Recombinant PSG17N-Myc-His, which consists of the leader peptide, N1 domain of PSG17 (formerly known as Cea 2), followed by the Myc and 6X His tags was generated using a baculovirus expression system and purified as described previously (6
). Transfections were performed with Lipofectamine 2000 (Invitrogen) and transfection efficiency was monitored using a plasmid encoding the green fluorescent protein.
The murine CD53 and CD82 cDNAs were amplified by reverse transcription (RT)-PCR from RAW cell RNA and the murine CD81 cDNA was amplified from pBluescript containing the CD81 cDNA. The primer sets used for the amplifications were: for CD53: 5′ATGGGCATGAGCAGCCTGAAA and 5′TCACAGCCCTAAAGCCTGGC; for CD82: 5′CAGAATGGGGGCAGGCTGTG and 5′CAGCAACCTCAGTACTTGGGG; for CD81: 5′ATGGGGGTGGAGGGCTGC and 5′TCAGTACACGGAGCTGTTCCGG. After amplification with Vent DNA polymerase, the cDNAs were cloned into pEF6/V5-His Topo (Invitrogen), colonies were probed with oligonucleotides specific for each tetraspanin and the correct orientation was confirmed. The murine CD151 cDNA in pcDNA3.1 Zeo was obtained from Dr. L. Ashman, The Hanson Centre for Cancer Research, Adelaide, Australia, and the murine CD63 cDNA in pcDNA3.1/GS was purchased from Research Genetics.
PSG17N-Myc-His–coated plates were prepared as follows. Bacteriological culture dishes were layered with 10 μg/ml goat anti–mouse IgG, extra serum absorbed (XSA; KPL) in phosphate coating solution (KPL). The plates were rinsed with PBS and blocked with BSA buffer (KPL). Anti-myc mAb (1 μg/ml) (Invitrogen) was added to each dish followed by the addition of PSG17N-Myc-His with washes after each step.
RAW 264.7 (American Type Culture Collection) cells were cultured in DMEM with high glucose, 5 mM sodium pyruvate (Irvine Scientific), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin B (PSA; Quality Biological), and 10% fetal bovine serum (FBS). Human embryonic kidney (HEK) 293T cells (Edge BioSystems) were cultured in DMEM, 10% FBS, 50 μg/ml gentamicin, 250 μg/ml G418 (Calbiochem), and PSA. HEK 293 EBNA cells (Invitrogen) were grown in DMEM, 10% FBS, PSA, and 250 μg/ml G418. Baby hamster kidney (BHK)–21 cells (American Type Culture Collection) were sustained in DMEM with 10% FBS and PSA. All cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Generation of the RAW 264.7 cDNA Library and Recovery of cDNA Clones by Panning.
RNA was extracted from RAW 264.7 cells using TRIzol (Invitrogen) and was used to generate the cDNA expression library in the PEAK10CV vector (Edge BioSystems). The unamplified library yielded ~4.3 × 106 primary transformants. For the first round of screening, 1.1 × 106 clones were plated on Luria-Bertani (LB) broth/agar plates with 100 μg/ml ampicillin (Sigma-Aldrich). After 16 h the plates were flooded with LB broth, the pooled bacteria were pelleted, and plasmid DNA was isolated.
Pooled purified plasmids from the library were transfected into HEK 293 EBNA cells. Positive transfectants were selected using 0.5 μg/ml puromycin (Edge BioSystems). At 72 h after selection, the cells were dislodged in PBS and 0.5 mM EDTA, and resuspended in binding buffer (PBS, 2% BSA). The detached cells were panned in PSG17N-Myc-His coated Petri dishes at 1.0–1.5 × 107 cells per dish. Non-adherent cells were removed by extensive washing with PBS/BSA and adherent cells were transferred to poly-L-lysine coated 96-well plates. An additional two rounds of panning were performed before the episomal plasmids were isolated and transformed into ElectroMAX™ DH10B cells (Invitrogen). Individual plasmids were purified and transfected into HEK 293T cells, after which the transfected cells were screened for their ability to bind PSG17N-Myc-His by ELISA (see below). Inserts from the plasmids that conferred PSG17N binding were sequenced with the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems).
Detection of PSG17 Binding to Transfected HEK 293T Cells by ELISA.
