ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 296, No. 2, August 1, pp. 698-703, 1992

Purification of Interleukin-1 ,d Converting Enzyme, the Protease That Cleaves the Interleukin-I ,d Precursor Shirley R. Kronheim, Amy Mumma, Teresa Greenstreet, Kirk Van Ness, Carl J. March, and Roy A. Black’

Paula J. Glackin,

Protein Chemistry Department, Immunex Corporation, 51 University Street, Seattle, Washington 98101

Received April 1, 1992, and in revised form May 20, 1992

We have purified the IL-l@ converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the p1 is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the Nterminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL- l/3 converting enzyme. 0 iv92 Academic Press, he.

Interleukin-1 (IL-l)’ is a lymphokine secreted by monocytes, which mediates a diverse array of biological activities (1). These include induction of thymocyte proliferation (2) and B-lymphocyte differentiation (3), fever induction (4), and roles in wound healing (2) and hematopoiesis (5). Interleukin-lfl (IL-l@), the predominant form of IL-l produced by human monocytes, is synthesized as an inactive 31,000-Da precursor which is cleaved after Asp-116 to yield mature IL-lp, a 17,500-Da protein, consisting of the 153 C-terminal residues of the prohormone (6). We and others have shown that THP-1 cells, a human mono&c cell line which can be induced to produce IL-l& contain a specific endoprotease that generates mature IL-l@ from the precursor (7, 8). This protease i To whom correspondence should be addressed. Fax: (206) 233-9733. * Abbreviations used: IL-l, interleukin-1; BSA, bovine serum albumin; DTT, dithiothreitol; SDS, sodium dodecyl sulfate, PVDF, polyvinyl difluoride; PTH, phenylthiohydantoin; PAGE, polyacrylamide gel electrophoresis. 698

cleaves only between aspartate and small, nonpolar amino acids (9, 10). Its profile of inhibitor sensitivity suggests that it is a cysteine protease with unusual characteristics (7). We now report a purification scheme for the IL-10 converting enzyme from the THP-1 cell line and the sequence of 21 of the first 23 amino acids of the protein. MATERIALS

AND METHODS

Protease assay. The assay was carried out as previously described by Black et al. (11). Since the IL-lp converting enzyme is salt sensitive, samples with a salt concentration >50 mM were desalted prior to assay. This was done by applying a 100~~1 sample mixed with 5 ~1 1% bovine serum albumin (BSA) on a prespun l-ml Bio-Gel P-6DG (Bio-Rad) column, which was equilibrated in 10 mM Tris-HCl, 5 mM dithiothreitol (DTT), pH 8.1, and centrifuging for 5 min at 2OOOg.The BSA was added to prevent nonspecific absorption to the Bio-Gel column. Five microliters (30 ng) purified recombinant IL-la precursor (12) was incubated with 10 ~1 sample for 60 min at 37°C. As a control, to check for endogenous IL-10 in the early stages of the purification, an aliquot of each sample was similarly incubated with 5 ~110 mM Tris-HCl, 5 mM DTT, pH 8.1. As a further control, 5 ~1 rIL-l/I precursor was incubated with 10 pl 10 mM Tris-HCl, pH 8.1. The incubations were terminated by the addition of sodium dodecyl sulfate (SDS) sample buffer and boiling for 5 min, followed by electrophoresis on 0.75-mm-thick SDS 14% polyacrylamide slab gels, using the discontinuous Tris-glycine system of Laemmli (13). Following electrophoresis, Western blotting was performed. The proteins were transferred from the gel onto nitrocellulose (Sartorius) and probed using a 20 pg/ml solution of a purified, IL-lb COOH-terminalspecific monoclonal antibody, 16F5 (12). After incubation with goat antimouse IgG-horseradish peroxidase (Bio-Rad), the blot was developed using horseradish peroxidase color developing reagent (Bio-Rad). Each gel contained prestained molecular weight standards (Bethesda Research Labs) and 100 ng purified recombinant mature IL-16 (17,500 Da) (14). Scanning densitometer. In order to estimate recoveries after each of the purification steps, pools of the active fractions were made and serially diluted. The pools and dilutions were then assayed as described above. The resulting Western blots were copiedonto transparency film (Scotch 3M No. 504) using a Konica copier and the copies scanned using a scanning densitometer (Hoefer Scientific Instruments GS-300). The absorbance units of the 17,500-Da bands were recorded. The absorbance units due to protease-generated mature IL-10 were calculated by subtracting the absorbance units of the endogenous IL-10 bands. This value was corrected for dilution and total volume of the pool. ooo3-9861/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

