Involvement of Caspase-1 and Caspase-3 in the Production and Processing of Mature Human Interleukin 18 in Monocytic THP.1 Cells
THP1 cell line
DOI:
10.1074/jbc.272.42.26595
Publication Date:
2002-07-26T14:48:08Z
AUTHORS (16)
ABSTRACT
Recently, human interleukin 18 (hIL-18) cDNA was cloned, and the recombinant protein with a tentatively assigned NH2-terminal amino acid sequence generated. However, natural hIL-18 has not yet been isolated, its cellular processing is therefore still unclear. To clarify this, we purified from cytosolic extract of monocytic THP.1 cells. Natural exhibited molecular mass 18.2 kDa, Tyr37. Biological activities were identical to those respect enhancement killer cell cytotoxicity interferon-γ production by peripheral blood mononuclear We also found two precursor (prohIL-18)-processing in cytosol These blocked separately caspase inhibitors Ac-YVAD-CHO Ac-DEVD-CHO. Further analyses partially enzymes revealed that one caspase-1, which cleaves prohIL-18 at Asp36-Tyr37 site generate mature hIL-18, other caspase-3, both Asp71-Ser72 Asp76-Asn77 biologically inactive products. results suggest are regulated enzymes, caspase-1 Interleukin (IL) 1The abbreviations used are: IL, interleukin; pro, precursor; h, human; ICE, interleukin-1β-converting enzyme; mAb, monoclonal antibody; pAb, polyclonal PCR, polymerase chain reaction; PB, phosphate buffer; IFN, interferon; NK cell, cell; ICA, IL-18-converting activity; IDA, IL-18-degrading hIL-18-CE, hIL-18-DE, CHAPS, 3-[(3-cholamidpropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PBMC, -18 (originally called IGIF, interferon-γ-inducing factor) novel cytokine multiple biological functions. In 1995 murine IL-18 liver extracts mice sensitized withPropionibacterium acnes followed elicitation lipopolysaccaride (1Okamura H. Nagata K. Komatsu T. Tanimoto Nukada Y. Tanabe F. Akita Torigoe Okura Fukuda S. Kurimoto M. Infect. Immun. 1995; 63: 3966-3972Crossref PubMed Google Scholar). The cloned libraries prepared livers endotoxin shock (2Okamura Tsutsui Yutsudo Hakura A. Hattori Namba Konishi Nature. 378: 88-91Crossref Scopus (2432) Using this as probe, normal library (3Ushio Micallef Fujii Ikeda Okamura J. Immunol. 1996; 156: 4274-4279PubMed based on homology expressed Escherichia coli, examined an 1 (IL-1) signature-like Scholar) reported similar IL-1 family fibroblast growth factor terms their trefoil structures (4Bazan J.F. Timans J.C. Kastelein R.A. 379: 591Crossref (265) Scholar, 5Murzin A.G. Lesk A.M. Chothia C. Mol. Biol. 1992; 223: 531-543Crossref (307) Despite similarities, IL-1β exhibit different 3Ushio 6Dinarello C.A. Blood. 87: 2095-2147Crossref Scholar), transmitted through specific receptors. 2Torigoe, K., Ushio, S., Okura, T., Kobayashi, Taniai, M., Kunikata, Murakami, Sanou, O., Kojima, H., Fujii, Ohta, Ikeda, Ikegami, Kurimoto, (1997) Chem., press. Genetic information suggested synthesized form (prohIL-18) no known signal peptide sequence. Therefore, proteolytic cleavage required for maturation like 7Gu Kuida Ku G. Hsiao Fleming M.A. Hayashi N. Higashino Nakanishi Flavell Sato V. Harding M.W. Livingston D.J. Su M.S.-S. Science. 1997; 275: 206-209Crossref (1031) 8Ghayur Banerjee Hugunin Butler D. Herzog L. Carter Quintal Sekut Talanian R. Paskind Wong W. Kamen Tracey Allen 386: 619-623Crossref (1055) Gu et al. (7Gu IL-1β-converting enzyme (ICE)/caspase-1 cleaved proIL-18 authentic site, Asp35-Asn36, active IL-18. had remained report, screened mRNA-expressing lines cell-free positively expressing Furthermore, identified hIL-18-processing same extract. One ICE/caspase-1, acts CPP32/caspase-3, degraded This first report identification existing Tetrapeptidyl ICE-like protease inhibitor CPP32-like Ac-DEVD-CHO, ICE- fluorogenic substrates Ac-YVAD-MCA Ac-DEVD-MCA, respectively, purchased Peptide Institute (Osaka, Japan). Neutralizing non-neutralizing anti-hIL-18 antibodies (mAbs) 125-2H (IgG) 25-2G raised against our laboratory. Human anti-precursor rabbit antibody (pAb) obtained Cistron (Pine Brook, NJ). Anti-human ICE-p20 subunit, anti-human CPP32, poly(ADP-ribose)polymerase goat pAbs, ICE-p10 subunit pAb Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Recombinant (His)6-tagged CMH-1, anti-CMH-1 antiserum (antibody 25) Vertex Pharmaceuticals (Cambridge, MA). 