Identification and Characterization of CPP32/ Mch2 Homolog 1, a Novel Cysteine Protease Similar to CPP32*

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1 (cid:98) converting enzyme (ICE) family of proteases with substrate specificity for Asp- X . CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding. Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not interleukin 1 (cid:98) precursor in vitro .

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1␤ converting enzyme (ICE) family of proteases with substrate specificity for Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding.

Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not interleukin 1␤ precursor in vitro.
Programmed cell death (apoptosis) plays an important role in embryonic development, homeostasis, and in diseases such as neurodegenerative disorders, autoimmune diseases, and cancer (for review, see Ellis et al. (1991)). Apoptosis has been characterized by a set of cellular events including cell shrinkage, chromatin condensation, and DNA fragmentation. It is becoming apparent that activation of proteases is a crucial event in the cellular execution of apoptosis (for review, see Martin and Green (1995)). Genetic studies in the nematode Caenorhabditis elegans have provided evidence that the Ced-3 gene is indispensable for cell death during worm development . The CED-3 protein shares 28% sequence identity with mammalian interleukin-1␤ (IL-1␤) 1 converting enzyme (ICE) that cleaves the inactive IL-1␤ precursor to the proinflammatory cytokine (Thornberry et al., 1992). ICE and CED-3 are members of a new cysteine protease family that also includes mammalian enzymes of Tx, ICE rel III, NEDD-2/ICH-1, CPP32, and MCH2 (Faucheu et al., 1995;Munday et al., 1995;Wang et al., 1994;Kumar et al., 1994;Fernandes-Alnemri et al., 1994, 1995. These cysteine proteases are composed of two subunits of approximately 20 kDa and 10 kDa derived from processing of precursor polypeptides. They share conserved amino acid residues for catalysis and binding of the P1 aspartate residue (Wilson et al., 1994;Walker et al., 1994). Overexpression of cysteine proteases in the Ice/Ced-3 gene family leads to apoptosis of transfected cells (Miura et al., 1993;Faucheu et al., 1995;Fernandes-Alnemri et al., 1994, 1995Gu et al., 1995a;, and ICE inhibitors block cell death of neurons deprived of neurotrophic factors (Gagliardini et al., 1994;Milligan et al., 1995). ICE or related proteases have also been implicated in Fas-and tumor necrosis factor ␣-mediated apoptosis (Kuida et al., 1995;Enari et al., 1995;Los et al., 1995;Miura et al., 1993), suggesting a physiological role for these cysteine proteases in apoptosis.
Recently, Nicholson et al. (1995) have identified CPP32 as a cysteine protease that is responsible for the cleavage of poly-(ADP-ribose) polymerase (PARP), a DNA repair enzyme that is cleaved from a 116-kDa polypeptide into 31-kDa and 85-kDa polypeptides at the onset of apoptosis and in nuclei treated with apoptotic cytosolic extracts (Kaufmann et al., 1993;Lazebnik et al., 1994). Darmon et al. (1995) found that cytotoxic T lymphocyte-specific granzyme B can process and activate CPP32, resulting in PARP cleavage in target cells, suggesting that granzyme B-induced apoptosis may be mediated by a protease activation cascade involving CPP32. Using polymerase chain reaction, Fernandes-Alnemri et al. (1995) identified a second human Ced-3 homolog, MCH2, that is 38% identical with CPP32. Overexpression of MCH2 in insect cells induces apoptosis, and purified MCH2 cleaves PARP in vitro, suggesting a possible role of MCH2 in vertebrate apoptosis. We recently cloned a cDNA encoding a 34-kDa polypeptide that is highly homologous to CPP32 and Mch2. In this report we describe the cloning, expression, and characterization of this new cysteine protease named CMH-1 (CPP32/Mch2 homolog).

