+ All Categories
Home > Documents > Generation of Biologically Active Interleukin- 18 by Proteolytic ...

Generation of Biologically Active Interleukin- 18 by Proteolytic ...

Date post: 01-Jan-2017
Category:
Upload: lethuan
View: 216 times
Download: 0 times
Share this document with a friend
6
THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Vol. 263, No. 19, Issue of July 5, pp. 9437-9442, 1988 Printed in U. S. A. Generation of Biologically Active Interleukin- 18 by Proteolytic Cleavage of the Inactive Precursor* (Received for publication, January 26, 1988) Roy A. Black$, Shirley R. Kronheim, Michael Cantrell, Michael C. Deeley, Carl J. March, Kathryn S. Prickett, Janis Wignall, Paul J. Conlon, David Cosman, Thomas P. Hopp, and Diane Y. Mochizuki From the Immunex Corporation, Seattle, Washington 98101 Interleukin-la (IL-18) is derived from an inactive precursor by proteolytic cleavage. To study IL-la processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incu- bation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated ma- terial showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these productsis the smallest one, and it co-migrates during electropho- resis with mature IL-la. Four purified known pro- teases were also tested for their effect on precursor IL- 18, and none of these products eo-migrated with the mature protein. Chymotrypsin and Staph ylococcue au- reus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded sub- stantially largerproducts,andthesehad little IL-1 activity. The products of three of the known proteases were identified byNHz-terminalsequencing.These results show conclusively that proteolysis of precursor IL-la generates biological activity and that the cleav- age must occur close to the mature NH2 terminus. IL-la’ and IL-10 are macrophage-derived polypeptide hor- mones involved in regulation of the immune system and in a wide range of other physiological responses (1-3). Directly or indirectly, IL-1 plays a role in the activation of hematopoietic stem cells and T-cells, in induction of fever and acute phase proteins, in degradation of cartilage, in inflammation, and in wound healing. Both of the IL-1s have a molecular mass of about 17,400 Da, but cloning of the cDNAs for these proteins indicated that the initial translation products should have molecular weights of about 31,000 (1,4). Subsequently, the intracellular forms of both IL-1s were indeed found to be of the larger size (E”).’ The extracellular, or mature, forms of IL-la and IL- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisemenf” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. The following abbreviations used are: IL-1, interleukin-1; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; r, recombinant; mAb, monoclonal antibody; PMSF, phenylmethanesul- fonyl fluoride; PBS, 20 mM sodium phosphate (pH 7.4), 140 mM NaCl; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. * Two groups have reported lower molecular weight intracellular IL-1 (8,9), but these observations may have been due to degradation after breakage of the cells (7). 1/3 consist of approximately the COOH-terminal halves of their precursors. In the case of IL-10, removal of the NH2- terminal half appears to be essential for biological activity: when IL-la and IL-10 mRNAs are translated in vitro, only the IL-la product is active (1) and binds to the IL-1 receptor (10). Generation of IL-lP activity thus requires processing of the precursor. Two further aspects of IL-1 processing also motivated this study. First, the precursor forms of both IL-1s lack a distin- guishable hydrophobic signal sequence, suggesting that they mayfollow an unusual secretory pathway (1). Second, the cleavage of the precursor occurs at an unusual site for hor- mone processing: the NH2-terminal alanine of mature IL-lP is preceded by the sequence valine-histidine-aspartate rather than by a pair of basic residues. It is thus of considerable interest to investigate the mechanisms involved in IL-1 proc- essing and secretion. There have been several reports of evidence for intermediate forms of IL-1 (7-9), but the actual means by which the precursors are processed to the mature forms remain unknown. Our approach to this problem has been to express the precursor of IL-10 in Escherichia coli, partially purify it, and use it as a substrate for extracts of monocyte and myeloid cell line proteases as well as for a number of purified common proteases. The products of the various digestions were analyzed by biological activity, by molecular weight, and by NH2-terminal sequence. EXPERIMENTAL PROCEDURES Expression of r-Precursor IL-lfi in E. coli-Recombinant precursor IL-10 was expressed in E. coli under the control of the phage X PL promoter and ~1857’” thermolabile repressor (11). Using standard recombinant DNA techniques, we constructed pLNIL-lBF (see Fig. 1) by ligating the following DNA segments: (1) 6160 base pairs of NcoI/HindIII-digested pLNIL-10 (12) containing the vector (confer- ring ampicillin resistance), codons 134-269 and 3’ noncoding regions of HuIL-10; (2) complementary synthetic oligonucleotides encoding residues 1-6 of IL-10 and NcoI and SstI complementary ends; and (3) a 380-base pair SstIIHindIII restriction fragment from plasmid IL-10-6 (1) encoding residues 7-133. The ligation mixture was trans- formed into the tetracycline-resistant host RRkpRK248cP (12) and correctly assembled plasmids were identified by restriction analysis of DNA isolated from transformants resistant to both ampicillin and tetracycline. Transformants containing pLNIL-lBF were tested for the produc- tion of precursor IL-10 by SDS-PAGE analysis (14) of cultures grown in super induction medium (15) to Am of 0.5 and derepressed for 1- 20 h by elevation of temperature from 30 to 42 “C. A protein of about 31,000 daltons was apparent in samples from pLNIL-lpF-containing cultures but not in control cultures lacking the IL-10 coding region. Immuno-dot blot analysis (16) with an anti-IL-10mAb and purified recombinant mature IL-10 (12) as standard indicated that the cul- tures contained approximately 2.5-5.0 pg/ml of r-precursor IL-la. Extraction of r-Precursor IL-10 from E. coli-Cell pellets from 2.5 liters of E. coli culture were resuspended in 20 ml of 30 mM Tris-HC1 9437
Transcript
Page 1: Generation of Biologically Active Interleukin- 18 by Proteolytic ...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 19, Issue of July 5, pp. 9437-9442, 1988 Printed in U. S. A.

