CPP32, a Novel Human Apoptotic Protein with Homology to Caenorhabditis elegans Cell Death Protein Ced-3 and Mammalian Interleukin-lP-converting Enzyme*

We have cloned a novel apoptotic gene from human Jurkat T-lymphocytes. The new gene encodes a 32-kDa putative cysteine protease (CPP32) with significant homology to Caenorhabditis elegans cell death protein Ced-3, mammalian interleukin-lp-converting enzyme (ICE), and the product of the mouse nedd2 gene. The CPP32 transcript is highly expressed and most abun- dant in cell lines of lymphocytic origin. Overexpression of CPP32 or ICE in Sf9 insect cells resulted in apoptosis. In addition, coexpression of recombinant p20 and pll derived from the parental full-length CPP32 sequence resulted in apoptosis in Sf9 cells. Our data suggest that similar to ICE, CPP32 is made of two subunits, p20 and pll, which form the active CPP32 complex. The apo- ptotic activity of CPP32 and its high expression in lymphocytes suggest that CPP32 is an important mediator of apoptosis in the immune system.

and structural homology (5, 6). ICE and its homologs are classified as a new class of cysteine proteases because they are different in sequence, structure, and substrate specificity from other known cysteine proteases (7-9). All three proteins have been shown to induce apoptosis when overexpressed in different cell types (4, 5). This apoptosis was inhibited by coexpression of the antiapoptotic protein Bc12 (4,5). Expression ofcrmA, a poxvirus-specific inhibitor of ICE (lo), inhibited ICE-induced apoptosis in fibroblasts (4). crmA also inhibited apoptosis of ganglion neuronal cells when introduced into these cells (11). Because crmA could inhibit other ICE homologs, it is not yet clear whether ICE or other members of this new class of cysteine proteases are involved in neuronal apoptosis.
Because of the importance of ICE-related cysteine proteases in apoptosis, we were interested in identifying other members of this important class of cysteine proteases. In this report we describe the cloning, expression, and partial characterization of a novel putative cysteine protease called CPP32. We show that CPP32 is related to Ced-3, ICE, and Nedd2 proteins and can cause apoptosis when expressed in Sf9 insect cells.

MATERIALS AND METHODS
Cloning of CPP32"Searching the GenBank expressed sequence tags (12) for sequences with homology to Ced-3 or ICE, we identified a human sequence of 399 bp (GenBank accession number T10341) with significant homology to Ced-3 and ICE. This sequence was then cloned by a combination of reverse transcription and polymerase chain reaction techniques (RT-PCR). Reverse transcription was performed on poly(A)+ RNA from the human T-lymphocyte cell line Jurkat using a 17-mer poly(T) primer and Moloney murine leukemia virus reverse transcriptase. The reverse transcription product was then used as a template for PCR using two specific primers, hcedl and hced2, derived hcedl, CAGAGGGGATCG'M'GTAGAAG hced2, GTTGCCACCTTTCG-from the GenBank T10341 sequence. Primer sequences were as follows: GTTAACC. The amplified DNA was blunt-ended with T4 DNA polymerase, phosphorylated with T4 polynucleotide kinase, and cloned into a SmaI-cut pBluescript I1 K S ' vector (Stratagene). The cloned cDNA was sequenced with T3 and T7 sequencing primers and found to match exactly the T10341 cDNA. This cDNAwas then excised from the vector, radiolabeled, and used to screen a Jurkat A Uni-ZAPTM XR cDNAlibrary constructed in our laboratory. Twenty X clones were selected, purified, and then rescued from the A Zap phage clones into the pBluescript I1 SKplasmid vector. The plasmid clones were characterized by restriction enzyme analysis and nucleotide sequencing.
Construction of Plasmids, Dansfer Vectors, and Recombinant Baculouiruses-The CPP32 cDNA was excised from the pBluescript I1 SKvector as a 1-kilobase cDNA fragment with EcoRI and PstI restriction enzymes and subcloned into an EcoRIIPstI-cut pVL1393 to generate the recombinant transfer vector pVL-CPP32. The cDNA fragment contains 57 bp of untranslated 5' sequence, the entire open reading frame, and 107 bp of untranslated 3' sequence. The cDNAs for the p20 and p l l subunits of CPP32 were generated by PCR using synthetic primers (p2OATG and p2OTGA for p20; pllATG and PllTAA for p l l ) and pVL-CPP32 as a template. Primer sequences were as follows: pPOATG, ATGGAGAACACTGAAAACTCAG, pSOTGA, GTCATCAT-CAACACCTCAGTCT pllATG, ATGGCGTGTCATAAAATACCAG; pllTAA, CCAACCAACCATITCTITAGTG.
The amplified DNA fragments were blunt-ended, phosphorylated, and then cloned in the SmaI site of p a 1 3 9 3 transfer vector under the polyhedrin promoter and designated as pVL-p20 and pVL-pll. Human ICE full-length cDNA was obtained by RT-PCR from Jurkat T-lymphocyte RNA and cloned into the BamHIIEcoRI sites of PVL1393.' All recombinant transfer vectors were then used to generate recombinant baculoviruses as described previously (13, 14). E. S. Alnemri, T. Fernandes-Alnemri, and G. Litwack, submitted for publication.