MATERIALS AND METHODS
Cloning of Cmh-1 cDNA-Sequence data base searching was performed using the Blast algorithm (Altschul et al., 1990). Initial multiple sequence alignments of known ICE family protein peptide sequences were generated by the MACAW program (Schuler et al., 1991) and later refined by the combination of the PILEUP algorithm (Genetics Computer Group, 1995) and pairwise comparison. A TBLASTN search of the GenBank TM EST data base maintained by the National Center for Biotechnology Information (NCBI) using the ICE peptide sequence revealed limited similarity with the sequence of a clone deposited by the WashU-Merck EST project with an accession number of T50828. The partial human cDNA clone 72778 corresponding to this EST sequence was obtained through the IMAGE Consortium (Lawrence Livermore National Laboratory). 5Ј rapid amplification of cDNA ends-PCR (polymerase chain reaction) generated a 1.0-kb fragment of the 5Ј region of complete cDNA encoding the novel ICE homolog. In short, a human spleen cDNA pool was ligated with a DNA adapter (Clontech), and PCR was performed using a primer to the 5Ј adapter (5Ј-CCATCCTAATAC-GACTCACTATAGGGC) and a primer to the EST T50828 sequence (5Ј-GCAAACTCTGTCAATTCACCC). A 0.8-kb SacI-HgiAI fragment containing the 5Ј coding region and a 1.55-kb HgiAI-KpnI fragment from the 72778 clone containing the 3Ј region were ligated together and subcloned into pBluescript SK (Stratagene) at SacI/KpnI to generate the full-length cDNA. The sequence of the novel cDNA named Cmh-1 was confirmed on both strands by ABI Prism Dye Deoxy Terminator sequencing using the ABI 373 DNA Sequencer.
Northern Blot Analysis of Cmh-1 mRNA-A human multiple tissue RNA blot (MTN blot, Clontech) containing 2 g/lane poly(A) ϩ RNA was * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be 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 TM /EMBL Data Bank with accession number(s) U40281.
hybridized to a 32 P-labeled full-length Cmh-1 cDNA probe. The blot was prewashed at room temperature and then washed at 0.1 ϫ SSC, 0.1% SDS at 42°C for 20 min and exposed to Kodak XAR-5 film.
Transient Expression and Analysis of Cmh-1 in COS Cells-A DNA fragment encoding the N-terminally T7-tagged CMH-1 lacking the first 23 amino acid residues was generated by PCR with primers 5Ј-GC-TGCAGTCTAGAGCTCCATGGCTAGCATGACTGGTGGACAGCAAA-TGGGTGCTAAGCCAGACCGGTCCTCG and 5Ј-GAATTCTAGATTC-TATTGACTGAAGTAGAGTTC. Amplified DNA was digested with XbaI and subcloned into the XbaI-cut pcDLSR␣ vector (Takebe et al., 1988). The C186S/W232A/R233E mutant was generated by site-directed mutagenesis using oligonucleotides 5Ј-CATTCAGGCTAGCCGAGGGAC and 5Ј-GCTATTACTCAGCTGAGAGCCCAGGA (Kunkel, 1985). Transient transfection of COS cells with the DEAE-dextran method was carried out as described by Gu et al. (1995a). Cells were harvested 24 h post-transfection for analysis of the expressed proteins by immunoblotting with an anti-T7 epitope monoclonal antibody (Novagen, Madison, WI). For detection of apoptosis by DNA laddering (Tsukada et al., 1995), COS cells (1 ϫ 10 6 ) were transfected with the tagged wild type or mutant Cmh-1 construct and harvested at 36 h post-transfection.