IL-IS

CONVERTING

ENZYME

Production of IL-l@ converting enzyme. THP-1 cells were obtained from the American type culture collection (TIB 202). The cells were propagated in RPM1 1640 low endotoxin medium (Whittaker Bioproducts) supplemented with 10% fetal calf serum and with penicillin, streptomycin, and glutamine. Prior to stimulation, cells were grown for 3 days in spinner flasks. Cells were harvested by centrifugation (12OOg, 20 min), the medium was decanted thoroughly, and the cells were resuspended in medium and stimulated, in spinner flasks, by addition of lipopolysaccharide, hydroxyurea, and silica, as described by Matsushima et al. (15). After 16 h, the stimulated cells were harvested by centrifugation (7OOg, 15 min), washed three times by resuspension in Hank’s balanced salt solution (about 1 liter per 2 X 10”’ cells) and recentrifugation for 10 min at 300g. Cells were resuspended in 10 mM Tris-HCl, 5 mM DTT, pH 8.1 (about 4X the volume of the packed cells). The suspension was frozen in dry ice/methanol and thawed at 37’C three times and the resulting lysate was stored in polypropylene bottles at -80°C until further use. Immediately prior to the first step of purification, the lysate was thawed and centrifuged for 20 min at 37,000g at 4°C. The supernatant, containing the solubilized protease activity, was removed and prepared for chromatography on DEAE-Sephacel, as described below. Purification of IL-l@ converting enzyme. All chromatographic steps were done at 4°C using a Pharmacia FPLC system. Buffers were prepared in distilled water and sterile filtered prior to use. The DEAE-Sephacel, hydroxyapatite, and blue agarose gels were pretreated with 0.1% TritonX 100 and 10% bovine calf serum to prevent nonspecific absorption of proteins to the gels. The blue agarose gel was initially washed with 8 M urea to remove any noncovalently absorbed dye. Chromatography fractions were assayed for protein concentration using the Bio-Rad protein assay (16) (Bio-Rad Laboratories, Richmond, CA) with ovalbumin as the standard and for protease activity using the assay described above. pH and conductivity were measured where appropriate. Fractions were also analyzed by polyacrylamide gel electrophoresis (PAGE) on 14% gels, followed by silver staining (17). Molecular weight estimates were made based on protein standards from Pharmacia Fine Chemicals. DEAE-Sephucel. Five hundred milliliters of solubilized protease activity from the THP-1 lysates (obtained from about 120 liters of cultured cells) was diluted 1:2 in 10 mM Tris-HCl, 5 mM DTT, pH 8.1, and the pH was adjusted to 8.1. This material was applied at a flow rate of 120 ml/h to a 20 X 4.4-cm column of DEAE-Sephacel (Pharmacia Fine Chemicals), which was preequilibrated in the above buffer. The column was then washed with 2 column vol of equilibration buffer and eluted with a linear gradient (3 column vol) ranging from 0 to 300 mM NaCl in 10 mM Tris-HCl, 5 mM DTT, pH 8.1. Fifteen-milliliter fractions were collected, analyzed for activity, and stored at 4°C until further purification (usually 5-7 h later). Hydroryapatite. The DEAE-Sephacel fractions containing IL-10 converting enzyme activity (90-100 ml) were pooled, diluted 1:2 in 50 mM potassium phosphate buffer, 5 mM DTT, pH 7.0, and applied at a flow rate of 60 ml/h to a 14 X 3-cm column of hydroxyapatite (HA Ultrogel, IBF Biotechnics), preequilibrated in the above buffer. The column was then washed with 2 column vol of equilibration buffer and eluted with a linear gradient (4 column vol), ranging from 50 to 200 mM potassium phosphate. Ten-milliliter fractions were collected, analyzed for activity, and stored at 4°C until further purification (usually 5-7 h later). Blue ragarose. The hydroxyapatite fractions containing IL-lj3 converting enzyme activity (70-90 ml) were pooled, diluted 1:3 in 10 mM Tris-HCl, 5 mM DTT, pH 8.1, and applied at a flow rate of 30 ml/h to a 20 X 1.6-cm column of blue agarose (GIBCO-BRL), which was preequilibrated in the above buffer. The column was then washed with 3 column vol of equilibration buffer and eluted with a linear gradient (5 column vol) ranging from 0 to 1 M NaCl in 10 mM Tris-HCl, 5 mM DTT, pH 8.1. Ten-milliliter fractions were collected, analyzed for activity, and stored at 4°C until further purification (usually 5-7 h later).