Generation of Biologically Active Interleukin- 18 by Proteolytic Cleavage of the Inactive Precursor*

(Received for publication, January 26, 1988)

Roy A. Black$, Shirley R. Kronheim, Michael Cantrell, Michael C. Deeley, Carl J. March, Kathryn S. Prickett, Janis Wignall, Paul J. Conlon, David Cosman, Thomas P. Hopp, and Diane Y. Mochizuki From the Immunex Corporation, Seattle, Washington 98101

Interleukin-la (IL-18) is derived from an inactive precursor by proteolytic cleavage. To study IL-la processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incu- bation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated ma- terial showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electropho- resis with mature IL-la. Four purified known pro- teases were also tested for their effect on precursor IL- 18, and none of these products eo-migrated with the mature protein. Chymotrypsin and Staph ylococcue au- reus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded sub- stantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NHz-terminal sequencing. These results show conclusively that proteolysis of precursor IL-la generates biological activity and that the cleav- age must occur close to the mature NH2 terminus.

IL-la’ and IL-10 are macrophage-derived polypeptide hor- mones involved in regulation of the immune system and in a wide range of other physiological responses (1-3). Directly or indirectly, IL-1 plays a role in the activation of hematopoietic stem cells and T-cells, in induction of fever and acute phase proteins, in degradation of cartilage, in inflammation, and in wound healing.

Both of the IL-1s have a molecular mass of about 17,400 Da, but cloning of the cDNAs for these proteins indicated that the initial translation products should have molecular weights of about 31,000 (1,4). Subsequently, the intracellular forms of both IL-1s were indeed found to be of the larger size (E”).’ The extracellular, or mature, forms of IL-la and IL-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisemenf” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. ’ The following abbreviations used are: IL-1, interleukin-1; SDS-

PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; r, recombinant; mAb, monoclonal antibody; PMSF, phenylmethanesul- fonyl fluoride; PBS, 20 mM sodium phosphate (pH 7.4), 140 mM NaCl; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

* Two groups have reported lower molecular weight intracellular IL-1 (8,9), but these observations may have been due to degradation after breakage of the cells (7).

1/3 consist of approximately the COOH-terminal halves of their precursors. In the case of IL-10, removal of the NH2- terminal half appears to be essential for biological activity: when IL-la and IL-10 mRNAs are translated in vitro, only the IL-la product is active (1) and binds to the IL-1 receptor (10). Generation of IL-lP activity thus requires processing of the precursor.

Two further aspects of IL-1 processing also motivated this study. First, the precursor forms of both IL-1s lack a distin- guishable hydrophobic signal sequence, suggesting that they may follow an unusual secretory pathway (1). Second, the cleavage of the precursor occurs at an unusual site for hor- mone processing: the NH2-terminal alanine of mature IL-lP is preceded by the sequence valine-histidine-aspartate rather than by a pair of basic residues. It is thus of considerable interest to investigate the mechanisms involved in IL-1 proc- essing and secretion. There have been several reports of evidence for intermediate forms of IL-1 (7-9), but the actual means by which the precursors are processed to the mature forms remain unknown. Our approach to this problem has been to express the precursor of IL-10 in Escherichia coli, partially purify it, and use it as a substrate for extracts of monocyte and myeloid cell line proteases as well as for a number of purified common proteases. The products of the various digestions were analyzed by biological activity, by molecular weight, and by NH2-terminal sequence.

EXPERIMENTAL PROCEDURES

Expression of r-Precursor IL-lfi in E. coli-Recombinant precursor IL-10 was expressed in E. coli under the control of the phage X PL promoter and ~1857’” thermolabile repressor (11). Using standard recombinant DNA techniques, we constructed pLNIL-lBF (see Fig. 1) by ligating the following DNA segments: (1) 6160 base pairs of NcoI/HindIII-digested pLNIL-10 (12) containing the vector (confer- ring ampicillin resistance), codons 134-269 and 3’ noncoding regions of HuIL-10; (2) complementary synthetic oligonucleotides encoding residues 1-6 of IL-10 and NcoI and SstI complementary ends; and (3) a 380-base pair SstIIHindIII restriction fragment from plasmid IL-10-6 (1) encoding residues 7-133. The ligation mixture was trans- formed into the tetracycline-resistant host RRkpRK248cP (12) and correctly assembled plasmids were identified by restriction analysis of DNA isolated from transformants resistant to both ampicillin and tetracycline.

Transformants containing pLNIL-lBF were tested for the produc- tion of precursor IL-10 by SDS-PAGE analysis (14) of cultures grown in super induction medium (15) to A m of 0.5 and derepressed for 1- 20 h by elevation of temperature from 30 to 42 “C. A protein of about 31,000 daltons was apparent in samples from pLNIL-lpF-containing cultures but not in control cultures lacking the IL-10 coding region. Immuno-dot blot analysis (16) with an anti-IL-10 mAb and purified recombinant mature IL-10 (12) as standard indicated that the cul- tures contained approximately 2.5-5.0 pg/ml of r-precursor IL-la.

