Yeast two-hybrid screening, cDNA cloning, and Northern blotting.
Yeast two-hybrid screening was performed on a human lung cDNA library (Clontech Laboratories Inc., Palo Alto, California, USA) using a Matchmaker System 2 (Clontech Laboratories Inc.) and a GAL4BD-TNFR1 extracellular domain fusion protein. Human ARTS-1 cDNA clones were isolated from a UniZAP XR phage cDNA library (Stratagene, La Jolla, California, USA) generated from NCI-H292 cells stimulated with 1 μM PMA for 24 hours and subjected to double-stranded automated fluorescent sequencing. Full-length ARTS-1 cDNA was generated by PCR of human lung poly(A+) mRNA (Clontech Laboratories Inc.) using Pfu Turbo (Stratagene) and the following primers: 5′-GCAAGAAGATGGTGTTTCTGCCCCTC-3′ (nucleotides 80–105) and 5′-TTACATACGTTCAAGCTTT-TCACT-3′ (nucleotides 2,890–2,913). Sequence analysis, including Kyte-Doolittle hydropathy prediction, was performed using MacVector 7.0 software (Accelrys, Burlington, Massachusetts, USA). The location of the putative hydrophobic transmembrane α-helical domain was predicted using several web-based analysis programs (MEMSAT2 [http://bioinf.cs.ucl.ac.uk/psipred/], SOSUI [http://sosui.proteome.bio.tuat.ac.jp.sosuiframe0.html], TMAP [http://www.mbb.ki.se/tmap/index.html], TMpred [http://www.ch.embnet.org/software/TMPRED_form.html], and TopPred2 [http://bioweb.pasteur.fr/seganal/interfaces/toppred.html]). A 32P-labeled probe was generated via random priming of gel-purified, full-length ARTS-1 cDNA and hybridized against a multiple tissue Northern blot (Clontech Laboratories Inc.) according to the manufacturer’s recommendations.
Anti–ARTS-1 serum generation.
A 17-amino-acid peptide (RGRNVHMKQEHYMKGSD), corresponding to amino acids 538–554 of the proposed ARTS-1 extracellular domain, was selected based upon its antigenicity and lack of homology with other protein sequences via BLAST homology search. New Zealand white rabbits were immunized with this peptide to generate anti–ARTS-1 serum (Research Genetics, Huntsville, Alabama, USA).
ARTS-1 immunoprecipitation and immunoblotting.
Human bronchial epithelial cells were obtained by fiberoptic bronchoscopy of normal research volunteers who had provided informed consent. The protocol for harvesting bronchial epithelial cells via bronchial brushings was approved by the National Heart, Lung, and Blood Institute Institutional Review Board for the protection of human subjects. Human bronchial epithelial cell lines were purchased from the American Type Culture Collection (Rockville, Maryland, USA). Primary cultures of normal human bronchial epithelial cells, umbilical vein endothelial cells (HUVECs), and fibroblasts were purchased from Clonetics Corp. (San Diego, California, USA). Crude membrane and cytosolic fractions were prepared from the NCI-H292 human pulmonary mucoepidermoid carcinoma cell line and HUVECs. Cells were isolated by scraping and disrupted by sonicating twice (for 10 seconds each time) in 50 mM Tris-HCl (pH 7.2), 150 mM NaCl (pH 7.2), 0.1% Triton X-100, and Complete protease inhibitor (Roche Diagnostics Corp., Indianapolis, Indiana, USA), followed by centrifugation at 1,000 g for 5 minutes to remove nuclei and debris. Cell lysates were centrifuged at 100,000 g for 1 hour and the crude membrane pellet was suspended by sonicating three times (for 2 seconds each time) in lysis buffer. For immunoprecipitation experiments, 200 μg of cell membrane fractions were incubated with 20 μg of a murine anti-human TNFR1 or TNFR2 monoclonal antibody (R&D Systems Inc., Minneapolis, Minnesota, USA), or with 1 μl of anti–ARTS-1 immune or preimmune serum. Incubation took place overnight at 4°C, followed by immunoprecipitation with protein A/G beads (Pierce Chemical Co., Rockford, Illinois, USA). Proteins were resolved via SDS-PAGE, electroblotted onto nitrocellulose membranes, and incubated with either ARTS-1 immune or preimmune serum diluted 1:20,000 or anti-TNFR1 or anti-TNFR2 monoclonal antibodies at a concentration of 2 μg/ml. Signals were detected by chemiluminescence using horseradish peroxidase–conjugated secondary antibodies. For immunoblotting experiments, 20 μg of cell membrane proteins per sample was used. For competitive immunoblotting experiments, 1 μl of anti–ARTS-1 serum was incubated with 1 mg of either the RGRNVHMKQEHYMKGSD peptide or BSA for 2 hours before immunoblotting. A goat anti-human TNF-α converting enzyme (TACE) polyclonal IgG antibody (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) was used at a concentration of 1 μg/ml for TACE immunoblotting experiments.