HEK 293T cells were seeded in poly-L-lysine coated 96-well plates at 5 × 104 cells per well and transiently transfected with plasmid DNA recovered after library screening as described above or encoding the CD53, CD63, CD81, CD82, or CD151 cDNAs. At 48 h after transfection, the cells were washed with binding buffer containing 0.01% sodium azide and PSG17N-Myc-His (10 μg/ml) or no ligand was added to each well. After 1 h of incubation at room temperature, the ligand was aspirated and the cells were washed five times with binding buffer without sodium azide. To detect binding of PSG17N-Myc-His, anti-myc-horseradish peroxidase (HRP)-conjugated mAb was added to the cells for 1 h at room temperature at a concentration of 1 μg/ml in binding buffer. Binding of the antibody to the ligand was detected after the addition of tetramethylbenzidine (TMB)-peroxidase substrate (KPL) followed by 2 N H2SO4. The color change was quantitated at 450 nm on a microplate spectrophotometer.
Competition experiments were performed in CD9-pEF6/V5-His (Invitrogen) transfected HEK 293T cells by adding increasing concentrations of anti-murine CD9 mAb KMC8.8 (BD PharMingen) or an isotype-matched control (rat IgG2a) for 1 h at room temperature before treatment with 5 μg/ml PSG17N. Absorbance was normalized to background binding of PSG17N in the presence of anti-CD9 mAb to empty plasmid transfected HEK 293T cells.
In Situ Rosetting Assay.
For the in situ rosetting assay, HEK 293T cells transiently transfected with empty vector or CD9-pEF6/V5-His were seeded at low density into poly-L-lysine coated 60-mm dishes (Becton Dickinson). Attached cells were washed in binding buffer (PBS-2% BSA). PSG17N-Myc-His (90 pM) or binding buffer alone was added to the dishes for 1 h at room temperature. The cells were washed four times with PBS to remove any unbound ligand before the addition of 1 μg/ml anti-myc mAb in binding buffer for 1 h at room temperature. As a control, the anti-myc mAb was omitted from some plates. Dishes were washed again with PBS before the addition of 15 μg of rabbit anti–mouse Ig coated beads (Bio-Rad Laboratories). Unbound beads were removed by washing extensively with PBS and receptor positive cells were viewed by microscopy.
HEK 293T or BHK-21 cells were transfected with murine CD9-pEF/V5-His or empty vector. 48 h after transfection, the cells were washed twice with wash buffer (PBS; 3% FBS; 0.01% sodium azide), before the addition of 10 μg PSG17N-Myc-His for 30 min at room temperature. After two washes, cells were sequentially incubated for 30 min on ice with 0.5 μg anti-myc mAb (Invitrogen), 0.5 μg biotin-labeled goat anti–mouse IgG2aκ (BD PharMingen) and 0.5 μg streptavidin- FITC (BD PharMingen) with two washes between each incubation. For inhibition experiments, 106 RAW 264.7 cells were preincubated with 1 μg of Fc block (BD PharMingen) and various concentrations of anti-CD9 mAb or isotype control mAb for 30 min on ice. After several washes, 2 μg of PSG17N-Myc-His were added to each tube followed by 0.7 μg of anti-myc mAb, and PE-labeled rat anti–mouse IgG1 (BD PharMingen). Binding of PSG17N-Myc-His to thioglycollate-induced peritoneal macrophages isolated from CD9-deficient mice or wild-type mice was determined after 24 h treatment of the macrophages with 10 ng/ml PMA followed by incubation with Fc block, PSG17N or a control myc-tagged protein, anti-myc mAb, and PE-labeled rat anti–mouse IgG1 as described above for the RAW cells. PSG17N binding to HEK 293T, BHK 21, RAW 264.7 cells and macrophages was analyzed by flow cytometry using an EPICS XL-MCL flow cytometer (Beckman Coulter) and the percent binding was determined with the System II Software program (Beckman Coulter). Overlays were produced with the WinList program (Verity Software House).
Alkaline Phosphatase Binding Assays.
The PSG17-alkaline phosphatase (PSG17-AP) fusion protein was generated by cloning the full length PSG17 cDNA into the AP-Tag 4 vector. CD9 binding assays with heat stable AP-PSG17 were performed as described by Flanagan and Cheng (22
). Briefly, HEK 293T cells transiently transfected with murine CD9 or empty vector were cultured in poly-L-lysine–coated six-well plates. Increasing concentrations (43, 86, and 129 nM) of AP-PSG17 or the AP control protein were added to each well in triplicate for 90 min at room temperature. The cells were thoroughly washed, and the concentration of bound protein was measured from cleared, heat-inactivated cell lysates with dephosphorylation of p-nitrophenyl phosphate, which was quantitated by absorption at 405 nm using an ELISA plate reader.