Extraction of r - P r e c u r s o r IL-10 from E. coli-Cell pellets from 2.5 liters of E. coli culture were resuspended in 20 ml of 30 mM Tris-HC1

9437

Page 2: Generation of Biologically Active Interleukin- 18 by Proteolytic ...

9438 Proteolytic Activation of IL-lP

asp ser arq g/v ser met ala glu val pro glu leu ala GAC TCT AGA GGATCC TAqGTAAGGAGqTTTAACC? ATG G,CA GAAGTA CCG F A G CTC,GCC; I -

' XboI TN S / D Nco I sst I J L" ""_ """ -- " " "-

" " T

"- " " __""

,- FIG. 1. Structure of plasmid pLNIL-1BF. pLNIL-lpF was derived from pLNIL-18 (12). See "Experimental Procedures" for details of construction. Abbreviations: X PL, bacteriophage X leftward promoter; [w, truncated X N gene; ZL-1, entire coding region of pre- cursor IL-lp; X til, h leftward terminator; ori, E. coli ColEl origin of replication; Ap', ampicillin resistance gene; S/D, the ribosome binding region as identified by Shine and Dalgarno (13); and T,, trans- lation terminator.

buffer (pH 9.5) containing 5 mM EDTA, 500 pg/ml of lysozyme, and 1 mM PMSF. The cell suspensions were homogenizedusing a Polytron homogenizer (Brinkmann Instruments), rapidly frozen in a Dry Ice/ methanol bath, and then thawed. Next, 200 ml of 30 mM Tris-HC1 buffer (pH 8.0) containing 150 mM NaCl and 1 mM PMSF was added to the suspensions, which were then homogenized until a uniform homogenate was obtained. The suspensions were incubated for 30 min at 4 "C, then centrifuged at 4 "C for 60 min at 3800 X g. The supernatant fractions were carefully decanted and filtered to remove any particulate matter. The pellets were re-extracted in 200 ml of 30 mM Tris-HC1 buffer (pH 8.0), containing 150 mM NaCl, 8 M urea, and 1 mM PMSF, as described above. Since both the Tris and the urea extracts contained substantial amounts of the r-precursor IL- 18, both were purified as described below.

Purificntion of r-Precursor ZL-lp-All chromatographic procedures were carried out at 4 "C. All fractions were assayed for protein concentration, and conductivity was measured where appropriate. After each chromatographic step, fractions were analyzed by SDS- PAGE (with a 10-20% gradient of polyacrylamide), followed by silver staining as described previously (17), and by Western blot using a mAb generated against purified mature IL-la.

Q-Sepharose (Pharmacia LKB Biotechnology 1nc.)-The extracts were diluted 1:4 in HZO, the pH was adjusted to 8.1, and the material was loaded at 100 ml/h onto a 25 X 2.5-cm Q-Sepharose column. For the Tris extract, the column was equilibrated in 10 mM Tris-HC1 (pH 8.1). For the urea extract, the column was equilibrated in 10 mM Tris-HC1 (pH 8.1), 2 M urea. The columns were washed with 8 column volumes of 10 mM Tris-HC1 (pH 8.1), and the bound proteins eluted with a linear gradient (three column volumes) ranging from 0 to 1.5 M NaCl in 10 mM Tris-HC1 (pH 8.1). Fractions of 7.5 ml were collected and stored at 4 "C until the next step of the purification.

Procion Red-Agarose (Bethesda Research Laboratories)-The Q- Sepharose fractions containing the r-precursor IL-lP (as determined by Western blot analysis) were pooled, diluted 1 : l O in 10 mM Tris- HCl (pH &I), and applied at 20 ml/h to a 25 X 2.5-cm column of Procion Red-agarose that had been equilibrated in 10 mM Tris-HC1 (pH 8.1). The column was washed with 4 column volumes of the starting buffer.

Phenyl-Sephurose CL-4B (Pbarmueia LKB Biotechnology ZncJ- Solid (NH4)2S04 was added to the pooled flow-through and wash of the Procion Red-agarose column to give a concentration of 0.2 M (NH4)2S0,. This solution was applied to a 20 X 5-cm column of phenyl-Sepharose CL-4B that had been equilibrated in 10 mM Tris- HC1 buffer (pH 8.1) containing 0.2 M (NH&S04. The column was washed with 3 column volumes of the starting buffer and then material was eluted initially with 4 column volumes of a decreasing linear gradient of (NH&SO,, generated with 0.2 and 0 M solutions in 10 ISIM Tris-HC1 buffer (pH 8.1). Finally, the material was eluted with 2 column volumes of 10 mM Tris-HC1 (pH 8.1). Fractions containing partially purified r-precursor IL-18 were pooled, dialyzed

against PBS, and stored at -70 "C until use. Assay of Biological Activity-IL-1 activity was measured by the

conversion assay (based on IL-1 induction of interleukin-2 activity) as previously described (18) except that the EL-4 6.1 ClO cell line (19) was used instead of LBRM-33-1A5 cells. The values reported are the averages of duplicates.

