©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of CPP32/Mch2 Homolog 1, a Novel Cysteine Protease Similar to CPP32 (*)

(Received for publication, November 8, 1995; and in revised form, November 22, 1995)

Judith A. Lippke Yong Gu Charlyn Sarnecki Paul R. Caron Michael S.-S. Su (§)

From the From Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1beta 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 1beta precursor in vitro.


INTRODUCTION

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 (Yuan et al., 1993). The CED-3 protein shares 28% sequence identity with mammalian interleukin-1beta (IL-1beta) (^1)converting enzyme (ICE) that cleaves the inactive IL-1beta 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, ICEIII, 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, 1995; Gu et al., 1995a; Tewari et al., 1995), 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 alpha-mediated apoptosis (Kuida et al., 1995; Enari et al., 1995; Los et al., 1995; Tewari and Dixit, 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 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). 5` rapid amplification of cDNA ends-PCR (polymerase chain reaction) generated a 1.0-kb fragment of the 5` 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 5` adapter (5`-CCATCCTAATACGACTCACTATAGGGC) and a primer to the EST T50828 sequence (5`-GCAAACTCTGTCAATTCACCC). A 0.8-kb SacI-HgiAI fragment containing the 5` coding region and a 1.55-kb HgiAI-KpnI fragment from the 72778 clone containing the 3` 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 µg/lane poly(A) RNA was hybridized to a P-labeled full-length Cmh-1 cDNA probe. The blot was prewashed at room temperature and then washed at 0.1 times 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 5`- GCTGCAGTCTAGAGCTCCATGGCTAGCATGACTGGTGGACAGCAAATGGGTGCTAAGCCAGACCGGTCCTCG and 5`-GAATTCTAGATTCTATTGACTGAAGTAGAGTTC. Amplified DNA was digested with XbaI and subcloned into the XbaI-cut pcDLSRalpha vector (Takebe et al., 1988). The C186S/W232A/R233E mutant was generated by site-directed mutagenesis using oligonucleotides 5`-CATTCAGGCTAGCCGAGGGAC and 5`-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 times 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-Gln) was constructed by introducing XhoI sites at the 5` and 3` ends of Cmh-1 cDNA by PCR using primers 5`-CGGCTCGAGGCTAAGCCAGACCGGTCCTCG and 5`-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 23-residue peptide (MGSSHHHHHHSSGLVPRGSHMLE, where LVPRGS represents a thrombin cleavage site) fused in-frame to the N terminus of CMH-1 starting at Ala, 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-beta-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 µg/ml leupeptin (Buffer A). Lysates were cleared by centrifugation at 100,000 times 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 S-labeled IL-1beta precursor and a truncated form of 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 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 ICE-like protein and 3`-untranslated region. Using rapid amplification of cDNA ends-PCR, we cloned the 5` end of the full-length cDNA from human spleen poly(A) mRNA and ligated it to the 3` cDNA fragment from the clone 72778 to generate the full-length 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 and His 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 the P1 position are identical with those found in ICE, including Arg and Arg, corresponding to ICE Arg and Arg, 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.


Figure 1: A, predicted amino acid sequence of the human Cmh-1. The conserved QACRG motif is underlined, and the catalytic residues His and Cys 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 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, ICEIII, 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.



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 pro-sequence 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 pcDLSRalpha (Takebe et al., 1988). To facilitate the detection of the expressed proteins by immunoblotting, a T7-epitope tag (MASMTGGQQMG, 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.


Figure 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*). B, induction of apoptosis in transfected cells by CMH-1 as evidenced by internucleosomal DNA fragmentation. COS cells were mock-transfected (lane 1), transfected with the tagged wild type CMH-1 (lane 2) or the mutant CMH-1 (lane 3), and harvested 36 h post-transfection. Chromosomal DNA was extracted from these cells and analyzed on a 1.8% agarose gel containing ethidium bromide. DNA size markers in kilobases are shown in lane MW.



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 (Nicholson et al., 1995; 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 ICE-like 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-Gln) 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 (Fig. 3). The N termini of the 12-kDa and 11-kDa polypeptides start at Ser and Ala 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-Ser and to a lesser extent at Asp-Ala. We propose that the 22-kDa and 12-kDa peptides represent the two subunits of the CMH-1 protease.