PURIFICATION

699

Sephadex G-75. The blue agarose fractions containing IL-l@ converting enzyme activity (70-90 ml) were pooled, concentrated on Centriprepconcentrators (Amicon) to a volume of 2 ml, and applied to a 95 X 2.5-cm column of Sephadex G-75 (Pharmacia Fine Chemicals) 5 mM DTT, pH 8.1. The column was equilibrated in 10 mM Tris-HCl, run at a flow rate of 20 ml/h. Four-milliliter fractions were collected. The same column was used in repeated experiments and the volume of buffer required to elute the protease was found to be constant. Thus, the active fractions could be pooled without analyzing for activity and immediately prepared for the next step of the purification. The column was calibrated with ferritin (400,000 Da), ovalbumin (43,000 Da), soybean trypsin inhibitor (21,000 Da), and DNP-aspartic acid (300 Da). Chromatofocusing. The Sephadex pool was concentrated on BSApretreated Centriprep-10 concentrators to a volume of 500 al. The pretreatment involved adding 15 ml 10 mM Tris-HCl, pH 8.1 containing 1% BSA to the Centripreps and centrifuging for 30 min, decanting the remaining solution, and washing thoroughly with 10 mM Tris-HCl, pH 8.1. The concentrated protease solution was mixed with 500 11125 mM Tris-acetate, 5 mM DTT, pH 8.3, and applied to a Mono P5/20 (Pharmacia Fine Chemicals) FPLC column preequilibrated in the Tris-acetate buffer. The column was eluted with Polybuffer 96: Polybuffer 74 (3:7, Pharmacia Fine Chemicals), pH 5.0, containing 5 mM DTT, at a flow rate of 15 ml/h. Thirty l-ml fractions were collected and analyzed for pH and protease activity. Nine-hundred microliters of each of the fractions was concentrated on BSA-pretreated Centriconconcentrators to a volume of 50 ~1 and loaded on a 14% polyacrylamide gel. Following electrophoresis, the proteins were transferred to a polyvinyl difluoride membrane (PVDF, Millipore, Immobilin-P), by electroblotting at 500 mA for 30 min (18). The PVDF membrane was subsequently stained with Coomassie blue. [‘4C]lodoacetate labeling. In some experiments, the concentrated chromatofocusing fractions were incubated with 0.5 mM [“Cliodoacetate (55 mCi/mmol, Amersham) for 10 min at 37°C prior to electrophoresis. PAGE, transfer to PVDF, and Coomassie blue staining were then carried out as described above. After drying, the PVDF membranes were placed in a film cassette with Kodak X-Omat film, and the film was exposed for 3 days at -80°C. N-Terminal amino acid sequencing. The Coomassie blue-stained protein band on the PVDF membrane that correlated with protease activity was excised from the membrane and loaded on an Applied Biosystems Model 477A protein sequencer (Applied Biosystems Inc., Foster City, CA). Phenylthiohydantoin (PTH) amino acids were analyzed by an on-line Applied Biosystems Model 120A PTH analyzer. Antibody production. To generate antibodies to the IL-lb converting enzyme, a peptide comprising the first 12 amino acids from the N-terminal sequence of the protein was synthesized. A Cys-Gly sequence was added to the C-terminus to facilitate conjugation to ovalbumin via a disulfide bridge. The peptide was synthesized on an Applied Biosystems 430A peptide synthesizer. After hydrogen fluoride cleavage of the peptide from the resin, the peptide was purified on a Vydac Crs HPLC column using a gradient from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid/H,O. Two milligrams of the above peptide was conjugated to 7.0 mg ovalbumin using m-maleimidobenzoyl-N-hydroxysuccinimide ester. A 3month-old female Staufland rabbit was immunized subcutaneously in four sites on the back, with 100 pg of the peptide-ovalbumin conjugate emulsified in complete Freund’s adjuvant. The animal was boosted every 3 weeks subcutaneously with 100 pg of the conjugate emulsified in incomplete Freund’s adjuvant. Ten milliliters of blood was drawn after 4 weeks and every 3 weeks thereafter. The bleeds were screened by a “dot blot” assay. Briefly, 25 ng of either ovalbumin or the ovalbumin-conjugate was applied to nitrocellulose membranes (Schleicher and Schuell) and allowed to dry. The membranes were incubated for 30 min in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, containing 3% BSA to block nonspecific binding sites. Dilutions of the rabbit bleeds were then applied on each spot where the ovalbumin or ovalbumin-conjugate had been applied and allowed to incubate for 60

