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-displayng 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.


Introduction
Cell surface proteins mediate intercellular recognition and signal transduction. In the immune system, the interaction of an antigenspecific T cell with an antigen presenting cell (APC) such as a B cell or a dendritic cell (DC) is mediated by the engagement of a diverse array of receptors on one cell, with ligands (or counter receptors) on the opposing cell, both of which are membrane proteins [1]. Such interactions between membrane proteins lead to various immune responses such as cytokine secretion, antibody production or the killing of target cells. In many cases, however, it has been difficult to detect these receptor-ligand interactions by conventional techniques, because the affinity of interaction between these membrane proteins is generally low (Kd,1 mM) [2]. It is likely that oligomerization of these proteins on the cell surface increases the avidity and stabilizes the interaction. Since it is difficult to reconstitute such oligomerization using the soluble monomeric form of these transmembrane proteins, several different systems have been attempted. One is the fusion of extracellular domain of membrane proteins and the Fc portion of immunoglobulin [3,4]. Another is the engraftment of chelator-lipid liposomes with the C-terminal hexahistidine-tagged extracellular domain of membrane proteins [5]. However, some membrane proteins may not retain the proper conformations required to bind ligand (or receptor) after such manipulations.
To detect membrane protein interaction, heterotypic cell adhesion assay has also been utilized. In this assay, two different types of cells individually expressing receptors or ligands on their surfaces are mixed, and heterotypic binding cells are detected either by microscopy or by labeling with a radioisotope or fluorochrome [6][7][8]. While this system allows detection of the interaction of native transmembrane proteins and their oligomers, endogenous receptor or other proteins may affect assays.
Meanwhile, there is accumulating evidence that heterologous membrane proteins are displayed on the extracellular baculovirus particles (budded virus, BV) (reviewed in [9]). We and other investigators have reported that membrane proteins such as cell surface receptors [10][11][12], a transporter [13], or enzymes [12,14] express on BV in biologically active form. Furthermore, the coexpression of multiple proteins results in the formation of functional protein complex on BV, as shown by the complex between G protein-coupled receptor and heterotrimeric G protein subunits [11] as well as the four-protein complex of c-secretase [14]. Since Sf9 insect cells, the host of baculovirus, are essentially free of the homologues of mammalian immune receptors or their ligands, this BV display system holds promise for its ability to provide a low background environment which would enable detection of the interaction between receptors and ligands.
In this study, we attempted to further utilize this BV display method to develop a system for detecting interactions between receptors and ligands, both of which are membrane proteins.

Detection of receptor-ligand interaction between membrane proteins displayed on BV
To test the application of the BV display system, we selected three representative membrane receptor-ligand pairs (CD2-CD58 [2], CD40-CD40 ligand [15], and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand [16]), all of which are important for the interaction and activation of immune cells. We infected Sf9 cells with recombinant baculoviruses, each containing human CD2, CD58 (CD2 ligand), mouse CD40, CD40 ligand (CD40L), mouse GITR, or GITR ligand (GITRL) cDNA, and recovered the BV fractions. Immunoblot analysis using antibodies against epitope tags confirmed the expression of these membrane proteins in the BV fractions ( Fig. 1). We sought to detect receptorligand interaction displayed on BV by ELISA system (illustrated in Fig. 2A). After CD2 (receptor)-displaying BV was immobilized in ELISA plate wells, CD58 (ligand)-displaying BV was added to the wells. We detected the binding of CD58-BV to the wells coated with CD2-BV by using a CD58-specific antibody ( Fig. 2B left panel). The binding of CD58-BV was dependent on CD2 because only minimal background binding to the wells coated with wild type BV was detected ( Fig. 2B left panel). Furthermore, preincubation of CD2-BV-coated wells with anti-CD2 antibody blocked the binding of CD58-BV ( Fig. 2B right panel). Similar specific binding was observed with the opposite combination, i.e., plate-bound CD58-BV and CD2-BV in solution (Fig. 2C). Specific interactions were also detected with mouse CD40 and CD40L ( Fig. 3A), and mouse GITR and GITRL (Fig. 3B). The interactions of these proteins were blocked by respective antibodies (data not shown).
Ligand-displaying BVs bind to receptor proteins expressed on the cell surface Next, we attempted to utilize the ligand protein-displaying BV as a tool to detect receptor expression on the cell surface. To this end, we detected the binding of BV to cells by flowcytometer using an antibody specific to baculoviral envelope protein gp64 and a fluorochrome-conjugated secondary antibody (illustrated in Fig. 4A). As shown in Fig. 4B, CD58-displaying BV bound to CD2-positive, but not CD2-negative Jurkat cells, a human T cell leukemia cell line. We also observed the binding of mouse CD40Ldisplaying BV to mouse splenic B cells, which express CD40. This binding was blocked by an anti-mouse CD40 monoclonal antibody (Fig. 4C). Furthermore, GITRL-displaying BV bound to GITR-expressing T cell hybridoma 18.3.5 cells (Fig. 4D).