Expression, Purification, and Characterization of Recombinant (His) 6 -tagged CMH-1 Protein-An expression plasmid for the N-terminally (His) 6 -tagged CMH-1(Ala 24 -Gln 303 ) was constructed by introducing XhoI sites at the 5Ј and 3Ј ends of Cmh-1 cDNA by PCR using primers 5Ј-CGGCTCGAGGCTAAGCCAGACCGGTCCTCG and 5Ј-GGGCTCGAGCTATTGACTGAAGTAGAGTTC and then ligating the resulting XhoI fragment into an XhoI-cut pET-15b-inducible Escherichia coli expression vector (Novagen). The resulting plasmid directs the synthesis of a polypeptide of 303 amino acids consisting of a 23residue peptide (MGSSHHHHHHSSGLVPRGSHMLE, where LVPRGS represents a thrombin cleavage site) fused in-frame to the N terminus of CMH-1 starting at Ala 24 , as confirmed by DNA sequencing and by N-terminal sequencing of the expressed proteins. E. coli strain BL21(DE3) carrying this plasmid was induced with 0.8 mM isopropyl-1-thio-␤-D-galactopyranoside, harvested, and lysed in a microfluidizor (Microfluidic, Watertown, MA) in a buffer containing 20 mM sodium phosphate (pH 6.8), 300 mM NaCl, 2 mM dithiothreitol, 10% glycerol, 0.4 mM phenylmethylsulfonyl fluoride, and 2.5 g/ml leupeptin (Buffer A). Lysates were cleared by centrifugation at 100,000 ϫ g for 30 min and the supernatant containing soluble CMH-1 was loaded onto a 0.5-ml nickel-NTA column (Qiagen) and washed with Buffer A extensively. The CMH-1 protein was eluted with 100 -200 mM imidazole in Buffer A to give a yield of approximately 5 mg of protein per liter of culture. The purified protein fraction contained three major polypeptides of approximately 34 kDa, 22 kDa, and 12 kDa. The N terminus of each of these peptides was analyzed using the ABI 477A Protein Sequencer. Cleavage of 35 Slabeled IL-1␤ precursor and a truncated form of 35 S-PARP by purified ICE or CMH-1 in vitro were carried out as described (Gu et al., 1995b).

RESULTS AND DISCUSSION
Cloning of Cmh-1, a Close Homolog of CPP32/Mch2-A TBLASTN search of the GenBank TM data base using the ICE peptide sequence revealed that an EST (expressed sequence tag) clone with the accession number T50828 showed limited sequence similarity. The clone corresponding to this sequence, human clone 72778, was obtained through the IMAGE Consortium, and its sequence was verified. The cDNA insert of clone 72778 contains a partial open reading frame for a novel ICElike protein and 3Ј-untranslated region. Using rapid amplification of cDNA ends-PCR, we cloned the 5Ј end of the full-length cDNA from human spleen poly(A) ϩ mRNA and ligated it to the 3Ј cDNA fragment from the clone 72778 to generate the fulllength cDNA.
The full-length cDNA contains a single large open reading frame encoding a polypeptide of 303 amino acids which we have named CMH-1 (Fig. 1). This protein was found to have the highest similarity to CPP32 and MCH2 with 52% and 31% identity, respectively. It shares limited overall similarity with other members of the Ice/Ced-3 gene family, including ICE itself which is 19% identical. However, there is strong similarity between CMH-1 and ICE in the region that comprises the core of the ICE structure as well as a number of residues surrounding the active site Cys 285 and His 237 in ICE (Wilson et al., 1994;Walker et al., 1994). In particular, all of the CMH-1 residues which line the putative substrate binding pocket at FIG. 1. A, predicted amino acid sequence of the human Cmh-1. The conserved QACRG motif is underlined, and the catalytic residues His 144 and Cys 186 are shown in bold. The autoprocessing aspartate sites between P21 and P11 subunits are circled. The putative cleavage for removal of prodomain occurs at Asp 23 which is boxed. B, sequence alignment of CMH-1 and human ICE homologs. The sequence of CMH-1 was aligned to other human ICE homologs. Residues which are conserved in more than 70% of the homologs are boxed. The prodomain of ICE and the putative prodomains of Tx, ICE rel III, and Ich1 are represented by dashes. The catalytic His and Cys are marked by asterisks. The location of residues which comprise the S1-S4 substrate side-chain binding pockets are marked by the numbers 1-4. Novel Ice/Ced-3 Family Protease CMH-1 1826 the P1 position are identical with those found in ICE, including Arg 87 and Arg 233 , corresponding to ICE Arg 179 and Arg 341 , strongly suggesting that CMH-1 has substrate specificity for Asp in this position as seen in other members of this family (Fig. 1B). There is much less sequence conservation in the binding pockets corresponding to the P3-P4 residues between CMH-1 and other homologs.
