©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of ICH-2, a Novel Member of the Interleukin-1-converting Enzyme Family of Cysteine Proteases (*)

Joanne Kamens (§) , Michael Paskind , Margaret Hugunin , Robert V. Talanian , Hamish Allen , David Banach , Nancy Bump , Maria Hackett , Cynthia G. Johnston , Ping Li , John A. Mankovich , Michele Terranova , Tariq Ghayur

From the (1)BASF Bioresearch Corporation, Worcester, Massachusetts 01605

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Interleukin-1 converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.


INTRODUCTION

ICE()is a member of a growing family of proteins involved in both cytokine maturation and apoptosis. ICE is an intracellular cysteine protease required for the proteolysis of the inflammatory cytokine interleukin-1 (IL-1) to its biologically active form(1, 2, 3, 4) . Unexpectedly, a role for ICE in programmed cell death, or apoptosis, was suggested by the cloning of ced-3, an invertebrate gene whose product is required for apoptosis during development in Caenorhabditis elegans, and the observation that the Ced-3 protein is 29% identical with ICE(5) . A role for ICE in apoptotic pathways was further supported by experiments showing that overexpression of ICE in a rat fibroblast cell line caused apoptosis, and this activity could be blocked by CrmA, a cowpox virus protein that is a selective inhibitor of ICE function(6) . Moreover, introduction of CrmA protein into chicken dorsal root ganglion cells or murine mammary epithelial cells blocked apoptosis induced by nerve growth factor deprivation or by the absence of extracellular matrix, respectively(7, 8) .

There is increasing evidence for an involvement of other ICE family members in apoptotic pathways. For example, a family member named Nedd-2 in mouse and ICH-1 in humans induced apoptosis when overexpressed in mammalian cells(9, 10) . Similarly, overexpression of CPP32, another ICE family member which was isolated from human cells, induced apoptosis in Sf9 insect cells(11) . Finally, another link between ICE family members and apoptosis was recently suggested by experiments showing that poly(ADP-ribose) polymerase, a protein that is processed in cells undergoing apoptosis, may be cleaved by an ICE-like chicken protease called prICE(12, 13) . Although ICE itself may participate directly in the diverse biological processes of cytokine maturation and apoptosis, it is also possible that in vivo, a related enzyme (or enzymes) plays a more pivotal role in one or both of these pathways.

Recently we generated ICE-deficient mice and observed that these mice are deficient in the production of mature IL-1, but develop normally (3). Thymocytes and macrophages from these animals undergo normal apoptosis with a variety of stimulatory signals. This suggests that despite its ability to induce apoptosis in vitro, ICE is not absolutely required for apoptosis in these murine cell types. To identify novel ICE-related proteins that may function in apoptosis or other biological processes, we have begun to clone human genes with sequence homology to human ICE (hICE). We report here the discovery of one such gene, ICH (ICE and Ced-3 homolog)-2, and a preliminary characterization of its protein product. This is the first demonstration and characterization of the protease activity of an ICE-related protein.


MATERIALS AND METHODS

Cloning of ICH-2

An oligo(dT) and random-primed human thymus cDNA library (Clontech) was screened with a 1,241-bp probe containing the entire human ICE coding sequence. The probe was generated by cleaving ICE5-1, a pGEM (Promega)-derived plasmid containing the human ICE coding sequence, with XhoI and BamHI. Hybridization of 4 10 plaque-forming units was performed overnight at 55 °C in 10% dextran sulfate, 0.1% sodium dodecyl sulfate (SDS), 1.25 Denhardt's solution, 5 SSC, 500 ng/ml poly(A), and 50 µg/ml sheared salmon sperm DNA. Filters were washed in 2 SSC, 0.1% SDS at 50 °C. Forty-three positive plaques were isolated, and phage inserts were sequenced. Five clones contained inserts with overlapping segments of the ICH-2 gene. One phage isolate (Th18-3) contained an approximately 1.5-kb insert in which there was a 1,131 bp open reading frame. Both strands of this phage insert were completely sequenced using an ABI automated sequencer.