700

KRONHEIM

min. After incubation with goat anti-rabbit IgG-horseradish peroxidase (Bio-Rad), the blot was developed using horseradish peroxidase color developing reagent (Bio-Rad). Zmmunoprecipitation. Ten microliters rabbit antiserum (from the bleed 10 weeks postimmunization), neat or diluted as described in Fig. 4, was incubated with 10 ~1 partially purified protease for 20 h at 4’C. A bleed of the same rabbit prior to immunization (normal rabbit serum) and two rabbit antisera raised against irrelevant peptides, representing portions of bactinicin and the IL-7 receptor, conjugated to ovalbumin were used as controls. (See Fig. 4 for details of dilutions.) Twenty microliters protein A-Sepharose CL-4B (Pharmacia) was added and incubated for 2 h on a rocker at 4’C. Samples were then centrifuged and the supernatants discarded. The protein A-Sepharose beads were washed three times with 10 mM Tris-HCl, pH 8.1, and then assayed for protease activity.

ET AL. WS ~--+---k-a a+-+-+

F

34 .44 .50 --+--+--+-t-t--I

.67

81

.95

l% -25.7 -18.4

FIG. 2. Blue agarose chromatography of hydroxyapatite pool of active fractions. An aliquot of each fraction was incubated with rIL-l@ precursor(+) or with buffer(-). Incubation mixtures were analyzed by Western blot. PRE, rIL-10 precursor; S, starting material; F, flowthrough; numbers indicate the molarity of NaCl in the fractions; MAT, mature IL-l@; MW, molecular weight standards (kDa).

RESULTS Samples being tested for IL-lfi converting enzyme activity were incubated with rIL-l/3 precursor and then analyzed by Western blots using an IL-l@ C-terminal-specific monoclonal antibody (12) (see Materials and Methods). As previously reported (7), two products are generated by the protease: a 17,500-Da protein which migrated to the same position as mature IL-l@ and a 28,000Da protein. We have previously shown that the 17,500Da protein results from cleavage after Asp-116 of the precursor and that the 28,000-Da protein results from a cleavage after Asp-27 of the precursor (7). In the initial purification steps, interpretation of the assay required accounting for endogenous mature IL-l/3 in the samples. The endogenous mature IL-lp is generated from the endogenous precursor during preparation of the cell lysates (data not shown). Thus, in the control samples (those incubated with buffer instead of with rIL-l/3 precursor), a band at 17,500 Da indicates the presence of endogenous mature IL-l& In those samples incubated with rIL-l/3 precursor, the appearance of a band at 17,500 Da, which is more intense than the corresponding band in the control sample, indicates protease activity converting precursor to mature IL-l@ in addition to the levels of endogenous IL-l/3. In order to assess the amount of

ii

------------a t-tS .Ol t-t-t-t-t-t-t-t-t-t-~ .03 .04 .05

.06

.07

.09

.12 .l4

.I6

.18 t-

35 -25.7 -18.4 -14.3

FIG. 1. DEAE-Sephacel chromatography of THP-1 cell lysates. An aliquot of each fraction was incubated with rIL-16 precursor(+) or with buffer(-). Incubation mixtures were analyzed by Western Blot. PRE, rIL-10 precursor; S, starting material; numbers indicate the molarity of NaCl in the fractions; MAT, mature IL-@; MW, molecular weight standards (kDa). The immunoreactive proteins of less than 31,000 Da in the rIL-l/3 precursor preparation were generated during fermentation and we have been unable to separate them from the full-length precursor

(12).

protease activity, the densitometric units of the endogenous IL-l@ band was subtracted from the units of the 17,500-Da band in the samples incubated with rIL-lb precursor. This was routinely done by eye, but can also be done using a scanning densitometer, as described above. The activity could also be monitored by the generation of the 28,000-Da band. This indicator of activity was particularly useful in the early stages of the purification, because the endogenous 28,000-Da protein was separated from the protease earlier in the procedure than was endogenous mature IL-lb. With some cell preparations, the protease was found to be completely solubilized simply by resuspending the cells in the low ionic strength buffer (see Materials and Methods). With other preparations, however, multiple cycles of freezing and thawing were required for maximal solubilization (data not shown). Since freezing and thawing of the cell suspension never reduced the activity, four freeze-thaw cycles were always carried out. The solubilized material was applied to a DEAESephacel column at pH 8.1 and the protease eluted from the column with 0.06-0.12 M NaCl (Fig. 1). This step removed 79% of the contaminating proteins, the bulk of which eluted with 0.14-0.25 M NaCl. This step was also useful in partially removing endogenous IL-lb, which eluted with 0.03-0.07 M NaCl, and endogenous IL-l@ precursor, which eluted with 0.09-0.17 M NaCl. Detection of the activity in the starting material is difficult due to the large amount of endogenous IL-1B at this stage. However, by diluting the sample, the protease-generated product becomes detectable above endogenous IL-l@ (data not shown). Hydroxyapatite chromatography removed the remaining endogenous IL-l& which eluted early in the gradient, prior to the protease. The protease eluted with 0.09-0.11 M potassium phosphate; 40% of the contaminating proteins eluted before the protease and 43% eluted after the protease. It was necessary to reduce the ionic strength of the hydroxyapatite pool for the protease to bind to the blue agarose column. The protease eluted from this column