Preparation of Membranes-HL-60 (20) and KG-1 (21) cells were cultured in RPMI 1640, supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine. Human peripheral blood monocytes were purified by Ficoll gradient separation and adherence to plastic. Cells were washed one time by resuspension at 4 "C in a solution containing 10 mM Hepes (pH 7.4), 10 mM MgC12, 140 mM NaCl, followed by centrifugation for 10 min at 200 X g. The washed cells were resuspended in the same buffer at 107-108 cells/ml and lysed by sonication (four 30-s bursts with a Braun-sonic 1510 soni- cator). EDTA was then added to 1 mM, and nuclei and unlysed cells were removed by centrifugation for 5 min at 700 X g. A crude membrane preparation was then obtained by subjecting the resulting supernatant fraction to centrifugation for 45 min at 35,000 rpm (with a TH641 rotor) in a Sorvall Model OTD65B ultracentrifuge. The pellet was generally resuspended in PBS at a density of 2 X 10' cell equivalents/ml. For the experiment shown in Fig. 3, the pellet was resuspended in a solution containing 20 mM sodium phosphate (pH 7.4, 1 M NaCl.

Proteolytic Treatment of Precursor IL-1P"When the biological activity or electrophoretic mobility of products was to be determined, 5 p1 of precursor IL-lP (about 50 pg/ml in PBS) was mixed with 10 pl of crude membranes or purified protease (15-75 pg/ml in PBS) and incubated at 37 "C for 30 min (unless indicated otherwise). The incubation was terminated by placing the samples on Dry Ice or by the addition of SDS sample buffer (14). Pancreatic elastase, a- chymotrypsin, and trypsin were from Worthington, and Stnphylococ- cus aureus protease was from Boehringer Mannheim. To generate products for NHZ-terminal sequencing, 0.5 ml of precursor IL-1P was incubated with 1 ml of purified protease at 37 "C for 30 min. PMSF was then added to 1 mM, and the samples were dialyzed against water. After dialysis, the samples were concentrated to dryness in a Speed- Vac concentrator and dissolved in SDS sample buffer. Following SDS-PAGE, the resulting protein bands were transferred to Poly- brene-coated fiber paper and visualized on a U.V. light box as de- scribed by Vandekerckhove et al. (22). The visualized bands were excised and stored under nitrogen until sequence analysis. Sequence analysis was performed on each excised band as described previously (1).

Western Blot Analysis of Proteolytic Products-SDS-PAGE was carried out with 12% polyacrylamide gels. The gels were placed in transfer buffer (0.192 M glycine, 0.025 M Tris-HC1 (pH 8.3), 20% v/v methanol), and protein was then electrophoresed onto nitrocellulose (Sartorius) in a Hoeffer transfer apparatus (1 h at maximum voltage). The nitrocellulose was subsequently placed in PBS containing 3%

Page 3: Generation of Biologically Active Interleukin- 18 by Proteolytic ...

Proteolytic Activation of IL-ID 9439

bovine serum albumin for a t least 15 min at room temperature. We used mAb 16F5 (see below) to probe the blot. IgG was added to a concentration of 9 pg/ml, and the incubation was continued for 30 min. The blot was then rinsed three times with PBS and placed in PBS, 3% bovine serum albumin with horseradish peroxidase-conju- gated goat-anti-mouse antibody (Bio-Rad) diluted 1:lOOO. After an hour at room temperature in this solution, the blot was rinsed three times with PBS and was developed with a solution obtained by mixing 6 mg of horseradish peroxidase developing reagent (Bio-Rad) dis- solved in 2 ml methanol and hydrogen peroxide (60 pl diluted into 10 ml of Tris-buffered saline).

Synthesis of IL-1PC Peptide-The synthetic peptide corresponding to the COOH terminus of IL-lP (IL-lPC, residues 255-269) was made by the Merrifield solid-phase method with N-a-tert-butyloxycar- bonyl-protected amino acids and standard side chain protection (23). After cleavage from the resin with hydrofluoric acid, the free peptide was purified by high performance liquid chromatography on a C18 Vydac column (1 X 25 cm) equilibrated in 0.1% trifluoroacetic acid and eluted with a gradient of acetonitrile containing 0.1% trifluoro- acetic acid. It was then conjugated to ovalbumin using glutaraldehyde. This conjugate is referred to as OVA-IL-l@C. Alternatively, a fatty acid-conjugated peptide, composed of the same amino acids coupled to an amino-terminal dipalmitoyl lysyl moiety, was prepared by the method of Hopp (24). This peptide is referred to as NDP-IL-1PC. It was purified by gel filtration on Bio-Gel P10 in 95% acetic acid/5% water and lyophilized.

IL-lPC Immunizations and Fusion-Female BALB/c mice were immunized subcutaneously with 25 Gg of the OVA-IL-lPC complex emulsified in complete Freund’s adjuvant and boosted subcutaneously at 3 weeks with the same dose emulsified in incomplete Freund’s adjuvant. Additional immunizations were carried out with NDP-IL- 1PC at 7 weeks (50 Gg, complete Freund’s adjuvant) and 9 weeks (25 pg, incomplete Freund’s adjuvant). A t approximately week 15, one mouse was challenged by intravenous injection of 100 pg of OVA-IL- 1PC in PBS. Four days later, the animal was bled, sacrificed, and used as donor for fusion to the NS-1 myeloma cell line. Fusions and hybridoma selection using hypoxanthine/aminopterin/thymidine me- dia were carried out as described previously (25). Monoclonal anti- bodies were screened for their specific reactivity with IL-1P. MAb 16F5 was found, by dot blot, to react with recombinant IL-l@ and not with recombinant IL-la or other lymphokines tested.