The P1 clones denoted DMPC-HFF#1-319-D4 and DMPC-HFF#1-618-B9 were isolated from the DuPont Merck Pharmaceutical Co. human foreskin fibroblast P1 library 1 by Genome Systems Inc. The following PCR primers derived from the human ICE coding sequence were used for the isolation of these clones: GACATGACTACAGAGCTGG and ACCACGGCAGGCCTGGAT.

Northern Blot Analysis

Human adult tissue Northern blot membranes were purchased from Clontech. Each lane contains 2 µg of pure poly(A) RNA. The ICH-2-specific probe was a 264-bp fragment consisting of bases 1-250 of the ICH-2 coding sequence plus a 14-bp tail introduced by the PCR reaction. PCR primers used to generate this fragment were CCCACTAGTTCCCTATGGCAGAAGGCAACCA and GGGATATTTGGTCTATGTT. The hICE-specific probe was a 347-bp fragment derived from bases 1 to 347 of the hICE coding sequence. The sequences of the PCR primers used to generate this fragment were CCCCTCGAGGCCATGGCCGACAAGGTCCTGAAGGAG and GGAAGAAAGTACTCCTTGAGAG. The human -actin control probe was supplied with the Northern blots (Clontech). Hybridization with the ICH-2 probe (25 ng of DNA, specific activity: 7 10 cpm/µg) was performed overnight at 65 °C in 5 SSPE (0.9 M NaCl, 0.5 M NaPO, 0.005 M EDTA, pH 7.7), 10 Denhardt's solution, 100 µg/ml sheared salmon sperm DNA, 50% formamide, 2% SDS and was followed by two washes at 60 °C in 2 SSC, 0.1% SDS. Hybridization with the ICE probe (50 ng of DNA, specific activity: 5 10 cpm/µg) was performed as above except the annealing temperature was 60 °C and the wash temperature was 55 °C.

Generation of Recombinant Baculoviruses

Recombinant transfer vectors were constructed by subcloning of PCR generated ICE and ICH-2 cDNAs into the BamHI and NotI cut baculovirus transfer vector pVL1393 (Invitrogen). The insert fragments containing the ICE and ICH-2 cDNAs were generated by PCR with a human ICE cDNA plasmid (14) or with ICH-2 phage clone Th18-3 (see above) as the respective templates. The 5` PCR primers for ICE were: GCCGGGATCCTATAAATATGGCCGACAAGGTCCTGAAGGAG (p45), CGGGATCCTATAAATATGCACCACCATCATCACCACGGATCTGGTCATATTGATGATGATGATAAGAACCCAGCTATGCCCACATCCTCAGGC (p32), GCCGGGATCCTATAAAATGAACCCAGCTATGCCCACATCCTCAGGC (P20). The 3` primers were CTGCGCGGCCGCATTTTAATGTCCTGGGAAGAGGTAG (p45 and p32), CTGCGCGGCCGCTTAATCTTTAAACCACACCACACCAGG (p20). The 5` PCR primers for ICH-2 were: CGGGATCC-TATAAATATGCACCACCATCATCACCACGGATCTGGTCATAT-TGATGATGATGATAAGGCAGAAGGCAACCACAGAAAAAAG (p44) and CGGGATCCTATAAATATGCACCACCATCATCACCACGGATCT-GGTCATATTGATGATGATGATAAGGCCCTCAAGCTTTGTCCTCAT (p30), and the 3` primer for both p44 and p30 was ATAGTTTAGCGGCCGCAATTTCAATTGCCAGGAAAGAGGTAG. These primers introduced restriction sites for subcloning as well as an NH-terminal polyhistidine sequence tag to p32 ICE and both forms of ICH-2. The correct clones were confirmed by restriction enzyme digestion and DNA sequencing.

