Matthew J. Peirce

Expression Profiling of Lymphocyte Plasma Membrane Proteins

The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.

Themis2/ICB1 Is a Signaling Scaffold That Selectively Regulates Macrophage Toll-Like Receptor Signaling and Cytokine Production

Background Thymocyte expressed molecule involved in selection 1 (Themis1, SwissProt accession number Q8BGW0) is the recently characterised founder member of a novel family of proteins. A second member of this family, Themis2 (Q91YX0), also known as ICB1 (Induced on contact with basement membrane 1), remains unreported at the protein level despite microarray and EST databases reporting Themis2 mRNA expression in B cells and macrophages. Methodology/Principal Findings Here we characterise Themis2 protein for the first time and show that it acts as a macrophage signalling scaffold, exerting a receptor-, mediator- and signalling pathway-specific effect on TLR responses in RAW 264.7 macrophages. Themis2 over-expression enhanced the LPS-induced production of TNF but not IL-6 or Cox-2, nor TNF production induced by ligands for TLR2 (PAM3) or TLR3 (poly I∶C). Moreover, LPS-induced activation of the MAP kinases ERK and p38 was enhanced in cells over-expressing Themis2 whereas the activation of JNK, IRF3 or NF-κB p65, was unaffected. Depletion of Themis2 protein by RNA inteference inhibited LPS-induced TNF production in primary human macrophages demonstrating a requirement for Themis2 in this event. Themis2 was inducibly tyrosine phosphorylated upon LPS challenge and interacted with Lyn kinase (P25911), the Rho guanine nucleotide exchange factor, Vav (P27870), and the adaptor protein Grb2 (Q60631). Mutation of either tyrosine 660 or a proline-rich sequence (PPPRPPK) simultaneously interrupted this complex and reduced by approximately 50% the capacity of Themis2 to promote LPS-induced TNF production. Finally, Themis2 protein expression was induced during macrophage development from murine bone marrow precursors and was regulated by inflammatory stimuli both in vitro and in vivo. Conclusions/Significance We hypothesise that Themis2 may constitute a novel, physiological control point in macrophage inflammatory responses.

Themis2 Is Not Required for B Cell Development, Activation, and Antibody Responses

Themis1 is a protein implicated in transducing signals from the TCR. Mice deficient in Themis1 show a strong impairment in T cell selection in the thymus and defective T cell activation. The related Themis2 protein is expressed in B cells where it associates with signaling proteins Grb2 and Vav1, and is tyrosine phosphorylated after BCR stimulation. Thus, it has been proposed that Themis2 may transduce BCR signals, and hence play important roles in B cell development and activation. In this article, we show that Themis2 is expressed in all developing subsets of B cells, in mature follicular and marginal zone B cells, and in activated B cells, including germinal center B cells and plasma cells. In contrast, B lineage cells express no other Themis-family genes. Activation of B cells leads to reduced Themis2 expression, although it remains the only Themis-family protein expressed. To analyze the physiological function of Themis2, we generated a Themis2-deficient mouse strain. Surprisingly, we found that Themis2 is not required for B cell development, for activation, or for Ab responses either to model Ags or to influenza viral infection.

Proteomic analysis of the lymphocyte plasma membrane using cell surface biotinylation and solution-phase isoelectric focusing.

Plasma membrane (PM) proteins are of particular interest to cell biologists because of their role in transducing information from the external environment to the cell interior, and because of their potential as therapeutic targets. The hydrophobicity and large size of these proteins renders their analysis by conventional proteomic approaches using 2D-electrophoresis problematic, limiting our ability to evaluate alterations of cell surface architecture as a function of varying physiological, pathological, or developmental state.In this chapter, we describe a simple method for enrichment and separation of plasma membrane proteins, prior to their identification by tandem mass spectrometry. Cell surface proteins are labeled with biotin using a reagent which does not enter the cell, purified by differential centrifugation and then affinity captured with streptavidin-agarose beads, before separation by a combination of solution-phase isoelectric focusing, and gradient gel electrophoresis, resulting in highly enriched membrane protein fractions suitable for characterization by mass spectrometry. We discuss the application of this protocol to the semiquantitative comparison of the plasma membrane proteins from resting and activated murine lymphocytes.

Mapping Lymphocyte Plasma Membrane Proteins

The pathological importance of tumor necrosis factor (TNF)-alpha in rheumatoid arthritis (RA) is now widely accepted. Ex vivo data from synovial cell cultures suggest that direct cell contact between activated T-cells and macrophages may be an important driver of macrophage TNF-alpha production in the RA joint. However, the ligand/receptor pairs driving this cell contact signal remain obscure. One reason for this is that plasma membrane (PM) proteins are resistant to systematic analysis using traditional proteomic approaches. In this chapter we present a method for the enrichment and resolution of PM proteins from murine T-cell hybridomas as a prelude to identification by tandem mass spectrometry. We used cell surface biotinylation, differential centrifugation and subsequent streptavidin affinity capture, followed by solution phase iso-electric focussing and tandem mass spectrometry to identify 75 PM proteins and make semiquantitative comparisons of resting and activated cells. The method is applicable to a wide variety of cell types.