Elution of IL-1 Activity from Polyacrylamide Gels-Gel lanes were sliced into 2-mm segments in the vicinity of mature IL-10 and 5-mm segments above and below this region. The slices were mashed in the medium used for the assay of IL-1 activity, and the suspension was left standing at 4 “C for 24 h. The polyacrylamide particles were then removed by centrifugation, and an aliquot of the supernatant fraction was taken for assay. About 5% of the activity applied to the gel was recovered.

Preparation of Recombinant Mature IL-lP-This material was prepared as described in Ref. 12.

RESULTS

Purification of r-Precursor IL-I@-In order to study the proteolytic activation of IL-lP, a large quantity of the precur- sor was required. We therefore cloned and expressed the precursor in E. coli, as described under “Experimental Pro- cedures.” Extraction of the E. coli cell suspension by freeze- thaw and lysozyme treatment resulted in the solubilization of 50% of the r-precursor IL-l@, and most of the remainder was extracted with 8 M urea. The extracts were diluted 1:4 to establish conditions under which the r-precursor IL-10 bound to the Q-Sepharose column: a reduction of the salt concentra- tion to less than 50 mM and, for the urea extract, a reduction of the urea concentration to 2 M. The r-precursor IL-1P eluted in a broad peak between 0.2 and 0.7 M NaCl. For the urea extract, this step was useful in removing the urea by extensive washing with 10 mM Tris-HC1 (pH 8.1) prior to elution. Following dilution of the Q-Sepharose pool 1: lO to lower the salt concentration to less than 0.01 M, passage through the Procion Red-agarose column removed 75% of the contami- nating proteins, while the r-precursor IL-1p flowed through the column without binding. The Procion Red-purified r- precursor IL-lP eluted from the phenyl-Sepharose column

with 0.10-0.05 M (NH4)&304. SDS-PAGE of an aliquot from each fraction, followed by silver staining, demonstrated that the r-precursor IL-lP extracted with Tris-HC1 was only about 50% pure, whereas the urea-extracted r-precursor IL-1@ was greater than 95% pure after this step.

In the course of this study we found that the bulk of the highly purified urea-extracted material was probably in a nonnative conformation, because it was almost totally de- graded upon incubation with either monocytic cell membranes or purified proteases, yielding only a trace of products detect- able by Western blot. However, urea-extracted material that eluted from the phenyl-Sepharose column at lower salt con- centrations, along with a number of major contaminants, and material that was extracted with Tris-HC1 showed very re- stricted proteolysis (see Fig. 3). The experiments reported below were performed with either partially purified Tris- extracted material or urea-extracted precursor eluted from the phenyl-Sepharose column at low salt concentrations.

Incubation of r-Precursor IL-I@ with Myeloid Cell Mem- branes-Because no mature IL-10 is found in the cytoplasm of monocytes (6), we reasoned that the processing activity may be located in the plasma membrane. We therefore used a crude membrane suspension from either of two human cell lines, which can produce IL-1, HL-60, or KG-1, as a source of proteolytic activity. Incubation of r-precursor IL-l@ with membranes from either cell line increased activity as a func- tion of time, yielding about a 500-fold increase after 60 min at 37 “C. Incubation at 30 “C yielded about half as great an increase (Fig. 2). No increase was observed when the precursor was incubated with PBS instead of membranes or with mem- branes from nonmyeloid cell lines. A Western blot following SDS-PAGE of the incubated material showed that the 31,000- Da precursor was broken down to three major products of lower molecular weight (designated A, B, and C), including one (band C) which migrated to the same position as mature IL-1@ (Fig. 3, lane 3). Similar results were obtained when human peripheral blood monocytes were used as the source of membranes (data not shown). Because the monoclonal antibody detection reagent was specific for the COOH-ter- minal sequence of mature IL-1@, all the observed products of 17,400 Da or greater should contain the entire sequence of the mature protein. To determine which fragment the activity was associated with, we subjected KG-1 membrane-digested precursor to SDS-PAGE, cut the gel into a number of seg- ments, and assayed the eluant from each segment for IL-1 activity. About 70% of the recovered activity was found to be

1 2 0 ’ ’ 8 1 I I

11

Time (min)

FIG. 2. Conversion of precursor IL- 18 to an active form by HL-60 membranes. HL-60 membranes and precursor IL-I@ were incubated together as described under “Experimental Procedures” for the indicated time periods at 37 (0) or 30 “C (0). The samples were then assayed for IL-1 activity.

Page 4: Generation of Biologically Active Interleukin- 18 by Proteolytic ...

9440 Proteolytic Activation of IL-ID

I 2 3 4 5 6 7 8

A B. C'

-43.0

-25.7

-18.4 -14.3

-6.2

FIG. 3. Western blot analysis of products generated by KG- 1 membranes and purified proteases. Material was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with an anti- body raised against the COOH terminus of IL-10. Lane 1, precursor IL-10; lane 2, mature IL-lp; lane 3, precursor IL-10 incubated with KG-1 membranes; lane 4, precursor IL-10 incubated with chymotryp- sin; lane 5, precursor IL-10 incubated with S. aureus protease; lane 6, precursor IL-10 incubated with elastase; lane 7, precursor IL-10 incubated with trypsin; lane 8, markers (kilodaltons).