To generate the recombinant baculovirus, the transfer vectors described above were used to cotransfect Sf9 cells with modified and linearized AcMNPV DNA using the BaculoGold transfection system (PharMingen). The cell culture supernatants containing baculovirus were harvested four days posttransfection and were plated for single plaques. Recombinant viral plaques were visually identified after neutral red staining (0.375 mg neutral red/ml of plating top agar) and PCR (primers GGATTATTCATACCGTCCCACCATC and GTGAGTTTTTGGTTCTTGCCGGGTCC) was performed on 10 µl of 100-µl single plaque suspensions in serum-free Grace's medium (Life Technologies, Inc.) to confirm insert size. After two rounds of replating, the purified recombinant viruses were used to generate high titer stocks. Sf9 cell infections were conducted at a multiplicity of infection of 5 in 60-mm dishes containing 3 10 cells, and DNA was analyzed at 48 h postinfection.

Expression of ICH-2 in Escherichia coli

DNA sequences encoding amino acids 105-377 of ICH-2 were PCR-subcloned into the unique EcoRI restriction site of the E. coli expression vector pMCH-1 using the following primers: GGGGAATTCATGGGTCATCATCATCATCATCA-TGGTAGCGGTCATATCGACGACGACGACAAGGCTCTGAAACTGT-GTCCGCATGAAGAGTTCCTGAGACTATG and GGGGGATCCTCTATTAATTGCCAGGAAAGAGGTAG. This subcloning placed the ICH-2 gene under the control of the inducible bacteriophage pL promoter and introduced an NH-terminal polyhistidine tag (six histidines) for purification on a nickel-chelating column(15) . An enterokinase cleavage site was also included to allow removal of the polyhistidine sequence. Amplification with these PCR primers resulted in the modification of the NH-terminal coding sequences of the 32-kDa encoding fragment to reflect E. coli codon usage. A silent mutation was also added near the NH terminus for removal of an EcoRI site to facilitate cloning.

The resultant plasmid, pMCH-1/N-His ICH-2 32 kDa, was transformed into E. coli strain MM294 (F-endA1 hsdR17 (r-m+) supE44 Thi-1)(16) which also contained a plasmid encoding the cI-857 temperature-sensitive pL promoter repressor. Growth of the transformed strain at 40 °C as described below caused induction of the pL promoter and expression of the 32-kDa N-His ICH-2 protein. An uninduced overnight culture (grown at 28 °C) of the transformed bacterial strain described above was used to inoculate (1:50) four 1-liter cultures. These were grown at 28 °C to an A value of 0.585 and then shifted to 40 °C. Cells were harvested and frozen 1 h after induction, a time point that in a small scale experiment gave a maximum of soluble protease activity (results not shown).

Purification of ICH-2

Cell pellets were resuspended in 100 ml of ice-cold lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 10% glycerol, 0.1% (w/v) CHAPS, 200 mM oxidized glutathione, 1 mM PMSF, 50 µM leupeptin, 1 µM pepstatin A). Cells were lysed in a microfluidizer and centrifuged at 11,500 g for 30 min at 4 °C. The lysate was diluted 1:1 with Buffer A (50 mM HEPES, 0.1 M NaCl, 10% (v/v) glycerol, pH 7.5) and passed over a 1-ml Hi-Trap chelating Sepharose column (Pharmacia Biotech Inc.) previously charged with 1 ml of 100 mM NiCl, washed with water, and equilibrated in Buffer A. The protein was eluted with a gradient to Buffer B (Buffer A plus 0.5 M imidazole). Fractions active in a protease assay (described below) were pooled and stored at -80 °C. Expression and purification of N-His ICE was analogous to that of N-His ICH-2, except that the purified protein was dialyzed against buffer A to remove excess imidazole before storage. The concentrations of N-His ICE and N-His ICH-2 were measured by Coomassie Plus Protein Assay (Pierce), the latter after desalting a sample on a Superdex 75 (Pharmacia) column.