Expression of Cmh-1 in Adult Human Tissues-We investigated the expression of Cmh-1 mRNA in various adult tissue by Northern analysis using a Cmh-1 specific cDNA probe. A 2.5-kb mRNA was detected in tissues including pancreas, lung, placenta, kidney, muscle, liver, and heart (data not shown). Notably, the expression of Cmh-1 was almost undetectable in brain. This ubiquitous pattern of expression in a variety of tissues is very similar to that observed for CPP32.
Proteolytic Activity and Induction of Apoptosis by CMH-1 in COS Cells-We first used transient expression in COS cells to determine if CMH-1 has the predicted cysteine protease activity. Based on the sequence alignment with CPP32, we estimated that the first 23 amino acids would represent the prosequence of CMH-1. A cDNA fragment encoding a CMH-1 protein lacking the first 23 amino acid residues was generated and subcloned into the COS cell expression vector pcDLSR␣ (Takebe et al., 1988). To facilitate the detection of the expressed proteins by immunoblotting, a T7-epitope tag (MASMTG-GQQMG, Tsai et al. (1992)) was fused to the N terminus of the protein. Immunoblotting of transfected COS cell lysates with an anti-T7 antibody detected two bands of approximately 34 kDa and 22 kDa, respectively. The 22-kDa band was absent in cells transfected with a mutant Cmh-1 cDNA with substitutions at the predicted active site residues (Fig. 2A). These results suggest that the T7-tagged truncated CMH-1 protein is capable of autoprocessing itself into subunits equivalent to the P20 and P10 polypeptides of ICE in COS cells.
To determine if CMH-1 protein expressed in COS cells may have other detectable protease activity, we co-expressed CMH-1 in COS cells with a truncated form of PARP, a known substrate for CPP32 and other ICE-like proteases both in vivo and in vitro Gu et al., 1995b). We found most of the 45-kDa PARP polypeptide was cleaved into a 31-kDa peptide when it is co-expressed with CMH-1, similar to the cleavage of PARP by ICE, Tx, and Nedd-2. This cleavage was abolished when the mutant CMH-1 was used, indicating that the cleavage is specific and that Cmh-1 encodes a protease that can either cleave PARP directly or activate an endogenous protease(s) that in turn cleaves PARP. A small amount of PARP cleavage was also observed when COS cells were transfected with PARP alone; this is most likely due to the presence of (an) endogenous ICE-like protease(s) in the cells.
Overexpression of the CMH-1 protein in COS cells also caused cells to round up, a sign of transfected cells undergoing apoptosis. To confirm this, we analyzed chromosomal DNA from cells transfected with the wild type Cmh-1 and found internucleosomal DNA fragmentation (Fig. 2B). DNA fragmentation was absent from mock-transfected cells or cells transfected with the mutant Cmh-1 (Fig. 2B). Thus, like other ICElike proteases, CMH-1 is capable of inducing apoptosis when overexpressed in cells.
Expression and Purification of Recombinant CMH-1-To further characterize the activity of CMH-1 protein, we expressed an N-terminally (His) 6 -tagged CMH-1(Ala 24 -Gln 303 ) in E. coli and purified the protein by Ni-affinity chromatography. The purified protein fraction contained three major polypeptides of approximately 34 kDa, 22 kDa, and 12 kDa (Fig. 3). An 11-kDa polypeptide was also present in some preparations. Immunoblotting with anti-(His) 6 -tag antibody and N-terminal sequencing analysis of these polypeptides indicated that the 34-kDa and 22-kDa polypeptides contain the (His) 6 -tag fused to the CMH-1 at Ala 24 (Fig. 3). The N termini of the 12-kDa and 11-kDa polypeptides start at Ser 199 and Ala 207 of CMH-1, respectively. Both residues are preceded by an aspartate, suggesting that 12-kDa and 11-kDa polypeptides are generated by processing of the CMH-1 protein at Asp 198 -Ser 199 and to a lesser extent at Asp 206 -Ala 207 . We propose that the 22-kDa and 12-kDa peptides represent the two subunits of the CMH-1 protease.