Application to expression cloning
Our results prompted us to explore the possibility of utilizing BV displaying membrane proteins as the probe for expression cloning of receptor (or ligand) cDNA. To this end, we transfected BaF/3 cells with a human T cell cDNA library using a retroviral transduction system. Cells bound to CD58-displaying BV were selected with an anti-viral gp64 antibody plus secondary antibodycoated magnetic beads. After magnetic sorting of the BV-bound cells three times, cells expressing human CD2 were enriched (Fig. 5A). The recovered cells were cloned by limiting dilution. PCR of cloned BaF/3 cell genomic DNA with retroviral vectorderived primers amplified the human CD2 cDNA sequence. Furthermore, flowcytometric analysis confirmed that these clones expressed human CD2 and that CD58-BV bound to these cells (Fig. 5B).

Discussion
Functional membrane proteins such as cell surface receptors, transporters, or enzymes, have been shown to heterologously express on BV [9-14, reviewed in 9]. In this study, we further applied this BV display system in an effort to detect interactions between membrane receptors and ligands in immune cells. We infected Sf9 cells with recombinant baculoviruses, individually encoding CD2, CD58, CD40, CD40L, GITR or GITRL, and recovered the BV fractions expressing these membrane proteins. We observed specific binding of CD58 (ligand)-displaying BV to CD2 (receptor)-displaying BV by ELISA (Fig. 2). Interactions were also detected between CD40 and CD40L, and between GITR and GITRL by this method (Fig. 3). We also demonstrated that membrane protein-displaying BV can be utilized to detect cells expressing their specific receptors (Fig. 4). Furthermore, we successfully cloned CD2 cDNA from an expression cDNA library by using CD58 (CD2 ligand)-displaying BV as the probe (Fig. 5). The BV display system described here is expected to have an advantage over other systems currently in use. It should be applicable to various membrane receptor proteins and their ligands (or counter receptors), which are also membrane proteins. Since membrane proteins displayed on BV have the capacity to move laterally on the surface of the viral membrane, they are able to form oligomers. Currently, the fusion of the extracellular domain of membrane proteins with the Fc portion of immunoglobulin (Fc-fusion protein) is commonly used as a technique to reconstitute dimer or pentamer structures and to detect receptors (or ligands) [3,4,17]. However, it is difficult to reconstitute   functional hetero-oligomeric complexes by this method. Furthermore, some proteins may not retain the proper conformation after such manipulation. Since heterologous membrane proteins express on BV in their native forms, it is highly likely that the BV display system reconstitutes functional hetero-oligomeric protein complexes. The low background of the intrinsic expression of these immune receptors or ligands in BV also provides a critically important advantage.
Recently several investigators have reported that they were able to attach retroviral or adenoviral particles to optical biosensor surfaces and then detect interaction with antibodies or ligands specific to the receptors displayed on the virus [18,19]. Baculoviral particles can also be immobilized on a solid surface, and proteins displayed on BV retain the ability to bind their ligands (or receptors) for at least several months at 4uC. The potential application to the development of a biochip sensor is another advantage of the BV display system.
In summary, we have developed a new method for detecting membrane protein interactions using the BV display system. The BV display system reported here is expected to be highly useful for the analysis of cell-to-cell interaction mechanisms important in the immune system, neural system, or developmental organ formation. It also can be applied to develop screening systems for therapeutic antibodies and/or compounds.

Recombinant baculovirus construction and Sf9 cell culture
The cDNAs for human CD2, CD58, and mouse CD40 were amplified by PCR from human lymph node and mouse spleen cDNA libraries (TAKARA Bio), respectively. The cDNA for mouse CD40L was purchased from ATCC. The cDNA for mouse GITR was amplified by RT-PCR from poly A-positive RNA isolated from an 18.3.5 T cell hybridoma, which expresses GITR [20]. The cDNA for mouse GITRL was cloned in our laboratory. The FLAG tag sequence was added to the 39-terminus of CD2 and 59-terminus of CD40L and GITRL cDNAs. The sequence for the HA tag was added to the 39-terminus of CD58, CD40, and GITR cDNAs. The nucleotide sequences of these DNA constructs were confirmed. The DNA fragments were subcloned into the baculoviral transfer vector pBlueBac4.5 (Invitrogen.). Recombinant baculoviruses were generated using a Bac-N-Blue system (Invitrogen) according to the manufacturers' instructions. Sf9 cells were cultured as described [11].

Preparation of the budded baculovirus (BV) fractions
Sf9 cells (2610 6 cells/ml) were infected with recombinant baculovirus at the multiplicity of infection (M.O.I.) of 5. Seventytwo hours after infection, the BV fraction was isolated from the culture supernatant of infected Sf9 cells as described [10,11]. The pellets of the BV fraction were resuspended in Tris-buffered saline (TBS) containing 1 mM EDTA, 50 mM E64, 2 mg/ml aprotinin, and 10 mg/ml leupeptin, and stored at 4uC. The expression of recombinant proteins in BV fraction was confirmed by SDSpolyacrylamide gel electrophoresis and Western blot analysis using an HRP-anti FLAG M2 antibody or an anti-HA antibody (F-7) plus HRP-goat anti mouse IgG. Samples were not heat-treated so as to minimize aggregation.