Generation of Labeled Cleavage Substrates

PCR primers 1 and 4 (see below) derived from the published human pro-IL-1 sequence (17) were used to isolate the full-length coding sequence in one step for subcloning into the XhoI and BamHI restriction sites of a T7 promoter containing plasmid derived from pSV (Clontech). The pro-IL-1 Asn-Val mutant was generated in a three-step PCR reaction. First primers 1 and 3 were used to generate the 5` mutant fragment, and primers 2 and 4 were used to generate the 3` mutant gene product. Second, the products of these reactions were combined, and overlapping extension PCR was performed to generate the assembled mutant product. Finally, the full-length mutant cDNA was amplified with primers 1 and 4 for subcloning into the vector described above. All PCR-generated fragments were sequenced after subcloning. PCR primers used were as follows: 1) CCCCTCGAGTCTGAAGCAGCCATGGCAGAAGTACCT, (2) GATAACCAGGCTTATGTGCACAACGTCCCTGTACGATC ACTGAACTGC, (3) GCAGTTCAGTGATCGTACAGGGACGTTGTGCACATAAGCCA CGTTATC, (4) CCCGGATCCGTACAGCTCTCTTTAGGAAGACACAAA (underlined bases indicate those mutated to introduce the amino acid changes). [S]Methionine-labeled protein substrates for in vitro cleavage assays were generated using a coupled in vitro transcription and translation system. 1 µg of plasmid DNA was added directly to TnT -coupled reticulocyte lysate (Promega). Reactions were allowed to proceed for 60 min at 30 °C, and the generated proteins were used immediately or stored at -20 °C. Cleavage assays were carried out in a reaction buffer of 100 mM HEPES, pH 7.0, 20% (v/v) glycerol, 5 mM dithiothreitol, 0.5 mM EDTA.

Enzymatic Activity Assays

Protease assays were conducted in a reaction buffer of 100 mM HEPES, pH 7.0, 20% (v/v) glycerol, 5 mM dithiothreitol, 0.5 mM EDTA. Enzyme samples of 0.5-1.0 µg of protein were diluted to 80 µl with reaction buffer and inhibitors (when present) and preincubated at 30 °C for 30 min. Reactions were initiated by addition of 20 µl of reaction buffer containing the substrate acetyl-Tyr-Val-Ala-Asp-p-nitroanilide (Ref. 18, Quality Controlled Biochemicals, Inc., Hopkinton, MA). Incubations were continued at 30 °C, and enzyme-catalyzed release of p-nitroanilide was monitored at 405 nm for 30 min in a microtiter plate reader (Molecular Devices). To estimate K values, substrate concentration was varied between 10 and 450 µM for N-His ICE and between 100 and 3,000 µM for N-His ICH-2. Substrate concentration was 500 µM for inhibitor studies, except as noted. All assays were performed in triplicate.


RESULTS AND DISCUSSION

Cloning of ICH-2

A human thymus cDNA library was screened under low stringency hybridization conditions with a human ICE gene probe. Of the 43 phage clones isolated, 5 contained ICH-2 sequences. The longest of these phage inserts contained a 1,131-bp open reading frame that was highly homologous to human ICE (Fig. 1A). Although ICE and ICH-2 are less homologous over the first 300 bases of their coding sequences (47%), they are very related in the regions encoding the mature form of ICE (67%). Analyses of genomic ICH-2 clones isolated in a similar screen of a human genomic library allowed us to determine the intron/exon structure of ICH-2. These data show that all of the intron positions are conserved between ICE (19) and ICH-2 with the exception of the second intron of ICE. This intron is absent in ICH-2, and the low level of sequence conservation between the second exon of ICH-2 and the corresponding region of the ICE gene suggests that the amino-terminal portions of these two genes may actually have different evolutionary ancestry. To demonstrate that the gene we have cloned is truly an expressed human gene, we performed reverse transcription-PCR on human thymus mRNA with ICH-2-specific primers. An approximately 1.1-kb product was generated, and its identity was confirmed by sequencing (data not shown).