42 hematopoietic cultured screening mRNA RPMI 1640 medium supplemented 10% heat-inactivated fetal bovine serum (Whittaker, Walkersville, MD), 100 units/ml penicillin, 50 μg/ml streptomycin. Cells under standard conditions humidified 5% CO2 air mixture 37 °C. obtain large amounts cells, anin vivo propagation method using immunosuppressed hamsters applied described previously (9Ando Ohta Sano O. Yamauchi Ando Jpn. Cancer Res. 1988; 79: 757-765Crossref (27) 10Fukuda Sanou Taniai Masaki Nakamura Sugimoto Lymphokine 7: 175-185Google Briefly, 5 × 105 cells transplanted subcutaneously into newborn injected intraperitoneally 0.1 ml anti-hamster thymocyte (J R Scientific, Inc., Woodland, Every 3 or 4 days after transplantation, these given intraperitoneally. After weeks, solid tumor masses harvested, rinsed thoroughly medium, small dispersed scissors then passed stainless steel meshes single suspension. Total RNAs isolated various ULTRASPEC-3 (Biotecx, Houston, TX) accordance supplier's instructions. reverse transcribed amplified PCR. thermocycle 30 35 cycles 94 °C min, 55 72 denaturation, annealing, extension, respectively. sequences primer pair 5′-GCTTGAATCTAAATTATTATCAGTC-3′ 5′-CAAATTGCATCTTATTATCATG-3′, produced 335-base product. PCR products run 3% agarose gels visualized ethidium bromide staining. All purification steps performed except DEAE-5PW chromatography, carried out room temperature. A hypotonic lysis (11Kostura M.J. Tocci Limjuco Chin Cameron P. Hillman Chartrain N.A. Schmidt J.A. Proc. Natl. Acad. Sci. U. 1989; 86: 5227-5231Crossref (464) pellets 1011 washed once 10 volumes buffer (20 mm Hepes, pH 7.4, KCl, 1.5 MgCl2, EDTA·2Na) collected centrifugation 1,000 g min. resulting resuspended frozen −20 until use. disrupted three freeze-thawing, debris removed 10,000 ×g. dialyzed (PB) 6.6. (7.2 liters) step loaded onto 3.6-liter DEAE-Sepharose Fast Flow column (Pharmacia Biotech Inc.) equilibrated PB washing buffer, eluted containing 0.2m NaCl 0.5 m NaCl. fractions activity 0.2 (8.0 pooled concentrated 1.0 liter hollow fiber membrane module AIP-1010 model (Asahi Chemical Industry, Tokyo, Japan) overnight 20 6.8. product 2 0.22-μm Millipak filter (Millipore, Bedford, MA), 550-ml (Tosoh, Tokyo) 6.8, buffer. Elution bed linear salt gradient (0–0.5 NaCl), 25-ml elute collected. 90–200 mmNaCl 44–54 membranes, Mini-module NM-3 Industry). affinity CNBr-activated Sepharose 4B (Pharmacia) mAb (125-2H) preparation 1-ml phosphate-buffered saline 7.2. extensively, sample glycine-HCl mNaCl 2.5. Eluted neutralized immediately 1.0m Tris. hIL-18-containing 4.2-ml phase ASAHIPAK C4P-50 4E (Showa Denko, Tokyo), 0.1% trifluoroacetic acid, Next, 24 increasing acetonitrile (0–90% acetonitrile). main peak fraction about 40% mapping hIL-18. IFN-γ assay As previous procedures, all (2.0 cells) above. Cell-free Miller (12Miller D.K. Ayala J.M. Egger L.A. Raju S.M. Yamin T.-T. Ding G.J.-F. Gaffney E.P. Howard A.D. Palyha O.C. Rolando Salley J.P. Thornberry Weidner J.R. Williams J.H. Chapman K.T. Jackson Kostura Molineaux Mumford Calaycay Chem. 1993; 268: 18062-18069Abstract Full Text PDF Granular ammonium sulfate added reach saturation. solution stirred precipitate centrifugation. supernatant brought 80% saturation sulfate, TGCD Tris, 7.8, glycerol, dithiothreitol (DTT)), fresh 16 h. poly(vinylidene fluoride) (Millipore). Half batches 45-ml 4.5 (0–0.25m NaCl). Fractions ICA (fractions 17–21 eluting NaCl) IDA 33–48 150–210 HGCD 7.2, DTT). preparations subjected further purification. 20-ml S-Sepharose Then 5-ml 6 (0–0.12 mKCl). 23–26 50–80 mmKCl microvolume concentration SMART system One-third 0.1-ml Mono S 25-μl KCl. 3–5 next process. 