CMH-1 Is a CPP32-like Cysteine Protease That Cleaves PARP but Not IL-1 ␤ Precursor-We further analyzed the protease activity of the purified CMH-1 protein using 35 Slabeled PARP or IL-1␤ precursor prepared by in vitro transcription and translation as substrates. We found that CMH-1 at less than 3 nM concentration almost completely cleaved the PARP substrate within 30 min (Fig. 4A). No cleavage of IL-1␤ precursor was observed under similar conditions, indicating that CMH-1, like CPP32, has a preferred substrate specificity for PARP. The efficiency of CMH-1 cleaving PARP appears to be significantly higher than that observed for ICE in cleaving the same substrate under identical conditions (Gu et al., 1995b) and is close to the activity of CPP32 against PARP 2 or ICE FIG. 2. PARP cleavage and induction of apoptosis by CMH-1 in COS cells. A, expression, autoprocessing, and PARP cleavage by CMH-1. COS cells were transfected with the tagged wild type or an active site mutant (C186S/W232A/R233E) CMH-1 expression plasmid, alone or in combination with a T7-tagged truncated PARP plasmid (Gu et al., 1995b) as indicated. Twenty-four hours later, the cells were harvested, and expressed proteins were analyzed by immunoblotting with an anti-T7 antibody. Molecular mass is indicated in kilodaltons. The unprocessed and the processed CMH-1 polypeptides were indicated along with the uncleaved truncated PARP (PARP(T)) and the tagged N-terminal fragment of cleaved PARP (PARP*). (His) 6 -tagged CMH-1(Ala 24 -Gln 303 ) was expressed and purified from E. coli soluble fractions by Ni-affinity chromatography as described under "Materials and Methods." Four l (2 g of total protein) of purified fraction were electrophoresed on 16% polyacrylamide gel. The proteins were detected either by Coomassie Blue staining (lane 1) or by immunoblotting with anti-(His) 6 -tag antibody (lane 2). N-terminal amino acid sequencing indicated that the 12-kDa and 11-kDa bands correspond to CMH-1 polypeptides starting at Ser 199 and Ala 207 , respectively.
Novel Ice/Ced-3 Family Protease CMH-1 1827 against IL-1␤ precursor (Gu et al., 1995b). This cleavage activity of CMH-1 was inhibited by the tetrapeptide inhibitor DEVD-CHO Wang et al., 1995) as well as the cysteine-alkylating reagents N-ethylmaleimide and iodoacetamide, but not by the ICE inhibitor YVAD-CHO (Thornberry et al., 1992;Gu et al., 1995b) or inhibitors of serine proteases or metalloproteases (Fig. 4B). Interestingly, CMH-1 also appears to be relatively insensitive to inhibition by crmA (cytokine response modifier A), a very potent ICE inhibitor synthesized by cowpox virus (Ray et al., 1992). At a crmA concentration of 16 g/ml which is about 50-fold higher than that required to completely inhibit an equivalent amount of ICE activity, 2 no inhibition of CMH-1 was observed. These results indicate that CMH-1 is a cysteine protease with properties closer to CPP32 than to ICE, consistent with its relative sequence identity to these two proteases. CPP32 has been implicated to play a major role in apoptosis and cytotoxic T lymphocyte-mediated cell killing as well as cellular regulation of sterol metabolism Darmon et al., 1995;Wang et al., 1995). CMH-1 and CPP32 share high amino acid similarity and conserved S1, S2, and S4 residues in the active site pocket. This sequence conservation might allow these two proteases to share an overlapping substrate profile and to have similar roles in apoptosis and other physiological processes. It is equally likely that CMH-1 protease has different preferred substrates that remain to be identified. The expression and purification of active CMH-1 protease should facilitate the characterization of the enzyme and its substrates.