Figure 1: Nucleotide sequence of ICH-2 and comparison of its predicted amino acid sequence with other ICE family members. A, DNA coding sequence of ICH-2 and its predicted amino acid sequence. The active site cysteine residue is boxed. Intron positions are indicated by arrows. B, protein sequence alignment of all human ICE family members and of the mouse and rat ICE sequences. Dotted lines indicate gaps introduced to allow optimal alignment of the sequences. Amino acids are numbered to the right of each sequence. The highly conserved pentapeptide containing the active site Cys is indicated in bold type. The catalytic Cys and His residues are indicated in bold type and are marked by an asterisk. The residues whose amino acid side chains form the P1 pocket are indicated in bold type and are marked with a bullet (4, 9-11, 23, 28).



Mature ICE is generated from its 404-amino acid precursor protein by removal of the NH-terminal 119 amino acid ``prodomain'' and of internal residues 298-316(4) , and so contains two subunits called p20 and p10. The crystal structure of mature human ICE complexed with peptidic inhibitors has been reported by ourselves and another group(14, 20) . These structures show that ICE is a tetramer of two p20 and two p10 subunits. The catalytic residues in the active site are Cys and His. Members of the ICE protease family have the unusual requirement for Asp in the P1 position(21, 22) . Four amino acid side chains form the P1 carboxylate binding pocket: Arg, Gln, Arg, and Ser. These four residues, as well as Cys and His are conserved in ICH-2, as well as in all ICE family members reported to date (Fig. 1B). The ICH-2 cDNA encodes a polypeptide with 53% amino acid identity to ICE over the entire length of the two proteins and 60% identity in the mature domains alone. ICH-2 is the most highly conserved homolog of ICE isolated to date; mature ICE has only 27% amino acid identity with ICH-1 and only 30% with CPP32.

Two bacteriophage P1 clones containing ICE sequences were analyzed using PCR and hybridization mapping. One of these clones also contains ICH-2 sequences, suggesting that ICH-2 is located in the vicinity of the ICE gene on human chromosome 11 band q22.2-q22.3 (data not shown)(19) .

Expression of ICH-2 in Human Tissues

To analyze the expression pattern of ICH-2, a human adult multitissue Northern blot was probed with short ICH-2- and ICE-specific probes (264 and 347 bp, respectively) derived from the less conserved NH-terminal regions of the coding sequences. Given the level of sequence conservation and size similarity of the mRNAs encoded by ICE family members, it was important to use carefully chosen, small probes to avoid cross-hybridization between family members. As has been observed, the ICE-specific probe hybridizes to three transcripts of 2.5, 1.7, and 0.5 kb(23) . Using an exon 6 ICE-specific probe, we do not detect the 0.5-kb message (data not shown). The ICH-2 probe hybridizes to a single 1.7-kb mRNA (Fig. 2). The level of ICH-2 expression may be lower than that of ICE, but the two genes show a very similar distribution pattern; under these hybridization conditions, ICE and ICH-2 messages can be detected in all tissues probed, with the exception of brain (although an ICE message has been detected in human brain(24) ). Two exceptions to the relatively conserved expression patterns are ovary and placenta; whereas ICE message is barely expressed in these tissues, there are appreciable levels of ICH-2.


Figure 2: mRNA expression pattern of ICH-2 in human tissues. Human adult multitissue Northern blots were hybridized sequentially with ICH-2, ICE, and actin-specific probes as described under ``Materials and Methods.'' Positions of size markers are indicated in kilobases. Note that caution should be used in comparing signal intensities on one blot to another as the actin control panel demonstrates clearly uneven loading.



ICH-2 Induces Apoptosis

As described above, all ICE family members reported to date have been shown to induce apoptosis when overexpressed in various cell lines. To determine if this was also true for ICH-2, we infected Sf9 insect cells with a recombinant baculovirus expressing either the full-length protein or a truncated version of ICH-2 which lacks the NH-terminal prodomain. Analysis of insect cells infected with these baculovirus constructs and constructs expressing full-length or truncated ICE showed that expression of these proteins caused Sf9 cells to exhibit the condensed morphology, cellular fragmentation, and internucleosomal DNA cleavage characteristic of cells undergoing apoptotic cell death (Fig. 3).