2.4-ml Superdex 200 80-μl 11–14. Biologically analysis polypeptides. 7.0 10-ml 25 mmBis-Tris 7.1. pool P Bis-Tris 7.1 8-ml polybuffer iminodiacetic 5.0, generates decreasing (pH 7.1–5.0). 5.8–6.0) 120-ml HGD (HGCD without CHAPS) 7.4. 29–35. 4.4-ml hydroxyapatite (Tonen, 0.5-ml m). 500 54 55, Amino portions processed forms (p18, p16, p15) determined automated sequencer 473A (Applied Biosystems, Foster City, proteins interest electrophoresis 15% SDS-PAGE transferred ProBlottTM membranes Biosystems). Coomassie Brilliant Blue staining excised direct sequencing. For mapping, digested clostripain (Sigma), fragments separated 2.5-ml ODS-120T (Tosoh) (0–70% acetonitrile) acid. detected wavelength 214 nm analyzed. 0.8-kilobase encoding ligated expression vector BCMGSNeO (13Karasuyama Kudo Melchers Exp. Med. 1990; 172: 969-972Crossref (300) transfected CHO-K1 electroporation, transfectants harvested days. analyzed immunoblotting hIL-18-specific (25-2G). ICE method. primers designed published hICE nucleotide (14Thornberry Bull H.G. Aunins Elliston K.O. Casano F.J. Lee T.D. Shively J.E. MacCross 356: 768-774Crossref (2242) COS-1 electroporation construct pCDM8 (Invitrogen, San Diego) Proteins extracted Dounce homogenizer. homogenate incubated h activate fractionation 75 chromatographies. quantities assessed two-site sandwich ELISA mAbs laboratory (15Taniguchi Nagaoka Kunikata Kayano Methods. (in press)Google bioactivity IFN-γ-inducing myelomonocytic line KG-1. 3K. Konishi, Tanabe, Taniguchi, Yamauchi, Tanimoto, Orita, submitted publication. KG-1 suspended seeded 96-well microplate. Samples wells induced culture supernatants ELISA. measured respectively Scholar,14Thornberry 18Nicholson D.W. Ali Vaillancourt C.K. Gallant Gareau Griffin P.R. Labelle Lazebnik Y.A. Munday Smulson M.E. Yu V.L. 376: 37-43Crossref (3816) substrate samples (10 μl) presence DTT microplates (100 7.4 (ICE-like assay) 6.5 0.5–1 (CPP32-like assay). levels generated 4-amino-2-methylcoumarin fluorescence plate reader (FluoroScan II, Labsystems Japan, 355 excitation 460 emission. proteases calculated normalized pmol formed/μl/min. identify hIL-18-producing lines, level constitutive transcription-PCR primers. lineages screened. highly such THP.1, KG-1, HBL-38, HL-60, some non-lymphocytic non-myelomonocytic MEG-01 TS9;22. B non-T, non-B BJAB, MOLP-2, NALM-20, weak observed. Interestingly, observed T (TableI).Table IConstitutive linesCell linesPCR cycles3035T DND-39−− HUT-78−− C5/MJ−− SAALT-3−− DND-41−− JM−− JURKAT−− PEER−− ED-S−−B NALM-6−− BALM-9−− BALM-10−− MKB-1−− Hair-M−− HPB-ALL−− DAUDI−− RAJI−− BAL-KHs−− BALL-1−− B372−− B373−− B374−− Ramos−+ MLB-1084−+ MOLP-2−++ BJAB−++Non-T, HDLM-2−− NALM-19−− NALM-20−++ NALM-24−++Myelomonocyte KCL-22−− U-937+++ ML-1+++ KU-812+++ THP.1++++ HL-60++++ KG-1+++++ HBL-38+++++Non-L, non-M L428−− HEL+++ MEG-01++++ TS9; 22++++The IL-18-specific studied. strength judged density band detected: very strongly expressed, +++; expressed; ++; weakly +; expression, −. Open table new tab Initially, chose four (THP.1, U-937, HBL-38) non-L, (HEL MEG-01) targets detection level. preliminary study, immunoblotting. Small U937 but little (data shown). mostly Next employed differentiation-inducing agents (lipopolysaccaride, retinoic A23187, phorbol myristate acetate) investigate whether reagents can augment secretion significant elevation intra- extracellular Based upon above mentioned results, selected starting material isolation purpose, numbers lysis. DEAE anion-exchange chromatography twice (Fig. 1, B) Subsequently, mAb-conjugated employed. kDa reducing C). overall yield ranged μg/1011 Direct sequencing affinity-purified polypeptide showed peptides coincided deduced 2;underlined). indicate derived proteolytically behind Asp36 residue manner homolog 2; vertical arrow) 2Okamura Scholar).Figure 2Amino alignment homolog. Alignment shown. Asterisks (*) denote residues sequences. arrow indicates NH2 terminus occurs. Partial natur
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