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FIG. 1. Nucleotide and predicted CPP32 isoform-a. A cDNA encoding a amino acid sequence of the human full-length human CPP32 was isolated from a Jurkat T-lymphocyte h Uni-ZAPTM XR cDNA library. The length of this cDNA is 2646 bp excluding the first 14 bp that are derived from the EcoRI cloning adaptor. The size of this cDNA is consistent with the observed size of the CPP32 mRNA (-2700 bp) by Northern blot analysis (Fig. 3). The single long open reading frame begins with an ATG at position 225 (Met-1) and terminates with TAA stop codon (asterisk) at position 1056 (His-277). An in-frame nonsense codon (TAG) in the 5'-noncoding region and seven consensus polyadenylation sites in the 3'noncoding region are underlined. The N termini of putative p20 and p l l sub-putative active site cysteine is boxed. The units of CPP32 are denoted by bold arrows. The putative aspartate cleavage sites between the p20 and p l l subunits are circled. The line above the sequence indicates the deleted sequence in CPP32 isoform-p. The two single base pair substitutions in isoform-p at nucleotide 395 and 794 are indicated above the sequence.
1 W V L T K G G C 9 C W I G G G G T G C T A T T G n ; A U ; C G G T T G T A W U L 102

307 A C~~~T A T C C C T~A T~~; G A T T A T C C~~T~T T T A~T A T A A T~T T~T~T A A~T T T~T A A A A~-~
G 408 Northern Blot Analysis of CPP32 mRNA--RNA samples (15 pgl sample) from different human cell lines were fractionated by electrophoresis in a 1.2% agarose gel containing 1.2% formaldehyde. The gel was blotted onto Duralon-W nylon membrane, and the membrane was then W cross-linked, prehybridized, and hybridized to 32P-labeled DNA probe at 42 "C. After hybridization the membrane was washed and exposed to x-ray film.

RESULTS AND DISCUSSION
Cloning of CPP32-One way to identify mammalian genes involved in apoptosis is to search for and isolate genes with homology to C. elegans ced-3 gene or mammalian ICE gene. Using the DNA sequence encoding the active site region of mammalian ICE or C. elegans Ced-3 to search the GenBank expressed sequence tags data base (121, we identified one short sequence under accession number T10341. Based on this sequence, a probe was prepared to screen a human Jurkat Tlymphocyte cDNA library. This resulted in the isolation of several cDNA clones. The sequence of one of these clones is shown in Fig. 1. This cDNA, termed isoform-a, contains a n open reading frame of 831 bp, which encodes a 277-amino acid protein with a predicted molecular mass of -32 kDa. The initiator methionine at nucleotide 225 conforms to the consensus Kozak translation initiation sequence (15) and is preceded by a n inframe termination codon at nucleotide 135. A second cDNA clone was also sequenced and found to contain a deletion of nucleotides 43-209 of the 5'-noncoding sequence. This resulted in a short 5'-noncoding region and the generation of a n inframe termination codon at position -21 relative to the ATG start codon. This deletion is probably due to alternative splicing of the parental CPP32 mRNA. This cDNA, termed as CPP32 isoform-0, also contains two base pair substitutions at nucleotide 395 and nucleotide 794. The substitution at nucleotide 395 did not alter the amino acid at that position. However, the substitution at nucleotide 794 changed the aspartate to glutamate at that position. These base pair substitutions could be a result of polymorphism in the CPP32 gene. A third cDNA was identified, which is similar in length to isoform-a cDNA, has no deletion in the 5'-noncoding region but contains the same two base pair substitutions found in isoform-p cDNA.
CPP32 Is a Novel Putative Cysteine Protease-A search of the SWISS-PROT data base revealed that the predicted CPP32 protein sequence is similar to the C. elegans Ced-3 protein (61, mammalian ICE, and mouse Nedd2 protein (5,7, 16) ( Fig. 2A). CPP32 is most similar to Ced-3: CPP32 shows 35% identity (58% similarity) with Ced-3,30% identity (53% similarity) with human ICE, and 30% identity (53% similarity) with mouse Nedd2 protein. Ced-3, ICE, or Nedd2 are less than 31% identical with one another. This suggests that CPP32 is more related to Ced-3 than to ICE or Nedd2 and could have a similar function to Ced-3. However, unlike Ced-3, ICE, or Nedd2, CPP32 lacks the long N terminus, which is most probably not required for activity. The highest degree of homology between the four proteins lies within and around the region that contains the highly conserved pentapeptide QACRG ( Fig. 2A). This pentapeptide contains the active site Cys-285 of active human ICE (7-9). In addition, ICE residues kg-179, His-237, and Arg-341, which are involved in substrate binding and catalysis (8,9) are conserved in all four proteins. The side chains of ICE residues Arg-179 and Arg-341 recognize the aspartate at P1 position of the substrate by direct charge-charge interaction (8,9). Based on these observations CPP32 could have the same substrate requirement as ICE, that is, it requires Asp in the P1 position. Whether CPP32 can cleave interleukin-lp remains to be established.
ICE is classified as a cysteine protease (7). The active form of ICE is generated after proteolytic cleavage of the ICE proenzyme p45 to generate two subunits with molecular mass values of 20 and 10 kDa, known as p20 and p10 subunits (Fig. 2 B ) (7). Two p20/p10 heterodimers associate with each other to form the active ICE tetrameric complex (8,9). Structural analysis of CPP32 based on its homology to ICE revealed that Asp-175 and Asp-181 are potential cleavage sites in CPP32. Proteolytic cleavage at these two sites would generate two polypeptides with molecular mass values of 20 and 11 kDa (Fig. 2B). These two polypeptides would be equivalent to the p20 and p10 subunits of ICE. As we have shown below, recombinant CPP32-p20 and CPP32-pll subunits can associate with each other to form active CPP32 complex. Although the N terminus of CPP32 contains three potential aspartate cleavage sites (Asp-9, Asp-28, and Asp-341, which are located outside the high homology re- The four known aspartate cleavage sites in ICE and the two potential aspartate cleavage sites in CPP32 are indicated. The two subunits of active ICE (p20 and p10) and the two putative subunits of active CPP32 (p20 and pll) are indicated by solid arrows.