Figure 3: Apoptotic DNA fragmentation in Sf9 cells expressing ICE or ICH-2. Genomic DNA was isolated 48 h postinfection from Sf9 cells infected with wild type baculovirus (lanes 1) or recombinant baculoviruses expressing the following proteins: p20 ICE (lane 2), p45 ICE (lane 3), p32 ICE (lane 4), p44 ICH-2 (lane 5), and p30 ICH-2 (lane 6). p45 ICE and p44 ICH-2 are full-length proteins, p32 ICE and p30 ICH-2 are truncated versions lacking the NH-terminal prodomains, and p20 ICE is the 20-kDa subunit of ICE alone (amino acids 120-297). DNA was analyzed on a 2.0% agarose gel. DNA size markers are shown (lane M).



Expression and Characterization of N-His ICH-2 Protein

The maturational processing of ICE, perhaps performed by ICE itself, is a result of cleavage after Asp residues 119, 297, and 316(4) . Asp and Asp are conserved in ICH-2 (Asp and Asp). The ICE Asp site is not perfectly conserved in ICH-2, but there is an Asp at position 104 which might define the cleavage site for removal of the prodomain and generation of a 32-kDa form of ICH-2. Therefore, to generate ICH-2 protein with cysteine protease activity, we subcloned the DNA sequences encoding amino acids 105-377 of ICH-2 into an E. coli expression vector under the control of an inducible pL promoter. By analogy with similar experiments on the ICE protein, this region of ICH-2 was expected to be expressed in E. coli and recovered as an active protease from the soluble fraction. As was done for the ICE protein (see below), an NH-terminal polyhistidine (N-His) sequence was introduced before the putative p32 coding region of ICH-2 to facilitate recovery of the protein. N-His ICE displays catalytic properties with peptidic substrates similar to those of native ICE.()N-His ICH-2 was expressed in E. coli and purified by nickel-chelating chromatography. Purified N-His ICH-2 migrated on an SDS-polyacrylamide gel as two bands with approximate molecular masses of 20 and 10 kDa (data not shown), suggesting that the N-His ICH-2 is proteolytically processed to subunits analogous to those of mature ICE during expression and/or purification. Purified N-His ICE also displayed major bands at 20 and 10 kDa. In addition, gel analysis revealed partial degradation, suggesting that a significant portion of the N-His ICE protein preparation was inactive (data not shown). NH-terminal sequencing of the 10-kDa band was identical to the ICH-2 sequence downstream of Asp, showing that this residue may serve as an analogous autocleavage site to Asp of ICE. Proteolytic activity of ICH-2 was analyzed in two ways; the N-His ICH-2 protein was tested for the ability to cleave whole protein substrates, and its enzymatic properties were studied in more detail using small peptide substrates.

ICE has been shown to cleave human pro-IL-1 at two processing sites: cleavage between Asp and Gly generates a 28-kDa intermediate, and cleavage between Asp and Ala generates the 17-kDa bioactive product(2, 25) . To investigate the cleavage reactions performed by the two proteases, we compared cleavage of an [S]methionine-labeled pro-IL-1 substrate protein by the purified N-His ICH-2 and N-His ICE proteins. N-His ICE processed all of the pro-IL-1 to the mature 17-kDa form by the 30-min time point, whereas incubation with N-His ICH-2 led to the generation of only small amounts of mature IL-1, even after 120 min (Fig. 4A). To compare the ability of the two enzymes to generate the 28-kDa intermediate form, equal amounts of N-His ICH-2 or N-His ICE were combined with a mutant pro-IL-1 protein in which Asp-Ala been changed to Asn-Val. N-His ICE and N-His ICH-2 are unable to cleave this mutated protein such that the 17-kDa mature form of IL-1 is generated (Fig. 4B). N-His ICE rapidly cleaves all of the mutant substrate to the 28-kDa form by 5 min; N-His ICH-2 performs this cleavage to completion by the 60-min time point. These data suggest that although ICE efficiently cleaves pro-IL-1 at both the Asp and Asp processing sites, ICH-2 demonstrates a pronounced difference in its activity on these two cleavage sites.


