A simple detection method for low-affinity membrane protein interactions by baculoviral display.

BACKGROUND: Membrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV). In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system. METHODOLOGY/PRINCIPAL FINDINGS: We infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA). Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L), and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand (GITRL). Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displaying BV and anti-gp64 antibody. CONCLUSIONS: We found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds

Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9

Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing. G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and is essential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals, called GLP/Eu-HMTase1. Here we show that GLP is also important for H3-K9 methylation of mouse euchromatin. GLP-deficiency led to embryonic lethality, a severe reduction of H3-K9 mono- and dimethylation, the induction of Mage-a gene expression, and HP1 relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency. Furthermore, we show that G9a and GLP formed a stoichiometric heteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo, likely through the formation of higher-order heteromeric complexes

Expression of heterologous membrane proteins in BV fraction confirmed by Western blot.

<p>Ten micrograms of each of the BV samples expressing CD2 or its ligand CD58 (A), CD40 or CD40L (B), and GITR or GITRL (C) were loaded in each lane. The blotted membranes were immuno-stained with either anti-FLAG or anti-HA antibodies according to the attached tag as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004024#s4" target="_blank">Materials and Methods</a>. The positions of the molecular mass marker proteins are indicated on the <i>left</i>.</p

Expression cloning of human CD2 by using CD58-displaying BV as the probe.

<p>(A) Enrichment of human CD2-positive cells from BaF/3 cells transfected with a human T cell cDNA library. The staining of cells with an FITC-labeled anti humanCD2 monoclonal antibody is shown. The thin line indicates cells incubated without anti-CD2 antibody. (B) Binding of anti-human CD2 antibody and CD58-displaying BV to BaF/3 cells isolated by magnetic sorting with CD58-BV. After the 3rd magnetic sorting and subcloning, cells were stained with an FITC-labeled anti-human CD2 monoclonal antibody and CD58-BV plus biotinylated anti-gp64 antibody and PE-streptavidin. Staining of a representative of three clones is shown.</p

Specific binding of ligand-displaying BV to cells expressing receptor.

<p>(A) Schematic view. X corresponds to CD2 (for B), CD40 (for C), or GITR (for D). Y corresponds to CD58 (for B), CD40L (for C), or GITRL (for D). (B) CD58-displaying BV binds to CD2-positive (top), but not to CD2-negative Jurkat cells (bottom). Left: binding of an anti-CD2 monoclonal antibody (clone RPA-2.10) plus a PE-anti mouse Ig-κ chain antibody. Right: binding of CD58-displaying BV plus an anti-gp64 antibody (clone A0505A) and a PE-anti mouse Ig-κ chain antibody. The thin line indicates cells incubated with the 2nd antibody only. (C) CD40-dependent binding of CD40L-displaying BV to mouse splenic B cells. BALB/c mouse splenocytes were incubated with CD40L-displaying BV and a biotinylated anti-gp64 monoclonal antibody (clone B8147A), and then were stained with FITC-labeled streptavidin and a PE-labeled anti-B220 antibody. CD40L-BV binding to the B cells (shown in the center lower panel) was blocked by pre-incubation with an anti-mouse CD40 monoclonal antibody (clone HM40-3) (the right upper panel) but not by control hamster IgG (right lower panel). (D) Binding of GITRL-displaying BV to GITR-positive cells. GITR-expressing mouse T cell hybridoma 18.3.5 cells were incubated with GITRL-displaying BV (right) or wild-type BV (left). Binding of BV was detected with a biotinylated anti-gp64 monoclonal antibody B8147A and PE-streptavidin. The thin line indicates cells incubated without BV.</p

Detection of specific interaction between CD40 and CD40L or GITR and GITRL displayed on BV.

<p>(A) Left panel: binding of CD40L-BV to the immobilized CD40-BV. Wells were coated with 0.25 µg/well of CD40-BV or wild-type BV. Binding was detected by using a biotinylated anti-mouse CD40L monoclonal antibody (clone MR1) and HRP-streptavidin. Right panel: binding of CD40-BV to the immobilized CD40L-BV. Wells were coated with 1 µg/well of CD40L-BV or wild-type BV. Binding was detected by using an anti-mouse CD40 monoclonal antibody (clone 3/23) and HRP-anti rat IgG+IgM. (B) Left panel: binding of GITRL-BV to the immobilized GITR-BV. Wells were coated with 1 µg/well of GITR-BV or wild-type BV. Binding was detected by using an anti-mouse GITRL monoclonal antibody (clone YGL386) and HRP-anti rat IgG+IgM. Right panel: binding of GITR-BV to the immobilized GITRL-BV. Wells were coated with 1 µg/well of GITRL-BV or wild-type BV. Binding was detected by using an anti-mouse GITR monoclonal antibody (clone DTA-1) and HRP-anti rat IgG+IgM. Filled circles indicate binding to BV displaying respective receptors (or ligands). Open circles indicate binding to wild-type BV.</p

Detection of specific interaction between CD2 and CD58 individually displayed on BV.

<p>(A) Schematic view of the ELISA system. Details are described in the text. X and Y correspond to CD2 and CD58 (or vice versa for C) respectively. (B) Left panel; binding of CD58-BV to immobilized CD2-BV (filled circles) or wild-type BV (open circles). Right panel: Blocking of CD58-BV binding to the plate-bound CD2-BV by pre-incubation of wells with an anti-CD2 antibody (filled circles) or control mouse IgG1 (open circles). (C) Left panel: binding of CD2-BV to the immobilized CD58-BV (filled circles) or wild-type BV (open circles). Right panel: blocking of CD2-BV binding of the plate-bound CD58-BV by pre-incubation of wells with an anti-CD58 antibody (filled circles) or control mouse IgG2a (open circles). Each well was coated with 0.5 µg of BV. Each data point represents detection in triplicate (error bar, 1 s.d.).</p