Enzyme-linked immunosorbent assay (ELISA)
The BV displaying human CD2 was diluted with TBS and adsorbed to the wells of a 96-well ELISA plate (Greiner Bio-One) overnight at 4uC. Wells were washed with phosphate-buffered saline (PBS) and blocked with TBS containing 40% BlockAce (Dainippon Sumitomo Pharma) for 1 h at room temperature. The BV displaying human CD58 was diluted with Hanks' Balanced Salt Solution (HBSS) containing 40% BlockAce, added to the wells, and incubated for 1 h at room temperature. Wells were washed with PBS, and incubated with a mouse monoclonal antibody specific to human CD58 (clone 1C3 (AICD58.6)). After being washed with PBS containing 0.01% Tween 20, wells were incubated with HRP-conjugated goat anti-mouse IgG, washed, and further reacted with the tetramethylbenzidine liquid substrate (Sigma). The reaction was terminated by the addition of 0.5 M H 2 SO 4 , and absorbance at 450 nm was quantitated using a 96well plate reader. Similar assays were carried out with the combination of human CD58-BV bound to the plate and CD2-BV in the solution. Binding between the BVs individually displaying mouse CD40 and CD40L, or GITR and GITRL was also detected by using a similar ELISA method.
For the blocking experiment, CD2-BV was immobilized to the ELISA plate and wells were blocked with BlockAce, as described above. After pre-incubating wells with a monoclonal anti-human CD2 antibody (clone RPA-2.10) for 15 min at room temperature, CD58-BV was added to the wells. Wells were incubated for 1 h at room temperature, washed, and reacted with a biotinylated anti-CD58 antibody (clone TS2/9.1.4.3), then, with HRP-conjugated streptavidin. Similar blocking assays were carried out with the combination of human CD58-BV bound to the plate and the CD2-BV in the solution using anti-human CD58 (clone 1C3 (AICD58.6)) and biotinylated anti-human CD2 (clone RPA-2.10) for the blocking and detection antibodies, respectively. Binding between the plate-bound mouse CD40-BV and mouse CD40L-BV in the liquid phase was also blocked by an anti-mouse CD40 monoclonal antibody (clone HM40-3). A biotinylated anti-mouse CD40L monoclonal antibody (clone MR1) was used as the detection antibody. Similar blocking assays were carried out with a combination of mouse CD40L-BV bound to the solid phase and CD40-BV in the liquid phase by using anti-mouse CD40L (clone MR1) and biotinylated anti-mouse CD40 (clone 3/23) for the blocking and detection antibodies respectively.

Flowcytometric analysis
Cells were washed with FACS buffer (PBS containing 1% fetal calf serum (FCS)) and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) containing 20% FCS at approximately 5610 6 cells/ml. One hundred microliters of cell suspension were incubated with BV for 30 min at 4uC, washed twice with FACS buffer, and incubated with an anti-baculoviral envelope protein gp64 monoclonal antibody (clone A0505A or B8147A) for 30 min on ice. The cells were washed, and then further incubated with FITC or PE-labeled rat anti-mouse Ig-k monoclonal antibody for 30 min on ice. The cells were washed again, resuspended in FACS buffer, and analyzed on a FACS Calibur flowcytometer (Becton Dickinson).

Expression cloning
A human T lymphocyte cDNA library was constructed as described [21] by using the pBabeX retroviral vector. Plat-E packaging cells [22] were transfected with the cDNA library using FuGene 6 (Roche Diagnostics). Two days later, culture supernatant containing retroviruses was collected and used to infect 2.4610 6 cells of mouse pro-B cell line BaF/3 (purchased from RIKEN BRC Cell Bank) in the presence of polybrene (8 mg/ml). After 4 h, fresh media, twice as much volume as that of the viral stock, was added and cells were cultured overnight. On the next day, cells were washed, resuspended in the fresh media, and cultured overnight once again. Two days after the day of infection, 8610 7 cells were washed with DMEM containing 20% FCS and incubated with 80 mg of hCD58-BV for 30 min at 4uC, washed again, then incubated with 10 mg of A0505A anti-baculoviral gp64 monoclonal antibody for 20 min at 4uC. Cells were further incubated with magnetic microbeads coated with goat anti-mouse IgG (Miltenyl Biotech). Cells bound with hCD58-BV were isolated by magnetic cell sorting using a MACS system (Miltenyl Biotech). After three time purification by magnetic sorting, isolated cells were cloned with limiting dilution. The cDNAs derived from the retroviral library were amplified from the genomic DNA of individual clones by PCR using upstream and downstream retroviral vector primers (59-CAGCCCTCACTCCTTCTC-39 and 59-CCCTTTTTCTGGAGACTAAAT-39).