S C~Y D T S L P F S V C~S C P P H K Q L R L S T~A~~~~~D N G D G P P C L L V K P C T P E F Y Q A . .
.... gion, we do not yet know whether active CPP32 is cleaved at these sites as well. Expression of CPP32 Tkanscript in Different Human Cell Lines-We examined the potential tissue distribution of CPP32 mRNAusing different human tumor cell lines. As shown in Fig.  3, CPP32 mRNA is detectable in all cell lines examined. Interestingly, CPP32 mRNA was highly expressed in cell lines of hematopoietic lineage such as lymphocytes and promyelocytes (Fig. 3, lanes 1-9). High expression was also observed in cell lines of brain and embryonic origins such asA173 (lane 14) and 293 (lane 151, respectively. In contrast, ICE mRNA was not detectable by Northern blot analysis in any of the lymphocyte cell lines described in Fig. 3. ICE mRNA was only detectable by RT-PCR in these cell lines (data not shown). A previous study has shown that peripheral blood Tand B-lymphocytes express substantially small amounts of ICE mRNA compared with peripheral blood monocytes and neutrophils (16). The high level of expression of CPP32 in cells of the immune system suggests that CPP32 could play an important role in regulation of apoptosis in the immune system. The immune system is one in which apoptosis occurs most frequently in response to many different stimuli.
Expression of CPP32 in Sfs Cells Induces Apoptosis-The baculovirus gene product p35 is an antiapoptotic protein that protects insect cells against baculovirus-induced apoptosis (17) cells were infected with a recombinant baculovirus expressing CPP32 under the polyhedrin promoter. Cells were also infected with the wild type virus and the recombinant ICE baculovirus as controls. Morphological, biochemical, and viability analyses revealed that cells infected with ICE or CPP32, but not with the wild type virus, had several characteristic signs of apoptosis including cytoplasmic membrane blebbing, nuclear fragmentation and condensation, and internucleosomal DNA cleavage (Fig. 4, lanes 2 and 3 ) . The viability of cells infected with ICE or CPP32 baculoviruses, was 50-8076 less than cells infected with the wild type virus a t 48 h post-infection.
The ability of CPP32 to induce apoptosis in Sf9 cells provided us with an opportunity to test whether active CPP32 is generated by proteolytic cleavage to p20 and p l l subunits (Fig. 2B). Recombinant p20 or p l l were expressed either individually or together in Sf9 cells using recombinant baculoviruses encoding the p20 or p l l subunits of CPP32. Cells were then examined for signs of apoptosis. Only cells coinfected with the p20 and p l l baculoviruses showed characteristic cytoplasmic membrane blebbing 24-48 h post-infection. This was associated with loss of viability and internucleosomal DNA cleavage (Fig. 4, lane 6). No signs of apoptosis were observed in cells expressing either p20 or p l l individually (lanes 4 and 5). Similar results also were obtained with recombinant p10 and p20 subunits of ICE.2 We also tested whether a heterodimer can be formed by coexpression of ICE-p2O and CPP32-pll or vice versa (i.e. CPP32-p20 and ICE-plO), and whether they could induce apoptosis in Sf9 cells.
These combinations were unable to cause apoptosis in Sf9 cells (data not shown). These observations suggest that a p20 subunit derived from ICE is unable to heterodimerize with the p l l from CPP32 and vice versa. Another possibility is that ICE-p20/ CPP32-pll or CPP32-p20/ICE-p10 heterodimers are inactive.
In conclusion, we have cloned a new member of the cysteine protease family. The structure of this new protein is similar to that of ICE, in that it is made up of two subunits derived from one precursor proenzyme. Similar to ICE and the other members of this family such as Ced-3 and Nedd2, this protein could be a mediator of apoptosis in human tissues, especially in cells of the immune system, where we have found it to be highly expressed. The cloning and characterization of CPP32 will aid the efforts to understand the function and regulation of this class of cysteine proteases and their participation in the molecular mechanism of apoptosis.