7.

al >

z 2 0 -

1 3 5 7 9 1 1 1 3 Slice Number

FIG. 4. Electrophoretic mobility of KG-1 membrane-gen- erated fragments possessing IL-1 activity. Precursor IL-lj3 was incubated with KG-1 membranes, and the mixture was then subjected to SDS-PAGE. The gel was sliced into a number of segments, and material was eluted from each segment and assayed for IL-1 activity (u). A parallel lane in which mature IL-10 was electrophoresed was treated similarly (0- - - -0). The numbers above the arrows indicate the molecular mass markers which migrated to the indicated positions. No activity was found a t positions above those shown.

associated with band C (Fig. 4). Based on the intensity of this band on the immunoblot, we estimate that its specific activity is about four orders of magnitude greater than that of the precursor and about one order of magnitude less than that of recombinant mature IL-lP.

Digestion of r-Precursor IL-IP with Purified Known Pro- teases-Since the observed increase in IL-1 activity could be due not solely to proteolytic cleavage but also to some other protein-modifying activity in the membranes, we investigated the effect of digesting r-precursor IL-lP with purified pro- teases. We also wanted to pursue the apparent correlation between larger products and lower specific activity.

Precursor protein was digested with elastase, trypsin, chy- motrypsin, or S. aureus protease, and the digest was then assayed for IL-1 activity. Elastase and trypsin digestion re- sulted in slight increases in activity, 7-fold and 10-fold, re- spectively, compared with the activity of undigested precursor. Staphylococcus protease yielded an increase of over 300-fold, and chymotrypsin digestion caused an increase of over 500-

TABLE I NH2-Terminal sequence of protease-generated products

Products were generated and processed as described under "Exper- imental Procedures." The resulting bands were isolated from a glass fiber blot as described by Vandekerckhove et a/. (22) and analyzed as described previously (1). Yields, in parentheses, are in picomoles. Background sequences greater than 5% of the observed main sequence level were not detected.

Cycle Trypsin" a-Chymotrypsin Elastase S. oureus

1 Ile (57.1) Val (24.3) Phe (29.1) Ala (21.4) 2 Val (54.3) His (14.7) Phe (32.3) Tyr (17.9) 3 Gly (39.7) Asp (22.4) Asp (27.4) Val (20.1) 4 Gly (41.3) Ala (23.9) Thr (16.2) His (11.1) 5 Tyr (45.1) Pro (11.6) Trp (14.1) Asp (18.7) 6 Thr (21.6) Val (12.3) Asp (20.7) Ala (19.1)

8 Gly (31.5) Ser (6.5) Glu (16.4) Val (13.4) 9 Ala (34.9) Leu (10.4) Ala (17.3) Arg (8.4)

10 Asn (28.7) Asn (9.2) Tyr (14.9) Ser (4.3) Sequence corresponds to that for bovine trypsin (26).

7 Cysb Arg (9.7) Asn (18.1) Pro (9.7)

b N o signal was detected at this cycle, as expected because no modifications were introduced prior to running the protein mixture on the gel used for blotting.

trypsln elostose oureus chyrnotryp~~n Staph

I , , I 1 I I . . .DKLR'K'MLV.. .EEPIFFDTWONEAYVHD~APV.. .

72 15 79 1 0 0 103 1111l3 117

mature ILIB

FIG. 5. Identification of cleavages in IL-lB precursor by various proteases. Precursor IL-10 was incubated with the indi- cated proteases, and the NHZ-terminal sequences of the resulting products were determined as described in the text. Solid arrows indicate cleavage sites deduced from the sequence analysis. Dashed arrows indicate the most likely cleavage sites with trypsin. Numbering is from the NHz-terminal methionine residue of the precursor (1).

fold. None of these enzymes had an effect on the IL-1 bioassay when added directly.

To analyze the products of these digestions, we again used Western blots probed with the anti-COOH-terminal mono- clonal antibody. This analysis showed that at the concentra- tions used in these experiments, each enzyme produced one predominant COOH-terminal-containing fragment. The chy- motrypsin-generated fragment was slightly larger than ma- ture IL-l& the Staphylococcus protease fragment was slightly larger again, the elastase product was about 18,500 Da3 and the trypsin product was about 25,000 Da (Fig. 3). To identify these fragments unambiguously, we determined their NH2- terminal sequences (Table I). This analysis indicated that chymotrypsin cleaves after Tyr-113, leaving three residues upstream from the mature NH2 terminus (see Fig. 5); Staph- ylococcus protease cleaves after Glu-111, leaving a 5-residue extension; and elastase cleaves after Ile-103, leaving a 13- residue extension. We were unable to sequence the product of trypsin digestion because it was contaminated with trypsin (see Table I); based on the known specificity of trypsin and the migration of this product relative to the others in SDS- PAGE, the cleavage probably occurs after Arg-75 or Lys-76, leaving a 41- or 40-residue extension.

