen expressed on tumor cells. Thus, although the diabody binds to all T-cells, only those in contact with PSMA-expressing cancer cells are activated. In conclusion, the PSMA x CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer.
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Low-valency, but Not Monovalent, Antigens Trigger the B-cell Antigen Receptor (BCR)
International Immunology.
Mar, 2010 |
Pubmed ID: 20145007 Antigen binding to the B-cell antigen receptor (BCR) leads to receptor triggering and B-lymphocyte activation. Here, we have probed the molecular requirements for BCR triggering in primary murine B cells using a set of defined soluble haptenated peptides. Bi- and trivalent haptens activated the BCR, as measured by protein phosphorylation, Ca(2+) influx, BCR down-modulation and CD69, CD86 and MHC class II up-regulation. In contrast, four distinct monovalent haptens were ineffective. Next, we used two different anti-idiotypic antibodies, which bind to the antigen-combining site of the BCR. Again, monovalent Fab fragments were ineffective, whereas bivalent antibodies could stimulate the BCR. These findings are compatible with ligand-induced clustering of monomeric BCRs or re-organization of BCR complexes within pre-formed BCR oligomers. Lastly, an increase in the valency of the haptenated peptides improved the activation potential, whereas variations in the distance between two haptens had no effect. This finding contributes to understand how the immune system can efficiently recognize structurally diverse antigens but still discriminate between foreign and self.
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Innate, Antigen-independent Role for T Cells in the Activation of the Immune System by Propionibacterium Acnes
European Journal of Immunology.
Sep, 2010 |
Pubmed ID: 20690177 Propionibacterium acnes is a human commensal but also an opportunistic pathogen. In mice, P. acnes exerts strong immunomodulatory activities, including formation of intrahepatic granulomas and induction of LPS hypersensitivity. These activities are dependent on P. acnes recognition via TLR9 and subsequent IL-12-mediated IFN-gamma production. We show that P. acnes elicits IL-12p40 and p35 mRNA expression in macrophages, and IFN-gamma mRNA in liver CD4(+) T cells and NK cells. After priming with P. acnes, CD4(+) T cells serve as the major IFN-gamma mRNA source. In the absence of CD4(+) T cells, CD8(+) T cells (regardless of antigenic specificity) or NK cells can produce sufficient IFN-gamma to induce the P. acnes-driven immune effects. Moreover, in the absence of alpha beta T cells, gamma delta T cells also enable the development of strongly enhanced TNF-alpha and IFN-gamma responses to LPS and intrahepatic granuloma formation. Thus, under microbial pressure, different T-cell types, independent of their antigen specificity, exert NK-cell-like functions, which contribute decisively to the activation of the innate immune system.
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A Leaky Mutation in CD3D Differentially Affects αβ and γδ T Cells and Leads to a Tαβ-Tγδ+B+NK+ Human SCID
The Journal of Clinical Investigation.
Oct, 2011 |
Pubmed ID: 21926461 T cells recognize antigens via their cell surface TCR and are classified as either αβ or γδ depending on the variable chains in their TCR, α and β or γ and δ, respectively. Both αβ and γδ TCRs also contain several invariant chains, including CD3δ, which support surface TCR expression and transduce the TCR signal. Mutations in variable chains would be expected to affect a single T cell lineage, while mutations in the invariant chains would affect all T cells. Consistent with this, all CD3δ-deficient patients described to date showed a complete block in T cell development. However, CD3δ-KO mice have an αβ T cell-specific defect. Here, we report 2 unrelated cases of SCID with a selective block in αβ but not in γδ T cell development, associated with a new splicing mutation in the CD3D gene. The patients' T cells showed reduced CD3D transcripts, CD3δ proteins, surface TCR, and early TCR signaling. Their lymph nodes showed severe T cell depletion, recent thymus emigrants in peripheral blood were strongly decreased, and the scant αβ T cells were oligoclonal. T cell-dependent B cell functions were also impaired, despite the presence of normal B cell numbers. Strikingly, despite the specific loss of αβ T cells, surface TCR expression was more reduced in γδ than in αβ T cells. Analysis of individuals with this CD3D mutation thus demonstrates the contrasting CD3δ requirements for αβ versus γδ T cell development and TCR expression in humans and highlights the diagnostic and clinical relevance of studying both TCR isotypes when a T cell defect is suspected.
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