Figure 4: Cleavage of ICE protein substrates in vitro. A, cleavage of wild type pro-IL-1. B, cleavage of mutant pro-IL-1 in which Asp-Ala has been mutated to Asn-Val. Equal amounts (20 µg for pro-IL-1 cleavage, 5 µg for mutant pro-IL-1 cleavage) of N-His ICE and N-His ICH-2 were combined with the indicated [S]methionine-labeled substrate protein. Samples were removed to denaturing loading buffer at the indicated time points and analyzed at the completion of the experiment on a 10-20% Tris-Tricine gradient polyacrylamide gel (Integrated Separation Systems). Positions of 31-kDa pro-IL-1 (pIL-1), the 28-kDa intermediate (28K), the 17-kDa mature IL-1 (mIL-1), and the 14-kDa prodomain are indicated with arrows.



The in vitro catalytic properties of N-His ICE and N-His ICH-2 were compared using the chromogenic peptide substrate acetyl-Tyr-Val-Ala-Asp-p-nitroanilide(18) . Plots of velocity versus substrate concentration (not shown) were fit to the Michaelis-Menten equation to obtain Kvalues which were 681 ± 84 µM for N-His ICH-2 and 83 ± 10 µM for N-His ICE. The tetrapeptide aldehyde inhibitor Ac-Tyr-Val-Asp-CHO (Ref. 4, Bachem Bioscience, Inc., King of Prussia, PA) also revealed differences in peptide binding to the enzymes. Using the substrate Ac-Tyr-Val-Ala-Asp-p-nitroanilide at concentrations equal to the measured K values for each enzyme, Ac-Tyr-Val-Asp-CHO displayed IC values of 38 and 748 nM for N-His ICE and N-His ICH-2, respectively. By contrast, small molecule inhibitors had similar effects on the two enzymes. Both were highly sensitive to the thiol reagents iodoacetamide and iodoacetic acid, confirming their classification as cysteine proteases, and both were resistant to the serine protease inhibitor PMSF (). E64(26) , an inhibitor of several cysteine proteases (27) with ICE being a notable exception(25) , was ineffective against both. The results of these small molecule inhibitor and peptide substrate/inhibitor studies argue that the enzymes share catalytic similarities but differ in their recognition of proteins in the P2-P4 positions.

We have identified a new member of the ICE cysteine protease family, a family with implicated or proven roles in both inflammation and apoptosis. The high degree of sequence conservation between ICE and ICH-2 suggests that the two proteases have similar or overlapping functions in vivo. However, it is clear that ICE and ICH-2 are not entirely redundant. ICH-2 is expressed in mice,()but apparently does not compensate for the lack of ICE protein in ICE-deficient mice, in the production of mature IL-1 or the response to lipopolysaccharide-mediated sepsis (3). We demonstrate here differences in the recognition of protein and peptide substrates and inhibitors by ICE and ICH-2, suggesting that ICE and ICH-2 may participate in quite different cellular functions or pathways.

  
Table: Inhibitor sensitivity of N-His ICH-2

Activity assays were performed in triplicate in the presence of inhibitors at the indicated concentrations as described under ``Materials and Methods.'' Results are expressed as percent of control (no inhibitor) reactions.



FOOTNOTES

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

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U25804.

§
To whom correspondence and reprint requests should be addressed: BASF Bioresearch Corp., Dept. of Immunology, 100 Research Dr., Worcester, MA 01605. Tel.: 508-849-2655; Fax: 508-831-3590; E-mail: kamens@biovax.dnet.basf-ag.de.

The abbreviations used are: ICE, interleukin-1-converting enzyme; hICE, human ICE; IL-1, interleukin-1; PCR, polymerase chain reaction; Sf9, Spodoptera frugiperda cells; AcMNPV, Autographa californica nuclear polyhedrosis virus; N-His, NH-terminal polyhistidine; PMSF, phenylmethylsulfonyl fluoride; bp, base pair(s); kb, kilobase(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

R. V. Talanian, unpublished results.

M. Terranova and J. Kamens, unpublished results.


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