To determine if the predominant products of chymotrypsin and Staphylococcus protease digestion are responsible for the activity these proteases generate, we subjected the digested material to SDS-PAGE, cut the gel lanes into a number of segments, and assayed the eluant from each segment for IL-

In some experiments elastase also generated a product slightly smaller than the chymotrypsin-generated fragment but larger than mature IL-lP.

Page 5: Generation of Biologically Active Interleukin- 18 by Proteolytic ...

Proteolytic Activation of IL-I@ 9441 1

U

>

OI

2 4 6 8 1 0 1 2 SLice Number

FIG. 6. Electrophoretic mobility of IL-1 activity produced by incubating precursor IL- 18 with chymotrypsin ( A ) and S. aureus protease (B) . Precursor IL-16 was incubated with the indicated protease and the mixture was then subjected to SDS-PAGE. The gel was sliced into a number of segments, and material was eluted from each segment and assayed for IL-1 activity (W). A parallel lane in which mature IL-lp was electrophoresed was treated similarly (0- - - 4). The numbers above the arrows indicate the molecular mass markers (kilodaltons) which migrated to the indicated positions. These positions are approximate due to the diffuse nature of the bands (see Fig. 3). No activity was found at positions above those shown.

1 activity. The activity generated by chymotrypsin digestion overlapped with the position of the mature IL-1p standard but ran slightly more slowly (Fig. 6A) , consistent with the Western blot and sequencing results. The activity generated by Staphylococcus protease digestion was more clearly dis- placed from the IL-1p standard (Fig. 6B), again consistent with the Western blot and the larger size predicted by se- quence analysis.

DISCUSSION

We have expressed and partially purified the precursor of IL-lp to study its proteolytic processing. Extraction of the protein with urea apparently yielded both a native form and a denatured form, and these forms were separated by phenyl- Sepharose chromatography. The form eluting with the lower salt concentration, as well as precursor extracted without urea, is native by the criteria that it yields discrete fragments upon incubation with proteases and, most importantly, that it yields a high level of IL-1 bioactivity upon digestion by certain protease preparations. As expected from previous work (1, lo), the intact precursor possesses virtually no biological activity.

Membrane preparations from either human myeloid cell lines or human peripheral blood monocytes contain proteases that generate three predominant products from the precursor (designated A, B, and C in Fig. 3). Most of the biological activity produced was associated with band C, which co- migrates with recombinant mature IL-1p. We do not know whether bands A and B are intermediates in IL-1P processing, are final products that occur in uiuo, although they have not been detected in monocyte cultures, or are artifacts of the in vitro conditions we have established. In uiuo, the precursor may be subject to more restricted proteolysis, if, for example, it emerges from the membrane in a certain orientation or in proximity to a particular protease. Moreover, the concentra- tion of the precursor is vastly higher in our in vitro system than it is in uiuo, possibly making it susceptible to proteases that might not normally act on it. Further work with cells in culture is required to determine whether there actually are intermediates or various final products such as A and B in IL-lp processing.

The cleavage pattern produced by the myeloid cell mem- branes probably results from the action of a number of pro- teases, and comparison with the products of known proteases suggests candidates for some of those involved. For example,

band B co-migrates with the product of chymotrypsin diges- tion and could be the product of cathepsin G, a chymotrypsin- like enzyme found in neutrophil granules (27); and band A, which migrates to a position similar or identical to the product of elastase digestion, may result from the action of neutrophil elastase (28). If bands A and B are genuine intermediates or alternative final forms, these observations suggest a role for the principal neutrophil proteases in IL-18 processing, al- though low levels of these enzymes have been reported in monocytes as well (29,30). Cathepsin L is a candidate for the band C-generating protease, because it can cleave between glutamate and alanine (31) and is secreted by activated mac- rophages (32). Future work will focus on isolating and iden- tifying the responsible enzymes from myeloid cell membranes. We know the enzymes involved are not ubiquitous because membranes from a number of nonmyeloid cell lines failed to generate significant IL-1 activity when incubated with the precursor.

The analysis of the products yielded by known proteases clearly shows that the precursor must be cleaved near the NH, terminus for full biological activity. Even the 5-residue extension resulting from digestion with S. aureus protease appears to decrease activity slightly, and the 13-residue exten- sion left by elastase digestion causes a drastic reduction in activity. This observation suggests that the 103 residues cut off by elastase serve some role other than that of sequestering IL-1 activity; these residues could comprise a separate hor- monal activity or they could be involved in secretion. Our results also raise the question of how the 13-residue extension left by elastase reduces activity. The additional residues might sterically block binding to the IL-1 receptor or they might affect the conformation of the COOH-terminal half of the precursor.

The use of precursor IL-1P as a substrate for proteolysis has thus provided insight into the means of activation of this widely active hormone and should enable us to identify and purify the protease(s) involved in its processing.

Acknowledgments-We thank Patrick Jenney, Teresa Metzger, Robin Ireland, Sue Tyler, Jennifer Slack, Dirk Anderson, Toby Hemenway, Paula Glackin, Aline Akelis, David Amberg, and David McMasters for their skillful assistance. We also thank Linda Troup for preparation of the manuscript.