ARTS-1 confocal immunofluorescence microscopy.
Nonfixed, nonpermeabilized bronchial brushings of normal human airway epithelial cells and frozen sections of normal human bronchi were used. Tissue samples of normal human bronchi were provided by the Cooperative Human Tissue Network (University of Virginia, Charlottesville, Virginia, USA), which is funded by the National Cancer Institute. Slides were blocked with 10% goat serum in PBS and incubated with antibodies diluted in 1.5% goat serum in PBS. The following antibodies were used: anti–ARTS-1 and preimmune rabbit serum (1:1,500 dilution), anti-TNFR1 extracellular domain (2 μg/ml murine monoclonal IgG2b; Santa Cruz Biotechnology Inc.), murine IgG2b isotype control (2 μg/ml; R&D Systems Inc.), Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG, and Alexa Fluor 633 F(ab′)2 fragment of goat anti-mouse IgG (4 μg/ml; Molecular Probes Inc., Eugene, Oregon, USA). Confocal immunofluorescence microscopy was performed using a Zeiss LSM 510 confocal microscope and 40× and 100× Plan-Neofluar objectives, respectively, for frozen sections of human bronchi and cytospins of bronchial brushings. Alexa Fluor 488 and Alexa Fluor 633 were imaged sequentially at a data depth of 8 bits using 488-nm and 633-nm excitation wavelength and LP 505 and LP 650 emission filters, with pinholes adjusted to produce an axial thickness of 4.0–5.0 μM.
Expression, purification, and characterization of recombinant ARTS-1.
The cDNA sequence for the putative ARTS-1 extracellular domain (amino acids 30–941) was cloned into the pGEX-6P-1 plasmid (Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). The glutathione s-transferase–ARTS-1 (GST–ARTS-1) fusion protein was isolated from the insoluble fraction of transformed BL21 E. coli by denaturation with 6 M urea in PBS and refolded by serial dialysis against PBS containing decreasing urea concentrations. The GST–ARTS-1 fusion protein was purified using a glutathione Sepharose 4B affinity column; purity was assessed by Coomassie brilliant blue–stained SDS-PAGE gels and by fast-performance liquid chromatography (FPLC) analysis using a Superose 6 HR 10/30 gel filtration column (Amersham Pharmacia Biotech).
A 20-amino-acid TNFR1 peptide substrate (TKLCLPQIENVKGTEDSGTT) containing the major and minor cleavage sites for TNFR1 shedding was synthesized by Sigma-Genosys (The Woodlands,Texas, USA) (4
). The TNFR1 peptide substrate was mixed with GST–ARTS-1 at a molar ratio of 4:1 in 50 μl of 50 mM Tris-HCl and 120 mM NaCl (pH 7.5) for 2 hours at either room temperature or 37°C. Samples were analyzed by HPLC (Hewlett-Packard, Palo Alto, California, USA) using a Jupiter C-18 column (Phenomenex Inc., Torrance, California, USA). Similarly, GST–ARTS-1 was incubated for 2 hours with a recombinant human TNFR1-Fc fusion protein (R&D Systems Inc.) containing the TNFR1 extracellular domain fused to the Fc region of human IgG1
. Endopeptidase activity was assessed by TNFR1 immunoblotting.
To assess whether GST–ARTS-1 possessed nonspecific endopeptidase activity, 5 μg of GST–ARTS-1 was incubated separately with 10 μg of either human albumin, BSA, rabbit myosin heavy chain, or human transferrin (Sigma-Aldrich, Milwaukee, Wisconsin, USA) overnight at 37°C. Samples were resolved by SDS-PAGE and stained with Coomassie brilliant blue.
