Reagent and plasmids
The following antibodies and reagents were used: human TLR9 and α-tubulin (eBioscience), mouse and human TLR9 (Imgenex), GFP (also detects YFP) (Invitrogen/Molecular Probes for immunoprecipitation and BD Clontech for immunoblotting), phospho- and total-p38 (Cell Signaling Technologies), biotin and Flag (Sigma), hemagglutinin for immuoprecipitation (HA) (ABM), HA for immunoblotting (Roche), cathepsin S (R&D) and secondary antibodies (Southern Biotech); Endoglycosidase H and Peptide N-glycosidase F (New England Biolabs); CpG DNAs (Eurofins MWG operon) 10104: 5′-TCGTCGTTTCGTCGTTTTGTCGTT-3′ and 2006 3′ Biotin: 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′; S. typhimurium
flagellin (Invivogen); Z-fafmk Cathepsin B ( CA-074 Me) and Cathepsin S inhibitor (Calbiochem), and Bafilomycin A1 (Tocris); 5X NF-κB-luciferase (Stratagene), and pSV-β-galactosidase (Promega). Mouse TLR9 was cloned into p3xFLAG-CMV-9 (Sigma). Soluble human TLR9 was cloned into pEYFP using the following primers: Forward: 5′-ATA TAT CTC GAG ATG GGT TTC TGC CGC AGC G- 3′, Reverse: 5′-ATA TAT GGA TCC GCC TCC TTG GCC TTG G- 3′. TLR9–4 was previously described [16
Cell culture and transfection
All cell lines except BJAB were cultured in DMEM with 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin, 10 mM Hepes, 1 mM sodium pyruvate and 10% low endotoxin FBS (complete DMEM). The human B cell line, BJAB, was cultured in complete RPMI 1640 medium. Cells routinely tested negative for mycoplasma by PCR. TLR9 stable cells were generated in HEK 293 cells and responded to CpG DNA as determined by 5X NF-κB-luciferase reporter assay. For transient transfection, cells were plated in 6 well plates and the following day were transfected with 4 μg DNA and 8 μl TransIT. After 36 hours the cells were lysed and immunoprecipitated with the indicated antibodies.
Phoenix cells were transfected with MIGR2-mTLR9-HA. Viral supernatant was collected and 0.5 ml added to cells in 12 well plates with 8 μg/ml polybrene and centrifugation at 1811 × g for 90 minutes at 32°C. After 48 hours, cells were lysed and immunoprecipitated with anti-HA antibody.
Luciferase reporter assay
HEK 293 cells were transfected as previously described [13
] and treated with CpG DNA or flagellin for 8 hours. Data were analyzed by determining the fold induction compared to unstimulated cells.
HEK 293 cells stably expressing TLR9-YFP were treated as indicated, washed once in PBS, and lysed for 15 minutes at 4°C in lysis buffer (137 mM NaCl, 20 mM Tris pH7.4, 1mM EDTA, 0.5% TritonX-100, protease inhibitor cocktail (Sigma) and 100 mM phenylmethylsulphonyl fluoride). Total protein was determined for clarified lysates using the BCA protein assay (Bio-Rad) and 2.0 – 2.5 mg of total protein was used for immunoprecipitation. Note that YFP is a variant of GFP and is recognized by the GFP anitbody. 25 μl of whole cell lysate was immunoblotted for anti-tubulin.
GFP immunoprecipitates from HEK 293 cells stably expressing TLR9-YFP were divided into three equal portions and were either untreated, or treated with endo H or PNGase F according to the manufacturer instructions (New England Biolabs) for two hours at 37°C and the reactions were stopped by adding 6X SDS-PAGE reduced sample buffer. The samples were analyzed by immunoblotting as described above.
Identification of the cleavage site for sTLR9
1×107 HEK 293 cells stably expressing TLR9-YFP per immunoprecipitation reaction (10 total) were lysed in one ml of lysis buffer each and immunoprecipitated with 5 μg/ml anti-TLR9 overnight at 4°C on a rotator. Protein A/G Sepharose was added for one hour at 4°C. After washing, each reaction was sequentially resuspended in 2X SDS-PAGE reduced sample buffer and boiled together for 5 minutes. The combined sample was separated by 8% SDS-PAGE and submitted to the Cornell Proteomics and Mass Spectrometry Core Laboratories Center for gel extraction, trypsin digestion and nano-liquid chromatography tandem mass spectrometry. Identified peptides had a confidence of >99%.
Mouse embryonic fibroblasts transiently transfected with Flag-tagged mouse TLR9 were cultured in cysteine and methionine free medium (Gibco) for one hour then pulsed for an additional hour with 0.1 mCi 35S-cysteine and 35S- methionine (Perkin-Elmer). The cells were washed two times in 1× HBSS and chased in complete medium for indicated times. The cells were lysed and immunoprecipitated with anti-FLAG and protein A/G sepharose beads for 4 hours. The immunoprecipitates were washed four times, eluted by boiling in 2X SDS-PAGE reduced sample buffer and separated by SDS-PAGE. The gel was soaked in enhancer (1M sodium salicylate) for 30 minutes and then visualized by autoradiography.
Protease enzyme activity
HEK 293 and RAW 264. 7 cells were lysed in RIPA lysis buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) and the pH was adjusted to pH 6.0 using 2N HCl. 100μl of the lysate was aliquotted into each well of a 96 well black walled plate (Costar). Cathepsin B and S substrate (Calbiochem) were added at the indicated concentration and the enzyme activity was measured by fluorometry at the indicated times(Gemini EM Microplate Reader, Molecular Devices). Substrate activity was calculated using the following excitation and emission criteria: Cathepsin B substrate (Excitation: 370, Emission: 440), Cathepsin S substrate (Excitation: 340, Emission: 405).
Cathespin s knockdown in HEK 293. Cathepsin S
shRNA and control shRNA were from Sigma-Aldrich (Catalog # SHCLNG-NM_004079). HEK 293 cells were transfected with the shRNAs and the cells were selected in 0.5mg/mL puromycin. Single cell clonal selection was performed and the knockdown of cathepsin S was confirmed by immunoblot.
HeLa cells were transfected with fluorescently labeled sTLR9 on a cover slip in a 24 well plate. After 48 hours the cells were fixed and imaged using a Leica SP5 confocal microscope.
CpG DNA pull-down
BJAB cells (6×107) were treated with 5μM of 3′ biotinylated CpG DNA, lysed and immunoprecipitated with anti-TLR9, anti-biotin antibodies, or beads alone for 4 hours. Protein A/G sepharose beads were added for an additional 12 hours at 4°C. After washing, 2X SDS-PAGE reduced sample buffer was added and proteins were separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted for TLR9 (N-terminus).
Data were analyzed using Instat, Graphpad prism software. Data were compared for significance using an unpaired t-test and were considered significant with p value of < 0.0001. Error bars represent mean ± standard deviation of triplicate samples. Data for cathepsin activity were analyzed by paired t-test.