REFERENCES 1. March, C. J., Mosley, B., Larsen, A., Cerretti, D. P., Braedt, G.,

Price, V., Gillis, S., Henney, C. S., Kronheim, S. R., Grabstein, K., Conlon, P. J., Hopp, T. P., and Cosman, D. (1985) Nature

2. Oppenheim, J. J., Kovacs, E. J., Matsushima, K., and Durum, S. K. (1986) Zmmunol. Today 7, 45-56

3. Mochizuki, D. Y., Eisenman, J. R., Conlon, P. J., Larsen, A. D., and Tushinski, R. J. (1987) Proe. Natl. Acud. Sei. U. S. A. 84,

4. Auron, P. E., Webb, A. C., Rosenwasser, L. J., Mucci, S. F., Rich, A., Wolff, S. M., and Dinarello, C. A. (1984) Proc. Natl. Acud. Sci. U. S. A. 81, 7907-7911

5. Giri, J. G., Lomedico, P. T., and Mizel, S. B. (1985) J. Zmmunol.

6. Bayne, E. K., Rupp, E. A., Limjuco, G., Chin, J., and Schmidt, J. A. (1986) J. Exp. Med. 163, 1267-1280

7. Auron, P. E., Warner, S. J. C., Webb, A. C., Cannon, J. G., Bernheim, H. A., McAdam, K. J . P. W., Rosenwasser, L. J., LoPreste, G., Mucci, S. F., and Dinarello, C. A. (1987) J. Zmmunol. 138, 1447-1456

8. Matsushima, K., Taguchi, M., Kovacs, E. J., Young, H. A., and Oppenheim, J. J. (1986) J. Zmmunol. 136,2883-2891

9. Lepe-Zuniga, J. L., Zigler, J. S., Jr., Zimmerman, M. L., and Gery, I. (1985) Mol. Zmmunol. 22, 1387-1392

10. Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A,, Cosman, D.,

315,641-647

5267-5271

134,343-349

Page 6: Generation of Biologically Active Interleukin- 18 by Proteolytic ...

9442 Proteolytic Activation of IL-lP Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Bid. Chem.

11. Maniatis, T., Ptashne, M., Backman, K., Kleid, D., Flashman, S., Jeffrey, A., and Mauer, R. (1975) Cell 5 , 109-113

12. Kronheim, S. R., Cantrell, M. A., Deeley, M. C., March, C. J., Glackin, P. J., Anderson, D. M., Hemenway, T., Merriam, J. E., Cosman, D., and Hopp, T. P. (1986) Biotechnology 4,1078- 1082

262,2941-2944

13. Shine, J., and Dalgarno, L. (1975) Nature 254,34-38 14. Laemmli, U. K. (1970) Nature 227, 680-685 15. Mott, J. E., Grant, R. A., Ho, Y.-S., and Platt, T. (1985) Proc.

Natl. Acad. Sci. U. S. A. 82, 88-92 16. Cantrell, M. A., Anderson, D., Price, V., Deeley, M., Libby, R.

T., Grabstein, K. H., Cerretti, D. P., Mochizuki, D. Y., Tush- inksi, R. J., and Cosman, D. (1987) in Recombinant Lympho- kines and Their Receptors (Gillis, S., ed) pp. 187-205, Marcel Dekker, Inc., New York

17. Kronheim, S. R., March, C . J., Erb, S. K., Conlon, P. J., Mochi- zuki, D. Y., and Hopp, T. P. (1985) J. Exp. Med. 161,490-502

18. Conlon, P. J. (1983) J. Immunol. 131, 1280-1282 19. MacDonald, H. R., Lees, R. K., and Bron, C. (1985) J. Zmmunol.

135.3944-3950

20.

21. 22.

23.

24. 25. 26.

27.

28.

29.

30.

31.

32.

Collins, S. J., Gallo, R. C., and Gallagher, R. E. (1977) Nature

Koeffler, H. P., and Golde, D. W. (1978) Science 200,1153-1154 Vandekerckhove, J., Bauw, G., Puype, M., Van Damme, J., and

Van Montague, M. (1985) Eur. J. Biochem. 152,9-19 Barany, G., and Merrifield, R. B. (1979) in The Peptides, Vol. 2

(Gross, E., and Meienhofer, J., eds) pp. 1-284, Academic Press, New York

270,347-349

HOPP, T. P. (1984) Mol. Immunol. 21, 13-16 Gillis, S., and Henney, C. S. (1981) J. ZmmumZ. 126,1978-1984 Mikes, O., Holeysovsky, V., Tomasek, V., and Sorm, F. (1966)

Tanaka, T., Minematsu, Y., Reilly, C. F., Travis, J., and Powers,

Stein, R. L., Strimpler, A. M., Hori, H., and Powers, J. C. (1987)

Senior, R. M., Campbell, E. J., Landis, J. A., Cox, F. R., Kuhn,

Senior, R. M., and Campbell, E. J. (1984) J. Immunol. 132 ,

Kargel, H.-J., Dettmer, R., Etzold, G., Kirschke, H., Bohley, P.,

Portnoy, D. A., Erickson, A. H., Kochan, J., Ravetch, J. V., and

Biochem. Biophys. Res. Commun. 24,346-352

J. C. (1985) Biochemistry 24,2040-2047

Biochemistry 26,1301-1305

C., and Koren, H. S. (1982) J. Clin. Inuest. 6 9 , 384-393

2547-2551

and Langner, J. (1980) FEES Lett. 114 , 257-260

Unkeless, J. C. (1986) J. Biol. Chem. 2 6 1 , 14697-14703


Recommended