Aminopeptidase activity of recombinant GST–ARTS-1 fusion protein was determined as the rate of amide bond hydrolysis of amino acid–p-nitroanilide substrates (Bachem, Torrance, California, USA) under conditions of constant enzyme activity throughout the assay. Amino acid–p-nitroanilides (final concentrations 0.25–8 mM) were incubated at room temperature with 24 pmol of GST–ARTS-1 fusion protein in 200 μl of 50 mM Tris-HCl and 120 mM NaCl (pH 7.5) for 1 hour. Reactions were terminated by addition of 800 μl of 1.4 M sodium acetate (pH 4.4). Amide bond hydrolysis was quantified by p-nitroanilide absorbance at 380 nm and corrected for spontaneous hydrolysis of the substrate by subtracting the absorbance of control incubations that were terminated at time zero. Kinetic constants were determined by Lineweaver-Burk analysis using six concentrations of each amino acid–p-nitroanilide substrate with triplicate assays. Correlation coefficients for each line were greater than 0.997. For TNF-α protease inhibitor (TAPI) inhibition studies, selected amino acid–p-nitroanilides (4 mM) were incubated at room temperature for 1 hour with 15 pmol of GST–ARTS-1 and either TAPI-0, TAPI-1, or TAPI-2 (6.25–100 μM).
ARTS-1 cell lines.
cDNAs encoding the full-length ARTS-1 coding sequence in the sense orientation and ARTS-1 bases 61–213 in the antisense orientation were cloned into the pTarget mammalian expression vector (Promega Corp., Madison, Wisconsin, USA). The ARTS-1 catalytic site mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene). NCI-H292 cells were stably transfected using FuGENE 6 (Roche Diagnostics Corp.) and maintained under selective pressure with geneticin (500 μg/ml). Cell lines were established from clones that had been generated by limiting dilutions and selected based upon enhanced or suppressed ARTS-1 membrane-associated protein expression, as determined by immunoblotting. HUVECs were transiently transfected for 4 days prior to collection of cell culture supernatants. TNFR1 and TNFR2 shedding into cell culture supernatants over a 24-hour period was assayed using a sandwich ELISA with a sensitivity of 7.8 pg/ml (R&D Systems Inc.). TAPI-0, TAPI-1, and TAPI-2 were purchased from Peptides International Inc. (Louisville, Kentucky, USA). Statistical analysis was performed using a paired Student t test with a Bonferroni correction for multiple comparisons. A P value of less than 0.05 was considered significant.
Total RNA was extracted using the RNeasy Maxi Kit (Qiagen Inc., Valencia, California, USA), and ribonuclease protection assays were performed with the RPA III kit (Ambion Inc., Austin, Texas, USA) using 60 μg of total RNA per sample. Antisense TNFR1 and GAPDH riboprobes were labeled using the DIG RNA labeling kit and detected with the DIG luminescent detection kit (Roche Diagnostics Corp.) The TNFR1 template, which was used to generate the 124-bp protected fragment corresponding to nucleotides 356–478 of TNFR1 mRNA (GenBank accession number NM001065), was verified by sequencing, while the GAPDH template used to generate the 316-bp protected fragment was purchased from Ambion Inc.
NCI-H292 cells were scraped in ice-cold lysis buffer (10 mM Tris at pH 7.5, 1 mM EDTA, 0.25 M sucrose, 0.8 mM AEBSF, 50 μg/ml antipain, 1.5 μM aprotinin, 83 μM chymostatin, 5.6 μM E-64, 2 μM leupeptin, 5 μM pepstatin, and 10 μM phosphoramidon) and lysed by 15 passes through a 25-gauge needle. Crude membranes were collected from postnuclear supernatants by centrifugation at 100,000 g for 1 hour, resuspended in lysis buffer, and underlaid with a discontinuous gradient containing 0.5 M, 0.86 M, 1.15 M, and 1.4 M sucrose in TE buffer. Following centrifugation at 175,000 g for 1.75 hours, the fractions and interfaces were collected from the top, and proteins were precipitated with 10% trichloroacetic acid and resuspended in gel loading buffer. Immunoblotting was performed as previously described, using monoclonal antibodies directed against TNFR1 (Santa Cruz Biotechnology Inc.), β-catenin, and GM130 (both from Becton, Dickinson and Co., Franklin Lakes, New Jersey, USA). Forty micrograms of protein per fraction was used on immunoblots to detect TNFR1 and β-catenin, while 200 μg of protein per fraction was used in studies to detect GM130.