IL-10

CONVERTING

ENZYME

701

PURIFICATION

FIG. 4. Immunoprecipitation of IL-10 converting enzyme with an antiserum made to its N-terminus. The protease was incubated with: 1, buffer; 2, buffer; 3, anti-N-terminal antiserum 1:lOO; 4, anti-N-terminal antiserum 1:250; 5, anti-N-terminal antiserum 1:500, 6, anti-N-terminal antiserum 1:lOOO; 7, normal serum 1:lOO; 8, anti-bactinicin antiserum 1:lOO; 9, anti-IL-7 receptor peptide antiserum 1:lOO. Protein A-Sepharose beads were then added to samples 2-9. The beads were subsequently washed and assayed for protease activity, along with an aliquot of sample 1. PRE, rIL-lfl precursor; MAT, mature IL-la; MW, molecular weight standards (kDa).

FIG. 3. Chromatofocusing chromatography of Sephadex G-75 pool of active fractions. A. Western blot. An aliquot of each fraction was incubated with rIt-10 precursor. Incubation mixtures were analyzed by Western blot. PRE, rIL-10 precursor; numbers indicate pH of the fractions; MAT, mature IL-la; MW, molecular weight standards (kDa). B. Coomassie blue staining. Each fraction (900 rl) was subjected to PAGE, followed by transfer to PVDF and Coomassie staining. MW, molecular weight standards (kDa); numbers indicate pH of the fractions.

with 0.50-0.68 M NaCl (Fig. 2); 82% of the contaminating proteins were removed in this step, 22% eluting earlier in the gradient and 60% remaining bound to the column. From analysis of several experiments, the protease was eluted from the Sephadex G-75 column with between 196 and 220 ml. The same volume was required to elute soybean trypsin inhibitor (data not shown), suggesting a molecular weight of approximately 21,000 Da. This step removed over 90% of the contaminating proteins. After the Sephadex G-75 step, more than 99.9% of the starting protein had been separated from the protease; however, PAGE of the fractions followed by silver staining still revealed several protein bands, none of which clearly correlated with activity (data not shown). The addition of a chromatofocusing step increased the specific activity of the protease a further 50-fold and enabled us to visualize a protein band that correlated well with activity. The Sephadex G-75 pool had to be concentrated prior to applying it to the chromatofocusing column. Since the protein concentration of the Sephadex G-75 pool was low (~30 pg/ml), it was found that pretreatment of the Centriprep-10 concentrators with BSA dramatically reduced loss of the protease during concentration (data not shown). Extensive rinsing of the treated

Centripreps prior to use prevented contamination of the samples with albumin. The protease eluted from the chromatofocusing column between pH 7.10 and 6.80 (Fig. 3A). Ninety percent of each fraction containing the protease was analyzed by PAGE, followed by transfer to PVDF and Coomassie blue staining. This analysis revealed five major bands, in the fractions with IL-lp converting enzyme activity, with molecular weights of approximately 47,000,45,000,31,000, 22,000, and 18,000 Da (Fig. 3B). The 22,000-Da protein correlated best with activity. The 18,000-Da protein, which was in two of the four active fractions, was eliminated from consideration because it was not labeled by [14C]iodoacetate, an inhibitor of the protease (7), whereas the 22,000-Da protein was labeled (data not shown). Although the IL-l/l protease was not homogeneous at this point, the 22,000-Da protein band was distinct enough from any contaminants to be cut out of the PVDF paper and sequenced. The sequence of 21 of the first 23 amino

TABLE

I

Summary of Purification

Step Cell lysate DEAE-Sephacel Hydroxyapatite Blue agarose Sephadex Chromatofocusing