Figure 3: Purification of recombinant (His)(6)-tagged CMH-1. (His)(6)-tagged CMH-1(Ala-Gln) was expressed and purified from E. coli soluble fractions by Ni-affinity chromatography as described under ``Materials and Methods.'' Four µl (2 µg 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 and Ala, respectively.



CMH-1 Is a CPP32-like Cysteine Protease That Cleaves PARP but Not IL-1 beta Precursor

We further analyzed the protease activity of the purified CMH-1 protein using S-labeled PARP or IL-1beta 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-1beta 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 against IL-1beta precursor (Gu et al., 1995b). This cleavage activity of CMH-1 was inhibited by the tetrapeptide inhibitor DEVD-CHO (Nicholson et al., 1995; 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 µg/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.


Figure 4: CMH-1 is a cysteine protease that cleaves PARP in vitro.A, CMH-1 specifically cleaves PARP but not IL-1beta precursor. Left panel, S-labeled PARP(T) prepared by in vitro transcription/translation (Gu et al., 1995) was incubated for 30 min at 37 °C with approximately 3 nM purified CMH-1 or buffer in the presence or absence of a 0.5 µM concentration of the tetrapeptide aldehyde inhibitor DEVD-CHO as described under ``Materials and Methods.'' Cleavage products were analyzed by SDS-PAGE and fluorography. PARP(T) denotes the uncleaved truncated form of PARP, and PARP* indicates the primary cleavage products (32 and 12 kDa). Right panel, S-labeled IL-1beta precursor was incubated alone or with purified recombinant ICE (3 nM) or CMH-1 (3 nM) proteins for 30 min at 37 °C and analyzed by SDS-PAGE and fluorography. Positions of IL-1beta precursor (pIL-1beta) and the cleavage product (IL-1beta) are indicated. B, effects of protease inhibitors on the PARP-cleavage activity of CMH-1 in vitro. Purified CMH-1 (3 nM) was incubated with S-PARP for 10 min at 37 °C in the presence of 0.5 µM DEVD-CHO, 1 µM YVAD-CHO, 16 µg/ml crmA, 5 mMN-ethylmaleimide, 5 mM iodoacetamide, 100 µML-1-chloro-3-(4-tosylamido)-7-amino-2-heptanone (TLCK), 100 µML-1-chloro-3-(4-tosylamido)-4-phenyl-2-heptanone (TPCK), 2 µg/ml aprotinin, or 5 mM EDTA as indicated. Cleavage products were analyzed by SDS-PAGE and fluorography.



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 (Nicholson et al., 1995; 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.


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(TM)/EMBL Data Bank with accession number(s) U40281[GenBank].

§
To whom correspondence should be addressed. Tel.: 617-576-3111; Fax: 617-499-7315; :su{at}vpharm.com.

(^1)
The abbreviations used are: IL-1beta, interleukin 1beta; ICE, interleukin 1beta converting enzyme; PARP, poly(ADP-ribose) polymerase; PCR, polymerase chain reaction; kb, kilobase(s); PAGE, polyacrylamide gel electrophoresis; YVAD-CHO, Ac-Tyr-Val-Ala-Asp-aldehyde; DEVD-CHO, Ac-Asp-Glu-Val-Asp-aldehyde; CMH-1, CPP32/Mch2 homolog 1; EST, expressed sequence tag.

(^2)
Y. Gu, J. A. Lippke, C. Sarnecki, and M. S.-S. Su, unpublished results.


ACKNOWLEDGEMENTS

We acknowledge the use of the NCBI blast server and thank the IMAGE Consortium for providing the initial cDNA clone. We thank Dr. David Pickup (Duke University) for providing the crmA clone. We would like to thank Brett O'Hare for DNA sequencing and oligonucleotide synthesis and Mark Fleming for the peptide N terminus sequence analysis. We thank Drs. M. Mullican and S. Harbeson for tetrapeptide aldehydes. We are grateful to Drs. J. Boger, V. Sato, D. Livingston, and P. McCaffrey for their comments on the manuscript.


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