Total protein (md 3660 781 127 23 0.75 0.01

Total activity (units) 9.67 6.14 3.72 3.33 2.33 1.55

X X X x X x

lo7 lo7 lo7 lo7 lo7 lo7

Yield (So) 100.0 63.5 38.5 34.4 24.1 16.0

Specific activity W/w) 2.64 7.86 2.93 1.45 3.10 1.55

X X x x x x

lo4 lo4 lo5 10” lo7 lo9

Note. IL-10 converting enzyme was purified from THP-1 lysates obtained from about 120 liters of cultured cells as described under Materials and Methods. Units of activity are based on the absorbance units of the protease-generated mature IL-10 detected by Western blot. Protein was measured using the Bio-Rad protein assay (16). These results represent the mean of four experiments.

702

KRONHEIM

acids was as follows: N-P-A-M-P-T-S-S-G-S-E-G-N-VK-L-A-X-L-E-X-A-Q. (X indicates that there was no interpretable signal at this cycle.) A FASTA search of the GCG and NBRF protein database (version 30.0, 9.91) found no significant homology with any known proteins (19). To obtain further evidence that the 22,000-Da protein is the protease, we immunized a rabbit with a peptide comprised of the first 12 amino acids of the N-terminal sequence of the protein and tested whether the resulting antiserum would immunoprecipitate the protease activity. By week 10 postimmunization, the titer of the antiserum to the peptide-ovalbumin conjugate (1:7000) was higher than to ovalbumin alone (1:700), suggesting that the serum contained antibodies to the peptide. This bleed was used in the immunoprecipitation experiments. In those samples where the protease was incubated with the rabbit antiserum to the peptide, protease activity was immunoprecipitated by the protein ASepharose, whereas in samples where the protease was incubated with normal rabbit serum or the irrelevant rabbit antisera, none of the protease activity was immunoprecipitated by the protein A-Sepharose (Fig. 4). This confirms that the 22,000-Da protein is indeed the protease. As a further control, the antiserum was preincubated with 500 ng of the peptide that was used for immunization for 24 h prior to incubation with the protease. In these samples, no protease activity was immunoprecipitated (data not shown). This result indicates that the antibodies responsible for the immunoprecipitation are those which recognize the Nterminal peptide. Table I summarizes the results of the procedures used to purify the IL-l/3 converting enzyme. The overall yield was 16.0% and the five chromatographic steps resulted in a 60,000-fold increase in specific activity. DISCUSSION This report presents the first method for purification of the human IL-l@ converting enzyme, a protease responsible for the processing of inactive IL-l@ precursor to active mature IL-l& and the first amino acid sequence data for the protease. With stimulated THP-1 cells as a source of the protease, we used five standard chromatographic columns to separate the protease from all but four contaminating proteins. Correlation of the activity with a single protein was achieved by subjecting the active fractions from the final chromatography step to PAGE. The sequence of the protein band of approximately 22,000 Da, which correlated with protease activity, showed no significant homology with any known proteins, indicating that we have identified a novel molecule. To confirm that the 22,000-Da protein is the protease, we synthesized a peptide based on the sequence of the protein, raised a rabbit antiserum to the peptide, and showed that the

ET AL.

antiserum immunoprecipitated IL-lp converting enzyme activity. Following the submission of this work for publication, it was reported that the converting enzyme is a heterodimer composed of the 22,000-Da protein described here and a lO,OOO-Da subunit (20). We would not have detected the smaller component because we used only reducing gels, and the bottom of the gels was obscured by the ampholines present in the fractions eluted from our final column. The purification protocol resulted in a 60,000-fold purification with a final yield of 16%. The 16% yield was in part due to loss of activity of the protease at 4°C. After only 24 h at 4°C there is a slight decrease in activity (data not shown). By using a rapid Western blot assay, we were able to perform this five-step purification procedure in 5 days, thus keeping the time-dependent loss of activity to a minimum. Future studies will focus on the enzyme’s catalytic mechanism, on the regulation of its activity, and on its localization within the cell. ACKNOWLEDGMENTS The authors thank the Bioresources Department for carrying out the cell culture work, Mr. Ron Hendrickson for synthesizing the peptide, the Hybridoma Department for generating antibodies, and Drs. Duke Virca and Paul Sleath for reviewing the manuscript.

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IL-1B CONVERTING

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18. Matsudaira,

P. (1987) J. Biol. Chem. 262,10035-10038.

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P., and Smithies, 0. (1984) Nucl. Acids Res.

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